Belén Climent

Role of Neural NO Synthase (nNOS) Uncoupling in the Dysfunctional Nitrergic Vasorelaxation of Penile Arteries from Insulin-Resistant Obese Zucker Rats

Objective Erectile dysfunction (ED) is considered as an early sign of vascular disease due to its high prevalence in patients with cardiovascular risk factors. Endothelial and neural dysfunction involving nitric oxide (NO) are usually implicated in the pathophysiology of the diabetic ED, but the underlying mechanisms are unclear. The present study assessed the role of oxidative stress in the dysfunctional neural vasodilator responses of penile arteries in the obese Zucker rat (OZR), an experimental model of metabolic syndrome/prediabetes. Methods and Results Electrical field stimulation (EFS) under non-adrenergic non-cholinergic (NANC) conditions evoked relaxations that were significantly reduced in penile arteries of OZR compared with those of lean Zucker rats (LZR). Blockade of NO synthase (NOS) inhibited neural relaxations in both LZR and OZR, while saturating concentrations of the NOS substrate L-arginine reversed the inhibition and restored relaxations in OZR to levels in arteries from LZR. nNOS expression was unchanged in arteries from OZR compared to LZR and nNOS selective inhibition decreased the EFS relaxations in LZR but not in OZR, while endothelium removal did not alter these responses in either strain. Superoxide anion production and nitro-tyrosine immunostaining were elevated in the erectile tissue from OZR. Treatment with the NADPH oxidase inhibitor apocynin or acute incubation with the NOS cofactor tetrahydrobiopterin (BH4) restored neural relaxations in OZR to levels in control arteries, while inhibition of the enzyme of BH4 synthesis GTP-cyclohydrolase (GCH) reduced neural relaxations in arteries from LZR but not OZR. The NO donor SNAP induced decreases in intracellular calcium that were impaired in arteries from OZR compared to controls. Conclusions The present study demonstrates nitrergic dysfunction and impaired neural NO signalling due to oxidative stress and nNOS uncoupling in penile arteries under conditions of insulin resistance. This dysfunction likely contributes to the metabolic syndrome-associated ED, along with the endothelial dysfunction also involving altered NO signalling.

Insulin resistance in penile arteries from a rat model of metabolic syndrome

BACKGROUND AND PURPOSE Metabolic and cardiovascular abnormalities accompanying metabolic syndrome, such as obesity, insulin resistance and hypertension, are all associated with endothelial dysfunction and are independent risk factors for erectile dysfunction. The purpose of the present study was to investigate the vascular effects of insulin in penile arteries and whether these effects are impaired in a rat model of insulin resistance and metabolic syndrome.

Signaling pathways involved in the H2O2-induced vasoconstriction of rat coronary arteries.

Hydrogen peroxide (H2O2) is an endogenous endothelium-derived hyperpolarizing factor released by flow and involved in the regulation of coronary blood flow. Because opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, the aim of this study was to assess whether H2O2 may act as a coronary vasoconstrictor and if so to determine the underlying signaling mechanisms. Intramyocardial arteries from male Wistar rats were mounted on microvascular myographs for simultaneous measurements of intracellular Ca(2+) ([Ca(2+)]i) and tension. On coronary arteries precontracted with the thromboxane A2 (TxA2) analogue U46619, H2O2 (1-300μM) elicited further moderate contractions in the proximal arterial segments and relaxed the more distal coronary branches, the contractions being markedly augmented in arteries depolarized by raising extracellular K(+). H2O2-elicited vasoconstriction on K(+)30-precontracted coronary arteries was blunted by catalase and significantly reduced by endothelial cell removal and by inhibitors of cyclooxygenase (COX) and of the TxA2 receptor (TP). H2O2 (50μM) increased by about 10-fold basal superoxide anion (O2(-)) production in coronary arteries measured by lucigenin-enhanced chemiluminescence, and H2O2-elicited contractions were reduced by the superoxide dismutase mimetic tempol and by NADPH oxidase inhibition. Furthermore, blockade of the ERK and p38 mitogen-activated protein (MAP) kinases significantly reduced the contractions elicited by high and low concentrations of peroxide, respectively, whereas Rho kinase inhibition nearly abolished these responses. H2O2 (50μM) elicited simultaneous and similar sustained increases in [Ca(2+)]i and tension that were blunted by blockade of voltage-dependent L-type channels, but resistant to the nonselective Ca(2+) channel blocker 2-aminoethoxydiphenyl borate. Moreover, endothelial cell removal reduced the increases in [Ca(2+)]i and contraction elicited by peroxide. The present data demonstrate that H2O2 is an endothelium-dependent vasoconstrictor in rat coronary arteries that activates smooth muscle Ca(2+) entry through L-type and non-L-type channels and various intracellular signaling pathways including the release of a COX-derived TP agonist, stimulation of the MAP and Rho kinase pathways, and production of NADPH oxidase-derived superoxide.

Mechanisms of relaxation by urocortin in renal arteries from male and female rats

Urocortin is a peptide recently identified, which is structurally related to the corticotropin‐releasing factor (CRF). To analyze the mechanisms that could be involved in its effect on renal arteries from male and female rats, the response to urocortin was studied in isolated segments, 2 mm long, of renal arteries from male and female rats. In renal artery segments precontracted with endothelin‐1 (1 nM), urocortin (1 pM–10 nM) produced concentration‐dependent relaxation, which was similar in the arteries from male and female rats. This relaxation was reduced by the antagonists of urocortin receptors astressin (1 μM) and α‐helical CRF(9–41) (1 μM) in arteries from both male and female rats. In renal arteries from female rats, the relaxation to urocortin was reduced by the inhibitor of adenyl cyclase SQ22536 (300 μM), by 8‐bromo‐cyclic‐ADP‐ribose (cADPR; 30 μM), an antagonist of the endogenous activator of sarcoplasmic Ca2+ channel cADPR and by ryanodine (1 μM), which produces depletion of sarcoplasmic Ca2+. In renal arteries from male rats, the relaxation to urocortin was increased by ryanodine, and was not modified by SQ22536 or 8‐bromo‐cADPR. These results suggest that the mechanisms involved in the relaxation to urocortin in renal arteries differ between female and male rats. In female rats, this relaxation may be mediated by the production of cyclic AMP (cAMP), synthesis of cADPR and release of sarcoplasmic Ca2+, whereas in male rats it is not mediated by cAMP.

Coronary reactivity to endothelin-1 during partial ischemia and reperfusion in anesthetized goats. Role of nitric oxide and prostanoids

To examine the coronary reactivity to endothelin-1 and its interaction with nitric oxide or prostanoids during partial coronary ischemia and reperfusion, left circumflex coronary artery flow was electromagnetically measured, and partial occlusion of this artery was induced for 60 min, followed by reperfusion in anesthetized goats (eight non-treated, six treated with N(w)-nitro-L-arginine methyl esther (L-NAME) and five treated with meclofenamate). During partial occlusion, coronary vascular conductance was reduced by 24-37% (P<0.01), and the coronary vasodilatation in response to acetylcholine (3-100 ng) and sodium nitroprusside (1-10 microg) was much decreased in every case; the vasoconstriction in response to endothelin-1 (1-10 microg) was depressed in non-treated animals, and this depression was reversed by L-NAME and was accentuated by meclofenamate. At 30 min of reperfusion coronary vascular conductance remained decreased by 22-27% (P<0.01), and the vasodilatation in response to acetylcholine (3-100 ng) and sodium nitroprusside (1-10 microg), as well as the vasoconstriction with endothelin-1 (1-10 microg), were as in the control and comparable in the three groups of animals. These results suggest: (a) that during ischemia, the coronary vasodilator reserve is greatly reduced, and the vasoconstriction with endothelin-1 is blunted, with preservation of the modulatory role of nitric oxide and involvement of vasoconstrictor prostanoids in this vasoconstriction, and (2) that during reperfusion, the coronary vasodilator reserve and the coronary reactivity to acetylcholine and endothelin-1 recover, but the modulatory role of nitric oxide in this reactivity may be attenuated.

Effects of diabetes on the vascular response to nitric oxide and constrictor prostanoids: gender and regional differences

To analyze the effects of diabetes mellitus on the vascular responsiveness to nitric oxide and thromboxane receptor stimulation, 2 mm long segments of basilar, coronary, renal and tail arteries from male and female, control (normoglycemic) and streptozotocin-induced diabetic rats, were prepared for isometric tension recording. In the segments at basal resting tension, the thromboxane analog U46619 (10(-9)-10(-5) M) produced concentration-dependent contraction, which was similar in arteries from male and female rats, and was reduced by diabetes in coronary arteries from male and in tail arteries from female rats. In the vascular segments precontracted with endothelin-1 (10(-9) M), acetylcholine (10(-9)-3 x 10(-5) M) produced concentration-dependent relaxation which was similar in all arteries from normoglycemic male and female rats, and was increased by diabetes in tail arteries from female, but not in those from male rats. In precontracted segments the nitric oxide donor sodium nitroprusside (10(-10)-10(-5) M) also produced concentration-dependent relaxation, which was higher in basilar arteries from normoglycemic females compared with males, and was increased by diabetes in tail arteries from female but not from male rats. These results suggest that diabetes may increase the relaxation to nitric oxide in tail arteries, and may reduce the contraction to thromboxane receptor activation in coronary and tail arteries in a gender-dependent way. These changes in vascular reactivity may be adaptative to the vascular alterations produced by diabetes.

Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

Emerging evidence suggests a selective up‐regulation of arginase I in diabetes causing coronary artery disease; however, the mechanisms behind this up‐regulation are still unknown. Activated p38 MAPK has been reported to increase arginase II in various cardiovascular diseases. We therefore tested the role of p38 MAPK in the regulation of arginase I and II expression and its effect on endothelial dysfunction in diabetes mellitus.

Upregulation of SK3 and IK1 Channels Contributes to the Enhanced Endothelial Calcium Signaling and the Preserved Coronary Relaxation in Obese Zucker Rats

Background and Aims Endothelial small- and intermediate-conductance KCa channels, SK3 and IK1, are key mediators in the endothelium-derived hyperpolarization and relaxation of vascular smooth muscle and also in the modulation of endothelial Ca2+ signaling and nitric oxide (NO) release. Obesity is associated with endothelial dysfunction and impaired relaxation, although how obesity influences endothelial SK3/IK1 function is unclear. Therefore we assessed whether the role of these channels in the coronary circulation is altered in obese animals. Methods and Results In coronary arteries mounted in microvascular myographs, selective blockade of SK3/IK1 channels unmasked an increased contribution of these channels to the ACh- and to the exogenous NO- induced relaxations in arteries of Obese Zucker Rats (OZR) compared to Lean Zucker Rats (LZR). Relaxant responses induced by the SK3/IK1 channel activator NS309 were enhanced in OZR and NO- endothelium-dependent in LZR, whereas an additional endothelium-independent relaxant component was found in OZR. Fura2-AM fluorescence revealed a larger ACh-induced intracellular Ca2+ mobilization in the endothelium of coronary arteries from OZR, which was inhibited by blockade of SK3/IK1 channels in both LZR and OZR. Western blot analysis showed an increased expression of SK3/IK1 channels in coronary arteries of OZR and immunohistochemistry suggested that it takes place predominantly in the endothelial layer. Conclusions Obesity may induce activation of adaptive vascular mechanisms to preserve the dilator function in coronary arteries. Increased function and expression of SK3/IK1 channels by influencing endothelial Ca2+ dynamics might contribute to the unaltered endothelium-dependent coronary relaxation in the early stages of obesity.

Relaxation by urocortin of rat renal arteries: effects of diabetes in males and females

OBJECTIVE Urocortin is a peptide structurally related to corticotropin releasing factor (CRF), and the present study was performed to examine the effects of diabetes mellitus on the relaxation by urocortin of renal arteries from males and females. METHODS The response to urocortin was studied in isolated segments, 2 mm long, from renal arteries, from male and female, control (normoglycemic) and streptozotocin-induced diabetic rats. RESULTS In the renal arterial segments precontracted with endothelin-1, urocortin produced concentration-dependent relaxation, that was not different between males and females. Diabetes reduced the relaxation in renal arteries from females but not in those from males. The potassium channel blocker charybdotoxin (10(-7) M) reduced the relaxation to urocortin of renal arteries from normoglycemic males and females. The cyclooxygenase inhibitor meclofenamate did not modify the relaxation to urocortin in renal arteries from normoglycemic males or females. The inhibitor of nitric oxide synthesis N(W)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) reduced the relaxation to urocortin in renal arteries from normoglycemic females, but not in renal arteries from normoglycemic males. Neither charybdotoxin, L-NAME or meclofenamate modified the relaxation to urocortin of renal arteries from diabetic females. CONCLUSION These results suggest that urocortin produces a marked vasodilation of renal arteries, which may be mediated by nitric oxide in females and by activation of potassium channels in both genders, and is reduced by diabetes in renal arteries from females.

Mechanisms of the protective effects of urocortin on coronary endothelial function during ischemia–reperfusion in rat isolated hearts

1 Urocortin is a vasodilator peptide related to corticotrophin‐releasing factor, which may protect endothelial function during coronary ischemia–reperfusion (I–R). The aim of this study was to study the mechanisms of this protective effect. 2 Hearts from Sprague–Dawley rats were isolated and perfused at constant flow and then exposed to 15 min global zero‐flow ischemia, followed by 15 min reperfusion. The relaxation to acetylcholine (10 nM–10 μM) was recorded after pre‐constriction of the coronary vasculature with U46619 (100–300 nM) in ischemic–reperfused or time‐control hearts. 3 After I–R, the coronary relaxation to acetylcholine was reduced and this reduction was attenuated by treatment with urocortin (10 pM), administered before ischemia and during reperfusion. 4 This urocortin‐induced improvement of the relaxation to acetylcholine was not modified by tetraethylammonium (10 mM), blocker of Ca2+ dependent‐potassium channels; glibenclamide (10 μM), blocker of KATP channels; Nw‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM), blocker of nitric oxide synthesis; or meclofenamate (10 μM), blocker of cyclooxygenase, but it was abolished by chelerythrine (3 μM), blocker of protein kinase C (PKC). 5 These results suggest that urocortin may protect coronary endothelial function during I–R by activation of PKC.