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

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Abstract
In a previous study, we demonstrated that a high concentration (≧1 µM)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 Ica(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 mM), ICa(L) was significantly inhibited by 27.9 ± 5.0 and 33.5 ± 4.8%, respectively. Forskolin (100 µM) 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 µM)and cGMP-dependent protein kinase (PK-G, 50 nM, pre-activated by 10 µM cGMP) significantly inhibited the peak amplitude of Ica(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 Ica(L)· The inhibition of Ica(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.

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