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 modulation of evoked release of endogenous noradrenaline in rabbit vas deferens by the use of muscarinic receptor agonists and subtype-prefering antagonists.Vasa deferentia of the rabbit were stimulated electrically by trains of 120 pulses delivered at 4 Hz or trains of 30 pulses at 1 Hz. The inhibition by muscarinic agonists of the stimulation-evoked 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 M₁ receptors inhibiting neurogenic twitch contractions in the rabbit vas deferens and with affinity data obtained at M₁(m₁)-M₄(m₅) 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 P₂ purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS) and indomethacin, indicating a tonic inhibition by endogenous A₁ and P₂ 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-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). In contrast, at a stimulation frequency of 1 Hz the evoked noradrenaline release was decreased by muscarinic agonists (EC₅₀): arecaidine propargyl ester (0.062 M), 4-Cl-McN-A-343 (0.32 M), 4-(4-fluorophenylcarbamoyloxy)-2-butynylN-methyl-pyrrolidinium tosylate (4-F-PyMcN⁺; 0.48 M) and methacholine (0.86 M). The affinity constants of most of the muscarinic antagonists atropine: pK_B = 9.47; (R)-trihexyphenidyl: pK_B = 9.18; pirenzepine: pA₂ = 7.68; methoctramine: pK_B = 6.90] are consistent with estimates of these antagonists at M₁(m₁) receptors determined in various 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 M₁, and against that of M₂ and M₃ receptors in the inhibition of evoked noradrenaline overflow. However, the high apparent pK_B of 8.30 for himbacine is not in accordance with an M₁ receptor; by contrast, it would be compatible with the presence of M₂ or M₄ receptors. The potencies of the tested muscarinic agonists and antagonists largely agree with those obtained for the inhibition of neurogenic twitch responses (0.05 Hz) in the rabbit vas deferens. In conclusion, the rabbit vas deferens is endowed with prejunctional muscarinic receptors mediating heteroinhibition of noradrenaline release that are probably of the same subtype as the putative M₁ receptors inhibiting neurogenic twitch contractions, and are not of the M₂, M₃ or m₅ subtype. Correspondence to: U. Grimm at the above address