CHARACTERIZATION OF THE PREJUNCTIONAL MUSCARINIC RECEPTORS MEDIATING INHIBITION OF EVOKED RELEASE OF ENDOGENOUS NORADRENALINE IN RABBIT ISOLATED VAS-DEFERENS

The aim of the present study was to characterize the prejunctional mod ulation of evoked release of endogenous noradrenaline in rabbit vas de ferens by the use of muscarinic receptor agonists and subtype-preferin g antagonists. Vasa deferentia of the rabbit were stimulated electrica lly by trains of 120 pulses delivered at 4 Hz or trains of 30 pulses a t 1 Hz. The inhibition by muscarinic agonists of the stimulation-evoke d overflow of endogenous noradrenaline in the absence and presence of antagonists was used to determine affinity constants for antagonists. These values were compared with those observed at putative M(1) recept ors inhibiting neurogenic twitch contractions in the rabbit vas defere ns and with affinity data obtained at M(1)(m(1))-M(4)(m(5)) receptors in functional studies and binding experiments. The evoked overflow of noradrenaline from sympathetic nerves was enhanced by the Al receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the P-2 purinoc eptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and indomethacin, indicating a tonic inhibition by endogenous A(1) and P-2 purinoceptor agonists and prostanoids, respectively. The stimulation-evoked overflow at 4 Hz was not sensitive to inhibition by the muscarinic agonists methacholine or 4-(4- lorophenylcarbamoyloxy) -2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). In contrast, at a stimulation frequency of I Hz the evoked noradrenaline release was de creased by muscarinic agonists (EC(50)): arecaidine propargyl ester (0 .062 mu M), 4-Cl-McN-A-343 (0.32 mu M), enylcarbamoyloxy)-2-butynyl-N- methyl-pyrrolidinium tosylate (4-F-PyMcN(+); 0.48 mu M) and methacholi ne (0.86 mu M). The affinity constants of most of the muscarinic antag onists [atropine: pK(B) = 9.47; (R)-trihexyphenidyl: pK(B) = 9.18; pir enzepine: pA(2) = 7.68; methoctramine: pK(B) = 6.90] are consistent wi th estimates of these antagonists at M,(m,) receptors determined in va rious functional and binding studies. The high antagonistic potency of pirenzepine and (R)-trihexyphenidyl and the agonistic activity of 4-F -PyMcN(+) argue for the involvement of M(1), and against that of M(2) and M(3) receptors in the inhibition of evoked noradrenaline overflow However, the high apparent pK(B) of 8.30 for himbacine is not in accor dance with an M(1) receptor; by contrast, it would be compatible with the presence of M(2) or M(4) receptors. The potencies of the tested mu scarinic agonists and antagonists largely agree with those obtained fo r the inhibition of neurogenic twitch responses (0.05 Hz) in the rabbi t vas deferens. In conclusion, the rabbit vas deferens is endowed with prejunctional muscarinic receptors mediating heteroinhibition of nora drenaline release that are probably of the same subtype as the putativ e M(1) receptors inhibiting neurogenic twitch contractions, and are no t of the M(2), M(3) or m(5) subtype.