Selective alkylation of rat urinary bladder muscarinic receptors with 4-DAMP mustard reveals a contractile function for the M2 muscarinic receptor

Our previous data indicate that M-3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective 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 receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M-3 receptors and 65% of M-2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M-3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M-2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M-2 subtype in bladder smooth muscle contraction.