MEASURING VOLUME PERTURBATION OF PROXIMAL TUBULAR CELLS IN PRIMARY CULTURE WITH 3 DIFFERENT TECHNIQUES

Osmotic cell volume perturbations of rabbit proximal tubule (PT) in pr imary culture were measured using three independent techniques. Automa tic cell thickness monitoring of PT monolayers revealed that cell volu me rapidly increased by 39 +/- 2% in hypotonic medium (150 mosM), whic h was followed by partial regulatory volume decrease (RVD). Subsequent incubation in hypertonic medium (500 mosM) rapidly decreased cell vol ume by 54 +/- 2% not followed by regulatory volume increase (RVI). Whe n cell volume in PT monolayers was derived from concentration changes in the trapped fluorescent dyes, fura 2 or 2',7'-bis(2-carboxyethyl)-5 (6)-carboxyfluorescein, osmotically induced cell volume changes appear ed much smaller (17 +/- 1 and 22 +/- 2% for similar hypo- and hyperton icity, respectively). However, changes in fluorescence intensity were most often not in agreement with anticipated cell volume changes. With the Coulter counter, a much larger shift in cell volume was observed in PT cell suspensions. In this situation, cell swelling in hypotonic medium amounted to 74 +/- 2% but was still followed by partial RVD. Hy pertonicity resulted in a decrease in cell volume of 42 +/- 3% not fol lowed by RVI. In conclusion, our study indicates that automatic cell t hickness monitoring of an epithelial cell layer cultured on a permeabl e support provides more reliable data than monitoring changes in fluor escence intensity of trapped dyes.