RAPID COMPUTERIZED ANALYSIS OF NA+/H+ EXCHANGE FLUX

The regulation of intracellular pH (pH(i)) is mediated by membrane transporters whose activity is directly controlled by pH(i). Therefore, transport rates must be compared at identical pH(i) values in functional studies of these transporters. This is conventionally performed using scatter plots showing initial rates of proton flux versus intracellular pH. We present justification for determining proton flux over a wide range of pH(i), by digitally smoothing a pH(i) trace and then directly taking the first derivative versus time of smoothed data. Compared to conventional least-squares analysis of initial rates, the derivative method generates much more information per experiment. Compared to other methods which fit pH(i) traces to a defined equation prior to rate calculation, the new method does not require that the pH(i) trace be well fit by any given mathematical function. The derivative technique is illustrated in an analysis of Na+/H+ exchange in Caco-2 cells. Intracellular pH is measured fluorometrically in cells loaded with BCECF (2',7'-bis[2-carboxyethyl]-5-(and-6)carboxyfluorescein). To validate the analysis of Na+/H+ exchange over an extended time range, we demonstrate that cellular acidification with NH4Cl does not change steady state Na+ content. We find that proton flux rates analyzed by the derivative method are equivalent to initial rates measured by least-squares analysis.