In the investigation process to know the endothelin (endothelin-1, ET-1) actions on coronary artery, we found that a small amount (picomolar concentration) of the peptide potentiates selectively the coronary smooth muscle contraction elicited by 5-hydroxytryptamine (5- HT). To uncover the mechanisms by which ET-1 potentiates 5-HT-induced coronary contraction, the vascular response to 5-HT plus ET-1 was pharmacologically analysed in relation to the possible role of protein kinase C(PKC). ET-1 (30-100pM) potentiated 5-HT-elicited coronary contraction 1.5- to 2-fold over the control contractile response. In contrast, simultaneous measurement of coronary muscles tension and intracellular Ca2+ concentrations ([Ca2+]i) using fura-2 showed that vascular smooth muscle ([Ca2+]i) changes due to 5-HT were in the same extent without any significant differences in the preparations treated with and without ET-1. The profile of ET-1-elicited potentiation of 5-HT-induced coronary contraction was basically similar to the effect of a tumor promoting phorbol ester, 12-deoxyphorbol 13-isobutylate (DPB, 3 nM), but not to that of BayK 8644 (10 nM), a dihydropyridine type of Ca2+ channel agonist. Ca2+ channel blockers (diltiazem, 10 μM; nicardipine, 100nM) abolished both muscle tension and [Ca2+]i changes induced by 5-HT alone. However, coronary contraction elicited by 5-HT plus ET-1 exhibited resistance to the treatment with Ca2+ channel blockers though they almost completely inhibited [Ca2+]i change. The contractile component remaining in the presence of Ca2+ channel blockers was abolished by putative PKC inhibitors (H-7, 5 μM; staurosporine, 10nM). ET-1 did not potentiate 5-HT-induced coronary contraction in the presence of staurosporine (10ºnM). Furthermore, in the preparation in which PKC was down-regulated by a 24-hr exposure to 4α-phorbol 12-myristate 13-acetate (PMA, 1 μM), 5-HT-induced coronary contraction was not potentiated by ET-1. Of the four PKC isoforms (PKCα, PKCβ1, PKCδ and PKCζ identified in coronary smooth muscle, only PKCδ was demonstrated to be substantially translocated from the cytosol to the membrane fraction by the stimulation with 5-HT plus ET-1. ET-1 is likely to potentiate 5-HT-induced coronary contraction through activation of PKC, and PKCδ is a key factor which mediates the potentiation of coronary smooth muscle tone. ET-1 may function as a deteriorating factor which induces enhanced coronary tone in conjuction not only with this platelet-derived autacoid but also with other biogenic amines.
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