Neuronally Released Acetylcholine Acts on the M₂ Muscarinic Receptor to Oppose the Relaxant Effect of Isoproterenol on Cholinergic Contractions in Mouse Urinary Bladder

Abstract

We investigated whether M(2) muscarinic receptor activation opposes isoproterenol-induced relaxation in mouse urinary bladder and whether endogenous acetylcholine acts through a similar M(2) mechanism. When measured in urinary bladder from M(3) receptor knockout mice, the muscarinic agonist oxotremorine-M elicited only very weak contractions. In the presence of alpha,beta-methylene ATP (30 microM) and isoproterenol (1 microM), however, oxotremorine-M elicited a robust contractile response. This response was completely absent in bladder from M(2)/M(3) double knockout mice, indicating that activation of the M(2) receptor inhibits the relaxant effect of isoproterenol on the contraction to alpha,beta-methylene ATP. Similar results were obtained when prostaglandin F(2alpha) (5 microM) was used as the contractile agent but not when serotonin was used. Electrical field stimulation of the urinary bladder from wild-type mouse elicited contractions that were inhibited 20% by atropine and 40% by desensitization with alpha,beta-methylene ATP. When measured in the presence of alpha,beta-methylene ATP to desensitize the purinergic component of contraction, isoproterenol exhibited moderately greater relaxant activity in field-stimulated bladder from the M(2) knockout mouse compared with that observed in wild-type bladder. This differential relaxant effect of isoproterenol was greatly increased in the presence of physostigmine. In contrast, no differential effects were noted for isoproterenol in similar experiments on bladders from M(3) knockout and M(2)/M(3) double knockout mice in the presence of physostigmine. Our results suggest that neuronally released acetylcholine acts on the M(2) muscarinic receptor to inhibit the relaxant effect of isoproterenol on the minor, cholinergic component of contraction in the field-stimulated mouse urinary bladder.

The M2-Muscarinic Receptor Inhibits the Development of Streptozotocin-Induced Neuropathy in Mouse Urinary Bladder

“…Isoproterenol was slightly more effective in relaxing EFS contractions in M 2 KO bladder compared with wild-type tissue (Fig. 6a), indicating that neuronally released acetylcholine acts on the M 2 receptor to oppose relaxant responses, as described previously, with other stimulation parameters (Ehlert et al, 2007). STZ treatment enhanced this effect (Fig.…”

“…It seems likely that the loss of function can be attributed to the loss of M 2 receptors, as described above, and not to changes in other mechanisms. We have previously reported no change in the sensitivity of the M 2 KO mouse urinary bladder to the contractile effects of mATP and prostaglandin F 2␣ and the relaxant effect of isoproterenol against KCl-and mATP-induced contractions (Ehlert et al, 2005(Ehlert et al, , 2007. Ito et al (2009) reported 35 and 34% decreases in total muscarinic receptors in bladder from M 4 and M 5 KO mice, suggesting that a loss of one subtype in a single KO mouse causes a loss of other subtypes.…”

“…Male M 3 KO mice rapidly develop distended urinary bladders and a complete loss of EFS bladder contraction, including the purinergic component, approximately 3 to 4 months of age (Matsui et al, 2000;Ehlert et al, 2007), presumably because of the near complete loss of postjunctional muscarinic receptor mediated-contraction. We have shown that the M 2 receptor causes a low-potency enhancement of M 3 receptor-mediated contractions of the urinary bladder (Ehlert et al, 2005).…”

“…The M 3 receptor acts through G q to mediate direct contraction in smooth muscle, whereas M 2 enhances M 3 receptor-mediated contraction and inhibits the relaxant effect of isoproterenol and forskolin on contractions elicited by other contractile receptors, including the M 3 (Ehlert et al, 2005(Ehlert et al, , 2007. In the mouse, the M 2 receptor also elicits a modest direct contraction of some smooth muscles, but direct M 2 contractions of the urinary bladder are quite small (Stengel et al, 2002;Matsui et al, 2003).…”

Cross-regulation between cardiac muscarinic acetylcholine receptors and β-adrenoceptors: lessons for use of knock-out mice

“…In a broader sense, such studies are a reminder that life-long knockout of one gene can lack expected or have unexpected consequences due to cross-regulation. On the other hand, studies based on systemic knockout mice, e.g., for the M 2 receptor (Ehlert et al 2007) and studies based on highly M 2 selective antagonists (Witte et al 2011) have yielded consistent findings on the interaction between the muscarinic and the β-adrenoceptor system, e.g., in the urinary bladder.…”