Mechanism of acetylcholine-induced inhibition of Ca current in bullfrog atrial myocytes.
The mechanism of the anti-beta-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 microM) 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 x 10(-6) M), as well as ISO, but it failed to block the effects of high doses of forskolin (greater than 5 x 10(-5) M). Intracellular 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, GTP gamma S (5 X 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 GTP gamma S even in the absence of ACh. Intracellular application of GTP gamma S 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). GTP gamma S (50 microM-1 mM) alone did not increase ICa significantly; however, when ISO was applied first, GTP gamma S (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 alpha-subunit of Gi which is coupled to the adenylate cyclase.
Nakajima
T
T
Department of Medical Physiology, University of Calgary School of Medicine, Alberta, Canada.
Wu
S
S
Irisawa
H
H
Giles
W
W
D000109:Acetylcholine / Q000494:pharmacology*; D000200:Action Potentials / Q000187:drug effects; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006325:Heart Atria / Q000166:cytology / Q000378:metabolism; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D011892:Rana catesbeiana; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism
D000067956:Adenylyl Cyclase Inhibitors; D011943:Receptors, Adrenergic, beta; D011976:Receptors, Muscarinic; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D007545:Isoproterenol; D000109:Acetylcholine; D002118:Calcium