| Backgrounds: Alcohol consumption is closely related with serious cardiovascular diseases,such as coronary artery diseases,hypertension,atherosclerosis and stroke.All these disorders are closely associated with vascular dysfunction.Clinical research found that ethanol could induce coronary spasm,or contract coronary artery through increasing sympathetic activity and catecholamines secretion,which lead to insufficient blood and oxygen supply of the myocardium and induce coronary artery diseases(such as angina,myocardial infarction,sudden coronary death etc.).Basic studies showed that acute ethanol intake contracted dog,pig and sheep coronary arteries.On the other hand,it was also reported that ethanol could relax porcine and guinea-pig coronary arteries.Clarifying the effects of ethanol on coronary artery is a prerequisite for the study of the relationship between ethanol and coronary artery disease.Unfortunately,reported results of the coronary myogenic responses to ethanol are inconsistent.Therefore,the effects of ethanol on coronary artery need further investigation.Experimental studies showed that chronic ethanol intake increased the vasoconstrictive response to endothelin-1 in anaesthetized rats and enhanced vasoconstrictive response of the mesenteric artery to phenylephrine.It was interestingly reported that there was a significant synergism between ethanol and hyperthermia in carotid artery vasoconstriction.Unfortunately,the direct clear evidence is scarce about whether and how endogenous vasoconstrictors affect the coronary vasomotor responsiveness to acute ethanol,although it is necessitated to study this synergism,because of persistent existence of these substances in vivo.Voltage-gated K+ channel(KV)is of great significance for maintenance of cell membrane potential and regulation of coronary arterial tone.Mitogen-activated protein kinases(MAPKs)play an important role in vascular contraction.Ethanol increased MAPKs expression in canine basilar arteries,canine cerebral middle arteries,rat mesenteric arteries and rat aortic vascular smooth muscle cells(VSMCs).MAPKs were involved in KV modulation in some cells including VSMCs.But until now,the effects of ethanol on KV function,MAPKs expression and their interaction in coronary arterial smooth muscle cell have not been clarified yet.The goals of the present study were to observe the effects of ethanol,alone and in the presence of endogenous vasoconstrictors,on rat coronary vasomotor responsiveness and to explore possible involvement of the RCASMC KV and MAPKs in ethanol-induced RCA contraction.This study may provide a strategy for prevention and treatment of ethanol-induced coronary artery diseases.Part 1 The vasotonic effect of ethanol in rat coronary arteryObjectives: 1.To observe the effects of ethanol on the quiescent and mildly stimulated rat coronary artery(RCA)rings.2.To observe the dependence of ethanol-induced contraction on extracellular Ca2+([Ca2+]o)influx and intracellular Ca2+([Ca2+]i)release in RCA rings stimulated with U46619.3.To observe the effects of MAPK pathway inhibitors on ethanol-induced contraction in mildly stimulated RCA rings.Methods:1.Measurement of arterial tension: All animals were anaesthetized with intraperitoneal administration of sodium pentobarbital(40 mg/kg)and then euthanized by exsanguination.After exsanguination,rat heart was removed and transferred quickly into 4? physiological salt solution(PSS)bubbled with 100% O2.RCAs were isolated carefully out of the surrounding connective tissue.The endothelium was denuded mechanically by carefully penetrating a fine stainless steel wire through the vessel lumen and rubbing gentle for several times.The denuded RCAs were cut into cylindrical rings(2 mm-long)and transversely mounted on a wire myograph(Multi Myograph System-610 M,DMT,Denmark)in the chamber containing 5 ml PSS bubbled with 100% O2 at 37?.The arterial tension was normalized according to standard procedures to a state equal to 80 mm Hg to permit optimal tension development.After equilibration for 1 h in the bath,the vessels were contracted repeatedly with 60 m M KCl.When the 60 m M KCl-induced contraction of the ring was repeatable,the rings were good at the vascular reactivity.The arterial rings were discarded in case that the contraction was not repeatable or not sustained,or the contraction was less than 2 m N.2.The concentration-contraction curves of ethanol were constructed by cumulative addition of ethanol(0.8,2.5 and 8.0 mg/ml)into the chamber in the absence or presence of 30 m M KCl,0.3 ?M U46619,or 3 n M ET-1(at these concentrations,the vasoconstrictors produced a contraction about 5-20% of 60 m M KCl-induced contraction).The contractive response to each concentration of ethanol was allowed to develop for 5-10 min,during which usually a relatively stable contraction plateau appeared.The tone change was presented as a percentage of 60 m M KCl-induced contraction.3.After 60 m M KCl-induced contraction which was used as a reference,the ring was rinsed with Ca2+-free PSS containing 0.5 m M EGTA for three times.When the tone of the ring restored to the baseline,ethanol 8.0 mg/ml,U46619(0.3 ?M)or U46619(0.3 ?M)+ ethanol(8.0 mg/ml)was added into the chamber to elicit a contraction dependent on [Ca2+]i release in [Ca2+]o-free PSS.When a relatively stable contraction plateau appeared,2.5 m M Ca Cl2 was added to the chamber to elicit a contraction dependent on [Ca2+]o influx.The tone change was presented as a percentage of 60 m M KCl-induced contraction.To further elucidate the contribution of [Ca2+]o influx through L-type voltage-gated Ca2+ channels to the ethanol-induced contraction,effect of nifedipine(0.1 ?M,a L-type voltage-gated Ca2+ channels blocker)on the contraction was observed.When the contraction induced by 0.3 ?M U46619 was sustained,ethanol(0.8,2.5 and 8.0 mg/ml)was added cumulatively into the chamber.When a relatively stable contraction of ethanol 8.0 mg/ml appeared,the ring was rinsed with PSS.After the tone of the ring restored to the baseline for 1 h,nifedipine was added to the chamber 10 min before the cumulative addition of ethanol(0.8,2.5 and 8.0 mg/ml)in the presence of 0.3 ?M U46619.4.In order to explore the possible mechanisms,the effects of MAPK pathway inhibitors on ethanol-induced RCA contraction were studied in stimulated RCA rings.When the contraction induced by 0.3 ?M U46619 or 30 m M KCl was sustained,ethanol(0.8,2.5 and 8.0 mg/ml)was added cumulatively into the chamber.When a relatively stable contraction of ethanol 8.0 mg/ml appeared,the ring was rinsed with PSS.After the tone of the ring restored to the baseline for 1 h,SB239063(1?M,a specific p38 MAPK inhibitor)or PD98059(3 ?M,a specific p44/42 MAPK inhibitor)was added to the chamber 10 min before the cumulative addition of ethanol(0.8,2.5 and 8.0 mg/ml)in the presence of 0.3 ?M U46619 or 30 m M KCl.The tone change was presented as a percentage of 60 m M KCl-induced contraction.Results: 1.In the quiescent RCA rings,ethanol(0.8,2.5,8.0 mg/ml)evoked a very small contraction(P>0.05).The contractions were 0.05 ± 0.02 m N,0.07 ± 0.01 m N,0.12 ± 0.03 m N,respectively,equal to 0.77 ± 0.21%,0.94 ± 0.12%,1.71 ± 0.32% of 60 m M KCl-induced contraction.The contractions induced by 0.3 ?M U46619,30 m M KCl or 3 n M ET-1 were 1.03 ± 0.10 m N,0.75 ± 0.03 m N,0.76 ± 0.03 m N,respectively,equal to 17.57 ± 2.46%,10.41± 1.71%,13.58 ± 1.06% of 60 m M KCl-induced contraction.Ethanol(0.8,2.5 and 8.0 mg/ml)strikingly contracted the stimulated RCA rings in a concentration-dependent manner.In the rings precontracted by U46619 0.3 ?M,the superimposed contractions induced by ethanol(0.8,2.5 and 8.0 mg/ml)were 1.66 ± 0.33 m N,3.41 ± 0.69 m N,5.68 ± 0.67 m N,respectively,equal to 28.02 ± 4.97 %,57.13 ± 8.01 % and 95.78 ± 7.14 % of 60 m M KCl-induced contraction.In the rings precontracted by 30 m M KCl or 3 n M ET-1,the contractions induced by 8.0 mg/ml ethanol were equal to 55.35 ± 7.51 % and 66.02 ± 7.85 % of 60 m M KCl-induced contraction respectively.2.Ethanol 8.0 mg/ml alone produced very small contractions in [Ca2+]o-free or 2.5 m M [Ca2+]o PSS solution.The contractions were 0.05 ± 0.01 m N,0.13 ± 0.03 m N,respectively,equal to 0.73 ± 0.21%,2.06 ± 0.57% of 60 m M KCl-induced contraction.Ethanol 8.0 mg/ml,in the presence of U46619 0.3 ?M,produced a small phasic contraction(0.43 ± 0.08 m N,6.68 ± 1.57% of 60 m M KCl-induced contraction)in [Ca2+]o-free PSS solution and a strong sustained tonic contraction(4.36 ± 0.89 m N,66.96 ± 8.18% of 60 m M KCl-induced contraction)upon restoration of 2.5 m M [Ca2+]o.To study the contribution of L-type voltage-gated Ca2+ channels to ethanol-induced contraction,the effect of nifedipine(0.1 ?M)was observed.Preincubation with nifedipine significantly depressed the contraction induced by ethanol(0.8,2.5 and 8.0 mg/ml)in the presence of U46619 in normal PSS(25.90 ± 4.2% vs 9.59 ± 3.55%,29.72 ± 4.38% vs 11.73 ± 2.51%,80.59 ± 12.08% vs 31.3 ± 4.26%,P<0.05).3.Preincubation with SB239063(1 ?M)or PD98059(3 ?M)significantly attenuated ethanol-induced contraction in the rings stimulated with either 0.3 ?M U46619 or 30 m M KCl.SB239063 decreased the contraction induced by ethanol(0.8,2.5 and 8.0 mg/ml)in the rings stimulated with 0.3 ?M U46619 from 27.20 ± 4.25% to 18.87 ± 3.11%,49.33 ± 8.05% to 26.61 ± 4.95%,98.76 ± 5.50% to 65.91 ± 7.50%(P<0.05).PD98059 decreased the contraction induced by ethanol(0.8,2.5 and 8.0 mg/ml)in 0.3 ?M U46619-stimulated rings by 39.90%,58.76%,59.94%,respectively.Preincubation with SB239063 decreased the contraction induced by ethanol(0.8, 2.5 and 8.0 mg/ml)in the rings stimulated with 30 m M KCl from 12.64 ± 2.82% to 10.40 ± 1.28%,16.97 ± 1.49% to 13.28 ± 1.15%,50.04 ± 4.25% to 27.62 ± 3.24%.PD98059 decreased the contraction induced by ethanol(0.8,2.5 and 8.0 mg/ml)in 30 m M KCl-stimulated rings by 19.18%,20.12%,48.85%,respectively.Conclusions: 1.Ethanol(0.8,2.5,8.0 mg/ml)only evoked a very small contraction in the quiescent RCA,but produced very strong and concentration-dependent contractions in the rings stimulated with low concentrations of U46619,KCl,or ET-1.2.Ethanol-induced contraction in mildly stimulated RCA rings,is mainly dependent on [Ca2+]o influx.L-type voltage-gated Ca2+ channels are possibly involved in ethanol-induced contraction.3.MAPK inhibitors(SB239063 and PD98059)markedly attenuated contractive response of the mildly stimulated RCA to ethanol,suggesting that activation of MAPK pathway is involved in the effects of ethanol on RCA.Part 2 Effects of ethanol on Kv currents of rat coronary artery smooth muscle cellsObjectives: 1.To observe the effects of ethanol on KV currents of RCASMCs.2.To observe the effects of MAPK pathway inhibitors on ethanol-induced reduction of RCASMC KV currents.Methods: 1.RCASMC isolation:Denuded RCAs were transferred into enzymatic solution I(cell separation solution containing 0.5 mg/ml papain,1 mg/ml bovine serum albumin and 1 mg/ml dithioerythritol).After incubation for 15-20 min bubbled with 100% O2 at 37?,the RCAs were transferred to enzymatic solution II(cell separation solution including 1 mg/ml bovine serum albumin,0.5 mg/ml collagenase F and 0.5 mg/ml collagenase H),and incubated for 6-10 min.After centrifugation,the supernatant was discarded and isolated RCASMCs were ready for electrical recording.2.The effects of ethanol on KV currents of RCASMCs were assessed with whole-cell patch clamp.(1)When KV currents were stable,ethanol(0.8,2.5 and 8.0 mg/ml)were added cumulatively into the cell bath to study the concentration-dependent effects of ethanol on RCASMCs.KV currents were recorded after ethanol was added into the bath for 2 min.(2)To study the effects of ethanol on KV currents in stimulated RCASMC,U46619 0.3 ?M was added to the bath 2 min before ethanol 8.0 mg/ml addition.(3)To study the effects of MAPK pathway inhibitors on ethanol-induced inhibition on RCASMC KV currents,specific inhibitors(SB239063 and PD98059)was added to the bath 2 min before ethanol addition.Results: 1.At a testing potential of +60 m V,the stable peak of KV currents was 810.73 ± 93.77 p A and current density was 59.57 ± 5.11 p A/p F.Ethanol(8 and 25 mg/ml)significantly decreased the maximal KV currents by 53.6 ± 6.11% and 56.6 ± 7.15% respectively without concentration-dependence,while ethanol 2.5 mg/ml did not affect KV currents.2.At a testing potential of +60 m V,0.3 ?M U46619 decreased the maximal KV currents by 29.39 ± 4.23%(42.06 ± 6.63 p A/p F,P<0.05)as compared with control.After ethanol was added into the bath,the maximal KV currents attenuated by 68.04 ± 7.12%(19.04 ± 2.88 p A/p F,P<0.05).3.At a testing potential of +60 m V,preincubation with SB239063 or PD98059 itself did not affect the normal KV currents,but abrogated KV current reduction induced by 8.0 mg/ml ethanol.Conclusions: 1.Ethanol inhibited KV currents of the myocyte freshly isolated from normal RCA without concentration-dependence.Ethanol could significantly reduce KV currents in mildly stimulated RCASMCs with U46619.2.SB239063 and PD98059 attenuated ethanol-induced inhibition on KV currents,suggesting that activation of MAPK pathway is involved in the effects of ethanol on KV currents of RCASMCs.Part 3 Effects of ethanol on KV protein expressions of rat coronary artery smooth muscle cellsObjectives: 1.To observe the effects of ethanol on KV1.1,KV1.2,KV1.5,KV9.3 protein expressions and p38,p44/42 phosphorylation levels of RCASMCs.2.To observe the effects of MAPK pathway inhibitors on ethanol-induced reduction of RCASMC KV1.1,KV1.2,KV1.5,KV9.3 protein expressions.Methods: 1.To study the effects of ethanol on KV protein expressions and phosphorylation of p38 and p44/42 proteins of RCASMCs,denuded RCAs cut from an animal were randomly assigned to 4 groups: ethanol 8.0 mg/ml,U46619 0.3 ?M,ethanol 8.0 mg/ml+U46619 0.3 ?M and control.Eight segments of each group were pooled in a well of 12-well plates and incubated at 37? in PSS.Each pooled sample was from eight separate animals and four samples were used for Western blot study(n=4).After incubation for 2 h in ethanol 8.0 mg/ml,U46619 0.3 ?M,ethanol 8.0 mg/ml+U46619 0.3 ?M or PSS(control),RCA samples were frozen in liquid nitrogen and stored at-80?.KV1.1,KV1.2,KV1.5,KV9.3 protein expressions and p38,p44/42 phosphorylation levels of RCASMCs were analyzed by Western blot.2.To observe the effects of MAPK pathway inhibitors on ethanol-induced reduction of RCASMC KV protein expressions,four denuded RCA segments cut from an animal were randomly assigned to 4 groups: ethanol 8.0 mg/ml,SB239063 1 ?M,ethanol 8.0 mg/ml+SB239063 1 ?M and control,or ethanol 8.0 mg/ml,PD98059 3 ?M,ethanol 8.0 mg/ml+PD98059 3 ?M and control.Eight segments of each group were pooled in a well of 12-well plates and incubated at 37? in PSS.Each pooled sample was from eight separate animals and four samples were used for Western blot study(n=4).For ethanol 8.0 mg/ml+SB239063 1 ?M and ethanol 8.0 mg/ml+PD98059 3 ?M group,SB239063 or PD98059 was added to the well 20 min before ethanol 8.0 mg/ml incubation.The remaining experimental procedures were the same with the above descriptions.KV1.1,KV1.2,KV1.5 and KV9.3 protein expressions of RCASMCs were analyzed by Western blot.Results: 1.Ethanol 8.0 mg/ml reduced the protein expressions of KV1.1,KV1.2,KV1.5 and KV9.3 by 34.58 ± 2.58%,31.46 ± 4.57%,26.23 ± 2.09%,35.61 ± 4.99% respectively(P<0.05).U46619 0.3 ?M reduced the protein expressions of KV1.1,KV1.2,KV1.5 and KV9.3 by 23.88 ± 3.46%,28.82 ± 4.38%,30.36 ± 3.64%,27.64 ± 3.31% respectively(P<0.05).Combination of U46619 and ethanol significantly decreased the protein expressions of KV1.1,KV1.2,KV1.5 and KV9.3 by 65.16 ± 7.47%,73.39 ± 6.88%,69.40 ± 5.33%,64.19 ± 6.95% respectively(P<0.05).Ethanol 8.0 mg/ml increased p38 MAPK and p44/42 MAPK phosphorylation by 122.68 ± 20.12%,52.05 ± 14.35% respectively(P<0.05).U46619 0.3 ?M increased p38 MAPK and p44/42 MAPK phosphorylation by 96.67 ± 13.25%,60.09 ± 14.69% respectively(P<0.05).Combination of U46619 and ethanol increased p38 MAPK and p44/42 MAPK phosphorylation by 441.07 ± 37.98%,442.63 ± 48.82% respectively(P<0.05).2.SB239063 and PD98059 itself did not affect the KV protein expressions,but abrogated KV protein expression reduction induced by ethanol 8.0 mg/ml.Conclusions: 1.Ethanol decreased the protein expressions of KV1.1,KV1.2,KV1.5 and KV9.3 and increased p38 MAPK and p44/42 MAPK phosphorylation.U46619+ethanol strengthened above effects.2.SB239063 and PD98059 abrogated KV protein expressions reduction induced by ethanol,suggesting that activation of MAPK pathway is involved in the effects of ethanol on KV protein expressions reduction of RCASMCs.Collective conclusions: 1.Ethanol produced very strong contractions in mildly stimulated RCA rings.2.Ethanol increased MAPK phosphorylation,inhibited KV currents,and decreased KV protein expressions.MAPK pathway inhibitors inhibited ethanol-induced RCA contraction,attenuated ethanol-induced inhibition on KV currents and KV protein expressions of RCASMCs.Taken together,our present results demonstrate that MAPK-mediated modulation of the myocyte voltage-gated K+ channels was involved in ethanol-induced rat coronary contraction. |
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