Protein kinase inhibitor H-89 reverses forskolin stimulation of cardiac L-type calcium current.
Calcium currents (ICa) and barium currents (IBa) were measured in freshly isolated single ferret ventricular myocytes, using the whole cell patch-clamp and perforated patch-clamp techniques with Na and K currents blocked by tetraethylammonium and Cs. The membrane potential (Em) dependence of activation and steady-state inactivation curves were determined using a Boltzmann relation, where E0.5 is the Em at half-maximal conductance. Forskolin (1 microM) increased the rate of ICa inactivation, especially in perforated patch, but slowed IBa inactivation. The acceleration is likely to be due to greater Ca-dependent inactivation of ICa, where the slowing of IBa inactivation may be due to protein kinase A-dependent slowing of Em-dependent inactivation. Forskolin (1-10 microM) also increased ICa amplitude by two- to threefold and shifted the E0.5 for both activation and inactivation to more negative potentials by 7-8 mV. The effect of forskolin on the amplitude of ICa could be reversed by an inhibitor of adenosine 3',5'-cyclic monophosphate-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1-10 microM). However, H-89 did not reverse the shift of E0.5 induced by forskolin. H-89 application by itself does not decrease basal ICa but does shift the E0.5 of both activation and inactivation to more negative values of Em. It is possible that H-89 reverses the shift induced by regulatory phosphorylation (due to forskolin) but induces a coincidental negative shift itself.
Yuan
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Department of Physiology, Loyola University Medical School, Maywood, Illinois 60153.
Bers
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D000818:Animals; D001464:Barium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D005576:Colforsin / Q000008:administration & dosage / Q000494:pharmacology*; D004553:Electric Conductivity; D004622:Embryo, Mammalian; D005289:Ferrets; D006321:Heart / Q000502:physiology*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D007668:Kidney; D007700:Kinetics; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides
D015220:Calcium Channels; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D005576:Colforsin; D001464:Barium; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide