Aims of study:
It has been reported that contraction of urinary bladder body is mediated via the smaller population of M3-receptors, but M2-mediated contraction has been demonstrated following M3-receptor inactivation and elevation of cAMP levels. The present study investigates the characterisation of muscarinic receptor subtypes in the bladder dome, bladder base and proximal urethra of the female pig.

Methods:
In receptor binding studies, displacement experiments using [3H]QNB with 4-DAMP (M3-selective antagonist) and methoctramine (M2-selective antagonist) determined the M2:M3 receptor ratio in membranes of pig bladder and urethra. In the functional studies in vitro, the affinity of these antagonists against carbachol induced contractions of tissue strips were also calculated in normal tissues and following selective M3-inactivation (incubation with 40µM 4-DAMP mustard in the presence of 1µM methoctramine to "protect" M2-receptors), precontraction with 50mM KCl and relaxation with isoprenaline (30µM).

Results:
In saturation binding studies, receptor density was significantly (p<0.05) more in bladder dome and base than in urethra, being 137.5±56.4, 130.5±25.7 and 44.1±13.2 fmol/mg protein, respectively. Dissociation constant (Kd) for [3H] QNB in bladder dome, base and urethra was similar, being 0.27±0.04, 0.27±0.11and 0.26±0.07 nM, respectively. In competition binding studies, displacement of [3H] QNB by 4-DAMP and methoctramine best fitted a 2-site model with Hill's slopes<1.0 in bladder dome and base, the high and low affinity site indicating M3 and M2 receptor, respectively, and an M2: M3 ratio of 3:1. In urethra, displacement of [3H] QNB by 4-DAMP and methoctramine best fitted 1-site model with Hill's slopes close to unity, the affinity indicating M2 receptor. On normal detrusor muscle strips in vitro, 4-DAMP had a high affinity in both bladder dome (n=12) and base (n=18), with Schild slopes close to unity, pKB value of 9.4±0.07 and 9.5±0.07,respecively. Methoctramine had a relatively low affinity in bladder dome (pKB=6.1±0.05, n=18). These results indicated that the M3-subtype mediates contraction of the bladder dome and base. 4-DAMP also had a high affinity in proximal urethra (pKB=9.46±0.15, n=9), however the Schild slope was less than unity (0.56±0.08). Methoctramine demonstrated pKB values of 6.90±0.14 with Schild slopes close to unity in urethra (n=12). These results suggested that the contraction of urethra was mediated by M3 and M2 receptors. In tissues where the M3-receptors had been inactivated and cAMP levels elevated, the affinity of 4-DAMP was significantly reduced in bladder dome (8.7±0.1, n=27,P<0.001) and base (8.5±0.08, n=12,P<0.0001) compared with normal tissues.

Conclusions:
Bladder dome and base have similar distribution of muscarinic receptor subtypes, the M3: M2 ratio being 3:1. Urethra appears to have predominantly M2 receptor. In vitro, the M3-subtype appears to mediate contraction of the normal pig bladder dome and base, and an involvement of M2-receptors in contraction was noted following selective M2-inactivation and cAMP elevation. Contraction of the pig urethra appears to be mediated by M2 and M3 receptors.