ENDOTHELIN ACTIVATES VOLTAGE-DEPENDENT CA2+ CURRENT BY A G-PROTEIN-DEPENDENT MECHANISM IN RABBIT CARDIAC MYOCYTES

1. Endothelin is a vasoactive peptide released from vascular endothelial cells which has potent cardiac inotropic effects. We examined the effect of endothelin on the verapamil-sensitive Ca2+ current (I(Ca)) in enzymatically dispersed rabbit ventricular myocytes. 2. Using the whole-cell voltage clamp technique with a standard dialysing pipette solution, the application of extracellular endothelin (20 nM) did not increase the peak I(Ca), but in fact caused a small reversible decline (903 +/- 109 pA without endothelin, 727 +/- 95 pA with endothelin (means +/- S.E.M., n = 14, P < 0.05)). 3. If GTP (100-mu-M) was added to the pipette solution, the extracellular application of endothelin (0.2 or 20 nM) caused a large, reproducible increase in peak I(Ca) (871 +/- 85 pA without endothelin, 1230 +/- 110 pA with 20 nM-endothelin (n = 10, P < 0.05). The endothelin enhancement of I(Ca) occurred after a delay of approximately 3-4 min at room temperature. 4. The GTP requirement for the endothelin effect on I(Ca) suggests that its effect may be mediated through a G protein-dependent pathway. To investigate this further, experiments were performed with pipette solutions containing guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S), a GDP analogue which inhibits G protein cycling. With the addition of GDP-beta-S (0.5-5.0 mM) to the pipette solution (along with 100-mu-M-GTP), the effect of endothelin on peak I(Ca) was blocked (1062 +/- 86 pA without endothelin, 1170 +/- 134 pA with endothelin (n = 11, P > 0.05)). 5. Incubation of myocytes with pertussis toxin (500 ng/ml) prevented the partial ACh-induced reversal of the isoprenolol enhancement of I(Ca). However, this identical treatment failed to block the endothelin enhancement of the voltage-dependent Ca2+ current (n = 4). 6. Taken together, these results confirm that while the effect of endothelin in rabbit cardiac ventricular myocytes is mediated through a G protein-dependent pathway, the G protein involved is pertussis toxin-insensitive.