Modulation of Ca²⁺ Channels by Cyclic Nucleotide Cross Activation of Opposing Protein Kinases in Rabbit Portal Vein

From the Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno.

Correspondence to Kathleen D. Keef, PhD, Anderson Medical Sciences Building, Reno, NV 89557. E-mail kathy{at}physio.unr.edu

Abstract—Cyclic nucleotides are known to modify voltage-gated (L-type) Ca²⁺ 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 cAMP- and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca²⁺ 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 µmol/L), significantly increased (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 inhibited by the PKA blocker 8-Bromo-2'-O-monobutyryladenosine-3',5'-monophosphorothioate, Rp-isomer (Rp 8-Br-MP cAMPS, 30 µmol/L) and enhanced by the PKG inhibitor ß-Phenyl-1,N²-etheno-8-bromoguanosine-3',5'-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 Ba²⁺ 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 µmol/L) elicited time-dependent effects, including an initial enhancement followed by suppression of Ba²⁺ currents. Ba²⁺ 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 Ca²⁺ 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.

Key Words: L-type Ca²⁺ channel • vascular smooth muscle • 8-bromo cAMP • 8-bromo cGMP • cGMP-dependent protein kinase

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