PRESYNAPTIC MUSCARINIC RECEPTOR SUBTYPES INVOLVED IN THE INHIBITION OF ACETYLCHOLINE AND NORADRENALINE RELEASE IN BOVINE CEREBRAL-ARTERIES

Experiments were performed in bovine cerebral arteries preincubated with [H-3]-choline or [H-3]-noradrenaline to analyze the presynaptic muscarinic receptors involved in inhibition of acetylcholine and noradrenaline release induced by electrical stimulation (4 Hz, 200 mA, 0.3 ms, 1 min). For this purpose, the actions of several muscarinic receptor antagonists on the H-3 overflow and on the carbacol-induced inhibition of this overflow were assessed. The evoked [H-3]-acetylcholine release and [H-3]-noradrenaline release were markedly reduced by the presence of tetrodotoxin, Ca2+-free medium, and the inhibitor of both choline transport and choline acetyltransferase, AF64A. Chemical sympathetic denervation with 6-hydroxydopamine (6-OHDA) decreased the uptake of [H-3]-noradrenaline, and AF64A reduced mainly the uptake of [H-3]-choline, but also of [H-3]-noradrenaline. Carbachol reduced the evoked [H-3]-noradrenaline and [H-3]-acetylcholine release; the IC50 values were 0.37 and 0.43-mu-mol/l, respectively. Atropine and 4-DAMP, but not AF-DX 116, methoctramine or pirenzepine, increased the evoked [H-3]-acetylcholine release. However, these muscarinic antagonists failed to modify the evoked [H-3]-noradrenaline release. Carbachol inhibited the release of both acetylcholine and noradrenaline. The inhibition was blocked by the antagonists. The rank orders of potency (based on plC50 values) were, in the case of [H-3]-acetylcholine release, atropine >4-DAMP>AF-DX 116 greater-than-or-equal-to pirenzepine greater-than-or-equal-to methoctramine, and, in the case of [H-3]-noradrenaline release, atropine >4-DAMP > AF-DX 116 greater-than-or-equal-to methoctramine greater-than-or-equal-to pirenzepine. These results suggest (1) that the prosynaptic receptors that modulate endogenous acetylcholine release are likely of the M3 subtype, whilst those involved on the effect of the exogenous agonist carbachol are Of M2 subtype, and (2) that those which inhibit noradrenaline release are probably a mixture of M2 and M3 subtypes as well. The autoinhibition of the acetylcholine release was funtionally active under our experimental conditions, while noradrenaline release does not appear to be modulated by muscarinic receptors in physiological conditions.