High spatial resolution studies of muscarinic neuroeffector junctions in mouse isolated vas deferens

It is acknowledged that neurotransmission in the mouse vas deferens is predominantly mediated by ATP and noradrenaline (NA) released from sympathetic nerves while cholinergic transmission in the rodent vas deferens is often overlooked despite early literature. Recently we have characterized a cholinergic component of neurogenic contraction of mouse isolated vas deferens. In the present paper, by confocal imaging of Ca(2+) dynamics we detected acetylcholine (ACh) action at muscarinic cholinergic neuroeffector junctions at high-resolution. Experiments were carried out in the presence of prazosin (100 nM) and alpha,beta methylene ATP (alpha,beta-MeATP) (1 microM) to inhibit responses to NA and ATP respectively. Exogenous ACh (10 microM) elicited Ca(2+) transients, an effect blocked by the muscarinic receptor antagonist, cyclopentolate (1 microM). Ca(2+) transients were evoked by electrical stimulation of intrinsic nerves in the presence of the cholinesterase inhibitor neostigmine (10 microM). Stimulation produced a marked increase in the frequency and number of Ca(2+) transients. Cyclopentolate reduced the frequency of occurrence of spontaneous and evoked events to control levels. The alpha(2)-adrenoceptor antagonist yohimbine (300 nM) did not affect the spontaneous Ca(2+) transients, but increased the frequency of occurrence of evoked transients, an effect inhibited by cyclopentolate. The postjunctional effects of neuronally-released ACh are limited by the action of cholinesterase. Release of ACh appears to be tonically inhibited by NA released from sympathetic nerve terminals through action at prejunctional alpha(2)-adrenoceptors. Tetrodotoxin (TTX, 300 nM) abolished the nerve-evoked Ca(2+) events, with no effect on Ca(2+) transients elicited by exogenous ACh. In conclusion, the presence of spontaneous and evoked cholinergic Ca(2+) transients in smooth muscle cells of the mouse isolated vas deferens has been revealed. These events are mediated by ACh acting at M(3) muscarinic receptors. This action stands in marked contrast to the lack of effect of neuronally-released NA on smooth muscle Ca(2+) dynamics in this tissue.