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 morphological distribution of pH regulatory mechanisms has been examined in a mouse mammary epithelial cell line (31EG4). Monolayers grown on permeable supports had tight junctions when grown 3-4 days in the presence of the lactogenic hormones dexamethasone (D, a synthetic glucocorticoid) and insulin (I), or in D, I, and prolactin (P), but there were no tight junctions in the absence of D. Microspectrofluorimetry of the pH-sensitive dye BCECF was used to measure pH (pHi) in cells mounted in a two-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 pHi recovery from an acid load induced by a pulse of NH3/NH4-containing solution. When monolayers were grown 3-4 d in the presence of P, D, and I, Na/H exchange was restricted to the basolateral membrane. In contrast, in the absence of P, Na/H exchange was present on both the apical and basolateral membranes. 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 morphological distribution. In all hormone conditions the antibody recognized a protein of approximately 110 kD (Western blot), and confocal immunofluorescence microscopy of this antibody and of an anti-ZO-1 (the marker of the tight junctions) antibody showed that the morphological distribution of the Na/H exchanger was similar to the functional distribution under all hormonal treatments. In addition, a putative Na/HCO3 cotransport system was monitored as a Na-dependent, amiloride-insensitive pHi recovery mechanisms that was inhibited by 200 microM H2DIDS. After treatment with D+I (but not with I alone) cotransport appeared exclusively on the basolateral membrane, and the polarized expression of this transporter was not altered by P. We conclude that when mammary cells are grown in D+I-containing media, the Na/H exchanger is expressed initially (i.e., after 3-4 d) on both the apical and basolateral membranes and later (5-6 d) on only the basolateral membrane. P (in the presence of D+I) selectively speeds this polarization, which is determined by polarized distribution of the exchanger to the apical and/or basal membrane and not by the activation and/or inactivation of transporters.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
M D
MD
Sjaastad
K S
KS
Zettl
G
G
Parry
G L
GL
Firestone
T E
TE
Machen
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D003907:Dexamethasone / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D011388:Prolactin / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers
D001639:Bicarbonates; D002352:Carrier Proteins; D007328:Insulin; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D003907:Dexamethasone; D011388:Prolactin; D012964:Sodium