Cholinergic innervation of the guinea-pig isolated vas deferens.

Recently, a cholinergic neurogenic component of contraction has been characterised in the aganglionic mouse vas deferens. In this paper, a cholinergic component of contraction in the guinea-pig vas deferens is characterised pharmacologically. A residual, tetrodotoxin-sensitive (TTX, 0.3 microM), neurogenic contraction was revealed after prolonged exposure (5 h) to the adrenergic neurone blocker bretylium (20 microM) or in the presence of prazosin (100 nM) and alpha,beta-methylene ATP (1 microM), a purinergic agonist which desensitizes P2X receptors. The bretylium-resistant component was potentiated by the acetylcholinesterase (AChE) inhibitor neostigmine (10 microM) and inhibited by the muscarinic-receptor (mAChR) antagonist cyclopentolate (1 microM). Nicotine (30 microM) enhanced the bretylium-resistant component. Neostigmine increased the second component of contraction in the presence of prazosin and alpha,beta-methylene ATP, whilst yohimbine (1 microM), an alpha(2) adrenergic receptor antagonist, enhanced both the first and second components of the electrically evoked contraction. These enhanced contractions were blocked by cyclopentolate in both cases. Nicotine enhanced the cholinergic component of contraction revealed by neostigmine but failed to have any detectable effects in the presence of cyclopentolate. Neostigmine alone increased the slow component of contraction which was reversed by cyclopentolate to control levels. The M(3) receptor-antagonist 4-DAMP (10 nM) markedly inhibited the cholinergic component of contraction to a level comparable with cyclopentolate. Laser microscopy has shown that neostigmine also increased the frequency of spontaneous Ca(2+) transients remaining in smooth muscle cells after perfusion with prazosin and alpha,beta-methylene ATP, an effect blocked by 4-DAMP. These experimental data show that there is a functional cholinergic innervation in the guinea-pig vas deferens whose action is limited by acetylcholinesterase, blocked by cyclopentolate and mediated through M3 receptors. Moreover, by blocking the cholinesterase, the increased amount of ACh generates spontaneous Ca(2+) transients in smooth muscle cells.

Cholinergic innervation of the mouse isolated vas deferens.

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 microM) 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 microM), reduced this residual component and the inhibition was reversed by the acetylcholinesterase inhibitor, neostigmine (1 and 10 microM). Nicotine (30 microM) 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 microM), a selective alpha(1)-adrenoceptor antagonist, and alpha,beta-methylene ATP (1 microM), a purinergic agonist that desensitise P2X receptors, neostigmine increased the hump component of contraction and yohimbine (0.3 microM), an alpha(2)-adrenoceptor antagonist, enhanced both components of the electrically evoked stimulation. The contraction was blocked by cyclopentolate (1 microM). 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 microM) and cyclopentolate (1 microM) to control levels. However, in control experiments, atropine or cyclopentolate did not detectably influence the delayed neurogenic contraction. Ach (10 microM) 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.