Evidence that M1 muscarinic receptors enhance noradrenaline release in mouse atria by activating protein kinase C.

1. The M1 selective muscarinic agonist, McNeil A 343, enhanced the electrically evoked release of noradrenaline from postganglionic sympathetic nerves in mouse atria. This has been found previously to be due to activation of muscarinic receptors of the M1 subtype, probably located on sympathetic nerve terminals. The present study investigated the signal transduction mechanisms involved in the release-enhancing effects of McNeil A 343. The release of noradrenaline from mouse atria was assessed by measuring the electrically-induced (3 Hz, 60 s) outflow of radioactivity from atria which had been pre-incubated with [3H]-noradrenaline. 2. 8-Bromo cyclic AMP in the presence of IBMX was used to enhance maximally S-I noradrenaline release through cyclic AMP-dependent mechanisms. However, the facilitatory effect of McNeil A 343 (10 microM) was not different from the effect in the absence of these drugs, suggesting that McNeil A 343 enhances noradrenaline release independently of the cyclic AMP system. Furthermore, the release-enhancing effect of McNeil A 343 (10 microM) on noradrenaline release was also not altered by the 5-lipoxygenase inhibitor, BW A4C. 3. The facilitatory effect of McNeil A 343 was not altered in the presence of drugs (trifluoperazine, W7, and calmidazolium) which inhibit calmodulin-dependent processes, suggesting that the mechanisms of action of McNeil A 343 does not depend on calmodulin. 4. It was considered likely that the facilitatory effect of McNeil A 343 on noradrenaline release may be due to activation of protein kinase C, since activators of protein kinase C enhance noradrenaline release. The facilitatory effect of McNeil A 343 was abolished by the non-selective protein kinase C inhibitor,K-252a. To investigate further the involvement of protein kinase C, mouse atria were chronically incubated (9-O h) with the protein kinase C activator, 4 beta-phorbol dibutyrate (1.0 microM) in order to down-regulate protein kinase C activity. In protein kinase C-down-regulated atria, the facilitatory effect of McNeil A 343 (30 microM) was abolished. Incubation with 4 alpha-phorbol dibutyrate which does not affect protein kinase C did not reduce the facilitatory effect of McNeil A 343. This provides evidence that activation of protein kinase C is involved in the signal transduction process of McNeil A 343.