MODULATION OF CA2-DEPENDENT MECHANISMS( CURRENT IN CANINE COLONIC MYOCYTES BY CYCLIC NUCLEOTIDE)

Regulation of Ca2+ currents by cyclic nucleotide-dependent mechanisms was studied in circular muscle cells isolated from canine proximal col on. Whole cell Ca2+ currents were recorded at 32 degrees C with the us e of amphotericin B-perforated patches. The effects of several agents known to increase levels of adenosine 3',5'-cyclic monophosphate (cAMP ) were tested. Vasoactive intestinal peptide (VIP) and isoproterenol ( 10(-7) to 10(-5) M) increased Ca2+ current in a concentration-dependen t manner. Forskolin (10(-7) M) and dibutyryl cAMP (10(-6) to 10(-5) M) also increased Ca2+ current. Higher concentrations of forskolin (10(- 6) to 10(-5) M) caused inhibition of Ca2+ current. Low concentrations (10(-5) to 10(-7) M) of dibutyryl cAMP or 8-bromo-cAMP caused concentr ation-dependent enhancement in Ca2+ current, and these effects were re versible on washout of the cAMP analogues. When the concentration of c AMP analogues was increased (10(-3) to 10(-4) M), we observed inhibiti on of Ca2+ current similar to the effects of forskolin. Membrane-perme able analogues of guanosine 3',5'-cyclic monophosphate produced exclus ively inhibitory effects. The nonspecific protein kinase inhibitor H-7 (up to 60 mu M) failed to block the effects of VIP, isoproterenol, an d forskolin, and it produced inhibitory effects on Ca2+ current, indep endent of agonist stimulation. The data suggest that low levels of cAM P may, via phosphorylation by protein kinase A, enhance L-type Ca2+ cu rrent, but higher concentrations of cAMP may ''cross over'' and activa te protein kinase G. Phosphorylation by protein kinase G appears to pr oduce a dominant inhibition of Ca2+ current.