Relative contribution of V-H+ATPase and NA+/H+ exchanger to bicarbonate reabsorption in proximal convoluted tubules of old rats

With aging, the kidney develops a progressive deterioration of several structures and functions. Proximal tubular acidification is impaired in old rats with a decrease in the activity of brush border Na+/H+ exchange and a fall of H-ion flux measured with micropuncture experiments. In the present work we evaluate the contribution of 5-N-ethyl-n-isopropyl amiloride- (EIPA) and bafilomycin-sensitive bicarbonate flux (JHCO3-) in proximal convoluted tubules of young and aged rats. We performed micropuncture experiments inhibiting the Na+/H+ exchanger with EIPA (10(-4) M) and the V-H+ATPase with bafilomycin (10(-6) M). We used antibodies against the NHE3 isoform of the Na+/H+ exchanger and the subunit E of the V-H+ATPase for detecting by Western blot the abundance of these proteins in brush border membrane vesicles from proximal convoluted tubules of young and old rats. The abundance of NHE3 and the V-H+ATPase was similar in 18-month-old and 3-month-old rats. The bicarbonate flux in old rats was 30% lower than in young rats. EIPA reduced by 60% and bafilomycin by 30% in young rats; in contrast, EIPA reduced by approximately 40% and bafilomycin by approximately 50% in old rats. The inhibited by bafilomycin was the same in young and old rats: 0.62 nmol.cm-2.s-1 and 0.71 nmol.cm-2.s-1, respectively. However, the EIPA-sensitive fraction was larger in young than in old rats: 1.26 nmol.cm-2.s-1 vs. 0.85 nmol.cm-2.s-1, respectively. These results suggest that the component more affected in bicarbonate reabsorption of proximal convoluted tubules from aged rats is the Na+-H+ exchanger, probably a NHE isoform different from NHE3.     

Effect of chronic inhibition of converting enzyme on proximal tubule acidification

The acute effect of angiotensin-converting enzyme inhibition (ACEi) on proximal convoluted tubule (PCT) function is well documented. However, the effect of chronic treatment is less known. The aim of this work was to evaluate the effect of chronic ACEi on PCT acidification (J(HCO(3)(-))). Rats received enalapril (10 mg.kg(-1).day(-1), added to the drinking water) during 3 mo. Micropuncture experiments were performed to measure the effect of chronic ACEi on J(HCO(3)(-)). Nitric oxide (NO.) synthesis in kidney cortex homogenates was assessed by quantifying the conversion of [(14)C]-L-arginine to [(14)C]-L-citrulline. Western blot analysis was performed to determine the abundances of V-H(+)ATPase and NHE3 isoform of the Na(+)/H(+) exchanger in proximal brush-border membrane vesicles (BBMV). Enalapril treatment induced an approximately 50% increase in J(HCO(3)(-)). Luminal perfusion with ethyl-isopropyl amiloride (EIPA) 10(-4)M or bafilomycin 10(-6)M decreased J(HCO(3)(-)) by approximately 60% and approximately 30%, respectively, in both control and enalapril-treated rats. The effect of EIPA and bafilomycin on absolute J(HCO(3)(-)) was larger in enalapril-treated than in control rats. Acute inhibition of NO. synthesis with N(G)-nitro-L-arginine methyl ester abolished the enalapril-induced increase in J(HCO(3)(-)). Cortex homogenates from enalapril-treated rats displayed a 46% increase in nitric oxide synthase (NOS) activity compared with those from untreated animals. Enalapril treatment did not affect the abundances of NHE3 and V-H(+)ATPase in BBMV. Our results suggest that PCT acidification is increased during chronic ACEi probably due to an increase in NO. synthesis, which would stimulate Na(+)/H(+) exchange and electrogenic proton transport.     

Decreased D-glucose transport across renal brush-border membrane vesicles from streptozotocin-induced diabetic rats

The uptake of Na(+)-dependent D-glucose by renal brush-border membrane vesicles (BBMV) isolated from streptozotocin-induced diabetic rats was decreased as compared with controls. Since a Vmax of 4.8 nmol/mg protein per 30 s in diabetic BBMV was significantly decreased as compared with that of controls (Vmax = 7.0 nmol/mg protein per 30 s) without changing an apparent affinity for D-glucose, the decrease in the Na(+)-dependent D-glucose uptake in diabetic rats is likely to be due to the reduction in the number of the transporter. These results are also confirmed by the binding study of [3H]phlorizin to diabetic BBMV. When the blood glucose level is lowered in diabetic rats by both the treatment with insulin and starvation, the decreased Na(+)-dependent D-glucose uptake is returned to control level. These results suggest that Na(+)-dependent D-glucose reabsorption through the apical membrane in proximal tubular kidney cells is dynamically regulated by the change in blood glucose level.     

Quantitation of the Na(+)-Pi cotransporter in renal cortical brush border membranes. [14C]phosphonoformic acid as a useful probe to determine the density and its change in response to parathyroid hormone.

To determine the density of Na(+)-Pi symporters in brush border membranes (BBM) from rat renal cortex, [14C] phosphonoformic acid [( 14C] PFA), a competitive inhibitor of Na(+)-Pi cotransport, was employed as a probe. The [14C]PFA binding was measured in BBM vesicles (BBMV) under equilibrated conditions (extra-vesicular Na+, K+, and H+ = intravesicular Na+, K+, and H+) to avoid modulatory effects of these solutes. BBMV were preincubated in media without or with addition of molar excess of Pi (greater than 20 times) to determine the Pi-protectable PFA-binding sites, and then [14C] PFA binding was determined. Only the [14C]PFA binding in the presence of Na+ displaceable by an excess of Pi was saturated and was independent of intravesicular volume of BBMV. This value denoted as "Pi-protectable Na(+)-[14C]PFA binding," was analyzed by Scatchard plot showing BmaxPFA = 375 +/- 129 pmol of PFA/mg protein, KDPFA = 158 +/- 18 microM; the Hill coefficient was congruent to 1. For Na(+)-dependent binding of [3H]phlorizin, in the same BBMV, Bmax = 310 +/- 37 pmol/mg protein and KD V 2.2 +/- 0.5 microM. BBMV prepared from cortex of thyroparathyroidectomized rats infused with phosphaturic doses of parathyroid hormone (PTH) were compared with vehicle-infused controls. Administration of PTH resulted in decrease of BmaxPFA (-38%) and of Na(+)-gradient-dependent uptake of 32Pi (-35%), but KDPFA was not changed. Neither BmaxPhl and KDPhl for Na(+)-phlorizin binding, nor the Na(+)-gradient-dependent uptake of [3H]D-glucose differed between PTH-treated and control rats. We conclude: (a) measurement of Pi-protectable Na(+)-[14C]PFA binding determines numbers and affinity of Na(+)-Pi symporters in renal BBMV; (b) the affinity of PFA for Na(+)-Pi symporter is similar to apparent affinity for Pi (KmPi), as determined from measurements of Na(+)-gradient-dependent 32Pi uptake by BBMV; (c) both Na(+)-Pi symporter and [Na+]D-glucose symporters are present within renal BBM in a similar range of density; (d) PTH decreases the number of Na(+)-Pi cotransporters in BBMV commensurate with the parallel decrease of Na(+)-gradient-dependent Pi transport, whereas the affinity of Na(+)-Pi symporters for Pi is not changed. These observations support the hypothesis that PTH decreases capacity for Na(+)-dependent Pi reabsorption by internalization of Na(+)-Pi symporters in BBM of renal proximal tubules.     

Prenatal programming of rat proximal tubule Na+/H+ exchanger by dexamethasone

Prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. Although an increase in tubular sodium reabsorption has been postulated to be a factor programming hypertension, this has never been directly demonstrated. The purpose of this study was to examine whether prenatal programming by dexamethasone affected postnatal proximal tubular transport. Pregnant Sprague-Dawley rats were injected with intraperitoneal dexamethasone (0.2 mg/kg) daily for 4 days between the 15th and 18th days of gestation. Prenatal dexamethasone resulted in an elevation in systolic blood pressure when the rats were studied at 7-8 wk of age compared with vehicle-treated controls: 131 +/- 3 vs. 115 +/- 3 mmHg (P < 0.001). The rate of proximal convoluted tubule volume absorption, measured using in vitro microperfusion, was 0.61 + 0.07 nl.mm(-1).min(-1) in control rats and 0.93+ 0.07 nl.mm(-1).min(-1) in rats that received prenatal dexamethasone (P < 0.05). Na(+)/H(+) exchanger activity measured in perfused tubules in vitro using the pH-sensitive dye BCECF showed a similar 50% increase in activity in proximal convoluted tubules from rats treated with prenatal dexamethasone. Although there was no change in abundance of NHE3 mRNA, the predominant luminal proximal tubule Na(+)/H(+) exchanger, there was an increase in NHE3 protein abundance on brush-border membrane vesicles in 7- to 8-wk-old rats receiving prenatal dexamethasone. In conclusion, prenatal administration of dexamethasone in rats increases proximal tubule transport when rats are studied at 7-8 wk old, in part by stimulating Na(+)/H(+) exchanger activity. The increase in proximal tubule transport may be a factor mediating the hypertension by prenatal programming with dexamethasone.     

Gbeta regulation of Na/H exchanger-3 activity in rat renal proximal tubules during development

The decreased natriuretic action of dopamine in the young has been attributed to decreased generation of cAMP by the activated renal D(1)-like receptor. However, sodium/hydrogen exchanger (NHE) 3 activity in renal brush-border membrane vesicles (BBMV) can be modulated independent of cytoplasmic second messengers. We therefore studied D(1)-like receptor regulation of NHE activity in BBMVs in 2-, 4-, and 12-wk-old (adult) rats. Basal NHE activity was least in 2-wk-old compared with 4- and 12-wk-old rats. D(1)-like agonist (SKF-81297) inhibition of NHE activity was also least in 2-wk-old (-1 +/- 9%, n = 3) compared with 4 (-15 +/- 5%, n = 6)- and 12 (-65 +/- 4%, n = 6)-wk-old rats. The decreased response to the D(1)-like agonist in BBMV was not caused by decreased D(1) receptors or NHE3 expression in the young. G(s)alpha, which inhibits NHE3 activity by itself, coimmunoprecipitated with NHE3 to the same extent in 2-wk-old and adult rats. G(s)alpha function was also not impaired in the young because guanosine 5'-O-(3-thiotriphosphate) decreased NHE activity to a similar extent in 4-wk-old and adult rats. Galpha(i-3) protein expression in BBMV also did not change with age. In contrast, Gbeta expression and the amount of Gbeta that coimmunoprecipitated with NHE3 in BBMV was greatest in 2-wk-old rats and decreased with age. Gbeta common antibodies did not affect D(1)-like agonist inhibition of NHE activity in adult rats (8%) but markedly increased it (48%)in 4-wk-old rats. We conclude that the decreased inhibitory effect of D(1)-like receptors on NHE activity in BBMV in young rats is caused, in part, by the increased expression and activity of the G protein subunit Gbeta/gamma. The direct regulation of NHE activity by G protein subunits may be an important step in the maturation of renal tubular ion transport.     

Impairment of Na/K-ATPase signaling in renal proximal tubule contributes to Dahl salt-sensitive hypertension

We have observed that, in renal proximal tubular cells, cardiotonic steroids such as ouabain in vitro signal through Na/K-ATPase, which results in inhibition of transepithelial (22)Na(+) transport by redistributing Na/K-ATPase and NHE3. In the present study, we investigate the role of Na/K-ATPase signaling in renal sodium excretion and blood pressure regulation in vivo. In Sprague-Dawley rats, high salt diet activated c-Src and induced redistribution of Na/K-ATPase and NHE3 in renal proximal tubules. In Dahl salt sensitive (S) and resistant (R) rats given high dietary salt, we found different effects on blood pressure but, more interestingly, different effects on renal salt handling. These differences could be explained by different signaling through the proximal tubular Na/K-ATPase. Specifically, in Dahl R rats, high salt diet significantly stimulated phosphorylation of c-Src and ERK1/2, reduced Na/K-ATPase activity and NHE3 activity, and caused redistribution of Na/K-ATPase and NHE3. In contrast, these adaptations were either much less effective or not seen in the Dahl S rats. We also studied the primary culture of renal proximal tubule isolated from Dahl S and R rats fed a low salt diet. In this system, ouabain induced Na/K-ATPase/c-Src signaling and redistribution of Na/K-ATPase and NHE3 in the Dahl R rats, but not in the Dahl S rats. Our data suggested that impairment of Na/K-ATPase signaling and consequent regulation of Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt-induced hypertension in the Dahl S rat.     

Different ionic conditions prompt NHE2 and NHE3 translocation to the plasma membrane

We tested whether NHE3 and NHE2 Na(+)/H(+) exchanger isoforms were recruited to the plasma membrane (PM) in response to changes in ion homeostasis. NHE2-CFP or NHE3-CFP fusion proteins were functional Na(+)/H(+) exchangers when transiently expressed in NHE-deficient PS120 fibroblasts. Confocal morphometry of cells whose PM was labeled with FM4-64 measured the fractional amount of fusion protein at the cell surface. In resting cells, 10-20% of CFP fluorescence was at PM and stable over time. A protocol commonly used to activate the Na(+)/H(+) exchange function (NH(4)-prepulse acid load sustained in Na(+)-free medium), increased PM percentages of PM NHE3-CFP and NHE2-CFP. Separation of cellular acidification from Na(+) removal revealed that only NHE3-CFP translocated when medium Na(+) was removed, and only NHE2-CFP translocated when the cell was acidified. NHE2/NHE3 chimeric proteins demonstrate that the Na(+)-removal response element resides predominantly in the NHE3 cytoplasmic tail and is distinct from the acidification response sequence of NHE2.     

Acute arterial hypertension inhibits proximal tubular fluid reabsorption in normotensive rat but not in SHR

The effect of acute arterial hypertension on proximal tubular fluid reabsorption was investigated in Sprague-Dawley rats and spontaneously hypertensive rats (SHR) by measuring proximal tubular flow with a nonobstructive optical method. Under control conditions, spontaneous tubular flow was oscillating at 0.02-0.03 Hz in Sprague-Dawley rats. Acute hypertension induced an immediate increase of mean tubular flow (50% increase after 20 min of hypertension) and augmentation of oscillatory amplitude. Acute hypertension did not alter single-nephron blood flow as measured by laser-Doppler velocimetry (n = 12), suggesting that the increase of tubular flow was due to inhibition of reabsorption but not increase of filtration. By contrast, spontaneous tubular flow was fluctuating aperiodically in SHR. Acute hype tension did not induce a continuous increase of tubular flow or an increase in amplitude of fluctuations (n = 15). When apical Na(+)/H(+) exchange activity of proximal tubule was monitored, acute hypertension did not alter the activity in SHR (n = 8), while similar procedures had been shown to inhibit apical Na(+)/H(+) exchange activity of proximal tubules by more than 40% in Sprague-Dawley rats. These observations suggest that acute hypertension inhibits proximal tubular fluid reabsorption by inhibiting apical Na(+)/H(+) exchange activity in Sprague-Dawley rats and that this mechanism is impaired in SHR.     

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H(+) absorption from the lumen. We thus examined the effect of inhibition of Na(+)/H(+) exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO(2) diffusion and HCO(3)(-) secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO(2) solution or pH 2.2 low CO(2) solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR(inh)-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO(2)]). Luminal high CO(2) solution increased CO(2) absorption and HCO(3)(-) secretion, accompanied by PV acidification and PV Pco(2) increase. During CO(2) challenge, CFTR(inh)-172 induced HCO(3)(-) absorption, while inhibiting PV acidification. S3226 reversed CFTR(inh)-associated HCO(3)(-) absorption. Luminal pH 2.2 challenge increased H(+) and CO(2) absorption and acidified the PV, inhibited by CFTR(inh)-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO(3)(-) secretion to absorption and inhibited PV acidification during CO(2) challenge, suggesting that HCO(3)(-) secretion helps facilitate CO(2)/H(+) absorption. Furthermore, CFTR inhibition prevented CO(2)-induced cellular acidification reversed by S3226. Reversal of increased HCO(3)(-) loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H(+) available to neutralize luminal HCO(3)(-) with consequent CO(2) absorption. NHE3, by secreting H(+) into the luminal microclimate, facilitates net transmucosal HCO(3)(-) absorption with a mechanism similar to proximal tubular HCO(3)(-) absorption.     

Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na(+)) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na(+)/H(+) exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na(+)/H(+) exchange activity by Na(+)-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na(+)/H(+) exchange activity by >30%. Moreover, the sgk2-mediated increase in Na(+)/H(+) exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na(+) transport through NHE3 in the proximal tubule.     

NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.     

Gene expression of Na+/H+ exchanger in zebrafish H+ -ATPase-rich cells during acclimation to low-Na+ and acidic environments

In mammalian nephrons, most of the Na(+) and HCO(3)(-) is reabsorbed by proximal tubular cells in which the Na(+)/H(+) exchanger 3 (NHE3) is the major player. The roles of NHEs in Na(+) uptake/acid-base regulation in freshwater (FW) fish gills are still being debated. In the present study, functional genomic approaches were used to clone and sequence the full-length cDNAs of the nhe family from zebrafish (Danio rerio). A phylogenetic tree analysis of the deduced amino acid sequences showed that zNHE1-8 are homologous to their mammalian counterparts. By RT-PCR analysis and double/triple in situ hybridization/immunocytochemistry, only zebrafish NHE3b was expressed in zebrafish gills and was colocalized with V-H(+)-ATPase but not with Na(+)-K(+)-ATPase, indicating that H(+)-ATPase-rich (HR) cells specifically express NHE3b. A subsequent quantitative RT-PCR analysis demonstrated that acclimation to low-Na(+) FW caused upregulation and downregulation of the expressions of znhe3b and zatp6v0c (H(+)-ATPase C-subunit), respectively, in gill HR cells, whereas acclimation to acidic FW showed reversed effects on the expressions of these two genes. In conclusion, both NHE3b and H(+)-ATPase are probably involved in Na(+) uptake/acid-base regulation in zebrafish gills, like mammalian kidneys, but the partitioning of these two transporters may be differentially regulated depending on the environmental situation in which fish are acclimatized.     

A phosphorylated phloretin derivative. Synthesis and effect on intestinal Na(+)-dependent phosphate absorption

2'-Phosphophloretin (2'-PP), a phosphorylated derivative of the plant chalcone, was synthesized. The effect of 2'-PP, on Na(+)-dependent phosphate uptake into intestinal brush-border membrane vesicles (BBMV) isolated from rabbit and rat duodenum and jejunum was examined. 2'-PP decreased Na(+)-dependent phosphate uptake into rabbit BBMV with an IC(50) of 55 nM and into rat BBMV with an IC(50) of 58 nM. 2'-PP did not affect Na(+)-dependent glucose, Na(+)-dependent sulfate, or Na(+)-dependent alanine uptake by rabbit intestinal BBMVs. 2'-PP inhibition of rabbit intestinal BBMV Na(+)-dependent phosphate uptake was sensitive to external phosphate concentration, suggesting that 2'-PP inhibition of Na(+)-dependent phosphate uptake was competitive with respect to phosphate. Binding of [(3)H]2'-PP to rabbit intestinal BBMV was examined. Binding of [(3)H]2'-PP was Na(+)-dependent with a K(0.5) for Na(+)(Na(+) concentration for 50% 2'-PP binding) of 30 mM. The apparent K(s) for Na(+)-dependent [(3)H]2'-PP binding to rabbit BBMVs was 58 nM in agreement with the IC(50) for 2'-PP inhibition of Na(+)-dependent phosphate uptake. These results indicate that 2'-PP bound to rabbit or rat intestinal BBMV Na(+)-phosphate cotransporter and inhibited Na(+)-dependent phosphate uptake. In rats treated with 2'-PP by daily gavage, the effect of 2'-PP on serum phosphate, serum glucose, and serum calcium was examined. In a concentration-dependent manner, 2'-PP reduced serum phosphate by 45% 1 wk after starting treatment. 2'-PP did not alter serum calcium or serum glucose. The apparent IC(50) for 2'-PP in vivo was 3 microM.     

Ouabain-stimulated trafficking regulation of the Na/K-ATPase and NHE3 in renal proximal tubule cells

We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate whether this mechanism is species-specific, ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular (22)Na(+) transport were compared in three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1). Ouabain-induced inhibition of transcellular (22)Na(+) transport is due to an ouabain-induced redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.     

Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).     

pH microdomains in oligodendrocytes

Oligodendrocytes (OLs) are cells that produce myelin in the central nervous system. Here we use ratiometric pH indicator dye to analyze intracellular pH in OLs in culture. The results reveal alkaline microdomains, which predominate in the perikaryon and proximal dendrites, and acidic microdomains, which predominate in distal dendrites. Spatial nonuniformity of pH is generated by differential subcellular distribution of Na(+)/H(+) exchanger (NHE), which is localized in a punctate distribution in the perikaryon and proximal processes, Na(+)/HCO(3)(-) cotransporter (NBC), which is localized in a punctate distribution in distal dendrites, and carbonic anhydrase isotype II (CAII), which is colocalized with either NHE or NBC. Inhibition of NHE activity by amiloride inhibits regeneration of alkaline microdomains after cytoplasmic acidification, whereas the inhibition of CAII activity with ethoxyzolamide inhibits acidification of dendrites. Fluorescence correlation spectroscopy analysis of CAII microinjected into OLs reveals freely diffusing protein throughout the cell as well as protein associated predominantly with NHE in the perikaryon and predominantly with NBC in the dendrites. Alkaline and acidic microdomains could be generated by transport metabolons consisting of CAII associated with NHE or NBC, respectively. This study provides the first evidence for pH microdomains in cells and describes a mechanism for how they are generated.     

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.