NHE3 Protein Expression Research Articles

Roles of Na+/H+ Exchanger Type 1 and Intracellular pH in Angiotensin II-Induced Reactive Oxygen Species Generation and Podocyte Apoptosis

A growing body of evidence suggests that podocyte apoptosis is a major cause of decreased podocyte number, which leads to albuminuria and glomerular injury. The aim of this study was to clarify the molecular mechanisms of angiotensin II (Ang II)-induced apoptosis in cultured mouse podocytes. We examined the effects of Ang II (100 nmol/L) on apoptosis, superoxide anions, and cytosol pH in podocytes. For intracellular pH measurements, image analysis was conducted using confocal laser microscopy after incubation with carboxyseminaphthorhodafluor-1. Superoxide anions and intracellular pH were elevated with Ang II treatment. Apoptotic cell numbers, as measured by TUNEL staining and caspase 3 activity, were also augmented in the Ang II–treated group. Pre-treatment with olmesartan (100 nmol/L, an Ang II type 1–receptor blocker), apocynin (50 μmol/L, NADPH oxidase inhibitor), or 5-N,N hexamethylene amiloride [30 μmol/L, Na+/H+ exchanger type 1 (NHE-1) inhibitor] abolished Ang II–induced podocyte apoptosis, whereas NHE-1 mRNA and protein expression was not affected by Ang II treatment. Moreover, Ang II increased NHE-1 phosphorylation. These results suggest that superoxide production, NHE-1 activation, and intracellular alkalization were early features prior to apoptosis in Ang II–treated mouse podocytes, and may offer new insights into the mechanisms responsible for Ang II–induced podocyte injury.

Lactobacillus acidophilus upregulates intestinal NHE3 expression and function

A major mechanism of electroneutral NaCl absorption in the human ileum and colon involves coupling of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchangers. Disturbances in these mechanisms have been implicated in diarrheal conditions. Probiotics such as Lactobacillus have been indicated to be beneficial in the management of gastrointestinal disorders, including diarrhea. However, the molecular mechanisms underlying antidiarrheal effects of probiotics have not been fully understood. We have previously demonstrated Lactobacillus acidophilus (LA) to stimulate Cl(-)/HCO3- exchange activity via an increase in the surface levels and expression of the Cl(-)/HCO3- exchanger DRA in vitro and in vivo. However, the effects of LA on NHE3, the Na(+)/H(+) exchanger involved in the coupled electroneutral NaCl absorption, are not known. Current studies were, therefore, undertaken to investigate the effects of LA on the function and expression of NHE3 and to determine the mechanisms involved. Treatment of Caco2 cells with LA or its conditioned culture supernatant (CS) for 8-24 h resulted in a significant increase in Na(+)/H(+) exchange activity, mRNA, and protein levels of NHE3. LA-CS upregulation of NHE3 function and expression was also observed in SK-CO15 cells, a human colonic adenocarcinoma cell line. Additionally, LA treatment increased NHE3 promoter activity, suggesting involvement of transcriptional mechanisms. In vivo, mice gavaged with live LA showed significant increase in NHE3 mRNA and protein expression in the ileum and colonic regions. In conclusion, LA-induced increase in NHE3 expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of probiotics.

Proximal tubule Na+/H+ exchanger activity in adult NHE8−/−, NHE3−/−, and NHE3−/−/NHE8−/− mice

NHE3 is the predominant Na(+)/H(+) exchanger on the brush-border membrane (BBM) of the proximal tubule in adults. However, NHE3 null mice still have significant renal BBM Na(+)/H(+) activity. NHE8 has been localized to the BBM of proximal tubules and is more highly expressed in neonates than adult animals. The relative role of NHE8 in adult renal H(+) transport is unclear. This study examined whether there was compensation by NHE8 in NHE3(-/-) mice and by NHE3 in NHE8(-/-) mice. NHE3(-/-) mice had significant metabolic acidosis, and renal BBM NHE8 protein abundance was greater in NHE3(-/-) mice than control mice, indicating that there may be compensation by NHE8 in NHE3(-/-) mice. NHE8(-/-) mice had serum bicarbonate levels and pH that were not different from controls. NHE3 protein expression on the BBM was greater in NHE8(-/-) mice than in wild-type mice, indicating that there may be compensation by NHE3 in NHE8(-/-) mice. Both BBM NHE3 and NHE8 protein abundance increased in response to acidosis. Blood pressure and Na(+)/H(+) exchanger activity were comparable in NHE8(-/-) mice to that of controls, but both were significantly lower in NHE3(-/-) mice compared with control mice. Compared with NHE3(-/-) mice, NHE3(-/-)/NHE8(-/-) mice had lower blood pressures. While serum bicarbonate was comparable in NHE3(-/-) mice and NHE3(-/-)/NHE8(-/-) mice, proximal tubule Na(+)/H(+) exchange activity was less in NHE3(-/-)/NHE8(-/-) mice compared with NHE3(-/-) mice. In conclusion, NHE3 is the predominant Na(+)/H(+) exchanger in adult mice. NHE8 may play a compensatory role in renal acidification and blood pressure regulation in NHE3(-/-) mice.

Blockade of peripheral and spinal Na+/H+ exchanger increases formalin-induced long-lasting mechanical allodynia and hyperalgesia in rats

The Na+/H+ exchanger (NHE) is involved in the regulation of intracellular pH and volume by mediating the electroneutral transport of H+ against an influx of Na+ ions. Since NHE1 regulates pH in neurons and astrocytes and it is expressed in nociceptive nerve fibers, it is likely that NHE may modulate neuronal excitability and pain transmission. The purpose of this study was to assess the participation of peripheral and spinal NHE in the secondary allodynia/hyperalgesia induced by formalin. In addition, we determined whether formalin injection modifies the expression of NHE1 in lumbar dorsal root ganglia (DRG) and dorsal spinal cord. Subcutaneous injection of 0.5% formalin into the dorsal surface of the hind paw produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-lasting bilateral secondary mechanical allodynia/hyperalgesia. Peripheral and intrathecal pre-treatment (−10min) with selective NHE inhibitors 5-(N,N-dimethyl)amiloride hydrochloride (DMA, 0.3–30μM), 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 0.3–30μM) and [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine dihydrochloride (zoniporide, 0.03–3μM) significantly increased 0.5% formalin-induced bilateral long-lasting secondary allodynia/hyperalgesia. Contrariwise, local peripheral or intrathecal post-treatment (day 6 postinjection) with these NHE inhibitors did not affect formalin-induced nociceptive behaviors. Formalin injection reduced NHE1 expression in ipsilateral and contralateral spinal dorsal horns from day 1 to 12. In addition, formalin diminished NHE1 protein expression in DRG at day 12. These results suggest that NHE1 plays a role in pain processing at peripheral and spinal levels in formalin-induced long-lasting nociceptive behaviors. Additionally, these results suggest that proteins involved in pH regulation could be targets for the development of new analgesic drugs.

Abstract 367: Exercising Exacerbates the Hypertrophic Response of Female Transgenic Mice Expressing Elevated Myocardial Na + /H + Exchanger Isoform 1

Cardiac hypertrophy (CH) is heart growth in response to environmental demands, and a variety of hormonal, paracrine and autocrine stimuli. It is a means to reduce stress on the ventricular wall. The Na+/H+ exchanger isoform 1 (NHE1) has been implicated in the development and progression of CH. To better understand the involvement of NHE1, male and female transgenic mice that express cardiac specific active NHE1 expression were studied. N-line mice expressed wild-type NHE1, and K-line mice expressed activated NHE1. NHE activity of adult ventricular cardiomyocytes and protein expression were elevated by approximately 2 and 3-fold in the N- and K-line mice vs. control. The K-line female mice assessed by echocardiography demonstrated significant global cardiac dysfunction. Left ventricular fractional cell shortening and ejection fraction were significantly decreased in the K-line mice (23.1 ± 3.8% and 45.2 ± 6.9% K-line vs. 36.5 ± 1.1% and 66.4 ± 1.5% control, respectively; p<0.05). The K-line female mice also exhibit myocardial remodeling. The heart weight to body weight ratio was significantly greater in the K-line mice (143 ± 10.0% of control; P<0.05). Cross sectional area (K-line 195.6 ± 16.4% of control; p<0.05) and interstitial fibrosis (K-line: 275.4 ± 11.6% of control; p<0.05) were also elevated. Increased expression of active NHE1 protein in male mice was also much more detrimental than expression of the wild type protein as was seen with the female transgenic mice. Therefore, the NHE1 induced hypertrophic effect was not gender dependent. However, NHE1 expression induced gender specific differences with exercise. Exercising exaggerated the HW/BW ratio in female mice expressing activated NHE1 compared to males. These results suggest that gender specific activation of NHE1 may be critical in promoting hypertrophy in females in comparison with males.

Coordinate adenosine A1 and A2A receptors regulation of the Na+/H+ exchanger 3 in ischemia/reperfusion injury

Acute kidney injury, due to ischemia/reperfusion injury (IRI), is associated with high morbidity and mortality and lacks specific therapeutic measures. Na+ absorption is severely compromised by IRI and both surface and total protein of the principal proximal tubule Na+ transporter, the Na+/H+ exchanger 3 (NHE3), are markedly decreased following IRI. We found that coordinate pharmacological activation of adenosine receptor subtype 1 (A1R) and 2A (A2AR) protected against IRI. Compared to vehicle‐treated ischemic rats, A1R agonist, given before ischemia, and A2AR agonist, given after ischemia, decreased the peak of plasma creatinine (Cr) and blood urea nitrogen (BUN) 24 hours after IRI and accelerated their reversal thereafter. We next examined how A1R and A2AR affect NHE3 protein expression in IRI. A1R agonist, given before ischemia, did not affect the drop in NHE3 surface, but blunted IRI‐induced reduction in NHE3 total protein. A2AR agonist, given after ischemia, reversed the decrease in NHE3 surface but did not affect loss of NHE3 total protein mediated by IRI. In summary, activation of A1R and A2AR synergistically reduced the peak and accelerated the reversal of Cr and BUN following IRI. A1R and A2AR act differently, but in conjunction, to preserve NHE3 protein and move it back to cell surface after IRI. Modulation of NHE3 protein expression may be a key mechanism by which A1R and A2AR mediate protection against IRI.

Altered Regulation of Renal Acid Base Transporters in Response to Ammonium Chloride Loading in Rats

The role of the kidney in combating metabolic acidosis has been a subject of considerable interest for many years. The present study was aimed to determine whether there is an altered regulation of renal acid base transporters in acute and chronic acid loading. Male Sprague-Dawley rats were used. Metabolic acidosis was induced by administration of NH4Cl for 2 days (acute) and for 7days (chronic). The serum and urinary pH and bicarbonate were measured. The protein expression of renal acid base transporters [type 3 Na+/H+ exchanger (NHE3), type 1 Na+/HCO3- cotransporter (NBC1), Na-K+ ATPase, H+-ATPase, anion exchanger-1 (AE-1)] was measured by semiquantitative immunoblotting. Serum bicarbonate and pH were decreased in acute acid loading rats compared with controls. Accordingly, urinary pH decreased. The protein expression of NHE3, H+-ATPase, AE-1 and NBC1 was not changed. In chronic acid loading rats, serum bicarbonate and pH were not changed, while urinary pH was decreased compared with controls. The protein expression of NHE3, H+-ATPase was increased in the renal cortex of chronic acid loading rats. These results suggest that unaltered expression of acid transporters combined with acute acid loading may contribute to the development of acidosis. The subsequent increased expression of NHE3, H+-ATPase in the kidney may play a role in promoting acid excretion in the later stage of acid loading, which counteract the development of metabolic acidosis.

Abstract 16723: Dopamine D 3 Receptor Decreases NHE3 Expression and Function by Inhibiting the Activity of the De-Ubiquitinylating Enzyme, USP48, in Renal Proximal Tubule Cells

Disruption of the dopamine D 3 receptor gene in mice (D 3 R -/- ) results in high blood pressure, salt sensitivity, decreased ability to excrete a sodium load and increased renal expression of sodium/hydrogen exchanger isoform 3 (NHE3) protein (180%, P<0.05) but not mRNA, and D 3 R blockade in wild-type mice increased (2 fold, P<0.04) NHE3 protein expression. We hypothesized that renal D 3 R regulates NHE3 expression/function. In human renal proximal tubular cells (hRPTCs), the colocalization and co-immunoprecipitation of D 3 R and NHE3 are increased by D 3 R stimulation (PD 128907, 1μM), associated to internalization of D 3 R and NHE3 (confocal microscopy) and inhibition (80%, P<0.05) of NHE3 dependent Na + transport. D 3 R stimulation also increases NHE3 ubiquitinylation and internalization of the ubiquitinylated NHE3 in the cytoplasm (150%, P<0.05) suggesting that D 3 R activation promotes NHE3 ubiquitinylation and internalization. To determine the mechanism of the D 3 R-mediated NHE3 ubiquitinylation we performed a yeast-two hybrid screening which showed that the 3 rd intracytoplasmic loop of human D 3 R interacts with human ubiquitin-specific peptidase 48 (USP48), a de-ubiquitinylating enzyme. D 3 R and USP48, and USP48 and NHE3 co-localized and co-immunoprecipitated in hRPTCs. USP48 accounted for ~30% of hRPTCs' total de-ubiquitinylating activity. D 3 R stimulation increased the interaction between D 3 R and USP48 and resulted in a 35% reduction in USP48 de-ubiquitinylating activity, indicating that D 3 R inhibits USP48 activity in hRPTCs. USP48 silencing increased the degradation of NHE3 (vehicle t 1/2 = 6.1 hr; USP48 siRNA t 1/2 = 12.9 hr; P<0.05) while over-expressing USP48 increased NHE3 half -life (t 1/2 = 21.8 hr; P<0.05 vs. vehicle) indicating that USP48 protects NHE3 from degradation via de-ubiquitinylation. Moreover, selective renal USP48 silencing in mice by subcapsular infusion of USP48 siRNA decreases NHE3 expression (25%, P<0.04) and systolic blood pressure measured under anesthesia (vehicle: 102±2 vs. siRNA 78±2 mmHg; P<0.03). Our data demonstrate that agonist activation of D 3 R promotes the degradation of NHE3 via the inhibition of USP48 and thus provide a novel mechanism by which the D 3 R engenders natriuresis and regulates blood pressure.

1069 POSTER Role of Sodium Hydrogen Exchanger Isoform 1 (NHE-1) in Integrins-mediated Migration of Human Breast Cancer Cells

Background: The Na-H exchangers (NHEs) are a family of membrane glycoproteins which transport H out of the cell in exchange for Na with a stoichiometry of 1:1. In mammalian cells, the NHE family consists of nine isoforms, NHE-1 to NHE-9. NHE-1, the first one of the isoforms to be cloned, is ubiquitously distributed. Several studies have shown both increased activity and protein of NHE-1 in transformed cells. Apart from its role as a principal regulator of intracellular pH (pHi) and cell volume, NHE-1 has been implicated in cell proliferation, transformation and migration. Cell migration is a multi-step process that requires spatial asymmetry which is stimulated by Rho GTPases, phosphoinositides and actin polymerization. Integrin family of receptors is responsible for cell surface interactions with extracellular matrix (ECM). NHE-1 may contribute to cell migration by: (a) affecting the cell volume, (b) regulating the intracellular pH and thereby the assembly and activity of cytoskeletal elements, (c) anchoring the cytoskeleton to the plasma membrane, and (d) by controlling cell adhesion. Disrupting NHE-1 function leads to impaired polarity of cells and their inability to migrate. Although NHE1 has been shown to affect cell migration through its various functions, a role for the exchanger in cell migration regulated by integrins has not been extensively studied. Fact that NHE1 binds several other proteins in the cytoplasmic regulatory domain, have led to the hypothesis that NHE1 can act as a plasma membrane scaffold that brings together many proteins so they can interact functionally. Thus, it is plausible to hypothesize the possible role NHE-1 might play by direct or indirect structural interaction with the assembly of cell adhesion molecules. Materials and Methods: 1. Inhibition of NHE1 activity (using ethyl-isopropyl-amiloride) and its effect on pHi and cell viability (Measurement of intracellular pH, MTT assay) 2. Effect of NHE1 silencing (using siRNA transfection) and its effect on cell viability (Western blot analysis, MTT assay) 3. Effect of NHE1 silencing and pharmacological inhibition of its activity on cell adhesion and motility. (In vitro cell adhesion assay, In vitro cell migration assay) Results: The data obtained shows that with either EIPA treatment or NHE-1 siRNA transfection, migratory capacity was impaired in MDA-MB231 human breast cancer cells. Interestingly, pharmacological inhibition of NHE-1 did not significantly reduce the integrins-dependent cell adhesion in these cells. However, down-regulation of NHE-1 protein expression had significant effect on integrins-mediated cell adhesion to fibronectin. Conclusion: This study shows that the effect of NHE-1 on integrinsdependent cell adhesion is independent of its activity. However, NHE-1 protein expression seems to be an important upstream event in the functional assembly of integrin receptors and may play an essential role in cancer cell adhesion to the extracellular matrix.

NHE-1 participates in isoproterenol-induced downregulation of SERCA2a and development of cardiac remodeling in rat hearts

Impaired Ca(2+) handling is one of the main characteristics in heart failure patients. Recently, we reported abnormal expressions of Ca(2+)-handling proteins in isoproterenol (ISO)-induced hypertrophied rat hearts. On the other hand, Na(+)/H(+) exchanger (NHE)-1 inhibitor has been demonstrated to exert beneficial effects in ischemic-reperfusion injury and in the development of cardiac remodeling. The aims of the present study are to investigate the role of NHE-1 on Ca(2+) handling and development of cardiac hypertrophy in ISO-infused rats. Male Wistar rats were randomly divided into vehicle [control (CTL)] and ISO groups without or with pretreatment with a selective NHE-1 inhibitor, BIIB-723. ISO infusion for 1 wk significantly increased the ratios of heart to body weight and left ventricle (LV) to body weight and collagen accumulation. All of these increases were antagonized by coadministration with BIIB-723. The ISO-induced significant increase in LV wall thickness was suppressed significantly (P < 0.05) by BIIB-723. ISO-induced decreases in cardiac stroke volume and a total mechanical energy per beat index, systolic pressure-volume area at midrange LV volume, were normalized by BIIB-723. The markedly higher expression of NHE-1 protein in the ISO group than that in CTL group was suppressed (P < 0.05) by BIIB-723. Surprisingly, ISO induced downregulation of the important Ca(2+)-handling protein sarcoplasmic reticulum Ca(2+)-ATPase 2a, the expression of which was also normalized by BIIB-723 without changes in phosphorylated phospholamban (PLB)/PLB expression. We conclude that NHE-1 contributes to ISO-induced abnormal Ca(2+) handling associated with cardiac hypertrophy. Inhibition of NHE-1 ameliorates cardiac Ca(2+)-handling impairment and prevents the development of cardiac dysfunction in ISO-infused rats.

Silencing of NHE-1 blunts the slow force response to myocardial stretch

Myocardial stretch induces a biphasic force response: a first abrupt increase followed by a slow force response (SFR), believed to be the in vitro manifestation of the Anrep effect. The SFR is due to an increase in Ca²⁺ transient of unclear mechanism. We proposed that Na⁺/H⁺ exchanger (NHE-1) activation is a key factor in determining the contractile response, but recent reports challenged our findings. We aimed to specifically test the role of the NHE-1 in the SFR. To this purpose small hairpin interference RNA capable of mediating specific NHE-1 knockdown was incorporated into a lentiviral vector (l-shNHE1) and injected into the left ventricular wall of Wistar rats. Injection of a lentiviral vector expressing a nonsilencing sequence (scramble) served as control. Myocardial NHE-1 protein expression and function (the latter evaluated by the recovery of pH(i) after an acidic load and the SFR) were evaluated. Animals transduced with l-shNHE1 showed reduced NHE-1 expression (45 ± 8% of controls; P < 0.05), and the presence of the lentivirus in the left ventricular myocardium, far from the site of injection, was evidenced by confocal microscopy. These findings correlated with depressed basal pH(i) recovery after acidosis [(max)dpH(i)/dt 0.055 ± 0.008 (scramble) vs. 0.009 ± 0.004 (l-shNHE1) pH units/min, P < 0.05], leftward shift of the relationship between J(H⁺) (H⁺ efflux corrected by the intrinsic buffer capacity), and abolishment of SFR (124 ± 2 vs. 101 ± 2% of rapid phase; P < 0.05) despite preserved ERK1/2 phosphorylation [247 ± 12 (stretch) and 263 ± 23 (stretch l-shNHE1) % of control; P < 0.05 vs. nonstretched control], well-known NHE-1 activators. Our results provide strong evidence to propose NHE-1 activation as key factor in determining the SFR to stretch.

Silencing of sodium/hydrogen exchanger in the heart by direct injection of naked siRNA

Cardiac Na(+)/H(+) exchanger (NHE1) hyperactivity is a central factor in cardiac remodeling following hypertension, myocardial infarction, ischemia-reperfusion injury, and heart failure. Treatment of these pathologies by inhibiting NHE1 is challenging because specific drugs that have been beneficial in experimental models were associated with undesired side effects in clinical practice. In the present work, small interference RNA (siRNA) produced in vitro to specifically silence NHE1 (siRNA(NHE1)) was injected once in vivo into the apex of the left ventricular wall of mouse myocardium. After 48 h, left ventricular NHE1 protein expression was reduced in siRNA(NHE1)-injected mice compared with scrambled siRNA by 33.2 ± 3.4% (n = 5; P < 0.05). Similarly, NHE1 mRNA levels were reduced by 20 ± 2.0% (n = 4). At 72 h, siRNA(NHE1) spreading was evident from the decrease in NHE1 expression in three portions of the myocardium (apex, medium, base). NHE1 function was assessed based on maximal velocity of intracellular pH (pH(i)) recovery (dpH(i)/dt) after an ammonium prepulse-induced acidic load. Maximal dpH(i)/dt was reduced to 14% in siRNA(NHE1)-isolated left ventricular papillary muscles compared with scrambled siRNA. In conclusion, only one injection of naked siRNA(NHE1) successfully reduced NHE1 expression and activity in the left ventricle. As has been previously suggested, extensive NHE1 expression reduction may indicate myocardial spread of siRNA molecules from the injection site through gap junctions, providing a valid technique not only for further research into NHE1 function, but also for consideration as a potential therapeutic strategy.

Different Natriuretic Responses in Obese and Lean Rats in Response to Nitric Oxide Reduction

Nitric oxide (NO) is an important regulator of renal sodium transport and participates in the control of natriuresis and diuresis. In obesity, the nitric oxide bioavailability was reportedly reduced, which may contribute to the maintenance of hypertension. The aim of this study was to determine the effect of NO depletion on renal sodium handling in a model of diet-induced obesity hypertension. Obese hypertensive (obesity-prone (OP)) and lean normotensive (obesity-resistant (OR)) Sprague-Dawley rats were treated with 1.2 mg/kg/day N(G)-nitro-L-arginine-methyl ester (L-NAME) for 4 weeks to inhibit NO synthesis. Acute pressure natriuresis and diuresis were measured in response to an increase in perfusion pressure. NHE3 and Na(+), K(+)-ATPase protein expression were measured by Western blot and NHE3 activity was determined as the rate of pH change in brush border membrane vesicles. NHE3 membrane localization was determined by confocal microscopy. L-NAME did not significantly attenuate the natriuretic and diuretic responses to increases in renal perfusion pressure (RPP) in OP rats while inducing a significant reduction in OR rats. Following chronic NO inhibition, NHE3 protein expression and activity and Na(+), K(+)-ATPase protein expression were significantly increased in the OR but not in the OP group. Immunofluorescence studies indicated that the increase in NHE3 activity could be, at least in part, due to NHE3 membrane trafficking. Obese hypertensive rats have a weaker natriuretic response in response to NO inhibition compared to lean rats and the mechanism involves different regulation of the apical sodium exchanger NHE3 expression, activity, and trafficking.

Abstract 2927: Regulation of Na+/H+ exchanger 1 (NHE1) expression by hyperactivation of Akt in PTEN-null mouse embryonic fibroblasts

Abstract The serine/threonine kinase, Akt, plays a key role in regulating the signaling of a multitude of biological processes including cell proliferation, differentiation, migration and survival. In addition, many human cancers commonly display aberrant hyperactivated Akt survival pathway, a phenomenon that is frequent in tumours that have impaired or deletion of the PTEN tumour suppressor gene. Interestingly, we have recently demonstrated that an increase in intracellular O2− could replace the removal of growth factors resulting in the activation of Akt through the inhibition of the tumour suppressor PTEN and inducing an increased expression of the Na+/H+ exchanger 1 (NHE1), mimicking the phenotype of PTEN-null mouse embryonic fibroblasts. The Na+/H+ exchanger 1 (NHE1) is a membrane antiporter that has been described to promote cell survival via a dual mechanisms- defending cell volume and pHi through Na+/H+ exchange and by functioning as a scaffold for the recruitment of a signalplex involving, PI3-Kinase, ERM and Akt. In the present study, we asked whether the hyperactivity of Akt found in cells that have lost the expression of PTEN could influence the expression of NHE-1. We show that the PTEN-null mouse embryonic fibroblast model displayed an increased expression of Na+/H+ exchanger 1 (NHE1) and hyperactivated Akt compared to its PTEN wildtype counterpart. Moreover the half-life of NHE-1 was shown to increase in PTEN-null fibroblasts compared to the wildtype mouse embryonic fibroblasts. Hence, we hypothesized that Akt hyperactivation could influence the stability of NHE-1 expression. Indeed, using drugs such as LY294002 and Wortmannin to inhibit the activation of AKT, we showed that activated Akt had an effect on NHE-1 expression as the decrease in NHE-1 expression correlated with the dephosporylation of Akt, both at the mRNA and protein levels. Similarly, by silencing Akt gene expression, a more prominent decrease in NHE-1 protein expression was observed. Taken together, our data suggest that hyperactivated Akt may play a crucial role in the stability of NHE-1 in PTEN null fibroblasts, a phenomenon not observed in wildtype fibroblasts. Results from these studies could establish that Akt may be involved in the regulation of NHE-1 expression in tumours that have lost the expression of PTEN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2927. doi:10.1158/1538-7445.AM2011-2927

Exercise-induced regulation of muscular Na+-K+ pump, FXYD1, and NHE1 mRNA and protein expression: importance of training status, intensity, and muscle type

It is investigated if exercise-induced mRNA changes cause similar protein expression changes of Na(+)-K(+) pump isoforms (α(1), α(2), β(1), β(2)), FXYD1, and Na(+)/K(+) exchanger (NHE1) in rat skeletal muscle. Expression was evaluated (n = 8 per group) in soleus and extensor digutorum longus after 1 day, 3 days, and 3 wk (5 sessions/wk) of either sprint (4 × 3-min sprint + 1-min rest) or endurance (20 min) running. Two hours after exercise on day 1, no change in protein expression was apparent in either training group or muscle, whereas sprint exercise increased the mRNA of soleus α(2) (4.9 ± 0.8-fold; P < 0.05), β(2) (13.2 ± 4.4-fold; P < 0.001), and NHE1 (12.0 ± 3.1-fold; P < 0.01). Two hours after sprint exercise, protein expression normalized to control samples was higher on day 3 than day 1 for soleus α(1) (41 ± 18% increase vs. 15 ± 8% reduction; P < 0.05), α(2) (64 ± 35% increase vs. 37 ± 12% reduction; P < 0.05), β(1) (17 ± 21% increase vs. 14 ± 29% reduction; P < 0.05), and FXYD1 (35 ± 16% increase vs. 13 ± 10% reduction; P < 0.05). In contrast, on day 3, soleus α(1) (0.1 ± 0.1-fold; P < 0.001), α(2) (0.2 ± 0.1-fold; P < 0.001), β(1) (0.4 ± 0.1-fold; P < 0.05), and β(2)-mRNA (2.9 ± 1.7-fold; P < 0.001) expression was lower than after exercise on day 1. After 3 wk of training, no change in protein expression relative to control existed. In conclusion, increased expression of Na(+)-K(+) pump subunits, FXYD1 and NHE1 after 3 days exercise training does not appear to be an effect of increased constitutive mRNA levels. Importantly, sprint exercise can reduce mRNA expression concomitant with increased protein expression.

The effect of progesterone on uterine fluid ph &amp;amp;endometrial nhe-1 protein expression in rats

A precise regulation of the uterine fluid volume and pH is essential for a successful embryo implantation. Progesterone has been reported to participate in uterine fluid volume regulation during this period, however its effect on the uterine fluid pH is unknown. As endometrial fluid absorption has been proposed to occur secondary to sodium (Na+) absorption under progesterone mediated effect, we therefore hy-pothesize that there may be a concomitant changes in fluid volume and pH if sodium-hydrogen exchanger (NHE), a protein responsible for both luminal Na+ absorption and H+ extrusion is involved. In view of these, our study aimed to investigate the possibility that progesterone affect the uterine fluid pH and endometrial NHE expression. Ovariectomised female Sprague- Dawley (SD) rats were treated with peanut oil (vehicle), oestradiol-3-benzoate and progesterone for three consecutive days. On the fourth day, in-vivo uterine perfusions were performed on anaesthetized rats. The collected perfusate were analyzed for the changes in pH. The effect of amiloride, a non-specific Na+-channel blocker on the pH was investigated. The expression of uterine NHE-1 protein was detected by Western blotting and immunohistochemistry. Our findings indicate that the fluid pH is the lowest in progesterone-treated group and amiloride ad-ministration significantly increased the pH in the same treatment group (p < 0.05). NHE-1 proteins were significantly expressed in the progesterone-treated group. In conclusion, progesterone induces a reduction of the uterine fluid pH and is amiloride-sensitive. The up-regulation of NHE-1 under a common proge-sterone effect may explain the role of this ex-changer in regulating the uterine fluid pH.

Loss of PDZ-adaptor protein NHERF2 affects membrane localization and cGMP- and [Ca2+]- but not cAMP-dependent regulation of Na+/H+exchanger 3 in murine intestine

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.

The effects of amiloride, a Na+–H+exchange inhibitor, on iliac artery stenosis after balloon injury in rabbitsThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

This study was designed to explore the effects of amiloride, a Na+-H+ exchange (NHE) inhibitor, on vessel stenosis by observing the expression of NHE-1 protein in vascular smooth muscle (VSM) after balloon injury and the effects of amiloride on VSM cell proliferation, migration, and excretion of extracellular matrices (ECMs). A total of 32 adult male New Zealand white rabbits were randomly divided into a balloon injury group (BG), an amiloride-treated group (AG), and a sham-operated group (SG). The left iliac artery was injured by inflating a 2.5 mm x 20 mm Foley catheter in BG and AG rabbits; in SG rabbits, the Foley catheter was inserted but not inflated. Amiloride (5 mg x kg(-1) x d(-1)) was injected intraperitoneally in AG and the same volume of distilled water was used in BG 3 days before balloon injury and for 28 days after the injury. The left iliac artery was stained by hematoxylin-eosin, alpha-actin, and Masson's trichrome to observe the vessel cava, neointima, media layer, and ECMs. NHE-1 proteins of the VSM were detected by Western blotting. A narrowing of the arterial cava, neointima formation, and thickened VSM layer were observed 28 days after balloon injury in BG and AG. However, in AG, the vessel cava was not as narrowed as that of BG and the intimal areas were to a lesser extent than in BG. In AG, the alpha-actin-positive areas and the ECM areas in the neointima were increased compared with SG, but to a lesser extent than in BG. The expression of NHE-1 protein in VSM was increased in BG and AG after balloon injury; however, the levels in AG were significantly less than in BG. In conclusion, VSM cell proliferation, migration, and excretion of ECMs contributed to vessel stenosis in the BG and AG rabbits. The expression of NHE-1 protein in VSM increased after balloon injury. Amiloride, an inhibitor of NHE-1, can limit the development of vessel stenosis through inhibition of VSM cell proliferation, migration, and excretion of ECMs.

Abstract 1266: TNF-alpha mediated down-regulation of Na+/H+ exchanger 1 (NHE1) in breast cancer MCF7 cells and its relevance to apoptosis

Abstract Intracellular alkalinization is a hallmark of cancer cells shown to confer resistance against caspase mediated apoptosis. Na+/H+ exchanger 1 (NHE1), an ion exchanger important in the maintenance of intracellular pH and cell volume homeostasis had been implicated in this intracellular alkalinization. Increased NHE1 protein level and activity has been found in many cancer cells types where increase in intracellular pH by constitutively active NHE1 confers resistance to apoptosis in these cells. Silencing of NHE1 protein in cancer cells results in reduced cell growth and increased in sensitivity to death triggers. TNF-α can induce H2O2 production in cells to mediate apoptosis with characteristic cell shrinkage. H2O2 had recently been shown to downregulate NHE1 expression and increase cells’ sensitivity to apoptosis. This study was undertaken to study the role of caspases and H2O2 in the regulation of NHE1 protein and gene expression by TNF-α. Results showed that TNF-α had both cytostatic and cytotoxic effects on MCF-7 cells with concurrent decrease in intracellular pH and NHE1 activity. NHE1 was found to be downregulated both at the protein and mRNA level following TNF-α treatment. Gelshift assay showed that transcription factors binding to the promoter of NHE1 could be blocked by pre-incubation with H2O2, suggesting that transcription of NHE1 could be redox-regulated. Indeed, we also show by using a luciferase reporter assay that NHE1 promoter is down-regulated in presence of H2O2. Various caspases were activated in TNF-α treated cells and the inhibition of caspases prevented the downregulation of NHE1. ROS was elevated in MCF-7 within 2 hours of treatment. Incubation of cells with catalase increased their survival in response to TNF-α, thus implicating H2O2 as a mediator of TNF-α induced cell death. Overexpression of catalase in MCF-7 reduced the downregulation of NHE1 by TNF-α. Our results suggest by TNF-α mediate cell death through decreasing NHE1 activity and down-regulating NHE1 expression in a ROS mediated manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1266.

Overexpression of the NHE1 isoform of the Na+/H+ exchanger causes elevated apoptosis in isolated cardiomyocytes after hypoxia/reoxygenation challenge

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH in the myocardium and other tissues. NHE1 is an important mediator of myocardial damage that occurs after ischemia-reperfusion injury. It has also been implicated in apoptotic damage in many tissues and its expression and activity are elevated in disease states in the myocardium. In this study, we examined the effect of additional exogenous NHE1 expression on isolated cardiomyocytes susceptibility to ischemia/reperfusion damage. Exogenous NHE1 elevated Na(+)/H(+) exchanger expression and activity when introduced into isolated cardiomyocytes through an adenoviral system. Isolated cardiomyocytes were subjected to simulated ischemia and reperfusion after infection with either control or NHE1-containing adenovirus. Cells were placed into an anaerobic chamber and effects of NHE1 expression after hypoxia/reoxygenation were examined. Hypoxia/reoxygenation increased caspase-3-like activity in controls, and the effect was greatly magnified in cells expressing NHE1 protein. It also elevated the percentage of apoptotic cardiomyocytes, which was also aggravated by expression of NHE1 protein. Hypoxia/reoxygenation also increased phospho-ERK levels. Elevated NHE1 expression was coincidental with increased expression of the ER stress protein, protein disulfide isomerase (PDI) and calreticulin (CRT). Our results demonstrate that increased NHE1 protein expression makes cells more susceptible to damage induced by hypoxia/reoxygenation in isolated cardiomyocytes. They suggest that elevated NHE1 in cardiovascular disease could predispose the human myocardium to enhanced apoptotic damage.