Physiological and morphological effects of poly-L-lysine on the toad bladder

Studies were carried out on the morphological and physiological effects of the binding of poly-L-lysine (polylysine; mol wt≊120,000) to the apical surface membrane of the toad bladder epithelium. Paired hemibladders were mounted in chambers and exposed to polylysine concentrations of 2, 8, or 80 μg/ml in the mucosal medium for periods of up to 2 hr. Radioautographs prepared after addition of(3)H-polylysine showed that the polymer was localized to the apical surface of the epithelium and in dense subapical masses in lysed cells. No significant morphological changes were seen in the epithelium by light or electron microscopy at polymer concentrations of 2 and 8 μg/ml. Exposure to 80 μg/ml lysed many epithelial cells, i.e., converted them to slightly swollen ghosts with pycnotic nuclei and empty cytoplasm, except for remnants of mitochondria and vesicular fragments of the endoplasmic reticulum. All of the superficial epithelial cells were lysed in stretched hemibladders. The plasma membranes of the lysed cells were uniformly thickened, and their intercellular attachments remained intact. In contracted hemibladders, lysed and normal-appearing cells were interspersed, and the number of lysed cells in the epithelium was proportional to the duration of exposure to high concentrations of the polycation. In parallel experiments, the effects of varying concentrations of polylysine on active Na(+) transport and osmotic flow of water were measured with and without vasopressin, aldosterone, or amphotericin B in the media. At a concentration of 2 μg/ml of polylysine in the mucosal bathing solutions, no change in the basal rate of Na(+) transport was seen, and the response to vasopressin was unimpaired. At a concentration of 8 μg/ml, there was a significant but small fall in electrical potential difference (PD) and in short-circuit current (SCC) and no interference with the response to vasopressin. At a concentration of 80 μg/ml, there was a rapid curvilinear fall in SCC to 54±4% of the baseline value and in PD to 21±3% of the baseline value in a 2-hr period. Simultaneous unidirectional isotope flux studies with(22)Na and(24)Na showed a more than twofold increase in the serosal to mucosal flux but no discrepancy between net flux and SCC. Despite the inhibitory action of the polymer, the stimulatory response in Na(+) transport to vasopressin, aldosterone, and amphotericin B was relatively preserved in that the percentage increase in SCC was the same in the polymer-treated and control hemibladders. The polycation produced a small but significant increase in osmotic water flow, and striking and irreversible inhibition of the water-flow response to vasopressin.