cyclic-gmp and linsidomine

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cell Line; Cells, Cultured; Cyclic GMP; Cytokines; DNA; DNA Damage; Free Radicals; Insulin; Insulin Secretion; Interleukin-1; Islets of Langerhans; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Rats; Vasodilator Agents

We compared the effects of L-arginine (L-ARG), the precursor of endogenous NO, on platelet aggregation and thromboxane A2 formation in vivo and in vitro. Human platelet-rich plasma (PRP) was anticoagulated with citrate (which decreases extracellular Ca2+) or with recombinant hirudin (which does not affect extracellular Ca2+). Two groups of 10 healthy male volunteers received intravenous infusions of L-ARG (30 g or 6 g, 30 min) or placebo. Blood was collected immediately before and at the end of the infusions for aggregation by ADP or collagen. Infusion of L-ARG inhibited ADP-induced aggregation in PRP anticoagulated with citrate by 37.5+/-6.3% (P < 0.05). In PRP anticoagulated with hirudin, aggregation was inhibited by 33.6+/-16.0% (P < 0.05). L-ARG infusion also inhibited platelet TXB2 formation and slightly, but not significantly decreased the urinary excretion rate of 2,3-dinor-TXB2; cGMP concentrations in PRP were significantly elevated during L-arginine infusion. In vitro preincubation with L-ARG (10 microM-2.5 mM) inhibited platelet aggregation in PRP anticoagulated with rhirudin, but not citrate. This effect was stereospecific for L-arginine, as D-arginine had no effect. It was dependent upon NO synthase activity, as indicated by increased cGMP levels in PRP. Moreover, both the NOS inhibitor L-NMMA and the inhibitor of soluble guanylyl cyclase ODQ antagonized the effects of L-ARG. Haemoglobin, an extracellular scavenger of NO, partly antagonized the antiplatelet effects of L-ARG. 8-Br-cyclic GMP and the exogenous NO donor linsidomine inhibited aggregation in PRP anticoagulated with citrate or r-hirudin. The inhibitory effects of L-ARG on platelet aggregation in vitro were paralleled by increased cyclic GMP levels; L-ARG also inhibited platelet TXB2 formation in PRP anticoagulated with r-hirudin, but not citrate. We conclude that the L-arginine/NO pathway is present in human platelets as a Ca2+-dependent anti-aggregatory pathway. In vivo the formation of NO from L-ARG by endothelial cells may contribute to the platelet-inhibitory effects of L-ARG. NO-releasing compounds like linsidomine inhibit platelet aggregation in vitro independent of extracellular Ca2+.

Topics: Adult; Antithrombins; Arginine; Blood Platelets; Cyclic GMP; Enzyme Inhibitors; Humans; In Vitro Techniques; Infusions, Intravenous; Male; Molsidomine; Nitric Oxide Synthase; Platelet Aggregation; Platelet Aggregation Inhibitors; Thromboxane A2; Thromboxane B2; Thromboxanes

We compared isosorbidedinitrate (ISDN) and 3-morpholinosydnonimine (SIN-1) as dilators of epicardial coronary arteries and inhibitors of ex vivo platelet aggregation in 23 patients referred for diagnostic coronary arteriography. After completion of the diagnostic study, the patient received graded intravenous (i.v.) infusions (0.5, 1.0, and 1.5 micrograms/kg/min) of either SIN-1 (n = 11) or ISDN (n = 12). Diameters of left anterior descending (LAD) and left ramus circumflex (RCX) coronary arteries were assessed by quantitative digital coronary arteriography before and 5 min after each infusion was started. SIN-1 required an infusion rate of 1.0 micrograms/kg/min to cause dilatation of proximal and middle segments of LAD and RCX. The highest infusion rate caused a modest decrease in mean arterial blood pressure (MAP). In these aspects, SIN-1 was equivalent to ISDN. In addition, blood was collected immediately before treatment and after infusion of the highest dose of ISDN or SIN-1. The sensitivity of platelet-rich plasma (PRP) to ADP and the thromboxane A2 (TXA2) mimetic U-46619 was determined in an aggregometer. The lesser responses to threshold concentrations of ADP and U-46619 and the slight shift in both concentration-response curves indicated that platelets of SIN-1-treated patients were slightly less sensitive to both stimuli as compared with platelets of ISDN-treated subjects. These ex vivo results suggest that SIN-1 may be superior to ISDN as an inhibitor of platelet activation.

Topics: Angina Pectoris; Blood Pressure; Coronary Vessels; Cyclic AMP; Cyclic GMP; Double-Blind Method; Female; Humans; Infusions, Intravenous; Isosorbide Dinitrate; Male; Middle Aged; Molsidomine; Platelet Aggregation; Platelet Aggregation Inhibitors; Vasodilator Agents

Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B-type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO-cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO(-) ) generation and PKG-1α nitration, while PKG-1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and mimicked by the ONOO(-) donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Peripheral lung extracts from 4-week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24 h (with exogenous activation of cGMP) revealed increased ONOO(-) levels, elevated PKG-1α nitration, and decreased kinase activity without changes in PKG-1α protein levels. However, in Shunt lambs treated with L-arginine or lambs administered polyethylene glycol conjugated-SOD (PEG-SOD) during inhaled NO exposure, ONOO(-) and PKG-1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG-1α nitration and subsequent attenuation of activity.

Topics: Administration, Inhalation; Animals; Animals, Newborn; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Free Radical Scavengers; Hypertension, Pulmonary; Metalloporphyrins; Molsidomine; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitric Oxide; Nitric Oxide Donors; Peroxynitrous Acid; Polyethylene Glycols; Protein Processing, Post-Translational; Pulmonary Artery; Pulmonary Circulation; Second Messenger Systems; Sheep; Spermine; Superoxide Dismutase; Vasodilation; Vasodilator Agents

Three different NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine hydrochloride (SIN-1) were used in order to investigate mechanisms of platelet inhibition through cGMP-dependent and -independent pathways both in human and rat. To this purpose, we also evaluated to what extent cGMP-independent pathways were related with the entity of NO release from each drug. SNP, GSNO and SIN-1 (100 μM) effects on platelet aggregation, in the presence or absence of a soluble guanylate cyclase inhibitor (ODQ), on fibrinogen receptor (α(IIb)β(3)) binding to specific antibody (PAC-1), and on the entity of NO release from NO donors in human and rat platelet rich plasma (PRP) were measured. Inhibition of platelet aggregation (induced by ADP) resulted to be greater in human than in rat. GSNO was the most powerful inhibitor (IC(50) values, μM): (a) in human, GSNO=0.52±0.09, SNP=2.83 ± 0.53, SIN-1=2.98 ± 1.06; (b) in rat, GSNO = 28.4 ± 6.9, SNP = 265 ± 73, SIN-1=108 ± 85. GSNO action in both species was mediated by cGMP-independent mechanisms and characterized by the highest NO release in PRP. SIN-1 and SNP displayed mixed mechanisms of inhibition of platelet aggregation (cGMP-dependent and independent), except for SIN-1 in rat (cGMP-dependent), and respectively lower or nearly absent NO delivery. Conversely, all NO-donors prevalently inhibited PAC-1 binding to α(IIb)β(3) through cGMP-dependent pathways. A modest relationship between NO release from NO donors and cGMP-independent responses was found. Interestingly, the species difference in NO release from GSNO and inhibition by cGMP-independent mechanism was respectively attributed to S-nitrosylation of non-essential and essential protein SH groups.

Topics: Animals; Blood Platelets; Cyclic GMP; Humans; Male; Molsidomine; Nitric Oxide Donors; Nitroprusside; Platelet Aggregation; Platelet Aggregation Inhibitors; Protein Conformation; Rats; Rats, Sprague-Dawley; Receptors, Fibrinogen; S-Nitrosoglutathione

To elucidate the mechanism by which local delivery of 3-morpholino-sydnonimine (SIN-1) affects intimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA).. Porcine coronary arteries were treated with PTCA and immediately afterwards locally treated for 5 minutes, with a selective cytosolic guanylate cyclase inhibitor, 1 H-(1,2,4)oxadiazole(4,3-alpha)quinoxaline-1-one (ODQ) + SIN-1 or only SIN-1 using a drug delivery-balloon. Arteries were angiographically depicted, morphologically evaluated and analyzed after one and eight weeks for actin, myosin and intermediate filaments (IF) and nitric oxide synthase (NOS) contents.. Luminal diameter after PCI in arteries treated with SIN-1 alone and corrected for age-growth was significantly larger as compared to ODQ + SIN-1 or to controls (p < 0.01). IF/actin ratio after one week in SIN-1 treated segments was not different compared to untreated segments, but was significantly reduced compared to ODQ + SIN-1 treated vessels (p < 0.05). Expression of endothelial NADPH diaphorase activity was significantly lower in untreated segments and in SIN-1 treated segments compared to controls and SIN-1 + ODQ treated arteries (p < 0.01). Restenosis index (p < 0.01) and intimal hyperplasia (p < 0.01) were significantly reduced while the residual lumen was increased (p < 0.01) in SIN-1 segments compared to controls and ODQ + SIN-1 treated vessels.. After PTCA local delivery of high concentrations of the NO donor SIN-1 for 5 minutes inhibited injury induced neointimal hyperplasia. This favorable effect was abolished by inhibition of guanylyl cyclase indicating mediation of a cyclic guanosine 3',5'-monophosphate (cGMP)-dependent pathway. The momentary events at the time of injury play crucial role in the ensuring development of intimal hyperplasia.

Topics: Actins; Analysis of Variance; Angioplasty, Balloon, Coronary; Animals; Coronary Angiography; Coronary Restenosis; Coronary Vessels; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Guanylate Cyclase; Hyperplasia; Intermediate Filaments; Molsidomine; Myosins; NAD(P)H Dehydrogenase (Quinone); NADP; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Oxadiazoles; Quinoxalines; Signal Transduction; Sus scrofa; Time Factors; Tunica Intima

Nitric oxide (NO) is a potent nonadrenergic, noncholinergic mediator of gastrointestinal smooth muscle. We aimed to investigate the effects of new NO/cyclic guanosine monophosphate (cGMP) pathway-affecting agents at the sheep sphincter of Oddi (SO) in vitro.. Sheep SO rings were mounted in organ baths and tested for isometric tension and cGMP levels in response to 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene; 3-morpholinosydnonimine hydrochloride (SIN-1); and BAY 41-2272 in the presence and absence of 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ).. 3,3-bis(Aminoethyl)-1-hydroxy-2-oxo-1-triazene; SIN-1; and BAY 41-2272 relaxed SO rings in a concentration-dependent manner. These relaxations were significantly decreased in the presence of ODQ (P < 0.05). All agents significantly increased the cGMP levels compared with the control group (P < 0.05). The increased cGMP levels in the 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene- and BAY 41-2272-treated groups were significantly different from both control and carbachol groups (P < 0.05), whereas the increase in the SIN-1 group was significantly different from all groups (P < 0.05). The cGMP levels were significantly lower in the presence of ODQ compared with its absence (P < 0.05).. The relaxation of SO rings by these agents may be via increasing the levels of cGMP. The additional increase produced by SIN-1 may be the combined effects of NO generation and activation of guanylyl cyclase.

Topics: Animals; Carbachol; Cyclic GMP; Dose-Response Relationship, Drug; Guanylate Cyclase; In Vitro Techniques; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Oxadiazoles; Pyrazoles; Pyridines; Quinoxalines; Sheep; Sphincter of Oddi; Triazenes

The nitric oxide synthase (NOS)/nitric oxide (NO) system integrates cellular biochemical machinery and energetics. In heart microenvironment, dynamic NO behaviour depends upon the presence of superoxide anions, haemoglobin (Hb), and myoglobin (Mb), being hemoproteins are major players disarming NO bioactivity. The Antarctic icefish, which lack Hb and, in some species, also cardiac Mb, represent a unique model for exploring Hb and Mb impact on NOS/NO function. We report in the (Hb(-)/Mb(-)) icefish Chaenocephalus aceratus the presence of cardiac NOSs activity (NADPH-diaphorase) and endothelial NOS (eNOS)/inducible NOS (iNOS) zonal immuno-localization in the myocardium. eNOS is localized on endocardium and, to a lesser extent, in myocardiocytes, while iNOS is localized exclusively in myocardiocytes. Confronting eNOS and iNOS expression in Trematomus bernacchii (Hb(+)/Mb(+)), C. hamatus (Hb(-)/Mb(+)) and C. aceratus (Hb(-)/Mb(-)) is evident a lower expression in the Mb-less icefish. NO signaling was analyzed using isolated working heart preparations. In T. bernacchii, L-arginine and exogenous (SIN-1) NO donor dose-dependently decreased stroke volume, indicating decreased inotropism. L-arginine-induced inotropism was NOSs-dependent, being abolished by NOSs-inhibitor NG-monomethyl-L-arginine (L-NMMA). A SIN-1-induced negative inotropism was found in presence of SOD. NOS inhibition by L-N5-N-iminoethyl-L-ornithine (L-NIO) and L-NMMA confirmed the NO-mediated negative inotropic influence on cardiac performance. In contrast, in C. aceratus, L-arginine elicited a positive inotropism. SIN-1 induced a negative inotropism, which disappeared in presence of SOD, indicating peroxynitrite involvement. Cardiac performance was unaffected by L-NIO and L-NIL. NO signaling acted via a cGMP-independent mechanism. This high conservation degree of NOS localization pattern and signaling highlights its importance for cardiac biology.

Topics: Animals; Antarctic Regions; Arginine; Blotting, Western; Cyclic GMP; Endocardium; Female; Fluorescent Antibody Technique; Heart Ventricles; Hemodynamics; Male; Molsidomine; Myocardium; NADPH Dehydrogenase; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; omega-N-Methylarginine; Perciformes; Signal Transduction; Stroke Volume

The nitric oxide (NO)-active drugs influence on the bioelectric activity of neurons of the pars reticulata of the substantia nigra was studied in urethane-anesthetized rats. A first group of animals was treated with 7-nitro-indazole (7-NI), a preferential inhibitor of neuronal NO synthase. In a second group of rats, electrophysiological recordings were coupled with microiontophoretic administration of Nomega-nitro-L-arginine methyl ester (L-NAME, a NO synthase inhibitor), 3-morpholino-sydnonimin-hydrocloride (SIN-1, a NO donor) and 8-Br-cGMP (a cell-permeable analogue of cGMP, the main second-messenger of NO neurotransmission). 7-NI and L-NAME caused a statistically significant decrease in the firing rate of most of the responsive cells, while application of SIN-1 and 8-Br-CGMP induced statistically significant excitatory effects. The results suggest a NO mediated excitatory modulation of the SNr neurons activity with a possible involvement of the cGMP pathway.

Topics: Action Potentials; Animals; Cyclic GMP; Down-Regulation; Enzyme Inhibitors; Iontophoresis; Male; Molsidomine; Neural Inhibition; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Rats; Rats, Wistar; Signal Transduction; Substantia Nigra; Up-Regulation

The cell signaling cascades that mediate pigment movements in crustacean chromatophores are not yet well established, although Ca(2+) and cyclic nucleotide second messengers are involved. Here, we examine the participation of cyclic guanosine monophosphate (cGMP) in pigment aggregation triggered by red pigment concentrating hormone (RPCH) in the red ovarian chromatophores of freshwater shrimp. In Ca(2+)-containing (5.5 mmol l(-1)) saline, 10 micromol l(-1) dibutyryl cGMP alone produced complete pigment aggregation with the same time course ( approximately 20 min) and peak velocity ( approximately 17 microm/min) as 10(-8) mol l(-1) RPCH; however, in Ca(2+)-free saline (9 x 10(-11) mol l(-1) Ca(2+)), db-cGMP was without effect. The soluble guanylyl cyclase (GC-S) activators sodium nitroprusside (SNP, 0.5 micromol l(-1)) and 3-morpholinosydnonimine (SIN-1, 100 micromol l(-1)) induced moderate aggregation by themselves ( approximately 35%-40%) but did not affect RPCH-triggered aggregation. The GC-S inhibitors zinc protoporphyrin IX (ZnPP-XI, 30 micromol l(-1)) and 6-anilino-5,8-quinolinedione (LY83583, 10 micromol l(-1)) partially inhibited RPCH-triggered aggregation by approximately 35%. Escherichia coli heat-stable enterotoxin (STa, 1 micromol l(-1)), a membrane-receptor guanylyl cyclase stimulator, did not induce or affect RPCH-triggered aggregation. We propose that the binding of RPCH to an unknown membrane-receptor type activates a Ca(2+)-dependent signaling cascade coupled via cytosolic guanylyl cyclase and cGMP to protein kinase G-phosphorylated proteins that regulate aggregation-associated, cytoskeletal molecular motor activity. This is a further example of a cGMP signaling cascade mediating the effect of a crustacean X-organ neurosecretory peptide.

Topics: Aminoquinolines; Animals; Brazil; Chromatophores; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Fresh Water; Guanylate Cyclase; Molsidomine; Nitroprusside; Palaemonidae; Pigments, Biological; Protoporphyrins; Signal Transduction

Impaired responsiveness to epinephrine and other catecholamines (CA) were previously reported in platelets of 20 approximately 30% healthy Japanese and Koreans. In the present study, the possible mechanisms of different responsiveness to CA in platelets of CA hypo-responders (CA-HY) and CA good-responders (CA-GR) were investigated. Increased platelet-leukocyte conjugate (PLC) formations were observed with whole blood of CA-GR than with that of CA-HY in both non-stimulated [mean fluorescence intensity (MFI) values: 1.33 +/- 0.26 vs. 1.16 +/- 0.19] and ADP (MFI: 5.54 +/- 3.46 vs. 2.15 +/- 1.13) or TRAP (MFI: 5.11 +/- 2.32 vs. 3.38 +/- 1.47) activated states. The platelets of CA-GR, when stimulated with ADP (10 microM), released approximately twice the amount of ATP than those of CA-HY (0.88 +/- 0.65 and 0.45 +/- 0.36 nmole, respectively). Nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels were significantly higher in non-stimulated PRP of CA-HY than in that of CA-GR (70.3 +/- 24.1 microM and 14.1 +/- 4.9 nM vs. 41.1 +/- 15.8 microM and 6.7 +/- 2.4 nM, respectively). The platelet-monocyte conjugation induced with either ADP or TRAP was significantly reduced in CA-GR with the addition of linsidomine, a NO donor, (MFI: 2.78 +/- 0.43 vs. 3.73 +/- 0.90, or 4.28 +/- 0.95 vs. 5.76 +/- 1.33, respectively). Moreover, the degree of platelet aggregation and the ATP secretion induced by epinephrine in CA-GR were significantly retarded with the addition of either linsidomine or 8-Bromo-cGMP (a cGMP analog) with more substantial effects on ATP release than aggregation. The results suggested that elevated NO and/or cGMP plasma levels may be responsible for the lower platelet aggregation and PLC formation observed in CA-HY than that in CA-GR.

Topics: Adenosine Triphosphate; Adult; Asian People; Blood Platelets; Catecholamines; Cyclic AMP; Cyclic GMP; Female; Humans; Leukocyte Common Antigens; Leukocytes; Male; Molsidomine; Nitrates; Nitric Oxide; Nitrites; Platelet Aggregation; Platelet Aggregation Inhibitors; Young Adult

The aim of this work was to investigate in the avascular heart of the frog Rana esculenta the influence of nitric oxide (NO) on ventricular systolic and diastolic functions by using a novel image analysis technique. The external volume variations of the whole ventricle were monitored during the heart cycle by video acquisition(visible light) and analysed by an appropriately developed software with a specific formula for irregular convex solids. The system, which measures the rate of volume changes and the ejection fraction, directly determined the volumetric behaviour of the working frog heart after stimulation or inhibition of NOS-NOcGMP pathway. End-diastolic volume (EDVext), end-systolic volume (ESVext), contraction and relaxation velocities (dV/dtsys and dV/dtdia, respectively), stroke volume (SV) and ejection fraction (EF), were measured before and after perfusion with NOS substrate (L-arginine), NO donor (SIN-1), cGMP analogue (8-Br-cGMP),NOS inhibitors (NG-monomethyl-L-arginine, L-NMMA; L-N(5)-(1-iminoethyl)-ornithine, L-NIO; 7-Nitroindazole,7-NI) and guanylyl cyclase inhibitor (ODQ). The results showed that NO reduces ventricular systolicfunction improving diastolic filling, while NOS inhibition increases contractility impairing ventricular filling capacity. The presence of activated eNOS (p-eNOS) was morphologically documented, further supporting that the mechanical activity of the ventricular pump in frog is influenced by a tonic release of NOS-generated NO.

Topics: Animals; Anura; Arginine; Cyclic GMP; Female; Heart Ventricles; In Vitro Techniques; Indazoles; Male; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; omega-N-Methylarginine; Perfusion; Serine; Software; Ventricular Function

The role of nitric oxide (NO)/cyclic GMP (cGMP) pathway in regulating detrusor smooth muscle (DSM) function is still to be elucidated. We have investigated the effects of NO donors and phosphodiesterase-5 (PDE5) inhibition on spontaneous excitations in DSM.. Multibundle DSM of the guinea-pig bladder generated spontaneous phasic contractions. The effects of sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), NO donors, 8-bromo-cyclicGMP (8-Br-cGMP), a cell-permeable cGMP analog and sildenafil, a PDE5 inhibitor on these contractions were examined. The effects of these agents on spontaneous action potentials were also studied using intracellular recording technique in single-bundle DSM.. SNP and SIN-1 enhanced spontaneous contractions in multibundle DSM and increased the frequency of spontaneous action potentials in single-bundle DSM. These excitatory effects were not antagonized by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor for guanylate cyclase, but were attenuated by cyclopiazonic acid (CPA), an inhibitor of sarco- and endoplasmic Ca ATPase (SERCA). 8-Br-cGMP invariably suppressed spontaneous contractility. Sildenafil inhibited spontaneous contractions in about 65% of multibundle DSM but had no effects on the remainder. In single-bundle DSM, sildenafil had no effect on spontaneous action potentials.. These results suggested that NO caused an enhancement of spontaneous contractions in DSM by accelerating spontaneous action potentials through cGMP-independent mechanisms, which may involve the Ca release from intracellular stores, whilst cGMP itself has inhibitory effects on DSM contractility. Sildenafil may indirectly suppress DSM contractility by diminishing synchronicity between functional units of DSM bundles without inhibiting excitability of DSM themselves.

Topics: Action Potentials; Animals; Calcium; Cyclic GMP; Electrophysiology; Female; Guinea Pigs; In Vitro Techniques; Indoles; Isometric Contraction; Male; Molsidomine; Muscle Contraction; Muscle, Smooth; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Phosphodiesterase Inhibitors; Piperazines; Purines; Signal Transduction; Sildenafil Citrate; Sulfones; Urinary Bladder

To investigate whether nitric oxide (NO) and/or cGMP protects the retina from chemical ischemia and underlie somatostatin's neuroprotective effects.. Eyecups of female Sprague-Dawley rats were incubated with PBS or the chemical ischemia mixture [iodoacetic acid (5 mM)/sodium cyanate (25 mM)] in the absence or presence of (1) arginine (0.05-2.0 mM), the substrate of nitric oxide synthase (NOS); (2) the NO donors sodium nitroprusside (SNP; 0.25-4.0 mM), 3-morpholinosydnonimine (SIN-1; 0.1, 0.3, 1.0 mM), SIN-1 (0.1 mM)/L-cysteine (5 mM, peroxynitrite scavenger), and NONOate (1, 5, 10 microM, slow NO releaser); (3) 8-Br-cGMP (0.1, 0.5, 1.0 mM); (4) BIM23014 (sst(2) receptor agonist; 1 microM), alone or in the presence of (5) the NOS inhibitor N(gamma)-monomethyl-L-arginine (NMMA; 0.5 mM); or (6) the guanylyl cyclase inhibitors 1H-[1,2,4]oxadiazolol [4,3-a]quinoxalin-1-one (ODQ;100 microM) and NS2028 (50 microM) for 60 minutes, at 5%CO(2)/air in 37 degrees C. The effect of SIN-1 (0.1, 0.3, 1.0, or 3.0 mM) on the retina was also examined. Subsequently, the eyecups were fixed and sectioned for choline acetyltransferase (ChAT) immunoreactivity and TUNEL staining.. Arginine and SNP had no effect on the chemical ischemia-induced toxicity. SIN-1, NONOate, and 8-Br-cGMP produced a concentration-dependent protective effect, as shown by ChAT immunoreactivity. TUNEL staining also confirmed the neuroprotective effect of these agents. L-cysteine partially reduced the SIN-1-induced protective effect. SIN-1 alone was toxic only at the highest concentration used (3 mM). NMMA, ODQ, and NS2028 reversed the protective effect of BIM23014.. The results suggest that a NO/peroxynitrite/cGMP mechanism may be important in the protection of the retina from ischemic insult. Furthermore, the NO/sGC/cGMP pathway is involved in the neuroprotective effects of sst(2) ligands against retinal ischemia.

Topics: Animals; Choline O-Acetyltransferase; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Fluorescent Antibody Technique, Indirect; In Situ Nick-End Labeling; Ischemia; Molsidomine; Nitric Oxide; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Retinal Diseases; Retinal Vessels; Somatostatin; Spermine

Within the striatum, the gaseous neurotransmitter nitric oxide (NO) is produced by a subclass of interneurons containing the neuronal NO synthase (nNOS). NO promotes the second messenger cGMP through the activation of the soluble guanyl cyclase (sGC) and plays a crucial role in the integration of glutamate (GLU) and DA transmission. The aim of this study was to characterize the impact of 6-hydroxyDA (6-OHDA) lesion of the rat nigrostriatal pathway on NO/cGMP system. In vivo extracellular single units recordings were performed under urethane anesthesia to avoid any potentially misleading contributions of cortically-driven changes on endogenous NO. Hence, no electrical extrastriatal stimulation was performed and great attention was paid to the effects of 3-morpholinosydnonimine (SIN-1, a NO donor), N(G)-nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) and Zaprinast (a PDE inhibitor) delivered by iontophoresis upon the main striatal phenotypes. The latter were operationally distinguished in silent medium spiny-like neurons (MSN), with negligible spontaneous activity but displaying glutamate-induced firing discharge at rest and spontaneously active neurons (SAN), representing to a large extent nonprojecting interneurons. SANs were excited by SIN-1 and Zaprinast while MSNs showed a clear inhibition during local iontophoretic application of SIN-1 and Zaprinast. In 6-OHDA animals, SIN-1-induced excitation in SANs was significantly increased (on the contrary, the inhibitory effect of L-NAME was less effective). Interestingly, in DA-denervated animals, a subclass of MSNs (40%) displayed a peculiar excitatory response to SIN-1. These findings support the notion of an inhibitory modulatory role exerted by endogenous NO on control striatal projection cells. In addition, these findings suggest a functional cross-talk between NO, spontaneously active interneurons, and projection neurons that becomes critical in the parkinsonian state.

Topics: Action Potentials; Analysis of Variance; Animals; Corpus Striatum; Cyclic GMP; Denervation; Dopamine; Dose-Response Relationship, Radiation; Electric Stimulation; Enzyme Inhibitors; Male; Molsidomine; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidopamine; Rats; Rats, Wistar; Signal Transduction; Sympatholytics

The structural elements of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling pathway have been described in the vestibular peripheral system. However, the functions of NO in the vestibular endorgans are still not clear. We evaluated the action of NO on the Ca(2+) currents in hair cells isolated from the semicircular canal crista ampullaris of the rat (P14-P18) by using the whole cell and perforated-cell patch-clamp technique. The NO donors 3-morpholinosydnonimine (SIN-1), sodium nitroprusside (SNP), and (+/-)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide (NOR-4) inhibited the Ca(2+) current in hair cells in a voltage-independent manner. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) prevented the inhibitory effect of SNP on the Ca(2+) current. The selective inhibitor of the soluble form of the enzyme guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), also decreased the SNP-induced inhibition of the Ca(2+) current. The membrane-permeant cGMP analogue 8-Br-cGMP mimicked the SNP effect. KT-5823, a specific inhibitor of cGMP-dependent protein kinase (PGK), prevented the inhibition of the Ca(2+) current by SNP and 8-Br-cGMP. In the presence of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent that prevents the S-nitrosylation reaction, the SNP effect on the Ca(2+) current was significantly diminished. These results demonstrated that NO inhibits in a voltage-independent manner the voltage-activated Ca(2+) current in rat vestibular hair cells by the activation of a cGMP-signaling pathway and through a direct action on the channel protein by a S-nitrosylation reaction. The inhibition of the Ca(2+) current by NO may contribute to the regulation of the intracellular Ca(2+) concentration and hair-cell synaptic transmission.

Topics: Animals; Calcium Channels, L-Type; Carbazoles; Cell Separation; Cyclic GMP; Data Interpretation, Statistical; Enzyme Inhibitors; Ethylmaleimide; Free Radical Scavengers; Hair Cells, Vestibular; In Vitro Techniques; Indoles; Molsidomine; Nitric Oxide; Nitroprusside; Patch-Clamp Techniques; Rats; Rats, Long-Evans; Signal Transduction

We investigated the role of NO (nitric oxide) in the isolated intestine of the sea water adapted eel, by testing the effect of various donors on I(sc) (short-circuit current), due to net Cl(-) absorption in the control conditions. We found that the endogenous NO-synthase substrate l-arginine as well as two different NO donors, SNP (sodium nitroprusside) and SIN-1 (3-morpholinosydnonimine), produced a slow and gradual decrease of I(sc). The effect of SNP was reduced by the pretreatment with ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one), a specific inhibitor of the soluble guanylyl cyclase, suggesting the involvement of cGMP (cyclic GMP) in some physiological actions of NO. The effect of the NO donors on I(sc) was similar to that observed when the tissues were perfused with solution in which the HCO(3)(-) buffer was substituted with Hepes buffer. In addition the NO donors produced a negligible effect on I(sc) when the tissues were perfused with Hepes buffer or in the presence of bilateral SITS(4-Acetoamido-4'-iso-thiocyanatostilbene-2,2'disulphonic acid), an inhibitor of the HCO(3)(-) transport mechanisms, operating on both cell membranes of the eel enterocyte and responsible for HCO(3)(-) uptake by the cell. Based on these observations we suggest that NO regulates I(sc) and hence the transepithelial ion transport indirectly by modulating the endocellular concentration of HCO(3)(-) and/or H(+). In addition it is likely that NO modulates the permeability of the paracellular pathway since SNP produced also an increase of the tissue conductance and a decrease of the magnitude of the dilution potential.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anguilla; Animals; Arginine; Bicarbonates; Chlorides; Cyclic GMP; Diffusion Chambers, Culture; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Intestinal Absorption; Intestinal Mucosa; Intestines; Membrane Potentials; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; omega-N-Methylarginine; Oxadiazoles; Permeability; Quinoxalines; Time Factors

We investigated the intracellular signalling pathways by which nitric oxide (NO) donors: diethylamine/NO (DEA/NO) and 3-morpholinosydnonimine (SIN-1) regulate the functional response of human neutrophils to activating stimuli.. The phosphorylation and nitration of signalling proteins, cyclic GMP level, neutrophil respiratory burst and adhesive activities and CD11b/CD18 molecule expression on neutrophils in the presence and absence of soluble guanylate cyclase inhibitors were determined.. NO donors showed strong inhibitory effect on activated neutrophils. NO donors nitrated the tyrosine residues in signalling proteins causing a decrease in tyrosine phosphorylation and neutrophils response to activation. Diethylamine/NO employed cyclic GMP as a signalling molecule in its action on neutrophils, whereas peroxynitrite anion donor affected neutrophil functions in a cGMP-independent manner. Moreover, we observed that peroxynitrite anion can overcome the nitric oxide molecule action.. We conclude that each NO donor depending on its concentration and chemical nature may act on different elements of neutrophil signalling pathways capable of inducing distinct neutrophil functions.

Topics: Animals; CD11b Antigen; CD18 Antigens; Cell Adhesion; Cyclic GMP; Guanylate Cyclase; Humans; Hydrazines; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Nitric Oxide; Nitric Oxide Donors; Respiratory Burst; Signal Transduction

We investigated whether the signal mechanism for relaxation may be affected by inflammation of the cat esophagus. Acute esophagitis was induced by perfusion with 0.1N HCI at a rate of 1 mL/min for 45 min over three consecutive days. We then isolated esophageal smooth muscle cells by enzymatic digestion with collagenase. We pre-contracted the isolated smooth cells with acetylcholine (ACh) (10(-5) M) and compared the agonist-induced relaxation of pre-con tracted normal cells with those of esophagitic cells. Vasoactive intestinal polypeptide (VIP) caused a dose-dependent relaxation in normal cells, and this curve was down shifted in esophagitic cells. Sodium nitroprusside (SNP) or SIN-1 (NO donor) produced dose-dependent relaxation in normal cells, which was not affected by esophagitis. 8-Br-cGMP (a cGMP ana log) also induced dose-dependent relaxation to a similar extent in both normal and esoph agitic cells. Forskolin (a cAMP activator) or db-cAMP (a cAMP analog) produced dose-dependent relaxation in normal cells, and this relaxation curve was down shifted in esoph agitic cells. Western blotting was used to determine what subtype of adenylyl cyclase was involved in the cAMP pathway. Western blot analysis of homogenates derived from esophageal smooth muscle using antibodies against adenylyl cyclase types II, III, IV and V/VI revealed the presence of type V and/or type VI only. This result suggests that relaxation via a cAMP-dependent pathway rather than a cGMP dependent-pathway is down regulated in cat acute esophagitis. This subsensitivity of the cAMP related pathway may be related to the activ ity of adenylyl cyclase V/VI.

Topics: Adenylyl Cyclases; Animals; Blotting, Western; Bucladesine; Cats; Cell Membrane Permeability; Colforsin; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Down-Regulation; Esophagitis; In Vitro Techniques; Isoenzymes; Molsidomine; Muscle Relaxation; Muscle, Smooth; Nitric Oxide Donors; Nitroprusside; Signal Transduction; Vasoactive Intestinal Peptide

The present study was designed to investigate the role of the sodium potassium adenosine triphosphatase (the Na(+)K(+) ATPase) in relaxation of bovine isolated bronchioles by a new NO donor, GEA 3175 (3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulphonyl]amino]-)hydroxide)). Bronchioles were mounted in a wire myograph for isometric tension recordings and contracted with 5-hydroxytryptamine (5-HT) or a K(+) rich solution. Concentration-dependent relaxations evoked by GEA 3175 were inhibited by ouabain or K(+) free solution. The guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3 microM) and ouabain (10 nM) reduced GEA 3175-evoked relaxations to the same extent without any additive effect. Iberiotoxin (10 nM), an inhibitor of large conductance Ca(2+)-activated K(+) channels inhibited GEA 3175-evoked relaxations to the same extent as ouabain. Combining ouabain and iberiotoxin completely abolished GEA 3175 relaxation. An inhibitor of protein kinase G (PKG), Rp-beta-phenyl-1,N(2)-etheno-8-bromo-guanosine-3'-5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPs), slightly reduced GEA 3175-induced relaxations. An inhibitor of cyclic AMP-dependent kinase (PKA), Rp-adenosine-3'-5'-cyclic phosphorothioate (Rp-cAMPs), inhibited the GEA 3175-induced relaxations to the same extent as ouabain. Inhibition of both PKG and PKA abolished GEA 3175 relaxation. The study provides evidence that the NO donor GEA 3175 causes guanylyl cyclase-dependent relaxations, taking place through cyclic GMP and cyclic AMP-dependent protein kinases followed by opening of large conductance Ca(2+)-activated K(+) channels and activation of smooth muscle Na(+)K(+) ATPase.

Topics: Animals; Bronchi; Calcium; Cattle; Colforsin; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Guanylate Cyclase; In Vitro Techniques; Molsidomine; Muscle Relaxation; Nitric Oxide; Nitric Oxide Donors; Ouabain; Oxadiazoles; Peptides; Potassium; Quinoxalines; S-Nitrosoglutathione; Serotonin; Sodium-Potassium-Exchanging ATPase; Thionucleotides; Triazoles

Peroxynitrite (ONOO(-)) is a compound formed by reaction of superoxide (O(2) (-)) with nitric oxide (NO) and is expected to possess characteristics of both O(2) (-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOO(-) in defense responses in plants, it has been very difficult to detect ONOO(-) in bioimaging due to its short half-life or paucity of methods for ONOO(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOO(-) in bioimaging, was used for intracellular ONOO(-) detection. ONOO(-) generation in tobacco BY-2 cells treated with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, occurred within 1 h and reached a maximum level at 6-12 h after INF1 treatment. Urate, a ONOO(-) scavenger, abolished INF1-induced ONOO(-) generation. It is well known that ONOO(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOO(-)-specific reaction. Western blot analysis using anti-nitrotyrosine antibodies recognized nitrotyrosine-containing proteins in 20 and 50 kDa bands in BY-2 protein extract containing SIN-1 [3-(4-morpholinyl) sydnonimine hydrochloride; an ONOO(-) donor]. These bands were also recognized in INF1-treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOO(-) detection and tyrosine nitration in defense responses in plants.

Topics: Algal Proteins; Aniline Compounds; Antibodies, Anti-Idiotypic; Cells, Cultured; Cyclic GMP; Fluoresceins; Fungal Proteins; Molsidomine; Nicotiana; Nitric Oxide; Peroxynitrous Acid; Phytophthora; Proteins; Reactive Oxygen Species; Superoxides; Tyrosine; Uric Acid

In the present study, we investigated the vasodilator properties of A-type, B-type and C-type natriuretic peptides (ANP, BNP and CNP respectively) and the NO (nitric oxide) donor sin-1 (3-morpholino-sydnonimine) in human by-pass grafts. In contrast with previous studies, the same vessel was used to demonstrate a direct link between cGMP production and functional relaxation. Remnants of the IMA (internal mammary artery) and SV (saphenous vein) were obtained from 82 patients undergoing coronary artery by-pass grafting. The responses to cumulative concentrations of ANP, BNP, CNP and sin-1 in vessel rings pre-contracted with a thromboxane A2 agonist (U46619) were measured in an organ bath. Additionally, intracellular cGMP production after single submaximal dose application of these drugs to vessel rings was determined by a RIA. ANP (P=0.001) and sin-1 (P<0.001) caused significant concentration-dependent relaxation of the IMA. In the SV, only sin-1 (P<0.001) induced marked concentration-dependent relaxation. At a single submaximal concentration, significant relaxation as well as intracellular cGMP production were found in response to ANP, BNP and sin-1 in the IMA. In contrast, in the SV, only sin-1 significantly induced cGMP production and relaxation. There was a moderate, but significant, correlation between intracellular cGMP net production and net relaxation in the IMA. In conclusion, ANP, as the most powerful relaxant of all the natriuretic peptides tested on the IMA, may be a possible alternative vasorelaxant to overcome peri-operative vasospasm in this artery. In contrast with sin-1, ANP and BNP were not effective vasorelaxants of the SV. Net relaxation in response to natriuretic peptides correlated with cGMP net concentrations in the IMA.

Topics: Adult; Aged; Aged, 80 and over; Atrial Natriuretic Factor; Coronary Artery Bypass; Cyclic GMP; Dose-Response Relationship, Drug; Female; Humans; Internal Mammary-Coronary Artery Anastomosis; Male; Mammary Arteries; Middle Aged; Molsidomine; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Natriuretic Peptides; Nitric Oxide Donors; Saphenous Vein; Tissue Culture Techniques; Vasodilation; Vasodilator Agents

We investigated the role of the NO/cGMP system in the vasodilatory response to hypercapnia after cortical spreading depression (CSD) in barbiturate anesthetized rats in vivo. Regional cerebral blood flow (rCBF) was measured by laser Doppler flowmetry (LDF). Hypercapnia (arterial pCO2 50-60 mm Hg) increased rCBF by 2.8+/-1.0%/mm Hg (n = 34). Fifteen minutes after CSD, resting rCBF was reduced to 87%, and rCBF response to hypercapnia was abolished (p < 0.001, n = 28). Within 1 h after CSD, only little restoration of vascular reactivity occurred. Topical application of the NO-donors S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN1), or spermine/NO complex (Sperm/NO), or of the cell permeable guanosine 3',5'-cyclic monophosphate (cGMP) analogue 8-Br-cGMP reestablished resting rCBF to values measured before CSD, and reversed CSD-induced attenuation of the cerebrovascular response to hypercapnia. Restoration of resting rCBF to pre-CSD level by the NO-independent vasodilator papaverine had no effect on the attenuated hypercapnic response. In conclusion, we have shown that the compromised vascular reactivity to hypercapnia after CSD can be reversed to normal reactivity by restoration of the basal NO or cGMP concentration in the cortex, suggesting a reduction of the cerebrovascular NO or cGMP concentration following CSD.

Topics: Analysis of Variance; Animals; Cerebral Cortex; Cerebrovascular Circulation; Cerebrovascular Disorders; Cortical Spreading Depression; Cyclic GMP; Drug Interactions; Hypercapnia; Laser-Doppler Flowmetry; Male; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Rats; Rats, Wistar; Spermine; Thionucleotides; Time Factors

The presence of nitric oxide (NO) synthase and of soluble guanylyl cyclase, the main NO-activated metabolic pathway, has been demonstrated in many cells of the subthalamic nucleus. In this study, the effects induced on the firing of 96 subthalamic neurons by microiontophoretically administering drugs modifying NO neurotransmission were explored in anaesthetized rats. Recorded neurons were classified into regularly and irregularly discharging on the basis of their firing pattern. Nomega-nitro-L-arginine methyl ester (L-NAME; a NO synthase inhibitor), 3-morpholino-sydnonimin-hydrocloride (SIN-1; a NO donor), S-nitroso-glutathione (SNOG; another NO donor) and 8-Br-cGMP (a cell-permeable analogue of cGMP, the main second-messenger of NO neurotransmission) were iontophoretically applied while performing single-unit extracellular recordings. The activity of most neurons was influenced in a statistically significant way: in particular, both current-related inhibitory L-NAME-induced effects (20/39 tested cells) and excitatory effects of SIN-1 (25/41 tested neurons), SNOG (19/32 tested cells) and 8-Br-cGMP (13/19 tested neurons) were demonstrated. Neither statistically significant differences between the responses of regularly and irregularly discharging cells, nor specific topographical clustering of responding neurons, were demonstrated. Neurons administered drugs oppositely modulating the NO neurotransmission often displayed responses to only one treatment. We hypothesize that NO neurotransmission could exert a modulatory influence upon subthalamic neurons, with a prevalent excitatory effect. However, in the light of the presence of some responses of opposite sign to the same drug displayed by different subthalamic neurons, more complex effects of NO neurotransmission could be suggested, probably due to interactions with other classical neurotransmitter systems.

Topics: Action Potentials; Analysis of Variance; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Iontophoresis; Male; Molsidomine; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Rats; Rats, Wistar; S-Nitrosoglutathione; Subthalamic Nucleus; Thionucleotides; Time Factors

Nitric oxide (NO) modulates the uptake and/or release of neurotransmitters through a variety of cellular mechanisms. However, the pharmacological and biochemical processes underlying these neurochemical effects of NO often remain unclear. In our study, we used immunocytochemical methods to study the effects of NO, cyclic guanosine monophosphate (cGMP), and peroxynitrite on the uptake and release of gamma-aminobutyric acid (GABA) and glycine in the turtle retina. In addition, we examined the involvement of glutamate receptors, calcium, and the GABA transporter in this GABA uptake and release. We also tested for interactions between the GABAergic and glycinergic systems. In general, we show that NO stimulated GABA release and inhibited glycine release. The NO-stimulated GABA release involved calcium-dependent or calcium-independent synaptic release or reversal of the GABA transporter. Some effects of NO on GABA release involved glutamate, cGMP, or peroxynitrite. NO promoted glycine uptake and inhibited its release, and this inhibition of glycine release was influenced by GABAergic modulation. These findings indicate that NO modulates the levels of the inhibitory transmitters GABA and glycine through several specific biochemical mechanisms in different retinal cell types and layers. Thus it appears that some of the previously described reciprocal interactions between GABA and glycine in the retina function through specific NO signaling pathways.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Cadmium; Citrulline; Cyclic GMP; DEET; Dizocilpine Maleate; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Free Radical Scavengers; GABA Antagonists; gamma-Aminobutyric Acid; Glycine; Immunohistochemistry; In Vitro Techniques; Molsidomine; Neural Inhibition; Nipecotic Acids; Nitric Oxide; Potassium; Retina; Silver Staining; Thiourea; Turtles; Vigabatrin

The potent vasomodulator adenosine (AD), thanks to the interaction with by A(1) and A(2) receptors, dilates systemic, coronary and cerebral vasculatures but exert a constrictor action in several vessels of respiratory organs. Recent investigations suggest that nitric oxide (NO) contributes to AD effects. In fish, both NO and AD induce atypical effects compared to mammals. Since there is very little information on the role of NO and its involvement in mediating the actions of AD in fish, we have analysed this question in the branchial vasculature of the elasmobranch Squalus acanthias and the teleost Anguilla anguilla using an isolated perfused head and a branchial basket preparation, respectively. In both dogfish and eel, AD dose-response curves showed a biphasic effect: vasoconstriction (pico to nanomolar range) and vasodilation (micromolar range). Both effects were abolished by the classic xanthine inhibitor theophylline (Theo) and also by specific antagonists of A(1) and A(2) receptor subtypes. To analyse the involvement of the NO/cGMP system in the AD responses, we tested a NOS inhibitor, l-NIO, and a specific soluble guanylate cyclase (sGC) blocker, ODQ. In both dogfish and eel preparations l-NIO abrogated all vasomotor effects of AD, whereas ODQ blocked the AD-mediated vasoconstriction without affecting the vasorelaxant response. This indicates that only AD-induced vasoconstriction is mediated by a NO-cGMP-dependent mechanism. By using the NO donor SIN-1, we showed a dose-dependent vasoconstrictory effect which was completely blocked by ODQ. These results provide compelling evidence that the vasoactive role of AD in the branchial circulation of S. acanthias and A. anguilla involves a NO signalling.

Topics: Adenosine; Anguilla; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Female; Hypoxia; In Vitro Techniques; Male; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Perfusion; Pressure; Signal Transduction; Squalus acanthias; Time Factors; Vasodilation

To test our hypothesis that the abnormally small efficacy of mu-opioid agonists in diabetic rats may be due to functional changes in the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway, we evaluated the effects of N-iminoethyl-L-ornithine, methylene blue, and 3-morpholino-sydnonimine on [D-Ala(2), NMePhe(4), Gly-ol(5)]enkephalin (DAMGO)-induced antinociception in both streptozotocin (STZ)-diabetic and nondiabetic rats. Animals were rendered diabetic by an injection of STZ (60 mg/kg intraperitoneally). Antinociception was evaluated by the formalin test. The mu-opioid receptor agonist DAMGO (1 microg per paw) suppressed the agitation response in the second phase. The antinociceptive effect of DAMGO in STZ-diabetic rats was significantly less than in nondiabetic rats. N-Iminoethyl-L-ornithine (100 microg per paw), an NO synthase inhibitor, or methylene blue (500 microg per paw), a guanylyl cyclase inhibitor, significantly decreased DAMGO-induced antinociception in both diabetic and nondiabetic rats. Furthermore, 3-morpholino-sydnonimine (200 microg per paw), an NO donor, enhanced the antinociceptive effect of DAMGO in nondiabetic rats but did not change in diabetic rats. These results suggest that the peripheral antinociceptive effect of DAMGO may result from activation of the L-arginine/NO/cGMP pathway and dysfunction of this pathway; also, events that are followed by cGMP activation may have contributed to the demonstrated poor antinociceptive response of diabetic rats to mu-opioid agonists.. This is the first study on the role of the nitric oxide (NO)/cyclic guanosine monophosphate pathway on [D-Ala(2), NMePhe(4), Gly-ol(5)]enkephalin (DAMGO)-induced peripheral antinociception and the effect of diabetes on this pathway. The study suggests a possible role of DAMGO as a peripherally-acting analgesic drug.

Topics: Analgesics, Opioid; Animals; Arginine; Blood Glucose; Cyclic GMP; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Formaldehyde; Guanylate Cyclase; Male; Methylene Blue; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Pain; Pain Measurement; Rats; Rats, Wistar

1. This study was designed to investigate the effects of the nitric oxide (NO) donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) on N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP, 1 x 10(-7) M)-induced human eosinophil chemotaxis, cyclic guanosine-3',5'-monophosphate (cGMP) levels, protein nitration and cytotoxicity. 2. Human eosinophils were exposed to SNP, SIN-1 and SNAP (0.001-1.0 mM) for either short (10 min) or prolonged (90 min) time periods. Exposition of eosinophils with these NO donors significantly inhibited the eosinophil chemotaxis irrespective of whether cells were exposed to these agents for 10 or 90 min. No marked differences were detected among them regarding the profile of chemotaxis inhibition. 3. Exposition of eosinophils to SNP, SIN-1 and SNAP (0.001-1.0 mM) markedly elevated the cGMP levels above basal levels, but the 90-min exposition resulted in significantly higher levels compared with the 10-min protocols (5.3+/-0.6 and 2.6+/-0.2 nM 1.5 x 10(6) cells(-1), respectively). The cGMP levels achieved with SNAP were greater than SNP and SIN-1. 4. The NO donors did not induce cell toxicity in any experimental condition used. Additionally, eosinophils exposed to SNP, SIN-1 and SNAP (1.0 mM each) either for 10 or 90 min did not show any tyrosine nitration in conditions where a strong nitration of bovine serum albumin was observed. 5. Our findings show that inhibitory effects of fMLP-induced human eosinophil chemotaxis by NO donors at short or prolonged exposition time were accompanied by significant elevations of cGMP levels. However, additional elevations of cGMP levels do not change the functional profile (chemotaxis inhibition) of stimulated eosinophils.

Topics: Adolescent; Adult; Blotting, Western; Cell Survival; Chemotaxis, Leukocyte; Cyclic GMP; Eosinophils; Female; Humans; In Vitro Techniques; Male; Middle Aged; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Nitric Oxide Donors; Nitroprusside; Penicillamine; Tetrazolium Salts; Thiazoles; Tyrosine

4-(Nitrooxy)-butyl-(S)-2-(6-methoxy-2-naphthyl)-propanoate (AZD3582) is a cyclooxygenase (COX)-inhibiting nitric oxide donator (CINOD). It donates nitric oxide (NO) in biological systems through as yet unidentified mechanisms. cGMP, a marker of intracellularly generated NO, was increased up to 27-fold over basal levels by AZD3582 (1-30microM) in LLC-PK1 kidney epithelial cells. A 5h pretreatment with glyceryl tinitrate (GTN, 0.1-1microM) attenuated the cGMP response to a subsequent challenge with AZD3582 or GTN. Similarly, AZD3582 (10-30microM) pretreatment reduced the increase in cGMP on subsequent incubation with AZD3582 or GTN. In contrast, cGMP stimulation by SIN-1, which releases NO independently of enzymatic catalysis, remained unimpaired in cells pretreated with GTN or AZD3582. Our results demonstrate that AZD3582 decreases the sensitivity of the guanylyl cyclase/cGMP system to GTN and vice versa. This suggests that bioactivation pathways for organic nitrates, which involve enzymatic catalysis, may be responsible for NO donation from AZD3582.

Topics: Animals; Cyclic GMP; Cyclooxygenase Inhibitors; LLC-PK1 Cells; Molsidomine; Naphthalenes; Naproxen; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Swine

This study tests the hypothesis that particulate (p) guanylyl cyclase (GC) and soluble (s) GC are involved in the distinct roles for the regulation of cGMP-PDE-cAMP signaling and of mechanical and secretory functions in the heart. Experiments were performed in perfused beating rabbit atria. C-type natriuretic peptide (CNP) and SIN-1, an NO donor, or BAY 41-2272 (BAY), a direct activator for sGC, were used to activate pGC and sGC, respectively. CNP and SIN-1 increased cGMP and cAMP efflux in a concentration-dependent manner. Increase in cAMP was a function of cGMP. The changes in cAMP efflux concentration in terms of cGMP were much more prominent in the atria treated with CNP than in the atria treated with SIN-1. Increase in cAMP efflux concentration was blocked by milrinone but not changed by EHNA. BAY increased cGMP but not cAMP in a concentration-dependent manner. CNP and SIN-1 decreased atrial stroke volume and myocytic ANP release. The decreases in terms of cGMP efflux concentration were much more prominent in the atria treated with CNP than in the atria treated with SIN-1 or BAY. Milrinone accentuated GC agonist-induced decreases in atrial stroke volume and ANP release. In the presence of ODQ, SIN-1 or BAY induced effects were not observed. These data suggest that pGC and sGC activations have distinct roles via cGMP-PDE3-cAMP signaling in the cardiac atrium: high and low gain switches, respectively, for the regulation of cAMP levels and contractile and secretory functions.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenine; Animals; Atrial Natriuretic Factor; Biological Transport; Cardiac Pacing, Artificial; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 2; Cyclic Nucleotide Phosphodiesterases, Type 3; Enzyme Inhibitors; Guanylate Cyclase; Heart Atria; Milrinone; Molsidomine; Myocardial Contraction; Myocytes, Cardiac; Natriuretic Peptide, C-Type; Nitric Oxide Donors; Oxadiazoles; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Pyrazoles; Pyridines; Quinoxalines; Rabbits; Receptors, Cytoplasmic and Nuclear; Second Messenger Systems; Soluble Guanylyl Cyclase; Stroke Volume

In the present investigation, we used standard patch clamp techniques to test whether nitric oxide (NO) generation has any role to play with either activation or inhibition of ATP-sensitive (KATP) channels in guinea-pig urinary bladder. We found that NO generation leads to activation of KATP channels through a cyclic guanosine monophosphate (c-GMP)-dependent protein kinase. 3-Morpholinosydnonimine (SIN, 100 microM) potentiated activation of an inward current in whole cell patch clamp experiments. Glibenclamide (10 microM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) inhibited the SIN-activated current. Both in cell-attached and in inside out patches, SIN (200 microM) potentiated KATP channel activity, and the increased channel activity in inside out patches was suppressed by glibenclamide (50 microM), ATP (1 mM) and (9s,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12,-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6] benzodiazocine-10-carboxylic acid, methyl ester (KT-5823, 10 nM). 8-Br-cGMP (100 microM) increased the KATP channel activity in cell-attached patches, and this was suppressed by glibenclamide (50 microM). These results suggest that the NO-c-GMP-PKG pathway contributes to activation of K(ATP) channels in guinea-pig urinary bladder myocytes.

Topics: Adenosine Triphosphate; Animals; Carbazoles; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Glyburide; Guinea Pigs; Indoles; Ion Channel Gating; Male; Membrane Potentials; Molsidomine; Myocytes, Smooth Muscle; Nitric Oxide; Nitric Oxide Donors; Oxadiazoles; Patch-Clamp Techniques; Potassium Channels; Quinoxalines; Signal Transduction; Urinary Bladder

To investigate how nitric oxide (NO) modulates short-circuit current (Isc) in isolated porcine ciliary processes.. Isc changes (Ussing-type chamber) induced either by the NO donors SNP or SIN-1, or by the cGMP analogue 8-pCPT-cGMP were assessed. The effect of inhibitors of guanylate cyclase (10 microM ODQ, 100 microM LY83583), protein kinase G (30 microM Rp-8-pCPT-cGMP, 3 microM KT 5823), protein kinase A (1 microM KT 5720), or protein kinase C (1 microM Go6983) on SNP- or 8-pCPT-cGMP-induced Isc changes were investigated. The effect of inhibitors of anion channel (100 microM niflumic acid, 1 mM DIDS, and 1 mM 9-AC), K+-channel (10 mM TEA, 10 mM BaCl2), Na+-channel blockers (1 mM amiloride), Na+-K+-2Cl- cotransporter inhibitor (0.5 mM bumetanide), or carbonic anhydrase inhibitor (1 mM acetazolamide) was studied. In Cl(-)- or HCO3(-)-free Krebs-Ringer solution, the effect of SNP- or 8-pCPT-cGMP-induced Isc changes was accessed.. SNP, SIN-1, or 8-pCPT-cGMP increased Isc with a change in the potential difference that became more negative toward the nonpigmented epithelium (aqueous) side. The Isc increase induced by SNP or SIN-1, but not by 8-pCPT-cGMP, was prevented by ODQ and LY83583. SNP- and 8-pCPT-cGMP-induced Isc increases were prevented by Rp-8-pCPT-cGMP or KT5823 (but not by KT5720 or Go6983), or by niflumic acid, DIDS, 9-AC, or acetazolamide (but not by TEA, BaCl2, amiloride, or bumetanide). The effect of SNP and 8-pCPT-cGMP was abolished in Cl(-)- and reduced in HCO3(-)-free solutions.. NO activates a guanylate cyclase-cGMP-protein kinase G pathway that appears to stimulate stroma-to-aqueous anionic transport, possibly Cl-, in porcine ciliary epithelium.

Topics: Animals; Anion Transport Proteins; Anions; Aqueous Humor; Carbonic Anhydrase Inhibitors; Chloride Channels; Ciliary Body; Corneal Stroma; Cyclic GMP; Electric Conductivity; Enzyme Inhibitors; Guanylate Cyclase; Ion Exchange; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Protein Kinase Inhibitors; Swine; Thionucleotides

The hepatic parasympathetic nerves and hepatic nitric oxide synthase (NOS) are involved in the secretion of a hepatic insulin sensitizing substance (HISS), which mediates peripheral insulin sensitivity. We tested whether binding of ACh to hepatic muscarinic receptors is an upstream event to the synthesis of nitric oxide (NO), which, along with the activation of hepatic guanylate cyclase (GC), permits HISS release. Male Wistar rats (8-9 wk) were anesthetized with pentobarbital sodium (65 mg/kg). Insulin sensitivity was assessed using a euglycemic clamp [the rapid insulin sensitivity test (RIST)]. HISS inhibition was induced by antagonism of muscarinic receptors (atropine, 3 mg/kg i.v.) or by blockade of NOS [NG-nitro-L-arginine methyl ester (L-NAME), 1 mg/kg intraportally (i.p.v.)]. After the blockade, HISS action was tentatively restored using a NOdonor [3-morpholynosydnonimine (SIN-1), 5-10 mg/kg i.p.v.] or ACh (2.5-5 microg.kg(-1).min(-1) .i.p.v.). SIN-1 (10 mg/kg) reversed the inhibition caused by atropine (RIST postatropine 137.7 +/- 8.3 mg glucose/kg; reversed to 288.3 +/- 15.5 mg glucose/kg, n = 6) and by L-NAME (RIST post-L-NAME 152.2 +/- 21.3 mg glucose/kg; reversed to 321.7 +/- 44.7 mg glucose/kg, n = 5). ACh did not reverse HISS inhibition induced by L-NAME. The role of GC in HISS release was assessed using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 nmol/kg i.p.v.), a GC inhibitor that decreased HISS action (control RIST 237.6 +/- 18.6 mg glucose/kg; RIST post-ODQ 111.7 +/- 6.2 mg glucose/kg, n = 5). We propose that hepatic parasympathetic nerves release ACh, leading to hepatic NO synthesis, which activates GC, triggering HISS action.

Topics: Acetylcholine; Animals; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Injections, Intravenous; Insulin Resistance; Liver; Male; Molsidomine; Muscarinic Antagonists; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Oxadiazoles; Portal Vein; Quinoxalines; Rats; Rats, Wistar; Signal Transduction

The role of nitric oxide (NO) in cardio-vascular homeostasis is now known to include allosteric redox modulation of cell respiration. An interesting animal for the study of this wide-ranging influence of NO is the cold-adapted Antarctic icefish Chionodraco hamatus, which is characterised by evolutionary loss of hemoglobin and multiple cardio-circulatory and subcellular compensations for efficient oxygen delivery. Using an isolated, perfused working heart preparation of C. hamatus, we show that both endogenous (L-arginine) and exogenous (SIN-1 in presence of SOD) NO-donors as well as the guanylate cyclase (GC) donor 8Br-cGMP elicit positive inotropism, while both nitric oxide synthase (NOS) and sGC inhibitors, i.e. L-NIO and ODQ, respectively, induce significant negative inotropic effects. These results therefore demonstrate that under basal working conditions the icefish heart is under the tonic influence of a NO-cGMP-mediated positive inotropism. We also show that the working heart, which has intracardiac NOS (shown by NADPH-diaphorase activity and immunolocalization), can produce and release NO, as measured by nitrite appearance in the cardiac effluent. These results indicate the presence of a functional NOS system in the icefish heart, possibly serving a paracrine/autocrine regulatory role.

Topics: Animals; Antarctic Regions; Arginine; Cardiotonic Agents; Cell Respiration; Cyclic GMP; Electrochemistry; Fluorescent Antibody Technique; Molsidomine; Myocardial Contraction; Myocardium; NADH Dehydrogenase; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Oceans and Seas; Ornithine; Perciformes

To evaluate the effects of the nitric oxide (NO)-donating compounds sodium nitroprusside (SNP), S-nitroso-glutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC), S-nitroso-N-acetylcysteine-ethylester (SNACET), and linsidomine (SIN-1) on the adrenergic tension of isolated human seminal vesicle strip preparations. The significance of the NO-cyclic guanosine monophosphate (cGMP) pathway in the regulation of smooth muscle tone in the human genitourinary tract has been well established. However, information on the significance of NO-mediated signal transduction in the functional control of the mammalian seminal vesicles is still sparse.. Seminal vesicle strip preparations were applied to an organ bath system under standard conditions. Tension was induced by the addition of 10 microM norepinephrine. After stable tension plateaus had been reached, the drugs were added in a cumulative manner (0.01 to 100 microM) and the isometric responses of the tissue registered. The effects of the compounds on the phasic contractility of the tissue preparations were also evaluated. The adenylyl cyclase-stimulating agent forskolin was used as a reference compound known to interfere with the cyclic adenosine monophosphate pathway.. Adrenergic tension was dose dependently attenuated by the drugs. The rank order of potency, from greater to lesser, was GSNO, SNAC, SNP, SIN-1, forskolin, and SNACET. The rank order (from greater to lesser) with regard to the inhibitory effects of the compounds on the frequency of phasic contractions of the tissue induced by the addition of norepinephrine was GSNO, SNAC, SNP, and SIN-1; the effects of SIN-1, forskolin, and SNACET on the frequency of contractions were nearly equipotent.. Our results strongly support the hypothesis that the contractility of the human seminal vesicle is under the control of the NO-cGMP pathway. This finding may give a rationale for the use of S-nitrosothiols, such as GSNO and SNAC, in the pharmacotherapy of hyperexcitatory disturbances of ejaculation (premature ejaculation).

Topics: Adrenergic alpha-Agonists; Aged; Colforsin; Cyclic GMP; Ejaculation; Erectile Dysfunction; Humans; In Vitro Techniques; Isometric Contraction; Male; Middle Aged; Molsidomine; Muscle Contraction; Muscle, Smooth; Nitric Oxide Donors; Nitroprusside; Norepinephrine; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione; Seminal Vesicles; Sexual Dysfunction, Physiological; Signal Transduction

Atrial natriuretic peptide (ANP) plays a key regulatory role in arterial blood pressure homeostasis. We recently generated mice with selective deletion of the ANP receptor, guanylyl cyclase-A (GC-A), in vascular smooth muscle (SMC GC-A knockout (KO) mice) and reported that resting arterial blood pressure was completely normal in spite of clear abolition of the direct vasodilating effects of ANP (Holtwick, R., Gotthardt, M., Skryabin, B., Steinmetz, M., Potthast, R., Zetsche, B., Hammer, R. E., Herz, J., and Kuhn M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7142-7147). The purpose of this study was to clarify mechanisms compensating for the missing vasodilator responses to ANP. In particular, we analyzed the effect of the endothelial, cGMP-mediated vasodilators C-type natriuretic peptide and nitric oxide (NO). In isolated arteries from SMC GC-A KO mice, the vasorelaxing sensitivity to sodium nitroprusside and the endothelium-dependent vasodilator, acetylcholine, was significantly greater than in control mice. There was no difference in responses to C-type natriuretic peptide or to the activator of cGMP-dependent protein kinase I, 8-para-chlorophenylthio-cGMP. The aortic expression of soluble GC (sGC), but not of endothelial NO synthase or cGMP-dependent protein kinase I, was significantly increased in SMC GC-A KO mice. Chronic oral treatment with the NO synthase inhibitor N(w)-nitro-l-arginine methyl ester increased arterial blood pressure, the effect being significantly enhanced in SMC GC-A KO mice. We conclude that SMC GC-A KO mice exhibit a higher vasodilating sensitivity to NO. This can be attributed to an enhanced expression of sGC, whereas the expression and/or activity levels of downstream cGMP-effector pathways are not involved. Increased vasodilating responsiveness to endothelial NO contributes to compensate for the missing vasodilating effect of ANP in SMC GC-A KO mice.

Topics: Acetylcholine; Administration, Oral; Animals; Aorta; Arteries; Blood Pressure; Blotting, Western; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Guanylate Cyclase; Mice; Mice, Knockout; Molsidomine; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Protein Structure, Tertiary; Receptors, Atrial Natriuretic Factor; Vasodilator Agents

1. Nitric oxide has been described either as pronociceptive or antinociceptive. In this investigation, using an electronic pressure-metre, the intradermal and the subcutaneous effects of prostaglandin E(2) (PGE(2)) and agents that mimic or inhibit the arginine/NO/cGMP pathway were compared. 2. The hypernociceptive effect of the intradermal injection of PGE(2) (100 ng) was immediate, peaking within 15-30 min and returning to basal values in 45-60 min. The subcutaneous injection of PGE(2) induced a hypernociception with a delayed peak (3 h) plateauing for 4-6 h. 3. Intradermal administration of 3-morpholino-sydnonimine-hydrochloride (SIN-1) enhanced, while its subcutaneous administration inhibited, subcutaneous hypernociception induced by PGE(2). This inhibition was prevented by ODQ (8 micro g) but not by NG-monomethyl-L-arginine (L-NMMA) (50 micro g). 4. Intradermal but not subcutaneous administration of L-arginine (1-100 micro g), SIN-1 (1-100 micro g) and dibutyrylguanosine 3':5'-cyclic monophosphate (db cGMP) (0.1-100 micro g) induced an early (15-30 min) dose-dependent hypernociceptive effect. Intradermal pretreatment with NG-monomethyl-L-arginine (L-NMMA; 50 micro g) inhibited the hypernociception induced by L-Arg (10 micro g), but not that induced by SIN-1 (10 micro g) or db cGMP (10 micro g). 5. Intradermal injection of ODQ (8 micro g) antagonized the hypernociception induced by L-arginine and SIN-1, but not that induced by db cGMP. 6. Considering (a) the different time course of intradermal and subcutaneous PGE(2)-induced hypernociception, (b) the opposite nociceptive effect of intradermal and subcutaneous administration of SIN-1 (db cGMP) as well as the arginine/NO/cGMP pathway, the existence of different subsets of nociceptive primary sensory neurons in which the arginine/NO/cGMP pathway plays opposing roles is suggested. This hypothesis would explain the apparent contradictory observations described in the literature.

Topics: Animals; Arginine; Bucladesine; Cyclic GMP; Dermis; Dinoprostone; Dose-Response Relationship, Drug; Hyperalgesia; Injections, Subcutaneous; Male; Molsidomine; Nitric Oxide; Nociceptors; omega-N-Methylarginine; Oxadiazoles; Pain Measurement; Quinoxalines; Rats; Rats, Wistar; Subcutaneous Tissue; Time Factors

In guinea pig, primate and man, nitric oxide (NO)-induced regulation of myometrial smooth muscle contraction is distinct from other smooth muscles because cyclic guanosine 3',5'-cyclic monophosphate (cGMP) accumulation is neither necessary nor sufficient to relax the tissue. To further our understanding of the mechanism of action of NO in myometrium, we employed the NO donors, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndonimine (SIN-1) proposed to relax airway smooth muscle by disparate mechanisms involving elevation in intracellular calcium ([Ca(2+)](i)) or cGMP accumulation, respectively. Treatment of guinea pig myometrial smooth muscle with either NO donor at concentrations thought to produce maximal relaxation of smooth muscles resulted in significant elevations in cGMP that were accompanied by phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein (VASP), shown here for the first time to be present and phosphorylated in myometrium. Stimulation of myometrial strips with oxytocin (OT, 1 microM) produced an immediate increase in contractile force that persisted in the continued presence of the agonist. Addition of SNAP (100 microM) in the presence of OT relaxed the tissue completely as might be expected of an NO donor. SIN-1 failed to relax the myometrium at any concentration tested up to 300 microM. In Fura-2 loaded myometrial cells prepared from guinea pig, addition of SNAP (100 microM) in the absence of other agonists caused a significant, reproducible elevation of intracellular calcium while SIN-1 employed under the same conditions did not. Our data further support the notion that NO action in myometrium is distinct from that in other smooth muscles and underscores the possibility that discrete regional changes in [Ca(2+)](i), rather than cGMP, signal NO-induced relaxation of the muscle.

Topics: Animals; Calcium; Calcium Signaling; Cell Adhesion Molecules; Cyclic GMP; Dose-Response Relationship, Drug; Female; Fura-2; Guinea Pigs; Microfilament Proteins; Molsidomine; Muscle Contraction; Muscle, Smooth; Myometrium; Nitric Oxide; Nitric Oxide Donors; Oxytocin; Phosphoproteins; Phosphorylation; S-Nitroso-N-Acetylpenicillamine; Up-Regulation

We investigated the potential involvement of peroxynitrite (ONOO(-)) in the modulation of calcium current (I(Ca)) in guinea pig ventricular myocytes with the whole-cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half-life of ONOO(-) at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O(2) (-)). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 microM) or SNAP (300 microM) increased basal I(Ca) (50.3 +/- 4.6%, n = 7 and 46.2 +/- 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 microM) also stimulated basal I(Ca) (44.6 +/- 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 microM), although separately ineffective on I(Ca), enhanced the current if applied together (33.5 +/- 0.7%, n = 7). The simultaneous donor of O(2) (-) and NO, SIN-1 (500 microM), also stimulated basal I(Ca) (22.8 +/- 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 microM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 microM), I(Ca) was still increased by SIN-1 (32.0 +/- 6.1%, n = 4 and 30.0 +/- 5.4%, n = 8). Both Manganese(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP, 100 microM) a ONOO(-) scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN-1 on I(Ca) (respectively -0.6 +/- 4.1%, n = 4 and 3.6 +/- 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN-1, while it was enhanced by blocking the NO-cGMP pathway with the NO synthase inhibitor L-NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP.

Topics: Animals; Calcium; Calcium Channels; Calcium Signaling; Cyclic AMP; Cyclic GMP; Guinea Pigs; Heart Ventricles; Metalloporphyrins; Molsidomine; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Donors; Peroxynitrous Acid; Pyrogallol; Superoxide Dismutase; Up-Regulation

We showed previously, using in vitro microdialysis, that activation of the nitric oxide (NO)/cyclic GMP pathway was the underlying mechanism of exogenous NO-induced dopamine (DA) secretion from PC12 cells. In this study, infusion of the potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1, 1.0 mM for 60 min) induced a long-lasting decrease in dialysate DA+3-methoxytyramine (3-MT) in dialysates from PC12 cell suspensions. Ascorbic acid (0.2 mM) co-infusion allowed SIN-1 to increase dialysate DA+3-MT. SIN-1+ascorbic acid effects were abolished by Ca(2+) omission. Infusion of high K(+) (75 mM) induced a 2.5-fold increase in dialysate DA+3-MT. The increase was inhibited by SIN-1 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mM) with SIN-1+high K(+) resulted in a 3.5 fold increase in dialysate DA+3-MT. The L-type Ca(2+) channel inhibitor nifedipine selectively inhibited the DA+3-MT increase pertaining to high K(+), while the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]-oxadiazolo[4,3]quinoxalin-1-one selectively inhibited the increase pertaining to SIN-1 effects. These results suggest that activation of the NO/sGC/cyclic GMP pathway is the underlying mechanism of extracellular Ca(2+)-dependent effects of SIN-1 on DA secretion from PC12 cells. Extracellular Ca(2+) entry occurs through nifedipine-insensitive channels. Ascorbic acid is a key determinant in modulating the distinct profiles of SIN-1 effects.

Topics: Animals; Antioxidants; Ascorbic Acid; Calcium; Calcium Channel Blockers; Cyclic GMP; Dialysis; Dopamine; Drug Interactions; In Vitro Techniques; Molsidomine; Nifedipine; Nitric Oxide; Nitric Oxide Donors; Oxadiazoles; PC12 Cells; Potassium; Rats; Signal Transduction; Time Factors

1. We examined the role of the NO/cyclic GMP (cyclic GMP) pathway in nitric oxide (NO)- and vasoactive intestinal peptide (VIP)-induced relaxation of feline lower oesophageal sphincter (LES). Furthermore, it was studied whether methylene blue, LY83583 and ODQ, which are soluble guanylate cyclase (sGC) inhibitors, could inhibit NO-induced relaxation. 2. The nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine (L-NNA) had no effect in sodium nitropruside (SNP)-induced relaxation, but 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1)-induced relaxation was decreased by the pretreatment of L-NNA, which showed that SIN-1, not SNP, could activate NOS to cause relaxation. Methylene blue and LY83583 did not inhibit the relaxation by SNP and SIN-1. However, the more specific sGC inhibitor ODQ blocked the relaxation induced by NO donors. 3. To identify the relationship of NOS, sGC and adenylate cyclase in VIP-induced relaxation, tissue were pretreated with L-NNA and ODQ and SQ22536. These inhibitors produced significant inhibition of this response to VIP. The adenylyl cyclase inhibitor SQ 22536 also inhibited relaxation by VIP. 4. In conclusion, our data showed that SNP- and SIN-1-induced relaxation was mediated by sGC. Of sGC inhibitors, methylene blue and LY83583 were not adequate for the examination of NO donor-induced feline LES smooth muscle relaxation. VIP also caused relaxation by the pathway involving NO and cGMP and cAMP.

Topics: Aminoquinolines; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cats; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Esophagogastric Junction; In Vitro Techniques; Indomethacin; Male; Methylene Blue; Molsidomine; Muscle Relaxation; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; omega-N-Methylarginine; Oxadiazoles; Quinoxalines; Signal Transduction; Vasoactive Intestinal Peptide

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arginine; Cyclic GMP; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Guanylate Cyclase; Isoenzymes; Lactones; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Oxadiazoles; Pain; Prostaglandin-Endoperoxide Synthases; Quinoxalines; Rats; Rats, Wistar; Sulfones; Uric Acid

Monocyte chemoattractant protein-1 (MCP-1) plays an important role in glomerulonephritis and nitric oxide (NO) exerts a variety of renal pathophysiological effects. We investigated the effect of exogenous NO on pro-inflammatory cytokine-induced MCP-1 expression in human mesangial cells and its signal transduction pathway. Cells were pretreated with NO donors such as 3-morpholino-sydnonimine (SIN-1) or nitroprusside, and then stimulated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta). MCP-1 expression of mRNA and protein were measured by Northern blot analysis and ELISA. NF-kappaB binding activity was determined by electrophoretic mobility shift assay. Degradation of IkappaB-alpha protein was assessed by Western blot analysis. SIN-1 inhibited TNF-alpha- or IL-1beta-induced MCP-1 mRNA expression in a dose-dependent manner and also suppressed the MCP-1 protein expression. Nitroprusside inhibited the MCP-1 mRNA expression as well. SIN-1 dose dependently inhibited the TNF-alpha- or IL-1beta-induced NF-kappaB binding activity and suppressed the TNF-alpha-induced degradation of IkappaB-alpha. Analogue of cGMP (8-bromo-cGMP) had no significant effect on TNF-alpha-induced MCP-1 mRNA expression and guanylate cyclase inhibitor (ODQ) also had no significant influence on the inhibitory effect of SIN-1. These results suggest that exogenous NO inhibits MCP-1 expression via suppression of NF-kappaB by reducing the degradation of IkappaB-alpha and through a cGMP-independent pathway.

Topics: Cells, Cultured; Chemokine CCL2; Cyclic GMP; Glomerular Mesangium; Guanylate Cyclase; Humans; I-kappa B Proteins; Interleukin-1; Molsidomine; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Protein Binding; RNA, Messenger; Signal Transduction; Tumor Necrosis Factor-alpha

1. Nitric oxide (NO) is a key modulator of cellular Ca(2+) signalling and a determinant of mitochondrial function. Here, we demonstrate that NO governs capacitative Ca(2+) entry (CCE) into HEK293 cells by impairment of mitochondrial Ca(2+) handling. 2. Authentic NO as well as the NO donors 1-[2-(carboxylato)pyrrolidin-1-yl]diazem-1-ium-1,2-diolate (ProliNO) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) suppressed CCE activated by thapsigargin (TG)-induced store depletion. Threshold concentrations for inhibition of CCE by ProliNO and DEANO were 0.3 and 1 micro M, respectively. 3. NO-induced inhibition of CCE was not mimicked by peroxynitrite (100 micro M), the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1, 100 micro M) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 1 mM). In addition, the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a] quinoxalin-1-one (ODQ, 30 micro M) failed to antagonize the inhibitory action of NO on CCE. 4. DEANO (1-10 micro M) suppressed mitochondrial respiration as evident from inhibition of cellular oxygen consumption. Experiments using fluorescent dyes to monitor mitochondrial membrane potential and mitochondrial Ca(2+) levels, respectively, indicated that DEANO (10 micro M) depolarized mitochondria and suppressed mitochondrial Ca(2+) sequestration. The inhibitory effect of DEANO on Ca(2+) uptake into mitochondria was confirmed by recording mitochondrial Ca(2+) during agonist stimulation in HEK293 cells expressing ratiometric-pericam in mitochondria. 5. DEANO (10 micro M) failed to inhibit Ba(2+) entry into TG-stimulated cells when extracellular Ca(2+) was buffered below 1 micro M, while clear inhibition of Ba(2+) entry into store depleted cells was observed when extracellular Ca(2+) levels were above 10 micro M. Moreover, buffering of intracellular Ca(2+) by use of N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)] bis [N-[25-[(acetyloxy) methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl] ester (BAPTA/AM) eliminated inhibition of CCE by NO, indicating that the observed inhibitory effects are based on an intracellular, Ca(2+) dependent-regulatory process. 6. Our data demonstrate that NO effectively inhibits CCE cells by cGMP-independent suppression of mitochondrial function. We suggest disruption of local Ca(2+) handling by mitochondria as a key mechanism of NO induced suppression of CCE.

Topics: Barium; Calcium; Cell Line; Cyclic GMP; Diethylamines; Dose-Response Relationship, Drug; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Oxadiazoles; Oxygen Consumption; Peroxynitrous Acid; Quinoxalines; Thapsigargin; Time Factors

Leukocyte adhesion to mesothelium is an important step during peritonitis, which is mediated by adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1). We investigated the effect of exogenous nitric oxide (NO) on VCAM-1 expression in cultured human peritoneal mesothelial cells and its signal transduction pathway. Mesothelial cells were exposed to tumor necrosis factor-alpha (TNF-alpha) in the presence or absence of NO donors, 3-morpholino-sydnonimine (SIN-1) and nitroprusside (NP). VCAM-1 mRNA and protein expression were measured by Northern blot analysis and flow cytometry. Nuclear factor-kappaB (NF-kappaB) binding activity was determined by electrophoretic mobility shift assay. Both SIN-1 and NP inhibited the TNF-alpha induced VCAM-1 mRNA expression in a dose dependent manner (0.25-2 mM). SIN-1 also suppressed the cell surface expression of VCAM-1 molecule. Furthermore, SIN-1 and NP inhibited the VCAM-1 mRNA expression induced by interleukin-1beta or lipopolysaccharide as well. NF-kappaB inhibitor, PDTC dose dependently inhibited the TNF-alpha induced VCAM-1 mRNA expression. SIN-1 inhibited the TNF-alpha- induced NF-kappaB binding activity. Analogue of cGMP (8-bromo-cGMP) had no significant effect on TNF-alpha-induced VCAM-1 mRNA expression and guanylate cyclase inhibitor (ODQ) also had no significant influence on the inhibitory effect of SIN-1. These results suggest that exogenous NO inhibits VCAM-1 expression via suppression of NF-kappaB through a cGMP-independent pathway.

Topics: Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Humans; Interleukin-1; Lipopolysaccharides; Molsidomine; NF-kappa B; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Peritoneum; Signal Transduction; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Information about the presence and effects of nitric oxide (NO) in fish vasculature is scant and contradictory. We have studied the NO/cGMP system in the branchial circulation of the teleost Anguilla anguilla using a branchial basket preparation under basal conditions and cholinergic stimulation. The effects of endogenous and exogenous NO were tested with L-arginine, the nitric oxide synthase (NOS) substrate, and the NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), respectively. L-arginine (from 10(-11) to 10(-6) M) and the NO donors (starting from 10(-14) M) caused dose-dependent vasoconstriction. Conversely, in the ACh-pre-contracted preparations both donors elicited vasodilation. SIN-1-induced vasoconstriction was due to NO generation: it was increased by superoxide dismutase (SOD) and blocked by NO scavenger hemoglobin. Pre-treatment with sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of SIN-1 and SNP. The stable cGMP analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br cGMP) induced dose-dependent vasoconstriction. Unexpectedly, three NOS inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-monomethyl-L-arginine (L-NMMA), L-N(5)-(1-iminoethyl) ornithine (L-NIO), caused mild vasoconstriction. ACh caused vasoconstriction, but at pico- and nanomolar concentrations it caused mild but significant vasodilation in 40% of the preparations. Both responses, blocked by atropine and pirenzepine, required an intact endothelium. The ACh-induced vasoconstriction was substantially independent of a NO-cGMP mechanism.

Topics: Acetylcholine; Animals; Arginine; Branchial Region; Cyclic GMP; Dose-Response Relationship, Drug; Eels; Endothelium, Vascular; Female; Gills; Hemoglobins; In Vitro Techniques; Male; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Vasoconstriction

We have examined the effects of the nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on Ca(2+) transients, L-type Ca(2+) current (I(Ca,L)), and cGMP/cAMP content in electrically-stimulated rat ventricular myocytes in the absence and presence of the beta-adrenergic stimulation with isoproterenol. SIN-1 had no effect at low concentrations, but decreased the amplitude of electrically-induced Ca(2+) transients at higher concentrations. SIN-1 attenuated the increase in Ca(2+) transients induced by isoproterenol in a concentration-dependent manner. SIN-1 Also reduced the amplitude of caffeine-induced Ca(2+) transients, and the increase in I(Ca,L) induced by isoproterenol. These effects of SIN-1 were associated with an increased cGMP and a decreased cAMP content in ventricular myocytes in either the absence or presence of isoproterenol. These data suggest that the inhibitory effect of SIN-1 on basal and beta-adrenergic stimulated Ca2+ signal in ventricular myocytes could be due to the depression in the SR function and I(Ca,L), possibly mediated by a cGMP/cAMP-dependent mechanism. Taken together, the present study supports the idea that NO acts as an inhibitory modulator of the cardiac function during pathological conditions associated with an abnormal production of NO such as septic shock.

Topics: Animals; Caffeine; Calcium; Calcium Channels; Cyclic AMP; Cyclic GMP; Drug Interactions; Heart Ventricles; In Vitro Techniques; Molsidomine; Myocardium; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Vasodilator Agents

Nitric oxide has been proposed to play an important role in neuronal development. We have previously shown that growth cones from an identified neuron, B5, in the snail Helisoma trivolvis, respond to nitric oxide (NO) donors by increasing the length of their filopodia within minutes of application (Van Wagenen and Rehder, 1999). This effect was mediated through a cGMP-induced increase in [Ca2+]i and resulted in an enlargement of the growth cone's action radius, suggesting that NO could function as a signaling molecule during neuronal pathfinding. We show here that NO functions as a specific rather than a general regulator of growth cone filopodia, because another identified neuron from the same ganglion, B19, failed to respond to NO with an increase in filopodial length. We found that, contrary to B5 neurons, B19 growth cones contained little or no soluble guanylyl cyclase (sGC) immunoreactivity, presumably preventing their response to NO. This hypothesis was supported by the finding that the sGC activator YC-1 (10 microM) had no effect on B19 filopodia but induced elongation of B5 filopodia. These results indicate that the effects of NO can be quite specific, and raise the interesting possibility that neurons could selectively tune in to NO by differentially expressing the target enzyme sGC in the appropriate cellular location during critical developmental stages. In addition, our NADPH-diaphorase staining and anti-NOS immunohistochemisty suggest that growth cones of B5 neurons, but not of B19 neurons, could be a source of NO, making NO a potential intra- and transcellular messenger.

Topics: Animals; Cells, Cultured; Cyclic GMP; Ganglia, Invertebrate; Growth Cones; Guanylate Cyclase; Molsidomine; NADPH Dehydrogenase; Nervous System; Nitric Oxide; Nitric Oxide Donors; Potassium Chloride; Snails

Low intrathecal (i.t.) doses of the nitric oxide (NO)-donor 3-morpholinosydnonimine (SIN-1) (0.1-2.0 microg/10 microl) reduced, while higher doses had no effect (5 or 100 microg/10 microl) or increased (10 and 20 microg/10 microl) the mechanical allodynia induced by chronic ligature of the sciatic nerve in rats. SIN-1 (0.1-100 microg/10 microl; i.t.) produced only antinociceptive effect in the rat tail flick test. The inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (4 microg/10 microl; i.t.), abolished the antinociceptive effects of SIN-1 in both tests and reduced the effect of high doses of SIN-1 in neuropathic rats. Hemoglobin (100 microg/10 microl; i.t.), a NO scavenger, inhibited the effect of low dose of SIN-1 and reduced the effect of high dose of SIN-1 in neuropathic rats. 8-Bromo-cGMP (125-500 microg/10 microl; i.t.), reduced the mechanical allodynia in neuropathic rats. The NO-synthase inhibitors, NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA) (75-300 microg/10 microl; i.t.) reduced the mechanical allodynia evoked by nerve injury and increased the tail-flick latency, respectively. These effects were reduced and inhibited, respectively, by previous i.t. ODQ. The effect of L-NOARG was enhanced in a non-significant manner by hemoglobin. These results indicate that SIN-1 and NO-synthase inhibitors reduce pain through a spinal mechanism that involves activation of guanylate cyclase. The effects of SIN-1 vary depending on the dose and pain model utilized, but its most sensitive effect seems to be antinociception. However, high doses of the NO-donor can intensify ongoing pain.

Topics: Animals; Chronic Disease; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hemoglobins; Injections, Spinal; Ligation; Male; Molsidomine; Nerve Compression Syndromes; Neuralgia; Nitric Oxide; Nitric Oxide Donors; Nitroarginine; Nociceptors; omega-N-Methylarginine; Oxadiazoles; Pain Measurement; Quinoxalines; Rats; Rats, Wistar; Sciatic Nerve

As nitric oxide is a gas, it cannot be stored and has to be synthesized as required. This suggests that it could be released wherever nitric oxide synthase (NOS) is activated and due to its unstable state will react with appropriate targets at this site of production. In both dissociated dorsal root ganglion (DRG) cultures and in acutely isolated, but intact, DRG, treatment with capsaicin or bradykinin caused cGMP synthesis, which could be blocked by NOS inhibitors. The cGMP was synthesized in cells different from those expressing the neuronal isoform of NOS (nNOS). In dissociated cultures many of the cells stimulated to produce cGMP were neurons, whereas in isolated ganglia they were always satellite glia cells. Surprisingly, the satellite glia cells surrounding the nNOS-containing neurons did not contain cGMP. Following nerve section in adult rats, many axotomized ganglion neurons expressed nNOS. Again in these axotomized ganglia, most cGMP was expressed in the satellite glia surrounding nNOS-negative neurons. However, an nNOS-selective inhibitor reduced the cGMP present in these axotomized ganglia, suggesting that the cGMP synthesized is stimulated by NO (nitrogen monoxide) produced by nNOS. In both dissociated cultures and axotomized ganglia, nNOS-containing processes were observed close to cGMP-positive cells. These observations lead to the suggestion that NO acts in a paracrine fashion when stimulating the synthesis of cGMP and may not be synthesized at all sites containing nNOS.

Topics: Animals; Animals, Newborn; Autocrine Communication; Axotomy; Bradykinin; Capsaicin; Carbon-Nitrogen Ligases; Cells, Cultured; Cyclic GMP; Female; Ganglia, Spinal; Immunohistochemistry; Male; Molsidomine; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oligodendroglia; Paracrine Communication; Rats; Rats, Wistar; Sciatic Nerve

To investigate the effect of nitric oxide (NO) on cardiac energy metabolism, isolated cardiomyocytes of Wistar rats were incubated in an Oxystat system at a constant ambient PO2 (25 mmHg) and oxygen consumption (VO2); free intracellular Ca(2+) (fura 2), free cytosolic adenosine [S-adenosylhomocysteine (SAH) method], and mitochondrial NADH (autofluorescence) were measured after application of the NO donor morpholinosydnonimine (SIN-1). In Na(+)-free medium (contracting cardiomyocytes), VO2 increased from 7.9 +/- 1.2 to 26.4 +/- 3.1 nmol x min(-1) x mg protein(-1). SIN-1 (100 micromol/l) decreased VO2 in contracting (-21 +/- 3%) and in quiescent cells (-24 +/- 7%) by the same extent. Inhibition of VO2 was dose dependent (EC(50): 10(-7) mol/l). S-nitroso-N-acetyl-penicillamine, another NO donor, also inhibited VO2, whereas SIN-1C (100 micromol/l), the degradation product of SIN-1, displayed no inhibitory effect. Intracellular Ca(2+) remained unchanged, and inhibition of protein kinases G, A, or C did not antagonize the effect of NO. Mitochondrial NADH increased with NO, indicating a reduced flux through the respiratory chain. In quiescent but not in contracting cardiomyocytes, NO significantly increased adenosine, indicating a reduced energy status. These data suggest the following. 1) NO decreases cardiac respiration, most likely via direct inhibition of the respiratory chain. 2) Whereas in quiescent cardiomyocytes the inhibition of aerobic ATP formation by NO causes reduction in energy status, contracting cells are able to compensate for the NO-induced inhibition of oxidative phosphorylation, maintaining energy status constant.

Topics: Adenosine; Alkaloids; Animals; Calcium; Carbazoles; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Electron Transport; Energy Metabolism; Enzyme Inhibitors; Indoles; Molsidomine; Muscle Fibers, Skeletal; Myocardium; NAD; Nitric Oxide; Nitric Oxide Donors; Oxygen Consumption; Penicillamine; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine

Leukocyte accumulation has been shown to be increased in sepsis. Moreover, in inducible nitric oxide synthase (iNOS) knockout mice, a further increase in leukocyte accumulation has been observed during sepsis, suggesting that nitric oxide (NO) may affect leukocyte/endothelial interaction. Accelerated peroxynitrite formation also occurs during sepsis. In the present study, the effect of peroxynitrite or NO on leukocyte adhesion to nitric oxide synthase (NOS)-inhibited or endotoxin-treated endothelium was examined. Bovine aortic endothelial cells were treated with either L-NAME or lipopolysaccharide (LPS) and interferon-gamma for 4 hr and subsequent leukocyte adhesion was measured. Both L-NAME and LPS treatment resulted in increased leukocyte adhesion compared with control. Neither a peroxynitrite donor, SIN-1, nor a direct NO donor, DETA-NO, had any effect on leukocyte adhesion to untreated endothelium. However, when the L-NAME or LPS-treated endothelial cells were treated simultaneously with either SIN-1 or DETA-NO, there was a significant reduction in leukocyte adhesion. Moreover, at the concentrations used in the present study, neither peroxynitrite nor NO showed harmful effects on normal cultured endothelial cells. These data demonstrating inhibition of leukocyte adhesion to endotoxin-treated endothelium suggest that peroxynitrite or NO may exert a beneficial effect during sepsis.

Topics: Animals; Cattle; Cell Adhesion; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Interferon-gamma; Leukocytes; Lipopolysaccharides; Molsidomine; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Triazenes

1. In myometrial strips from near-term non-labouring human uterus, addition of oxytocin (OT) evoked dose-dependent (10 - 3000 nM) phasic contractions that were antagonized by atosiban (1 microM) and relaxed by addition of the nitric oxide donor S-nitroso L-cysteine (Cys-NO). In near-term labouring myometrium, however, addition of OT was ineffective at raising additional tone. 2. In both labouring and non-labouring tissue, Cys-NO mediated relaxation of spontaneous or OT-induced contractions (IC(50)=1 microM) was unaffected by prior addition of the guanylyl cyclase (GC) inhibitors ODQ (1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one; 1 microM), or methylene blue (MB; 10 microM). 3. Elevation of intracellular cyclic GMP accompanying 30 microM Cys-NO addition in non-labouring tissue (7.5 fold) or in labouring tissues (2.5 fold) was completely blocked in tissues that had been pre-treated with ODQ or MB. 4. Charybdotoxin (ChTx), iberiotoxin (IbTx) and kaliotoxin (KalTx) all shifted the Cys-NO inhibition curve to the right and reduced the degree of relaxation produced by maximal Cys-NO treatment (100 microM in non-labouring tissue; in labouring tissue, KalTx prevented Cys-NO mediated relaxation in both stimulated and unstimulated tissue. 5. Addition of the NO-donor S-nitroso N-acetyl penicillamine (SNAP) produced a dose-dependent relaxation of pregnant myometrium while 3-morpholinosyndonimine (SIN-1) did not. The failure of SIN-1 to relax OT-induced contractions was not due to a failure of the donor to stimulate myometrial GC. 6. We demonstrate that despite the ability of NO to stimulate myometrial GC in pregnant uterine muscle, relaxations are independent of cyclic GMP action. Effects of K(+)-channel inhibitors suggests that NO-induced relaxation in human uterine smooth muscle may be subserved by direct or indirect activation of one or more calcium-activated K(+)-channels.

Topics: Charybdotoxin; Cyclic GMP; Cysteine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Guanylate Cyclase; Humans; In Vitro Techniques; Labor, Obstetric; Molsidomine; Muscle Relaxation; Muscle, Smooth; Myometrium; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Oxadiazoles; Oxytocin; Penicillamine; Peptides; Pregnancy; Quinoxalines; S-Nitrosothiols; Scorpion Venoms; Time Factors; Uterus

Topics: Acetylcholine; Animals; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Guanylate Cyclase; In Vitro Techniques; Isometric Contraction; Methylene Blue; Mice; Molsidomine; Nitric Oxide Donors; Pregnancy; Uterine Contraction; Uterus

Nitric oxide (NO) is thought to play an important role in the regulation of neonatal pulmonary vasculature. It has been suggested that neonates with pulmonary hypertension have a defective NO pathway. Therefore, we measured in 1-day-old piglets exposed to hypoxia (fraction of inspired O(2) = 0.10) for 3 or 14 days to induce pulmonary hypertension 1) the activity of NO synthase (NOS) via conversion of L-arginine to L-citrulline and the concentration of the NO precursor L-arginine in isolated pulmonary vessels, 2) the vasodilator response to the NO donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) and the cGMP analog 8-bromo-cGMP in isolated perfused lungs, and 3) the production of cGMP in response to SIN-1 in isolated perfused lungs. After 3 days of exposure to hypoxia, endothelial NOS (eNOS) activity was unaffected, whereas, after 14 days of hypoxia, eNOS activity was decreased in the cytosolic fraction of pulmonary artery (P < 0.05) but not of pulmonary vein homogenates. Inducible NOS activity was decreased in the cytosolic fraction of pulmonary artery homogenates after both 3 (P < 0.05) and 14 (P < 0.05) days of hypoxia but was unchanged in pulmonary veins. Pulmonary artery levels of L-arginine were unaffected by hypoxic exposure. After 3 days of exposure to hypoxia, the reduction in the dilator response to SIN-1 (P < 0.05) coincided with a decrease in cGMP production (P < 0.005), suggesting that soluble guanylate cyclase activity may be altered. When the exposure was prolonged to 14 days, dilation to SIN-1 remained decreased (P < 0.05) and, although cGMP production normalized, the dilator response to 8-bromo-cGMP decreased (P < 0.05), suggesting that, after prolonged exposure to hypoxia, cGMP-dependent mechanisms may also be impaired. In conclusion, neonatal hypoxia-induced pulmonary hypertension is associated with multiple disruptions in the NO pathway.

Topics: Animals; Animals, Newborn; Arginine; Blood Vessels; Cyclic GMP; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Pulmonary Circulation; Swine; Vasodilation; Vasodilator Agents

1. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP- and vasoactive intestinal polypeptide (VIP)/adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-signalling pathways in the gastric fundus of wild type and cGKI-deficient mice. 2. Using immunohistochemistry, similar staining patterns for NO-synthase, cyclic GMP- and VIP-immunoreactivities were found in wild type and cGKI-deficient mice. 3. In isolated, endothelin-1 (3 nM - 3 microM)-contracted, muscle strips from wild type mice, electrical field stimulation (1 - 16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI-deficient mice only the slowly developing relaxation was observed. 4. The responses to the NO donor, SIN-1 (10 nM - 100 microM), and to 8-Br-cyclic GMP (10 nM - 100 microM) were markedly impaired in strips from cGKI-deficient mice, whereas the responses to VIP (0.1 nM - 1 microM) and forskolin (0.1 nM - 1 microM) were similar to those in wild type mice. 5. These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP-pathway can exert their effects independently of cGKI.

Topics: Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Electric Stimulation; Female; Fluorescent Antibody Technique; Immunohistochemistry; Male; Mice; Mice, Knockout; Molsidomine; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; Receptor Cross-Talk; Signal Transduction; Stomach; Vasoactive Intestinal Peptide

We tested the hypothesis that increasing myocardial cyclic GMP would attenuate cyclic AMP induced positive inotropy and O2 consumption, in part, through changes in cyclic AMP and that renal hypertension-induced cardiac hypertrophy (HYP) would alter this relationship. Anesthetized, open chest rabbits (N = 48) were divided into four groups of control (CON) and HYP animals which received vehicle (VEH), isoproterenol 10(-6)M (ISO), 3-morpholinosyndnonimine 10(-4)M, (SIN-1), or a combination of ISO+SIN-1. Coronary blood flow (microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption in both subepicardium (EPI) and subendocardium (ENDO). Left ventricular change in wall thickness (%) was increased significantly by ISO in both CON (16 +/- 4 to 31 +/- 6) and HYP (17 +/- 2 to 24 +/- 3). Percent change in wall thickness was similar in the CON, SIN-1, and ISO+SIN-1 groups. Myocardial O2 consumption (ml O2/min/100 g) was increased by ISO in CON (10.3 +/- 1.0 to 13.6 +/- 2.0 EPI; 10.9 +/- 1.0 17.1 +/- 1.7 ENDO) and HYP (8.2 +/- 1.4 to 12.3 +/- 2.2 EPI; 6.6 +/- 1.4 to 14.8 +/- 1.8 ENDO). Oxygen consumption was unaffected by SIN-1 in CON and HYP animals. ISO+SIN-1 caused attenuated ISO-induced increases in O2 consumption in CON in EPI and ENDO, and in EPI in HYP. Cyclic GMP (pmol/g) was unchanged by ISO in CON and HYP, and increased by SIN-1 in CON (8.1 +/- 1.3 to 19.2 +/- 2.3 EPI) and HYP (9.1 +/- 1.5 to 12.8 +/- 2.0 EPI). Cyclic GMP remained elevated with ISO+SIN-1 in both groups. Cyclic AMP (pmol/g) was increased significantly by ISO in CON (496 +/- 43 to 725 +/- 106 EPI; 534 +/- 44 to 756 +/- 148 ENDO) and insignificantly in HYP (435 +/- 50 to 566 +/- 35 EPI; 497 +/- 51 to 583 +/- 47 ENDO). Cyclic AMP levels were unaffected by SIN-1 in either group. Isoproterenol induced increases in cyclic AMP were blunted by ISO+SIN-1 in CON (496 +/- 43 to 537 +/- 59 EPI) and not affected in HYP. The current study demonstrated attenuation of cyclic AMP mediated increased inotropy and O2 consumption by increasing cyclic GMP, which appeared, in part, related to cyclic GMP-induced reduction in cyclic AMP. This effect of cyclic GMP on cyclic AMP was not observed in myocardial hypertrophy.

Topics: Animals; Cardiomegaly; Cardiotonic Agents; Cyclic AMP; Cyclic GMP; Isoproterenol; Molsidomine; Myocardial Contraction; Oxygen Consumption; Rabbits; Vasodilator Agents

We compared the effects of two redox forms of nitric oxide, NO(+) [liberated by S-nitroso-N-acetyl-penicillamine (SNAP)] and NO. [liberated by 3-morpholinosydnonimine (SIN-1) in the presence of superoxide dismutase], on cytosolic concentration of Ca(2+) ([Ca(2+)](i); single cells) and tone (intact strips) obtained from human main stem bronchi and canine trachealis. SNAP evoked a rise in [Ca(2+)](i) that was unaffected by removing external Ca(2+) but was markedly reduced by depleting the internal Ca(2+) pool using cyclopiazonic acid (10(-5) M). Dithiothreitol (1 mM) also antagonized the Ca(2+) transient as well as the accompanying relaxation. SNAP attenuated responses to 15 and 30 mM KCl but not those to 60 mM KCl, suggesting the involvement of an electromechanical coupling mechanism rather than a direct effect on the contractile apparatus or on Ca(2+) channels. SNAP relaxations were sensitive to charybdotoxin (10(-7) M) or tetraethylammonium (30 mM) but not to 4-aminopyridine (1 mM). Neither SIN-1 nor 8-bromoguanosine 3',5'-cyclic monophosphate had any significant effect on resting [Ca(2+)](i), although both of these agents were able to completely reverse tone evoked by carbachol (10(-7) M). We conclude that NO(+) causes release of internal Ca(2+) in a cGMP-independent fashion, leading to activation of Ca(2+)-dependent K(+) channels and relaxation, whereas NO. relaxes the airways through a cGMP-dependent, Ca(2+)-independent pathway.

Topics: 4-Aminopyridine; Animals; Bronchi; Calcium; Calcium-Transporting ATPases; Charybdotoxin; Cyclic GMP; Dithiothreitol; Dogs; Enzyme Activation; Free Radicals; Guanylate Cyclase; Humans; In Vitro Techniques; Molsidomine; Muscle, Smooth; Nitric Oxide; Nitric Oxide Donors; Oxidation-Reduction; Penicillamine; Potassium Channels; Potassium Chloride; Tetraethylammonium; Trachea

Inducible heme oxygenase (HO-1) has recently been recognized as an antioxidant and cytoprotective gene. By use of Western blotting, cell viability analysis, and antisense technique, the present study investigates the involvement of HO-1 in endothelial protection induced by the clinically used nitric oxide (NO) donor molsidomine (specifically, its active metabolite 3-morpholinosydnonimine [SIN-1]) and the second messenger cGMP. In bovine pulmonary artery endothelial cells, SIN-1 and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at 1 to 100 micromol/L induced the synthesis of HO-1 protein in a concentration-dependent fashion up to 3-fold over basal levels. HO-1 induction by SIN-1 was inhibited in the presence of the NO scavenger phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4, 3-a]quinoxalin-1-one. 8-Bromo-cGMP (1 to 100 micromol/L) and dibutyryl cGMP (1 to 100 micromol/L) as well as the activator of particulate guanylyl cyclase atrial natriuretic peptide (1 to 100 nmol/L) produced increases in HO-1 protein similar to those produced by SIN-1. SIN-1 and 8-bromo-cGMP increased heme oxygenase activity (bilirubin formation). Cytoprotection by NO donors was abrogated in the presence of the heme oxygenase inhibitor tin protoporphyrin IX. Pretreatment of cells with a phosphorothioate-linked HO-1 antisense oligonucleotide prevented protection by SIN-1 or 8-bromo-cGMP against tumor necrosis factor-alpha cytotoxicity, whereas sense and scrambled HO-1 were without effect under these conditions. Our results show for the first time that HO-1 is a cGMP-sensitive endothelial gene and establish conclusively a causal relationship between HO-1 induction and endothelial protection by the NO/cGMP system. By targeting cytoprotective HO-1, NO donors may therefore be expected to induce antioxidant, antiatherogenic, and anti-inflammatory effects.

Topics: Animals; Cattle; Cell Survival; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Induction; Heme Oxygenase (Decyclizing); Molsidomine; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Pulmonary Artery; Second Messenger Systems

To better understand the mechanism(s) underlying nitric oxide (. NO)-mediated toxicity, in the presence and absence of concomitant oxidant exposure, postmitotic terminally differentiated NT2N cells, which are incapable of producing. NO, were exposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1). Exposure to SIN-1, which generated peroxynitrite in the range of 25-750 nM/min, produced a concentration- and time-dependent delayed cell death. In contrast, a critical threshold concentration (>440 nM/min) was required for. NO to produce significant cell injury. Examination of cells by electron microscopy shows a largely necrotic injury after peroxynitrite exposure but mainly apoptotic-like morphology after. NO exposure. Cellular levels of reduced thiols correlated with cell death, and pretreatment with N-acetylcysteine (NAC) fully protected from cell death in either PAPA/NO or SIN-1 exposure. NAC given within the first 3 h posttreatment further delayed cell death and increased the intracellular thiol level in SIN-1 but not. NO-exposed cells. Cell injury from. NO was independent of cGMP, caspases, and superoxide or peroxynitrite formation. Overall, exposure of non-. NO-producing cells to. NO or peroxynitrite results in delayed cell death, which, although occurring by different mechanisms, appears to be mediated by the loss of intracellular redox balance.

Topics: Acetylcysteine; Animals; Cell Death; Cell Differentiation; Cell Line; Cell Survival; Cyclic GMP; Hydrazines; Molsidomine; Necrosis; Neurons; Nitrates; Nitric Oxide; Nitric Oxide Donors; Oxidants; Sulfhydryl Compounds

Mammalian vestibular organs have two types of hair cell, type I and type II, which differ morphologically and electrophysiologically. Type I hair cells alone express an outwardly rectifying current, I(K, L), which activates at relatively negative voltages. We used whole cell and patch configurations to study I(K,L) in hair cells isolated from the sensory epithelia of rat semicircular canals. I(K,L) was potassium selective, blocked by 4-aminopyridine, and permeable to internal cesium. It activated with sigmoidal kinetics and was half-maximally activated at -74.5 +/- 1.6 mV (n = 35; range -91 to -50 mV). It was a very large conductance (91 +/- 8 nS at -37 mV; 35 nS/pF for a cell of average size). Patch recordings from type I cells revealed a candidate ion channel with a conductance of 20-30 pS. Because I(K,L) was activated at the resting potential, the cells had low input resistances (R(m)): median 25 MOmega at -67 mV versus 1.3 GOmega for type II cells. Consequently, injected currents comparable to large transduction currents (300 pA) evoked small (

Topics: 4-Aminopyridine; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cyclic GMP; Gluconates; Guanylate Cyclase; Hair Cells, Vestibular; Membrane Potentials; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Patch-Clamp Techniques; Penicillamine; Potassium; Potassium Channel Blockers; Rats; Rats, Long-Evans; Semicircular Canals; Signal Transduction

NO-Aspirin (NCX-4016) releases nitric oxide (NO) in biological systems through as yet unidentified mechanisms. In LLC-PK1 kidney epithelial cells, a 5-h pretreatment with glyceryl trinitrate (GTN, 0.1-1 microM) significantly attenuated the cyclic GMP response to a subsequent challenge with both NO-aspirin or GTN. Similarly, NO-aspirin (10-100 microM) was found to induce tolerance to its own cyclic GMP stimulatory action and to that of GTN. In contrast, cyclic GMP stimulation by the spontaneous NO donor SIN-1, which releases NO independently of enzymatic catalysis, remained unimpaired in cells pretreated with GTN or NO-aspirin. The observed cross-tolerance between NO-aspirin and GTN cells indicates that bioactivation pathways of organic nitrates, which have been shown to involve cytochrome P450, may also be responsible for NO release from NO-aspirin. Prolonged treatment with NO-aspirin causes down-regulation of the cellular cyclic GMP response, suggesting that tolerance may occur during therapy with NO-aspirin.

Topics: Animals; Aspirin; Cell Line; Cyclic GMP; Cyclic N-Oxides; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Epithelial Cells; Free Radical Scavengers; Imidazoles; Kidney; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Protein Binding; Swine

Mesangial cells (MC), grown on extracellular matrix (ECM) protein-coated plates and stretched, proliferate and produce ECM, recapitulating in vivo responses to increased glomerular capillary pressure (Pgc). Transduction of strain involves mitogen-activated protein kinases (MAPK), and we have shown that p38 MAPK is activated by strain in MC. Because in vivo studies show that nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing Pgc, we studied whether NO attenuated stretch-induced p38 activation in MC. Increasing p38 activation occurred with increasing stretch, maximally at 10 min at -27-kPa vacuum. Cyclic strain increased nuclear translocation of phosphorylated p38 by immunofluorescent microscopy and nuclear protein binding to nuclear factor-kappaB (NF-kappaB) consensus sequences by mobility shift assay. Both events were largely abrogated by the p38 inhibitor SB-203580. The NO donors 3-morpholinosydnonimine, S-nitroso-N-acetylpenicillamine, and 8-bromoguanosine 3',5'-cyclic monophosphate, a stable cGMP analog, prevented p38 activation and nuclear translocation. Thus strain induces p38 activity and translocation to the nucleus and p38-dependent increases in nuclear protein binding to NF-kappaB. This pathway is attenuated by the NO donors or a cGMP analog.

Topics: Animals; Biological Transport; Cells, Cultured; Cyclic GMP; Enzyme Activation; Enzyme Inhibitors; Fluorescent Antibody Technique; Glomerular Mesangium; Imidazoles; Mitogen-Activated Protein Kinases; Molsidomine; Nitric Oxide; Nitric Oxide Donors; p38 Mitogen-Activated Protein Kinases; Penicillamine; Pyridines; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Stress, Mechanical

The functional role of nitric oxide (NO) and the guanylate cyclase/cGMP second messenger system was investigated in the mouse bladder. Electrical field stimulation and the NO-donor 3-morpholino-sydnonimin hydrochloride (SIN-1) did not induce relaxation of the carbachol-precontracted bladder. However, sodium nitroprusside (10(-3) M) was found to enhance the contractile response to electrical field stimulation by 24+/-6% (n=8; P<0.05) without affecting the contractile response to carbachol. The enhancement of bladder contractility evoked by sodium nitroprusside was inhibited by the guanylate cyclase inhibitor 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalime-1-one (ODQ; 10(-6) M). Incubation of bladder strips with SIN-1 and sodium nitroprusside caused an increase in cGMP accumulation as measured by radioimmunoassay. Immunohistochemical studies showed cGMP-immunoreactivity in nerve fibres and in stromal cells, but not in smooth muscle bundles after exposure to NO-donors. The results show that NO-donors have no inhibitory effect on smooth muscle tone in the mouse bladder, but that NO may have a functional role as an excitatory neuromodulator. The targets of endogenous NO in the bladder may be the demonstrated cGMP-positive structures, i.e., nerves and stromal cells.

Topics: Animals; Arginine; Carbachol; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Female; In Vitro Techniques; Mice; Mice, Inbred BALB C; Molsidomine; Muscle Relaxation; Muscle, Smooth; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Phosphodiesterase Inhibitors; Piperazines; Piperidines; Purines; Quinazolines; Sildenafil Citrate; Sulfones; Urethra; Urinary Bladder

In some fish blood vessels, the existence of a NO (nitric oxide) system has been reported. We examined the possibility that this NO system acts as an endothelium-derived relaxing factor (EDRF) in carp aorta using the carp aorta alone and in a combined carp-rat aorta donor-detector system. Use of the typical NO stimulating agent in mammal acetylcholine (ACh) only induced constriction of the carp aorta. This response was not modified by denudation or by NO synthesis inhibition with N-nitro-L-arginine methyl ester. Neither the indirect NO stimulating agents bradykinin and histamine nor the direct NO releasers sodium nitroprusside (SNP) and SIN-1 induced vasorelaxation. Both SNP and ACh elevated the cGMP concentration in rat aorta, but not in carp aorta. In the aorta combination set-up, where carp served as a NO donor and rat aorta served as a NO detector, no relaxation of the rat aorta was observed. The calcium ionophore A23187, a known EDRF producer in mammals, induced relaxation of carp aorta through an endothelium- and cyclooxygenase-dependent mechanism. These results indicate that carp aorta does not produce NO as an EDRF nor does it respond to exogenously supplied NO. The major EDRF in carp is apparently a product(s) of cyclooxygenase metabolism.

Topics: Acetylcholine; Animals; Aorta; Calcimycin; Carps; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; In Vitro Techniques; Ionophores; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Phenylephrine; Rats; Rats, Wistar; Vasodilator Agents

Nitric oxide (NO) has concentration-dependent biphasic myocardial contractile effects. We tested the hypothesis, in isolated rat hearts, that NO cardiostimulation is primarily non-cGMP dependent. Infusion of 3-morpholinosydnonimine (SIN-1, 10(-5) M), which may participate in S-nitrosylation (S-NO) via peroxynitrite formation, increased the rate of left ventricular pressure rise (+dP/dt; 19 +/- 4%, P < 0.001, n = 11) without increasing effluent cGMP or cAMP. Superoxide dismutase (SOD; 150 U/ml) blocked SIN-1 cardiostimulation and led to cGMP elaboration. Sodium nitroprusside (10(-10)-10(-7) M), an iron nitrosyl compound, did not augment +dP/dt but increased cGMP approximately eightfold (P < 0.001), whereas diethylamine/NO (DEA/NO; 10(-7) M), a spontaneous NO. donor, increased +dP/dt (5 +/- 2%, P < 0.05, n = 6) without augmenting cGMP. SIN-1 and DEA/NO +dP/dt increase persisted despite guanylyl cyclase inhibition with 1H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-1-one (10(-5) M, P < 0.05 for both donors), suggesting a cGMP-independent mechanism. Glutathione (5 x 10(-4) M, n = 15) prevented SIN-1 cardiostimulation, suggesting S-NO formation. SIN-1 also produced SOD-inhibitable cardiostimulation in vivo in mice. Thus peroxynitrite and NO donors can stimulate myocardial contractility independently of guanylyl cyclase activation, suggesting a role for S-NO reactions in NO/peroxynitrite-positive inotropic effects in intact hearts.

Topics: Animals; Cyclic GMP; Diethylamines; Drug Combinations; Enzyme Inhibitors; Glutathione; In Vitro Techniques; Male; Molsidomine; Myocardial Contraction; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Nucleotides, Cyclic; Oxadiazoles; Oxidation-Reduction; Quinoxalines; Rats; Rats, Wistar; Superoxide Dismutase

Nitric oxide (NO) donors increase heart rate (HR) through a guanylyl cyclase-dependent stimulation of the pacemaker current I(f), without affecting basal I(Ca-L). The activity of I(f)is known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca(2+). We examined the role of cGMP-dependent signaling pathways and intracellular Ca(2+)stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1-100 micromol/l 3-morpholino-sydnonimine (SIN-1; with superoxide dismutase, n=6) or diethylamine-NO (DEA-NO, n=8) was significantly attenuated by blockers of the cGMP-inhibited phosphodiesterase (PDE3; trequinsin, milrinone or Ro-13-6438, n=22). In addition, the rate response to DEA-NO or sodium nitroprusside (SNP) was significantly reduced following inhibition of PKA (KT5720 or H-89, n=15) but not PKG (KT5728 or Rp-8-pCPT-cGMPs, n=16). Suppression of sarcoplasmic (SR) Ca(2+)release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 micromol/l and 60 micromol/l, n=16) significantly reduced the chronotropic response to 1-100 micromol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 micromol/l SNP significantly increased diastolic and peak Ca(2+)fluorescence (+13+/-1% and +28+/-1%, n=6, P<0.05). Our findings are consistent with a functionally significant role of cAMP/PKA signaling (via cGMP inhibition of PDE3) and SR Ca(2+)in mediating the positive chronotropic effect of NO donors.

Topics: Animals; Calcium; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guinea Pigs; Heart Atria; Heart Rate; Hydrazines; Indoles; Isoquinolines; Male; Milrinone; Models, Biological; Molsidomine; Nitric Oxide Donors; Nitrogen Oxides; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Platelet Aggregation Inhibitors; Pyrroles; Quinazolines; Ryanodine; Sarcoplasmic Reticulum; Signal Transduction; Spectrometry, Fluorescence; Sulfonamides; Superoxide Dismutase; Tetrahydroisoquinolines; Thionucleotides; Time Factors; Vasodilator Agents

Topics: Animals; Coronary Circulation; Cyclic GMP; Heart; Molsidomine; Myocardial Contraction; Myocardium; Nitric Oxide; Nitric Oxide Donors

Our recent work has suggested that the nitric oxide/guanosine 3', 5'-cyclic monophosphate (NO/cGMP) signal transduction system contributes to central sensitization of spinothalamic tract (STT) neurons in part by influencing the descending inhibition of nociception resulting from stimulation in the periaqueductal gray. This study was designed to examine further whether activation of the NO/cGMP cascade reduces the inhibition of the activity of STT neurons mediated by spinal inhibitory amino acid (IAA) receptors. Responses of STT cells to noxious cutaneous stimuli were inhibited by iontophoresis of glycine and GABA agonists in anesthetized monkeys. Administration of 8-bromoguanosine-3',5'-cyclophosphate sodium (8-bromo-cGMP), a membrane permeable analogue of cGMP, either by microdialysis or by iontophoresis reduced significantly the IAA-induced inhibition of wide dynamic range (WDR) STT cells in the deep layers of the dorsal horn. The reduction in inhibition lasted for up to 1-1.5 h after the cessation of drug infusion. In contrast, IAA-induced inhibition of WDR STT cells in the superficial dorsal horn and high-threshold (HT) cells in superficial or deep layers was not significantly changed during 8-bromo-cGMP infusion. Iontophoresis of 8-bromo-cGMP onto STT cells produced the same actions as produced by microdialysis of this agent, but the effect was not as long-lasting nor as potent. Finally, an attenuation of the IAA receptor-mediated inhibition of STT cells produced by iontophoretic release of a NO donor, 3-morpholinosydnonimine, could be blocked by pretreatment of the spinal cord with a guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. These results suggest that an increased spinal cGMP level contributes to the sensitization of WDR STT neurons in the deep dorsal horn in part by down-regulating spinal IAA receptors. However, no evidence is provided in this study that the NO/cGMP cascade regulates IAA receptors on HT and superficial WDR neurons. Combined with the preceding studies, our data support the view that NO and cGMP function in the same signal transduction cascade and play an important role in central sensitization.

Topics: Animals; Cyclic GMP; Enzyme Inhibitors; gamma-Aminobutyric Acid; Glycine; Guanylate Cyclase; Iontophoresis; Macaca fascicularis; Male; Microdialysis; Molsidomine; Neural Inhibition; Neurons; Nitric Oxide Donors; Receptors, GABA; Receptors, Glycine; Spinothalamic Tracts

Nitric oxide (NO) has been proposed to play an important role during neuronal development. Since many of its effects occur during the time of growth cone pathfinding and target interaction, we here test the hypothesis that part of NO's effects might be exerted at the growth cone. We found that low concentrations of the NO-donors DEA/NO, SIN-1, and SNP caused a rapid and transient elongation of filopodia as well as a reduction in filopodial number. These effects resulted from distinct changes in filopodial extension and retraction rates. Our novel findings suggest that NO could play a physiological role by temporarily changing a growth cone's morphology and switching its behavior from a close-range to a long-range exploratory mode. We subsequently dissected the pathway by which NO acted on growth cones. The effect of NO donors on filopodial length could be blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase (sGC), indicating that NO acted via sGC. Supporting this idea, injection of cyclic GMP (cGMP) mimicked the effect of NO donors on growth cone filopodia. Moreover, application of NO-donors as well as injection of cGMP elicited a rapid and transient rise in intracellular calcium in growth cones, indicating that NO acted via cGMP to elevate calcium. This calcium rise, as well as the morphological effects of SIN-1 on filopodia, were blocked by preventing calcium entry. Given the role of filopodia in axonal guidance, our new data suggest that NO could function at the neuronal growth cone as an intracellular and/or intercellular signaling molecule by affecting steering decisions during neuronal pathfinding.

Topics: Animals; Calcium; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Growth Cones; Guanylate Cyclase; Hydrazines; Molsidomine; Mutagens; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroprusside; Oxadiazoles; Pseudopodia; Quinoxalines; Snails; Sulfhydryl Reagents

Using an established cell culture model, the present study investigates whether linsidomine (SIN-1), a spontaneous donor of nitric oxide and active metabolite of the antianginal drug molsidomine, induces tolerance to its own cyclic GMP stimulatory action or shows a diminished response after tolerance induction with glyceryl trinitrate.. Incubations with nitric oxide donors were carried out in LLC-PK1 kidney epithelial cells. Intracellular levels of cyclic GMP, the vasodilatory second messenger of nitric oxide, were determined by radioimmunoassay.. A 5-h preincubation with glyceryl trinitrate (0.01-100 microM) led to complete inhibition of a subsequent cyclic GMP stimulation by glyceryl trinitrate but left the cyclic GMP response to SIN-1 unaltered. Similarly, cyclic GMP elevations by the spontaneous nitric oxide donors sodium nitroprusside and spermine NONOate were not affected after pretreatment with glyceryl trinitrate. Moreover, pretreatment with SIN-1 (1-1000 microM) had no significant effect on SIN-1-dependent cyclic GMP stimulation.. Our results show that in LLC-PK1 cells, SIN-1 is free of tolerance induction and not cross-tolerant to glyceryl trinitrate. This may be due to the spontaneous nitric oxide release from SIN-1, which in contrast to nitric acid esters does not require enzymatic bioactivation and may therefore be unaffected by nitrate tolerance.

Topics: Animals; Cyclic GMP; Dose-Response Relationship, Drug; Drug Tolerance; Guanylate Cyclase; LLC-PK1 Cells; Molsidomine; Mutagens; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroglycerin; Nitroprusside; Spermine; Swine; Vasodilator Agents

The diffusible intercellular messenger nitric oxide may have a modulatory role in the thalamus and this action may be mediated via activation of soluble guanylate cyclase. In order to investigate this possibility, we applied the cyclic-GMP analogue 8-Bromo-cyclic-GMP (8-Br-cGMP) onto neurones in the ventrobasal and lateral geniculate nuclei of the thalamus in anaesthetised rats, and compared its effects with those of a nitric oxide donor. 8-Br-cGMP enhanced the responses of neurones to iontophoretically applied NMDA and AMPA. Furthermore, somatosensory and visual responses of ventrobasal and lateral geniculate neurones were enhanced to 274+/-76% and 217+/-69% of control values, respectively. These effects were similar to those seen with nitric oxide donors in this study and previous work from this laboratory. When applied to thalamic neurones in an in vitro slice preparation, 8-Br-cGMP caused a membrane depolarisation associated with a decrease in input resistance. These findings indicate that activation of guanylate cyclase can cause a membrane depolarisation of thalamic neurones in vitro, and that this effect is sufficient to enhance action responses to ionotropic glutamate receptor stimulation via either exogenous agonists or sensory stimulation.

Topics: Action Potentials; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Cyclic GMP; Evoked Potentials, Visual; Excitatory Amino Acid Agonists; Geniculate Bodies; Guanylate Cyclase; In Vitro Techniques; Molsidomine; N-Methylaspartate; Neurons, Afferent; Nitric Oxide; Nitric Oxide Donors; Rats; Rats, Wistar

1. The cardiac effects of the NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP) and 3-morpholino-sydnonimine (SIN-1) were studied in frog fibres to evaluate the contribution of cyclic GMP-dependent mechanisms. 2. SNP and SNAP (0.1-100 microM) reduced the force of contraction in a concentration-dependent manner in atrial and ventricular fibres. This effect was associated with a reduction in the time to peak (TTP) and the time for half-relaxation of contraction (T). 3. SIN-1 (100 microM) also reduced the force of contraction in two-thirds of the atrial fibres. However, it exerted a positive inotropic effect in the remaining atrial fibres, as well as in most ventricular fibres. 4. The guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM) antagonized the negative inotropic effects of SIN-1 (50 microM) and SNAP (25 microM) but had no effect on the positive inotropic response to SIN-1 (100 microM). 5. In the presence of SIN-1, superoxide dismutase (SOD, 50-200 U ml-1) either potentiated the negative inotropic effect or turned the positive inotropic effect of the drug into a negative effect. SOD had no effects when applied alone or in the presence of SNAP. 6. 6-Anilino-5,8-quinolinedione (LY 83583, 3-30 microM), a superoxide anion generator also known as a cyclic GMP-lowering agent, exerted a positive inotropic effect, which was antagonized by SOD (200-370 U ml-1) but not by ODQ (10 microM). 7. We conclude that SNP, SNAP and SIN-1 exert cyclic GMP-dependent negative inotropic effects, which are attributed to the generation of NO. In addition, SIN-1 and LY 83583 exert cyclic GMP-independent positive inotropic effects, which require the generation of superoxide anion.

Topics: Aminoquinolines; Animals; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Half-Life; Heart; Heart Atria; Heart Ventricles; In Vitro Techniques; Molsidomine; Muscle Fibers, Skeletal; Myocardial Contraction; Nitric Oxide Donors; Nitroprusside; Penicillamine; Rana esculenta; S-Nitroso-N-Acetylpenicillamine

Heme-binding protein 23 (HBP23) is a cytosolic protein that binds the prooxidant heme with high affinity and has been implicated in the cellular protection against reactive oxygen species (ROS). Because lipopolysaccharide (LPS) stimulates macrophages to produce large amounts of ROS the gene expression of HBP23 was analyzed during treatment with LPS in cultured rat Kupffer cells (KC). HBP23 was constitutively expressed in KC and up-regulated on the protein and messenger RNA (mRNA) level by LPS with a time response distinct from that of TNFalpha, but in coordination with that of heme oxygenase-1 (HO-1), which is the inducible isoform of the rate-limiting enzyme of heme degradation. A parallel up-regulation of HBP23 and HO-1 mRNA by LPS was also observed in cultured peritoneal macrophages and peripheral blood monocytes. HBP23 mRNA induction by LPS occurred on the transcriptional level as indicated by blocking with actinomycin D. The induction of HBP23 mRNA expression by LPS was preceded by that of the inducible nitric oxide synthase (iNOS) and the production of nitrite in KC. Treatment with the NOS inhibitor NG-monomethyl L-arginine prevented HBP23 mRNA induction by LPS, which was reversed by an excess of L-arginine. Both the nitric oxide (NO)-donor S-nitroso-N-acetylpenicillamine and the peroxynitrite donor SIN-1 increased HBP23 mRNA expression. HBP23 mRNA induction by LPS was down-regulated by interleukin 10 and transforming growth factor beta1 with a NO-independent mechanism. LPS-stimulated KC exhibited marked protection against the cytotoxicity mediated by H2O2. The data suggest that NO and peroxynitrite are major mediators of the LPS-dependent up-regulation of HBP23 in KC.

Topics: Animals; Carrier Proteins; Cell Survival; Cells, Cultured; Cyclic GMP; Cytochrome-B(5) Reductase; Gene Expression Regulation; Heme-Binding Proteins; Hemeproteins; Kinetics; Kupffer Cells; Lipopolysaccharides; Male; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; omega-N-Methylarginine; Penicillamine; Protein Biosynthesis; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Transcription, Genetic

Recent studies have shown that nitric oxide (NO) modulates K+-channel activity which play an important role in controlling vascular tone. The formation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) has also been recognized to be associated with the vasodilatory effect of NO. Both cyclic GMP and NO increase whole-cell K+-current by activating Ca2+-activated K+-channels (K(Ca)-channels). Here, we show evidence that activators of soluble guanylyl cyclase sodium nitroprusside or 3-morpholino-sydnonimine (SIN-1), and an analogue of cyclic GMP 8-bromo-cyclic GMP enhance the relaxation induced by cromakalim which is blocked by glibenclamide (a specific inhibitor of ATP-sensitive K+-channels [K(ATP)-channels]), and partially attenuated by methylene blue (an inhibitor of cyclic GMP formation). However, this is not due to the increase of cyclic GMP level by cromakalim itself because the relaxation induced by cromakalim is not associated with the changes of cyclic GMP level formed in the aortic smooth muscle. Thus, it is most likely that cyclic GMP also modulates activity of K(ATP)-channels, in addition to K(Ca)-channels, in the rat aorta.

Topics: Adenosine Triphosphate; Animals; Aorta, Thoracic; Apamin; Calcium; Charybdotoxin; Cromakalim; Cyclic GMP; Endothelium, Vascular; Glyburide; In Vitro Techniques; Male; Methylene Blue; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroprusside; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Inbred WKY

In the present work, we demonstrated that chemically different nitric oxide (NO)-releasing compounds inhibit tumor necrosis factor alpha (TNF-alpha)-induced polymorphonuclear leukocyte adhesion to endothelial cells in vitro. Two mesoionic oxatriazole derivatives GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3(3, 4-dichlorophenyl)-chloride) and GEA 3175 (1,2,3,4-oxatriazolium, -3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt) were compared to the earlier-known NO donor SIN-1 (3-morpholino-sydnonimine). GEA 3162 (3-10 microM) and GEA 3175 (10-30 microM) inhibited human polymorphonuclear leukocyte adhesion to B(4) endothelial cells in a dose-dependent manner being more potent than SIN-1. In the present model, leukocytes rather than endothelial cells seemed to be the target of the effect of NO. Flow cytometric analysis showed that NO-releasing compounds did not alter TNF-alpha induced CD11/CD18 surface expression in polymorphonuclear leukocytes. The inhibitory action of NO-releasing compounds on adhesion paralleled with the increased synthesis of cGMP in polymorphonuclear leukocytes. Analogues of cGMP inhibited polymorphonuclear leukocyte adhesion indicating a role for cGMP in the action of NO donors. The results suggest that exogenous NO in the form of NO-releasing compounds inhibits polymorphonuclear leukocyte adhesion to endothelial cells, which may be implicated in the regulation of leukocyte migration and leukocyte-mediated tissue injury.

Topics: Animals; CD11 Antigens; CD18 Antigens; Cell Adhesion; Coculture Techniques; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Humans; Molsidomine; Neutrophils; Nitric Oxide Donors; Rabbits; Thionucleotides; Triazoles; Tumor Necrosis Factor-alpha

1. We report opposite inotropic effects of NO donors in frog cardiac fibres. The negative effect, elicited by either 3-morpholino-sydnonimine (SIN-1) or S-nitroso-N-acetyl-penicillamine (SNAP), involved cyclic GMP (cGMP) production. However, SIN-1, unlike SNAP, could elicit a positive effect, in a superoxide dismutase (SOD)-sensitive manner. SIN-1, unlike SNAP, can release both NO and superoxide anion, the precursors of peroxynitrite (OONO-). The role of these messengers was examined. 2. Catalase did not reduce the positive inotropic effect of SIN-1. Thus, a conversion of superoxide anion into hydrogen peroxide was not involved in this effect. In addition, catalase did not modify the negative effects of SIN-1 plus SOD, or SNAP plus SOD. 3. LY 83583, a superoxide anion generator, elicited a positive inotropic effect, like SIN-1. The effect of LY 83583 was additive to the negative effects of SIN-1 or SNAP, and to the positive effect of SIN-1. Thus, superoxide anion generation, per se, did not account for the positive effect of SIN-1. 4. Authentic peroxynitrite (OONO-), but not mock-OONO- (negative control plus decomposed OONO-), exerted a dramatic positive inotropic effect in cardiac fibres. The effect of OONO- was larger in atrial fibres, as compared with ventricular fibres. 5. The positive effect of OONO- was not additive with that of SIN-1, suggesting a common mechanism of action. In contrast, the effects of either OONO- or SIN-1 were additive with the negative inotropic effect of SNAP. Furthermore, the effect of OONO-, like that of SIN-1, was not antagonized by 1H-[1,2,4]xidiazolo[4, 3-a]quinoxaline-1-one (ODQ; 10 microM), the guanylyl cyclase inhibitor. 6. The positive inotropic effects of SIN-1 and OONO- were not modified by hydroxyl radical scavengers, such as dimethyl-thio-urea (DMTU; 10 mM). 7. The positive inotropic effect of SIN-1 (100 microM) was abolished in sodium-free solutions, a treatment that eliminates the activity of the sodium-calcium exchanger. In contrast, the effect of SIN-1 was unchanged by a potassium channel inhibitor (tetraethyl-ammonium, 20 mM), or a sodium-potassium pump inhibitor (ouabain 10 microM). 8. We conclude that OONO- is a positive inotropic agent in frog cardiac fibres. The generation of OONO- accounts for the positive inotropic effect of SIN-1. OONO- itself was responsible for the positive inotropic effect, and appeared to modulate the activity of the sodium-calcium exchanger.

Topics: Aminoquinolines; Animals; Atrial Function; Catalase; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Heart Atria; Heart Ventricles; Hydroxyl Radical; Molsidomine; Muscle Fibers, Skeletal; Myocardial Contraction; Myocardium; Nitrates; Nitric Oxide Donors; Oxadiazoles; Oxidants; Quinoxalines; Rana esculenta; Sodium; Sodium-Calcium Exchanger; Ventricular Function

In the present work, the vasorelaxant effect of dioclein, a new flavonoid isolated from Dioclea grandiflora (Leguminoseae), was investigated in the rat aorta. Dioclein induced a concentration-dependent relaxation in vessels pre-contracted with phenylephrine (IC(50)=1.3+/-0.3 microM), a response which was abolished after endothelium removal. Neither indomethacin (10 microM), an inhibitor of cyclo-oxygenase, nor atropine (1 microM), an antagonist of muscarinic receptors, modified the effect of dioclein. Dioclein (30 microM) induced a significant increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in aortic rings with endothelium. The nitric oxide (NO) synthase inhibitor, N(G)-nitro-L-arginine-methyl-ester (L-NAME, 300 microM), strongly inhibited or abolished the relaxing effect and rise in cyclic GMP levels induced by dioclein. Furthermore, dioclein (30 microM) had no effect on the endothelium-independent relaxation produced by the NO donor, 3-morpholino-sydnonimine (SIN-1), while superoxide dismutase (100 U ml(-1)) significantly potentiated it. These results indicate that, in the rat aorta, dioclein induces a NO- and endothelium-dependent vasorelaxant effect, which is associated with cyclic GMP elevation. This vasorelaxation likely results from enhanced synthesis of NO rather than enhanced biological activity of NO.

Topics: Animals; Aorta, Thoracic; Brazil; Cyclic GMP; Endothelium, Vascular; Flavanones; Flavonoids; In Vitro Techniques; Male; Molsidomine; Muscle Relaxation; Nitric Oxide; Phenylephrine; Plant Roots; Plants, Medicinal; Rats; Rats, Wistar; Vasoconstrictor Agents; Vasodilator Agents

The effects of nitric oxide (NO) on human neutrophil chemotactic responses and release of interleukin (IL)-8 was studied. Neutrophils exposed to chemoattractants (IL-8, FMLP, leukotriene B4, and C5a) failed to show increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP), an indicator of NO production. Although NO increased cGMP in neutrophils, neither of two NO donors (sodium nitroprusside and 3-morpholino-sydonimine) nor a NO synthase inhibitor (N omega-nitro-L-arginine) altered FMLP- or IL-8-elicited neutrophil chemotaxis (P > .25 for all). However, lipopolysaccharide-induced IL-8 production was increased in a dose-dependent manner by a combination of sodium nitroprusside and N-acetylcysteine (P = .03) or by S-nitrosoglutathione (P = .004). NO-augmented IL-8 release was not reproduced by treating neutrophils with dibutyryl-cGMP. Up-regulation of IL-8 release by NO was associated with increased IL-8 mRNA levels (P = .009). These data suggest that NO does not directly affect neutrophil chemotaxis but may indirectly alter chemotactic responses by increasing IL-8 production via a cGMP-independent pathway.

Topics: Acetylcysteine; Cells, Cultured; Chemotaxis; Complement C5a; Cyclic GMP; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; Ferricyanides; Glutathione; Humans; Interleukin-8; Leukotriene B4; Lipopolysaccharides; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Nitroso Compounds; omega-N-Methylarginine; RNA, Messenger; S-Nitrosoglutathione; Up-Regulation

Several ion channels are reportedly redox responsive, but the molecular basis for the changes in activity is not known. The mechanism of nitric oxide action on the cardiac calcium release channel (ryanodine receptor) (CRC) in canines was explored. This tetrameric channel contains approximately 84 free thiols and is S-nitrosylated in vivo. S-Nitrosylation of up to 12 sites (3 per CRC subunit) led to progressive channel activation that was reversed by denitrosylation. In contrast, oxidation of 20 to 24 thiols per CRC (5 or 6 per subunit) had no effect on channel function. Oxidation of additional thiols (or of another class of thiols) produced irreversible activation. The CRC thus appears to be regulated by poly-S-nitrosylation (multiple covalent attachments), whereas oxidation can lead to loss of control. These results reveal that ion channels can differentiate nitrosative from oxidative signals and indicate that the CRC is regulated by posttranslational chemical modification(s) of sulfurs.

Topics: Animals; Calcium; Cyclic GMP; Cysteine; Dithiothreitol; Dogs; Electric Conductivity; Ethylmaleimide; Glutathione; Liposomes; Mercaptoethanol; Molsidomine; Myocardium; Nitric Oxide; Nitrosation; Nitroso Compounds; Oxidation-Reduction; Proteolipids; Ryanodine Receptor Calcium Release Channel; S-Nitrosoglutathione; S-Nitrosothiols; Sulfhydryl Compounds

1. The effect of diclofenac (10-100 microM) on vanadate-induced contraction of rat uterus in calcium-free buffer containing EDTA and the modification of this response by pertussis toxin (50 micrograms/ml), Rp-cAMPS (10 microM), W-7 (10 and 60 microM), L-NMMA (10 and 100 microM) and D-NMMA (100 microM) has been assessed. The effects of sodium nitroprusside (10 microM-1 mM), 3-morpholinosydnonimine (SIN-1; 0.1-100 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ; 0.1-100 microM) and 8-BrcGMP (10 microM to 1 mM) on vandate-evoked contraction were also studied. 2. Diclofenac produced dose-dependent relaxation of vanadate (0.3 mM)-induced contraction (EC50:17.3 +/- 1.8 microM, n = 11). This effect was significantly (P < 0.05) reduced by pertussis toxin (EC50: 37.4 +/- 4.5 microM, n = 6) and Rp-cAMPS (EC50:36.3 +/- 3.1 microM, n = 6). 3. The calmodulin inhibitor W-7 (1-100 microM) relaxed, in a concentration-dependent way, the vanadate contraction (EC50:67.0 +/- 18 microM). W-7 (10 and 60 microM) did not modify the relaxation elicited by diclofenac, which suggests that calmodulin inhibition and the increase of cAMP are two different actions of diclofenac. 4. The action of diclofenac was antagonized (P < 0.05) by L-NMMA (100 microM) and ODQ (1 and 100 microM) but not by D-NMMA (100 microM), which suggests the involvement of NO-synthase in this effect. 5. Sodium nitroprusside (1 mM) relaxed the vanadate contraction by only 31.7 +/- 1.04% (n = 7) and SIN-1 by 27.1 +/- 1.2% (n = 6). This suggests that, under the present experimental conditions, both NO donors were ineffective. However, 8-BrcGMP (EC50:327 +/- 71 microM, n = 7) relaxed this contraction up to 58.7 +/- 1.89%. Rp-cAMPS (10 microM) did not modify the 8-BrcGMP effect. Thus, a partial contribution of cGMP to inhibitor effect of drugs on rat uterus was possible. 6. The association between L-NMMA plus ODQ, L-NMMA plus Rp-cAMPS and ODQ plus Rp-cAMPS did not produce more displacement than L-NMMA, Rp-cAMPS or ODQ alone. This suggests the involvement of NO and cyclic nucleotides in the relaxant effect of diclofenac in rat uterus.

Topics: Adenylyl Cyclases; Animals; Cyclic GMP; Cyclooxygenase Inhibitors; Diclofenac; Drug Interactions; Enzyme Inhibitors; Female; GTP-Binding Proteins; Molsidomine; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Nucleotides, Cyclic; omega-N-Methylarginine; Oxadiazoles; Pertussis Toxin; Quinoxalines; Rats; Rats, Wistar; Sensitivity and Specificity; Sulfonamides; Uterine Contraction; Uterus; Vanadates; Vasodilator Agents; Virulence Factors, Bordetella

This study was designed to determine the effect of subarachnoid hemorrhage (SAH) on potassium (K+) channels involved in relaxations of cerebral arteries to nitrovasodilators. The effects of K+ channel inhibitors on relaxations to 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP) were studied in rings of basilar arteries obtained from untreated dogs and dogs exposed to SAH. The levels of cyclic GMP were measured by radioimmunoassay. In rings without endothelium, concentration-dependent relaxations to SIN-1 (10(-9)-10(-4) mol/L) and SNP (10(-9)-10(-4) mol/L) were not affected by SAH, whereas increase in cyclic GMP production stimulated by SIN-1 (10(-6) mol/L) was significantly suppressed after SAH. The relaxations to SIN-1 and SNP were reduced by charybdotoxin (CTX: 10(-7) mol/L), a selective Ca(2+)-activated K+ channel inhibitor, in both normal and SAH arteries; however, the reduction of relaxations by CTX was significantly greater in SAH arteries. By contrast, the relaxations to these nitrovasodilators were not affected by glyburide (10(-5) mol/L), an ATP-sensitive K+ channel inhibitor, in both normal and SAH arteries. These findings suggest that in cerebral arteries exposed to SAH, CA(2+)-activated K+ channels may play a compensatory role in mediation of relaxations to nitric oxide. This may help to explain mechanisms of relaxations to nitrovasodilators in arteries with impaired production of cyclic GMP.

Topics: 4-Aminopyridine; Animals; Basilar Artery; Charybdotoxin; Cyclic GMP; Dogs; Female; Humans; Male; Molsidomine; Nitroprusside; Potassium Channels; Subarachnoid Hemorrhage; Uridine Triphosphate; Vasodilation; Vasodilator Agents

The nitric oxide/cyclic 3',5'-guanosine monophosphate (NO/cGMP) signaling pathway has been implicated in certain forms of developmental and adult neuronal plasticity. Here we use whole-mount immunocytochemistry to identify components of this pathway in the nervous system of postembryonic lobsters as they develop through metamorphosis. We find that the synthetic enzyme for NO (nitric oxide synthase, or NOS) and the receptor for this transmitter (NO-sensitive soluble guanylate cyclase) are broadly distributed in the central nervous system (CNS) at hatching. In the brain, NOS immunoreactivity is intensified during glomerular development in the olfactory and accessory lobes. Whereas only a few neurons express NOS in the CNS, many more neurons synthesize cGMP in the presence of NO. NO-sensitive guanylate cyclase activity is a stable feature of some cells, while in others it is regulated during development. In the stomatogastric nervous system, a subset of neurons become responsive to NO at metamorphosis, a time when larval networks are reorganized into adult motor circuits. cGMP accumulation was occasionally detected in the nucleus of many cells in the CNS, which suggests that cGMP may have a role in transcription. Based on these findings, we conclude that the NO/cGMP signaling pathway may participate in the development of the lobster nervous system. Furthermore, NO may serve as a modulatory neurotransmitter for diverse neurons throughout the CNS.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Central Nervous System; Cyclic GMP; Female; Ganglia, Invertebrate; Larva; Molsidomine; Nephropidae; Nerve Net; Nerve Tissue Proteins; Neuronal Plasticity; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Penicillamine; Signal Transduction; Smell

The possible coexistence of nitric oxide (NO) and acetylcholine in the rat major pelvic ganglion (MPG) was examined by double immunohistochemistry using antisera raised against NO synthase (NOS) and choline acetyltransferase (ChAT). The smooth muscle responses of the isolated bladder and urethra were recorded after bilateral cryoganglionectomy of the MPG, focusing on the possible development of denervation supersensitivity. In the MPG, NOS immunoreactivity (ir) was seen in a large number of cell bodies, but it was not as abundant as ChAT-ir cell bodies. Double immunolabeling showed that all NOS-ir cell bodies also displayed ChAT-ir. In ganglionectomized bladders, the electrical field stimulation (EFS)-evoked contractile response was markedly reduced. When compared with control bladders, detrusor strips from ganglionectomized rats were more sensitive to carbachol as revealed by a lower negative logarithm of the drug concentration eliciting 50% relaxation (6.5 +/- 0.04 vs. 5.9 +/- 0.07). In the urethra, the NO-mediated relaxant response to EFS was practically abolished by ganglionectomy, whereas no difference was found in sensitivity to 3-morpholinosydnonimine hydrochloride (SIN-1). SIN-1 produced an equal increase in tissue levels of guanosine 3',5'-cyclic monophosphate in urethral preparations from control and ganglionectomized rats. The results suggest that the NOS-ir nerves that mediate inhibition of rat urethral smooth muscle tone originate from the MPG and contain ChAT. No denervation supersensitivity to nitrergic stimulation was observed in the urethra after ganglionectomy.

Topics: Acetylcholine; Animals; Carbachol; Cyclic GMP; Electric Stimulation; Female; Ganglia; Ganglionectomy; Immunohistochemistry; Molsidomine; Muscle Contraction; Muscle, Smooth; Nitric Oxide; Nitric Oxide Synthase; Parasympathomimetics; Rats; Rats, Sprague-Dawley; Urethra; Urinary Bladder; Urinary Tract

Effects of zaprinast, an inhibitor of guanosine 3', 5'-cyclic monophosphate (cGMP)-specific phosphodiesterase, and methylene blue, an inhibitor of soluble guanylate cyclase, on the negative chronotropic response to CD-832, a novel dihydropyridine derivative with a nitrate moiety, and nifedipine were examined with isolated guinea-pig right atria in the presence and absence of isoproterenol. CD-832 and nifedipine produced concentration-dependent negative chronotropic effects both in the absence and presence of isoproterenol. In the absence of isoproterenol, the concentration-response curves for CD-832 and nifedipine were neither potentiated by zaprinast nor inhibited by methylene blue. In the presence of isoproterenol (10[-8] M), zaprinast produced a three-fold leftward shift of the concentration-response curve for CD-832, while methylene blue produced a three-fold rightward shift. The concentration-response curve for nifedipine was not affected by these agents. SIN-1, a nitric oxide (NO) donor, had no chronotropic effect in the absence of isoproterenol, but had a concentration-dependent negative chronotropic effect in the presence of isoproterenol: the beating rate decreased to values close to that in the absence of isoproterenol. These findings suggest that NO-cGMP mediated pathway is involved in the negative chronotropic actions of CD-832 under beta-adrenergic stimulation.

Topics: Animals; Calcium Channel Blockers; Cyclic GMP; Female; Guinea Pigs; Heart Rate; In Vitro Techniques; Isoproterenol; Male; Molsidomine; Niacinamide; Nifedipine; Nitric Oxide

The gas radical, nitric oxide (NO), is a key signalling molecule in the cardiovascular, nervous and immune systems. Recently it has been found that it is produced by both the osteoblast and osteoclast and that it has major effects in producing osteoclast detachment and exerting a tonic inhibition of bone resorption. This detaching effect is mediated by a rapid increase in cGMP following calcium-triggered e-NOS activation during normal bone resorption. This effect is not reproduced in vitro by 8-bromo-cGMP but is seen with the newer rapidly permeant 8-pCPT-cGMP. However the inhibition of bone resorption by SIN-1 in vitro is not mediated solely by cGMP but depends on other factors still unidentified. Superoxide anions alone produces both osteoclast detachment and inhibition of resorption. Both of these actions may be mediated at least in part by peroxynitrite which has the same effect as NO alone on osteoclast detachment.

Topics: Animals; Animals, Newborn; Bone Resorption; Calcium; Catalase; Cells, Cultured; Cyclic GMP; Enzyme Activation; Hydrogen Peroxide; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Osteoclasts; Rats; Rats, Sprague-Dawley; Superoxides; Thionucleotides

Interactions of mesangial cells (MCs) with components of the extracellular matrix (ECM) profoundly influence the MC phenotype, such as attachment, contraction, migration, survival and proliferation. Here, we investigated the effects of exogenous nitric oxide (NO) on the process of MC adhesion to ECM molecules. Incubation of rat MCs with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) dose- and time-dependently inhibited MC adhesion and spreading on various ECM substrata, being more pronounced on collagen type I than on collagen type IV, laminin or fibronectin. In contrast, SNAP did not inhibit MC adhesion to L-polylysine-coated plates. The inhibitory effects of SNAP were reduced by hemoglobin and enhanced by superoxide dismutase. The anti-adhesive action of SNAP was mimicked not only by other NO donors but also by 8-bromo-cGMP, and significantly reversed by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ). Moreover, SNAP and 8-bromo-cGMP decreased the adhesion-induced phosphorylation of focal adhesion kinase (pp125FAK). In the presence of SNAP or 8-bromo-cGMP, adherent MCs exhibited disturbed organization of alpha-actin filaments and reduced numbers of focal adhesions, as shown by immunocytochemistry. In additional experiments with adherent MCs, it was found that exposure to SNAP or 8-bromo-cGMP for 12 and 24 hours induced detachment of MCs. The results indicate that exogenous NO interferes with the establishment and maintenance of MC adhesion to ECM components. This inhibitory NO effect is mediated predominantly by cGMP-signaling. Disturbance of MC attachment to ECM molecules could represent an important mechanism by which NO affects MC behavior in vitro and in vivo.

Topics: Animals; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Collagen; Cyclic GMP; Cytoskeleton; Extracellular Matrix; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Glomerular Mesangium; Glutathione; Molsidomine; Nitric Oxide; Nitroprusside; Nitroso Compounds; Penicillamine; Protein-Tyrosine Kinases; Rats; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione; Signal Transduction

Studies were conducted to clarify the effects of nitric oxide donors NOR 3 ((+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide, FK409), SIN-1 (3-morpholinosydnonimine) and SNAP (S-nitroso-N-acetylpenicillamine) on the accumulation of cGMP and cAMP and Ca2+ mobilization as well as ketogenesis from oleate in isolated rat hepatocytes. NOR 3 caused inhibition of ketogenesis from oleate along with stimulation of cGMP accumulation in rat hepatocytes, whereas SIN-1 and SNAP exerted no effect on ketogenesis despite their marked stimulation of cGMP accumulation. Although the nitric oxide trapping agent, carboxy-PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), antagonized the stimulation by NOR 3 of cGMP accumulation, it failed to modulate the anti-ketogenic action of NOR 3. Furthermore, neither 8-bromoguanosine-3',5'-cyclic monophosphate nor N2,2'-O-dibutyrylguanosine-3',5'-cyclic monophosphate mimicked the anti-ketogenic action of NOR 3. It is concluded in the present study that NOR 3-induced inhibition of ketogenesis in rat hepatocytes is not mediated by cGMP. The present study revealed that the remaining structure of NOR 3 from which nitric oxide had been spontaneously released had no anti-ketogenic action. We first and clearly demonstrated that nitrite production was dramatically enhanced when NOR 3 was incubated in the presence of rat hepatocytes. The mechanism whereby NOR 3 inhibits ketogenesis in rat hepatocytes will be discussed.

Topics: Adenosine Triphosphate; Animals; Benzoates; Calcium; Cells, Cultured; Cyclic GMP; Imidazoles; Lactic Acid; Liver; Male; Molsidomine; Nitric Oxide; Nitro Compounds; Oleic Acid; Penicillamine; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Sodium Nitrite; Vasodilator Agents

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth mu

Topics: Acetylcholine; Animals; Apamin; Calcimycin; Carotid Arteries; Cell Membrane; Cyclic GMP; Glyburide; In Vitro Techniques; Membrane Potentials; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Penicillamine; Peptides; Potassium Channel Blockers; Rabbits

We tested the hypothesis that increasing cyclic GMP with nitric oxide (NO) would reduce cardiac myocyte metabolism less than other forms of guanylate cyclase stimulation. The steady state O2 consumption (VO2) of a suspension of ventricular myocytes in 2.0 mM Ca2+ isolated from hearts of New Zealand white rabbits was measured in a glass chamber using Clark-type oxygen electrode. The cellular cyclic GMP levels, determined by radioimmunoassay, were increased by (1) adding 3-morpholinosydnonimine (SIN-1, 10(-8)-10(-5) M) and nitroprusside (10(-8)-10(-5) M), NO donors-soluble guanylate cyclase stimulators; (2) carbon monoxide (CO, 1.5 x 10(-8)-1.5 x 10(-6) M), soluble guanylate cyclase stimulator and (3) guanylin (10(-8)-10(-5) M), particulate guanylate cyclase stimulator. The baseline myocyte cyclic GMP level was 86 +/- 13 fmol/10(5) myocytes with a corresponding VO2 of 268 +/- 21 nl O2/min per 10(5) myocytes. An inverse relationship between cellular cyclic GMP levels and VO2 existed in these myocytes. The regression equations for the four treatments were: VO2 = -0.45 x [cyclic GMP] + 294.4, r = 0.94 for SIN-1; VO2 = -1.46 x [cyclic GMP] + 444.7, r = 0.96 for CO; VO2 = -1.25 x [cyclic GMP] + 389.1, r = 0.84 for guanylin and VO2 = -0.55 x [cyclic GMP] + 322.8. r = 0.79 for nitroprusside. The regression lines of the two NO donors were parallel. A similar result was also evident for the regressions of CO and guanylin. However, the slopes of both the SIN-1 and nitroprusside regression line were significantly less steep than that of either the CO or guanylin lines. Therefore, VO2 is reduced less for a similar increase in cyclic GMP with NO donors compared to direct stimulation with CO or guanylin. These results suggest that NO has metabolic effects on myocytes in addition to its stimulatory effects on cellular cyclic GMP.

Topics: Animals; Cells, Cultured; Cyclic GMP; Enzyme Activation; Guanylate Cyclase; Heart; Heart Ventricles; Molsidomine; Myocardium; Nitric Oxide; Oxygen Consumption; Rabbits

We investigated the role of K+ channels and intracellular Ca2+ stores in the relaxations induced by the NO donor 3-morpholinosydnonimine (SIN-1) and 8-bromo-cGMP (8-BrcGMP), 8-(4-chlorophenylthio)-cGMP (pCPT-cGMP), and alpha, beta-methylene-ATP in isolated segments of rat ileum. The inhibitory responses to SIN-1 and the cGMP analogs were not influenced by the K+ blockers apamin, charybdotoxin, iberiotoxin, or glibenclamide, whereas relaxations induced by alpha,beta-methylene-ATP were abolished by apamin and tetraethylammonium. The NO-donor SIN-1 and the cGMP analogs were able to inhibit contractions induced by activation of L-type Ca2+ channels (BAY-K-8644), by carbachol (CCh), and by cyclopiazonic acid (CPA), a blocker of sarcoplasmic Ca2+-ATPase. However, the inhibition of the combined CPA and CCh response was reduced and the dose-response curve of SIN-1 shifted to the right. Intracellular Ca2+ stores were emptied by incubation in Ca2+-free buffer and repetitive stimulation with CCh or BAY-K-8644. After restoration of extracellular Ca2+, the inhibitory effect of SIN-1 and pCPT-cGMP was only attenuated, whereas in the additional presence of CPA, the inhibitory effect of SIN-1 was blocked and the effect of 8-BrcGMP reduced. Thus depleting intracellular Ca2+ stores attenuated the effect of SIN-1 and 8-BrcGMP, suggesting an involvement of functional Ca2+ stores.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenosine Triphosphate; Animals; Apamin; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Carbachol; Charybdotoxin; Cyclic GMP; Egtazic Acid; Glyburide; Ileum; In Vitro Techniques; Indoles; Kinetics; Male; Molsidomine; Muscle Contraction; Muscle, Smooth; Peptides; Potassium Channels; Rats; Rats, Wistar; Tetraethylammonium; Tetrodotoxin; Thionucleotides

The addition of nitric oxide (NO), in the form of either donor compounds or nitric oxide gas, inhibits hormone-stimulated cAMP accumulation in N18TG2 cells. Hormone receptors and Gs are not targets of NO because forskolin-stimulated cAMP accumulation is also inhibited. The inhibitory effect of NO is not altered by pretreatment of cells with pertussis toxin, indicating that Gi is not mediating the effect of NO. cAMP accumulation in these cells is not altered by cell incubation with Ca++ ionophore or calmidazolium, indicating that calmodulin is not the target for NO. Experiments also rule out changes in phosphodiesterase or cGMP as mediators of the effect of NO. Cell incubation with superoxide dismutase in the presence or absence of catalase indicate that nitric oxide is the reactive species. The inhibitory action of nitric oxide is readily reversed, allowing full recovery of hormone and forskolin stimulation within 20 min of incubation in the absence of nitric oxide. The sum of the data indicate that NO targets either the adenylyl cyclase itself, or a regulatory component distinct from G proteins or calmodulin, to inhibit activation of the enzyme.

Topics: Adenylyl Cyclase Inhibitors; Animals; Calmodulin; Colforsin; Cyclic AMP; Cyclic GMP; GTP-Binding Protein alpha Subunits, Gi-Go; Mice; Molsidomine; Neuroblastoma; Nitric Oxide; Tumor Cells, Cultured

1. Inhibition of inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca2+ release by cGMP was examined in intact rat megakaryocytes, by using a combination of single cell fluorescence microscopy to monitor intracellular free calcium ([Ca2+]i) and flash photolysis of caged second messengers. 2. Sodium nitroprusside (SNP), a nitric oxide (NO) donor, and the hydrolysis-resistant cGMP analogue 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (pCPT-cGMP) inhibited Ca2+ release induced by photolysis of caged IP3. Neither of them affected the rate of Ca2+ removal from the cytoplasm following photolysis of caged Ca2+. 3. Photolysis of the caged NO donor 3-morpholinosydnonimine (SIN-1) during agonist-induced [Ca2+]i oscillations inhibited Ca2+ release without affecting the rate of Ca2+ uptake and/or extrusion. 4. We conclude that the inhibition of IP3-induced Ca2+ release is the principal mechanism of NO-cGMP-dependent inhibition of [Ca2+]i mobilization. 5. IPG, a specific peptide inhibitor of cGMP-dependent protein kinase (cGMP-PK), blocked the inhibitory effect of pCPT-cGMP, indicating that the inhibition of IP3-induced Ca2+ release by pCPT-cGMP is mediated by cGMP-PK. However, the simultaneous application of both IPG and IP20, a specific peptide inhibitor of cAMP-dependent protein kinase (cAMP-PK), was required to block the inhibitory effect of SNP. These data strongly suggest that NO-cGMP-dependent inhibition of [Ca2+]i mobilization is mediated via the activation of both cGMP-PK and cAMP-PK.

Topics: Animals; Bone Marrow Cells; Calcium; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Kinetics; Megakaryocytes; Models, Biological; Molsidomine; Nitric Oxide Donors; Nitroprusside; Photolysis; Rats; Rats, Wistar; Second Messenger Systems; Thionucleotides

Clinically, nitric oxide (NO*) is widely used as a pulmonary vaso- and bronchodilator agent. However, the precise molecular mechanisms by which NO. induces smooth muscle relaxation are not well established. It has been suggested that NO. relaxes airway smooth muscle (ASM) via a 3',5'-cyclic guanosine monophosphate (cGMP)-dependent pathway, and our previous work has shown that Ca2+-activated K+ (KCa) channels are susceptible to cGMP-dependent protein kinase (PKG)-dependent phosphorylation (A. Alioua, J. P. Huggins, and E. Rousseau. Am. J. Physiol. 1995;268:L1057-L1063). To assess whether KCa channels are also directly activated by NO. or one of its derivatives such as peroxynitrite, the activity of these channels was measured upon fusion of sarcolemmal vesicles derived from bovine tracheal smooth muscle cells into planar lipid bilayers (PLB). It was found that in the absence of adenosine triphosphate (ATP), cGMP, and cGMP-dependent protein kinase, NO* donors such as 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) or 3-morpholinosydnonimine hydrochloride (SIN-1) in the presence of superoxide dismutase (SOD), added on either side of the bilayer, caused a concentration- dependent increase in the open probability (Po) of KCa channels without altering their unitary conductance. Release of NO*, which was measured by chemiluminescence analysis in parallel experiments, affected the gating behavior of KCa channels in the presence of SOD and ethyleneglycol-bis-(beta-aminoethyl ether)- N,N'-tetraacetic acid (EGTA) by reducing the mean closed times and increasing the number and duration of short open events. PAPA NONOate, a true NO. donor, had similar effects in the presence of ethylenediaminetetraacetic acid (EDTA), a heavy-metal chelator, and K-urate, a peroxynitrite scavenger. Addition of either 5 mM dithiothreitol (DTT) or 5 mM reduced glutathione (GSH), as well as 5 mM N-ethylmaleimide (NEM)-an alkylating agent-to the trans (intracellular) side of an experimental chamber slightly increased channel Po but prevented further channel activation by NO* donors. However, neither DTT nor GSH was able to reverse the effect of NO*. In contrast to SIN-1, DTT had no effect when added to the cis (extracellular) side of the chamber. This suggests that the effect of NO* is most likely due to a chemical modification (nitrothiosylation) of intracellular sulfhydryl group(s). Neither PAPA NONOate (NO*), nor SIN-1 had any effect on sarcolemmal Cl- channels rec

Topics: Animals; Bronchi; Calcium; Charybdotoxin; Cyclic GMP; Dithiothreitol; Ethylmaleimide; Glutathione; Humans; Hydrazines; Lipid Bilayers; Male; Molsidomine; Muscle, Smooth; Nitric Oxide; Potassium Channels; Rats; Rats, Sprague-Dawley

We investigated whether nitric oxide (NO) exerts an inhibition on its own synthesis in the gastric myenteric plexus in rats. Nonadrenergic, noncholinergic relaxations in response to transmural electrical stimulation (TS) were markedly antagonized by NG-nitro-L-arginine methyl ester, (10(-4) M) and abolished by tetrodotoxin (10(-6) M). Pretreatment with various NO donors (3-morpholino-sydnonymide [SIN-1 (3 x 10(-7) to 3 x 10(-6) M)], S-nitroso-N-acetylpenicillamine (10(-6) to 10(-5) M), sodium nitroprusside (10(-8) to 3 x 10(-8) M) and 8-bromoquanosine 3', 5'-cyclic monophosphate [8-bromo-cGMP (10(-6) to 3 x 10(-6) M)]) significantly inhibited TS-evoked nonadrenergic, noncholinergic relaxations in a dose-dependent manner. In contrast, vasoactive intestinal polypeptide (10(-8) M)-induced relaxations were not affected by SIN-1 or 8-bromo-cGMP. TS evoked a significant increase in 3H-citrulline formation, which was completely abolished by calcium-free medium, NG-nitro-L-arginine methyl ester, (10(-4) M) and tetrodotoxin (10(-6) M). 3H-citrulline formation evoked by TS was significantly inhibited by SIN-1 (10(-7) to 10(-5) M) and 8-bromo-cGMP (10(-7) to 10(-5) M) in a dose-dependent manner. The inhibitory effect of SIN-1 was partially prevented by 1H-[1,2, 4]oxadiazolo[3,4-a]quinoxalin-1-one (10(-5) M), a guanylate cyclase inhibitor. We conclude that NO synthesis in the gastric myenteric plexus is negatively regulated by NO and cGMP. This suggests an autoregulatory feedback mechanism of NO synthesis in the gastric myenteric plexus.

Topics: Animals; Calcium Channels; Citrulline; Cyclic GMP; Dose-Response Relationship, Drug; Gastric Mucosa; Male; Molsidomine; Muscle Relaxation; Myenteric Plexus; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Rats; Rats, Sprague-Dawley; Stomach; Vasoactive Intestinal Peptide

The aim of the present study was to test the hypothesis that bradykinin-stimulated release of nitric oxide (NO) and/or prostacyclin from endothelium blocks myocyte hypertrophy in vitro. Angiotensin II increased [3H]phenylalanine incorporation by 21 +/- 2% in myocytes cocultured with endothelial cells; this was abolished by bradykinin in the presence of endothelial cells. Bradykinin increased cytosolic concentrations of cGMP by 29 +/- 4% in myocytes cocultured with endothelial cells. This was abolished by inhibition of NO synthase and by a cyclooxygenase inhibitor. Angiotensin II also increased [3H]phenylalanine incorporation by 28 +/- 3% in myocytes cultured in the absence of endothelial cells. This effect of angiotensin II in monoculture was abolished by donors of NO but not by bradykinin. Neither the stable analog of prostacyclin (iloprost) nor the prostacyclin second messanger analog 8-bromo-cAMP (8-BrcAMP) blocked the effect of angiotensin II. Furthermore, 8-BrcAMP and iloprost individually increased [3H]phenylalanine incorporation. The antihypertrophic effects of bradykinin are critically dependent on endothelium-derived NO.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Angiotensin II; Animals; Bradykinin; Cardiomegaly; Cells, Cultured; Coculture Techniques; Cyclic GMP; Endothelium, Vascular; Heart; Iloprost; Indomethacin; Male; Molsidomine; Myocardium; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; omega-N-Methylarginine; Phenylalanine; Protein Biosynthesis; Rats; Rats, Sprague-Dawley

In functional experiments, the nitric oxide (NO) donor N-morpholino-N-nitroso-aminoacetonitrile or the cGMP analog 8-(4-chlorophenylthio)-cGMP caused a concentration-dependent, tetrodotoxin-resistant relaxation of precontracted strips from rat small intestine. The inhibitory effect of both substances was completely blocked at lower concentrations and was significantly attenuated at higher concentrations by the selective cGMP-dependent protein kinase (cGK) antagonist KT-5823 (1 microM). cGK-I was identified by immunohistochemistry in circular and longitudinal muscle, lamina muscularis mucosae, and smooth muscle cells of the villi and in fibroblast-like cells of the small intestine. Additionally, there was staining of a subpopulation of myenteric and submucous plexus neurons. Double staining for neuronal NO synthase (nNOS) and cGK-I demonstrated a colocalization of these two enzymes. Western blot analysis of smooth muscle preparations and isolated nerve terminals demonstrated that these structures predominantly contain the cGK-Ibeta isoenzyme, whereas the cGK-Ialpha expression is about threefold less. The isoform cGK-II was entirely confined to mucosal epithelial cells. These results show that cGK-I is expressed in different muscular structures of the small intestine and participates in the NO-induced relaxation of gastrointestinal smooth muscle. The presence of cGK-I in NOS-positive enteric neurons further suggests a possible neuronal action site.

Topics: Alkaloids; Animals; Carbazoles; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Guinea Pigs; Indoles; Intestinal Mucosa; Intestine, Small; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth; Myenteric Plexus; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Polymerase Chain Reaction; Pyrroles; Rats; Rats, Wistar; Thionucleotides

1. The effects of oxatriazole-type (GEA 3162 and GEA 5624) nitric oxide (NO) donors on mitogenesis and proliferation were studied in vascular smooth muscle cell (VSMC) culture. The effects of the GEA-compounds were compared with well-known NO-donors 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). 2. All NO-donors released NO and increased the production of cyclic GMP concentration-dependently. The production of cyclic GMP was inhibited by the guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). 3. The NO-donors inhibited basal and serum-induced DNA synthesis concentration-dependently. The GEA-compounds were needed in concentrations 10 times lower than SIN-1 and SNAP. GEA 3162, SIN-1 and SNAP were also able to inhibit serum-induced cell proliferation. GEA 5624 was ineffective. The antimitogenic effect of NO-donors was not reduced by inhibiting the guanylate cyclase. 4. These results suggest that NO inhibits serum-induced DNA synthesis and proliferation of VSMC by a cyclic GMP-independent mechanism. The oxatriazole-type NO-donor GEA 3162 was found to be a more potent inhibitor of mitogenesis and cell proliferation than SIN-1 and SNAP.

Topics: Animals; Cell Division; Cells, Cultured; Cyclic GMP; DNA; Male; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitrites; Penicillamine; Rats; Rats, Wistar; Signal Transduction; Triazoles; Urea

The exposure of rat mesangial cells to cytokines promoted activation of the p42/p44 mitogen-activated protein kinase (MAPK). We identified a rapid and delayed phase of MAPK activation with distinctive activity increases at 5 to 15 min and 15 to 24 h. Rapid and late MAPK activation were attenuated by the redox-modulating agent N-acetylcysteine. Specifically, late-phase activation coincided with endogenous nitric oxide (NO) generation and in turn was suppressed by the NO synthase-blocking compounds diphenyliodonium or nitroarginine methyl ester. By using NO-liberating agents such as S-nitrosoglutathione and 3-morpholinosydnonimine, we investigated intermediary signaling elements of NO in promoting MAPK activation. Early and transient activation at 5 min was suppressed by the soluble guanylyl cyclase-blocking agent 1H-(1,2,4)-oxdiazolo-(4,3-alpha)-6-bromoquinoxazin-1-one (NS 2028) and, moreover, was mimicked by the lipophilic cyclic GMP (cGMP) analogue 8-bromo-cGMP. In contrast, NO-mediated activation achieved within hours was unrelated to cGMP signaling. Late and persistent MAPK activation, induced by NO donors or endogenously generated NO, was found in association with inhibition of phosphatase activity. In vitro dephosphorylation of activated and immunoprecipitated p42/p44 by cytosolic phosphatases was sensitive to the readdition of NO and was found to be inhibited in cytosol of S-nitrosoglutathione-stimulated cells. Also, cells that had been exposed to cytokines for 24 h revealed a blocked phosphatase activity, which was successfully attenuated by the NO synthase inhibitor nitroarginine methyl ester and, therefore, was NO mediated. Conclusively, NO affects p42/p44 MAPK in rat mesangial cells twofold: rapid activation is cGMP mediated, whereas late activation is transmitted via inhibition of tyrosine dephosphorylation.

Topics: Acetylcysteine; Animals; Biphenyl Compounds; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cyclic GMP; Enzyme Activation; Free Radical Scavengers; Glomerular Mesangium; Glutathione; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molsidomine; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Onium Compounds; Oxadiazoles; Oxazines; Phosphorylation; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; Rats; S-Nitrosoglutathione; Second Messenger Systems

Using cultured LLC-PK1 cells the present study investigates mechanisms leading to nitrate tolerance and its reversal. A 5-h pretreatment with glyceryl trinitrate (GTN, 0.01-100 microM) resulted in desensitization of the intracellular cyclic GMP response to a subsequent 10-min challenge with GTN (1 microM). The spontaneous donor of nitric oxide (NO) spermine NONOate, which releases NO independently of enzymatic catalysis, did not induce tolerance to its own cyclic GMP stimulatory effect and remained fully effective in GTN-tolerant cells. Tolerant cells regained sensitivity to GTN after a 30-h incubation in media. Recovery of the cyclic GMP response was blocked in the presence of cycloheximide (10 microM) suggesting that de novo protein synthesis is necessary for tolerance reversal. Our results demonstrate that nitrate tolerance is specific for nitric acid esters and possibly due to down-regulation of enzymes involved in bioactivation of, and NO generation from, organic nitrates.

Topics: Animals; Cell Line; Cyclic GMP; Cycloheximide; Kinetics; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroglycerin; Nitroprusside; Protein Biosynthesis; Spermine; Vasodilator Agents

The effect of two nitric oxide (NO) donors, SIN-1 and DEA/NO, as well as of the inactive SIN-1 derivative molsidomin, was studied on locus coeruleus (LC) neurons in a slice preparation using intracellular recordings. In addition, the effect of the guanylate cyclase inhibitor ODQ was analysed. Furthermore, the effect of NO donors on cyclic guanosine monophosphate (GMP) levels in the LC was studied using the indirect immunofluorescence technique, and the expression of soluble guanylyl cyclase with in situ hybridization. In 36 of 66 LC neurons extracellular application of SIN-1 and DEA/NO caused a hyperpolarization and a decrease in apparent input resistance. In almost 20% of neurons SIN-1 increased the firing rate. No effect could be recorded with the brain-inactive SIN-1 derivative molsidomin. The membrane permeable cGMP analogue 8-bromo-cGMP imitated the action of SIN-1. The hyperpolarizing effect of SIN-1 and DEA/NO was attenuated by preincubation with the guanylyl cyclase inhibitor ODQ. The immunohistochemical analysis revealed lack of cGMP immunostaining in non-stimulated slices, whereas SIN-1 dramatically increased this staining in about 40% of the LC neurons, and these neurons were all tyrosine hydroxylase positive, that is noradrenergic. A large proportion of the LC neurons expressed soluble guanylyl cyclase mRNA. The present and previous results suggest that NO, released from a small number of non-noradrenergic neurons in the LC, mainly has an inhibitory influence on many noradrenergic neurons, by upregulating cGMP levels via stimulation of soluble guanylyl cyclase. As nitric oxide synthase is present only in a small number of non-noradrenergic neurons (Xu et al., 1994), a few neurons may influence a large population of noradrenergic LC neurons, which in turn may control activity in many regions of the central nervous system.

Topics: Animals; Cyclic GMP; Excitatory Postsynaptic Potentials; Fluorescent Antibody Technique, Indirect; Hydrazines; In Situ Hybridization; In Vitro Techniques; Locus Coeruleus; Male; Molsidomine; Neural Pathways; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley

The nitrovasodilator 3-morpholinosydnonimine (SIN-1) slowly decomposes to release both nitric oxide (NO) and superoxide (O2-) and thereby produces peroxynitrite (ONOO-), a powerful oxidant which has been proposed to mediate the toxic actions caused by NO. Indeed, ONOO has been shown to cause neuronal death and it has been proposed to occur in different disorders of the CNS such as brain ischaemia, AIDS-associated dementia, amyothrophic lateral sclerosis, etc. We have found that SIN-1 was only slightly toxic to 1-week-old rat cortical neurones in primary culture (LC50=2.5+/-0.5 mM). Superoxide dismutase (SOD; 100 U/ml) significantly increased SIN-1-induced toxicity, an effect that was enhanced in the presence of HbO2, abolished by catalase and accompanied by the formation of hydrogen peroxide (H2O2). We have also found that 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylate cyclase, enhances cell death induced by SIN-1 (0.2-0.5 mM) + SOD (100 U/ml) in a concentration-dependent way (EC50=0.073+/-0.004 microM). Simultaneously, ODQ inhibits the elevation of cyclic GMP concentrations induced by SIN-1 + SOD in cortical cells (IC50=0.022+/-0.014 microM). Finally, we have also shown that the cyclic GMP mimetic, 8-bromo-cyclic GMP reverses the potentiating effect induced by ODQ on SIN-1 + SOD-induced neuronal death and inhibits the neurotoxicity induced by H2O2 (100 microM). Taken together, these data suggest that H2O2 is the species responsible for the potentiation by SOD of SIN-1-induced cell death and that cyclic GMP elevations confer selective cytoprotection against this H2O2-mediated component of cell death.

Topics: Animals; Cell Death; Cyclic GMP; Drug Synergism; Enzyme Inhibitors; Hydrogen Peroxide; Molsidomine; Neurons; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; Superoxide Dismutase; Vasodilator Agents

The inhibitory innervation of guinea-pig urethral smooth muscle was investigated histochemically and functionally. The distribution of immunoreactivities to haem oxygenases (HO), neuronal NO synthase (nNOS), and vasoactive intestinal polypeptide (VIP) was studied, and the functional effects of the corresponding putative transmitters, CO, NO, and VIP, were assessed. HO-2 immunoreactivity was found in all nerve cell bodies of intramural ganglia, localized between smooth muscle bundles in the detrusor, bladder base and proximal urethra. About 70% of the ganglionic cell bodies were also NOS-immunoreactive (IR), whereas a minor part was VIP-IR. Some ganglion cells exhibiting tyrosine hydroxylase (TH) activity were demonstrated. Rich numbers of NOS-IR varicose nerve terminals could be found innervating the smooth muscle of the urethra, whereas VIP-IR terminals were less numerous. A rich number of TH-IR terminals were observed. The bladder showed a similar distribution of nerves, although only a few number of TH-IR nerves could be found. In bladder preparations exposed to sodium nitroprusside, cGMP-IR cells could be seen, forming an interconnecting network with long spindle-shaped processes. The cGMP-IR cells were especially abundant in the outer smooth muscle layers of the bladder, but less numerous in the urethra. In urethral strip preparations, electrical field stimulation evoked long-lasting frequency-dependent relaxations. The relaxations were not inhibited by the NO-synthesis inhibitor, L-NOARG, or enhanced by the NO-precursor, L-arginine. The haem precursor, 5-aminolevulinic acid (5-ALA), or the inhibitor of guanylate cyclase, ODQ, did not affect the urethral relaxations. Exogenously applied NO, SIN-1, and VIP relaxed the preparations by approximately 50%, whereas the relaxation evoked by exogenous CO was minor. These results suggest that CO probably is not involved in non-adrenergic, non-cholinergic inhibitory control of the guinea-pig urethra, where a non-NO/cGMP mediated relaxation seems to be predominant.

Topics: Animals; Carbon Monoxide; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Female; Fluorescent Antibody Technique; Ganglia, Autonomic; Guinea Pigs; Heme Oxygenase (Decyclizing); Immunohistochemistry; Molsidomine; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Sodium-Potassium-Exchanging ATPase; Urethra; Urinary Bladder; Vasoactive Intestinal Peptide; Xanthenes

Nitric oxide (NO), including NO free radicals (*NO) and peroxynitrite (OONO-), modulates the release of neurotransmitters from neuronal tissues. Although we reported that S-nitroso-cysteine stimulated noradrenaline release in brain slices, we now show that only S-nitroso-cysteine inhibits noradrenaline release from PC12 cells. S-Nitroso-cysteine inhibited, in a dose-dependent manner (up to 0.6 mM), the Ca2+ -dependent [3H]noradrenaline release induced by ionomycin, adenosine 5'-O-(3-thiotriphosphate), or high K+, from PC12 cells labeled with [3H]noradrenaline. Sodium nitroprusside, S-nitroso-N-acetylpenicillamine, and 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene, which specifically release NO free radicals in neutral buffer, had minimal effects on [3H]noradrenaline release, although they markedly stimulated cyclic GMP accumulation. 3-Morpholinosydonimine, which releases peroxynitrite, had no effect on either [3H]noradrenaline release or cyclic GMP accumulation. S-Nitroso-cysteine inhibited phorbol 12-myristate 13-acetate- and mastoparan (wasp venom toxin)-induced [3H]noradrenaline release. These findings suggest that 1) S-nitroso-cysteine, but not other NO donors, inhibits some common process occurring during noradrenaline release in PC12 cells, 2) neither NO radicals, peroxynitrite, nor cyclic GMP mediate the inhibitory effects of S-nitroso-cysteine in PC12 cells.

Topics: Animals; Calcium; Cyclic GMP; Cysteine; Molsidomine; Nitric Oxide; Nitroprusside; Nitroso Compounds; Norepinephrine; PC12 Cells; Rats; S-Nitrosothiols

Hydra feeding response is a very primitive olfactory-like behavior present in a multicellular organism. We investigated the role of nitric oxide (NO) in the induction and control of hydra feeding response. Under basal conditions, hydra specimens produce detectable amounts of nitrite (NO2-), the breakdown product of NO. When hydra were incubated with reduced glutathione (GSH), the typical activator of feeding response, an increase of basal NO production was observed. This effect was inhibited by glutamic or alpha-aminoadipic acids, two GSH antagonists, which block GSH-induced feeding response, and by the NO synthase (NOS) inhibitor L-NAME. Moreover, we found that hydra possess a calcium-dependent (but calmodulin-independent) NOS isoform. By using exogenous NO donors and NOS inhibitors, we demonstrated that NO stimulus can participate both in triggering tentacular movements and in recruiting neighbor tentacles during hydra feeding response. By using dbt2-cGMP, an analog to cGMP, we observed that the NO effect was independent of cGMP pathway. Our results strongly implicate NO involvement in hydra very primitive feeding behavior, thus confirming its preservation throughout evolution.

Topics: Animals; Calcium; Calmodulin; Cyclic GMP; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Feeding Behavior; Hydra; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Nitroprusside; Olfactory Pathways

Recently we described K+ channels in the basolateral membrane of principal cells of rat cortical collecting duct (CCD) which are regulated by a cGMP-dependent protein kinase (Pflugers Arch 429:338-344, 1995). We examined the effects of the NO-liberator sodium nitroprusside (SNP) on single channel activity and membrane voltage (Vm) in principal cells of rat CCD, and on transepithelial voltage, lumen-to-bath Na+ fluxes, and osmotic water permeability in isolated perfused rat CCD tubules. While in patch clamp experiments SNP (10 microM) hyperpolarized principal cells from -54 +/- 10 mV to -71 +/- 5 mV (N = 5) and increased the activity of the described K+ channels from 0.05 +/- 0.03 to 0.45 +/- 0.14 (N = 5) in cell-attached and from 0.04 +/- 0.02 to 0.25 +/- 0.05 (N = 4) in excised patch clamp experiments, it had no effect on basal or AVP-dependent transepithelial voltage, Na+ fluxes, or the osmotic water permeability. In addition, neither 50 microM SIN-1, another liberator of NO, nor 1 mM L-NAME, an inhibitor of the NO-synthase, changed Vm significantly. Furthermore, in cGMP-assays SNP failed to increase intracellular cGMP in CCD segments. Thus, we conclude that in the rat CCD transport is not regulated via the NO-pathway and that SNP acts as an cGMP independent activator of K+ channels in the basolateral membrane of these cells.

Topics: Animals; Arginine Vasopressin; Cyclic GMP; Enzyme Inhibitors; Female; In Vitro Techniques; Kidney Cortex; Kidney Tubules, Collecting; Membrane Potentials; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Perfusion; Potassium Channels; Rats; Rats, Wistar

The effect of guanosine 3',5'-cyclic monophosphate (cGMP) on cytosolic Ca2+ dynamics and associated alterations in macromolecule permeability was investigated in cultured monolayers of aortic endothelial cells. Addition of the membrane-permeable cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 5 x 10(-4)M) or activators of the soluble (3-morpholinosydnonimine, 10(-5) M) or the particulate guanylyl cyclase (atrial natriuretic peptide, 10(-7) M) to unstimulated monolayers led to a decrease in permeability (8-BreGMP: 62 +/- 8% of control) without affecting low basal cytosolic Ca2+ concentration ([Ca2+]i, 87 +/- 8 nM). In contrast, under conditions of elevated [Ca2+]i (503 +/- 95 nM) and increased permeability (155 +/- 7% of control) induced by 10(-6) M ionomycin, 8-BrcGMP, 3-morpholinosydnonimine, or atrial natriuretic peptide provoked a further increase in permeability (8-BrcGMP: 255 +/- 27%). These agents failed to increase permeability when added before or after the ionomycin-triggered transitory rise in [Ca2+]i. The increase in permeability in response to 8-BrcGMP was due to a secondary further rise in [Ca2+]i (758 +/- 87 nM), which was abolished in the absence of extracellular Ca2+, indicating influx of exogenous Ca2+ as the cause. Changes in [Ca2+]i and permeability were inhibited, in the presence of the Rp diastereomer of 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphothioate (2 x 10(-5) M), an inhibitor of the cGMP-dependent protein kinase. These findings show that, depending on [Ca2+]i, cGMP can play opposite roles in endothelial permeability in one and the same cell preparation.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Calcium; Capillary Permeability; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Extracellular Space; Guanylate Cyclase; Macromolecular Substances; Molsidomine; Serum Albumin; Stimulation, Chemical; Swine

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, N(G)-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall.

Topics: Animals; Basilar Artery; Calcimycin; Chelating Agents; Cyclic GMP; Dogs; Egtazic Acid; Enzyme Inhibitors; Gene Expression; Immunohistochemistry; In Vitro Techniques; Ionophores; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; omega-N-Methylarginine; Recombinant Proteins; Transfection; Uridine Triphosphate; Vasodilation; Vasodilator Agents

We tested the hypothesis that increasing myocardial cyclic GMP levels would reduce myocardial O2 consumption and that renal hypertension (One Kidney-One Clip, 1K1C)-induced cardiac hypertrophy would change this relationship. Four groups of anesthetized open-chest New Zealand white rabbits (N = 26) were utilized. Either vehicle or 3-morpholinosydnonimine (SIN-1) (10(-4) M, a guanylate cyclase activator) was topically applied to the left ventricular surface of control or 1K1C rabbits. Coronary blood flow (radioactive microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption. Myocardial cyclic GMP levels were determined by radioimmunoassay. Guanylate cyclase activity was measured by conversion of GTP to cyclic GMP. 1K1C rabbits had a greater heart weight-to-body weight ratio (3.29 +/- 0.15) than controls (2.63 +/- 0.19). Systolic blood pressure was higher in 1K1C rabbits than in controls. In control rabbits, cyclic GMP levels (pmoles/g) were higher in SIN-1-treated (EPI: 7.5 +/- 1.6; ENDO: 8.1 +/- 1.5) than in vehicle-treated animals (EPI: 5.4 +/- 0.4; ENDO: 5.6 +/- 0.6). This effect was enhanced in 1K1C rabbits, with cyclic GMP levels in the SIN-1-treated group (EPI: 11.9 +/- 1.3; ENDO: 13.0 +/- 1.5) almost double those observed in the vehicle-treated group (EPI: 6.3 +/- 0.8; ENDO: 7.7 +/- 1.1). There were no significant differences in basal or maximally stimulated guanylate cyclase activity between controls and 1K1C rabbits. Myocardial O2 consumption (ml O2/min/100 g) was significantly less in the EPI region of control animals treated with SIN-1 (7.2 +/- 1.2) than in the same region of controls treated with vehicle (9.1 +/- 2.0). Myocardial O2 consumption was also significantly less in SIN-1-than vehicle-treated 1K1C animals (SIN-1-treated: EPI: 6.9 +/- 0.8; ENDO: 6.2 +/- 0.7; vehicle-treated: EPI: 10.0 +/- 0.8; ENDO: 12.5 +/- 3.0). There was no significant difference in O2 consumption between control and 1K1C animals after treatment with SIN-1. Thus, there was a greater elevation in cyclic GMP in 1K1C rabbits, but this did not result in a corresponding greater depression in O2 consumption. This suggests that cyclic GMP plays a role in the control of myocardial metabolism, and that the sensitivity of myocardial O2 consumption to changes in cyclic GMP is reduced by renal hypertension-induced cardiac hypertrophy.

Topics: Animals; Blood Gas Analysis; Cardiomegaly; Coronary Circulation; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Hemodynamics; Hypertension, Renovascular; Molsidomine; Myocardium; Oxygen; Rabbits

Since recent studies demonstrate that vascular smooth muscle cells synthesize two distinct guanylate cyclase-stimulatory gases, NO and CO, we examined possible regulatory interactions between these two signaling molecules. Treatment of rat aortic smooth muscle cells with the NO donors, sodium nitroprusside, S-nitroso-N-acetyl-penicillamine, or 3-morpholinosydnonimine, increased heme oxygenase-I (HO-1) mRNA and protein levels in a concentration and time-dependent manner. Both actinomycin D and cycloheximide blocked NO-stimulated HO-1 mRNA and protein expression. Nuclear run-on experiments demonstrated that NO donors increased HO-1 gene transcription between 3- and 6-fold. In contrast, NO donors had no effect on the stability of HO-1 mRNA. Incubation of vascular smooth muscle cells with the membrane-permeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, failed to induce HO-1 gene expression. Treatment of vascular smooth muscle cells with NO donors also stimulated the production and release of CO, as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. Finally, incubating vascular smooth muscle cells with interleukin-1 beta and tumor necrosis factor-alpha induced NO synthesis and also significantly increased the level of HO-1 protein. The cytokine-stimulated production of both NO and HO-1 protein in smooth muscle cells was blocked by the NO synthase inhibitor methyl-L-arginine. These results demonstrate that exogenously administered or endogenously released NO stimulates HO-1 gene expression and CO production in vascular smooth muscle cells. The ability of NO to induce HO-catalyzed CO release from vascular smooth muscle cells provides a novel mechanism by which NO might modulate soluble guanylate cyclase and, thereby, vascular smooth muscle cell and platelet function.

Topics: Animals; Carbon Monoxide; Cells, Cultured; Cyclic GMP; Cycloheximide; Cytokines; Dactinomycin; Enzyme Inhibitors; Gene Expression Regulation; Heme Oxygenase (Decyclizing); Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Penicillamine; Rats; RNA, Messenger; S-Nitroso-N-Acetylpenicillamine; Time Factors; Transcription, Genetic

Carboxy-PTIO reacts rapidly with NO to yield NO2 and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1, Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC50 = 36 +/- 5 microM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC50 of 0.12 +/- 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC50 of 0.11 +/- 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger.

Topics: Animals; Benzoates; Cattle; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Free Radical Scavengers; Imidazoles; Kinetics; Molsidomine; Nitrates; Nitric Oxide; Nitrites; Serum Albumin, Bovine; Swine; Tyrosine

Electrical stimulation of cerebellar parallel fibers (PF) increases cerebellar blood flow (BFcrb), a response that is attenuated by glutamate receptor antagonists and NO synthase (NOS) inhibitors. We investigated whether administration of NO donors could counteract attenuation by NOS inhibitors of vasodilation produced by PF stimulation. In halothane-anesthetized rats the cerebellar cortex was exposed and superfused with Ringer solution. PF were stimulated with microelectrodes (100 microA, 30 Hz), and BFcrb was recorded by a laser-Doppler probe. During Ringer superfusion, PF stimulation increased BFcrb by 56 +/- 7% and hypercapnia by 72 +/- 5% (n = 5). Superfusion with the nonselective NOS inhibitor N-nitro-L-arginine (L-NNA, 1 mM) reduced resting BFcrb and attenuated the response to PF stimulation (-47 +/- 5%) and hypercapnia (-46 +/- 7%; PCO2 = 50-60 mmHg). After L-NNA, superfusion with the NO donors 3-morpholinosydnonimine (100 microM, n = 5) or S-nitroso-N-acetyl-penicillamine (5 microM, n = 5) reestablished resting BFcrb (P > 0.05 vs. before L-NNA) and reversed L-NNA-induced attenuation of the response to hypercapnia (P > 0.05 vs. before L-NNA) but not PF stimulation (P > 0.05 vs. after L-NNA). Similar results were obtained when NOS activity was inhibited with the inhibitor of neuronal NOS 7-nitroindazole (50 mg/kg i.p.). Like NO donors, the guanosine 3',5'-cyclic monophosphate analog 8-bromoguanosine 3',5'-cyclic monophosphate (n = 5), administered after L-NNA, restored resting BFcrb and counteracted inhibition of the response to hypercapnia but not PF stimulation. In contrast to NO donors and 8-bromoguanosine 3',5'-cyclic monophosphate, the NO-independent vasodilator papaverine (100 microM, n = 5) had no effect on attenuation of responses to PF stimulation or hypercapnia. Thus NO donors are unable to reverse the effect of NOS inhibition on vasodilation produced by PF stimulation. The data support the hypothesis that the vascular response to PF stimulation, at variance with hypercapnia, requires NOS activation and NO production. Thus NO plays an obligatory role in vasodilation produced by increased functional activity in cerebellar cortex.

Topics: Animals; Blood Pressure; Carbon Dioxide; Cerebellum; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Glutamic Acid; Hyperemia; Indazoles; Male; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxygen; Papaverine; Partial Pressure; Penicillamine; Rats; Rats, Sprague-Dawley; Regional Blood Flow; S-Nitroso-N-Acetylpenicillamine; Vasodilation

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 microM) inhibited degranulation (beta-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 microM) did not inhibit, but at high concentrations (1000-2350 microM), it potentiated FMLP-induced beta-glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 microM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 microM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.

Topics: Acetonitriles; Calcium; Cell Degranulation; Cyclic GMP; Humans; Molsidomine; Morpholines; Neutrophils; Nitrates; Nitric Oxide

The mechanisms underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide (NO) and cyclic GMP (cGMP) are still not completely understood. The present study was designed to determine the role of potassium channels in the relaxations to NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), as well as 8-bromo-3',5' -cGMP (a synthetic analogue of cGMP) and zaprinast (a selective cGMP phosphodiesterase inhibitor).. Rings of canine middle cerebral asteries without endothelium were suspended in Krebs-Ringer bicarbonate solution for isometric tension recording. The levels of cGMP were measured by radioimmunoassay. Relaxations to NO donors 8-bromo-cGMP and zaprinast were studied in the presence and in the absence of K+ channel blockers charybdotoxin (large-conductance Ca(2+)-activated K+ channels), glyburide (ATP-sensitive K+ channels), 4-aminopyridine (delayed rectifier K+ channels), and BaCl2 (multiple types of K+ channels).. Concentration-dependent relaxations caused by NO donors (SIN-1 and SNP) were significantly reduced in arteries treated with BaCl2 (3 x 10(-4) mol/L) or charybdotoxin (3 x 10(-8) mol/L). Relaxations to 8-bromo-cGMP were not affected by the same concentrations of BaCl2 and charybdotoxin; however, they were reduced by higher concentrations of BaCl2 (3 x 10(-3) mol/L) or charybdotoxin (10(-7) mol/L). Zaprinast-induced relaxations were significantly reduced by BaCl2 (3 x 10(-4) mol/L) or charybdotoxin (3 x 10(-8) mol/L). Glyburide (10(-5) mol/L) and 4-aminopyridine (10(-3) mol/L) did not alter the relaxations to SIN-1 or SNP. The production of cGMP stimulated by SIN-1 in the vascular smooth muscle was not affected by BaCl2 (3 x 10(-3) mol/L) or charybdotoxin (10(-7) mol/L).. These results indicate that in canine middle cerebral arteries, a significant portion of relaxations to NO liberated from nitrovasodilators is mediated by large-conductance Ca(2+)-activated K+ channels. Other K+ channels, sensitive to BaCl2, may also be involved in the mechanism of relaxations induced by NO.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; 4-Aminopyridine; Animals; Barium Compounds; Cerebral Arteries; Charybdotoxin; Chlorides; Cyclic GMP; Diltiazem; Dogs; Glyburide; In Vitro Techniques; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Potassium Channels; Purinones; Vasodilation; Vasodilator Agents

Nitric oxide (NO) increases 3',5'-cyclic guanosine monophosphate (cGMP) in vascular smooth muscle and increases cerebral blood flow (CBF). In early stages of cerebral ischemia, NO plays a beneficial role in sustaining CBF. Subarachnoid hemorrhage (SAH), one of the main causes of ischemia, may impair vascular reactivity to NO. To test the hypothesis, 48 h after SAH was induced in rats, we examined the CBF response to the NO donor, SIN-1 (3-morpholinosydnonimine). We measured CBF by laser-Doppler flowmetry in association with: (1) intracarotid injection (for 30 min) of SIN-1 (1.5 mg/kg), 8-bromo-cGMP (7.5 mg/kg), papaverin (1.5 mg/kg) or vehicle; (2) cortical superfusion (for 90 min) of SIN-1 (10(-5) M) or vehicle through the cranial window. Hypotension produced by these vasodilators was controlled with phenylephrine. Vehicle alone did not change CBF throughout the measurement. Intracarotid infusion of SIN-1 (n = 6/group) increased CBF up to 128.6 +/- 3.9% and 111.9 +/- 2.9% in the control group and the SAH group, respectively. SAH significantly attenuated the response (P < 0.05, ANOVA). SAH did not affect the CBF increases elicited by intracarotid administration of cGMP or papaverin, or cortical superfusion of SIN-1. We conclude that during chronic vasospasm SAH disturbs the pathway between NO release and cGMP production in large cerebral arteries. The impairment accounts for the fragility of the brain in the face of ischemia following SAH.

Topics: Animals; Brain; Cerebral Arteries; Cerebral Cortex; Cerebrovascular Circulation; Cyclic GMP; Injections, Intra-Articular; Male; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide; Papaverine; Phenylephrine; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Subarachnoid Hemorrhage; Time Factors; Vasodilator Agents

We investigated the chronotropic effect of increasing concentrations of sodium nitroprusside (SNP, n = 8) or 3-morpholinosydnonimine (SIN-1, n = 6) in isolated guinea pig spontaneously beating sinoatrial node/atrial preparations. Low concentrations of NO donors (nanomolar to micromolar) gradually increased the beating rate, whereas high (millimolar) concentrations decreased it. The increase in rate was (1) enhanced by superoxide dismutase (50 to 100 U/mL, n = 6), (2) prevented by the guanylyl cyclase inhibitors 6-anilino-5,8-quinolinedione (5 mumol/L, n = 6) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 mumol/L, n = 6), and (3) mimicked by 8-bromo-cGMP (n = 6) with no additional positive chronotropic effect of SIN-1 (n = 5). The response to 10 mumol/L SNP (n = 28) or 50 mumol/L SIN-1 (n = 16) was unaffected by IcaL antagonism with nifedipine (0.2 mumol/L) but was abolished after blockade of the hyperpolarization-activated inward current (I(f)) by Cs+ (2 mmol/L) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (1 mumol/L). The effect on I(f) was further evaluated in rabbit isolated patch-clamped sinoatrial node cells (n = 21), where we found that 5 mumol/L SNP or SIN-1 caused a reversible Cs(+)-sensitive increase in this current (+130% at -70 mV and +250% at -100 mV). In conclusion, NO donors can affect pacemaker activity in a concentration-dependent biphasic fashion. Our results indicate that the increase in beating rate is due to stimulation of I(f) via the NO-cGMP pathway. This may contribute to the sinus tachycardia in pathological conditions associated with an increase in myocardial production of NO.

Topics: Animals; Cyclic GMP; Data Interpretation, Statistical; Female; Guinea Pigs; Heart Rate; In Vitro Techniques; Ion Channels; Male; Molsidomine; Nitric Oxide; Nitroprusside; Rabbits; Sinoatrial Node; Superoxide Dismutase; Time Factors; Vasodilator Agents

L2 cells, a cloned pneumocyte-derived cell line, express voltage-dependent L-type Ca2+ channels, causing transient depolarizing spikes of the membrane potential (Vm) [P. Dietl, T. Haller, B. Wirleitner, H. Völkl, F. Friedrich, and J. Striessing. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L873-L883, 1995]. In this study, we examined the effect of nitric oxide (NO)- and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent cell signaling on the activity of L-type Ca2+ channels. Using conventional microelectrodes, spontaneous depolarizations (SD) of Vm by activation of these channels are regularly seen in the presence of 10 mM bath Sr2+. The NO donors sodium nitroprusside (SNP; 1 mM), 3-morpholinosydnonimine (SIN-1; 100 microM), as well as S-nitroso-N-acetyl-D,L-penicillamine (SNAP; 10 microM) caused a significant reduction of the frequency of Sr(2+)-induced SD. These effects were completely reversed by 6-anilino-5,8-quinolinequinone (10 microM), an inhibitor of the soluble guanylyl cyclase, and could be mimicked by 8-bromoguanosine 3'5'-cyclic monophosphate (8-BrcGMP; 100 microM). Perforated patch-clamp experiments revealed that 8-BrcGMP exerted a significant decrease of the depolarization-induced L-type Sr2+ current in the majority of tested cells. Consistent with the dependency of these NO-mediated effects on cGMP, incubation of L2 cells with SNP, SIN-1, and SNAP lead to a pronounced increase of cellular cGMP concentration. We conclude that the NO donors inhibit the activity of L-type Ca2+ channels in L2 cells via a cGMP-dependent pathway. In the alveoli, this might occur under conditions associated with the release of NO.

Topics: Aminoquinolines; Animals; Calcium Channels; Calcium Channels, L-Type; Cell Line; Cyclic GMP; Enzyme Inhibitors; Epithelium; Guanylate Cyclase; Kinetics; Membrane Potentials; Microelectrodes; Molsidomine; Nitroprusside; Penicillamine; Pulmonary Alveoli; Rats; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Strontium; Time Factors

The relaxation responses to nicorandil, nitroglycerin (NTG), and cromakalim were compared in isolated dog large (>1.5 mm inside diameter) and small (<0.3 mm inside diameter) epicardial coronary arteries. Nicorandil and NTG produced more potent relaxing effects in large coronary arteries. In contrast, cromakalim produced greater relaxation in small arteries. No significant differences were observed in the nitric oxide (NO)-induced response after treatment with superoxide dismutase. The responses to 8-bromo-cyclic guanosine monophosphate (cGMP), SIN-1, and atrial natriuretic peptide did not differ in arteries of different sizes. Treatment with L-cysteine had no significant effect on the relaxation responses to NTG in both large and small coronary arteries. Oxyhemoglobin and glibenclamide inhibited relaxation induced by nicorandil in large and small coronary arteries. Oxyhemoglobin had a greater suppressive effect on the response to nicorandil in large coronary arteries than in small coronary arteries. Methylene blue inhibited the response to nicorandil in large coronary arteries. These findings suggest that nicorandil behaves predominantly as a nitrate in large epicardial coronary arteries rather than small epicardial arteries and that this difference between large and small coronary arteries with regard to the nitrate action of nicorandil may be the result of a pathway in which nicorandil is converted to NO.

Topics: Animals; Atrial Natriuretic Factor; Benzopyrans; Coronary Vessels; Cromakalim; Cyclic GMP; Dogs; Female; Glyburide; In Vitro Techniques; Male; Methylene Blue; Molsidomine; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Niacinamide; Nicorandil; Nitric Oxide; Nitroglycerin; Nitroprusside; Oxyhemoglobins; Pyrroles; Vasodilator Agents

Nitric oxide (NO) has proven benefits in treating adult respiratory distress syndrome (ARDS). The protective mechanism remains unclear, but cyclic guanosine monophosphate (cGMP)-dependent signal transduction pathways have been suggested. Our laboratory has implicated polymorphonuclear neutrophil (PMN) priming and subsequent activation in the pathogenesis of postinjury ARDS and has shown that NO inhibits superoxide anion production from activated PMNs. More recently, the pivotal role of elastase in PMN-mediated tissue injury has been emphasized. Consequently, our study hypothesis was that NO attenuates platelet-activating factor (PAF) priming for elastase release through a cGMP-dependent pathway in human PMNs.. PMNs isolated from human volunteers were preincubated with the NO donor 3-morpholinosydnonimine hydrochloride (SIN-1; 10(-6) to 10(-2) mol/L), cGMP (10(-3) mol/L), or the cell-permeable cGMP analog dibutyryl-cGMP (10(-3) mol/L) for 10 minutes. The cells were then primed with platelet-activating factor (PAF) 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (200 nmol/L), which was followed by N-formyl-methionyl-leucyl-phenylalanine (fMLP) activation (1 mumol/L). Elastase release was measured by the cleavage of N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (AAPV-pNA).. NO (through SIN-1) significantly attenuated elastase release from PAF-primed PMNs in a dose-dependent manner. Cell-permeable dibutyryl-cGMP attenuated PMN elastase release similar to NO, but cGMP (not cell-permeable) had no effect.. NO attenuates elastase release from PAF-primed PMNs through an intracellular cGMP-dependent signal transduction pathway. These findings may partially explain the beneficial effects of NO in the treatment of postinjury ARDS and add to our knowledge of the complex intracellular pathways involved in NO/PMN interactions.

Topics: Adult; Analysis of Variance; Cyclic GMP; Dibutyryl Cyclic GMP; Humans; In Vitro Techniques; Kinetics; Leukocyte Elastase; Models, Biological; Molsidomine; Neutrophils; Nitric Oxide; Platelet Activating Factor; Signal Transduction; Substrate Specificity

Cytosolic guanylyl cyclases (GTP pyrophosphate-lyase [cyclizing; EC 4.6.1.2]), primary receptors for nitric oxide (NO) generated by NO synthases, are obligate heterodimers consisting of an alpha and a beta subunit. The alpha1/beta1 form of guanylyl cyclase has the greatest activity and is considered the universal form. An isomer of the beta1 subunit, i.e., beta2, has been detected in the liver and kidney, however, its role is not known. In this study, we investigated the function of beta2. Immunoprecipitation experiments showed that the beta2 subunit forms a heterodimer with the alpha1 subunit. NO-stimulated cGMP formation in COS 7 cells cotransfected with the alpha1 and beta2 subunits was approximately 1/3 of that when alpha1 and beta1 subunits were cotransfected. The beta2 subunit inhibited NO-stimulated activity of the alpha1/beta1 form of guanylyl cyclase and NO-stimulated cGMP formation in cultured smooth muscle cells. Our results provide the first evidence that the beta2 subunit can regulate NO sensitivity of the alpha1/beta1 form of guanylyl cyclase. Northern analysis for guanylyl cyclase subunits was performed on RNA from kidneys of Dahl salt-sensitive rats, which have been shown to have decreased renal sensitivity to NO. Compared to the Dahl salt-resistant rat, message for beta2 was increased, beta1 was decreased, and alpha1 was unchanged. These results suggest a molecular basis for decreased renal guanylyl cyclase activity, i.e. , an increase in the alpha1/beta2 heterodimer, and decrease in the alpha1/beta1 heterodimer.

Topics: Animals; Blood Pressure; Blotting, Northern; Blotting, Western; Cell Line; COS Cells; Cyclic GMP; Guanylate Cyclase; Kidney; Male; Molsidomine; Muscle, Smooth; Nitric Oxide; Rats; Rats, Inbred Strains; Recombinant Fusion Proteins; RNA, Messenger; Transfection; Vasodilator Agents

We hypothesize that the interaction between protein kinase C (PKC) and nitric oxide (NO) plays a role in the modulation of cerebral vascular tone, and the disturbance of this interaction following subarachnoid hemorrhage (SAH) results in vasospasm. To prove this hypothesis with direct evidence, PKC activities of smooth muscle cells of canine basilar arteries in the control and in the SAH groups were measured by an enzyme immunoassay method. N omega-nitro-L arginine (L-NA), an inhibitor of NO production, enhanced PKC activity. This enhancement was inhibited neither by 8-bromo-guanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) nor SIN-1, a NO releasing agent. PKC activity in the SAH was significantly higher than in the control; however, no further enhancement was produced with L-NA. In the SAH, PKC activity was not inhibited either by 8-bromo-cGMP or SIN-1. We conclude that NO maintains an appropriate vascular tone through inactivation of PKC, and that this effect is disturbed following SAH, resulting in PKC-dependent vascular contraction, such as vasospasm. On the other hand, once PKC has been activated, NO precursors do not inhibit PKC. These facts indicate NO inactivates PKC through the inhibition of phosphatidylinositol breakdown.

Topics: Animals; Basilar Artery; Cyclic GMP; Dogs; Enzyme Activation; Enzyme Inhibitors; Female; Immunoenzyme Techniques; Ischemic Attack, Transient; Male; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Protein Kinase C; Reference Values; Subarachnoid Hemorrhage

In cultured endothelial cells, incubation with TNF-alpha (50 ng/ml) for 72 h markedly reduced viability of endothelial cells. A 6-h pre-incubation with the nitric oxide (NO) donor linsidomine (SIN-1, 10-150 microM) protected endothelial cells in a concentration-dependent manner and increased viability by up to 59% of control. The unmetabolized parent compound molsidomine and the NO-free metabolite of SIN-1 3-morpholinoiminoacetonitrile (SIN-1C) were without cytoprotective effect. Cytoprotection by SIN-1 was completely abolished by the NO scavenger 2-phenyl-4,4,5,5, -tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 30 microM). A cytoprotective effect comparable to SIN-1 was observed when preincubating the cells with dibutyryl cyclic GMP (10-100 microM). Moreover, no protection by SIN-1 occurred in the presence of cycloheximide (1 microM) or 1H--1,2,4-oxadiazole-4, 3-a-quinoxalin-1-one (ODQ, 0.1 microM), a selective inhibitor of soluble guanylyl cyclase. Tin protoporphyrin-IX (SnPP, 25 microM), an inhibitor of heme oxygenase, was found to attenuate SIN-1-induced cytoprotection. Our results demonstrate that SIN-1 produces a long-term endothelial protection against cellular injury by TNF-alpha, presumably via a cyclic GMP-dependent pathway leading to up-regulation of protective proteins such as heme oxygenase.

Topics: Acetonitriles; Animals; Cattle; Cells, Cultured; Cyclic GMP; Drug Interactions; Endothelium, Vascular; Heme Oxygenase (Decyclizing); Molsidomine; Morpholines; Nitric Oxide; Reactive Oxygen Species; Tumor Necrosis Factor-alpha; Vasodilator Agents

We examined the role of endogenous NO in the autonomic regulation of atrioventricular (AV) nodal function by studying spontaneous action potentials (SAPs) and L-type Ca2+ current (ICa-L) in isolated single AV nodal cells from adult rabbit hearts. Both the perforated and the membrane-ruptured patch-clamp techniques in the whole-cell configuration were used under conditions known to alter NO production. Three NO donors, 3-morpholinosydnonimine (SIN-1, 0.1 mmol/L), S-nitroso-acetylcysteine (0.1 mmol/L), and sodium nitroprusside (0.1 mmol/L), suppressed the beta-adrenergic agonist isoproterenol (ISO, 1 mumol/L)-stimulated increase in ICa-L. SIN-1 also decreased the frequency and amplitude of SAPs. In cells in which ICa-L had been previously attenuated by the muscarinic agonist carbamylcholine (CCh, 1 mumol/L), SIN-1 had no additive effect. CCh activated an acetylcholine-sensitive outward K+ current (IK(ACh)) in AV nodal cells, in addition to the ICa-L inhibition. Intracellular dialysis with the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA, 0.5 mmol/L) blocked CCh-induced, but not SIN-1-induced, ICa.L attenuation. However, intracellular dialysis with methylene blue (20 mumol/L), which inhibits NO-mediated activation of guanylyl cyclase and cGMP production, blocked the effects of both CCh and SIN-1 on ICa-L. In these cells, neither L-NMMA nor methylene blue affected the CCh-activated IK(ACh). Direct application of cGMP (10 mumol/L) via internal dialysis significantly inhibited ISO-stimulated ICa-L. In AV nodal cells internally perfused with either a nonhydrolyzable cAMP analogue, 8-Br-cAMP (0.5 mmol/L), or a high concentration of cAMP (0.5 mmol/L), CCh did not inhibit, ICa-L but still activated IK(ACh). CCh-induced ICa-L attenuation could be abolished or quickly reversed by the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (20 mumol/L). However, CCh still significantly suppressed ISO-stimulated ICa-L after the cGMP-inhibited PDE isozyme (PDE3) had been selectively inhibited by milrinone (5 mumol/L). Immunohistochemical staining identified the presence of the endothelial constitutive NO synthase (ecNOS or NOS3) in both single AV nodal cells in vitro and in cryostat sections of AV nodal tissue in situ. These results demonstrate that endogenous NO is involved in the muscarinic cholinergic attenuation of ICa-L in AV nodal cell; the mechanism likely involves the cGMP-stimulated PDE.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Action Potentials; Animals; Arginine; Atrioventricular Node; Calcium Channels; Carbachol; Cyclic GMP; Immunohistochemistry; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Rabbits; Receptors, Muscarinic

Cardiac endothelium releases a number of factors that may modulate performance of underlying cardiac muscle. Nitric oxide (NO), which accounts for the biological activity of the vascular endothelium-derived relaxing factor and relaxes vascular smooth muscle by elevating intracellular cGMP, may be involved in this cardiac modulation.. We examined the myocardial contractile effects of the NO-releasing nitrovasodilators sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1), and S-nitroso-N-acetyl-penicillamine (SNAP); of a cGMP analogue, 8-bromo-cGMP; and of the cGMP-phosphodiesterase inhibitor zaprinast in isolated cat papillary muscle. Modulation of these effects by endocardial endothelium (EE) and by cholinergic and adrenergic stimulation was also investigated. Concentration-response curves with addition of NO-releasing nitrovasodilators (SNP, SIN-1, SNAP) and 8-bromo-cGMP resulted in a biphasic inotropic response. Although administration of low concentrations induced a positive inotropic effect, higher concentrations induced a negative inotropic effect. Both NO-induced positive and negative inotropic effects were attenuated by methylene blue, suggesting a role for cGMP. The response to high concentrations of 8-bromo-cGMP was shifted to the right in muscles with damaged EE, whereas cholinergic stimulation shifted the curve leftward. Zaprinast caused a monophasic concentration-dependent positive inotropic effect; damaging the EE shifted the terminal portion of the curve upward. Concomitant cholinergic or adrenergic stimulation modified the response to zaprinast into a negative inotropic response.. NO and cGMP induced a concentration-dependent biphasic contractile response. The myocardial contractile effects of NO and cGMP were modulated by the status of EE and by concomitant cholinergic or adrenergic stimulation.

Topics: Animals; Cats; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Molsidomine; Myocardial Contraction; Nitric Oxide; Nitroprusside; Penicillamine; Purinones; S-Nitroso-N-Acetylpenicillamine

Left ventricular hypertrophy (LVH) produced by aortic valve plication leads to increased myocardial cyclic GMP. We tested whether this was a result of increased soluble guanylate cyclase activity or nitric oxide (NO) synthase and its functional consequences. We used the nitric oxide donor 3-morpholino-sydnonimine (SIN-1) or the NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME) in 12 control and 12 LVH anesthetized open-chest mongrel dogs. L-NAME (6 mg/kg) or SIN-1 (1 microgram/kg per min) was infused into the left anterior descending coronary artery and regional segment work and cyclic GMP levels were determined. In vitro myocardial guanylate cyclase sensitivity (0.43 +/- 0.04 to 0.28 +/- 0.04 mM [EC50]) and maximal activity (10.1 +/- 2.9 to 25.5 +/- 6.5 pmol/mg protein per min) were significantly increased in LVH as compared with control animals in response to nitroprusside stimulation, but cyclic GMP-phosphodiesterase activity was similar. In LVH dogs, basal cyclic GMP was significantly elevated in vivo when compared with controls. Treatment of dogs with SIN-1 resulted in a significant increase in cyclic GMP in control (1.09 +/- 0.12 to 1.48 +/- 0.19 pmol/gram) and a greater increase in the LVH group (1.78 +/- 0.16 to 3.58 +/- 0.71 pmol/g). L-NAME had no effect on myocardial cyclic GMP levels in control or LVH dogs. Segment work decreased in the control group after SIN-1 (1,573 +/- 290 to 855 +/- 211 grams x mm/min). LVH dogs showed no decrement in work as a result of treatment with SIN-1. L-NAME did not cause significant changes in myocardial cyclic GMP, O2 consumption, or work in either control or LVH dogs, but vascular effects were evident. SIN-1 increased cyclic GMP, and with greater effect on LVH; however, this resulted in a decrement in function only in the control group. The greater increased cyclic GMP in LVH dogs is not related to increased NO production, but is related to significantly higher sensitivity and maximal activity of soluble myocardial guanylate cyclase.

Topics: Animals; Arginine; Cyclic GMP; Dogs; Female; Guanylate Cyclase; Hypertrophy, Left Ventricular; Male; Molsidomine; NG-Nitroarginine Methyl Ester; Oxygen Consumption

Many inflammatory diseases are associated with a hypoproliferative anaemia. Patients with this anaemia often present with serum erythropoietin (EPO) concentrations that are too low for the degree of their anaemia. Proinflammatory cytokines, in addition to their inhibitory effects on proliferation of erythroid progenitors, could contribute to the pathogenesis of this anaemia by reducing EPO production. Because several cytokines stimulate nitric oxide (NO) synthase we propose that nitric oxide might mediate the suppression of EPO production during inflammation. In order to test this hypothesis we investigated the effects of NO donors on 24-h hypoxia-induced EPO production in the hepatocellular carcinoma cell line HepG2. Following application of the NO donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), EPO production was dose-dependently reduced: compared to the untreated control EPO production was lowered by 89% with SNP (1000 microM), by 66% with SIN-1 (1000 microM), and by 72% with SNAP (500 microM). In contrast, 8-bromo-cGMP did not inhibit EPO formation. Since pyrogallol (300 microM) and H2O2 (250 microM) showed a comparable suppression of EPO synthesis, we propose that NO might affect EPO production either by a similar direct influence on the cellular redox state or via increasing the cellular content of reactive oxygen species.

Topics: Carcinoma, Hepatocellular; Cyclic GMP; Cytokines; Erythropoietin; Humans; Hydrogen Peroxide; Liver Neoplasms; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Penicillamine; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Tumor Cells, Cultured

Numerous functional studies establish the role of nitric oxide (NO) as a neuromodulator in the central nervous system which affects synaptic transmission. However, there are only a few reports indicating a direct and postsynaptic effect of nitric oxide on the electrical activity of neurons in the central nervous system. The aim of this study was to characterize the effect of nitric oxide on spontaneously active neurons in spinal cord slices using an extracellular recording technique. Because in the lumbar rat spinal cord the NO producing enzyme NO-synthase is primarily located in the superficial dorsal horn (laminae I+II) and around the central canal (lamina X), we restricted our recordings to these areas. While the majority of neurons increased their electrical activity during superfusion with the NO-donor sodium nitroprusside (SNP) in lamina X, neurons in laminae I+II were mainly inhibited by SNP. The excitatory and the inhibitory effects were dose-dependent and reversible and were mimicked by other NO-donors and membrane permeable cyclic guanosine monophosphate (8Br-cGMP) on the same neurons. The spinal cord slice preparation contains functional NO-synthase (NOS), because selective blockade of NOS increased the spontaneous activity of those neurons from laminae I+II which were inhibited by SNP and this effect could be reversed by superfusion with the natural substrate for NOS, L-arginine. It is concluded that NO can activate and inhibit the activity of spinal cord neurons by raising cGMP levels and that these effects are lamina specific. A general consequence of our results is that the NO-induced production of cGMP alone does not allow any prediction about an excitatory or inhibitory effect of NO on the discharge rate of neurons. Thus the NO mediated increase and decrease in neuronal activity is probably the result of intracellular mechanisms downstream from the production of cGMP which results in the activation or inhibition of different ion channels on neurons in laminae I+II and X.

Topics: Animals; Cyclic GMP; Dose-Response Relationship, Drug; Electrophysiology; Enzyme Inhibitors; Histocytochemistry; Male; Molsidomine; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; omega-N-Methylarginine; Organ Culture Techniques; Penicillamine; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Spinal Cord; Vasodilator Agents

The study was carried out to assess the effect of nitric oxide (NO) generation or inhibition of NO synthase on the cardiac response to beta- and alpha 1-adrenergic agonists. In addition, we determined the effects of the cell-permable analogue of cGMP, 8-bromo-cGMP (8Br-cGMP).. Experiments were done in electrically-paced isolated perfused rat hearts as well as in ventricular myocytes. Hearts were exposed to either the beta-adrenoceptor agonist, isoproterenol (0.1 microM), or the alpha 1-adrenoceptor agonist, phenylephrine (2 microM in the presence of equimolar concentrations of propranolol), either with each drug alone or in the presence of the NO donors S-nitrosoacetylpenicillamine (SNAP, 10 microM) and 3-morpholino-sydnonimine (SIN-1, 10 microM), the NO synthase inhibitor L-NAME (10 microM) or 8Br-cGMP (50 microM). These concentrations of SNAP and 8Br-cGMP increase tissue cGMP levels approximately 3-fold after 15 min treatment. Myocardial contractility was assessed by determining left ventricular pressure with a fluid-filled balloon inserted into the left ventricle. Similar experiments were performed in myocytes in which cell shortening and intracellular calcium transients were determined although concentrations of isoproterenol and phenylephrine in myocytes were higher (1 and 5 microM, respectively) than those used in isolated hearts in order to achieve optimum responses.. In isolated hearts isoproterenol increased developed pressure by about 50%, which was totally prevented by SNAP and SIN-1 and unaffected by L-NAME. 8Br-cGMP, however, did not significantly diminish the positive inotropic effect of isoproterenol. Phenylephrine increased developed pressure of isolated hearts by about 30%, but this was totally unaffected by either SNAP, SIN-1 or 8Br-cGMP. In myocytes, isoproterenol significantly increased the calcium transient by more than 50% and cell shortening by about 70%. Both effects were significantly attenuated by SNAP, SIN-1 and 8Br-cGMP but unaffected by L-NAME. Phenylephrine significantly increased cell shortening and the calcium transient, but these responses were unaffected either by SNAP or 8Br-cGMP.. The present study demonstrate that NO as well as guanylate cyclase inhibitors and, to a lesser extent, 8Br-cGMP attenuate beta-receptor-mediated cardiac responses and supports the concept that NO serves as an endogenous regulator of beta-mediated effects of catecholamines in the heart. In addition, our findings suggest that the antiadrenergic effects of NO are restricted to these receptors but likely do not involve alpha 1-mediated effects.

Topics: Adrenergic alpha-Agonists; Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Calcium; Cell Size; Cells, Cultured; Cyclic GMP; Isoproterenol; Male; Molsidomine; Myocardial Contraction; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Penicillamine; Perfusion; Phenylephrine; Propranolol; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine; Stimulation, Chemical; Vasodilator Agents

The basal release of vasoactive intestinal polypeptide (VIP) from freshly prepared enriched synaptosomes was 159.1 +/- 17.3 fmol/mg protein (100%), which constituted 2.5% of the total VIP content. Basal VIP release was reduced by 65% by removal of external Ca2+. Release of VIP was stimulated by depolarization with KCl (65 mM, 143%) and in the presence of veratridine (10(-6) M, 184%), monensin (10(-5) M, 131%), and the Ca2+ ionophore A-23187 (10(-6) M, 160%). Stimulation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanisms using isoproterenol (10(-6)-10(-4) M) and forskolin (10(-6) and 10(-5) M) had no stimulatory influence on VIP release. In contrast, sodium nitroprusside (10(-4) M, 198%), the nitric oxide (NO) donor 3-(morpholino)sydnonimine (10(-4) M, 155%), and the guanosine 3',5'-cyclic monophosphate (cGMP) analogue 8-bromo cGMP (10(-4) M, 196%) caused a significant release of VIP. L-Arginine (10(-3) M, 246%) also caused a significant increase of VIP release that was antagonized by the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (5 x 10(-4) M, 131%), which had no effect when given alone. The results demonstrate that VIP can be released from enriched synaptosomes by Ca(2+)-dependent mechanisms by NO agonists or NO-dependent mechanisms. It is speculated that this VIP release is induced by a presynaptic stimulatory mechanism of NO and this effect could enhance or contribute to the action of NO.

Topics: Animals; Arginine; Calcium; Cell Fractionation; Cyclic GMP; Intestine, Small; Membrane Potentials; Molsidomine; NADPH Dehydrogenase; Nerve Endings; Nitric Oxide; Nitroprusside; Protein Kinase C; Rats; Rats, Wistar; Synaptosomes; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide

This study tested whether nitric oxide modulates the expression of intercellular adhesion molecule-1 (ICAM-1) expression in glomerular mesangial cells. In an enzyme-linked immunosorbent assay, interleukin 1 beta (IL-1 beta; 10 ng/mL) increased ICAM-1 molecule expression on cultured rat mesangial cell surface in a time-dependent manner. Addition of the nitric oxide donors 3-morpholino-sydnonimine (SIN-1) or sodium nitroprusside significantly suppressed IL-1 beta-induced ICAM-1 molecule expression in a dose-dependent manner. The inhibitory effect of SIN-1 was abolished in the presence of a nitric oxide scavenger hemoglobin, but not in the presence of superoxide dismutase or pyrrolidine dithiocarbamate. Addition of 8-bromo-cyclic GMP showed no significant effect on IL-1 beta-induced ICAM-1 expression. In Northern blot analysis, the expression of ICAM-1 mRNA was barely detected in unstimulated cells, whereas ICAM-1 gene transcripts were clearly expressed after exposure to IL-1 beta for 3 h, and addition of SIN-1 decreased IL-1 beta-induced ICAM-1 mRNA accumulation. These results suggest that nitric oxide suppresses ICAM-1 expression in IL-1 beta-stimulated mesangial cells, independent of cGMP formation.

Topics: Animals; Antibodies, Monoclonal; Cells, Cultured; Cyclic GMP; Enzyme-Linked Immunosorbent Assay; Free Radical Scavengers; Glomerular Mesangium; Intercellular Adhesion Molecule-1; Interleukin-1; Male; Molsidomine; Nitric Oxide; Rats; Rats, Sprague-Dawley; RNA, Messenger

1. The effects of nitric oxide (NO) releasing substances, sodium nitroprusside, 3-morpholino sydnonimine (SIN-1) and a novel oxatriazole derivative, GEA 3162, on blood pressure and heart rate were studied after peripheral or central administration in anaesthetized normotensive Wistar rats. 2. Given as cumulative intravenous injections, both nitroprusside and GEA 3162 (24-188 nmol kg-1) induced short-lasting and dose-dependent decreases in mean arterial pressure, while SIN-1 decreased blood pressure only slightly even after larger doses (94-3000 nmol kg-1). Heart rate increased concomitantly with the hypotensive effect of the NO-releasing substances. 3. Cumulative intracerebroventricular administration of GEA 3162 (24-188 nmol kg-1) induced a dose-dependent hypotension with slight but insignificant increases in heart rate. In contrast, intracerebroventricular nitroprusside induced little change in blood pressure, while a large dose of SIN-1 (3000 nmol kg-1, i.c.v.) slightly increased mean arterial pressure. However, intracerebroventricular nitroprusside and SIN-1 increased heart rate at doses that did not significantly affect blood pressure. 4. To determine whether the cardiovascular effects of GEA 3162 were attributable to an elevation of cyclic GMP levels, pretreatments with methylene blue, a putative guanylate cyclase inhibitor, were performed. This substance failed to attenuate the cardiovascular effects of peripherally or centrally administered GEA 3162, suggesting that the effects were independent of guanylate cyclase. 5. In conclusion, the centrally administered NO-donor, GEA 3162, induced a dose-dependent. hypotensive response without significant changes in heart rate. Furthermore, intracerebroventricular injections of nitroprusside and SIN-1 increased heart rate without affecting blood pressure. These results suggest that NO released by these drugs may affect central mechanisms involved in cardiovascular regulation independently of cyclic GMP.

Topics: Animals; Blood Pressure; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Heart Rate; Injections, Intravenous; Injections, Intraventricular; Male; Molsidomine; Nitric Oxide; Nitroprusside; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Triazoles

1. The NO donor 3-morpholino-sydnonimine (SIN-1; 0.01-10 microM) evoked concentration-dependent relaxation of rat isolated mesenteric arteries pre-constricted with phenylephrine (1-3 microM). The relaxation to SIN-1 was not significantly different between endothelium-intact or denuded arterial segments or segments in which basal nitric oxide (NO) synthesis was inhibited (n = 8; P > 0.05). In contrast, the membrane permeable analogue of guanosine 3':5'-cyclic monophosphate (cyclic GMP), 8-Br-cyclic GMP (0.01-1 mM), was much less effective in relaxing intact than denuded arterial segments or intact arterial segments pre-incubated with NO synthase blockers (n = 4; P < 0.01). 2. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min) alone, did not alter SIN-1-evoked relaxation in any tissues (n = 5; P > 0.05). However, in parallel experiments, ODQ almost completely inhibited both basal and SIN-1-stimulated production of cyclic GMP in both the presence and absence of NO synthase blockers (n = 6; P < 0.01) indicating that full relaxation to SIN-1 can be achieved in the absence of an increase in cyclic GMP. 3. Exposure of endothelium-intact arterial segments to the potassium channel blocker charybdotoxin (50 nM; 10 min), significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 90% (n = 5; P < 0.01). In contrast, in arterial segments in which either the endothelial cell layer had been removed or basal NO synthesis inhibited, relaxation to SIN-1 was not reduced in the presence of charybdotoxin (n = 6; P > 0.05). However, in the presence of NO synthase blockers and L-arginine (300 microM) together, charybdotoxin did significantly inhibit SIN-1-evoked relaxation to a similar extent as intact tissues (maximum response induced by around 80%; n = 4; P < 0.01). 4. Pre-incubation with apamin (30 nM; 10 min) or glibenclamide (10 microM; 10 min) did not alter SIN-1-evoked relaxation of phenylephrine-induced tone in any tissues (n = 4 and n = 6, respectively; P > 0.05). However, in the presence of either ODQ and apamin, or ODQ and glibenclamide, SIN-1-evoked relaxation was significantly attenuated in intact arterial segments and segments in which NO synthesis was blocked. 5. Exposure of intact arterial segments to charybdotoxin and apamin, in the presence of NO synthase blockers, also significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 80% (n = 4; P < 0.01). 6. Addition of superoxide dismutas

Topics: Animals; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Male; Mesenteric Arteries; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Potassium Channels; Rats; Rats, Wistar; Superoxide Dismutase; Vasodilator Agents

Endogenously released nitric oxide (NO) in airways might contribute to physiological bronchodilation; induced production of NO might play a role in the pathogenesis of asthma, although it could also be a compensatory mechanism to other factors that cause bronchoconstriction or inflammation. To investigate the efficacy of NO donors on bronchial tone, the bronchorelaxing efficacies of NO donors, new experimental GEA compounds 3268 and 5145 (oxatriazole sulphonylamides) were compared with those of sodium nitroprusside and SIN-1 (3-morpholinosydnonimine) and to the standard beta 2-adrenergic agonist, salbutamol, in bronchi of guinea-pigs and rats in-vitro. Their relaxing effects were also studied in rat mesentery arteries to compare the selectivity for airways. The capacity of the NO donors to produce nitrites and nitrates was assayed by the Griess reaction. The novel NO donors GEA 3268 and GEA 5145 were more potent bronchorelaxing agents than the old NO donors sodium nitroprusside and SIN-1. In guinea-pig bronchi, however, salbutamol was most potent. In rat bronchi the GEA compounds induced the strongest relaxation effect when compared with the old NO donors or with salbutamol. The airway selectivity of the drugs studied decreased in the order of salbutamol, SIN-1, GEA 5145, GEA 3268, sodium nitroprusside. The nitrites and nitrates produced spontaneously did not correlate with the efficacy of the relaxants. The results obtained suggest that NO is only partly responsible for the relaxation and the potency is dependent on the animal species and constricting agents used.

Topics: Albuterol; Animals; Bronchodilator Agents; Cyclic GMP; Female; Guinea Pigs; In Vitro Techniques; Male; Mesenteric Arteries; Molsidomine; Nitric Oxide; Nitroprusside; Rats; Rats, Wistar; Triazoles

There is ample evidence that nitric oxide (NO) is an important neurotransmitter in many tissues of the urogenital tract. The aim of the present study was to examine the possible role of NO in ureteral relaxation. Human ureteral rings were mounted in organ bath chambers and precontracted in KCl. Increasing doses of the NO donor linsidomine (SIN-1) were added with and without prior blockade of the NO/cGMP pathway by methylene blue and protein kinase (PK) inhibitors Rp-8-pCPT-cGMPS and RP-8-CPT-cAMPS. Electrical filed stimulation (EFS) was done before and after incubation with L-NOARG (NG-nitro-L-arginine) and TTX (tetratodoxin). For detection of neuronal NO synthase (NOS), ureters were stained immunohistochemically. Ureteral strips were dose dependently relaxed by SIN-1; preincubation with methylene blue and protein kinase G inhibitor significantly reduced the SIN-1-induced relaxations. No effects of L-NOARG and TTX on EFS-induced tone alterations were found. NOS-positive neuronal axons and nerve-ending-like structures were found in the muscular layers. Our in vitro findings suggest that ureteral relaxation may involve the NO pathway.

Topics: Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Humans; Immunohistochemistry; In Vitro Techniques; Molsidomine; Muscle Relaxation; Muscle Tonus; Muscle, Smooth; Nitric Oxide; Potassium Chloride; Thionucleotides; Ureter

Leukocyte adhesion to vascular endothelium is a crucial step in the early stages of atherosclerosis, which may be mediated by the interaction of adhesion molecules expressed on the surfaces of both cell types. In this study, we investigated the effects of nitric oxide (NO) on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). ICAM-1 and VCAM-1 protein and mRNA expression were determined by cellular ELISA and Northern blot analysis, respectively. Both ICAM-1 and VCAM-1 expression were increased markedly by interleukin-1 beta (IL-1 beta). This IL-1 beta-mediated induction of ICAM-1 and VCAM-1 expression was significantly inhibited in the presence of a NO donor 3-morpholino-sydnonimine (SIN-1) in a dose-dependent manner. The inhibitory effect of SIN-1 was abolished in the presence of a NO scavenger haemoglobin, while addition of 8-bromo-cGMP showed no significant effect on IL-1 beta-induced ICAM-1 or VCAM-1 expression. Northern blot analysis showed that IL-1 beta markedly increased ICAM-1 and VCAM-1 mRNA expression, while SIN-1 decreased the accumulation of these transcripts induced by IL-1 beta. These results suggest that NO could prevent the focal adhesion and accumulation of leukocytes through the inhibition of ICAM-1 and VCAM-1 expression in endothelial cells.

Topics: Cell Adhesion; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; Molsidomine; Nitric Oxide; RNA, Messenger; Umbilical Veins; Vascular Cell Adhesion Molecule-1

The effects of nitric oxide on evoked acetylcholine (ACh) release were studied at two identified cholinergic neuro-neuronal synapses of the nervous system of the mollusc Aplysia californica. The NO-donor, 3-morpholinosydnonimine (SIN-1), decreased the amplitude of evoked inhibitory postsynaptic currents (buccal ganglion) and potentiated that of evoked excitatory postsynaptic currents (abdominal ganglion). SIN-1 acted by modulating the number of ACh quanta released. 8Br-cGMP mimicked the effects of NO on ACh release in both types of synapses thus pointing to the involvement of a NO-sensitive guanylate cyclase. Presynaptic voltage-dependent Ca2+ and K+ (IA and late outward rectifier) currents were not modified by SIN-1 suggesting another final target for NO/cGMP. The labelling of a NO-synthase by immunostaining in several neurones as well as the modulation of ACh release by L-arginine indicate that an endogenous NO-synthase is involved in the modulation of synaptic efficacy in both buccal and abdominal ganglia.

Topics: Acetylcholine; Action Potentials; Animals; Aplysia; Arginine; Calcium Channels; Cholinergic Agonists; Cholinergic Antagonists; Cyclic GMP; Ganglia, Invertebrate; Ganglia, Parasympathetic; Guanylate Cyclase; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Potassium Channels; Synapses; Synaptic Transmission

The biochemical signaling pathways involved in nitric oxide (NO)-mediated cholinergic inhibition of L-type Ca2+ current (ICa[L]) were investigated in isolated primary pacemaker cells from the rabbit sinoatrial node (SAN) using the nystatin-perforated whole-cell voltage clamp technique. Carbamylcholine (CCh; 1 microM), a stable analogue of acetylcholine, significantly inhibited ICa(L) after it had been augmented by isoproterenol (ISO; 1 microM). CCh also activated an outward K+ current, IK(ACh). Both of these effects of CCh were blocked completely by atropine. Preincubation of the SAN cells with L-nitro-arginine methyl ester (L-NAME; 0.2-1 mM), which inhibits NO synthase (NOS), abolished the CCh-induced attenuation of ICa(L) but had no effect on IK(ACh). Coincubation of cells with both L-NAME and the endogenous substrate of NOS, L-arginine (1 nM), restored the CCh-induced attenuation of ICa(L), indicating that L-NAME did not directly interfere with the muscarinic action of CCh on ICa(L). In the presence of ISO the CCh-induced inhibition of ICa(L) could be mimicked by the NO donor 3-morpholino-sydnonimine (SIN-1; 0.1 mM). SIN-1 had no effect on its own or after a maximal effect of CCh had developed, indicating that it does not inhibit ICa(L) directly. SIN-1 failed to activate IK(ACh), demonstrating that it did not activate muscarinic receptors. Both CCh and NO are known to activate guanylyl cyclase and elevate intracellular cGMP. External application of methylene blue (10 microM), which interferes with the ability of NO to activate guanylyl cyclase, blocked the CCh-induced attenuation of ICa(L). However, it also blocked the activation of IK(ACh), suggesting an additional effect on muscarinic receptors or G proteins. To address this, a separate series of experiments was performed using conventional whole-cell recordings with methylene blue in the pipette. Under these conditions, the CCh-induced attenuation of ICa(L) was blocked, but the activation of IK(ACh) was still observed. Methylene blue also blocked the SIN-1-induced decrease in ICa(L). 6-anilino-5,8-quinolinedione (LY83583; 30 microM), an agent known to decrease both basal and CCh-stimulated cGMP levels, prevented the inhibitory effects of both CCh and SIN-1 on ICa(L), but had no effect on the activation of IK(ACh) by CCh. In combination, these results show that CCh- and NO-induced inhibition of ICa(L) is mediated by cGMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Aminoquinolines; Animals; Arginine; Carbachol; Cardiotonic Agents; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Enzyme Inhibitors; GTP-Binding Proteins; Guanylate Cyclase; Heart Rate; Isoproterenol; Methylene Blue; Molsidomine; Muscarinic Agonists; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Platelet Aggregation Inhibitors; Rabbits; Receptors, Cholinergic; Receptors, Muscarinic; Sinoatrial Node; Thionucleotides

1. Recent studies have suggested that the generation of nitric oxide (NO) and hydrogen peroxide (H2O2) by islet NO synthase and monoamine oxidase, respectively, may have a regulatory influence on insulin secretory processes. We have investigated the pattern of insulin release from isolated islets of Langerhans in the presence of various pharmacological agents known to perturb the intracellular levels of NO and the oxidation state of SH-groups. 2. The NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) dose-dependently increased L-arginine-induced insulin release. D-Arginine did not influence L-arginine-induced insulin secretion. However, D-NAME which reportedly has no inhibitory action on NO synthase, modestly increased L-arginine-induced insulin release, but was less effective than L-NAME. High concentrations (10 mM) of D-arginine as well as L-NAME and D-NAME could enhance basal insulin release. 3. The intracellular NO donor, hydroxylamine, dose-dependently inhibited insulin secretion induced by L-arginine and L-arginine+L-NAME. 4. Glucose-induced insulin release was increased by NO synthase inhibition (L-NAME) and inhibited by the intracellular NO donor, hydroxylamine. Sydnonimine-1 (SIN-1), an extracellular donor of NO and superoxide, induced a modest suppression of glucose-stimulated insulin release. SIN-1 did not influence insulin secretion induced by L-arginine or the adenylate cyclase activator, forskolin. 5. The intracellular 'hydroperoxide donor' tert-butylhydroperoxide in the concentration range of 0.03-3 mM inhibited insulin release stimulated by the nutrient secretagogues glucose and L-arginine. Low concentrations (0.03-30 microM) of tert-butylhydroperoxide, however enhanced insulin secretion induced by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). 6. Islet guanosine 3':5'-cyclic monophosphate (cyclic GMP) content was not influenced by 10 mML-arginine or tert-butylhydroperoxide at 3 or 300 micro M but was markedly increased (14 fold) by a high hydroxylamine concentration (300 micro M). In contrast, islet adenosine 3':5'-cyclic monophosphate (cyclicAMP) content was increased (3 fold) by L-arginine (10 mM) and (2 fold) by tert-butylhydroperoxide(300 micro M).7. Our results strongly suggest that NO is a negative modulator of insulin release induced by the nutrient secretagogues L-arginine and glucose. This effect is probably not mediated to any major extent by the guanylate cyclase-cyclic GMP system but may rather be

Topics: 1-Methyl-3-isobutylxanthine; Amino Acid Oxidoreductases; Animals; Arginine; Colforsin; Cyclic AMP; Cyclic GMP; Female; Glucose; Hydrogen Peroxide; Hydroxylamine; Hydroxylamines; In Vitro Techniques; Insulin; Islets of Langerhans; Mice; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Peroxides; Reactive Oxygen Species; tert-Butylhydroperoxide; Vasodilator Agents

LLC-PK1 epithelial cells and RFL-6 fibroblasts secreted both cyclic AMP (cAMP) and cyclic GMP (cGMP) when costimulated with forskolin and 3-morpholinosydnonimine (a chemical nitric oxide generator). Intracellular cAMP levels as high as 1100 and 12,000 pmol/10(6) cells were achieved for the two cell types, respectively. These levels were high enough to reach approximately 50% saturation of the cAMP transporter and inhibited transport of cGMP to an equal extent, suggesting that the two cyclic nucleotides compete for a common transport system. The rates of secretion of cGMP and cAMP from LLC-PK1 cells increased in proportion to their rates of synthesis as concentrations of stimulant were varied, but increased only 25% relative to intracellular concentrations in response to inhibition of phosphodiesterases by 3-isobutylmethylxanthine. It is proposed that secretion of cyclic nucleotides is not simply proportional to the total intracellular pool in these cells, but rather is coupled to synthesis. In support of this model, oxyhemoglobin was used to trap nitric oxide and block activity of guanylate cyclase in cells treated with 3-morpholinosydnonimine. As a result, secretion of cGMP ceased within 1 min, whereas intracellular levels decreased slowly over 60 min. Probenecid [p-(dipropylsulfamoyl)benzoic acid] is a nonselective antagonist of anion transport that inhibited secretion of cAMP in both cell types but, unexpectedly, blocked synthesis of cGMP, and this was reflected in direct inhibition of soluble guanylate cyclase in cell lysates. Two heat-stable, high molecular weight factors that confer sensitivity to probenecid were identified, and these factors increased the sensitivity of guanylate cyclase to nitric acid by an order of magnitude.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cell Line; Cyclic AMP; Cyclic GMP; Epithelium; Fibroblasts; Guanylate Cyclase; Molsidomine; Nitric Oxide; Oxyhemoglobins; Probenecid; Swine

The involvement of cyclic nucleotides and of phosphodiesterase activities in IL-4-induced IgE production and release of the soluble form of the low affinity receptor for IgE (sCD23) by normal human peripheral blood mononuclear cells (PBMC) was evaluated. PBMC were stimulated by a suboptimal dose of IL-4 (10 ng/ml) cAMP inducers, adrenaline (ADR) and cholera toxin (CTx), which were found to potentiate IL-4-induced IgE production and sCD23 release after 12 days of culture. In the presence of an optimal dose of IL-4 (30 ng/ml), both ADR and CTx inhibited the production of both IgE and sCD23. In the presence of a chemical cGMP inducer, Sin-1, the production of IgE induced by 10 ng/ml IL-4 appeared to be potentiated whereas in the same experimental situation the sCD23 production was decreased. Sin-1 was found to inhibit the production of both IgE and sCD23 as effectively as cAMP inducers when an optimal dose of IL-4 was used. Since Sin-1 is a nitric oxide (NO) generating compound, we evaluated the possible involvement of the L-arginine metabolic pathway using a competitive inhibitor of L-arginine, NG-monomethyl-L-arginine (LNMMA). In the presence of 1 mM LNMMA both IgE and sCD23 production induced by either a sub-optimal or an optimal dose were partially inhibited (from 50 to 80% inhibition depending on the donor). The generation of cAMP and cGMP in the cells is controlled by cyclic nucleotide phosphodiesterases (CN-PDE), so we evaluated the effect of a CN-PDE inhibitor, isobutyl-methyl xanthine (IBMX), on the IL-4-induced IgE and sCD23 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; Arginine; B-Lymphocytes; Cholera Toxin; Cyclic AMP; Cyclic GMP; Drug Synergism; Epinephrine; Gene Expression Regulation; Humans; Immunoglobulin E; Interleukin-4; Leukocytes, Mononuclear; Molsidomine; Nitric Oxide; omega-N-Methylarginine; Receptors, IgE; Recombinant Fusion Proteins

Human platelets suspended in plasma or buffer were incubated with low concentrations of the nitric oxide (NO)-donor 3-morpholino-syndnonime (SIN-1; 100 nM to 1 microM) and the stable prostacyclin analogue iloprost (50 or 100 pM) and analyzed for cyclic nucleotide levels and protein phosphorylation. SIN-1 and iloprost synergistically stimulated the phosphorylation of rap1B and the 50 kDa vasodilator-stimulated phosphoprotein. SIN-1 stimulated platelet cyclic GMP and cAMP-levels and enhanced the increase in cyclic AMP elicited by iloprost. It was found that the mechanism underlying the synergistic phosphorylation of the 50 kDa protein and rap1B was different: synergistic phosphorylation of the 50 kDa protein seemed to be mediated by activation of both protein kinases A and G, whereas the synergistic rap1B phosphorylation could be attributed entirely to activation of protein kinase A. Measurement of rap1B phosphorylation might be a useful tool to monitor the action of systemically applied prostacyclin-analogues and nitrovasodilators in pharmacological studies.

Topics: Autoradiography; Blood Platelets; Blotting, Western; Cells, Cultured; Cyclic AMP; Cyclic GMP; Drug Synergism; GTP-Binding Proteins; Humans; Iloprost; Molsidomine; Nitric Oxide; Phosphorylation; Platelet Aggregation Inhibitors; rap GTP-Binding Proteins

Nitric oxide (NO) donors were used to investigate the effect of NO on and the role of cyclic GMP in the regulation of human natural killer (NK) cell function. NO-producing drugs, molsidomine and its metabolite 3-morpholinesydnonimine (SIN-1), inhibited NK cell-mediated cytotoxicity significantly at 0.04-5 mM. At 1 mM, SIN-1 completely inhibited NK cell activity while molsidomine decreased NK cell-mediated cytolysis by 35% of the control value. These data suggest that NO from exogenous NO-donors may down-regulate NK cell cytotoxic function. The stimulatory effect of interferon-gamma (IFN-gamma) on human NK cell-mediated killing could not overtake the NK cell inhibition induced by the NO releasing drugs, indicating different modes of action for IFN-gamma and SIN-1. The results in the present study also showed that SIN-1 (1 mM) stimulated cyclic GMP production 37-fold in NK cells. In the presence of 0.5 mM IBMX, a phosphodiesterase inhibitor, the increase in cyclic GMP was even more pronounced, demonstrating a relation between cyclic GMP stimulation and NK cell inhibition by SIN-1. Further evidence for mediation via cyclic GMP was provided by the finding that methylene blue (20 microM), an inhibitor of soluble guanylate cyclase, decreased both the inhibition of SIN-1-induced NK cell cytotoxicity as well as cyclic GMP formation. Moreover, membrane-penetrating cyclic GMP and its analogues inhibited NK cell-mediated cytolysis significantly. Molsidomine was without effect on cyclic GMP levels. Our data indicate that cyclic GMP may play a role in human NK cell regulation and suggest that the inhibitory effect of cGMP may be elicited by NO.

Topics: 1-Methyl-3-isobutylxanthine; Adult; Cyclic GMP; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Humans; Interferon-gamma; Killer Cells, Natural; Methylene Blue; Molsidomine; Nitric Oxide; Tumor Cells, Cultured

The molecular mechanism of the synergistic platelet inhibition by activators of adenylate cyclase and guanylate cyclase in human platelets was investigated. The adenylate cyclase activators iloprost and prostaglandin E1 and the guanylate cyclase activator 3-morpholino-syndnonimine (SIN-1) dose-dependently inhibited thrombin-induced aggregation of washed human platelets. Furthermore, SIN-1 at a concentration inhibiting platelet aggregation by only 10% shifted the IC50 values of iloprost and prostaglandin E1 by one order of magnitude to the left, indicating a synergistic action of adenylate cyclase and guanylate cyclase activators. Iloprost and prostaglandin E1 dose-dependently elevated platelet cAMP without a significant influence on cGMP. In contrast, the platelet cGMP level was dose-dependently elevated by SIN-1. In addiiton, SIN-1 markedly increased cAMP level induced by low concentrations of adenylate cyclase activators (0.1-0.3 nM iloprost or 10-150 nM prostaglandin E1). In contrast, the rise in cAMP induced by higher adenylate cyclase activator concentrations (3 nM iloprost or 30 microM prostaglandin E1) was significantly reduced in the presence of SIN-1. The same biphasic mode of action of SIN-1 was observed with forskolin, an adenylate cyclase stimulator acting receptor independently, indicating a prostacyclin-receptor independent mechanism. The cAMP elevating effect of SIN-1 in the presence of low prostanoid concentrations was completely abolished by piroximone, a selective inhibitor of phosphodiesterase type III. Therefore, the inhibition of phosphodiesterase III by cGMP seems to be the mechanism for the elevation of cAMP levels by SIN-1 in the presence of low concentration of adenylate cyclase activators in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Alprostadil; Blood Platelets; Cyclic AMP; Cyclic GMP; Drug Synergism; Enzyme Activation; Guanylate Cyclase; Humans; Iloprost; In Vitro Techniques; Molsidomine; Nitric Oxide; Platelet Aggregation; Platelet Aggregation Inhibitors

The presence of soluble guanylate cyclase in the pineal and its regulation by adrenergic pathways has been well documented. Recent evidence points to adrenergically stimulated nitric oxide generation as a mechanism for coupling this pathway. To what extent nitric oxide (NO) signalling can influence adrenergically stimulated melatonin synthesis has not been investigated. Cyclic guanosine 3',5'-monophospate (cGMP) signal transduction in the bovine pineal has also received little attention. We describe in the present report: 1) a dose-dependent elevation of cGMP in response to the nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1), 2) a dose-dependent inhibition of melatonin synthesis by SNP and SIN-1, but not by 8-Br-cGMP in both bovine and rat pineal cell cultures, which is not due to cytotoxicity as judged by two different approaches, and 3) immunohistochemical evidence for the presence of nitric oxide synthase (NOS) (EC 1.14.23.-) in the intact bovine pineal gland and in cultured bovine pinealocytes. These data support the view that NOS is a component of the cGMP-generating system in mammalian pinealocytes. Although NO-donor molecules are also potent activators of cGMP accumulation, they may have other important actions in the pineal, namely the inhibition of adrenergic-stimulated melatonin synthesis. As SNP and SIN-1 exerted this inhibitory effect on cells regardless of whether they were stimulated by isoproterenol, forskolin or 8-Br-cAMP it would appear that NO-donors can act 'downstream' from the receptor/adenylate cyclase level.

Topics: Animals; Cattle; Cells, Cultured; Cyclic GMP; Isoproterenol; Melatonin; Molsidomine; Nitric Oxide; Nitroprusside; Pineal Gland; Rats; Stimulation, Chemical

To determine the effect of nitric oxide (NO) on sperm motility in vitro.. Normal human sperm separated by centrifugation through a discontinuous Percoll gradient and subsequent swim-up were incubated for up to 24 hours with NO donors, with and without the known NO quencher hemoglobin, as well as with agents that raise intracellular cyclic 3',5'-guanosine monophosphate (cGMP). Sperm respiration was determined by a tetrazolium-formazan spectrophotometric assay.. Andrology laboratory.. Absolute sperm motility and respiration.. Sperm incubated with the NO donors 1 mM nitroprusside, 100 to 125 microM 3-morpholinosydnonimine, and 25 to 125 microM pure nitric oxide gas dissolved in buffer were inhibited in motility in a dose-dependent fashion. The inhibition could be reversed by the NO quencher hemoglobin. Agents that raise cellular cGMP (dibutyryl cGMP or 8-bromo-cGMP) did not inhibit motility. Nitric oxide inhibited sperm respiration, as measured by the tetrazolium-formazan assay.. Nitric oxide reduces sperm motility, possibly by a mechanism involving inhibition of cellular respiration independent of an elevation of intracellular cGMP. Nitric oxide elaborated in the female or male genital tract in vivo could adversely influence sperm function and fertility.

Topics: Cyclic GMP; Hemoglobins; Humans; Male; Molsidomine; Nitric Oxide; Nitroprusside; Reactive Oxygen Species; Sperm Motility

1. We have investigated the correlation between relaxation and changes in cyclic nucleotide content of human tracheal smooth muscle (HTSM) in vitro following inhibitory non-adrenergic non-cholinergic (i-NANC) neural bronchodilator responses evoked by electrical field stimulation (EFS), and compared these with changes seen with sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and vasoactive intestinal peptide (VIP). The effects of N omega-nitro-L-arginine methyl ester (L-NAME), Methylene Blue and alpha-chymotrypsin (alpha-CT) were studied. 2. EFS (10 Hz, 1 ms, 40 V for 30 s) evoked a time-dependent relaxation accompanied by a concurrent rise in cGMP, both of which were maximal at 30 s and unaffected by epithelium removal. Levels of cAMP were more variable than those of cGMP and were not significantly changed at any time point. 3. SIN-1 (1 mM) and SNP (100 microM) also produced time-dependent relaxations which were maximal between 2 and 8 min, accompanied by concomitant rises in cGMP; however, these changes were larger than those associated with i-NANC relaxations. cAMP levels were unchanged at all time points. 4. EFS-evoked i-NANC relaxations and cGMP increases (time, t = 30 s) were inhibited by L-NAME. The effects were partially reversed by L-arginine (1 mM), but not by D-arginine. D-NAME and alpha-CT (2 u ml-1) had no effect on either relaxation or cGMP accumulation. Tetrodotoxin (TTX, 3 microM) inhibited both relaxation and cGMP accumulation. 5. VIP (1 microM) also produced a time-dependent relaxation associated with a concurrent rise in cAMP levels with no change in cGMP levels. 6. Methylene Blue (10 microM) partially inhibited EFS (10 Hz)-evoked i-NANC relaxation and cGMP accumulation, and almost completely inhibited both relaxation and cGMP accumulation evoked by SIN-1 (1 mM). Methylene Blue had no significant effect on relaxation or cGMP accumulation evoked by SNP (100 microM). 7. Neural i-NANC relaxations in HTSM are associated with a concurrent selective accumulation of cGMP which is unaffected by epithelium removal. This is inhibited in a stereoselective manner by L-NAME and mimicked by SNP and SIN-1; however, cGMP accumulation was greatly increased with SNP and SIN-1 suggesting compartmentalized changes in cGMP content. VIP also caused relaxation associated with an increase of cAMP; however, no evidence was found for VIP being involved in i-NANC relaxation. Hence nitric oxide (NO), or a NO-containing complex, appears to mediate i-NANC

Topics: Adenosine Monophosphate; Adolescent; Adult; Arginine; Bronchoconstriction; Child; Chymotrypsin; Cyclic GMP; Electric Conductivity; Epithelium; Female; Humans; Male; Methylene Blue; Middle Aged; Molsidomine; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Tetrodotoxin; Time Factors; Trachea; Vasoactive Intestinal Peptide; Vasodilator Agents

1. The present study investigates whether or not chronic feeding of rats with a diet enriched in fish oil affects the reactivity of balloon-injured carotid arteries. The left carotid arteries were injured in vivo by the repeated passage of a balloon catheter. Both the right (control artery) and the left carotid arteries were excised 24 h after the injury, and suspended in organ chambers for the measurement of changes in isometric tension in the presence of indomethacin. 2. Phenylephrine evoked similar concentration-contraction curves in the right (control) carotid arteries without endothelium from control and fish oil-fed rats. Balloon injury decreased the contractility of carotid arteries to phenylephrine in both types of rats and the pEC50 for phenylephrine was significantly decreased in balloon-injured arteries from control rats compared to those obtained in arteries from fish oil-fed rats (pEC50 7.59 +/- 0.1 and 7.28 +/- 0.06, respectively) while maximal contractions were similar (1.93 +/- 0.15 g and 1.79 +/- 0.12 g, respectively). 3. The treatment of control right carotid arteries without endothelium with either NG-nitro-L-arginine (an inhibitor of nitric oxide synthase) or superoxide dismutase (which protects nitric oxide from degradation) did not affect significantly the contractions to phenylephrine in either group. In these preparations, methylene blue (an inhibitor of soluble guanylate cyclase) decreased slightly but significantly maximal contractions to phenylephrine in both groups. The treatment of balloon-injured carotid arteries with NG-nitro-L-arginine or methylene blue partly restored contractions to phenylephrine in arteries from both types of rat. Superoxide dismutase further depressed the contractility to the alpha l-adrenoceptor agonist in balloon-injured arteries from control diet-fed rats but had no effect in balloon-injured preparations from fish oil-fed rats.4. 3-Morpholino-sydnonimine (SIN-1, a donor of nitric oxide) evoked similar concentration-dependent relaxations in control and balloon-injured carotid arteries from both types of rat.5. Balloon injury caused an increase in the tissue content of cyclic GMP in carotid arteries from control diet-fed rats. This production of cyclic GMP was abolished by N0-nitro-L-arginine. Superoxide dismutase potentiated significantly the production of cyclic GMP caused by balloon injury in control but not in fish oil-fed rats.6 These observations confirm that in vivo balloon injury causes the pro

Topics: Analysis of Variance; Animals; Arginine; Blood Pressure; Body Weight; Carotid Arteries; Carotid Artery Injuries; Catheterization; Cyclic GMP; Endothelium, Vascular; Fatty Acids; Fish Oils; Indomethacin; Isometric Contraction; Male; Methylene Blue; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Phenylephrine; Rats; Rats, Wistar; Superoxide Dismutase; Vascular Resistance; Vasodilator Agents

The study shows that endothelial cells from human umbilical veins have a soluble guanylate cyclase which can be activated by sodium nitroprusside (SNP), SIN-1 (3-morpholinosydnonimine) and S35b (4-methyl-3-phenylsulfonylfuroxan). Cells which were pretreated with these compounds showed an inhibition of thrombin-induced arachidonic acid release, PGI2 formation, PAF synthesis and PMNL adhesion. Endothelial guanylate cyclase can also be activated by nitric oxide (NO) which is generated in endothelial cells upon stimulation with thrombin or ionomycin. It is suggested that endogenously produced NO might control cell activation and endothelial function through a cGMP-dependent mechanism.

Topics: Arachidonic Acid; Cell Adhesion; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Epoprostenol; Guanylate Cyclase; Humans; Molsidomine; Nitric Oxide; Nitroprusside; Oxadiazoles; Platelet Activating Factor; Thrombin; Umbilical Veins

The aim of the present study was to investigate, whether nitric oxide (NO) modifies prostacyclin synthesis in endothelial cells. Two different NO-donors: SIN-1 (3-morpholino sydnonimine) and GEA 3175 (4-aryl-substituted oxatriazol derivative), and the NO-synthesis inhibitor; L-NAME were used. Endothelial cells were incubated with the tested compounds with or without Ca ionophore A23187 stimulation. SIN-1 (> 33 microM) and GEA 3175 (> 1 microM) increased the endothelial cGMP levels independently of A23187 stimulation. SIN-1 did not influence prostacyclin synthesis. GEA 3175 (> 33 microM) increased prostacyclin synthesis up to 2-fold, when incubated without A23187. GEA 3175 with A23187 induced about 30% inhibition in prostacyclin synthesis. L-NAME decreased unstimulated prostacyclin synthesis and this inhibition was reversed by GEA 3175. Obviously NO is able to modulate prostacyclin synthesis, however, much higher concentrations are needed than those to increase cGMP synthesis.

Topics: Animals; Arginine; Calcimycin; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Epoprostenol; Female; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Wistar; Triazoles

The effect of congeners of nitrogen monoxide (NO) on iron (Fe) uptake from 59Fe-125I-transferrin (Tf) and release of 59Fe from prelabelled cells have been investigated in SK-MEL-28 human melanoma cells, human K562 cells and mouse MDW-4 cells. These studies have been initiated as it has been suggested that the tumoricidal effects of NO may be mediated by its acting to release Fe from cells (Hibbs et al., 1984 Biochem. Biophys. Res. Commun. 123, 716-723; Hibbs et al., 1988 Biochem. Biophys. Res. Commun. 157, 87-94). The nitrosonium ion (NO+) generator, sodium nitroprusside (SNP), decreased 59Fe uptake by melanoma cells to 57% of the control without decreasing 125I-Tf uptake after a 4-h incubation with 59Fe-125-Tf (1.25 microM). Longer incubations up to 24 h decreased 59Fe uptake and also 125I-Tf uptake. Two breakdown products of SNP, ferricyanide and cyanide, had no effect on 59Fe uptake. In addition, photolysis of the SNP solution prevented the inhibition of 59Fe uptake, suggesting that NO was the active agent. Two nitric oxide (NO.) producing agents, 3-morpholinosydnonimine (SIN), and S-nitroso-N-acetylpenicillamine (SNAP), also decreased 59Fe uptake from 59Fe-125I-Tf. Superoxide dismutase increased the efficacy of SIN, and the NO-scavenger, oxyhaemoglobin, prevented the inhibition of 59Fe uptake mediated by SNAP, again suggesting that NO was the active agent. Furthermore, dialysis studies demonstrated that none of the NO-generating agents could remove 59Fe from 59Fe-125I-Tf, suggesting that the decrease in cellular Fe uptake observed was not due to NO releasing Fe from the Fe-binding sites of Tf. Despite the ability of NO-producing agents at inhibiting 59Fe uptake by cells, they could not remove significant amounts of 59Fe from melanoma cells prelabelled with either 59Fe-citrate or 59Fe-125I-Tf. Similar data were obtained using K562 and MDW-4 cells. Interestingly, the NO+ generating agent, SNP, had no effect on [3H]thymidine uptake. However, when SNP was converted to an NO. generator by the addition of 1 mM ascorbate, its effect was similar to the NO. generator, SNAP, markedly reducing [3H]thymidine incorporation to 33% of the control value. The addition of unlabelled diferric Tf (0.625 microM) to SNAP ameliorated its inhibitory effect on cellular [3H]thymidine uptake, suggesting that the interaction of NO. with Fe was of importance in the inhibition observed. The results are discussed in the context of the cytostatic potential of NO via its binding to F

Topics: Animals; Cell Line; Cyclic GMP; Humans; Iodine Radioisotopes; Iron; Iron Radioisotopes; Melanoma; Mice; Molsidomine; Nitric Oxide; Nitrites; Nitroprusside; Thymidine; Transferrin

Electrophysiological studies suggest that activation of large-conductance Ca-activated K channels (KCa) with nitric oxide (NO) causes hyperpolarization and relaxation of smooth muscle. We determined whether KCa blockers decreased relaxation to the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydonimine-hydrochloride (SIN-1) in isolated segments from main pulmonary artery (MPA), its left branch (LPA), aorta (Ao), carotid artery (CA), and trachea (Tr). NO donors caused concentration-dependent relaxation of tissues precontracted with histamine whereas the inactive carrier molecule C88-3934 was without effect. The rank order profiles of SNAP and SIN-1 sensitivity were CA = Ao = MPA > LPA = Tr. Compared with histamine, 80 mM KCl precontraction caused variable reductions in tissue sensitivity and maximum relaxation to SNAP. The KCa antagonists charybdotoxin, iberiotoxin, and tetraethylammonium decreased sensitivity to SNAP and SIN-1 2- to 11-fold in MPA, LPA, and Tr, with variable shifts in Ao and CA. The effect of iberiotoxin was not altered by removing the endothelium or epithelium. Furthermore, charybdotoxin or iberiotoxin did not alter basal or SNAP-stimulated guanosine 3',5'-cyclic monophosphate content. Glibenclamide, noxiustoxin, and leiurotoxin I, antagonists of ATP-dependent, delayed rectifier, and small-conductance KCa channels, respectively, had no effect. In conclusion, antagonists of KCa decrease NO donor-mediated relaxation of pulmonary arterial and tracheal smooth muscle.

Topics: Animals; Calcium; Cyclic GMP; Guinea Pigs; In Vitro Techniques; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth; Muscle, Smooth, Vascular; Nitric Oxide; Penicillamine; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Trachea

Cardiac Ca2+ current (ICa) was shown to be regulated by cGMP in a number of different species. Recently, we found that the NO-donor SIN-1 (3-morpholino-sydnonimine) exerts a dual regulation of ICa in frog ventricular myocytes via an accumulation of cGMP. To examine whether NO also regulates Ca2+ channels in human heart, we investigated the effects of SIN-1 on ICa in isolated human atrial myocytes. An extracellular application of SIN-1 produced a profound stimulatory effect on basal ICa at concentrations > 1 pM. Indeed, 10 pM SIN-1 induced a approximately 35% increase in ICa. The stimulatory effect of SIN-1 was maximal at 1 nM (approximately 2-fold increase in ICa) and was comparable with the effect of a saturating concentration (1 microM) of isoprenaline, a beta-adrenergic agonist. Increasing the concentration of SIN-1 to 1-100 microM reduced the stimulatory effect in two thirds of the cells. The stimulatory effect of SIN-1 was not mimicked by SIN-1C, the cleavage product of SIN-1 produced after liberation of NO. This suggests that NO mediates the effects of SIN-1 on ICa. Because, in frog heart, the stimulatory effect of SIN-1 on ICa was found to be due to cGMP-induced inhibition of cGMP-inhibited phosphodiesterase (cGI-PDE), we compared the effects of SIN-1 and milrinone, a cGI-PDE selective inhibitor, on ICa in human. Milrinone (10 microM) induced a strong stimulation of ICa (approximately 150%), demonstrating that cGI-PDE controls the amplitude of basal ICa in this tissue. In the presence of milrinone, SIN-1 (0.1-1 nM) had no stimulatory effect on ICa, suggesting that the effects of SIN-1 and MIL were not additive. We conclude that NO may stimulate ICa in human atrial myocytes via inhibition of the cGI-PDE.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Anura; Calcium Channels; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Female; Heart; Heart Atria; Humans; Isoproterenol; Kinetics; Male; Membrane Potentials; Middle Aged; Milrinone; Molsidomine; Nitric Oxide; Patch-Clamp Techniques; Pyridones; Time Factors; Vasodilator Agents

The influence of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent dilators on autoregulatory responses (AR) of arcuate arteries (ArcA) and afferent arterioles at early sites and at juxtaglomerular sites (JAA) was assessed by videomicroscopy using in vitro blood-perfused juxtamedullary nephron preparations. AR were quantified as fractional changes in luminal diameter induced by doubling blood perfusion pressure (60-120 mmHg). Baseline AR ranged from 17 +/- 2% to 21 +/- 2% in ArcA and from 24 +/- 2% to 34 +/- 4% in JAA. Direct perivascular applications of increasing concentrations of 8-bromo-cGMP (8-BrcGMP, 10 microM to 1 mM), of the NO donors sodium nitroprusside (10 microM to 1 mM) and 3-morpholino-sydnonimine chlorhydrate (SIN1; 10 microM to 1 mM), and of rat atrial natriuretic factor (ANF, 0.1 nM to 10 nM) dose- and pressure-dependently dilated all vessels at 60 mmHg. Concomitantly, AR values were dose-dependently reduced or reversed to pressure-induced dilations. During application of 8-BrcGMP and NO donors, the segmental gradient of sensitivity of AR was ArcA > JAA; the opposite gradient was found with ANF (i.e., JAA > ArcA). The present results demonstrate that compounds known to utilize the cGMP-signaling pathway act as modulators of AR along the juxtamedullary preglomerular vasculature.

Topics: Animals; Atrial Natriuretic Factor; Cyclic GMP; Dose-Response Relationship, Drug; Homeostasis; Kidney Glomerulus; Male; Molsidomine; Nitroprusside; Rats; Rats, Sprague-Dawley; Renal Circulation; Vasodilator Agents

The relaxation to bradykinin in canine coronary arteries is mediated by endothelium-derived nitric oxide (NO) and hyperpolarizing factor (EDHF). Desensitization to the kinin was induced by incubation of canine coronary arteries with endothelium with 10(-8) M bradykinin for 30 min. After washout, tissues were contracted with prostaglandin F2 alpha, and concentration-relaxation curves to bradykinin were obtained in control and desensitized arteries treated with indomethacin. After desensitization, there was a shift to the right of the concentration-relaxation curves to bradykinin. However, the elevation in guanosine 3',5'-cyclic monophosphate (cGMP) levels evoked by bradykinin was similar in both groups of tissues. The curves to bradykinin obtained in the presence of NG-nitro-L-arginine (an NO synthase inhibitor) were depressed, whereas those obtained in arteries contracted with potassium (to eliminate the EDHF-mediated relaxation) were not affected by the desensitization. Addition of NG-nitro-L-arginine, oxyhemoglobin, or methylene blue before the desensitization procedure preserved, whereas 3-morpholinosydnonimine (SIN-1, a donor of NO) and 8-bromoguanosine 3',5'-cyclic monophosphate impaired, the EDHF-mediated relaxation to bradykinin. Thus the selective impairment of the EDHF-dependent relaxation to bradykinin may be mediated by NO, acting mainly through increased production of cGMP.

Topics: Animals; Arginine; Arteries; Bradykinin; Coronary Vessels; Cyclic GMP; Dogs; Drug Resistance; In Vitro Techniques; Methylene Blue; Molsidomine; Nitroarginine; Oxyhemoglobins; Potassium; Vasodilation; Vasodilator Agents

The biochemical mechanism of bile canalicular contraction is similar to that of smooth muscle contraction. Contraction follows inositol-1,4,5-trisphosphate (InsP3)-dependent Ca2+ release, which activates actin-myosin interactions. Nitric oxide is a myorelaxant through the actions of 5'-cyclic guanosine monophosphate (cGMP) and is produced in hepatocytes exposed to endotoxin and cytokines. The aim of this study was to investigate the effect of nitric oxide on canalicular contraction and to determine the mechanism by which cGMP interferes with the contractile signal.. The canalicular motility in rat hepatocyte doublets was measured by microscopic image analysis, and intracellular Ca2+ was measured by fluorescence microscopy. cGMP and InsP3 were determined by radio-immunoassay and high-pressure liquid chromatography. Ca2+ release from liver homogenate was measured by filtration and superfusion assays.. Compounds that release nitric oxide stimulated hepatocellular production of cGMP and prevented agonist-induced contraction by inhibiting the increase in intracellular Ca2+. The cGMP analogue bromo-cGMP prevented contraction and the increase in Ca2+. Bromo-cGMP marginally decreased InsP3 production. cGMP blocked InsP3-dependent Ca2+ release from internal stores.. These findings suggest that nitric oxide interferes with Ca2+ signals by cGMP-mediated inhibition of the InsP3 receptor/Ca2+ channel and that hepatocellular production of nitric oxide may be cholestatic by impairing canalicular motility.

Topics: Animals; Bile Canaliculi; Calcium; Calcium Channel Blockers; Cyclic GMP; Inositol 1,4,5-Trisphosphate; Male; Molsidomine; Muscle Contraction; Nitric Oxide; Nitroprusside; Rats; Signal Transduction; Vasodilator Agents

1. The aim of the present studies was to examine the effects of nitric oxide donors on arrhythmias induced by coronary artery occlusion and reperfusion, and on cardiac cyclic nucleotides. Experiments were performed in pentobarbitone-anaesthetized rats prepared for occlusion of the left coronary artery. 2. Sodium nitroprusside (0.1, 0.3 and 1 microgram kg-1 min-1) had no significant effects on the incidence of ventricular tachycardia, total ventricular fibrillation or the mortality resulting from 25 min of acute myocardial ischaemia when compared with values in controls. In addition, there was no alteration in the number of ventricular premature beats that occurred in survivors. 3. 3-Morpholinosydnonimine-N-ethylcarbamide (SIN-1, 10, 20 and 40 micrograms kg-1 min-1) caused marked hypotension but did not alter the incidence or severity of ischaemia-induced arrhythmias. In rats subject to abrupt reperfusion after 5 min of myocardial ischaemia, lower doses of SIN-1 (1, 3 and 10 micrograms kg-1 min-1) still caused significant reductions in systolic and diastolic blood pressure but were devoid of antiarrhythmic activity. 4. In separate experiments in sham-operated rats, sodium nitroprusside (1 microgram kg-1 min-1), isosorbide dinitrate (30 and 60 micrograms kg-1 min-1) and SIN-1 (20 and 40 micrograms kg-1 min-1) had no significant effects on cardiac cyclic GMP content. 5. These results indicate that nitric oxide donors do not alter arrhythmias induced by acute coronary artery occlusion or reperfusion in anaesthetized rats. Although increases in total cardiac cyclic GMP could not be detected, the results suggest that, at least in the rat, cyclic GMP does not influence these arrhythmias.

Topics: Anesthesia; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Cyclic AMP; Cyclic GMP; Hemodynamics; Isosorbide Dinitrate; Male; Molsidomine; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitroprusside; Rats; Rats, Wistar; Vasodilator Agents

The mechanisms by which guanosine 3',5'-cyclic monophosphate (cGMP) modulates the contraction induced by ATP were investigated in small mesenteric resistance arteries of the rat. The nitric oxide donors 3-morpholinosydnonimine (SIN-1, 10 microM) and sodium nitroprusside (SNP, 10 microM) increased cGMP but not adenosine 3',5'-cyclic monophosphate (cAMP) content of the tissue. SIN-1, SNP, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 100 microM) inhibited the myosin light chain phosphorylation and the contractile response to ATP. Both effects were completely reversed by the selective inhibitor of cGMP protein kinase, Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (30 microM). The sensitivity to Ca2+ of arteries permeabilized with Staphylococcus aureus alpha-toxin (4,000 hemolytic units/ml) was not affected by 8-BrcGMP. The two nitric oxide donors and 8-BrcGMP decreased the rise in intracellular Ca2+ induced by ATP. The vasodilator agents abolished the contractile response to the exogenous calcium in vessels that were exposed to 3 mM ATP after depletion of intracellular Ca2+ stores. Thapsigargin (1 microM), an inhibitor of the sarcoplasmic reticulum Ca(2+)-adenosinetriphosphatase, reversed the inhibitory effect of the vasodilator agents when the contraction induced by ATP was elicited in the presence of the Ca2+ entry blocker nitrendipine (1 microM) or in Ca(2+)-free medium. These results show that cGMP inhibits ATP-induced contraction by decreasing intracellular Ca2+ concentration in small resistance arteries. They indicate that this effect results from decreased Ca2+ influx and enhanced Ca2+ sequestration through a thapsigargin-sensitive pump via activation of a cGMP protein kinase.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium-Transporting ATPases; Cyclic AMP; Cyclic GMP; In Vitro Techniques; Male; Mesenteric Arteries; Molsidomine; Myosins; Nitrendipine; Nitroprusside; Phosphorylation; Rats; Rats, Wistar; Terpenes; Thapsigargin; Vascular Resistance; Vasoconstriction

The interaction of three antiplatelet drugs was studied in vitro: aspirin, an inhibitor of the cyclooxygenase pathway of platelet activation; iloprost, a stable analog of prostacyclin that increases platelet cAMP; and the nitrix oxide donors SIN-1 and sodium nitroprusside (SNP), which both raise platelet cGMP. Platelet adhesion and aggregation evoked by collagen/ADP were measured in anticoagulated blood under physiological flow conditions using the new Thrombostat. Aggregation was also measured in platelet-rich plasma (PRP) upon stimulation by a low (2.5 micrograms/ml) and high (20 micrograms/ml) dose of collagen, ADP, or thrombin-receptor activating peptide (TRAP). We found a synergism between iloprost and aspirin in inhibiting platelet adhesion/aggregation in flowing blood and aggregation of PRP stimulated by collagen. The mean inhibitory concentrations (IC50) of iloprost in the presence of aspirin were much lower (0.7 nM and 0.5 nM in flowing blood and low-dose collagen-stimulated PRP, respectively) than in the absence of aspirin (3 and 3.6 nM, respectively). Synergism between SIN-1 and aspirin was observed in inhibiting platelet activation in flowing blood but was much less pronounced in inhibiting collagen-induced aggregation of PRP. SIN-1/SNP and iloprost synergistically inhibited the aggregation of PRP induced by collagen as well as platelet adhesion/aggregation in blood. We found that two protein substrates of cAMP- and cGMP-dependent protein kinases, rap1B and a 50 kD protein, were associated with the functional synergism between SIN-1 and iloprost and were synergistically phosphorylated by platelet treatment with both iloprost and SIN-1. Platelet inhibition by SIN-1, iloprost, and aspirin was synergistic when measured in blood. In contrast, only additive effects of SIN-1 and iloprost were observed when platelet aggregation was measured in aspirin-treated PRP stimulated by ADP, TRAP, or collagen. Our study defines the basis for a more effective antiplatelet therapy using a combination of cGMP- and cAMP-elevating and cyclooxygenase-inhibiting drugs. The results also emphasize the importance of using various methods for the evaluation of antiplatelet drugs.

Topics: Adenosine Triphosphate; Aspirin; Blood Platelets; Cyclic AMP; Cyclic GMP; Drug Interactions; Humans; Iloprost; In Vitro Techniques; Iodine Radioisotopes; Molsidomine; Nitric Oxide; Nitroprusside; Phosphorylation; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Platelet Aggregation Inhibitors

In the presence of 3-isobutyl-methylxanthine (IBMX), induction of cyclic 3',5'-guanosine monophosphate (GMP) production in human washed platelets (HWP) by nitric oxide donors (NOD) is followed by its accumulation in the surrounding medium in a time- and concentration-dependent manner. Thirty minutes incubation of HWP with 3-morpholino-sydonimine (SIN-1, 10 microM) at 37 degrees C resulted in a 4.6-fold increase of cyclic GMP in platelets, whereas in the extracellular medium the increase was 17.6-fold. Similar results were obtained when other NOD such as S-nitroso-N-acetylpenicyllamine (SNAP) and 3-(2-methoxy-5-chlorophenyl)oxatriazol-5-imine (GEA 3184) and the selective phosphodiesterase inhibitor, zaprinast (M&B 22948, 10 microM), were used. Probenecid (1-300 microM), an inhibitor of organic anion transport, or ouabain (1-300 microM), an inhibitor of Na+/K+ adenine triphosphate (ATP)-ase had no effect on cyclic GMP production or extrusion after stimulation with SIN-1. Significantly prostaglandin A1 (PGA1) and prostaglandin D2 (PGD2) inhibited the efflux of cyclic GMP from platelets induced by SNAP (10 microM) in a concentration-dependent fashion, with an IC50 of 63 +/- 16 and 143 +/- 17 microM, respectively. These studies suggest that the extrusion of cyclic GMP from human platelets after activation of soluble guanylate cyclase by NOD may contribute to the control of cyclic GMP levels in platelets with potential physiological and therapeutic consequences.

Topics: 1-Methyl-3-isobutylxanthine; Blood Platelets; Cells, Cultured; Culture Media; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Space; Guanylate Cyclase; Humans; Ion Transport; Molsidomine; Nitric Oxide; Ouabain; Penicillamine; Phosphodiesterase Inhibitors; Platelet Aggregation Inhibitors; Probenecid; Prostaglandin D2; Prostaglandins; Prostaglandins A; Purinones; S-Nitroso-N-Acetylpenicillamine; Sodium-Potassium-Exchanging ATPase; Time Factors; Triazoles

The effects of the K+ channel blockers, apamin, tetraethylammonium and 4-aminopyridine, upon the relaxations of the isolated rat proximal duodenum induced by nitregic nerve activation, nitric oxide (NO), the NO donor 3-morpholinosydnonimine (SIN-1) and Br-cyclic GMP were determined. The effects of the guanylate cyclase inhibitors, cystamine and N-methylhydroxylamine, on NO-, SIN-1- and nitrergic nerve-induced responses were also investigated. Apamin inhibited nitrergic nerve-, NO-and SIN-1-induced relaxations but did not affect those induced by Br-cGMP. Tetraethylammonium and 4-aminopyridine as well as cystamine and N-methylhydroxylamine failed to affect the relaxations caused by any of the agents tested. These findings indicate that, in the rat proximal duodenum, nitrergic nerve activation as well as exogenous nitric oxide cause relaxation through a cGMP-independent, apamin sensitive mechanism.

Topics: 4-Aminopyridine; Animals; Apamin; Cyclic GMP; Cystamine; Duodenum; Hydroxylamines; In Vitro Techniques; Male; Molsidomine; Muscle Relaxation; Nitric Oxide; Rats; Rats, Wistar; Tetraethylammonium Compounds

1. The relaxant effect of KRN2391, suggested to act both as a nitrate and a K+ channel opener, was investigated in the rabbit lower urinary tract and compared with the effects of the NO-donor SIN-1 and the K+ channel opener levcromakalim. 2. KRN2391 10(-4) M was able to relax precontracted urethral preparations by 87 +/- 4%. Corresponding values for levcromakalim 10(-4) M and SIN-1 10(-4) M were 58 +/- 8% and 103 +/- 2%, respectively. The -logEC50 values for KRN2391, SIN-1 and levcromakalim were 6.0 +/- 0.1, 4.9 +/- 0.2 and 5.8 +/- 0.2. The relaxant effect of KRN2391 on the bladder was small (29 +/- 3%). 3. The levels of cyclic GMP in the urethral preparations were significantly increased after administration of KRN2391 10(-4) M and SIN-1 10(-4) M, but not after levcromakalim 10(-4) M, the levels measured being 9.9 +/- 2.2, 20.9 +/- 5.1, and 5.2 +/- 1.0, compared to the control value, 3.7 +/- 0.5 pmol/mg protein. The levels of cyclic AMP were, however, not changed. 4. The relaxations, caused by KRN2391 in the urethral preparations, were accompanied by a hyperpolarization (14 +/- 4 mV) of the membrane potential. 5. Methylene blue 3 x 10(-5) M and glibenclamide 10(-5) M significantly reduced the relaxant effect of KRN2391 in the urethral smooth muscle. 6. We suggest that in the rabbit lower urinary tract, KRN2391 acts mainly as an NO-donor.

Topics: Animals; Benzopyrans; Cromakalim; Cyclic GMP; Electrophysiology; Female; Hypoglycemic Agents; Molsidomine; Muscle Relaxation; Muscle, Smooth; Pyridines; Pyrroles; Rabbits; Urethra; Vasodilator Agents

Nitric oxide (NO) donors such as sodium nitroprusside (SNP, 0.01-1 micrograms) or 3-morpholino-sydnonimine (SIN-1, 0.1-10 micrograms) administered intracerebroventricularly (i.c.v) produced a dose-dependent potentiation of beta-endorphin-induced antinociception assessed by the tail-flick test in ICR mice. The same i.c.v. treatment with SNP or SIN-1 did not affect the antinociception induced by mu-, delta-, or kappa-opioid receptor agonists. The goal of the present study was to determine if the potentiation of the beta-endorphin-induced antinociception by NO donors is mediated by the activation of NO-cGMP system. Co-administration of hemoglobin (30-120 micrograms) or methylene blue (1.25-5 micrograms), but not N omega-nitro-L-arginine (1-5 micrograms) given i.c.v. dose-dependently attenuated the potentiating effects of SNP or SIN-1 on beta-endorphin-induced antinociception. However, the same i.c.v. treatments of mice with hemoglobin, methylene blue or N omega-nitro-L-arginine did not directly affect the i.c.v. administered beta-endorphin-induced antinociception. On the other hand, the treatment of mice with a combination of NO donor (SNP, 0.1 micrograms or SIN-1, 1 microgram) and zaprinast (a cGMP phosphodiesterase inhibitor, 1 microgram) further potentiated beta-endorphin-induced antinociception. These results indicate that the potentiating effect of SNP or SIN-1 on beta-endorphin-induced antinociception is mediated by the increased production of NO-cyclic GMP in the brain. However, the NO-cGMP system is not directly involved in the beta-endorphin-induced antinociception.

Topics: Analgesics; Analgesics, Opioid; Animals; beta-Endorphin; Cyclic GMP; Dose-Response Relationship, Drug; Drug Synergism; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Molsidomine; Nitric Oxide; Nitroprusside; Pain Measurement; Rats

1. The modulation of the guanosine 3':5'-cyclic monophosphate (cyclic GMP)- and adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase activities by the diastereomers of 8-bromo-beta phenyl-1, N2-ethenoguanosine 3':5'-cyclic monophosphorothioate, ((Rp)- and (Sp)-8-bromo-PET-cyclic GMPS) was investigated by use of purified protein kinases. In addition, the effects of (Rp)-8-bromo-PET-cyclic GMPS on protein phosphorylation in intact human platelets and on [3H]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries were also studied. 2. Kinetic analysis with purified cyclic GMP-dependent protein kinase (PKG) type I alpha and I beta, which are expressed in the rat tail artery, revealed that (Rp)-8-bromo-PET-cyclic GMPS is a competitive inhibitor with an apparent Ki of 0.03 microM. The activation of purified cyclic AMP-dependent protein kinase (PKA) type II was antagonized with an apparent Ki of 10 microM. 3. In human platelets, (Rp)-8-bromo-PET-cyclic GMPS (0.1 mM) antagonized the activation of the PKG by the selective activator 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate (8-pCPT-cyclic GMP; 0.2 mM) without affecting the activation of PKA by (Sp)-5, 6-dichloro-1-beta-D-ribofurano-sylbenzimidazole- 3':5'-cyclic monophosphorothioate ((Sp)-5,6-DCl-cyclic BiMPS; 0.1 mM). 4. (Rp)-8-bromo-PET-cyclic GMPS was not hydrolysed by the cyclic GMP specific phosphodiesterase (PDE) type V from bovine aorta but potently inhibited this PDE. 5. The corresponding sulphur free cyclic nucleotide of the two studied phosphorothioate derivatives, 8-bromo-beta-phenyl-1, N2-ethenoguanosine-3':5'-cyclic monophosphate (8-bromo-PET-cyclic GMP), had no effect on electrically-induced [3H]-noradrenaline release but concentration-dependently decreased the stimulation-induced vasoconstriction. (Rp)-8-bromo-PET-cyclic GMPS (3 microM) shifted the vasoconstriction response to the right without affecting stimulation evoked tritium overflow. 6. The NO donor, 3-morpholinosydnonimine (SIN-1) relaxed rat tail arteries precontracted with phenylephrine (1 microM). The SIN-1 concentration-relaxation curve was shifted in a parallel manner to the right by (Rp)-8-bromo-PET-cyclic GMPS, suggesting that the relaxation was mediated by a cyclic GMP/PKG-dependent mechanism. 7. The [3H]-noradrenaline release-enhancing effect and stimulation-induced decrease in vasoconstriction of forskolin were unaffected by (Rp)-8-bromo-PET-cycl

Topics: Animals; Base Sequence; Blood Platelets; Cattle; Cell Adhesion Molecules; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Humans; In Vitro Techniques; Microfilament Proteins; Molecular Sequence Data; Molsidomine; Muscle, Smooth, Vascular; Norepinephrine; Phosphoproteins; Phosphorylation; Rats; Stereoisomerism; Thionucleotides; Vasoconstriction; Vasodilator Agents

Differentiation of murine erythroleukemia (MEL) cells induced by hexamethylene bisacetamide (HMBA) and DMSO was inhibited by several structurally unrelated nitric oxide (NO)-releasing agents and two membrane-permeable cGMP analogues. Since the effect of the NO-releasing agents was augmented by a cGMP phosphodiesterase inhibitor, at least some of their effect appeared to be mediated by activation of cytosolic guanylate cyclase. The drugs did not globally block differentiation since hemin-induced differentiation was undisturbed. In HMBA-treated cells, the NO-releasing agents and cGMP analogues reduced beta-globin and delta-aminolevulinate synthetase mRNA expression and inhibited the late down-regulation of c-myb mRNA that is required for HMBA-induced differentiation of MEL cells; the regulation of c-myc mRNA was not changed by the drugs. Nuclear run-off analyses showed that the drugs inhibited the HMBA-induced changes in beta-globin and c-myb transcription rates, and transient transfection of a reporter gene construct demonstrated that the drugs inhibited HMBA-inducible enhancer function of the alpha-globin control region, which contains binding sites for the erythroid transcription factors NF-E2 and GATA-1. The NO-releasing agents and cGMP analogues largely prevented HMBA-induced increases in DNA binding of NF-E2, whereas DNA binding of GATA-1 and SP-1 was not affected. The inhibition of erythroid gene expression by NO and cGMP analogues may be physiologically important under conditions of high NO production by endothelial cells and macrophages, i.e. during acute or chronic inflammation.

Topics: 5-Aminolevulinate Synthetase; Acetamides; Animals; Base Sequence; Cell Differentiation; Cell Line; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Cyclic GMP; Dimethyl Sulfoxide; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; GATA1 Transcription Factor; Gene Expression Regulation, Neoplastic; Globins; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Molsidomine; NF-E2 Transcription Factor; NF-E2 Transcription Factor, p45 Subunit; Nitric Oxide; Nuclear Proteins; Oligonucleotide Probes; Oncogenes; Phosphodiesterase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Purinones; Recombinant Proteins; RNA, Messenger; Thionucleotides; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

A role for nitric oxide (NO) in the regulation of hypothalamic neurohormone secretion has been suggested. The aim of the present study was to establish a direct involvement of this novel intracellular regulatory molecule in the control of GnRH release. For this purpose, the GT1-1 GnRH-secreting continuous cell line was treated with various agents that can modify the endogenous NO synthase activity or, alternatively, with substances that can liberate NO, mimicking an increased concentration of this molecule in the cell. Treatment of GT1-1 cells with increasing concentrations of L-arginine, the direct precursor of NO, produced a marked reduction of norepinephrine-stimulated GnRH release despite a lack of effect on basal secretion. Similarly, the NO donors SIN-1 and acidified NaNO2 potently reduced basal as well as KCl-stimulated GnRH secretion. Conversely, sodium nitroprusside caused a significant inhibition of KCl-stimulated, but not basal, GnRH secretion. Addition of these agents to GT1-1 cells resulted in a marked increase in intracellular cGMP accumulation. Addition of the NO synthase inhibitors N-nitro-L-arginine and N-nitro-L-arginine methyl ester stimulated basal GnRH secretion without modifying norepinephrine- or KCl-stimulated release. In addition, treatment of GT1-1 cells with both L-arginine analogs produced a significant inhibition of the basal cGMP concentration. Together, these data suggest an inhibitory role for NO in the control of GnRH secretion from GT1-1 cells.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cell Line; Cyclic GMP; Gonadotropin-Releasing Hormone; Mice; Mice, Transgenic; Molsidomine; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Norepinephrine; Sodium Nitrite

We sought to determine whether the attenuation of the hypercapnic cerebrovasodilation associated with inhibition of nitric oxide synthase (NOS) can be reversed by exogenous NO. Rats were anesthetized (halothane) and ventilated. Neocortical cerebral blood flow (CBF) was monitored by a laser-Doppler probe. The NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME; 40 mg/kg iv) reduced resting CBF [-36 +/- 5% (SE); P < 0.01, analysis of variance] and attenuated the increase in CBF elicited by hypercapnia (partial pressure of CO2 = 50-60 mmHg) by 66% (P < 0.01). L-NAME reduced forebrain NOS catalytic activity by 64 +/- 3% (n = 10; P < 0.001). After L-NAME, intracarotid infusion of the NO donor 3-morpholinosydnonimine (SIN-1; n = 6) increased resting CBF and reestablished the CBF increase elicited by hypercapnia (P > 0.05 from before L-NAME). Similarly, infusion of the guanosine 3',5'-cyclic monophosphate (cGMP) analogue 8-bromo-cGMP (n = 6) reversed the L-NAME-induced attenuation of the hypercapnic cerebrovasodilation. The NO-independent vasodilator papaverine (n = 6) increased resting CBF but did not reverse the attenuation of the CO2 response. SIN-1 did not affect the attenuation of the CO2 response induced by indomethacin (n = 6). The observation that NO donors reverse the L-NAME-induced attenuation of the CO2 response suggests that a basal level of NO is required for the vasodilation to occur. The findings are consistent with the hypothesis that NO is not the final mediator of smooth muscle relaxation in hypercapnia.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cerebrovascular Circulation; Cyclic GMP; Hypercapnia; Male; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Papaverine; Rats; Rats, Sprague-Dawley; Vasodilation

Nitric oxide (NO) is a messenger molecule that is produced from L-arginine by NO synthase (NOS). Some NOS isoforms are present in cells constitutively, whereas others can be induced by cytokines. Recent evidence suggests that NO inhibits intracellular pH regulation by the vacuolar H(+)-adenosinetriphosphatase (ATPase) in macrophages, which contain an inducible form of NOS. The vacuolar H(+)-ATPase is involved in proton secretion in intercalated cells in the collecting duct. We have therefore examined the effect of NO on bafilomycin-sensitive H(+)-ATPase activity in individual cortical collecting ducts (CCD) microdissected from collagenase-treated kidneys of normal rats using a fluorometric microassay. Incubation of CCD with the NO donors, sodium nitroprusside (0.1 and 1 mM) or 3-morpholino-sydnonimine hydrochloride (SIN-1, 30 microM), caused a dose-dependent decrease in H(+)-ATPase activity. Incubation of CCD with lipopolysaccharide (LPS) and interferon-gamma, which induces NOS in macrophages, decreased H(+)-ATPase activity by 85%. This effect was prevented by simultaneous incubation with N omega-nitro-L-arginine, a competitive inhibitor of NOS, indicating that the decrease in H(+)-ATPase activity was caused by NO production. Incubation with 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP) also inhibited H(+)-ATPase activity, suggesting that NO may exert its effect in the CCD via activation of guanylyl cyclase and production of cGMP. Immunohistochemistry using antibodies to the macrophage-type NOS revealed strong labeling of intercalated cells in the CCD, confirming the presence of NOS in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Oxidoreductases; Animals; Anti-Bacterial Agents; Arginine; Cyclic GMP; Enzyme Induction; Ethylmaleimide; Female; In Vitro Techniques; Interferon-gamma; Kidney Tubules, Collecting; Kinetics; Lipopolysaccharides; Macrolides; Macrophages; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley; Vacuoles

Recent studies demonstrate that nitric oxide and cyclic guanosine 3',5'-monophosphate may mediate hyperalgesia induced by N-methyl-D-aspartate at the level of the spinal cord. One possible mechanism for this action is that nitric oxide increases transmitter release from the primary afferent nociceptors that synapse in the dorsal horn of the spinal cord. To address this possibility, we investigated whether various nitric oxide donors and cyclic guanosine 3',5'-monophosphate could alter the release of substance P and calcitonin gene-related peptide from rat sensory neurons in culture. Sodium nitroprusside (100 nM to 100 microM) had little effect on basal release of either peptide, but it significantly increased the release of substance P and calcitonin gene-related peptide induced by 50 nM capsaicin. In contrast, sodium nitroprusside did not alter release evoked by 100 nM bradykinin or 30 mM KCl. Two other nitric oxide-donating compounds, S-nitroso-N-acetylpenicillamine and 3-morpholinosydnonimine did not enhance resting or capsaicin-evoked peptide release, although they induced a marked elevation in the intracellular cyclic guanosine 3',5'-monophosphate levels. Pretreating the cultures with 8-bromo-cyclic guanosine 3',5'-monophosphate, (0.5 or 0.1 mM for 30 or 60 min) did not result in the enhancement of capsaicin-induced release from sensory neurons. Moreover, pretreating the cells with the nitric oxide synthase inhibitor, NG-nitro-L-arginine (100 microM), abolished the rise in cyclic guanosine 3',5'-monophosphate induced by capsaicin without altering capsaicin-stimulated release of either peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcitonin Gene-Related Peptide; Capsaicin; Cells, Cultured; Cyclic GMP; Molsidomine; Neurons, Afferent; Neuropeptides; Nitric Oxide; Nitroprusside; Rats; Rats, Sprague-Dawley; Substance P

Exposure of rat aortic rings without endothelium to interleukin-1 beta for 5 h significantly attenuated the contractions due to phenylephrine and increased the tissue content of guanosine 3',5'-cyclic monophosphate (cyclic GMP) due to the induction of nitric oxide synthase. The presence of pyrrolidine dithiocarbamate, a specific inhibitor of nuclear transcription factor kappa B activation, during the exposure of the rings to interleukin-1 beta prevented these responses to interleukin-1 beta. Rat aortic rings which had been incubated for 5 h with interleukin-1 beta in the absence and presence of pyrrolidine dithiocarbamate prior to the organ chamber experiment had a similar concentration-dependent relaxation curve for acetylcholine in rings with endothelium, and for 3-morpholino-sydnonimine (SIN-1) in rings without. Pyrrolidine dithiocarbamate applied acutely did not alter the tone elicited by phenylephrine in rings with or without endothelium and had no effect on the subsequent relaxation induced by acetylcholine in rings with endothelium or by SIN-1 in rings without endothelium. These observations suggest that pyrrolidine dithiocarbamate prevents the interleukin-1 beta-mediated expression of the inducible nitric oxide synthase without affecting the activity of the constitutive enzyme in the rat aorta.

Topics: Acetylcholine; Amino Acid Oxidoreductases; Analysis of Variance; Animals; Antioxidants; Aorta, Thoracic; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Induction; Interleukin-1; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; NADPH Dehydrogenase; Nitric Oxide Synthase; Pyrrolidines; Radioimmunoassay; Rats; Rats, Wistar; Thiocarbamates; Vasodilator Agents

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Calcium; Cells, Cultured; Cerebellar Cortex; Cyclic GMP; Electron Spin Resonance Spectroscopy; Enzyme Activation; Epilepsy; Feedback; Free Radicals; Glutamic Acid; Guanylate Cyclase; Kindling, Neurologic; Mice; Molsidomine; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Receptors, N-Methyl-D-Aspartate

The effects of Trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a vitamin E analogue, (60-900 microM) and SIN-1 (3-morpholino sydnonimine), a nitric oxide donor, (30-3000 microM) on arachidonic acid metabolism and on cyclic GMP formation in calcium ionophore A23187 (calcimycin)-stimulated human polymorphonuclear leukocytes were investigated. Trolox C elicited a dose dependent decrease in leukotriene B4 levels and increase in prostaglandin E2 levels but did not affect cyclic GMP levels. SIN-1 dose dependently inhibited leukotriene B4 and stimulated prostaglandin E2 and cyclic GMP formation. Dibutyryl cyclic GMP did not affect the formation of leukotriene B4 and prostaglandin E2. Trolox C (180 microM), which itself had no effect on cyclic GMP levels, enhanced the effect of SIN-1 (100 microM) on cyclic GMP levels more than 5-fold. The effects of SIN-1 on arachidonic acid metabolism seem to be independent of cyclic GMP and are probably due to nitric oxide. In this experimental model both Trolox C and SIN-1 have similar actions on the prostaglandin/leukotriene ratio, and Trolox C potentiates the SIN-1-induced increase in cyclic GMP levels.

Topics: Antioxidants; Arachidonic Acid; Calcimycin; Centrifugation, Density Gradient; Chromans; Cyclic GMP; Dibutyryl Cyclic GMP; Dinoprostone; Dose-Response Relationship, Drug; Guanylate Cyclase; Humans; Leukotriene B4; Molsidomine; Neutrophils; Radioimmunoassay; Vasodilator Agents; Vitamin E

Application of NMDA, or agents releasing nitric oxide (NO), onto the dendrites of hippocampal granule cells increased the levels of the mRNA encoding MAP2, a cytoskeletal component induced during periods of neurite outgrowth. Furthermore, local increases in the hybridisation signal in the molecular layer, representing dendritic MAP2 mRNA, occurred independently of changes in MAP2 mRNA levels in the cell body layer. The selective modulation of MAP2 mRNA in dendrites reveals a mechanism allowing a sustained stimulation of dendritic outgrowth to be confined to those regions of a neuron's dendritic arbour local to glutamate receptor stimulation.

Topics: Animals; Base Sequence; Cyclic GMP; Cytoskeleton; Dendrites; Gene Expression Regulation; Hippocampus; Male; Microtubule-Associated Proteins; Molecular Sequence Data; Molsidomine; N-Methylaspartate; Nerve Tissue Proteins; Nitric Oxide; Nitroprusside; Rats; Rats, Wistar; Receptors, Glutamate; RNA, Messenger

This study was designed to determine if nitric oxide (NO) has direct effects on heart rate or if it is involved in the chronotropic actions of adrenergic or cholinergic stimulation. Right atria were isolated from hearts of adult male rats, bathed in Krebs-Henseleit buffer (37 degrees C), and used to monitor spontaneous rate. For comparison, ring segments of thoracic aorta were also suspended in the Krebs-Henseleit solution and used to examine vascular actions of various agents. The dose-dependent chronotropic effects of acetylcholine (10(-7)-10(-3) M) and norepinephrine (10(-8)-3 x 10(-4) M) in right atria were not affected by pretreatment with 10(-4) M N-nitro-L-arginine or 10(-3) M N-nitro-L-arginine-methyl ester, inhibitors of L-arginine-derived NO production. SIN-1 (3-morpholino-sydnonimine), an agent which releases NO in aqueous solution, elicited a dose-dependent (0.3-100 microM) vasorelaxation in aortic preparations constricted with 60 mM KCl; the ED50 value for this effect was increased by pretreatment with methylene blue (10 microM) and LY-83,583 (6-(phenylamino)-5,8- quinolinedione; 1 and 3 microM), compounds which inhibit NO-induced stimulation of guanylate cyclase. SIN-1 produced a negative chronotropic effect in right atria; however, this action was not observed at concentrations less than 300 microM and was not antagonized by methylene blue or LY-83,583. 8-Bromo cyclic GMP produced a dose-dependent (10-3000 microM) decrease in KCl-induced tension in aortic rings. In right atria, 8-bromo cyclic GMP elicited a positive chronotropic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Aminoquinolines; Animals; Aorta, Thoracic; Arginine; Cyclic GMP; Guanylate Cyclase; Heart Atria; Heart Rate; In Vitro Techniques; Male; Methylene Blue; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroarginine; Norepinephrine; Parasympathetic Nervous System; Rats; Rats, Sprague-Dawley; Sympathetic Nervous System; Vasodilator Agents

The aim of this study was to investigate whether short-term treatment with nitric oxide donors could mimic cytokine inhibition of insulin secretion. We tested the nitric oxide generating compounds 3-morpholinosydnonimine (SIN-1), S-nitroso-N-penicillamine (SNAP), S-nitrosoglutathione and hydroxylamine for their ability to inhibit insulin secretion, raise cyclic GMP and lower cyclic AMP levels in isolated rat islets of Langerhans and the insulin-secreting cell lines HIT-T15 and RINm5F. In islets, all nitric oxide donors inhibited glucose-induced insulin secretion and raised cyclic GMP levels. SIN-1 and S-nitrosoglutathione also reduced cyclic AMP, while SNAP and hydroxylamine had no effect. Insulin secretion in HIT-T15 cells was inhibited by SIN-1, SNAP and hydroxylamine and in RINm5F cells by hydroxylamine. Inhibition of HIT-T15 and RINm5F cell insulin secretion was not accompanied by an increase in cyclic GMP levels. The degree of inhibition of insulin secretion was unrelated to the extent of release of nitric oxide by the compounds as measured by nitrite and nitrate production. More effective inhibition by S-nitrosoglutathione and hydroxylamine versus SIN-1 and SNAP may be related to intracellular versus extracellular site of nitric oxide generation.

Topics: Animals; Cell Line; Cyclic AMP; Cyclic GMP; Drug Interactions; Female; Glutathione; Hydroxylamine; Hydroxylamines; Insulin; Insulin Secretion; Islets of Langerhans; Molsidomine; Nitric Oxide; Nitroso Compounds; Penicillamine; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione

The relaxing effects of several specific and nonspecific inhibitors of phosphodiesterases (PDE) on rabbit isolated corpus cavernosum (CC) and spongiosum (CS) were investigated. Preparations were mounted in organ baths, and isometric tension was recorded. The results were compared with the effects of direct administration of analogs of the second messenger cyclic nucleotides and the effects of forskolin, a direct stimulator of adenylate cyclase, and the nitric oxide donor 3-morpholinosydnonimine (SIN 1). All drugs relaxed the phenylephrine-induced contractions in CC and CS in a dose-dependent fashion. In CC and CS, type III (SK&F 95654) and type V (zaprinast and dipyramidole) PDE inhibitors, as well as the nonspecific inhibitors papaverine and trequinsin, showed no differences in IC50. The type IV inhibitor rolipram relaxed CC and CS at significantly lower concentrations (p < 0.005) than any other PDE inhibitor, and in CC the type III and IV inhibitor zardaverine was more potent (p < 0.05) than SK&F 95654. SIN 1 stimulates guanylate cyclase and effectively inhibits contractions in CC and CS. Activation of adenylate cyclase by forskolin also was highly effective (p < 0.005). It is concluded that PDE inhibition constitutes an effective relaxing mechanism in rabbit CC and CS. The marked effects of the different types of PDE inhibitors support the importance of cyclic guanosine 3',5'-monophosphate and cyclic adenosine 3',5'-monophosphate in smooth muscle relaxation in erectile tissue.

Topics: Animals; Colforsin; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Molsidomine; Muscle Tonus; Muscle, Smooth; Penis; Phenylephrine; Phosphodiesterase Inhibitors; Rabbits

Application of N-methyl-D-aspartate on to the dendrites of hippocampal granule cells dramatically decreased prodynorphin messenger RNA levels in the affected cells while increasing proenkephalin messenger RNA levels. Sin-1 molsidomine (an agent which releases nitric oxide) and 8-bromo-cyclic GMP were similarly effective, and the actions of sin-1 molsidomine were blocked by inhibition of cyclic GMP-dependent protein kinase. Since, in this region, dynorphins act to inhibit potentiation of synaptic transmission, while enkephalins have excitatory effects, this switch in opioid gene expression is likely to have a prolonged effect on the efficiency of the mossy fibre synapses. In addition, the results demonstrate a powerful role for nitric oxide in the long-term regulation of hippocampal excitability.

Topics: 2-Amino-5-phosphonovalerate; Animals; Base Sequence; Cyclic GMP; Dendrites; Enkephalins; Gene Expression Regulation; Hippocampus; In Situ Hybridization; Molecular Sequence Data; Molsidomine; N-Methylaspartate; Nitric Oxide; Nitroprusside; Protein Precursors; Rats; RNA, Messenger; Vasodilator Agents

Recent studies of long-term potentiation (LTP) in the CA1 region of the hippocampus have demonstrated that nitric oxide (NO) may be involved in some forms of LTP and have suggested that postsynaptically generated NO is a candidate to act as a retrograde messenger. However, the molecular target(s) of NO in LTP remain to be elucidated. The present study examined whether either of two potential NO targets, a soluble guanylyl cyclase or an ADP-ribosyltransferase (ADPRT; EC 2.4.2.31) plays a role in LTP. The application of membrane-permeant analogs of cGMP did not produce any long-lasting alterations in synaptic strength. In addition, application of a cGMP-dependent protein kinase inhibitor did not prevent LTP. We found that the CA1 tissue from hippocampus possesses an ADPRT activity that is dramatically stimulated by NO and attenuated by two different inhibitors of mono-ADPRT activity, phylloquinone and nicotinamide. The extracellular application of these same inhibitors prevented LTP. Postsynaptic injection of nicotinamide failed to attenuate LTP, suggesting that the critical site of ADPRT activity resides at a nonpostsynaptic locus. These results suggest that ADP-ribosylation plays a role in LTP and are consistent with the idea that an ADPRT may be a target of NO action.

Topics: 2-Amino-5-phosphonovalerate; Animals; Cyclic GMP; Dibutyryl Cyclic GMP; Evoked Potentials; Hippocampus; In Vitro Techniques; Long-Term Potentiation; Male; Molsidomine; N-Methylaspartate; NAD; Niacinamide; Nitric Oxide; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Vasodilator Agents

Nitric oxide (NO) synthesized by vascular endothelial cells is a potent vasodilator substance. The actions of NO extend well beyond its vasodilatory properties, and increasingly, NO has been recognized as an important signal for intercellular and intracellular communication. Recently, NO has been implicated in the regulation of vascular and blood-brain barrier permeability. NO has also been shown to modulate ion channels in excitable cells, thus affecting neuronal firing. We report the results of patch-clamp experiments that show a modulatory action of NO as well as cGMP and cAMP on a hyperpolarization-activated current (Iha) carried by both Na+ and K+ ions in blood-brain barrier endothelial cells. Iha was recorded in cells dialyzed with 0.2 mmol/L GTP-gamma-S to inhibit a large inwardly rectifying potassium current. This ionic current and its modulation by NO may play a role in the regulation of the transport of ions, nutrients, and other molecules to the brain and serve as an integral part of the blood-brain barrier. The modulation of Iha by a cyclic guanosine nucleotide may also explain previous reports suggesting a role for NO in the regulation of blood-brain barrier function.

Topics: Animals; Blood-Brain Barrier; Brain; Cell Membrane; Cells, Cultured; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Guanosine 5'-O-(3-Thiotriphosphate); Male; Models, Cardiovascular; Molsidomine; Nitric Oxide; Nitroprusside; Rats; Rats, Sprague-Dawley; Tetraethylammonium; Tetraethylammonium Compounds; Vasodilator Agents

To examine the hypothesis that nitric oxide (NO) acts as an autocrine, antiproliferative regulator and that exogenous NO donor inhibitors the proliferation of cultured rat mesangial cells.. The cellular effects of endogenous and exogenous NO were studied in rat mesangial cells in a two-dimensional culture of early mesangial cell passages. The proliferation of mesangial cells was determined by cell-counting and uptake of [3H]-thymidine. NO generation was induced by incubation with interleukin-1 beta (5 u/ml) or bacterial lipopolysaccharide (10 micrograms/ml) for 24 h. NO release by mesangial cells was assessed by measuring the accumulation of nitrite, the major stable end-product of NO, in mesangial cell supernates. In addition, cyclic GMP (cGMP) formation was measured by radioimmunoassay as an indicator for NO generation.. The formation of nitrite and cGMP was significantly increased after incubation of mesangial cells with interleukin-1 beta or lipopolysaccharide. This effect was greatly reduced by an inhibitor of NO synthesis. NG-monomethyl-L-arginine (L-NMMA; 0.1 mmol/l). The NO donor 3-morpholino-sydnonimine-HCl also increased the cGMP concentrations in the mesangial cells. The proliferation of mesangial cells was analysed in growth-arrested and mitogen-stimulated (platelet-derived growth factor, platelet-derived growth factor plus ATP and fetal calf serum) mesangial cells in the presence and absence of L-NMMA and the NO synthase substrate L-arginine (1 mmol/l). At 48 h platelet-derived growth factor (50 ng/ml), and platelet-derived growth factor (50 ng/ml) plus ATP (0.1 mmol/l) and fetal calf serum 5% each significantly increased the uptake of [3H]-thymidine in mesangial cells. These effects were not altered in the presence of L-NMMA or L-arginine. Pretreatment with interleukin-1 beta or with lipopolysaccharide also failed to affect the uptake of [3H]-thymidine in resting or proliferating mesangial cells. 3-Morpholino-sydnonimine-HCl (10(-3) to 10(-6) mol/l) did not suppress the mitogen-induced proliferation of mesangial cells, even when it was administered three times a day.. The present findings support recent observations that interleukin-1 beta and lipopolysaccharide strongly induce NO production in mesangial cells, as is shown indirectly by the greatly increased formation of nitrite and cGMP. However, these effects were not associated with antiproliferative action on mitogen-stimulated mesangial cells. Similarly, the exogenous NO donor 3-morpholino-sydnonimine-HCl induced cGMP formation but failed to inhibit proliferation of mesangial cells when used at a non-toxic dose. Our observations do not support the contention that the formation of NO and cGMP constitutes an autocrine downregulating mechanism in the control of the growth of mesangial cells. It remains to be seen what pathophysiological role the induction of NO release plays in the regulation of the behaviour of mesangial cells, e.g. during an inflammatory response to glomerular injury.

Topics: Animals; Arginine; Cell Division; Cells, Cultured; Cyclic GMP; Glomerular Mesangium; Interleukin-1; Lipopolysaccharides; Molsidomine; Nitric Oxide; omega-N-Methylarginine; Rats; Rats, Sprague-Dawley

1. Platelet activation in vivo occurs in healthy pregnancy and is more pronounced in pre-eclampsia. 2. This study has investigated: (i) the inhibitory potency of the nitric oxide donors 3-morpholinosydnonimine and sodium nitroprusside, on the platelet release reaction in vitro in non-pregnant, healthy pregnant and pre-eclamptic women; (ii) the concentration of cyclic GMP during incubation of washed platelets with sodium nitroprusside in a separate group of non-pregnant, healthy pregnant and pre-eclamptic women. 3. The half-maximal inhibitory concentration of sodium nitroprusside, in the presence of a phosphodiesterase inhibitor, for inhibition of the platelet release reaction was lower in the pre-eclamptic subjects than in the non-pregnant subjects (P < 0.05). 4. Several of the pre-eclamptic women were studied again postnatally. The half-maximal inhibitory concentrations of sodium nitroprusside and 3-morpholinosydnonimine were higher in the postnatal than in the antenatal sample (P < 0.02). 5. Peak platelet cyclic GMP responses to sodium nitroprusside were significantly higher in the pre-eclamptic women than in the healthy pregnant and non-pregnant women. 6. These results suggest that platelets are more sensitive to the inhibitory effects of nitric oxide donors in pre-eclampsia.

Topics: Adolescent; Adult; Blood Platelets; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Female; Humans; Molsidomine; Nitric Oxide; Nitroprusside; Platelet Activation; Pre-Eclampsia; Pregnancy

The presence of nitric oxide synthase (NOS) in the retina, the constitutive isoform in photoreceptor outer segments and the inducible form in retinal pigmented epithelial (RPE) cells, has been demonstrated, but the role of the free radical NO produced, remains unknown. We have investigated the effect of NO on the process of rod outer segment (ROS) phagocytosis. Using an in vitro assay for phagocytosis in primary cultures of bovine RPE cells, we demonstrate that NO released by SIN-1 (3-morpholinosydnonimine) in the culture medium inhibits the phagocytosis of ROS. Furthermore, endogenous NO, produced by RPE cells cotreated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), is also able to decrease RPE cell phagocytic activity. This effect depends upon the continuous presence of NO during the assay and is abolished by the scavenging of NO by hemoglobin or by the inhibition of NO synthase activity by L-arginine analog, NG-monomethyl-L-arginine. Pretreatment of ROS with SIN-1 failed to impair subsequent phagocytosis, demonstrating that NO directly affects the RPE cells ability to phagocytose ROS. The inhibitory effect of NO is cGMP independent, since 8-bromo-cGMP does not modify this process. This decrease of ROS phagocytosis by RPE cells caused by NO may occur as a result of retinal inflammation, and could lead to photoreceptor degeneration.

Topics: Animals; Cattle; Cells, Cultured; Cyclic GMP; Interferon-gamma; Lipopolysaccharides; Molsidomine; Nitric Oxide; Phagocytosis; Pigment Epithelium of Eye; Rod Cell Outer Segment

Nitrovasodilators relax airway smooth muscle by both guanosine 3',5'-cyclic monophosphate (cGMP)-dependent and cGMP-independent mechanisms and by mechanisms that reduce cytosolic calcium concentration ([Ca2+]i). This study was conducted to determine the relative importance of these mechanisms in relaxation of canine tracheal smooth muscle (CTSM) induced by 3-morpholinosydnonimine (SIN-1). We measured 1) the effect of SIN-1 on force, [cGMP]i, and [Ca2+]i, and 2) the ability of methylene blue (MB) to antagonize SIN-1-induced relaxation and cGMP accumulation. The ratio of fura 2 emission fluorescence intensities due to excitation at 340- and 380-nm wavelengths (F340/F380) was used as an index of [Ca2+]i. In strips contracted with 0.3 microM acetylcholine (ACh, n = 8) or 24 mM KCl (n = 8), SIN-1 (1-100 microM) caused a concentration-dependent decrease in force which was correlated with a concentration-dependent increase in [cGMP]i. MB (10 microM) proportionally attenuated both relaxation and cGMP accumulation. In fura 2-loaded strips contracted with 0.3 microM ACh (n = 7) or 30 mM KCl (n = 7), reductions in force induced by SIN-1 (1-100 microM) were accompanied by decreases in F340/F380. These findings suggest that in CTSM contracted with ACh or KCl, SIN-1 causes relaxation which appears to be mediated by cGMP-dependent mechanisms that reduce [Ca2+]i.

Topics: Animals; Calcium; Cyclic AMP; Cyclic GMP; Cytosol; Dogs; Female; In Vitro Techniques; Male; Methylene Blue; Molsidomine; Muscle Relaxation; Muscle, Smooth; Osmolar Concentration; Trachea

In this study, we analysed the implication of superoxide (O2-.) and nitric oxide (NO.) free radicals and their resulting product peroxynitrite (ONOO-) in the neuronal death induced by the activation of the glutamatergic receptor of the N-methyl-D-aspartate (NMDA) subtype using cultured cerebellar granule cells. The NOl donor SIN-1 (3-morpholinosydnonimine N-ethylcarbamide), at concentrations which produced a much higher guanylate cyclase activation (i.e. NO. concentration) than NMDA, was not neurotoxic and did not increase the NMDA-induced neuronal death. The absence of involvement of NO. in NMDA-induced neuronal death was confirmed by the ineffectiveness of L-NG-nitroarginine (L-Narg) as a neuroprotective compound. Electron paramagnetic resonance (EPR) experiments, using 5,5-dimethyl pyrroline 1-oxide (DMPO) as a spin trap, indicated that NMDA receptor stimulation led to the generation of O2-. from at least 15-30 min. The generation of O2-. by xanthine (XA)-xanthine oxidase (XO) induced a neuronal death similar to that of NMDA. XA-XO-induced neuronal death was suppressed by addition of either superoxide dismutase (SOD) plus catalase (CAT), or DMPO in the incubation medium. In contrast, NMDA-induced neuronal death was widely blocked by DMPO and other spin trap compounds, but not by SOD +/- CAT. XA-XO-induced neuronal death was not potentiated by SIN-1 indicating that ONOO- is not more toxic than O2-. in our neuronal model.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Catalase; Cell Death; Cells, Cultured; Cerebellum; Cyclic GMP; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Mice; Molsidomine; N-Methylaspartate; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Receptors, N-Methyl-D-Aspartate; Superoxide Dismutase; Superoxides; Xanthine Oxidase

When islets were cultured with interleukin-1 beta (1 or 100 pmol/l) for 12 h in arginine-containing medium, cyclic GMP levels were increased 1.6- and 4.5-fold respectively. The arginine analogue, N-omega-nitro-L-arginine methyl ester, which blocks nitric oxide formation and partially reverses inhibition of insulin secretion by 100 pmol/l interleukin-1 beta, largely, but not completely, blocked generation of cyclic GMP. Treatment of islets with 100 pmol/l interleukin-1 beta for 12 h significantly decreased islet cyclic AMP generation in the absence of isobutylmethylxanthine (from 13.1 +/- 0.7 to 9.3 +/- 0.8 fmol/micrograms islet protein), this fall was arginine-dependent and may have resulted from an effect on a cyclic AMP phosphodiesterase, since it was masked if isobutylmethylxanthine was present. Isobutylmethylxanthine (0.4 mmol/l) reduced the inhibitory potency of interleukin-1 beta in 15 h slightly but significantly from 80.5 to 59.0%. The morpholinosydnonimine SIN-1, which is a nitric oxide donor, inhibited insulin secretion, raised islet cyclic GMP and lowered cyclic AMP; its effects were similar to those of interleukin-1 beta. However, 6-anilinoquinoline-5,8-quinone, [LY83583 (1-10 mumol/l)], inhibited insulin secretion, and significantly decreased cyclic GMP while 8-bromocyclic GMP stimulated insulin secretion. Both low- and high-dose interleukin-1 beta treatment give a large arginine-dependent and a small, yet significant, arginine-independent increase in cyclic GMP. The inhibitory effect of SIN-1 or interleukin-1 beta on insulin secretion seems to depend to a small extent on decreased islet cyclic AMP, though sustained increases in nitric oxide or depleted islet GTP may directly affect the secretory process.

Topics: 1-Methyl-3-isobutylxanthine; Aminoquinolines; Animals; Antihypertensive Agents; Arginine; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Female; Insulin; Insulin Secretion; Interleukin-1; Islets of Langerhans; Kinetics; Molsidomine; NG-Nitroarginine Methyl Ester; Rats; Rats, Sprague-Dawley

The effects of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on the L-type Ca2+ current (ICa) were examined in frog ventricular myocytes under basal and phosphorylated conditions. SIN-1 was found to exert insignificant effects on basal ICa but to induce a biphasic action on stimulated ICa. Indeed, in the nanomolar range of concentrations (0.1-10 nM), SIN-1 induced a pronounced (approximately 40%) stimulation of ICa elevated by a non-maximal concentration of forskolin (0.3 microM). However, the stimulatory effects of SIN-1 on ICa were not additive with those of maximal concentrations (10 microM) of forskolin or intracellular cAMP. In contrast, at higher concentrations (100 nM to 1 mM), SIN-1 strongly reduced ICa (by up to 85%) which had been previously stimulated by cAMP, forskolin, or isoprenaline. All the effects of SIN-1 appeared to be mediated by the liberation of NO since they were suppressed by methylene blue and LY83583 and were not mimicked by SIN-1C, a metabolite of SIN-1. The stimulatory or inhibitory effects of SIN-1 were absent, respectively, in the presence of milrinone (10 microM) or when the hydrolysis-resistant cAMP analog 8-bromo-cAMP was used instead of cAMP to stimulate ICa. In addition to its effects on ICa, SIN-1 induced a dose-dependent stimulation of guanylyl cyclase activity in the cytosolic and membrane fractions of frog ventricle. The membrane form of guanylyl cyclase displayed a higher sensitivity to SIN-1 than the cytosolic form, which correlated with SIN-1 sensitivity of ICa. Our data suggest that the activatory and inhibitory effects of NO donors on ICa result from an inhibition of the cGMP-inhibited cAMP-phosphodiesterase and an activation of the cGMP-stimulated cAMP-phosphodiesterase, respectively, both linked to the activation of guanylyl cyclase, possibly a membrane form of the enzyme.

Topics: Animals; Calcium Channels; Colforsin; Cyclic GMP; Enzyme Activation; Guanylate Cyclase; Heart Ventricles; In Vitro Techniques; Isoproterenol; Molsidomine; Nitric Oxide; Phosphoric Diester Hydrolases; Rana esculenta

Nitric oxide, a free-radical gas produced endogenously by several mammalian cell types, has been implicated as a diffusible intercellular messenger subserving use-dependent modification of synaptic efficacy in the mature central nervous system. It has been suggested on theoretical grounds that nitric oxide might play an analogous role during the establishment of ordered connections by developing neurons. We report here that nitric oxide rapidly and reversibly inhibits growth of neurites of rat dorsal root ganglion neurons in vitro. In addition, we show that exposure to nitric oxide inhibits thioester-linked long-chain fatty acylation of neuronal proteins, possibly through a direct modification of substrate cysteine thiols. Our results demonstrate a potential role for nitric oxide in the regulation of process outgrowth and remodelling during neuronal development, which may be effected at least in part through modulation of dynamic protein fatty acylation in neuronal growth cones.

Topics: Acylation; Animals; Cells, Cultured; Cyclic GMP; Dibutyryl Cyclic GMP; Ganglia, Spinal; Hemoglobins; Molsidomine; Nerve Crush; Nerve Tissue Proteins; Neurites; Neurons; Nitric Oxide; Palmitic Acid; Palmitic Acids; PC12 Cells; Rats; Sciatic Nerve; Time Factors; Vasodilator Agents

This study investigates the mechanisms of platelet inhibition by the nitrate esters isosorbide dinitrate, isoidide dinitrate, isomannide dinitrate, isosorbide 2-mononitrate and isosorbide 5-mononitrate as compared to the spontaneous nitric oxide (NO)-donor linsidomine, the active metabolite of molsidomine. Nitrates and linsidomine dose-dependently inhibited aggregation, ATP secretion and thromboxane formation of washed human platelets at a rank order of potency, identical with that for stimulation of cyclic GMP in cultured rat lung fibroblasts. While linsidomine (0.1 mM) caused a 3-fold platelet cGMP elevation, there was a weak (< or = 30%) but significant cGMP stimulation by organic nitroesters, which was tightly correlated with inhibition of platelet aggregation (r = 0.926, P = 0.008). Zaprinast (2 microM) potentiated, while methylene blue (1 microM) and oxyhemoglobin (10 microM) reversed the antiaggregatory effects. Linsidomine (0.5 microM-0.1 mM) dose-dependently released NO in a cell-free system. No spontaneous NO release was detected with organic nitroesters (0.1 mM). These data suggest that, to some extent, bioactivation of organic nitroesters occurs in platelets, resulting in platelet inhibition via the NO/cGMP system.

Topics: Adenosine Triphosphate; Animals; Anisotropy; Blood Platelets; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Esters; Fibroblasts; Humans; Lung; Molsidomine; Nitrates; Nitric Oxide; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Thromboxanes

This study evaluated the physiological effects of compounds that alter guanosine 3',5'-cyclic monophosphate (cGMP) on the increase in vascular protein clearance induced by nitric oxide (NO) synthesis inhibition in the feline small intestine. A lymphatic vessel draining the small bowel was cannulated; vascular protein clearance and intestinal blood flow were measured. N omega-nitro-L-arginine methyl ester (L-NAME), the NO inhibitor, was infused (0.5 mumol/min) into the superior mesenteric artery. Vascular protein clearance increased approximately 4.6-fold, whereas blood flow decreased to 50% of control. Elevation of cGMP by 1) cytosolic guanylate cyclase activation with a NO donor (SIN 1) or 2) a cGMP analogue, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) completely prevented the rise in microvascular permeability associated with L-NAME. Moreover, these compounds reduced (almost 90%) baseline vascular protein clearance, whereas inhibition of cytosolic guanylate cyclase with methylene blue significantly increased this parameter. Atrial natriuretic factor (ANF) has been reported to increase tissue cGMP levels and microvascular permeability. In this study, ANF did indeed increase intestinal microvascular permeability however this occurred independent of changes in intestinal cGMP levels. These data support a role for cGMP associated with NO-induced microvascular permeability alterations and raise the possibility that ANF has a cGMP-independent effect on microvascular permeability within the intestine.

Topics: Animals; Arginine; Atrial Natriuretic Factor; Capillary Permeability; Cats; Cyclic GMP; Intestines; Methylene Blue; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Regional Blood Flow

Low output heart failure induces abnormalities of endothelium dependent vasodilation, but the mechanisms responsible for this remain unclear. As blood flow can alter endothelial cell function, in particular nitric oxide (NO) release, the activity of endothelium derived relaxing factor (EDRF) was investigated in a rat model of high output heart failure.. The thoracic aorta upstream of an aorto-caval fistula in rats was submitted to hormonal changes (similar to those in heart failure) and to high blood flow (opposite to that found in low output heart failure). Functional and biochemical arterial properties were studied in aorto-caval fistula rats and in sham operated rats three months after operation. The vascular responses were studied by exposing aortic segments from fistula and sham operated rats to increasing concentrations of agonists. Aortic cyclic guanosine monophosphate (cGMP) concentration was assessed as an index of NO synthase activity. The effect of NO synthase blockade on functional and biochemical arterial properties was also studied.. Plasma atrial natriuretic factor (ANF) was increased in fistula rats compared to sham operated rats. The concentrations of acetylcholine or the calcium ionophore A23187 required to produce 10% and 50% maximum relaxation (EC10 and EC50) were similar in the two groups. Relaxation in response to low concentrations of Sin-1 (an NO donor) was shifted rightwards in fistula rats and EC10 was greater than in the controls. The aortic cGMP concentration was higher in aorto-caval fistula rats than in sham operated rats (p = 0.008). The differences between aorto-caval fistula rats and sham operated rats were probably the result of increased basal EDRF-NO release in the former, since NO synthase blockade abolished the differences in both aortic cGMP and the dose-response curve to Sin-1.. The arterial wall upstream of a chronic aorto-caval fistula has increased cGMP content and hyposensitivity to Sin-1, which may be due to enhanced basal EDRF-NO release. These changes, strikingly different from those found in the low output heart failure, suggest that haemodynamic rather than neuroendocrine factors play a determinant role in the altered vasodilator response in heart failure.

Topics: Acetylcholine; Animals; Atrial Natriuretic Factor; Calcimycin; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Heart Failure; Male; Molsidomine; Nitric Oxide; Rats; Rats, Wistar; Vasodilation

The molecular mechanism(s) underlying the vasodilator activity of CAS 936 (3-(cis-2,6-dimethylpiperidino)-N-(4-methoxybenzoyl)-sydn oni mine) and its metabolites 3-(cis-2,6-dimethylpiperidino)-sydnonimine (C87 3754) and N-(cis-2,6-dimethylpiperidino)-N-nitroso-2-aminoacetonitrile (C873786) was investigated. These compounds were tested for their relaxant activity in isolated rabbit arterial segments, activation of purified soluble guanylyl cyclase and release of nitric oxide (NO) in vitro and in vivo. C873754 and C873786 inhibited the noradrenaline-induced contraction and increased the cyclic GMP content of endothelium-denuded rabbit aortic and femoral segments, whereas CAS 936 was without effect. Similarly, both metabolites, but not CAS 936, activated purified soluble guanylyl cyclase (EC50 about 30 microM) and released NO in buffered aqueous solutions, as detected by electron spin resonance (esr) spectrometry. Both in vitro and in vivo an accumulation of NO was detected by esr spectrometry in vascular tissues exposed to the metabolites of CAS 936, whereas a significant release of NO from CAS 936 was only detected in the isolated rabbit liver, but not in vascular tissue. It is conceivable, therefore, that the metabolites of CAS 936 appearing in the systemic circulation after hepatic biotransformation induce vasodilatation by release of NO and activation of soluble guanylyl cyclase in vascular smooth muscle. Moreover, the activation of soluble guanylyl cyclase in vitro by the metabolites of CAS 936 was significantly enhanced by co-incubation with certain particulate fractions from bovine aortic endothelial and smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blood Vessels; Cattle; Cyclic GMP; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Female; In Vitro Techniques; Male; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide; Nitroso Compounds; Piperidines; Rabbits; Swine; Sydnones; Vasodilator Agents

Experiments were designed to determine the role of the L-arginine pathway in endothelium-dependent relaxations to vasopressin. The effects of L-arginine analogues NG-nitro-L-arginine (L-NNA), NG-nitro-L-arginine methyl ester (L-NAME), and NG-monomethyl-L-arginine (L-NMMA) on basal and vasopressin-induced activity of nitric oxide synthase were studied in isolated canine basilar arteries. Rings with and without endothelium were suspended for isometric tension recording in Krebs-Ringer bicarbonate solution bubbled with 94% O2-6% CO2 (37 degrees C, pH 7.4). Radioimmunoassay was used to determine the level of guanosine 3',5'-cyclic monophosphate (cGMP). All experiments were performed in the presence of indomethacin, a cyclooxygenase inhibitor. L-NAME and L-NMMA caused endothelium-dependent contractions and inhibited basal production of cGMP. In contrast, L-NNA did not affect basal tone or basal production of cGMP. L-Arginine analogues inhibited relaxations to vasopressin but did not affect relaxations to a nitric oxide donor, molsidomine (SIN-1). The effects of L-NNA, L-NAME, and L-NMMA were reversed in the presence of L-arginine. The relaxations to vasopressin were associated with an increase of cGMP levels in the arterial wall. This effect of vasopressin was inhibited in the presence of L-NNA. These studies suggest that the relaxations to vasopressin are mediated by activation of the endothelial L-arginine pathway, leading to increased production of nitric oxide, with subsequent activation of guanylate cyclase in smooth muscle cells. In canine basilar artery, L-NAME and L-NMMA are nonselective inhibitors of both basal and stimulated production of nitric oxide, whereas L-NNA selectively inhibits vasopressin-induced activation of the L-arginine pathway.

Topics: Animals; Arginine; Basilar Artery; Cyclic GMP; Dogs; Endothelium, Vascular; In Vitro Techniques; Molsidomine; Nitric Oxide; Nitroarginine; Vasodilation; Vasopressins

Recent data have demonstrated that inhibition of nitric oxide synthesis exacerbated the mucosal injury associated with reperfusion of the postischemic intestine. In this study, using a feline 1-h intestinal ischemia followed by reperfusion model, we tested the possibility that exogenous sources of nitric oxide may prevent the reperfusion-induced mucosal barrier disruption and examined the mechanisms involved. Mucosal barrier integrity was assessed by determining 51Cr-EDTA clearance from blood to lumen. Intestinal blood flow and resistance were also determined. Reperfusion after 1 h of ischemia significantly increased 51Cr-EDTA clearance (0.05 +/- 0.01 to 0.35 +/- 0.07 ml.min-1.100 g-1) and decreased intestinal blood flow by 50%. Exogenous sources of nitric oxide including SIN-1, CAS-754, and nitroprusside as well as exogenous L-arginine all reduced reperfusion-induced mucosal barrier dysfunction without improving intestinal blood flow. Inhibition of endogenous nitric oxide with NG-nitro-L-arginine methyl ester between 1 and 2 h of reperfusion further augmented the rise in mucosal permeability associated with ischemia-reperfusion. Addition of the permeable analogue of guanosine 3',5'-cyclic monophosphate, 8-bromoguanosine 3',5'-cyclic monophosphate, improved reperfusion-induced intestinal blood flow significantly but did not provide protection against mucosal barrier disruption associated with the first hour of ischemia-reperfusion. Exogenous sources of nitric oxide can reduce reperfusion-induced mucosal barrier dysfunction independent of alterations in intestinal blood flow.

Topics: Animals; Cats; Cyclic GMP; Intestinal Mucosa; Intestines; Ischemia; Molsidomine; Nitric Oxide; Nitroprusside; Permeability; Regional Blood Flow; Reperfusion Injury; Sydnones

1. The study was designed to test the hypothesis that nitric oxide (NO)-releasing compounds increase guanosine 3':5'-cyclic monophosphate (cyclic GMP) production in human polymorphonuclear leucocytes (PMNs) and concomitantly inhibit PMN functions, i.e. leukotriene B4 (LTB4) synthesis, degranulation, chemotaxis and superoxide anion (O2-) release. The effects of two new NO-releasing compounds, GEA 3162 and GEA 5024 were compared to 3-morpholino-sydnonimine (SIN-1) and S-nitroso-N-acetyl-penicillamine (SNAP). 2. GEA 3162 and GEA 5024 (1-100 microM) inhibited Ca ionophore A23187-induced LTB4 and beta-glucuronidase release, chemotactic peptide FMLP-induced chemotaxis and opsonized zymosan-triggered chemiluminescence dose-dependently in human PMNs. SIN-1 and SNAP were weaker inhibitors. 3. Cellular cyclic GMP production was increased after exposure to NO-donors concomitantly with the inhibition of PMN functions. No alterations in the levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were detected. 4. The results suggest that NO, possibly through increased cyclic GMP, inhibits the activation of human PMNs and may thus act as a local modulator in inflammatory processes.

Topics: Cell Degranulation; Chemotaxis, Leukocyte; Cyclic AMP; Cyclic GMP; Glucuronidase; Humans; In Vitro Techniques; Leukotriene B4; Luminescent Measurements; Molsidomine; Neutrophils; Nitric Oxide; Penicillamine; S-Nitroso-N-Acetylpenicillamine; Superoxides; Triazoles

This study aimed to examine the role of nitric oxide (NO) in the regulation of renin secretion from renal juxtaglomerular (JG) cells. Using primary cultures of mouse renal JG cells, we found that sodium nitroprusside (SNP) and 3-morpholino-sydnonimin-hydrochloride (SIN-1), two structurally different liberators of NO, led to a transient inhibition during the first hour followed by a marked dose-dependent stimulation of renin secretion lasting for an additional 20 h. This stimulatory effect was blunted by methylene blue (50 microM) and was reversible within minutes after removal of the NO liberators. SNP and SIN-1 also stimulated guanylate cyclase activity in the cultures with a maximum within the first hour of incubation. Increasing intracellular guanosine 3',5'-cyclic monophosphate levels by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (100 microM) or by atrial natriuretic peptide (10 nM) decreased basal renin secretion but did not inhibit the effect of SNP. The stimulatory effect of SNP was not related to adenosine 3',5'-cyclic monophosphate levels in the JG cells and was blunted after chelation of extracellular calcium by 2 mM ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N'N'-tetraacetic acid. Taken together, our findings suggest that liberators of NO have two effects on renin secretion from isolated JG cells: an inhibitory effect mediated by stimulation of soluble guanylate cyclase activity and a stimulatory effect mediated by an as yet unknown pathway that requires extracellular calcium.

Topics: Animals; Cell Separation; Cyclic AMP; Cyclic GMP; Juxtaglomerular Apparatus; Mice; Molsidomine; Nitric Oxide; Nitroprusside; Renin

The effects of the NO-donor 3-morpholinosydnonimin (SIN-1) on isometric tension, cyclic guanosine 3',5'-monophosphate (cyclic GMP) accumulation and neuronal release of 3H-noradrenaline were investigated in rabbit isolated corpus cavernosum (CC), and compared to the actions of sodium nitroprusside (SNP) and the cyclic GMP-specific phosphodiesterase inhibitor zaprinast. SIN-1, zaprinast and SNP concentration dependently relaxed rabbit CC preparations contracted by 1 microM. phenylephrine. All the drugs were highly effective, and the order of potency was SNP > zaprinast > SIN-1. SIN-1 had a biphasic effect on contractions evoked by electrical field stimulation of nerves: at low concentrations (1 and 10 microM.), SIN-1 inhibited the contractions, while at concentrations > or = 100 microM., the contractions were again increased. There were no changes in baseline tension. Electrically evoked contractions were inhibited by zaprinast in a concentration-dependent manner. Compared with controls, 1 mM. SIN-1 caused a significant (p < 0.05) increase in both the basal efflux and in the electrically induced release of 3H from CC preparations incubated with 3H-noradrenaline. SIN-1, zaprinast and SNP increased tissue levels of cyclic GMP. There was no positive correlation between cyclic GMP accumulation and the relaxant effects of the drugs. The effects of SIN-1 and SNP on the tissue content of cyclic GMP were not significantly affected by methylene blue, an inhibitor of soluble guanylate cyclase. It may be concluded that SIN-1, zaprinast and SNP are effective in relaxing isolated penile erectile tissue, and this effect is associated with an increase in the tissue content of cyclic GMP via pathways not sensitive to methylene blue. However, additional mechanisms beside stimulation of adrenergic neurotransmission and activation of guanylate cyclase in the smooth muscle cell seem to participate in the action of SIN-1 on rabbit penile erectile tissue.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Male; Molsidomine; Nitric Oxide; Nitroprusside; Penile Erection; Penis; Purinones; Rabbits; Vasodilator Agents

The effects of sodium nitroprusside (SNP) and 3-morpholino sydnonimine (SIN-1), isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN), and molsidomine (the inactive precursur of SIN-1) on monocyte chemotaxis and cyclic GMP (cGMP) concentration were studied. SNP and SIN-1 inhibited monocyte N-formyl-methionyl-leucyl-phenylalanine-stimulated migration and increased cGMP concentrations in a dose-dependent (> 10(-5) mol.l-1) and time-dependent manner. Furthermore, 8-bromo cGMP inhibited monocyte chemotaxis in a dose-dependent fashion. In contrast, ISDN, GTN and molsidomine did not alter monocyte migration or cGMP concentration. These results support earlier observations that nitric oxide inhibits monocyte function in vitro via a cGMP-mediated mechanism. The differential effects of the spontaneous and thiol-dependent NO-donating nitrovasodilators on monocyte function suggests that monocytes, like platelets, are not able to directly metabolise ISDN and GTN. If similar observations can be made in vivo, it is possible that certain nitrovasodilators might be used therapeutically to inhibit monocyte function, for example during atherogenesis.

Topics: Cell Migration Inhibition; Chemotaxis, Leukocyte; Cyclic GMP; Humans; Isosorbide Dinitrate; Molsidomine; Monocytes; Nitroglycerin; Nitroprusside; Vasodilator Agents

This study was designed to determine vasodilator activities of two endothelium-derived relaxing factors: prostacyclin (PGI2) and nitric oxide (NO) in human umbilical arteries. Isolated vessel segments were contracted by submaximal concentrations of serotonin and bradykinin. These contractions were enhanced after inhibition of prostaglandin formation by the cyclooxygenase inhibitor indomethacin and after removal of the endothelium, both resulting in a pronounced decrease in PGI2 formation. Contractions remained unchanged after treatment of the vessels with nitro-L-arginine, a selective inhibitor of endogenous NO biosynthesis. The efficacy of inhibition of NO biosynthesis was established by a more than 60% reduction in cyclic GMP accumulation. Even inhibition of stimulated NO formation by histamine did not change vascular tone. These data suggest that PGI2 rather than NO is an endothelium-derived relaxing factor in human umbilical arteries.

Topics: Arginine; Bradykinin; Cyclic GMP; Epoprostenol; Female; Histamine; Humans; Iloprost; In Vitro Techniques; Indomethacin; Molsidomine; Muscle Contraction; Nitric Oxide; Nitroarginine; Serotonin; Umbilical Arteries; Vasodilation

Most platelet agonists activate and elevate the cytosolic free calcium concentration in human platelets through receptor-dependent mechanisms that are antagonized by cAMP- and cGMP-elevating agents. Nitrovasodilators such as nitroprusside and endothelium-derived relaxing factor are potent cGMP-elevating platelet inhibitors. In the present study, the role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of ADP- and thrombin-evoked calcium elevation and activation of human platelets was investigated. Preincubation of platelets with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP; a membrane-permeant selective activator of the cGMP-dependent protein kinase that does not significantly affect cGMP-regulated phosphodiesterases) inhibited the thrombin-induced phosphorylation mediated by myosin light chain kinase and protein kinase C. Nitrovasodilator-induced protein phosphorylation in human platelets was distinct from that induced by cAMP-elevating prostaglandins and could be mimicked by 8-pCPT-cGMP. Preincubation of human platelets with nitrovasodilators or 8-pCPT-cGMP inhibited the ADP- and thrombin-evoked calcium elevation in the presence and absence of external calcium. Nitrovasodilators and 8-pCPT-cGMP also inhibited the agonist-induced Mn2+ influx, but stopped-flow experiments indicated that the ADP receptor-operated cation channel was not significantly inhibited. These results suggest that in human platelets nitrovasodilators inhibit the agonist-induced calcium mobilization from intracellular stores and the secondary store-related calcium influx but not the ADP receptor-operated cation channel. The results also suggest that these nitrovasodilator effects are mediated by cGMP and the cGMP-dependent protein kinase.

Topics: Adenosine Diphosphate; Alprostadil; Blood Platelets; Calcium; Cell Compartmentation; Cyclic GMP; Cytoplasm; Humans; In Vitro Techniques; Ion Channel Gating; Kinetics; Molsidomine; Nitroprusside; Phosphoproteins; Protein Kinases; Receptors, Cell Surface; Thrombin; Vasodilator Agents

We studied the effects of nitric oxide (NO)-producing agents on N-methyl-D-aspartate (NMDA) receptor activation in cultured neurons. 3-Morpholino-sydnonimine (SIN-1) blocked both NMDA-induced currents and the associated increase in intracellular Ca2+. The actions of SIN-1 were reversible and suppressed by hemoglobin. A degraded SIN-1 solution that did not release NO was unable to block NMDA receptors. This showed that the SIN-1 effects were due to NO and not to another breakdown product. Similar results were obtained with 1-nitrosopyrrolidine (an NO-containing drug) and with NO released from NaNO2. Pretreatment with hemoglobin potentiated NMDA-induced effects, demonstrating that endogenous NO modulates NMDA receptors. Since NMDA receptor activation induces NO synthesis, these results suggest a feedback inhibition of NMDA receptors by NO under physiological condition.

Topics: Animals; Calcium; Cyclic GMP; In Vitro Techniques; Mice; Molsidomine; N-Methylaspartate; N-Nitrosopyrrolidine; Nitric Oxide; Nitroso Compounds; Receptors, N-Methyl-D-Aspartate; Sodium Nitrite

In response to inflammatory agents such as thrombin, cultured endothelial cells produce platelet-activating factor (PAF), which has been linked with most inflammatory and immune processes, and is a potent coronary constrictor. Sodium nitroprusside (SNP) and SIN-1 (3-morpholinosydnonimine), which spontaneously release the free radical nitric oxide (NO), cause direct relaxation of blood vessels and inhibition of platelet aggregation by activating soluble guanylate cyclase. In the present study we report that in human umbilical vein endothelial cells (HUVEC) these compounds stimulate the production of cGMP and inhibit thrombin-induced PAF synthesis in a concentration-dependent manner. 8-bromo-cGMP, a permeant non-hydrolysable analogue of cGMP, mimics the inhibitory effect of NO-generating vasodilators. PAF synthesis requires phospholipase A2-mediated hydrolysis of membrane precursors to lyso-PAF, which is in turn converted into PAF by an acetyltransferase. The thrombin-elicited activation of both enzymes is inhibited in a dose-dependent way in HUVEC pretreated with SNP and SIN-1. The inhibitory effect of SNP and SIN-1 on the thrombin-mediated PAF synthesis suggests a new mechanism of action whereby the endogenous NO can affect vascular tone and endothelium-dependent intercellular adhesion. Moreover, PAF production in endothelial cells appears to be an important target for the pharmacological action of nitrovasodilators.

Topics: Acetyltransferases; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Humans; Molsidomine; Nitroprusside; Platelet Activating Factor; Thrombin

The effects of 1 mM L-cysteine on sodium nitrite-, 3-morpholinosydnonimine (SIN-1)- and acetylcholine-induced relaxation and cyclic GMP accumulation were studied in isolated noradrenaline-precontracted rat mesenteric arterial rings. L-Cysteine augmented the relaxation and cyclic GMP increase induced by sodium nitrate and SIN-1 but not those induced by acetylcholine. The effects of L-cysteine on relaxation were independent of the presence of intact endothelium. The results suggest that L-cysteine protects exogenously released nitric oxide.

Topics: Acetylcholine; Animals; Cyclic GMP; Cysteine; Drug Synergism; Endothelium, Vascular; In Vitro Techniques; Male; Mesenteric Arteries; Molsidomine; Nitric Oxide; Rats; Rats, Inbred Strains; Sodium Nitrite; Vasodilation

The relaxant effect of the vasodilator drug, nicorandil, was studied in circular strips of bovine coronary arteries. To differentiate between relaxation caused by cyclic GMP (cGMP) and by hyperpolarization, the influence of cGMP was blocked with methylene blue and that of hyperpolarization with the inhibitor of ATP-dependent K+ channels, glibenclamide. Methylene blue and glibenclamide inhibited nicorandil-induced relaxation to similar extents. Cromakalim-induced relaxation but not that due to sodium nitroprusside (nitroprusside-Na) was inhibited by glibenclamide. Methylene blue inhibited the relaxation caused by nitroprusside-Na but not that due to cromakalim. The different modes of action of the two components of relaxation caused by nicorandil were studied in agonist-agonist interaction experiments. The interaction between nicorandil and nitroprusside-Na or 3-morpholino-sydnonimine (SIN-1) was overadditive in the absence of glibenclamide but additive, i.e. competitive, in the presence of glibenclamide. The interaction of nicorandil with cromakalim or pinacidil was overadditive in the absence of methylene blue but additive, i.e. competitive, in the presence of methylene blue. The results show that nicorandil relaxes smooth muscle through two independent mechanisms: ATP-dependent activation of K+ channels and stimulation of guanylyl cyclase resulting in increases in cGMP.

Topics: Animals; Benzopyrans; Cattle; Coronary Vessels; Cromakalim; Cyclic GMP; Drug Interactions; Glyburide; Guanidines; In Vitro Techniques; Methylene Blue; Molsidomine; Muscle Relaxation; Niacinamide; Nicorandil; Nitroprusside; Pinacidil; Potassium Channels; Pyrroles; Sensitivity and Specificity; Vasodilator Agents

In order to investigate possible effects of endothelium-derived relaxing factor (EDRF or NO.) on platelet phospholipase A2 activity, human platelets labelled with [3H]arachidonic acid ([3H]AA) were stimulated with thrombin (0.5 IU/ml) in the absence or in the presence of sin-1, a vasodilator and platelet inhibitor releasing NO. by spontaneous decomposition at physiological pH. Sin-1 promoted a dose-dependent inhibition of [3H]AA liberation, which was identical in the presence or in the absence of 1 mM Ca2+ in the external medium, suggesting that a reduction of Ca2+ influx was not responsible for this metabolic effect. Using fura-2 as a fluorescent Ca2+ indicator, sin-1 was found to inhibit similarly both Ca2+ influx and Ca2+ mobilization, the latter effect being directly related to a reduction of inositol 1,4,5-tris phosphate production by phospholipase C. However, comparison of cytoplasmic free calcium concentrations ([Ca2+]i) and of [3H]AA liberation attained by platelets treated under various experimental conditions indicated the lack of a direct relationship between [Ca2+]i and platelet phospholipase A2 activity. The effects of sin-1 on [3H]AA liberation could be reproduced by a membrane-permeant analogue of cGMP (8-bromo cyclic GMP), with no evidence of additional effects of sin-1 under these conditions. These data bring further support to the view that Ca2+, although being a necessary cofactor of intracellular phospholipase A2, is not the only regulator of the enzyme. Owing to the multiple effects of this drug on various events involved in membrane-signal transduction (Ca2+ influx, phospholipase C and phospholipase A2 activation), it is suggested that sin-1 inhibits platelet function at an early step of signal transduction, probably by elevating cGMP through a direct effect of NO. on cytosolic guanylate cyclase.

Topics: Arachidonic Acid; Blood Platelets; Calcium; Cyclic GMP; Humans; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Molsidomine; Nitric Oxide; Phospholipases A; Phospholipases A2; Thrombin

1. In superfused precontracted strips of rabbit aorta, methylene blue (MeB) or pyocyanin (Pyo, 1-hydroxy-5-methyl phenazinum betaine) at concentrations of 1-10 microM inhibited relaxations induced by endothelium-derived relaxing factor (EDRF), glyceryl trinitrate (GTN), S-nitroso-N-acetyl-penicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1). However, the vasorelaxant actions of sodium nitroprusside (NaNP) or sodium nitrite (NaNO2) were enhanced by MeB or Pyo. Oxyhaemoglobin (HbO2, 1 microM) inhibited the activities of EDRF and all of the nitrovasodilators studied. Vascular preparations were not relaxed by Pyo unless pretreated with NaNP (0.05-10 microM). 2. In bathed, precontracted rings of rabbit aorta, Pyo (10 microM) produced a shift to the left of the cumulative concentration-response curve for NaNP (0.01-10 microM). The rise in guanosine-3':5'-cyclic monophosphate (cyclic GMP) content of aortic tissue was also enhanced. 3. The vasorelaxant potency of NaNP (30 microM) at pH 5-8 and at 37 degrees C remained unchanged over 2.5 h while a solution of SNAP (30 microM) progressively lost its biological activity over 60 min. The in vitro degradation of the biological activity of SNAP was accelerated by MeB (150 microM) or Pyo (150 microM), whereas the vasorelaxant potency NaNP (30 microM) was doubled when incubated with MeB or Pyo. 4. In human platelet-rich plasma, MeB or Pyo (0.3-3.0 microM) uncovered an anti-aggregatory action of subthreshold concentrations of NaNP (4-8 microM). This was abrogated by HbO2 (10 microM).5. We conclude that MeB or Pyo differ from HbO2 in their mode of interaction with nitrovasodilators.HbO2 scavenges nitric oxide that is released from all types of nitrovasodilators. MeB and Pyo exert a similar action towards organic nitrovasodilators (e.g. SNAP, SIN-1). However, the pharmacological actions of inorganic nitrovasodilators (e.g. NaNP or NaNO2) are potentiated by MeB and Pyo owing to facilitation of the intracellular release of nitric oxide from the inorganic nitrovasodilators.

Topics: Animals; Aorta; Cyclic GMP; Drug Interactions; In Vitro Techniques; Methylene Blue; Molsidomine; Nitric Oxide; Nitroglycerin; Nitroprusside; Penicillamine; Platelet Aggregation; Pyocyanine; Rabbits; S-Nitroso-N-Acetylpenicillamine; Sodium Nitrite; Vasodilator Agents

The role of the L-arginine-NO-cGMP pathway in morphine-induced central analgesia was investigated in two nociceptive tests: PGE2-induced hind paw hyperalgesia and tail-flick. The central analgesic effect of morphine was potentiated by MY5445, a specific cGMP phosphodiesterase inhibitor. I.c.v. injections of morphine or carbachol caused dose-dependent analgesia, which was prevented by methylene blue, an inhibitor of guanylate cyclase. The NO synthase inhibitor, N-iminoethyl-L-ornithine, prevented carbachol-induced analgesia, but did not affect morphine-induced analgesia. Our results suggest that activation of cGMP may underlies analgesia induced by morphine and carbachol. The activation of guanylate cyclase by carbachol seems to depend on the L-arginine-NO pathway, but that caused by morphine remains to be further characterized.

Topics: Analgesia; Animals; Arginine; Brain; Carbachol; Cyclic GMP; Dinoprostone; Male; Molsidomine; Morphine; Nitric Oxide; Ornithine; Phthalazines; Rats; Rats, Wistar

1. The possible roles of the L-arginine-NO pathway and of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in regulating the prejunctional release of noradrenaline and neurogenic vasoconstriction were investigated in the perfused rat tail artery. 2. In the presence of N omega-nitro-L-arginine methyl ester (L-NAME; 30 microM), an inhibitor of NO formation, the vasoconstrictor responses to perivascular nerve stimulation (24 pulses at 0.4 Hz, 0.3 ms, 200 mA) and to exogenous noradrenaline (1 microM) were significantly enhanced, whereas the stimulation-evoked tritium overflow from [3H]-noradrenaline preloaded arteries was not modified. The vasoconstriction enhancing effect of L-NAME was prevented by L-arginine (1 mM) but not D-arginine (1 mM) and was abolished by removal of the endothelium. 3. The NO donor, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1; 0.1-30 microM), and the cyclic GMP phosphodiesterase inhibitor, zaprinast (0.1-30 microM) both induced a concentration-dependent inhibition of the electrical field stimulation-induced vasoconstriction, while atrial natriuretic peptide (ANP; 100 nM) produced only a slight decrease of the vasoconstrictor response. Methylene blue (3 microM), a known inhibitor of soluble guanylate cyclase increased the electrical field stimulation-induced vasoconstriction. SIN-1 and methylene blue when administered simultaneously, antagonized each others effect. None of the compounds tested (SIN-1, zaprinast, ANP or methylene blue) had any significant effect on the stimulation-evoked [3H]-noradrenaline overflow. 4. 8-Bromo-cyclic GMP, a potent activator of cyclic GMP-dependent protein kinase, markedly and concentration-dependently (3-300 microM) increased [3H]-noradrenaline overflow but decreased field stimulation-induced vasoconstriction. Dibutyryl-cyclic GMP (100 JM), a weak activator of cyclic GMP-dependent protein kinase, affected neither the pre- nor the postjunctional response to electrical field stimulation.5. These data show that an NO-like substance of endothelial origin, derived from L-arginine, attenuates vasoconstriction in the rat tail artery, whether neurally-induced or evoked by exogenous noradrenaline.Since noradrenaline release was unaltered by compounds modifying NO production, this NO-like compound acted through a postjunctional mechanism. The lack of prejunctional effects of both soluble and membrane-associated guanylate cyclase activators, despite a large effect of 8-bromo-cyclic GMP,suggests that endogenou

Topics: Animals; Arginine; Arteries; Atrial Natriuretic Factor; Cyclic GMP; Electric Stimulation; Endothelium, Vascular; In Vitro Techniques; Male; Methylene Blue; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Norepinephrine; Purinones; Rats; Tail; Vasoconstriction; Vasodilator Agents

The effects of exogenous guanosine 5'-triphosphate (GTP), guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), cysteine and Trolox C, a water soluble vitamin E analogue, were studied on basal and nitrovasodilator-induced cyclic GMP formation in isolated human lymphocytes. Incubation of lymphocytes in the presence of GTP (0.1 mM) and GTP gamma S (0.1 mM) increased cyclic GMP more than twofold. SIN-1 and sodium nitroprusside dose-dependently increased cyclic GMP, but nitroglycerin and sodium nitrite were ineffective. GTP and GTP gamma S potentiated SIN-1 and sodium nitroprusside-induced cyclic GMP formation. In the presence of GTP and GTP gamma S, nitroglycerin, but not sodium nitrite, was able to increase lymphocyte cyclic GMP. Cysteine (1 mM) enhanced cyclic GMP formation induced by sodium nitroprusside and nitroglycerin. Trolox C (0.1 mM) potentiated SIN-1-induced cyclic GMP formation. These results indicate that exogenous GTP and GTP gamma S enhance guanylate cyclase stimulation by spontaneous nitric oxide releasers and nitroglycerin in lymphocytes. Cysteine, a redox-compound and Trolox C, an antioxidant, have different effects on guanylate cyclase activation by nitric oxide releasers, SIN-1 and sodium nitroprusside.

Topics: Chromans; Cyclic GMP; Cysteine; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylate Cyclase; Humans; L-Lactate Dehydrogenase; Lymphocytes; Molsidomine; Nitroprusside; Vasodilator Agents

The effects of nitric oxide-releasing compounds on Dictyostelium discoideum cell development and guanylyl cyclase activity were studied. The addition of SNP (sodium nitroprusside) or SIN-1 (3-morpholino-syndnonimine) to starved cells inhibited their differentiation and aggregation in a concentration-dependent manner. In contrast to mammalian systems, SNP did not significantly affect guanylyl cyclase activity in cell lysates of D. discoideum, nor did it stimulate cGMP production in intact cells. The results suggest that the inhibitory effects of NO on D. discoideum cell aggregation are through a mechanism independent of an effect on guanylyl cyclase activity.

Topics: Animals; Antihypertensive Agents; Cyclic AMP; Cyclic GMP; Dictyostelium; Guanylate Cyclase; Molsidomine; Morphogenesis; Nitric Oxide; Nitroprusside; Vasodilator Agents

The possibility that calmodulin inhibitors impair the constitutive but not the inducible nitric oxide synthase(s)-mediated inhibitions of tone was investigated in the rat aorta. The endothelium-dependent relaxations evoked by acetylcholine, ATP and the calcium ionophore A23187 (which are mediated by the constitutive nitric oxide synthase) were inhibited by calmodulin inhibitors [calmidazolium, W-7 and (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, hydrochloride, fendiline] and by an inhibitor of nitric oxide synthase, nitro L-arginine. Nitro L-arginine but not calmidazolium reduced the inhibitory influence of the endothelium on the concentration-contraction curves evoked by phenylephrine. Treatment of aortic rings without endothelium with interleukin-1 beta inhibited the contractions to phenylephrine by inducing nitric oxide synthase activity. Nitro L-arginine but not calmidazolium restored the contractility of the aortic rings. The relaxations evoked by a donor of nitric oxide, 3-morpholino-sydnonimine, were minimally affected by calmidazolium and nitro L-arginine. The basal tissue content in, and the production of, guanosine 3',5' cyclic monophosphate evoked by acetylcholine in rings with endothelium were inhibited by calmidazolium and nitro L-arginine. The production of cyclic GMP evoked by interleukin-1 beta in rings without endothelium was inhibited by nitro L-arginine but not by calmidazolium. These observations indicate that calmodulin inhibitors inhibit the constitutive but not the inducible nitric oxide synthase(s) in the rat aorta.

Topics: Amino Acid Oxidoreductases; Animals; Aorta; Arginine; Calmodulin; Cyclic GMP; Drug Interactions; Endothelium, Vascular; Fendiline; Imidazoles; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Inbred Strains; Vasodilator Agents

The present study investigated the underlying mechanisms associated with the loss of responsiveness of veins grafted into the arterial circulation. In particular, the possibility that the altered response is related to modifications of the biologic properties of the vascular smooth muscle, of the endothelial cells or both was tested. Segments of jugular veins were grafted in the reverse position into the carotid arteries in rabbits. After 4 weeks the patent vein grafts and unoperated veins were removed, and endothelium-dependent (acetylcholine) and endothelium-independent (nitric oxide, SIN-1 [the active metabolite of molsidomine32]) relaxations were studied in vitro. In unoperated veins, acetylcholine, nitric oxide, and SIN-1 induced a concentration-dependent relaxation in the presence and absence of the endothelium, respectively. These relaxations were associated with a time-dependent accumulation of guanosine 3':5'-cyclic monophosphate (cyclic GMP). Both relaxation and production of cyclic GMP were inhibited by methylene blue and hemoglobin. Unstimulated veins with endothelium had a significantly higher content of cyclic GMP than did preparations without endothelial cells. This difference was abolished by hemoglobin and methylene blue. In vein grafts acetylcholine induced only minor endothelium-dependent relaxations, whereas nitric oxide and SIN-1 evoked concentration-dependent relaxations in preparations without endothelium, which were shifted significantly to the right compared to unoperated veins. In vein grafts the endothelium-mediated production of cyclic GMP (basal and stimulated by acetylcholine) was significantly reduced when compared to that in unoperated veins, and that evoked by SIN-1 was not different.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Carotid Arteries; Cyclic GMP; Endothelium, Vascular; Hemoglobins; In Vitro Techniques; Jugular Veins; Male; Molsidomine; Muscle Relaxation; Nitric Oxide; Rabbits; Vasodilator Agents; Veins

In the central nervous system, glutamate receptor activation and other stimuli can lead to the cellular production of nitric oxide (NO), an activator of the cyclic GMP-synthesising enzyme, soluble guanylate cyclase. Four 'nitrovasodilators' which yield NO were tested for their ability to elevate cGMP levels in rat cerebellar slices. Nitroprusside (NP), SIN-1, S-nitroso-N-penicillamine (SNAP) and hydroxylamine all caused very large (up to 300-fold) increments. Their threshold concentrations were between 1 and 30 microM. SNAP was the most potent (EC50 approximately 50 microM) followed by hydroxylamine (200 microM) and SIN-1 (1 mM), the latter compound having the highest efficacy. No maximal response to NP was evident at concentrations up to 10 mM. Slices could be challenged a second time with NP (300 microM) with no evidence of a change in sensitivity. The NO-donors are likely to be valuable for studying the functions of NO in brain tissue; however, the concentrations of NP, SNAP and SIN-1 required to elevate cGMP in the slices are orders of magnitude higher than those needed to stimulate guanylate cyclase activity in broken cell preparations, suggesting that rapid inactivation of NO takes place in the intact tissue.

Topics: Animals; Cerebellum; Cyclic GMP; Hydroxylamine; Hydroxylamines; In Vitro Techniques; Molsidomine; Nitric Oxide; Nitroprusside; Penicillamine; Rats; S-Nitroso-N-Acetylpenicillamine; Vasodilator Agents

Topics: Analgesia; Animals; Arginine; Cyclic GMP; Male; Methylene Blue; Molsidomine; Morphine; Nitric Oxide; Ornithine; Phthalazines; Rats; Rats, Inbred Strains

Nitric oxide (NO) is a novel messenger molecule that is produced following glutamate receptor activation and which stimulates cyclic GMP (cGMP) formation. To determine if the mode of action of NO is predominantly intra- or inter-cellular in intact brain tissue, we tested the ability of haemoglobin (Hb), a NO-binding protein that remains extracellular, to inhibit cGMP accumulation induced by glutamate receptor agonists in adult rat cerebellar slices. Responses to agonists acting selectively on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors were inhibited by Hb (IC50 approximately 20 microM) suggesting that NO functions predominantly as a mediator of communication between cells. The effects of NO-donating drugs (e.g. nitroprusside) were also inhibited by Hb, implying that they yield NO extracellularly.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Cerebellum; Cyclic GMP; Drug Interactions; Glutamates; Glutamic Acid; Hemoglobins; Hydroxylamine; Hydroxylamines; Ibotenic Acid; Kainic Acid; Molsidomine; N-Methylaspartate; Nitric Oxide; Nitroprusside; Penicillamine; Rats; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; S-Nitroso-N-Acetylpenicillamine

Nitrovasodilators increase both cyclic GMP and cyclic AMP in isolated platelets (Maurice DH, Haslam RJ. Mol Pharmacol 1990;37:671-81). To determine whether this occurs in blood, platelet cyclic[3H]GMP and cyclic [3H]AMP were measured in prelabeled rabbit platelets resuspended in modified Tyrode's solution or citrated blood. In the former medium, increases in cyclic [3H]nucleotides in response to nitroprusside (NP) and 3-morpholinosydnonimine (SIN-1) were maximal by 1 min; in blood, maximal increases were observed only after 10 min and were much smaller. In blood, SIN-1 was more effective than the same concentration of NP. After 10 min, 100 microM SIN-1 increased platelet cyclic[3H )GMP by 475 +/- 58% and cyclic[3H]AMP by 29 +/- 7% (means +/- SEM, 18 experiments). Supraadditive increases in platelet cyclic [3H]AMP in blood were observed when SIN-1 was combined with prostaglandin E1 (PGE1). Thus, after 10 min, SIN-1 (100 microM), PGE1 (20 nM), and SIN-1 + PGE1 increased cyclic[3H]AMP by 25 +/- 7, 35 +/- 6, and 130 +/- 17%, respectively (four experiments). In the same experiments, release of platelet [14C]serotonin by platelet-activating factor (PAF) was inhibited by 22 +/- 5, 2 +/- 2, and 61 +/- 5%, respectively. Increases in platelet cyclic[3H]GMP with SIN-1 were unaffected by PGE1. These results suggest that although cyclic GMP may mediate the effects of SIN-1 alone on platelet function, cyclic AMP mediates the synergistic action of SIN-1 and PGE1. M&B 22,948 (a selective cyclic GMP phosphodiesterase inhibitor) enhanced the increases in platelet cyclic[3H]GMP and cyclic[3H]AMP caused by SIN-1 and also increased the associated inhibition of [14C]serotonin release. M&B 22,948 also augmented the synergistic increases in cyclic[3H]AMP and inhibition of platelet function caused by SIN-1 + PGE1. The results show that a selected nitrovasodilator (e.g., SIN-1), a prostaglandin and a cyclic GMP phosphodiesterase inhibitor can exert synergistic effects on platelets in blood. This may be relevant to the pharmacologic management of thromboembolic disease.

Topics: Alprostadil; Animals; Blood Platelets; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Drug Synergism; Epoprostenol; Male; Molsidomine; Nitric Oxide; Purinones; Rabbits; Serotonin; Vasodilator Agents

We investigated the possible involvement of reduced nitric oxide (NO) formation in development of nitrate tolerance in an intact organ circulation. NO formation was measured spectrophotometrically on-line in the coronary effluent of Langendorff hearts of rabbits. Short-term (3 min) infusion of glyceryl trinitrate (GTN, 40 microM) or a sydnonimine (SIN-1, 2.3 microM), the active metabolite of molsidomine, into the coronary inflow tract resulted in a decrease in coronary vascular resistance and NO release into the coronary effluent. Pretreatment with 250 microM GTN for 30 min resulted in considerably reduced NO formation and coronary vasodilation, whereas NO release and coronary vasodilation subsequent to SIN-1 remained unchanged. In hearts pretreated with 250 microM SIN-1 for 30 min, there was no effect on GTN- or SIN-1-induced vasodilation and NO release. Studies of cyclic GMP formation in rat lung fibroblasts further indicated that GTN bioconversion rather than desensitization of the soluble guanylate cyclase is involved in GTN tolerance. These data suggest metabolic, endothelium-independent NO release from GTN during passage through the coronary circulation. This NO release is reduced in nitrate-tolerant cells and appears to be the major cause of nitrate tolerance in intact circulatory systems.

Topics: Animals; Bradykinin; Cell Line; Coronary Vessels; Cyclic GMP; Drug Tolerance; Fibroblasts; Heart; Iloprost; Lung; Molsidomine; Myocardium; Nitrates; Nitric Oxide; Nitroglycerin; Rabbits; Rats; Vasodilator Agents

The effects of exogenous guanosine 5'-triphosphate (GTP) and guanosine on nitroglycerin-, sodium nitrite- and SIN-1-induced guanosine 3',5'-cyclic monophosphate (cyclic GMP) accumulation and smooth muscle relaxation were studied using endothelium-denuded rat mesenteric artery rings precontracted with noradrenaline. Preincubation of contracted artery rings with GTP (100 microM) or guanosine (100 microM) before eliciting relaxations with nitrovasodilators significantly shifted the dose-response curves of nitrocompounds to the left and augmented the increases in cyclic GMP. GTP and guanosine alone also induced cyclic GMP accumulation in pre-contracted artery rings. These effects of GTP and guanosine on nitrovasodilator responses were not related to the preincubation period (0-30 min). The present results raise the possibility of a cell membrane site of action for GTP and guanosine, which mediates the activation of soluble guanylate cyclase and leads to increased nitrovasodilator-induced cyclic GMP accumulation and arterial smooth muscle relaxation.

Topics: Animals; Cyclic GMP; Guanosine; Guanosine Triphosphate; In Vitro Techniques; Male; Molsidomine; Muscle, Smooth, Vascular; Nitroglycerin; Rats; Rats, Inbred Strains; Sodium Nitrite; Vasodilation

The osteoclast is unique in its ability to resorb bone, and excessive osteoclastic activity has been implicated in osteoporosis, Paget disease of bone, rheumatoid arthritis, and the growth of metastases in bone. The activity of this cell is controlled by the main circulating inhibitor, calcitonin, in association with locally produced modulators. We show that nitric oxide (NO) may be an important member of the latter group. NO is produced by the vascular endothelium and nervous system and is involved in both neurotransmission and the regulation of blood pressure. However, our results show that the autocoid is also a potent inhibitor of osteoclast function. NO (30 microM) produced a decrease to approximately 50% of the original osteoclast spread area. Similar effects were also produced by 3-morpholinosydnonimine or sodium nitroprusside, reagents that spontaneously release NO. These shape changes were associated with a reduction of bone resorption after a 24-hr incubation of isolated osteoclasts on devitalized bone slices. NO is thought to act by stimulating guanylate cyclase, with a consequent increase in cyclic GMP, but a different mode of action is likely in the osteoclast since dibutyryl or 8-bromo cyclic GMP have no effect. It should be noted that calcitonin can produce similar changes in shape and activity but is associated with an increase in osteoclast intracellular calcium and cessation of membrane movement; neither of these is produced by NO, suggesting that its mode of action is different. The abundance of NO-producing endothelial cells in bone marrow and their proximity to osteoclasts suggests that marrow endothelial cells may play a physiological role in the regulation of osteoclastic activity.

Topics: Animals; Animals, Newborn; Bone Resorption; Calcitonin; Calcium; Cell Membrane; Cells, Cultured; Cyclic GMP; Cytosol; Dibutyryl Cyclic GMP; Ionomycin; Kinetics; Molsidomine; Nitric Oxide; Osteoclasts; Rats; Rats, Inbred Strains; Vasodilator Agents

Different nitrovasodilators were used to assess the role of cyclic GMP in the regulation of polymorphonuclear leukocyte (PMN) function. Molsidomine and its metabolites, 3-morpholinosydnonimine (SIN-1) and N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A) at 0.01-1 mM, inhibited lysosomal enzyme release from PMN stimulated by 30 nM formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). At 1 mM, molsidomine, SIN-1 and SIN-1A decreased beta-glucuronidase release by 19, 37 and 46% of the control, respectively. Glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) showed no effect on beta-glucuronidase release from PMN. At 1 mM, SIN-1A, SIN-1 and SNP in the presence of 0.5 mM isobutylmethylxanthine (IBMX) stimulated cyclic GMP 21-, 9- and 14-fold, respectively, demonstrating a relation between cyclic GMP stimulation and neutrophil inhibition by the molsidomine metabolites. GTN and unmetabolized molsidomine were without effect on cyclic GMP levels. The hypothesis of an inhibitory effect of cyclic GMP on neutrophil function was further supported by the attenuation of SIN-1-induced inhibition of enzyme release by methylene blue (10 microM), an inhibitor of soluble guanylate cyclase. Moreover, 8-bromo cyclic GMP and dibutyryl cyclic GMP, 1 mM, decreased beta-glucuronidase release from FMLP-stimulated PMN by 12 and 44% of the control, respectively. These data demonstrate that cyclic GMP is an inhibitory second messenger in human PMN and suggest that this action of SIN-1 may be of considerable interest under conditions of platelet/PMN activation, e.g. during myocardial ischemia.

Topics: 1-Methyl-3-isobutylxanthine; Cyanides; Cyclic AMP; Cyclic GMP; Glucuronidase; Humans; In Vitro Techniques; Macrophage Activation; Methylene Blue; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitroglycerin; Nitroprusside; Nitrosamines; Vasodilator Agents

This study investigates the action of nitrovasodilators on f-metleu-phe (FMLP)-stimulated LTB4 release and intracellular cyclic nucleotide levels in human polymorphonuclear leukocytes (PMN). Sodium nitroprusside, and the molsidomine (MOL) metabolites SIN-1 and SIN-1A potently inhibited LTB4 release and increased cGMP levels. No significant effects on LTB4 release or cGMP accumulation were observed in the presence of molsidomine or glyceryl trinitrate. None of the compounds tested affected cAMP levels. It is suggested that nitrovasodilators (i) inhibit LTB4 release from human PMN via enhanced cGMP and (ii) that this inhibition requires the presence of an active metabolite, probably nitric oxide.

Topics: Cyclic GMP; Humans; Leukotriene B4; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitroglycerin; Nitrosamines; Vasodilator Agents

To find out whether 3-morpholino-sydnonimine (SIN 1), the active metabolite of molsidomine, exerts its antiaggregatory effects not only in vitro but also in vivo, we tested ex vivo aggregation before and after intravenous application of molsidomine in healthy volunteers. We also measured plasma levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) as SIN 1, the bioactive metabolite of molsidomine, becomes effective via activation of soluble guanylate cyclase. In eight out of ten subjects molsidomine had an inhibitory effect on platelet aggregation and a higher threshold concentration of platelet-activating factor was required after molsidomine application to induce irreversible aggregation. Despite the effect on platelets, plasma cyclic GMP levels did not increase. These results suggest that the nitric oxide-containing SIN 1 inhibits platelet aggregation not only in vitro but also in vivo and that this property can be a beneficial effect in antianginal therapy.

Topics: Adult; Cyclic GMP; Female; Humans; Male; Molsidomine; Platelet Aggregation; Platelet Aggregation Inhibitors; Prodrugs; Vasodilator Agents

We investigated the roles of cyclic GMP and cyclic AMP in the inhibition of rabbit platelet aggregation and degranulation by two nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1; the active metabolite of molsidomine), with particular reference to the synergistic interaction of these drugs with prostaglandin E1 (PGE1). Changes in platelet cyclic [3H]GMP and cyclic [3H]AMP were measured by rapid and sensitive prelabeling techniques, the validity of which were confirmed by radioimmunoassays. Incubation of the platelets with 0.1 to 10 microM SNP alone for 0.5 min caused progressively greater inhibitions of platelet function associated with large dose-dependent increases in cyclic [3H]GMP and 1.4- to 3.0-fold increases in cyclic [3H]AMP. However, addition of SNP with the adenylate cyclase activator, PGE1, at a concentration of the latter that had little effect alone, caused much larger increases in cyclic [3H]AMP and greatly enhanced the inhibition of platelet aggregation. SIN-1 had effects similar to those of SNP, although it was less active. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) diminished the increases in cyclic [3H]AMP caused by SNP or SIN-1 in both the presence and absence of PGE1 but reduced the inhibition of platelet function caused by the nitrovasodilators only in the presence of PGE1. These results suggest that, although cyclic GMP may mediate the inhibition of rabbit platelet function by high concentrations of nitrovasodilators added alone, the synergistic interaction of lower concentrations with PGE1 depends on an enhanced accumulation of cyclic AMP. Synergistic effects on cyclic [3H]AMP accumulation were also observed on incubation of platelets with SNP and adenosine, another activator of adenylate cyclase. Hemoglobin, which binds nitric oxide, blocked or reversed the increases in both cyclic [3H]GMP and cyclic [3H]AMP in platelets caused by the nitrovasodilators added either alone or with PGE1. Cilostamide, a selective inhibitor of platelet low Km cyclic AMP phosphodiesterase, had effects on platelet cyclic [3H]AMP accumulation identical to those of SNP, suggesting that the action of the latter depends on inhibition of the same enzyme. M&B 22,948, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in both cyclic [3H]GMP and cyclic [3H]AMP caused by SNP. A hyperbolic relationship was found between the increases in cyclic [3H]GMP and cyclic [3H]AMP caused by differe

Topics: Adenosine; Adenylyl Cyclases; Alprostadil; Animals; Blood Platelets; Cyclic AMP; Cyclic GMP; Enzyme Activation; Ferricyanides; Hemoglobins; In Vitro Techniques; Kinetics; Molsidomine; Nitroprusside; Platelet Activating Factor; Platelet Aggregation; Platelet Aggregation Inhibitors; Quinolones; Rabbits; Radioimmunoassay; Vasodilator Agents

A previous study revealed that elevation of platelet cyclic GMP induced by a pharmacological activator of soluble guanylate cyclase, 3-morpholinosydnonimine (SIN-1), induced a major inhibition of Ca2+ influx caused by thrombin, as detected by monitoring the fluorescence of the Ca2+ indicator quin-2. In contrast, activation of phospholipase C as well as Ca2+ mobilization presumably promoted by inositol-1,4,5-trisphosphate was less affected by SIN-1 treatment. In the present study, the effects of SIN-1 on Ca2+ influx have been investigated in more detail using platelets loaded with millimolar concentrations of quin-2. Under these conditions, Ca2+ entry from the medium into the platelet cytoplasm could be followed either by detecting fluorescence quenching by Mn2+ or by determination of 45Ca2+ uptake. Both events were inhibited by SIN-1 in a dose-dependent manner. Furthermore, the inhibition of 45Ca2+ uptake and of fluorescence increase observed in the presence of extracellular Ca2+ displayed remarkably parallel dose-response curves, suggesting that elevation of cyclic GMP brought about by SIN-1 inhibits the opening of "receptor-operated channels" whose precise nature remains to be determined.

Topics: Biological Transport; Blood Platelets; Calcium; Cyclic GMP; Enzyme Activation; Fluorescence; Guanylate Cyclase; Humans; In Vitro Techniques; Molsidomine; Solubility; Thrombin; Vasodilator Agents

We compared in vitro the effects of molsidomine, its active metabolite SIN-1, sodium nitroprusside, and the organic nitrates nitroglycerin, isosorbide-5-mononitrate, and isosorbide-2,5-dinitrate on platelet aggregation induced by platelet activating factor and on the activity of soluble guanylate cyclase. In addition, the effects of molsidomine and of isosorbide-5-mononitrate on ex vivo platelet function were studied. In vitro, SIN-1 and sodium nitroprusside were about 100-fold more potent activators of platelet guanylate cyclase and inhibitors of platelet activating factor-induced aggregation than the other agents. In contrast, in ex vivo experiments, not only molsidomine but also isosorbide-5-mononitrate inhibited platelet activating factor-induced aggregation. These data indicate that molsidomine, SIN-1, and organic nitrates can in vivo, like endothelium-derived relaxing factor, inhibit platelet aggregation and exert antithrombotic properties, although nitrates apparently cannot be converted in platelets to active metabolites. Since the antiaggregatory properties are observed when platelet activating factor is used as an aggregant, and since platelet activating factor-induced aggregation is only weakly influenced by inhibitors of cyclo-oxygenase, this effect might be useful clinically.

Topics: Cyclic GMP; Enzyme Activation; In Vitro Techniques; Molsidomine; Nitrates; Nitric Oxide; Platelet Activating Factor; Platelet Aggregation; Platelet Aggregation Inhibitors; Vasodilator Agents

SIN-1, the metabolite of molsidomine, caused a dose-dependent inhibition of the aggregation of rabbit platelets induced by adenosine diphosphate (ADP) and the stable thromboxane mimetic U-46619. Molsidomine was inactive in this respect. In the presence of a threshold concentration of prostacyclin, the antiaggregating activity of SIN-1 became more pronounced. As a result, the dose-response curve of SIN-1 was shifted to the left. These findings suggest that SIN-1 could be of therapeutic value to suppress platelet aggregation during atherosclerotic disease when the endogenous supply of endothelium-derived relaxing factor is compromised.

Topics: Animals; Cyclic GMP; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; In Vitro Techniques; Molsidomine; Nitric Oxide; Platelet Aggregation; Platelet Aggregation Inhibitors; Rabbits; Vasodilator Agents

Molsidomine and its vasoactive metabolite SIN-1 elicit anti-ischemic properties by a therapeutically useful pattern of different vasoactive actions combined with a cyclic GMP-mediated inhibition of platelet adhesion and aggregation. Following molsidomine or SIN-1, local venodilatation and an increase in total effective vascular compliance (as an integrated parameter of venodilatation) were observed. This reduction in preload caused a decrease in myocardial oxygen consumption. Molsidomine caused a slowly progressing dilatation (still present and significant after 4 h), while SIN-1 caused an immediate increase in diameter of the large coronary arteries. The cyclic GMP-mediated dilator effects were accompanied by similar increases in platelet cyclic GMP levels (in the effluent of perfused hearts), indicating an increased potential against platelet activation, adhesion, and aggregation. The combined effect on cardiac preload, conductance of the epicardial arteries, and inhibition of platelet activation improves the ratio between myocardial oxygen supply and demand and suppression of ischemic events.

Topics: Animals; Blood Platelets; Coronary Vessels; Cyclic GMP; Dogs; Dose-Response Relationship, Drug; Hemodynamics; Molsidomine; Platelet Aggregation Inhibitors; Vasodilator Agents

Topics: Cyclic GMP; Drug Tolerance; Endothelium, Vascular; Enzyme Activation; Humans; Molsidomine; Nitric Oxide; Platelet Activation; Vasodilator Agents

The release of NO from SIN-1, the active metabolite of molsidomine, was measured in vitro in Langendorff-perfused rabbit hearts. NO in the coronary effluent was determined on-line using the oxyhemoglobin technique. Left ventricular and coronary perfusion pressure were also recorded continuously. Glyceryl trinitrate and iloprost were used as reference compounds. Infusion of SIN-1 or glyceryl trinitrate into the coronary inflow resulted in a significant and dose-dependent NO release. An apparently identical response was seen when SIN-1 was infused into the coronary effluent while the response to glyceryl trinitrate was greatly reduced or abolished. The glyceryl trinitrate-induced coronary vasodilation was only slightly diminished in presence of oxyhemoglobin whereas the response to SIN-1 was abolished. This is explained by complete scavenging of NO by oxyhemoglobin within the vessel lumen. In isolated porcine aortic endothelial cells, SIN-1 induced a significant and dose-dependent increase in cyclic GMP, whereas glyceryl trinitrate was ineffective. This would argue against biotransformation of glyceryl trinitrate to NO by endothelial cells. Finally, glyceryl trinitrate-tolerant heart preparations exhibited a considerably reduced or even undetectable release of NO, whereas the response to SIN-1 was unchanged.

Topics: Animals; Cells, Cultured; Coronary Circulation; Coronary Vessels; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Activation; Iloprost; Molsidomine; Nitric Oxide; Nitroglycerin; Oxyhemoglobins; Rabbits; Vasodilator Agents

The molecular mechanism of tolerance development to nitrovasodilators, most prominent with nitroglycerin, associated with desensitization of guanylate cyclase is still unclear. Nitric oxide (NO) appears to be the common denominator of this group of drugs that leads to guanylate cyclase activation, followed by increases in levels of cyclic GMP and relaxation. It was therefore decided to study whether NO itself, which causes some tolerance, interferes with the actions of (a) SIN-1 and sodium nitroprusside, both of which are thought to act directly by NO formation, which explains why they cause little tolerance; and (b) with the actions of nitroglycerin, which stimulates cyclic GMP formation only in the presence of cysteine and causes pronounced (large) tolerance. Experiments were performed in circular strips of isolated de-endothelialized bovine coronary artery by measuring isotonic changes in length and cyclic GMP determined by radioimmunoassay. When the strips were treated with submaximal effective concentrations of NO, some tolerance was observed, as shown by moderate attenuation of the rises in cyclic GMP, and a rightward shift of the dose-response curve of the relaxing effects by a dose factor of 10 (DF = 10). Exposure to nitroglycerin, SIN-1, or sodium nitroprusside rendered the strips cross-tolerant to NO to a comparable extent as NO itself, suggesting that under these conditions the NO component of all of these drugs that caused similar tolerance is displayed. When the strips were treated with NO and subsequently challenged with nitroglycerin, SIN-1, or sodium nitroprusside, the NO cross-tolerance was uniformly lower than the tolerance to the challenging agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cattle; Coronary Vessels; Cyclic GMP; Drug Tolerance; Enzyme Activation; In Vitro Techniques; Molsidomine; Nitric Oxide; Nitroglycerin; Nitroprusside; Vasodilator Agents

The effects of 3-morpholinosydnonimine (SIN-1) were studied in isolated canine basilar arteries. Rings with and without endothelium were suspended in Krebs-Ringer bicarbonate solution bubbled with 95% O2-5% CO2. Changes in isometric force were recorded. SIN-1, nitric oxide, and sodium nitroprusside caused concentration-dependent relaxations of control preparation contracted by uridine-5'-triphosphate. The removal of endothelium augmented the relaxation to the nitrovasodilators. Increasing concentrations of SIN-1 reduced endothelium-dependent contractions to the calcium ionophore A23127 and arachidonic acid. The results of the present study suggest that SIN-1 causes relaxation and prevents endothelium-dependent contractions in cerebral arteries. The inhibitory effect of SIN-1 is reduced by the presence of endothelium possibly by interaction with endothelium-derived relaxing factors and/or superoxide anions produced in endothelial cells. The inhibition of endothelium-dependent contractions could be due in part to chemical interactions between endothelium-derived contracting factor (superoxide anion) and the nitric oxide liberated by SIN-1.

Topics: Animals; Basilar Artery; Cyclic GMP; Dogs; In Vitro Techniques; Molsidomine; Muscle Relaxation; Nitric Oxide; Nitroprusside; Vasodilator Agents

We studied the influence of endothelium-derived relaxing factor (EDRF) on sydnonimine (SIN-1)-induced vasodilatation and the accumulation of cyclic GMP in the rabbit femoral artery. The potency of SIN-1 to elicit vasodilatation in norepinephrine-contracted femoral arteries was significantly enhanced in the absence of the endothelium or following impairment of the synthesis of EDRF with gossypol or NG-nitro-L-arginine, whether the application of SIN-1 was intra- or extraluminal. The increase in cyclic GMP in the femoral segments by a combination of SIN-1 and endothelium-derived relaxant factor (released by the endothelium of either the rabbit thoracic aorta or the femoral artery) was significantly less than the sum of the increases in cyclic GMP induced by each agent alone. In contrast, stimulation of purified soluble guanylate cyclase by submaximal concentrations of SIN-1 was additive with the effect of EDRF, released from acetylcholine-stimulated rabbit aortas. This indicates the absence of a direct interaction between the factor and SIN-1 at the level of soluble guanylate cyclase. The interaction seems to be specific for cyclic GMP-mediated responses, since cyclic AMP-induced dilatations elicited by isoproterenol were not affected by the presence of the endothelium. The results indicate that the endothelium can modulate the vascular reactivity to SIN-1. This modulation may be mediated either by EDRF or by another endothelial substance that alters the metabolism or the action of cyclic GMP in vascular smooth muscle.

Topics: Animals; Aorta, Thoracic; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Guanylate Cyclase; In Vitro Techniques; Isoproterenol; Molsidomine; Nitric Oxide; Rabbits; Vasodilation; Vasodilator Agents

The purpose of the present investigations was to determine whether or not SIN-1, a metabolite of molsidomine that spontaneously releases nitric oxide, stimulates the production of adenosine-3',5'-cyclic monophosphate (cyclic AMP) and of guanosine-3',5'-cyclic monophosphate (cyclic GMP) in endothelial cells. All experiments were performed on first or second passage cultured porcine aortic endothelial cells. SIN-1 induced a time- and concentration-dependent accumulation of cyclic GMP but not of cyclic AMP. The production of cyclic GMP evoked by SIN-1 but not evoked by human alpha-natriuretic polypeptide was inhibited by treatment of the cells with either methylene blue (an inhibitor of soluble guanylate cyclase) and hemoglobin (a scavenger of nitric oxide). These data suggest that SIN-1 enhances the activity of soluble guanylate cyclase, which in turn induces the accumulation of cyclic GMP in endothelial cells. This response is probably due to the spontaneous release of nitric oxide, which is a potent activator of soluble guanylate cyclase.

Topics: Animals; Aorta, Thoracic; Cells, Cultured; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Heme; Methylene Blue; Molsidomine; Rodentia; Vasodilator Agents

Topics: Arachidonic Acid; Arachidonic Acids; Cyclic GMP; Glucuronidase; Humans; In Vitro Techniques; Molsidomine; Neutrophils; Nitrosamines; Vasodilator Agents

We studied the effect of nitroglycerin (NTG), endothelium-derived relaxing factor (EDRF), sydnonimine SIN-1, and sodium nitroprusside (SNP) on vascular tone, cyclic GMP content and activity of soluble guanylate cyclase (GC) (in homogenates) of tolerant (1 h 0.55 mM NTG) and non-tolerant (1 h vehicle) de-endothelialized rabbit aortae (RA) as well as on cyclic GMP content of cultured smooth muscle cells (SMC) from RA. Nitrate tolerance significantly attenuated NTG-induced vasodilation of precontracted (1.0 microM norepinephrine) RA, increase in cyclic GMP in RA and SMC, and activation of guanylate cyclase in homogenates as compared to controls. In contrast, vasodilation and cyclic GMP increases to NNP, SIN-1, and EDRF (from cultured bovine aortic endothelial cells) were not affected in RA and SMC, despite desensitization of guanylate cyclase to activation with SNP and SIN-1 in homogenized tolerant RA.. A desensitization of soluble guanylate cyclase to activation with NO can be demonstrated under non-physiological conditions (disrupted cells) in homogenates from nitrate tolerant RA. However, in intact cells (in situ or in culture) soluble guanylate cyclase is not desensitized to EDRF, SIN-1 or SNP. Therefore reduced generation of NO from NTG because of impaired biotransformation of NTG must be regarded as the basis of nitrate tolerance.

Topics: Animals; Aorta; Cattle; Cells, Cultured; Cyclic GMP; Drug Tolerance; Endothelium, Vascular; Guanylate Cyclase; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide; Nitroglycerin; Nitroprusside; Prodrugs; Rabbits; Vascular Resistance; Vasodilator Agents

The effects of sin 1, a metabolite of an antianginal agent, molsidomine, were investigated on human platelet activation induced by thrombin. This drug promoted a slight inhibition of serotonin release in a medium containing 1 mM Ca2+ or 1 mM EGTA (from 63% to 46% and from 57% to 41% of total serotonin secretion, respectively, with the highest dose used). Under these conditions, Ca2+ movements, monitored by quin 2 fluorescence, were markedly impaired. The most pronounced effect was towards Ca2+ influx, which presented a rapid inhibition with low doses. In the presence of external calcium, thrombin raised cytoplasmic free Ca2+ concentration from 100 nM to 1277 nM. This was reduced to 466 nM and 175 nM with 10(-7) M and 10(-4) M sin 1, respectively. Ca2+ mobilization from internal stores was less inhibited, since cytoplasmic free Ca2+ movements, sin 1 was tested on [32P] phosphatidic acid synthesis resulting from phospholipase C activation induced by thrombin. Phosphatidic acid labelling displayed a maximal inhibition of 43-50% with the highest doses of sin 1 (10(-4) M-10(-3) M) with or without Ca2+ in the incubation medium. However, this effect appeared much more sensitive to sin 1 in the presence of external Ca2+ (25% at 10(-7) M sin 1 with external Ca2+ against 12% at the same sin 1 concentration with EGTA). This discrepancy might be explained by the difference of cGMP level obtained when platelets were treated by sin 1 in the presence or in the absence of Ca2+ in the medium. This study shows that the major target of sin 1 via cGMP is not platelet phospholipase C as previously described, but inhibition of Ca2+ influx through plasma membrane.

Topics: Blood Platelets; Calcium; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Humans; In Vitro Techniques; Molsidomine; Phosphatidic Acids; Serotonin; Thrombin; Type C Phospholipases

In the uterine smooth muscle, SIN-1 stimulated cGMP accumulation independently of the presence of Ca2+ and activated the soluble form of guanylate-cyclase through mechanisms apparently similar to those involved in the stimulations evoked by NO-containing compounds. These activations appear different from those induced by hydroperoxy-unsaturated fatty acids and which contribute to the carbachol-mediated cGMP accumulation. SIN-1 did not influence the rise in cAMP of the biosynthesis of PG1(2) and 12-HETE due to exogenous arachidonic acid. By contrast, SIN-1 markedly inhibited the increased synthesis of PG1(2) induced by the ionophore A23187 which was due to a prior, Ca2+-dependent, liberation of endogenous arachidonic acid. The data suggests an interference of SIN-1 with the generation and/or the expression of the Ca2+ signal.

Topics: Animals; Arachidonic Acids; Cyclic AMP; Cyclic GMP; Epoprostenol; Female; Guanylate Cyclase; Guinea Pigs; Molsidomine; Myometrium

Present evidence indicates that the active metabolite SIN-1 of the prodrug molsidomine dilates vascular smooth muscle and inhibits platelet aggregation by a direct stimulatory effect on soluble guanylate-cyclase in the cytosol of vascular smooth muscle cells and platelets, respectively. Evidence from studies in bovine coronary arteries is presented to proof the causal relation between SIN-1 induced rises in cGMP and relaxation under a variety of pharmacological conditions. In contrast to organic nitrates, SIN-1, which activates guanylate-cyclase in vitro independently of the presence of added cysteine, does not cause tolerance. Tolerance most pronounced with nitroglycerin appears to be due to a direct inactivating effect of this drug on guanylate-cyclase.

Topics: Animals; Cyclic GMP; Molsidomine; Muscle, Smooth, Vascular; Nitrates; Platelet Aggregation; Vasodilation

SIN-1, a metabolite of the vasodilating drug molsidomine, was found to stimulate dose dependently (0.01-1 mM) soluble guanylate cyclase from bovine coronary arteries up to 100-fold the control value. The stimulatory effect of SIN-1 increased with rising concentrations of MnC1(2) or MgC1(2) and was diminished in the presence of methylene blue or ferricyanide. The time course of SIN-1-induced guanylate cyclase stimulation was characterized by a lag phase which was not observed after preincubation of the enzyme with SIN-1. In contrast to nitroglycerin and sodium nitroprusside, SIN-1 did not require the presence of cysteine or other thiols to stimulate guanylate cyclase. The results presented in this study provide further evidence that SIN-1 exerts its dilating effect on coronary vessels via direct stimulation of guanylate cyclase.

Topics: Animals; Cations, Divalent; Cattle; Coronary Vessels; Cyclic GMP; Ferricyanides; Guanylate Cyclase; In Vitro Techniques; Methylene Blue; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Nitroprusside; Oxadiazoles; Sodium Nitrite; Sulfhydryl Compounds; Sydnones

Nitrovasodilators relax vascular smooth muscle by one common mechanism, the activation of soluble guanylate cyclase leading to increased formation of cGMP. The considerable differences in potency between various nitrovasodilators appear, at least in part, to be due to the different pathways of their transformation into activators of guanylate cyclase such as nitrous oxide or nitrosothiol. Major differences were also found in the ability of these compounds to induce tolerance in isolated bovine coronary artery strips. Although the mechanism of tolerance development is still not clarified, it appears likely that cysteine deficiency may be responsible for this phenomenon since this thiol appears to be required for the transformation of certain nitrovasodilators (e.g. nitroglycerin) into stimulators of guanylate cyclase and also in the final step of activation of this enzyme.

Topics: Animals; Cattle; Coronary Vessels; Cyclic GMP; Drug Tolerance; Guanylate Cyclase; In Vitro Techniques; Isosorbide Dinitrate; Molsidomine; Nitrates; Nitroglycerin; Vasodilation

The mode of action of the in vitro active metabolites SIN-1 and SIN-1A of the vasodilator prodrug molsidomine was studied in bovine coronary artery strips. Both compounds increased cyclic GMP levels in close association with, but prior to their relaxing action. Relaxation and rises in cyclic GMP by SIN-1 were potentiated by M & B 22,948, an inhibitor of cyclic GMP phosphodiesterase and attenuated by methylene blue, a dye that inhibits activation of guanylate cyclase by SIN-1 and various nitrovasodilators. A single significant correlation between rises in cGMP and relaxation was obtained for both SIN compounds and various nitrovasodilators. Relaxation by SIN-1A was independent of the presence of endothelium and was not affected by various inhibitors of arachidonic acid metabolism. In contrast to nitroglycerin, SIN-1 did not induce substantial tolerance nor were its actions reduced in artery strips that were tolerant to nitroglycerin. The results indicate that SIN-1A relaxes coronary smooth muscle by a direct stimulant effect on soluble guanylate cyclase in vascular smooth muscle cells.

Topics: Animals; Cattle; Coronary Vessels; Cyclic GMP; Dose-Response Relationship, Drug; Drug Synergism; Drug Tolerance; Endothelium; In Vitro Techniques; Methylene Blue; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Oxadiazoles; Phosphodiesterase Inhibitors; Sydnones; Time Factors; Vasodilation

Molsidomine is enzymatically metabolized in the liver to SIN-1 and readily converted into the active metabolite SIN-1A, which carries a free nitroso group. Evidence obtained in isolated circular strips from bovine coronary arteries indicates that SIN-1 increases cyclic guanosine monophosphate in close association with its relaxant effects in coronary strips under various pharmacologic conditions, suggesting that cyclic guanosine monophosphate mediates relaxation. Various nitrovasodilators act by the same mechanism, which is stimulation of guanylate cyclase. In this study the effect of nitroglycerin depended on the presence of a special thiol, cysteine, whereas SIN-1 was active also in the absence of cysteine. Cysteine deficiency was found to be associated with tolerance. After prolonged exposure to the drug, tolerance toward nitroglycerin developed in coronary strips that was antagonized by cysteine. SIN-1 produced no significant tolerance and was also fully active in nitroglycerin-tolerant strips. We conclude that SIN-1 relaxes vascular smooth muscle by direct stimulation of guanylate cyclase, whereas nitroglycerin probably must be converted into a cyclase stimulator by a cysteine-dependent reaction.

Topics: Animals; Cattle; Coronary Vessels; Cyclic GMP; Drug Tolerance; In Vitro Techniques; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Oxadiazoles; Sydnones; Vasodilator Agents

In vitro experiments on precontracted canine coronary arteries were performed to study the direct relaxant effects of molsidomine (MOLS) and its active metabolite, SIN-1, and to determine if there is a relationship between effect and cGMP level elevations. The effects of MOLS and SIN-1 were compared with those of a classic vasodilator, nitroglycerin (NTG). At equimolar doses (10(-6)M) SIN-1 exerted greater relaxant effect than NTG (80 +/- 2% and 60 +/- 5%, respectively) in spite of the fact that it produced less of an increase in cyclic guanosine monophosphate (cGMP) levels. cGMP levels fell rapidly after they peaked, but relaxation was maintained. cGMP elevation preceded the induction of relaxation by NTG but not that induced by SIN-1. Relaxation occurred faster after NTG than after SIN-1. Since SIN-1 has a greater relaxant effect than NTG in spite of the fact that SIN-1 induces less of an increase in cGMP levels and the fact that the peak elevation does not precede the onset of relaxation, the causal nexus between GMP level elevation and relaxation effect after sydnonimines should be challenged.

Topics: Animals; Coronary Vessels; Cyclic AMP; Cyclic GMP; Dogs; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Male; Molsidomine; Muscle Contraction; Muscle, Smooth, Vascular; Nitroglycerin; Oxadiazoles; Sydnones; Vasodilator Agents