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1.
Initiation of intestinal Na+-glucose cotransport results intransient cell swelling and sustained increases in tight junction permeability. Since Na+/H+ exchange has beenimplicated in volume regulation after physiological cell swelling, wehypothesized that Na+/H+ exchange might also berequired for Na+-glucose cotransport-dependent tightjunction regulation. In Caco-2 monolayers with activeNa+-glucose cotransport, inhibition ofNa+/H+ exchange with 200 µM5-(N,N-dimethyl)- amiloride induced 36 ± 2% increases in transepithelial resistance (TER). Evaluation using multiple Na+/H+ exchange inhibitors showed thatinhibition of the Na+/H+ exchanger 3 (NHE3)isoform was most closely related to TER increases. TER increases due toNHE3 inhibition were related to cytoplasmic acidification becausecytoplasmic alkalinization with 5 mM NH4Cl prevented bothcytoplasmic acidification and TER increases. However, NHE3 inhibitiondid not affect TER when Na+-glucose cotransport wasinhibited. Myosin II regulatory light chain (MLC) phosphorylationdecreased up to 43 ± 5% after inhibition ofNa+/H+ exchange, similar to previous studiesthat associate decreased MLC phosphorylation with increased TER afterinhibition of Na+-glucose cotransport. However, NHE3inhibitors did not diminish Na+-glucose cotransport. Thesedata demonstrate that inhibition of NHE3 results in decreased MLCphosphorylation and increased TER and suggest that NHE3 may participatein the signaling pathway of Na+-glucosecotransport-dependent tight junction regulation.

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2.
Cytoplasmic pH (pHi) was evaluated duringNa+-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pHi increased by 0.069 ± 0.002 within 150 s after initiation of Na+-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na+-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa+/H+ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa+-glucose cotransport with an ED50 of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pHi increases at <500 µM.Na+-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pHi increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na+-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa+-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na+ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

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3.
ETB receptor activation leads to activation and phosphorylation of NHE3   总被引:1,自引:0,他引:1  
In OKP cellsexpressing ETB endothelinreceptors, activation ofNa+/H+antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation ofNa+/H+exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation incells expressing ETB receptors butnot in cells expressing ETAreceptors. Receptor specificity was not due to demonstrable differencesin receptor-specific activation of tyrosine phosphorylation pathways orinhibition of adenylyl cyclase. Phosphorylation was associated with adecrease in mobility on SDS-PAGE, which was reversed by treatingimmunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation wasfirst seen at 5 min and was maximal at 15-30 min. Phosphorylationwas maximal with 109 MET-1. Phosphorylation occurred on threonine and serine residues atmultiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKPcells on multiple threonine and serine residues.ETB receptor specificity, timecourse, and concentration dependence are all similar betweenET-1-induced increases in NHE3 activity and phosphorylation, suggestingthat phosphorylation plays a key role in activation.  相似文献   

4.
Intestinal neutral NaCl absorption, which is made up ofbrush-border (BB)Na+/H+exchange linked to BBCl/HCO3exchange, is up- and downregulated as part of digestion and diarrhealdiseases. Glucocorticoids stimulate ileal NaCl absorption and BBNa+/H+exchange. Intestinal BB contains twoNa+/H+exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbitileal BBNa+/H+exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BBNa+/H+exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ~50% to ilealNa+/H+exchange. Glucocorticoids (methylprednisolone) increase BBNa+/H+exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times),without an effect on NHE2 activity. Thus ileal BBNa+/H+exchange in animals treated with glucocorticoids is 69% via NHE3. Aquantitative Western analysis for NHE3 was developed, using as aninternal standard a fusion protein of the COOH-terminal 85 amino acidsof NHE3 and maltose binding protein. Glucocorticoid treatment increasedthe amount of BB NHE3. The quantitative Western analysis showed thatNHE3 makes up 0.018% of ileal BB protein in control rabbits and0.042% (2.3 times as much) in methylprednisolone-treated rabbits.Methylprednisolone treatment did not alter the amount of ileal BB NHE2protein. NHE3 turnover number was estimated to be 458 cycles/s underbasal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thusmethylprednisolone stimulates ileal BBNa+/H+exchange activity only by an effect on NHE3 and not on NHE2; it does soprimarily by increasing the amount of BB NHE3, although it alsoincreases the NHE3 turnover number.

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5.
Since thediscovery of the first intracellular Na+/H+exchanger in yeast, Nhx1, multiple homologs have been cloned andcharacterized in plants. Together, studies in these organismsdemonstrate that Nhx1 is located in the prevacuolar/vacuolarcompartment of cells where it sequesters Na+ into thevacuole, regulates intravesicular pH, and contributes to vacuolarbiogenesis. In contrast, the human homolog of Nhx1, Na+/H+ exchanger isoform 6 (NHE6), has beenreported to localize to mitochondria when transiently expressed as afusion with green fluorescent protein. This result warrantsreevaluation because it conflicts with predictions from phylogeneticanalyses. Here we demonstrate that when epitope-tagged NHE6 istransiently expressed in cultured mammalian cells, it does notcolocalize with mitochondrial markers. It also does not colocalize withmarkers of the lysosome, late endosome, trans-Golgi network,or Golgi cisternae. Rather, NHE6 is distributed in recyclingcompartments and transiently appears on the plasma membrane. Theseresults suggest that, like its homologs in yeast and plants, NHE6 is anendosomal Na+/H+ exchanger that may regulateintravesicular pH and volume and contribute to lysosomal biogenesis.

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6.
The plasma membrane Na+/H+ exchanger NHE1 has an established function in intracellular pH and cell volume homeostasis by catalyzing electroneutral influx of extracellular Na+ and efflux of intracellular H+. A second function of NHE1 as a structural anchor for actin filaments through its direct binding of the ezrin, radixin, and moesin (ERM) family of actin-binding proteins was recently identified. ERM protein binding and actin anchoring by NHE1 are necessary to retain the localization of NHE1 in specialized plasma membrane domains and to promote cytoskeleton-dependent processes, including actin filament bundling and cell-substrate adhesions. This review explores a third function of NHE1, as a plasma membrane scaffold in the assembly of signaling complexes. Through its coordinate functions in H+ efflux, actin anchoring, and scaffolding, we propose that NHE1 promotes protein interactions and activities, assembles signaling complexes in specialized plasma membrane domains, and coordinates divergent signaling pathways. hydrogen ion efflux; intracellular pH; molecular scaffold  相似文献   

7.
The relevance of nongenomic pathways to regulation of epithelial function by aldosterone is poorly understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO3 absorption in the renal medullary thick ascending limb (MTAL) through a nongenomic pathway. Here, we examined the transport mechanism(s) responsible for this regulation, focusing on Na+/H+ exchangers (NHE). In the MTAL, apical NHE3 mediates H+ secretion necessary for HCO3 absorption; basolateral NHE1 influences HCO3 absorption by regulating apical NHE3 activity. In microperfused rat MTALs, the addition of 1 nM aldosterone rapidly decreased HCO3 absorption by 30%. This inhibition was unaffected by three maneuvers that inhibit basolateral Na+/H+ exchange and was preserved in MTALs from NHE1 knockout mice, ruling out the involvement of NHE1. In contrast, exposure to aldosterone for 15 min caused a 30% decrease in apical Na+/H+ exchange activity over the intracellular pH range from 6.5 to 7.7, due to a decrease in Vmax. Inhibition of HCO3 absorption by aldosterone was not affected by 0.1 mM lumen Zn2+ or 1 mM lumen DIDS, arguing against the involvement of an apical H+ conductance or apical K+-HCO3 cotransport. These results demonstrate that aldosterone inhibits HCO3 absorption in the MTAL through inhibition of apical NHE3, and identify NHE3 as a target for nongenomic regulation by aldosterone. Aldosterone may influence a broad range of epithelial transport functions important for extracellular fluid volume and acid-base homeostasis through direct regulation of this exchanger. thick ascending limb; acid-base transport; epithelial Na+ transport; kidney  相似文献   

8.
NHE1/SLC9A1 is a ubiquitous isoform of vertebrate Na+/H+ exchangers (NHEs) functioning in maintaining intracellular concentrations of Na+ and H+ ions. Calcineurin homologous protein-1 (CHP1) binds to the hydrophilic region of NHE1 and regulates NHE1 activity but reportedly does not play a role in translocating NHE1 from the endoplasmic reticulum to the plasma membrane. However, an antiport function of NHE1 requiring CHP1 remains to be clarified. Here we established CHP1-deficient chicken B lymphoma DT40 cells by gene targeting to address CHP1 function. CHP1-deficient cells showed extensive decreases in Na+/H+ activities in intact cells. Although NHE1 mRNA levels were not affected, NHE1 protein levels were significantly reduced not only in the plasma membrane but in whole cells. The expression of a CHP1 transgene in CHP1-deficient cells rescued NHE1 protein expression. Expression of mutant forms of CHP1 defective in Ca2+ binding or myristoylation also partially decreased NHE1 protein levels. Knockdown of CHP1 also caused a moderate decrease in NHE1 protein in HeLa cells. These data indicate that CHP1 primarily plays an essential role in stabilization of NHE1 for reaching of NHE1 to the plasma membrane and its exchange activity. membrane protein; transporter; antiporter; quality control; degradation  相似文献   

9.
Squalamine, anendogenous molecule found in the liver and other tissues ofSqualus acanthias, hasantibiotic properties and causes changes in endothelial cell shape. Thelatter suggested that its potential targets might include transportproteins that control cell volume or cell shape. The effect of purifiedsqualamine was examined on clonedNa+/H+exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120fibroblasts. Squalamine (1-h pretreatment) decreased the maximalvelocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 µg/ml, respectively) and alsoincreasedK'[H+]i.Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitoryeffect of squalamine was 1) timedependent, with no effect of immediate addition and maximum effect with1 h of exposure, and 2) fullyreversible. Squalamine pretreatment of the ileum for 60 min inhibitedbrush-border membrane vesicleNa+/H+activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at theconcentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of thebrush-border NHE isoform NHE3 and not NHE1 or NHE2,2) acts in a nontoxic and fullyreversible manner, and 3) has adelayed effect, indicating that it may influence brush-borderNa+/H+exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator.

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10.
The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering the intraocular pressure (IOP), a function that is mediated by the bilayer ocular ciliary epithelium (CE), in conjunction with the trabecular meshwork. ANP regulates Na+/H+ exchanger (NHE) activity, and inhibitors of NHE have been shown to lower IOP. We examined whether NPs influence the NHE activity of the CE, which is comprised of pigmented (PE) and nonpigmented (NPE) epithelial cells, by directly recording the rate of intracellular pH (pHi) recovery from its inner NPE cell layer. NPs inhibited, in a dose-dependent manner (1–100 nM), the rate of pHi recovery with the order of potency CNP > ANP > BNP, indicative that this inhibition is mediated by the presence of NPR type B receptors. 8-Bromo-cGMP (8-BrcGMP), a nonhydrolyzable analog of cGMP, mimicked NPs in inhibiting the rate of Na+-dependent pHi recovery. In contrast, ethylisopropyl amiloride (EIPA, 100 nM) or amiloride (10 µM) completely abolished the pHi recovery by NHE. 18-Glycyrrhetinic acid (18-GA), a gap junction blocker, attenuated the inhibitory effect of CNP on the rate of pHi recovery, suggesting that NHE activity in both cell layers of the CE is coregulated. This interpretation was supported, in part, by the coexpression of NHE-1 isoform mRNA in both NPE and PE cells. The mechanism by which the inhibitory effect of NPs on NHE-1 activity might influence the net solute movement or fluid transport by the bilayer CE remains to be determined. Na+/H+ exchanger type 1; intracellular pH; aqueous humor  相似文献   

11.
Most vital cellular functions aredependent on a fine-tuned regulation of intracellular ion homeostasis.Here we have demonstrated, using COS cells that were untransfected ortransfected with wild-type rat ouabain-resistantNa+-K+-ATPase, that partial inhibition ofNa+-K+-ATPase has a dramatic influence oncell attachment to fibronectin. Ouabain dose-dependently decreasedattachment in untransfected cells and in cells expressing wild-typeNa+-K+-ATPase, but not in cells expressingouabain-insensitive Na+-K+-ATPase, whereasinhibition of Na+-K+-ATPase by loweringextracellular K+ concentration decreased attachment in allthree cell types. Thirty percent inhibition ofNa+-K+-ATPase significantly attenuatedattachment. Na+-K+-ATPase inhibition caused asustained increase in the intracellular Ca2+ concentrationthat obscured Ca2+ transients observed in untreated cellsduring attachment. Inhibitors of Ca2+ transporterssignificantly decreased attachment, but inhibition ofNa+/H+ exchanger did not. Ouabain reduced focaladhesion kinase autophosphorylation but had no effect on cell surfaceintegrin expression. These results suggest that the level ofNa+-K+-ATPase activity strongly influences cellattachment, possibly by an effect on intracellular Ca2+.

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12.
We recently reported that Na+/H+ exchanger isoform 1 (NHE1) activity in astrocytes is stimulated and leads to intracellular Na+ loading after oxygen and glucose deprivation (OGD). However, the underlying mechanisms for this stimulation of NHE1 activity and its impact on astrocyte function are unknown. In the present study, we investigated the role of the ERK1/2 pathway in NHE1 activation. NHE1 activity was elevated by 75% in NHE1+/+ astrocytes after 2-h OGD and 1-h reoxygenation (REOX). The OGD/REOX-mediated stimulation of NHE1 was partially blocked by 30 µM PD-98059. Increased expression of phosphorylated ERK1/2 was detected in NHE1+/+ astrocytes after OGD/REOX. Moreover, stimulation of NHE1 activity disrupted not only Na+ but also Ca2+ homeostasis via reverse-mode operation of Na+/Ca2+ exchange. OGD/REOX led to a 103% increase in intracellular Ca2+ concentration ([Ca2+]i) in NHE1+/+ astrocytes in the presence of thapsigargin. Inhibition of NHE1 activity with the NHE1 inhibitor HOE-642 decreased OGD/REOX-induced elevation of [Ca2+]i by 73%. To further investigate changes of Ca2+ signaling, bradykinin-mediated Ca2+ release was evaluated. Bradykinin-mediated intracellular Ca2+ transient in NHE1+/+ astrocytes was increased by 84% after OGD/REOX. However, in NHE1–/– astrocytes or NHE1+/+ astrocytes treated with HOE-642, the bradykinin-induced Ca2+ release was increased by only 34%. Inhibition of the reverse mode of Na+/Ca2+ exchange abolished OGD/REOX-mediated Ca2+ rise. Together, our data suggest that ERK1/2 is involved in activation of NHE1 in astrocytes after in vitro ischemia. NHE1-mediated Na+ accumulation subsequently alters Ca2+ homeostasis via Na+/Ca2+ exchange. intracellular pH; cortical astrocytes; sodium/calcium exchange; intracellular sodium ion  相似文献   

13.
The protein responsible for the Na+/Li+ exchange activity across the erythrocyte membrane has not been cloned or isolated. It has been suggested that a Na+/H+ exchanger could be responsible for the Na+/Li+ exchange activity across the erythrocyte membrane. Previously, we reported that in the trout erythrocyte, the Li+/H+ exchange activity (mediated by the Na+/H+ exchanger βNHE) and the Na+/Li+ exchange activity respond differently to cAMP, DMA (dimethyl-amiloride) and O2. We concluded that the DMA insensitive Na+/Li+ exchange activity originates from a different protein. To further examine these findings, we measured Li+ efflux in fibroblasts expressing the βNHE as the only Na+/H+ exchanger. Moreover, the internal pH of these cells was monitored with a fluorescent probe. Our findings indicate that acidification of fibroblasts expressing the Na+/H+ exchanger βNHE, induces a Na+ stimulated Li+ efflux activity in trout erythrocytes. This exchange activity, however, is DMA sensitive and therefore differs from the DMA insensitive Na+/Li+ exchange activity. In these fibroblasts no significant DMA insensitive Na+/Li+ exchange activity was found. These results support the hypothesis that the trout erythrocyte Na+/Li+ exchange activity is not mediated by the Na+/H+ exchanger (βNHE) present in these membranes. Received: 6 December 1996/Revised: 11 August 1997  相似文献   

14.
The cDNAencoding theNa+/H+exchanger (NHE) from Amphiumaerythrocytes was cloned, sequenced, and found to be highly homologous to the human NHE1 isoform (hNHE1), with 79% identity and 89%similarity at the amino acid level. Sequence comparisons with otherNHEs indicate that the Amphiumatridactylum NHE isoform 1 (atNHE1) islikely to be a phylogenetic progenitor of mammalian NHE1. The atNHE1protein, when stably transfected into the NHE-deficient AP-1 cell line(37), demonstrates robustNa+-dependent proton transportthat is sensitive to amiloride but not to the potent NHE1 inhibitorHOE-694. Interestingly, chimeric NHE proteins constructed by exchangingthe amino and carboxy termini between atNHE1 and hNHE1 exhibited drugsensitivities similar to atNHE1. Based on kinetic, sequence, andfunctional similarities between atNHE1 and mammalian NHE1, we proposethat the Amphiuma exchanger shouldprove to be a valuable model for studying the control of pH and volumeregulation of mammalian NHE1. However, low sensitivity of atNHE1 to theNHE inhibitor HOE-694 in both nativeAmphiuma red blood cells (RBCs) and intransfected mammalian cells distinguishes this transporter from itsmammalian homologue.  相似文献   

15.
Infection withhuman cytomegalovirus (HCMV) causes an enlargement (cytomegaly) ofhuman fibroblasts (MRC-5). As a first step toward determining whethersolute uptake, mediated in part by Na+/H+exchange, is responsible for the development of cytomegaly, we studiedthe effects of HCMV infection on intracellular pH(pHi) regulation (nominalCO2/concn = 0) by comparing cytomegalic cells with mock-infected cells.Seventy-two hours after HCMV infection of MRC-5 cells we observed thefollowing changes relative to mock-infected cells: restingpHi is 0.1-0.2 pH unit morealkaline; the intrinsic buffering power of the cytoplasm was reduced by~40-50%; acid-loadingH+-equivalent fluxes were reduced;and there were alterations of Na+/H+exchanger (NHE) properties, including an alkaline shift of the pHi dependence of activity, areduction of the apparent affinity for extracellularNa+, and an increase of theapparent maximum velocity and a large increase in stimulation by ahyperosmotic challenge. These results indicate that HCMV infectionexerts a profound effect on functional properties of the NHE, onacid-loading mechanisms, and on intrinsic cellular buffering power.These effects are consistent with a role for the NHE in the developmentof cytomegaly.

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16.
Glucocorticoids stimulate the intestinal absorption of Na+ and water partly by regulation of the Na+/H+ exchanger 3 (NHE3). Previous studies have shown both genomic and nongenomic regulation of NHE3 by glucocorticoids. Serum and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be part of this cascade, where phosphorylation of NHE3 by SGK1 initiates the translocation of NHE3 to the cell surface. In the present work, we examined a series of changes in SGK1 and NHE3 induced by glucocorticoids using human colonic Caco-2 and opossum kidney cells. We found that dexamethasone rapidly stimulated SGK1 mRNAs, but a significant change in protein abundance was not detected. Instead, there was an increase in SGK1 kinase activity as early as at 2 h. An increase in NHE3 protein abundance was not detected until 12 h of dexamethasone exposure, although the transport activity was significantly stimulated at 4 h. These data demonstrate that the changes of SGK1 precede those of NHE3. Chronic regulation (24 h) of NHE3 was blocked completely by prevention of protein synthesis with cycloheximide or actinomycin D and by the glucocorticoid receptor blocker RU486. The acute effect of dexamethasone was similarly abrogated by RU486, but was insensitive to cycloheximide and actinomycin D. Similarly, the stimulation of SGK1 activity by dexamethasone was blocked by RU486 but not by actinomycin D. Together, these data show that the acute effect of glucocorticoids on NHE3 is mediated by a glucocorticoid receptor dependent mechanism that activates SGK1 in a nongenomic manner. Na+/H+ exchanger 3; serum and glucocorticoid-inducible kinase 1  相似文献   

17.
Protein kinase D inhibits plasma membrane Na+/H+ exchanger activity   总被引:3,自引:0,他引:3  
The regulation of plasma membraneNa+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel proteinkinase C- and phorbol ester-regulated kinase, was investigated. Todetermine the effect of PKD on NHE activity in vivo, intracellular pH(pHi) measurements were made inCOS-7 cells by microepifluorescence using the pH indicator cSNARF-1.Cells were transfected with empty vector (control), wild-type PKD, orits kinase-deficient mutant PKD-K618M, together with green fluorescentprotein (GFP). NHE activity, as reflected by the rate of acid efflux(JH), wasdetermined in single GFP-positive cells following intracellularacidification. Overexpression of wild-type PKD had no significanteffect on JH(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control atpHi 7.0). In contrast,overexpression of PKD-K618M increasedJH (5.31 ± 0.57 mM/min at pHi 7.0;P < 0.05 vs. control). Transfectionwith these constructs produced similar effects also in A-10 cells,indicating that native PKD may have an inhibitory effect on NHE in bothcell types, which is relieved by a dominant-negative action ofPKD-K618M. Exposure of COS-7 cells to phorbol ester significantlyincreased JH in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M(because basal JHwas already near maximal). A fusion protein containing the cytosolicregulatory domain (amino acids 637-815) of NHE1 (the ubiquitousNHE isoform) was phosphorylated in vitro by wild-type PKD, but with lowstoichiometry. These data suggest that PKD inhibits NHE activity,probably through an indirect mechanism, and represents a novel pathwayin the regulation of the exchanger.

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18.
We investigated the role of intracellular Mg2+(Mgi2+) on the ATP regulation ofNa+/Ca2+ exchanger in squid axons and bovineheart. In squid axons and nerve vesicles, the ATP-upregulated exchangerremains activated after removal of cytoplasmic Mg2+, evenin the absence of ATP. Rapid and complete deactivation of theATP-stimulated exchange occurs upon readmission ofMgi2+. At constant ATP concentration, the effectof intracellular Mg2+ concentration([Mg2+]i) on the ATP regulation of exchangeris biphasic: activation at low [Mg2+]i,followed by deactivation as [Mg2+]i isincreased. No correlation was found between the above results and thelevels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] measured innerve membrane vesicles. Incorporation ofPtdIns(4,5)P2 into membrane vesicles activates Na+/Ca2+ exchange in mammalian heart but not insquid nerve. Moreover, an exogenous phosphatase prevents MgATPactivation in squid nerves but not in mammalian heart. It is concludedthat 1) Mgi2+ is an essentialcofactor for the deactivation part of ATP regulation of the exchangerand 2) the metabolic pathway of ATP upregulation of theNa+/Ca2+ exchanger is different in mammalianheart and squid nerves.

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19.
The mechanism of light-dependent active transport of pyruvatein C4 mesophyll chloroplasts has not been clarified, particularlyin Na+-type C4 species, in which the pyruvate uptake into mesophyllchloroplasts is enhanced by illumination or by making a Na+gradient (Na+-jump) across the envelope in the dark. We re-investigatedhere the effect of Na+ on the active transport of pyruvate inmesophyll chloroplasts of Panicum miliaceum, a Na+-type C4 species,by comparing the rate of pyruvate uptake at various externalpHs under four conditions; in the light and dark together with/withoutNa+-jump: (1) At neutral pH, the rate of pyruvate uptake inthe dark was enhanced by Na+-jump but scarcely by illumination.(2) While the enhancement effect by Na+-jump was independentof external pH, that by illumination increased greatly at pHover 7.4, and the effects of light and Na+ at the alkaline pHwere synergistic. (3) The light-enhanced pyruvate uptake wasrelated to stromal alkalization induced by illumination. Infact, pyruvate uptake was induced by H+-jump in the medium frompH 8.0 to 6.7. (4) Stromal pH was lowered by the addition ofK+-pyruvate and more by Na+-pyruvate into the medium at pH 7.8in the light. (5) However, the pH and ATP levels in the stromawere not affected by Na+-jump. Thus, we discussed possibility that besides pyruvate/Na+ cotransportat neutral pH in the medium, pyruvate/H+ cotransport enhancedby the presence of Na+ operates in mesophyll chloroplasts ofNa+-type C4 species at alkaline medium. 1Present address: Biological Resources Division, Japan InternationalResearch Center for Agricultural Sciences (JIRCAS), Ministryof Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba,305 Japan  相似文献   

20.
Apoptosis is a complex process essential for normal tissue development and cellular homeostasis. While biochemical events that occur late in the apoptotic process are better characterized, early physiological changes that initiate the progression of cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to growth factor withdrawal, experienced a rapid and transient rise in cytosolic pH. We found that the protein responsible was the pH-regulating, plasma membrane protein Na+/H+ exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of the stress-activated kinase, p38 MAP kinase. In the current study, we examined how NHE1 is activated during apoptosis. We identified the phosphorylation sites on NHE1 that regulate its alkalinizing activity in response to a cell death stimulus. Performing targeted mutagenesis, we observed that substitution of Ser726 and Ser729 for alanines produced a mutant form of NHE1 that did not alkalinize in response to an apoptotic stimulus, and expression of which protected cells from serum withdrawal- induced death. In contrast, substitution of Ser726 and Ser729 for glutamic acids raised the basal pH and induced susceptibility to death. Analysis of serine phosphorylation showed that phosphorylation of NHE1 during apoptosis decreased upon mutation of Ser726 and Ser729. Our findings thus confirm a necessary function for NHE1 during apoptosis and reveal the critical regulatory sites that when phosphorylated mediate the alkalinizing activity of NHE1 in the early stages of a cell death response. pH; sodium hydrogen exchanger; mitogen-activated protein kinase  相似文献   

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