Muscarinic autoinhibition of acetylcholine release in mouse atria is not transduced through cyclic AMP or protein kinase C.

1. The present study investigated the second messenger pathways that may mediate muscarinic receptor autoinhibition of acetylcholine release in mouse atria. The stimulation-induced (S-I) outflow of radioactivity from mouse isolated atria incubated with [3H]-choline was Ca(2+)-dependent and tetrodotoxin-sensitive and was used as an index of neuronal acetylcholine release. 2. The cell permeable analogue of cyclic AMP, 8-bromocyclic AMP (1 x 10(-3)M) enhanced the S-I outflow of radioactivity (33%), lower concentrations having no effect. Similarly, the adenylate cyclase activator forskolin (1 x 10(-5)M) had a small facilitatory effect on acetylcholine release. On the other hand the phosphodiesterase inhibitor 3-isobutylmethylxanthine (1 x 10(-4)M) had no effect on the S-I outflow of radioactivity. Together these results suggest that the adenylate cyclase/cyclic AMP system does not have an appreciable role in the modulation of acetylcholine release. 3. The protein kinase C activator phorbol dibutyrate (0.1-3 x 10(-6)M) enhanced the S-I acetylcholine release (maximally by 45%). The effects of phorbol dibutyrate were attenuated by the protein kinase inhibitor staurosporine (1 x 10(-7)M), which by itself had no effect on the S-I outflow of radioactivity. This latter result suggests that there is no tonic activation of protein kinase C during acetylcholine release. 4. Atropine (1 x 10(-7)M) markedly enhanced (232%) the S-I outflow of radioactivity, presumably by preventing feedback inhibition on acetylcholine release through prejunctional muscarinic receptors. This effect is unlikely to involve adenylate cyclase or protein kinase C since it was far greater than the effects of activation of either system with forskolin and phorbol dibutyrate, respectively. Furthermore, the facilitatory effect of atropine was not attenuated by staurosporine, which although a protein kinase C inhibitor, is also an effective inhibitor of cyclic AMP dependent protein kinase (protein kinase A).