Dependent pH Recovery Research Articles

Gene Variants in Sodium Bicarbonate Cotransporter NBCe2 Increase Sodium and Bicarbonate Transport in Human Renal Proximal Tubule

RationaleThe sodium bicarbonate cotransporter NBCe2 (encoded by SLC4A5) is involved in the regulation of renal tubular sodium bicarbonate transport. We hypothesized that human SLC4A5 single nucleotide polymorphisms (SNPs, rs10177833 and rs7571842) increase NBCe2 expression and renal proximal tubule (RPT) sodium transport and may be a cause of salt sensitivity of blood pressure.ObjectiveTo characterize the function of wild‐type (WT) and homozygous variants (HV) of SLC4A5.Methods and ResultsThe expressions of NBCe2 mRNA and protein were not different between RPT cells (RPTCs) carrying WT (n=4) or HV SLC4A5 (n=6) before or after dopaminergic or angiotensin (II and III) stimulation. However, total transcellular (luminal to basolateral) RPTC sodium transport, NHE3 protein expression, and Cl−/HCO3− exchanger activity were higher in HV than WT SLC4A5. Bicarbonate‐dependent pH recovery after an intracellular acid load, as well as NBCe2 mRNA and protein, was increased when intracellular sodium was elevated (5.2±1.1 mM) in HV SLC4A5 compared with WT SLC4A5 (pH recovery Vmax 0.22±0.02 and Km 6.39±0.81 in HV vs Vmax 0.28±0.02 and Km 4.30±0.53 in WT, P<0.05, P<0.05, n=3). The increased NBCe2‐specific bicarbonate‐dependent pH recovery in HV was verified in stable SLC4A5 shRNA cell lines (n=8/12/group). RPTCs carrying HV SLC4A5 had increased binding of HNF4A to SLC4A5 DNA (1.52±0.10‐fold, N=3, P<0.05, t‐test), using purified HNF4A and ChIP and oligo binding assays. Additionally, the difference between HV and WT in bicarbonate‐dependent pH recovery was abolished by two HNF4A antagonists. RPTCs isolated from human urine of salt‐sensitive subjects who are HV for SLC4A5 had increased bicarbonate dependent pH recovery, relative to salt‐resistant subjects who are WT for SLC4A5 (n=4/group).ConclusionsNBCe2 activity is stimulated by an increase in intracellular sodium and is hyper‐responsive in RPTCs carrying HV SLC4A5 through an aberrant HNF4A‐mediated mechanism, which may be a key mechanism in the etiology of human salt sensitivity.

Abstract 116: Characterization of a Sodium Responsive Human Sodium Bicarbonate Transporter NBCe2 in Human Proximal Tubule

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure (BP), which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT), another site of bicarbonate transport. The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by siRNA and Western blot, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and HEK293 cells. We immune-localized (peptide pre-absorbable) NBCe2 protein in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and in isolated RPTC apical membrane. NBCe2 was primarily found in the Golgi while NBCe1, another electrogenic member of the same family, was primarily found at the basolateral membrane, under basal conditions. Following a short-term increase in intracellular sodium, NBCe2 expression increased at the apical membrane in cultured slices of human kidney (1.75±0.15 fold increase over basal, N=3, p<0.05) and polarized, immortalized RPTCs (0.08±0.015 vs 0.03±0.006 control, NBCe2/CD13 RFU, N=3, p<0.05), while the basolateral localization of NBCe1 was decreased (30.7±5.4% over basal, N=3, p<0.05). DIDS (electrogenic sodium bicarbonate inhibitor, 500 μM, 10 min) inhibitable bicarbonate dependent pH recovery in the presence of EIPA (NHE inhibitor,100 nM, 10 min) after ammonium chloride prepulse (20 mM, 5 min) was higher in the cells cultured under high salt conditions (26.7±7.2% over basal, N=3, p<0.05). Thus, NBCe2 appears to be important in apical sodium and bicarbonate cotransport under high salt conditions. Future studies will examine the role of NBCe2 in mediating increased renal sodium transport in human salt sensitivity of BP.

Effects of MS-31-038, a novel Na(+)-H+ exchange inhibitor, on the myocardial infarct size in rats after postischemic administration.

The abilities of 2-(2-methylphenyl)-5,7-dimethoxy-4-quinolyl carbonylguanidine dihydrochloride (CAS 181048-29-3, MS-31-050) and 2-phenyl-8-(2-methoxyethoxy)-4-quinolyl carbonylguanidine bismethanesulfonate (CAS 181048-36-2, MS-31-038) in inhibiting Na(+)-H+ exchange, ischemia- and reperfusion-induced injury were determined and compared with those of 4-isopropyl-3-methylsulfonylbenzoyl guanidine methanesulfonate (CAS 159138-81-5, IMGM), a selective inhibitor of Na(+)-H+ exchange. MS-31-050 and IMGM exhibited comparable inhibitory effects on Na(+)-dependent pH recovery and antiarrhythmic effects during ischemia in anesthetized rats. In rats subjected to ischemia and reperfusion, MS-31-050 (10 mg/kg i.v.) significantly reduced the infarct size when given prior to the onset of ischemia. However, postischemic treatment with either MS-31-050 or IMGM failed to protect reperfused hearts. In contrast, MS-31-038 reduced the infarct size dramatically from 65.4 +/- 7.4% in control to 29.9 +/- 11.6% at 3 mg/kg and 9.8 +/- 3.4% at 10 mg/kg even when administered before the onset of reperfusion. These results suggest the beneficial effects of Na(+)-H+ exchange inhibitors on myocardial ischemia/reperfusion injury.

Stimulation of electrogenic intestinal dipeptide transport by the glucocorticoid dexamethasone

According to recent in vitro experiments, the peptide transporter PepT2 is stimulated by the serum- and glucocorticoid-inducible kinase SGK1. The present study explored the contribution of SGK1 to the regulation of electrogenic intestinal peptide transport. Intestinal PepT1 was expressed in Xenopus oocytes, and peptide transport was determined by dual electrode voltage clamping. Peptide transport in intestinal segments was determined utilizing Ussing chamber. Cytosolic pH (pH( i )) was determined by BCECF fluorescence and Na(+)/H(+) exchanger activity was estimated from Na(+)-dependent pH recovery (pH ( i )) following an ammonium pulse. In PepT1-expressing Xenopus oocytes, coexpression of SGK1 enhanced electrogenic peptide transport. Intestinal transport and pH( i ) of untreated mice were similar in SGK1 knockout mice (sgk1 ( -/- )) and their wild-type littermates (sgk1 ( +/+ )). Glucocorticoid treatment (4 days 10 microg/g body weight (bw)/day dexamethasone) increased peptide transport in sgk1 ( +/+ ) but not in sgk1 (-/-) mice. Irrespective of dexamethasone treatment, luminal peptide (5 mM glycyl-glycine) led to a similar early decrease of pH( i ) in sgk1 (-/-) and sgk1 (+/+) mice, but to a more profound and sustained decline of pH( i ) in sgk1 (-/-) than in sgk1 ( +/+ ) mice. In the presence and absence of glycyl-glycine, pH ( i ) was significantly enhanced by dexamethasone treatment in sgk1 ( +/+ ) mice, an effect significantly blunted in sgk1 ( -/- ) mice. During sustained exposure to glycyl-glycine, pH ( i ) was significantly larger in sgk1 (+/+) mice than in sgk1 (-/-) mice, irrespective of dexamethasone treatment. In conclusion, basal intestinal peptide transport does not require stimulation by SGK1. Glucocorticoid treatment stimulates both Na(+)/H(+) exchanger activity and peptide transport, effects partially dependent on SGK1. Moreover, chronic exposure to glycyl-glycine stimulates Na(+)/H(+) exchanger activity, an effect again involving SGK1.

Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO(3) transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO(3) transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO(3)(-) currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO(3)-dependent pH recovery from a CO(2)-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO(2)/HCO(3)(-)-free solution that was abolished by Cl(-) removal from the bath. In the presence of CO(2)/HCO(3)(-), however, the outward current produced by D555E decreased only slightly after Cl(-) removal. Starting from a Cl(-)-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO(2)/HCO(3)(-). The substitution of Asp(555) with an asparagine also produced a Cl(-) current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO(3)(-). Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp(555) plays a role in HCO(3)(-) selectivity.

IRBIT, Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3, Binds Na+/H+ Exchanger NHE3 and Activates NHE3 Activity in Response to Calcium

Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.

Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse

In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-inducible kinase (SGK)1 on the activity of the Na+/H+ exchanger (NHE3). SGK1 requires activation by phosphoinositide-dependent kinase (PDK)1, which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study was performed to explore the role of PDK1 in the regulation of NHE3 activity. Because mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing approximately 20% of PDK1 (pdk1(hm)) were compared with their wild-type littermates (pdk1(wt)). NHE3 activity in the intestine and PDK1-overexpressing HEK-293 cells was estimated by utilizing 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence for the determination of intracellular pH. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium prepulse (DeltapH(NHE)). The pH changes after an ammonium pulse allowed the calculation of cellular buffer capacity, which was not significantly different between pdk1(hm) and pdk1(wt) mice. DeltapH(NHE) was in pdk1(hm) mice, only 30 +/- 6% of the value obtained in pdk1(wt) mice. Conversely, DeltapH(NHE) was 32 +/- 7% larger in PDK1-overexpressing HEK-293 cells than in HEK-293 cells expressing the empty vector. The difference between pdk1(hm) and pdk1(wt) mice and between PDK1-overexpressing and empty vector-transfected HEK cells, respectively, was completely abolished in the presence of the NHE3 inhibitor S3226 (10 microM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in the intestine, pointing to a role of PDK1-dependent signaling in the regulation of NHE-mediated electrolyte transport.

Intestinal function of gene-targeted mice lacking serum- and glucocorticoid-inducible kinase 1

In vitro experiments have revealed the ability of serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate intestinal Na(+)-coupled glucose cotransporter 1 (SGLT1) and intestinal Na(+)/H(+) exchanger 3 (NHE3). The present study explored the contribution of SGK1 to the regulation of intestinal transport in vivo. SGK1 transcript levels were determined by real-time PCR and glucose-induced currents (I(g)) reflecting SGLT1 activity by Ussing chamber experiments. BCECF fluorescence was utilized for the determination of Na(+)-dependent pH recovery from an ammonium pulse (DeltapH(NHE)) reflecting NHE activity. As a result, intestinal SGK1 transcript levels were significantly enhanced by a 4-day treatment with 10 microg.mg body wt(-1).day(-1) dexamethasone (Dex). I(g) was, under control conditions, virtually identical in sgk1 knockout mice (sgk1(-/-)) and their wild type littermates (sgk1(+/+)). A 4-day treatment with Dex, however, increased I(g) approximately threefold in sgk1(+/+) mice but not in sgk1(-/-) mice. DeltapH(NHE) was similar in sgk1(-/-) and sgk1(+/+) mice before treatment. Dex increased DeltapH(NHE) approximately threefold in sgk1(+/+) mice and approximately twofold in sgk1(-/-)mice, an effect significantly blunted in the presence of the specific NHE3 blocker S-3226 (10 microM). According to Western blot analysis, Dex significantly enhanced SGLT1 and NHE3 protein abundance in brush-border membranes of sgk1(+/+) mice but not of sgk1(-/-)mice. In conclusion, basic functions of SGLT1 and NHE3 in the intestine do not require stimulation by SGK1. However, the effects of glucocorticoids on SGLT1 are fully, and on NHE3 partially, dependent on SGK1.

Aldosterone Stimulates Activity and Surface Expression of NHE3 in Human Primary Proximal Tubule Epithelial Cells (RPTEC)

The steroid hormone aldosterone is a major regulatorof extracellular volume and blood pressure.Aldosterone effectors are for example the epithelialNa+ channel (ENaC), the Na+-K+-ATPase and theproximal tubule Na+/H+ exchanger isoform 3 (NHE3).The aim of this study was to investigate whetheraldosterone acts directly on proximal tubule cells tostimulate NHE3 and if so whether the EGF-receptor(EGFR) is involved. For this purpose, primary humanrenal proximal tubule cells were exposed toaldosterone. NHE3 activity was determined from Na+-dependent pH-recovery, NHE3 surface expressionwas determined by biotinylation and immunoblotting.EGFR-expression was assessed by ELISA. pHi-measurements revealed an aldosterone-inducedincrease in NHE3 activity, which was inhibited by themineralocorticoid receptor blocker spironolactone andby the EGFR-kinase inhibitor AG1478. Immunoprecipitationand immunoblot analysis showed analdosterone-induced increase in NHE3 surface expression, which was also inhibited byspironolactone and AG1478. Furthermore,aldosterone enhanced EGFR-expression.In conclusion, aldosterone stimulates NHE3 in humanproximal tubule cells. The underlying mechanismsinclude AG1478 inhibitable kinase and are paralleledby enhanced EGFR expression, which could becompatible with EGF-receptor-pathway-dependentsurface expression and activity of NHE3 in humanprimary renal proximal tubule epithelial cells.

NBCn1 is a basolateral \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Na}^{+}\mathrm{-HCO}_{3}^{-}\) \end{document} cotransporter in rat kidney inner medullary collecting ducts

Primary cultures of rat inner medullary collecting duct (IMCD) cells Na(+) dependently import HCO(3)(-) across the basolateral membrane through an undefined transport protein. We used RT-PCR, immunoblotting, and immunohistochemistry to identify candidate proteins for this basolateral Na(+)-HCO(3)(-) cotransport. The mRNA encoding the electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1 was detected as the only Na(+)-HCO(3)(-) cotransporter in the rat inner medulla (IM) among the five characterized Na(+)-dependent HCO(3)(-) transporters. The mRNA of a yet uncharacterized transporter-like protein, BTR1, was also present in the IM, but its expression in microdissected tubules seemed restricted to the thin limbs of Henle's loop. Immunoblotting confirmed the presence of NBCn1 as an approximately 180-kDa protein of the rat IM. Anti-NBCn1 immunolabeling was confined to the basolateral plasma membrane domain of IMCD cells in the papillary two-thirds of the IM. Consistent with the presence of NBCn1, IMCD cells possessed stilbene-insensitive, Na(+)- and HCO(3)(-)-dependent pH recovery after acidification, as assessed by fluorescence microscopy using a pH-sensitive intracellular dye. In furosemide-induced alkalotic rats, NBCn1 protein abundance was decreased in both the IM and inner stripe of outer medulla (ISOM) as determined by immunoblotting and immunohistochemistry. In contrast, NBCn1 abundance in the IM and ISOM was unchanged in NaHCO(3)-loaded animals, and the NBCn1 abundance increased only in the ISOM after NH(4)Cl loading. In conclusion, NBCn1 is a basolateral Na(+)-HCO(3)(-) cotransporter of IMCD cells and is differentially regulated in IMCD and medullary thick ascending limb.

The NHX Family of Na+-H+ Exchangers in Caenorhabditis elegans

Na+-H+ exchangers prevent cellular acidification by catalyzing the electroneutral exchange of extracellular sodium for an intracellular proton. To date, seven Na+-H+ exchangers have been identified in mammals, and although several members of this family have been extensively studied and characterized, it is clear that there are major gaps in our understanding with respect to the remaining family members. To initiate the study of Na+-H+ exchangers in a genomically defined and genetically tractable model system, we have cloned the complete cDNAs and analyzed splice site variation for nine putative homologs from the nematode Caenorhabditis elegans, which we have called NHX-1 through -9. The expression patterns and cellular distributions of the NHX proteins were determined using transcriptional and translational promoter-transgene fusion constructs to green fluorescent protein. Four of the putative exchangers were expressed at the cell surface, whereas five of the exchangers were associated with the membranes of intracellular organelles. Individual isoforms were expressed exclusively in the intestine, seam cells, hypodermal cells of the main body syncytium, and the excretory cell, all of which are polarized epithelial cells, suggesting a role for these proteins in epithelial membrane transport processes in the nematode. Other isoforms were found to express either ubiquitously or in a pan-neural pattern, suggesting a more conserved role in cell pH regulation or neuronal function. Finally, we show that recombinant NHX-4, the ubiquitous nematode Na+-H+ exchanger, mediates Na+-dependent pH recovery after intracellular acidification. NHX-4 has a K(a) for Na+ of approximately 32 mm, is not Cl- -dependent, and is relatively insensitive to the amiloride analog EIPA.

Determination of Basolateral Na+/H+ Exchange Activity in MDCK Cells Using a Multiwell–Multilabel Reader

Na+/H+ exchangers (NHE) are important membrane transport proteins involved in transepithelial transport and cellular pH homeostasis. NHE-1, important for cellular pH and volume homeostasis, is expressed in the basolateral membrane of epithelial cells. We evaluated the use of a multiwell–multilabel reader to investigate basolateral NHE-1 in confluent MDCK cells and compared the results with data obtained using an imaging system equipped with a filter perfusion system. Using the multiwell–multilabel reader we obtained virtually the same values for steady-state pH and NHE-1 activity under control conditions compared to the imaging system. With both setups Na+-dependent pH recovery after an acid load occurred virtually only after basolateral addition of Na+. Furthermore, Na+-dependent pH recovery was reduced by >80% in the presence of the NHE-1 inhibitor HOE642. In addition, we detected an almost identical increase of NHE-1 activity with the two methods after stimulation of protein kinase C using phorbol myristate acetate. In summary, our data indicate that multiwell–multilabel readers are suitable to investigate physiology and regulation of basolateral NHE. Thus, multiwell–multilabel readers offer a valid and convenient alternative to investigate basolateral transporters.

Molecular cloning, tissue distribution, and functional expression of the human Na(+)/H(+) exchanger NHE2.

In the present report, we describe the cloning of a human colonic cDNA that describes the full-length Na(+)/H(+) exchanger (NHE) 2 coding region. The human NHE2 (hNHE2) cDNA encodes for a polypeptide of 812 amino acids with a 90% overall identity to both rabbit and rat NHE2 isoforms. In comparison with SLC9A2, recently reported as the human NHE2, the hNHE2 polypeptide is 115 amino acids longer in the NH(2)-terminal end and shows only an 84% DNA nucleotide sequence identity. Northern blot analysis revealed that hNHE2 message has an uneven tissue distribution, with high levels in the skeletal muscle, colon, and kidney and lower levels in the testis, prostate, ovary, and small intestine. Protein expression studies with hNHE2 clone showed that a 75-kDa protein was expressed. Stable expression of transfected full-length hNHE2 cDNA in Na(+)/H(+) exchange-deficient LAP1 cells exhibited Na(+)-dependent pH recovery after an acid prepulse that was inhibited by 0.1 mM amiloride. These data indicate that this cDNA is the true human NHE2 cDNA and that the encoded protein is capable of catalyzing Na(+)/H(+) exchange activity.

Expression of transfected human Na+/H+ exchanger (NHE-1) in the the basolateral membrane of opossum kidney cells.

Some epithelial cells have Na+/H+ exchanger (NHE) activity in both apical and basolateral membranes. Amiloride-sensitive NHE-1 is generally identified in the basolateral membrane. The renal cell line, OK7a, targets amiloride-resistant NHE predominantly to the apical membrane. It is controversial whether the transfected NHE-1 is targeted preferentially to the basolateral membrane in OK7a cells, when human NHE-1 is chronically expressed under control of constitutively active promoters. We tried to identify the membranes in which the transfected human NHE-1 could be detected following acute expression in OK7a cells. We have always observed small Na(+)-dependent pH recovery in the basolateral membrane in OK7a cells. It is, however, controversial whether or not OK7a cells express NHE activity in the basolateral membrane. We also characterized Na(+)-dependent pH recovery in the basolateral membrane. It was not inhibited by [4,4'diisothiocyanatostilbene-2,2'-disulfonic acid] (DIDS), [4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid] (SITS), or contralateral amiloride. Li+ but not K+, chol+, or NMG+ could replace Na+. These results are consistent with the presence of the NHE in the basolateral membrane. NHE activities were predominant in the apical membrane and those in both membranes were resistant to amiloride analogs. After stable transfection with human NHE-1 in a vector utilizing the metallothionein promoter, overnight induction with Zn(2+)increased the NHE activity and its sensitivity to amiloride only in the basolateral membrane in OK7a cells. We conclude that the transfected human NHE-1 is exclusively targeted to the basolateral membrane of OK7a cells during acute induction.

Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells.

Several isoforms of Na+/H+ exchanger (NHE-1-5) have been identified. LLC-PK1 clone 4 (CL4) expresses the amiloride-sensitive type of NHE predominantly in the basolateral membrane, which is believed to be NHE-1. It is not clear whether CL4 expresses NHE in the apical membrane and which side of NHE is encoded by the NHE-1 mRNA. Using acidified CL4 cells on the filter membrane, we examined Na(+)-dependent pH recovery of the apical and basolateral membranes separately. Na+ applied to the apical membrane recovered cell pH. Na(+)-dependent pH recovery in the apical membrane was not inhibited by SITS, DIDS, or contralateral amiloride. Li+ but not K+, chol+, or NMG+ could replace Na+. These data are consistent with the presence of NHE in the apical membrane. Transfection with an antisense oligonucleotide corresponding to the 5' terminal site of NHE-1 cDNA of CL4 decreased NHE activity in the basolateral membrane but not in the apical membrane. We conclude that CL4 expresses NHE activities in both apical and basolateral membranes and that NHE-1 mRNA encodes NHE only in the basolateral membrane.

Glucocorticoids stimulate Na+/H+ antiporter in OKP cells.

Previous studies have demonstrated that systemic administration of glucocorticoids stimulates proximal tubule acidification in part by increasing Na+/H+ antiporter activity; however, these studies could not exclude the possibility that changes in Na+/H+ antiporter activity were secondary to glucocorticoid-induced hemodynamic changes. The present study examined the effect of dexamethasone on Na+/H+ antiporter activity in quiescent OKP cells. Na+/H+ antiporter activity was assayed as the initial rate of Na(+)-dependent pH recovery from an acid load. Intracellular pH was measured using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Dexamethasone produced a dose- and time-dependent stimulation of Na+/H+ antiporter activity in OKP cells. Dexamethasone produced a 24% stimulation in Na+/H+ antiporter activity at 10(-9) M and an approximately 40% stimulation of Na+/H+ antiporter activity at both 10(-8) and 10(-6) M. The effect of 10(-6) M dexamethasone was seen within 4 h of incubation and was due to an increase in maximal velocity (Vmax, 3.03 vs. 1.79 pH units/min) with no change in the affinity constant for sodium (KNa, 47.2 vs. 42.0 mM). The stimulatory effect of dexamethasone on Na+/H+ antiporter activity was blocked by cycloheximide and was not observed with 10(-8) M aldosterone. These data demonstrate a direct effect of glucocorticoids to stimulate Na+/H+ antiporter activity in OKP cells.

Stimulation by thyroid hormone of Na+-H+ exchange activity in cultured opossum kidney cells

Cultured opossum kidney (OK) cells were used to determine whether thyroid hormone has a direct stimulatory effect on Na(+)-H+ exchange activity in an intact cellular preparation. Na+ uptake or intracellular pH recovery was measured in confluent monolayer cells following acid loading with NH4Cl. Triiodo-L-thyronine (T3) had no effect on cell number or protein and DNA contents but stimulated amiloride-sensitive Na+ uptake in a dose- and time-dependent manner. Maximal stimulation of Na+ uptake was observed at 10(-7) M T3 and 10(-6) M L-thyroxine (T4) with half-maximal effects at 10(-9) M T3 and 3 x 10(-8) M T4. The T3 specific binding capacity of OK cells was 96 +/- 15 fmol/mg DNA with a KD of 1.1 +/- 0.2 x 10(-9) M T3. Neither T3 nor T4 had any effect on amiloride-insensitive Na+ uptake. In kinetic studies of Na+ uptake, T3 increased the Vmax from 123 +/- 22 to 157 +/- 24 nmol.mg-1.min-1 without changing the Michaelis-Menten kinetics (Km) for Na+ (21 +/- 1 in control and 22 +/- 4 mM in T3-treated cells). Studies of intracellular pH (pHi) showed that the resting pHi and the buffering capacity were unaffected by T3. However, after an acid load, OK cells treated with T3 exhibited a greater rate of Na(+)-dependent pH recovery than untreated control cells. These results indicate that thyroid hormone can stimulate Na(+)-H+ exchange activity directly in renal cell line without apparent changes in pHi, cellular hypertrophy, or hyperplasia.