Contraction Of Rabbit Basilar Artery Research Articles

Altered expression of connexin43 and its possible role in endothelin - 1 - induced contraction in rabbit basilar artery

Objective To investigate the role of connexin43 in ET- 1 -induced contraction in rabbit basilar artery.Methods The ET - 1 - induced contraction without or with carbenoxolone was studied with an isometric tension system.The expression of connexin43 protein in ET - t stimulated basilar arteries was studied with Western blot.Scrape/scratch method was used to analyze the function of gap junction in cultured rabbit cerebrovascular smooth muscle ceils.Results ET - 1 produced a concentration - dependent contraction.Carbenoxolone inhibited ET- 1 induced contraction.The connexin43 protein level was increased in ET- 1stimulated basilar arteries.Carbenoxolone decreased the connexin43 protein level increased by ET - 1.Cells treated with ET - 1 appeared positive communication and the dye transfer was increased in a time - dependent fashion.Carbenoxolone suppressed the ET - 1 - induced increasement of dye transfer.Conclusions The enhancement of gap junction intercellular communication is activated by ET - 1 via modulating the expression of connexin43,and plays an important role in the pathogenesis of cerebral vasospasm.Inhibition of vascular GJIC as a means of reducing cerebral vasospasm after SAH may have therapeutic advantage. Key words: Connexin43 ; Gap junction ; ET - 1 ; Cerebral vasospasm

Altered expression of connexin43 and its possible role in endothelin-1-induced contraction in rabbit basilar artery

Background and purpose: Endothelin-1 (ET-1) plays an important role in the pathogenesis of cerebral vasospasm. Gap junction participates in the pathologic processes of cerebrovascular diseases and may play an important role. The aim of this study is to investigate the role of connexin43 in ET-1-induced contraction in rabbit basilar artery.Methods: The ET-1-induced contraction without or with carbenoxolone was studied with an isometric tension system. Expression of connexin43 protein in ET-1–stimulated basilar arteries was studied with Western blot. Scrape/scratch method was used to analyse the function of gap junction in cultured rabbit cerebrovascular smooth muscle cells.Results: (1) ET-1 produced a concentration-dependent contraction. Carbenoxolone inhibited ET-1-induced contraction; (2) connexin43 protein level is increased in ET-1-stimulated basilar arteries. Carbenoxolone decreases the connexin43 protein level increased by ET-1; (3) cells treated with ET-1 appeared positive communicate and the dye transfer was increased in a time-dependent fashion; 4 carbenoxolone suppressed the ET-1-induced increases in dye transfer.Conclusion: The enhancement of gap junction intercellular communication is activated by ET-1 via modulating the expression of connexin43, and plays an important role in the pathogenesis of cerebral vasospasm. Inhibiting vascular GJIC as a means of reducing cerebral vasospasm after SAH may be of therapeutic advantage.

Chloride Efflux is Involved in ET-1 and 5-HT-Induced Contraction in Rabbit Basilar Artery

Chloride (Cl-) efflux induces depolarization and contributes to contraction of cerebral arteries. We tested the effect of endothelin-1 and 5-hydroxytryptamine on isometric tension in rabbit basilar artery by inhibition of Na+-K+-2Cl- co-transporter and Cl-/HCO3- exchanger to decrease Cl-, by decreasing extracellular Cl- concentration, and by blocking Cl- channels using Cl- channel inhibitors. We have made the following observations: (i)endothelin-1 and 5-hydroxytryptamine produced contraction in the normal Cl- Krebs-Henseleit bicarbonate solution (123 mM Cl-); (ii)inhibition of Na+-K+-2Cl- co-transporter with bumetanide abolished the contractions; (iii) bicarbonate-free solution with HEPES reduced contractions to 5-hydroxytryptamine and endothelin-1; (iv) substitution of extracellular Cl- with methanesulfonate acid (MS- 113 mM, Cl- 10 mM) enhanced peak contraction to 5-hydroxytryptamine and endothelin-1 and decreased plateau contraction to 5-hydroxytryptamine, but did not affect the plateau contraction to endothelin-1 and KCl; and (v) blockade of Ca2+-dependent Cl- channel with niflumic acid and non-selective Cl- channel with 5-nitro-2-(3-phenylpropylamino) benzoic acid and indanyloxyacetic acid-94, R- (+)-methylindazone (R- (+)-IAA-94)decreased contractions to endothelin-1 and 5-hydroxytryptamine.However, removal of endothelium attenuated the effect of Cl-channel inhibitors. In conclusion, Cl- channels and Cl- flux are involved in endothelin-1-induced and 5-hydroxytryptamine-induced contraction in rabbit basilar artery. Cl- channel blockers might exert additional effects by releasing vasodilatation agents from endothelial cells.

Mechanisms underlying diabetes enhancement of endothelin-1-induced contraction in rabbit basilar artery

The influence of alloxan-induced diabetes on the reactivity of rabbit basilar artery to endothelin-1 was examined. Endothelin-1 induced concentration-dependent contraction of basilar arteries that was higher in diabetic than in control rabbits. Endothelium removal produced a higher enhancement of the endothelin-1-induced contraction in control than in diabetic rabbits. N G-nitro- l-arginine ( l-NOArg) enhanced the maximal contraction induced by endothelin-1 in control rabbits and potentiated this response in diabetic rabbits. Endothelin ET A receptor antagonist, cyclo( d-Asp-Pro- d-Val-Leu- d-Trp) (BQ-123), inhibited endothelin-1-induced contraction in both rabbit groups. Endothelin ET B receptor antagonist, 2,6-Dimethylpiperidinecarbonyl-γ-Methyl-Leu-N in-(Methoxycarbonyl)- d-Trp- d-Nle (BQ-788), enhanced endothelin-1-induced contraction in control rabbits and decreased the potency of endothelin-1 in diabetic rabbits. Sodium nitroprusside-induced relaxation of basilar arteries was lower in diabetic than in control rabbits. These results suggest that mechanisms underlying rabbit basilar artery hyperreactivity to endothelin-1 include decreased endothelial modulation of endothelin-1-induced contraction, with impaired endothelial endothelin ET B receptor activity; decreased sensitivity to nitric oxide (NO) in vascular smooth muscle; and enhanced participation of muscular endothelin ET A and ET B receptors.

Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery.

This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ET(A) and ET(B) receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ET(A), but not ET(B), receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 microM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.

Characterization of Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery.

This study attempted to characterize Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery using whole-cell patch-clamp and measurement of intracellular free Ca2+ concentration. Endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in addition to the voltage-operated Ca2+ channel (VOCC). These channels can be discriminated using Ca2+ channel blockers, SK&F 96365 and LOE 908. Tension study was conducted to clarify the Ca2+ channels involved in endothelin-1-induced contraction of basilar artery. Endothelin-1-induced basilar artery contraction is fully dependent on extracellular Ca2+ influx. Based on sensitivity to nifedipine, an L-type VOCC blocker, VOCCs have a minor role in endothelin-1-induced contraction. Both LOE 908 and SK&F 96365 inhibit endothelin-1-induced contraction in a concentration-dependent manner, and their combination abolished it. The median inhibitory concentrations of these blockers for endothelin-1-induced contraction correlated well with those of the endothelin-1-induced [Ca2+]i responses. Thus, the inhibitory action of these blockers on endothelin-1-induced contraction may be mediated by blockade of NSCC-1, NSCC-2, and the SOCC. Extracellular Ca2+ influx through NSCC-1, NSCC-2, and SOCC may be essential for endothelin-1-induced basilar artery contraction.

Manipulation of chloride flux affects histamine-induced contraction in rabbit basilar artery.

Cl(-) efflux induces depolarization and contraction of smooth muscle cells. This study was undertaken to explore the role of Cl(-) flux in histamine-induced contraction in the rabbit basilar artery. Male New Zealand White rabbits (n = 16) weighing 1.8-2.5 kg were euthanized by an overdose of pentobarbital sodium. The basilar arteries were removed for isometric tension recording. Histamine produced a concentration-dependent contraction that was attenuated by the H(1) receptor antagonist chlorpheniramine (10(-8) M) but not by the H(2) receptor antagonist cimetidine (3 x 10(-6) M) in normal Cl(-) Krebs-Henseleit bicarbonate solution (123 mM Cl(-)). The histamine-induced contraction was reduced by the following manipulations: 1) inhibition of Na(+)-K(+)-2Cl(-) cotransporter with bumetanide (3 x 10(-5) and 10(-4) M), 2) bicarbonate-free HEPES solution to disable Cl(-)/HCO exchanger, and 3) blockade of Cl(-) channels with the use of niflumic acid, 5-nitro-2-(3-phenylpropylamino) benzoic acid, and indoleacetic acid 94 R-(+)-methylindazone. In addition, substitution of extracellular Cl(-) (10 mM) with methanesulfonate acid (113 mM) transiently enhanced histamine-induced contraction. Manipulation of Cl(-) flux affects histamine-induced contraction in the rabbit basilar artery.

Role of Cl- current in endothelin-1-induced contraction in rabbit basilar artery.

Cl- efflux induces depolarization and contraction of smooth muscle cells. This study was undertaken to explore the role of Cl- channels in endothelin-1 (ET-1)-induced contraction in rabbit basilar artery. Male New Zealand White rabbits (n = 26), weighing 1.8-2.5 kg, were euthanized by an overdose of pentobarbital. The basilar arteries were removed for isometric tension recording. ET-1 produced a concentration-dependent contraction of the rabbit basilar artery in the normal Cl- Krebs-Henseleit bicarbonate buffer (123 mM Cl-). The ET-1-induced contraction was reduced by the following manipulations: 1) inhibition of Na+-K+-2Cl- cotransporter with bumetanide (3 x 10(-5) and 10(-4) M), 2) bicarbonate-free solution to disable Cl-/HCO exchanger, and 3) preincubation of rings with the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and indanyloxyacetic acid 94. The ET-1-induced contraction was enhanced by substitution of extracellular Cl- (10 mM) with methanesulfonic acid (113 mM). Cl- channels are involved in ET-1-induced contraction in the rabbit basilar artery.

Role of sarcoplasmic reticulum in regulation of tonic contraction of rabbit basilar artery.

Superficial sarcoplasmic reticulum (SR) regulates smooth muscle force development directly by Ca(2+) release and removal to and from the cytoplasm (Somlyo and Somlyo. J Cardiovasc Pharmacol 8, Suppl 8: S42-S47, 1986) by buffering Ca(2+) influx and contributing to Ca(2+) extrusion (Mueller and van Breemen. Nature 281: 682-683, 1979) and indirectly by releasing Ca(2+) near Ca(2+)-activated K(+) channels (K(Ca)) to hyperpolarize the plasma membrane (Bolton and Imaizumi. Cell Calcium 20: 141-152, 1996 and Nelson et al. Science 270: 633-637, 1995). In the rabbit basilar artery, relative contributions of direct effects and those mediated through activation of K(Ca) were evaluated by measuring force and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to the SR-depleting agents thapsigargin and ryanodine and the large conductance K(Ca) (BK(Ca)) blockers iberiotoxin (IbTX) and tetraethylammonium ion (TEA). A large contraction was observed in response to K(Ca) blockade with either 3 mM TEA or 100 nM IbTX and also after addition of 10 microM ryanodine or 2 microM thapsigargin. When K(Ca) was blocked first with TEA or IbTX, subsequent addition of thapsigargin or ryanodine also increased force. Measurements of fura 2 fluorescence showed parallel increases in [Ca(2+)](i) in response to sequential blockade of sarco(endo)plasmic reticulum Ca(2+)-ATPase and K(Ca) regardless of the order of application. It appears that a significant fraction of K(Ca) remains activated in the absence of SR function and that SR contributes to relaxation after blockade of K(Ca). We found that depletion of SR before stimulating Ca(2+) influx through voltage-gated Ca(2+) channels markedly reduced force development rate and that thapsigargin abolished this effect. We conclude that the SR of rabbit cerebral arteries modulates constriction by direct and indirect mechanisms.

In vitro and in vivo uroselectivity of B8805-033, an antagonist with high affinity at prostatic alpha1A- vs. alpha1B- and alpha1D-adrenoceptors.

We have investigated the pharmacological properties of B8805-033 [(+/-)- 1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl] amino]-2,4(1H,3H)-pyrimidinedione], a new alpha1A-adrenoceptor (AR) selective antagonist. In radioligand binding studies, B8805-033 was 150- to 1200-fold selective for alpha1A-ARs (pKi rat cerebral cortex 8.70, cloned human receptor 7.71) relative to alpha1B-ARs (pKi rat cerebral cortex 5.60, rat liver 5.39, cloned human receptor 5.16) and alpha1D-ARs (pKi cloned human receptor 5.49). B8805-033 inhibited noradrenaline (NA) induced contractions mediated by alpha1A-ARs in rat vas deferens and rabbit and human prostate (pA2 7.62-8.40) much more potently than those mediated by alpha1B-ARs in guinea pig and mouse spleen or by alpha1D-ARs in rat aorta and pulmonary artery (pA2 5.21-5.52). With the exception of a high agonist affinity at 5-HT1A receptors (pKi 9.74 in pig cortex, pD2 6.82 for contraction of rabbit basilar artery) and a moderate to low affinity at histamine H1-receptors (pA2 6.74) and beta1-ARs (pA2 5.75), B8805-033 did not interact with a number of other neurotransmitter receptors (pKi or pA2<5.0). From the i.v. doses of B8805-033 to either inhibit the urethral pressure response to NA by 50% (29 nmol/kg) or to evoke a fall in diastolic blood pressure by 25% (1.54 micromol/kg) in anaesthetized dogs, an urethral/ vascular selectivity ratio of 52 was obtained, far exceeding that found for the nearly unselective prazosin (ratio 1.8). We conclude that B8805-033 is a highly alpha1A-AR selective antagonist, which may potentially be useful as pharmacological tool to investigate alpha1-AR heterogeneity and in the treatment of benign prostatic hyperplasia.

Relaxant effect of U0126 in hemolysate-, oxyhemoglobin-, and bloody cerebrospinal fluid-induced contraction in rabbit basilar artery.

It has been suggested that mitogen-activated protein kinase (MAPK) is involved in cerebral vasospasm after subarachnoid hemorrhage. The present study was undertaken to explore the inhibitory effect of U0126, a novel MAPK inhibitor, in the contraction of the rabbit basilar artery by 3 spasmogens: hemolysate, oxyhemoglobin, and bloody cerebrospinal fluid (CSF) from patients with vasospasm. The contraction and relaxation of rabbit basilar arteries were measured by isometric tension. MAPK immunoprecipitation was assessed by Western blot analysis. (1) Pretreatment of the rabbit basilar arteries with U0126 reduced contractions to hemolysate, oxyhemoglobin, or bloody CSF applied subsequently. (2) In the absence of endothelial cells, U0126 produced an inhibitory effect similar to the contractions induced by hemolysate, oxyhemoglobin, or bloody CSF. (3) U0126 relaxed the sustained contraction induced by hemolysate, oxyhemoglobin, or bloody CSF. (4) Hemolysate, oxyhemoglobin, and bloody CSF enhanced MAPK immunoprecipitation. (5) U0126 reduced MAPK immunoprecipitation induced by hemolysate, oxyhemoglobin, and bloody CSF. (6) Hemolysate, oxyhemoglobin, and bloody CSF significantly increased MAPK activity in the rabbit basilar artery. (7) U0126 abolished the effect of hemolysate, oxyhemoglobin, or bloody CSF on MAPK activation. This study demonstrated a role of MAPK in the contraction of rabbit basilar arteries by hemolysate, oxyhemoglobin, and bloody CSF. MAPK inhibitor U0126 may be useful in the treatment of cerebral vasospasm.

Effect of endothelin-converting enzyme inhibitors on big endothelin-1 induced contraction of rabbit basilar artery.

In 1988, a novel family of endothelium derived extremely potent vasoconstrictor peptides, named endothelins, were isolated from cultured porcine endothelial cells. ET-1, a 21-amino acid peptide, is synthesized by vascular endothelial or smooth muscle cells by specific proteolytic cleavage at Trp-21-Val-22 of a precursor 38 amino-acid peptide, big ET-1 [6]. When applied to the adventitial side of blood vessels, endothelins cause a dose dependent, long lasting vasoconstriction, similar to cerebral vasospasm following aneurysmal SAH. Increased levels of big ET-1, ET-1 and ET-3 were detected in the cerebrospinal fluid and plasma of patients after aneurysmal SAH [5]. In cerebral vessels, endothelins bind to two different receptor subtypes. The ETA receptor is present in vascular smooth muscle cells and mediates vasoconstriction. The ETB receptor is present in brain, endothelium, and smooth muscle cells and mediates endothelium dependent vasodilatation, but can also cause vasoconstriction.

Mechanism of endothelin-1-induced contraction in rabbit basilar artery.

Endothelin-1 (ET-1) is suggested to be a major cause of cerebral vasospasm after subarachnoid hemorrhage. However, the mechanism of ET-1-induced contraction in cerebral arteries remains unclear. This study was undertaken to demonstrate the possible role of protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) in ET-1-induced contraction. PD-98059, damnacanthal, wortmannin, AG-490, genistein, calphostin C, and staurosporine were used to inhibit, or relax, the ET-1-induced contraction of basilar artery, studied with an isometric tension system. Immunoprecipitation of MAPK in ET-1-stimultated rings of basilar artery without or with the above inhibitors was studied with Western blot. (1) ET-1 produced concentration-dependent contraction and MAPK immunoprecipitation in rabbit basilar artery by activation of ET(A) but not ET(B) receptors. (2) MAPK inhibitors PD-98059 and U-0126 produced dose-dependent inhibition of ET-1-induced contraction. (3) The Src tyrosine kinase inhibitor damnacanthal, the phosphatidylinositol-3 kinase inhibitor wortmannin, and the Janus tyrosine kinase(2) inhibitor AG-490 abolished ET-1-induced contraction. (4) The PKC inhibitor staurosporine but not calphostin C abolished ET-1-induced contraction, and the PTK inhibitor genistein partially reduced ET-1-induced contraction. (5) In arteries precontracted by ET-1, PD-98059, U-0126, wortmannin, AG-490, genistein, and staurosporine produced concentration-dependent relaxation. (6) ET-1 induced a biphasic and time-dependent MAPK immunoprecipitation. (7) PD-98059, U-0126, genistein, AG-490, and damnacanthal, but not staurosporine or wortmannin, abolished the effect of ET-1 on MAPK immunoreactivity. This study demonstrated that MAPK may be involved in ET-1-induced contraction in rabbit basilar artery. MAPK is downstream of PTK, Src, and Janus tyrosine kinase pathways but may not be downstream of phosphatidylinositol-3 kinase pathways. The possible involvement of PKC in ET-1-induced contraction requires further investigation. Inhibition of these pathways may offer alternative treatment for ET-1-induced contraction and cerebral vasospasm.

Mitogen-activated protein kinase plays an important role in hemolysate-induced contraction in rabbit basilar artery.

Mitogen-activated protein kinase (MAPK) is an important signaling factor in the vascular proliferation and contraction, the two features of cerebral vasospasm following subarachnoid hemorrhage. We studied the possible involvement of MAPK in hemolysate-induced signal transduction and contraction in rabbit basilar artery. Isometric tension was used to record the contractile response of rabbit basilar artery to hemolysate. Western blots using antibodies for MAPK were conducted. 1) Hemolysate produced a concentration-dependent contraction of rabbit basilar artery. Pre-incubation of arteries with MAPK kinase inhibitor PD-98059 markedly reduced the contraction induced by hemolysate. PD-98059 also relaxed, in a concentration-dependent fashion, the sustained contraction induced by hemolysate (10%). 2) Hemolysate produced a time-dependent elevation of MAPK immunoreactivity in Western blot in rabbit basilar artery. MAPK was enhanced 3 min after hemolysate exposure and the effect reached maximum at 5 min. The immunoreactivity of MAPK decayed slowly with time, but the level of MAPK was still higher than the basal level even at two hours after exposure to hemolysate. 3) Pre-incubation of arteries with MAPK kinase inhibitor PD-98059 abolished the effect of hemolysate on MAPK immunoreactivity. Hemolysate produced contraction of rabbit basilar artery possibly by activation of MAPK. MAPK inhibitors may be useful in the treatment of cerebral vasospasm.

Mitogen-activated protein kinase mediation of hemolysate-induced contraction in rabbit basilar artery.

Mitogen-activated protein kinase (MAPK) is an important signaling factor in vascular proliferation and contraction, which are the two features of cerebral vasospasm that follow subarachnoid hemorrhage. The authors studied the possible involvement of MAPK in hemolysate-induced signal transduction and contraction in rabbit basilar artery (BA). Isometric tension was used to record the contractile response of rabbit BA to hemolysate, and Western blots were obtained using antibodies for MAPK. The following results are reported. 1) Hemolysate produced a concentration-dependent contraction of rabbit BA; however, preincubation of arteries with the MAPK kinase (MEK) inhibitor PD-98059 markedly reduced this contraction. The administration of PD-98059 also relaxed, in a concentration-dependent fashion, the sustained contraction induced by 10% hemolysate. 2) The Janus tyrosine kinase 2 inhibitor AG-490, preincubated with arterial rings, reduced the contractile response to hemolysate but failed to relax the sustained contraction induced by this agent. The Src-tyrosine kinase inhibitor damnacanthal and the phosphatidylinositol 3-kinase inhibitor wortmannin failed to reduce hemolysate-induced contraction. 3) Hemolysate produced a time-dependent elevation of MAPK immunoreactivity as seen on Western blots of rabbit BA. The MAPK was enhanced 1 minute after hemolysate exposure and the effect reached maximum levels at 5 minutes. The immunoreactivity of MAPK decayed slowly over time, but the level of this kinase was still higher than the basal level, even at 2 hours after exposure to hemolysate. Preincubation of arteries with the MEK inhibitor PD-98059 abolished the effect of hemolysate on MAPK immunoreactivity. Hemolysate produced contraction of rabbit BA, possibly by activation of MAPK, and therefore MAPK inhibitors may be useful in the treatment of cerebral vasospasm.

Vasodilation Effects and Action Mechanisms of TMB-8 on Basilar Artery in Rabbits.

BACKGROUND: 8-(N,N'-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) is a potent Ca(2+)-antagonist that can prevent/treat ischemic stroke and inhibit the contractility of smooth, skeletal, and cardiac muscles. Further studies are warranted to elucidate the efficacy of TMB-8 on rabbit basilar artery preparation and its action mechanisms on vascular smooth muscle cell cultures. METHODS AND RESULTS: Effects of TMB-8 on the contractility of rabbit's basilar artery in vitro and those on intracellular free Ca(2+) concentrations, [Ca(2+)](i), were studies with isolated organ bath and Fura-2 methods. Histamine-induced concentration-response curves were shifted by TMB-8 in a mixed manner whereas those of norepinephrine and KCl were shifted in a non-competitive manner. In the presence of nifedipine or in a Ca(2+)-free medium, 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (10 µM) induced an immediate transient contraction in rabbit basilar artery, whereas ryanodine showed a slow, weak, sustained contraction, followed by a weak, sustained relaxation. TMB-8 (30 µM) significantly inhibited these contractions of BHQ and ryanodine. Further, aminophylline enhanced the inhibitory action of TMB-8 on vasocontractions, suggesting that TMB-8's inhibitory actions may be related to the increase of cAMP level. The muscle contraction induced by BHQ was enhanced by pretreatment of the artery ring with TMB-8 for 15 minutes and then TMB-8 was rinsed out. These results indicate that TMB-8 pretreatment can increase Ca(2+) sequestration into sarcoplasmic reticulum, which leads to a larger subsequent Ca(2+) release by BHQ. KCl-induced increase of [Ca(2+)](i) in vascular smooth muscle cells was reduced when the cells were bathed in the medium containing nifedipine. TMB-8 made further reduction on KCl-induced [Ca(2+)](i) increase in nifedipine-containing solution, which had already blocked the voltage-operated Ca(2+) entry. CONCLUSION: These results indicate that (a) TMB-8 can enhance Ca(2+) sequestration into sarcoplasmic reticulum, which leads to a larger amount of Ca(2+) that can be released by BHQ; (b) TMB-8 can inhibit KCl-induced muscle contraction caused by the reduction of [Ca(2+)](i) through saturation of Ca(2+) inside the sarcoplasmic reticulum rather than a direct blockade of Ca(2+)-influx at cell membrane site; and (c) TMB-8 increases cAMP, which enhances Ca(2+) uptake into the sarcoplasmic reticulum.

The direct inhibitory action of phentolamine on the contraction of rabbit basilar artery.

The effects of phentolamine on rabbit basilar artery were studied and compared with those on other smooth muscles. The drug showed a dose-dependent depressing effect on high-potassium-induced contracture of rabbit basilar artery. The depressing effect was stronger on tonic than on phasic contraction. When phentolamine was applied during high-potassium-induced tonic contraction, dose-dependent relaxation was observed. The degree of phentolamine-induced-relaxation of high-potassium-induced tonic contraction was not affected by surgical denervation of the basilar artery, although catecholamine content was almost completely lost, as indicated by chemical and histochemical experiments. The relaxed tension caused by phentolamine was reversed by addition of Ca in a dose-dependent manner. Verapamil also showed a dose-dependent relaxing effect on high-potassium-induced contracture, with this relaxed tension being reversed by the addition of Ca in a dose-dependent manner. The relaxing effect of phentolamine on rabbit basilar artery was stronger than on rabbit aorta or guinea-pig taenia coli. Phentolamine depressed serotonin- or histamine-induced contraction at a concentration more than 10 times higher than the concentration for depression of noradrenaline-induced contraction. The depression of high-potassium-induced contracture and reversal by additional Ca, especially in denervated preparations, indicates that phentolamine acts directly on smooth muscle in a similar manner to that of Ca antagonists.