Werber A H, Morgan R A, Zhou P, Yang C
Department of Pharmacology and Neuroscience A136, Albany Medical College, NY 12208, USA.
Alcohol. 1997 Jul-Aug;14(4):351-60. doi: 10.1016/s0741-8329(96)00183-8.
The intracellular mechanisms mediating vasoconstriction by ethanol are poorly understood. This investigation was designed to provide evidence on the role of protein kinase C (PKC) and calmodulin in vasoconstriction by ethanol. We studied helically cut strips of rat aorta that were exposed to ethanol before and in the presence of the PKC inhibitors calphostin C (79, 239, and 798 nM) or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7, 10 microM), and the calmodulin inhibitor, trifluoperazine (TFP, 10 microM). To test for the specificity of the PKC inhibitors, we measured the responses of aortas to potassium and phorbol 12-myristate 13-acetate (PMA) in the absence and presence of calphostin C and H7. To test for the specificity of TFP, we measured the responses of aortas to serotonin, potassium, PMA, and the thromboxane A2 mimic. 9,11-dideoxy-11 alpha, 9 alpha-epoxy-methanoprostaglandin F2a (U46619), in the absence and presence of TFP. We also studied the effect of the combination of calphostin C and TFP on constriction of the aorta by ethanol. We also measured the importance of intracellular and extracellular calcium in constriction of the aorta by ethanol. Force generation was measured before, and then during exposure of the strips to calcium-free buffer with EGTA, or calcium-free buffer with EGTA plus caffeine. We found that both PKC inhibitors antagonized vasoconstriction by ethanol and PMA. However, H7 antagonized contractions by potassium, but calphostin C did not. We found that TFP caused 99 +/- 1% inhibition of maximum contraction to serotonin, 90 +/- 4% inhibition of maximum contraction to potassium, 63 +/- 6% inhibition of maximum contraction to PMA, and 8 +/- 5% inhibition of maximum contraction to U46619. TFP caused a 22 +/- 8% inhibition of contraction to ethanol. The combination of TFP and calphostin C antagonized vasoconstriction by ethanol to a degree similar to that of calphostin C alone. We also found that contractions to ethanol were only 16 +/- 7% of control values in a calcium-free plus EGTA buffer. Contractions to ethanol were 0 +/- 1% of control values in calcium-free buffer with EGTA plus caffeine. We conclude that: 1-vasoconstriction by ethanol is, at least in part, mediated by PKC; 2-constriction by ethanol is mediated to a minimal extent by calmodulin, and 3-part of the constriction by ethanol of the aorta is mediated by a caffeine-sensitive pool of intracellular calcium.
乙醇介导血管收缩的细胞内机制尚不清楚。本研究旨在为蛋白激酶C（PKC）和钙调蛋白在乙醇介导的血管收缩中的作用提供证据。我们研究了螺旋切割的大鼠主动脉条，这些主动脉条在暴露于乙醇之前以及在存在PKC抑制剂钙泊三醇C（79、239和798 nM）或1-(5-异喹啉磺酰基)-2-甲基哌嗪（H7，10 μM）以及钙调蛋白抑制剂三氟拉嗪（TFP，10 μM）的情况下。为了测试PKC抑制剂的特异性，我们测量了主动脉在不存在和存在钙泊三醇C和H7的情况下对钾和佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）的反应。为了测试TFP的特异性，我们测量了主动脉在不存在和存在TFP的情况下对5-羟色胺、钾、PMA和血栓素A2类似物9,11-二脱氧-11α,9α-环氧-甲撑前列腺素F2α（U46619）的反应。我们还研究了钙泊三醇C和TFP联合使用对乙醇诱导的主动脉收缩的影响。我们还测量了细胞内和细胞外钙在乙醇诱导的主动脉收缩中的重要性。在将主动脉条暴露于含乙二醇双四乙酸（EGTA）的无钙缓冲液或含EGTA加咖啡因的无钙缓冲液之前和期间测量肌力产生。我们发现两种PKC抑制剂均能拮抗乙醇和PMA介导的血管收缩。然而，H7能拮抗钾诱导的收缩，但钙泊三醇C不能。我们发现TFP对5-羟色胺最大收缩的抑制率为99±1%，对钾最大收缩的抑制率为90±4%，对PMA最大收缩的抑制率为63±6%，对U46619最大收缩的抑制率为8±5%。TFP对乙醇收缩的抑制率为22±8%。TFP和钙泊三醇C联合使用对乙醇介导的血管收缩的拮抗程度与单独使用钙泊三醇C相似。我们还发现，在含EGTA的无钙缓冲液中，对乙醇的收缩仅为对照值的16±7%。在含EGTA加咖啡因的无钙缓冲液中，对乙醇的收缩为对照值的0±1%。我们得出结论：1-乙醇介导的血管收缩至少部分由PKC介导；2-乙醇介导的收缩在最小程度上由钙调蛋白介导；3-乙醇诱导的主动脉收缩部分由对咖啡因敏感的细胞内钙池介导。
