RESUMEN
PURPOSE: Type 3 muscarinic receptors, which are present in the bladder wall, are important for bladder function. However, their role in the context of the urothelium is not well defined. Understanding the role of type 3 muscarinic receptors has been limited by the lack of specific type 3 muscarinic receptor antibodies. Thus, we identified a specific type 3 muscarinic receptor antibody and investigated the site of type 3 muscarinic receptors in sham operated and obstructed guinea pig bladders. MATERIALS AND METHODS: The specificity of 4 commercially available type 3 muscarinic receptor antibodies was determined. Immunohistochemistry was then done in bladder tissue from sham operated and obstructed guinea pig bladders. RESULTS: One of the 4 antibodies examined had the needed specificity in terms of blocking peptide and Western blot characterization. Using this antibody type 3 muscarinic receptor immunoreactivity was associated with muscle cells, nerves and interstitial cells. Four types of interstitial cells were identified, including suburothelial, lamina propria, surface muscle and intramuscular interstitial cells. In the obstructed model the bladder wall was hypertrophied and there was nerve fiber loss. The number of lamina propria, surface muscle and intramuscular interstitial cells was increased but not the number of suburothelial interstitial cells. Also, surface muscle interstitial cells appeared to form clusters or nodes with type 3 muscarinic receptor immunoreactivity. CONCLUSIONS: Nerve loss and the up-regulation of interstitial cells with type 3 muscarinic receptor immunoreactivity may underlie major functional changes in the pathological bladder. This indicates that type 3 muscarinic receptor specific anticholinergic drugs may affect not only the detrusor muscle, as previously thought, but also interstitial cells and nerve fibers.
Asunto(s)
Anticuerpos/inmunología , Receptor Muscarínico M3/inmunología , Receptor Muscarínico M3/metabolismo , Obstrucción del Cuello de la Vejiga Urinaria/inmunología , Obstrucción del Cuello de la Vejiga Urinaria/metabolismo , Vejiga Urinaria/inmunología , Vejiga Urinaria/metabolismo , Animales , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Cobayas , Inmunohistoquímica , Masculino , Vejiga Urinaria/cirugía , Obstrucción del Cuello de la Vejiga Urinaria/cirugíaRESUMEN
OBJECTIVE: To identify the cells expressing the M(3) 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 microm) were then cut and stained with antibodies to the type 3 muscarinic receptor (M(3)), the interstitial cell marker vimentin and the nonspecific nerve marker PGP 9.5. The specificity of the antibody to the M(3) receptor was established using the complementary blocking peptide and Western blot analysis of human embryonic kidney (HEK) cells transfected to express the M(3) receptor protein. RESULTS The M(3) antibody pre-incubated with its blocking peptide showed no immunohistochemical staining. Investigating this antibody using HEK cells transfected to express the M(3) receptor protein and control HEK cells showed a single band in the transfected cells and no band in the control cells. M(3) receptor immunoreactivity (M(3)-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 M(3)-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 M(3)-IR. Few nerve fibres were associated with the M(3)-IR Su-ICs. The M(3)-IR Su-ICs were most numerous and prominent in the lateral wall. The number of M(3)-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 M(3)-IR. A population of umbrella cells in the lateral wall also showed weak punctate M(3)-IR. CONCLUSIONS Using a well-characterized M(3) antibody, these results show for the first time that the M(3) 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.