Endothelium-dependent vasorelaxation to the AMPK activator AICAR is enhanced in aorta from hypertensive rats and is NO and EDCF dependent

Activation of AMP-activated protein kinase (AMPK) induces vasorelaxation in arteries from healthy animals, but the mechanisms coordinating this effect are unclear and the integrity of this response has not been investigated in dysfunctional arteries of hypertensive animals. Here we investigate the mechanisms of relaxation to the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) in isolated thoracic aorta rings from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Although AICAR generated dose-dependent (10(-6)-10(-2) M) relaxation in precontracted WKY and SHR aortic rings with (E(+)) or without (E(-)) endothelium, relaxation was enhanced in E(+) rings. Relaxation in SHR E(+) rings was also enhanced at low [AICAR] (10(-6) M) compared with that of WKY (57 ± 8% vs. 3 ± 2% relaxation in SHR vs. WKY E(+)), but was similar and near 100% in both groups at high [AICAR]. Pharmacological dissection showed that the mechanisms responsible for the endothelium-dependent component of relaxation across the dose range of AICAR are exclusively nitric oxide (NO) mediated in WKY rings, but partly NO dependent and partly cyclooxygenase (COX) dependent in SHR vessels. Further investigation revealed that ACh-stimulated COX-endothelium-derived contracting factors (EDCF)-mediated contractions were suppressed by AICAR, and this effect was reversed in the presence of the AMPK inhibitor Compound C in quiescent E(+) SHR aortic rings. Western blots demonstrated that P(Thr(172))-AMPK and P(Ser(79))-acetyl-CoA carboxylase (indexes of AMPK activation) were elevated in SHR versus WKY E(+) rings at low AICAR (～2-fold). Together these findings suggest that AMPK-mediated inhibition of EDCF-dependent contraction and elevated AMPK activation may contribute to the enhanced sensitivity of SHR E(+) rings to AICAR. These results demonstrate AMPK-mediated vasorelaxation is present and enhanced in arteries of SHR and suggest that activation of AMPK may be a potential strategy to improve vasomotor dysfunction by suppressing enhanced endoperoxide-mediated contraction and enhancing NO-mediated relaxation.     

autoregulatory role of endothelium-derived nitric oxide (NO) on Lipopolysaccharide-induced vascular inducible NO synthase expression and function

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.     

Alpha-lactorphin and beta-lactorphin improve arterial function in spontaneously hypertensive rats

alpha-lactorphin (Tyr-Gly-Leu-Phe) lowers blood pressure in conscious adult SHR. This tetrapeptide is originally released from milk protein alpha-lactalbumin by enzymatic hydrolysis. In order to evaluate the antihypertensive mechanisms of alpha-lactorphin, the effects of the tetrapeptide on vascular function were investigated in (30-35 weeks old) spontaneously hypertensive rats (SHR) with established hypertension and age-matched normotensive Wistar-Kyoto (WKY) rats in vitro. In addition, we studied the vascular effects of another structurally related tetrapeptide, beta-lactorphin (Tyr-Leu-Leu-Phe), which originates from milk protein beta-lactoglobulin. Endothelium-dependent relaxation to acetylcholine (ACh) was reduced in mesenteric arterial preparations of SHR as compared to those of WKY. In SHR, the ACh-induced relaxation was augmented by alpha-lactorphin or beta-lactorphin. The role of nitric oxide (NO) is suggested, since this improvement was abolished by the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Simultaneous potassium channel inhibitor tetraethylammonium (TEA) elicited no additional effect on the ACh-induced relaxation. The cyclooxygenase inhibitor diclofenac did not attenuate the augmented ACh relaxation induced by alpha-lactorphin or beta-lactorphin, suggesting that endothelial vasodilatory prostanoids were not involved in the effect of the tetrapeptides. Endothelium-independent relaxation to the NO donor sodium nitroprusside (SNP) was augmented in mesenteric arterial preparations of SHR by simultaneous beta-lactorphin. The tetrapeptides did not alter vascular responses in mesenteric arteries from WKY. In conclusion, both alpha-lactorphin and beta-lactorphin improved vascular relaxation in adult SHR in vitro. The beneficial effect of alpha-lactorphin was directed towards endothelial function, whereas beta-lactorphin also enhanced endothelium-independent relaxation.     

A macrocyclic nitrosyl ruthenium complex is a NO donor that induces rat aorta relaxation.

The vasorelaxation induced by a nitrosyl macrocyclic ruthenium complex, proposed as a new nitric oxide (NO) carrier, was studied in rat isolated aorta. The compound trans-[RuCl([15]aneN4)NO]2+ was characterized by elemental analysis, UV-visible spectrum, and infrared spectrum. Based on the electrochemical process, the reduction of the compound was followed by NO release, which was also observed using norepinephrine as a reducing agent and NO released was analyzed by a sensor. Vasorelaxation induced by this NO donor was studied and compared to those obtained with sodium nitroprusside (SNP). The relaxation induced by the compound was concentration-dependent in denuded rat aortas and occurred only in pre-contracted arteries with norepinephrine. The macrocyclic compound induced relaxation with a similar efficacy as SNP, although the potency of SNP was slightly greater. The time to reach maximum relaxation (595 s) was longer than that of SNP (195 s). Relaxation was completely abolished by oxyhemoglobin, a known NO scavenger.     

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F(1α) (6-keto-PGF(1α)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1α) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS.     

Link between free radicals and protein kinase C in glucose-induced alteration of vascular dilation

Development of vascular complications in diabetes has been linked to the quality of glucose regulation and characterized by endothelial dysfunction. The exact mechanism behind vascular complications in diabetes is poorly understood. However, alteration of nitric oxide (NO) biosynthesis or bioactivity is strongly implicated and the mechanism behind such alterations is still a subject for research investigations. In the present study, we tested the hypothesis that glucose-induced attenuation of vascular relaxation involves protein kinase C (PKC)-linked generation of free radicals. Vascular relaxation to acetylcholine (ACh; 10(-9)-10(-5) M), isoproterenol (10(-9)-10(-5) M), or NO donor, sodium nitropruside (SNP; 10(-9)-10(-6) M) was determined in phenylephrine (PE, 10(-7) M) pre-constricted aortic rings from Sprague-Dawley rats in the presence or absence of 30 mM glucose (30 min), L-nitro-arginine methyl ester (L-NAME; 10(-4) M for 15 min), a NO synthase inhibitor, or xanthine (10(-5) M), a free radical generator. ACh dose-dependently caused relaxation that was attenuated by L-NAME, glucose, or xanthine. Pre-incubation (15 min) of the rings with vitamin C (10(-4) M), an antioxidant or calphostin C (10(-6) M), a PKC inhibitor, restored the ACh responses. However, high glucose had no significant effects on SNP or isoproterenol-induced relaxation. ACh-induced NO production by aortic ring was significantly reduced by glucose or xanthine. The reduced NO production was restored by pretreatment with vitamin C or calphostin C in the presence of glucose, but not xanthine. These data demonstrate that oxidants or PKC contribute to glucose-induced attenuation of vasorelaxation which could be mediated via impaired endothelial NO production and bioavailability. Thus, pathogenesis of glucose-induced vasculopathy involves PKC-coupled generation of oxygen free radicals which inhibit NO production and selectively inhibit NO-dependent relaxation.     

Reduction of asymmetric dimethylarginine involved in the cardioprotective effect of losartan in spontaneously hypertensive rats

Previous studies have indicated that nitric oxide synthase (NOS) inhibitors can induce an increase of blood pressure and exacerbate myocardial injury induced by ischemia and reperfusion, whereas angiotensin II receptor antagonists protect the myocardium against injury induced by ischemia and reperfusion. Isolated hearts from male spontaneously hypertensive rats (SHR) or male Wistar-Kyoto rats (WKY) were subjected to 20 min global ischemia and 30 min reperfusion. Heart rate, coronary flow, left ventricular pressure, and its first derivatives (+/-dP/dt(max)) were recorded, and serum concentrations of asymmetric dimethylarginine (ADMA) and NO and the release of creatine kinase in coronary effluent were measured. The level of ADMA was significantly increased and the concentration of NO was decreased in SHR. Ischemia and reperfusion significantly inhibited the recovery of cardiac function and increased the release of creatine kinase, and ischemia and reperfusion-induced myocardial injury in SHR was aggravated compared with WKY. Vasodilation responses to acetylcholine of aortic rings were decreased in SHR. Treatment with losartan (30 mg/kg) for 14 days significantly lowered blood pressure, elevated the plasma level of NO, and decreased the plasma concentration of ADMA in SHR. Treatment with losartan significantly improved endothelium-dependent relaxation and cardiac function during ischemia and reperfusion in SHR. Exogenous ADMA also aggravated myocardial injury induced by ischemia and reperfusion in isolated perfused heart of WKY, as shown by increasing creatine kinase release and decreasing cardiac function. The present results suggest that the protective effect of losartan on myocardial injury induced by ischemia and reperfusion is related to the reduction of ADMA levels.     

Nitric oxide relaxes rat tail artery smooth muscle by cyclic GMP-independent decrease in calcium sensitivity of myofilaments

The effects of authentic nitric oxide (NO, 10(-6) M) and NO-donors such as sodium nitroprusside (SNP, 10(-5) M) and glyceryl trinitrate (GTN, 10(-4) M) on contractile force and free intracellular calcium level ([Ca2+]i) were studied on precontracted with high potassium chloride (KCl, 70 mM) isolated rings of rat tail artery. The sensitivity of contractile myofilaments to Ca2+ was measured using chemically permeabilized (alpha-toxin, beta-escin, Triton X-100) vascular rings. [Ca2+]i and contractile activity were measured simultaneously. The relationship of [Ca2+]i and tension developed was studied in endothelium-denuded rings and controlled calcium response was evaluated in both endothelium-denuded and permeabilized vascular rings. Both authentic NO and NO-donors decreased [Ca2+]i and high potassium-induced tension with a different time course. Inhibitor of soluble guanylyl cyclase (sGC) LY83583 (10(-5) M) did not affect SNP-induced relaxation whereas the other sGC inhibitor ODQ (10(-6) M) attenuated SNP-induced relaxation. Both inhibitors had no effect on NO- and SNP-induced reduction in [Ca2+]i. On the contrary, GTN induced neither relaxation nor decrease in [Ca2+]i on application of both LY83583 and ODQ. Tail artery rings permeabilized with alpha-toxin, beta-escin, but not with Triton X-100 were relaxed by authentic NO and NO-donors, but to a less extent than non-permeabilized rings. Dithioerythritol (DTE, 5 x 10(-3) M) that maintains sulfhydryl (SH) groups in reduced state preventing their nitrosylation attenuated NO-induced relaxation in both non-permeabilized and permeabilized tail artery rings. The cyclic heptapeptide mycrocystin-LR (MC-LR) (10(-5) M), an inhibitor of type 1 and 2A phosphatases, induced sustained increase in tension of beta-escin permeabilized rings in low Ca2+ (10(-8) M) solution. The tension was not affected by authentic NO and SNP. We conclude that authentic NO and SNP relax rat tail artery smooth muscle (SM) in the presence of inhibitors of sGC via cyclic guanosine monophosphate (cGMP)-independent pathway, whereas relaxation induced by GTN is inhibited. The data demonstrate that cGMP-dependent pathway in vascular smooth muscle is ubiquitous, but not the only way of relaxation induced by NO. NO can modulate vascular tone directly by reducing sensitivity of contractile myofilaments to [Ca2+]i and may involve activation of protein phosphatase(s).     

Iptakalim ameliorates relaxation to acetylcholine in thoracic aortic rings impaired by microvesicles derived from hypoxia/reoxygenation-treated HUVECs

Objective: To investigate the effect of Iptakalim(Ipt) preventing injury of endothelial microvesicles(EMVs) derived from hypoxia/reoxygenation(H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism. Methods: H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope(TEM). Thoracic aortic rings of rats were incubated with 10~(-7)-10~(-3 )mol/L Ipt and co-cultured with 10 μg/ml H/R-EMVs for 4 hours, and their endothelium- dependent relaxation in response to acetylcholine(ACh) was recorded in vitro. The nitric oxide(NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase(e NOS), phosphorylated e NOS(p-e NOS, Ser-1177), serine/threonine kinas(Akt) and phosphorylated Akt(p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting. Results: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles(100–1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner(P0.05, P0.01). The expression of total e NOS(t-e NOS) and total Akt(t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-e NOS and p-Akt increased after treated with Ipt(P0.01). Conclusion: Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-e NOS and p-Akt expression.     

Comparative effects of melatonin and vitamin E in restoring aortic relaxation in pancreatectomized rats

In a previous study we reported the efficacy of melatonin to restore the decreased relaxation response to acetylcholine (ACh) or to sodium nitroprusside (SNP) in aortic rings of rats turned hyperglycemic by subtotal pancreatectomy. The effect was amplified by pre-incubation in a high (44 mmol/l) glucose solution, a situation that resulted in oxidative stress. We hereby compare the effect of another antioxidant, vitamin E, with that of melatonin on ACh response in intact aortic rings or on SNP response in endothelium-denuded aortic rings obtained from pancreatectomized or sham-operated rats. Dose-response curves to ACh or SNP were performed in the presence or absence of melatonin or vitamin E (10-5 mol/1) in 10 or 44 mmol/1 glucose medium. Melatonin was more effective than vitamin E in restoring ACh- or SNP-induced relaxation of aortic rings in a high glucose medium. The differences between the two antioxidants may rely on the ability of melatonin to diffuse readily into intracellular compartments.     

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.     

Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors

We tested the hypothesis that previously demonstrated gender differences in ACh-induced vascular relaxation could involve diverse Na(+)-K(+)-ATPase functions. We determined Na(+)-K(+)-ATPase by measuring arterial ouabain-sensitive 86Rb uptake in response to ACh. We found a significant increase of Na+ pump activity only in aortic rings from female rats (control 206 +/- 11 vs. 367 +/- 29 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.01). Ovariectomy eliminated sex differences in Na(+)-K(+)-ATPase function, and chronic in vivo hormone replacement with 17beta-estradiol restored the ACh effect on Na(+)-K(+)-ATPase. Because ACh acts by enhancing production of NO, we examined whether the NO donor sodium nitroprusside (SNP) mimics the action of ACh on Na(+)-K(+)-ATPase activity. SNP increased ouabain-sensitive 86Rb uptake in denuded female arteries (control 123 +/- 7 vs. 197 +/- 12 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.05). Methylene blue (an inhibitor of guanylate cyclase) and KT-5823 (a cGMP-dependent kinase inhibitor) blocked the stimulatory action of SNP. Exposure of female thoracic aorta to the Na+/K+ pump inhibitor ouabain significantly decreased SNP-induced and ACh-mediated relaxation of aortic rings. At the molecular level, Western blot analysis of arterial tissue revealed significant gender differences in the relative abundance of catalytic isoforms of Na(+)-K(+)-ATPase. Female-derived aortas exhibited a greater proportion of alpha2-isoform (44%) compared with male-derived aortas. Furthermore, estradiol upregulated the expression of alpha2 mRNA in male arterial explants. Our results demonstrate that enhancement of ACh-induced relaxation observed in female rats may be in part explained by 1) NO-dependent increased Na(+)-K(+)-ATPase activity in female vascular tissue and 2) greater abundance of Na(+)-K(+)-ATPase alpha2-isoform in females.     

Effect of diabetes on aortic nitric oxide synthesis in spontaneously hypertensive rats; does captopril modulate this effect?

Nitric oxide (NO) is a potent regulator in the cardiovascular system; it is generated by the nitric oxide synthase (NOS) family of proteins. NO produced in endothelial cells plays a crucial role in vascular functions. The aim of this study was to clarify the effect of diabetes on aortic NO synthesis in a model of genetic hypertension and determine whether captopril modulates this effect. Diabetes was induced in ten weeks old spontaneously hypertensive rats (SHR) by streptozotocin injection. The rats were allocated into 3 groups: control group 1, non-diabetic SHR; group 2, diabetic SHR; group 3, diabetic SHR group receiving captopril at 80 mg/kg in drinking water for 4 weeks. Mean blood pressure (MBP) was measured once a week by tail-cuff method. Aortic NO metabolities (nitrite/nitrate) and endothelial NOS (NOS-3) were assayed by Griess reaction and by immunoblotting and immunohistochemistry, respectively. There was a significant decrease in nitrite/nitrate (NOx) in aortas of diabetic SHR compared with controls. The decrease of aortic NOx in diabetic SHR was accompanied by a decrease in NOS-3 expression. Captopril treatment reduced MBP without affecting either NOx level or NOS-3 expression in aortas of diabetic SHR. We conclude that STZ-induced diabetes decreased NO in aortas of SHR that may reflect endothelial cell dysfunction; captopril administration decreased MBP without affecting NO level in aortas of diabetic SHR which suggest that the blood pressure-lowering effects of captopril were independent of NO.     

Vascular relaxation mediated by hydroxylamines and oximes: their conversion to nitrites and mechanism of endothelium dependent vascular relaxation

Hydroxylamines (R-NHOH) and oximes (R = NOH) relax rat aortic rings independent of the presence of the endothelium. The relaxation is inhibited by methylene blue, an inhibitor of soluble guanylate cyclase and by hemoglobin, an inhibitor of the endothelium dependent relaxing factor (EDRF). Both the oximes and hydroxylamines generate NO/NO2- ions on treatment with iodine in glacial acetic acid. However, there is no correlation between relaxation and NO/NO2- formation. Compared to hydroxylamines, the oximes are less potent relaxing agents and not efficiently converted to NO/NO2- ions. We suggest that endothelium dependent relaxation is associated with a hydroxylamine like compound and is not directly related to NO.     

Endothelium negatively modulates the vascular relaxation induced by nitric oxide donor, due to uncoupling NO synthase

Nitrosyl ruthenium complexes have been characterized as nitric oxide (NO) donors that induce relaxation in the denuded rat aorta. There are some differences in their vascular relaxation mechanisms compared with sodium nitroprusside. This study investigates whether the endothelium could interfere with the [Ru(terpy)(bdq)NO]³⁺-TERPY-induced vascular relaxation, by analyzing the maximal relaxation (Emax) and potency (pD₂) of TERPY. Vascular reactivity experiments showed that the endothelium negatively modulates (pD₂: 6.17 ± 0.07) the TERPY relaxation in intact rat aortic rings compared with the denuded rat aorta (pD₂: 6.65 ± 0.07). This effect is abolished by a non-selective NO-synthase (NOS) inhibitor L-NAME (pD₂: 6.46 ± 0.10), by the superoxide anion () scavenger TIRON (pD₂: 6.49 ± 0.08), and by an NOS cofactor BH₄ (pD₂: 6.80 ± 0.10). The selective dye for (DHE) shows that TERPY enhances concentration in isolated endothelial cells (intensity of fluorescence (IF):11258.00 ± 317.75) compared with the basal concentration (IF: 7760.67 ± 381.50), and this enhancement is blocked by L-NAME (IF: 8892.33 ± 1074.41). Similar results were observed in vascular smooth muscle cells (concentration of superoxide after TERPY: 2.63 ± 0.17% and after TERPY + L-NAME: −4.63 ± 0.14%). Considering that TERPY could induce uncoupling NOS, thus producing , we have also investigated the involvement of prostanoids in the negative modulation of the endothelium. The non-selective cyclooxygenase (COX) inhibitor indomethacin and the selective tromboxane (TXA₂) receptor antagonist SQ29548 reduce the effect of the endothelium on TERPY relaxation (pD₂ INDO: 6.80 ± 0.17 and SQ29548: 6.85 ± 0.15, respectively). However, a selective prostaglandin F_2α receptor antagonist (AH6809) does not change the endothelium effect. Moreover, TERPY enhances the concentration of TXA₂ stable metabolite (TXB₂), but this effect is blocked by L-NAME and TIRON. The present findings indicate that TERPY induces uncoupling of eNOS, enhancing concentration. This enhancement in concentration induces COX activation, producing TXA₂, which negatively modulates the rat aorta relaxation induced by the NO donor TERPY.     

L-carnitine and propionyl-L-carnitine improve endothelial dysfunction in spontaneously hypertensive rats: different participation of NO and COX-products

L-carnitine and propionyl-L-carnitine are supplements to therapy in cardiovascular pathologies. Their effect on endothelial dysfunction in hypertension was studied after treatment with either 200 mg/kg of L-carnitine or propionyl-L-carnitine during 8 weeks of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Endothelial function was assessed in aortic rings by carbachol-induced relaxation (CCh 10(-8) to 10(-4) M) and factors involved were characterized in the presence of the inhibitors: L-NAME, indomethacin, the TXA2/PGH2 Tp receptor antagonist ICI-192,605 and the thromboxane synthetase inhibitor-Tp receptor antagonist, Ro-68,070. The effect on phenylephrine-induced contractions was also observed. To identify the nature of vasoactive COX-derived products, enzyme-immunoassay of incubation media was assessed. Involvement of reactive oxygen species was evaluated by incubating with superoxide dismutase and catalase. Nitric oxide production was evaluated by serum concentration of NO2+NO3.Treatment with both compounds improved endothelial function of rings from SHR without blood pressure change. Propionyl-L-carnitine increased NO participation in WKY and SHR. L-carnitine reduced endothelium-dependent responses to CCh in WKY due to an increase of TXA2 production. In both SHR and WKY, L-carnitine enhanced concentration of PGI2 and increased participation of NO. Results in the presence of SOD plus catalase show that it might be related to antioxidant properties of L-carnitine and propionyl-L-carnitine. Comparison between the effect of both compounds shows that both may reduce reactive oxygen species and increase NO participation in endothelium-dependent relaxations in SHR. However, only L-carnitine was able to increase the release of the vasodilator PGI2 and even enhanced TXA2 production in normotensive rats.     

The hypothesis of the main role of H2S in coupled sulphide-nitroso signalling pathway

As a part of the nitroso signalling pathway, nitroso-compounds serve as stores and carriers of NO; as part of the sulphide signalling pathway, bound sulfane-sulphur compounds serve as stores and carriers of H2S. Here we hypothesise a coupled sulphide-nitroso signalling pathway, in which H2S plays a main role. H2S releases NO from the endogenous S-nitroso-compounds nitroso-cysteine, nitroso-acetylcysteine and nitroso-albumin. Relaxation of noradrenaline-precontracted aortic rings by H2S is also enhanced in the presence of nitroso-albumin, which may implicate the involvement of the nitroso signalling pathway. Pretreatment of albumin, cysteine, N-acetylcysteine and lipids with H2S results in binding of sulphur to these compounds creating thus new-modified sulphur compounds that release NO from nitroso-compounds directly and/or through released H2S, which suggests sulphide-nitroso signalling pathway participation. This hypothesis is supported by the observation that the pretreatment of noradrenaline-precontracted aortic rings with H2S significantly enhanced relaxation induced by nitroso-glutathione in the absence of H2S. We assume that the NO release from nitroso-compounds directly by H2S or indirectly by the H2S-induced sulphur-bound compounds represents coupled sulphide-nitroso signalling, which may explain some of the numerous biological effects of H2S that are shared with NO.     

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca²⁺ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca²⁺ mobilization study shows that l-NAME inhibited the reduction of Ca²⁺-concentration in VSM cells and reduced the increase in Ca²⁺-concentration in endothelial cells induced by SNP. This enhancement in Ca²⁺-concentration in the endothelial cells is due to a voltage-dependent Ca²⁺ channels activation. The present findings indicate that the relaxation and [Ca²⁺]_i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.     

Involvement of Na(+)/Ca(2+) exchanger in endothelial NO production and endothelium-dependent relaxation

Endothelial nitric oxide (NO) synthase (eNOS) is controlled by Ca(2+)/calmodulin and caveolin-1 in caveolae. It has been recently suggested that Na(+)/Ca(2+) exchanger (NCX), also expressed in endothelial caveolae, is involved in eNOS activation. To investigate the role played by NCX in NO synthesis, we assessed the effects of Na(+) loading (induced by monensin) on rat aortic rings and cultured porcine aortic endothelial cells. Effect of monensin was evaluated by endothelium-dependent relaxation of rat aortic rings in response to acetylcholine and by real-time measurement of NO release from cultured endothelial cells stimulated by A-23187 and bradykinin. Na(+) loading shifted the acetylcholine concentration-response curve to the left. These effects were prevented by pretreatment with the NCX inhibitors benzamil and KB-R7943. Monensin potentiated Ca(2+)-dependent NO release in cultured cells, whereas benzamil and KB-R7943 totally blocked Na(+) loading-induced NO release. These findings confirm the key role of NCX in reverse mode on Ca(2+)-dependent NO production and endothelium-dependent relaxation.     

Menadione induces endothelial dysfunction mediated by oxidative stress and arylation

Our previous studies showed that menadione causes endothelial dysfunction which results in decreased relaxation and increased contraction of blood vessels. This investigation examined the role of two possible mechanisms (oxidative stress and arylation) in menadione-induced endothelial dysfunction. Menadione increased superoxide anion generation in aortic rings in a dose-dependent manner. Superoxide dismutase (SOD), reversed the inhibitory effects of menadione on vascular relaxation. The relaxation induced by the NO donor, sodium nitroprusside, was inhibited by menadione pretreatment in a dose-dependent manner. Endothelial nitric oxide synthase activity (eNOS) was suppressed by menadione. Menadione resulted in a dose-dependent reduction of cGMP levels accumulated by acetylcholine. This reduction of cGMP levels was blocked by SOD treatment, suggesting that superoxide anion generated by menadione could play a role in the inhibition of the nitric oxide pathway. Evidence supporting a possible role for arylation in impaired vascular relaxation was suggested by the observation that benzoquinone, which does not induce oxidative stress in aortic rings, inhibited acetylcholine-induced vascular relaxation to the same extent as menadione. Collectively, these results suggest that menadione can cause endothelial dysfunction in blood vessels by the inhibition of the nitric oxide pathway via superoxide anion generation and that arylation activity may also be another important mechanism.