Modulation of Ca Channels by Cyclic Nucleotide Cross Activation of Opposing Protein Kinases in Rabbit Portal Vein

Cyclic nucleotides are known to modify voltage-gated (L-type) Ca channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMPand cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca channel function by using both conventional and perforated-patch–clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 mmol/L), significantly increased (14% to 16%) peak Ba currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca channel currents were significantly inhibited by the PKA blocker 8-Bromo-29-O-monobutyryladenosine-39,59-monophosphorothioate, Rp-isomer (Rp 8-Br-MP cAMPS, 30 mmol/L) and enhanced by the PKG inhibitor b-Phenyl-1,Netheno-8-bromoguanosine-39,59-monophosphorothioate, Rp-isomer (Rp-8-Br PET cGMPS, 10 nmol/L). In the presence of Rp 8-bromo PET cGMPS (10 to 100 nmol/L), both 8-bromo cAMP (0.1 mmol/L) and 8-bromo cGMP (0.1 mmol/L) enhanced Ba currents (13% to 39%). The excitatory effect of 8-bromo cGMP was blocked by Rp 8-bromo MB-cAMPS. Both 8-bromo cAMP (0.05 mmol/L) and forskolin (10 mmol/L) elicited time-dependent effects, including an initial enhancement followed by suppression of Ba currents. Ba currents were also enhanced when cells were dialyzed with the catalytic subunit of PKA. This effect was reversed by the PKA blocker KT 5720 (200 nmol/L). Our results suggest that cAMP/PKA stimulation enhances and cGMP/PKG stimulation inhibits L-type Ca channel activity in rabbit portal vein myocytes. Our results further suggest that both cAMP and cGMP have a primary action mediated by their own kinase as well as a secondary action mediated by the opposing kinase. (Circ Res. 1998;82:557-565.)