We have used intracellular recording to investigate the existence of a functional link between muscarinic presynaptic acetylcholine (ACh) autoreceptors, the intracellular serine-threonine kinases-mediated transduction pathways and transmitter release in the motor nerve terminals of adult rats. We found the following. (1) Transmitter release was reduced by the M1 muscarinic acetylcholine receptor (mAChR) blocker pirenzepine and enhanced by the M2 blocker methoctramine. The unselective mAChR blocker atropine increased ACh release, which suggests the unmasking of another parallel release-potentiating mechanism. There are therefore two opposite, though finely balanced, M1–M2 mAChR-operated mechanisms that tonically modulate transmitter release. (2) Both M1 and M2 mechanisms were altered when protein kinase C (PKC), protein kinase A (PKA) or the P/Q-type calcium channel were blocked. (3) Both PKC and PKA potentiated release when they were specifically stimulated [with phorbol 12-myristate 13-actetate (PMA) and Sp-8-Br cAMPs, respectively], and both needed the P/Q channel. (4) In normal conditions PKC seemed not to be directly involved in transmitter release (the PKC blocker calphostin C did not reduce release), whereas PKA was coupled to potentiate release (the PKA blocker H-89 reduced release). However, when an imbalance of the M1–M2 mAChRs function was experimentally produced with selective blockers, an inversion of the kinase function occurred and PKC could then stimulate transmitter release, whereas PKA was uncoupled. (5) The muscarinic function may be explained by the existence of an M1-mediated increased PKC activity-dependent potentiation of release and an M2-mediated PKA decreased activity-dependent release reduction.
These findings show that there is a precise interrelation pattern of the mAChRs, PKC and PKA in the control of the neurotransmitter release.