Gender comparison of muscarinic receptor expression and function in rat and human urinary bladder: differential regulation of M₂ and M₃ receptors?

Abstract

Since symptoms of bladder dysfunction occur more frequently in women than in men and since muscarinic receptors are the physiologically most important system to mediate bladder contraction, we have compared the number, subtype distribution and function of muscarinic receptors in bladders from male and female rats. Muscarinic receptor function was also assessed in bladder strips from male and female human bladder. Male and female rats expressed a similar number of muscarinic receptors (144±5 vs. 140±6 fmol/mg protein in saturation radioligand binding). While competition binding curves for the moderately M₂-selective methoctramine were not consistently better fitted by a two-site model, most competition curves for the M₃-selective darifenacin were biphasic and yielded 29±10% and 31±7% high affinity sites (corresponding to M₃ receptors) in male and females, respectively. Immunoreactivity of α-subunits of the G-proteins G_q/11, G_i1/2, G_i3 and G_s did not significantly differ between both genders. The muscarinic receptor agonist carbachol similarly stimulated inositol phosphate accumulation in bladder slices from male and female rats with calculated maximum responses of 69±17 and 77±18% over basal and pEC₅₀ values of 4.90±0.45 and 4.40±0.46, respectively. While darifenacin inhibited carbachol-stimulated inositol phosphate formation approximately 100-fold more potently than methoctramine, each antagonist was similarly potent in both genders. Carbachol concentration-dependently contracted bladder strips with a pEC₅₀ of 5.66±0.05 and 5.72±0.06 and maximum effects of 4.3±0.1 and 4.2±0.2 mN/mg wet weight in male and female rats, respectively. The contractile effect of carbachol was concentration-dependently antagonised by the non-selective atropine (1–30 nM), the M₁-selective pirenzepine (1–30 M), the M₂-selective methoctramine (1–10 µM) and the M₃-selective darifenacin (10–100 nM), with the latter exhibiting a partly unsurmountable antagonism. The overall potency of all four antagonists suggested that contraction was mediated predominantly if not exclusively by M₃ receptors with no appreciable differences between both male and female rats. Similarly, the maximum effects (4.4±0.6 vs. 4.4±2.4 mN/mg) and pEC₅₀ (6.07±0.05 vs. 6.32±0.14) of carbachol did not differ between genders in bladder samples from 25 consecutive patients. We conclude that number und function of muscarinic receptors and the relative roles of their M₂ and M₃ subtypes do not differ between urinary bladders of male and female rats; at least with regard to overall muscarinic responsiveness this situation appears to be similar in humans.