nitroarginine and 6-anilino-5-8-quinolinedione

We have investigated the effect of oxidatively-modified low density lipoproteins (ox-LDL) on the contractility of rabbit trabecular smooth muscle. Low density lipoproteins (LDL) were isolated from fresh human plasma pooled from multiple donors and oxidized by exposure to copper. Corpus cavernosum strips from New Zealand White rabbits were studied in organ chambers for isometric tension measurement. Corporeal strips in which moderate tone was induced by phenylephrine, contracted when exposed to ox-LDL, but not when exposed to either native LDL (nLDL) or LDL protected from oxidation by butylated hydroxytoleune (BHT-LDL). Removal of the endothelium, or treatment of the corporeal strips with N(omega)-nitro-L-arginine (nitric oxide synthase inhibitor), methylene blue of LY83583 (guanylate cyclase inhibitors/superoxide producing agents), did not prevent ox-LDL-induced contraction. ox-LDL, dose-dependently, enhanced the contractile response of corporeal strips to low and moderate concentrations by phenylephrine. nLDL had no significant effect on phenylephrine-induced contraction of corporeal strips. ox-LDL, nLDL or BHT-LDL had no effect on relaxation induced by the endothelium-dependent dilator, acetylcholine, or the nitric oxide donor, nitroprusside. In conclusion, this present study demonstrates significant pro-contractile effects of ox-LDL on corporeal smooth muscle, this effect is independent of the endothelium or the nitric oxide/cGMP pathway. The pro-contractile effect of ox-LDL may interfere with penile smooth muscle relaxation, necessary for the initiation and maintenance of penile erection.

Topics: Aminoquinolines; Animals; Butylated Hydroxytoluene; Copper; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Lipoproteins, LDL; Male; Muscle Contraction; Muscle, Smooth; Nitric Oxide Synthase; Nitroarginine; Penis; Phenylephrine; Potassium; Rabbits

1. We have investigated the effects of varying flow velocity (U) upon permeability to potassium ions (PK) of single perfused mesenteric venules in anaesthetised rats. PK was estimated using a development of the single bolus microperfusion technique at chosen flow velocities in the range of 300 to 6000 microm s-1. 2. In an initial study on 12 vessels, there was a strong positive correlation between PK and U. This was described by the relation: PK = 0.0053U + 8.86, where PK and U are both expressed in micrometres per second (microm s-1). 3. The addition of the nitric oxide (NO) synthase inhibitors (20 micromol l-1) N G-monomethyl-L-arginine (L-NMMA) and N G-nitro L-arginine (L-NNA) to the superfusate abolished the positive correlation between PK and U. The addition of D-NNA (20 micromol l-1) did not change the relation between PK and U where the median value for the slope of the relation was 57.7 (+/- 58.7 interquartile (IQR)) x 10-4 (n = 4). The addition of L-arginine (200 micromol l-1) restored the relation between PK and U where the slope of the relation was increased from 3.9 (+/- 16.3 IQR) x 10-4 to 69.2 (+/- 13.5 IQR) x 10-4 (n = 7). 4. The addition of the guanylate cyclase inhibitor LY83583 (10 micromol l-1) abolished the positive correlation between PK and U (n = 6). 5. Our data suggest that the flow modulates the potassium permeability through the walls of single perfused rat mesenteric venules via a NO-cGMP-dependent process.

Topics: Algorithms; Aminoquinolines; Anesthesia; Animals; Biological Transport, Active; Calibration; Diffusion; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Indicators and Reagents; Male; Mesenteric Veins; Microelectrodes; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Perfusion; Permeability; Potassium; Rats; Rats, Sprague-Dawley

Maximum velocity of shortening, Vo, was measured by the method of Edman [J Physiol (Lond) 291:143-159, 1979] on extensor digitorum longus muscles of a mouse in vitro at 20 degreesC. Blockers of nitric oxide synthase, 10 mM nitro-l-arginine or 1 mM 7-nitroindazole, reduced Vo by 18% and 22%, respectively. On removal of the inhibitor, Vo returned to the control value. It was found that 10 mM nitro-d-arginine, an enantiomer of nitro-l-arginine inactive against nitric oxide synthase, did not affect Vo. A donor of nitric oxide, 0.1 mM nitroprusside, increased Vo by 15%. It removed the inhibition caused by nitro-l-arginine. Another donor of nitric oxide, 1 microM (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), increased Vo by 8%. An inhibitor of cGMP synthase, 0.01 mM Ly-83583, decreased Vo by 18%. An analogue of cGMP, 0.1 mM 8-bromo-cGMP, increased Vo by 17%. A general inhibitor of phosphodiesterases, 0.02 mM 3-isobutyl-1-methylxanthine (IBMX), increased Vo by 17%. An inhibitor specific of cGMP phosphodiesterase, 0.01 mM dipyridamole, increased Vo by 8%. The maximal isometric force (F0) was not modified by the drugs, except by 7-nitroindazole and Ly-83583, which depressed F0 by 12%. The cGMP level in tetanized muscles decreased by 12-27% in the presence of blockers of nitric oxide synthase. We conclude that the level of intracellular nitric oxide modulates Vo through the cGMP pathway.

Topics: 1-Methyl-3-isobutylxanthine; Aminoquinolines; Animals; Biomechanical Phenomena; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Skeletal; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Penicillamine; Phosphodiesterase Inhibitors; Stereoisomerism

Sodium nitroprusside (SNP) (10(-9)-10(-6) mol) and carbachol (10(-9)-10(-6) mol) induced dose-dependent vasodilatation in the perfused rabbit isolated ovarian vascular bed. Carbachol, but not SNP-induced vasodilatation was abolished by treatment with CHAPS (4. 7 mg ml-1, 30 s) to remove the endothelium. Carbachol-induced responses were also significantly attenuated by LY 83583 (10(-5) M) and methylene blue (3x10(-5) M). L-NOARG (10(-5) M) reduced carbachol-induced vasodilatation. None of these compounds affected SNP-induced vasodilator responses. Both SNP- and carbachol-induced vasodilatation were attenuated by raising the [K+] in the Krebs' solution to 40 mM. The responses were also reduced by TEA (20 mM) but not by glibenclamide. It was therefore concluded that SNP induced cGMP-independent vasodilator responses in the perfused rabbit ovarian vascular bed. This vasodilator response involved membrane hyperpolarisation since it was lost in high [K+] Krebs' solution. (c) 1998 The Italian Pharmacological Society.

Topics: Aminoquinolines; Animals; Carbachol; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Female; Guanylate Cyclase; In Vitro Techniques; Methylene Blue; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Nitroprusside; Ovary; Rabbits; Vasodilation

Nitric oxide (NO) is known to be synthesized from L-arginine in a reaction catalyzed by NO synthase. Liver cytochrome P-450 enzymes also catalyze the oxidative cleavage of C==N bonds of compounds containing a -C(NH2)==NOH function, producing NO in vitro. The present study was designed to investigate whether there was evidence of a similar pathway for the production of NO in tracheal smooth muscle cells. Formamidoxime (10(-2) to 10(-4) M), a compound containing -C(NH2)==NOH, relaxed carbachol-contracted tracheal rings and increased intracellular cGMP in cultured tracheal smooth muscle cells, whereas L-arginine had no such effect. NO was detectable in the medium containing cultured tracheal smooth muscle cells when incubated with formamidoxime. Ethoxyresorufin (10(-7) to 10(-4) M), an alternate cytochrome P-450 substrate, inhibited formamidoxime-induced cGMP accumulation as well as tracheal ring relaxation in cultured tracheal smooth muscle cells. The NO synthase inhibitors Nomega-nitro-L-arginine (10(-3) M) and NG-monomethyl-L-arginine (10(-3) M) had no effect on formamidoxime-induced cGMP accumulation. These results suggest that NO can be synthesized from formamidoxime in tracheal smooth muscle cells, presumably by a reaction catalyzed by cytochrome P-450.

Topics: Aminoquinolines; Animals; Cells, Cultured; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Hydroxylamines; In Vitro Techniques; Isometric Contraction; Male; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Oxazines; Oximes; Rats; Rats, Inbred Lew; Trachea

Relaxations induced by electrical field stimulation and acetylcholine were compared with those induced by acidified sodium nitrite, sodium nitroprusside, S-nitrosoglutathione and S-nitroso-N-acetyl-D,L-penicillamine in the mouse corpus cavernosum precontracted with phenylephrine. NG-nitro-L-arginine inhibited electrical field stimulation- or acetylcholine-induced relaxation, but was ineffective on relaxations caused by the other stimuli. Hydroquinone and pyrogallol had no inhibitory action on the relaxations caused by any stimulus except acidified sodium nitrite. Incubation of the tissue with diethyldithiocarbamic acid significantly inhibited the relaxations induced by all stimuli except papaverine. In the tissues pre-treated with diethyldithiocarbamic acid, superoxide dismutase, hydroquinone and pyrogallol failed to yield restore or further inhibit the relaxations in response to electrical field stimulation or acetylcholine. LY 83583 (6-anilino-5,8-quinolinedione) and hydroxocobalamin clearly inhibited the relaxant responses to electrical field stimulation, acetylcholine, S-nitrosoglutathione and acidified sodium nitrite whereas there was significant enhancement of the relaxation produced by S-nitroso-N-acetyl-D,L-penicillamine. These findings suggest that the relaxant factor released from non-adrenergic non-cholinergic nerves or endothelial cells in mouse cavernosal tissue may be a superoxide anion-resistant nitric oxide-containing molecule and that S-nitrosoglutathione rather than S-nitroso-N-acetyl-D,L-penicillamine could be a suitable candidate for this.

Topics: Acetylcholine; Aminoquinolines; Animals; Chelating Agents; Ditiocarb; Electric Stimulation; Enzyme Inhibitors; Hematinics; Hydrogen-Ion Concentration; Hydroquinones; Hydroxocobalamin; In Vitro Techniques; Male; Mercaptoethanol; Mice; Muscle Relaxation; Muscle, Smooth; Mutagens; Nitric Oxide; Nitroarginine; Nitroprusside; Nitroso Compounds; Penis; Pyrogallol; S-Nitrosothiols; Sodium Nitrite; Vasodilator Agents

We have shown that endogenous nitrogen oxides (NOx) modulate excitation-contraction coupling in diaphragm. Because cyclic GMP (cGMP) is a second messenger for nitric oxide (NO) inhibition of smooth muscle contraction, we rested the hypothesis that NO acts via cGMP in diaphragm. Fiber bundles from rat diaphragm were studied in vitro. Immunohistochemical analysis using a cGMP-specific monoclonal antibody confirmed the presence of cGMP in the subsarcolemmal region, near nitric oxide synthase (NOS). cGMP measured by ELISA in control muscle (0.27 pmol/mg +/- 0.01 SE) was significantly increased by the NO donor S-nitroso-N-acetylcysteine 1 mM (0.55+/-0.05; N = 6; P < 0.001). Contractile studies showed that the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) 10 mM increased submaximal (40 Hz) tetanic force (P < 0.0001). L-NNA effects were exaggerated by the guanylate cyclase inhibitor LY83583 5-10 microM; force at 40 Hz was increased (P < 0.001). L-NNA effects were partially reversed by 8-bromo-cGMP 1 mM (8-Br-GMP; a cell-permeable cGMP analogue; P < 0.0001) or dipyridamole 10 microM (DPM; a phosphodiesterase inhibitor; P < 0.0001). 8-Br-GMP and DPM produced more-complete L-NNA reversal in combination (P < 0.0001). We conclude that cGMP functions as a second messenger by which NO inhibits diaphragm contraction.

Topics: Aminoquinolines; Animals; Cyclic GMP; Diaphragm; Dipyridamole; Enzyme Inhibitors; Guanylate Cyclase; Male; Muscle Contraction; Nitric Oxide; Nitroarginine; Phosphodiesterase Inhibitors; Rats; Rats, Sprague-Dawley; Second Messenger Systems

The diffusible messenger carbon monoxide (CO) has been proposed to mediate endogenous cyclic guanosine 3',5'-monophosphate (cGMP) formation and sensory adaptation in vertebrate olfactory receptor neurons (ORNs). We have identified and characterized a long-lasting form of odor response adaptation (LLA) that operates at the level of isolated salamander ORNs and does not require any interactions from other cells. Manifestations of LLA are seen in reduced amplitude and prolonged kinetics of the cAMP-mediated excitatory odor response and the generation of a persistent current component that lasts for several minutes and is attributable to cyclic nucleotide-gated (CNG) channel activation by cGMP. Because these effects can be mimicked by micromolar amounts of exogenous cGMP or CO, we applied various inhibitors of cGMP formation. LLA is abolished selectively by heme oxygenase inhibitors known to prevent CO release and cGMP formation in ORNs, whereas odor excitation remains unaffected. In contrast, blockers of nitric oxide synthase are unable to eliminate LLA. Several controls rule out a contribution of nonspecific actions to the effects of CO inhibitors. The results indicate that endogenous CO/cGMP signals contribute to olfactory adaptation and underlie the control of gain and sensitivity of odor transduction. The findings offer a mechanism by which a single, brief odor stimulus can be translated into long-lasting intracellular changes that could play an important role in the perceptual adaptation to odors, and explain the longstanding puzzle that the olfactory CNG channels can be gated by both cAMP and cGMP.

Topics: Acclimatization; Ambystoma; Aminoquinolines; Animals; Cadmium; Carbon Monoxide; Cyclic GMP; Cyclohexanols; Enzyme Inhibitors; Eucalyptol; In Vitro Techniques; Menthol; Monoterpenes; Nitroarginine; Odorants; Olfactory Receptor Neurons; omega-N-Methylarginine; Reaction Time; Second Messenger Systems; Sulfonamides; Terpenes

It has been proposed that capacitative Ca influx into both pancreatic acinar cells and HT-29 colonic cells is regulated by stimulation of nitric oxide synthase (NOS). NO, in turn, controls cGMP levels through effects on guanylate cyclase. We tested this possibility by measuring Ca (and Ba) entry into human embryonic kidney 293 cells and into 293 cells that had been transfected with the neuronal NOS gene (293/NOS). 293 cells had undetectable levels of NOS, while 293/NOS cells exhibited very high levels [Bredt D.S., Ferris C.D., Snyder S.H. Nitric oxide synthase regulatory sites. J Biol Chem 1992; 267: 10976-10981]. Ca (or Ba) entry into single cells was measured as the rate of increase of the Fura-2 fluorescence ratio (digital imaging microscopy) during rapid changes from Ca-free (or Ba-free) to Ca- (or Ba-) containing solutions (using high K to depolarize the membrane potential). cGMP levels (EIA method) were measured to correlate to rates of Ca entry. 100 microM ATP caused release of Ca from internal stores, but no sustained plateau due to Ca entry in either 293 or 293/NOS cells. Cyclopiazonic acid (CPA, which inhibits the Ca pump of the internal store, allowing Ca to leak from the store) caused apparent Ca entry to increase 5-10-fold from similar, low levels in both 293 and 293/NOS cells. CPA-stimulated Ca entry was unaffected by the NOS inhibitor N-nitro-L-arginine (L-NA) in either 293 or 293/NOS cells. In 293 cells [cGMP] was low; ATP and CPA both increased [cGMP] by 2-fold, and the guanylate cyclase inhibitor LY83583 and L-NA decreased [cGMP] by 50-75%. [cGMP] was 20-fold higher in 293/NOS cells than in 293 cells; these [cGMP] were not affected by ATP and CPA, but were effectively decreased by 80-90% by L-NA and by LY83583. Thus, [cGMP] and Ca or Ba entry showed no relationship to each other: Ca entry was small into cells in which [cGMP] was either low (resting 293, CPA + L-NA or CPA + LY83583), intermediate (ATP-treated 293) or high (resting 293/NOS). Similarly, Ca entry was high into cells in which [cGMP] was low (CPA + L-NA- or CPA + LY83583-treated 293), intermediate (CPA-treated 293 and CPA + L-NA-treated 293/NOS) or high (CPA- or ATP-treated 293/NOS). We conclude that, as in most other non-excitable cells, Ca entry into 293 cells is stimulated by loss of Ca from the store but, unlike pancreatic and colonic cells, this capacitative Ca entry does not appear to be regulated by NO and cGMP. Therefore, although capacitative entry across the plasma m

Topics: Adenosine Triphosphate; Aminoquinolines; Barium; Calcium; Calcium-Transporting ATPases; Cell Line; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Humans; Indoles; Kidney; Membrane Potentials; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Potassium; Transfection

Pituitary adenylate cyclase-activating peptide (PACAP) receptors and their signaling pathways were characterized in dispersed rabbit gastric muscle cells. 125I-PACAP-27 and 125I-vasoactive intestinal peptide (VIP) binding to muscle cells were inhibited equally by PACAP and VIP (mean inhibitory concentration 0.8 to 1.3 nM) and desensitized to the same extent (70-80%) by exposure to either peptide. PACAP, like VIP, increased cytosolic free Ca2+ and the formation of L-[3H]citrulline, NO-3/NO-2, guanosine 3',5'-cyclic monophosphate (cGMP), and adenosine 3'5'-cyclic monophosphate (cAMP) and induced relaxation (mean effective concentration 1.8 +/- 0.1 nM) that was partly inhibited by NG-nitro-L-arginine (L-NNA), VIP-(10-28), and PACAP 6-38. L-[3H]citrulline and cGMP formation were blocked by nifedipine, L-NNA, and pertussis toxin (PTx), implying activation of a G protein-coupled, Ca(2+)-calmodulin-dependent nitric oxide (NO) synthase. PACAP-induced relaxation was inhibited to the same extent (46-49%) by nifedipine, L-NNA, PTx, and the protein kinase G inhibitor KT-5823; the inhibition reflected the component of relaxation mediated by the NO-cGMP pathway. The residual relaxation was abolished by the protein kinase A inhibitor H-89. The pattern of inhibition of all responses was identical to that observed with VIP. Desensitization with VIP or PACAP abolished cAMP formation but had no effect on L-[3H]citrulline and cGMP formation induced by either peptide. Receptor protection with VIP or PACAP preserved fully all responses (L-[3H]citrulline, cGMP, and cAMP formation and relaxation) to either peptide. The complete cross-competition, cross-desensitization, cross-antagonism, and cross-protection of receptors by either VIP or PACAP are consistent with interaction of both peptides with the same receptors; the receptors consist of two classes, each coupled to a distinct signaling pathway.

Topics: Adenylate Cyclase Toxin; Alkaloids; Aminoquinolines; Animals; Calcium; Carbazoles; Cells, Cultured; Citrulline; Cyclic AMP; Cyclic GMP; Cytosol; Enzyme Inhibitors; GTP-Binding Proteins; Guanylate Cyclase; Indoles; Isoquinolines; Kinetics; Muscle Relaxation; Muscle, Smooth; Neuropeptides; Neurotransmitter Agents; Nifedipine; Nitric Oxide Synthase; Nitroarginine; Pertussis Toxin; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Kinase Inhibitors; Rabbits; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Signal Transduction; Stomach; Sulfonamides; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

We have investigated the differences between the nitric oxide synthase inhibitor (NOSI), L-NMMA, and the guanylate cyclase inhibitors (GCI), methylene blue and LY 83583, in their abilities to increase vasoconstrictor responses in vitro and in vivo. In rat small mesenteric arterial rings, 1 h exposure to the NOSI, L-NMMA (100 microM), and the GCI, methylene blue (10 microM), alone or in combination with L-NMMA, caused a significant reduction in the maximum relaxation to ACh in mesenteric arteries pre-contracted with the thromboxane mimetic U46619 (10 microM). Hence, both NOSI and GCI inhibit endothelium-dependent relaxations to ACh in rat small mesenteric artery. However, 1 h exposure to L-NMMA and L-NNA (both 100 microM), but not methylene blue (10 microM), significantly increased the contractile response to U-46619 (10 microM) in rat small mesenteric artery. It was decided to investigate further this difference between NOSI and methylene blue. In rat small mesenteric arterial rings, L-NMMA (10 microM) and LY 83583 (1-10 microM) significantly increased the contractile response to KCl (40 mM) or to noradrenaline (10 microM), when administered during the contraction. However, methylene blue (1-10 microM) increased the contractile response to KCl but not noradrenaline. In rat aortic rings, L-NMMA (100 microM), methylene blue (1-10 microM) and LY 83583 (1-10 microM) significantly increased the contractile response to KCl (40 mM) or to noradrenaline (1 microM). In the pithed rat preparation, L-NMMA (40.3 micromol kg(-1), i.v.) significantly increased the pressor response both to bolus injection of noradrenaline (3.13 nmol kg[-1]) and to spinal pressor nerve stimulation. However, methylene blue (3.13-15.6 micromol kg[-1]) or LY 83583 (4.0-40.0 micromol kg[-1]), failed to affect pressor responses to either NA or pressor nerve stimulation. Hence, there are differences between NOSI and GCI in their abilities to increase vasoconstrictor responses, especially when comparing responses in vitro and in vivo. This suggests that nitric oxide has actions in addition to activation of guanylate cyclase to modulate vasoconstrictor responses, presumably by membrane hyperpolarisation, and that this action may be more important in vivo.

Topics: Acetylcholine; Aminoquinolines; Animals; Aorta; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Male; Mesenteric Arteries; Methylene Blue; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Rats; Rats, Wistar; Vasoconstriction

In the present study, we describe a role for cyclic GMP (cGMP) in the signalling pathway that leads from alpha-adrenergic receptor activation to intracellular Ca2+ mobilization in rat lacrimal acinar cells. The alpha-adrenergic agonist, phenylephrine, stimulates intracellular Ca2+ release which is blocked by inhibitors of guanylate cyclase and cGMP-dependent protein kinase Ia. The membrane-permeable cGMP analogues, dibutyryl-cGMP and 8-bromo-cGMP, potentiate ( approximately 5-fold) the Ca2+ response to submaximal phenylephrine stimulation. In contrast, the same cGMP analogues have no effect on cyclic ADP-ribose-evoked Ca2+ release from permeabilized lacrimal acinar cells. Collectively, these findings suggest that cGMP, via cGMP-dependent protein kinase I alpha , is required for intracellular Ca2+ release following alpha-adrenergic receptor activation in lacrimal acinar cells.

Topics: Acetylcholine; Adenosine Diphosphate Ribose; Aminoquinolines; Animals; Arginine; Calcium; Cells, Cultured; Cyclic ADP-Ribose; Cyclic GMP; Dibutyryl Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Kinetics; Lacrimal Apparatus; Male; Nitric Oxide Synthase; Nitroarginine; Phenylephrine; Rats; Rats, Wistar

The aim of this study was to investigate the involvement of nitric oxide (NO) in the nonadrenergic, noncholinergic (NANC) relaxation of the urinary bladder of the Atlantic cod, Gadus morhua. NADPH diaphorase-reactive nerve cells, presumed to be able to produce NO, were found in the vesicular nerve. The cells occurred alone and in ganglia together with stained and unstained cells. The effect of inhibitors of NO synthesis on the relaxation was examined in vitro in isolated muscle preparations. NG-nitro-L-arginine methyl ester (10(-4) M) and NG-nitro-L-arginine (L-NNA; 10(-4) M) decreased the electrically induced relaxation to 32 +/- 6 (n = 8) and 28 +/- 6% (n = 8) of the control, respectively. L-Arginine (10(-3) M) increased the relaxation to 152 +/- 24% (n = 8), without affecting the inhibition by L-NNA. The beta-adrenoceptor antagonist propranolol together with L-arginine analogues abolished the relaxation in 7 of 11 preparations. The NO donor sodium nitroprusside (NaNP) caused a concentration-dependent relaxation of the bladder, with a maximal effect obtained at 10(-4) M. LY-83583 (10(-5) M), a guanylate cyclase inhibitor, decreased both the electrically (n = 8) and the NaNP (10(-6) M, n = 9)-induced relaxation to 69 +/- 5 and 20 +/- 4% of the control, respectively. Together these findings suggest that NO is involved in the NANC regulation of the motility of the urinary bladder of the Atlantic cod.

Topics: Aminoquinolines; Animals; Arginine; Electric Stimulation; Enzyme Inhibitors; Female; Fishes; Guanylate Cyclase; Histocytochemistry; Male; NADPH Dehydrogenase; Nervous System Physiological Phenomena; Neural Inhibition; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Urinary Bladder

In this study we tested the hypothesis that lactate, independent of changes in pH, can affect skeletal muscle blood flow through arteriolar dilation that may be mediated by guanosine 3',5'-cyclic monophosphate. Isolated, cannulated, and pressurized first-order rat cremaster skeletal muscle arterioles were studied in a chamber containing Krebs-bicarbonate buffer under no-flow conditions. At pH 7.4 and PO2 of 65 Torr, neutralized lactic acid (lactate) and pyruvic acid (pyruvate) caused arteriolar dilation over the 1-10 mM concentration range. This response to lactate was not altered by 10(-5) M indomethacin, 10(-4) M NG-nitro-L-arginine, or removal of the endothelium. However, responses to 1 and 3 mM pyruvate were significantly inhibited by 100% by endothelium removal, and the response to 10 mM pyruvate was inhibited by 71%. The relaxation of endothelium-denuded arterioles to lactate was inhibited by 10 microM methylene blue, 10 microM LY-83583, hypoxia (PO2 7-10 Torr), and diphenyliodonium, an inhibitor of superoxide-producing flavo-protein enzymes. In contrast, arteriolar dilation to the acidification of the Krebs buffer to pH 7.15, produced by increasing the CO2 concentration of the gas mixture from 5 to 10%, was not inhibited by methylene blue. These results are consistent with lactate-induced skeletal muscle arteriolar dilation being dependent on H2O2-mediated activation of vascular smooth muscle guanylate cyclase and independent of endothelium-derived mediators.

Topics: Aminoquinolines; Animals; Arterioles; Cattle; Cyclic GMP; Cyclooxygenase Inhibitors; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Hydrogen Peroxide; Hydrogen-Ion Concentration; Image Processing, Computer-Assisted; In Vitro Techniques; Indomethacin; Lactic Acid; Male; Methylene Blue; Muscle, Skeletal; Nitric Oxide Synthase; Nitroarginine; Placenta; Pulmonary Artery; Rats; Rats, Wistar; Regional Blood Flow; Vasodilation

We have investigated the possible roles of cyclic GMP (cGMP) in initiating or regulating capacitiative Ca2+ entry in rat pancreatic acinar cells. In medium containing 1.8 mM external Ca2+, thapsigargin activated Ca2+ entry and slightly but significantly increased intracellular cGMP concentration. This rise in cGMP levels was prevented by pretreating the cells with the guanylate cyclase inhibitor, LY-83583, or by omitting Ca2+ during stimulation by thapsigargin or methacholine. LY-83583 and NG-nitro-L-arginine (L-NA, an inhibitor of NO synthase) both had a small inhibitory effect on Ca2+ entry when they were added after thapsigargin in Ca2(+)-containing medium, and they reduced by 32 and 48% respectively the thapsigargin-induced capacitative Ca2+ entry when added to the cells during a 20 min preincubation period. However, neither dibutyryl cGMP (Bt2cGMP) nor sodium nitroprusside, an NO mimic, affected either basal intracellular Ca2+ concentration [Ca2+]i or thapsigargin-induced capacitative Ca2+ entry. Further, the inhibitory effects observed after preincubation with LY-83583 or L-NA could not be prevented by preincubation with Bt2cGMP, nor could they be reversed by adding Bt2cGMP, 8-bromo-cGMP or sodium nitroprusside acutely after activation of capacitative Ca2+ entry by thapsigargin. Finally, pretreatment of cells with LY-83583 or L-NA did not affect Ca2+ signalling in response to 1 microM methacholine, including the pattern of [Ca2+]i oscillations. In conclusion, in pancreatic acinar cells, the rise in cellular cGMP levels appears to depend on, rather than cause, the increase in [Ca2+]i with agonist stimulation.

Topics: Aminoquinolines; Animals; Arginine; Biological Transport; Calcium; Calcium Channels; Calcium-Transporting ATPases; Cyclic GMP; Enzyme Inhibitors; Fura-2; Guanylate Cyclase; Methacholine Chloride; Muscarinic Agonists; Nitric Oxide Synthase; Nitroarginine; Pancreas; Rats; Rats, Sprague-Dawley; Terpenes; Thapsigargin

A possible role of the nitric oxide (NO)/cGMP pathway in the regulation of Ca2+ entry into HT29/B6 human colonic epithelial cells was investigated using digital image processing of Fura-2 fluorescence and immunoblotting for nitric oxide synthase (NOS). We tested the hypothesis that Ca2+ store depletion causes increased NOS activity and [NO], which is stimulatory to Ca2+ entry by increasing guanylate cyclase (GC) and [cGMP]. Cells were incubated in 95 mM K(+)-containing solutions to depolarize the cell membrane potential and thereby exclude effects of NO and CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium nitroprusside (SNP, 0.5 microM and 30 microM), a NO donor, only slightly raised intracellular [Ca2+] ([Ca2+]i) in resting cells, but in 100 microM carbachol-stimulated cells the sustained, elevated Ca2+ plateau (reflecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 microM SNP, while 5, 10 or 30 microM SNP either had no effect or were inhibitory. Pretreatment of cells with the NOS inhibitor N-nitro-L-arginine (1 mM) reduced carbachol-stimulated Ca2+ entry, and simultaneous treatment with 0.5 microM (but not 30 microM) SNP restored Ca2+ influx. 8-Br-cGMP (1 mM) had little effect on [Ca2+]i or on rates of Ca2+ or Ba2+ influx into resting cells, but there were large effects on cells in which capacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 microM). The GC inhibitor LY83583 (10 microM) reduced carbachol-stimulated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particulate fraction of cells. The data are consistent with the notion that Ca2+ entry into HT29/B6 cells is regulated by endothelial NOS/NO and GC/cGMP, but effects are most pronounced in store-depleted cells. Thus, NO and cGMP appear to potentiate the action of messengers released from the store during the emptying process, but NO and cGMP have only small effects of their own to open the Ca2+ channel in the plasma membrane. High [SNP] appeared to be inhibitory while low [SNP] was stimulatory, indicating that a precise range of [NO] may be required for effective stimulation of Ca2+ entry.

Topics: Amino Acid Oxidoreductases; Aminoquinolines; Arginine; Barium; Biological Transport; Calcium; Carbachol; Colon; Colonic Neoplasms; Cyclic GMP; Epithelium; Guanylate Cyclase; Humans; Indoles; Intestinal Mucosa; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Second Messenger Systems; Tumor Cells, Cultured

The mechanism of activation of the agonist-stimulated Ca2+ entry pathway in the plasma membrane is not known. To determine the role of nitric-oxide synthase (NOS) and cGMP in the regulation of this pathway, we used intact and streptolysin O (SLO)-permeable pancreatic acini and measured the relationship between Ca2+ release from internal stores, the NO metabolic pathway, generation of cGMP, and activation of Ca2+ entry. We found that agonist- or thapsigargin (Tg)-activated Ca2+ entry is inhibited by L-NA, a specific inhibitor of NOS, and by LY83583, an inhibitor of guanylyl cyclase. Inhibition of Ca2+ entry by inhibition of NOS was reversed by the NO releasing molecules NO2- and sodium nitroprusside (SNP) and by Bt2cGMP. Inhibition of Ca2+ entry by inhibition of guanylyl cyclase was reversed by Bt2cGMP, but not by the NO releasing agents. The use of L-NA-treated cells and different concentrations of SNP revealed that cGMP has a dual effect on Ca2+ entry. Increasing cGMP up to 10-fold above control activated Ca2+ entry. Further increase in cGMP up to 80-fold above control inhibited Ca2+ entry in a concentration-dependent manner. Measurement of cellular cGMP in intact cells showed that carbachol, Tg, and NO2- increased cGMP to similar levels. The effects of carbachol and Tg were inhibited by L-NA and LY83586, whereas the effect of NO2- was inhibited only by LY83583. SLO-permeabilized cells were shown to be agonist-competent in that the agonist induced Ca2+ release from the inositol 1,4,5-trisphosphate (IP3) pool and activated a NO-dependent generation of cGMP. These cells were used to study the regulation of NOS by Ca2+ and by Ca2+ content of the internal stores. When internal stores were maintained loaded with Ca2+, increasing medium [Ca2+] up to 2.5 microM only modestly increased NOS activity. In contrast, the depletion of Ca2+ from internal stores markedly increased NOS activity independent of medium [Ca2+]. Thus, NOS senses both cytosolic [Ca2+]i and internal store Ca2+ load. We propose that activation of Ca2+ entry involves an agonist-mediated Ca2+ release from internal stores which activates a cellular pool of NOS to generate cGMP, which then modulates Ca2+ entry pathway in the plasma membrane. This mechanism can explain the capacitative nature of Ca2+ entry. The biphasic effect of cGMP provides the cells with a negative feedback mechanism which inhibits Ca2+ entry during periods of high cell [Ca2+]i. This could allow oscillatory behavior of Ca2+ entry

Topics: Amino Acid Oxidoreductases; Aminoquinolines; Animals; Arginine; Biological Transport; Calcium; Cyclic GMP; Enzyme Activation; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Pancreas; Rats; Signal Transduction

1. The vasodilator effects of hydralazine in vitro, using the Krebs' perfused human placental lobule was studied. Single placental lobules were bilaterally perfused (maternal and fetal sides 5 mL/min each, 95% O2, 5% CO2, 37 degrees C) and changes in fetal arterial pressure (FAP) and venous outflow (VO) were recorded. 2. Submaximal vasoconstriction was induced by KCl (20-50 mmol/L), which increased basal FAP from 22.8 +/- 1.7 to 91.3 +/- 3.9 mmHg (n = 9, P < 0.001), and decreased VO from 4.1 +/- 0.6 to 0.2 +/- 0.1 mL/min (n = 6, P < 0.01). 3. Hydralazine caused vasodilatation (IC50 1.9 mmol/L, n = 9) and increased VO in the presence of KCl-induced vasoconstriction. 4. Infusion of N omega-nitro-L-arginine (100 mumol/L) to block nitric oxide synthase caused the basal FAP to increase from 30.9 +/- 5.9 to 47.4 +/- 6.7 (n = 6, P < 0.05) and significantly potentiated hydralazine-induced vasodilatation (n = 7, P < 0.05). 5. The soluble guanylate cyclase inhibitor LY 83583 (6-anilino-5,8-quinolinedione) (1 mumol/L) significantly antagonized the vasodilatation produced by hydralazine (n = 5, P < 0.05). 6. Thus, Hydralazine appears to activate guanylate cyclase, leading to increased cyclic GMP in fetal arterial vascular smooth muscle to cause vasorelaxation. No evidence was obtained to suggest that hydralazine exerted its action by either releasing nitric oxide from endothelial cells in the placenta or acting as a nitric oxide donor.

Topics: Adolescent; Adult; Amino Acid Oxidoreductases; Aminoquinolines; Arginine; Blood Pressure; Dose-Response Relationship, Drug; Embryonic and Fetal Development; Female; Guanylate Cyclase; Heart Rate, Fetal; Humans; Hydralazine; In Vitro Techniques; Linear Models; Nitric Oxide Synthase; Nitroarginine; Perfusion; Placenta; Potassium Chloride; Pregnancy; Vasoconstriction; Vasodilation

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

Thapsigargin induced endothelium-dependent relaxation and cGMP production in rat thoracic aorta, and these effects were inhibited by nitric oxide (NO) pathway inhibitors, a calmodulin inhibitor and removal of Ca2+, suggesting that NO is involved in the thapsigargin-induced relaxation. Thapsigargin may deplete Ca2+ stores in the endothelial cells by inhibiting the Ca(2+)-ATPase, a Ca2+ pump, which in turn triggers influx of extracellular Ca2+, leading to activation of constitutive NO synthase and resultant NO generation. The NO thus formed may activate soluble guanylate cyclase to produce cGMP in the vascular smooth muscle.

Topics: Aminoquinolines; Animals; Aorta, Thoracic; Arginine; Calcium; Calcium-Transporting ATPases; Cyclic GMP; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Rats; Rats, Wistar; SRS-A; Terpenes; Thapsigargin

Vasoactive intestinal peptide release and L-[3H]citrulline production were examined in ganglia isolated from the myenteric plexus of guinea-pig intestine. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperizinium stimulated vasoactive intestinal peptide release and L-[3H]citrulline production; the latter was considered an index of nitric oxide production. Both vasoactive intestinal peptide release and L-[3H]citrulline production were abolished by tetrodotoxin, hexamethonium, and the nitric oxide synthase inhibitor, NG-nitro-L-arginine. Inhibition of vasoactive intestinal peptide release by NG-nitro-L-arginine was reversed by L-arginine but not by D-arginine. Exogenous nitric oxide stimulated vasoactive intestinal peptide release whereas exogenous vasoactive intestinal peptide had no effect on L-[3H]citrulline production. The pattern of stimulation by nitric oxide and inhibition by NG-nitro-L-arginine implied that vasoactive intestinal peptide release is facilitated by and may be dependent on nitric oxide production. Consistent with this notion, vasoactive intestinal peptide release in response to either 1,1-dimethyl-4-phenylpiperizinium or nitric oxide was abolished by KT 5823, an inhibitor of cyclic GMP-dependent protein kinase activity and by LY83583, an inhibitor of soluble guanylate cyclase activity. The study provides the first direct evidence of nitric oxide production from enteric ganglia.

Topics: Alkaloids; Aminoquinolines; Animals; Arginine; Carbazoles; Citrulline; Dimethylphenylpiperazinium Iodide; Ganglia, Autonomic; Guinea Pigs; Hexamethonium; Hexamethonium Compounds; Ileum; In Vitro Techniques; Indoles; Kinetics; Muscle, Smooth; Myenteric Plexus; Nitric Oxide; Nitroarginine; Protein Kinase Inhibitors; SRS-A; Tetrodotoxin; Tritium; Vasoactive Intestinal Peptide

Small arteries of the mesenteric arcade from Wistar rats display rhythmic oscillations superimposed on the tonic contractile response when exposed to submaximal doses of noradrenaline. We have previously shown that mechanical removal of the endothelium abolishes these oscillations. In the present study different methods to eliminate or modify the influence of the endothelium were used in order to further characterize the mechanisms behind rhythmic contractions in these vessels. Endothelium was removed either mechanically or chemically by perfusing the vessels with 0.3% CHAPS. The absence of functional endothelium enhanced noradrenaline sensitivity and simultaneously abolished oscillations in tension and membrane potential, but did not affect resting membrane potential. The rhythmic activity was also reduced or abolished by exposure to haemoglobin, methylene blue, LY83583 or L-NNA. Indomethacin and propranolol were without effect. Sodium nitroprusside or the permeant analogue of cyclic GMP, 8-bromo cyclic GMP, restored rhythmic activity in precontracted endothelium-denuded vessels. The data suggest that release of nitric oxide from the endothelium, and subsequent generation of cyclic GMP in the smooth muscle, activates oscillations in membrane potential and tension; the oscillator itself appears to be located within the smooth muscle cells.

Topics: Aminoquinolines; Animals; Arginine; Arteries; Electrophysiology; Endothelium, Vascular; Hemoglobins; In Vitro Techniques; Male; Methylene Blue; Nitroarginine; Periodicity; Rats; Rats, Wistar; Splanchnic Circulation; Vasoconstriction

The mechanism of action of vasoactive intestinal peptide (VIP) was examined in isolated gastric and taenia coli muscle cells and compared with that of nitric oxide (NO), sodium nitroprusside (SNP), and isoproterenol. In gastric muscle cells, VIP stimulated NO production, increased adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels, and induced relaxation in a concentration-dependent fashion. The NO synthase inhibitor NG-nitro-L-arginine abolished NO and cGMP production and partly inhibited relaxation. The soluble guanylate cyclase inhibitor LY 83583 abolished cGMP production and partly inhibited relaxation. (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], a preferential inhibitor of cAMP-dependent protein kinase (cAK), and KT5823, a preferential inhibitor of cGMP-dependent protein kinase (cGK), partly inhibited relaxation separately and abolished relaxation in combination. The pattern implied that VIP induced relaxation by activation of cAK and by NO-mediated stimulation of cGMP and activation of cGK. In taenia coli muscle cells, VIP did not increase NO production or cGMP levels: relaxation was accompanied by an increase in cAMP and was partly inhibited by (R)-p-cAMPS and KT5823 and abolished by a combination of both inhibitors. Isoproterenol increased only cAMP levels in both cell types, which induced relaxation by activating cAK at low concentrations of agonist and both cAK and cGK at high concentrations in a pattern identical to that observed with VIP in taenia coli muscle cells. SNP and NO increased only cGMP levels in both cell types, which induced relaxation by activating cGK only. We conclude that cAK and cGK can be activated separately and mediate relaxation independently.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aminoquinolines; Animals; Arginine; Cell Separation; Colon; Cyclic AMP; Cyclic GMP; Digestive System; Digestive System Physiological Phenomena; Enzyme Activation; Gastric Fundus; Guinea Pigs; Isoproterenol; Muscle Relaxation; Muscles; Nitrates; Nitric Acid; Nitroarginine; Nitroprusside; Protein Kinase C; SRS-A; Vasoactive Intestinal Peptide

Hypoxia causes complex changes in vascular tone of isolated blood vessels. This study was performed in rings with and without endothelium of rat abdominal aortas and canine coronary arteries suspended in organ chambers for isometric tension recording. In both aortic and coronary rings with endothelium precontracted with a half-maximal concentration of phenylephrine or prostaglandin F2 alpha, respectively, hypoxia induced transient relaxations (20 +/- 2 and 15 +/- 3%, respectively); removal of the endothelium prevented the response in aortas, but not coronary arteries. The transient hypoxic relaxation was followed in both preparations by endothelium-dependent contractions (EDCs). Hypoxic relaxations were prevented by indomethacin (10 microM) in canine arteries, but not in rat aortas. The inhibitor of nitric oxide (NO) synthase, N omega-nitro-L-arginine (30 microM), inhibited hypoxic relaxations in intact rat aortas, but left those in coronary arteries unchanged. Similar results were obtained with methylene blue and LY 83583. In preparations without endothelium, sodium nitroprusside (30 nM) elicited a reappearance of hypoxic relaxations in the rat but not the dog coronary artery. Thus, hypoxic relaxation is mediated by a prostaglandin in the dog coronary artery, but by endothelium-derived NO in the rat aorta. As the response was dependent on the level of contraction, this suggests that the release or action of NO decreases with increasing tone.

Topics: Aminoquinolines; Animals; Aorta, Abdominal; Arginine; Coronary Vessels; Dinoprost; Dogs; Endothelium, Vascular; In Vitro Techniques; Methylene Blue; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Norepinephrine; Oxygen; Rats; SRS-A; Vascular Resistance; Vasodilation

Our laboratory has previously described in isolated 1- to 4-mm calf pulmonary arteries, an endothelium-independent contraction to hypoxia that appears to involve the removal of a H2O2-elicited guanosine 3',5'-cyclic monophosphate (cGMP)-mediated relaxation. In this study, we examined the effects of changes in O2 tension (PO2) on isolated endothelium-intact and endothelium-denuded calf pulmonary resistance arteries of approximately 200 microns in diameter. Resistance arteries precontracted with U46619 were found to undergo a contraction when exposed to a PO2 of 24-27 Torr (hypoxia) from a Po2 of 150 Torr (O2 atmosphere). This contraction was significantly larger in endothelium-intact than endothelium-removed arteries. In the intact artery, 30 microM nitro-L-arginine (NLA), an inhibitor of the biosynthesis of nitric oxide-like activators of guanylate cyclase, increased tone under O2 atmosphere and reduced the contraction to hypoxia to the level observed in the endothelium-removed artery. Reoxygenation caused a relaxation, which was not dependent on the endothelium or inhibited by NLA. The inhibitor of guanylate cyclase activation, LY83583 (10 microM), increased tone under O2 atmosphere, eliminated the contraction to hypoxia, and inhibited the relaxation to reoxygenation, whereas indomethacin (10 microM) did not alter these responses. Thus modulation of a cGMP mechanism, not involving the endothelium or metabolism of arginine, is a primary mediator of responses to changes in O2 tension, and the endothelium appears to cause an enhancement of the contraction to hypoxia via suppression by hypoxia of the tonic generation of an arginine-derived relaxing factor.

Topics: Acetylcholine; Aminoquinolines; Animals; Arginine; Cattle; Cyclic GMP; Endothelium, Vascular; Hypoxia; Indomethacin; Nitroarginine; Oxygen; Partial Pressure; Prostaglandin Antagonists; Pulmonary Artery; Vascular Resistance; Vasoconstriction; Vasodilation

We have demonstrated previously that in response to hypoxia, isolated rat pulmonary arteries show an initial endothelium-dependent relaxation followed by an endothelium-independent transient contraction. In the presence of increased extracellular Ca2+, both of these responses were enhanced in endothelium-intact arteries. Nitro-L-arginine, a blocker of the biosynthesis of endothelium-derived relaxing factor (EDRF), abolished the initial endothelium-dependent relaxation and Ca(2+)-induced enhancement of hypoxic contraction in endothelium-intact arteries but did not alter responses in endothelium-denuded vessels. Inhibition of prostaglandin formation with indomethacin had no effect on the hypoxia-elicited responses. Preincubation with LY 83583, an inhibitor of guanylate cyclase activation, abolished the initial hypoxia-elicited relaxation and subsequent contraction. M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor, decreased tone under O2 but not under N2, causing an apparent enhancement of the contraction to hypoxia. Thus the modulation of hypoxic responses by the endothelium is dependent on changes in EDRF production, and a decrease in smooth muscle cGMP not involving an EDRF mechanism appears to mediate the endothelium-independent hypoxic contraction observed in the isolated rat pulmonary artery.

Topics: Aminoquinolines; Animals; Arginine; Cyclic GMP; Hypoxia; In Vitro Techniques; Indomethacin; Male; Nitroarginine; Pulmonary Artery; Purinones; Rats; Rats, Inbred Strains

1. The aim of this investigation was to study the relationship between contractile responsiveness, activation of the L-arginine pathway and tissue levels of guanosine 3':5'cyclic monophosphate (cylic GMP) in aortic rings removed from rats 4 h after intraperitoneal administration of bacterial endotoxin (E. coli. lipopolysaccharide, LPS, 20 mg kg-1). 2. LPS-treatment resulted in a reduction of the sensitivity and maximal contractile response to noradrenaline (NA). 3. Depression of the maximal contractile response was restored to control by 6-anilo-5,8-quinolinedione (LY 83583, 10 microM), which prevents activation of soluble guanylate cyclase. 4. Cyclic GMP levels in tissue from LPS-treated rats were 2 fold greater than cyclic GMP levels detected in tissue from control (saline-treated) rats. The LPS-induced increase in cyclic GMP content was observed both in the presence and absence of functional endothelium. 5. Addition of L-arginine 1 mM) to maximally contracted aortic rings produced significantly relaxation of rings from LPS-treated rats but not rings from control animals. In the LPS-treated group, addition of L-arginine was also associated with a significant increase in cyclic GMP content. L-Arginine had no effect on the cyclic GMP content of control rings. D-Arginine (1 mM) was without effect. 6. In rings from LPS-treated rats, NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), an inhibitor of nitric oxide (NO) production, increased the contractile response to NA and prevented the LPS-induced increase in cyclic GMP content. In control rings, L-NAME increased the NA sensitivity only when the endothelium remained intact and reduced the cyclic GMP content of these rings to that of control endothelium-denuded rings. 7. These results demonstrate that LPS-induced hyporeactivity to NA occurs secondarily to activation of the L-arginine pathway and subsequent activation of soluble guanylate cyclase in vascular tissue. In addition they suggest that LPS induces the production of an NO-like relaxing factor in non-endothelial cells.

Topics: Aminoquinolines; Animals; Arginine; Cyclic GMP; Endotoxins; Enzyme Activation; Guanylate Cyclase; In Vitro Techniques; Lipopolysaccharides; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Rats; Rats, Inbred Strains; SRS-A

Release of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase co-incubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 +/- 0.5-fold (from 31 +/- 9 to 153 +/- 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 +/- 15% (with 2 microM free calcium; EC50 0.3 microM). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 microM) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation NG-nitro-L-arginine (1 mM) and by LY 83583 (1 microM), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells.

Topics: Aminoquinolines; Animals; Arginine; Calcium; Calcium Chloride; Chromatography, Gas; Cytosol; Endothelium, Vascular; Guanylate Cyclase; In Vitro Techniques; NADP; Nitric Oxide; Nitroarginine; Swine