Huacong Chen

Impaired mucin synthesis and bicarbonate secretion in the colon of NHE8 knockout mice

Sodium/hydrogen exchanger 8 (NHE8), the newest member of the SLC9 family, is expressed at the apical membrane of the epithelial cells in the intestine and the kidney. Although NHE8 has been shown to be an important player for intestinal sodium absorption early in development, its physiological role in the intestine remains unclear. Here, we successfully created a NHE8 knockout (NHE8(-/-)) mouse model to study the function of this transporter in the intestinal tract. Embryonic stem cells containing interrupted NHE8 gene were injected into mouse blastocyst to produce NHE8(+/-) chimeras. NHE8(-/-) mice showed no lethality during embryonic and fetal development. These mice had normal serum sodium levels and no signs of diarrhea. Apically expressed NHE2 and NHE3 were increased in the small intestine of the NHE8(-/-) mice in compensation. The number of goblet cells and mucin (MUC)-positive cells in the colon was reduced in NHE8(-/-) mice along with mucosal pH, MUC2 expression as well as downregulated in adenoma (DRA) expression. Therefore, the role of NHE8 in the intestine involves both sodium absorption and bicarbonate secretion.

Somatostatin stimulates intestinal NHE8 expression via p38 MAPK pathway

Diarrhea is a common manifestation of gastrointestinal disorders. Diarrhea-induced losses of fluid and electrolyte could lead to dehydration and electrolyte imbalances, resulting in significant morbidity and mortality, especially in children living in developing countries. Somatostatin, a peptide hormone secreted by D-cells, plays an important role in regulating motility and intestinal Na(+) absorption. Although octreotide, a somatostatin analog, is used to treat diarrhea, its mechanisms of action are unclear. Here we showed that octreotide increased brush-border membrane Na(+)/H(+) exchanger 8 (NHE8) expression in the small intestine to the exclusion of other NHEs that participate in Na(+) absorption. The same effect also occurred in human intestinal cells (Caco-2). We found that the increase of NHE8 expression by somatostatin required p38 mitogen-activated protein kinase (MAPK) activation. Furthermore, the somatostatin receptor SSTR2 antagonist CYN154806 could abolish somatostatin-induced NHE8 expression and p38 MAPK phosphorylation. Thus our data provided the first concrete evidence indicating that somatostatin stimulates intestinal Na(+) absorption by increasing intestinal NHE8 expression through the SSTR2-p38 MAPK pathway.

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.

NHE2X3 DKO mice exhibit gender-specific NHE8 compensation

NHE8, the newest member of the sodium/hydrogen exchanger family, is expressed in the epithelial cells of the intestine and the kidney. Intestinal expression of NHE8 is significantly higher than that of NHE2 and NHE3 at a young age, suggesting that NHE8 is an important player for intestinal sodium absorption during early development. The current study was designed to explore if NHE8 plays a compensatory role for the loss of NHE2 and NHE3 function in NHE2X3 double-knockout (NHE2X3 DKO) mice. We further explored the regulatory mechanism(s) responsible for the change in NHE8 expression in NHE2X3 DKO mice. We found that >95% of NHE2X3 DKO mice survived through weanling. However, only 60% of male NHE2X3 DKO mice and 88% of female NHE2X3 DKO mice survived to 6 wk of life. We also found that the expression of NHE8 in wild-type female mice was higher compared with wild-type male mice after puberty. In NHE2X3 KDO mice, NHE8 expression was increased in females but not in males. Using Caco-2 cells as a model of the small intestine, we showed that testosterone inhibited endogenous NHE8 expression by reducing NHE8 mRNA synthesis, whereas estrogen had no effect on NHE8 expression. Thus our data show for the first time that intestinal NHE8 has a compensatory role in NHE2X3 DKO mice and this regulation is gender-dependent.

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.

NHE8 plays important roles in gastric mucosal protection

Sodium/hydrogen exchanger (NHE) 8 is an apically expressed membrane protein in the intestinal epithelial cells. It plays important roles in sodium absorption and bicarbonate secretion in the intestine. Although NHE8 mRNA has been detected in the stomach, the precise location and physiological role of NHE8 in the gastric glands remain unclear. In the current study, we successfully detected the expression of NHE8 in the glandular region of the stomach by Western blotting and located NHE8 protein at the apical membrane in the surface mucous cells by a confocal microscopic method. We also identified the expression of downregulated-in-adenoma (DRA) in the surface mucous cells in the stomach. Using NHE8(-/-) mice, we found that NHE8 plays little or no role in basal gastric acid production, yet NHE8(-/-) mice have reduced gastric mucosal surface pH and higher incidence of developing gastric ulcer. DRA expression was reduced significantly in the stomach in NHE8(-/-) mice. The propensity for gastric ulcer, reduced mucosal surface pH, and low DRA expression suggest that NHE8 is indirectly involved in gastric bicarbonate secretion and gastric mucosal protection.

Disruption of NHE8 expression impairs Leydig cell function in the testes

Multiple sodium/hydrogen exchanger (NHE) isoforms are expressed in the testes, and they play various roles in cell volume regulation, intracellular pH regulation, and fluid absorption. NHE8, the most recently characterized NHE family member, is detected in the Leydig cells in humans and mice in great abundance by immunohistochemistry in the current study. Male mice lacking NHE8 expression were infertile. Despite having intact male reproductive organs, male NHE8-/- mice have smaller testes and lacked spermatozoon in the seminiferous tubules and the epididymis. At the age of 39 wk, few spermogonia were seen in the testis in NHE8-/- mice. Although male NHE8-/- mice have normal serum levels of luteinizing hormone and follicle-stimulating hormone, serum testosterone level was significantly reduced. These mice have decreased expression of luteinizing hormone receptor in the testes. In NHE8 small-interfering RNA-transfected mouse Leydig cells (MLTC-1), silencing of NHE8 decreased the expression of luteinizing hormone receptor by ∼70%. Moreover, loss of NHE8 function in Leydig cells resulted in disorganized luteinizing hormone receptor membrane distribution. Therefore, male infertility in NHE8-/- mice is at least partially due to the disruption of luteinizing hormone receptor distribution and consequent low testosterone production, which leads to Sertoli cell dysfunction. Our work identified a novel role of NHE8 in male fertility through its effect on modifying luteinizing hormone receptor function.

1 Loss of NHE8 Expression Results in Gastric Ulcers in Mice

NHE8 is a member of the SLC9 family. This Na+/H+ exchanger is expressed on the apical membrane of intestinal epithelial cells. Although NHE8 has been shown to be important for intestinal sodium absorption early in development, its physiological role in the gastrointestinal tract remains unknown. By using NHE8 knockout (NHE8KO) mouse model, we discovered that NHE8 plays an important role in protecting the gastric mucosa against gastric acid. Stomach tissue was isolated from NHE8KO mice and their wild-type littermates. Localization of NHE8 expression in the stomach was detected using Immunohistochemistry. Mucosal surface pH was measured using a surface pH meter. Histology of the stomach of NHE8KOmice was also analyzed. RNA was isolated and used for PCR to detect the expression of PAT1 and DRA. Total tissue lysate was prepared for Western detection of NHE8, PAT1 and DRA. Immunohistochemistry indicated that NHE8 is expressed on the apical membrane of gastric epithelial cells. Histological evaluation showed no abnormality of the stomach in NHE8KO mice. Interestingly, higher incidence of gastric bleeding was observed in NHE8KO mice. The appearance of ulcers was seen in the glandular stomach. Mucosal surface pH measurement indicated that NHE8KO mice had decreased mucosal surface pH compared to wild-type mice (3.72 ± 0.13 in NHE8KO vs. 4.26 ± 0.09 in wild-type). Lower surface pH of NHE8KO gastric mucosa suggests that HCO3secretion may be altered. Therefore, we studied the expression of PAT1 and DRA in NHE8KO mice. Indeed, PAT1 and DRA mRNA expression in the stomach of NHE8KO mice were significantly reduced by ~30% and ~90%, respectively, in NHE8KO mice compared to their wild-type littermates. At the protein expression level, PAT1 protein abundance was reduced by ~70% and DRA protein abundance was nearly abolished in NHE8KO mice. Thus NHE8 may play an important role in mucosal protection by participating bicarbonate secretion.

T1732 Inhibition of NHE8 Expression by Dexamethasone

with this activity. A combination of four subunits reached highest glucogenesis indicating a broader spectrum of glucosidase activities than any individual subunit alone. Some alphaLDx was not completely available for enzyme hydrolysis, and residues are considered either slowly digestible or resistant to human enzyme digestion. This study for the first time shows the direct role of individual mucosal glucosidase subunits in starch digestion and reveals the potential of producing slow glucose release dextrins.

W1778 Epidermal Growth Factor Effect on NHE8 in the Intestine

G A A b st ra ct s acetylcholine (ACh)-induced vasodilation in pressurized mesenteric arteries (diameter: 170250 microns).Methods: Isobaric preparations of 4th-order mesenteric arteries from SpragueDawley rats were used. The arterial diameter was continuously recorded using video microscopy. The effect of Chrm1-4, COX, NOS and potassium channel inhibitors on ACh-induced vasodilation was investigated. The role of Chrm on pressure-induced vascular tone (20-100 mm Hg) and phenylephrine sensitivity was also determined. Only arteries that developed myogenic tone were used. Results: Arteries spontaneously developed >20% reduction in passive diameter. ACh-induced dose-dependent (0.001-10 μM) vasodilation (ANOVA: P<0.05; log EC50: -6.49) was blocked by atropine (non-selective Chrm inhibitor;1 μM) and 4-DAMP (Chrm3-selective inhibitor; 0.1 μM; log EC50: -3.98) (P<0.05 for both). Pirenzepine (Chrm1-selective inhibitor; 1 μM; log EC50: -5.78), methoctramine (Chrm2-selective inhibitor; 1 μM; log EC50: -6.21) and tropicamide (Chrm4-selective inhibitor; 1 μM; log EC50: -5.73) had no effect on ACh-mediated vasodilation. ACh also stimilated dose-dependent vasodilation in arteries incubated with indomethacin (INDO) and L-NAME (COX and NOS inhibitors; 10 and 300 μM, respectively; log EC50: -6.53; ANOVA: P<0.05). Glibenclamide (KATP blocker; 10 μM), 4-aminopyridine (KV blocker; 1 mM), BaCl (KIR blocker; 30 μM) had no effect while tetraethylammonium (KCa blocker; 10 mM) attenuated INDOand LNAME-resistant vasodilation (P<0.05). Intraluminal application of apamin plus charybdotoxin (SKCa and IKCa blockers respectively; 50 nM each) completely abolished INDOand L-NAME-resistant ACh-mediated vasodilation (P<0.05). Additionally, 4-DAMP blocked INDOand L-NAME-resistant ACh-mediated vasodilation (P<0.05) but had no effect on pressure-induced myogenic tone or phenylephrine dose-response. Conclusions:These data indicate that, in small mesenteric arteries in rats, (i) Chrm3 plays a key role in ACh-mediated vasodilation but has no role in regulating basal vascular tone; (ii) Non-prostaglandin and non-NO mechanisms play an important role in Chrm3-mediated vasodilation that is likely induced by EDHF. We conclude that Chrm3 activation induces EDHF-mediated vasodilation in rat small mesenteric arteries.