NHE3 mRNA Research Articles

Expression of Na+-H+ exchanger 1 in human gastric carcinoma tissue and its clinical significance

To determine the expression of Na+/H+ exchanger 1(NHE1) in human gastric carcinoma tissue and to investigate the association between NHE1 expression and clinicopathological characteristics. The expressions of NHE1 mRNA and protein were detected in both gastric carcinoma tissue (n=60) and adjacent gastric mucosa tissue (n=30) by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The association between the expression and the clinicopathological characteristics was analyzed. The relative expression levels of NHE1 mRNA and protein in gastric carcinoma tissue were 0.786+/-0.291 and 1.442+/-0.175, which were significantly higher than those in adjacent gastric mucosa tissue (0.369+/-0.052 and 0.348+/-0.029) (P<0.01). The expression of NHE1 mRNA was positively correlated with NHE1 protein in the gastric carcinoma tissue (r=0.264, P<0.05). The expressions of NHE1 mRNA and protein were associated with the depth of invasion, lymph node metastasis, and TNM staging (P<0.05). However, no statistical difference was found in age, gender, and tumor differentiation (P>0.05). The expression levels of NHE1 mRNA and protein are significantly up-regulated in gastric carcinoma tissue, which may be involved in the development of gastric carcinoma.

Tumor necrosis factor-α represses the expression of NHE2 through NF-κB activation in intestinal epithelial cell model, C2BBe1

High levels of proinflammatory cytokines are linked to pathogenesis of diarrhea in inflammatory bowel disease (IBD). Na(+) absorption is compromised in IBD. The studies were designed to determine the effect of tumor necrosis factor-α (TNF-α) on the expression and activity of NHE2, a Na(+) /H(+) exchanger (NHE) that is involved in transepithelial Na(+) absorption in intestinal epithelial cells. NHE2 regulation was examined in TNF-α-treated C2BBe1 cells by reverse-transcription polymerase chain reaction (RT-PCR), reporter gene assays, and Western blot analysis. NHE isoform activities were measured as ethyl-isopropyl-amiloride- and HOE694-sensitive (22) Na-uptake. In vitro and in vivo protein-DNA interactions were assessed by gel mobility shift assays and chromatin immunoprecipitation studies. TNF-α treatment of C2BBe1 cells led to repression of NHE2 promoter activity, mRNA, and protein levels; and inhibited both NHE2 and NHE3 mediated (22) Na-uptake. 5'-deletion analysis of the NHE2 promoter-reporter constructs identified basepair -621 to -471 as the TNF-α-responsive region (TNF-RE). TNF-α activated NF-κB subunits, p50 and p65, and their DNA-binding to a putative NF-κB motif within TNF-RE. Mutations in the NF-κB motif abolished NF-κB-DNA interactions and abrogated TNF-α-induced repression. Ectopic overexpression of NF-κB resulted in repression of NHE2 expression. Two functionally distinct inhibitors of NF-κB blocked the inhibitory effect of TNF-α. The human NHE2 isoform is a direct target of transcription factor NF-κB. TNF-α-mediated activation of NF-κB decreases the expression and activity of NHE2 in the intestinal epithelial cell line, C2BBe1. These findings implicate NF-κB in the modulation of Na(+) absorption during intestinal inflammatory conditions such as IBD where a high level of TNF-α is detected.

Transcriptional inhibition of intestinal NHE8 expression by glucocorticoids involves Pax5

Sodium/hydrogen exchangers (NHEs) are a family of proteins that transport sodium ions into the cells by moving protons out of the cells. They play a major role in sodium absorption, cell volume regulation, and intracellular pH regulation. Three out of nine identified NHEs (NHE2, NHE3, and NHE8) are expressed on the apical membrane of intestinal epithelial cells. Glucocorticoids have been found to regulate NHE3 function in the intestine, but it is unknown if they have a similar function on NHE8 expression. Interestingly, high glucocorticoid levels in the intestine coincide chronologically with the change from high expression of NHE8 to high expression of NHE3. Studies were performed to explore the role of glucocorticoids on NHE8 expression during intestinal maturation. Brush-border membrane vesicles were isolated from intestinal epithelia, and Western blotting was performed to determine NHE8 protein expression of suckling male rats treated with methylpredisolone. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcription factors involved in glucocorticoid-mediated regulation. Our results showed that the expression of NHE8 mRNA and protein was decreased in glucocorticoid-treated rats and human intestinal epithelial cells (Caco-2). The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by glucocorticoid treatment. GMSAs suggested that the reduction in promoter activity in the presence of glucocorticoids was due to enhanced transcription factor Pax5 binding on the NHE8 proximal promoter region. In conclusion, this study showed that glucocorticoids inhibit NHE8 gene expression by increasing Pax5 binding on NHE8 gene promoter, suggesting an important role for Pax5 during intestinal maturation.

S1286 Enhanced Expression of Vacuolar H+-ATPase in Native Human Hepatic Carcinoma

Background&Aims: Maintenance of intracellular pH is crucial to normal cell function, as many cellular processes have a narrow pH optimum. Tumor cells possess high-glycolytic activity and produce acidic metabolites. Moreover, tumor cells often exist in a hypoxic microenvironment with a lower extracellular pH than that of surrounding normal cells. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. In this study, we made an investigation on expressions of H+-regulated channels/ exchangers in native human hepatic carcinoma. Methods: The mRNA expressions of Na+/ H+ exchanger (NHE), H+-K+ ATPase, and vacuolar H+-ATPase (V-ATPase) subunit c were examined by real time PCR. The protein expressions were analyzed by Western Blot. The protein expression and intracellular distribution of V-ATPase subunit c was further analyzed by immunohistochemistry. Results: The expressions of mRNA and protein of H+-K+ ATPase were not detected in native human hepatic carcinoma and normal hepatic tissues. The mRNA and protein expression levels of NHE1 and NHE3 were similar in native human hepatic carcinoma and normal hepatic tissues. However, the mRNA and protein expression levels of V-ATPase subunit c in native human hepatic carcinoma were markedly increased, compared with normal hepatic tissues. Immunohistochemistry analysis further confirmed the enhanced expression of V-ATPase subunit c on the plasma membrane of hepatic carcinoma cells. Conclusion: The results demonstrated that the expression of V-ATPase in human hepatic carcinoma is markedly increased, indicating V-ATPase is possibly involved in the regulation of intracellular pH of hepatic carcinoma cells. V-ATPase may represent a potential target for a novel pharmacotherapy of hepatic carcinoma.

S1288 Pre-Operative Biliary Drainage in Periampullary Malignancy Does Not Increase the Rates of Postoperative Complication

Background&Aims: Maintenance of intracellular pH is crucial to normal cell function, as many cellular processes have a narrow pH optimum. Tumor cells possess high-glycolytic activity and produce acidic metabolites. Moreover, tumor cells often exist in a hypoxic microenvironment with a lower extracellular pH than that of surrounding normal cells. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. In this study, we made an investigation on expressions of H+-regulated channels/ exchangers in native human hepatic carcinoma. Methods: The mRNA expressions of Na+/ H+ exchanger (NHE), H+-K+ ATPase, and vacuolar H+-ATPase (V-ATPase) subunit c were examined by real time PCR. The protein expressions were analyzed by Western Blot. The protein expression and intracellular distribution of V-ATPase subunit c was further analyzed by immunohistochemistry. Results: The expressions of mRNA and protein of H+-K+ ATPase were not detected in native human hepatic carcinoma and normal hepatic tissues. The mRNA and protein expression levels of NHE1 and NHE3 were similar in native human hepatic carcinoma and normal hepatic tissues. However, the mRNA and protein expression levels of V-ATPase subunit c in native human hepatic carcinoma were markedly increased, compared with normal hepatic tissues. Immunohistochemistry analysis further confirmed the enhanced expression of V-ATPase subunit c on the plasma membrane of hepatic carcinoma cells. Conclusion: The results demonstrated that the expression of V-ATPase in human hepatic carcinoma is markedly increased, indicating V-ATPase is possibly involved in the regulation of intracellular pH of hepatic carcinoma cells. V-ATPase may represent a potential target for a novel pharmacotherapy of hepatic carcinoma.

S1289 Photodynamic Therapy for Unresectable Hepatocellular Carcinoma With Bile Duct Invasion and Obstructive Jaundice

Background&Aims: Maintenance of intracellular pH is crucial to normal cell function, as many cellular processes have a narrow pH optimum. Tumor cells possess high-glycolytic activity and produce acidic metabolites. Moreover, tumor cells often exist in a hypoxic microenvironment with a lower extracellular pH than that of surrounding normal cells. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. In this study, we made an investigation on expressions of H+-regulated channels/ exchangers in native human hepatic carcinoma. Methods: The mRNA expressions of Na+/ H+ exchanger (NHE), H+-K+ ATPase, and vacuolar H+-ATPase (V-ATPase) subunit c were examined by real time PCR. The protein expressions were analyzed by Western Blot. The protein expression and intracellular distribution of V-ATPase subunit c was further analyzed by immunohistochemistry. Results: The expressions of mRNA and protein of H+-K+ ATPase were not detected in native human hepatic carcinoma and normal hepatic tissues. The mRNA and protein expression levels of NHE1 and NHE3 were similar in native human hepatic carcinoma and normal hepatic tissues. However, the mRNA and protein expression levels of V-ATPase subunit c in native human hepatic carcinoma were markedly increased, compared with normal hepatic tissues. Immunohistochemistry analysis further confirmed the enhanced expression of V-ATPase subunit c on the plasma membrane of hepatic carcinoma cells. Conclusion: The results demonstrated that the expression of V-ATPase in human hepatic carcinoma is markedly increased, indicating V-ATPase is possibly involved in the regulation of intracellular pH of hepatic carcinoma cells. V-ATPase may represent a potential target for a novel pharmacotherapy of hepatic carcinoma.

S1287 Clinical Outcome and Prognostic Factors of Extrahepatic Cholangiocarcinoma After Radical Resection: Comparison Based on Tumor Location

Background&Aims: Maintenance of intracellular pH is crucial to normal cell function, as many cellular processes have a narrow pH optimum. Tumor cells possess high-glycolytic activity and produce acidic metabolites. Moreover, tumor cells often exist in a hypoxic microenvironment with a lower extracellular pH than that of surrounding normal cells. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. In this study, we made an investigation on expressions of H+-regulated channels/ exchangers in native human hepatic carcinoma. Methods: The mRNA expressions of Na+/ H+ exchanger (NHE), H+-K+ ATPase, and vacuolar H+-ATPase (V-ATPase) subunit c were examined by real time PCR. The protein expressions were analyzed by Western Blot. The protein expression and intracellular distribution of V-ATPase subunit c was further analyzed by immunohistochemistry. Results: The expressions of mRNA and protein of H+-K+ ATPase were not detected in native human hepatic carcinoma and normal hepatic tissues. The mRNA and protein expression levels of NHE1 and NHE3 were similar in native human hepatic carcinoma and normal hepatic tissues. However, the mRNA and protein expression levels of V-ATPase subunit c in native human hepatic carcinoma were markedly increased, compared with normal hepatic tissues. Immunohistochemistry analysis further confirmed the enhanced expression of V-ATPase subunit c on the plasma membrane of hepatic carcinoma cells. Conclusion: The results demonstrated that the expression of V-ATPase in human hepatic carcinoma is markedly increased, indicating V-ATPase is possibly involved in the regulation of intracellular pH of hepatic carcinoma cells. V-ATPase may represent a potential target for a novel pharmacotherapy of hepatic carcinoma.

Epidermal growth factor inhibits intestinal NHE8 expression via reducing its basal transcription

Sodium/hydrogen exchangers (NHEs) play a major role in Na(+) absorption, cell volume regulation, and intracellular pH regulation. Of the nine identified mammalian NHEs, three (NHE2, NHE3, and NHE8) are localized on the apical membrane of epithelial cells in the small intestine and the kidney. Although the regulation of NHE2 and NHE3 expression has been extensively studied in the past decade, little is known about the regulation of NHE8 gene expression under physiological conditions. The current studies were performed to explore the role of epidermal growth factor (EGF) on NHE8 expression during intestinal maturation. Brush-border membrane vesicles (BBMV) were isolated from intestinal epithelia, and Western blot analysis was performed to determine NHE8 protein expression of sucking male rats treated with EGF. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcriptional factors involved in EGF-mediated regulation. Our results showed that intestinal NHE8 mRNA expression was decreased in EGF-treated rats and Caco-2 cells, and NHE8 protein abundance was also decreased in EGF-treated rats. The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by EGF treatment. This could be explained by reduced binding of transcription factor Sp3 on the NHE8 basal promoter region in the presence of EGF. Pretreatment with MEK1/2 inhibitor UO-126 could prevent EGF-mediated inhibition of NHE8 gene expression. In conclusion, this study showed that EGF inhibits NHE8 gene expression through reducing its basal transcription, suggesting an important role of EGF in regulating NHE expression during intestinal maturation.

Na+/H+ Exchanger NHE3 is differentially regulated in renal proximal tubules of pre‐hypertensive and hypertensive spontaneously hypertensive rats

The present work aimed to measure NHE3 in vivo transport activity and to define the mechanisms underlying NHE3 regulation in young pre‐hypertensive (5‐week‐old, systolic blood pressure (SBP) = 92 ± 2 mmHg) and adult hypertensive spontaneously hypertensive rats (SHR) (14‐week‐old, SBP = 187 ± 4 mmHg). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo microperfusion. NHE3‐mediated bicarbonate reabsorption was stimulated in 5‐week‐old SHR (44 ± 5%, P&lt;0.01) and reduced in 14‐week‐old SHR (57 ± 4%, P&lt;0.001) compared to Wistar Kyoto (WKY) rats at corresponding age. Stimulated NHE3 transport activity in young pre‐hypertensive SHR was associated with enhanced NHE3 microvillus expression, lower levels of phosphorylation at Ser552 and increased association with the enzyme dipeptidyl peptidase IV (DPPIV). Conversely, adult hypertensive SHR showed decreased NHE3 microvillus membrane content, higher relative ratio of phosphorylated Ser552, and lower amount of the NHE3‐DPPIV complex. No differences on renal cortical NHE3 protein content and mRNA levels were found between SHR and its WKY counterpart. We conclude that NHE3 is differentially regulated before and after development of hypertension in SHR by mechanisms that involve post‐translational modifications, subcellular redistribution and assembly with interacting proteins. Supported by FAPESP and CNPq

Testosterone down‐regulates sodium‐hydrogen exchanger isoform 8 (NHE8) expression

NHE8 is the newest member of the sodium‐hydrogen exchanger family. It is expressed in intestinal and renal epithelial cells. We have shown that NHE8 is important for intestinal sodium absorption early in life and that its expression is regulated by many physiological factors. Since studies have shown that certain genes are regulated differently by gender, we investigated if NHE8 is one of these genes. Therefore, we compared NHE8 expression in male and female mice. RNA and brush‐border membrane protein were isolated from the intestinal mucosa of mice. Real‐time PCR was used to determine NHE8 mRNA levels, and Western blot was used to determine NHE8 protein levels. We performed the same experiments on Caco2 cells to study the effect of sex hormones on NHE8 expression. Our results showed that at four weeks of age, RNA and protein expression of NHE8 was similar between male and female mice, but at eight weeks of age, RNA and protein expression of NHE8 was significantly reduced by ~50% in male mice compared to female mice. In Caco2 cells, estrogen (100nM, 18 hours) had no effect whereas testosterone (100nM, 18 hours) inhibited NHE8 expression at RNA and protein levels that are comparable with the in vivo observations. In conclusion, our results suggest that NHE8 expression differs between male mice and female mice. The difference is likely due to testosterone's inhibitory effect. Our study was supported by NIH grant R01DK073638.

Preserved Na+/H+ exchanger isoform 3 expression and localization, but decreased NHE3 function indicate regulatory sodium transport defect in ulcerative colitis†

A major causative factor of diarrhea in ulcerative colitis (UC) patients is the loss of Na(+) absorptive capacity of the inflamed colonic mucosa. Potential contributing mechanisms include reduced driving force for active transport, and impaired expression, mislocalization, or defective transport function of Na(+) absorptive proteins. We therefore studied the expression, brush border membrane (BBM) localization, and transport capacity of the major intestinal Na(+) absorptive protein, the Na(+)/H(+) exchanger isoform 3 (NHE3) in biopsies from UC patients. In UC and control biopsies, inflammation was graded histologically, NHE3, tumor necrosis factor alpha (TNF-alpha), villin, as well as other housekeeping genes were analyzed by quantitative real-time polymerase chain reaction (PCR), BBM localization of NHE3 determined by immunohistochemistry, and confocal microscopy. Na(+) absorptive capacity was assessed by (22)Na(+) isotope fluxes and NHE3 transport activity measured microfluorometrically in BCECF-loaded surface colonocytes within isolated crypts. In mildly, moderately, and severely inflamed sigmoid colon of UC patients, neither NHE3 mRNA expression nor the abundance of NHE3 in the BBM was significantly altered compared to other structural components of the BBM. However, Na(+) absorption was strongly reduced by approximately 80% and acid-activated NHE3 transport activity was significantly decreased in the surface cells of sigmoid colonic crypts even in moderately inflamed mucosa. In the colonic mucosa of patients with active UC, NHE3 transport capacity was found significantly decreased despite correct NHE3 location and abundance in the brush border, independent of current treatment. These findings suggest functional NHE3 transport as a novel factor for inflammatory diarrhea in UC patients.

Molecular characterization and expression analysis of Na +/H + exchanger (NHE)-1 and c-Fos genes in sea bass ( Dicentrarchus labrax, L) exposed to acute and chronic hypercapnia

Hypercapnia (increased blood CO 2 pressure), due to an increasing of aquatic P CO2, is a common event under conditions of intensive aquaculture and in fish a complex set of physiological and biochemical alterations are employed to cope with this environmental stress. Many of these adjustments depend to a large extent on changes in the expression of genes that encode for physiologically relevant proteins. In this study we report first on the molecular cloning and characterization of c-Fos and Na +/H + exchanger-1 (NHE-1) cDNAs in sea bass ( Dicentrarchus labrax). Both sequences were deposited in the GenBank with accession no. DQ838581 for c-Fos and EU180587 for NHE-1. We then utilized the real-time PCR technology to monitor dynamic changes in levels of c-Fos and NHE-1 transcripts in response to acute (1 h at 71.42 ± 14.85 mg/l of free CO 2) and chronic (96 h at 47.56 ± 4.99 mg/l of free CO 2) hypercapnia. The c-Fos mRNA abundance was significantly increased in brain in response to both acute and chronic hypercapnia conditions, whereas NHE-1 mRNA abundance was downregulated during acute hypercapnia in gill and kidney, and upregulated in kidney during chronic hypercapnia exposure. These findings suggest that both genes are involved in the response to hypercapnia in sea bass and may be good biomarkers capable of providing useful information about fish welfare.

886 Do Mutations of NHE3 At the Site of Poorly Functioning NHE3 Polymorphisms Contribute to Diarrhea of Congenital Na Diarrhea?

of NHE3 from the membrane and diarrhea. Aim: We investigated the expression, brush border membrane (BBM) localization, and transport function of the major intestinal sodium absorptive transporter, the Na+/H+ exchanger NHE3, as well as the expression of its regulatory PDZ-adapter proteins NHERF1 and PDZK1, TNF-alpha, actin, and a variety of housekeeping genes, in the small and large intestine of mice after induction of a CD45RBhigh transfer colitis in the acute phase of diarrhea. Results: After colitis induction, TNF-alpha levels were increased both in the inflamed colon and microscopically normal-appearing small intestine. NHE3 mRNA expression was up regulated in the colon and unaltered in the small intestine, NHE3 protein expression and localization in the brush border membrane (BBM) was not altered, but acid-activated NHE3 transport rates in small intestinal villous and colonic surface cells were severely decreased, and fluid absorption In Vivo was decreased in the small intestine. PDZK1 mRNA expression was down regulated, whereas that of NHERF1 was not altered. Glucocorticoid treatment of colitic mice for 4 days decreased colonic mucosal cytokine expression and increased PDZK1 expression as well as fluid absorption. In order to investigate whether the down regulation of PDZK1 and the disturbed NHE3 transport activity is causally related, we studied PDZK1 protein expression and NHE3 transport activity in colonic surface enterocytes in the intestine of PDZK1 +/mice, and found PDZK1 content reduced by 60% and NHE3 transport activity significantly decreased. Conclusion: We suggest that during immune-mediated intestinal inflammation, NHE3 BBM protein abundance is normal, but the regulation of its transport activity is disturbed. PDZK1 down regulation during inflammation may be one factor responsible for inflammation-associated NHE3 dysfunction.

Morphological adaptation with preserved proliferation/transporter content in the colon of patients with short bowel syndrome

In short bowel syndrome (SBS), although a remaining colon improves patient outcome, there is no direct evidence of a mucosal colonic adaptation in humans. This prospective study evaluates morphology, proliferation status, and transporter expression level in the epithelium of the remaining colon of adult patients compared with controls. The targeted transporters were Na+/H+ exchangers (NHE2 and 3) and oligopeptide transporter (PepT1). Twelve adult patients with a jejuno-colonic anastomosis were studied at least 2 yr after the last surgery and compared with 11 healthy controls. The depth of crypts and number of epithelial cells per crypt were quantified. The proliferating and apoptotic cell contents were evaluated by revealing Ki67, PCNA, and caspase-3. NHE2, NHE3, PepT1 mRNAs, and PepT1 protein were quantified by quantitative RT-PCR and Western blot, respectively. In patients with SBS compared with controls, 1) hyperphagia and severe malabsorption were documented, 2) crypt depth and number of cells per crypt were 35% and 22% higher, respectively (P < 0.005), whereas the proliferation and apoptotic levels per crypt were unchanged, and 3) NHE2 mRNA was unmodified; NHE3 mRNA was downregulated near the anastomosis and unmodified distally, and PepT1 mRNA and protein were unmodified. We concluded that, in hyperphagic patients with SBS with severe malabsorption, adaptive colonic changes include an increased absorptive surface with an unchanged proliferative/apoptotic ratio and well-preserved absorptive NHE2, NHE3, and PepT1 transporters. This is the first study showing a controlled nonpharmacological hyperplasia in the colon of patients with SBS.

NHE1, NHE3, and NHE5 mRNA expression in brainstem and cortex of Sprague‐Dawley rat

In brainstem CO2 chemosensitive neurons, an increase in CO2 (hypercapnia) leads to a maintained reduction in intracellular pH (pHi) while in non‐chemosensitive neurons, pHi recovery is observed. Although the precise mechanisms for this difference in pHi regulation remain to be determined, it has been suggested that different isoforms of the Na+/H+‐exchanger (NHE) may be responsible. To begin to explore this possibility, real‐time RT‐PCR was used to examine mRNA expression for three NHE isoforms ‐ NHE1 (Slc9a1), NHE3 (Slc9a3), and NHE5 (Slc9a5) ‐ in brainstem and cortex (non‐chemosensitive control) of 21 day‐old SD rat. Preliminary data indicate that each of the NHE isoforms is present in both brainstem and cortex. To further investigate the distribution within the medulla, mRNA expression levels for the NHE3 and NHE5 in dorsal (dM) versus ventral (vM) medulla were compared. These analyses revealed that NHE5 mRNA levels are ~4‐fold higher in the vM than in the dM (when normalized to Actb), but NHE3 mRNA levels are similar between the two regions. Additional studies are needed to further evaluate the relative abundance of different NHE mRNAs in chemosensitive areas such as the NTS/DMV, VLM, the caudal medullary raphe, and the LC. To date, however, our data suggest that differential expression of NHE isoforms may participate in pHi regulation in chemosensitive and/or non‐chemosensitive neurons. Supported by NS045321.

The effect of short‐term, low‐salinity acclimation on gill NHE, AE1 and HAT expression in the longhorn sculpin, Myoxocephalus octodecemspinosus

We previously reported that the marine, longhorn sculpin, Myoxocephalus octodecemspinosus, can acclimate to 20% SW for days with no change in plasma osmolality, ion concentrations, or hematocrit level. Sculpin, however, lose ions during acclimation to 10% SW for 24 and 72 h and this reaches a lethal level after 6 days. During these acclimations, the sculpin is incapable of down‐regulating gill NKCC or CFTR, suggesting that they cannot turn off their gill ion secreting mechanisms. The counterpart to this is whether they can upregulate the transporters necessary for ion uptake during acclimation to low salinity water, such as the Na+/H+ exchangers (NHE), anion exchanger 1 (AE1) or H+‐ATPase (HAT), and thus maintain proper ion and water balance. We determined that sculpin upregulate gill NHE3 mRNA and NHE2c, while significantly down regulating AE1 during acclimation to 20% SW, and have undetectable levels of NHE2b during acclimation to 10% SW. There were no significant changes in HAT or NHE8. These transporters were immunolocalized in the sculpin gill, and we determined that during acclimation to 10 or 20% SW they are distributed in different aspects of the ion transporting cells of the gill. We conclude that longhorn sculpin are incapable of properly regulating gill ion transporters; thus, this is a physiological barrier from living in low‐salinity (below 10% SW) or fresh water.

Angiotensin II directly regulates intestinal epithelial NHE3 in Caco2BBE cells

BackgroundAngiotensin II (AII) effects on intestinal Na+ transport may be multifactorial. To determine if AII might have a direct effect on intestinal epithelial Na+ transport, we investigated its actions on Na+ transport in human intestinal epithelial Caco2BBE cells.ResultsAII increased apical (brush border) sodium-hydrogen exchanger (NHE)-3, but not NHE2, activity within one hour. Similarly, only apical membrane NHE3 abundance increased at 1–2 hours without any change in total NHE3 protein abundance. From 4–48 hours, AII stimulated progressively larger increases in apical NHE3 activity and surface abundance, which was associated with increases in NHE3 protein expression. At 4–24 hours, NHE3 mRNA increases over baseline expression, suggesting increased gene transcription. This was supported by AII induced increases in rat NHE3 gene promoter-reporter activity. AII induction of NHE3 was blocked by the AII type I receptor antagonist losartan. Acute changes in AII-induced increases in NHE3 exocytosis were blocked by a phospholipase C inhibitor, an arachidonic acid cytochrome P450 epoxygenase inhibitor, as well as phosphatidylinositol 3 kinase (PI3K) inhibitors and Akt inhibitor, partially blocked by a metalloproteinase inhibitor and an EGF (epidermal growth factor) receptor kinase inhibitor, but not affected by an inhibitor of MEK-1 (MAPKK-1, mitogen activated protein kinase kinase-1).ConclusionWe conclude that angiotensin II has a direct role in regulating intestinal fluid and electrolyte absorption which may contribute to its overall effects in regulation systemic volume and blood pressure. AII activates several key signaling pathways that induce acute and chronic changes in NHE3 membrane trafficking and gene transcription.

Na+/H+ exchanger isoform NHE3 and NHERF adaptor protein expression, localization and function in the small and large intestine of CD45RB high transfer colitis

BackroundThe molecular mechanisms for inflammatory diarrhea are multifactorial and incompletely understood.AimWe investigated the expression, localization and function of the major intestinal sodium absorptive transporter, the Na+/H+ exchanger NHE3, as well as its regulatory PDZ‐adapter proteins NHERF1 and PDZK1, in the small and large intestine of mice after induction of a CD45RBhigh transfer colitis.ResultsAfter colitis induction, TNF‐alpha levels were increased both in the inflamed colon and microscopically normal‐appearing small intestine. NHE3 mRNA expression was upregulated in the colon and unaltered in the small intestine, NHE3 protein expression and localization in the brush border membrane (BBM) was not altered, but acid‐activated NHE3 transport rates in small intestinal villous and colonic surface cells were severely decreased, and fluid absorption in vivo was decreased in the small intestine. PDZK1 mRNA expression was downregulated, whereas that of NHERF1 was not altered. Glucocorticoid treatment of colitic mice for 4 days decreased colonic mucosal cytokine expression and increased PDZK1 expression as well as fluid absorption.ConclusionWe suggest that during immune‐mediated intestinal inflammation, NHE3 BBM protein abundance is normal, but the regulation of its transport activity is disturbed. PDZK1 downregulation during inflammation may be one factor responsible for inflammation‐associated NHE3 dysfunction

Transcriptional regulation of the human Na +/H + exchanger NHE3 by serotonin in intestinal epithelial cells

Serotonin (5-HT) decreases NHE2 and NHE3 activities under acute conditions in human intestinal epithelial cells. Here, we have investigated the effects of 5-HT on expression of the human NHE3 gene and the mechanisms underlying its transcriptional regulation in differentiated C2BBe1 cells. Treatment of the human intestinal epithelial cell line, C2BBe1, with 5-HT (20 μM) resulted in a significant decrease in NHE3 mRNA and protein expression. In transient transfection studies, 5-HT repressed the NHE3 promoter activity by ∼55%. The repression of the NHE3 promoter activity in response to 5-HT was accompanied by reduced DNA-binding activity of transcription factors Sp1 and Sp3 to the NHE3 promoter without alteration in their nuclear levels. Pharmacological inhibitors of protein kinase C reversed the inhibitory effect of 5-HT on the promoter activity. Our data indicate that 5-HT suppresses the transcriptional activity of the NHE3 promoter and this effect may be mediated by PKCα and modulation of DNA-binding affinities of Sp1 and Sp3.

HL-1 mouse cardiomyocyte injury and death after simulated ischemia and reperfusion: roles of pH, Ca2+-independent phospholipase A2, and Na+/H+ exchange

The Ca(2+)-independent phospholipase A(2) VI (iPLA(2)-VI) and the Na(+)/H(+) exchanger isoform 1 (NHE1) are highly pH-sensitive proteins that exert both protective and detrimental effects in cardiac ischemia-reperfusion. Here, we investigated the role of extracellular pH (pH(o)) in ischemia-reperfusion injury and death and in regulation and function of iPLA(2)-VI and NHE1 under these conditions. HL-1 cardiomyocytes were exposed to simulated ischemia (SI; 0.5% O(2), 8 mM K(+), and 20 mM lactate) at pH(o) 6.0 and 7.4, with or without 4 or 8 h of reperfusion (SI/R). Cytochrome c release and caspase-3 activation were reduced after acidic compared with neutral SI, whereas necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of iPLA(2)-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet not after neutral, SI. The isoform-selective enantiomers R- and S-BEL both mimicked the effect of racemic BEL after acidic SI. In contrast, inhibition of NHE activity by EIPA had no significant effect on necrosis after SI. Both neutral and acidic SI were associated with a reversible loss of F-actin and cortactin integrity. Inhibition of iPLA(2)-VI disrupted F-actin, cortactin, and mitochondrial integrity, whereas inhibition of NHE slightly reduced stress fiber content. iPLA(2)-VIA and NHE1 mRNA levels were reduced during SI and upregulated in a pH(o)-dependent manner during SI/R. This also affected the subcellular localization of iPLA(2)-VIA. Thus, the mode of cell death and the roles and regulation of iPLA(2)-VI and NHE1 are at least in part determined by the pH(o) during SI. In addition to having clinically relevant implications, these findings can in part explain the contradictory results obtained from previous studies of iPLA(2)-VIA and NHE1 during cardiac I/R.