CULTURE-INDUCED ALTERATIONS IN ALVEOLAR TYPE-II CELL NA+ CONDUCTANCE

Changes in Na+ transport in rat alveolar type II (ATII) cells during c ulture were quantified and related to alterations in spatial distribut ion of proteins antigenically related to amiloride-sensitive Na+ chann els. Adult rat ATII cells were cultured for periods ranging from 24 to 96 h. When patch clamped in the whole cell mode, both freshly isolate d and cultured ATII cells exhibited outwardly rectified Na+ currents. At 0 and 24 h in culture, these currents were equally inhibited by ami loride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',-4'-amiloride (inhibi tory constant approximately 1 muM). These conductive pathways were equ ally permeable to Na+ and K+. Immunocytochemical localization at 0 or 24 h in culture revealed the presence of plasma membrane antigenic sit es; after 48 h, the appearance of intracellular antigenic sites increa sed significantly. A single band of molecular mass 135 kDa in membrane proteins of freshly isolated ATII cells was recognized in Western blo ts; at 48 h in culture, two lower bands with molecular masses of 75 an d 65 kDa were detected in either membrane or cytoplasmic proteins. Pho tolabeling with 2'-methoxy-5'-nitrobenzamil showed that the 135-, 75-, and 65-kDa bands contained amiloride-binding sites. These results sug gest the presence of low amiloride affinity conductive pathways in fre shly isolated and cultured ATII cells. Culturing ATII cells resulted i n internalization and possible breakdown of these pathways and decreas ed Na+ transport.