REGULATION OF L-TYPE CALCIUM CHANNELS BY CYCLIC-NUCLEOTIDES AND PHOSPHORYLATION IN SMOOTH-MUSCLE CELLS FROM RABBIT PORTAL-VEIN

In a previous study, we demonstrated that a high concentration (greate r than or equal to mu M) of isoproterenol (ISO) produced a dual effect on L-type Ca2+ current (I-Ca(L)) in vascular smooth muscle (VSM) cell s from the portal vein: an initial stimulatory action followed by a su stained inhibition. The first stimulatory phase was fast (presumably m ore direct) and may reflect G-protein gating of the Ca2+ channels. The second inhibitory phase was slower (presumably more indirect) and may be mediated by the adenylate cyclase/cAMP pathway. In order to define further the mechanism for the ISO inhibition of I-Ca(L), the effects of cyclic nucleotides and their related protein kinases were examined in freshly isolated single smooth muscle cells from the rabbit portal vein using the whole-cell voltage clamp technique. To isolate I-Ca(L), the pipette solution contained high Cs+ (to block K+ outward current) , and the bath contained physiological salt solution. Upon extracellul ar application of membrane-permeable cAMP and cGMP analogs (8-Br-cAMP and 8-Br-cGMP, 3 mM), I-Ca(L) was significantly inhibited by 27.9 +/- 5.0 and 33.5 +/- 4.8%, respectively. Forskolin (100 mu M) also depress ed I-Ca(L) The protein kinase inhibitor, H-7, prevented the inhibitory effects of both cyclic nucleotides and forskolin. In addition, intrac ellular application (via the patch pipettes) of cAMP-dependent protein kinase (PK-A, catalytic subunit; 1.76 mu M) and cGMP-dependent protei n kinase (PK-G, 50 nM, pre-activated by 10 mu M cGMP) significantly in hibited the peak amplitude of I-Ca(L) by 45.5 +/- 10 and 43.2 +/- 6.2% , respectively. These results indicate that, in portal vein VSM cells, phosphorylation of the Ca2+ channel protein, or of an associated regu latory protein, by PK-A and PK-G depresses I-Ca(L) The inhibition of I -Ca(L) by cyclic nucleotides may decrease the intracellular Ca2+ conce ntration and contractility, and therefore contributes to their vasodil atory effects. Thus, Ca2+ channel phosphorylation may provide an impor tant mechanism for the cyclic nucleotide-dependent actions of some vas odilators.