Localization of ecto-ATPase in rat kidney and isolated renal cortical membrane vesicles

Abstract

Brush-border (BBMV) and basolateral membrane vesicles (BLMV) from rat renal cortex exhibit an ecto-ATPase activity that is distinct from other ATPases. We have examined the cellular and regional distribution of this enzyme in rat kidney using antibodies against rat liver ecto-ATPase. In isolated vesicles, the distribution shown by biochemical assays of ATPase activity was confirmed by immunocytochemistry and Western blotting. Indirect immunofluorescence and immunogold labeling showed that brush borders of the S1 and S3 segments of the proximal tubule (PT) were stained, but the S2 segment was negative. Staining was most intense in the S3 segment. The luminal membrane of the initial part of the thin descending limb of Henle also showed a marked staining. Surprisingly, basolateral plasma membranes of PT had no detectable staining. However, the plasma membrane of endothelial cells was heavily stained, both in larger vessels and in peritubular capillaries. Using an antibody against rat thrombomodulin, a marker for endothelial cell plasma membranes, we showed that preparations of BBMV, BLMV, and endocytic vesicles are all contaminated with these membranes. This may explain, at least partially, the biochemically measured ecto-ATPase activity in renal cortical membrane vesicles. Finally, no specific staining in the kidney was found using polyclonal antipeptide antibodies against the "long form" of liver ecto-ATPase, either by immunocytochemistry or by Western blotting. This indicates either that there is no long isoform of the ecto-ATPase in the kidney or that the intracellular domains of the long form are different in the two tissues.

Role of microwave heating in antigen retrieval in cryosections of the formalin-fixed mammalian tissues

“…Previous studies showed localization of CAM105 in the BBM of S1 and S3, but not S2 segments of the rat kidney proximal tubules, and also in the plasma membrane of peritubular capillaries (Sabolić et al, 1992a). The present study was performed on cryosection of the kidney cortex, thus showing the protein expression in proximal tubule S1 segments and peritubular capillaries ( Fig.…”

“…The commercial antibodies against the following proteins were used: Na/KATPase (monoclonal anti-peptide antibody against the α1 subunit of the human protein, Santa Cruz Biotechnology, CA, USA), metallothionein (monoclonal antibody against the polymerized MT1 and MT2 horse holoproteins; Dako North America, CA, USA), actin (monoclonal anti-peptide antibody; Millipore, MA, USA), and α-tubulin (monoclonal antibody against the see urchin filament; Sigma, MO, USA). The non-commercial antibodies against the following proteins were used: megalin (polyclonal antibody against the rat holoprotein; characterized in Abbate et al, 1994 andSabolić et al, 2002), CAM105 (polyclonal antibody against the rat holoprotein; characterized in Sabolić et al, 1992a), AQP1; polyclonal antibody against the rat holoprotein; characterized in Sabolić et al, 1992b), and V-ATPase (polyclonal anti-peptide antibody against the 31 kDa ("E") subunit;…”

“…In these experiments, cryosections of the PFA-fixed rat kidney or liver tissues underwent the above-listed unmasking protocols, followed by immunostaining of proteins known to be largely localized in the specific cell membrane domains of the nephron epithelium and hepatocytes: cell adhesion molecule CAM105, an ectoprotein expressed in the luminal (apical/brush-border (BBM)) membrane of the proximal tubules and the plasma membrane of peritubular capillaries (Sabolić et al, 1992a), megalin, a glycoprotein expressed in the BBM and subapical vesicles of the proximal tubules (Abbate et al, 1994;Kerjaschki & Farquhar, 1983;Sabolić et al, 2002), Na/K-ATPase, an integral membrane protein expressed in the contraluminal (basolateral (BLM)) cell membrane of the proximal and other tubules (Kashgarian et al, 1985;Sabolić et al, 1999) and in the hepatocyte sinusoidal membrane, and AQP1, a transmembrane protein expressed in both luminal and contraluminal membranes of the proximal tubules and thin descending limbs (Nielsen et al, 1993;Sabolić et al, 1992b).…”

Fluorescence imaging of extracellular purinergic receptor sites and putative ecto-ATPase sites on isolated cochlear hair cells

“…These membranebound enzymes with their ATP hydrolyzing site on the extracellular surface have been found on a wide range of cell types and membrane preparations, including cholinergic nerve terminals (Zimmermann et al, 1986) endothelial cells (see Gordon, 1986, for review), renal cortical membrane vesicles (Sabolic et al, 1992) hepatocytes (Lin, 1989) and smooth muscle cells (see Cusack et al, 1988, for review). In conjunction with other ectonucleotidases, ecto-ATPases are responsible for initiating the complete hydrolysis of ATP to adenosine (Zimmermann et al, 1986), but are not associated with membrane transport (Lin, 1990).…”

Identification of the human NHE-1 form of Na(+)-H+ exchanger in rabbit renal brush border membranes.

“…Ab-I detected a doublet band of an approximate molecular mass of > 90 kD in both membrane fractions. A recent report by Sabolic et al ( 18) using rat BBM and BLM indicates that both membrane preparations may be contaminated with endothelial cell membranes.…”