M3 muscarinic receptor expression on suburothelial interstitial cells

OBJECTIVE To identify the cells expressing the M3 muscarinic receptor subtype in the lamina propria of the bladder. MATERIALS AND METHODS The bladders from five normal guinea pigs were isolated and fixed in 4% paraformaldehyde. Tissues sections (10 µm) were then cut and stained with antibodies to the type 3 muscarinic receptor (M3), the interstitial cell marker vimentin and the nonspecific nerve marker PGP 9.5. The specificity of the antibody to the M3 receptor was established using the complementary blocking peptide and Western blot analysis of human embryonic kidney (HEK) cells transfected to express the M3 receptor protein. RESULTS The M3 antibody pre‐incubated with its blocking peptide showed no immunohistochemical staining. Investigating this antibody using HEK cells transfected to express the M3 receptor protein and control HEK cells showed a single band in the transfected cells and no band in the control cells. M3 receptor immunoreactivity (M3‐IR) was detected primarily on a dense network of vimentin‐positive (vim+) cells lying immediately below the urothelium, i.e. the suburothelial interstitial cells (Su‐ICs). The M3‐IR was punctate and appeared to be located on the cell surface. The diffuse network of cells in the remaining regions of the lamina propria showed no M3‐IR. Few nerve fibres were associated with the M3‐IR Su‐ICs. The M3‐IR Su‐ICs were most numerous and prominent in the lateral wall. The number of M3‐IR/vim+ cells diminished towards the bladder base and were absent in the bladder urethral junction. In the base and urethral junction there were vim+ cells that were not M3‐IR. A population of umbrella cells in the lateral wall also showed weak punctate M3‐IR. CONCLUSIONS Using a well‐characterized M3 antibody, these results show for the first time that the M3 muscarinic receptor in the lamina propria is located specifically on the Su‐ICs. The physiological role of these cells is unknown and consequently the significance of what appears to be a cholinergic signalling system is unclear. Previously published data showed that these cells respond to nitric oxide and atrial natriuretic peptide with an increase in cGMP and possibly prostaglandin. All of these observations, taken together, suggest that the Su‐ICs receive multiple inputs and that they must be part of a complex signalling system in this region of the bladder wall.