Regulation of L-Type Calcium Channels by Cyclic Nucleotides and Phosphorylation in Smooth Muscle Cells from Rabbit Portal Vein

Abstract

In a previous study, we demonstrated that a high concentration (≧1 µ M)of isoproterenol (ISO) produced a dual effect on L-type Ca²⁺ current (Ica(L)) in vascular smooth muscle (VSM) cells from the portal vein: an initial stimulatory action followed by a sustained inhibition. The first stimulatory phase was fast (presumably more 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 Ic_a(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 Ica(L), the pipette solution contained high Cs⁺ (to block K⁺ outward current), and the bath contained physiological salt solution. Upon extracellular application of membrane-permeable cAMP and cGMP analogs (8-Br-cAMP and 8-Br-cGMP, 3 m M), I_Ca(L) was significantly inhibited by 27.9 ± 5.0 and 33.5 ± 4.8%, respectively. Forskolin (100 µ M) also depressed Ica(L)· The protein kinase inhibitor, H-7, prevented the inhibitory effects of both cyclic nucleotides and forskolin. In addition, intracellular application (via the patch pipettes) of cAMP-dependent protein kinase (PK-A, catalytic subunit; 1.76 µ M)and cGMP-dependent protein kinase (PK-G, 50 n M, pre-activated by 10 µ M cGMP) significantly inhibited the peak amplitude of Ic_a(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 regulatory protein, by PK-A and PK-G depresses Ic_a(L)· The inhibition of Ic_a(L) by cyclic nucleotides may decrease the intracellular Ca²⁺ concentration and contractility, and therefore contributes to their vasodilatory effects. Thus, Ca²⁺ channel phosphorylation may provide an important mechanism for the cyclic nucleotide-dependent actions of some vasodilators.