Sodium-hydrogen Exchanger Isoform Research Articles

Transcriptional Regulation of the Rat NHE3 Gene: FUNCTIONAL INTERACTIONS BETWEEN GATA-5 AND Sp FAMILY TRANSCRIPTION FACTORS

Expression of sodium-hydrogen exchanger isoform 3 (NHE3) in the intestinal and renal epithelium plays a critical role in sodium absorption and acid/base homeostasis. To decipher rat NHE3 gene regulation, its cis-acting regulatory elements and associated transcription factors were characterized by transient transfection of Caco-2, IEC-6, Qt6, and Drosophila SL2 cells. Deletion and mutational analyses demonstrated that the atypical TATA box located at bp -26/-31 was not necessary for promoter activity, and that a -20/+8-bp fragment represents a functional initiator. Within the 81-bp upstream region, three Sp transcription factor binding sites were critical because their mutation drastically reduced promoter activity. The roles of Sp1 and Sp3 were further demonstrated by electromobility shift assay and by transactivation of the NHE3 promoter in SL2 cells by forced expression of Sp1 and Sp3. Both of these transcription factors were found to act synergistically with GATA-5 bound to a GATA box in exon 1 (+20/+23 bp). These studies demonstrate that rat NHE3 promoter is initiator-driven and controlled mainly by Sp1 and Sp3, which functionally interact with GATA-5. This interaction represents a novel regulatory mechanism, which is likely to participate in a gradient of intestinal gene expression along the crypt-villus axis.

NHERF-1 uniquely transduces the cAMP signals that inhibit sodium–hydrogen exchange in mouse renal apical membranes

Sodium–hydrogen exchanger regulatory factor isoform-1 (NHERF-1) and NHERF-2 are two structurally related PDZ-domain-containing protein adapters that effectively transduce cyclic AMP (cAMP) signals that inhibit NHE3, the sodium–hydrogen exchanger isoform present at the apical surface of kidney and gut epithelia. The mouse renal proximal tubule expresses both NHERF isoforms, suggesting their redundant functions as regulators of renal electrolyte metabolism. To define the role of NHERF-1 in the physiological control of NHE3, we analyzed NHE3 activity in isolated brush border membrane (BBM) preparations from renal proximal tubules of wild-type (WT) and NHERF-1 (−/−) mice. Basal Na +–H + exchange was indistinguishable in BBMs from WT and NHERF-1 (−/−) mice (0.96±0.08 and 0.95±0.10 nmol/mg protein/10 s, respectively). Activation of membrane bound cAMP-dependent protein kinase (PKA) by cAMP inhibited NHE3 activity in WT BBMs (0.55±0.07 nmol/mg protein/10 s or 40±9%, P<0.01) but had no discernible effect on Na +–H + exchange in the NHERF-1 (−/−) BBM (0.97±0.07 nmol/mg protein/10 s; P=not significant). This was associated with a significant decrease in cAMP-stimulated phosphorylation of NHE3 immunoprecipitated from solubilized NHERF-1 (−/−) BBMs. As the protein levels for NHE3, NHERF-2, PKA and ezrin were not changed in the NHERF-1 (−/−) BBMs, the data suggest a unique role for NHERF-1 in cAMP-mediated inhibition of NHE3 activity in the renal proximal tubule of the mouse.

Effects of the NHE-1 inhibitor cariporide alone or together with the P2Y 12 antagonist AR-C 69331 MX on CD62p expression and formation of platelet–leukocyte aggregates

The sodium–hydrogen exchanger isoform 1 (NHE-1) contributes to platelet activation at elevated pH. Effects of NHE-1 inhibitors on platelet degranulation and formation of proinflammatory and procoagulatory platelet–leukocyte aggregates (PLA) and possible interactions with P2Y 12 inhibitors—which also affect platelet degranulation—have not been investigated. Whole blood from healthy human subjects was incubated with the NHE-1 inhibitor cariporide and the P2Y 12 inhibitor AR-C 69331 MX at clinically reasonable concentrations, in the presence of normal pH or in a propionate model to activate the NHE-1 (approximately pH 7.0). The degranulation marker CD62p, the expression of the activated GPIIb/IIIa receptor (PAC-1), and formation of platelet–leukocyte (monocyte) aggregates (PLA) was assessed by flow cytometry. Cariporide at concentrations up to 20 μg/ml had no effects on ADP- (5 μM) or TRAP- (2 μM) induced CD62p expression or PLA formation at normal pH. At pH 7.0 and stimulation with ADP, PLA decreased from 64±24% (control) to 47±23% under cariporide at 2 μg/ml ( p<0.05), and the MFI of PLA (i.e. the platelet mass attached at monocytes) decreased from 547±203 to 360±96 units ( p<0.05). PAC-1 MFI decreased from 66±23 to 34±18 units ( p<0.05) after ADP and from 74±29 to 42±17 units ( p<0.05) after TRAP, respectively. AR-C 69331 MX (10 nM) had inhibitory effects on all parameters irrespectively of the pH, and the combination of both agents at pH 7.0 shows additive effects. In conclusion, our investigation points to—perhaps clinically relevant—effects of NHE-1 inhibition on the degranulation of platelets and formation of platelet–leukocyte aggregates.

EGF regulation of rat intestinal sodium hydrogen exchanger isoform 2 (NHE 2)

EGF regulation of rat intestinal sodium hydrogen exchanger isoform 2 (NHE 2)

EGF regulation of rat intestinal sodium hydrogen exchanger isoform 2 (NHE 2)

EGF regulation of rat intestinal sodium hydrogen exchanger isoform 2 (NHE 2)

Sodium–hydrogen exchangers and sodium–bicarbonate co-transporters: ontogeny of protein expression in the rat brain

We used western blotting to examine the developmental profiles (at embryonic day 16 and postnatal days 1, 13, 23, 33 and 105) of protein expression for three sodium–hydrogen exchanger isoforms (1, 2 and 4) and for a sodium–bicarbonate co-transporter in three CNS regions (cortex, cerebellum and brainstem–diencephalon). In microsomal preparations, sodium–hydrogen exchanger isoform 1 and sodium–bicarbonate co-transporter protein expression in the CNS increases gradually from embryonic day 16 (25–40% of the adult level) to postnatal day 105. In contrast, sodium–hydrogen exchanger isoform 2 and 4 expression reaches a maximum (three to 20 times the adult level) at around three to four weeks of age. There is significant regional heterogeneity in the expression of sodium–hydrogen exchanger and sodium–bicarbonate co-transporter proteins in the rat CNS. Sodium–hydrogen exchanger isoform 1 was highly expressed in the brainstem–diencephalon, whereas the sodium–bicarbonate co-transporter was robustly expressed in the cerebellum and brainstem–diencephalon. These data indicate that the expression of sodium–hydrogen exchanger and sodium–bicarbonate co-transporter proteins varies as a function of both development and specific brain region.

Sp1-mediated transcriptional regulation of the rat sodium hydrogen exchanger isoform 2 (NHE-2) gene promoter

The sodium hydrogen exchanger isoform 2 (NHE-2) is an apical membrane transporter which is highly expressed in the intestine. The regulation of NHE-2 under physiological and pathophysiological conditions is important in maintenance of electrolyte and acid-base balance. We have recently cloned the promoter of rat NHE-2 and identified two cis-elements required for osmotic response of rat NHE-2 gene in mIMCD-3 cells (Bai, et al., Am. J. Physiol. 46:RII12-9, 1999). To further investigate the mechanism controlling the activity of NHE-2 promoter, its regulatory elements and transcription factors were characterized in mIMCD-3 cells and Drosophila Schneider cells. Transient transfection of luciferase reporter gene constructs containing different lengths of NHE-2 promoter indicated that the minimal promoter was localized to the region of (-)21 to (+ )101 (as related to transcription initial site). Within this putative promoter region, two Sp1 elements were found to overlap in the (-)21 to (+)1 bp region. The introduction of mutations within these SpI elements and the use of SpI antisera in electrophoretic mobility shift assays demonstrated that Spl bound to this region of the NHE-2 promoter. Moreover, these in vitro studies indicated that both putative SpI response elements were utilized. Transient transfection assays using NHE-2 promoter constructs that incorporated mutations of the SpI elements clearly demonstrated that binding of Spl to the NHE-2 promoter was absolutely required for optimal levels of reporter gene expression. Expression of luciferase reporter gene linked to the (-)21 and (-)289 NHE-2 promoter regions was up-regulated by coexpression of Sp I expression vector in Drosophila Schneider cells, which lack the native SpI transcription factor. Therefore, these studies provide strong evidence that Sp I plays a central role in regulating basal levels of NHE-2 transcription. This study was supported by NIDDK Grant 2ROIDK41274-1O. 3054

Expression of the Na ‐H exchanger isoform‐1 and cyclooxygenases in human placentas: Their implications in preeclampsia

The sodium hydrogen exchanger isoform, NHE-1 plays an important role in electrolyte and water homeostasis. These functions are compromised in pregnancies complicated with preeclampsia. At present it is not known whether NHE-1 expression is altered during preeclampsia. In the present study the placental level of NHE-1 protein was measured using immunoblotting. Since prostaglandins regulate the secretory and absorptive functions, the levels of prostaglandin E-2 as well as the expression of cyclooxygenase-1 and -2 were also estimated. The amount of NHE-1 protein and cyclooxygenase-2 was reduced in preeclamptic placentas, whereas the level of cyclooxygenase-1 remained unaltered. In contrast, prostaglandin E-2 concentration was higher in preeclampsia. Suppression of NHE-1 might render the placenta with impaired uptake of water and electrolytes and therefore may be involved in the pathogenesis of preeclampsia. While prostaglandin E-2 may play a role in preeclampsia, these findings discount the induction of cyclooxygenase-genes for this increase.

Altered expression of the Na+/H+ exchanger isoform-3 in experimental colitis: effect of garlic.

The sodium-hydrogen exchanger isoform, NHE-3 is essential for the absorption of sodium and water from intestine. Whether this protein plays any role in inflammatory bowel disease is less understood. To address this issue, NHE-3 mRNA and protein levels were estimated in the terminal ileum and colon of the rats having colitis induced with trinitrobenzenesulphonic acid (TNBS). The effect of garlic (Allium sativum) was also evaluated on the expression of NHE-3. The animals were treated with garlic extract intraperitoneally starting 2 h before the TNBS administration until day 4 post-TNBS administration and were sacrificed on day 5. In control animals, the levels of NHE-3 in colon was higher than the ileum. As a result of colitis, the levels of NHE-3 protein and mRNA increased both in the colon and terminal ileum. Garlic treatment of the colitic animals resulted in a selective suppression of NHE-3 in the terminal ileum. Colitis caused an induction of the myeloperoxidase activity, the marker of inflammation in the colon but not in the ileum. These findings suggest that induction of NHE-3 is not primarily due to inflammation. Selective suppression of this protein in ileum by garlic may cause loss of sodium chloride and water during colitis.

NHE-1 is the sodium–hydrogen exchanger isoform present in erythroid cells

Erythrocyte sodium hydrogen exchanger (NHE) represents one of a limited number of sodium entry pathway in erythrocytes. At least five NHE isoforms have been identified, differing in tissue specificity, regulatory characteristics, and pharmacological sensitivities. Although physiological characteristics of erythrocyte NHE suggest that the widely expressed NHE-1 isoform may be present, evidence is not conclusive and does not exclude the existence of other isoforms. In this study, Northern blot and reverse transcription–polymerase chain reaction (RT–PCR) analyses were used to test for five NHE isoforms in erythroid cells. Blood from patients with sickle cell disease was depleted of white blood cells (WBC) by passage through leukocyte filters and cellulose column. RT–PCR performed on WBC depleted reticulocyte RNA using a NHE-1 primer set yielded product a of expected size, the sequence of which was identical to the published human NHE-1 sequence. Northern blot analysis of the reticulocyte RNA using a 1.6 kb probe revealed a message of approximately 5.0 kb in size. RT–PCR analysis of rat kidney RNA using primers specific for NHE isoforms -2, -3, -4 and rat brain RNA using primer specific for NHE-5 isoform yielded products of expected size, whereas WBC depleted RNA under identical conditions yielded no products. These results identify the erythroid isoform of the sodium–hydrogen exchanger as NHE-1.

Sodium-hydrogen exchanger isoform 3 (NHE-3) is not expressed in the jejunal mucosa of 2 patients with microvillus inclusion disease

Sodium-hydrogen exchanger isoform 3 (NHE-3) is not expressed in the jejunal mucosa of 2 patients with microvillus inclusion disease

Phylogenetically conserved sequences in the promoter of the rabbit sodium-hydrogen exchanger isoform 1 gene( NHE1/ SLC9 A1)

NHE1 (gene symbol SLC9 A1) encodes an isoform of the amiloride-sensitive Na +-H + exchanger that is present in many cells and the basolateral membrane of renal epithelia. Expression of NHE1 is modulated in response to chronic metabolic acidosis, growth factors, and phorbol esters. To begin examining the molecular basis for this regulation, a rabbit genomic clone that contained 6 kb of 5′ flanking region, the first exon, and a portion of the first intervening sequence of NHE1 was obtained. The principal transcription start site in native rabbit tissues was located 798 by 5' to the initiation codon. The sequence of the proximal 5′ flanking region of rabbit NHE1 was similar to the human sequence and contained a TATA-box, (G + C)-boxes, homopyrimidine direct repeats, and a putative AP-1 site. When ligated to luciferase and transfected into porcine renal epithelial cells (LLC-PK 1), 708 by of proximal 5′ flanking region exhibited orientation-dependent promoter activity.

Activity and density of the Na+/H+ antiporter in normal and transformed human lymphocytes and fibroblasts.

The turnover number for the sodium-hydrogen exchanger isoform 1 (NHE-1) has been determined in human lymphocytes and MRC5 fibroblasts and in their virally transformed counterparts. Using fluorometric methods, we have determined the intracellular pH and Na+/H+ antiport activity of these cells. Intracellular pH was elevated in both lines of transformed cells. In contrast, Na+/H+ antiport activity was apparently unchanged in simian virus 40-transformed MRC5 fibroblasts (MRC5 SV1 TV1 28.9 +/- 5.2 mM/min compared with MRC5 fibroblasts 26.5 +/- 5.3 mM/min) but slightly increased in Epstein-Barr virus-transformed lymphoblasts (16.7 +/- 1.0 mM/min compared with lymphocytes 13.5 +/- 2.3 mM/min, P < 0.05). With the use of specific antisera to NHE-1, viral transformation was associated with a decreased number of NHE-1 molecules per cell in fibroblasts (from 441,504 +/- 53,428 to 64,745 +/- 7,151 sites/cell, P < 0.001) but an increased number in lymphocytes (from 14,066 +/- 3,100 to 22,474 +/- 4,050 sites/cell, P < 0.01). The NHE-1 density per cell yielded very similar turnover numbers for NHE-1 in the untransformed cells (lymphocytes, 3,161 +/- 833 cycles/s; MRC5 fibroblasts, 3,026 +/- 441 cycles/s), which were significantly elevated in the transformed cells (lymphoblasts, 8,471 +/- 1,177 cycles/s; MRC5 SV1 TV1, 10,521 +/- 2,299 cycles/s, P < 0.001 compared with untransformed cells). We conclude that viral transformation has different effects on Na+/H+ antiport activity and NHE-1 density per cell in different cell types, but the turnover number of NHE-1 is significantly increased after viral transformation, which correlates with the increased proliferation rate of these transformed cells.

Sodium???hydrogen antiporter protein in normotensive Wistar???Kyoto rats and spontaneously hypertensive rats

To examine the mechanism of increased Na-H antiport activity in tissues of the spontaneously hypertensive rat (SHR) by measuring the amount of sodium-hydrogen exchanger isoform 1 (NHE-1) in cultured vascular and striated muscle cells, and in ex vivo tissue extracts of membranes from the brain, heart, kidney and skeletal muscle. A polyclonal rabbit antibody was raised against a fusion protein consisting of a section of the carboxyl tail of NHE-1 and beta-galactosidase. Cell extracts were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and proteins were transferred to supported nitrocellulose. NHE-1 was detected by Western blotting and quantified by densitometry. Cultured aortic and striated muscle cells from SHR contained similar amounts of NHE-1 on Western blots to those from control Wistar-Kyoto (WKY) rat cells. Ex vivo extracts of crude membranes from SHR tissues also contained quantities of NHE-1 similar to those from WKY rat tissues. The increased Na-H antiport activity observed in SHR cells in vitro and in vivo is not due to an increased amount of NHE-1 protein in SHR cells. This suggests that in this model of hypertension the increased transport activity results from an increased turnover number per NHE-1 molecule.