HORMONAL-REGULATION OF THE POLARIZED FUNCTION AND DISTRIBUTION OF NA H EXCHANGE AND NA/HCO3 COTRANSPORT IN CULTURED MAMMARY EPITHELIAL-CELLS/

The time course for development of polarized function and morphologica l distribution of pH regulatory mechanisms has been examined in a mous e mammary epithelial cell line (31EG4). Monolayers grown on permeable supports had tight junctions when grown 3-4 days in the presence of th e lactogenic hormones dexamethasone (D, a synthetic glucocorticoid) an d insulin (I), or in D, I, and prolactin (P), but there were no tight junctions in the absence of D. Microspectrofluorimetry of the pH-sensi tive dye BCECF was used to measure pH (pH(i)) in cells mounted in a tw o-sided perfusion chamber to distinguish pH regulatory activity at the apical and basolateral membranes. Na/H exchange was assayed as the Na -dependent, amiloride-sensitive component of pH(i) recovery from an ac id load induced by a pulse of NH3/NH4-Containing solution. When monola yers were grown 3-4 d in the presence of P, D, and I, Na/H exchange wa s restricted to the basolateral membrane. In contrast, in the absence of P, Na/H exchange was present on both the apical and basolateral mem branes. After 5-6 days, in the presence or absence of P, Na/H exchange was present only on the basolateral membrane. An antibody to the NHE- 1 isoform of the Na/H exchanger was used to determine its morphologica l distribution. In all hormone conditions the antibody recognized a pr otein of approximately 110 kD (Western blot), and confocal immunofluor escence microscopy of this antibody and of an anti-ZO-1 (the marker of the tight junctions) antibody showed that the morphological distribut ion of the Na/H exchanger was similar to the functional distribution u nder all hormonal treatments. In addition, a putative Na/HCO3 cotransp ort system was monitored as a Na-dependent, amiloride-insensitive pH(i ) recovery mechanism that was inhibited by 200 muM H2DIDS. After treat ment with D+I (but not with I alone) cotransport appeared exclusively on the basolateral membrane, and the polarized expression of this tran sporter was not altered by P. We conclude that when mammary cells are grown in D+I-containing media, the Na/H exchanger is expressed initial ly (i.e., after 3-4 d) on both the apical and basolateral membranes an d later (5-6 d) on only the basolateral membrane. P (in the presence o f D+I) selectively speeds this polarization, which is determined by po larized distribution of the exchanger to the apical and/or basal membr ane and not by the activation and/or inactivation of transporters. Sin ce the Na/HCO3 cotransporter (which requires D+I for expression and is unaffected by P) is expressed only in the basolateral membrane, we su ggest that polarization of different pH regulatory mechanisms may be i ndependently regulated in mammary epithelial cells.