ICS 1999, Denver
Informally discussed posters
Pharmacology
The Role of M2-Muscarinic receptors in pig contraction

T. Yamanishi, R. Chess-Williams, C.R. Chapple, K. Yasuda
Department of Biomedical Science, University of Sheffield, Department of Urology, Royal Hallamshire Hospital, Sheffield, UK and Dokkyo University, Japan.

Aims of Study: Several studies have identified the M₃-receptor as being the predominant muscarinic receptor subtype responsible for contraction of the bladder to muscarinic agonists in vitro. However in many species the density of the M₂-receptor subtype is greater than that of the M₃-subtype. Recently it has been shown that in the rat bladder, where the M₂:M₃ ratio is about 9:1, an M₂-mediated contraction can be demonstrated following M₃-receptor inactivation and elevation of cAMP levels (recontraction). ¹ This study examines whether a similar role for M₂-receptors can be demonstrated in the pig bladder, where the M₂:M₃ ratio is closer to that reported for the human bladder (i.e.3:1).

Methods: Pig detrusor strips (dome region) were set up in aerated Krebs solution at 37 °C and concentration-response curves (CRCs) obtained to carbachol in the absence and presence of the M₃-selective antagonist 4-DAMP and M₂-selective antagonist methoctramine. Similar experiments were performed on tissues following selective M₃-inactivation (incubation of tissues with 40nM 4DAMP mustard for 60 min in the presence of 1µM methoctramine to protect M₂-receptors), precontraction with 50 mM KCL and relaxation with isoprenaline (30µM) or forsklin (1µM). Parallel control experiments were used to correct for time-dependent changes in tissue sensitivity. Antagonist affinity (PKb values) and Schild plots were constructed form shifts of CRCs to carbachol.

Results: On normal detrusor muscle strips in vitro, 4-DAMP and methoctramine caused parallel rightward shifts of CRCs without a change in maximum responses, and yielded a mean PKb value of 9.6 (n=4) and 6.1 (n=6), respectively, and a Schild slope similar to unity (0.94±0.12 and 0.89±0.15, respectively). In tissues where the M₃-receptors had been inactivated and cAMP levels elevated, a re-contraction was obtained to carbachol. 4DAMP again caused rightward shifts or CRCs, but the antagonist was less potent, the mean PKb value being 8.72 (n=9) significantly lower (p<0.01) than in the normal tissues. Methoctramine also caused rightward shifts or CRCs, and the antagonist was more potent, the mean PKb value being 7.06 (n=6) signigicantly higher (p<0.001) than in the normal tissues.

Conclusions: The data in normal tissues suggest that in the pig, as in other species, responses were mediated solely via the M₃-receptor. The re-contraction after M₃-inactivation with protection of M₂-receptor appears to be mediated by M₂-receptor.

Table: Effects of antagonists on CRCs obtained to carbachol

Reference:

Hedge S.S. et al., (1997)Br. J. Pharmachol 120, 1409-1418.