INTRACELLULAR MECHANISMS OF CONSTRICTION OF RAT AORTA BY ETHANOL

The intracellular mechanisms mediating vasoconstriction by ethanol are poorly understood. This investigation was designed to provide evidenc e on the role of protein kinase C (PKC) and calmodulin in vasoconstric tion by ethanol. We studied helically cut strips of rat aorta that wer e exposed to ethanol before and in the presence of the PKC inhibitors calphostin C (79, 239, and 798 nM) or 1-(5-isoquinolinylsulfonyl)-2-me thylpiperazine (H7, 10 mu M), and the calmodulin inhibitor, trifluoper azine (TFP, 10 mu M). To test for the specificity of the PKC inhibitor s, we measured the responses of aortas to potassium and phorbol 12-myr istate 13-acetate (PMA) in the absence and presence of calphostin C an d H7. To test for the specificity of TFP, we measured the responses of aortas to serotonin, potassium, PMA, and the thromboxane A(2) mimic, 9,11-dideoxy-11 alpha,9 alpha-epoxy-methanoprostaglandin F-2 alpha (U4 6619), in the absence and presence of TFP. We also studied the effect of the combination of calphostin C and TFP on constriction of the aort a by ethanol. We also measured the importance of intracellular and ext racellular calcium in constriction of the aorta by ethanol. Force gene ration was measured before, and then during exposure of the strips to calcium-free buffer with EGTA, or calcium-free buffer with EGTA plus c affeine. We found that both PKC inhibitors antagonized vasoconstrictio n by ethanol and PMA. However, H7 antagonized contractions by potassiu m, but calphostin C did not. We found that TFP caused 99 +/- 1% inhibi tion of maximum contraction to serotonin, 90 +/- 4% inhibition of maxi mum contraction to potassium, 63 +/- 6% inhibition of maximum contract ion to PMA, and 8 +/- 5% inhibition of maximum contraction to U46619. TFP caused a 22 +/- 8% inhibition of contraction to ethanol. The combi nation 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 ca lcium-free plus EGTA buffer. Contractions to ethanol were 0 +/- 1% of control values in calcium-free buffer with EGTA plus caffeine. We conc lude that: 1- vasoconstriction by ethanol is, at least in part, mediat ed 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. (C ) 1997 Elsevier Science Inc.