[H-3]Noradrenaline release was studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Acetylcholine elicited a concentration- and time-dependent increase in H-3 outflow which was half-maximal at about 300 mu M and within 5 s. The overflow induced by 10 s exposure to 300 mu M acetylcholine was reduced by the nicotinic antagonist hexamethonium, but increased by the muscarinic antagonist atropine. Cd2+ (300 mu M) prevented the overflow evoked by electrical field stimulation, but reduced acetylcholine-induced overflow by less than 50%. Removal of extracellular Ca2+ abolished stimulation-evoked tritium overflow, irrespective of the stimulus, The selective alpha(2)-adrenoceptor agonist UK 14,304 inhibited acetylcholine-evoked overflow to a significantly smaller extent (approximate to 25% maximal inhibition) than electrically induced overflow (greater than or equal to 45% maximal inhibition). These inhibitory effects were antagonized by the alpha(2)-adrenoceptor antagonist yohimbine. Noradrenaline (0.1 mu M) reduced acetylcholine-evoked overflow to the same extent as did UK 14,304 (0.1 mu M). UK 14,304 had no effect when H-3 overflow was evoked by acetylcholine in the presence of 300 mu M Cd2+. Currents through nicotinic acetylcholine receptors and voltage-activated Ca2+ currents were studied with the whole-cell variant of the patch-clamp technique. UK 14,304 reduced nicotinic acetylcholine receptor currents and voltage-activated Ca2+ currents with similar potency and efficacy. Yohimbine, however, antagonized only the inhibition of voltage-activated Ca2+ currents, but not the effects of UK 14,304 on nicotinic receptor currents. Furthermore, yohimbine per se reduced currents through nicotinic receptors. Noradrenaline (10 mu M) inhibited voltage-dependent Ca2+ currents just as did UK 14,304 (10 mu M), but failed to reduce currents through nicotinic acetylcholine receptor channels. Cd2+ (300 mu M) abolished voltage-activated Ca2+ currents and reduced nicotinic acetylcholine receptor currents by 65%. These results indicate that acetylcholine evokes noradrenaline release from rat sympathetic neurons by activation of nicotinic receptors and restricts this release via muscarinic receptors. The acetylcholine-induced transmitter release is based on two mechanisms, one involving and the other one bypassing voltage-dependent Ca2+ channels, alpha(2)-Adrenoceptor activation reduces voltage-activated Ca2+ currents and affects exclusively the component of acetylcholine-induced release which involves voltage-dependent Ca2+ channels. These results support the hypothesis that voltage-activated Ca2+ channels are the sole site of autoinhibitory alpha(2)-adrenergic effects on transmitter release from rat sympathetic neurons. The inhibitory effects of alpha(2)-adrenoceptor agonists and antagonists on currents through nicotinic acetylcholine receptors are not mediated by an alpha(2)-adrenoceptor.
