Endothelin-1 and photoreleased diacylglycerol increase L-type Ca2+ current by activation of protein kinase C in rat ventricular myocytes

1. The amphotericin B-perforated whole-cell patch clamp technique was used to determine the modulation of L-type Ca2+ channels by protein kinase C (PKC)-mediated pathways in adult rat ventricular myocytes. 2. Application of 10 nM endothelin-1 (ET-1) increased peak Ca2+ current (I-Ca) by 28.2 +/- 2.5% (n = 13) and slowed current decay, These effects were prevented by the endothelin receptor antagonist; PD145065 (10 mu M) and by the PKC inhibitor chelerythrine (8 mu M). To establish if direct activation of PKC mimicked the ET-1 effect, the active and inactive phorbol esters (phorbol-12-myristate-13-acetate and 4 alpha-phorbol-12,13-didecanoate) were tested. Both phorbol esters (100 nM) resulted in a small (similar to 10 %) increase in I-Ca, suggesting PKC-independent effects. 4. Bath application of dioctanoylglycerol (diC(8)), a diacylglycerol (DAG) analogue which is capable of directly activating PKC, caused a gradual decline in peak I-Ca (50.4 +/- 6.2%, n = 5) and increased the rate of current decay. These effects were unaffected by the PKC inhibitor chelerythrine (8 mu M). 5. Intracellular photorelease of caged diC(8) with 3 or 10 s exposure to UV light produced a concentration-dependent increase in peak I-Ca (20.7 +/- 8.5% (n = 8) for 3 s UV and 60.8 +/- 11.4% (n = 13) for 10 s UV), which could be inhibited by chelerythrine. 6. Our results demonstrate that both ET-1 and intracellularly photoreleased diC(8) increase I-Ca by a PKC-mediated pathway which is in direct contrast to the PKC-independent inhibition of I-Ca produced by bath-applied diC(8). We conclude that specific cellular pools of DAG are crucially important; in the regulation of I-Ca by PKC.