The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent,but can couple to an H+ conductance

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT- PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time- course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH- sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.     

Apical NHE isoforms differentially regulate butyrate-stimulated Na absorption in rat distal colon

Bicarbonate and butyrate stimulate electroneutral Na absorption via apical membrane Na-H exchange (NHE) in rat distal colon. cAMP downregulates NHE-3 isoform and inhibits HCO3-dependent, but not butyrate-dependent, Na absorption. This study sought to determine whether 1) the apical membrane NHE-2 and NHE-3 isoforms differentially mediated HCO3- and butyrate-dependent Na absorption, and 2) cAMP had different effects on NHE-2 and NHE-3 isoforms. The effect of specific inhibitors of NHE-2 and NHE-3 isoforms (50 microM HOE 694 and 2 microM S3226, respectively) on unidirectional 22Na transepithelial fluxes performed across isolated mucosa from rat distal colon under voltage-clamp conditions was examined. HCO3 stimulation of Na absorption was inhibited by EIPA, a nonspecific inhibitor of all NHE isoforms, by S3226 and dibutyryl cAMP but not by HOE 694. In contrast, butyrate stimulation of Na absorption was not altered by dibutyryl cAMP and was not inhibited by HOE 694 in the absence of dibutyryl cAMP, but in the presence of dibutyryl cAMP was HOE694 sensitive. In contrast, S3226 inhibited butyrate-stimulated Na absorption in the absence of dibutyryl cAMP, but not in its presence. We conclude that 1) HCO3-stimulated Na absorption is mediated solely by NHE-3 isoform, whereas butyrate-stimulated Na absorption is mediated by either NHE-3 or NHE-2 isoform, and 2) dibutyryl cAMP selectively inhibits NHE-3 isoform but stimulates NHE-2 isoform. Dibutyryl cAMP does not inhibit butyrate-stimulated Na absorption as a result of its differential effects on NHE-2 and NHE-3 isoforms.     

Regulation of DRA and AE1 in rat colon by dietary Na depletion

Two distinct Cl/anion exchange activities (Cl/HCO(3) and Cl/OH) identified in apical membranes of rat distal colon are distributed in cell type-specific patterns. Cl/HCO(3) exchange is expressed only in surface cells, whereas Cl/OH exchange is localized in surface and crypt cells. Dietary Na depletion substantially inhibits Cl/HCO(3) but not Cl/OH exchange. We determined whether anion exchange isoforms (AE) and/or downregulated in adenoma (DRA) are expressed in and related to apical membrane anion exchanges by examining localization of AE isoform-specific and DRA mRNA expression in normal and Na-depleted rats. Amplification of AE cDNA fragments by RT-PCR with colonic mRNA as template indicates that AE1 and AE2 but not AE3 mRNAs are expressed. In situ hybridization study revealed that AE1 mRNA is expressed predominantly in surface but not crypt cells. In contrast, AE2 polypeptide is expressed in basolateral membranes and DRA protein is expressed in apical membranes of both surface and crypt cells. AE1 mRNA is only minimally present in proximal colon, and DRA mRNA abundance is similar in distal and proximal colon. Dietary Na depletion reduces AE1 mRNA abundance but did not alter DRA mRNA abundance. This indicates that AE1 encodes surface cell-specific aldosterone-regulated Cl/HCO(3) exchange, whereas DRA encodes aldosterone-insensitive Cl/OH exchange.     

Characterization of apical membrane Cl-dependent Na/H exchange in crypt cells of rat distal colon

A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cells of rat distal colon. The presence of Cl is required for both outward proton gradient-driven Na uptake in apical membrane vesicles (AMV) and Na-dependent intracellular pH recovery from an acid load in the crypt gland. The present study establishes that Cl-dependent outward proton gradient-driven (22)Na uptake 1) is saturated with increasing extravesicular Na concentration with a Michaelis constant (K(m)) for Na of approximately 24.2 mM; 2) is saturated with increasing outward H concentration gradient with a hyperbolic curve and a K(m) for H of approximately 1.5 microM; 3) is inhibited by the Na/H exchange (NHE) inhibitors amiloride, ethylisopropylamiloride, and HOE-694 with an inhibitory constant (K(i)) of approximately 480.2, 1.1, and 9.5 microM, respectively; 4) is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor at low concentration and a Cl channel blocker at high dose, and by 5-nitro-2(3-phenylpropylamino)benzoic acid, a Cl channel blocker, with a K(i) of approximately 280.6 and 18.3 microM, respectively; and 5) substantially stimulated Cl-NHE activity by dietary Na depletion, which increases plasma aldosterone and inhibits NHE in surface cell AMV. These properties of Cl-NHE are distinct from those of NHE1, NHE2, and NHE3 isoforms that are present in colonic epithelial cells; thus these results suggest that the colonic crypt cell Cl-NHE is a novel NHE isoform.     

NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice

The mechanism of apical Na(+)-dependent H(+) extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pH(i)) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na(+) stimulated similar rates of pH(i) recovery in cells at the base of distal colonic crypts of wild-type or Na(+)/H(+) exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 microM HOE694 (NHE2 inhibitor) blocked 68-75% of the pH(i) recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 microM), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 microM), inhibited pH(i) recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na(+) leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na(+)-dependent pH(i) recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pH(i) recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na(+)-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.     

Non-genomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon

Studies from our laboratory have demonstrated rapid ( < 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC activity and a PKC-dependent Ca(2+) entry through L-type Ca(2+) channels specifically by mineralocorticoids in distal colon. Aldosterone directly stimulates the activity of the PKC alpha isoform (but not PKC delta, PKC epsilon and PKC zeta) in a cell-free assay system containing only purified commercially available enzyme, appropriate substrate peptide, co-factors and lipid vesicles. The primary ion transport target of the non-genomic signal transduction cascade elicited by aldosterone in epithelia is the Na(+)-H(+) exchanger. In isolated colonic crypts, aldosterone produced a PKC alpha sensitive intracellular alkalinisation within 1 min of hormone addition. Intracellular alkalinisation upregulates an ATP-dependent K(+) channel, which is involved in K(+) recycling to maintain the electrical driving force for Na(+) absorption, while inhibiting a Ca(2+) -dependent K(+) channel, which generates the charge balance for Cl(-) secretion. The non-genomic response to aldosterone in distal colon appears to enhance the capacity for absorption while down-regulating the potential for secretion. We have also demonstrated rapid (< 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC alpha activity, and a PKC delta- and PKA-dependent Ca(2+) entry through di-hydropyridine-blockable Ca(2+) channels specifically by 17beta-estradiol in distal colon. These rapid effects are female gender specific and are insensitive to inhibitors of the classical estrogen receptor (ER). 17 beta-Estradiol directly stimulated the activity of both PKC delta and PKC alpha (but not PKC epsilon or PKC zeta) in a cell-free assay system. E2 rapidly inhibited basolateral K(Ca) channel activity which would be expected to result in an acute inhibition of Cl(-) secretion. Physiological concentrations of E2 (0.1-10 nM) reduced both basal and secretagogue-induced Cl(-) secretion. This anti-secretory effect of E2 is sensitive to PKC inhibition, intracellular Ca(2+) chelation, and is female gender specific and insensitive to inhibitors of the classical ER. These observations link rapid non-genomic activation of second messengers with a rapid gender-specific physiological effect in the whole tissue. Aldosterone and E2 differ in their protein kinase signal transduction and both hormones stimulate specific PKC isoforms indicating both common and divergent signalling systems for salt-retaining steroid hormones. The physiological function of non-genomic effects of aldosterone and estradiol is to shift the balance from net secretion to net absorption in a pluripotential epithelium.     

Apical Na+/H+ exchange near the base of mouse colonic crypts

Colonic crypts can absorb fluid, but the identity of the absorptive transporters remains speculative. Near the crypt base, the epithelial cells responsible for vectorial transport are relatively undifferentiated and often presumed to mediate only Cl- secretion. We have applied confocal microscopy in combination with an extracellular fluid marker [Lucifer yellow (LY)] or a pH-sensitive dye (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to study mouse colonic crypt epithelial cells directly adjacent to the crypt base within an intact mucosal sheet. Measurements of intracellular pH report activation of colonocyte Na+/H+ exchange in response to luminal or serosal Na+. Studies with LY demonstrate the presence of a paracellular fluid flux, but luminal Na+ does not activate Na+/H+ exchange in the nonepithelial cells of the lamina propria, and studies with LY suggest that the fluid bathing colonocyte basolateral membranes is rapidly refreshed by serosal perfusates. The apical Na+/H+ exchange in crypt colonocytes is inhibited equivalently by luminal 20 microM ethylisopropylamiloride and 20 microM HOE-694 but is not inhibited by luminal 20 microM S-1611. Immunostaining reveals the presence of epitopes from NHE1 and NHE2, but not NHE3, in epithelial cells near the base of colonic crypts. Comparison of apical Na+/H+ exchange activity in the presence of Cl- with that in the absence of Cl- (substitution by gluconate or nitrate) revealed no evidence of the Cl--dependent Na+/H+ exchange that had been previously reported as the sole apical Na+/H+ exchange activity in the colonic crypt. Results suggest the presence of an apical Na+/H+ exchanger near the base of crypts with functional attributes similar to those of the cloned NHE2 isoform.     

Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism.

A variety of cell types regulate their volume in anisotonic media by stimulating Na+/H+ exchange. Like growth factors, osmotic challenge activates the Na+/H+ antiport by increasing its sensitivity to intracellular [H+]. To investigate the molecular mechanism underlying this shift in pH sensitivity, the antiporter of 32P-labeled human bladder carcinoma cells and of Chinese hamster ovary cells was immunoprecipitated using antibodies raised against the cytosolic domain of the NHE-1 isoform of the Na+/H+ exchanger. Unlike the effects of growth promoters, activation of the antiport during volume regulation was not associated with increased phosphorylation. The possible coexistence of multiple antiporter isoforms was considered. The cytosolic alkalosis normally elicited by hypertonic media was found to be absent in Na+/H+ exchange-deficient fibroblasts. Responsiveness to osmotic challenge was restored by stable transfection of these cells with the cDNA encoding NHE-1. In these transfectants, phosphorylation of the antiporter was also unaffected during osmotic activation. The unchanged phosphate content of the antiporter might be explained by dephosphorylation of one site with concomitant phosphorylation at a different site. However, this possibility appears unlikely since phosphoamino acid analysis revealed that serine was the only residue phosphorylated in immunoprecipitated antiports of both control and osmotically stimulated cells. Moreover, phosphopeptide maps of control and hypertonically activated antiports were identical. These findings reveal a novel mode of activation of Na+/H+ exchange not requiring direct phosphorylation of the antiporter. We propose the existence of dual control of Na+/H+ exchange by phosphorylation-dependent and -independent mechanisms.     

Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.     

Isolation and characterization of a cDNA encoding the putative distal colon H+,K(+)-ATPase. Similarity of deduced amino acid sequence to gastric H+,K(+)-ATPase and Na+,K(+)-ATPase and mRNA expression in distal colon, kidney, and uterus.

A series of Northern blot hybridization experiments using probes derived from the rat gastric H+,K(+)-ATPase cDNA and the human ATP1AL1 gene revealed the presence of a 4.3-kilobase mRNA in colon that seemed likely to encode the distal colon H+,K(+)-ATPase, the enzyme responsible for K+ absorption in mammalian colon. A rat colon library was then screened using a probe from the ATP1AL1 gene, and cDNAs containing the entire coding sequence of a new P-type ATPase were isolated and characterized. The deduced polypeptide is 1036 amino acids in length and has an Mr of 114,842. The protein exhibits 63% amino acid identity to the gastric H+,K(+)-ATPase alpha-subunit and 63% identity to the three Na+,K(+)-ATPase alpha-subunit isoforms, consistent with the possibility that it is a K(+)-transporting ATPase. Northern blot analyses show that the 4.3-kilobase mRNA is expressed at high levels in distal colon; at much lower levels in proximal colon, kidney, and uterus; and at trace levels in heart and forestomach. The high mRNA levels in distal colon and the similarity of the colon pump to both gastric H+,K(+)- and Na+,K(+)-ATPases suggest that it is the distal colon H+,K(+)-ATPase. Furthermore, expression of its mRNA in kidney raises the possibility that the enzyme also corresponds to the H+,K(+)-ATPase that seems to play a role in K+ absorption and H+ secretion in the distal nephron.     

Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.

Biochemical and pharmacological data support the existence of multiple forms of the Na/H exchanger (NHE). Two isoforms, termed NHE-1 and NHE-2, have recently been isolated from rabbit ileal villus epithelial cells (Tse, C. M., Ma, A. I., Yang, V. W., Watson, A. J. M., Levine, S., Montrose, M. H., Potter, J., Sardet, C., Pouysségur, J., and Donowitz, M. (1991) EMBO J. 10, 1957-1967; Tse, C. M., Watson, A. J. M., Ma, A. I., Pouysségur, J., and Donowitz, M. (1991) Gastroenterology 100, A258). To identify additional molecular forms of the exchanger, rat brain, heart, kidney, stomach, and spleen cDNA libraries were screened for their presence using an NHE-1 cDNA probe under low stringency hybridization conditions. cDNAs encoding rat NHE-1 and two structurally related proteins, designated NHE-3 and NHE-4, have been isolated. Based on the deduced amino acid sequences, NHE-1, -3, and -4 are similar in size, having relative molecular masses of 91,506, 92,997, and 81,427, respectively. Overall, the proteins exhibit approximately 40% amino acid identity to each other and have similar hydropathy profiles, suggesting that they have the same transmembrane organization. The predicted N-terminal transmembrane regions of the three proteins, which span between 453 and 503 amino acids, exhibit the highest degree of identity (45-49%). In contrast, the C-terminal cytoplasmic regions, which span between 247 and 378 amino acids, exhibit very low amino acid identity (24-31%). Tissue distribution studies reveal that the NHE-1 mRNA is present at varying levels in all tissues examined, whereas NHE-3 and NHE-4 mRNAs exhibit a more limited distribution. NHE-3 mRNA is expressed at high levels in colon and small intestine, with significant levels also present in kidney and stomach. NHE-4 mRNA is most abundant in stomach, followed by intermediate levels in small intestine and colon and lesser amounts in kidney, brain, uterus, and skeletal muscle. These data suggest that the molecular basis for the functional diversity of the Na/H exchanger in mammals is based, at least in part, on expression of multiple members of a gene family.     

The Na+-driven Cl-/HCO3- exchanger. Cloning, tissue distribution, and functional characterization

The Na(+)-driven Cl(-)/HCO(3)(-) exchanger is an important regulator of intracellular pH in various cells, but its molecular basis has not been determined. We show here the primary structure, tissue distribution, and functional characterization of Na(+)-driven chloride/bicarbonate exchanger (designated NCBE) cloned from the insulin-secreting cell line MIN6 cDNA library. The NCBE protein consists of 1088 amino acids having 74, 72, and 55% amino acid identity to the human skeletal muscle, rat smooth muscle, and human kidney sodium bicarbonate cotransporter, respectively. The protein has 10 putative membrane-spanning regions. NCBE mRNA is expressed at high levels in the brain and the mouse insulinoma cell line MIN6 and at low levels in the pituitary, testis, kidney, and ileum. Functional analyses of the NCBE protein expressed in Xenopus laevis oocytes and HEK293 cells demonstrate that it transports extracellular Na(+) and HCO(3)(-) into cells in exchange for intracellular Cl(-) and H(+), thus raising the intracellular pH. Thus, we conclude that NCBE is a Na(+)-driven Cl(-)/HCO(3)(-) exchanger that regulates intracellular pH in native cells.     

The Na+/H+ exchanger isoform 1

The Na+/H+ exchanger (NHE) isoform 1 is a ubiquitously expressed integral membrane protein which regulates intracellular pH in mammalian cells. Nine isoforms of the Na+/H+ exchanger have been identified. The isoform first discovered has two domains: an N-terminal membrane domain containing approximately 500 amino acids and a C-terminal regulatory domain containing approximately 315 amino acids. The exchanger, which resides in the plasma membrane, exchanges an intracellular proton for an extracellular sodium, thereby regulating intracellular pH. It is involved in cell growth and differentiation, cell migration, and regulation of sodium fluxes. The Na+/H+ exchanger plays an important role in myocardial damage during ischemia and reperfusion and has recently been implicated as a mediator of cardiac hypertrophy. Inhibitors of the Na+/H+ exchanger, which may prove useful in the clinical treatment of these conditions, are currently being developed and clinical trials are underway.     

Involvement of the Na+-independent Cl-/HCO3- exchange (AE) isoform in the compensation of myocardial Na+/H+ isoform 1 hyperactivity in spontaneously hypertensive rats

Enhanced activity of Na+/H+ isoform 1 (NHE-1) and the Na+-independent Cl-/HCO3- exchange (AE) is a feature of the hypertrophied myocardium in spontaneously hypertensive rats (SHR). The present study explored the possibility that sustained intracellular acidosis due to increased myocardial acid loading through AE causes NHE-1 enhancement. To this aim, SHR were treated for 2 weeks with a rabbit polyclonal antibody against an AE3 isoform that was recently developed and proven to have inhibitory effects on myocardial AE activity. We then compared the AE activity in the left ventricle papillary muscles isolated from untreated SHR with antiAE3-treated SHR; AE activity was measured in terms of the rate of intracellular pH recovery after an intracellular alkali load was introduced. AE activity was diminished by approximately 70% in SHR treated with the antiAE3 antibody, suggesting that the AE3 isoform is a major carrier of acid-equivalent influx in the hypertrophied myocardium. However, the antibody treatment failed to normalize NHE-1 activity that remained elevated in the myocardium of normotensive rats. The data therefore rule out the possibility that NHE-1 hyperactivity in hypertensive myocardium was due to sustained intracellular acidosis induced by increased AE activity that characterizes SHR myocardial tissue.     

The cardiac Na-H exchanger: a key downstream mediator for the cellular hypertrophic effects of paracrine, autocrine and hormonal factors.

The major mechanism by which the heart cell regulates intracellular pH is the Na(+)-H(+) exchanger (NHE) with the NHE-1 isoform as the primary cardiac subtype. Although NHE-1 has been implicated in mediating ischemic injury, more recent evidence implicates the antiporter as a key mediator of hypertrophy, which is produced by various autocrine, paracrine and hormonal factors such as endothelin-1, angiotensin II, and alpha(1) adrenoceptor agonists. These agonists activate the antiporter via phosphorylation-dependent processes. NHE-1 inhibition is likely conducive to attenuating the remodelling process after myocardial infarction. These effects probably occur independently of infarct size reduction and involve attenuation of subsequent postinfarction heart failure. As such, inhibitors of NHE offer substantial promise for clinical development that will attenuate acute responses to myocardial postinfarction and chronic pos t infarction, which evolve toward heart failure. The regulation of NHE-1 is discussed as is its potential role in mediating cardiomyocyte hypertrophy.