STUDIES ON THE KINETICS OF NA+/H+ EXCHANGE IN OK CELLS - INTRODUCTION OF A NEW DEVICE FOR THE ANALYSIS OF POLARIZED TRANSPORT IN CULTURED EPITHELIA

The present study describes a new perfusion technique-based on the use of a routine spectrofluorometer - which enables fluorometric evaluation of polarity, regulation and kinetics of Na+/H+ exchange at the level of an intact monolayer. Na+/H+ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na+/H+ exchange activity was measured by monitoring changes in intracellular pH (pH(i)) after an acid load, using the pH-sensitive dye 2'7'-bis (carboxyethyl) 5-6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pH(i) recovery mechanism, which is Na+ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pH(i) = 7.68; pH(o) = 7.4) where Na+/H+ exchange is not detectable, transport rate increased as pH(i) decreased. A positive cooperativity characterized the interaction of internal H+ with the exchanger, and suggests multiple H+ binding sites. In contrast, extracellular [Na+] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na+ was 19 mM at an intracellular pH of 7.1 and 60 mM at an intracellular pH of 6.6. Inhibition of Na+/H+ exchange activity by EIPA was competitive with respect to extracellular [Na+] and the K(i) was 3.4-mu-M. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pH(i) and processes associated with their polarized expression and regulation.