Basal Nitric Oxide Release Research Articles

Advanced glycation end products quench nitric oxide in vitro.

Nitric oxide (NO) is known as an important mediator of endothelial function. The aim of this investigation was to evaluate the influence of mediators of retinal pathology - vascular endothelial growth factor (VEGF) and advanced glycation end products (AGEs) - on NO release from choroidal endothelial cells (CEC) and retinal pigment epithelial (RPE) cells to elucidate the complex role of NO. Bovine CEC were stimulated using VEGF (1, 10, and 100 ng/ml), and RPE cells were exposed to interferon-gamma (IFN-gamma 100 U/ml) and lipopolysaccharide (LPS 1 micro g/ml). NO release into the media was assessed by an amperometric NO sensor. The influence of AGEs (10 and 100 micro g/ml) on NO release from CEC and RPE cells was investigated. The competitive NO synthase inhibitor L(omega)-nitro-L-arginine methyl ester (L-NAME 2 nmol) was used to pretreat the cells 2 h before NO measurement. Unstimulated CEC produced low basal levels of NO in vitro (39.1+/-13.9 nmol), and unstimulated RPE cells produced minimal basal levels of NO (15.7+/-7.0 nmol). Exposure of CEC to VEGF for 30 min resulted in a dose-dependent rise of NO in the medium, which was significantly inhibited by L-NAME. Stimulation of RPE cells with IFN-gamma and LPS resulted in a rise of NO in the bath to 125.9+/-18.5% of basal values. Basal NO release from CEC, and stimulated NO release from RPE cells, was significantly reduced by AGE treatment and L-NAME. These data demonstrate that AGEs formed from the nonenzymatic glycation of proteins with reducing sugars quench NO activities in vitro. The results implicate AGEs as important modulators of NO activity and may be relevant to the impairment of endothelial functions observed in diabetes and aging.

Medroxyprogesterone acetate does not antagonize estrogen-induced increases in endothelium-dependent vasodilation: potential clinical implications

Objective: Determine the effect of combined 17β-estradiol benzoate (E 2) and medroxyprogesterone acetate (MPA) administration on endothelium-dependent vasorelaxation to acetylcholine (ACh). Design: Prospective, ex vivo study. Setting: Academic research laboratory. Animal(s): Mature female rats. Intervention(s): Ovariectomized rats received one of the following interventions daily for 3 days: [1] corn oil via IM injection, [2] E 2 (20 μg/kg IM), or [3] E 2 (20 μg/kg IM) and MPA (10 mg/kg IM). Main Outcome Measure(s): Basal release of nitric oxide (NO) and endothelium-dependent and endothelium-independent vasodilation from thoracic aortas obtained from each group. Result(s): Estradiol treatment potentiated the endothelium-dependent relaxation to ACh when compared with the control group. Administration of MPA with E 2 did not antagonize the beneficial effect of E 2 on endothelium-dependent relaxation. Estradiol treatment alone or in combination with MPA did not affect endothelium-independent vasodilation as compared with the case of the control group. Administration of E 2 resulted in increased basal NO release (assessed indirectly by measuring the constrictor response to N G-nitro- l-arginine [methyl ester (L-NAME)]) when compared with the case of the control group, and the addition of MPA to E 2 did not alter the effect of E 2 on basal NO release. Conclusion(s): Estradiol potentiates endothelium-dependent relaxant responses and increases basal endothelial NO release. Medroxyprogesterone acetate does not antagonize these effects of E 2.

Altered L-arginine metabolism results in increased nitric oxide release from uraemic endothelial cells.

Results regarding the nitric oxide (NO) system in uraemia are contradictory. L-arginine, the precursor of NO, is also metabolized by arginase to form ornithine and urea. In the present study, endothelial NO production and arginine metabolism in uraemia were assessed. In addition an in vivo model was used to examine excess consumption of NO in uraemia. NO and amino acid measurements were made from basal and stimulated (by bradykinin) uraemic and control endothelial cells. Reverse-transcriptase PCR was used to assess endothelial NO synthase (eNOS) and inducible NOS (iNOS) expression. Finally, aortae of uraemic rats were stained for nitrotyrosine (a marker of peroxynitrite). Basal uraemic cells produced more NO than the control cells. L-arginine levels were greater in uraemic (supernatants/cells), but ornithine levels were higher in control (supernatants/cells). Following stimulation, NO levels in supernatants were similar, but the rise in NO production was greater in control compared with uraemic cells; l-arginine levels still remained higher in uraemic supernatants/cells. Differences in ornithine concentration (supernatants/cells) disappeared following bradykinin stimulation, due to a rise in ornithine levels in the uraemic group. There was no difference in eNOS expression, nor was iNOS detected in either group. Only aortae from uraemic rats showed evidence for nitrotyrosine staining. These studies demonstrated increased basal NO release in uraemic endothelial cells, perhaps by inhibition of arginase and hence diversion of arginine to the NO pathway. The increased NO produced under basal conditions may be inactive due to excessive consumption, resulting in peroxynitrite formation. Interestingly, bradykinin appears to restore arginase activity in uraemia, resulting in normalization of NO production.

Gender differences in contractile and relaxing responses of aortic rings from lipopolysaccharide-treated rats

Aortic rings from male and female Sprague-Dawley rats were placed in organ chambers filled with Krebs solution, and the isometric tension was recorded. There were no gender differences in responses to potassium chloride or phenylephrine in either intact or endothelium-denuded rings from control animals. Treatment with 10 mg/kg of lipopolysaccharide (LPS) attenuated the contractile response to potassium chloride in intact, but not in endothelium-denuded rings, and suppressed the phenylephrine contractions in rings both with and without endothelium from male and female rats. Endothelium-dependent and -independent relaxation by acetylcholine and the nitric oxide donor diethylamine/NO, respectively, did not depend on gender and were equally suppressed by LPS treatment. Treatment with LPS accentuated the effects of oxyhemoglobin in rings with and without endothelium and this was significantly larger in rings from females compared to males. Thus, gender differences were present in the effect of LPS on vascular responses. LPS induced a greater increase in basal release of nitric oxide and a greater inhibition of depolarization by potassium chloride, but not receptor-activated contractions, in aortic rings from female versus male rats.

Increased basal nitric oxide release despite enhanced free radical production in hypertension

Although in hypertension a defect in stimulated nitric oxide (NO) is well established, little is known about basal NO levels. Thus, we measured directly in vessels from normotensive [Wistar-Kyoto (WKY)] rats and spontaneously hypertensive rats (SHR) both basal and stimulated NO production using a novel technique [4,5-diaminofluorescein (DAF-2) fluorescence]. Isolated vessels were exposed to the fluorescent probe DAF-2. After the technique was validated with increasing doses of acetylcholine in the presence and absence of NG-nitro-L-arginine methyl ester (l-NAME), we measured NO production in vessels from WKY rats and SHR in the same experimental setting. Finally, to explore the impact of reactive oxygen species (ROS) on NO release, we analysed the effect of an antioxidant, such as ascorbic acid, on basal and stimulated NO in aortic rings of WKY rats and SHR. Aortic rings from SHR exhibited a higher basal NO production and a lower responsiveness to agonist-induced NO release as compared with those observed in WKY rats. Also in resistance vessels such as mesenteric arteries, basal NO production was higher in hypertension. In hypertensive rats, ascorbic acid was able to further increase basal NO release and recovered the impaired stimulated NO production, whereas no effect was detected in normotensive rats. Our data reveal an increased basal NO availability in hypertension despite the increased production of ROS, suggesting a greater complexity in hypertensive endothelial dysfunction when the analysis is focused on direct NO measurement.

Flow rate-perfusion pressure relationships in situ in the uterine circulation of pregnant rats

Objective: Our purpose was to study the effects of inhibition of nitric oxide synthesis on perfusion pressure and flow rate-perfusion pressure relationships in the rat uterine circulation in situ. Study Design: Nonpregnant, midpregnant (day 14), and late pregnant (day 21) Sprague-Dawley rats were studied. The vascular bed of the intact uterus and its contents were isolated and perfused with Krebs buffer (37°C, pH ~7.4, 2% dextran and indomethacin, 10−5 mol/L) through a cannula inserted into the abdominal aorta close to the iliac artery bifurcation, and perfusion pressure was monitored. After equilibration, the flow rate was increased from 1 mL/min to 8 or 16 mL/min, in the absence and presence of Nω-nitro-L-arginine methyl ester (L-NAME), phenylephrine, or both. Results: The flow rate-perfusion pressure relationship in midpregnant rats (n = 9) was not significantly different from that in late pregnant rats (n = 12), but the latter was significantly greater than the relationship in nonpregnant animals (n = 5). L-NAME did not influence perfusion pressure and flow rate-perfusion pressure relationships in any of the groups. However, L-NAME enhanced the phenylephrine-induced and flow rate-induced increase in perfusion pressure in the vascular beds from nonpregnant and midpregnant animals, and to a lesser extent in late pregnant rats. L-arginine did not influence perfusion pressure or the flow rate-perfusion pressure relationship in any group. Conclusions: Vasoconstriction produced by phenylephrine uncovers basal release of endotheliumderived nitric oxide. Vasoconstriction increases perfusion pressure responses to increases in flow rate in the uterine vascular beds of nonpregnant, midpregnant, and late pregnant rats. The release of endothelial nitric oxide in the uterine vascular beds depends on the basal contractile state of the vasculature. (Am J Obstet Gynecol 2002;186:1022-6.)

Interactive effects of growth hormone and oestrogen on vascular responses in hypophysectomised female rats.

Growth hormone (GH) and oestrogen (E(2)) are associated with beneficial effects on the cardiovascular system and it is therefore of great interest to study their interactive effects on haemodynamics and vascular function. Female hypophysectomised (Hx) rats were treated for seven days with GH, E(2) or a combination of the hormones. Systolic blood pressure (SBP), heart rate (HR) and plasma insulin-like growth factor-I (IGF-I) were measured. Contractile properties and endothelial function were studied in isolated resistance arteries using the wire-myograph technique. Hypophysectomy, per se, caused a fall in SBP and HR, while vascular adrenergic reactivity (sensitivity to applied noradrenaline) was enhanced. Impaired acetylcholine-induced relaxation and basal release of nitric oxide, suggests endothelial dysfunction after Hx. After supplementation with GH, SBP remained low while HR increased towards the control level. GH increased plasma IGF-I, but had no effect on vascular contractility or endothelial responses. E(2) replacement resulted in blunted plasma IGF-I, while the vascular adrenergic and serotonergic responses were reinforced. Endothelial function was not improved after E(2) treatment. When GH and E(2) were given in combination, the GH-induced increase in body weight, plasma IGF-I levels and HR were counteracted by E(2). Moreover, the anticipated reinforcement of the vascular serotonergic response by E(2) was reduced. Neither E(2) nor GH+E(2) affected SBP. The results suggest that GH and E(2) might have interactive effects on haemodynamic and metabolic parameters, but not on the contractility or endothelial function of resistance arteries, in Hx female rats.

Anti-atherosclerotic effect of beta-blocker with nitric oxide-releasing action on the severe atherosclerosis.

It is not completely understood whether nitric oxide donors and beta-adrenoceptor antagonists have anti-atherosclerotic effects. The anti-atherosclerotic effects of beta-adrenergic receptor antagonists and nitric oxide donors on severe atherosclerosis induced by cholesterol and inhibition of nitric oxide synthesis were determined. Six groups of New Zealand white male rabbits were treated for 10 weeks, under the following regimens: group I: high-cholesterol diet (HCD) (standard diet plus 0.5% cholesterol); group II: HCD plus N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase; group III: HCD plus L-NAME and isosorbide dinitrate; group IV: HCD plus L-NAME and nitroglycerin; group V: HCD plus L-NAME and nipradilol (beta-blocker with nitric oxide-releasing action); and group VI: HCD plus L-NAME and atenolol (beta-blocker). Serum lipid levels did not differ among the six groups. Blood pressure and heart rates were slightly decreased in groups V and VI. The atherosclerotic area and aortic cholesterol increased in L-NAME-treated animals but not in animals in group V. The endothelium-dependent relaxations and basal nitric oxide release were impaired in the L-NAME treatment group, though not in group V, in comparison with those in group I. cGMP in the aorta was increased in groups III, IV, and V as compared with that in group II. Endothelial nitric oxide synthase mRNA was decreased in the aortae of L-NAME-treated rabbits and increased in aortae in group V, in comparison with that in group I. Conclusively, nipradilol, beta-blocker with nitric oxide-releasing action, in contrast to the other beta-blockers and nitric oxide donors, showed a successful anti-atherosclerotic effect through the restoration of nitric oxide bioavailability and possible interaction with oxygen radicals.

Lack of impairment of nitric oxide-mediated responses in a rat model of high-renin hypertension.

1. Angiotensin (Ang) II triggers the expression of a pro- oxidant phenotype in the vascular wall, suggesting that activation of the renin-angiotensin system (RAS) causes endothelial dysfunction in various pathological situations, such as hypertension. However, this hypothesis has been mostly tested in a setting of exogenous administration of AngII. 2. We tested the hypothesis of a role for endogenous activation of the RAS leading to oxidant stress and endothelial dysfunction in a high-renin model of hypertension (i.e. two-kidney, one-clip hypertension) in rats. One month after clipping or sham surgery, aorta were isolated from untreated rats or rats treated by the angiotensin AT1 receptor antagonist irbesartan (10 mg/kg per day). Mesenteric artery segments were also isolated from normotensive or hypertensive rats. 3. Hypertension reduced the relaxations to acetylcholine but did not affect the ratio of contractions to phenylephrine in the presence compared with the absence of a nitric oxide (NO) synthase inhibitor, used as an index of basal release of NO. 4. The free radical scavenger tempol reduced the contractions to phenylephrine in the absence, but not in the presence, of an inhibitor of NO synthesis. This index of free radical-mediated degradation of NO was not affected by hypertension. In parallel, hypertension did not affect the expression of p22phox, a component of the free radical generating enzyme reduced nicotinamide adenine dinucleotide phosphate oxidase. 5. Chronic treatment with the AT1 receptor antagonist decreased blood pressure, moderately improved the response to acetylcholine, but did not affect basal NO release in hypertensive rats, although it did increase basal NO release in normotensive rats. 6. Thus, this model of hypertension is characterized by an impaired stimulated NO release but not of basal NO release in isolated arteries. Furthermore, there was no functional evidence of an increased oxidative stress-mediated impairment of NO release. This is not in favour of a direct link between activation of the RAS and development of endothelial dysfunction in experimental hypertension.

Gender differences in contractile and relaxing responses of aortic rings from lipopolysaccharide-treated rats

Aortic rings from male and female Sprague-Dawley rats were placed in organ chambers filled with Krebs solution, and the isometric tension was recorded. There were no gender differences in responses to potassiumchloride or phenylephrine in either intact or endothelium-denuded rings from control animals. Treatment with 10 mg/kg of lipopolysaccharide (LPS) attenuated the contractile response to potassium chloridein intact, but not in endothelium-denuded rings, and suppressed the phenylephrine contractions in rings both with and without endothelium from male and female rats. Endothelium-dependent and -independentrelaxation by acetylcholine and the nitric oxide donor diethylamine/NO, respectively, did not depend on gender and were equally suppressed by LPS treatment. Treatment with LPS accentuated the effects ofoxyhemoglobin in rings with and without endothelium and this was significantly larger in rings from females compared to males. Thus, gender differences were present in the effect of LPS on vascular responses.LPS induced a greater increase in basal release of nitric oxide and a greater inhibition of depolarization by potassium chloride, but not receptor-activated contractions, in aortic rings from female versusmale rats.

Effects of antihypertensive drugs on endothelial dysfunction: clinical implications.

Essential hypertension is associated with endothelial dysfunction, which is caused mainly by the production of oxygen-free radicals that can destroy nitric oxide (NO), and impair its beneficial and protective effects on the vessel wall. In prospective studies, endothelial dysfunction is associated with increased incidence of cardiovascular events. Antihypertensive drugs show contrasting effects in terms of improvement or restoration of endothelial function. Little evidence is available with beta-blockers. Whereas treatment with atenolol has a negative effect in peripheral subcutaneous and muscle microcirculation, insufficient evidence is available to establish whether new compounds such as nebivolol, which activates the L-Arginine--NO pathway, and carvedilol, which has strong antioxidant activity, can improve endothelial function in patients with hypertension. Calcium channel antagonists, particularly the dihydropyridines, can reverse impaired endothelium-dependent vasodilation in different vascular districts, including the subcutaneous, epicardial, renal and forearm circulation. However, conflicting results are found in the brachial artery. In the forearm circulation, nifedipine and lacidipine can improve endothelial dysfunction by restoring NO availability through a mechanism probably related to an antioxidant effect. ACE inhibitors, on the other hand, seem to improve endothelial function in subcutaneous, epicardial, brachial and renal circulation, whereas they are ineffective in potentiating the blunted response to acetylcholine in the forearm of patients with essential hypertension. They can also selectively improve endothelium-dependent vasodilation to bradykinin, an effect not mediated by restoring NO availability but probably related to hyperpolarisation. Recent evidence suggests angiotensin II AT(1)-receptor antagonists can restore endothelium-dependent vasodilation to acetylcholine in subcutaneous microcirculation but not in that of the forearm muscle. Evidence concerning the effect of these drugs on the brachial artery in patients with atherosclerosis is positive. However, treatment with an AT(1)-receptor antagonist can improve basal NO release and decrease the vasoconstrictor effect of endogenous endothelin-1. In conclusion, despite the considerable evidence that impaired endothelium-dependent vasodilation can be improved by appropriate antihypertensive treatment, no clinical data exist demonstrating that the reversal of endothelial dysfunction is associated with a reduction in cardiovascular events. In the near future, large scale clinical trials are required to demonstrate that treatment of endothelial dysfunction can lead to better prognosis in patients with essential hypertension.

Raloxifene enhances nitric oxide release in rat aorta via increasing endothelial nitric oxide mRNA expression

We report the modulatory effects of chronic oral LY139481 (raloxifene) on basal release of nitric oxide (NO) and mRNA levels of endothelial NO synthase (eNOS) in rat thoracic aorta. Constrictor dose–response curves to phenylephrine were generated before and after pretreatment with N ω-nitro- l-arginine methyl ester ( l-NAME), an inhibitor of NO synthase. Aortic segments were obtained from four groups of rats gavaged orally for 21 days: (i) ovariectomized, (ii) sham, (iii) ovariectomized estradiol-treated, and (iv) ovariectomized raloxifene-treated. Intact aortic rings from sham rats and ovariectomized rats receiving raloxifene and estrogen showed a greater potentiation of the phenylephrine responses after l-NAME. Semi-quantitative reverse transcription-polymerase chain reaction indicated a gender-based difference in eNOS mRNA expression in thoracic aorta. Moreover, we demonstrated that eNOS mRNA expression in the upper thoracic aorta was significantly higher in treatment groups. These results show that chronically administered raloxifene is exerting a potentially important vasculo-protective effect by stimulating eNOS expression.

Endothelial dysfunction and stroke.

Endothelial dysfunction, intended as the complex multifaced pathological product of different vasculotoxic agents or injuries, is viewed today as an attractant intermediate phenotype of cardiovascular diseases with usually long and unpredictable natural history. Furthermore, endothelial dysfunction may not only represent a vascular disease marker, but may actually play an important pathogenetic role, leading to progression of the disease and unfavourable outcomes. Among these vascular diseases, cerebrovascular accidents, namely stroke, clearly represent a paradigmatic example of the potential role of dysfunctional endothelium. In fact, in the world's growing elderly population few diseases are more dreaded than stroke. With an increasing incidence and mortality of 30%, stroke carries the threat of death or long-term disability and suffering. Endothelium produces nitric oxide (NO) under basal conditions and in response to a variety of vasoactive stimuli in large cerebral arteries and in the cerebral microcirculation. In addition to exerting a tonic dilator effect on the cerebral circulation, basal release of NO may protect cerebral endothelium by inhibiting aggregation of platelets and leukocytes. In this paper, we analyse current evidence suggesting that endothelial dysfunction can play a role in the pathogenesis of ischaemic stroke.

Direct Measurement of VEGF-Induced Nitric Oxide Production by Choroidal Endothelial Cells

Vascular endothelial growth factor (VEGF) and nitric oxide (NO) seem to be involved in the process of angiogenesis, but their interactions are not clearly understood. The aim of this study was to investigate the influence of VEGF on NO production of choroidal endothelial cells (CEC) and its importance in angiogenesis. Experiments were performed using cultured bovine CEC. Basal NO release of unstimulated CEC was measured and compared to NO release of VEGF-stimulated CEC (1, 10, and 100 ng/ml). Further, cells were pretreated with Nω-nitro-l-arginine methyl ester (l-NAME, 1 and 2 mM) and incubated with and without VEGF (10 ng/ml) to investigate the effect of blocking NO synthase. NO release into the medium was assessed by an amperometric NO sensor. To show the importance of NO in angiogenesis, proliferation and migration of CEC were measured after VEGF stimulation and in the presence or absence of l-NAME (1 and 2 mM). Unstimulated CEC continuously produced low levels of NO. Stimulation of the cells with VEGF resulted in a dose-dependent increase in NO release. The time course after stimulation with 10 ng/ml VEGF was characterized by a prompt initial rise up to 140% of unstimulated levels and a subsequent sustained increase over 120 min. Pretreatment with l-NAME attenuated the VEGF-induced response. l-NAME incubation alone led to a reduction in basal NO release. l-NAME also significantly diminished the VEGF-enhanced CEC proliferation and migration.The results demonstrate that VEGF enhances the formation of NO in cultured CEC. The blockade of NO production reduces CEC proliferation and migration, an effect which may be important for controlling angiogenesis, especially in reducing neovascularization in age-related macular degeneration in the eye.

Homocysteine-induced inhibition of nitric oxide production in platelets: a study on healthy and diabetic subjects.

The molecular mechanisms involved in the platelet activation observed in hyperhomocysteinemia are not known. We aimed to discover if homocysteine concentrations are associated with abnormal platelet nitric oxide production in healthy and diabetic subjects. The study cohort included 28 patients with Type I (insulin-dependent) diabetes mellitus, 30 patients with Type II (non-insulin-dependent) diabetes mellitus, and 34 healthy subjects. Homocysteine plasma concentrations were measured by high-performance liquid chromatography. Platelet nitric oxide production was measured using a nitric oxide meter before and after a 3-h incubation with 100 micromol/l homocysteine. Stimulation experiments were done in vitro by the addition of alpha-thrombin (0.2 U/ml). Basal platelet nitric oxide production was lower in diabetic patients than in healthy subjects. Nitric oxide release was reduced by in vitro homocysteine incubation, being lower in platelets from diabetic patients than in platelets from control subjects. Thrombin increased nitric oxide synthesis in platelets from healthy subjects both in the presence and absence of homocysteine. In diabetic subjects thrombin increased nitric oxide release in the absence of homocysteine. But in the presence of homocysteine the response was reduced. An inverse relation was found between plasma homocysteine levels and basal platelet nitric oxide release in diabetic and healthy subjects. Homocysteine could exert its atherogenic action in healthy and diabetic subjects partly by inhibiting platelet nitric oxide production with the subsequent increased platelet activation and aggregation.

HMG-CoA reductase inhibitor stabilizes rabbit atheroma by increasing basal NO and decreasing superoxide.

Male rabbits fed a 0.5% cholesterol diet for 8 wk were divided into three groups. Group 1 was hypercholesterolemic; group 2 was fed a regular diet for an additional 12 wk; and group 3 was fed a regular diet with simvastatin (5 mg x kg(-1) x day(-1)). Simvastatin treatment reduced the atherosclerotic area and total and esterified cholesterol concentrations in the thoracic aorta. Tone-related basal nitric oxide (NO) release was highest in group 3. Acetylcholine-induced, NO-dependent relaxation was improved in group 3 compared with group 2. Amount of endothelial nitric oxide synthase (eNOS) mRNA in vessels increased in group 1, compared with normal aorta, and decreased in group 2; however, it did not decrease in group 3. The amount of O released from vessels increased in group 1 and group 2 compared with normal rabbits; however, it decreased in group 3, especially in the endothelial cells. Peroxynitrite determined by nitrotyrosine staining decreased in group 3. Additionally, the arteries of rabbits fed a regular diet with or without simvastatin were investigated. The aorta from simvastatin-treated group showed increase of tone-related basal NO release and eNOS mRNA and decrease of O release. Taken together, upregulation of eNOS and decrease of O treatment were observed in vivo in the process of the sufficient stabilization of atheroma following simvastatin.

Basal tonic release of nitric oxide coupled to cGMP production regulates the vascular reactivity of the mesenteric bed

To reveal a basal production of nitric oxide (NO) and guanosine 3′,5′ cyclic monophosphate (cGMP) in the rat arterial mesenteric bed, mesenteries were perfused in the absence and in the presence of selective blockers of the l-arginine cascade. Endothelium removal or inhibition of NO synthase significantly reduced the release of NO and tissue cGMP. A significant correlation between these messengers was shown. Blockade of soluble guanylyl cyclase with 0.3–10 μM 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ) only reduced basal cGMP production; 1–100 nM sildenafil (Sild), an inhibitor of phosphodiesterase V, increased basal tissue cGMP without modifying the release of NO. Acetylcholine (0.01–10 μM) caused a concentration-dependent rise in NO and cGMP evoking a proportional vasodilatation, demonstrating the interdependence between these messengers and vascular reactivity. Endothelium removal or NO synthase blockade reduced the acetylcholine-induced increase of messengers and the vasodilatation. ODQ attenuated only the increase in cGMP and the vasodilatation, while sildenafil increased cGMP without significantly altering luminal NO release. The present results highlight a tonic release of NO and its involvement in endothelial-smooth muscle signaling; NO and cGMP are determinants of vascular reactivity.

Nitric oxide in the control of renal hemodynamics and excretory function

Experimental evidence has now been amassed to indicate that inhibition of nitric oxide (NO) synthase reduces total or regional renal blood flow by approximately 25 to 30% and markedly increases the renal vascular resistance, demonstrating that basal release of NO helps to maintain the relatively low vascular resistance that is characteristic for the kidney. It has been demonstrated that intraarterial administration of NO synthase inhibitors causes marked reductions in sodium excretion without changes in filtered load and suppressed the arterial pressure-induced natriuretic responses in the kidney. We also demonstrated that a constant rate infusion of a NO donor in dogs pretreated with a NOS inhibitor resulted in increases in sodium excretion but failed to restore the slope of the relation between arterial pressure and sodium excretion, suggesting that an alteration in intrarenal NO production rate during changes in arterial pressure is involved in the mediation of pressure natriuresis. Further experiments in dogs performed in our laboratory have confirmed that there is a direct relationship between changes in arterial pressure and intrarenal NO activity measured using NO-sensitive microelectrodes in the renal tissue. These arterial pressure-induced changes in intrarenal NO activity were seen positively correlated with the changes in urinary excretion rates of sodium. Collectively, these data suggest that acute changes in arterial pressure alter intrarenal NO production, which inhibits tubular sodium reabsorption to manifest the phenomenon of pressure natriuresis.

Caveolin-1 peptide exerts cardioprotective effects in myocardial ischemia-reperfusion via nitric oxide mechanism.

Caveolin-1 is a protein constituent of cell membranes. The caveolin-1 scaffolding region (residues 82-101) is a known inhibitor of protein kinase C. Inhibition of protein kinase C results in maintained nitric oxide (NO) release from the endothelium, which attenuates cardiac dysfunction after ischemia-reperfusion (I/R). Therefore, we hypothesized that the caveolin-1 scaffolding region of the molecule, termed caveolin-1 peptide, might attenuate postischemia polymorphonuclear neutrophil (PMN)-induced cardiac dysfunction. We examined the effects of caveolin-1 peptide in isolated ischemic (20 min) and reperfused (45 min) rat hearts reperfused with PMNs. Caveolin-1 peptide (165 or 330 microg) given intravenously 1 h before I/R significantly attenuated postischemic PMN-induced cardiac dysfunction, as exemplified by left ventricular developed pressure (LVDP) (P < 0.01) and the maximal rate of developed pressure (+dP/dt(max)) (P < 0.01), compared with I/R hearts obtained from rats given 0.9% NaCl. In addition, caveolin-1 peptide significantly reduced cardiac PMN infiltration from 195 +/- 5 PMNs/mm2 in untreated hearts to 103 +/- 5 and 60 +/- 5 PMNs/mm2 in hearts from 165 and 330 microg caveolin-1 peptide-treated rats, respectively (P < 0.01). PMN adherence to the rat coronary vasculature was also significantly reduced in rats given either 165 or 330 microg caveolin-1 peptide compared with rats given 0.9% NaCl (P < 0.01). Moreover, caveolin-1 peptide-treated rat aortas exhibited a 2.2-fold greater basal release of NO than vehicle-treated aortas (P < 0.01), and this was inhibited by NG-nitro-L-arginine methyl ester. These results provide evidence that caveolin-1 peptide significantly attenuated PMN-induced post-I/R cardiac contractile dysfunction in the isolated perfused rat heart, probably via enhanced release of endothelium-derived NO.

Valsartan improves vascular endothelial dysfunction in essential hypertension

Vascular endothelial dysfunction can be demonstrated in essential hypertension, and reversing this abnormality is an attractive therapeutic objective. We tested whether this could be achieved by blocking the AT1 receptor and compared this with calcium channel blockade. 12 hypertensives [mean age 60 (SD 8) years] with a mean ambulatory blood pressure of 154(10)/97(6) mmHg were randomized following a 2-week placebo (baseline) run-in period in a single blind crossover fashion to 8 week treatment period with either valsartan or amlodipine, separated by 2-week placebo washout period. Forearm venous occlusion plethysmography and intra-arterial infusions of acetylcholine (ACh) and NG-monomethyl-L-arginine (L-NMMA) were used to assess stimulated and basal endothelium-dependent nitric oxide (NO) release, respectively. Sodium nitroprusside (SNP) was used as endothelium-independent NO (control vasodilator). Valsartan and amlodipine each lowered the clinic blood pressure to the same extent (Table). The vasodilatory response to ACh was significantly increased with valsartan but remained unchanged with amlodipine. The response to SNP was not different between treatments suggesting that the endothelium-independent pathway was unaffected. Valsartan and amlodipine similarly increased the vasoconstrictive response to L-NMMA (Table). p<0.05 versus baseline; p<0.001 versus baseline.[table] p<0.05 versus baseline; p<0.001 versus baseline.[table] Valsartan, an AT1 receptor antagonist, increased both stimulated and basal endothelial nitric oxide release whereas for the same degree of blood pressure control, amlodipine had no effect on stimulated NO release. Thus, AT1 receptor blockade may offer superior vascular protection in hypertension. Table legend: SBP/DBP systolic and diastolic blood pressure (SD); FBF=Forearm Blood Flow (SEM)