Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG2b, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, taken together with results in the following paper [Wu, J.-S.R., & Lever, J.E. (1987) Biochemistry (following paper in this issue)], provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter.     

Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger

The aim of this study was to identify and purify the Na+-H+ exchanger from rabbit renal brush border membranes by use of affinity chromatography. Triton-solubilized membranes were equilibrated with an affinity matrix consisting of the amiloride analogue A35 (5-N-(3-aminophenyl)amiloride) covalently coupled to Sepharose CL-4B beads through a triglycine spacer arm. The matrix was then washed extensively with buffer and sequentially eluted with buffer, buffer containing 5 mM amiloride, and 1% sodium dodecyl sulfate (SDS). Eluates were concentrated and subjected to SDS-polyacrylamide gel electrophoresis. The silver-stained gel revealed a 25-kDa protein that was not visible in the initial solubilized brush border membrane extract, was not eluted from the affinity matrix by buffer alone, but was eluted with 5 mM amiloride. A subsequent elution with 1% SDS did not release any more of the 25-kDa protein, indicating that it had been completely eluted from the affinity matrix by amiloride. The presence of 5 mM amiloride during equilibration of the solubilized brush border extract with the affinity matrix completely blocked adsorption of the 25-kDa protein. The relative abundance of this protein correlated closely with Na+-H+ exchange activity when preparations of cortical brush border membrane vesicles, outer medullary brush border membrane vesicles, and cortical basolateral membrane vesicles were compared. Moreover, binding of the protein to the affinity matrix was inhibited by amiloride and amiloride analogues with a rank order identical to that for inhibition of Na+-H+ exchange activity. These findings strongly suggest that the 25-kDa protein is a structural component of the Na+-H+ exchanger.     

Inhibitory effect of local anesthetics on Na+/H+ antiporter in brush border membrane-reconstituted vesicles

The effects of three local anesthetics, lidocaine, dibucaine, and tetracaine, on Na+/H+ antiporter activity were examined in brush border membrane-reconstituted vesicles. Lidocaine at 10 microM inhibited H+ efflux in the presence of an inward Na+ gradient, suggesting that this anesthetic specifically inhibits the Na+/H+ antiporter. On the other hand, dibucaine and tetracaine decreased H+ efflux even in the absence of a Na+ gradient.     

Photoaffinity labeling by [3H]-N5-methyl-N5-isobutylamiloride of proteins which cofractionate with Na+/H+ antiport activity

N5-Methyl-N5-isobutylamiloride (MIA) is one of a series of 5-N-substituted amiloride analogues which exhibit high affinity and specificity for inhibition of Na+/H+ antiport. Amiloride-sensitive [3H]MIA binding to renal brush border membranes exhibited a Kd of 250 nM and a Bmax of 8.6 pmol/mg of protein. Specific binding was optimal at pH 7.5 and inhibited in the presence of Na+ and Li+. Inhibition by amiloride exhibited biphasic kinetics. After resolution of solubilized membranes by high-pressure liquid chromatography, MIA binding activity cofractionated together with Na+/H+ antiport activity, measured after reconstitution in asolectin vesicles, into a major and a minor peak. When fractions containing the major peak of Na+/H+ antiport activity were incubated with [3H]MIA and then photolyzed with a mercury arc lamp, covalent incorporation of label into polypeptides of apparent molecular mass 81 and 107 kDa was observed. These photolabeled bands were also observed in intact brush border membranes in addition to labeled polypeptides of apparent molecular mass 60 and 46 kDa, respectively. Labeling was inhibited by amiloride, reduced in the presence of Na+, and not observed in the absence of photolysis. These data point to the 81- and 107-kDa polypeptides as candidates for identification as components of a Na+/H+ antiport system in renal brush border membranes.     

Purification and reconstitution of a 75-kilodalton protein identified as a component of the renal Na+/glucose symporter

A 75-kilodalton (kDa) protein was purified from solubilized renal brush border membranes by using high-pressure liquid chromatography (HPLC) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Functional and immunological properties identified the 75-kDa protein as a component of the Na+/glucose symport system. The purified protein was specifically recognized by a monoclonal antibody that functionally interacts with the Na+/glucose symporter. Na+-dependent phlorizin binding activity was associated with fractions containing the 75-kDa protein during HPLC fractionation on the anion exchanger Mono-Q and was greatly increased after reconstitution into egg yolk phosphatidylcholine vesicles. The final purified preparation contained glucosamine and a blocked N-terminus.     

Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8-Br-cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29-18 and Caco-2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta-galactosidase and the C-terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI-AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the 'housekeeping' Na+/H+ exchanger.     

Na+/H+ antiporters

Na+/H+ antiports or exchange reactions have been found widely, if not ubiquitously, in prokaryotic and eukaryotic membranes. In any given experimental system, the multiplicity of ion conductance pathways and the absence of specific inhibitors complicate efforts to establish that the antiport observed actually results from the activity of a specific secondary porter which catalyzes coupled exchanged of the two ions. Nevertheless, a large body of evidence suggests that at least some prokaryotes possess a delta psi-dependent, mutable Na+/H+ antiporter which catalyzes Na+ extrusion in exchange for H+; in other bacterial species, the antiporter my function electroneutrally, at least at some external pH values. The bacterial Na+/H+ antiporter constitutes a critical limb of Na+ circulation, functioning to maintain a delta mu Na+ for use by Na+-coupled bioenergetic processes. The prokaryotic antiporter is also involved in pH homeostasis in the alkaline pH range. Studies of mutant strains that are deficient in Na+/H+ antiporter activity also indicate the existence of a relationship, e.g., a common subunit or regulatory factor, between the Na+/H+ antiporter and Na+/solute symporters in several bacterial species. In eukaryotes, an electroneutral, amiloride-sensitive Na+/H+ antiport has been found in a wide variety of cell and tissue types. Generally, the normal direction of the antiport appears to be that of Na+ uptake and H+ extrusion. The activity is thus implicated as part of a complex system for Na+ circulation, e.g., in transepithelial transport, and might have some role in acidification in the renal proximal tubule. In many experimental systems, the Na+/H+ antiport appears to influence intracellular pH. In addition to a role in general pH homeostasis, such Na+-dependent changes in intracellular pH could be part of the early events in a variety of differentiating and proliferative systems. Reconstitution and structural studies, as well as detailed analysis of gene loci and products which affect the antiport activity, are in their very early stages. These studies will be important in further clarification of the precise structural nature and role(s) of the Na+/H+ antiporters. In neither prokaryotes nor eukaryotes systems is there yet incontrovertible evidence that a specific protein carrier, that catalyzes Na+/H+ antiport, is actually responsible for any of the multitude of effects attributed to such antiporters. The Na+-H+ exchange might turn out to be side reactions of other porters or the additive effects of several conductance pathways; or, as appears most likely in at least some bacteria and in renal tissue, the antiporter may be a discrete, complex carr     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.     

Methyl isobutyl amiloride: a new probe to assess the number of Na-H antiporters

We measured the binding of [3H]-5-(N-methyl-N-isobutyl) amiloride (MIA) to purified rabbit renal brush border membranes. MIA binding was protein, temperature and time dependent with optimal binding at pH 8.0 or above. At low pH MIA binding was inhibited, suggesting competition between H+ ions and MIA for the MIA binding site. There was 70-80% specific binding which reached a plateau at 30 min and remained stable thereafter for 150 min. Scatchard analysis revealed one family of binding sites with Bmax of 3.4 +/- 0.4 pmoles/mg protein and Kd of 30.5 +/- 2.3 nM. MIA inhibited the Vmax of the Na-H antiporter (assessed by acridine orange quenching) in a dose dependent fashion with 100% inhibition at MIA concentration of 10(-3) M and this inhibition was greater than that of amiloride. We conclude that MIA, a potent inhibitor of the Na-H antiporter, displays a high percentage of specific binding to renal brush border membranes and can be used to assess the number of the Na-H antiporters.     

Reconstitution, identification, purification, and immunological characterization of the 110-kDa Na+/Ca2+ antiporter from beef heart mitochondria.

The mitochondrial Na+/Ca2+ antiporter plays a key role in the physiological regulation of intramitochondrial Ca2+, which in turn attunes mitochondrial enzymes to the changing demands of the cell for ATP. We have now purified the Na+/Ca2+ antiporter from beef heart mitochondria by assaying detergent-solubilized chromatography fractions for reconstitutive activity. Na+ and Ca2+ transport were assayed using the fluorescent probes, sodium-binding benzofuran isophthalate and Fura-2, respectively. This approach enabled us to identify Na+/Ca2+ exchange activity with a 110-kDa inner membrane protein that catalyzed Na(+)-dependent Ca2+ transport and Ca(2+)-dependent Na+ transport. A new finding was that the Na+/Ca2+ antiporter also catalyzed Na+/Li+ exchange in the absence of Ca2+. All modes of transport were electroneutral and were inhibited by diltiazem and tetraphenylphosphonium cation. Monospecific polyclonal antibodies to the 110-kDa protein inhibited Na+/Ca2+ and Na+/Li+ exchange in the reconstituted system and recognized 110-kDa proteins in mitochondrial membranes isolated from rat heart, liver, and kidney.     

Evidence from 23Na NMR studies for the existence of sodium-channels in the brush border membrane of the renal proximal tubule

A fast Na+-exchange is shown to be present in isolated renal brush border membranes. The lower limit of the rate constant for this process, calculated from the 23Na-NMR spectrum is 580 sec-1. The actual exchange rate may be higher. A fast 7Li exchange is also shown to be present in the isolated membrane vesicles. The characteristic overshoot of the Na+ dependent D-glucose cotransport and Na+/H+ antiport can be demonstrated. The fact that neither treatment with papain, nor lowering of the temperature to 5 degrees C affected the 23Na-NMR spectra obtained in the renal brush border membrane vesicles is consistent with the possibility that the fast Na+-exchange occurs through a channel mechanism.     

Identification, purification, and characterization of a stilbenedisulfonate binding glycoprotein from canine kidney brush border membranes. A candidate for a renal anion exchanger

Canine renal brush border membrane proteins that bind stilbenedisulfonate inhibitors of anion exchange were identified by affinity chromatography. A 130-kDa integral membrane glycoprotein from brush border membrane was shown to bind specifically to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate immobilized on Affi-Gel 102 resin. The bound protein could be eluted effectively with 1 mM 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS). The 130-kDa protein did not bind to the affinity resin in the presence of 1 mM BADS or when the solubilized extract was covalently labeled with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). This protein was labeled with [3H]H2DIDS, and the labeling was prevented by BADS. The 130-kDa protein did not cross-react with antibody raised against human or dog erythrocyte Band 3 protein. The 130-kDa protein was accessible to proteinase K and chymotrypsin digestion in vesicles but not to trypsin. The 130-kDa protein was sensitive to endo-beta-N-acetylglucosaminidase F treatment both in the solubilized state and in brush border membrane vesicles showing that it was a glycoprotein and that the carbohydrate was on the exterior of the vesicles. This glycoprotein was resistant to endo-beta-N-acetylglucosaminidase H treatment suggesting a complex-type carbohydrate structure. The protein bound concanavalin A, wheat germ agglutinin, and Ricinus communis lectins, and it could be purified using wheat germ agglutinin-agarose.     

Reconstitution of Na+/H(+)-antiporter of bovine renal brush-border membrane into proteoliposomes and detection of a 110 kDa protein cross-reactive with antibodies against a human Na+/H(+)-antiporter partial peptide in antiport-active fractions after partial fractionation.

Bovine renal brush-border membranes were solubilized by 1.6% sodium cholate. Na+/H(+)-antiporter was recovered in the supernatant after centrifugation at 160,000 x g for 1 h and was successfully reconstituted into proteoliposomes by a cholate-dialysis procedure. The reconstituted Na+/H(+)-antiporter showed a pH-gradient dependent and amiloride-sensitive 22Na+ uptake very similar to that of brush-border membrane vesicles. Factors affecting the efficiency of reconstitution as well as the stability of the solubilized antiporter at various temperatures were studied. Sodium cholate-solubilized brush-border membrane proteins were fractionated by Sephacryl S-400 and DEAE-Toyopearl chromatography, and fractions containing reconstitutively active Na+/H(+)-antiporter were identified. A 110 kDa peptide cross-reactive with a polyclonal antibody against a C-terminal peptide (22-amino acid residues) of human Na+/H(+)-antiporter was consistently found on the immunoblot of the active fractions. A closely similar peptide was also detected in human placental membranes by this antibody. These results strongly suggest that the 110 kDa protein is responsible for Na+/H(+)-antiporter activity.     

Effect of angiotensin-II on renal Na+/H+ exchanger-NHE3 and NHE2

The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na+/H+ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of 22Na+ study in the presence of 50 microM HOE-694 revealed that Na+ uptake mediated by NHE3 was increased approximately 88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na+ uptake by stimulating NHE3 mRNA and protein content.     

Isolation and properties of a mutant of Escherichia coli possessing defective Na+/H+ antiporter

A mutant of Escherichia coli with defective Na+/H+ antiporter was isolated. The rationale for its isolation was that cells possessing defective Na+/H+ antiporter, which is essential for establishment of a Na+ gradient, could not grow with a carbon source that was taken up with Na+. The mutant had no appreciable Na+/H+ antiporter activity, but its K+/H+ antiporter and Ca2+/H+ antiporter activities were normal. Judging from the reversion frequency, the defect seems to be due to a single mutation. The mutant could not grow at alkaline pH. Therefore, the Na+/H+ antiporter, but not the K+/H+ antiporter or the Ca2+/H+ antiporter, seems to be responsible for pH regulation in alkaline medium. This mutant will be useful for cloning the Na+/H+ antiporter gene and for detection of Na+-substrate cotransport systems.     

Differential regulation of Na+/H+ antiporter gene expression in vascular smooth muscle cells by hypertrophic and hyperplastic stimuli

The Na+/H+ antiporter is a ubiquitous transmembrane protein that plays a vital role in cell growth via regulation of intracellular Na+ and H+. In vascular smooth muscle cells (VSMC), vasoconstrictors and mitogens rapidly activate the antiporter, suggesting that both should have growth promoting effects. Indeed, angiotensin II increases VSMC protein and volume (hypertrophy), but does not increase cell number (hyperplasia). In the present work we investigated whether alterations in the steady state levels of Na+/H+ antiporter mRNA might differentiate these VSMC growth responses. Differences in function of the Na+/H+ antiporter appeared likely because exposure of growth-arrested VSMC for 24 h to 100 nM angiotensin II decreased intracellular pH from 7.08 to 6.99, while exposure to 10% calf serum caused an increase to 7.18. Simultaneous measurement of Na+/H+ antiporter mRNA levels, using the human c28 cDNA, revealed a 25-fold increase in response to serum (as well as to platelet-derived and fibroblast growth factors), but no change in response to angiotensin II. All agonists increased mRNA levels of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase approximately 3-fold. The increase in Na+/H+ antiporter mRNA induced by serum was first apparent within 2 h and peaked 24 h after treatment. These results demonstrate that expression of Na+/H+ antiporter mRNA in VSMC is dependent on growth state: hyperplastic agonists (serum, platelet-derived and fibroblast growth factor) increase the steady state levels of Na+/H+ antiporter mRNA while a hypertrophic agonist (angiotensin II) does not.     

Inhibition and labeling of the rat renal Na+/H+-exchanger by an antagonist of muscarinic acetylcholine receptors

A covalently binding label for muscarinic acetylcholine receptors, propylbenzilylcholine mustard (PrBCM), irreversibly inhibits the Na+/H+ exchanger in rat renal brush-border membrane vesicles. Substrates of the antiporter, Na+ and Li+, as well as inhibitors, amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and propranolol, protect the antiporter from inactivation by PrBCM. With [3H]PrBCM a band with an app. Mr of 65 kDa is predominantly labeled. Amiloride protects this band from labeling with [3H]PrBCM and [14C]-N,N'-dicyclohexylcarbodiimide (DCCD) proving its identity with the renal Na+/H+ exchanger. Our data reveal a specific interaction of PrBCM with the Na+/H+ exchanger and suggest structural relations between antiporter and receptors.     

Na+/H+ antiporter activity in hamster embryos is activated during fertilization

This study characterized the activation of the regulatory activity of the Na+/H+ antiporter during fertilization of hamster embryos. Hamster oocytes appeared to lack any mechanism for the regulation of intracellular pH in the acid range. Similarly, no Na+/H+ antiporter activity could be detected in embryos that were collected from the reproductive tract between 1 and 5 h post-egg activation (PEA). Activity of the Na+/H+ antiporter was first detected in embryos collected at 5.5 h PEA and gradually increased to reach maximal activity in embryos collected at 7 h PEA. Parthenogenetically activated one-cell and two-cell embryos demonstrate Na+/H+ antiporter activity, indicating that antiporter activity is maternally derived and initiated by activation of the egg. The inability of cycloheximide, colchicine, or cytochalasin D to affect initiation of antiporter activity indicates that antiporter appearance is not dependent on the synthesis of new protein or recruitment of existing protein to the cell membrane. In contrast, incubation of one-cell embryos with sphingosine did inhibit the appearance of Na+/H+ antiporter activity, showing that inhibition of normal protein kinase C activity is detrimental to antiporter function. Furthermore, incubation of oocytes with a phorbol ester which stimulates protein kinase C activity induced Na+/H+ antiporter activity in oocytes in which the activity was previously absent. Incubation with an intracellular calcium chelator also reduced the appearance of antiporter activity. Taken together, these data indicate that the appearance of Na+/H+ antiporter activity following egg activation may be due, at least in part, to regulation by protein kinase C and intracellular calcium levels.