nitroarginine and calmidazolium

Shear stress or vasocontriction causes endothelial nitric oxide (NO) release resulting in the regulation of vascular smooth muscle tone in small resistance arteries. Generation of NO is inhibited by nitric oxide synthase (NOS) inhibitors. In this study, we investigated the effect of residual NO, released even in the presence of NOS inhibitors, on the membrane depolarization and phenylephrine-induced contractions of smooth muscle. For this purpose, we used hydroxocobalamin (HC), an NO scavenger, in the presence of NOS inhibitiors, Nω-nitro- L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in mesenteric arteries isolated from rats. Phenylephrine (0,01-10 μM), an α

Topics: Adrenergic alpha-1 Receptor Agonists; Animals; Benzoates; Free Radical Scavengers; Hydroxocobalamin; Imidazoles; Male; Membrane Potentials; Mesenteric Arteries; Muscle Contraction; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Phenylephrine; Protein Kinase Inhibitors; Rats, Wistar

Carbon monoxide (CO) and nitric oxide (NO) can be involved in the regulation of cerebral circulation. Inhibition of production of either one of these gaseous intercellular messengers inhibits newborn pig cerebral arteriolar dilation to the excitatory amino acid glutamate. Glutamate can increase NO production. Therefore, the present study tests the hypothesis that NO, which is increased by glutamate, stimulates the production of CO by cerebral microvessels. Experiments used freshly isolated cerebral microvessels from piglets that express only heme oxygenase-2 (HO-2). CO production was measured by gas chromatography-mass spectrometry. Although inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-l-arginine (l-NNA) did not alter basal HO-2 catalytic activity or CO production, l-NNA blocked glutamate stimulation of HO-2 activity and CO production. Furthermore, the NO donor sodium nitroprusside mimicked the actions of glutamate on HO-2 and CO production. The action of NO appears to be via cGMP because 8-bromo-cGMP mimics and 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) blocks glutamate stimulation of CO production and HO-2 catalytic activity. Inhibitors of neither casein kinase nor phosphotidylinositol 3-kinase altered HO-2 catalytic activity. Conversely, inhibition of calmodulin with calmidazolium chloride blocked glutamate stimulation of CO production and reduced HO-2 catalytic activity. These data suggest that glutamate may activate NOS producing NO that leads to CO synthesis via a cGMP-dependent elevation of HO-2 catalytic activity. These results are consistent with the findings in vivo that either HO or NOS inhibition blocks cerebrovascular dilation to glutamate in piglets.

Topics: Animals; Animals, Newborn; Carbon Monoxide; Cerebrovascular Circulation; Enzyme Inhibitors; Guanylate Cyclase; Imidazoles; Microcirculation; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Oxadiazoles; Quinoxalines; Swine

Shear stress and tyrosine phosphatase inhibitors have been shown to activate the endothelial NO synthase (eNOS) in a Ca2+/calmodulin-independent manner. We report here that isometric contraction of rabbit aorta activates eNOS by a pharmacologically identical pathway. Endothelium-intact aortic rings were precontracted under isometric conditions up to 60% of the maximal phenylephrine-induced tone. The NO synthase inhibitor NGnitro-L-arginine (L-NA) and the soluble guanylyl cyclase inhibitor NS 2028 induced an additional contraction, the amplitude of which depended on the level of precontraction. The maximal production of NO by isometrically contracted aortic rings (as estimated by the increase in cGMP in detector smooth muscle cells in a superfusion bioassay) was observed during the initial phase of isometric contraction and was greater than that detected following the application of acetylcholine. The supplementary L-NA-induced increase in vascular tone was inhibited by the nonselective kinase inhibitor staurosporine and the tyrosine kinase inhibitors erbstatin A and herbimycin A. Another tyrosine kinase inhibitor, genistein, the calmodulin antagonist calmidazolium, and the selective protein kinase C inhibitor, Ro 31-8220, had no effect. Coincident with the enhanced NO formation during isometric contraction was an increase in the tyrosine phosphorylation of endothelial proteins, which also correlated with the level of precontraction. Thus, isometric contraction activates eNOS via a Ca2+-independent, tyrosine kinase inhibitor-sensitive pathway and, like shear stress, seems to be an independent determinant of mechanically induced NO formation.

Topics: 1-Methyl-3-isobutylxanthine; Acetylcholine; Animals; Aorta, Thoracic; Benzoquinones; Calcium; Calmodulin; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Female; Genistein; Hydroquinones; Imidazoles; In Vitro Techniques; Indoles; Isometric Contraction; Lactams, Macrocyclic; Male; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Oxadiazoles; Oxazines; Quinones; Rabbits; Rifabutin; Staurosporine

The present study was aimed to determine the effect of calmodulin inhibitors on the relaxant response of isolated dog and monkey cerebral arteries to vasodilator nerve stimulation, which is hypothesized to be mediated by nitric oxide (NO) from nerve endings. The relaxations caused by nerve stimulation by electrical pulses in endothelium-denuded arteries were attenuated by treatment with calmidazolium and W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride) and were abolished by NG-nitro-L-arginine, an inhibitor of nitric oxide synthase, and tetrodotoxin. The calmodulin inhibitors also attenuated the relaxations caused by nicotine and substance P, which were endothelium-independent and -dependent, respectively, but did not influence the relaxant response to NO. It is concluded that calmodulin is required for activation of the NO synthase present in the vasodilator nerve as well as that in the endothelium.

Topics: Animals; Arginine; Calmodulin; Cerebral Arteries; Dogs; Electric Stimulation; Endothelium, Vascular; Female; Imidazoles; In Vitro Techniques; Isometric Contraction; Macaca; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nicotine; Nitric Oxide; Nitroarginine; Substance P; Sulfonamides

1. Endothelin-3 (ET-3) at concentrations below those which caused contraction (30 nM) elicited endothelium-dependent relaxation followed by rebound contraction in rat isolated thoracic aorta. 2. Endothelin-1 also relaxed the rat aorta with a similar potency. 3. The nitric oxide (NO) synthase inhibitor, NG-nitro L-arginine, the radical scavenger, haemoglobin and the soluble guanylate cyclase inhibitor, methylene blue, each inhibited the ET-3-induced relaxation. 4. The calmodulin inhibitor, calmidazolium, considerably attenuated the relaxation caused by ET-3 without affecting that to nitroprusside. 5. Concentrations of ET-3 that were necessary to induce the relaxation also caused concentration-dependent elevation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels. 6. NG-nitro L-arginine, haemoglobin, methylene blue, calmidazolium and removal of the endothelium completely abolished ET-3-stimulated cyclic GMP production. 7. These results suggest that ET-3 triggers NO formation possibly via ETB receptors on the endothelium to activate soluble guanylate cyclase, which in turn stimulates cyclic GMP production and smooth muscle relaxation. The enzyme contributing to the NO formation may be of the calcium/calmodulin-dependent, constitutive type.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Arginine; Calmodulin; Cyclic GMP; Endothelins; Hemoglobins; Imidazoles; In Vitro Techniques; Male; Methylene Blue; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Rats; Rats, Wistar

1. The role of calmodulin in endothelium-dependent relaxations in the canine coronary artery, was investigated by use of the inhibitor of calmodulin, calmidazolium. 2. The endothelium-dependent relaxations to adenosine diphosphate (ADP) and nebivolol, a beta-adrenoceptor antagonist, in control solution, and to bradykinin in high potassium solution (to inhibit endothelium-dependent hyperpolarization), were abolished by nitro-L-arginine (30 microM), an inhibitor of nitro oxide-synthase. Calmidazolium (10 microM) did not inhibit these relaxations. 3. Calmidazolium did not affect the endothelium-independent relaxations to SIN-1, an exogenous donor of nitric oxide (NO). 4. The relaxations to bradykinin and to the calcium ionophore A23187 in control solution were inhibited to a small extent by calmidazolium (10 microM). 5. Bradykinin and A23187 induced relaxations in the presence of nitro-L-arginine (30 microM) that were abolished by calmidazolium (10 microM) but not affected by glibenclamide (10 microM), an inhibitor of ATP-sensitive K+ channels. 6. The endothelium-independent relaxations to lemakalim, an ATP-sensitive K+ channel opener, were not affected by calmidazolium (10 microM) but were inhibited by glibenclamide (10 microM). 7. These results suggest that calmidazolium does not inhibit the endothelium-dependent relaxations due to endothelium-derived NO in the canine coronary artery but inhibits either the production of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells or its effects on vascular smooth muscle cells. Furthermore these results suggest that EDHF contributes to endothelium-dependent relaxations in the canine coronary artery.

Topics: Adenosine Diphosphate; Adrenergic beta-Antagonists; Animals; Arginine; Benzopyrans; Bradykinin; Calcimycin; Calmodulin; Coronary Vessels; Cromakalim; Dogs; Endothelium, Vascular; Ethanolamines; Imidazoles; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Nebivolol; Nitric Oxide; Nitroarginine; Pyrroles; 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

A NO synthase (NOS, EC 1.14.23) was isolated from human cerebellum by two sequential chromatography steps, that is affinity chromatography on 2'5'ADP sepharose and size exclusion chromatography on Superose 6. Human NOS migrated as a single band of 160 kDa on SDS/PAGE. The enzyme was Ca2+/calmodulin-regulated and NADPH/tetrahydrobiopterin (BH4)-dependent, which are characteristics of a type I NOS previously isolated from rat cerebellum. Antisera raised against purified rat cerebellar NOS crossreacted specifically with a 160 kDa protein in crude supernatant fraction of human cerebellum and purified human NOS but not in crude supernatant fraction of the temporal lobe. These findings provide evidence that nitrinergic signal transduction through conversion of L-arginine to L-citrulline and NO does also occur in humans and NO may function as a neurotransmitter in the human central nervous system.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Calmodulin; Cerebellum; Chromatography, Affinity; Chromatography, Gel; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Humans; Imidazoles; Kinetics; Molecular Weight; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Rats; Temporal Lobe; Trifluoperazine