nitroarginine and 3-(5--hydroxymethyl-2--furyl)-1-benzylindazole

This study tested the hypothesis that nitric oxide (NO) synthase inhibition in mice would result in hypertension characterized by increased agonist-induced vasoconstrictor responsiveness and attenuated endothelium-dependent vasodilation. Administration of N-nitro-L-arginine (L-NNA), an NO synthase inhibitor (1 g/L, 4 weeks), via drinking water to mice resulted in significant elevations in blood pressure. Phenylephrine-induced contraction was significantly increased in aortic rings from L-NNA-treated mice compared with rings from control mice. Aortic rings from control mice showed a concentration-dependent relaxation to acetylcholine whereas those obtained from L-NNA-treated mice showed a biphasic response, contracting at lower concentrations while relaxing at higher concentrations. Aortic rings from L-NNA-treated mice had decreased relaxation to acetylcholine and increased sensitivity to sodium nitroprusside compared with control rings. The relaxation induced by an NO-independent soluble guanylyl cyclase activator was not different between groups. In aortic rings from control and L-NNA-treated mice pre-contracted with phenylephrine, the administration of L-NNA to the organ bath caused additional and sustained contraction. When compared with the contraction induced by phenylephrine, L-NNA-induced contraction in aorta from control mice was significantly higher than that in aorta from L-NNA-treated mice. We conclude that mice treated with L-NNA develop hypertension and that a reduction in NO availability is responsible for the changes observed in vascular reactivity.

Topics: Acetylcholine; Animals; Aorta; Blood Pressure; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Activators; Enzyme Inhibitors; Heart Rate; Hypertension; In Vitro Techniques; Indazoles; Male; Mice; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Phenylephrine; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

1 In this study we examined the endothelium-dependent effect of YC-1 - a benzyl indazole derivative which directly activates soluble guanylyl cyclase (sGC) - on vascular relaxation and nitric oxide (NO) and guanosine-3',5'-cyclic monophosphate (cyclic GMP) in endothelial cells. 2 In preconstricted rat aortic rings with intact endothelium, YC-1 produced a concentration-dependent relaxation. However, the concentration response curve was shifted rightward to higher concentrations of YC-1, when (i) the aortas were pre-treated with L-NG-nitroarginine methylester (L-NAME) or (ii) the endothelium was removed. 3 Incubation of bovine aortic endothelial cells (BAEC) with YC-1 produced a concentration-dependent NO synthesis and release as assessed using a porphyrinic microsensor. Pre-incubating cells with L-NAME or with 8-bromo-cyclic GMP decreased this effect indicating that the YC-1 stimulation of NO synthesis is due to an activation of nitric oxide synthase, but not to an elevation of cyclic GMP. No direct effect of YC-1 on recombinant endothelial constitutive NO synthase activity was observed. 4 The YC-1 stimulated NO release was reduced by 90%, when extracellular free calcium was diminished. 5 In human umbilical vein endothelial cells (HUVEC), YC-1 stimulated intracellular cyclic GMP production in a concentration- and time-dependent manner. Stimulation of cyclic GMP was greater with a maximum concentration of YC-1 compared to calcium ionophore A23187. Similar effects were observed in BAEC and rat microvascular coronary endothelial cells (RMCEC). 6 When HUVEC and RMCEC were pre-treated with L-NG-nitroarginine (L-NOARG), the maximum YC-1 stimulated cyclic GMP increase was reduced by >/=50%. 7 These results indicate, that beside being a direct activator of sGC, YC-1 stimulates a NO-synthesis and release in endothelial cells which is independent of elevation of cyclic GMP but strictly dependent on extracellular calcium. The underlying mechanism needs to be determined further.

Topics: Animals; Aorta, Thoracic; Bradykinin; Calcimycin; Cattle; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Activation; Guanylate Cyclase; Humans; In Vitro Techniques; Indazoles; Ionophores; Male; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroarginine; Rats; Rats, Wistar; Solubility; Time Factors

This study examined the mechanism through which nitric oxide inhibits the release of acetylcholine and excitatory motor neurotransmission in the guinea-pig ileum. The selective inhibitor of nitric oxide-sensitive guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), concentration-dependently enhanced both basal release (-log EC50: 6.8) and electrically (10 Hz)-evoked release (-log EC50: 6.0) of [3H]acetylcholine from longitudinal muscle-myenteric plexus preparations preincubated with [3H]choline. The increase by ODQ of basal release appeared to be exocytotic since it was prevented by tetrodotoxin (300 nM) and absence of calcium from the superfusion medium. In addition, ODQ (1 microM) increased the electrically-evoked tachykininergic and cholinergic muscle contractions as measured in the presence of scopolamine (100 nM) or of the neurokinin-1 receptor antagonist CP 99994 (100 nM), respectively. The nitric oxide synthase inhibitor L-N(G)-nitro-arginine (100 microM) behaved similar to ODQ and increased cholinergic and tachykininergic motor neurotransmission. The nitric oxide-independent activator of soluble guanylyl cyclase, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole, concentration-dependently inhibited the electrically evoked acetylcholine release (-log EC50: 6.0) and longitudinal muscle contractions (-log EC50: 5.7). ODQ (10 microM) antagonized the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole. The results suggest that endogenous nitric oxide tonically activates soluble guanylyl cyclase in myenteric neurons which leads to inhibition of the release of the excitatory transmitters acetylcholine and substance P. ODQ prevents the effects of nitric oxide and thus facilitates cholinergic and tachykininergic motor neurotransmission in the guinea-pig ileum.

Topics: Acetylcholine; Animals; Depression, Chemical; Electric Stimulation; Enzyme Inhibitors; Female; Guanylate Cyclase; Guinea Pigs; Ileum; Indazoles; Male; Motor Neurons; Muscle Contraction; Muscle, Smooth; Myenteric Plexus; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxadiazoles; Quinoxalines; Synaptic Transmission