Isolation and separation of toad bladder epithelial cells

The epithelium of the urinary bladder ofBufo marinus is composed of 5 cell types, i.e., granular (Gr), mitochondria-rich (MR) and goblet (G) cells which face the urinary lumen, microfilament-rich (MFR) and undifferentiated cells (Un) located basally. The epithelium was dissociated by collagenase and EGTA treatment. Fractionation of dispersed cells by isopycnic centrifugation on dense serum albumin solutions yielded 4 fractions: (i) a very light fraction ( $\bar \rho \simeq 1.025$ ) enriched in MR and MFR cells; (ii) a light fraction ( $\bar \rho \simeq 1.045$ ) enriched in vacuolated Gr cells; (iii) a heavy fraction ( $\bar \rho \simeq 1.065$ ) composed essentially of aggregated Gr cells, and (iv) a pellet ( $\bar \rho \simeq 1.085$ ) enriched in G and undifferentiated cells. Recoveries were based on cell counts and DNA measurements. DNA content per cell was 13.2 pg±0.9 (n=37). From 1 g fresh tissue, 62±5×10⁶ (n=10) cells were recovered before isopycnic centrifugation of which about 70% excluded Trypan blue. After centrifugation, 90 to 95% of the cells excluded the vital dye and ∼3⁹×10⁶ cells were recovered from the gradient. Cell metabolism in each fraction was estimated by oxygen consumption measurements in absence or presence of ouabain, acetazolamide, and dinitrophenol. The consumption was threefold higher in the very light and light fractions when compared to the heavy and pellet fractions. Ouabain sensitive oxygen consumption (QO₂) represented 12 to 35% of the total O₂ consumption depending on the cell fraction, and acetazolamide sensitive QO₂ varied from −0.8% in the heavy fractions to 20% in the lighter fractions. DNP increased QO₂ in all fractions by 20 to 50%. Finally, the cells were able to reaggregate and form junctional complexes upon addition of calcium to the medium.