Alterations in perivascular innervation function in mesenteric arteries from offspring of diabetic rats.

BACKGROUND AND PURPOSE: We have reported that exposure to a diabetic intrauterine environment during pregnancy increases blood pressure in adult offspring, but the mechanisms involved are not completely understood. This study was designed to analyse a possible role of perivascular sympathetic and nitrergic innervation in the superior mesenteric artery (SMA) in this effect. EXPERIMENTAL APPROACH: Diabetes was induced in pregnant Wistar rats by a single injection of streptozotocin. Endothelium-denuded vascular rings from the offspring of control (O-CR) and diabetic rats (O-DR) were used. Vasomotor responses to electrical field stimulation (EFS), NA and the NO donor DEA-NO were studied. The expressions of neuronal NOS (nNOS) and phospho-nNOS (P-nNOS) and release of NA, ATP and NO were determined. Sympathetic and nitrergic nerve densities were analysed by immunofluorescence. KEY RESULTS: Blood pressure was higher in O-DR animals. EFS-induced vasoconstriction was greater in O-DR animals. This response was decreased by phentolamine more in O-DR animals than their controls. L-NAME increased EFS-induced vasoconstriction more strongly in O-DR than in O-CR segments. Vasomotor responses to NA or DEA-NO were not modified. NA, ATP and NO release was increased in segments from O-DR. nNOS expression was not modified, whereas P-nNOS expression was increased in O-DR. Sympathetic and nitrergic nerve densities were similar in both experimental groups. CONCLUSIONS AND IMPLICATIONS: The activity of sympathetic and nitrergic innervation is increased in SMA from O-DR animals. The net effect is an increase in EFS-induced contractions in these animals. These effects may contribute to the increased blood pressure observed in the offspring of diabetic rats.

El ejercicio aeróbico moderado evita la disfunción endotelial y neuronal producida por una dieta rica en grasas en arteria mesentérica de rata / Aerobic exercise training avoids endothelial and neuronal dysfunction produced by a high fat diet in rat mesenteric artery

Objetivo: Determinar si la obesidad inducida por una dieta rica en grasa (HFD) está asociada con modificaciones en las funciones endotelial o neuronal, y los efectos del entrenamiento aeróbico moderado en estos cambios. Materiales y métodos: : Se utilizaron: (i) ratas control (dieta estándar); (ii) ratas alimentadas con una dieta HFD durante ocho semanas, y (iii) ratas HFD sometidas a un entrenamiento aeróbico moderado. Se analizaron las respuestas vasomotoras a acetilcolina (ACh) y estimulación eléctrica (EE), el efecto de L-NAME sobre dichas respuestas, la respuesta vasodilatadora al donante de óxido nítrico (NO) DEA-NO, las liberaciones de NO y de O2.- y la expresión de nNOS y eNOS. Resultados: La ingesta de la dieta HFD disminuyó la respuesta vasodilatadora a ACh e incrementó la respuesta vasoconstrictora a EE. El efecto del L-NAME fue menor en ambos casos en ratas HFD. Las liberaciones de NO endotelial y neuronal fueron disminuidas en ratas HFD. La liberación de O2.- solo aumentó en arterias de ratas HFD con endotelio. La vasodilatación a DEA-NO disminuyó sólo en arterias HFD con endotelio. HFD no modificó la expresión de eNOS, pero disminuyó la expresión de nNOS. Todos estos cambios fueron evitados por el entrenamiento aeróbico moderado. Conclusión: La práctica de ejercicio aeróbico moderado evitó la disfunción de la inervación nitrérgica perivascular y endotelial inducidas por una dieta HFD, evitando el desarrollo de mecanismos que favorecen la hipertensión (AU)

Objective: We investigated whether high-fat diet (HFD)-induced obesity was associated with modifications on endothelial or innervation functions, and the possible effects of aerobic exercise training on these changes. Methods: (i) Control rats (standard diet); (ii) rats fed a HFD for 8 weeks; and (iii) HFD rats submitted to an aerobic exercise training were used. Vasomotor responses to acetylcholine (ACh) and electric field stimulation (EFS), the effect of L-NAME in these responses, vasomotor responses to nitric oxide (NO) donor DEA-NO, NO and O2.- releases, and nNOS and eNOS expression were analysed. Results: : HFD decreased ACh vasodilatation and increased EFS-induced contraction. The effect of L-NAME was lower in both cases in HFD segments. Both endothelial and neuronal NO releases were decreased in HFD. O2.- release was augmented only in endothelium-intact HFD arteries. DEA-NO was decreased only in endothelium- intact segments from HFD. HFD decreased nNOS and did not modify eNOS expressions. All the modifications described were avoided after training. Conclusion: Aerobic exercise training avoided endothelial and nitrergic innervation dysfunction induced by a HFD, thus avoiding the development of mechanisms which lead to hypertension (AU)

Factors involved in rosuvastatin induction of insulin sensitization in rats fed a high fat diet.

BACKGROUND AND AIM: To investigate whether rosuvastatin can improve insulin sensitivity in overweight rats having a high fat diet (HFD). The potential mechanisms involved in this action were evaluated, including SIRT-1, other factors involved in glucose metabolism and stress signaling pathways. METHODS AND RESULTS: Male Wistar rats (n = 30) were divided into three groups: (i) rats fed a standard diet (3.5% fat); (ii) rats fed a HFD (33.5% fat); and (iii) rats fed a HFD and treated with rosuvastatin (15 mg/kg/day). Evolution: 7 weeks. HFD rats showed increased body, epididymal and lumbar adipose tissue weights. Plasma levels of cholesterol, triglycerides, VLDL, glucose and insulin and leptin/adiponectin ratio were higher in HFD rats, and rosuvastatin treatment reduced them. SIRT-1, p53, PGC-1α, PPAR-γ and GLUT-4 protein levels in white adipose tissue (WAT) were lower, and JNK was higher in HFD rats compared to controls. Rosuvastatin treatment normalized expression of these mediators. Endothelium-dependent relaxation was reduced in mesenteric rings from HFD rats compared to controls and rosuvastatin enhanced it in HFD rats. CONCLUSION: Rosuvastatin treatment reduced insulin resistance without affecting body weight or WAT loss in HFD rats. Reduction of leptin and JNK, and enhancement of SIRT-1, p53, PGC-1α, PPAR-γ and GLUT-4 expression in WAT could contribute to insulin sensitization. Normalization of SIRT-1 expression in WAT could be considered a key novel mechanism that aids in explaining the beneficial effects of rosuvastatin on the amelioration of glucose metabolism and the arrangement of multiple signaling pathways participating in insulin resistance in overweight HFD rats.

Relevance of vascular peroxisome proliferator-activated receptor γ coactivator-1α to molecular alterations in atherosclerosis.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is emerging as a novel factor that plays a critical role in integrating signalling pathways in the control of cellular and systemic metabolism. We investigated the role of vascular expression of PGC-1α and related factors, such as sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ (PPARγ) and adiponectin, during the atherosclerotic process. Endothelial function, vascular superoxide anion production and inflammatory mediators were also evaluated. This study was carried out in male New Zealand rabbits fed a diet containing 0.5% cholesterol and 14% coconut oil for 8 weeks. Animals developed mixed dyslipidaemia and atherosclerotic lesions, which were associated with endothelial dysfunction, aortic overproduction of superoxide anions and inflammation. Expression of PGC-1α, SIRT1, PPARγ and adiponectin was reduced (P<0.05) in aorta from atherosclerotic rabbits. Levels of PGC-1α were correlated negatively (P<0.05) with total cholesterol levels, aortic superoxide anion production and tumour necrosis factor-α expression, and positively (P<0.05) with maximal relaxation in response to acetylcholine. The observed results suggest that PGC-1α could be considered to be a link between the main atherosclerotic processes (endothelial dysfunction, oxidation and inflammation) and alterations of other factors involved in vascular wall integrity, such as SIRT1, PPARγ and adiponectin.

Endothelium modulates vasoconstrictor response to prostaglandin I2 in rat mesenteric resistance arteries: interaction between EP1 and TP receptors.

BACKGROUND AND PURPOSE: Prostacyclin (PGI(2)) is usually described as an endothelium-derived vasodilator, but it can also induce vasoconstriction. We studied the vasomotor responses to PGI(2) in resistance arteries and the role of thromboxane (TP) and prostaglandin E(2) (EP) receptors in this effect. EXPERIMENTAL APPROACH: Mesenteric resistance arteries were obtained from Sprague-Dawley rats. Vasomotion to PGI(2) was studied in segments of these arteries with and without endothelium and in presence of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), the potassium channel blockers apamin plus charybdotoxin, the non-selective EP receptor antagonist AH6809, the selective TP receptor antagonist SQ29548 or the EP(1) receptor antagonist SC19220. PGI(2)-induced NO release was analysed in the absence or presence of SQ29548, AH6809 or SC19220. KEY RESULTS: PGI(2) caused contractions in arterial segments that were increased by endothelium removal, L-NAME or L-NAME plus apamin plus charybdotoxin and abolished by SQ29548. In segments with endothelium, AH6809 or SC19220 almost abolished the contractions to PGI(2); this effect was prevented by L-NAME, L-NAME plus apamin plus charybdotoxin or by endothelium removal. PGI(2) induced NO release that was inhibited by the prostacyclin receptor (IP receptor) antagonist, RO1138452, and increased by SQ29548, SC19220 and AH6809. The increase in NO release induced by these separate drugs was inhibited by RO1138452. CONCLUSIONS AND IMPLICATIONS: PGI(2) activated the TP receptor in mesenteric resistance arteries and produced vasoconstriction, which the endothelium modulated through TP and EP(1) receptors. PGI(2) also released endothelium-derived hyperpolarizing factor and, through IP receptor activation, induced NO release, which in turn, was antagonized by TP and EP(1) receptor activation.

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation.

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca2+-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.

Chronic ouabain treatment increases the contribution of nitric oxide to endotheliumdependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclinand endothelium-dependent hyperpolarizing factor to endothelium-dependentvasodilation induced by acetylcholine in rat aorta from control and ouabain-inducedhypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omeganitro-L-arginine methyl esther (L-NAME) inhibited the vasodilator response toacetylcholine in segments from both groups but to a greater extent in segments fromouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higherin segments from ouabain-treated rats. Preincubation with the prostacyclin synthesisinhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacininhibited the vasodilator response to acetylcholine in aortic segments from bothgroups. The Ca2+-dependent potassium channel blocker charybdotoxin inhibited thevasodilator response to acetylcholine only in segments from control rats. Theseresults indicate that hypertension induced by chronic ouabain treatment is accompaniedby increased endothelial nitric oxide participation and impaired endotheliumdependenthyperpolarizing factor contribution in acetylcholine-induced relaxation.These effects might explain the lack of effect of ouabain treatment on acetylcholineresponses in rat aorta (AU)

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Aldosterone induces endothelial dysfunction in resistance arteries from normotensive and hypertensive rats by increasing thromboxane A2 and prostacyclin.

BACKGROUND AND PURPOSE: The present study was designed to assess whether cyclooxygenase-2 (COX-2) activation is involved in the effects of chronic aldosterone treatment on endothelial function of mesenteric resistance arteries (MRA) from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). EXPERIMENTAL APPROACH: Relaxation to acetylcholine was measured in MRA from both untreated and aldosterone-treated strains. Vasomotor responses to prostacyclin and U46619 were also analysed. Release of 6-oxo-prostaglandin (PG)F1alpha and thromboxane B2 (TxB2) was determined by enzyme immunoassay. COX-2 protein expression was measured by western blot. KEY RESULTS: Aldosterone reduced acetylcholine relaxation in MRA from both strains. In MRA from both aldosterone-treated strains the COX-1/2 or COX-2 inhibitor (indomethacin and NS-398, respectively), TxA2 synthesis inhibitor (furegrelate), prostacyclin synthesis inhibitor (tranylcypromine) or TxA2/ PGH2 receptor antagonist (SQ 29 548), but not COX-1 inhibitor SC-560, increased acetylcholine relaxation. In untreated rats this response was increased only in SHR. Prostacyclin elicited a biphasic vasomotor response: lower concentrations elicited relaxation, whereas higher concentrations elicited contraction that was reduced by SQ 29 548. Aldosterone increased the acetylcholine-stimulated production of 6-oxo-PGF(1alpha) and TxB2 in MRA from both strains. COX-2 expression was higher in both strains of rats treated with aldosterone. CONCLUSIONS AND IMPLICATIONS: Chronic treatment with aldosterone impaired endothelial function in MRA under normotensive and hypertensive conditions by increasing COX-2-derived prostacyclin and thromboxane A2. As endothelial dysfunction participates in the pathogenesis of many cardiovascular disorders we hypothesize that anti-inflammatory drugs, specifically COX-2 inhibitors, could ameliorate vascular damage in patients with elevated aldosterone production.

Orchidectomy increases the formation of non-endothelial thromboxane A2 and modulates its role in the electrical field stimulation-induced response in rat mesenteric artery.

The aim of this study was to analyze whether endogenous male sex hormones influence the release of thromboxane A2(TXA2) and its role in the electrical field stimulation (EFS)-induced response, as well as the mechanism involved. For this purpose, endothelium-denuded mesenteric arteries from control and orchidectomized male Sprague-Dawley rats were used to measure TXA2 release; EFS-induced response, nitric oxide (NO), norepinephrine (NA), and prostaglandin (PG) I2 release were also measured in the presence of the TXA2 synthesis inhibitor furegrelate. Orchidectomy increased basal and EFS-induced TXA2 release. Furegrelate decreased the EFS-induced contraction in arteries from control rats, but did not modify it in arteries from orchidectomized rats. The EFS-induced neuronal NO release and vasodilator response were increased by furegrelate in arteries from control rats, but were not modified in arteries from orchidectomized rats. Furegrelate did not modify the EFS-induced NA release or vasoconstrictor response in arteries from either control or orchidectomized rats. The EFS-induced PGI2 release was not modified by furegrelate in arteries from control rats, but was increased in arteries from orchidectomized rats. The results of the present study show that endogenous male sex hormone deprivation i) increases non-endothelial TXA2 release and ii) regulates the effect of endogenous TXA2 on the EFS-induced response through different mechanisms that, at the least, involve the NO and PGI2 systems. In arteries from control rats, inhibition of TXA2 formation decreases the EFS-induced response by increasing neuronal NO release. In arteries from orchidectomized rats, the EFS-induced response is unaltered after the inhibition of TXA2 formation, by increasing PGI2 release.

Pathophysiology and therapeutic possibilities of calcitonin gene-related peptide in hypertension.

Calcitonin gene related peptide (CGRP), a 37 amino acid neuropeptide, is the most potent vasodilator known. Participation of CGRP in hypertension and related diseases, such as preeclampsia or vasospasm after subarachnoid haemorrage, is one of the most studied topics. In this review we summarize the published roles of CGRP in pathophysiology of hypertension in humans and in experimental models. We also discuss the effects of direct administration of CGRP in the treatment of hypertension and of anti-hypertensive drugs that enhance the release or response of endogenous calcitonin gene-related peptide: angiotensin converting enzyme inhibitors, selective antagonists for the angiotensin II receptor, beta-blockers, magnesium sulphate for preeclampsia and rutaecarpine, as well as the possibilities using CGRP in gene therapy for prevention of vasospasm after subarachnoid haemorrage.

Aldosterone and its blockade: a cardiovascular and renal perspective.

Aldosterone not only contributes to salt and water homeostasis, but also exerts direct cardiovascular and renal effects. Numerous experimental and clinical studies indicate that aldosterone participate in cardiac alterations associated with hypertension, heart failure, diabetes and other pathological entities. It is important to mention that dietary salt is a key factor in aldosterone-mediated cardiovascular damage, since damage was more evident in animals on a high-salt diet than animals on a low salt diet. A pathophysiological action of aldosterone involves development of extracellular matrix and fibrosis, inflammation, stimulation of reactive oxygen species production, endothelial dysfunction, cell growth and proliferation. Many studies showed local extra-adrenal production of aldosterone in brain blood vessel, and the heart, which contribute in an important manner to the pathological actions of this mineralocorticoid. Several studies such as RALES, EPHESUS, 4E and others, recently showed that mineralocorticoid-receptor (MR) antagonists, alone or in combination with ACE inhibitors or ARBs, reduced the risk of progressive target organ damage and hospitalization in patients with hypertension and heart failure. These clinical benefits support the therapeutic usefulness of MR antagonists.

Pathophysiology and therapeutic possibilities of calcitonin gene-related peptide in hypertension

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Calcitonin gene related peptide (CGRP), a 37 amino acid neuropeptide, is themost potent vasodilator known. Participation of CGRP in hypertension and relateddiseases, such as preeclampsia or vasospasm after subarachnoid haemorrage, is one ofthe most studied topics. In this review we summarize the published roles of CGRPin pathophysiology of hypertension in humans and in experimental models. We alsodiscuss the effects of direct administration of CGRP in the treatment of hypertensionand of anti-hypertensive drugs that enhance the release or response of endogenouscalcitonin gene-related peptide: angiotensin converting enzyme inhibitors, selectiveantagonists for the angiotensin II receptor, beta-blockers, magnesium sulphate forpreeclampsia and rutaecarpine, as well as the possibilities using CGRP in gene therapyfor prevention of vasospasm after subarachnoid haemorrage

Orchidectomy increases beta-adrenoceptor activation-mediated neuronal nitric oxide and noradrenaline release in rat mesenteric artery.

BACKGROUND/AIMS: A previous study has demonstrated that endogenous male sex hormones do not alter neuronal nitric oxide (NO) release in rat mesenteric artery. However, the regulatory role of endogenous male sex hormones on noradrenaline (NA) release in rat mesenteric artery is not known. The present study was designed to analyze whether endogenous male sex hormones influence the NA release induced by electrical field stimulation (EFS), as well as the possible modification in NA and neuronal NO release by presynaptic beta-adrenoceptor activation. METHODS: For this purpose, mesenteric arteries from control and orchidectomized male Sprague-Dawley rats were used. Basal and EFS-induced neuronal NO and NA release, as well as the contractile effect induced by EFS, was measured. RESULTS: Basal and EFS-induced neuronal NO and NA release were similar in arteries from control and orchidectomized rats. The beta-adrenoceptor agonist clenbuterol did not modify EFS-induced neuronal NO and NA release in arteries from control rats. In contrast, in arteries from orchidectomized animals, clenbuterol increased both neuronal NO and NA release; this increase was prevented by incubation with the beta-adrenoceptor antagonist propranolol. However, the contractile response elicited by EFS was not modified by clenbuterol in either group of rats. CONCLUSIONS: These results show that orchidectomy does not alter the EFS-induced NA release. What is more, activation of presynaptic beta-adrenoceptors does not modify EFS-induced NA and neuronal NO release in arteries from control rats although it increases the release of both neurotransmitters in arteries from orchidectomized rats. Despite these modifications, the EFS-induced contractile response is preserved in arteries from orchidectomized rats.

Aging alters neuronal nitric oxide release from rat mesenteric arteries: role of presynaptic beta-adrenoceptors.

This study examines the influence of aging on the neuronal nitric oxide (NO) and noradrenaline (NA) release elicited by electrical field stimulation (EFS; 200 mA, 0.3 ms, 1-16 Hz, 30 s) in endothelium-denuded mesenteric arteries from young and old rats, as well as the influence of the presynaptic beta-adrenoceptors in that release. EFS induced frequency-dependent contractions. N(G)-nitro-l-arginine methyl ester (l-NAME) only enhanced EFS-elicited contractions in arteries from young rats. Capsaicin did not alter the EFS-induced contractions in either age group. Clenbuterol did not modify the contraction elicited by EFS in arteries from young or old rats either. A subsequent addition of l-NAME also induced an increase in the EFS-induced response in arteries from both age groups. In old rats, the presence of propranolol did not alter the response induced by EFS, and the subsequent addition of clenbuterol or clenbuterol plus l-NAME did not affect this response. In precontracted segments, sodium nitroprusside or clenbuterol induced similar relaxation in both age groups. None of the drugs used altered the response to exogenous NA or basal tone. In arteries preincubated with [(3)H]NA, EFS induced (3)H release, which remained unmodified in the presence of clenbuterol or propranolol in young rats, whereas clenbuterol increased the (3)H overflow in old rats, and this effect was abolished by propranolol. These drugs did not alter the basal (3)H efflux and indicate that in rat mesenteric arteries EFS induces NA release in both age groups, and only NO release in young animals. Activation of presynaptic beta-adrenoceptors increased NA and probably NO release in aged rats.

Angiotensin II increases neurogenic nitric oxide metabolism in mesenteric arteries from hypertensive rats.

We investigated, in mesenteric arteries from hypertensive rats (SHR), the possible changes in neurogenic nitric oxide (NO) release produced by angiotensin II (AII), and the possible mechanisms involved in this process. In deendothelialized segments the NO synthase inhibitor N(G)-nitro-L-arginine (L-NAME, 10 microM) increased the contractions caused by electrical field stimulation (EFS, 200 mA, 0.3 ms, 1-16 Hz, for 30 s). AII (0.1 nM) enhanced the response to EFS, which was unmodified by the subsequent addition of L-NAME. The AII antagonist receptor saralasine (0.1 microM) prevented the effect of AII, and the subsequent addition of L-NAME restored the contractile response. SOD (25 u/ml) decreased the reponse to EFS and the subsequent addition of L-NAME increased this response. AII did not modify the decrease in EFS response induced by SOD, and the addition of L-NAME increased the response. None of these drugs altered the response to exogenous noradrenaline (NA) or basal tone except SOD, which increased the basal tone, an effect blocked by phentolamine (1 microM). In arteries pre-incubated with [3H]-NA, AII did not modify the tritium efflux evoked by EFS, which was diminished by SOD. AII did not alter basal tritium efflux while SOD significantly increased it. These results suggest that EFS of SHR mesenteric arteries releases neurogenic NO, the metabolism of which is increased in the presence of AII by the generation of superoxide anions.

Mechanisms of blockade by the novel migraine prophylactic agent, dotarizine, of various brain and peripheral vessel contractility.

The novel antimigraineur, dotarizine, inhibited 5-HT (5 hydroxytryptamine)-evoked contractions of rabbit vertebral, aorta, femoral and mesenteric arteries, with IC(50)s of 1.35, 1.40, 0.52 and 1.09 microM, respectively. Flunarizine had little effect on these contractions, while ketanserin was more potent (IC(50)s of 0.17 microM for vertebral, 0.22 microM for aorta, 0.05 microM for femoral and 0.03 microM for mesenteric arteries). At 10 microM, dotarizine caused 40% blockade of K(+)-evoked contractions of rabbit aorta, and 70% inhibition of 5-HT-evoked responses; these values were 30% and 20% for 10 microM flunarizine. Contractions of rabbit aorta elicited by noradrenaline, angiotensin II or prostaglandin F(2alpha) were not affected by 10 microM dotarizine or flunarizine. Ketanserin shifted to the right, in parallel, the concentration-response curves for 5-HT in rabbit aorta; however, dotarizine caused a non-competitive type of blockade, increasing the maximum 5-HT contraction at 30 nM and decreasing it at 3 and 30 microM. K(+)-evoked contractions of rabbit aorta were halved by 3 microM dotarizine in a voltage-independent manner; flunarizine caused a delayed-type, non-reversible post-drug blockade, and exhibited some voltage-dependence. Blockade by nifedipine was voltage-dependent and fully reversible. Ca(2+)-evoked contractions of depolarised bovine middle cerebral arteries were blocked by 1--3 microM dotarizine in a non-surmountable manner. Contraction of these vessels evoked by electrical stimulation was blocked 50% and 70% by 1 and 3 microM dotarizine, respectively. Dotarizine (1--3 microM) also inhibited to a similar extent the K(+)-evoked [(3)H]noradrenaline release from cultured rat sympathetic neurones. These data suggest that the mechanism of blockade by dotarizine of cerebral vessels contractility has three components: (i) presynaptic inhibition of noradrenaline release; (ii) blockade of postsynaptic vascular 5-HT receptors; (iii) blockade of Ca(2+)entry into the vascular smooth muscle cell cytosol. The compound does not affect the vascular receptors for noradrenaline, angiotensin II or prostaglandin F(2alpha).

Increase in neurogenic nitric oxide metabolism by endothelin-1 in mesenteric arteries from hypertensive rats.

We investigated, in mesenteric arteries from hypertensive rats (SHRs), the possible changes in neurogenic nitric oxide (NO) release produced by endothelin-1 (ET-1), and the mechanisms involved in this process. The contractile response induced by electrical field stimulation (EFS; 200 mA, 0.3 ms, 1-16 Hz, for 30 s) in deendotheliumized mesenteric segments was abolished by tetrodotoxin and phentolamine. The NO synthase inhibitor N(G)-nitro-L-arginine (L-NAME, 10 microM) increased the contractions caused by EFS. ET-1 enhanced the contraction induced by EFS, which was unaltered by the subsequent addition of L-NAME. The ETA antagonist-receptor BQ-123 (1 microM) inhibited the effect of ET-1 on EFS response, whereas the ETB antagonist-receptor BQ-788 (3 microM) partially blocked it, and the subsequent addition of L-NAME restored the contractile response in both cases. SOD (25 unit/ml) decreased the response to EFS, and the subsequent addition of L-NAME increased this response. ET-1 did not modify the decrease in EFS response induced by SOD, and the addition of L-NAME increased the response. None of these drugs altered the response to exogenous noradrenaline (NA) or basal tone except SOD, which increased the basal tone, an effect blocked by phentolamine (1 microM). In arteries preincubated with [3H]NA, ET-1 did not modify the tritium efflux evoked by EFS, which was diminished by SOD. ET-1 did not alter basal tritium efflux, whereas SOD significantly increased the efflux. These results suggest that EFS of SHR mesenteric arteries releases neurogenic NO, the metabolism of which is increased in the presence of ET-1 by the generation of superoxide anions.

Influence of hypertension on nitric oxide synthase expression and vascular effects of lipopolysaccharide in rat mesenteric arteries.

1. Experiments were designed to investigate the effects of the inducible nitric oxide synthase (iNOS) stimulator, lipopolysaccharide (LPS), on noradrenaline (NA) responses and on NOS activity and its expression in intact mesenteric resistance arteries (MRAs) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. In MRAs from WKY, LPS (10 microg ml(-1); 1-5 h) reduced the vasoconstrictor responses to NA (0.1 - 30 microM) in the presence, but not in the absence of L-arginine (L-Arg, 10 microM). However, in SHR arteries, LPS induced an incubation-time dependent reduction of NA responses in the absence, as well as the presence, of L-Arg. The LPS inhibitory effect was reduced by the non-specific NOS inhibitor L-N(G)-nitroarginine methyl ester (L-NAME, 100 microM) and the selective iNOS inhibitor, aminoguanidine (100 microM). 3. L-NAME alone similarly shifted the concentration-response curve to NA leftward in arteries from both strains, while aminoguanidine had no effect. L-Arg shifted the curve to NA rightward only in SHR MRAs. 4. Basal activity of both iNOS and constitutive NOS (conversion of [(3)H]-L-Arg to [(3)H]-L-citrulline) was similar in arteries from both strains. After 5 h incubation with LPS, only iNOS activity in arteries from SHR was increased. 5. Basal iNOS protein expression was undetectable; basal endothelial (eNOS) protein expression was similar in arteries from both strains, while neuronal (nNOS) was greater in arteries from SHR. LPS induced iNOS protein expression, that was higher in arteries from SHR than in those from WKY. 6. These results indicate that NO production, via iNOS induction, is greater than in those from MRAs from SHR to WKY.

Role of protein kinase C in electrical-stimulation-induced neuronal nitric oxide release in mesenteric arteries from hypertensive rats.

This study examines the influence of hypertension on neuronal nitric oxide (NO) release and its modulation by protein kinase C (PKC). For this purpose, mesenteric segments without endothelium were obtained from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs), and neurogenic NO release induced by electrical field stimulation (EFS) was examined in these segments. EFS induced frequency-dependent contractions. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and the sensorial neurotoxin capsaicin increased EFS-induced contractions in SHR segments, but did not affect these contractions in segments from WKY rats. In segments from SHRs, the increase in EFS-induced response by capsaicin was further increased by the combination of capsaicin and L-NAME. EFS-induced contractions in SHR arteries were unaltered by the protein synthesis inhibitor cycloheximide or by 2-amine-5,6-dihydro-6-methyl-4H-1,3-tiazine (AMT), an inhibitor of inducible NO synthase, and increased by the guanylate cyclase inhibitor Methylene Blue. In these arteries, capsaicin plus the PKC inhibitor calphostin C increased the contractions elicited by EFS; the addition of L-NAME did not affect this increase. Phorbol 12,13-dibutyrate (PDBu) did not modify the response to EFS in these arteries pretreated with capsaicin, although a combination of PDBu and L-NAME was effective. These results indicate that, in mesenteric arteries, EFS induces the release of NO from perivascular nitrergic nerves and of neuropeptides from sensory nerves, but only in hypertensive rats. The NO released is synthesized by constitutive neuronal NO synthase in a manner that is positively modulated by PKC, an enzyme that seems to be activated in hypertension.

Regulation of beta-adrenoceptor-mediated relaxation of the rat aorta is modulated by endogenous ovarian hormones.

The aim of this study was to assess the influence of the endogenous status of ovarian hormones on the relaxation induced by the beta-adrenoceptor agonists isoprenaline (isoproterenol) and dobutamine in thoracic aorta segments, precontracted with noradrenaline, from age-matched (13-week-old) virgin (oestrus) and ovariectomized (OVX) prepubertal female Wistar rats. Isoprenaline-induced relaxation was decreased in intact aortic segments from OVX rats compared with that in segments from oestrus rats. Relaxation was significantly reduced by endothelium removal, 1 micromol/l propranolol or 100 micromol/l N(G)-nitro-L-arginine methyl ester (L-NAME). The beta(1)-adrenoceptor agonist dobutamine induced less relaxation in intact arteries from oestrus rats than did isoprenaline, and dobutamine-induced relaxation was markedly less in intact segments from OVX compared with oestrus rats. This dobutamine-induced relaxation was abolished by endothelium removal, and reduced by 1 micromol/l propranolol, 100 micromol/l L-NAME or 1 micromol/l yohimbine. Cholera toxin (an activator of the stimulatory G-protein G(s)) caused relaxation in intact arteries from oestrus rats; this relaxation was decreased by both deprivation of ovarian hormones and endothelium removal. Forskolin (a direct activator of the catalytic subunit of adenylate cyclase) and sodium nitroprusside (a nitric oxide donor and cGMP-dependent vasodilator agonist) induced similar endothelium-independent relaxation in arteries from both oestrus and OVX rats. These results suggest that the relaxation elicited by endothelial beta-adrenoceptor activation in the rat thoracic aorta is impaired by deprivation of female ovarian hormones; this impairment is caused, at least in part, by decreases in both the endothelial release of NO and G(s) function.