Acetylcholine inhibits Ca2+ current by acting exclusively at a site proximal to adenylyl cyclase in frog cardiac myocytes

1. The effects of acetylcholine (ACh) on the L-type Ca2+ current (I-Ca) stimulated by isoprenaline (Iso) or forskolin (Fsk) were examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion. 2. The exposure of one half of the cell to 1 mu M Iso produced a half-maximal increase in I-Ca since a subsequent application of Iso to the other half induced an additional effect of nearly the same amplitude. Similarly, addition of 1 mu M ACh to only one half of a cell exposed to Iso on both halves reduced the effect of Iso by only similar to 50%. 3. When 10 mu M Iso or 30 mu M Fsk were applied to a Ca2+-free solution on one half of the cell, I-Ca was increased in the remote part of the cell where adenylyl cyclase activity was not stimulated. However, addition of ACh (3-10 mu M) to the remote part had no effect on I-Ca, while addition of ACh to the part of the cell exposed to Iso or Fsk strongly antagonized the stimulatory effects of these drugs. 4. Our data demonstrate that ACh regulates I-Ca by acting at a site proximal to adenylyl cyclase in frog ventricular cells. We conclude that the muscarinic regulation of I-Ca does not involve any additional cAMP-independent mechanisms occurring downstream from cAMP generation.