cyclic-gmp and chelerythrine

A reduction of the nitric oxide (NO) action in vascular smooth muscle cells (VSMC) could play a role in the vascular damage induced by the glycaemic excursions occurring in diabetic patients; in this study, we aimed to clarify whether a short-term incubation of cultured VSMC with high glucose reduces the NO ability to increase cGMP and the cGMP ability to phosphorylate VASP at Ser-239. We observed that a 180 min incubation of rat VSMC with 25 mmol/L glucose does not impair the NO-induced cGMP increase but reduces VASP phosphorylation in response to both NO and cGMP with a mechanism blunted by antioxidants. We further demonstrated that high glucose increases radical oxygen species (ROS) production and that this phenomenon is prevented by the PKC inhibitor chelerythrine and the NADPH oxidase inhibitor apocynin. The following sequence of events is supported by these results: (i) in VSMC high glucose activates PKC; (ii) PKC activates NADPH oxidase; (iii) NADPH oxidase induces oxidative stress; (iv) ROS impair the signalling of cGMP, which is involved in the antiatherogenic actions of NO. Thus, high glucose, via oxidative stress, can reduce the cardiovascular protection conferred by the NO/cGMP pathway via phosphorylation of the cytoskeleton protein VASP in VSMC.

Topics: Acetophenones; Animals; Antioxidants; Benzophenanthridines; Cell Adhesion Molecules; Cells, Cultured; Cyclic GMP; Glucose; Male; Microfilament Proteins; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; Nitric Oxide; Oxidative Stress; Phosphoproteins; Phosphorylation; Protein Kinase C; Rats; Rats, Zucker; Reactive Oxygen Species; Serine

Vasomotion is linked to the rapid oscillations of intracellular calcium levels. In rat pulmonary artery, this activity can manifest as a slow periodic on-off pattern, the timing of which depends on the type and intensity of pharmacological stimuli employed. In this study, we have sought to characterize a slow-wave vasomotor activity pattern induced in isolated arterial ring preparations by simultaneous exposure to the α(1) -adrenoceptor agonist phenylephrine (1-10 nm) and the L channel agonist S(-)-Bay K 8644 (3-20 nm). Treated tissues responded with a stable on-off pattern of vasomotion persisting for >5 h at 5-6 cycles/h. In intact rings, this response was suppressed by methacholine and restored or enhanced by N(ω) -nitro-l-arginine methyl ester. Analogous inhibitory effects were obtained with high Mg(2+) , 8-Br-cGMP (but not 8-Br-cAMP), riluzole, ryanodine, chelerythrine, and fasudil. Pinacidil (30 nm) increased off-cycle length without change in slow-wave amplitude. Conversely, tetraethylammonium (1.0-3.0 mm) augmented the latter without affecting periodicity. Carbenoxolone (10 μm) abolished slow-wave activity, while raising basal tone and inducing random phasic activity. In endothelium-denuded rings, the threshold of agonist-induced slow-wave vasomotion was lowered and a similar inhibitory effect obtained with carbenoxolone. In conclusion, the slow-wave pattern of vasomotion described here is (i) subject to inhibitory modulation by endothelial NO and an array of voltage-gated and leak K conductances yet to be fully characterized; (ii) dependent on Ca(2+) from both extracellular and sarcoendoplasmatic sources; (iii) controlled by kinase (Rho and PKC)-mediated regulation of myosin light chain phosphatase; and (iv) synchronized via intermyocyte gap junctions.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; 8-Bromo Cyclic Adenosine Monophosphate; Adrenergic alpha-1 Receptor Agonists; Animals; Benzophenanthridines; Calcium Channel Agonists; Calcium Signaling; Carbenoxolone; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Endothelium, Vascular; Gap Junctions; In Vitro Techniques; Male; Methacholine Chloride; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Phenylephrine; Pinacidil; Potassium Channels; Protein Kinase C; Protein Kinase Inhibitors; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Riluzole; Ryanodine; Tetraethylammonium; Vasomotor System

Nitric oxide (NO) is a biologically active molecule which has been reported to protect the heart against ischemia and reperfusion injury in different species. This study aimed to test the hypothesis that nitric oxide may induce the expression of heat shock protein 72 (HSP72) which may protect the heart against ischemia.. Rabbits were given intravenous saline or S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, or Zaprinast, an inhibitor of cyclic guanosine monophosphate (GMP)-phosphodiesterase, which may increase myocardial cyclic GMP content. Twenty-four hours later, the rabbits were either sampled to measure HSP72, or induced with a 30-minute coronary occlusion followed by a 120-minute reperfusion, and then the infarct size was measured. Meanwhile, chelerythrine (CHE, an inhibitor of protein kinase C) was given intravenously 5 minutes before SNAP injection and the effect on HSP72 expression and infarct size was determined.. Twenty-four hours after pretreatment, immunoblotting showed HSP72 expression increased in the SNAP group compared with control groups, and this was blocked by CHE. Myocardial infarct size in the SNAP group was smaller than that of the control group ((32.4 +/- 5.8)% vs (51.1 +/- 4.7)%, P < 0.05). Pretreated with CHE abolished the infarct size-limiting effect of SNAP ((46.0 +/- 5.1)%). Pretreatment with Zaprinast neither induced HSP72 expression nor reduced infarct size ((55.4 +/- 5.4)%).. NO induced HSP72 expression and a delayed protection to the heart via the activities of protein kinase C by a cyclic GMP-independent pathway.

Topics: Animals; Benzophenanthridines; Cyclic GMP; Hemodynamics; HSP72 Heat-Shock Proteins; Male; Myocardial Infarction; Myocardial Ischemia; Nitric Oxide; Nitric Oxide Donors; Phosphodiesterase Inhibitors; Protein Kinase C; Purinones; Rabbits; S-Nitroso-N-Acetylpenicillamine

Carperitide, a synthetic alpha-human atrial natriuretic peptide (ANP) is a newly developed drug for the treatment of heart failure. However, effects of carperitide on susceptibility to ischemia reperfusion injury are left to be determined. Isolated rat hearts were subjected to Langendorff perfusion. Six hearts received 0.1 microM of carperitide for 10 min, 6 hearts received 1 mM of a NO synthetase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) for 5 min before the infusion of carperitide, 6 hearts received 0.02 microM of a PKC synthetase inhibitor chelerythrine chloride for 5 min before the infusion of carperitide, 6 hearts received 100 microM of a selective mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-dehydroxydecanoate (5HD) before the infusion of carperitide, 6 hearts received 10 microM of a soluble guanylate cyclase inhibitor methylene blue for 5 min before the infusion of carperitide, and 6 hearts served as a control with no drug infusion. All hearts were then subjected to 20 min of global ischemia followed by 120 min of reperfusion. Left ventricular pressures and coronary flow were measured throughout the experiment and infarct size was detected at the end of experiment. Both plasma and tissue cGMP levels were also determined. The results showed: (1) Carperitide significantly reduced infarct size compared to control (26.1 +/- 2.8 vs. 42.7 +/- 2.3%, carperitide vs. control, p < 0.05). This effect was reversed by L-NAME, chelerythrine and 5HD, but not methylene blue. (2) Plasma cGMP levels were increased in carperitide-treated group. This effect was reversed by L-NAME (0.16 +/- 0.03 vs. 1.04 +/- 0.09* vs. 0.28 +/- 0.02 nmol/L, control vs. carperitide vs. L-NAME, *p < 0.01 vs. control). We conclude that preischemic infusion of carperitide exerts cardioprotective effects possibly through NO-PKC dependent pathway followed by mitochondrial KATP channel activation.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Atrial Natriuretic Factor; Benzophenanthridines; Cyclic GMP; Enzyme Inhibitors; Heart; Ischemia; Ischemic Preconditioning, Myocardial; Methylene Blue; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phenanthridines; Potassium Channel Blockers; Potassium Channels; Protein Kinase C; Rats; Reperfusion; Time Factors; Tissue Distribution

The aim of this study was to investigate the role of nitric oxide (NO) in a cellular model of early preconditioning (PC) in cultured neonatal rat ventricular myocytes. Cardiomyocytes "preconditioned" with 90 min of stimulated ischemia (SI) followed by 30 min reoxygenation in normal culture conditions were protected against subsequent 6 h of SI. PC was blocked by N(G)-monomethyl-L-arginine monoacetate but not by dexamethasone pretreatment. Inducible nitric oxide synthase (NOS) protein expression was not detected during PC ischemia. Pretreatment (90 min) with the NO donor S-nitroso-N-acetyl-L,L-penicillamine (SNAP) mimicked PC, resulting in significant protection. SNAP-triggered protection was completely abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but was unaffected by chelerythrine or the presence of glibenclamide and 5-hydroxydecanoate. With the use of RIA, SNAP treatment increased cGMP levels, which were blocked by ODQ. Hence, NO is implicated as a trigger in this model of early PC via activation of a constitutive NOS isoform. After exposure to SNAP, the mechanism of cardioprotection is cGMP dependent but independent of protein kinase C or ATP-sensitive K(+) channels. This differs from the proposed mechanism of NO-induced cardioprotection in late PC.

Topics: Alkaloids; Animals; Animals, Newborn; Anti-Infective Agents; Benzophenanthridines; Cells, Cultured; Cyclic GMP; Dexamethasone; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Glucocorticoids; Glyburide; Heart Ventricles; Hypoglycemic Agents; Ischemic Preconditioning; Muscle Fibers, Skeletal; Myocardial Ischemia; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; omega-N-Methylarginine; Oxadiazoles; Penicillamine; Phenanthridines; Potassium Channels; Protein Kinase C; Quinoxalines; Rats; Rats, Sprague-Dawley

An elastin-derived peptide with an average molecular mass of 25 kDa was shown to induce monocyte chemotaxis at the optimal concentration of 10(-1) micrograms/ml. Homologous deactivation test showed that monocytes exposed to the elastin-derived peptide at 10(-1) micrograms/ml lost their chemotactic responsiveness when reexposed to the same stimulus. In conjunction with chemotactic response to the elastin-derived peptide, intracellular guanosine 3', 5'-monophosphate (cGMP) levels were enhanced but intracellular adenosine 3', 5'-monophosphate (cAMP) levels were not. The monocyte migration induced by the elastin-derived peptide was inhibited by cGMP dependent protein kinase (PKG) inhibitor, but not by cAMP dependent protein kinase inhibitor and protein kinase C inhibitor. These results suggest that the elastin-derived peptide induces monocyte chemotaxis by increasing the level of cGMP, followed by activating PKG.

Topics: Alkaloids; Animals; Benzophenanthridines; Carbazoles; Cattle; Chemotaxis, Leukocyte; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Drug Synergism; Elastin; Humans; Indoles; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Phenanthridines; Time Factors

We have previously reported that the serine protease plasmin generated during contact activation of human plasma triggers biosynthesis of leukotrienes (LTs) in human peripheral monocytes (PMs), but not in polymorphonuclear neutrophils (PMNs). We now show that purified plasmin acts as a potent chemoattractant on human monocytes, but not on PMNs. Human plasmin or plasminogen activated with urokinase, but not active site-blocked plasmin or plasminogen, elicited monocyte migration across polycarbonate membranes. Similarly, stimulation of monocytes with plasmin, but not with active site-blocked plasmin or plasminogen, induced actin polymerization. As assessed by checkerboard analysis, the plasmin-mediated monocyte locomotion was a true chemotaxis. The plasmin-induced chemotactic response was inhibited by the lysine analog trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), which prevents binding of plasmin/ogen to the appropriate membrane binding sites. In addition, active site-blocked plasmin inhibited monocyte migration triggered by active plasmin. Further, plasmin-induced monocyte chemotaxis was inhibited by pertussis toxin (PTX) and 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG) and chelerythrine, two structurally unrelated inhibitors of protein kinase C (PKC). Plasmin, but not active site-blocked plasmin or plasminogen, triggered formation of cyclic guanosine monophosphate (cGMP) in monocytes. LY83583, an inhibitor of soluble guanylyl cyclase, inhibited both plasmin-induced cGMP formation and the chemotactic response. The latter effect could be antagonized by 8-bromo-cGMP. In addition, KT5823 and (Rp)-8-(p-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate [(Rp)-8-pCPT-cGMPs], two structurally unrelated inhibitors of cGMP-dependent protein kinase, inhibited plasmin-mediated monocyte chemotaxis. Thus, beyond being a stimulus for lipid mediator release, plasmin is a potent and specific chemoattractant for human monocytes acting via a cGMP-dependent mechanism. Therefore, plasmin represents a proinflammatory activator for human monocytes.

Topics: Alkaloids; Aminoquinolines; Benzophenanthridines; Binding Sites; Carbazoles; Chemotactic Factors; Chemotaxis; Cyclic GMP; Enzyme Inhibitors; Fibrinolysin; Glyceryl Ethers; Guanylate Cyclase; Humans; Indoles; Lysine; Male; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Organ Specificity; Pertussis Toxin; Phenanthridines; Plasminogen; Protein Kinase C; Signal Transduction; Thionucleotides; Tranexamic Acid; Urokinase-Type Plasminogen Activator; Virulence Factors, Bordetella

The role of Ca2+ and protein kinase C (PKC) activity in the release of immunoreactive endothelin-1 (ET-1) from cultured porcine aortic endothelial cells of first or second passage has been studied. ET-1 accumulation within cells and secretion into cell-conditioned medium over 3 and/or 5 hr was measured. Confluent cells incubated in medium containing 1.8 mM Ca2+ (control condition) accumulated and released ET-1 in a time-dependent way. Reducing intracellular free Ca2+ concentration ([Ca2+]i) by adding the Ca2+ entry blockers NiCl2 (0.5 mM) and amiloride (1 mM) or the Ca2+ chelator EGTA (5 mM) to the incubation medium reduced ET-1 secretion to between 50 and 30% of controls, respectively (P < 0.01). To determine the effect of high [Ca2+]i on ET-1 release, cells were incubated with thapsigargin (10-1000 nM) or Ca2+ ionophore A23187 (1 microM) which raised [Ca2+]i progressively from 190 nM (control) to > 1 microM. Both agents reduced ET-1 secretion in a concentration-dependent manner to between 50 and 20% of controls (P < 0.01). Intracellular levels of ET-1 were also reduced at both low and high [Ca2+]i (P < 0.01). In the presence of the PKC inhibitors chelerythrine (50 microM) and H-7 (60 microM), basal ET-1 secretion was reduced to below 20% of controls (P < 0.01). The PKC activator phorbol 12-myristate 13-acetate (0.4 microM) stimulated ET-1 release 1.4-fold (P < 0.01) and its effect was abolished by EGTA (5 mM). Increased [Ca2+]i stimulated the production and release of cyclic guanosine-3',5'-monophosphate, but basal ET-1 secretion rates correlated poorly with nucleotide levels. These data indicate that: (i) at resting [Ca2+]i concentrations, ET-1 release is close to maximal and is reduced at lower and higher concentrations, resulting in a bell-shaped relationship between [Ca2+]i and ET-1 release; and (ii) basal ET-1 release is largely determined by Ca(2+)-dependent PKC activity.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Benzophenanthridines; Biological Transport; Calcium; Cells, Cultured; Cyclic GMP; Endothelins; Endothelium, Vascular; Isoquinolines; Phenanthridines; Piperazines; Protein Kinase C; Swine; Tetradecanoylphorbol Acetate

The cellular regulation mechanism of Na-K-Cl cotransport was studied in dispersed acinar cells of the guinea pig nasal gland by a microfluorimetric imaging method using the Na(+)-sensitive dye sodium-binding benzofuran isophthalate. Addition of 1 micron acetylcholine (ACh) induced an immediate increase in intracellular Na+ concentration ([Na+]i) by 36.7 +/- 9.9 mM, which was almost completely abolished by the addition of atropine. The increased [Na+]i after cholinergic stimulation was due to the external (Cl-)-dependent cotransport system (about 80% of the total Na+ influx) and the dimethyl amiloride-sensitive (Na+)-H+ exchange system (of about 20%). The ACh-induced increase in [Na+]i was dependent on extracellular Ca2+ and was prevented by pretreatment with 8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate or O-O'-bis(2-aminophenyl)ethyleneglycol-N, N, N', N'-tetraacetic acid tetraacetoxymethylester. Addition of 1 microns ionomycin mimicked the ACh-induced increase in [Na+]i which was dependent on external Cl-. Moreover, both a calmodulin antagonist trifluoperazine and a myosin light chain kinase inhibitor ML-7 reduced the ACh-induced response in [Na+]i. However, the following treatment did not affect the basal [Na+]i nor the ACh-induced increase in [Na+]i: (i) addition of dibutyryl cAMP, 8-Br-cGMP, or phorbol 12-myristate 13-acetate, (ii) pretreatment of protein kinase inhibitors, H-89, H-8, H-7 or chelerythrine, (iii) prevention of cytosolic Cl- efflux by the addition of diphenylamine-2-carboxylic acid or, (iv) prevention of cytosolic K+ efflux by the addition of charybdotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Biological Transport; Bucladesine; Bumetanide; Calcium; Calmodulin; Carrier Proteins; Charybdotoxin; Cholinergic Fibers; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Exocrine Glands; Furosemide; Guinea Pigs; Ionomycin; Ionophores; Isoquinolines; Nasal Cavity; Phenanthridines; Protein Kinase Inhibitors; Protein Kinases; Second Messenger Systems; Signal Transduction; Sodium; Sodium-Potassium-Chloride Symporters; Sulfonamides; Tetradecanoylphorbol Acetate; Trifluoperazine; Vasoactive Intestinal Peptide