L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation

The formation of nitric oxide (NO) from L-arginine by vascular endothelial cells and its relationship to endothelium-dependent relaxation of vascular rings was studied. The release of NO, measured by bioassay or chemiluminescence, from porcine aortic endothelial cells stimulated with bradykinin was enhanced by infusions of L-, but not D-arginine. The release of 15NO, determined by high resolution mass spectrometry, from L-guanidino 15N (99%) arginine was also observed, indicating that NO is formed from the terminal guanidino nitrogen atom(s) of L-arginine. L-NG-monomethyl arginine (L-NMMA), but not D-NMMA, inhibited both the generation of NO by endothelial cells in culture and the endothelium-dependent relaxation of rabbit aortic rings. Both these effects were reversed by L-arginine. These data indicate that L-arginine is the physiological precursor for the formation of NO which mediates endothelium-dependent relaxation.     

Accumulated endogenous NOS inhibitors,decreased NOS activity,and impaired cavernosal relaxation with ischemia

We examined whether endogenous inhibitors of nitric oxide (NO) synthesis are involved in the impaired cavernosal relaxation with ischemia in rabbits. Two weeks after cavernosal ischemia caused by partial vessel occlusion, endothelium-dependent and electrical field stimulation (EFS)-induced neurogenic NO-mediated relaxations, but not sodium nitroprusside (SNP)-induced relaxation, were significantly impaired in the isolated corpus cavernosum. The Ca(2+)-dependent NO synthase (NOS) activity and the basal and stimulated cGMP productions with carbachol or EFS were significantly decreased after ischemia. Supplementation of excess L-arginine partially recovered both of the impaired relaxations. The contents of N(G)-monomethyl-L-arginine (L-NMMA) and asymmetric N(G), N(G)-dimethyl-L-arginine (ADMA) but not L-arginine and symmetric N(G),N'(G)-dimethyl-L-arginine (SDMA) were increased in the cavernosal tissues after ischemia. Authentic L-NMMA and ADMA but not SDMA concentration dependently inhibited both relaxations without affecting the relaxation produced by SNP in the control. Excess L-arginine abolished the inhibition with L-NMMA and ADMA. These results suggest that the impaired NO-mediated cavernosal relaxations after ischemia are closely related to the decreased NOS activity and the increased accumulation of L-NMMA and ADMA.     

Mg(2+)-induced endothelium-dependent relaxation of blood vessels and blood pressure lowering: role of NO

In vitro extracellular Mg(2+) concentration ([Mg(2+)](0)) produces endothelium-dependent and endothelium-independent relaxations in rat aorta in a concentration-dependent manner. These relaxant effects of Mg(2+) on intact rat aortic rings, but not denuded rat aortic rings, were suppressed by either N(G)-monomethyl-L-arginine (L-NMMA), N(omega)-nitro-L-arginine methyl ester (L-NAME), or methylene blue. The inhibitory effects of L-NMMA and L-NAME could be reversed partly by L-arginine. [Mg(2+)](0)-induced dilatation in vivo in rat mesenteric arterioles and venules was almost completely inhibited by N(G)-nitro-L-arginine and L-NMMA. Removal of extracellular Ca(2+) concentration ([Ca(2+)](0)) or buffering intracellular Ca(2+) concentration in endothelial cells, with 10 microM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, markedly attenuated the relaxant effects of Mg(2+). Mg(2+) produced nitric oxide (NO) release from the intact aortic rings in a concentration-dependent manner. Removal of [Ca(2+)](0) diminished the increased NO release induced by elevated levels of [Mg(2+)](0). In vivo infusion of increasing doses (1-30 microM/min) of MgSO(4), directly into the femoral veins of anesthetized rats, elicited significant concentration-dependent sustained increases in serum total Mg and concomitant decreases in arterial blood pressure. Before and after employment of various doses of MgSO(4), intravenous administration of either L-NMMA (10 mg/kg) or L-NAME (10 mg/kg) increased (i.e., reversed) the MgSO(4)-lowered blood pressure markedly, and intravenous injection of L-arginine restored partially the increased blood pressure effects of both L-NMMA and L-NAME. Our results suggest that 1) small blood vessels are very dependent on NO release for Mg(2+) dilatations and 2) the endothelium-dependent relaxation induced by extracellular Mg(2+) is mediated by release of endothelium-derived relaxing factor-NO from the endothelium, and requires Ca(2+) and formation of guanosine 3',5'-cyclic monophosphate.     

Relationship between flow rate and NO production in postnatal mesenteric arteries

We studied mesenteric arterial arcades from 3- and 35-day-old swine to determine the relationship between perfusate flow rate and release of nitric oxide (NO) into mesenteric effluent. Mesenteric arterial arcades were perfused under controlled-flow conditions with a peristaltic pump using warm oxygenated Krebs buffer. Basal rates of NO production were 43.6 +/- 4.2 vs. 12.1 +/- 2.5 nmol/min in 3- vs. 35-day-old mesentery during perfusion at in vivo flow rates (9 vs. 20 ml/min, respectively). Rate of NO production was directly related to flow rate over a wide range of flows (5-40 ml/min) in 3- but not 35-day-old mesentery. Both age groups demonstrated a brisk, albeit brief, increase in NO production in response to infusion of NO-dependent vasodilator substance P (10(-8) M/min). Tyrosine kinase inhibitor herbimycin A and L-arginine analog L-NMMA significantly attenuated flow-induced increase in NO production, and phosphatase inhibitor phenylarsine oxide increased magnitude of flow-induced increase in NO production in 3-day-olds. Removal of extracellular Ca(2+) and depletion of intracellular Ca(2+) stores (Ca(2+)-free Krebs with EGTA plus thapsigargin) had no effect on NO production in either group. Thus, basal rate of NO production is greater in mesenteric arterial arcades from 3- than from 35-day old swine, a direct relationship between flow rate and NO production rate is present in mesentery from 3- but not 35-day-olds, and phosphorylation events are necessary for this interaction to occur.     

Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated L-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-L-arginine (L-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and L-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for L-arginine is 3.14 microM with a Vmax of 0.14 micromol mg-1 min-1, whereas Ki values of 0.9 microM and 1.1 microM were determined for ADMA and L-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 microM level of L-arginine, MA levels of >10 microM were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 microm. Similar effects were observed in isolated vessels where 5 microm ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and L-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. L-arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, approximately 4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury.     

A nitric oxide synthesis inhibitor decreased prostaglandin production in rat mesenteric vasculature

Endothelial cells synthesize and release nitric oxide (NO) and prostacyclin (PGI₂) which are involved in the regulation o f vascular tone and blood pressure. Our objective was to evaluate the effects of inhibiting NO synthesis on vascular prostaglandin (PG) and cyclic nucleotide production, as well as the pressor response to norepinephrine (NE). Isolated mesenteric arterial beds were perfused with Krebs-Henseleit solution containing 100 μM N^G-monomethyl-L-arginine (L-NMMA), 100 μM L-arginine (LA), or vehicle. After a 30 min equilibration 0.1, 0.5, 1, or 5 μM NE was infused into the superior mesenteric artery and the perfusion pressure was monitored. The basal perfusion pressure did not differ significantly between groups. The pressure-response curve was shifted to the right in the L-NMMA group vs. the LA and control groups. Perfusion was similarly performed with a Krebs-Henseleit solution containing 100 μM L-NMMA, LA, D-arginine, or vehicle. Perfusates were collected before and after NE infusion for the measurement of PGE₂, 6-keto-PGF_1α, TxB₂, cAMP, and cGMP. In the L-NMMA group the release of PGE₂ and 6-keto-PGF1α was decreased, and the release of cGMP was prevented. Production of cAMP did not differ between the four groups before NE infusion, and NE increased cAMP release in the L-NMMA group and controls. The results indicate that inhibition of NO synthesis by L-NMMA enhanced the pressor response to NE, possibly mediated by the decreased cGMP and PGI₂ production in resistance vessels.     

Role of nitric oxide in the airway response to exercise in healthy and asthmatic subjects.

A role of nitric oxide (NO) has been suggested in the airway response to exercise. However, it is unclear whether NO may act as a protective or a stimulatory factor. Therefore, we examined the role of NO in the airway response to exercise by using N-monomethyl-L-arginine (L-NMMA, an NO synthase inhibitor), L-arginine (the NO synthase substrate), or placebo as pretreatment to exercise challenge in 12 healthy nonsmoking, nonatopic subjects and 12 nonsmoking, atopic asthmatic patients in a double-blind, crossover study. Fifteen minutes after inhalation of L-NMMA (10 mg), L-arginine (375 mg), or placebo, standardized bicycle ergometry was performed for 6 min using dry air, while ventilation was kept constant. The forced expiratory volume in 1-s response was expressed as area under the time-response curve (AUC) over 30 min. In healthy subjects, there was no significant change in AUC between L-NMMA and placebo treatment [28.6 +/- 17.0 and 1.3 +/- 20.4 (SE) for placebo and L-NMMA, respectively, P = 0.2]. In the asthmatic group, L-NMMA and L-arginine induced significant changes in exhaled NO (P < 0.01) but had no significant effect on AUC compared with placebo (geometric mean +/- SE: -204.3 +/- 1.5, -186.9 +/- 1.4, and -318.1 +/- 1.2%. h for placebo, L-NMMA, and L-arginine, respectively, P > 0.2). However, there was a borderline significant difference in AUC between L-NMMA and L-arginine treatment (P = 0.052). We conclude that modulation of NO synthesis has no effect on the airway response to exercise in healthy subjects but that NO synthesis inhibition slightly attenuates exercise-induced bronchoconstriction compared with NO synthase substrate supplementation in asthma. These data suggest that the net effect of endogenous NO is not inhibitory during exercise-induced bronchoconstriction in asthma.     

Role of nitric oxide in lower esophageal sphincter relaxation to swallowing.

Studies were performed in the opossum to define the role of the L-arginine-nitric oxide (NO) pathway in lower esophageal sphincter (LES) relaxation to swallowing and vagal stimulation in viv and intramural nerve stimulation in vitro. In vivo, L-NAME, a water soluble NO synthase (NOS) inhibitor, caused antagonism of LES relaxation due to reflex-induced swallowing. L-NAME (20 mg/kg i.v.) reduced the amplitude of swallow induced relaxation from 88% to 28%. LES relaxation due to electrical stimulation of peripheral end of decentralized vagus nerve was also antagonized. The effects of L-NAME were reversed by L-arginine, but not by D-arginine. L-NAME treatment did not antagonize LES relaxation to intravenous administration of isoproterenol. In vitro, NO and sodium nitroprusside (SNP) caused a decrease in the sphincter tone. The relaxing effect caused by NO and SNP was not antagonized by tetrodotoxin or omega-conotoxin. Inhibitors of NO synthase, L-NMMA and L-NNA, caused slight increase in the spontaneous resting LES tone and concentration-dependent antagonism of electrical field stimulation (EFS) induced LES relaxation. L-NNA (10(-4)M) abolished EFS induced LES relaxation at low frequencies (less than 5 Hz) and antagonized the relaxation to a value 20% of the control at 20 Hz. The antagonistic action of L-NMMA and L-NNA was unaffected by D-arginine but was reversed by L-arginine. The inhibitory effect of NO, SNP, or two other putative inhibitory neurotransmitters (VIP and CGRP) on the LES was not antagonized by L-NNA. These studies show that inhibitors of NO synthase selectively antagonize LES relaxation to all three modes of intramural inhibitory nerve stimulation including physiological swallowing. These studies suggest that the L-arginine-nitric oxide pathway is involved in physiological relaxation of the LES.     

Nitric oxide-dependent and -independent mechanisms in the relaxation elicited by acetylcholine in fetal rat aorta

The aim of the present study was to analyze the mechanisms involved in the relaxation induced by 1 microM acetylcholine (ACh) in aortic segments from fetal rats at term precontracted with 3 microM prostaglandin F2alpha (PGF2alpha) and incubated with 1 microM indomethacin. The endothelium-dependent relaxation caused by ACh was reduced by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.1 mM), such an effect was reversed by 0.1 mM L-arginine (L-Arg). After precontraction of segments with 50 mM KCl the relaxant response to ACh was smaller than that after precontraction with PGF2alpha; this reduction was increased by L-NMMA, whereas L-NMMA plus L-Arg potentiated the relaxation. Thiopentone sodium (0. 1 mM), ouabain (10 microM), tetraethylammonium (TEA, 0.5 mM) and apamin (1 microM), inhibitors of cytochrome P450 monooxygenases, Na+ pump, Ca2+-activated (KCa) and small-conductance (SKCa) K+ channels, respectively, reduced the relaxation to ACh, which was unaffected by charybdotoxin (0.1 microM) and glibenclamide (1 microM), inhibitors of large-conductance BKCa and ATP-sensitive K+ channels. The L-NMMA/indomethacin-resistant relaxation to ACh was markedly reduced by thiopentone sodium, and similarly decreased by either ouabain or TEA. The endothelium-independent relaxation induced by exogenous NO (10 microM) in segments precontracted with PGF2alpha was unaltered by ouabain, glibenclamide, TEA and after precontraction with 50 mM KCl, and potentiated by L-NMMA. The potentiation of NO responses by L-NMMA was also observed in segments precontracted with KCl. These results suggest that ACh relaxes the fetal rat aorta by endothelial release of both NO and endothelium-derived hyperpolarizing factor (EDHF), a metabolite derived from cytochrome P450 monooxygenases, that hyperpolarizes smooth muscle cells by activation of KCa, essentially SKCa channels, and Na+ pump. It seems that when the effect of EDHF is abolished, the formation of NO could be increased.     

L-NG-monomethyl arginine inhibits the vasodilating effects of low dose of endothelin-3 on rat mesenteric arteries

We have reported that low doses of endothelin-3 (ET-3) elicited continuous vasodilation of rat mesenteric arteries, which is possibly related to endothelium-derived relaxing factor (EDRF). In order to clarify whether or not the vasodilating effects of ET-3 are associated with EDRF, we examined the effects of L-NG-monomethyl arginine (L-NMMA), an analog of L-arginine, on low-dose ET-3 induced vasodilation of rat mesente-Hc arteries. Infusion of 50 microM L-NMMA inhibited the vasodilation induced by 10(-13) M ET-3 and rather elicited an increase in perfusion pressure, which itself was decreased by infusion of 150 microM L-arginine. In the presence of 50 microM L-NMMA, 10(-13) M ET-3 did not elicit any vasodilation of the mesenteric arteries preconstricted with NE, in which 150 microM L-arginine, but not D-arginine, caused considerable vasodilation. These data suggest that the vasodilating effects of low doses of ET-3 are associated with EDRF as an endothelium-derived nitric oxide.     

Absence of implication of L-arginine/nitric oxide pathway on neuronal cell injury induced by L-glutamate or hypoxia.

L-glutamate, N-methyl-D-aspartate (NMDA), kainate, quisqualate and sodium nitroprusside increased cyclic GMP (cGMP) level on rat whole brain cell culture. The accumulation of cGMP evoked by L-glutamate was inhibited by a NMDA antagonist MK-801, an inhibitor of guanylate cyclase methylene blue and two nitric oxide (NO) synthase inhibitors NG-monomethyl-L-arginine (L-NMMA) and L-NG-nitroarginine (NO2Arg). The inhibition of L-NMMA on cGMP level was reversed partially by addition of L-arginine. Although MK-801 was able to protect cells from neuronal injury induced by L-glutamate or by 5 h hypoxia, L-NMMA and NO2Arg were ineffective. The present study suggests that cGMP elevation mediated by NO following activation by L-glutamate is not involved in neuronal cell injury.     

Nitric oxide from endothelium and smooth muscle modulates responses to sympathetic nerve stimulation: implications for endotoxin shock.

The influence of nitric oxide (NO) on vascular responses to transmural stimulation (TNS) of noradrenergic nerves was studied in isolated rings of rat iliac arteries. TNS produced frequency-dependent contractions in all vessels. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) significantly enhanced TNS responses in intact vessels, but not in those in which the endothelium had been removed. However, in endothelium-denuded rings incubated for 8 hours, L-NMMA increased the contractions induced by nerve stimulation, an effect which was prevented by treatment with dexamethasone or cycloheximide, and enhanced by incubation with lipopolysaccharide and gamma-interferon. Addition of L-arginine reversed the effect of L-NMMA in intact rings; however, it significantly decreased below control values TNS-induced contractions in vessels without endothelium. The results indicate that a) the arterial response to noradrenergic nerve stimulation is modulated by NO originating either in endothelial cells or in smooth muscle cells after induction of NO synthase activity, and b) once NO synthase is induced, the limiting step in NO production is the availability of the substrate L-arginine. An overproduction of vascular NO in the presence of endotoxin or other inflammatory stimuli may prevent the vascular response to sympathetic stimuli and contribute to the vasodilation observed in inflammation or endotoxic shock.     

Relaxation of bovine coronary artery and activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside and a carcinogenic nitrosoamine.

The principal objective of this study was to test the hypothesis that nitroprusside relaxes vascular smooth muscle via the reactive intermediate, nitric oxide (NO), and that the biologic action of NO is associated with the activation of guanylate cyclase. Nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and NO elicit concentration-dependent relaxation of precontraced helical strips of bovine coronary artery. Nitroprusside, MNNG and NO also markedly activate soluble guanylate cyclase from bovine coronary arterial smooth muscle and, thereby, stimulate the formation of cyclic GMP. Three heme proteins, hemoglobin, methemoglobin and myoglobin, and the oxidant, methylene blue, abolish the coronary arterial relaxation elicited by NO. Similarly, these heme proteins, methylene blue and another oxidant, ferricyanide, markedly inhibit the activation of coronary arterial guanylate cyclase by NO, nitroprusside and MNNG. The following findings support the view that certain nitroso-containing compounds liberate NO in tissue:heme proteins, which cannot permeate cells, inhibit coronary arterial relaxation elicited by NO, but not by nitroprusside or MNNG; the vital stain, methylene blue, inhibits relaxation by NO, nitroprusside and MNNG; heme proteins and oxidants inhibit guanylate cyclase activation by NO, nitroprusside and MNNG in cell-free mixtures. The findings that inhibitors of NO-induced relaxation of coronary artery also inhibit coronary arterial guanylate cyclase activation suggest that cyclic GMP formation may be associated with coronary arterial smooth muscle relaxation.     

Epithelium-dependent regulation of airways smooth muscle function. A histamine-nitric oxide pathway.

The airway epithelium is responsible for the production of a number of arachidonic acid and non-prostanoid inhibitory factors. Epithelium synthesises nitric oxide (NO) which may be important in regulating the function of airways smooth muscles. We studied in vitro the effect of histamine (100 nM-100 microM) which increases the NO release on rabbit airway smooth muscles induced by 80 mM KC1 in the presence or not of 10(-5) Methylene blue (MB) (inactivator of guanylate cyclase) or N(G)-monomethyl L-arginine (L-NMMA), a NOS inhibitor. All experiments were done in tracheal muscle strips from 28 rabbits with epithelium and after epithelium removal. The additional use of histamine (1 microM) on KC1 contraction induced a relaxation of 10% of the initial contraction. The additional use of L-NMMA decreased the relaxation to 5% of initial contraction. MB rather than L-NMMA increased the contraction significantly (p<0.01). Epithelium removal increased the contraction induced by KC1 (80 mM) and histamine (1 microM) by about 30% (p<0.001). NO release especially from epithelium regulates the airways smooth muscle functions. Damage to the epithelium may contribute to an increase in airways sensitivity, observed in asthma.     

Blockade of nitric oxide synthase potentiates the suppression of vasodilators by norepinephrine in the hepatic artery.

We have previously shown that nitric oxide (NO) and adenosine suppress vasoconstriction induced by norepinephrine infusion and sympathetic nerve stimulation in the hepatic artery and superior mesenteric artery. NO is involved in the control of basal vascular tone in the superior mesenteric artery but not the hepatic artery. The vasodilation induced by adenosine is inhibited by NO in the superior mesenteric artery but not in the hepatic artery. Based on these known interactions of catecholamines, adenosine, and NO, the objective of this study was to test the hypothesis that NO modulates the interaction between vasoconstrictors and vasodilators in the hepatic artery. We examined the ability of norepinephrine to suppress adenosine-mediated vasodilation and the role of NO in this interaction. Hepatic arterial blood flow and pressure were monitored in pentobarbital-anesthetized cats. The maximum hepatic arterial vasoconstrictor response to norepinephrine infusion was potentiated by blockade of NO production using Nomega-nitro-L-arginine methyl ester (L-NAME), and the potentiation was reversed by L-arginine. The maximum dilator response to adenosine was only slightly suppressed (14.0+/-5.8%, P < 0.05) by norepinephrine infusion; however, after the NO blockade, the suppression by norepinephrine of the vasodilation induced by adenosine was substantially potentiated (45.2+/-9.1%, P < 0.05). Similar results were obtained for isoproterenol-induced vasodilation. We conclude that the interaction between these vasodilators and norepinephrine was modulated by NO which inhibited the vasoconstriction and the suppression of vasodilators caused by norepinephrine and that in the absence of NO production, norepinephrine-induced constriction and the ability to antagonize dilation is substantially potentiated.     

NW-nitro L-arginine benzyl ester, a potent irreversible inhibitor of endothelium dependent relaxation

In the rat aorta preparation, we have used the nitro-arginine derivative, NW-nitro L-arginine benzyl ester (NABE), as a probe to investigate the relationship between L-arginine and the endothelium derived relaxing factor (EDRF). We find NABE to be a potent endothelium dependent vasoconstrictor and inhibitor of relaxation. The effect of NABE is irreversible and not removed by subsequent washing. The vasoconstrictor effect of NABE differs from other EDRF inhibitors like NG-monomethyl L-arginine (L-NMMA) in that it is not antagonized by pre-treatment with excess L-arginine. In contrast, like other EDRF inhibitors, at high concentration NABE exhibits vasodilation which is antagonized by methylene blue. We suggest that the previous reports on the antagonism between L-arginine and the putative EDRF inhibitors like L-NMMA are due to their structural similarities rather than to externally added L-arginine acting as a substrate for EDRF synthesis.     

Nitric oxide regulates retinal vascular tone in humans

The purpose of the present study was to investigate the contribution of basal nitric oxide (NO) on retinal vascular tone in humans. In addition, we set out to elucidate the role of NO in flicker-induced retinal vasodilation in humans. Twelve healthy young subjects were studied in a three-way crossover design. Subjects received an intravenous infusion of either placebo or NG-monomethyl-L-arginine (L-NMMA; 3 or 6 mg/kg over 5 min), an inhibitor of NO synthase. Thereafter, diffuse luminance flicker was consecutively performed for 16, 32, and 64 s at a frequency of 8 Hz. The effect of L-NMMA on retinal arterial and venous diameter was assessed under resting conditions and during the hyperemic flicker response. Retinal vessel diameter was measured with a Zeiss retinal vessel analyzer. L-NMMA significantly reduced arterial diameter (3 mg/kg: -2%; 6 mg/kg: -4%, P < 0.001) and venous diameter (3 mg/kg: -5%; 6 mg/kg: -8%, P < 0.001). After placebo infusion, flicker induced a significant increase in retinal vessel diameter (P < 0.001). At a flicker duration of 64 s, arterial diameter increased by 4% and venous diameter increased by 3%. L-NMMA did not abolish these hyperemic responses but blunted venous vasodilation (P = 0.017) and arterial vasodilation (P = 0.02) in response to flicker stimulation. Our data indicate that NO contributes to basal retinal vascular tone in humans. In addition, NO appears to play a role in flicker-induced vasodilation of the human retinal vasculature.     

Nitric oxide mediates protective effect of endothelin receptor antagonism during myocardial ischemia and reperfusion

Endothelin (ET) receptor antagonism protects from ischemia-reperfusion injury. We hypothesized that the cardioprotective effect is related to nitric oxide (NO) bioavailability. Buffer-perfused rat and mouse hearts were subjected to ischemia and reperfusion. At the onset of ischemia, the rat hearts received vehicle, the dual endothelin type A/type B (ETA/ETB) receptor antagonist bosentan (10 microM), the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 microM), the combination of bosentan and L-NMMA or the combination of bosentan, L-NMMA, and the NO substrate L-arginine (1 mM). Hearts from wild-type and endothelial NO synthase (eNOS)-deficient mice received either vehicle or bosentan. Myocardial performance, endothelial function, NO outflow, and eNOS expression were monitored. Bosentan significantly improved myocardial function during reperfusion in rats and in wild-type mice, but not in eNOS-deficient mice. The functional protection afforded by bosentan was inhibited by L-NMMA, whereas it was restored by L-arginine. Myocardial expression of eNOS (immunoblotting) increased significantly in bosentan-treated rat hearts compared with vehicle hearts. Recovery of NO outflow during reperfusion was enhanced in the bosentan-treated rat heart. The endothelium-dependent vasodilator adenosine diphosphate increased coronary flow by 18 +/- 9% at the end of reperfusion in the bosentan group, whereas it reduced coronary flow by 7 +/- 5% in the vehicle group (P < 0.001). The response to the endothelium-independent dilator sodium nitroprusside was not different between the two groups. In conclusion, the dual ETA/ETB receptor antagonist bosentan preserved endothelial and cardiac contractile function during ischemia and reperfusion via a mechanism dependent on endothelial NO production.     

Release of nitric oxide from human vascular smooth muscle cells

It has recently been shown that nitric oxide (NO) or a labile NO-releasing compound is produced in endothelial cells. In the present study we measured the NO-release from human vascular smooth muscle cells in culture. The cells released an average 2.2 x 10(-9) moles nitric oxide per 10(8) cells in ten minutes with a large variation between different cell lines and passages without stimulators. The NO-release was markedly reduced by the inhibitor of NO-formation NG-momomethyl-L-arginine (L-NMMA, 10(-5) M) to 3% of the control levels (p less than 0.02, n = 4), but unaffected by acetylcholine, bradykinin or endothelin -1, -2 or -3. In a microperfusion system the compound released from human vascular smooth muscle cells caused profound relaxation of isolated rat mesenteric resistance arteries. Thus, human vascular smooth muscle cells in culture produce and release biologically active NO from L-arginine.     

Pregnancy-associated increase in rat systemic arteries endothelial nitric oxide production diminishes vasoconstrictor but does not enhance vasodilator responses

Late pregnancy in rats is characterized by a decrease in arterial pressure and in isolated arterial vessels response to vasoconstrictors. In uterine arteries the pregnancy-associated attenuation of the response to vasoconstrictors has been attributed to an increase in basal and agonist-induced endothelial NO production. However, the role of NO in pregnancy-associated changes of systemic arteries reactivity to vasoactive agents remains to be fully elucidated. We examined whether pregnancy influences the reactivity of systemic arteries to vasodilator or vasoconstrictor agents through NO-dependent mechanisms. Thoracic aortic rings and mesenteric arterial bed of late pregnant rats showed refractoriness to phenylephrine-induced vasoconstriction that was abolished by NO synthase inhibition. The potency of L-NNA to enhance tension of aortic rings preconstricted with phenylephrine (10–20% of their maximal response) was significantly lower in preparations from pregnant animals. In phenylephrine-contracted aortas and mesenteric bed, the effects of the endothelium-dependent vasodilators acetylcholine, A23187 and bradykinin, were not influenced by pregnancy. Similarly, pregnancy did not affect the vasodilator responses of adenosine, isoproterenol, capsaicin, nitroprusside, forskolin, and Hoe234 in the mesenteric bed. NO synthase activity measured by determining the conversion of L−[³H]-arginine to L−[³H]-citrulline in aorta and mesenteric arteries homogenates was not altered by pregnancy. These findings show that endothelial-dependent and -independent vasodilators action as well as NO synthase activity in systemic arteries is uninfluenced by pregnancy, whereas pregnancy-associated hyporeactivity of systemic arteries to vasoconstrictors is related to an enhanced endothelial NO production either spontaneous or elicited directly or indirectly by vasoconstrictor agents. This interpretation implies that the enhanced NO production observed in systemic arteries during late pregnancy involves cellular pathways other than the ones involved in the response to endothelium-dependent vasodilators such as acetylcholine.