Resistive properties of the epithelial membranes of the urinary bladder of the toad, Bufo marinus, determined using the fluorescent dye, RH160

A technique is described for quantitative epifluorescence studies of the apical membrane of the epithelial cells of the urinary bladder of the toad, Bufo marinus, using the lipid-soluble dye, RH160. When the urinary bladder is appropriately mounted, fluorescence signals, in response to a transepithelial voltage pulse, can be recorded from the epithelium immediately after the addition of the dye to the mucosal bath, and for some hours subsequently. The optical signal, recorded as the change in fluorescence in response to a transepithelial voltage pulse, as a fraction of resting fluorescence, was found to be a linear function of the applied voltage over the range +/- 200 mV, and was approximately 3% for a 100 mV change in transepithelial potential. The signal was enhanced by amiloride (10 mumol.l-1), reduced by bretylium (5 mmol.l-1) and abolished in the presence of nystatin (730 U.ml-1). Calculations based on these data permitted estimation of the fractional resistance of the apical membrane, which was found to be 0.85 under control conditions. Apical membrane resistance was 8.6 k omega.microF, and the basolateral membrane resistance was 1.5 k omega.microF. These findings support the conclusion that the apical membrane of toad urinary bladder epithelial cells is of high resistance, thus resembling other sodium-transporting epithelia.