The distribution of single P2x1-receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder

The distribution of purinergic (P-2x1) receptors on smooth muscle cells in relation to autonomic nerve varicosities in the rat urinary bladder has been determined using immunofluorescence and confocal microscopy. P-2x1 receptors were visualized using rabbit polyclonal antibodies against the extracellular domain of the P-2x1 receptor, and varicosities were visualized using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P-2x1 receptor clusters were observed on the smooth muscle cells of the detrusor, namely, a large ellipse of mean long axis 1.23 +/- 0.21 mu m and short axis 0.92 +/- 0.17 mu m and a smaller spherical cluster with a mean diameter of 0.40 +/- 0.04 mu m. The latter occured in much greater numbers than the former in selected areas, with a density as high as 0.8 per mu m(2) or two orders of magnitude more than the larger-sized clusters. The large clusters are in general located beneath varicosities, with only 4.5% of P-2x1 clusters not possessing an overlying varicosity. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P-2x1 and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P-2x1 receptor clusters. On occasions, two or more small SV2-labelled varicosities about 0.7 mu m in diameter each with a receptor patch were found juxtaposed to each other; these might represent the splitting up of a single large varicosity. These observations are discussed in relation to the identity of the autonomic neuromuscular junction.