Heterogeneity of release-regulating muscarinic receptors in rat sympathetic neurons: evidence for inhibitory presynaptic M1 receptors

Of the 5 known subtypes of mAChRs, M2, M3, and M4 have been reported to act as inhibitory presynaptic receptors in the nervous system, in general, and in sympathetic neurons, in particular. M1 receptors, in contrast, have rather been viewed as facilitatory presynaptic receptors. In superior cervical ganglion (SCG) neurons, M1 receptors are well known to inhibit KCNQ channels. Previously, we were able to show that non-presynaptic M1 receptors in SCG neurons enhance noradrenaline release through an inhibition of KCNQ channels. However, M1 receptors also mediate an inhibition of voltage-activated Ca2+ channels, which represents the predominant mechanism of presynaptic inhibition. Hence, presynaptic M1 receptors may exert inibitory presynaptic modulation. To test this possibility, we performed experiments on rat superior cervical ganglion neurons. In primary cultures tritium overflow was assayed to investigate the release of [3H]noradrenaline, and the perforated patch-clamp technique was employed to record Ca2+ currents. The muscarinic agonist oxotremorine M transiently enhanced 3H outflow and reduced electrically evoked release, once the stimulatory effect had faded. The stimulatory effect was enhanced by pertussis toxin and was abolished by blocking M1 receptors, by opening KCNQ channels, and by preventing action potential propagation. The inhibitory effect, in contrast, was not altered by preventing action potentials or by opening KCNQ channels, but was reduced by pertussis toxin. The inhibition remaining after pertussis toxin treatment was abolished by blockage of M1 receptors or inhibition of phospholipase C. When [3H]noradrenaline release was triggered independently of voltage-activated Ca2+ channels, oxotremorine M failed to cause any inhibition. The inhibition of Ca2+ currents by oxotremorine M was reduced by pertussis toxin and then abolished by the blockage of M1 receptors. This demonstrates that M1, in addition to M2, M3, and M4, receptors mediate presynaptic inhibition in sympathetic neurons using phospholipase C to close voltage-activated Ca2+ channels. In addition, our results contradict the widely accepted concept that all inhibitory presynaptic receptors restrict transmitter release through a direct inhibition of Ca2+ channels via G protein βγ subunits and offer an alternative mechanism.