Muscarinic receptor transcript and protein density in hypertrophied and atrophied rat urinary bladder

Abstract

Aims: Our previous studies showed that bladder hypertrophy shifts the muscarinic receptor subtype mediating contraction from M(3) towards M(2) along with increased M(2) and decreased M(3) protein concentration. We quantified mRNA for M(1) through M(5) receptors to determine whether the changes in M(2) and M(3) protein levels was due to changes in transcription.

Methods: Bladder hypertrophy was induced by bladder outlet obstruction (BOO), major pelvic ganglion electrocautery (DEN), and major pelvic ganglion decentralization (DEC). Bladder atrophy was induced by ureteral diversion (DIV). Additional groups included denervated and diverted (DEN-DIV), sham operated (SHAM), and normal (NOR) controls. Transcripts were quantified using a multiplex ribonuclease protection assay (RPA) and receptor protein density was determined by immunoprecipitation. Receptor transcripts were expressed per unit total RNA.

Results: Although all five receptor subtype transcripts were detected in all experimental groups, the densities of M(1), M(4), and M(5) were much lower than for the M(2) and M(3) subtype. There were more M(2) receptor transcripts than all the others, consistent with M(2) protein determinations. M(2) transcripts were significantly increased in DEN and BOO bladders. Surprisingly, M(3) transcripts were also significantly increased in BOO. There was a significant correlation (r=0.98, P<0.001) between protein density and transcript density for the M(2) but not the M(3) receptor among the different experimental groups.

Conclusions: Changes in mRNA concentration are reflected by changes in protein density for the M(2) receptor but not for the M(3) receptor. Extrapolation of functional effects from transcript density data is invalid for M(3) mediated bladder contractions.

Fig. 1

Example of results from multiplex ribonuclease protection assay (RPA). Lane 1 is 2,000 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 bands are quantified using Optiquant software. 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 forM₁, M₄, M₅, M₂, M₃, L32, and GAPDH, respectively.

Fig. 2

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 to sham, **P < 0.01 compared to sham, ⁺P < 0.05 compared to normal, ⁺⁺P < 0.01 compared to normal.

Fig. 3

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

Fig. 4

Density of M₂ and M₃ muscarinic receptor transcriptswhen normalized to either GAPDH (panel A) or L32 (panel B).

Fig. 5

Correlation between transcript density and protein density for the M₂ (panels A and C) or the M₃ (panels B and D) receptor when normalized to either L32 (panels A and B) or GAPDH (panels C and D).