Hypertrophy changes the muscarinic receptor subtype mediating bladder contraction from M3 toward M2

Abstract

Major pelvic ganglion electrocautery (MPGE) and spinal cord injury in the rat induce bladder hypertrophy and a change in muscarinic receptor subtypes mediating bladder contraction from predominantly M3 to a combination of M2 and M3. To determine whether this is a result of bladder hypertrophy or denervation, we studied the following groups: sham-operated controls, urinary diversion (DIV), MPGE together with urinary diversion (DIV-DEN), bilateral MPGE (DEN), bladder outlet obstruction (BOO), and MPG decentralization (MPGDEC). The degree of bladder denervation was determined by the maximal carbachol response normalized to the response to electric field stimulation. Receptor subtype density was determined by immunoprecipitation. The affinity of subtype-selective muscarinic antagonists for inhibition of carbachol-induced contractions was used to determine the subtype-mediating contraction. DEN, MPG-DEC, and BOO bladders were hypertrophic whereas DIV bladders were atrophic compared with sham operated. Bladder contraction in sham-operated, DIV, and DIV-DEN was mediated by the M3 receptor subtype, whereas the M2 subtype participated in contraction in the DEN, MPG-DEC, and BOO groups. The hypertrophied bladders had an increase in total and M2 receptor density while all experimental groups showed a reduction in M3 receptor density. Thus bladder hypertrophy, independent from bladder denervation, causes a shift in the muscarinic receptor subtype mediating bladder contraction from M3 toward M2.

Fig. 1

Time course of denervation-induced changes in the maximal carbachol response and the nerve-evoked responses to electric field stimulation (EFS) of 8 V, 30 Hz, 1 ms presented as means ± SE. There were no significant differences in the carbachol-evoked maximum (Carb Max). However, by 24 h the nerve-evoked contractions were significantly reduced. *Significantly different from control, P < 0.05.

Fig. 2

DIV, urinary diversion; DIV-DEN, major pelvic ganglion (MPG) electrocautery (MPGE) together with urinary diversion; DEN, bilateral MPGE; BOO, bladder outlet obstruction; MPG-DEC; MPG decentralization. A: effect of experimental pathologies on bladder hypertrophy presented as means ± SE (mg/g). *DEN, BOO, and MPG-DEC are significantly greater than sham operated while DIV is significantly less than sham operated (n = 9–24 rats/group).B: nerve-evoked contractile response to EFS of 8 V, 30 Hz, 1 ms presented as means ± SE. *DIV-DEN and DEN are significantly less than sham operated. #DIV-DEN is significantly greater than DEN (n = 40–60 muscle strips/group). C: maximal carbachol contractile response presented as means ± SE. *BOO is significantly greater than every other group (n = 40–60 muscle strips/group).

Fig. 3

Effect of experimental pathologies on the potency of carbachol to induce contraction presented as means ± SE. *DEN is significantly less than sham operated, DIV, and MPG-DEC (n = 7–36 muscle strips/group).

Fig. 4

Effect of experimental pathologies on the density of urinary bladder muscarinic receptor subtypes. M₂ and M₃ receptors were labeled with [³H]QNB and solubilized as described in Luthin et al. (19). Data shown are average fmol (±SE) of receptor/mg solubilized protein from sham operated, DIV, DIV-DEN, DEN, BOO, and MPG-DEC (n = 4–5 determinations from 6–18 bladders). Protein concentration in the solubilized receptor preparation was ~8% of the protein concentration in the crude homogenate. Compared with filtration binding, ~50% of the muscarinic receptors were solubilized (data not shown). Group differences were determined using ANOVA with a post hoc Newman-Keuls test. For total receptor density all groups are different from each other. For M₂ receptor density, all groups are different from each other. For M₃ receptor density, sham operated is different from DIV, DIV-DEN, DEN, and BOO.

Fig. 5

Correlation of muscarinic receptor subtype density with functional denervation. For each individual muscle strip the ratio of the carbachol maximum:EFS-induced contraction was determined. The average of the individual muscle strips for each experimental group for functional denervation is plotted vs. the immunoprecipitation data for M₂ receptor density (A) and M₃ receptor density (B). The ratio of the average carbachol maximum to the average EFS contraction displayed in Fig. 2 does not equal the average of the individual ratios for the strips. The density of M₂ receptors correlates with functional denervation. No correlation between the M₃ receptor density and functional denervation is seen.