POLARIZED DISTRIBUTION OF NA-H+ ANTIPORT ACTIVITY IN RAT ALVEOLAR EPITHELIAL-CELLS()

In this study, we investigated the polarized distribution of Na+-H+ an tiport activity in alveolar epithelial cell monolayers. Rat alveolar t ype II pneumocytes were grown on detachable tissue culture-treated Nuc lepore filters. The membrane filters, with their adherent intact alveo lar epithelial cell monolayers, were mounted in a cuvette designed to contain two fluid compartments separated by the monolayer. Cells were loaded with the pH-sensitive dye 2',7'-biscarboxyethyl-5,6-carborrylfl uorescein and intracellular pH (pH(i)) measured spectrofluorometricall y. Monolayers were studied at ambient temperature on days 3-4 in cultu re, coincident with the development of high tissue resistance (R(T) gr eater than or equal to 2000 Omega.cm(2)). Cells were incubated in HCO3 --free Na+ buffer [tin mM) 140 NaCl, 6 HEPES, pH 7.4] and acidified by NH3 prepulse. Rates of realkalinization (J(H+)) were calculated as th e product of the initial rate of recovery (dpH(i)/dt) and the intracel lular buffer capacity (beta(i)). Under control conditions, recovery oc curred with an initial J(H+) Of 28.4 mM/min. When 100 mu M dimethylami loride (DMA), an amiloride analogue with enhanced specificity for inhi biting the Na+-H+ antiporter, was present in the basolateral fluid, re covery was inhibited by > 90%. Conversely, when the monolayers were ac idified in Na+ buffer containing DMA. (100 mu M) in the apical fluid, acidification and recovery were identical to control. Recovery from ac idification was inhibited by basolateral DMA with a one-half maximal i nhibitory concentration (IC50) of 100 nm and by basolateral amiloride with an IC50 of 10 mu m. Recovery was completely inhibited by omission of Na+ from the basolateral fluid, but omission of Na+ from apical fl uid had no effect. We conclude that Na+-H+ antiport activity is locate d exclusively on the basolateral surface of these alveolar epithelial cell monolayers, where it most likely represents the high-amiloride af finity isoform of the Na+-H+ antiporter, NHE-1. The Na+-H+ antiporter, asymmetrically distributed to the basolateral surface of the polarize d alveolar epithelium, contributes to intracellular homeostasis in alv eolar pneumocytes and may also play a role in signal transduction in t hese cells.