Measuring volume perturbation of proximal tubular cells in primary culture with three different techniques

Osmotic cell volume perturbations of rabbit proximal tubule (PT) in primary culture were measured using three independent techniques. Automatic cell thickness monitoring of PT monolayers revealed that cell volume rapidly increased by 39 +/- 2% in hypotonic medium (150 mosM), which was followed by partial regulatory volume decrease (RVD). Subsequent incubation in hypertonic medium (500 mosM) rapidly decreased cell volume by 54 +/- 2% not followed by regulatory volume increase (RVI). When 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 appeared much smaller (17 +/- 1 and 22 +/- 2% for similar hypo- and hypertonicity, 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. Hypertonicity resulted in a decrease in cell volume of 42 +/- 3% not followed by RVI. In conclusion, our study indicates that automatic cell thickness monitoring of an epithelial cell layer cultured on a permeable support provides more reliable data than monitoring changes in fluorescence intensity of trapped dyes.