Rate-limiting Steps in the Beta-Adrenergic Stimulation of Cardiac Calcium Current
Abstract
Fast-flow perfusion and flash photolysis of caged compounds were used to study the activation kinetics of L-type calcium current (/ca) in frog cardiac myocytes. Rapid exposure to isoproterenol (Iso) for 1 s or ~ 1 min produced similar kinetics of increase in /Ca with an initial lag period of ~ 3 s, followed by a monophasic rise in current with a half-time of ~ 20 s. Epinephrine, as well as caged Iso, produced increases with similar kinetics. The fact that/ca increased significantly even after short Iso applications suggests that agonist binding to the receptor is rapid and that the increase in/ca is independent of free agonist. To dissect the kinetic contributions of various steps in the cAMP-phosphorylation cascade, the kinetics of the responses to caged cAMP and caged GTP~/S and fast perfusion of forskolin, acetylcholine, and propranolol were compared. The response to caged cAMP exhibited no lag period, but otherwise increased at a rate similar to that produced by Iso and reached a peak at ~ 40 s after flash photolysis. This suggests that the lag period itself is due to a step before cAMP accumulation, but that activation of protein kinase and phosphorylation of the calcium channel are relatively slow. A lag period was also observed when /Ca was stimulated by flash photolysis of caged GTP~/S and when adenylyl cyclase was activated directly by rapid perfusion with forskolin. The lag period observed with forskolin may be due to slow binding of forskolin. The lag period was not due to the time required for cAMP to reach a threshold concentration, because a similar lag was observed in response to Iso in cells having/ca previously stimulated submaximally by internal perfusion with a low concentration of cAMP. These results suggest that the lag period can be attributed to a step associated with activation of adenylyl cyclase and cAMP accumulation.
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