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

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

SABOLIC,

OGNJEN

CULIC,

SUE-HWA

LIN,

AND

DENNIS

BROWN

Renal Unit, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129; and Department of Molecular Pathology, University of Texas, The M. D. Anderson Cancer Center, Houston, Texas 77030 Saboli6, Ivan, Ognjen &lie, Sue-Hwa Lin, and Dennis Brown. Localization of ecto-ATPase in rat kidney and isolated renal cortical membrane vesicles.Am. J. Physiol. 262 (Rend Fluid Electrolyte Physiol. 31): F217-F228,1992.-Brush-border (BBMV) and basolateralmembranevesicles(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 usingantibodies against rat liver ecto-ATPase. In isolated vesicles,the distribution shown by biochemical assaysof ATPase activity was confirmed by immunocytochemistry and Western blotting. Indirect immunofluorescenceand immunogold labeling showed that brush borders of the Sl and S3 segmentsof 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 descendinglimb of Henle alsoshoweda marked staining. Surprisingly, basolateral plasmamembranesof PT had no detectablestaining. However, the plasma membraneof endothelial cells was heavily stained, both in larger vesselsand in peritubular capillaries. Using an antibody against rat thrombomodulin, a marker for endothelial cell plasmamembranes,we showedthat preparations of BBMV, BLMV, and endocytic vesiclesare all contaminated with these membranes.This may explain, at least partially, the biochemically measuredecto-ATPase activity in renal cortical membrane vesicles.Finally, no specific staining in the kidney was found usingpolyclonal antipeptide antibodiesagainst 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 domainsof the long form are different in the two tissues. immunofluorescence;immunogold; endothelial cells; proximal tubule; anti-ecto-adenosinetriphosphataseantibodies

(ATPase) is a plasma membrane enzyme with a nucleotide-splitting site localized on the outside of the cell surface (8, 17, 25, 27, 28, 38). The enzyme is 1) able to hydrolyze nonspecifically different nucleotides, 2) equally active with Ca2+ and Mg+, 3) poorly sensitive to many common inhibitors of various intracellular ATPases, and 4) resistant to proteolysis. An enzyme with similar characteristics, i.e., the Ca2+-MgQtimulated nucleotidase, has been found in plasma membranes from liver, kidney, pancreas, vascular endothelium, rat mammary gland, neuroblastoma, glial cells, and intestine, as well as in polymorphonuclear leukocytes and granulocytes, blood platelets, and several cultured cell lines (6, 8, 12, 17, 19, 24, 25, 27-29, 32, 38, 39). The wide-spread occurrence of an ecto-ATPase in various cells indicates that this enzyme may serve important functions, but its possible cellular role has not been clarified. Its putative identity with a Ca2+-transporting ATPase has recently been disproven (28). The only ecto-ATPase enzyme so far isolated, cloned, and expressed in cultured mammalian cells is that from

ECTO-ADENOSINETRIPHOSPHATASE

the liver plasma membrane (25, 27). The amino acid sequence of the liver enzyme was deduced from an analysis of cDNA clones and a genomic clone and was predicted to be a 519-amino acid protein with a calculated molecular mass of 57,388 Da. The enzyme is highly glycosylated (16 potential asparagine-linked glycosylation sites) and has a relative mobility in sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) of -100 kDa (25,27). The cytoplasmic, COOHterminal domain (71 amino acids) has two phosphorylation consensus sequences, for adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase and for tyrosine kinase, whereas at the external, NH,-terminal domain there are two consensus sequences for nucleotide-binding domains (27). Indirect immunofluorescence studies with antibodies against purified ectoATPase holoprotein localized the enzyme mainly to the canalicular pole of hepatocyte plasma membranes. However, recent studies with antibodies against the holoenzyme and with antipeptide antibodies against specific intracellular COOH-terminal domains revealed the existence of long and short ecto-ATPase isoforms in the hepatocyte plasma membrane, with the latter shorter for 61 amino acids at the COOH-terminal domain (26; unpublished observations). Studies on isolated rabbit kidney tubules localized the activity of a Ca2+- Mg+-ATPase along the entire nephron, with the highest activities measured in proximal and collecting tubules (21). An ATPase with similar characteristics has been measured in both brush-border and basolateral membranes from various mammalian kidneys (6,22,39), including the rat (8,38). In some publications (21, 22), it has been suggested that the enzyme may function in transmembrane Ca2+ transport. Recently, we have measured an ATPase activity with nucleotide specificity that resembles that of an ectoATPase in preparations of both brush-border (BBMV) and basolateral membrane vesicles (BLMV) isolated from rat renal cortex (38). However, because of the highly polarized nature of proximal tubule epithelial cells, the presence of similar activities of the same enzyme in both kinds of plasma membranes is an unusual and unexpected finding. Therefore, to resolve this and previous contradictory observations, we used specific anti-ectoATPase antibodies to study by immunocytochemical techniques the localization of this enzyme in frozen sections of the rat kidney and in membrane vesicles isolated from kidney cortex. MATERIALS AND METHODS Antibodies. Polyclonal antibodies hss were generated by injecting rabbits with SDS-PAGE-purified liver ecto-ATPase

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Fig. 1. Western blot of ecto-ATPase in membrane vesicles isolated from rat renal cortex and in tissue homogenates of rat kidney zones. Blotting was performed with polyclonal antibody against liver ectoATPase holoprotein (A,&. BBMV, brush-border membrane vesicles; BLMV, basolateral membrane vesicles; EV, endocytic vesicles; OS, outer stripe; and IS, inner stripe. holoprotein (25). Polyclonal antipeptide antibodies against COOH-terminal portions of liver ecto-ATPase were prepared by injecting rabbits with peptides coupled to keyhole limpet hemocyanin (25); anti-C1 (peptide sequence, CDLTEHKPSTSSHNLGP; amino acid positions 460-475), anti-& (peptide sequence, CDSPNKVDDVSYSVLNFNAQ; amino acid positions 478-496), and anti-C3 (peptide sequence, CQSKRPTSASSSPTETVYSVVKKK; amino acid positions 497-519) antibodies were prepared against peptide sequences within the cytoplasmic, COOH-terminal portion of the liver ecto-ATPase. Polyclonal antibody (rabbit serum) against rat thrombomodulin holoprotein was a gift from Dr. D. M. Stern, Columbia University, New York, NY. Preparation of kidney homogenates and isolation of membrane vesicles. All experiments were performed on Sprague-Dawley

rats (200-250 g body wt). The kidney zones (the cortex, outer and inner stripesof the outer medulla, and inner medulla) were dissectedmanually. BBMV were isolated by the Mg-EGTAaggregation method of Biber et al. (2). Cortical BLMV and endocytic vesicleswere isolated by the differential and Percoll density gradient centrifugation method of Scalera at al. (36) and Sabolic and Burckhardt (34), respectively. Comparedwith the homogenate,BBMV were enriched in a luminal membrane marker enzyme activity, leucine aminopeptidase (LAP) (EC 3.4.11.1), 16.8 f 0.27 times, and in the activity of Na+-K+ATPase (EC 3.6.1.3), a marker for contraluminal membrane, 1.5 f 0.29 times (n = 3). BLMV were enriched in LAP and Na+-K+-ATPase activities 1.2 + 0.27 and 14.6 + 1.41 times (n = 3), respectively. Endocytic vesicles were enriched in LAP activity 2.7 + 0.1 times and in N-ethylmaleimide (NEM)sensitiveATPase activity (proton-pumping ATPase) 35.3 f 0.6 times (n = 4). LAP activity was measuredcalorimetrically at ambient temperature with L-leucine-4-nitroanilide as a substrate under the conditions describedin the commercialkit (kit no. 5364;Merck, Darmstadt, FRG). Ouabain-sensitiveNa+-K+ATPase activity was followed spectrophotometrically at am-

bient temperature with the coupled ATP-regenerating system of Penefsky and Bruist (33). The isolatedvesicleswere washed two times with KC1 buffer containing (in mM) 300 mannitoi, 100 KCl, 5 MgS04, and 5 N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid (HEPES)/tris(hydroxymethyl)aminomethane (Tris), pH 7.4, diluted with the same buffer to a protein concentration of 10mg/ml, and either usedimmediately in the experiments or kept in liquid nitrogen until further use. Total plasmamembranesfrom lung were obtained from the pellet following differential centrifugation of lung homogenate between 2,500 and 48,000g. The homogenization step in this and preceding isolations was performed in the presenceof 1 mM phenylmethylsulfonyl fluoride. Pi liberation assay of ATPase actiuity. The ecto-ATPase activity in preparations of BBMV and BLMV was measured by modifying previously describedP, liberation assays(8,9,35, 38). Vesicleswere diluted with ice-cold KC1 buffer to a protein concentration of 0.5 mg/ml. The reaction mixture in a total volume of 0.2 ml contained (in mM) 150 KCI, 5 MgSO1, 50 HEPES-Tris, pH 7.4, 1 levamisole, 2 ouabain, 0.5 vanadate, and 1 NEM, as well as 5 pg/ml oligomycin and 10 pg vesicle protein. The inhibitors were present to abolish the activity of alkaline phosphatase, Na+-K+-ATPase, mitochondrial H+ATPase, Ca’+-ATPase, and the vacuolar-type H+-ATPase, respectively (8, 35, 38). After preincubation of the mixture at 37°C for 10 min, the reaction was started by adding a stock solution of Tris-ATP (final concentration, 5 mM). The reaction wasterminated after 15 min by addition of 1 ml of an ice-cold stop solution (2.9% ascorbicacid, 0.45% ammoniummolybdate, and 2.86% SDS in 0.48 N HCl). Further processingof the samplesand calorimetric measurementof liberated P, were performed asdescribedby Dean at al. (9). The NEM-sensitive ATPase (H+-ATPase) in endocytic vesicleswas measuredby the samemethod except the amount of vesicle protein in the assaywas20 pg. The NEM-sensitive ATPase wasthe difference in ATPase activity in the absenceand presenceof 1 mM NEM. Western blotting. Aliquots of homogenatesand isolatedmembranes were solubilized by boiling for 5 min in samplebuffer [1% SDS, 30 mM Tris .HCl, pH 6.8, 5% 2+mercaptoethanol, and 12% (vol/vol) glycerol]. Proteins (100 and 150 pg/lane in thrombomodulin and ecto-ATPase studies, respectively) were separated by electrophoresis through 10% Laemmli SDSPAGE, and transferred to Immobilon membrane (Millipore, Bedford, MA). The membraneswere briefly stained with Coomassieblue to check the efficiency of the transfer. Destained membranes were blocked in blotting buffer containing 5% nonfat dry milk, 0.15 M NaCl, 1% Triton X-100, and 20 mM Tris. HCl, pH 7.4, followed by incubation with anti-ecto-ATPaseor anti-thrombomodulin antibodies diluted l:l,OOO in blotting buffer at room temperature overnight. The membranes were then washedby several changesof blotting buffer, incubated for 60 min with l:l,OOOdilution in blotting buffer of goat anti-rabbit IgG conjugated to alkaline phosphatase (Vector Laboratories, Burlingame, CA), washedagain, and stained for alkaline phosphataseactivity with the BCIP/NBT phosphate substrate system (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Tissue preparation and immunoflwrescence microscopy. Rats were anesthetizedwith Inactin (Byk-Gulden Pharmaceuticals). The kidneys and liver were perfused via the abdominal aorta, first with Hanks’ balanced salt solution for l-2 min and then with a fixative containing 2% paraformaldehyde, 10 mM sodium periodate, and 75 mM lysine (29) for 10 min. Organswere removed, sliced,and kept overnight in the samefixative at 4°C followed by washing (3 times) with phosphate-buffered saline (PBS). The tissue slices were then kept in PBS containing 0.02% sodiumazide at 4°C until further use. To prepare 3-pm frozen sectionsfor indirect immunofluorescence study, tissue fragments were equilibrated in PBS con-


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Fig. 2. Localization of ecto-ATPase on ultrathin frozen sections of membrane vesicles with protein A-gold technique. Sections were incubated with anti-ectoATPase antibodies (A,& or preimmune serum, followed by protein A-gold. Some of gold particles, representing location of antigenic sites, are indicated with small arrows. When incubated with preimmune serum. BBMV (A) and BLMV (C) show no specific labeling. When specific anti-ecto-ATPase antibody was used, gold particles were located on external side of membranes of many of vesicles in BBMV and BLMV preparations (B and D, respectively). Bar = 0.25 pm.

30% sucroseat room temperature for 60 min, embedded in OCT medium (Miles, Elkhart, IN), frozen in liquid nitrogen, and sectionedusing a Reichert Frigocut cryostat. Sectionswere placed on gelatin-coated glassslidesand kept in PBS for 10 min. To study the ecto-ATPase, the liver and kidney sections were incubated at room temperature for 90 min with sera (diluted 1:200 with PBS) raised against holoprotzein (A,& or COOH-terminal portions of liver ecto-ATPase (anti-&, antiCz, and anti-C,), followed by washing three times for 5 min in PBS containing 2.7% NaCl. The washingwith a high-salt buffer decreasednonspecific binding of antibodies. The sectionswere then incubated for 60 min with fluoresceinatedgoat anti-rabbit

taining

immunoglobulin antibody (8.3 pg/ml in PBS) (Calbiochem), followed by washing three times for 5 min in PBS containing 2.7% NaCl. Sections were finally mounted in 60% glycerol in 0.2 M Tris.HCl, pH 8.0, containing 2% n-propyl gallate to retard quenching of the fluorescence signal. Sections were examined and photographed with a Nikon FKA photomicroscope (Donsanto, Natick, MA) equipped for epifluorescence and with Kodak T-Max film. Control sectionswere incubated with preimmune seruminstead of immune serum. To study the distribution of thrombomodulin in the kidney, tissue sections were incubated with anti-thrombomodulin serum (diluted 1:lOO with PBS) at room temperature for 60


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Fig. 3. Localization by indirect immunofluorescence of ecto-ATPase by &9 (A), anti-C1 (B), anti-C2 (C), and anti-C3 (D) antipeptide antibodies in cryostat sections of rat liver. b9 heavily labels plasma membrane in bile canaliculi and weakly labels sinusoidal plasma membranes. Antipeptide antibodies label only plasma membrane in bile canaliculi. min, followed by washingin PBS, incubation with fluorescein-

ated goat anti-rabbit immunoglobulin antibody for 60 min, and washing. The sections were further processedas described above for ecto-ATPase study. Electron microscopyof isolatedvesiclesand tissueslices.The preparation and negative staining with uranyl acetate of ultrathin frozen sectionsof isolated vesiclesand kidney tissue were performed according to the method of Tokuyasu (37). Briefly, vesicleswere fixed with 0.5% glutaraldehyde, washedwith PBS, and embeddedin 3% agar. The kidneys were fixed asdescribed above and cut into small blocks. The small solid agar blocks with vesiclesand the pieces of kidney tissue were infiltrated with 2.3 M sucroseovernight. The blocks were frozen in liquid nitrogen, and ultrathin sections(60 nm) were cut on a Reichert FC4D ultracryomicrotome and mounted on carbon/Parlodioncoated copper grids. The specimen-containing grids were first washedwith PBS containing 20 mM glycine three times for 5 min, incubated with the seraagainstecto-ATPase (diluted with PBS, 1:lOO)for 30 min, and washedwith PBS containing 0.1% bovine serumalbumin (BSA) four times for 3 min. Thereafter, the grids were incubated with protein A-gold solution (diluted 1:lOO with PBS containing 1% BSA) for 30 min, followed by washingin PBS containing 1% BSA (once for 5 min) and PBS alone (3 times for 5 min). The sampleson the grids werefurther fixed with 1% glutaraldehyde (in PBS) for 5 min, washedwith water twice for 5 min, and stained with 2% uranyl acetate (in water) for 5 min. Thereafter, the grids were destainedwith and embeddedin 2% methyl cellulose,dried, and viewed on a Philips CM10 electron microscope. Control sections were incubated with preimmune seruminstead of immune serum.

In accord with previous findings (38), in isolated rat renal cortical BBMV and BLMV we found a significant activity of an ATPase that is insensitive to levamisole, ouabain, oligomycin, vanadate, and NEM. The activity of this ATPase in BBMV and BLMV was 655 f 11.7 (n = 11) and 585 -I 45.6 nmol Pi emin-’ - mg protein-’ (n = 5), respectively. Because isolated BBMV are impermeant to ATP and are largely oriented with right side out (3, 14) and because in our previous publications we showed that this ATPase in both kinds of membranes exhibits a broad (non)specificity for trinucleotides similar to that of ecto-ATPases in other mammalian cells and tissues, we assumed that this ATPase belongs to the ecto-ATPase family (8, 38). To confirm this, equal amounts of BBMV and BLMV proteins were subjected to SDS-PAGE, transferred onto Immobilon, and immunoblotted using antiserum against liver ecto-ATPase holoprotein (As,&. In liver plasma membranes, this serum labels a protein with a relative molecular mass (M,) of -100 kDa (25). As shown in Fig. 1, with A669 a broad protein band of similar M, is stained in both BBMV and BLMV, with the intensity of staining stronger in BBMV. When the serum was preincubated with purified ecto-ATPase, no specific staining was seen on Western blots (data not shown). Furthermore, with sera against three different peptide sequences of the cytoplasmic COOH-terminal portion of liver ecto-ATPase (anti-C, anti-&, and anti-&), i.e., sera that had been previously shown to label liver ecto-ATPases in PAGE analysis (26, 27), on Western blots of the kidney membranes no protein bands were stained (not shown). To further localize ecto-ATPase in BBMV and BLMV, we prepared ultrathin frozen sections of the vesicles and incubated them with preimmune or immune serum against ecto-ATPase, followed by protein A-gold and staining with uranyl acetate. As shown in Fig. 2 (A-D), negatively stained vesicles are sharply delineated by a membrane bilayer. In both BBMV and BLMV (Fig. 2, B and D, respectively), the intensity of gold particle labeling seen with the specific antibody was much greater than the low background level seen with preimmune serum (Fig. 2, A and C). Where present, gold particles were localized at the external domain of the vesicle membrane (Fig. 2, arrows). However, in preparations of both kinds of vesicles, more than 50% of the vesicles remained unlabeled. Further localization of ecto-ATPase in renal membranes was investigated by indirect immunofluorescence and protein A-gold labeling in cryostat sections of various kidney regions. However, to prove a positive action of anti-ecto-ATPase antibodies in our hands, we first performed indirect immunofluorescence staining of 3-pm sections of the liver tissue. In accord with previous findings (25), antibody A669 labeled strongly bile canalicular membrane, whereas the sinusoidal membrane was faintly stained (Fig. 3A). Sera anti-&, anti-&, and anti-& labeled only bile canalicular membrane (Fig. 3, B, C, and D, respectively), thus indicating a selective distribution of ecto-ATPase isoforms in different plasma membrane domains of the liver cell. These studies were not further elaborated.


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Fig. 4. A: localization by indirect immunofluorescence of ecto-ATPase by A669in a cryostat section of rat kidney cortex. Proximal tubules show heterogeneous labeling of brush border. There is heavy labeling of brush border in Sl segment, identified by its continuity with glomerulus (G), whereas basolateral membranes of this segment show no detectable staining (thin arrows). Other proximal tubule profiles show either an intermediate level of staining or no staining of brush border. Endothelial cells of glomerular capillaries are weakly stained (G), whereas those of peritubular capillaries are heavily labeled (thick arrows). Cytoplasmic fluorescence seen in proximal tubules represents lysosomal autofluorescence and was also found in control sections. Bar = 40 pm. B: ultrathin frozen sections of brush border from Sl segment of proximal tubule labeled with anti-ecto-ATPase antibodies, followed by protein A-gold. Gold particles are distributed on extracellular side of microvillar membrane (arrows). C: with preimmune serum only a low level of background labeling was detectable. Bar = 0.5 pm.

Before performing experiments on frozen kidney sections, we screened homogenates prepared from manually dissected kidney zones for the presence of ecto-ATPase in Western blots. As shown in Fig. 1, a protein band of M, of -100 kDa is labeled with anti-ecto-ATPase antibodies (As& in all four kidney zones. The intensity of the staining is highest in the cortex and outer stripe of outer medulla, followed by inner stripe of outer medulla. In the homogenate from inner medulla, staining was also present, but it was weak. As the amount of protein on the blots was the same in all lanes, the staining intensity corresponds to relative abundance of ecto-ATPase in the various kidney regions. In similar experiments (data not

shown), no staining with antisera (anti-&, anti-C&, and anti-C&) against cytoplasmic COOH-terminal portions of the long form of liver ecto-ATPase was detectable in any region. Indirect immunofluorescence labeling with Ass9 in cryostat sections of the superficial rat kidney cortex is shown in Fig. 4A. As identified by its continuity with the glomerulus, the brush border in the Sl segment of the proximal tubule was strongly stained. The brush border in other proximal tubule profiles was stained weakly or not at all. Basolateral membranes in all proximal tubules were unstained. The endothelial cell plasma membrane in glomerular capillaries was stained weakly, whereas


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Fig. 5. A: localization by indirect immunofluorescence of ecto-ATPase by bs in tubules in a cryostat section of an intermediate part of rat kidney cortex. In proximal tubule (PT), neither brushborder nor basolateral membranes (thin arrows) are labeled. Cortical distal tubule and collecting duct are also unstained (asterisks). Endothelial cells of peritubular capillaries are heavily labeled (arrowheads). Bar = 20 pm. B: ultrathin frozen sections of brush border from S2 segment of proximal tubule labeled with anti-ecto-ATPase antibodies, followed by protein A-gold. Gold particles are rare, and labeling is not different from that with preimmune serum (not shown). Bar = 0.5 pm.

that in peritubular capillaries was heavily stained. Labeling by anti-ecto-ATPase antibodies followed by protein A-gold showed numerous gold particles localized at the external domain of the microvillar plasma membrane in brush borders of the Sl segment (Fig. 4B). Only a low background labeling was observed in cryosections incubated with preimmune serum and protein A-gold (Fig. 4C). Many proximal tubule segments, probably S2 regions, showed no indirect immunofluorescence staining by AGG9, neither of brush-border nor basolateral membranes (Fig. 5A). Distal tubules and cortical collecting ducts were also unstained. However, the plasma membrane of endothelial cells in peritubular capillaries was strongly stained. As shown in Fig. 5B, the labeling of the brush border in

S2 segment epithelial cells by anti-ecto-ATPase antibodies followed by protein A-gold was poor and was not different from the labeling observed in sections incubated with preimmune serum (not shown). Cryostat sections of the outer stripe of the outer medulla showed a very strong labeling by anti-ecto-ATPase antibodies of the brush border in S3 segments of the proximal tubule and a relatively weaker labeling of endothelial cells in peritubular capillaries (Fig. 6A). The basolateral membranes of the epithelial cells were unstained. Labeling with anti-ecto-ATPase antibodies and protein A-gold in ultrathin frozen sections of brush border in S3 segments of the proximal tubule is shown in Fig. 6B. The labeling with gold particles at the external domains of the microvillar membrane was more extensive


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Fig. 6. A: localization by indirect immunofluorescence of ecto-ATPase by h9 in S3 segments of proximal tubule. Brush border is heavily stained by antiecto-ATPase antibodies, whereas basolateral membrane is unstained. Plasma membrane of endothelial cells in peritubular capillaries is stained, but less than in superficial cortex (compare with Fig. 4-4). Bar = 40 am. B: ultrathin frozen sections of brush border from S3 segment of proximal tubule labeled with anti-ecto-ATPase antibodies, followed by protein A-gold. Gold particles label extracellular side of microvillar membrane (arrows). Number of gold particles is much greater than in Sl segment (compare Fig. 4B). C: level of background labeling with preimmune serum is low. Bar = 0.5 pm.

than in the Sl segment (compare to Fig. 4B). No significant labeling was found in sections incubated with preimmune serum (Fig. 6C). In addition to endothelial cells of peritubular capillaries, the endothelium in larger renal vessels was also strongly stained with anti-ecto-ATPase antibodies, as shown by indirect immunofluorescence of an arteriole from rat renal cortex (Fig. 7). The preceding indirect immunofluorescence experiments indicate that the basolateral membranes of proximal tubule epithelial cells are not labeled with our antibodies. This result was confirmed by examining the basolateral side of proximal tubule cells labeled with

protein A-gold. As shown in Fig. 8A, no gold particles were found on proximal tubule basolateral membranes. On the contrary, the plasma membrane of endothelial cells was heavily labeled. Sections incubated with nonimmune serum exhibited only a low background labeling (Fig. 8B). By the same technique, the staining of basolateral membrane in other tubules could also not be observed (not shown). In cryostat sections of the border between the outer and inner stripe of the kidney medulla, we found a bright immunofluorescence staining of brush borders in terminal S3 segments of the proximal tubule and a sharp staining of the luminal membrane of the initial part of


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Fig. 7. Localization by indirect immunofluorescence of ecto-ATPase by A669in arteriole in cryostat section of rat renal cortex. Endothelium of renal arteriole (A), as well as endothelium of peritubular capillaries, is strongly labeled. Labeled and nonlabeled proximal tubules are also indicated (PT). Bar = 20 pm.

the descending thin limb (DTL); the transition between these two tubules is indicated by an arrow in Fig. 9A. More distal segments of the DTL were not stained. Immunogold labeling of ultrathin frozen sections at the transition between proximal tubule and DTL showed mmerous gold particles at the external surface of the microvillar plasma membrane in the terminal cell of the proximal tubule S3 segment and at the external surface of the apical plasma membrane of the initial DTL cell (Fig. 9C). As shown in Fig. 9B, bundles of endothelial cells that belong to vasa recta and which spread deeply into papilla were stained. The identity of these cells as vasa recta endothelium was proven 1) by electron microscopic immunocytochemistry following incubation of sections with A669and secondary antibody conjugated with horseradish peroxidase and 2) by the extensive labeling of these structures with antibodies against thrombomodulin, an endothelial cell plasma membrane marker (see below, data not shown). With the use of polyclonal sera against peptides derived from the COOH-terminal portion of the long liver ecto-ATPase form (anti-C, anti-&, and anti-C!& no specific staining of renal epithelial and endothelial cells was detectable (data not shown). By both indirect immunofluorescence and protein Agold immunocytochemistry, a significant labeling of intracellular membrane vesicles in the proximal tubule was not apparent, indicating that either the ecto-ATPase in renal cortical endosomes was not present or the methods were not sensitive enough to detect the enzyme in these vesicles. We therefore isolated endocytic vesicles from rat renal cortex as previously described (34). In the absence of NEM, the isolated endosomes exhibited an ATPase activity of 147 rt 8.2 nmol Pi - min-’ . mg protein-l (n = 6), which dropped to 84 f 5.1 nmol Pi-mine’. mg protein- ’ in the presence of 1 ‘mM NEM. As demonstrated previously by biochemical assay in isolated endosomes (35) and by immunolocalization of the H+ pump

Fig. 8. A: ultrathin frozen section of basolateral side of proximal tubule epithelium labeled with anti-ecto-ATPase antibodies (A&, followed by protein A-gold. No gold particles are found on basolateral membrane (arrowheads). However, endothelial cell of peritubular capillary is heavily labeled (arrows). B: only a low background labeling is found in sections incubated with preimmune serum. Bar = 0.5 pm.

in proximal tubule cells (4), the NEM-sensitive portion of the ATPase activity reflects the H+-ATPase present in these vesicles. The NEM-insensitive ATPase in endosomal preparations probably represents ecto-ATPase activity. Indeed, Western blots of endosomal membrane proteins revealed the presence of ecto-ATPase in endosomal vesicles, although it was less abundant than in the BBMV (Fig. 1). The preceding immunocytochemical observations indicate that in the kidney cortex an ecto-ATPase is detectable in endothelial cell plasma membranes and in luminal membranes of some proximal tubule segments, but not in contraluminal and endosomal membranes. In all preparations of isolated cortical membrane vesicles, however, a significant ecto-ATPase activity is readily measurable. Thus the ecto-ATPase activity in isolated BBMV may reflect the intrinsic property of these membranes but may also be partially due to contamination by endothelial cell plasma membranes. On the contrary, the ecto-ATPase activity in isolated BLMV may be due either to contaminating endothelial cell plasma membranes or proximal tubule brush-border membranes, or both. To estimate the contamination of membrane vesi-


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Fig. 9. A and B: localization by indirect immunofluorescence of ecto-ATPase by &g in cryostat section of rat kidney outer medulla. Brush border of S3 segment of proximal tubules is heavily stained (A and B). Luminal membrane of initial portion of thin descending limb (A, arrowhead), and endothelial cells of vasa recta (B, arrows) are also labeled. Bar = 20 pm (A) and 40 brn (B). C: ultrathin frozen section of transition between S3 segment of proximal tubule and descending thin limb of Henle (DTL) labeled with anti-ecto-ATPase antibodies, followed by protein A-gold. Gold particles are localized at external side of plasma membrane microvilli in proximal tubule S3 segment and luminal membrane of a DTL cell (DTL, arrows). Bar = 0.5 pm.

cles isolated from kidney cortex by endothelial cell plasma membranes, we studied by Western blot the presence of thrombomodulin in vesicle preparations. Thrombomodulin is a glycoprotein with an apparent molecular mass of 75 and ,105 kDa when analyzed on SDS-PAGE in nonreduced and reduced conditions, respectively (11). Distribution studies in rabbit tissues have indicated that thrombomodulin is a specific marker of endothelial cell plasma membranes (10). Indeed, using polyclonal antibodies (rabbit serum) against rat thrombomodulin, we performed indirect immunofluorescence studies in frozen sections of rat kidney cortex (Fig. 10) and found extensive staining of endothelial cell plasma membranes in glomeruli, arteries, and peritubular capillaries. Plasma membranes of kidney tubules, including the brush-border and basolateral membranes of the prox-

imal tubule, were not stained. A similar staining pattern, i.e., staining only of endothelial cell plasma membranes, was also observed in other kidney regions (not shown). On Western blots of plasma membranes from lung, the tissue with a high content of thrombomodulin (lo), two major bands of 75 and 105 kDa (representative for nonreduced and reduced thrombomodulin forms, respectively) were detected (Fig. 11). Similar bands were stained in total kidney cortex homogenate and in isolated renal cortical BBMV, BLMV, and endocytic vesicles. In all fractions from the kidney the 75-kDa band was stronger than 105-kDa band, indicating an incomplete reduction of thrombomodulin under our experimental conditions. The intensity of 75-kDa band was strong in brush-border membranes, intermediate in endocytic vesicles, and the weakest in basolateral membranes. An


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Fig. 10. Localization by immunofluorescence of thrombomodulin by anti-thrombomodulin antibodies in frozen section of rat kidney cortex. Plasma membranes of endothelial cells in glomeruli (G), renal artery (arrow), and peritubular capillaries are strongly stained. Brush-border membranes in proximal tubule cells (asterisks), basolateral membranes in same cells (arrowhead), as well as plasma membranes in other tubules, are not stained. Bar = 40 nm.

4105 -75

29*

18, Fig. 11. Western blot of thrombomodulin in rat lung plasma membranes, kidney cortex homogenate (KCH), and membrane vesicles isolated from rat renal cortex. Bands with M, of 75 and 105 kDa correspond to nonreduced and reduced forms of thrombomodulin, respectively.

additional 68-kDa band, stained weakly in lung plasma membranes and kidney cortex homogenate and strongly in brush-border membranes, may be a degradation product of the 75-kDa form or may represent a soluble short form of thrombomodulin found in the plasma and urine (20) that may bind to isolated membranes during their preparation.

By using a biochemical assay and immunocytochemical labeling with specific antibodies, we examined the localization of ecto-ATPase in rat renal tissue and membrane vesicles isolated from rat kidney cortex. In accord with previous biochemical findings (6,8, 19, 38, 39), the presence of ecto-ATPase in preparations of renal cortical BBMV and BLMV was confirmed by 1) measuring enzyme activity by the Pi liberation assay, 2) Western immunoblotting of membrane proteins with anti-ecto-ATPase antibodies, and 3) labeling of ultrathin frozen sections of membrane vesicles by anti-ecto-ATPase antibodies followed by protein A-gold. Whereas the indirect immunofluorescence and protein A-gold labeling studies on frozen kidney sections clearly confirmed the presence of ecto-ATPase in the brush border of many but not all proximal tubules, staining of basolateral membranes was not detectable. On the contrary, in all kidney zones we observed a strong staining of the plasma membrane of endothelial cells in peritubular capillaries and larger vessels. We therefore conclude that the presence of ecto-ATPase in preparations of BLMV may be an artifact due to a significant contamination by membranes that contain intrinsic ecto-ATPase activity. Possible candidates for these contaminating membranes could be either proximal tubule brush-border membranes and/or plasma membranes from endothelial cells of peritubular blood vessels and capillaries. The enrichment factor of brush border enzyme LAP in preparations of BLMV is -1.2, i.e., 14 times smaller than in BBMV preparations. Hence, if the ecto-ATPase activity in BLMV preparations originated solely from contaminating proximal tubule luminal membranes, then the ecto-ATPase activity in preparations of BLMV and BBMV should differ by a factor of 14. The measured activity of ecto-ATPase in BLMV, however, was only 10% smaller than in BBMV, thus indicating that the preparations of BLMV may be contaminated by different types of plasma membranes (notably from endothelial cells). However, Western blots with anti-thrombomodulin antibodies indicated only a modest contamination of basolateral membrane preparations with thrombomodulin, a marker for endothelial cell plasma membrane, that cannot totally account for the relatively high ecto-ATPase activity in contraluminal vesicle preparations. Thus we conclude that, whereas a part of the ecto-ATPase activity in BLMV is due to immunologically related ecto-ATPases from contaminating proximal tubule brush-border membranes and endothelial cell plasma membranes, some of the measured enzyme activity may result from an immunologically distinct ecto-ATPase. This enzyme may share a similar sensitivity to inhibitors and specificity to nucleotides and could derive from either the basolateral membrane itself or from some other as yet unidentified contaminating membranes. Indeed, recent data indicate that kinetically different forms of ecto-ATPases may exist in preparations of BBMV (8). By Western blotting and Pi liberation, ecto-ATPase activity was also found in preparations of renal cortical endocytic vesicles. The enrichment factor for LAP of 2.7 indicates that these vesicles are also contaminated by proximal tubule luminal membranes. This enrichment


LOCALIZATION

OF

ECTO-ATPASE

factor is about six times smaller than the enrichment of Similarly, the activity of NEM-insensitive ATPase in preparations of isolated endocytic vesicles is comparatively smaller (~7.5 times) than in BBMV. Because the endocytic vesicles are oriented with the cytoplasmic side out and because the ATP-splitting site of the intrinsic ecto-ATPase in the vesicle membrane is most probably inaccessible to ATP, measured ecto-ATPase activity in endosomal preparations may be partly explained by the presence of contaminating right-side-out and opened BBMV. However, Western blots of endosomal membranes with antithrombomodulin antibodies showed that these vesicles are also contaminated with endothelial cell plasma membranes that may contribute to the total ecto-ATPase activity in these vesicle preparations. The indirect immunofluorescence and protein A-gold labeling studies clearly showed the presence of ectoATPase in brush borders of Sl and more abundantly in S3 segments, whereas staining in S2 segments of the proximal tubule was absent. This explains the limited labeling by protein A-gold in some, but not all, vesicles in preparations of isolated BBMV; vesicles isolated from kidney cortex originate from both ecto-ATPase-positive Sl and . ecto-ATPase -negative S2 proximal tubule segments. The staining was present also in luminal membranes of the initial part of the DTL, whereas other parts of the renal tubule remained unstained. However, Western blots of isolated BBMV with anti-thrombomodulin antibodies clearly showed that these preparations are strongly contaminated with endothelial cell plasma membranes, which may be responsible for at least a part of the immunologically related ecto-ATPase activity. We cannot exclude the possibility that endothelial cell plasma membranes also contain LAP, a brush-border membrane marker, and thus copurify with membrane vesicles from proximal tubule cells. This assumption is supported by previous histochemical demonstration of LAP in cultivated aortic endothelial cells (18). The function of ecto-ATPase in these cells is unknown. The ecto-ATPase in vascular endothelial and other nonpolar cells may serve to degrade ATP and other nucleotides released into the blood by exocytosis from the same cells, platelets, neurons, and cells from adrenal medulla (17). The released nucleotides, primarily ATP and ADP, influence many biological processes including platelet aggregation, vascular tone, neurotransmission, cardiac function, and muscle contraction (17). These actions may be mediated by an increase in permeability for cations (5,13) and Na+-H+ exchanger activity (23) of the plasma membrane, depolarization of the cell membrane (5), and by the increased release of Ca2+ from intracellular stores (16). Some of these effects may be mediated by P2purinergic receptors present at the plasma membrane, but others are nonspecific. Therefore the function of ecto-nucleotidases may be to terminate the effects of adenine nucleotides on cells or to regulate these effects via a phosphatase activity (28). Moreover, based on some similarities in nucleotide specificity of ecto-ATPases and P2-purinergic receptors in some cells, some authors have suggested that the ecto-ATPase itself may be the P2purinergic receptor (28). There is no direct evidence for any e&o -ATPase func-

LAP activity in isolated BBMV.

IN

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KIDNEY

F227

tion in the kidney brush border. Recently, we demonstrated that rat ren .a1 cortical BBMV possess a cascade of ectonucleotidases, including the ecto-ATPase and ecto-%nucleotidase, capable of hydrolyzing various adenine nucleotides to adenosine and Pi (8). The substrates for ectonucleotidases may be either filtered or may enter tubular fluid by exocytosis from proximal tubule cells. The released products, of which adenosine is an important regulator of various renal functions, may thereafter be reabsorbed by separate transport systems present in the proximal tubule luminal membrane. However, recent immunocytochemical findings by Gandhi et al. (15), which show that ecto-5’-nucleotidase is present not only in proximal tubule brush borders but also in the luminal membrane of the collecting duct intercalated cells, indicate that some degradation products of ecto-ATPase action in the proximal tubule, notably 5’-nucleotide, may have some function in the distal nephron and are finally degraded and possibly reabsorbed in the collecting duct. From that point of view, a high ecto-ATPase activity in the proximal tubule S3 segment and initial DTL may serve to generate distally active nucleotides. However, our present finding of ecto-ATPase in brush borders of Sl and S3, but not S2 segments, of the proximal tubule and in the luminal membrane of the initial DTL suggests some additional function(s) for ecto-ATPase in the renal tubule. Indeed, recent data by Aurivillius at al. (1) and Lin et al. (26) revealed that the liver ecto-ATPase is immunologically and structurally identical to a cell adhesion molecule termed cellCAM105. The cell-CAM105, with an apparent M, of 105 kDa, has been purified from rat liver plasma membranes, and its distribution in the liver is similar to that of ectoATPase (31). It consists of two structurally similar highly glycosylated polypeptide chains and is involved in cellcell adhesion of adult rat hepatocytes in vivo (31). By immunohistochemical techniques the cell-CAM.105 has been localized in many rat organs and cells and is highly concentrated in epithelial cells with densely packed brush borders, such as bile canaliculi, small intestinal epithelium, and renal proximal tubule cells (31 ). This finding raises questions concerning the possible role of ecto-ATPase/ceKCAM105 in the regulation of cell-cell interaction mediated by ATP and other nucleotides at the level of microvilli of the proximal tubule. However, the peculiar distribution of ecto-ATPase in the brush border of some but not all proximal tubule segments and the presence of this enzyme in the initial segment of DTL, which has no brush border, precludes any general conclusion. In view of the finding of long and short forms of ectoATPases in liver plasma membranes (26; unpubl ished observations ), we tested for the presence of a long form in the kidney by using antipeptide antibodies against the cytoplasmic COOH-terminal portion of ecto-ATPase. This portion is absent in the short form of the liver enzyme (26; unpublished observations). We could not demonstrate significant staining in the kidney with these antibodies. Because staining with antibodies against the holoenzyme was positive and because it was negative with antibodies against COOH-terminal portion of the long form, we conclude that the kidney has either only a short form or that the intracellular domains of the long


F228

LOCALIZATION

ecto-ATPase ent.

OF

ECTO-ATPASE

isoforms in the liver and kidney are differ-

We thank Dr. G. Andres for the suggestion to use thrombomodulin as a marker for endothelial cell plasma membranes and Dr. D. Stern for sending us the anti-thrombomodulin antibodies. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38452. Address for reprint requests: I. Sabolic, Renal Unit, Massachusetts General Hospital, 149 13th St., 8th Floor, Charlestown, MA 02129. Received 27 June 1991; accepted in final form 3 October 1991. REFERENCES 1. Aurivillius, and

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Isolated membrane vesicles in the evaluation of the nature, localization, and regulation of renal transport processes.

proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney.

sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex.

Transport of glutamine by rat kidney brush-border membrane vesicles.

Hypoxanthine uptake in isolated rat renal cortical tubule fragments.

DA1 receptor mediated regulation of Na(+)-H+ antiport activity in rat renal cortical brush border membrane vesicles.

Biosynthesis of basement membrane matrix by isolated rat renal glomeruli.

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles.

Ion dependence of cystine and lysine uptake by rat renal brush-border membrane vesicles.

Isolation of lumenal and contralumenal plasma membrane vesicles from kidney.

Localization of adenylate cyclase in Dictyostelium discoideum. II. Cytochemical studies on whole cells and isolated plasma membrane vesicles.

Renal handling and effects of S(+)-ibuprofen and R(-)-ibuprofen in the rat isolated perfused kidney.

Active transport of L-glutamate by membrane vesicles isolated from rat brain.

Chloroquine, a novel inhibitor of amino acid transport by rat renal brush border membrane vesicles.

Effect of dietary phosphate intake on phosphate transport by isolated rat renal brush-border vesicles.

Cytoplasmic membrane vesicles of Escherichia coli. II. Orientation of the vesicles studied by localization of enzymes.

Nucleoside transport in brush border membrane vesicles from human kidney.

Characterization of alpha 2-adrenoceptors in the rat: proximal tubule, renal membrane and whole kidney studies.

Taurocholate--sodium co-transport by brush-border membrane vesicles isolated from rat ileum.

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Na(+)-dependent biotin transport into brush-border membrane vesicles from rat kidney.

Phenylalanine uptake in isolated renal brush border vesicles.

Proline and glycine uptake by renal brushborder membrane vesicles.

Plasma membrane vesicles isolated from epimastigote forms of Trypanosoma cruzi.