Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase.

The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.

Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes.

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.

Preparation and use of monoclonal antibodies to Na+,K+-ATPase.

Application of monoclonal antibody techniques to the study of Na+,K+-ATPase offers a unique opportunity to investigate the molecular basis of sodium potassium transport. As a highly specific tool it can help define the transport process of a molecular, epithelial, and organ level by allowing biochemical, physiological, and morphological approaches to use a single probe. With the use of proper screening procedures, a highly specific reagent can be developed with great utility for a variety of different technical approaches.

Sodium-hydrogen exchangers and sodium-bicarbonate co-transporters: ontogeny of protein expression in the rat brain.

We used western blotting to examine the developmental profiles (at embryonic day 16 and postnatal days 1, 13, 23, 33 and 105) of protein expression for three sodium-hydrogen exchanger isoforms (1, 2 and 4) and for a sodium-bicarbonate co-transporter in three CNS regions (cortex, cerebellum and brainstem-diencephalon). In microsomal preparations, sodium-hydrogen exchanger isoform 1 and sodium-bicarbonate co-transporter protein expression in the CNS increases gradually from embryonic day 16 (25-40% of the adult level) to postnatal day 105. In contrast, sodium-hydrogen exchanger isoform 2 and 4 expression reaches a maximum (three to 20 times the adult level) at around three to four weeks of age. There is significant regional heterogeneity in the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins in the rat CNS. Sodium-hydrogen exchanger isoform 1 was highly expressed in the brainstem-diencephalon, whereas the sodium-bicarbonate co-transporter was robustly expressed in the cerebellum and brainstem-diencephalon. These data indicate that the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins varies as a function of both development and specific brain region.

Molecular biology of renal Na(+)-H+ exchangers.

Na(+)-H+ exchangers are plasma membrane proteins that are responsible for Cl- and HCO3- reabsorption and regulation of intracellular pH in several nephron segments. Recently, a cDNA encoding a human Na(+)-H+ exchanger of unknown tissue origin was cloned, and Northern blot analysis revealed that structurally similar transcripts were expressed in rabbit kidney and porcine renal epithelial cells (LLC-PK1). To clone these renal transcripts we employed cDNA library screening and the polymerase chain reaction (PCR). We obtained 2.5 kb and 1.4 kb cDNAs corresponding to the 5' untranslated region and the membrane-associated domain of a rabbit renal Na(+)-H+ exchanger. From LLC-PK1 cells we obtained 1.5 kb and 1.3 kb cDNAs encoding the membrane-associated and cytoplasmic domains. The sequences of cDNAs from these three species were very similar and suggested a high degree of evolutionary conservation. Immunolocalization of synthetic oligopeptides derived from the deduced amino acid sequences indicated that the cloned cDNAs encoded the amiloride-sensitive form of the Na(+)-H+ exchanger present in basolateral membranes of epithelia. cDNAs were also used to study regulation of Na(+)-H+ exchanger gene expression in the kidney, and we found that metabolic acidosis stimulated both the transport rate and steady-state transcript levels of the basolateral Na(+)-H+ exchanger in LLC-PK1 cells.

The pilo-Ruffini complex: a non-sinus hair and associated slowly-adapting mechanoreceptor in primate facial skin.

A spray-type of nerve ending identified as a Ruffini corpuscle closely associated with a non-sinus hair has been defined in terms of its histologic, ultrastructural and physiologic parameters. The hair and its associated mechanoreceptor, termed a pilo-Ruffini complex, responds as a slowly adapting (SA) mechanoreceptor, whereas most non-sinus hair-associated mechanoreceptors are rapidly adapting. Morphologically, the terminal nerve fibers branch repeatedly within a unique connective tissue matrix, and the neurite and associated connective tissue matrix forms a collar around the hair follicle. This receptor, on the basis of its organization, is interpreted as corresponding to the corpuscle or end organ of Ruffini.

Sodium-hydrogen exchange isoform expression in blood cells: implications for studies in diabetes mellitus.

There have been many reports of increased Na-H exchange (NHE) activity in the peripheral blood cells (erythrocytes, lymphocytes and platelets) of patients with diabetes mellitus compared to nondiabetic controls. This raised NHE activity has been hypothesized to reflect increased NHE activity in kidney and vascular smooth muscle. Raised NHE activity in these tissues could play a pathophysiological role in mediating hypertension, vascular smooth muscle cell proliferation and progressive renal impairment. It is now known that there are at least five NHE isoforms, but a specific study examining expression of NHE isoforms in peripheral blood cells has not been reported. This study used specific antisera to NHE isoforms 1, 3 and 4 to examine NHE expression by immunoblot analysis. Erythrocyte, lymphocyte and platelet membranes from both rabbit and rat were separated by standard methods. A monoclonal antibody to NHE-1 reacted with a 100-110 kDa band in rabbit and rat platelets and lymphocytes (identical to that observed in basolateral-enriched renal cortical vesicles) and a 100 kDa band in rabbit and rat erythrocytes. In both species, the intensity of the staining was greatest in platelet membranes. A polyclonal antibody to NHE-3, the isoform present on the apical membranes of renal proximal tubule, showed no evidence of staining in any of the peripheral blood cell preparations. Similarly there was no evidence of expression of NHE-4 in the peripheral blood cell preparations. Peripheral blood cells express NHE-1, which likely accounts for amiloride-sensitive Na-H exchange in these cells, playing a role in cell volume and pH regulation. However, there is no evidence that there is expression of NHE-3 or NHE-4 in peripheral blood cells. These data have implications for studies in hypertension and diabetes mellitus which measure peripheral blood cell Na-H exchange and hypothesize regarding a direct pathophysiological role for this increased activity.

Regulated intramembrane proteolysis of megalin: linking urinary protein and gene regulation in proximal tubule?

Regulated intramembrane proteolysis (RIP) represents an evolutionarily conserved process linking receptor function with transcriptional regulation. Best characterized by the Notch signaling pathway, RIP involves regulated ectodomain shedding followed by gamma-secretase-mediated release of the C-terminal, cytosolic domain. The C-terminus in turn translocates to the nucleus where it interacts with other proteins to regulate expression of specific genes. Recent studies in our laboratory have shown that megalin, a scavenger receptor in proximal tubule, is subjected to RIP in a manner very similar to that of Notch. We showed that megalin in subjected to protein kinase C-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-associated C-terminal fragment (MCTF). The MCTF in turn forms the substrate for gamma-secretase. These data implicate megalin as a central element of a Notch-like signaling pathway linking protein reabsorption and gene regulation in proximal tubule. The likelihood that megalin processing plays an important role in the progression of proteinuric kidney disease is discussed.

Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments.

To obtain a highly specific reagent that could be utilized for ultrastructural localization of Na,K-ATPase, monoclonal antibodies were produced using microsomal preparations of outer renal medulla of dog and rat enriched for Na,K-ATPase. The monoclonal antibody (C62.4) raised against dog antigen, immunoprecipitated a 96,000 Dalton protein from membranes labeled either with 35S methionine or 3H NAB ouabain. Na,K-ATPase, Na-ATPase, and KpNPPase activity were 25, 60, and 100% maximal after reaction with C62.4. Na,K-ATPase activated with SDS was inhibited, but Na,K-ATPase in tight right-side-out membrane vesicles was not. C62.4 inhibited ouabain binding in the presence of Na,K, and Mg, but did not inhibit ouabain binding in the presence of Mg and Pi. Labeling of broken membranes was readily seen using C62.4 labeled with colloidal gold. Intact right-side-out vesicles showed no evidence of labeling, demonstrating that the antibody is directed to an epitope of the cytoplasmic domain of the enzyme. Differential localization of C62.4 along the nephron was identified. Glomeruli showed no significant antibody binding except by occasional cells in the mesangial regions. Only basal lateral membranes of cells from all tubule segments labeled with C62.4. There was no evidence of specific apical labeling. The thick ascending limb of Henle's loop demonstrated the greatest concentration of antibody binding. In the cortical and outer medullary collecting duct, only principal cells showed abundant antibody binding. Intercalated cells showed no detectable evidence of antibody binding on any surface. These studies demonstrate that Na,K-ATPase is localized exclusively to the basal lateral membrane of renal tubular epithelial cells and varies in density and distribution in different nephron segments.

Ontogeny of rabbit renal cortical NHE3 and NHE1: effect of glucocorticoids.

The neonatal proximal tubule has a lower rate of bicarbonate absorption and Na+/H+ antiporter activity than the proximal tubule of adult animals. Two isoforms of the Na+/H+ antiporter have been localized to the proximal tubule. NHE3 is located on the apical membrane, whereas NHE1, the isoform found on most mammalian cells, is present on the basolateral membrane. The Na+/H+ antiporter isoforms that increase with renal maturation are unknown. The purpose of the present study was to examine the maturation of rabbit renal cortical NHE3 and NHE1 mRNA and protein abundance and to determine whether the rate of maturation of these isoforms was affected by glucocorticoids. Renal cortex from neonatal rabbits (1 wk) had approximately one-fourth the NHE3 mRNA and protein abundance as that from adult animals. Renal cortical NHE1 mRNA and protein abundance did not change significantly during maturation. Glucocorticoids have been shown to accelerate the maturation of neonatal bicarbonate absorption and apical membrane Na+/H+ antiporter activity. Daily subcutaneous administration of dexamethasone starting at 4 days of age (10 micrograms/100 g body wt) for 3 days and 2 h before being killed resulted in a twofold increase in NHE3 mRNA abundance and a threefold increase in NHE3 protein abundance. NHE1 mRNA and protein abundance were unaffected. These data show that there is selective maturation of NHE3 during renal cortical development, which can be accelerated by administration of glucocorticoids.

Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies.

The Na-K-Cl cotransporter (NKCC) is present in most animal cells where it functions in cell volume homeostasis and epithelial salt transport. We developed six monoclonal antibodies (designated T4, T8, T9, T10, T12, and T14) against a fusion protein fragment encompassing the carboxy-terminal 310 amino acids of the human colonic NKCC. These T antibodies selectively recognized putative NKCC proteins in a diverse variety of animal tissues. Western blot analysis of membranes isolated from 23 types of cells identified single bands of immunoreactive protein ranging in mass from 146 to 205 kDa. The amount of immunoreactive protein detected in these cells correlated with loop diuretic binding site density. Proteins identified previously as Na-K-Cl cotransporters by loop diuretic photoaffinity labeling were mutually recognized by multiple T antibodies. Most of the T antibodies effectively immunoprecipitated the denatured form of the NKCC protein. Immunocytochemical studies on the rabbit parotid gland demonstrated that NKCC is restricted to the basolateral margin of the acinar cells and absent from the ducts, in accord with the central role of Na-K-Cl cotransport in chloride secretion. In the rabbit kidney, NKCC was localized to the apical membrane of thick ascending limb cells, consistent with its role in chloride reabsorption.

Active (9.6 s) and inactive (21 s) oligomers of NHE3 in microdomains of the renal brush border.

We have previously shown that Na(+)-H(+) exchanger isoform NHE3 exists as both 9.6 and 21 S (megalin-associated) oligomers in the renal brush border. To characterize the oligomeric forms of the renal brush border Na(+)-H(+) exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a nonmicrovillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter colocalized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 coprecipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9.6, while the S value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na(+) gradient caused generation of an inside acid pH gradient in the microvilli, indicating Na(+)-H(+) exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states: a 9.6 S active form present in microvilli and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na(+)-H(+) exchange activity may be mediated by shifting the distribution between these forms of NHE3.

Membrane topology of NHE3. Epitopes within the carboxyl-terminal hydrophilic domain are exoplasmic.

Experimental data indicate that the relatively hydrophilic carboxyl-terminal domains of Na+-H+ exchangers mediate the regulation of transporter activity through interactions with cytoskeletal effectors. It has therefore been assumed that this entire domain lies on the cytoplasmic surface of the plasma membrane. The purpose of the present study was to determine the membrane orientation of the COOH-terminal 131 amino acids of Na+-H+ exchanger isoform NHE3 by use of three monoclonal antibodies that recognize at least two distinct epitopes within this region. Enzyme-linked immunosorbent assay studies demonstrated binding of these monoclonal antibodies (mAbs) to intact right-side-out renal brush border membrane vesicles in the absence of detergent. Moreover, when coupled to an affinity matrix to isolate membrane vesicles, the anti-NHE3 mAbs bound structures that were morphologically identical to intact microvilli. To confirm the identity of the exoplasmic antigen bound by the antibodies, immunoprecipitation studies were performed. Intact right-side-out brush border membrane vesicles were incubated with the mAbs in the absence of detergent. The membranes were pelleted, supernatant with unbound antibody was removed, the pellet was solubilized, and then immunoprecipitation with secondary antibody was performed. Immunoblot analysis indicated that NHE3 was precipitated after binding of the mAbs to intact membranes. Finally, the localization of the mAb epitopes was determined using high resolution immunocytochemistry. Ultrathin cryosections of rat kidney were labeled with the mAbs and bound antibody detected with the colloidal gold technique. Labeling was restricted to the exoplasmic surface of microvilli of the proximal tubule. Taken together, these findings indicate that epitopes within the carboxyl terminus of the Na+-H+ exchanger isoform NHE3 are exposed to the outside of the plasma membrane.

Changes in membrane surfaces of collecting duct cells in potassium adaptation.

Chronic potassium loading results in an increased capacity of the distal nephron to secrete potassium. The cellular mechanism for this adaptation has been correlated to an increase in the activity of sodium-potassium-ATPase. Because adaptation may be dependent on the greater availability of potassium pumps in the basolateral membrane, a stereologic analysis of the membrane surface area was performed to determine whether the apparent increase in pump sites was due to an increase in cell membrane surface. With potssium adaptation, the number of microplicated cells in the outer medulla was reduced from 31 to 18%. There was a marked increase in the basolateral infolding of principal cells, and membrane surface increased by 32%. In papillary collecting duct cells, the basolateral membrane surface was unchanged but the surface density of the luminal membrane increased by 50%. These observations suggest that amplification of the basolateral cell membrane to increase the number of potassium pump sites per cell plays an important role in the mechanism of potassium adaptation in the outer medulla. A different mechanism involving the luminal membrane operates in the papillary collecting duct. Structural alterations in cell membrane surfaces are thus related to the regulation of the epithelial transport of electrolytes.

Specific association of megalin and the Na+/H+ exchanger isoform NHE3 in the proximal tubule.

We investigated whether the renal brush border Na+/H+ exchanger NHE3 exists in assemblies with other proteins in native kidney membranes. To this end we generated monoclonal antibodies (mAbs) against affinity purified NHE3 protein complexes. Hybridomas were selected based on ability to immunoprecipitate NHE3. One of the resulting mAbs (10A3) labeled a high molecular mass (>200 kDa) protein and stained primarily the coated pit region of the proximal tubule in a manner similar to that described for megalin (gp330). We then confirmed that both mAb 10A3 and a known anti-megalin mAb immunoprecipitated and immunoblotted the same protein, namely megalin. mAb 10A3 specifically co-precipitated NHE3 but not villin or NaPi-2 from solubilized renal membranes, indicating specificity of the NHE3-megalin interaction. When immunoprecipitations were performed using either 10A3 or anti-NHE3 mAb 2B9 after separation of solubilized renal proteins by sucrose velocity gradient centrifugation, we found that NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form, and that when NHE3 assembles with megalin, epitopes within the carboxyl-terminal 131 amino acids of NHE3 are blocked. Taken together, these findings indicate that a significant pool of NHE3 exists as a multimeric complex with megalin in the brush border of the proximal tubule.

Cellular ultrastructure of Amphiuma distal nephron: effects of exposure to potassium.

The cellular ultrastructure of the renal distal nephron of the salamander, Amphiuma means, was examined by electron-microscopic and stereological techniques before and after exposure to potassium in the ambient environment. The distal nephron of Amphiuma is composed of three ultrastructurally distinct segments: early distal (or diluting segment), late distal, and collecting tubule. The early distal tubule structurally resembles the mammalian thick ascending limb of Henle's loop. Large renin-like granules are present in the smooth muscle cells of the afferent arteriole in the vicinity of the early distal tubule, suggesting the presence of a rudimentary juxtaglomerular apparatus. Late distal tubules are composed of one large cell type characterized by extensive basal membrane invaginations, often extending to the luminal membrane. Collecting tubules contain principal and intercalated cells that are ultrastructurally similar to cells of the mammalian cortical collecting tubule. Exposure to potassium had no effect on the ultrastructure of early distal cells but led to a sharp increase in the basolateral membrane surface density of principal cells in the collecting tubule (1.17 +/- 0.08-1.63 +/- 0.13 micron2/micron3). Potassium adaptation leads to a similar structural response in the mammalian collecting tubule. Since Amphiuma collecting tubules can be isolated and perfused in vitro and impaled with ion- and voltage-sensitive microelectrodes, the observed structural adaptation suggests that the collecting tubule may be a useful preparation to study the cellular mechanisms of potassium adaptation.

Immunocytochemical characterization of Na(+)-H+ exchanger isoform NHE-1 in rabbit kidney.

We have recently isolated cDNAs encoding a Na(+)-H+ exchanger isoform, referred to as NHE-1, from rabbit kidney and LLC-PK1 cells. To identify the NHE-1 protein and to establish its cellular and subcellular localization in the rabbit kidney, we prepared antibodies to a NHE-1 fusion protein. cDNA encoding the COOH-terminal 41 amino acids of NHE-1 was subcloned into a maltose-binding protein vector and the purified fusion protein (FP347A) used to immunize guinea pigs. To identify the NHE-1 protein, we performed Western blot analysis against membrane fractions prepared from rabbit renal cortex. Anti-FP347A antibody specifically reacted with a polypeptide with an apparent molecular mass of 100-110 kDa that was enriched in basolateral membrane fractions. When indirect immunofluorescence was performed on semithin (0.5 micron) cryosections of paraformaldehyde-lysine-periodate-fixed rabbit kidney, anti-FP347A specifically stained the basolateral plasma membrane of cells of the proximal tubule, thick ascending limb, and distal convoluted tubule. Anti-FP347A similarly stained connecting tubule cells and principal cells. No staining was detected on the apical membrane of any cells of the rabbit nephron. We conclude that NHE-1 is a 100- to 110-kDa protein expressed on the basolateral membrane of multiple nephron segments.

gp330 associates with a 44-kDa protein in the rat kidney to form the Heymann nephritis antigenic complex.

Using antibodies isolated from glomeruli of nephritic rats we have previously identified a 330-kDa cell surface glycoprotein (gp330) as a major pathogenic antigen of Heymann nephritis (HN), an experimental model of human membranous glomerulonephritis. Recently, we have isolated a cDNA clone, C14, encoding a polypeptide that contains a pathogenic epitope of HN responsible for the initiation of the disease. Subsequently, another protein, alpha 2-macroglobulin receptor-associated protein (alpha 2-MRAP), which is a subunit of the receptor for human alpha 2-macroglobulin/low density lipoprotein receptor-related protein (LRP), was shown to possess a high degree of sequence homology to the C14 protein (C14p). In this report, we have investigated the relationship between gp330, C14p, and alpha 2-MRAP. Immunoprecipitation studies demonstrate that gp330 forms a heterodimeric association with a 44-kDa polypeptide that is stable to detergent extraction and long-term centrifugation. Further, immunoblotting analysis on the purified complex indicates that the 44-kDa associated protein shares immunological identity to C14p and alpha 2-MRAP. In addition, antibodies eluted from glomeruli of HN rats and antibodies to a C14 fusion protein immunoprecipitated gp330 and the 44-kDa protein, demonstrating that the epitopes responsible for the initial events of HN are accessible within the complex. Based on these data, three models are proposed to explain how pathogenic epitopes in the gp330-44-kDa, HN antigenic complex may be presented at the cell surface and initiate the onset of HN.

Ion transport and structure of urinary bladder epithelium of Amphiuma.

The urinary bladder of Amphiuma exhibits stable transport properties and an electrical potential difference in vitro. The lumen is significantly negative to the serosa and under short-circuited conditions flux rations for Na and Cl of 5.92 +/- 0.42 and 1.81 +/- 0.20, respectively, were observed. The close agreement between the short-circuit current and net Na flux suggests that most, if not all, of the current is carried by Na. Both ouabain and amiloride decreased the short-circuit current and the mucosal-to-serosal (M leads to S) flux of Na. Furosemide caused a transient increase in the M leads to S flux of Na and Cl but ADH was without effect. In bladders that had high transmural resistance, a net movement of K in the M leads to S direction under short-circuited conditions with flux ratios of up to 2 could be observed. The epithelium of the Amphiuma bladder consists of three cell types: granular cells, basal cells, and mitochondria-rich cells. No goblet cells are present. The mitochondria-rich cells comprise less than 5% of the population of the surface epithelium in Amphiuma in contrast to other amphibian bladders, where it accounts for up to 25% of the population.

Role of NHE3 in mediating renal brush border Na+-H+ exchange. Adaptation to metabolic acidosis.

The aims of the present study were to estimate the fraction of renal brush border membrane Na+-H+ exchange activity mediated by the isoform NHE3 and to evaluate whether the increased brush border Na+-H+ exchange observed in metabolic acidosis is due to increased expression of NHE3 protein. Compared with other isoforms, NHE3 is known to have a unique profile of sensitivity to pharmacologic inhibitors, including relative resistance to amiloride analogs and HOE694. We therefore assessed the inhibitor sensitivity of pH gradient-stimulated 22Na uptake in renal brush border vesicles isolated from normal rats. The I50 values for amiloride (30 microM), dimethylamiloride (10 microM), ethylisopropylamiloride (6 microM), and HOE694 (>100 microM) were markedly dissimilar from those reported for NHE1 and NHE2 but were nearly identical to reported values for NHE3. Na+-H+ exchange activity in renal brush border vesicles isolated from rats with 5 days of NH4Cl-induced metabolic acidosis was increased 1.5-fold compared with control rats, with no change in inhibitor sensitivity. Western blot analysis indicated that NHE3 protein expression was greater in brush border membranes from acidotic compared with control rats. We conclude that virtually all measured Na+-H+ exchange activity in brush border membranes from control and acidotic rats is mediated by NHE3 and that metabolic acidosis causes increased expression of renal brush border NHE3 protein.