REGULATION OF CALCIUM-CHANNEL CURRENT IN A7R5 VASCULAR SMOOTH-MUSCLE CELLS BY CYCLIC-NUCLEOTIDES

In vascular smooth muscle (VSM) cells, the slow inward calcium current (I-Ca) may be regulated by phosphorylation of the calcium channel pro tein or of associated regulatory proteins. We investigated the role of several protein kinase systems in the regulation of I-Ca in cultured A7r5 cells, a clonal cell line derived from rat aorta. The perforated- patch voltage-clamp technique was used to record whole cell I-Ca. To i solate the I-Ca, the pipette contained high Cs+ and the bath contained 140 mM tetraethylammonium to block potassium currents. Ba2+ was used as the charge carrier. In control experiments, I-Ca was stable for at least 15 min. Compared with 23 +/- 3% in the time-control group (i.e., run-down; n = 10), 3 mM 8-bromoadenosine 3',5'-cyclic monophosphate ( 8-BrcAMP) inhibited peak I-Ca by 53 +/- 3% (n = 9) within 15 min. Simi larly, 3 mM 8-bromo-guanosine 3',5'-cyclic monophosphate (8-BrcGMP) in hibited I-Ca by 59 +/- 4 (n = 11). Application of 30 mu M forskolin in hibited I-Ca by 58 +/- 9% (n = 6) within 5 min (compared with 4 +/- 3% for the 5-min time control). Forskolin also shifted the reversal pote ntial to the left, suggesting a stimulation of an outward current. In the presence of the protein kinase inhibitor, 1-(5-isoquinolinylsulfon yl)-2-methylpiperazine, the same dose of forskolin had no effect (n = 7). The water-soluble analogue of forskolin (L-858051, 30 mu M) decrea sed I-Ca by 72 +/- 11% (n = 9) and reduced the outward current compone nt. These results suggest that, in A7r5 cells, cAMP and cGMP both modu late whole cell I-Ca, and the data are consistent with a phosphorylati on-dependent mechanism. The observed inhibition of I-Ca by the cyclic nucleotides presumably lowers calcium influx and cell excitability and therefore causes vasodilation. Thus channel phosphorylation may be an important mechanism for the cyclic nucleotide-dependent actions of so me vasodilators.