Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.     

Depletion of cellular cholesterol enhances macrophage MAPK activation by chitin microparticles but not by heat-killed Mycobacterium bovis BCG

When macrophages phagocytose chitin (N-acetyl-d-glucosamine polymer) microparticles, mitogen-activated protein kinases (MAPK) are immediately activated, followed by the release of Th1 cytokines, but not IL-10. To determine whether phagocytosis and macrophage activation in response to chitin microparticles are dependent on membrane cholesterol, RAW264.7 macrophages were treated with methyl-beta-cytodextrin (MBCD) and stimulated with chitin. These results were compared with the corresponding effects of bacterial components including heat-killed (HK) Mycobacterium bovis bacillus Calmette-Guèrin (BCG) and an oligodeoxynucleotide (ODN) of bacterial DNA (CpG-ODN). The MBCD treatment did not alter chitin binding or the phagocytosis of chitin particles 20 min after stimulation. At the same time, however, chitin-induced phosphorylation of cellular MAPK was accelerated and enhanced in an MBCD dose-dependent manner. The increased phosphorylation was also observed for chitin phagosome-associated p38 and ERK1/2. In contrast, CpG-ODN and HK-BCG induced activation of MAPK in MBCD-treated cells at levels comparable to, or only slightly more than, those of control cells. We also found that MBCD treatment enhanced the production of tumor necrosis factor-alpha (TNF-alpha) and the expression of cyclooxygenase-2 (COX-2) in response to chitin microparticles. In neither MBCD- nor saline-treated macrophages, did chitin particles induce detectable IL-10 mRNA synthesis. CpG-ODN induced TNF-alpha production, and COX-2 expression were less sensitive to MBCD treatment. Among the agonists studied, our results indicate that macrophage activation by chitin microparticles was most sensitive to cholesterol depletion, suggesting that membrane structures integrated by cholesterol are important for physiological regulation of chitin microparticle-induced cellular activation.     

Alpha-thrombin, epidermal growth factor, and okadaic acid activate the Na+/H+ exchanger, NHE-1, by phosphorylating a set of common sites.

The ubiquitous and amiloride-sensitive Na+/H+ exchanger (NHE-1), a plasma membrane phosphoglycoprotein that regulates intracellular pH, is rapidly activated by growth factors. We showed previously that epidermal growth factor (EGF), alpha-thrombin, or serum stimulates Na+/H+ exchange activity in growth-arrested Chinese hamster lung fibroblasts (ER22 cells) in a time-dependent manner which correlates with increased phosphorylation of NHE-1 at serine residues (Sardet, C., Counillon, L., Franchi, A., and Pouysségur, J. (1990) Science 247, 723-726). Here we show that the tumor promoter, okadaic acid, a potent in vivo inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), stimulates Na+/H+ exchange in G0-arrested ER22 cells and in exchanger-deficient fibroblasts transfected with the human NHE-1 cDNA. Okadaic acid effects are maximal at 1 microM (EC50 = 500 nM), detected in 2 min, complete within 15-20 min, and are additives when combined with EGF or alpha-thrombin. Parallel to the pHi-induced rise, okadaic acid alone or together with growth factors stimulated the phosphorylation of NHE-1. More importantly tryptic phosphopeptide maps of NHE-1, immunoprecipitated from cells treated with EGF, alpha-thrombin, or okadaic acid, show a common pattern of phosphorylation. This pattern consists of five major 32P-labeled peptides (P1-P5) present in lower amounts in resting cells. One of them, P5, barely detectable in resting cells is increased up to 15-fold in mitogen-stimulated cells. Taken together these results reinforce the notion that phosphorylation of NHE-1 controls the set point value of the exchanger and suggest that: (i) the proximate step in Na+/H+ exchange activation is mediated by as yet unidentified growth factor-activatable serine "NHE-1 kinase(s)" and (ii) this NHE-1 kinase(s), partly active in resting cells, integrate signals from receptor tyrosine kinases and G protein-coupled receptors.     

A close association of the ganglioside-specific sialidase Neu3 with caveolin in membrane microdomains

The ganglioside-specific sialidase Neu3 has been suggested to play essential roles in regulation of cell surface functions because of its major localization in the plasma membrane and strict substrate preference for gangliosides involved in signal transduction. Here we show that human Neu3 sialidase is enriched in caveolae microdomains and closely associates with caveolin like other caveolin-binding signaling molecules. Using HeLa cells and Neu3-transfected COS-1 cells, endogenous and exogenous Neu3 was found to co-concentrate caveolin-1 in low density Triton X-100-insoluble membrane fractions on sucrose density gradients of the respective cell extracts, as assessed by enzyme activity assays and immunoblotting with a monoclonal antibody to human Neu3. The presence of a putative caveolin-binding motif within Neu3 prompted us to determine whether Neu3 binds to caveolin-1. In transfectants expressing a polyhistidine-tagged form of Neu3, caveolin-1 co-eluted with Neu3 on affinity column chromatography. A mutation with a single amino acid change in the caveolin-binding motif led to inhibition of recruitment of the sialidase to the microdomain, accompanied by reduction of the enzyme activity. Neu3 also failed to associate with caveolin-enriched microdomains by cholesterol depletion with beta-cyclodextrin (with concomitant decrease of the sialidase activity), whereas Neu3 was activated by increased caveolin-1 expression. The tight association of Neu3 with caveolin-1 was supported further by co-immunoprecipitation of Neu3 by anti-caveolin-1 antibody. These results strongly suggest that Neu3 functions as a caveolin-related signaling molecule within caveolin-rich microdomains.     

Cannabinoid receptor CB1 activates the Na+/H+ exchanger NHE-1 isoform via Gi-mediated mitogen activated protein kinase signaling transduction pathways

We previously showed that the cannabinoid receptor CB1 stably transfected in Chinese hamster ovary cells was constitutively active and could be inhibited by the inverse agonist SR 141716A. In the present study, we demonstrate that the cannabinoid agonist CP-55940 induced cytosol alkalinization of CHO-CB1 cells in a dose- and time-dependent manner via activation of the Na+/H+ exchanger NHE-1 isoform. By contrast, the inverse agonist SR 141716A induced acidification of the cell cytosol, suggesting that the Na+/H+ exchanger NHE-1 was constitutively activated by the CB1 receptor. CB1-mediated NHE1 activation was prevented by both pertussis toxin treatment and the specific MAP kinase inhibitor PD98059. NHE-1 and p42/p44 MAPK had a similar time course of activation in response to the addition of CP-55940 to CHO-CB1 cells. These results suggest that CB1 stimulates NHE-1 by G(i/o)-mediated activation of p42/p44 MAP kinase and highlight a cellular physiological process targeted by CB1.     

Focal localization of the NHE-1 isoform of the Na+/H+ antiport: assessment of effects on intracellular pH.

Na+/H+ exchange (antiport) is a major pathway for the regulation of intracellular pH. Antiport activity is stimulated when suspended cells adhere to the substratum. In this report, immunofluorescence was used to study the subcellular localization of the ubiquitous NHE-1 isoform of the antiport. NHE-1 was not distributed homogeneously on the surface of the cells. Instead, antiports were found to accumulate along the border of lamellipodia and near the edge of finer processes. Dual immunofluorescence experiments demonstrated that vinculin, talin and F-actin are concentrated at sites of NHE-1 accumulation. A mutated construct of NHE-1 lacking residues 566-635 of the cytosolic domain also accumulated near marginal lamellae. In contrast, the focal distribution observed in adherent cells was not detectable in cells grown in suspension. Fluorescence ratio imaging was used to define the functional consequences of focal accumulation of NHE-1. In the steady state, the pH was virtually identical throughout the cytosol. Moreover, no pH gradients were found to develop when cells recovered from an acid load by activation of Na+/H+ exchange. This is probably because of the presence of high concentrations of mobile buffers in the cytosol. The focal accumulation of antiporters near the cell margins may be involved in stimulation by adherence and/or generation of local osmotic gradients.     

Role of sodium/hydrogen exchanger isoform 1 in microglial activation and proinflammatory responses in ischemic brains

Our recent study reveals that Na?/H? exchanger isoform 1 (NHE-1) mediates H? extrusion during "respiratory bursting", which is important for microglial activation. In the present study, we further investigated whether NHE-1 plays a role in proinflammatory activation of microglia in vivo using a mouse model of transient focal cerebral ischemia and reperfusion (I/R). Activated microglial cells were identified by their expression of two microglial marker proteins (CD11b and Iba1) as well as by their transformation from a "ramified" to an "amoeboid" morphology. An immediate increase in activated microglial numbers was detected in the ipsilateral ischemic core area of NHE-1?/? brains at 1 hour (h) I/1 h R, which gradually decreased during 6-24 h I/R. This was followed by a sharp rise in microglial activation in the peri-infarct area and an increase in proinflammatory cytokine formation at 3 day after I/R. Interestingly, HOE 642 (a potent NHE-1 inhibitor) -treated or NHE-1 heterozygous (NHE-1?/?) mice exhibited less microglia activation, less NADPH oxidase activation, or a reduced proinflammatory response at 3-7 day after I/R. Blocking NHE-1 activity also significantly decreased microglial phagocytosis in vitro. In contrast, astrogliosis formation in the peri-infarct area was not affected by NHE-1 inhibition. Taken together, our results demonstrate that NHE-1 protein was abundantly expressed in activated microglia and astrocytes. NHE-1 inhibition reduced microglial proinflammatory activation following ischemia.     

Cholesterol and the biosynthesis of glycosphingolipids are required for sperm activation in Caenorhabditis elegans

Ejaculated mammalian sperm must acquire fertilization capacity after residing into the female reproductive tract, a process collectively known as capacitation. Cholesterol efflux was required for sperm maturation. Different from flagellated sperm, C. elegans sperm are crawling cells. C. elegans sperm are highly enriched with cholesterol though this animal species lacks biosynthetic pathway for cholesterol and its survival requires an exogenous cholesterol supply. The low abundance of cholesterol in C. elegans lipid extract is thought insufficient to form lipid microdomains ubiquitously in this organism. We present evidence that cholesterol is enriched in the plasma membrane of C. elegans spermatids and that cholesterol- and glycosphingolipids (GSLs)-enriched membrane microdomains (lipid microdomains) mediate sperm activation. Disruption of sperm lipid microdomains by acute manipulation of cholesterol in vitro blocks the sperm activation. Restriction of cholesterol uptake also results in the abnormal sperm activation in both males and hermaphrodites. Manipulation of the integrity of lipid microdomains by targeting the biosynthesis of GSLs inhibits sperm activation and the inhibition can be rescued by the addition of exogenous GSLs. The cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, which are exclusively found in lipid microdomains, also affects sperm activation. We conclude that localized signaling mediated by lipid microdomains is critical for worm sperm activation. Lipid microdomains composed of cholesterol and GSLs have been observed in flagellated sperm of several animal species, thus cholesterol, before its efflux from the plasma membrane, might be needed to assemble into a platform for some more important upstream signal sorting during spermatogenesis than was previously thought.     

Cholesterol and the biosynthesis of glycosphingolipids are required for sperm activation in Caenorhabditis elegans

Ejaculated mammalian sperm must acquire fertilization capacity after residing into the female reproductive tract, a process collectively known as capacitation. Cholesterol efflux was required for sperm maturation. Different from flagellated sperm, C. elegans sperm are crawling cells. C. elegans sperm are highly enriched with cholesterol though this animal species lacks biosynthetic pathway for cholesterol and its survival requires an exogenous cholesterol supply. The low abundance of cholesterol in C. elegans lipid extract is thought insufficient to form lipid microdomains ubiquitously in this organism. We present evidence that cholesterol is enriched in the plasma membrane of C. elegans spermatids and that cholesterol- and glycosphingolipids (GSLs)-enriched membrane microdomains (lipid microdomains) mediate sperm activation. Disruption of sperm lipid microdomains by acute manipulation of cholesterol in vitro blocks the sperm activation. Restriction of cholesterol uptake also results in the abnormal sperm activation in both males and hermaphrodites. Manipulation of the integrity of lipid microdomains by targeting the biosynthesis of GSLs inhibits sperm activation and the inhibition can be rescued by the addition of exogenous GSLs. The cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, which are exclusively found in lipid microdomains, also affects sperm activation. We conclude that localized signaling mediated by lipid microdomains is critical for worm sperm activation. Lipid microdomains composed of cholesterol and GSLs have been observed in flagellated sperm of several animal species, thus cholesterol, before its efflux from the plasma membrane, might be needed to assemble into a platform for some more important upstream signal sorting during spermatogenesis than was previously thought.     

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent,but can couple to an H+ conductance

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT- PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time- course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH- sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.     

A role for hTRPC1 and lipid raft domains in store-mediated calcium entry in human platelets

We have previously suggested that store-mediated Ca2+ entry (SMCE) in human platelets may be activated by a secretion-like coupling model, involving de novo coupling of the type II inositol 1,4,5-trisphosphate receptor (IP(3)RII) to the putative Ca2+ entry channel, hTRPC1. In other cells, hTRPC1 has been reported to be associated with cholesterol-rich lipid raft domains (LRDs) in the plasma membrane. Here we have shown that hTRPC1 is largely associated with detergent-resistant platelet membranes, from which it is partially released when the cells are depleted of cholesterol by treatment with methyl-beta-cyclodextrin (MBCD). MBCD treatment inhibited thapsigargin (TG)-evoked SMCE in a concentration-dependent manner, reducing it to 38.1+/-4.1% at a concentration of 10mM. Similarly, the Ca2+ entry evoked by thrombin (1unit/ml) was reduced to 48.2+/-4.5% of control following MBCD (10mM) treatment. Thrombin- and TG-evoked coupling between IP(3)RII and hTRPC1 was also reduced following cholesterol depletion. These results suggest that hTRPC1 is associated with LRDs in human platelets and that these domains are important for its participation in SMCE.     

Membrane microdomain formation is crucial in epiboly during gastrulation of medaka

Membrane microdomain (microdomain) was isolated from early gastrula embryos. The isolated microdomain was characterized by enrichment of cholesterol and sphingomyelin, and by the presence of huge glycoproteins containing Lewis X structure. Importance of the microdomain in the progress of epiboly was assessed using methyl beta-cyclodextrin (MBCD) and C2-ceramide that disrupt microdomains through different mechanisms. Both reagents efficiently disrupted the microdomain structure and concomitantly impaired epiboly. Interestingly, when embryos pretreated with MBCD, a cholesterol-binding molecule, were exogenously supplemented with cholesterol, the embryos underwent not only reconstitution of the microdomain, but also complete restoration to the normal epiboly. Thus, normal or impaired development is reversibly controlled by the cholesterol-dependent formation or disruption of microdomains. The most typical phenotype of the microdomain-disrupted embryos is detachment of cells from the blastoderm, suggesting that a major contribution of microdomains to epiboly is cell adhesion of blastodermal cells.     

Limited cholesterol depletion causes aggregation of plasma membrane lipid rafts inducing T cell activation

Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10–20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca²⁺. Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD–cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.     

Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism.

A variety of cell types regulate their volume in anisotonic media by stimulating Na+/H+ exchange. Like growth factors, osmotic challenge activates the Na+/H+ antiport by increasing its sensitivity to intracellular [H+]. To investigate the molecular mechanism underlying this shift in pH sensitivity, the antiporter of 32P-labeled human bladder carcinoma cells and of Chinese hamster ovary cells was immunoprecipitated using antibodies raised against the cytosolic domain of the NHE-1 isoform of the Na+/H+ exchanger. Unlike the effects of growth promoters, activation of the antiport during volume regulation was not associated with increased phosphorylation. The possible coexistence of multiple antiporter isoforms was considered. The cytosolic alkalosis normally elicited by hypertonic media was found to be absent in Na+/H+ exchange-deficient fibroblasts. Responsiveness to osmotic challenge was restored by stable transfection of these cells with the cDNA encoding NHE-1. In these transfectants, phosphorylation of the antiporter was also unaffected during osmotic activation. The unchanged phosphate content of the antiporter might be explained by dephosphorylation of one site with concomitant phosphorylation at a different site. However, this possibility appears unlikely since phosphoamino acid analysis revealed that serine was the only residue phosphorylated in immunoprecipitated antiports of both control and osmotically stimulated cells. Moreover, phosphopeptide maps of control and hypertonically activated antiports were identical. These findings reveal a novel mode of activation of Na+/H+ exchange not requiring direct phosphorylation of the antiporter. We propose the existence of dual control of Na+/H+ exchange by phosphorylation-dependent and -independent mechanisms.     

Effects of methyl-beta-cyclodextrin-mediated cholesterol depletion in porcine sperm compared to somatic cells

In this study, the use of methyl-beta-cyclodextrin (MBCD) to support capacitation of sperm cells was studied. Sperm were incubated with MBCD or alternatively capacitated in an in vitro fertilization medium. The effects of these incubations on phospholipid scrambling (using merocyanin), cholesterol depletion, GM-1 localization (using cholera-toxin B (CTX)), and membrane deterioration were assessed. For comparison, this was also tested in MBCD-treated MDCK cells. In MDCK cells, upto 71% of cholesterol was depleted, which coincided with a more diffuse CTX staining without any obvious effects on cell viability. In sperm, a similar depletion of 53% cholesterol was found after a 10 mM MBCD treatment. However, no merocyanin response was observed in viable sperm after MBCD treatments (indicating a lack of membrane changes associated with sperm capacitation). In contrast to MDCK, cells >1 mM MBCD caused plasma membrane disintegration and rendered sperm immotile. At higher concentrations also acrosome disruption was noted. CTX staining was absent at < 0.1 mM MBCD incubations but appeared at higher MBCD levels and was found to be specific for deteriorated cells that showed morphological signs of acrosome disruption. No significant plasma membrane deterioration, acrosome disruption, and sperm immotility nor CTX staining and only a modest (< 15%) cholesterol depletion were observed in conventionally capacitated sperm, where 40% of the intact sperm showed merocyanin staining. Taken together, the results indicate that membranes of sperm are more sensitive to MBCD-mediated cholesterol depletion than MDCK cells and that the use of MBCD to support sperm capacitation cannot be recommended due to its spermicidal effects.     

Hypertonicity activates Na+/H+ exchange through Janus kinase 2 and calmodulin

The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells. NHE-1 may be an important pathway for Na(+) entry during volume restoration, yet the molecular mechanisms underlying the osmotic regulation of NHE-1 are poorly understood. In the present study we conducted a screen for important signaling molecules that could be involved in hypertonicity-induced activation of NHE-1 in CHO-K1 cells. Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Inhibitors of Ca(2+)/calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated this activation, whereas neither calcium chelation nor inhibitors of protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and Ca(2+)/calmodulin-dependent enzymes had significant effects. Hypertonicity also resulted in the rapid tyrosine phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an inhibitor of Jak2. Immunoprecipitation studies showed that hypertonicity stimulates the assembly of a signaling complex that includes CaM, Jak2, and NHE-1. Formation of the complex could be blocked by AG490. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells in large part through the following pathway: hypertonicity --> Jak2 phosphorylation and activation --> tyrosine phosphorylation of CaM --> association of CaM with NHE-1 --> NHE-1 activation.     

Na+/H+ exchange inhibitors: a new class of cardioprotectors

The Na+/H+ exchanger (NHE) extrudes intracellular H+ in exchange for Na+ in an electroneutral process. Of the 6 mammalian exchanger isoforms identified to date, the NHE-1 is believed to be the molecular homologue of the sarcolemma Na+/H+ transporter. The exchanger is activated primarily by a reduction in intracellular pH, although such activation is subject to modulation by a variety of endogenous mediators (catecholamines, thrombin, endothelin) through receptor-mediated mechanisms. A large body of animal studies using both in vitro and in vivo models indicates that the inhibition of the sarcolemma NHE-1 attenuates myocardial injury in ischemia and reperfusion. Cardioprotective effects of NHE-1 inhibition involve a reduced susceptibility to severe ventricular arrhythmia, augmentation of contractile function recovery, and limitation of infarction size during reperfusion. Such protection is likely to arise partly from attenuation of "Ca2+ overload" in ischemic cardiomyocytes, which has been causally linked with all these pathologic phenomena. A marked benefit that has been observed with cariporide (HOE-642) and its structurally related congener HOE-694 in patients with acute myocardial infarction and in cardiac surgery demonstrates that selective NHE-1 inhibitors represent a novel and effective class of cardioprotectors.     

Na+/H+ exchange subtype 1 inhibition during extracellular acidification and hypoxia in glioma cells

Lactacidosis is a common feature of ischaemic brain tissue, but its role in ischaemic neuropathology is still not fully understood. Na(+)/H(+) exchange, a mechanism involved in the regulation of intracellular pH (pH(i)), is activated by low pH(i). The role of Na(+)/H(+) exchange subtype 1 was investigated during extracellular acidification and subsequent pH recovery in the absence and presence of (4-isopropyl-3-methylsulphonyl-benzoyl)-guanidine methanesulfonate (HOE642, Cariporid), a new selective and powerful inhibitor of the Na(+)/H(+) exchanger subtype 1 (NHE-1). It was compared for normoxia and hypoxia in two glioma cell lines (C6 and F98). pH(i) was monitored by fluorescence spectroscopy using the intracellularly trapped pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Alterations in glial cell metabolism were characterized using high-resolution (1)H, (13)C and (31)P NMR spectroscopy of perchloric acid extracts. NHE-1 contributed to glial pH regulation, especially at pathologically low pH(i) values. NHE-1 inhibition with HOE642 during acidification caused exacerbated metabolic disorders which were prolonged during extracellular pH recovery. However, NHE-1 inhibition during hypoxia protected the energy state of glial cells.