Decreased expression of neuronal nitric oxide synthase contributes to the endothelial dysfunction associated with cigarette smoking in human.

Endothelial nitric oxide synthase (eNOS) malfunctioning has been proposed to contribute to the endothelial damage produced by cigarette. Besides eNOS, neuronal NOS (nNOS) is also expressed in most vascular tissues and plays an important role in the endothelium-dependent vascular relaxation. We hypothesize that nNOS may contribute to the endothelium dysfunction produced by cigarette in smokers. Vascular function was assessed in human resistance mesenteric arteries using a wire myograph, the level of protein expression by Western blot, eNOS and nNOS localization by immunofluorescence. Measurement of NO was assessed by fluorescence microscopy. Arteries of smokers showed impaired endothelium-dependent vascular relaxation in response to acetylcholine. Pharmacological nonselective blockade of NOS with l-NAME and selective nNOS blockade with inhibitor 1 reduced the relaxation of the mesenteric artery of both smokers and nonsmokers. Interestingly, the inhibitory effect of NOS inhibitors was greater in nonsmokers than in smokers. The expression of total nNOS and eNOS and the level of phosphorylation at eNOS-pSer1177 were reduced in arteries of smokers as compared with nonsmokers. No differences between groups were observed in the expression of total COX-1, COX-2, catalase and SOD-1. Immunofluorescence analysis showed the presence of nNOS in the vascular endothelium in both groups. Acetylcholine-induced NO production was impaired in arteries from smokers as compared to nonsmokers. Selective inhibition of nNOS caused a decreased in NO production, which was greater in nonsmokers than in smokers. Our data show that a decrease in nNOS expression contributes to the endothelial dysfunction caused by cigarette smoking in human.

Neuronal nitric oxide synthase contributes to the normalization of blood pressure in medicated hypertensive patients.

Neuronal nitric oxide synthase (nNOS) is expressed in the cardiovascular system and besides NO, generates H2O2. nNOS has been proposed to contribute to the control of blood pressure in healthy humans. The aim of this study was to verify the hypothesis that nNOS can contribute to the control of vascular relaxation and blood pressure in hypertensive patients undergoing drug treatment. The study was conducted in resistance mesenteric arteries from 63 individuals, as follows: 1) normotensive patients; 2) controlled hypertensive patients (patients on antihypertensive treatment with blood pressure normalized); 3) uncontrolled hypertensive patients (patients on antihypertensive treatment that remained hypertensive). Only mesenteric arteries from uncontrolled hypertensive patients showed impaired endothelium-dependent vasorelaxation in response to acetylcholine (ACh). Selective nNOS blockade with inhibitor 1 and catalase, which decomposes H2O2, decreased vasorelaxation in the three groups. However, the inhibitory effect was greater in controlled hypertensive patients. Decreased eNOS expression was detected in both uncontrolled and controlled hypertensive groups. Interestingly nNOS expression and ACh-stimulated H2O2 production were greater in controlled hypertensive patients, than in the other groups. ACh-stimulated NO production was lower in controlled hypertensive when compared to normotensive patients, while uncontrolled hypertensive patients showed the lowest levels. Catalase and nNOS blockade inhibited ACh-induced H2O2 production. In conclusion, nNOS-derived H2O2 contributes to the endothelium-dependent vascular relaxation in human resistance mesenteric arteries. The endothelial dysfunction observed in uncontrolled hypertensive patients involves decreased eNOS expression and NO production. The normalization of vascular relaxation and blood pressure in controlled hypertensive patients involves increased nNOS-derived H2O2 and NO production.

Neuronal Nitric Oxide Synthase in Vascular Physiology and Diseases.

The family of nitric oxide synthases (NOS) has significant importance in various physiological mechanisms and is also involved in many pathological processes. Three NOS isoforms have been identified: neuronal NOS (nNOS or NOS 1), endothelial NOS (eNOS or NOS 3), and an inducible NOS (iNOS or NOS 2). Both nNOS and eNOS are constitutively expressed. Classically, eNOS is considered the main isoform involved in the control of the vascular function. However, more recent studies have shown that nNOS is present in the vascular endothelium and importantly contributes to the maintenance of the homeostasis of the cardiovascular system. In physiological conditions, besides nitric oxide (NO), nNOS also produces hydrogen peroxide (H2O2) and superoxide ([Formula: see text]) considered as key mediators in non-neuronal cells signaling. This mini-review highlights recent scientific releases on the role of nNOS in vascular homeostasis and cardiovascular disorders such as hypertension and atherosclerosis.

Dihydrogoniothalamin, an Endothelium and NO-Dependent Vasodilator Drug Isolated from Aniba panurensis.

Dihydrogoniothalamin is a styrylpyrone isolated from the leaves of Aniba panurensis. The present work aimed at investigating the vasorelaxant activity of dihydrogoniothalamin and its underlying mechanism of action in the rat aorta. Dihydrogoniothalamin (0.01-100 µM) induced a concentration-dependent vasodilatation of aortas precontracted with phenylephrine. Endothelium removal or pretreatment of the preparation with NG nitro-L-arginine-methyl-ester abolished the vasodilator response for dihydrogoniothalamin. Pretreatment with calmidazolium did not affect the vasodilator response of dihydrogoniothalamin. On the other hand, wortmannin, a nonselective inhibitor of phosphatidylinositol 3-kinases, and protein kinase B inhibitor IV significantly shifted the concentration-response curve of dihydrogoniothalamin to the right and reduced its maximal effect. A nonselective antagonist of estrogen receptors, ICI 182,780, and a selective antagonist of estrogen receptor α, methyl-piperidino-pyrazole, were able to reduce the relaxation induced by dihydrogoniothalamin, but no effect was observed in the presence of the selective antagonists of estrogen receptor ß and G protein-coupled receptor 30, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), and G-15, respectively. Dihydrogoniothalamin also increased the phosphorylation of the activation sites of endothelial nitric oxide synthase and protein kinase B. The present results led us to conclude that dihydrogoniothalamin is a vasodilator drug acting in an endothelium- and nitric oxide-dependent manner through a mechanism involving the activation of nitric oxide synthase via the phosphatidylinositol 3-kinase/protein kinase B pathway, partially by stimulation of estrogen receptor α.

Mechanism of the antihypertensive and vasorelaxant effects of the flavonoid tiliroside in resistance arteries.

Hypertension is a leading cause of death and disability globally, and its prevalence continues to accelerate. The cardiovascular effects of the flavonoid tiliroside have never been reported. In this work, using complementary in vivo and in vitro approaches, we describe the antihypertensive effect of tiliroside and the underlying mechanisms involved in the reduction of blood pressure. Tiliroside (1, 5 or 10 mg/kg) induced a dose-dependent long-lasting decrease in blood pressure in conscious DOCA-salt hypertensive rats that was accompanied by an increased heart rate. Tiliroside also induced a concentration-dependent vasodilation of mesenteric resistance arteries precontracted with phenylephrine. Removal of the endothelium or pretreatment of the preparation with L-NAME or indomethacin did not modify the vasodilator response for tiliroside. When vessels were precontracted with a high K⁺ (50 mM) solution, tiliroside exhibited a vasodilator effect similar to that observed in vessels precontracted with phenylephrine. Experiments carried out in nominally Ca²âº-free solution showed that tiliroside antagonized CaCl2-induced contractions. Moreover, tiliroside reduced the rise in intracellular Ca²âº concentration induced by membrane depolarization in vascular smooth muscle cells. Finally, tiliroside decreased the voltage-activated peak amplitude of the L-type Ca²âº channel current in freshly dissociated vascular smooth muscle cells from mesenteric arteries. Altogether, our results point to an antihypertensive effect of tiliroside due to a reduction in peripheral resistance through blockage of voltage-activated peak amplitude of the L-type Ca²âº channel in smooth muscle cells.

Increased expression of endothelial iNOS accounts for hyporesponsiveness of pulmonary artery to vasoconstrictors after paraquat poisoning.

Paraquat is a toxic herbicide that induces severe acute lung injury (ALI) and pulmonary hypertension in humans. Although vascular disorders are present and contribute to increased mortality in ALI patients, there is little data available on vascular responsiveness after toxic exposure to paraquat. We aimed to evaluate the vascular response of isolated pulmonary arteries from rats treated with a dose of paraquat that induces ALI. Paraquat treatment did not modify the relaxant response of pulmonary artery to acetylcholine, but greatly reduced phenylephrine-induced contraction. Removal of the endothelium, inhibition of nitric oxide synthase (NOS) with L-NAME or selective inhibition of inducible NOS (iNOS) with L-NIL, restored contraction of vessels from paraquat poisoned rats to the same level as those not exposed to paraquat. The basal production of NO and expression of iNOS were increased in endothelium-intact but not in endothelium-denuded vessels from paraquat-poisoned rats. Expression of endothelial NOS was not modified. Our findings suggest that paraquat poisoning increases endothelial iNOS expression and basal NO production decreasing responsiveness of pulmonary artery to vasoconstrictors. Thus, our results do not support the hypothesis that pulmonary hypertension in paraquat-induced ALI is mediated by a reduction in endothelial NO production or increased contractility of pulmonary artery.

Complexation with beta-cyclodextrin confers oral activity on the flavonoid dioclein.

Dioclein is a flavonoid reported to have many beneficial effects on the cardiovascular system such as vasorelaxant, hypotensive, antioxidant and antiarrythmogenic activities. However, use as pharmaceuticals is limited due to the lack of oral activity and low water solubility. In this work, intending to improve its oral activity, we performed a 1:1 inclusion complex (IC) between dioclein and beta-cyclodextrin (beta-CD). The IC was characterized by nuclear magnetic resonance and infrared spectroscopy and its vasodilator and hypotensive effects were evaluated in mice. The inclusion of dioclein in beta-CD increased the water solubility 44% compared to free dioclein. The IC (2.5mgkg(-1)) produced a higher and long lasting change in systolic blood pressure (SBP) after intraperitoneal administration compared to free dioclein. When given orally, free dioclein (10mgkg(-1)) showed no hypotensive effect while the IC induced a pronounced decrease in SBP. The in vitro vasodilator effect of dioclein was unchanged by its inclusion in beta-CD showing that the IC does not change the interaction between dioclein and its cellular targets. In conclusion, our results show that the new complex prepared by inclusion of dioclein in beta-CD improves the hypotensive effect of the flavonoid by increasing its bioavailability and enables dioclein to be effective after oral administration. The mechanism underling the increase in bioavailability is probably a consequence of a protective effect of beta-CD against in vivo biodegradation by enzymes and possibly increased water solubility.

Activation of nitric oxide modulator effect by isometric contraction in rat resistance arteries.

The participation of nitric oxide (NO) as a modulator of the isometric contraction, as well as the underlying mechanism, was investigated in rat small mesenteric arteries. In the presence of a functional endothelium, L-NAME and L-NA similarly increased the contractions induced by phenylephrine, dependently on the level of contraction. However, no effect was observed in the absence of a functional endothelium. In the presence of selective inhibitors of protein kinase C (PKC) and phosphatidyl-inositol 3-kinase (PI3K), calphostin-C and wortmannin, respectively, the effect of L-NAME was not modified. The same observation was done in the presence of the tyrosine kinase inhibitors, genistein and tyrphostin A-23. However, in the presence of a Ca2+-independent tyrosine kinase inhibitor, erbstatin-A, and on the presence of a non-selective kinase inhibitor, staurosporine, a strong reduction was observed. Our results suggest that protein kinases are involved in the activation of nitric oxide modulator effect on isometric contractions in resistance arteries.

Mechanisms involved in the vasodilator effect of the flavanol floranol in rat small mesenteric arteries.

The mechanism underlying the vasodilator effect of the flavonoid floranol was studied in rat small mesenteric arteries. Floranol produced a concentration-dependent vasorelaxant effect in endothelium-containing and endothelium-denuded vessels pre-contracted with phenylephrine, which was more potent in endothelium-intact vessels. In endothelium-intact mesenteric arteries, l-NAME but not indomethacin produced a shift to the right in the vasorelaxant effect of floranol. In endothelium-denuded vessels TEA and BaCl2 did not change the floranol-induced vasorelaxation. When endothelium-denuded vessels were pre-contracted with 50 mM KCl, floranol induced a vasorelaxant effect comparable with phenylephrine pre-contracted vessels. We conclude that floranol is a new vasodilator compound in rat small mesenteric arteries. Part of this effect is dependent on endothelial nitric oxide (NO) and part is dependent on the inhibition of voltage-dependent calcium channels in the smooth muscle cells.