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Recently, a population of nerves has been described in the aganglionic mouse vas deferens, in which electrically evoked contractions were insensitive to high concentrations of the adrenergic neurone blocker, bretylium. In this paper, the pharmacology of this nerve-evoked contraction has been examined in more detail.Bretylium (20 μM) revealed, after 5 h exposure, a new residual neurogenic contraction (20 stimuli at 10 Hz) that was tetrodotoxin-sensitive.The muscarinic antagonist, cyclopentolate (0.1 and 1 μM), reduced this residual component and the inhibition was reversed by the acetylcholinesterase inhibitor, neostigmine (1 and 10 μM).Nicotine (30 μM) enhanced the residual component revealed by bretylium, suggesting that there are prejunctional nicotinic receptors (nAchRs) influencing acetylcholine (Ach) release.In the presence of prazosin (0.1 μM), a selective α1-adrenoceptor antagonist, and α,β-methylene ATP (1 μM), a purinergic agonist that desensitise P2X receptors, neostigmine increased the hump component of contraction and yohimbine (0.3 μM), an α2-adrenoceptor antagonist, enhanced both components of the electrically evoked stimulation. The contraction was blocked by cyclopentolate (1 μM).In the absence of bretylium, neostigmine alone increased the hump component of contraction in a frequency-dependent manner. This increase was reversed by atropine (1 μM) and cyclopentolate (1 μM) to control levels. However, in control experiments, atropine or cyclopentolate did not detectably influence the delayed neurogenic contraction.Ach (10 μM) induced a contraction in the mouse vas deferens, either when applied alone or in the presence of neostigmine.Thus, it has been demonstrated unequivocally that the mouse vas deferens is innervated by functional cholinergic nerves, whose action is terminated by cholinesterase. Furthermore, Ach release can be enhanced by activation of prejunctional nAchRs presumably located on the cholinergic nerve terminals.British Journal of Pharmacology (2005) 146, 927–934. doi:10.1038/sj.bjp.0706357; published online 19 September 2005 [ABSTRACT FROM AUTHOR]

Background and purpose:The antidysrhythmic bretylium is useful experimentally because it selectively abolishes neurotransmitter release from sympathetic peripheral nerve terminals. Its mechanism of action seemed settled, but recent results from optical monitoring of single terminals now suggests a new interpretation.Experimental approach:Orthograde transport of a dextran-conjugated Ca2+ indicator to monitor Ca2+ in nerve terminals of mouse isolated vas deferens with a confocal microscope. In some experiments, local neurotransmitter release was detected by monitoring neuroeffector Ca2+ transients (NCTs) in adjacent smooth muscles, a local measure of purinergic transmission. Sympathetic terminals were identified with catecholamine fluorescence (UV excitation) or post-experiment immunohistochemistry.Key results:Bretylium (10 μM) abolished NCTs at 60/61 junctions over the course of 2 h, indicating effective abolition of neurotransmitter release. However, bretylium did not abolish the field stimulus-induced Ca2+ transient in most nerve terminals, but did increase both action potential delay (by 2±0.4 ms) and absolute refractory period (by 4±2 ms). Immunohistochemistry demonstrated that 85–96% of terminals orthogradely filled with a dextran-conjugated fluorescent probe contained Neuropeptide Y (NPY). A formaldehyde–glutaraldehyde-induced catecholamine fluorescence (FAGLU) technique was modified to allow sympathetic terminals to be identified with a Ca2+ indicator present. Most terminals contained catecholamines (based on FAGLU) or secrete ATP (as NCTs in adjacent smooth muscle cells are abolished).Conclusions and implications:Bretylium can inhibit neurotransmitter release downstream of Ca2+ influx without abolishing the nerve terminal action potential. Bretylium-induced increases in the absolute refractory period permit living sympathetic terminals to be identified.British Journal of Pharmacology (2008) 153, 831–839; doi:10.1038/sj.bjp.0707623; published online 10 December 2007 [ABSTRACT FROM AUTHOR]