Regulation of bladder muscarinic receptor subtypes by experimental pathologies

Abstract

1 The M3 muscarinic receptor subtype is widely accepted as the receptor on smooth muscle cells that mediates cholinergic contraction of the normal urinary bladder and other smooth muscle tissues, however, we have found that the M2 receptor participates in contraction under certain abnormal conditions. The aim of this study was to determine the effects of various experimental pathologies on the muscarinic receptor subtype mediating urinary bladder contraction. 2 Experimental pathologies resulting in bladder hypertrophy (denervation and outlet obstruction) result in an up-regulation of bladder M2 receptors and a change in the receptor subtype mediating contraction from M3 towards M2. Preventing the denervation-induced bladder hypertrophy by urinary diversion prevents this shift in contractile phenotype indicating that hypertrophy is responsible as opposed to denervation per se. 3 The hypertrophy-induced increase in M2 receptor density and contractile response is accompanied by an increase in the tissue concentrations of mRNA coding for the M2 receptor subtype, however, M3 receptor protein density does not correlate with changes in M3 receptor tissue mRNA concentrations across different experimental pathologies. 4 This shift in contractile phenotype from M3 towards M2 subtype is also observed in aged male Sprague-Dawley rats but not females or either sex of the Fisher344 strain of rats. 5 Four repeated, sequential agonist concentration response curves also cause this shift in contractile phenotype in normal rat bladder strips in vitro, as evidenced by a decrease in the affinity of the M3 selective antagonist p-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD). 6 A similar decrease in the contractile affinity of M3 selective antagonists (darifenacin and p-F-HHSiD) is also observed in bladder specimens from patients with neurogenic bladder as well as certain organ transplant donors. 7 It is concluded that although the M3 receptor subtype predominantly mediates contraction under normal circumstances, the M2 receptor subtype can take over a contractile role when the M3 subtype becomes inactivated by, for example, repeated agonist exposures or bladder hypertrophy. This finding has substantial implications for the clinical treatment of abnormal bladder contractions.

Figure 1

The effect of various experimental pathologies on bladder hypertrophy (panel a), nerve-evoked bladder contractions by carbachol (panel b) and cholinergic receptor stimulated contractions (panel c). Data is presented as mean ± SEM. Top panel (a) *DEN, BOO and DEC are significantly greater than sham while DIV is significantly less than sham (n = 9–24 rats per group). Middle panel (b): contractile response to electric field stimulation of 8 V, 30 Hz, 1 ms, *DIV-DEN and DEN are significantly less than sham. # DIV-DEN is significantly greater than DEN (n = 40–60 muscle strips per group). Bottom panel (c): The maximal contractile response to carbachol, *BOO is significantly greater than every other group (n = 40–60 muscle strips per group).

Figure 2

The effect of various experimental pathologies on the density of muscarinic receptor subtypes determined by immunoprecipitation. M₂ and M₃ receptors were labelled with [³H]-QNB, solubilized and immunoprecipitated as described previously (Luthin, Harkness, Artymyshyn & Wolfe, 1988). Data shown are average fmoles ± SEM of receptor mg⁻¹ solubilized protein from sham operated, DIV, DIV-DEN, DEN, BOO and DEC (n = 4–5 triplicate determinations from six to 18 bladders). The concentration of protein in the solubilized receptor preparation was approximately 8% of the protein concentration in the crude homogenate. As compared to filtration binding, approximately 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 and M₂ receptor density all groups are statistically significantly different from each other. For M₃ receptor density, the sham group is statistically significantly different from DIV, DIV-DEN, DEN and BOO.

Figure 3

Correlation between M₂ (panel a) and M₃ (panel B) muscarinic receptor subtype density with functional denervation. Functional denervation was evaluated for each individual muscle strip using the ratio of the carbachol maximum contractile response to the electric field stimulation (EFS) induced maximal contractile response. The higher this ratio is the greater degree of functional denervation. The average of the individual muscle strips for each experimental group for functional denervation is plotted vs. the immunoprecipitation data (shown in Fig. 2) 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 shows a statistically significant correlation with functional denervation, however, no correlation between the M₃ receptor density and functional denervation is seen.

Figure 4

Development of a multiplex ribonuclease protection assay for quantitative determination of tissue muscarinic receptor subtype mRNA concentration. Representative phosphor image from a polyacrylamide electrophoresis gel. Lane 1 is 2000 dpm of undigested probe while lane 2 shows the amount of probe protected by 20 μg of total RNA isolated from rat bladder. Images are visualized by use of a phospho imager and the individual bands are quantified using Optiquant software (results shown in Fig. 5). The undigested probe lengths are 314, 285, 259, 209, 168, 141, and 124 nucleotides, while the protected probe lengths are 285, 256, 230, 180, 139, 113, and 96 nucleotides for M₁, M₄, M₅, M₂, M₃, L32 and GAPDH respectively.

Figure 5

Density of transcripts for housekeeping genes L32 and GAPDH (panel a) and M₂ and M₃ muscarinic receptors (panel b). The density of transcripts for both L32 and GAPDH change significantly as a result of the experimental conditions. The density of transcripts coding for the M₂ muscarinic receptor subtype increase significantly compared to sham in DEN and BOO while the M₃ transcript density is increased in BOO and decreased in DIV bladders. NOR (normal, n = 9), SHAM (n = 11), DIV (n = 9), DEN–DIV (n = 8), DEN (n = 9), BOO (n = 6), DEC (n = 6). *P < 0.05 compared with sham, **P < 0.01 compared to sham, +P < 0.05 compared with normal, ++P < 0.01 compared with normal.

Figure 6

Correlation between M₂ (top panel) and M₃ (bottom panel) muscarinic receptor protein density and transcript density. The density of M₂ receptor protein, as determined by subtype selective immunoprecipitation (shown in Fig. 2), positively correlates with the density of M₂ RNA as determined by RPA (top panel). However, there is no correlation between the density of M₃ receptor protein and M₃ receptor RNA (bottom panel).

Figure 7

Affinity of muscarinic receptor subtype selective antagonists against carbachol-induced bladder contraction in young and old Sprague–Dawley rats. Blocks with horizontal hatching indicates the M₂ affinity range for each antagonist and vertical hatching indicates the M₃ affinity range. In the old male SD rats the experimentally determined affinity of the M₃ selective antagonists and are closer to (darifenacin) or within (p-fluoro-hexahydro-sila-diphenidol) the M₂ affinity range of these antagonists.

Figure 8

Subtype selective antagonist affinity in young and old rats of the Fisher strain.

Figure 9

Effect of four sequential carbachol concentration response curves (CRCs) on the affinity of the antagonist p-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) in normal Sprague–Dawley rats. Results are shown as mean ± SEM for 16 strips (first to fourth CRC) and eight separate strips with and without 1 μm p-F-HHSiD added after the fourth CRC (larger symbols). Symbols without error bars indicate that the SEM is less than the size of the symbol.

Figure 10

Affinity of subtype selective antimuscarinics for inhibition of carbachol-induced contraction of urinary bladder muscle strips in vitro from spinal injury patients. The shaded areas represent the affinity ranges of antimuscarinics (darifenacin, methoctramine and p-fluoro-hexahydro-sila-diphenidol) for M₂ receptors (horizontal hatching) and M₃ receptors (vertical hatching) reported in the literature (Caulfield, 1993; Hegde et al., 1997). The symbols refer to the affinities which were determined by Schild analysis of the bladder tissue obtained from the individual patients.

Figure 11

Affinity of subtype selective antimuscarinics for inhibiting carbachol-induced contraction of urinary bladder muscle strips in vitro from organ donors. Notation is the same as indicated for Fig. 10.