Alterations in the Vasoreactivity of Hypertensive Rat Aortic Rings: Role of Nitric Oxide and Superoxide Radicals

Objectives:Present study was undertaken to investigate involvement of nitric oxide (NO) and superoxide radicals in the modulation of vasoreactivity in a model of renal hypertension

Method:Hypertension was induced in the male Sprague Dawley rats by aortic banding just above the left kidney. Relaxation or contraction following cumulative addition of acetylcholine (Ach, 1 × 10^-8to 1 x^-5M) or phenylephrine (PE, 1 × 10^-8to 1 × 10^-5mol/1) was studied in the aortic rings obtained from sharn operated normotensive, hypertensive and captopril pretreated rats. Ach and PE responses were taken in the presence or absence of NO synthase inhibitor (L-NAME; 1 × 10^-5and 1 × 10^-4mol/1). Spontaneous release of NO from the aortic rings was evaluated by studying the inhibition of adenosine phosphate stundated platelet aggregation, while superoxide radcals were estunated by cytochrome c reduction method

Results:Ach induced vasorelaxation in PE precontracted rings was impaired followmg 8 wk alter aorbc bandmg, while spontaneous release of NO remained unaffected. Captopril pretreatment restored the aortic ring responsiveness to Ach. An increase in the superoxide radicalgeneration and PE induced contraction following L-NAME treatment in the hypertensive rat aortic rings was observed

Conclusion:Attenuation in the Ach induced NO release and augmentation in the superoxide radcal generation seems to play an important role in the modulation of vasoreactivity following renal hypertension in rats     

Lipoxygenase-dependent mechanisms in hypertension

This study was designed to examine the contribution of lipoxygenase products to mechanisms of vascular contraction and elevated blood pressure in rats with aortic coarctation-induced hypertension. In cytosolic fractions of aortae taken from hypertensive rats, 12-lipoxygenase protein was increased as compared to normotensive controls. Aortic rings from hypertensive, but not from normotensive rats, exhibited a basal tone which was reduced 74+/-12 and 71+/-22%, respectively, by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC, 10(-5) mol/L) and 5,8,11-eicosatriynoic acid (ETI, 10(-5) mol/L). CDC (8 mg/kg s.c.) did not affect the blood pressure of normotensive rats but decreased that of hypertensive rats from 182+/-6 to 151+/-10 mm Hg. The blood pressure lowering effect of CDC was blunted in hypertensive rats pretreated with indomethacin or antibodies against 5,6-dihydro-prostaglandin I2. These data suggest contribution of lipoxygenase-derived products to mechanisms underlying aortic smooth muscle basal tone and elevated blood pressure in rats with aortic coarctation-induced hypertension. The vasodepressor effect of CDC depends on a mechanism involving vasodilatory prostaglandins.     

Role of K+ channels in EDHF-dependent relaxation induced by acetylcholine in canine coronary artery

Summary To identify the K⁺ channels responsible for endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation, we studied the effects of various K⁺ channel blockers on acetylcholine-induced relaxation, which persists even in the presence of both an inhibitor of nitric oxide synthase and that of cyclooxygenase, in canine coronary artery rings. A nonselective K⁺ channel blocker, tetrabutylammonium (TBA), a large and intermediate conductance Ca²⁺-activated K⁺ channel blocker, charybdotoxin (CTX), and a voltage-dependent K⁺ channel blocker, 4-aminopyridine (4-AP), significantly inhibited this residual relaxation. A combined treatment with CTX and 4-AP almost completely blocked the relaxation. Neither a large (iberiotoxin) nor a small (apamin) conductance Ca²⁺-activated K⁺ channel blocker blocked the relaxation. We also investigated effects of K⁺ channel blockers on basal tone to determine whether or not EDHF is involved in regulating basal tone. TBA and CTX substantially raised basal tone to a greater degree in endothelium-intact preparations than in endothelium-denuded preparations. These results indicate that EDHF may exert its relaxing action through intermediate conductance Ca²⁺-activated and voltage-dependent K⁺ channels in canine coronary arteries. In addition, EDHF may play a role in maintaining basal vascular tone. This study was supported in part by a Grant-in-Aid for Scientific Research (B07457167) from the Ministry of Education, Science and Culture of Japan.     

Nebivolol ameliorates asymmetric dimethylarginine-induced vascular response in rat aorta via β3 adrenoceptor-mediated mechanism

Background: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, induces endothelial dysfunction. Nebivolol, a highly selective β₁-adrenergic receptor (AR) blocker, is the only beta-blocker known to induce vascular production of nitric oxide. Objective: The present study was designed to evaluate the effect and mechanism of nebivolol on ADMA-induced vascular response in rat aorta in vitro. Methods: In vitro, the effects of nebivolol and ADMA on resting tone or contraction induced by phenylephrine (PE, 10^?6?mol/L) and relaxation induced by acetylcholine (Ach, 10^?10–10^?5?mol/L) were evaluated. Results: ADMA in a concentration-dependent manner increased the resting and PE-induced tone and reduced Ach-induced relaxation. Nebivolol inhibited the ADMA-induced enhancements in tone and reversed the effects of ADMA on Ach-induced relaxation. These effects of nebivolol were blocked by selective β₃ receptor blocker cyanopindolol (1?μM), but not by selective β₂ receptor blocker butoxamine (50?μM). Conclusions: Nebivolol ameliorates the ADMA-induced vascular responses in rat aorta, at least in part, by mechanisms involving β₃ adrenoceptor.     

Relationships between dopamine-induced changes in cytosolic free calcium concentration ([Ca2+]i) and rate of prolactin secretion

This study was undertaken to investigate the relationship between dopamine (DA) induced changes in the cytosolic calcium concentration ([Ca²⁺]_i) and the rate of prolactin secretion using GH₄ZR₇, a rat pituitary cell line, which express only one subtype of D₂ receptor. GH₄ZR₇ cells were loaded with Fluo-3, a fluorescent Ca²⁺ indicator, and then perifused with two different doses of DA (10⁻⁷ mol/L and 5×10⁻⁴ mol/L). We monitored changes in [Ca²⁺]_i and rate of prolactin release simultaneously by attaching a spectrofluorometer to a dynamic perifusion system. DA has stimulatory and inhibitory effect on prolactin secretion in GH₄ZR₇ cells; 10⁻⁷ mol/L DA slightly increased [Ca²⁺]_i and stimulated prolactin release, whereas 5×10⁻⁴ mol/L DA decreased [Ca²⁺]_i and inhibited prolactin secretion. When the cells were pretreated with pertussis toxin (PTX), 10⁻⁷ mol/L DA had no significant change in [Ca²⁺]_i while stimulating prolactin release, and 5×10⁻⁴ mol/L DA reduced [Ca²⁺]_i without having any significant effect on the rate of prolactin secretion. The results of this study demonstrate that changes in [Ca²⁺]_i do not always correlate with the rate of prolactin release from lactotrophs. The dissociation between [Ca²⁺]_i and prolactin release is somewhat expected considering the diverse role of [Ca²⁺]_i and post-[Ca²⁺]_i events, which can change the rate of prolactin release.     

Inhibition of nitric oxide activity by arginine analogs in human renal arteries

Background:Plasma levels of endogenous guanidine compounds are increased in various pathologic conditions, including chronic renal failure. In the present study we tested the effects of some of these compounds on basal and stimulated nitric oxide activity in human renal arteries.Methods:Rings from human renal arteries were obtained from 22 patients undergoing nephrectomy. The rings were suspended in organ baths for isometric recording of tension. We then studied the effects of N^G-monomethyl-l-arginine (L-NMMA), N^G,N^G-dimethyl-l-arginine (asymmetrical dimethylarginine [ADMA]), aminoguanidine (AG), and methylguanidine (MG) on artery rings under basal and stimulated conditions.Results:In precontracted arteries, L-NMMA (1 μmol/L to 1 mmol/L) and ADMA (1 μmol/L to 3 mmol/L) caused concentration- and endothelium-dependent contractions (median effective concentrations [EC₅₀] = 13.3 μmol/L and 17.5 μmol/L, respectively; Emax = 15 ± 4% and 17 ± 4% of the response to 100 mmol/L KCl, respectively). Aminoguanidine (0.01 to 3 mmol/L) and MG (0.01 to 3 mmol/L) produced endothelium-independent contractions (Emax = 9 ± 3% and 16 ± 2% of the response to 100 mmol/L KCl, respectively). l-arginine (1 mmol/L) but not d-arginine (1 mmol/L) prevented the contractions by L-NMMA and ADMA, but did not change contractions induced by AG and MG. In precontracted arteries, the relaxation to acetylcholine was decreased but not abolished by L-NMMA and ADMA. The remaining relaxation was reduced by charybdotoxin (0.1 μmol/L) and tetraethylammonium (1 mmol/L).Conclusions:The results demonstrate that L-NMMA and ADMA reduce basal and stimulated nitric oxide activity in human renal arteries. An increase in the plasma concentrations of methylarginines associated with renal disease should be considered as a risk factor for endothelial dysfunction and abnormal vasomotor tone in human renal arteries.     

Inhibition of CO<Subscript>2</Subscript> production from glucose by arginine in brain slices of rats

In the present study we evaluated the in vivo effect of arginine on CO₂ production from glucose in a medium with physiological and high extracellular K⁺ concentrations. We also tested the influence of the nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME), on the effects elicited by arginine in order to investigate the possible participation of NO and/or its derivatives on the effects of arginine on CO₂ production from glucose. Sixty-day-old rats were treated with a single intraperitoneal injection of saline (control; group I), arginine (0.8 g/kg; group II), L-NAME (2.0 mg/kg; group III) or arginine (0.8 g/kg) plus L-NAME (2.0 mg/kg; group IV) and were killed 1 h later. Results showed that arginine administration inhibited CO₂ production from glucose at physiological extracellular K⁺ concentration and L-NAME prevented such effect. In contrast, arginine administration had no effect on CO₂ production from glucose at high extracellular K⁺ concentration. Based on these data, we also investigated the in vitro effect of arginine on CO₂ production from glucose in a medium with physiological extracellular K⁺ concentration in hippocampus slices. Results showed that arginine (0.1–1.5 mM) when added to the incubation medium did not alter CO₂ production from glucose in hippocampus slices of untreated rats. In addition, we also demonstrated that arginine inhibits Na⁺, K⁺-ATPase activity. The data indicate that the reduction of CO₂ production by arginine was probably mediated by NO and/or its derivatives, which could act inhibiting the activity of Na⁺, K⁺-ATPase. The results suggest that arginine impairs energy metabolism in hippocampus slices of rats.     

Chronic Ethanol Feeding Potentiates α1‐Adrenoceptor Responsiveness in SHR Aortas

Previous studies including ours demonstrated a hypotensive response to ethanol in spontaneously hypertensive rats (SHRs). In this study, we investigated whether this hypotensive effect of ethanol involves alterations in vascular α₁‐adrenergic receptor responsiveness. The contractile responses to the α₁‐receptor agonist phenylephrine were evaluated in aortic rings obtained from pair‐fed SHRs receiving liquid diet with or without ethanol (2.5% or 5%, w/v) for 3 months. The responses were measured in aortas with and without endothelium to determine the role of the endothelium in the observed responses. The liquid diet intake was similar in the control and ethanol groups throughout the study whereas the body weight was significantly reduced by ethanol. Cumulative addition of phenylephrine (1 × 10^?9–1 × 10^?4 M) caused concentration‐related contractile responses. These responses were significantly reduced after endothelium denudation suggesting a role for the endothelium in the modulation of α₁‐receptor responsiveness. Ethanol (2.5% and 5%) caused significant and concentration‐related increases in the contractile responses elicited by phenylephrine but not KCl. The maximum contraction (E_max) caused by phenylephrine in rings obtained from SHRs treated with 2.5% and 5% ethanol amounted to 413.6 ± 26.3 and 513.0 ± 46.7 mg tension/mg tissue, respectively, compared with 383.6 ± 35.2 mg tension/mg tissue in control rings. The enhancement of α₁ contractions by ethanol was virtually abolished in rings pretreated with the α₁‐receptor antagonist prazosin, suggesting upregulation of α₁‐receptors in aortas of ethanol‐fed rats. Endothelium denudation also abolished ethanol‐evoked increases in phenylephrine contractions. These findings suggest that chronic ethanol feeding upregulates aortic α₁‐receptors, which may be a consequence of chronic α₁‐receptor blockade by ethanol. The latter may account, at least in part, for the hypotensive response elicited by ethanol in SHRs.     

Calcium Supplementation Is Associated With Endothelium Dependent Attenuation of Vascular Smooth Muscle Reactivity in Normotensive Pregnant and Nonpregnant Rats

The study tests the hypothesis that the blood pressure lowering effect of a high calcium diet is mediated through attenuation of vascular reactivity and examined the mechanisms involved in both normotensive pregnant and nonpregnant rats. The contractile responses of aortic rings of Wistar rats fed on high (1.7%, 2.1%) and normal (0.9%) calcium diets to phenylephrine, angiotensin II, KCl, and CaCl₂ were studied. The relaxations to acetylcholine and potassium chloride, as well as the effects of endothelial denudation, pretreatment with indomethacin (10⁻⁶ mol/L), methylene blue (10⁻⁶ mol/L), and calcium free solution on the responses to phenylephrine were also examined.In both pregnant and nonpregnant rats, the contractile responses of aortic rings of animals fed a high calcium diet to all the agents were significantly attenuated, compared with those of controls. After endothelial denudation, or treatment with methylene blue, but not with indomethacin, the responses of the rings to phenylephrine were enhanced and not different from similarly treated rings from rats on a normal calcium diet. There was no difference in the contractile responses to phenylpehrine in calcium free solution. The relaxation to acetylcholine, but not to potassium chloride, was enhanced in rings from rats on a high calcium diet. The diminution in reactivity was not associated with corresponding changes in sensitivity of the tissues.It is concluded that in normotensive rats a high calcium diet is associated with diminished vascular smooth muscle reactivity that is endothelium dependent, and involves increased stimulation of the nitric oxide–guanylate cyclase pathway but not of the sodium–potassium ATPase or prostacyclin.     

Nitric oxide production in arterial vessels of cirrhotic rats

Indirect evidence exists implicating vascular nitric oxide in the pathogenesis of arterial vasodilation in cirrhosis. In the current study, a coincubation assay to estimate the vascular nitric oxide production was developed and the nitric oxide production by arterial segments of cirrhotic and control rats was assessed. In the assay, measurement of reporter monolayer cell-associated cGMP levels allows the influence of nitric oxide released by arterial segments to be determined. RFL-6 cells served as reporter cells. Nitric oxide production was determined in thoracic aorta and mesenteric arteries of 22 control rats, 10 cirrhotic rats without ascites, and 12 cirrhotic rats with ascites. Basal and bradykinin-stimulated (10⁻⁶ mol/L) intracellular content of nitric oxide-dependent cGMP was significantly higher in RFL-6 cells coincubated with aortic segments of cirrhotic rats with (21.3 ± 3.6 pmol/10⁵ cells, P < .05 and 44.7 ± 7.0 pmol/10⁵cells, P < .025) and without ascites (15.3 ± 3.0 pmol/10⁵cells, P < .05 and 43.2 ± 7.6 pmol/10⁵cells, P < .05) than in those incubated with aortic segments of control rats (9.7 ±1.3 and 19.5 ± 2.5 pmol/10⁵cells). RFL-6 cells exposed to bradykinin-stimulated mesenteric arterial segments of cirrhotic rats also showed increased cGMP content (ascitic: 2.73 ± 0.31 pmol/10⁵cells, P < .005; nonascitic: 2.58 ± 0.51 pmol/10⁵cells, P < .025) compared with cells exposed to control mesenteric arterial segments (1.28 ± 0.15 pmol/10⁵cells). No differences between cirrhotic and control vessels were observed after endothelium denudation. These results indicate that basal and bradykinin-stimulated vascular nitric oxide production is higher in cirrhotic rats with and without ascites than in control rats in and that the endothelial lining is the site where vascular L-arginine nitric oxide pathway activation takes place in experimental cirrhosis.     

Basal release of endothelium-derived nitric oxide at site of spasm in patients with variant angina

Objectives.The aim of this study was to investigate the basal release of nitric oxide at spastic sites in patients with variant angina.Background.We previously reported that endothelium-dependent dilator responses to acetylcholine, substance P and bradykinin are preserved at the site of coronary artery spasm. However, it is not known whether the basal release of endothelium- derived nitric oxide is altered at the spastic site.Methods.The effects of intracoronary N^G-monomethyl-l-arginine (l-NMMA, an inhibitor of nitric oxide synthesis) at cumulative doses of 50, 100 and 200 μmol on basal coronary artery tone were investigated in eight patients with variant angina and normal coronary angiograms and in eight control subjects. The lumen diameters of large epicardial coronary arteries were assessed by quantitative coronary arteriography.Results.Coronary spasm was provoked by the intracoronary administration of acetylcholine in all patients with variant angina. l-NMMA did not alter the arterial pressure and heart rate but significantly decreased the coronary artery diameter at spastic and nonspastic sites. Constrictive responses to l-NMMA were significantly greater (p < 0.01) at the spastic site (constriction by 200 μmol, 22 ± 7%, mean ± SD) than at the nonspastic site (10 ± 7%). Constrictive responses to l-NNMA at the nonspastic site in patients with variant angina were comparable to those in the control subjects.Conclusions.These findings support the hypothesis that the basal release of nitric oxide may not be decreased at the spastic site in patients with variant angina.     

内源性一氧化氮在哮喘大鼠气道高反应性中的作用

目的　利用一氧化氮合成前体Ｌ精氨酸（ＬＡｒｇ） 和一氧化氮合酶抑制剂亚硝基Ｌ精氨酸甲酯（ＬＮＡＭＥ）研究内源性一氧化氮在哮喘大鼠气道高反应性中的作用,探讨支气管哮喘的发病机制。方法　用卵白蛋白作为致敏原制备哮喘大鼠模型,建立大鼠离体气管环张力的测定方法,并用ＬＡｒｇ、ＬＮＡＭＥ和ＬＡｒｇ＋ ＬＮＡＭＥ孵育离体气管环,观察气管环乙酰胆碱浓度反应曲线和最大收缩反应的变化,同时观察去上皮对哮喘大鼠气道反应性的影响。结果　哮喘大鼠（１０ 只） 离体气管环经ＬＮＡＭＥ１０５ ｍｏｌ／Ｌ孵育后对乙酰胆碱的最大收缩反应从孵育前（１２４±３９） ｍｇ 上升到（１８７ ±５３） ｍｇ,孵育前、后最大收缩反应比较差异具有显著性（ Ｐ＜ ０．０１）,浓度反应曲线上移,而ＬＡｒｇ 可以逆转ＬＮＡＭＥ的作用,单用ＬＡｒｇ ２×１０５ ｍｏｌ／Ｌ和ＬＡｒｇ１０３ ｍｏｌ／Ｌ孵育气管环,对哮喘大鼠气管环的最大收缩反应和浓度反应曲线与对照组比较,差异无显著性（ Ｐ＞ ０．０５） 。去上皮哮喘大鼠气管环的反应性与上皮完整气管环比较差异有显著性（ Ｐ＜ ０．００５） ,而ＬＡｒｇ、ＬＮＡＭＥ＋ ＬＡｒｇ 和ＬＮＡＭＥ孵育去上     

Adenosine A2A receptor agonists with potent antiplatelet activity

Selected adenosine A_2A receptor agonists (PSB-15826, PSB-12404, and PSB-16301) have been evaluated as new antiplatelet agents. In addition, radioligand-binding studies and receptor-docking experiments were performed in order to explain their differential biological effects on a molecular level. Among the tested adenosine derivatives, PSB-15826 was the most potent compound to inhibit platelet aggregation (EC₅₀ 0.32 ± 0.05 µmol/L) and platelet P-selectin cell-surface localization (EC₅₀ 0.062 ± 0.2 µmol/L), and to increase intraplatelets cAMP levels (EC₅₀ 0.24 ± 0.01 µmol/L). The compound was more active than CGS21680 (EC₅₀ 0.97±0.07 µmol/L) and equipotent to NECA (EC₅₀ 0.31 ± 0.05 µmol/L) in platelet aggregation induced by ADP. In contrast to the results from cAMP assays, K_i values determined in radioligand-binding studies were not predictive of the A_2A agonists’ antiplatelet activity. Docking studies revealed the key molecular determinants of this new family of adenosine A_2A receptor agonists: differences in activities are related to π-stacking interactions between the ligands and the residue His264 in the extracellular loop of the adenosine A_2A receptor which may result in increased residence times. In conclusion, these results provide an improved understanding of the requirements of antiplatelet adenosine A_2A receptor agonists.     

Field stimulation-induced noradrenaline release from guineapig atria is modulated by prejunctional α2-adrenoceptors and protein kinase C

Summary Guinea-pig left atria were loaded with 10 Ci 7-[³H]noradrenaline, and noradrenaline release from sympathetic nerve endings was then elicited by refractory period field stimulation. When one pulse of 0.2 ms duration was applied during each refractory period, the resulting transmitter release was halved by 3×10^–7 mol/l of the ₂-adrenoceptor agonist clonidine and increased about 2.5-fold by either 3×10^–7 mol/l of the ₂-adrenoceptor antagonist idazoxan, 5×10^–3 mol/l of the potassium channel blocker tetraethylammonium chloride (TEA) or 3×10^–7 mol/l phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC). Phorbol-12-myristate-13-acetate-4-O-methylether, a compound which does not stimulate PKC, was ineffective. The stimulatory effect of PMA was antagonized by 7×10^–5 mol/l of the PKC inhibitor polymyxin B. No significant transmitter release was observed when either PMA or TEA was applied together with 10^–7 mol/l of the sodium channel blocker tetrodotoxin. Combinations of either idazoxan and TEA or PMA and TEA caused greater increased of the noradrenaline release than any individual drug given alone. Thus, different mechanisms of action seem to mediate the increase of noradrenaline release by action potential prolongation on the one hand and activation of PKC or inhibition of ₂-adrenoceptors on the other hand. In contrast, the effects of idazoxan and PMA were not additive which suggests a common mechanism of action. In atria pretreated for 10 min with 10^–4 mol/l N-ethylmaleimide, an alkylating agent which inactivates G_i-proteins, neither idazoxan nor PMA caused a significant increase of the stimulation-induced transmitter release, while TEA was still effective. When a train of four pulses, lasting 0.05 ms each, was applied during each refractory period, the resulting transmitter release was not modified by idazoxan or PMA, but was significantly increased by TEA. From these results, a scheme is proposed which, links the regulation of noradrenaline release by prejunctional ₂-adrenoceptors and protein kinase C via an influence on a common inhibitory G_i-protein.     

Effects of the omega-3 fatty acids on vascular tone in hypercholesterolaemia and balloon arterial injury

Dietary polyunsaturated fatty acids (ω-3 PUFAs) are associated with reduced coronary heart disease (CHD) risk. To define mechanisms, we investigated the vasoactive properties of these fatty acids in a rat model of hypercholesterolaemia (HC) and balloon arterial injury. Mature Wistar Kyoto (WKY) rats were fed a control (C), HC-inducing diet or a HC diet after aortic balloon injury (HC+EI) for 3 weeks. Serum cholesterol (mg/dL) was increased in HC (793±31) and HC+EI (857±36) as compared to C (85±3, p≤0.001). Cumulative concentration-response curves to norepinephrine (NE, 10⁻⁹ to 10⁻⁵ mol/L) followed by docosahexaenoic (DHA) or eicosapentaenoic (EPA) (1 to 100 μmol/L) were generated in isolated aortic rings (intact and de-endothelialised, E-). Overall, contractions to NE (10⁻⁹ to 10⁻⁵ mol/L) in intact rings were diminished in HC and HC+EI groups than were apparent in the C group (p≤0.001). De-endothelialised rings exhibited much greater NE contractile responses as compared to intact rings from each of the 3 groups. Endothelium-dependent relaxations to acetylcholine (10⁻⁷ to 10⁻⁴ mol/L) were significantly decreased in the HC+EI group. Relaxant responses to the ω-3 PUFAs, DHA and EPA (1–100 μmol/L) were similar in intact rings, but enhanced in E- rings among the 3 groups. De-endothelialised ring responses were −8.6 to −35.5% (C), −8.4 to −30.5% (HC), −7.8 to −25.6% (HC+EI) to DHA (1–100 μmol/L) and −6.8 to −339% (C), −4.8 to =t-40.8% (HC), −8.9 to −34.6% (HC+EI) to EPA (1–100 μmol/L). In areas of aortic intimal hyperplasia, lipid deposition quantified spectrophotometrically using oil red O (ORO) was greater in HC+EI (0.539 μmol ORO/mm² aortic tissue, p<0.001) as compared to C (0.292) and HC (0.315) groups. We conclude that (1) aortic contractile properties to NE are altered in rats with HC and HC+EI, (2) dietary HC after aortic injury leads to intimal hyperplasia/lipid deposition and impaired endothelium-dependent relaxations in WKY rats, and (3) ω-3 PUFAs have vasorelaxant properties in HC and HC+EI rat aorta. These properties may be beneficial in CHD.     

Effect of terlipressin on in vitro vascular hyporeactivity of portal hypertensive rats

Backgrounds/Aims: Isolated vessels of portal hypertensive rats exhibit decreased responsiveness to vasoconstrictors. The vasopressin analogue terlipressin analogue terlipressin is used in the treatment of portal hypertension since it is known to reduce portal pressure, an effect that is thought to arise from splanchnic vasoconstriction via stimulation of vasoconstrictor V₁ receptors. This study assessed the effect of terlipressin on the in vitro vascular reactivity of portal hypertensive rats to the α-adrenoceptor agonist methoxamine.Methods: Portal hypertension was produced by portal vein ligation. Sham-operated rats served as controls. In isolated perfused mesentric arteries of portal vien ligated and sham-operated rats pressor responses to methoxamine (3 nmol-3 μmol) were determined in the absence and presence of the nitric oxide synthase inhibitor N^g-nitro-L-arginine methyl ester (L-NAME; 100 μM), terlipressin or the selective V₂ receptor agonist desmopressin (each 0.5 μM). In addition, the direct pressor properties of terlipressin (3 pmol-100 nmol) were compared to arginine vasopressin (3 pmol-1 nmol) in vessels of normal rats.Results: Mesenteric vessels of portal vein ligated rats were markedly hyporeactive to methoxamine, even in the presence of L-NAME. Terlipressin alone reduced and in combination with L-NAME abolished the difference in reactivity to methoxamine between the portal vein ligated and sham-operated groups, while desmopressin was ineffective. Arginine vasopressin potently contracted vessels of normal rats with a threshold dose of 10 pmol and was maximally effective at 300 pmol. In contrast, terlipressin failed to produce pressor responses up to 100 nmol.Conclusions: Hyporeactivity of mesenteric vessels of portal vein ligated rats to methoxamine is predominantly independent of nitric oxide. Terlipressin alone ameliorates and in combination with L-NAME abolishes the hyporesponsiveness to methoxamine presumably by inhibiting the nitric oxide-independent mechanism that underlies the reduced responsiveness to methoxamine in portal hypertension. This effect of terlipressin appears to be independent of stimulation of V₂ as well as vasoconstrictor V₁ receptors.     

The vascular endothelium masks the persistent inhibition of rat thoracic arterial tone induced by S-nitrosoglutathione

Aim

In endothelium-denuded arteries, the nitric oxide (NO) donor S-nitrosoglutathione (GSNO) induced a persistent hypo-reactivity to vasoconstrictors, and low-molecular weight thiols such as N-acetyl cysteine (NAC) produced a relaxant effect. These effects were attributed to the formation of vascular NO stores. In arteries with a functional endothelium, such long-lasting effects on arterial tone have not been well characterised. In this study, we proposed to examine the possibility of storing exogenous NO when the vascular endothelium is still able to produce its own NO.

Methods

For this purpose, changes in isometric tension of isolated arteries were assessed in organ chambers, and nitrosothiol formation was characterised by confocal microscopy.

Results

In rat aortic rings with endothelium pre-exposed to GSNO, the contractile response to norepinephrine (NE) was not attenuated in comparison with control rings, but NAC induced a relaxant effect. However, an attenuation of the response to NE was observed in GSNO-exposed, intact aortic rings after inhibition of NO synthase by N^w-nitro-L-arginine methylester (L-NAME) or in GSNO-denuded rings.The relaxing effects of NAC were due to the mobilisation of NO from nitrosothiols after nitrosylation of protein SH residues. Moreover, the hypo-reactivity to NE and the relaxant effect of NAC were abolished by 1H-[1,2,4] oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, and partially by the K⁺-sensitive channel inhibitor tetra-ethyl-ammonium (TEA).

Conclusion

These data show that endothelium-derived NO masked the persistent effect of GSNO in rat thoracic aorta. However, the ability of GSNO to form releasable NO stores without altering the vascular tone can be particularly useful in preventing endothelial dysfunction in which NO formation decreases.     

V2-receptor–mediated relaxation of human renal arteries in response to desmopressin

The effects of deamino-8-d-arginine vasopressin (desmopressin), a V₂ receptor antidiuretic agonist, were studied in isolated rings from branches of renal arteries obtained from 22 patients undergoing nephrectomy. The rings were suspended in organ bath chambers for isometric recording of tension. In precontracted rings with norepinephrine (10⁻⁶ to 3 × 10⁻⁶ mol/L), desmopressin (10⁻¹¹ to 3 × 10⁻⁷ mol/L) caused endothelium-dependent relaxation (81% ± 4% reversal of the initial contraction in arteries with endothelium; 20% ± 4% in arteries without endothelium; P < .05). The relaxation to desmopressin in rings with endothelium was reduced significantly by indomethacin (10⁻⁶ mol/L) and unaffected by the inhibitor of nitric oxide synthase N^G-nitro-l-arginine methyl ester (10⁻⁴ mol/L). Two V₁ receptor antagonists (a peptidic and a nonpeptidic) had no effect on desmopressin-induced relaxation. However, V₂ receptor antagonists (three peptidic and a nonpeptidic) reduced significantly (P < .05) the maximal response to desmopressin. The results of this study show that desmopressin exerts powerful endothelium-dependent relaxation of human renal arteries, probably through stimulation of V₂-like receptors that may bring about the release of dilating prostaglandins.     

Nitric oxide generation mediated by β-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus

Aims/hypothesis Type 2 diabetic patients have been shown to have reduced basal platelet nitric oxide synthase activity, which is a possible contributor to the vascular complications seen in the disease. We investigated platelet nitric oxide generation stimulated by -adrenoceptors and adenylyl cyclase in Type 2 diabetic patients and control subjects.Methods Platelets isolated from blood taken from nine Type 2 diabetic patients and nine healthy control subjects of similar age were treated with isoproterenol 1 µmol/l, forskolin 1 µmol/l or vehicle. Platelet nitric oxide synthase activity was measured by L-[³H]-arginine to L-[³H]-citrulline conversion, cyclic GMP content by radioimmunoassay, and nitric oxide synthase type 3 expression by western blotting.Results Basal platelet nitric oxide synthase activity was lower in diabetic patients than in control subjects (0.01±0.02 pmol L-citrulline/10⁸ platelets, compared with 0.12±0.05; p<0.05), although no corresponding difference was seen in basal platelet cyclic GMP (0.61±0.39 and 0.13±0.22 pmol cyclic GMP/10⁸ platelets respectively; p=0.37). In control subjects isoproterenol 1 µmol/l and forskolin 1 µmol/l increased platelet nitric oxide synthase activity (to 0.27±0.08 and 0.27±0.07 pmol L-citrulline/10⁸ platelets respectively; p<0.05 for each in comparison with basal) and cyclic GMP (to 1.84±0.41 and 1.86±0.48; p<0.05 for each in comparison with basal). This effect was not achieved in diabetic patients. Isoproterenol- and forskolin-stimulated cyclic GMP correlated inversely with plasma glucose and HbA_1c. Platelet nitric oxide synthase type 3 expression was not different in control and diabetic subjects and was not changed by acute exposure of platelets to isoproterenol.Conclusions/interpretation Nitric oxide generation stimulated by -adrenoceptors and adenylyl cyclase is impaired in platelets of people with Type 2 diabetes mellitus, with no corresponding change in nitric oxide synthase type 3 expression. It is possible that this impairment contributes to the thrombotic and atherosclerotic complications of Type 2 diabetes.Abbreviations AR -adrenoceptors - GFP gel-filtered platelets - L-NAME N^G-nitro-L-arginine methyl ester - L-NMMA N^G-monomethyl-L-arginine - NO nitric oxide - NOS2 nitric oxide synthase type 2 - NOS3 nitric oxide synthase type 3     

Up-regulation of A 2B adenosine receptor in A 2A adenosine receptor knockout mouse coronary artery

In this study, we looked into possible compensatory changes of other adenosine receptors (ARs) in A_2A genetic knockout mice (A2AKO) as well as the functional role of nitric oxide (NO) in A_2A AR-mediated vasodilation. Gene expression of ARs from coronary arteries of A_2A AR wild type mice (A2AWT) and A2AKO was studied using real-time PCR. Functional studies were carried out in isolated heart and isolated coronary artery preparations. A_2B AR was found to be 4.5 fold higher in A2AKO than in A2AWT, while A_2A AR expression was absent in A2AKO. There was no difference in A₁ and A₃ ARs between WT and KO animals. The concentration-relaxation curve for adenosine-5′-N-ethylcarboxamide (NECA, non-selective AR agonist) in isolated coronary arterial rings in A2AKO was shifted to the left when compared to A2AWT. The concentration-response curve for A_2B selective agonist (BAY 60-6583) was also shifted to the left in A2AKO hearts. L-NAME, a non-specific NO synthase inhibitor, did not affect baseline coronary flow (CF) until the concentration reached 10 µM in A2AWT (76.32 ± 11.35% from baseline, n = 5). In A2AKO, the CF decreased significantly by L-NAME only at a higher concentration (100 µM, 93.32 ± 5.8% from baseline, n = 5). L-NMA (1 µM, n = 4), another non-specific NO synthase inhibitor, also demonstrated similar results in decreasing CF (59.66 ± 3.23% from baseline in A2AWT, while 81.76 ± 8.91% in A2AKO). It was further demonstrated that the increase in CF by 100 µM NECA was significantly blunted with 10 µM L-NAME (377.08 ± 25.23% to 305.41 ± 30.73%, n = 9) in A2AWT but not in A2AKO (153.66 ± 22.7% to 143.88 ± 36.65%, n = 5). Similar results were also found using 50 nM of CGS-21680 instead of NECA in A2AWT (346 ± 22.85 to 277 ± 31.39, n = 6). No change in CF to CGS-21680 was noted in A_2AAKO. Our data demonstrate, for the first time, that coronary A_2B AR was up-regulated in mice deficient in A_2A AR. We also provide direct evidence supporting a role for NO in A_2A AR-mediated coronary vasodilation. The data further support the role for A_2A AR in the regulation of basal coronary tone through the release of NO.