Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport and cause congenital hypophosphatemia.

The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg495His or Arg495Cys variants within the internal PDZ motif. The wild-type internal 494TRL496 PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a 494AAA496 substitution blocked hormone-inhibitable phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg495His or Arg495Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast with WT NPT2A, NPT2A Arg495His, or Arg495Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg495 changes the electrostatics, preventing phosphorylation of the upstream Thr494, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxy-terminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

Deletion of the sodium/hydrogen exchanger 6 causes low bone volume in adult mice.

The sodium/hydrogen exchanger 6 (NHE6) localizes to recycling endosomes, where it mediates endosomal alkalinization through K+/H+ exchange. Mutations in the SLC9A6 gene encoding NHE6 cause severe X-linked mental retardation, epilepsy, autism and corticobasal degeneration in humans. Patients with SLC9A6 mutations exhibit skeletal malformations, and a previous study suggested a key role of NHE6 in osteoblast-mediated mineralization. The goal of this study was to explore the role of NHE6 in bone homeostasis. To this end, we studied the bone phenotype of NHE6 knock-out mice by microcomputed tomography, quantitative histomorphometry and complementary ex vivo and in vitro studies. We detected NHE6 transcript and protein in both differentiated osteoclasts and mineralizing osteoblasts. In vitro studies with osteoclasts and osteoblasts derived from NHE6 knock-out mice demonstrated normal osteoclast differentiation and osteoblast proliferation without an impairment in mineralization capacity. Microcomputed tomography and bone histomorphometry studies showed a significantly reduced bone volume and trabecular number as well as an increased trabecular space at lumbar vertebrae of 6 months old NHE6 knock-out mice. The bone degradation marker c-terminal telopeptides of type I collagen was unaltered in NHE6 knock-out mice. However, we observed a reduction of the bone formation marker procollagen type 1 N-terminal propeptide, and increased circulating sclerostin levels in NHE6 knock-out mice. Subsequent studies revealed a significant upregulation of sclerostin transcript expression in both primary calvarial cultures and femora derived from NHE6 knock-out mice. Thus, loss of NHE6 in mice causes an increase of sclerostin expression associated with reduced bone formation and low bone volume.

FengLiao affects gut microbiota and the expression levels of Na+/H+ exchangers, aquaporins and acute phase proteins in mice with castor oil-induced diarrhea.

The severe side effects of chemosynthetic anti-diarrhea drugs have created an interest in low-toxic alternative plant-derived compounds. FengLiao consists of Polygonum hydropiper Linn. and Daphniphyllum calycinum Bench., and is widely used in China to treat diarrhea due to low levels of toxicity. In this study, the effects of FengLiao were analyzed in a castor oil-induced diarrhea model, using the anti-diarrhea drug, loperamide, as the positive control. The effects were evaluated using stool characteristics and the expression levels of various diarrhea-related factors in the jejunum and liver, as well as changes in the microbiota of the jejunum. The symptoms of diarrhea and stool consistency were improved through FengLiao and loperamide treatment. Furthermore, FengLiao down-regulated alpha 1-acid glycoprotein (AGP) and C-reactive protein (CRP) levels, and up-regulated transferrin (TRF) mRNA levels in the liver, and down-regulated Aquaporin 3 (AQP3) and Na+/H+ exchanger isoform 8 (NHE8) expression in the epithelial cells of the jejunum. It also increased the relative abundance of Bifidobacterium, Aerococcus, Corynebacterium_1 and Pseudomonas, and lowered the Firmicutes/Bacteroidetes (F/B) ratio, which maintained the balance between immunity and intestinal health. Taken together, FengLiao alleviated castor oil-induced diarrhea by altering gut microbiota, and levels of jejunum epithelial transport proteins and acute phase proteins.

A gain of function paradox: Targeted therapy for glioblastoma associated with abnormal NHE9 expression.

Glioblastoma (GBM) is the most frequent and inevitably lethal primary brain cancer in adults. It is recognized that the overexpression of the endosomal Na+ /H+ exchanger NHE9 is a potent driver of GBM progression. Patients with NHE9 overexpression have a threefold lower median survival relative to GBM patients with normal NHE9 expression, using available treatment options. New treatment strategies tailored for this GBM subset are much needed. According to the prevailing model, NHE9 overexpression leads to an increase in plasma membrane density of epidermal growth factor receptors (EGFRs) which consequently enhances GBM cell proliferation and migration. However, this increase is not specific to EGFRs. In fact, the hallmark of NHE9 overexpression is a pan-specific increase in plasma membrane receptors. Paradoxically, we report that this gain of function in NHE9 can be exploited to effectively target GBM cells for destruction. When exposed to gold nanoparticles, NHE9 overexpressing GBM cells accumulated drastically high amounts of gold via receptor-mediated endocytosis, relative to control. Irradiation of these cells with near-infrared light led to apoptotic tumour cell death. A major limitation for delivering therapeutics to GBM cells is the blood-brain barrier (BBB). Here, we demonstrate that macrophages loaded with gold nanoparticles can cross the BBB, deliver the gold nanoparticles and effect the demise of GBM cells. In combination with receptor tyrosine kinase inhibition, we show this approach holds great promise for a new GBM-targeted therapy.

Genome-Wide Identification of Na+/H+ Antiporter (NHX) Genes in Sugar Beet (Beta vulgaris L.) and Their Regulated Expression under Salt Stress.

Salinity is one of the major environment factors that limits the growth of plants and the productivity of crops worldwide. It has been shown that Na+ transporters play a central role in salt tolerance and development of plants. The objective of this study was to identify Na+/H+ antiporter (NHX) genes and investigate their expression patterns in sugar beet (Beta vulgaris L.) subjected to various concentrations of NaCl. A total of five putative NHX genes were identified and distributed on four chromosomes in sugar beet. Phylogenetic analysis revealed that these BvNHX genes are grouped into three major classes, viz Vac- (BvNHX1, -2 and -3), Endo- (BvNHX4), and PM-class NHX (BvNHX5/BvSOS1), and within each class the exon/intron structures are conserved. The amiloride-binding site is found in TM3 at N-terminus of Vac-class NHX proteins. Protein-protein interaction (PPI) prediction suggested that only BvNHX5 putatively interacts with calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK), implying it might be the primary NHX involved in CBL-CIPK pathway under saline condition. It was also found that BvNHX5 contains one abscisic acid (ABA)-responsive element (ABRE), suggesting that BvNHX5 might be involved in ABA signal responsiveness. Additionally, the qRT-PCR analysis showed that all the BvNHX genes in both roots and leaves are significantly up-regulated by salt, and the transcription levels under high salinity are significantly higher than those under either low or moderate salinity. Taken together, this work gives a detailed overview of the BvNHX genes and their expression patterns under salt stress. Our findings also provide useful information for elucidating the molecular mechanisms of Na+ homeostasis and further functional identification of the BvNHX genes in sugar beet.

Potassium resistance of halotolerant and alkaliphilic Halomonas sp. Y2 by a Na+-induced K+ extrusion mechanism.

In most halophiles, K+ generally acts as a major osmotic solute for osmotic adjustment and pH homeostasis. However, strains also need to extrude excessive intracellular K+ to avoid its toxicity. In the halotolerant and alkaliphilic Halomonas sp. Y2, an Na+-induced K+ extrusion process was observed when the cells were confronted with high extracellular K+ pressure and supplementation by millimolar Na+ ions. Among three mechanosensitive channels (KefA) and two K+/H+ antiporters founded in the genome of the strain, ke1 displayed around 3-5-fold upregulation to ion stress at pH 8.0, while much higher upregulation of Ha-mrp was observed at pH 10.0. Compared to the growth of wild-type Halomonas sp. Y2, deletion of these genes from the strain resulted in different growth phenotypes in response to the osmotic pressure of potassium. In combination with the transcriptional response of these genes, we proposed that the KefA channel of Ke1 is the main contributor to the K+-extrusion process under weak alkalinity, while the Mrp system plays critical roles in alleviating K+ contents at high pH. The combination of these strategies allows Halomonas sp. Y2 to grow over a range of extracellular pH and ion concentrations, and thus protect cells under high osmotic stress conditions.

Expressing class I wheat NHX (TaNHX2) gene in eggplant (Solanum melongena L.) improves plant performance under saline condition.

Brinjal or eggplant (Solanum melongena L.) is an important solanaceous edible crop, and salt stress adversely affects its growth, development, and overall productivity. To cope with excess salinity, vacuolar Na+/H+ antiporters provide the best mechanism for ionic homeostasis in plants under salt stress. We generated transgenic eggplants by introducing wheat TaNHX2 gene that encodes a vacuolar Na+/H+ antiporter in to the eggplant genome via Agrobacterium-mediated transformation using pBin438 vector that harbors double35S:TaNHX2 to confer salinity tolerance. Polymerase chain reaction and southern hybridization confirmed the presence and integration of TaNHX2 gene in T1 transgenic plants. Southern positive transgenic eggplants showed varied levels of TaNHX2 transcripts as evident by RT-PCR and qRT-PCR. Stress-inducible expression of TaNHX2 significantly improved growth performance and Na+ and K+ contents from leaf and roots tissues of T2 transgenic eggplants under salt stress, compared to non-transformed plants. Furthermore, T2 transgenic eggplants displayed the stable leaf relative water content and chlorophyll content, proline accumulation, improved photosynthetic efficiency, transpiration rate, and stomatal conductivity than the non-transformed plants under salinity stress (200 mM NaCl). Data showed that the T2 transgenic lines revealed that reduction in MDA content, hydrogen peroxide, and oxygen radical production associated with the significant increase of antioxidant enzyme activity in transgenic eggplants than non-transformed plants under salt stress (200 mM NaCl). This study suggested that the TaNHX2 gene plays an important regulatory role in conferring salinity tolerance of transgenic eggplant and thus may serve as a useful candidate gene for improving salinity tolerance in other vegetable crops.

Exogenous Pi supplementation improved the salt tolerance of maize (Zea mays L.) by promoting Na+ exclusion.

The mechanism of phosphate (Pi)-mediated salt tolerance in maize is poorly understood. In this study, the effects of Pi (H2PO4-) on the salt tolerance of two contrasting genotypes was investigated in a pot experiment. We discovered that the application of 3 mM Pi could alleviate salt injury caused by 200 mM NaCl. High amounts of compatible solutes and low amounts of reactive oxygen species (ROS) were also observed under Pi application. Consistent with the increased tolerance, the total number of roots and the growth of shoots increased to relieve salt stress. This phenomenon could be associated with the observed increased expression of nitrate transporters. Furthermore, the seedlings presented a negative relationship between sodium (Na+) and Pi (low Na+ content and high Pi content), which is related to the genes ZmNHX1, ZmPHT1;8, and ZmPHT1;9, indicating that the exclusion of Na+ was promoted by high Pi uptake. However, high Na+ and low potassium (K+) efflux were detected in the roots, and these were positively correlated with two K+ transporters. These observations indicate that Na+ exclusion was directly induced by high K+ retention rather than Pi absorption. We conclude that maize salt tolerance increased in response to Pi application by promoting Na+ exclusion.

Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer.

The degree of intrinsic and interpatient phenotypic heterogeneity and its role in tumor evolution is poorly understood. Phenotypic drifts can be transmitted via inheritable transcriptional programs. Cell-type specific transcription is maintained through the activation of epigenetically defined regulatory regions including promoters and enhancers. Here we have annotated the epigenome of 47 primary and metastatic estrogen-receptor (ERα)-positive breast cancer clinical specimens and inferred phenotypic heterogeneity from the regulatory landscape, identifying key regulatory elements commonly shared across patients. Shared regions contain a unique set of regulatory information including the motif for transcription factor YY1. We identify YY1 as a critical determinant of ERα transcriptional activity promoting tumor growth in most luminal patients. YY1 also contributes to the expression of genes mediating resistance to endocrine treatment. Finally, we used H3K27ac levels at active enhancer elements as a surrogate of intra-tumor phenotypic heterogeneity to track the expansion and contraction of phenotypic subpopulations throughout breast cancer progression. By tracking the clonality of SLC9A3R1-positive cells, a bona fide YY1-ERα-regulated gene, we show that endocrine therapies select for phenotypic clones under-represented at diagnosis. Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients.

Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells.

BACKGROUND/AIMS: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs). Na+/H+-exchanger 3 (NHE3) has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear. METHODS: In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3) levels, transmission electron microscopy (TEM), tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62) mRNA and protein levels, and autophagy-related gene (ATG) 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes) to red (autolysosomes) LC3 puncta. RESULTS: Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment. CONCLUSIONS: These results demonstrate that NHE (most likely NHE3) may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.

Salt supplementation ameliorates developmental kidney defects in COX-2-/- mice.

Deficiency of cyclooxygenase-2 (COX-2) activity in the early postnatal period causes impairment of kidney development leading to kidney insufficiency. We hypothesize that impaired NaCl reabsorption during the first days of life is a substantial cause for nephrogenic defects observed in COX-2-/- mice and that salt supplementation corrects these defects. Daily injections of NaCl (0.8 mg·g-1·day-1) for the first 10 days after birth ameliorated impaired kidney development in COX-2-/- pups resulting in an increase in glomerular size and fewer immature superficial glomeruli. However, impaired renal subcortical growth was not corrected. Increasing renal tubular flow by volume load or injections of KCl did not relieve the renal histomorphological damage. Administration of torsemide and spironolactone also affected nephrogenesis resulting in diminished glomeruli and cortical thinning. Treatment of COX-2-/- pups with NaCl/DOCA caused a stronger mitigation of glomerular size and induced a slight but significant growth of cortical tissue mass. After birth, renal mRNA expression of NHE3, NKCC2, ROMK, NCCT, ENaC, and Na+/K+-ATPase increased relative to postnatal day 2 in wild-type mice. However, in COX-2-/- mice, a significantly lower expression was observed for NCCT, whereas NaCl/DOCA treatment significantly increased NHE3 and ROMK expression. Long-term effects of postnatal NaCl/DOCA injections indicate improved kidney function with normalization of pathologically enhanced creatinine and urea plasma levels; also, albumin excretion was observed. In summary, we present evidence that salt supplementation during the COX-2-dependent time frame of nephrogenesis partly reverses renal morphological defects in COX-2-/- mice and improves kidney function.

Effect of exogenous selenium supply on photosynthesis, Na+ accumulation and antioxidative capacity of maize (Zea mays L.) under salinity stress.

The mechanism of selenium-mediated salt tolerance has not been fully clarified. This study investigated the possible role of selenium (Se) in regulating maize salt tolerance. A pot experiment was conducted to investigate the role of Se (0, 1, 5 and 25 µM Na2SeO3) in photosynthesis, antioxidative capacity and ion homeostasis in maize under salinity. The results showed that Se (1 µM) relieved the salt-induced inhibitory effects on the plant growth and development of 15-day-old maize plants. Se application (1 µM) also increased the net photosynthetic rate and alleviated the damage to chloroplast ultrastructure induced by NaCl. The superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were increased, and ZmMPK5, ZmMPK7 and ZmCPK11 were markedly up-regulated in the roots of Se-treated plants, likely contributing to the improvement of antioxidant defence systems under salinity. Moreover, 1 µM Se increased K+ in the shoots while decreasing Na+ in the roots, indicating that Se up-regulates ZmNHX1 in the roots, which may be involved in Na+ compartmentalisation under salinity. The findings from this single experiment require repetition together with measurement of reactive oxygen species (ROS), but nevertheless suggest that exogenous Se alleviates salt stress in maize via the improvement of photosynthetic capacity, the activities of antioxidant enzymes and the regulation of Na+ homeostasis.

Variation in tissue Na(+) content and the activity of SOS1 genes among two species and two related genera of Chrysanthemum.

BACKGROUND: Chrysanthemum, a leading ornamental species, does not tolerate salinity stress, although some of its related species do. The current level of understanding regarding the mechanisms underlying salinity tolerance in this botanical group is still limited. RESULTS: A comparison of the physiological responses to salinity stress was made between Chrysanthemum morifolium 'Jinba' and its more tolerant relatives Crossostephium chinense, Artemisia japonica and Chrysanthemum crassum. The stress induced a higher accumulation of Na(+) and more reduction of K(+) in C. morifolium than in C. chinense, C. crassum and A. japonica, which also showed higher K(+)/Na(+) ratio. Homologs of an Na(+)/H(+) antiporter (SOS1) were isolated from each species. The gene carried by the tolerant plants were more strongly induced by salt stress than those carried by the non-tolerant ones. When expressed heterologously, they also conferred a greater degree of tolerance to a yeast mutant lacking Na(+)-pumping ATPase and plasma membrane Na(+)/H(+) antiporter activity. The data suggested that the products of AjSOS1, CrcSOS1 and CcSOS1 functioned more effectively as Na (+) excluders than those of CmSOS1. Over expression of four SOS1s improves the salinity tolerance of transgenic plants and the overexpressing plants of SOS1s from salt tolerant plants were more tolerant than that from salt sensitive plants. In addition, the importance of certain AjSOS1 residues for effective ion transport activity and salinity tolerance was established by site-directed mutagenesis and heterologous expression in yeast. CONCLUSIONS: AjSOS1, CrcSOS1 and CcSOS1 have potential as transgenes for enhancing salinity tolerance. Some of the mutations identified here may offer opportunities to better understand the mechanistic basis of salinity tolerance in the chrysanthemum complex.

Protective effect of ultrafiltered XinMaiJia extract against H2O2-induced injury in human umbilical vein endothelial cells through NHE1 downregulation.

We examined the protective effects of ultrafiltered XinMaiJia (XMJ) extract in a hydrogen peroxide (H2O2)-induced injury model in human umbilical vein endothelial cells (HUVECs) and determined the corresponding changes in the Na(+)-H(+) exchanger (NHE1) protein content and NHE1 gene expression. H2O2-induced HUVECs were treated with different concentrations of XMJ extract and the corresponding changes in morphology, activity, membrane permeability, biochemical indicators, cytokine concentration, NHE1 protein content, and NHE1 gene expression were determined. H2O2 significantly promoted HUVEC injury, whereas ultrafiltered XMJ extract significantly improved the morphological changes in injured HUVECs, increased their activity, and decreased NHE1 gene and protein expression, as well as limited the decrease in membrane permeability and expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, interleukin (IL)-1, IL-6, and nuclear factor-kB. Ultrafiltered XMJ extract inhibited H2O2-induced HUVEC injury by inhibiting NHE1 activity.

Comparison of effect of sex hormone manipulation during neonatal period, on mRNA expression of Slc9a4, Nr3c2, Htr5b and Mas1 in hippocampus and frontal cortex of male and female rats.

It has long been known that spatial memory and the ability to navigate through space are sexually dimorphic traits among mammals, and numerous studies have shown that these traits can be altered by means of sex hormone manipulation. Hippocampus, the main organ involved in this kind of memory, has specific signature genes with high expression level compared to other regions of the brain. Based on their expression levels and the role that products of these genes can play in processes like signal transduction, mediation of hormone effects and long term potentiation, these genes can be considered as genes necessary for routine tasks of hippocampus. Male and female rat pups were injected with estradiol and testosterone respectively. at early stage of their lives to examine the effect of sex hormone manipulation on mRNA expression of Slc9a4, Nr3c2, Htr5b and Mas1 using comparative quantitative real-time polymerase chain reaction. The results showed that expressions of these genes are strongly influenced by sex hormones in both the frontal cortex and hippocampus, especially in male hippocampus, in which expression of all genes were up-regulated. Htr5b was the only gene that was affected only in the males. Expression of Mas1 was contrary to expectations, showed stronger changes in its expression in cortex than in hippocampus. Nr3c2 was down regulated in all samples but up regulated in male hippocampus, and Slc9a4 also showed a huge up-regulation in male hippocampus compared to other samples.

Na+/H+ exchanger regulatory factor 1 (NHERF1) directly regulates osteogenesis.

Bone formation requires synthesis, secretion, and mineralization of matrix. Deficiencies in these processes produce bone defects. The absence of the PDZ domain protein Na(+)/H(+) exchange regulatory factor 1 (NHERF1) in mice, or its mutation in humans, causes osteomalacia believed to reflect renal phosphate wasting. We show that NHERF1 is expressed by mineralizing osteoblasts and organizes Na(+)/H(+) exchangers (NHEs) and the PTH receptor. NHERF1-null mice display reduced bone formation and wide mineralizing fronts despite elimination of phosphate wasting by dietary supplementation. Bone mass was normal, reflecting coordinated reduction of bone resorption and formation. NHERF1-null bone had decreased strength, consistent with compromised matrix quality. Mesenchymal stem cells from NHERF1-null mice showed limited osteoblast differentiation but enhanced adipocyte differentiation. PTH signaling and Na(+)/H(+) exchange were dysregulated in these cells. Osteoclast differentiation from monocytes was unaffected. Thus, NHERF1 is required for normal osteoblast differentiation and matrix synthesis. In its absence, compensatory mechanisms maintain bone mass, but bone strength is reduced.

Berberine increases the expression of NHE3 and AQP4 in sennosideA-induced diarrhoea model.

Berberine, a compound isolated from Chinese Goldthread Rhizome, has been widely used as a non-prescription drug to treat diarrhoea in China. Previous studies have demonstrated multiple pharmacological activities for berberine, including its significant role in antimicrobial activity. However, its effect on ion exchange and water transfer remains unclear. The present study aims to explore the effect of berberine on the expression of Na(+)/H(+) exchanger3 (NHE3) and aquaporin4 (AQP4) in both diarrhoea mouse model induced by sennosideA and human intestinal epithelium cell line (HIEC). Semi-quantitative RT-PCR, immunohistochemistry and western blotting were adopted to detect the mRNA and protein expression levels of NHE3 and AQP4. Furthermore, the absorption of berberine and the PKC activity were detected by HPLC and PepTag® Assay to elucidate the underlying mechanisms. It was shown that the expression levels of NHE3 and AQP4 were significantly increased in the diarrhoea mice treated with berberine compared with the untreated diarrhoea mice. Similarly, the expression levels of NHE3 and AQP4 were strikingly enhanced in HIEC co-treated with sennosideA and berberine compared with samples treated with sennosideA only. We also found the maximal absorption of berberine to be approximately 0.01%. In addition, no significant change of PKC activity was observed in the different HIEC treated groups. These results showed that berberine was able to increase the expression of NHE3 and AQP4, suggesting that berberine might exhibit its anti-diarrhoeal effect partially by enhancing the absorption of Na(+) and water.

Expression of wheat Na(+)/H(+) antiporter TNHXS1 and H(+)- pyrophosphatase TVP1 genes in tobacco from a bicistronic transcriptional unit improves salt tolerance.

Abiotic stress tolerance of plants is a very complex trait and involves multiple physiological and biochemical processes. Thus, the improvement of plant stress tolerance should involve pyramiding of multiple genes. In the present study, we report the construction and application of a bicistronic system, involving the internal ribosome entry site (IRES) sequence from the 5'UTR of the heat-shock protein of tobacco gene NtHSF-1, to the improvement of salt tolerance in transgenic tobacco plants. Two genes from wheat encoding two important vacuolar ion transporters, Na(+)/H(+) antiporter (TNHXS1) and H(+)-pyrophosphatase (TVP1), were linked via IRES to generate the bicistronic construct TNHXS1-IRES-TVP1. Molecular analysis of transgenic tobacco plants revealed the correct integration of the TNHXS1-IRES-TVP1construct into tobacco genome and the production of the full-length bicistronic mRNA from the 35S promoter. Ion transport analyses with tonoplast vesicles isolated from transgenic lines confirmed that single-transgenic lines TVP1cl19 and TNHXS1cl7 had greater H(+)-PPiase and Na(+)/H(+) antiport activity, respectively, than the WT. Interestingly, the co-expression of TVP1 and TNHXS1 increased both Na(+)/H(+) antiport and H(+)-PPiase activities and induced the H(+) pumping activity of the endogenous V-ATPase. Transgenic tobacco plants expressing TNHXS1-IRES-TVP1 showed a better performance than either of the single gene-transformed lines and the wild type plants when subjected to salt treatment. In addition, the TNHXS1-IRES-TVP1 transgenic plants accumulated less Na(+) and more K(+) in their leaf tissue than did the wild type and the single gene-transformed lines. These results demonstrate that IRES system, described herein, can co-ordinate the expression of two important abiotic stress-tolerance genes and that this expression system is a valuable tool for obtaining transgenic plants with improved salt tolerance.

The Arabidopsis Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproduction.

Intracellular Na(+)/H(+) (NHX) antiporters have important roles in cellular pH and Na(+), K(+) homeostasis. The six Arabidopsis thaliana intracellular NHX members are divided into two groups, endosomal (NHX5 and NHX6) and vacuolar (NHX1 to NHX4). Of the vacuolar members, NHX1 has been characterized functionally, but the remaining members have largely unknown roles. Using reverse genetics, we show that, unlike the single knockouts nhx1 or nhx2, the double knockout nhx1 nhx2 had significantly reduced growth, smaller cells, shorter hypocotyls in etiolated seedlings and abnormal stamens in mature flowers. Filaments of nhx1 nhx2 did not elongate and lacked the ability to dehisce and release pollen, resulting in a near lack of silique formation. Pollen viability and germination was not affected. Quantification of vacuolar pH and intravacuolar K(+) concentrations indicated that nhx1 nhx2 vacuoles were more acidic and accumulated only 30% of the wild-type K(+) concentration, highlighting the roles of NHX1 and NHX2 in mediating vacuolar K(+)/H(+) exchange. Growth under added Na(+), but not K(+), partly rescued the flower and growth phenotypes. Our results demonstrate the roles of NHX1 and NHX2 in regulating intravacuolar K(+) and pH, which are essential to cell expansion and flower development.

High dose vitamin D3 attenuates the hypocalciuric effect of thiazide in hypercalciuric rats.

Thiazide is known to decrease urinary calcium excretion. We hypothesized that thiazide shows different hypocalciuric effects depending on the stimuli causing hypercalciuria. The hypocalciuric effect of hydrochlorothiazide (HCTZ) and the expression of transient receptor potential vanilloid 5 (TRPV5), calbindin-D(28K), and several sodium transporters were assessed in hypercalciuric rats induced by high calcium diet and vitamin D(3). Urine calcium excretion and the expression of transporters were measured from 4 groups of Sprague-Dawley rats; control, HCTZ, high calcium-vitamin D, and high calcium-vitamin D with HCTZ groups. HCTZ decreased urinary calcium excretion by 51.4% in the HCTZ group and only 15% in the high calcium-vitamin D with HCTZ group. TRPV5 protein abundance was not changed by HCTZ in the high calcium-vitamin D with HCTZ group compared to the high calcium-vitamin D group. Protein abundance of NHE3, SGLT1, and NKCC2 decreased in the hypercalciuric rats, and only SGLT1 protein abundance was increased by HCTZ in the hypercalciuric rats. The hypocalciuric effect of HCTZ is attenuated in high calcium and vitamin D-induced hypercalciuric rats. This attenuation seems to have resulted from the lack of HCTZ's effect on protein abundance of TRPV5 in severe hypercalciuric condition induced by high calcium and vitamin D.