Changes In NHE Activity Research Articles

Short‐ and Long‐term Regulation of Intestinal Na+/H+ exchange by TLR4 in intestinal epithelial cells

The ability of recognizing microbiological infections is largely dependent on toll‐like receptors (TLR). TLR4 is expressed in the cell membrane and is activated by bacterial lipopolysaccharide, one of components of the outer membrane of Gram‐negative bacteria.AimsTo evaluate the effect of TLR4 activation by Monophosphoryl Lipid A (MPLA) on Na+/H+ exchanger (NHE) activity in T84 cells as well the signal transduction set in motion.Material and methodsNHE activity and kinetics were measured using fluorimetric assays and protein expression was determined by western blot analysis.ResultsShort‐ and long‐term MPLA (0.01 to 50 µg/ml) exposure induced an NHE1 concentration dependent inhibition (st ‐4.9±5 to ‐25.2±5 %; lt ‐3.9± to ‐31.9±4 %) without any effect over NHE membrane protein expression. The cell signal triggered AC3, PKA, PLC and PKC signal molecules. Long‐term exposure induced changes in NHE activity but affecting different NHE isoforms depending on MPLA concentration, lower MPLA induced NHE3 inhibition and higher MPLA concentrations induced NHE1 inhibition. In both cases AC3, PKA, PLC and PKC are involved. That is, the final effector of NHE1 was PCK and for NHE3 was PKA. MPLA induced an impaired recover of pHi and consequent acidification of intracellular media.ConclusionsActivation of TLR4 exerts marked inhibition of both NHE1 and NHE3 activity in intestinal epithelial cells. Transduction mechanisms set into motion during short‐ and long‐term TLR4‐mediated inhibition of NHE1 activity involves AC and PKA. However, short‐ and long‐term TLR4 activation might use different signalling pathways.

Changes in the activity of defense mechanisms against induced acidosis during meiotic maturation in mouse oocytes

Previous work has indicated that although activity of the HCO3-/Cl- exchanger (AE), which regulates intracellular alkalosis, is high in the germinal vesicle (GV) stage, the oocyte is inhibited as it progresses through meiotic maturation. In this study, we aimed to investigate the defense mechanisms against acidosis during the meiotic maturation stages. Intracellular pH (pHi) was recorded using a microspectrofluorometric technique, and Na+/H+ (NHE) and Na+-dependent HCO3-/Cl- exchanger (NDCBE) activity were determined by measuring the recovery rate from induced acidosis. Additionally, SLC9A1 (for NHE) and SLC4A8 (for NDCBE) gene transcription levels were determined by real-time PCR. The recovery rate of first meiotic prophase (GV) oocytes was high, but it decreased during the meiotic metaphase II (MII) stage in HCO3--free medium; it became high again at the pronuclear zygote (PN) stage. Recovery rate was significantly inhibited by 5-(N-ethyl-N-isopropyl) amiloride and cariporide or in the absence of extracellular Na+, implicating NHE, specifically NHE1 activity. Moreover, the level of SLC9A1 transcription correlated with the observed changes in NHE activity. The changes in NHE activity during meiotic maturation displayed a similar pattern to that of AE. The recovery rate from acidosis was significantly higher in MII stage oocytes and PN zygotes in HCO3--containing medium; however, the increase was significantly inhibited in Na+-free medium or 4,4'-diisocyanatostilbene-2,2'-disulfonic acid. Furthermore, changes in the transcription of SLC4A8 during meiotic maturation were concordant with the level of exchanger activity. These results indicate that NDCBE activity is present in mouse oocytes and zygotes, and that this activity exhibits a different pattern than that of AE and NHE during meiotic maturation.

Molecular cloning, tissue distribution and ontogenetic expression of sodium proton exchanger isoform 2 (NHE-2) mRNA in the small intestine of pigs

Molecular cloning, tissue distribution and ontogenetic regulation of sodium/proton exchanger isoform 2 (NHE-2) mRNA expression were evaluated in the pig small intestine during postnatal development. The 2872-bp porcine full cDNA sequence of the NHE-2 (EF672046) cloned in this study showed 80% and 70% homology with known human and mouse gene sequence, respectively. Hydrophobic prediction suggests 13 putative membrane-spanning domains within porcine NHE-2. The porcine NHE-2 mRNA was detected in the brain, liver, kidney, heart, lung, small intestine and muscle. The small intestine had the highest NHE-2 mRNA abundance and the brain, lung and liver had the lowest NHE-2 mRNA abundance (P < 0.05). Along the longitudinal axis, the duodenum had the highest NHE-2 mRNA abundance and the ileum and colon had the lowest NHE-2 mRNA abundance (P < 0.05). The NHE-2 mRNA level was increased from day 1 to day 26 in the duodenum (P < 0.05) and dropped dramatically on day 30 (P < 0.05). There is no difference between day 1 and day 7 (P > 0.05). After day 30, the NHE-2 mRNA level remained the same except on day 90 (P > 0.05). The mRNA expression of NHE-2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages and growing stages of life, which may contribute to changes in NHE activity.

Molecular Cloning, Tissue Distribution and Ontogenetic Expression of Sodium Proton Exchanger (NHE2) mRNA in the Small Intestine of Pigs

Molecular cloning, tissue distribution and ontogenetic regulation of sodium/proton exchanger isoform 2 (NHE2) mRNA expression were evaluated in the pig small intestine during postnatal development. The 2872‐bp porcine full cDNA sequence of the NHE2 (EF672046) cloned in this study showed 80% and 70% homology with known human and mouse gene sequence, respectively. Hydrophobic prediction suggests 13 putative membrane‐spanning domains within porcine NHE2. The porcine NHE2 mRNA was detected in brain, liver, kidney, heart, lung, small intestine and muscle. The small intestine had the highest NHE2 mRNA abundance and the brain, lung and liver had the lowest NHE2 mRNA abundance (P &lt; 0.05). Along the longitudinal axis, the duodenum had the highest NHE2 mRNA abundance and the ileum and colon had the lowest NHE2 mRNA abundance (P &lt; 0.05). The NHE2 mRNA level was increased from the day 1 to day 26 in the duodenum (P &lt; 0.05) and dropped dramatically on day 30 (P &lt; 0.05). There is no difference between day 1 and day 7 (P &gt; 0.05). After day 30, the NHE2 mRNA level stayed at the same except on day 90 (P &gt; 0.05). The mRNA expression of NHE2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages and growing stages of life, which may contribute to changes in NHE activity.

Acute and Chronic Regulation of the Renal Na+/H+ Exchanger NHE3 in Rats with STZ-Induced Diabetes mellitus

Background: Early stages of diabetic nephropathy are characterized by alterations of glomerular filtration, increased tubular sodium and water reabsorption, and systemic volume expansion, which may be a major cause for the development of hypertension. As a significant fraction of renal salt and water transport is mediated by the proximal tubular Na⁺/H⁺ exchanger NHE3, we investigated its regulation in rats with STZ-induced diabetes mellitus. Methods: Male Sprague-Dawley rats were injected +/– streptozotocin (STZ, 60 mg/kg), and sacrificed after 2, 7 or 14 days. Renal cortical BBM vesicles were prepared to measure Na⁺/H⁺ exchange (NHE) activity and NHE3 protein abundance. Cortical NHE3 mRNA was extracted to perform Northern blot analysis. Pharmacological inhibitors were used in vivo and in vitro in order to identify isoform specificity conferring changes in NHE activity mediated by the diabetic milieu. Results: Compared to control rats, STZ rats were clearly hyperglycemic at all time points studied. NHE activity was significantly increased by 40 and 37% in diabetic rats after 7 and 14 days, respectively, but not after 2 days. The increase in Na⁺/H⁺ exchange activity was not inhibited by HOE-642 (3 µM). Administration of exogenous insulin to diabetic rats resulted in lower blood sugars, but not NHE activity. Moreover, serum glucose concentration did not correlate with NHE activity in any subgroup nor in all animals analyzed together. However, in STZ rats supplemented with exogenous insulin NHE activity was positively correlated with serum insulin concentrations (r = 0.86, p < 0.01). In vivo, the increase in NHE activity induced by STZ could be completely inhibited when rats were fed 6 ppm of HOE-642 with the diet over 14 days. The changes in Na⁺/H⁺ exchange activity were not paralleled by changes in NHE3 protein or mRNA abundance in diabetic rats at any of the time points investigated. Conclusions: These results suggest that proximal tubular Na/H exchange activity is modified in the early stage of diabetes mellitus.

Expression of the Na+/H+ exchanger regulatory protein family in genetically hypertensive rats.

To examine a possible involvement of a regulatory protein of Na+/H+ exchanger (NHE) in the increased renal NHE activity in spontaneously hypertensive rats (SHR), we investigated mRNA expression of inhibitory members of the NHE regulatory protein family, NHERF1 and NHERF2, in the kidney. Prehypertensive 4-week-old and hypertensive 11-week-old SHR and age-matched Wistar-Kyoto (WKY) rats were used to determine the changes in NHE activity and NHERF family expression in the kidney. Dahl salt sensitive (DS) and resistant rats were also used to examine whether these changes are specific for SHR. mRNA expression in the kidney was quantified by RNase protection assay. The NHE activity in primary cultured proximal tubular cells was measured as Na-dependent pHi recovery rate by the NH4Cl prepulse technique with 2'7'-bis-(2-carboxyethyl)-5.6-carboxyfluorescein (BCECF). NHERF1 mRNA expression was significantly decreased in both prehypertensive and hypertensive SHR in comparison with age-matched WKY rats, whereas NHERF2 mRNA expression was significantly increased in SHR only in the hypertensive period. Antihypertensive treatment did not abolish these changes seen in control SHR. On the other hand, hypertensive DS rats fed a high-salt diet showed significant decreases in NHE activity and NHE3 mRNA expression compared with normotensive DS rats fed a low-salt diet, without significant changes in NHERF1 and NHERF2 mRNA expression. These results suggest that decreased expression of NHERF1 may be related to the enhanced NHE activity in SHR and that these changes are likely to be genetically determined, whereas the increased NHERF2 expression may be induced as a compensatory mechanism.

Gestational profile of Na +/H + exchanger and Cl −/HCO 3− anion exchanger mRNA expression in placenta using real-time QPCR

The onset of maternal blood flow (10–12 weeks gestation) results in increased oxygenation of the placenta. We investigated whether the expressions of Na +/H + exchanger (NHE) and Cl −/HCO 3 − anion exchanger (AE), thought to have an important role in maintaining intracellular pH of the syncytiotrophoblast and fetal pH homeostasis, are altered at the same time as this increase in blood flow. Real-time quantitative PCR was used to examine steady state levels of NHE (NHE1, 2, 3) and AE (AE1, 2) mRNA expression in early (6–9 weeks) and late (10–13 weeks) first trimester and full-term (38–40 weeks) placentas. β-Actin, IF2B and GAPDH mRNA was also measured. None of the genes showed a significant difference in expression between the early and late first trimester groups. However, NHE2 ( p<0.001) and GAPDH ( p<0.05) mRNA expression significantly increased 18- and 3.7-fold between early first trimester and term. In conclusion, this study provides additional evidence that GAPDH is an unsuitable housekeeping gene for normalization of transcript levels in placenta. The expression of NHE and AE in the villous placenta is not altered concomitant with the onset of maternal blood flow. However, NHE2 transcripts appear to be gestationally regulated, which may contribute to changes in NHE activity.

Effects and interaction, of cariporide and preconditioning on cardiac arrhythmias and infarction in rat in vivo.

1. Although Na+-H+ exchange (NHE) inhibitors are reported to protect the myocardium against ischaemic injury, NHE activation has also been proposed as a potential mechanism of ischaemic preconditioning-induced protection. This study was performed to test any modifiable effect of cariporide, an NHE inhibitor, on cardioprotective effects of preconditioning. 2. Anaesthetized rats were subjected to 30 min of coronary artery occlusion and 150 min of reperfusion. The preconditioning (PC) was induced by 3 min of ischaemia and 10 min of reperfusion (1PC) or three episodes of 3 min ischaemia and 5 min reperfusion (3PC). Cariporide (0.3 mg kg(-1)) an NHE inhibitor, was administered 30 min (cari(30)) or 45 min (cari(45)) before coronary ligation (n=8-11 for each group). 3. Ventricular arrhythmias during 30 min ischaemia and infarct size (measured by triphenyltetrazolium (TTC) and expressed as a per cent area at risk (%AAR)) were determined. Cari(30) reduced ventricular fibrillation (VF) incidence and infarct size (from 45 to 0% and 34+/-4 to 9+/-2%; each P<0.05), whereas cari(45) did not. Likewise, 3PC reduced these variables (to 0% and 10+/-2%; P<0.05 in each case) whereas 1PC did not. Moreover, subthreshold preconditioning (1PC) and cariporide (cari(45)), when combined, reduced VF incidence and infarct size (to 0% and 15+3%; each P<0.05 ). 4. In conclusion, changes in NHE activity do not seem to be responsible for the cardioprotective action of ischaemic preconditioning. Protective effects of NHE inhibition and subthreshold preconditioning appear to act additively.

Angiotensin II and the maturation of renal cortical Na+/H+ exchanger activity during fetal life in sheep.

The postnatal rise in renal Na+ reabsorption is associated with an increase in proximal tubule apical membrane Na+/H+ exchanger (NHE) activity in sheep. Inasmuch as circulating angiotensin II (ANG II) levels increase immediately after birth and ANG II is known to upregulate NHE activity in the adult proximal tubule, we postulated that ANG II plays a role in mediating maturational changes in NHE activity. We therefore studied the effects of ANG II infusion (10 micrograms/h) for 24 h on renal cortical NHE activity in chronically instrumented, twin ovine fetuses (129 +/- 2 days gestation, term is 145 days, n = 10 pairs); one twin of each pair served as a control. After 24 h, the fetuses were killed and brush-border membrane vesicles (BBMV) were prepared from the renal cortices. Postinfusion plasma ANG II levels were significantly higher and plasma renin activities were significantly lower in treated fetuses compared with controls. Kinetic analysis revealed an increase in NHE activity after ANG II treatment; however, the difference was not statistically significant: maximal velocity (in nmol.s-1.mg protein-1) control 1.65 +/- 0.50, treated 2.31 +/- 0.66 (P = 0.11, n = 9 pairs); Michaelis constant control 8.29 +/- 1.17 mM, treated 9.84 +/- 1.26 mM (P = 0.11). Northern blots of total RNA from the cortices of these animals were hybridized to a D-[32P]UTP-labeled antisense RNA probe prepared from a 1.3-kb rat NHE3 cDNA fragment. There were no differences between the groups in NHE3 mRNA levels (32P counts were control 413 +/- 54, treated 340 +/- 46). ANG II does not appear to play an important role in the regulation of NHE activity in the proximal tubule of the near-term sheep fetus.

Glucose-induced changes in activity and phosphorylation of the Na +/H + exchanger, NHE-1, in vascular myocytes from Wistar-Kyoto and spontaneously hypertensive rats

Increased Na +/H + exchanger (NHE) activity has been demonstrated in cells from patients with hypertension and diabetic nephropathy. Vascular myocytes from the spontaneously hypertensive rat (SHR) also exhibit increased NHE activity as compared with cells from the normotensive Wistar Kyoto rat (WKY). The interaction of increased glucose concentrations with NHE activity is unclear. The effect of glucose on NHE activity, NHE-1 (isoform 1) protein expression, and phosphorylation of cultured vascular myocytes from these rat strains was thus investigated. NHE activity was determined fluorometrically with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). A rabbit NHE-1—specific polyclonal antibody was used (1) to measure NHE-1 abundance in Western blots of cell extracts and (2) for immunoprecipitating 32P-labeled NHE-1. Cells from SHR exhibited increased NHE activity and NHE-1 phosphorylation as compared with cells from WKY, with similar NHE-1 protein content per cell. Incubation in 25 mmol · L −1 glucose for 24 hours led to increased NHE activity only in WKY cultures, with no change in NHE-1 protein but a concomitantly reduced NHE-1 phosphorylation. Changes in NHE activity in WKY cells were reversed by inhibition of protein kinase C. Incubation of SHR cells with 25 mmol · L −1 glucose did not enhance the increased NHE activity or NHE-1 phosphorylation present in these cells. Thus, high glucose levels have disparate effects on NHE activity and NHE-1 phosphorylation in cells from different rat strains. The glucose-induced increase in NHE-1 turnover number in WKY cells is not mediated by an increase in its direct phosphorylation, but is dependent on protein kinase C.

Mutational analysis of transmembrane histidines in the amiloride-sensitive Na+/H+ exchanger.

The histidine-reactive reagent, diethyl pyrocarbonate (DEPC) inhibits the human amiloride-sensitive Na+/H+ exchanger (NHE1) in stably transfected fibroblasts. NHE1 was protected by cimetidine and amiloride from DEPC, and DEPC inhibition was reversed with hydroxylamine, suggesting a role for critical histidine groups in NHE activity. We replaced the histidines (H) in putative transmembrane domains (H35, H120, H349) with glycine (G) using site-directed mutagenesis. There was no significant change in NHE activity of the H120G; H349G; H120,349G; and H35,120,349G mutants compared with wild type. The 50% inhibition concentration values for amiloride, ethyl isopropyl amiloride (EIPA), and cimetidine of the H349G mutant were significantly increased compared with the wild-type NHE1. We also examined the DEPC effect on the transport activity of the triple histidine mutant (H35,120,349G) and found that NHE1 activity was still inhibited by DEPC with reversal by hydroxylamine and protected by amiloride and cimetidine. Kinetic analysis of DEPC inhibition indicated that two "critical" histidine residues are required for NHE transport activity. Substitutions of H349 with asparagine (N), glutamine (Q), serine (S), tyrosine (Y), valine (V), leucine (L), and phenylalanine (F) were also examined. There were no changes in NHE activity of these mutants compared with wild type. The H349G and H349L mutants became more resistant to amiloride, whereas the H349Y and H349F mutants became more sensitive to amiloride. The H349S (mimics NHE3) and H349Y (mimics NHE4) mutations had only modest effects on amiloride sensitivity. These results indicate that H349 affects the interaction of NHE1 with its inhibitors, even though substitutions at this site, per se, do not appear to explain the differences in amiloride sensitivity between different NHE isoforms. Despite clear-cut effects of the H349G mutation on the competitive interaction of NHE1 with cimetidine and EIPA, this mutation did not affect the affinity of NHE1 for its cationic substrates (Na+, Li+).

Glucose-Induced Changes in Turnover of Na+/H+ Exchanger of Immortalized Lymphoblasts From Type I Diabetic Patients With Nephropathy

Increased cellular Na+/H+ exchanger (NHE) activity has been demonstrated in type I diabetic patients with nephropathy. Such patients also have a previous history of poor glycemic control. The interaction between hyperglycemia and changes in NHE activity remains obscure. Therefore, we examined the effects of media containing 5 and 25 mmol/l glucose on the increased NHE activity and turnover number in Epstein-Barr virus-transformed lymphoblasts from patients with diabetic nephropathy compared with normoalbuminuric diabetic and nondiabetic control subjects. NHE activity was determined fluorometrically, and NHE isoform 1 (NHE-1) density was measured with specific polyclonal antibodies. In the presence of 5 mmol/l glucose, cells from patients with diabetic nephropathy exhibited higher NHE activity with intracellular pH clamped to 6.0 compared with diabetic and nondiabetic control subjects (P < 0.005 for both), due to a higher turnover number of NHE-1. Incubation in 25 mmol/l glucose for 48 h caused an increase in NHE activity (P < 0.001) and turnover number (P < 0.01) in the diabetic nephropathy group only, with no significant change in the diabetic or nondiabetic control groups. The rate constants for cell proliferation and NHE activity or turnover number were correlated when cells were cultured in 5 mmol/l glucose (r = 0.34 and 0.32, respectively; P < 0.05) or 25 mmol/l glucose media (r = 0.66 and 0.65, respectively; P < 0.001). We conclude that only lymphoblasts from the diabetic nephropathy group show an increase in NHE activity and turnover number under conditions mimicking hyperglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)