Epidermal growth factor (EGF) increases Na+ uptake in several cell types through an electroneutral, amiloride-sensitive pathway putatively identified as Na+/H+ countertransport. We have previously shown that EGF rapidly activates amiloride-sensitive net Na+ influx in the A431 human epidermoid carcinoma cell line. We also describe the presence of transmembrane, amiloride-sensitive Na+/H+ exchange in A431 cells using a new fluorescence technique for the measurement of intracellular pH (pH(i)) based on the incorporation of fluorescein-dextran into the cell cytoplasm. The low pK(a) of fluorescein (~6.4) prevented the direct assessment of the inferred, EGF-induced cytoplasmic alkalinization, mediated by stimulated Na+/H+ exchange. In this paper, 4',5'-dimethylfluorescein (pK(a) 6.75) was coupled to dextran, allowing increased pH sensitivity of the fluorescence assay in the physiological range. Using this improved assay, basic features of pH(i) regulation in A431 cells are documented, including the role of Na+/H+ exchange and Na+-linked (Cl-/HCO3- exchange in acid extrusion. We directly demonstrate a rapid elevation of pH(i) by addition of EGF as well as by serum in A431 cells. The pH(i) increase is half-maximal at 5-10 ng/ml of EGF, is dependent on external Na+, independent of external Ca2+, and inhibited by millimolar amiloride. EGF and serum also enhance Na+/H+ exchange-mediated cytosolic acidification when the transmembrane Na+ concentration gradient favors Na+ efflux from the cells. An alkaline pH(i) shift, caused by activation of Na+/H+ exchange, may be an important primary event in the mechanism of EGF action.
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1983|