Mechanisms involved in UTP-induced contraction in isolated rat aorta

The mechanisms of UTP-induced contractions in the rat aorta strips were studied. These were only partially inhibited in a Ca(2+)-free medium or by incubation with verapamil or nifedipine. Successive challenges did not decrease the magnitude of the contraction in the absence of external Ca(2+). Quin 2(acetoxymethyl) ester (Quin 2AM), 8-(N,N-diethylamino)octyl 3,4,5-trimetoxybenzoate (TMB-8), thapsigargin and ryanodine inhibited these contractions. The participation of protein kinase C is also very likely, since downregulation by the phorbol 12,13 dibutyrate (PDB) decreased UTP-induced contraction, and staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) antagonized UTP-induced contractions and relaxed UTP-induced tonic contractions. Therefore, different pools of intracellular Ca(2+) and protein kinase C seem to participate in UTP-induced contraction and in the mechanisms of maintenance in a Ca(2+)-free medium.     

Chlorzoxazone inhibits contraction of rat thoracic aorta

Chlorzoxazone has been reported to activate the intermediate-conductance, Ca(2+)-activated K(+) channels in aortic endothelial cells and to relax the artery. The aim of the present study was to characterize the chlorzoxazone-induced relaxation of rat thoracic artery. Chlorzoxazone did not affect the tension of the thoracic artery rings at rest, but relaxed the precontraction induced by 1 muM noradrenaline in an endothelium independent manner. Preincubation with chlorzoxazone also antagonized the contraction induced by 1 microM noradrenaline or 25 mM KCl. The chlorzoxazone-induced relaxation of the thoracic artery pre-contracted by noradrenaline was suppressed by 5 mM tetraethylammonium, 75 mM ethanol and 2 microM paxilline, but not by 2 microM clotrimazole. Chlorzoxazone relaxed the 4-aminopyridine-induced contraction. The pattern of chlorzoxazone-induced relaxation was different from that of verapamil, the L-type Ca(2+) channel blocker. The inhibition of the noradrenaline-induced contraction by chlorzoxazone was attenuated when chlorzoxazone treatment was prolonged to 4 h. No difference in the contraction-relaxation was found between the artery rings from normal rats and those from rats that received 100 mg/kg chlorzoxazone for 7 days. We conclude that chlorzoxazone abolishes the contraction of rat thoracic artery induced by noradrenaline and that the effect of chlorzoxazone is endothelium independent and also not mediated by intermediate-conductance, Ca(2+)-activated K(+) channels.     

Mechanisms underlining gender differences in Phenylephrine contraction of normoglycaemic and short-term Streptozotocin-induced diabetic WKY rat aorta

The female gender reduces the risk, but succumbs more to cardiovascular disease. The hypothesis that short-term (8weeks) Streptozotocin-induced diabetes could produce greater female than male vascular tissue reactivity and the mechanistic basis were explored. Aortic ring responses to Phenylephrine were examined in age- and sex-matched normoglycaemic/diabetic rats. The normoglycaemic male tissue contracted significantly more than the normoglycaemic female and the male/female diabetic tissues. Endothelial-denudation, l-NAME or MB reversed these differences suggesting an EDNO-cGMP dependence. 17β-oestradiol exerted relaxant effect on all endothelium-denuded (and normoglycaemic endothelium-intact male) tissues, but not endothelium-intact normoglycaemic female. The greater male tissue contraction is attributable to absent 17β-oestradiol-modulated relaxation. Indomethacin blockade of COX attenuated male normoglycaemic and female diabetic tissue contraction (both reversed by l-NAME), but augmented diabetic male tissue contraction. These data are consistent with the raised contractile TXA(2) and PGE(2) in normoglycaemic male and diabetic female tissues, and the relaxant PGI(2) in diabetic male (and female). The higher levels of PGI(2) in the normoglycaemic and diabetic female perhaps explain their greater relaxant response to Acetylcholine compared to the respective male. In conclusion, there is an endothelium-dependent gender difference in the effect of short term diabetes on vascular tissue reactivity which is COX mediated.     

Mechanism of human urotensin II-induced contraction in rat aorta

Urotensin II induced sustained contraction with an EC(50) value of 2.29 +/- 0.12 nM in rat aorta. Urotensin II (100 nM) transiently increased cytosolic Ca(2+) level ([Ca(2+)](i)), followed by a small sustained phase superimposed with rhythmic oscillatory change. In the presence of verapamil and La(3+), the [Ca(2+)](i) oscillation was completely inhibited, although a small transient increase in [Ca(2+)](i) remained. The urotensin II-induced contraction was also partially inhibited by verapamil and La(3+). Combined application of verapamil, La(3+), and thapsigargin completely inhibited the increase in [Ca(2+)](i) with only partial inhibition of the contraction elicited by urotensin II. Urotensin II increased myosin light chain (MLC) phosphorylation to a level greater than that induced by 72.7 mM KCl (high K(+)). Pretreatment with Go6983 (PKC inhibitor), U0126 (MEK inhibitor), or SB203580 (p38MARK inhibitor) partially inhibited the urotensin II-induced contraction with no effects on the high K(+)-induced contractions. Wortmannin (MLC kinase inhibitor) only partially inhibited urotensin II-induced contraction, although it completely inhibited the high K(+)-induced contraction. These results suggest that urotensin II-induced contraction is mediated by the Ca(2+)/calmodulin/MLC kinase system and modulated by the Ca(2+) sensitization mechanisms to increase MLC phosphorylation. In addition, activations of PKC, p38MAPK, and ERK1/2 modulate the contractility mediated by urotensin II in rat aorta.     

Characterization of histamine-induced contraction in rat isolated aorta.

High concentrations of histamine (greater than 10 microM) contract rat aortic rings and the effect is greatly enhanced when the endothelium is removed. The present study was aimed at characterizing the histamine-induced contractions of de-endothelialized rat aortic rings. These contractions were poorly inhibited by the histamine H1-receptor antagonist, mepyramine (1 and 10 microM) and insensitive to the histamine H2-receptor antagonist, cimetidine (10 microM), and to the cyclooxygenase inhibitor, indomethacin (5 microM). In contrast, the alpha-adrenoceptor antagonists, prasozin and pentholamine, antagonized these contractions in a concentration-dependent manner (respective apparent pKB values 9.7 and 7.9) and nifedipine (3 microM) reduced them by about 75%. Pretreatment of de-endothelialized rings with 8-bromo-cyclic GMP and of intact rings with methylene blue resulted in respective inhibition and enhancement of histamine-induced contractions, quite similarly to the effects in the presence and in the absence of endothelium, respectively. Histamine elicited endothelium-dependent relaxation of aortic rings precontracted by prostaglandin F2 alpha. This relaxation was abolished in the presence of mepyramine (1 microM). However, mepyramine failed to mimic the enhancing effect of endothelium removal on histamine-induced contractions of resting aortic rings. It is concluded that, in rat aorta, (1) contractions induced by high concentrations of histamine (greater than 10 microM) are probably mediated by alpha 1-adrenoceptors; and (2) spontaneous, but not histamine-stimulated, release of endothelium-derived relaxing factor is mainly involved in the modulation of histamine-induced contractions.     

Effects of glyceollin I on vascular contraction in rat aorta

The present study was undertaken to investigate the molecular mechanisms by which glyceollin I inhibits vascular contraction in rat aorta. Rat aortic rings were treated with either glyceollin I or vehicle when vascular contraction reached plateaus. We measured the activity of GTP-RhoA and Rho GTPase-activating protein (RhoGAP) and the phosphorylation level of the myosin light chain (MLC₂₀), myosin phosphatase targeting subunit 1 (MYPT1), and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Glyceollin I reduced vascular contraction whether endothelium is present or denuded. Glyceollin I reduced vascular contraction induced by NaF, U46619, phenylephrine, or PDBu. Blockers of K⁺ channels did not affect the vasorelaxation induced by glyceollin I. Glyceollin I reduced activation of RhoA as well as phosphorylation level of MLC₂₀. Glyceollin I also reduced phosphorylation of MYPT1 and CPI17 induced by NaF but not PDBu. However, glyceollin I had no direct effect on RhoGAP activation in vitro. Glyceollin I reduced vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway.     

Leu5]enkephalin inhibits norepinephrine-induced contraction of rat aorta

Leucine-enkephalin produces a dose related decrease in the contractile response of spirally cut strips of rat aorta to norepinephrine (NE). The maximal effect (55.0 +/- 1.0 percent reduction in developed tension) occurs at a leucine-enkephalin concentration of 10(-10) M. The attenuation of contractile response is reversed by naloxone.     

Oxygen-derived free radical-induced vasoconstriction by thromboxane A2 in aorta of the spontaneously hypertensive rat

This study was performed to clarify the mechanism of vasoconstriction induced by oxygen-derived free radicals in spontaneously hypertensive rats. The isometric tension of aortic rings from spontaneously hypertensive rats and Wistar-Kyoto rats was measured in Krebs-Henseleit solution. Oxygen-derived free radicals were generated by mixing xanthine and xanthine oxidase. The removal of endothelium enhanced the contractions induced by oxygen-derived free radicals. The inhibition of nitric oxide production with NG-nitro-L-arginine methyl ester (10(-4) M) enhanced the contractions. Treatment with the thromboxane A2 (TXA2) synthetase inhibitor OKY-046 (10(-4) M) or RS-5186 (10(-4) M) markedly reduced the contractions. Treatment with the cyclooxygenase inhibitor indomethacin (10(-5) M) and a TXA2/prostaglandin H2 (PGH2) receptor antagonist, ONO-3708 (10(-6) M), completely abolished the oxygen-derived free radical-induced contractions. In contrast, treatment with the PGI2 synthetase inhibitor tranylcypromine (10(-4) M) did not attenuate the oxygen-derived free radical-induced contractions. Whether endothelium was present or not, the release of TXB2, PGE2, and 6-keto-PGF1alpha, but not PGF2alpha, was increased by the production of oxygen-derived free radicals. Catalase and the hydroxyl radical scavenger deferoxamine plus mannitol markedly inhibited the oxygen-derived free radical-induced contractions. These results suggest that oxygen-derived free radical-induced vasoconstriction in spontaneously hypertensive rat aorta is caused by TXA2 and PGH2 released in smooth muscle.     

Mechanisms underlying pervanadate-induced contraction of rat cremaster muscle arterioles

The current study examined the role of extracellular Ca2+, calmodulin and myosin light-chain kinase (MLCK) in pervanadate-induced constriction of cannulated, pressurized rat cremaster arterioles. Pervanadate (0.03-100 microM) induced a concentration-dependent constriction of arterioles that was significantly attenuated (P<0.05) by the tyrosine kinase inhibitor tyrphostin 47 (30 microM). The L-type voltage-sensitive Ca2+ channel antagonists verapamil (10 microM) and nifedipine (1 microM) dilated vessels possessing myogenic tone but had no demonstrable effect on pervanadate constriction, while a higher concentration of nifedipine (10 microM) reduced constriction by approximately 50%. Pervanadate-induced contractions were reduced by the calmodulin inhibitor W-7 (N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide, 50 microM) and the MLCK inhibitor ML-7 (1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine, 10 or 30 microM). Removal of extracellular Ca2+ abolished the contractile effect of pervanadate. Measurement of changes in arteriolar wall [Ca2+] using the Ca2+ sensitive dye Fura-2 showed that pervanadate did not increase [Ca2+] during arteriolar constriction. These observations suggest that pervanadate-induced contraction of smooth muscle in the cremaster arteriole involves Ca2+/calmodulin-dependent myosin phosphorylation and possibly sensitization of the contractile apparatus to Ca2+.     

Uridine adenosine tetraphosphate induces contraction and relaxation in rat aorta

Uridine adenosine tetraphosphate (Up(4)A) has been recently reported as an endothelium-derived vasoconstrictor and plasma levels of this dinucleotide are increased in juvenile hypertensive subjects. This study aimed to evaluate the vascular actions of Up(4)A, typify the putative purinergic receptors that might mediate these effects and characterize the intracellular signaling pathways that may govern Up(4)A responses. Up(4)A induced a modest endothelium-dependent relaxation of rat aortic rings contracted with phenylephrine. From baseline, Up(4)A induced concentration-dependent contractions that were significantly potentiated by endothelium removal or nitric oxide synthase inhibition. The contractile response induced by Up(4)A was not tachyphylactic and was significantly reduced in the presence of P1 or P2X receptor antagonists, L-type Ca(2+) channel blocker and Rho-kinase inhibitor. Up(4)A-induced contraction apparently involves superoxide anion formation since it was significantly reduced by treatment with apocynin or tempol. This study presents the unique findings that the endogenous compound Up(4)A is able to induce relaxation in addition to contraction of rat aorta. Up(4)A-induced contraction is modulated by nitric oxide production, mediated by P1 and P2X receptor activation, and involves L-type Ca(2+) channels, Rho-kinase pathway and superoxide formation.     

Rapid effect of progesterone on the contraction of rat aorta in-vitro

Progesterone induced rapid relaxation of KCl-induced contraction of rat aortic rings. The relaxant effect of progesterone on aortic rings was concentration-dependent (over the range of 10(-10) to 10(-5) M) and partially dependent on the endothelium. Application of a nitric oxide (NO) synthase antagonist N(G)-monomethyl-L-arginine (L-NMMA, 10(-5) M) after progesterone treatment partially inhibited the relaxant effects of progesterone. This suggested that part of the effect was through the production of nitric oxide. Washing out the steroid hormone in the bath solutions could quickly reverse the inhibitory effects of progesterone on phasic tension generation in aortic rings. Five minutes after washout, the tension generation in aortic rings was completely restored. Cultured endothelial cells from rat aorta increased release of NO into culture media in response to a 60-min exposure to progesterone. Aldosterone and dexamethasone were also tested, and failed to relax KCl-induced contraction of aortic rings. These data suggest that the vascular effects of progesterone are not mediated by a genomic action of this steroid, and that the vascular effects are mediated partially through endothelial NO production.     

Atrial peptides inhibit protein kinase C-mediated contraction in rat aorta

The effects of atriopeptin III on phorbol ester-induced contraction were examined in aorta from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Precontracted SHR aorta was less responsive than WKY aorta to atriopeptin III-induced relaxation. Additionally, SHR aorta had significantly greater basal and phorbol ester-stimulated protein kinase C (PKC) activity than WKY aorta. The altered PKC response as well as hyporesponsiveness to atriopeptin III in SHR aorta may contribute to an altered vascular response and to the pathogenesis of hypertension.     

Microtubule depolymerization facilitates contraction of rat aorta via activation of Rho-kinase

This study tests the hypothesis that microtubule (MT) depolymerization facilitates contraction of rat aorta via activation of Rho-kinase. Aortic rings from Sprague-Dawley rats were placed in a muscle bath for the measurement of isometric force generation. Bath temperature was decreased from 37 to 10-20 degrees C (30 min), inducing MT depolymerization. Some vessels were treated with nocodazole (10(-5) M) or colchicine (10(-8)-10(-5) M) to stabilize the MTs in the depolymerized state, and the remaining vessels were treated with dimethyl sulfoxide (DMSO: vehicle). Warming of vessels to 37 degrees C induced a significantly greater contraction in nocodazole- and colchicine-treated vessels as compared with controls, and this increase was blocked by pretreatment with taxol (10(-5) M; a MT stabilizing agent) [force (mg): NOC 1159 +/- 93; COL 1138 +/- 69; DMSO 578 +/- 14; TAX + NOC 526 +/- 43; TAX + COL 538 +/- 90]. Following the sustained contraction in response to rewarming, Rho-kinase inhibition with Y-27632 (10(-5) M) relaxed nocodazole- and colchicine-treated rings to a significantly greater extent as compared to DMSO-treated vessels (percent relaxation: NOC 64 +/- 2; COL 65 +/- 5; DMSO 33 +/- 5). These results support the hypothesis that MT depolymerization facilitates contraction of rat aorta via activation of Rho-kinase.     

Mechanisms of vasorelaxation to testosterone in the rat aorta

We have investigated the role of endothelium-derived relaxing factors, K(+) channels and steroid receptors in vasorelaxation to testosterone in the rat aorta. Testosterone (1 nM-mM) caused acute concentration-dependent vasorelaxation. Both indomethacin (10 microM) and flurbiprofen (10 microM) uncovered relaxant responses to testosterone. The action of indomethacin was inhibited by endothelial removal. N(G)-nitro-L-arginine methyl ester (L-NAME, 300 microM) had no effects on testosterone-induced responses. In the presence of indomethacin, the vasorelaxant potency of testosterone was reduced by depolarization with 60 mM KCl or charybdotoxin (100 nM), but not by glibenclamide (10 microM), 4-aminopyridine (1 mM) or barium chloride (30 microM). The responses to testosterone were not inhibited by flutamide (10 microM) or mifepristone (30 microM). Pre-treatment of the aorta with testosterone (100 microM) inhibited CaCl(2)-induced contraction. In the present study, we have demonstrated that testosterone causes acute vasorelaxations, which are modulated via endothelium-derived prostanoids. The responses uncovered by cyclooxygenase inhibitors are due to the activation of K(Ca) channels, while at higher concentrations, testosterone inhibits Ca(2+) influx.     

Mechanisms of diethylstilbestrol-induced relaxation in rat aorta smooth muscle

The mechanisms of diethylstilbestrol (1 to 30 microM)-induced relaxation on noradrenaline (30 nM)-raised tone in the rat aorta smooth muscle were studied. Neither the increase of calcium content in the medium (3, 6 and 9 mM) nor Bay K 8644 (3, 10 and 100 nM) reversed diethylstilbestrol relaxation. Tamoxifen (3 microM), the quaternary derivate (tamoxifen ethyl bromide, 3 microM), actinomycin D (30 microM), cycloheximide (100 microM), Rp-cAMPS (30 microM), TPCK (1 microM) and difluoromethylornithine (1 mM) inhibited diethylstilbestrol-induced relaxation. Incubation with 2 microg/ml pertussis toxin, propranolol (1 microM), H-7 (10 microM), 2',3'- and 2',5'-dideoxiadenosine (10 and 30 microM, respectively) and methylene blue (10 microM) did not modify diethylstilbestrol-induced relaxation. Our results showed that presumably an activation of membrane mechanisms, protein kinase A activation, genomic mechanisms and polyamine synthesis might participate in diethylstilbestrol-elicited relaxation in addition to the increase in K(ATP) permeability, as previously described. Actinomycin D produces a synergistic effect, with tamoxifen, difluoromethylornithine and glibenclamide antagonizing the effect of diethylstilbestrol. In the case of the association of actinomycin D and glibenclamide, the antagonism of relaxation is complete. The fact that tamoxifen- and difluoromethylornithine-dependent mechanisms participate in diethylstilbestrol relaxation inhibited by glibenclamide suggests that two transduction pathways are involved in the relaxation. Therefore, K(ATP) channels and genomic mechanisms, both modulated by cyclic AMP (cAMP)-dependent mechanisms, are associated with diethylstilbestrol relaxation.     

Mechanisms of methyclothiazide-induced inhibition of contractile responses in rat aorta

Methyclothiazide (MCTZ), a thiazide diuretic, inhibits the contractile response induced by norepinephrine in aortic rings from 12-week-old spontaneously hypertensive rats (SHR). Although not modified by indomethacin, this inhibition was attenuated by either mechanical removal of the endothelium or N omega-nitro-L-arginine (NOLA) treatment. These results suggest that the MCTZ effects on the norepinephrine-evoked vascular response are mediated by an endothelium-dependent mechanism involving endothelium-dependent relaxing factor (EDRF)/nitric oxide (NO) release. MCTZ was also found to alter the contractile response induced by the addition of Ca(2+) to a depolarizing solution, and this inhibitory effect was partially abolished by NOLA application. Our data led us to propose that MCTZ relaxes aortic rings, resulting in an endothelium-dependent relaxation phenomenon that could even be reinforced under high-K(+) depolarizing conditions.     

银杏内酯B对离体大鼠胸主动脉收缩活动的影响

目的:研究银杏内酯B对大鼠离体胸主动脉血管环收缩活动的影响.方法:采用离体血管灌流的方法,观察银杏内酯B对不同浓度氯化钾、去甲肾上腺素引起的大鼠胸主动脉收缩反应的影响,以收缩率为指标.结果:银杏内酯B高浓度(0.1 g·L-1)和中浓度(0.03 g·L-1)对KCl(60 mmol·L-1)引起的胸主动脉收缩具有拮抗作用(P<0.05,P<0.01),并能使20 mmol·L-1～60 mmol·L-1氯化钾对胸主动脉的收缩作用减弱或明显减弱(P<0.05,P<0.01);银杏内酯B高浓度(0.1 g·L-1)和中浓度(0.03 g·L-1)对去甲肾上腺素(10-5 mol·L-1)引起的大鼠胸主动脉收缩具有拮抗作用(P<0.05,P<0.01),并能使0.1～10 μmol·L-1 NE对胸主动脉的收缩作用减弱或明显减弱(P<0.05,P<0.01).结论:银杏内酯B对氯化钾、去甲肾上腺素引起的大鼠离体胸主动脉血管环收缩活动有明显拮抗作用.     

Mechanisms of vasorelaxation induced by N-allylsecoboldine in rat thoracic aorta

N-Allylsecoboldine was shown to be the most effective of several boldine derivatives that were tested for their vasorelaxing effect on the rat aorta. In KCl (60 mmol/l) medium, Ca²⁺ (0.03–3 mmol/l)-induced vasoconstriction was inhibited, concentration-dependently, by N-allylsecoboldine. The IC₅₀ for N-allylsecoboldine was calculated to be about 4 mol/l (for a Ca²⁺ concentration of 1 mmol/1). The vasorelaxant effect on KCl-induced responses was more pronounced at 60 mmol/l KCl than at 15 mmol/1 KCI. Contraction of rat aorta in response to phenylephrine (0.01-100 mol/l) was concentration-dependently inhibited by N-allylsecoboldine and by verapamil (3–30 mol/l), while contraction in response to B-HT 920, serotonin or PGF₂ was not affected. This relaxing effect of N-allylsecoboldine persisted in endothelium-denuded aorta. In cultured A 10 vascular smooth muscle cells, N-allylsecoboldine and verapamil displaced the binding of [³H]-prazosin (K _i values = 0.4±0.2 and 0.6±0.2 mol/l, respectively). The increase of inositol monophosphate caused by phenylephrine in rat aorta was completely suppressed by chloroethylclonidine, but only slightly inhibited by N-allylsecoboldine and by verapamil. Glibenclamide or charybdotoxin did not affect the relaxation induced by N-allylsecoboldine of aortic rings precontracted with phenylephrine. Neither the cGMP nor the cAMP content was changed by N-allylsecoboldine. We conclude that N-allylsecoboldine relaxes the rat aorta by blocking Ca²⁺ channels and that it also has an antagonistic effect at ₁-adrenoceptors. Correspondence to: C.M. Teng at the above address