Effects of protein kinase inhibitors on canine Purkinje fibre pacemaker depolarization and the pacemaker current i(f).

1. The effects of the protein kinase inhibitors H‐7 and H‐8 were investigated on diastolic depolarization of the action potential with microelectrodes and on the pacemaker current if with the two‐microelectrode voltage clamp in canine cardiac Purkinje fibres. 2. Both 200 microM‐H‐7 and 100 microM‐H‐8 had no significant effect on the slope of diastolic depolarization but eliminated the actions of isoprenaline (1 microM). 3. We examined the actions of H‐7 and H‐8 on if in the presence and absence of isoprenaline. H‐7 (200 microM) shifted the pacemaker current if in the negative direction on the voltage axis, whereas 100 microM‐H‐8 had no significant effect by itself. Both 200 microM‐H‐7 and 100 microM‐H‐8 can reverse or prevent the actions of isoprenaline (1‐5 microM) on if. 4. We applied activators of the cyclic AMP cascade down‐stream to the beta‐receptor, to further evaluate where H‐7 and H‐8 might be exerting their effects. When exposing Purkinje fibres to an adenylyl cyclase activator (forskolin, 10‐50 microM), a phosphodiesterase inhibitor (IBMX, 100 microM) and a permeable cyclic AMP analogue (8‐chlorophenylthio‐cyclic AMP, 200 microM‐1 mM), the amplitude of if was increased. H‐7 and H‐8 at 100‐200 microM eliminated each of these actions. 5. These results suggest that a phosphorylation process is involved in the modulation of the pacemaker current, if, in Purkinje fibres. The different actions of H‐7 and H‐8 on basal if suggest the hypothesis that other protein kinases, possibly protein kinase C, might also be involved in regulating basal phosphorylation of if in Purkinje fibres.