Involvement of Na+/H+ exchanger in the calcium signaling in epimastigotes of Trypanosoma cruzi

We studied the effect of Na(+) extracellular on Ca(2+) mobilization from intracellular store evoked by carbachol in Trypanosoma cruzi. We report that slow component of Ca(2+) signaling evoked by agonist is dependent on extracellular Na(+) but not on InsP(3) increase. Moreover, this Ca(2+) signaling progressively increased when pH of the medium changed from 7.0 to 7.8. In addition, we found that it was regulated by PKC. The agonist was also able to induce the alkalinization of the acidic compartment, and both Ca(2+) signaling and alkalinization were inhibited by the EIPA-inhibitor of the Na(+)/H(+) exchanger. These results demonstrated the alkalinization of acidic vacuoles and PKC are involved in the triggering of the epimastigote Ca(2+) signaling.     

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.     

The role of Na+/H+ exchanger in serotonin secretion from porcine blood platelets

This study was undertaken to evaluate whether a link exists between the activation of protein kinase C (PKC), operation of Na(+)/H(+) exchanger (NHE), cell swelling and serotonin (5-HT) secretion in porcine platelets. Activation of platelets by thrombin or phorbol 12-myristate 13-acetate (PMA), a PKC activator, initiated a rapid rise in the activity of Na(+)/H(+) exchanger and secretion of 5-HT. Both thrombin- and PMA-evoked activation of Na(+)/H(+) exchanger was less pronounced in the presence of ethyl-isopropyl-amiloride (EIPA), an NHE inhibitor, and by GF 109203X, a PKC inhibitor. Monensin (simulating the action of NHE) caused a dose-dependent release of 5-HT that was not abolished by GF 109203X or EGTA. Lack of Na(+) in the suspending medium reduced thrombin-, PMA-, and monensin-evoked 5-HT secretion. GF 109203X nearly completely inhibited 5-HT release induced by PMA-, partly that induced by thrombin, and had no effect on 5-HT release induced by monensin. EIPA partly inhibited 5-HT release induced by thrombin and nearly totally that evoked by PMA. Electronic cell sizing measurements showed an increase in mean platelet volume upon treatment of cells with monensin, PMA or thrombin. The PMA- and thrombin-evoked rise in mean platelet volume was strongly reduced in the presence of EIPA. As judged by optical swelling assay monensin and PMA produced a rapid rise in platelet volume. The swelling elicited by PMA was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Hypoosmotically evoked platelet swelling did not affect platelet aggregation but significantly potentiated thrombin-evoked release of 5-HT and ATP. Taken together, these results show that in porcine platelets PKC may promote 5-HT secretion through the activation of NHE. It is hypothesized that enhanced Na(+)/H(+) antiport may result in a rise in cell membrane tension (due to cell swelling) which in turn facilitates fusion of secretory granules with the plasma membrane leading to 5-HT secretion.     

Ammonia excretion via Rhcg1 facilitates Na⁺ uptake in larval zebrafish, Danio rerio, in acidic water

The involvement of a Na(+)/H(+) exchanger (NHE) in mediating Na(+) uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na(+) uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na(+) uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na(+) uptake in fish reared in acidic water (pH ～ 4.0). A role for NHE in Na(+) uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na(+)/H(+) exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na(+) uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na(+) uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na(+) uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H(+)-ATPase) significantly reduced Na(+) uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na(+) uptake in larval zebrafish in acidic water: one in which Na(+) uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H(+)-ATPase.     

Functional analysis of acidic amino acids in the cytosolic tail of the Na+/H+ exchanger

The mammalian Na(+)/H(+) exchanger is a membrane protein with a C-terminal regulatory cytosolic domain and an N-terminal membrane domain. Na(+)/H(+) exchanger isoform 1 (NHE1) possesses a conserved amino acid sequence of seven consecutive acidic residues in the distal region of the cytosolic tail. We examined the structural and functional role of this acidic sequence. In human NHE1, varying mutations of the sequence (753)EEDEDDD(759) resulted in defective NHE1 activity. Mutation of the core acid sequence, (755)DED(757), or of the entire sequence caused a decrease in the activity of NHE1 in response to acute acid load. This was not due to changes in Na(+) affinity but rather due to decreased maximum velocity of the protein and delayed activation. Mutation of the target sequence did not affect the ability of the cytoplasmic domain to bind carbonic anhydrase II or tescalcin but did affect calmodulin binding. Mutation of the acidic domain also caused altered sensitivity to trypsin and changes in size of the protein in gel-filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results demonstrate that the acidic sequence is critical in maintaining proper conformation of the cytosolic domain, calmodulin binding, and in maintenance of Na(+)/H(+) exchanger activity.     

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.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

The Na+/H+ exchanger cytoplasmic tail: structure,function, and interactions with tescalcin

We characterized the regulatory cytoplasmic tail of the Na(+)/H(+) exchanger using a histidine-tagged protein containing the C-terminal 182 amino acids (His182). Both tescalcin and calmodulin, two Na(+)/H(+) exchanger binding proteins, bound to the His182 protein. Cascade blue was used to label the His182 protein. Calcium caused an increase in fluorescence, suggesting exposure of the label on the protein to a more hydrophilic environment. Decreasing external pH caused a transient increase in cascade blue fluorescence, followed by a decrease in fluorescence of the cascade blue labeled Na(+)/H(+) exchanger C-terminus. Tescalcin caused a decrease in fluorescence by labeled His182 protein, and calcium reversed this effect. Expression of tescalcin in vivo inhibited activity of the Na(+)/H(+) exchanger when there was an intact C-terminus of the protein. We examined the CD spectra of His182 in the presence and absence of tescalcin. The C-terminal amino acids demonstrated a very small amount of alpha-helical structure and much more beta-sheet and beta-turn. This was not greatly affected by the presence of tescalcin, but calcium caused an increase in the amount of beta-structure and a decrease in the unstructured proportion of the protein. Sedimentation equilibrium analysis demonstrated that the C-terminal 182 amino acids exist predominantly as a monomer. The results suggest that the C-terminus of the Na(+)/H(+) exchanger exists primarily as a monomeric protein that binds regulatory tescalcin and can change conformation depending on pH and calcium. Conformation changes in this region of the protein may be responsible for altering the pH sensitivity of the intact Na(+)/H(+) exchanger.     

The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.     

Activation of Na+/H+ exchanger-directed protein kinases in the ischemic and ischemic-reperfused rat myocardium

The activity of the Na(+)/H(+) exchanger has been implicated as an important contributing factor in damage to the myocardium that occurs during ischemia and reperfusion. We examined regulation of the protein in ischemic and reperfused isolated hearts and isolated ventricular myocytes. In isolated myocytes, extracellular signal-regulated kinases were important in regulating activity of the exchanger after recovery from ischemia. Ischemia followed by reperfusion caused a strong inhibitory effect on NHE1 activity that abated with continued reperfusion. Four major protein kinases of size 90, 55, 44, and 40 kDa phosphorylated the Na(+)/H(+) exchanger. The Na(+)/H(+) exchanger-directed kinases demonstrated dramatic increases in activity of 2-10-fold that was induced by 3 different models of ischemia and reperfusion in intact hearts and isolated myocytes. p90(rsk) was identified as the 90-kDa protein kinase activated by ischemia and reperfusion while ERK1/2 was identified as accounting for some of the 44-kDa protein kinase phosphorylating the Na(+)/H(+) exchanger. The results demonstrate that MAPK-dependent pathways including p90(rsk) and ERK1/2 and are important in regulating the Na(+)/H(+) exchanger and show their dramatic increase in activity toward the Na(+)/H(+) exchanger during ischemia and reperfusion of the myocardium. They also show that ischemia followed by reperfusion have important inhibitory effects on Na(+)/H(+) exchanger activity.     

Discovery of a novel potent Na+/Ca2+ exchanger inhibitor: design, synthesis and structure-activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives

Design, synthesis and structure-activity relationships for 3,4-dihydro-2(1H)-quinazolinone derivatives with the inhibitory activities of the Na(+)/Ca(2+) exchanger are discussed. These studies based on lead compound 1a lead to the discovery of a structurally novel and highly potent inhibitor against the Na(+)/Ca(2+) exchanger 4f (SM-15811), which directly inhibited the Na(+)-dependent Ca(2+) influx via the Na(+)/Ca(2+) exchanger in cardiomyocytes with a high potency.     

Increased Na(+)/H(+) exchanger isoform 1 activity in spontaneously hypertensive rats: lack of mutations within the coding region of NHE1

Enhanced Na(+)/H(+) exchange, measured as amiloride derivative-sensitive Na(+) and H(+) fluxes in cells with a preliminary acidified cytoplasm (Deltamu(H+)-induced Na(+)/H(+) exchange), is one of the most prominent intermediate phenotypes of altered vascular smooth muscle cell (VSMC) function in spontaneously hypertensive rats (SHR). Analysis of Na(+)/H(+) exchange in F(2) hybrids of SHR and normotensive rats seems to be the most appropriate approach in the search for the genetic determinants of abnormal activity of this carrier. However, the measurement of Deltamu(H+)-induced Na(+)/H(+) exchange is hardly appropriate for precise analysis of the carrier's activity in VSMC derived from several hundred F(2) hybrids. To overcome this problem, we compared the rate of (22)Na influx under baseline conditions and in Na(+)-loaded (ouabain-treated) VSMC. The dose-dependency of the rate of Deltamu(H+)-induced H(+) efflux as well as of (22)Na influx in control and ouabain-treated cells on ethylisopropylamiloride (EIPA) concentration were not different (K(0.5) approximately 0.3 microM), suggesting that these ion transport pathways are mediated by the same carrier. EIPA-sensitive (22)Na influx in Na(+)-loaded cells was approximately 6-fold higher than in ouabain-untreated VSMC and was increased by 50-70% in two different substrains of SHR. About the same increment of EIPA-sensitive (22)Na influx in Na(+)-loaded VSMC was observed in 5- to 6-week-old SHR (an age at which hypertension has not yet developed) as well as in stroke-prone SHR (SHRSP) with severe hypertension, indicating that the heightened activity of Na(+)/H(+) exchange is not a consequence of long-term blood pressure elevation. To examine whether or not the augmented activity of Na(+)/H(+) exchange in SHR is caused by mutation of NHE1, i.e. the only isoform of this carrier expressed in VSMC, we undertook single-stranded conformational polymorphism analysis of 23 NHE1 cDNA fragments from SHR and SHRSP and sequencing of the 456-2421 NHE1 cDNA fragment. This study did not reveal any mutation in the entire coding region of NHE1. The lack of mutation in the coding region of NHE1 indicates that the augmented activity of the ubiquitous Na(+)/H(+) exchanger in primary hypertension is caused by altered regulation of carrier turnover number or/and its plasma membrane content.     

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.     

Na(+)-dependent pH regulation by the amitochondriate protozoan parasite Giardia intestinalis

Giardia intestinalis is a pathogenic fermentative parasite, which inhabits the gastrointestinal tract of animals and humans. G. intestinalis trophozoites are exposed to acidic fluctuations in vivo and must also cope with acidic metabolic endproducts. In this study, a combination of independent techniques ((31)P NMR spectroscopy, distribution of the weak acid pH marker 5,5-dimethyl-2,4-oxazolidinedione (DMO) and the fluorescent pH indicator 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF)) were used to show that G. intestinalis trophozoites exposed to an extracellular pH range of 6.0--7.5 maintain their cytosolic pH (pH(i)) within the range 6.7--7.1. Maintenance of the resting pH(i) was Na(+)-dependent but unaffected by amiloride (or analogs thereof). Recovery of pH(i) from an intracellular acidosis was also Na(+)-dependent, with the rate of recovery varying with the extracellular Na(+) concentration in a saturable manner (K(m) = 18 mm; V(max) = 10 mm H(+) min(-1)). The recovery of pH(i) from an acid load was inhibited by amiloride but unaffected by a number of its analogs. The postulated involvement of one or more Na(+)/H(+) exchanger(s) in the regulation of pH(i) in G. intestinalis is discussed.     

Dimerization is crucial for the function of the Na+/H+ exchanger NHE1

The Na(+)/H(+) exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H(+) concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na(+) or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range.     

Vacuolar H(+)-ATPase localized in plasma membranes of malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of parasitized erythrocytes

Recent biochemical studies involving 2',7'-bis-(2-carboxyethyl)-5, 6-carboxylfluorescein (BCECF)-labeled saponin-permeabilized and parasitized erythrocytes indicated that malaria parasite cells maintain the resting cytoplasmic pH at about 7.3, and treatment with vacuolar proton-pump inhibitors reduces the resting pH to 6.7, suggesting proton extrusion from the parasite cells via vacuolar H(+)-ATPase (Saliba, K. J., and Kirk, K. (1999) J. Biol. Chem. 274, 33213-33219). In the present study, we investigated the localization of vacuolar H(+)-ATPase in Plasmodium falciparum cells infecting erythrocytes. Antibodies against vacuolar H(+)-ATPase subunit A and B specifically immunostained the infecting parasite cells and recognized a single 67- and 55-kDa polypeptide, respectively. Immunoelectron microscopy indicated that the immunological counterpart of V-ATPase subunits A and B is localized at the plasma membrane, small clear vesicles, and food vacuoles, a lower extent being detected at the parasitophorus vacuolar membrane of the parasite cells. We measured the cytoplasmic pH of both infected erythrocytes and invading malaria parasite cells by microfluorimetry using BCECF fluorescence. It was found that a restricted area of the erythrocyte cytoplasm near a parasite cell is slightly acidic, being about pH 6.9. The pH increased to pH 7.3 upon the addition of either concanamycin B or bafilomycin A(1), specific inhibitors of vacuolar H(+)-ATPase. Simultaneously, the cytoplasmic pH of the infecting parasite cell decreased from pH 7.3 to 7.1. Neither vanadate at 0.5 mm, an inhibitor of P-type H(+)-ATPase, nor ethylisopropylamiloride at 0.2 mm, an inhibitor of Na(+)/H(+)-exchanger, affected the cytoplasmic pH of erythrocytes or infecting parasite cells. These results constitute direct evidence that plasma membrane vacuolar H(+)-ATPase is responsible for active extrusion of protons from the parasite cells.     

Phosphoinositide 3-kinase accelerates calpain-dependent proteolysis of fodrin during hypoxic cell death

We have shown recently that phosphoinositide 3-kinase (PI 3-kinase) accelerates the hypoxia-induced necrotic cell death of H9c2, derived from rat cardiomyocytes, by enhancing metabolic acidosis. Here we show the downstream events of acidosis that cause hypoxic cell death. Hypoxia induces the proteolysis of fodrin, a substrate of calpain. Intracellular Ca(2+) chelation by BAPTA, and the addition of SJA6017, a specific peptide inhibitor of calpain, also reduces cell death and fodrin proteolysis, indicating that Ca(2+) influx and calpain activation might be involved in these events. The overexpression of wild type PI 3-kinase accelerates fodrin proteolysis, while dominant-negative PI 3-kinase reduces it. Both (N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger, and KB-R7943, an inhibitor of the Na(+)/Ca(2+) exchanger, reduce hypoxic cell death and fodrin proteolysis. The depletion of intracellular Ca(2+ )stores by thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, also reduces cell death and fodrin proteolysis, indicating that Ca(2+ )release from intracellular Ca(2+ )stores might be also involved. These results indicate that PI 3-kinase might accelerate hypoxic cell death by enhancing the calpain-dependent proteolysis of fodrin.     

pH regulation in the intracellular malaria parasite, Plasmodium falciparum. H(+) extrusion via a V-type H(+)-ATPase

The mechanism by which the intra-erythrocytic form of the human malaria parasite, Plasmodium falciparum, extrudes H(+) ions and thereby regulates its cytosolic pH (pH(i)), was investigated using saponin-permeabilized parasitized erythrocytes. The parasite was able both to maintain its resting pH(i) and to recover from an imposed intracellular acidification in the absence of extracellular Na(+), thus ruling out the involvement of a Na(+)/H(+) exchanger in both processes. Both phenomena were ATP-dependent. Amiloride and the related compound ethylisopropylamiloride caused a substantial reduction in the resting pH(i) of the parasite, whereas EMD 96785, a potent and allegedly selective inhibitor of Na(+)/H(+) exchange, had relatively little effect. The resting pH(i) of the parasite was also reduced by the sulfhydryl reagent N-ethylmaleimide, by the carboxyl group blocker N,N'-dicyclohexylcarbodiimide, and by bafilomycin A(1), a potent inhibitor of V-type H(+)-ATPases. Bafilomycin A(1) blocked pH(i) recovery in parasites subjected to an intracellular acidification and reduced the rate of acidification of a weakly buffered solution by parasites under resting conditions. The data are consistent with the hypothesis that the malaria parasite, like other parasitic protozoa, has in its plasma membrane a V-type H(+)-ATPase, which serves as the major route for the efflux of H(+) ions.     

Alterations of Na(+) homeostasis in hepatocyte reoxygenation injury

Reperfusion injury represents an important cause of primary graft non-function during liver transplantation. However, the mechanism responsible for cellular damage during reoxygenation has not yet been completely understood. We have investigated whether changes in intracellular Na(+) distribution might contribute to cause hepatocyte damage during reoxygenation buffer after 24 h of cold storage. Hepatocyte reoxygenation resulted in a rapid increase in cellular Na(+) content that was associated with cytotoxicity. Na(+) accumulation and hepatocyte death were prevented by the omission of Na(+) from the incubation medium, but not by the addition of antioxidants. Blocking Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) co-transporter by, respectively, 5-(N,N-dimethyl)-amiloride or omitting HCO(3)(-) from the reoxygenation medium significantly decreased Na(+) overload and cytotoxicity. Stimulation of ATP re-synthesis by the addition of fructose also lowered Na(+) accumulation and cell death during reoxygenation. A significant protection against Na(+)-mediated reoxygenation injury was evident in hepatocytes maintained in an acidic buffer (pH 6.5) or in the presence of glycine. The cytoprotective action of glycine or of the acidic buffer was reverted by promoting Na(+) influx with the Na(+)/H(+) ionophore monensin. Altogether, these results suggest that Na(+) accumulation during the early phases of reoxygenation might contribute to liver graft reperfusion injury.