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Introduction:The bladder urothelium is multilayered. Ability to functionally study urothelial cells while preserving in situ location would represent an advance in bladder urothelial biology.
Materials & Methods:Mice were cardiac perfused with PBS. Bladders were excised. Urothelial sheets were dissected off with a microscope (5x magnification). Sheets were stained with H&E. In separate experiments, single cell electrophysiology was performed by placing urothelial sheets in Ringer’s bath solution, with either basal or apical surface down. Using 40x magnification, individual urothelial cells from different layers were identified. Potassium currents on these cells were measured in situ using single channel patch−clamp technique.
Results:Histology revealed that sheets were free of lamina propria and smooth muscles. The proportion of apical cells, compared to proportion of intermediate and basal cells, with measureable potassium currents was considerably higher (69% of apical versus 16% of intermediate and 18% of basal cells). Of active patches detected in apical cells, 100% of these patches showed a 43 pS current conductance. For intermediate and basal cells, 75−83% demonstrated a 43 pS current and 17−25% demonstrated a 22 pS current. Single cells could also be individually microdissected completely off the urothelium.
Conclusions:A novel approach was developed in which individual cells within the multilayered urothelium were identified and functionally studied in situ. Membrane potassium conductances were different from different layers. Single cells from an identified layer can be harvested off the urothelium. This technique allows investigators to study cellular functions while preserving location within the urothelium.