Selective alkylation of rat urinary bladder muscarinic receptors with 4-DAMP mustard reveals a contractile function for the M-2 muscarinic receptor

Our previous data indicate that M-3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by sele ctive alkylation of M-3 receptors with 4-DAMP mustard and suggest that the M-2 receptor subtype may be involved in inhibition of beta-adrenergic recep tor induced relaxation, therefore, allowing recontraction. Alkylation resul ted in 85% of M-3 receptors and 65% of M-2 receptors unable to bind radioli gand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and d irect carbachol contractions were inhibited by antagonists with affinities consistent with M-3 receptor mediated contraction. In contrast, the affinit ies of antagonists for inhibition of carbachol induced recontractions follo wing isoproterenol stimulated relaxation in the presence of 90 mM KCl, indi cated a contractile function for the M-2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role fo r the M-2 subtype in bladder smooth muscle contraction.