A BICARBONATE-DEPENDENT PROCESS INHIBITABLE BY DISULFONIC STILBENES AND A NA+/H+ EXCHANGE MEDIATE NA-22+ UPTAKE INTO CULTURED BOVINE CORNEAL ENDOTHELIUM

Abstract

22Na+ uptake into confluent monolayers of cultured bovine corneal endothelial cells was studied in the presence of ouabain (10-4 M) to inhibit active Na extrusion. In bicarbonate saline, uptake was reduced to a similar degree either by amiloride (10-3 M) or by 4-acetamido-4''-isothiocyanostilbene-2,2''-disulfonic acid (SITS) (10-3 M). A further reduction was obtained with SITS-pretreated cells in the presence of amiloride. SITS-sensitive uptake was further characterized in saline containing both ouabain (10-4 M) and amiloride (10-3 M). It was absolutely dependent on bicarbonate, which could not be substituted by other plasma membrane permeable buffers (50 mM acetate or 25 mM glycodiazine). It was a saturable function of both bicarbonate and Na concentration. Half-maximal fluxes occurred between 3 and 7 mM HCO3 (at 151 mM Na) and between 35 and 60 mM Na (at 28 mM HCO3). Uptake into Na-depleted cells was reduced as opposed to Na-rich cells, and SITS-sensitive 22Na+ efflux out of 22Na+-loaded cells into Na-free medium was less than efflux into sodium saline, indicating trans-stimulation by Na. The amiloride-sensitive pathway was studied in the absence of bicarbonate to inhibit uptake via the SITS-sensitive pathway. 22Na+ uptake into Na-depleted cells increased steeply with extracellular pH in the range between pH 6 and 8 and could be largely blocked by 10-3, but not by 10-5 M amiloride. Bovine corneal endothelial cells probably possess at least 2 distinct pathways for Na uptake, amiloride sensitive 22Na+ fluxes being mediated by a Na+/H+ antiport, while the SITS-sensitive process is probably identical to a bicarbonate-sodium cotransport system postulated earlier from electrophysiological studies.