MODULATION OF CA2-NUCLEOTIDE CROSS ACTIVATION OF OPPOSING PROTEIN-KINASES IN RABBIT PORTAL-VEIN( CHANNELS BY CYCLIC)

Cyclic nucleotides are known to modify voltage-gated (L-type) Ca2+ cha nnel activity in vascular smooth muscle cells, but the exact mechanism (s) underlying these effects is not well defined. The purpose of the p resent study was to investigate the modulatory role of the cAMP- and c GMP-dependent protein kinase (PKA and PKG, respectively) pathways in C a2+ channel function by using both conventional and perforated-patch-c lamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 mu mol/L), significantly inc reased (14% to 16%) peak Ba2+ currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba2+ currents (23% to 31%). In contrast , 8-bromo cGMP inhibited Ba2+ currents at all concentrations tested (0 .01 to 1 mmol/L). Basal Ca2+ channel currents were significantly inhib ited by the PKA blocker O-monobutyryladenosine-3',5'-monophosphorothio ate, Rp-isomer (Rp 8-Br-MP cAMPS, 30 mu mol/L) and enhanced by the PKG inhibitor etheno-8-bromoguanosine-3'5'-monophosphorothioate, Rp-isome r (Rp-8-Br PET cGMPS, 10 nmol/L). In the presence of Rp 8-bromo PET cG MPS (10 to 100 nmol/L), both 8-bromo cAMP (0.1 mmol/L) and 8-bromo cGM P (0.1 mmol/L) enhanced Ba2+ currents (13% to 39%). The excitatory eff ect of 8-bromo cGMP was blocked by Rp 8-bromo MB-cAMPS. Both 8-bromo c AMP (0.05 mmol/L) and forskolin (10 mu mol/L) elicited time-dependent effects, including an initial enhancement followed by suppression of B a2+ currents. Ba2+ currents were also enhanced when cells were dialyze d with the catalytic subunit of PKA. This effect was reversed by the P KA blocker KT 5720 (200 nmol/L). Our results suggest that cAMP/PKA sti mulation enhances and cGMP/PKG stimulation inhibits L-type Ca2+ channe l activity in rabbit portal vein myocytes. Our results further suggest that both cAMP and cGMP have a primary action mediated by their own k inase as well as a secondary action mediated by the opposing kinase.