Role of cAMP, cGMP, and protein kinase C in regulation of calcium current through the L-type calcium channels in the electroexcitable membrane of smooth muscle cells

Abstract

Regulation of calcium current through L-type calcium channels (I Ca,L) of the guinea pigtaenia coli smooth muscle cell (SMC) membrane by cyclic nucleotides and protein kinase C (PKC) was studied using a voltage-clamp technique with intracellular dialysis or membrane patch perforation with amphotericin B. Non-selective blockers of serine/threonine kinase, staurosporine and H-7 reduced theI Ca,L amplitude in a dose-dependent manner. Dose-dependent suppression ofI Ca,L was also produced by a selective PKC blocker, chelerythrine, and a cAMP-and cGMP-dependent protein kinase (PKA, PKG), blocker H-8. Forskolin, which increases the intracellular level of cAMP, as well as membrane-permeant cAMP analogs, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, exerted complex effects onI Ca,L. The latter increased at their concentrations below 10 μM and decreased at their higher concentrations. 8-Bromo-cGMP reducedI Ca,L in all cases. Addition of 50 μM GTPγS to the micropipette solution caused a marked and slowly developing increase inI Ca,L. 8-Bromo-cAMP (1 μM) increasedI Ca,L by 30%, both in the control and during the action of GTPγS. The blockade of PKC by 10 μM chelerythrine removed the effect of GTPγS onI Ca,L. The results suggest that basal activity of L-type calcium channels in SMC of the guinea pigtaenia coli depends on PKC- and PKA-dependent phosphorylation. PKC can increase theI Ca,L amplitude provided G proteins are activated. cAMP at low concentrations likewise increasesI Ca,L (probably through activation of PKA). PKG apparently mediatesI Ca,L drops evoked by cAMP at high concentrations and by cGMP.