IFN-gamma downregulates expression of Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells.

Diarrhea associated with inflammatory bowel diseases has traditionally been attributed to stimulated secretion. The purpose of this study was to determine whether chronic stimulation of intestinal mucosa by interferon-gamma (IFN-gamma) affects expression and function of the apical membrane Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and Caco-2/bbe (C2) cells. Confluent C2 cells expressing NHE2 and NHE3 were treated with IFN-gamma for 2, 24, and 48 h. Adult rats were injected with IFN-gamma intraperitoneally for 12 and 48 h. NHE2 and NHE3 activities were measured by unidirectional (22)Na influx across C2 cells and in rat brush-border membrane vesicles. NHE protein and mRNA were assessed by Western and Northern blotting. IFN-gamma treatment of C2 monolayers caused a >50% reduction in NHE2 and NHE3 activities and protein expression. In rats, region-specific, time- and dose-dependent reductions of NHE2 and NHE3 activities, protein expression, and mRNA were observed after exposure to IFN-gamma. Chronic exposure of intestinal epithelial cells to IFN-gamma results in selective downregulation of NHE2 and NHE3 expression and activity, a potential cause of inflammation-associated diarrhea.

SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human intestinal C2/bbe cells.

Short-chain fatty acids (SCFA), produced by colonic bacterial flora fermentation of dietary carbohydrates, promote colonic Na absorption through mechanisms not well understood. We hypothesized that SCFA promote increased expression of apical membrane Na/H exchange (NHE), serving as luminal physiological cues for regulating colonic Na absorptive capacity. Studies were performed in human colonic C2/bbe (C2) monolayers and in vivo. In C2 cells exposed to butyrate, acetate, proprionate, or the poorly metabolized SCFA isobutyrate, apical membrane NHE3 activity and protein expression increased in a time- and concentration-dependent manner, whereas no changes were observed for NHE2. In contrast, no significant changes in brush-border hydrolase or villin expression were noted. Analogous to the in vitro findings, rats fed the soluble fiber pectin exhibited a time-dependent increase in colonic NHE3, but not NHE2, protein, mRNA, and brush-border activity. These changes were region-specific, as no changes were observed in the ileum. We conclude that luminal SCFA are important physiological cues for regulating colonic Na absorptive function, allowing the colon to adapt to chronic changes in dietary carbohydrate and Na loads.

Regulation of intestinal epithelial brush border Na(+)/H(+) exchanger isoforms, NHE2 and NHE3, in C2bbe cells.

Until recently, studies to characterize the intestinal epithelial Na(+)/H(+) exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na(+)/H(+) exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH(i) only approximate Na(+)/H(+) exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used (22)[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca(++) involves changes to both K(Na) and V(max). In contrast, the same two second messengers inhibit NHE3 by a decrease in V(max) exclusively. Phorbol ester activation of protein kinase C alters both V(max) and K(Na) of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways.

A unique Na+/H+ exchanger, analogous to NHE1, in the chicken embryonic fibroblast.

We report the characterization of an Na+/H+ exchanger (NHE) in embryonic fibroblasts (SL-29 cells) of the chicken, a terrestrial vertebrate, where Na+ conservation is important. This exchanger is electroneutral, has a single Na+ binding site, and is highly sensitive to amiloride (IC50 2 microM), dimethyl amiloride (350 nM), and ethyl-isopropyl amiloride (25 nM). It is stimulated by serum, transforming growth factor-alpha, hypertonicity, and okadaic acid. Although these features make it resemble mammalian NHE1, other characteristics suggest distinct differences. First, in contrast to mammalian NHE1 it is inhibited by cAMP and shows a biphasic response to phorbol esters and a highly variable response to increased intracellular Ca2+ concentration. Second, whereas full-length human and rat NHE1 cDNA probes recognize a 4.8-kb transcript in rat tissues, they recognize only a 3.9-kb transcript in chicken tissues. An antibody against amino acids 631-746 of human NHE1 sequence fails to recognize a protein in SL-29 cells. Rat NHE2 and NHE3 probes do not recognize any transcript in chicken fibroblasts. The SL-29 exchanger differs markedly from the previously characterized chicken intestinal apical exchanger in its amiloride sensitivity and regulation by phorbol esters. These results suggest that a modified version of mammalian NHE1 is present in chicken tissues and imply that another functionally distinct Na+/H+ exchanger is expressed in aves.

Expression of multiple Na+/H+ exchanger isoforms in rat parotid acinar and ductal cells.

Several members of the Na+/H+ exchanger gene family (NHE1, NHE2, NHE3, and NHE4) with unique functional properties have been cloned from rat epithelial tissues. The present study examined the molecular and pharmacological properties of Na+/H+ exchange in rat parotid salivary gland cells. In acinar cells superfused with a physiological salt solution (145 mM Na+), Na+/H+ exchanger activity was inhibited by low concentrations of the amiloride derivative ethylisopropyl amiloride (EIPA; IC50 = 0.014 +/- 0.005 microM), suggesting the expression of amiloride-sensitive isoforms NHE1 and/or NHE2. Semiquantitative RT-PCR confirmed that NHE1 transcripts are most abundant in this cell type. In contrast, the intermediate sensitivity of ductal cells to EIPA indicated that inhibitor-sensitive and -resistant Na+/H+ exchanger isoforms are coexpressed. Ductal cells were about one order of magnitude more resistant to EIPA (IC50 = 0.754 +/- 0.104 microM) than cell lines expressing NHE1 or NHE2 (IC50 = 0.076 +/- 0.013 or 0.055 +/- 0.015 microM, respectively). Conversely, ductal cells were nearly one order of magnitude more sensitive to EIPA than a cell line expressing the NHE3 isoform (IC50 = 6.25 +/- 1.89 microM). Semiquantitative RT-PCR demonstrated that both NHE1 and NHE3 transcripts are expressed in ducts. NHE1 was immunolocalized to the basolateral membranes of acinar and ductal cells, whereas NHE3 was exclusively seen in the apical membrane of ductal cells. Immunoblotting, immunolocalization, and semiquantitative RT-PCR experiments failed to detect NHE2 expression in either cell type. Taken together, our results demonstrate that NHE1 is the dominant functional Na+/H+ exchanger in the plasma membrane of rat parotid acinar cells, whereas NHE1 and NHE3 act in concert to regulate the intracellular pH of ductal cells.

Membrane-limited expression and regulation of Na+-H+ exchanger isoforms by P2 receptors in the rat submandibular gland duct.

1. Cell-specific reverse transcriptase-polymerase chain reaction (RT-PCR), immunolocalization and microspectrofluorometry were used to identify and localize the Na+-H+ exchanger (NHE) isoforms expressed in the submandibular gland (SMG) acinar and duct cells and their regulation by basolateral and luminal P2 receptors in the duct. 2. The molecular and immunofluorescence analysis showed that SMG acinar and duct cells expressed NHE1 in the basolateral membrane (BLM). Duct cells also expressed NHE2 and NHE3 in the luminal membrane (LM). 3. Expression of NHE3 was unequivocally established by the absence of staining in SMG from NHE3 knockout mice. NHE3 was expressed in the LM and in subluminal regions of the duct. 4. Measurement of the inhibition of NHE activity by the amiloride analogue HOE 694 (HOE) suggested expression of NHE1-like activity in the BLM and NHE2-like activity in the LM of the SMG duct. Several acute and chronic treatments tested failed to activate NHE activity with low affinity for HOE as expected for NHE3. Hence, the physiological function and role of NHE3 in the SMG duct is not clear at present. 5. Activation of P2 receptors resulted in activation of an NHE-independent, luminal H+ transport pathway that markedly and rapidly acidified the cells. This pathway could be blocked by luminal but not basolateral Ba2+. 6. Stimulation of P2U receptors expressed in the BLM activated largely NHE1-like activity, and stimulation of P2Z receptors expressed in the LM activated largely NHE2-like activity. 7. The interrelation between basolateral and luminal NHE activities and their respective regulation by P2U and P2Z receptors can be used to co-ordinate membrane transport events in the LM and BLM during active Na+ reabsorption by the SMG duct.

Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe.

We examined the regulation of the Na+/H+ exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinal C2/bbe cells, which spontaneously differentiate and have little basal apical NHE activity. Unidirectional apical membrane 22Na+ influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of 5-(N,N-dimethyl)amiloride-inhibitable flux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHE activity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulated by serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na+ influx), NHE2 and NHE3 exhibited similar patterns of regulation, suggesting that the second messenger effects observed were not secondary to effects on cell pH. Thus, in contrast to their expression in nonepithelial cells, NHE2 and NHE3 expressed in an epithelial cell line behave similarly to endogenously expressed intestinal apical membrane NHEs. We conclude that physiological regulation and function of epithelium-specific NHEs are dependent on tissue-specific factors and/or conditional requirements.

Aldosterone stimulates intestinal Na+ absorption in rats by increasing NHE3 expression of the proximal colon.

Na+ retention by the colon in response to salt deprivation is mediated in part by the resulting secondary hyperaldosteronism. We show that experimental hyperaldosteronism, to levels seen with salt deprivation, causes an increase in the selective expression and activity of NHE3, an apically located isoform of the Na+/H+ exchange family that functions in transepithelial Na+ absorption. The effect of aldosterone on NHE3 expression is tissue specific, occurring in intestine and not in kidney. Within the intestine, these effects are regional, being observed only in proximal colon, and different in distribution from that observed with glucocorticoids, where the predominant effect occurs in ileum. Although glucocorticoids are well known to exert many effects via regulation of transcript levels, the present study demonstrates that aldosterone stimulates intestinal Na+ absorption by increasing cellular NHE3 expression, a response that is tissue and region specific.

Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney.

We present evidence that tissue distribution of two highly conserved Na+/H+ exchanger isoforms, NHE2 and NHE4, differs significantly from previously published reports. Riboprobes unique to each of these antiporters, from 5' (noncoding and coding) and 3' coding regions, were used to analyze mRNA from adult rat kidney and intestine by ribonuclease protection assay and in situ hybridization. In contrast to earlier work that concluded that both NHE2 and NHE4 were expressed throughout the intestine and in the kidney, our data show that there is no NHE2 message in the kidney and NHE4 is not expressed in small or large intestine. Analyses of intestinal epithelial and kidney membrane proteins by an NHE2-specific antibody identified a doublet at < 90 kDa in intestine but not in kidney. NHE2 is highly expressed in the Na(+)-absorptive epithelium of jejunum, ileum, and ascending and descending colon. NHE4 mRNA message is found in the inner medulla of the kidney as previously reported (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao, and E. B. Chang. J. Biol. Chem. 269: 29704-29709, 1994) and not in the intestine. From these data, we speculate that neither NHE2 nor NHE4 has a role in renal Na+ absorption. NHE2 is likely involved in gut Na+ absorption, whereas NHE4 may have a specialized role in cell volume rectification of inner medullary collecting duct cells. Knowledge of the correct tissue and cell-specific distribution of these two antiporters should help significantly in understanding their physiological roles.

An avian sodium-hydrogen exchanger.

Intestinal sodium transporters, such as the Na+/H+ exchanger (NHE) are important for Na+ conservation in land birds. In mammals, at least five isoforms of the exchanger, NHEs 1-5, have been cloned, with NHE-1 occurring in epithelial basolateral and nonepithelial cell membranes and NHE-3 being restricted to epithelial apical/brush border membranes. We had demonstrated earlier that chicken intestinal brush border membranes possess NHE activity that functionally resembles mammalian NHE-3. In this study, we used mammalian NHE-1 and NHE-3 probes to examine if chicken enterocytes possess these transporters. Antisera against rat NHE-3 recognized a 97 kDa protein in chicken intestinal brush border membrane, while a NHE-3 cDNA probe failed to recognize any transcript. A NHE-1 antibody failed to recognize any protein in brush border or basolateral membrane, while a NHE-1 cDNA probe recognized a 3.9 kb transcript. Thus, there is more than one NHE isoform in chicken intestine, and our results suggest a novel avian NHE family.

Inverse relationship between membrane lipid fluidity and activity of Na+/H+ exchangers, NHE1 and NHE3, in transfected fibroblasts.

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of approximately 50% in the specific activities of both NHE1 and NHE3. Only Vmax was affected; KNa was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4 degrees C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.

Characterization of the rat Na+/H+ exchanger isoform NHE4 and localization in rat hippocampus.

The kinetics of the Na+/H+ exchanger (NHE) isoform NHE4 were studied by measuring 22Na+ fluxes in stably transfected NHE-deficient fibroblasts. Unlike NHE1, NHE2, and NHE3, activation of this isoform is dependent on hyperosmolarity-induced cell shrinkage. It is virtually inactive at isosmolarity and most active at 490 mosM. When induced by cell shrinkage, NHE4 exhibits a sigmoidal response to increasing extracellular Na+ concentrations, suggesting allosteric or cooperative binding kinetics. In comparison, NHE1 and -3 exhibit hyperbolic velocity vs. extracellular Na+ concentration responses at both iso- and hyperosmolar conditions. Unlike NHE1 and NHE4, hyperosmolarity-induced cell shrinkage inhibits NHE3 activity in transfected fibroblasts, reducing maximum velocity by 40%, with no effect on binding affinity to extracellular Na+.NHE4 is relatively insensitive to inhibition by amiloride analogues in the order 5-(N,N-dimethyl)amiloride > 5-(N,N-hexamethylene)amiloride ride > amiloride > 5-(N-ethyl-N-isopropyl)amiloride. Time-dependent inhibition of activity by cytochalasin D suggests a relationship between the actin cytoskeleton and regulation by cell shrinkage. By in situ hybridization of fixed tissues, NHE4 mRNA was found to be highly expressed in the cavi amnoni fields of rat hippocampus. The kinetics of this exchanger, when considered with its unusual tissue distribution in renal inner medullary collecting tubules and hippocampus, are-consistent with NHE4 having a specialized role in cell functions.

Biosynthesis and cell surface delivery of the NHE1 isoform of Na+/H+ exchanger in A6 cells.

The Na+/H+ exchanger isoform NHE1 is localized to the basolateral membrane of renal and intestinal epithelia. We examined the plasma membrane distribution, biosynthesis, and cell surface delivery of NHE1 in A6 epithelia. NHE1 was localized to the basolateral membrane. Studies of NHE1 biosynthesis with a pulse-chase protocol demonstrated that a core glycosylated, endoglycosidase H-sensitive, 90-kDa NHE1 was present 0-5 h into the chase period and that mature 110-kDa NHE1 was present 1-24 h into the chase period. Studies of plasma membrane delivery of newly synthesized NHE1 demonstrated that the 90-kDa NHE1 was detected at both apical and basolateral membranes 2-5 h into the chase period. The 110-kDa NHE1 was observed at the basolateral membrane 5-24 h into the chase period. These results suggest that NHE1 is expressed primarily at the basolateral membrane of A6 cells, that core glycosylated NHE1 is delivered to the plasma membrane in a nonpolarized manner, and that nature 110-kDa NHE1 is delivered to the basolateral membrane.

Glycosylation of the Na+/H+ exchanger isoform NHE-3 is species specific.

The glycosylation of Na+/H+ exchanger isoform NHE-3 was studied in brush border membrane (BBM) vesicles isolated from rabbit, dog, and rat kidney cortex. Western blot analyses were performed against BBM proteins by using polyclonal antibodies to an NHE-3 fusion protein. In rabbit kidney, NHE-3 antibody recognized a band with approximately 95 kd molecular mass. Treatment of rabbit cortical BBM with glycopeptidase F, at 16 U/ml, for 4 or 16 hours increased the apparent mobility of NHE-3 to 84 and 82 kd, respectively. Incubation of rabbit BBM proteins for 16 hours with endoglycosidase H, at 0.1 U/ml, did not alter the apparent mobility of NHE-3. Deglycosylation of NHE-3 with glycopeptidase F did not affect acid-stimulated, amiloride-sensitive sodium 22 influx in BBM vesicles as compared with that in controls (p > 0.05). Immunoblot analysis against BBM proteins from canine kidney cortex demonstrated the presence of an approximately 83 to 92 kd protein. Treatment of canine BBM with glycopeptidase F or endoglycosidase H or F for 16 hours did not alter the apparent mobility of NHE-3, suggesting that canine renal NHE-3 is not glycosylated. Treatment of canine kidney BBM with glycopeptidase F did not affect acid-stimulated 22Na+ influx as compared with that in controls (p > 0.05). Immunoblot analysis against BBM proteins from rat kidney cortex demonstrated the presence of a sharp band at 90 kd. Treatment of rat BBM with glycopeptidase F or endoglycosidase H or F for 16 hours did not alter the apparent mobility of NHE-3, suggesting that rat renal NHE-3 is not glycosylated. The above experiments suggest that NHE-3 glycosylation in mammalians is species specific and that glycosylation does not affect the exchanger activity.

Inhibition of glycosylation decreases Na+/H+ exchange activity, blocks NHE-3 transport to the membrane, and increases NHE-3 mRNA expression in LLC-PK1 cells.

Recent studies have shown that NHE-3 is the luminal Na+/H+ exchanger isoform in cultured renal proximal tubule cells LLC-PK1 and OK (J Biol Chem 1994; 269:15613-8). The purpose of the current experiments was to study the role of NHE-3 glycosylation on antiporter activity in LLC-PK1 cells. Treatment of LLC-PK, cells with 1.5 microgram/ml tunicamycin for 24 hours, which blocks glycosylation in the endoplasmic reticulum, significantly decreased antiporter activity as asses sed by acid-stimulated sodium 22 uptake (9.52 +/- 1.0 nmol/mg protein in control cells vs 5.85 +/- 0.7 nmol/mg protein in tunicamycin-treated cells, p < 0.01, n = 4) and sodium-dependent pHi recovery from an acid load (0.46 +/- 0.05 pH/min in control cells vs 0.35 +/- 0.04 pH/min in tunicamycin-treated cells, p < 0.02, n = 6). Lactate dehydrogenase (LDH) concentration in the medium was the same in both groups (p > 0.05), indicating that the inhibitory effect of tunicamycin was not caused by cell toxicity. Northern hybridization of poly(A)+ RNA from LLC-PK1 cells illustrated that in tunicamycin-treated cells, NHE-3 mRNA expression increased threefold over control cells. Immunoblots of luminal membranes from control LLC-PK, cells with specific NHE-3 antiserum showed a doublet at 94 to 95 kd and a band at 90 kd. Luminal membranes from tunicamycin-treated cells showed only one strong band at 95 kd. NHE-3 immunoblots of whole cell extract from tunicamycin-treated cells showed that in addition to the 95 kd protein, an 87 kd band was also detected. These results are consistent with the possibility that the two bands in the 94 and 90 kd areas became deglycosylated and did not reach the membrane in the presence of tunicamycin. We conclude that glycosylation of the Na+/H+ exchanger isoform NHE-3 is essential for antiporter activity in LLC-PK, cells. The results further suggest that glycosylation of NHE-3 mediates the translocation and insertion of this exchanger in the plasma membrane.

Differential regulation of Na+/H+ exchange and H(+)-ATPase by pH and HCO3- in kidney proximal tubules.

This study examines the effects of acute in vitro acid-base disorders on Na+/H+ and H(+)-ATPase transporters in rabbit kidney proximal tubules (PT). PT suspensions were incubated in solutions with varying acid base conditions for 45 min and utilized for brush border membrane (BBM) vesicles preparation. BBM vesicles were studied for Na+/H+ exchange activity (assayed by 22Na+ influx) or abundance (using NHE-3 specific antibody) and H(+)-ATPase transporter abundance (using antibody against the 31 kDa subunit). The Na+/H+ exchanger activity increased by 55% in metabolic acidosis (pH 6.5, HCO3- 3 mM) and decreased by 41% in metabolic alkalosis (pH 8.0, HCO3- 90 mM). The abundance of NHE-3 remained constant in acidic, control, and alkalotic groups. H(+)-ATPase abundance, however, decreased in metabolic acidosis and increased in metabolic alkalosis by 57% and 42%, respectively. In PT suspensions incubated in isohydric conditions (pH 7.4), Na+/H+ exchanger activity increased by 29% in high HCO3- group (HCO3- 96 mM) and decreased by 16% in the low HCO3- groups (HCO3- 7 mM. The NHE-3 abundance remained constant in high, normal, and low [HCO3-] tubules. The abundance of H(+)-ATPase, however, increased by 82% in high [HCO3-] and decreased by 77% in the low [HCO3-] tubules. In PT suspensions incubated in varying pCO2 and constant [HCO3-], Na+/H+ exchanger activity increased by 35% in high pCO2 (20% pCO2, respiratory acidosis) and decreased by 32% in low pCO2 (1.5% pCO2, respiratory alkalosis) tubules. The NHE-3 abundance remained unchanged in high, normal, and low pCO2 tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

A unique sodium-hydrogen exchange isoform (NHE-4) of the inner medulla of the rat kidney is induced by hyperosmolarity.

Membrane sodium-hydrogen exchangers (NHEs), found in virtually all cell types, appear to have diverse and essential roles in regulating cellular pH and mediating vectorial transport by epithelial cells. However, the functional and physiological role of the recently cloned isoform NHE-4 remains unknown. Unlike other Na-H exchanger isoforms, NHE-4 transfected into NHE-deficient mutant fibroblasts demonstrated no amiloride-inhibitable sodium uptake, under basal or acid-loaded isoosmotic conditions. By immunoblot analysis, only the NHE-4 transfectants synthesized a 100-kDa protein, which cross-reacted to polyclonal antibody made to an NHE-4 fusion protein. However, when cells were subjected to acute hyperosmolar cell shrinkage conditions, amiloride-sensitive NHE activity was readily detected at 420 mosm, exhibiting maximal activity at 490 mosm. By in situ hybridization, NHE-4 expression in the rat kidney was found to be limited to the inner renal medullary collecting tubules, the region of highest tissue osmolarity fluctuations in the body. We conclude that NHE-4 is an unusual isoform of sodium-hydrogen exchangers that may play a specialized supplementary role in cell volume regulation.

Localization of the Na+/H+ exchanger isoform NHE-3 in rabbit and canine kidney.

The distribution and subcellular localization of Na+/H+ exchanger isoform NHE-3 was studied in rabbit and canine kidney using polyclonal antibodies to an NHE-3 fusion protein. Western blot analyses were performed against microsomal, brush-border, and basolateral membranes isolated from rabbit kidney cortex, outer medulla, and inner medulla. Immunoblots indicated that NHE-3 antibody recognized a strong band with 95-100 kDa molecular mass in cortical microsomes. Subcellular localization studies showed that NHE-3 was expressed in brush-border membranes of kidney cortex. Expression of NHE-3 in the medullary regions was studied by immunoblot analysis of NHE-3 antibody against the microsomal membranes from the outer and inner medulla. NHE-3 antibody specifically labelled a 95-100 kDa protein in outer but not inner medulla. Subcellular localization studies demonstrated that NHE-3 is localized to the brush-border membranes of the outer medulla. Immunoblot analysis against brush-border membranes from canine kidney cortex and outer medulla demonstrated the presence of an 83-90 kDa protein. The above experiments suggest that NHE-3 in rabbit kidney is a 95-100 kDa protein and is expressed in brush-border membranes of the cortex and outer medulla. The canine kidney NHE-3 is a 83-90 kDa protein and is expressed in brush-border membranes of the cortex and outer medulla. Based on its subcellular localization, we conclude that NHE-3 may be involved in vectorial Na+ and HCO3- transport and pHo regulation.

Glucocorticoids regulate Na+/H+ exchange expression and activity in region- and tissue-specific manner.

Na+/H+ exchangers (NHEs) are integral membrane proteins that exchange Na+ for H+ across membranes. Four isoforms have been cloned (NHE-1-4). NHE-3 localizes to the apical domain, and its expression is increased in dexamethasone-treated rats by Northern analysis. This stimulatory effect on expression is region and tissue specific, being present in ileum and proximal colon, but not in jejunum, distal colon, or kidney. The increase in transcript expression in ileum correlates with an increase in protein expression by immunoblotting. Changes in apical Na+/H+ exchange activity, as measured by 22Na uptake into brush-border membrane vesicles, correlate with expression differences, with significant increases observed in ileum and proximal colon. In situ hybridization showed NHE-3 mRNA only in villus and absorptive cells of control rats, the pattern not being altered by dexamethasone treatment. This suggests that dexamethasone does not increase expression by inducing crypt cells to express NHE-3 prematurely. We conclude that glucocorticoids selectively increase intestinal NHE-3 activity in a region-specific manner and that this effect also appears to be tissue specific.

Effect of high osmolality on Na+/H+ exchange in renal proximal tubule cells.

Na+/H+ exchanger isoform and the effect of high osmolality on its function was studied in cultured renal epithelial cells (LLC-PK1 and OK). Using NHE-3-specific antibody, immunoblots of luminal membranes from LLC-PK1 and OK cells specifically labeled proteins with molecular masses 90 and 95 kDa, indicating that NHE-3 is the isoform expressed on the luminal membranes of these epithelia. Proximal tubular suspensions from rabbit kidney cortex were incubated in control (310 mosm/liter) or high osmolality (510 mosm/liter) medium for 45 min and utilized for brush border membrane vesicle preparation. Influx of amiloride-sensitive 22Na+ at 10 s (pHo 7.5, pHi 6.0) into brush border membrane vesicles was 37% lower in the high osmolality group (p < 0.03). LLC-PK1 or OK cells were grown to confluence and examined for Na+/H+ exchange activity. An increase in medium osmolality to 510 mosm following acid loading decreased the 5-min uptake of the amiloride-sensitive 22Na+ in LLC-PK1 and OK cells (p < 0.04 and < 0.03 for LLC-PK1 cell OK cells, respectively). An increase in medium osmolality to 510 mosm in vascular smooth muscle cells, which express NHE-1, produced 45 and 64% stimulation of the amiloride-sensitive 22Na+ influx at base-line pHi and acid-loaded condition, respectively (p < 0.03 and < 0.01). Down-regulation of protein kinase C by preincubation with phorbol 12-myristate 13-acetate or inhibition of Ca(2+)-calmodulin-dependent protein kinase (calmodulin-kinase II) by N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W-7) in LLC-PK1 cells did not block the inhibitory effect of high osmolality on Na+/H+ exchange activity. We conclude that renal proximal tubule epithelial cells express Na+/H+ exchange isoform NHE-3 on their luminal membranes and that hyperosmolality decreases transporter activity during cell acidification. This inhibitory effect might be unique to the NHE-3 isoform, since vascular smooth muscle cells which express NHE-1 exhibit an increase in Na+/H+ exchange activity in response to high osmolality.