Neurotransmitter release evoked by alpha-latrotoxin in the smooth muscle of the female pig urethra

Neuronal regulation of smooth muscle tone in the female pig urethra has mainly been studied in vitro using electrical field stimulation (EFS) of nerves. Excitatory control is considered to be exerted by released noradrenaline, whereas inhibitory control is non-adrenergic non-cholinergic (NANC), and mediated by nitric oxide (NO), and an as yet unidentified agent. We investigated the functional and morphological effects of alpha-latrotoxin (alphaLTX), a spider neurotoxin believed to cause massive release of vesicle-stored neurotransmitters, on spontaneously developed urethral smooth muscle tone. The effects were compared to those of EFS and high potassium. In the presence of the NO-synthesis inhibitor N(omega)-nitro-L-arginine (L-NOARG: 0.3 mM) both alphaLTX and EFS evoked contractions. After treatment with scopolamine and phentolamine, no contraction was observed, and under these conditions alphaLTX and EFS induced relaxation. At low frequencies (<12 Hz), the EFS-induced relaxations were rapid, whereas at higher frequencies (>12 Hz), they were biphasic, consisting of a rapid first phase followed by a more long-lasting second phase. L-NOARG abolished the relaxations at low frequencies, as well as the first phase of the biphasic relaxation. The second phase was not affected by treatment with L-NOARG, but 0.1 microM omega-conotoxin GVIA, blocker of N-type voltage-operated calcium- channels (VOCCs), markedly reduced or abolished the response. In the presence of L-NOARG or omega-conotoxin GVIA, the alphaLTX-induced relaxation was significantly decreased, and the combination of L-NOARG and omega-conotoxin GVIA further reduced or abolished the relaxation. In preparations-treated with tetrodotoxin or scorpion venom, believed to inactivate nerves by acting on sodium channels, alphaLTX and EFS had no effects. alphaLTX-induced relaxation was not associated with changes in cyclic GMP or cyclic AMP content. High (80 mM) potassium solution induced a triphasic response of the preparation. A transient relaxation was followed by a restoration of tone, and then by a persistent relaxation. The persistent relaxation was slightly reduced by scorpion venom or L-NOARG, but reduced by 50% by a combination of L-NOARG and omega-conotoxin GVIA. Ultrastructural analysis of the urethral circular smooth muscle layer revealed a moderate amount of nerve profiles supplying the smooth muscle. In control preparations, the nerve profiles contained both small synaptic vesicles and large dense core vesicles. alphaLTX caused a major loss of both types of vesicle. The present data suggest that alphaLTX has the ability to release not only adrenergic and cholinergic transmitters, but also NANC mediators of relaxation, including NO, from nerve terminals in the urethra.