Characterization of the purinergic receptor subtype on guinea‐pig suburothelial myofibroblasts

OBJECTIVE To identify particular purinoceptor subtypes by immunohistochemical labelling, as a layer of suburothelial myofibroblasts has been identified in the urinary bladder, and these cells respond to exogenous ATP by generating an intracellular Ca 2+ transient, but the particular purinoceptor that responds to ATP is unclear. MATERIALS AND METHODS Tissue sections and isolated cells from the urothelial layer of the guinea‐pig bladder were used. Preparations were labelled with primary antibodies to the intermediate‐filament protein, vimentin, or the purinoceptors P2X 3 , P2Y 1 , P2Y 2 , P2Y 4 and P2Y 6 . For single‐labelling we used a secondary antibody tagged with the fluorescent marker Cy3, and for double‐labelling also a secondary antibody tagged with fluorescein isothiocyanate or Cy2. Images were examined using a confocal microscope, with an argon (488 nm) or helium‐neon (543 nm) laser. RESULTS Vimentin‐labelling was confined to the suburothelial layer and appeared as discrete signals. Isolated cells labelled with vimentin and strongly for the P2Y 6 antibody. There was weaker staining for P2X 3 , P2Y 2 and P2Y 4 , but none to P2Y 1 . With frozen sections there was P2Y 6 labelling in the urothelial and suburothelial layer. CONCLUSION The predominant purinoceptor in suburothelial myofibroblasts, from these labelling studies, is the P2Y 6 subtype. However, there was weaker labelling to other subtypes, suggesting multiple receptor subtypes or heterogeneity of receptor subunits. The consequences of there being multiple purinoceptor subtypes in the suburothelial space with respect to sensory signalling are discussed.