MECHANISM OF ACETYLCHOLINE-INDUCED INHIBITION OF CA CURRENT IN BULLFROG ATRIAL MYOCYTES

The mechanism o f the anti-fl-adrenergic action of acetylcholine (ACh) on Ca current, Ica, was examined using the tight-seal, whole-cell voltage clamp technique in single atrial myocytes from the bullfrog. Both isoproterenol (ISO) and forskolin increased Ica dose dependently. After Ica had been enhanced maximally by ISO (10-6 M), subsequent application of forskolin (50 µM) did not further increase Ica, suggesting that ISO and forskolin increase Ica via a common biochemical pathway, possibly by stimulation of adenylate cyclase. ACh (10-5 M) completely inhibited the effect of low doses of forskolin (2 × 10-6 M), as well as ISO, but it failed to block the effects of high doses of forskolin (>5 × 10-5 M). IntraceUular application of cyclic AMP (cAMP) also increased Ica. ACh (10-5 M) failed to inhibit this cAMP effect, indicating that the inhibitory action of ACh occurs at a site proximal to the production of cAMP. ACh (10-5 M) also activated an inwardly rectifying K+ current Ik(ACh). Intracellular application of a nonhydrolyzable GTP analogue, GTPγS (5 × 10-4 M), activated Ik(ACh) within several minutes; subsequent application of ACh (10-5 M) did not increase Ik(ACh) further. These results demonstrate that a GTP-binding protein coupled to these K+ channels can be activated maximally by GTPγS even in the absence of ACh. Intracellular application of GTPγS also strongly inhibited the effect of ISO on Ica in the absence of ACh. Pertussis toxin (IAP) completely prevented both the inhibitory effect of ACh on Ica and the ACh-induced activation of Ik(ACh). GTPγS (50 µM-1 mM) alone did not increase Ica significantly; however, when ISO was applied first, GTPγS (5 x 10-4 M) gradually inhibited the ISO effect on Ica. These results indicate that ACh antagonizes the effect of ISO on Ica via a GTP-binding protein (Gi and/or Go). This effect may be mediated through a direct inhibition by the cz-subunit of Gi which is coupled to the adenylate cyclase. © 1990, Rockefeller University Press., All rights reserved.