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. 2001 Jan 2;98(1):154–159. doi: 10.1073/pnas.98.1.154

Figure 3.

Figure 3

Differentiation-dependent expression and apical secretion of plasminogen activators by cell- and organ-cultured bovine urothelium. (A–C) mRNA levels (relative to glyceraldehyde-3-phosphate dehydrogenase as assessed by Northern blot, ●) and PA activities (zymography, ▴) of uroplakin III (A), tPA (B), and uPA (C) in cultured bovine urothelial cells (17). Cells were harvested at 25% confluence (lane 1), 50% (lane 2), 100% (lane 3), 3 days postconfluence (pc) (lane 4), 6 days pc (lane 5), and 9 days pc (lane 6). Loading was normalized on the basis of cell numbers. Note the differentiation-dependent expression of UPIII and tPA and, less strikingly, of uPA. (D) Stimulation of PA secretion. Cultured bovine urothelial cells were treated with a control medium (lanes 1 and 4), 1 μM calcium ionophore A23187 (lanes 2 and 5), and 1 mM 8-Br-cAMP (lanes 3 and 6). Numbers above denote the percentages of PA activities in conditioned media (lanes 1–3) vs. cell lysates (lanes 4–6). (E) Polarized secretion of PAs. Bovine urothelial cells were cultured on a Nucleopore filter (Transwell, 0.4-mm pore size; Costar). A and BL denote the apical and basal-lateral compartments, respectively. The media were collected after 24 h in a control medium (E) or, after 15 min, in the presence of 1 mM of cAMP (F). Note that a great majority of the PAs were secreted apically and that the polarity was not affected by cAMP stimulation. (G) Secretion of tPA and uPA by organ-cultured bovine bladder mucosa into the overlying culture medium. Samples were 20 μg of total protein extract of in vivo bovine urothelium (lane 1), 15 μl of fresh bovine urine (lane 2); 20 μl of a medium that had been conditioned by organ-cultured bovine urothelium for 3 h (lane 3); 20 μl of medium conditioned in the presence of 5 μg/ml brefeldin A, a secretory inhibitor (lane 4) (32); 5 μg of total protein extract of 5-day postconfluent cell-cultured bovine urothelium (lane 5); and 20 μl of the culture medium that had been conditioned for 24 h by 5-day postconfluent cells (lane 6). Note the secretion of tPA and uPA by organ-cultured urothelium and its inhibition by BFA. (H) Detection of PP5 in the same samples as in G by immunoblotting. Note the detection of PP5 in urothelium, fresh urine, and in a medium conditioned by organ-cultured bovine urothelium; also note the absence of PP5 in cultured urothelial cells.