Monitoring of sodium:proton exchange in isolated hepatocytes by electronic cell sizing

Abstract

To investigate volume-regulating processes in the hepatocyte, a rapid and precise method of measuring cell volume in isolated hepatocytes was devised which uses a Coulter Counter equipped with a Channelyzer. Isolated hepatocytes exhibit a volume-decreasing mechanism (potassium channel) which is triggered by cell volume increases as small as 10%. Cell volume increases in the hepatocyte may be mediated by activity of the Na:H exchanger. To examine Na:H exchange-mediated cell volume increases, without apparent interference by the volume-decreasing mechanism, acetate was substituted for chloride in the incubation medium. Hepatocytes placed in a medium containing sodium acetate at an acidic pH exhibit a continuous amiloride-sensitive swelling. A simple procedure was devised for estimating Na:H exchanger set-point by electronic cell sizing. In a sodium acetate medium, the internal pH equilibrates with the external pH. By placing cells in sodium acetate medium of various pH values and measuring the rate of amiloride-sensitive swelling, an estimate of Na:H exchanger set-point can be obtained. By this method, the exchanger was estimated to cease activity above an intracellular pH of 7.2. This method could be useful for identification of stimuli that might promote cell enlargement by raising the exchanger set-point. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate tetraacetic acid raises the set-point of the exchanger in the isolated hepatocytes, resulting in exchanger activity at normal cellular pH, and at the same time promotes hepatocyte swelling. Exchanger activation via a kinase C-mediated mechanism is one possible way that hepatocyte enlargement may occur.