title: Biochemical evidence for the presence of an amiloride binding protein in adult alveolar type II pneumocytes.
abstract: An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.
authors.affiliation: Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
authors.forename: Y
authors.initials: Y
authors.lastname: Oh
authors.forename: S
authors.initials: S
authors.lastname: Matalon
authors.forename: T R
authors.initials: TR
authors.lastname: Kleyman
authors.forename: D J
authors.initials: DJ
authors.lastname: Benos
mesh_terms: D000345:Affinity Labels; D000584:Amiloride / Q000031:analogs & derivatives / Q000737:chemistry; D006631:Amine Oxidase (Copper-Containing); D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D016676:Macrophages, Alveolar / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D015222:Sodium Channels / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D008565:Membrane Proteins; D015222:Sodium Channels; C076676:2'-methoxy-5'-nitrobenzamil; D000584:Amiloride; C064150:6-bromobenzamil; D006631:Amine Oxidase (Copper-Containing); C065976:AOC1 protein, human