Gap junctions and connexin expression in human suburothelial interstitial cells

Objective To determine whether suburothelial interstitial cells of the human bladder express gap junctions, and if so, to establish their extent and composition, using immunocytochemistry, confocal microscopy and electron microscopy. Materials and methods Bladder tissue was obtained at cystectomy; the tissue was: (i) frozen for cryosectioning and Northern blot analysis; (ii) fixed and embedded for standard thin‐section electron microscopy; and (iii) processed using low‐denaturation conditions in Lowicryl for immunogold‐label electron microscopy. Cryosections were immunofluorescently labelled using antibodies against connexins 43, 40 and 45, vimentin, desmin and c‐Kit ligand, and examined by confocal microscopy. Double labelling was used to determine the spatial relationship of labelling for connexin43 with that of vimentin and desmin. Thin‐section electron microscopy was used to investigate interstitial cell ultrastructure and permit unequivocal identification of gap junctions, and immunogold labelling of Lowicryl sections was applied to localize connexin43. Results Immunoconfocal microscopy showed prominent labelling for the gap junction protein, connexin43, in a suburothelial band of cells that was also strongly positive for vimentin. The connexin43/vimentin‐positive cells showed only weak labelling for desmin and c‐Kit ligand, and were immunonegative for connexins 40 and 45. Northern blotting showed a corresponding abundance of connexin43 transcript in the mucosal layer but not the detrusor layer of the bladder wall. Electron microscopy revealed abundant gap junctions, recognized by their pentalaminar structure, between the cell processes of interstitial cells in the suburothelial zone. That these interstitial cell gap junctions were the source of the connexin43 immunolabelling observed by immunoconfocal microscopy was confirmed by immunogold labelling in sections of Lowicryl‐embedded tissue examined by electron microscopy. Conclusion A network of interstitial cells, extensively linked by connexin43‐containing gap junctions, is located beneath the urothelium in human bladder. As gap junctions provide pathways for direct cell‐to‐cell communication, the interstitial cellular network may operate as a functional syncytium, integrating signals and responses in the bladder wall.