Differences in effects of forskolin and an analog on calcium currents in cardiac myocytes suggest intra- and extracellular sites of action.
The effects of forskolin (FO) and a water-soluble derivative of FO, L858051 (7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino)-butyryl] forskolin), were compared on calcium currents (ICa) studied by the whole-cell patch-clamp technique in frog ventricular cardiac myocytes. Both FO and L858051 increased ICa, with half-times of 160 +/- 20 sec and 343 +/- 22 sec, respectively. The stimulation was blocked by internal perfusion with inhibitors of protein kinase A. The EC50 for stimulation of ICa was 0.3 microM for FO and 1.0 microM for L858051. The maximal stimulated current was the same for both drugs, 20.3 microA/cm2 and 23.1 microA/cm2, respectively. Internal perfusion with 30-500 microM guanylyl 5'-imidodiphosphate [Gpp(NH)p] suppressed ICa stimulation by low concentrations of FO or L858051. This suppression was due to a rightward shift in the concentration-response curve, with increases in the EC50 values to 11.4 microM for FO and 28.4 microM for L858051. Isoproterenol (ISO) was ineffective in increasing ICa after the FO-stimulated ICa had been reduced by Gpp(NH)p and FO had been washed out. In contrast, after the L858051-stimulated current had been reduced by Gpp(NH)p, ISO stimulated ICa significantly. This stimulation was blocked by inhibitors of protein kinase A and was due to a positive effect of L858051 not shared by FO. A brief application of L858051 after Gpp(NH)p had blocked the ISO response restored the ISO response for at least 30 min. This effect was mimicked by internal perfusion with low concentrations of L858051. We conclude that the ability of brief exposure of L858051, but not FO, to restore the response to ISO after Gpp(NH)p is due to the accumulation of L858051 intracellularly, due to its hydrophilicity. Because internal L858051 and FO are very ineffective in stimulating adenylyl cyclase, whereas internal L858051 can restore the ISO response blocked by Gpp(NH)p, we propose that FO compounds can affect adenylyl cyclase at two sites, one site that is accessible only from the extracellular side that stimulates catalytic activity and another that is accessible from the intracellular side that increases beta-agonist efficacy in the presence of Gpp(NH)p.
Hartzell
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Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322.
Budnitz
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D000818:Animals; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D005576:Colforsin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004224:Diterpenes; D004305:Dose-Response Relationship, Drug; D005071:Evoked Potentials / Q000187:drug effects; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D006352:Heart Ventricles; D007700:Kinetics; D011892:Rana catesbeiana; D013015:Sotalol / Q000494:pharmacology
D015220:Calcium Channels; D004224:Diterpenes; C055627:L 858051; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D013015:Sotalol