Na(+)/H(+) exchange in mosquito Malpighian tubules

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na(+)/H(+) exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 microM 5-(N-ethyl-n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na(+)/H(+) exchange. Steady-state intracellular pH was measured microfluorometrically by using 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na(+) or in the presence of 100 microM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na(+)/H(+) exchanger by using the NH(4)Cl pulse technique resulted in a rate of recovery from the NH(4)Cl-induced acute acid load of 8.7 +/- 1.0 x 10(-3) pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na(+) or in the presence of 100 microM EIPA were 0.7 +/- 0.6 and -0.3 +/- 0.3 x 10(-3) pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na(+)/H(+) exchanger.     

Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.     

Altered cardiac Na(+)/H(+) exchange in phospholipase D-treated sarcolemmal vesicles

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.     

EGF promotes gastric mucosal restitution by activating Na(+)/H(+) exchange of epithelial cells

This study was conducted to determine whether the contributions of epidermal growth factor (EGF) to gastric mucosal restitution after injury are mediated by stimulation of Na(+)/H(+) exchangers in surface mucous cells (SMC). Intact sheets of guinea pig gastric mucosae were incubated in vitro. Intracellular pH (pH(i)) in SMC was measured fluorometrically, using 2',7'- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Restitution after Triton X-100-induced injury was evaluated by recovery of electrical resistance. At neutral luminal pH, exogenous EGF (ex-EGF) increased pH(i) and enhanced restitution in the absence but not in the presence of serosal HCO. During exposure to luminal acid, ex-EGF not only prevented intracellular acidosis but also promoted restitution. These effects of ex-EGF were blocked by serosal amiloride or anti-EGF-receptor antibody. In the absence of ex-EGF, restitution was inhibited by replacement of luminal and serosal solutions with fresh solutions and was blocked more completely by serosal anti-EGF-receptor antibody. These results suggest that both endogenous and ex-EGF contribute to restitution via basolateral EGF receptors, with effects mediated, at least in part, by stimulation of basolateral Na(+)/H(+) exchangers.     

Rapamycin sensitive ROS formation and Na(+)/H(+) exchanger activity in dendritic cells

Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na(+)/H(+) exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 μM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 μM) and partially reversed by additional application of tert-butylhydroperoxide (10 μM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.     

Increased vacuolar Na(+)/H(+) exchange activity in Salicornia bigelovii Torr. in response to NaCl

Shoots of the halophyte Salicornia bigelovii are larger and more succulent when grown in highly saline environments. This increased growth and water uptake has been correlated with a large and specific cellular accumulation of sodium. In glycophytes, sensitivity to salt has been associated with an inability to remove sodium ions effectively from the cytoplasm in order to protect salt-sensitive metabolic processes. Therefore, in Salicornia bigelovii efficient vacuolar sequestration of sodium may be part of the mechanism underlying salt tolerance. The ability to compartmentalize sodium may result from a stimulation of the proton pumps that provide the driving force for increased sodium transport into the vacuole via a Na(+)/H(+) exchanger. In current studies, increased vacuolar pyrophosphatase activity (hydrolysis of inorganic pyrophosphate and proton translocation) and protein accumulation were observed in Salicornia bigelovii grown in high concentrations of NaCl. Based on sodium-induced dissipation of a pyrophosphate-dependent pH gradient in vacuolar membrane vesicles, a Na(+)/H(+) exchange activity was identified and characterized. This activity is sodium concentration-dependent, specific for sodium and lithium, sensitive to methyl-isobutyl amiloride, and independent of an electrical potential. Vacuolar Na(+)/H(+) exchange activity varied as a function of plant growth in salt. The affinity of the transporter for Na(+) is almost three times higher in plants grown in high levels of salt (K(m)=3.8 and 11.5 mM for plants grown in high and low salt, respectively) suggesting a role for exchange activity in the salt adaptation of Salicornia bigelovii.     

Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate

The carrier-mediated, electroneutral exchange of Na(+) for H(+) across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na(+)/H(+) exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP(2), in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP(2)-binding motifs. Fusion proteins encoding these motifs bound PIP(2) in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP(2)-binding domains had greatly reduced transport capability, implying that association with PIP(2) is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP(2).     

Na(+)/H(+) exchange inhibition-induced cardioprotection in dogs: effects on neutrophils versus cardiomyocytes

Numerous studies have examined the effect of Na(+)/H(+) exchanger (NHE) inhibition on the myocardium; however, the effect of NHE-1 inhibition on neutrophil function has not been adequately examined. An in vivo canine model of myocardial ischemia-reperfusion injury in which 60 min of left anterior descending coronary artery occlusion followed by 3 h of reperfusion was used to examine the effect of NHE-1 inhibition on infarct size (IS) and neutrophil function. BIIB-513, a selective inhibitor of NHE-1, was infused before ischemia. IS was expressed as a percentage of area at risk (IS/AAR). NHE-1 inhibition significantly reduced IS/AAR and reduced neutrophil accumulation in the ischemic myocardium. NHE-1 inhibition attenuated both phorbol 12-myristate 13-acetate- and platelet-activating factor-induced neutrophil respiratory burst but not CD18 upregulation. Furthermore, NHE-1 inhibition directly protected cardiomyocytes against metabolic inhibition-induced lactate dehydrogenase release and hypercontracture. This study provides evidence that the cardioprotection induced by NHE-1 inhibition is likely due to specific protection of cardiomyocytes and attenuation of neutrophil activity.     

Na(+)/H(+) exchange inhibition prevents endothelial dysfunction after I/R injury

Whereas inhibition of the Na(+)/H(+) exchanger (NHE) has been demonstrated to reduce myocardial infarct size in response to ischemia-reperfusion injury, the ability of NHE inhibition to preserve endothelial cell function has not been examined. This study examined whether NHE inhibition could preserve endothelial cell function after 90 min of regional ischemia and 180 min of reperfusion and compared this inhibition with ischemic preconditioning (IPC). In a canine model either IPC, produced by one 5-min coronary artery occlusion (1 x 5'), or the specific NHE-1 inhibitor eniporide (EMD-96785, 3.0 mg/kg) was administered 15 min before a 90-min coronary artery occlusion followed by 3 h of reperfusion. Infarct size (IS) was determined by 2,3,5-triphenyl tetrazolium chloride staining and expressed as a percentage of the area-at-risk (IS/AAR). Endothelial cell function was assessed by measurement of coronary blood flow in response to intracoronary acetylcholine infusion at the end of reperfusion. Whereas neither control nor IPC-treated animals exhibited a significant reduction in IS/AAR or preservation of endothelial cell function, animals treated with the NHE inhibitor eniporide showed a marked reduction in IS/AAR and a significantly preserved endothelial cell function (P < 0.05). Thus NHE-1 inhibition is more efficacious than IPC at reducing IS/AAR and at preserving endothelial cell function in dogs.     

Demonstration of a Na(+)/H(+) exchanger NHE1 in fresh bovine corneal endothelial cell basolateral plasma membrane.

Apical and basolateral plasma membranes of fresh bovine corneal endothelial cells were isolated using positively charged polyacrylamide beads. Marker enzyme assays demonstrated that the isolated apical and basolateral plasma membrane domains could be isolated and separated with relative purity. Western blotting with a polyclonal anti-NHE1 antibody detected a protein of 70 kDa in the basolateral plasma membrane isolate. NHE1 immunoreactivity was not detected in the apical membrane sample. This suggests that the Na(+)/H(+) exchanger, NHE1, is strictly localised to the basolateral membrane of fresh bovine corneal endothelial cells.     

Effects of omega-3 polyunsaturated fatty acids on cardiac sarcolemmal Na(+)/H(+) exchange

Myocardial ischemia-reperfusion activates the Na(+)/H(+) exchanger, which induces arrhythmias, cell damage, and eventually cell death. Inhibition of the exchanger reduces cell damage and lowers the incidence of arrhythmias after ischemia-reperfusion. The omega-3 polyunsaturated fatty acids (PUFAs) are also known to be cardioprotective and antiarrhythmic during ischemia-reperfusion challenge. Some of the action of PUFAs may occur via inhibition of the Na(+)/H(+) exchanger. The purpose of our study was to determine the capacity for selected PUFAs to alter cardiac sarcolemmal (SL) Na(+)/H(+) exchange. Cardiac membranes highly enriched in SL vesicles were exposed to 10-100 microM eicosapentanoic acid (EPA) or docosahexanoic acid (DHA). H(+)-dependent (22)Na(+) uptake was inhibited by 30-50% after treatment with > or =50 microM EPA or > or =25 microM DHA. This was a specific effect of these PUFAs, because 50 microM linoleic acid or linolenic acid had no significant effect on Na(+)/H(+) exchange. The SL vesicles did not exhibit an increase in passive Na(+) efflux after PUFA treatment. In conclusion, EPA and DHA can potently inhibit cardiac SL Na(+)/H(+) exchange at physiologically relevant concentrations. This may explain, in part, their known cardioprotective effects and antiarrhythmic actions during ischemia-reperfusion.     

Na(+)/H(+) exchange inhibition reduces hypertrophy and heart failure after myocardial infarction in rats

We investigated the effect of sodium/hydrogen exchange inhibition (NHE-1) on hypertrophy and heart failure after coronary artery ligation (CAL) in the rat. Animals were subjected to occlusion (or sham) of the left main coronary artery and immediately administered a control diet or one consisting of the NHE-1 inhibitor cariporide for 13-15 wk. Hearts were separated by small [30% of LV) infarcts. CAL depressed change in left ventricular increase in pressure over time (LV +dP/dt) in small and large infarct groups by 18.8% (P < 0.05) and 34% (P < 0.01), respectively, whereas comparative values for the cariporide groups were 8.7% (not significant) and 23.1% (P < 0.01), respectively. LV end-diastolic pressure was increased by 1,225% in the control large infarct group but was significantly reduced to 447% with cariporide. Cariporide also significantly reduced the degree of LV dilation in animals with large infarcts. Hypertrophy, defined by tissue weights and cell size, was reduced by cariporide, and shortening of surviving myocytes was preserved. Infarct sizes were unaffected by cariporide, and the drug had no influence on either blood pressure or the depressed inotropic response of infarcted hearts to dobutamine. These results suggest an important role for NHE-1 in the progression of heart failure after myocardial infarction.     

Na(+)/H(+) exchange subtype 1 inhibition reduces endothelial dysfunction in vessels from stunned myocardium

Myocardial ischemia and reperfusion cause myocyte and vascular dysfunction, frequently termed "stunning." We hypothesized that inhibiting the Na(+)/H(+) exchanger subtype 1 isoform (NHE(1)) during ischemia and reperfusion limits myocardial and coronary microvascular stunning. Anesthetized rats completed 2 x 10-min coronary artery occlusions separated by 5-min of reperfusion, followed by 15 or 60 min of reperfusion. Vehicle (saline) or the NHE(1) inhibitor cariporide (HOE-642) was administered 15 min before ischemia and was continued throughout each protocol. After reperfusion, hearts were excised, and the reactivity of resistance arteries (internal diameter, approximately 120 microm) was assessed. The first derivative of left ventricular (LV) pressure, LV developed pressure, and LV systolic wall thickening were depressed (P < 0.05) similarly in vehicle- and cariporide-treated rats during ischemia and after 15 or 60 min of reperfusion compared with sham-operated animals that were not exposed to ischemia (i.e., controls). In vessels obtained after 15 min of reperfusion, the maximal response to acetylcholine-induced relaxation (10(-8)-10(-4) M) was blunted (P < 0.05) in vessels from vehicle- (approximately 35%) and cariporide-treated rats (approximately 55%) compared with controls (approximately 85%). However, the percent relaxation to acetylcholine was greater (P < 0.05) in cariporide-treated rats compared with vehicle-treated rats. Maximal contractile responses to endothelin-1 (10(-11)-10(-7) M) were increased (P < 0.05) similarly in vehicle- and cariporide-treated rats compared with controls. Relaxation to sodium nitroprusside (10(-4) M) was not different among groups. Results were similar in vessels obtained from animals after 60 min of reperfusion. These findings suggest that NHE(1) inhibition before coronary occlusion lessens ischemia-induced microvascular dysfunction for 15-60 min after reperfusion but does not alter myocardial contractile function in the area at risk.     

Na(+)/H(+) exchange inhibition attenuates hypertrophy and heart failure in 1-wk postinfarction rat myocardium

Na(+)/H(+) exchange (NHE) represents a major mechanism for intracellular pH regulation, particularly in the ischemic myocardium. NHE has also been shown to be important in the regulation of cell proliferation and growth. We examined whether inhibition of NHE results in an attenuation of early postinfarction myocyte remodeling responses in the rat. Male Sprague-Dawley rats were randomized to receive either a control diet or an identical diet supplemented with the NHE inhibitor cariporide. After 1 wk, animals were anesthetized, subjected to ligation of the left main coronary artery, and maintained for an additional week, after which time they were anesthetized and intraventricular pressures were obtained. Hearts were removed, and myocytes were isolated to obtain cell dimensions and determine the response to isoproterenol. Body, heart, and lung weights were obtained. Coronary artery ligation in control animals resulted in a significant elevation in left ventricular end-diastolic pressure, as well as increased heart weight- and lung weight-to-body weight ratios, both of which were abrogated by cariporide. Cell length and area significantly increased by 14 and 19.2%, respectively, whereas cell width increased by 4.1% (P > 0.05). These cells exhibited a significant hyporesponsiveness to the positive inotropic responses to isoproterenol at the lower drug concentrations (3 and 10 nM). A <1% dimensional change occurred in myocytes from cariporide-fed animals, and the hyporesponse to isoproterenol was reversed. Cariporide had no effect on infarct size or blood pressure. These studies suggest that the early adaptive hypertrophic response of surviving myocytes is dependent on NHE activity. As such, it is attractive to suggest that NHE inhibition could be an effective therapeutic strategy for prevention of postinfarction remodeling, independent of infarct size or afterload reduction.     

Identification of a pH regulated Na(+)/H(+) antiporter of Methanococcus jannaschii

The genome of the hyperthermophilic archaeon Methanococcus jannaschii contains three Na(+)/H(+) antiporter related genes Mj0057, Mj1521 and Mj1275. Comparative sequence alignments revealed that Mj0057 and Mj1521 belong to the NhaP family whereas Mj1275 is a member of the NapA family. The genes were cloned and expressed in the double mutant Escherichia coli strain Frag144 (DeltanhaA, DeltanhaB) to analyze their capability of mediating DeltapH driven Na(+) flux in everted vesicles. From the tested clones only Mj0057 displayed Na(+) (Li(+))/H(+) antiporter activity. The transport was pH dependent and occurred at pH 7.0 and below. At pH 6.0 the apparent K(m) values for Na(+) and Li(+) were approximately 10 and 2.5 mM, respectively.