AJP - Cell Physiology, Vol 262, Issue 5 C1228-C1238, Copyright © 1992 by American Physiological Society
Immunocytochemical and functional characterization of Na+ conductance in adult alveolar pneumocytes
S. Matalon, K. L. Kirk, J. K. Bubien, Y. Oh, P. Hu, G. Yue, R. Shoemaker, E. J. Cragoe Jr and D. J. Benos
Department of Anesthesiology, University of Alabama, Birmingham 35233.
The purpose of this study was to document the existence, assess the spatial
localization, and characterize some of the transport properties of proteins
antigenically related to epithelial Na+ channels in freshly isolated rabbit
and rat alveolar type II (ATII) cells. ATII cells, isolated by elastase
digestion of lung tissue and purified by density-gradient centrifugation,
were incubated with polyclonal antibodies raised against Na+ channel
protein purified from beef kidney papilla (NaAb), followed by a secondary
antibody (goat antirabbit immunoglobulin G conjugated to fluorescein
isothiocyanate). Rat ATII cells exhibited specific staining with NaAb at
the level of the plasma membrane, which, in most cells, colocalized with
that of the lectin Maclura pomiferra agglutinin, an apical surface marker.
In Western blots, NaAb specifically recognized a 135 +/- 10-kDa protein in
rat ATII membrane vesicles. When patch clamped in the whole cell mode using
symmetrical solutions (150 mM Na+ glutamate), ATII cells exhibited
outwardly rectified Na+ currents that were diminished by amiloride (10-100
microM) instilled into the bath solution. Ion substitution studies showed
that the conductive pathways were three times more permeable to Na+ than
K+. Amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride were
equally effective in diminishing 22Na+ flux into rabbit and rat ATII cells
(45% inhibition at 100 microM, with IC50 of approximately 1 microM for all
inhibitors). Tetraethylammonium chloride (10 mM) or BaCl2 (2 mM),
well-known K+ channel blockers, had no effect on 22Na+ uptake. These
results indicate that ATII cells express an amiloride-sensitive Na+
conductance, probably a channel, with a lower affinity for amiloride and
its structural analogues than the well-established amiloride-sensitive Na+
channels found in bovine renal papila and cultured amphibian A6 kidney
cells.
