共查询到20条相似文献,搜索用时 31 毫秒
1.
Churchill GC Lurtz MM Louis CF 《American journal of physiology. Cell physiology》2001,281(3):C972-C981
The quantitative effects of Ca2+signaling on gap junctional coupling in lens epithelial cells have beendetermined using either the spread of Mn2+ that is imagedby its ability to quench the fluorescence of fura 2 or the spread ofthe fluorescent dye Alexa Fluor 594. Gap junctional coupling wasunaffected by a mechanically stimulated cell-to-cell Ca2+wave. Furthermore, when cytosolic Ca2+ concentration(Ca ) increased after the addition of the agonistATP, coupling was unaffected during the period thatCa was maximal. However, coupling decreasedtransiently ~5-10 min after agonist addition whenCa returned to resting levels, indicating that thistransient decrease in coupling was unlikely due to a direct action ofCa on gap junctions. An increase inCa mediated by the ionophore ionomycin that wassustained for several minutes resulted in a more rapid and sustaineddecrease in coupling (IC50 ~300 nM Ca2+, Hillcoefficient of 4), indicating that an increase in Ca alone could regulate gap junctions. Thus Ca increases that occurred during agonist stimulation and cell-to-cell Ca2+ waves were too transient to mediate a sustaineduncoupling of lens epithelial cells. 相似文献
2.
Lyn- and ERK-mediated vs. Ca2+-mediated neutrophil O2- responses with thermal injury 总被引:1,自引:0,他引:1
Fazal Nadeem; Al-Ghoul Walid M.; Schmidt Megan J.; Choudhry Mashkoor A.; Sayeed Mohammed M. 《American journal of physiology. Cell physiology》2002,283(5):C1469
We evaluated thedependency of neutrophil O production on PTK-Lyn andMAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn wasassessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessedby Western blot analysis. O production was measuredby isoluminol-enhanced luminometry. Imaging technique was employed tomeasure neutrophil [Ca2+]i in individualcells. Thermal injury caused marked upregulation of Lyn and ERK1/2accompanying enhanced neutrophil O production.Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478)before burn injury caused complete inhibition of the respective kinaseactivation. Both AG556 and AG1478 produced an ~66% inhibition inO production. Treatment with diltiazem (DZ) producedan ~37% inhibition of O production withoutaffecting Lyn or ERK1/2 activation with burn injury. Ca2+mobilization was upregulated with burn injury but not affected bytreatment of burn rats with AG556. Unlike the partial inhibition ofburn-induced O production by AG556, AG1478, or DZ,platelet-activating factor antagonist (PAFa) treatment of burn ratsproduced near complete inhibition of O production.PAFa treatment also blocked activation of Lyn. The findings suggestthat the near complete inhibition of O production byPAFa was a result of blockade of PTK as well as Ca2+signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result ofpotentiation of Ca2+-linked and -independent signalingtriggered by inflammatory agents such as PAF. 相似文献
3.
Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells 总被引:1,自引:0,他引:1
HCO -dependentfluid secretion by the corneal endothelium controls corneal hydrationand maintains corneal transparency. Recently, it has been shown thatmRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO transport have not been reported. Immunoblotting for CFTR showed asingle band at ~170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirectimmunofluorescence confocal microscopy indicated that CFTR locates tothe apical membrane. Relative changes in apical and basolateralchloride permeability were estimated by measuring the rate offluorescence quenching of the halide-sensitive indicator6-methoxy-N-ethylquinolinium iodide during Clinflux in the absence and presence of forskolin (FSK). Apical andbasolateral Cl permeability increased 10- and 3-fold,respectively, in the presence of 50 µM FSK. FSK-activated apicalchloride permeability was unaffected by H2DIDs (250 µM);however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 µM) and glibenclamide (100 µM) inhibited activated Clfluxes by 45% and 30%, respectively. FSK-activated basolateral Cl permeability was insensitive to NPPB, glibenclamide,or furosemide but was inhibited 80% by H2DIDS.HCO permeability was estimated by measuring changesin intracellular pH in response to quickly lowering bath[HCO ]. FSK (50 µM) increased apicalHCO permeability by twofold, which was inhibited42% by NPPB and 65% by glibenclamide. BasolateralHCO permeability was unaffected by FSK. Genistein(50 µM) significantly increased apical HCO andCl permeability by 1.8- and 16-fold, respectively. When50 µM genistein was combined with 50 µM FSK, there was no furtherincrease in Cl permeability; however,HCO permeability was reduced to the control level.In summary, we conclude that CFTR is present in the apical membrane ofbovine corneal endothelium and could contribute to transendothelialCl and HCO transport. Furthermore,there is a cAMP-activated Cl pathway on the basolateralmembrane that is not CFTR. 相似文献
4.
Novak D Quiggle F Artime C Beveridge M 《American journal of physiology. Cell physiology》2001,281(3):C1014-C1022
We utilized HRP.1 cells derived from midgestation ratplacental labyrinth to determine that the primary pathway for glutamate uptake is via system X , a Na+-dependenttransport system. Kinetic parameters of system X activity were similar to those previously determined in rat and humanplacental membrane vesicle preparations. Amino acid depletion caused asignificant upregulation of system X activity at 6, 24, and 48 h. This increase was reversed by the addition ofglutamate and aspartate but not by the addition of -(methylamino)isobutyric acid. Immunoblot analysis of the three transport proteins previously associated with systemX activity indicated a trend toward an increase inGLT1, EAAC1, and GLAST1 immunoreactive protein contents by 48 h;cell surface expression of the same was enhanced by 24 h.Inhibition analysis suggested key roles for EAAC1 and GLAST1 in basalanionic amino acid transfer, with an enhanced role for GLT1 underconditions of amino acid depletion. In summary, amino acid availabilityas well as intracellular metabolism regulate anionic amino acid uptake into this placental cell line. 相似文献
5.
Teixeira CC Mansfield K Hertkorn C Ischiropoulos H Shapiro IM 《American journal of physiology. Cell physiology》2001,281(3):C833-C839
An elevation in inorganic phosphate(Pi) concentration activates epiphyseal chondrocyteapoptosis. To determine the mechanism of apoptosis,tibial chondrocytes were treated with Pi, andnitrate/nitrite (NO /NO ) levelswere determined. Pi induced a threefold increase in the NO /NO concentration; inhibitorsof nitric oxide (NO) synthase activity and Pi transportsignificantly reduced NO /NO levels and prevented cell death. Furthermore, a dose-dependent increasein cell death was observed after exposure of chondrocytes toS-nitrosoglutathione. Pi increased caspase 3 activity 2.7-fold. Both caspase 1 and caspase 3 inhibitors protectedchondrocytes from Pi-induced apoptosis.Pi caused a significant decrease in the mitochondrialmembrane potential, while NO synthase inhibitors maintainedmitochondrial function. While Pi caused thiol depletion, inhibition of Pi uptake or NO generation served tomaintain glutathione levels. The results suggest that NO serves tomediate key metabolic events linked to Pi-dependentchondrocyte apoptosis. 相似文献
6.
Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea 总被引:2,自引:0,他引:2
Jayaraman S Song Y Verkman AS 《American journal of physiology. Cell physiology》2001,281(5):C1504-C1511
Airway surface liquid (ASL) pH hasbeen proposed to be important in the pathophysiology of cysticfibrosis, asthma, and cough. Ratio image analysis was used to measurepH in the ASL after staining with the fluorescent pH indicator2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-dextran. ASL pH in bovine airway cell cultures grown at anair-liquid interface was 6.98 ± 0.06 in the absence and 6.81 ± 0.04 in the presence of HCO /CO2. Steady-state ASL pH changed in parallel to changes in bath pH and wasacidified by Na+ or Cl replacement but wasnot affected by the inhibitors amiloride, glibenclamide, or4,4'-dinitrostilbene-2,2'-disulfonic acid. In response to suddenacidification or alkalization of the ASL by ~0.4 pH units byHCl/NaOH, ASL pH recovered to its initial value at a rate of 0.035 pHunits/min (HCO ) and 0.060 pH units/min(+HCO ); the pH recovery rate was reduced byamiloride and H2DIDS. In anesthetized mice in which thetrachea was surgically exposed for measurement of BCECF-dextranfluorescence through the translucent tracheal wall, ASL pH was7.14 ± 0.01. ASL pH was sensitive to changes in blood pH createdby metabolic (HCl or NaHCO3 infusion) or respiratory (hyperventilation, hypoventilation) mechanisms. ASL pH is thus primarily determined by basolateral fluid pH, andH+/OH transport between the ASL andbasolateral fluid involves amiloride-sensitive Na+/H+ exchange and stilbene-sensitiveCl/HCO exchange. The rapid response ofASL pH to changes in systemic acid-base status may contribute to airwayhypersensitivity in asthma and other airway diseases. 相似文献
7.
Carlin Ryan W.; Quesnell Rebecca R.; Zheng Ling; Mitchell Kathy E.; Schultz Bruce D. 《American journal of physiology. Cell physiology》2002,283(4):C1033
This study focused on the role ofsodium-bicarbonate cotransporter (NBC1) in cAMP-stimulated iontransport in porcine vas deferens epithelium. Ion substitutionexperiments in modified Ussing chambers revealed that cAMP-mediatedstimulation was dependent on the presence of Na+,HCO , and Cl for a full response.HCO -dependent current was unaffected byacetazolamide, bumetanide, or amiloride but was inhibited bybasolateral 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.Na+-driven, HCO -dependent,stilbene-inhibitable anion flux was observed across the basolateralmembrane of selectively permeabilized monolayers. Results ofradiotracer flux studies suggest a4,4'-dinitrostilbene-2,2'-disulfonate-sensitive stoichiometry of 2 baseequivalents per Na+. Antibodies raised against rat kidneyNBC epitopes (rkNBC; amino acids 338-391 and 928-1035)identified a single band of ~145 kDa. RT-PCR detected NBC1 message inporcine vas deferens epithelia. These results demonstrate that vasdeferens epithelial cells possess the proteins necessary for thevectoral transport of HCO and that these mechanismsare maintained in primary culture. Taken together, the results indicatethat vas deferens epithelia play an active role in male fertility andhave implications for our understanding of the relationship betweencystic fibrosis and congenital bilateral absence of the vas deferens. 相似文献
8.
Signaling by eNOS through a superoxide-dependent p42/44 mitogen-activated protein kinase pathway 总被引:2,自引:0,他引:2
Wang Weihan; Wang Shuibang; Nishanian Ervant V.; Del Pilar Cintron Ana; Wesley Robert A.; Danner Robert L. 《American journal of physiology. Cell physiology》2001,281(2):C544
Expression ofendothelial nitric oxide synthase (eNOS) in transfected U-937 cellsupregulates phorbol 12-myristate 13-acetate (PMA)-induced tumornecrosis factor- (TNF-) production through a superoxide(O )-dependent mechanism. Because mitogen-activatedprotein kinases (MAPK) have been shown to participate in both reactiveoxygen species signaling and TNF- regulation, their possible role ineNOS-derived O signal transduction was examined. Aredox-cycling agent, phenazine methosulfate, was found to bothupregulate TNF- (5.8 ± 1.0 fold; P = 0.01) andincrease the phosphorylation state of p42/44 MAPK (3.1 ± 0.2 fold; P = 0.01) in PMA-differentiated U-937 cells. AlthoughS-nitroso-N-acetylpenicillamine, a nitric oxide(NO) donor, also increased TNF- production, NO exposure led tophosphorylation of p38 MAPK, not p42/44 MAPK. Upregulation of TNF-production by eNOS transfection was associated with increases inactivated p42/44 MAPK (P = 0.001), whereas levels ofphosphorylated p38 MAPK were unaffected. Furthermore, cotransfectionwith Cu/Zn superoxide dismutase, which blocks TNF- upregulation byeNOS, also abolished the effects on p42/44 MAPK. Expression ofGln361eNOS, a mutant that produces O but not NO, still resulted in p42/44 MAPK phosphorylation. In contrast, twoNADPH binding site deletion mutants of eNOS that lack oxidase activityhad no effect on p42/44 MAPK. Finally, PD-98059, a p42/44 MAPK pathwayinhibitor, blocked TNF- upregulation by eNOS (P = 0.02).Thus O produced by eNOS increases TNF- productionvia a mechanism that involves p42/44 MAPK activation. 相似文献
9.
Neurotrophins are expressed in the adult kidney, but theirsignificance is unclear. We showed previously that nerve growth factor(NGF) inhibits HCO absorption in the rat medullarythick ascending limb (MTAL) via an extracellular signal-regulatedkinase (ERK)-dependent pathway. Here we examined whether otherneurotrophic factors affect MTAL HCO absorption.Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor had no effect. In contrast, neurotrophin-3 (NT-3,0.7 nM) inhibited HCO absorption by 40%(half-maximal inhibition at ~0.4 nM). Inhibition by NT-3 was additiveto inhibition by NGF. Inhibitors of ERK activation that blockinhibition by NGF had no effect on inhibition by NT-3. In contrast,8-bromo-cAMP or forskolin pretreatment blocked inhibition by NT-3 butnot NGF. Inhibition by NT-3 was also blocked by the specific proteinkinase A (PKA) inhibitor myristoylated PKI(14-22) amide and by vasopressin, which inhibits HCO absorption via cAMP. Inhibitors of phosphatidylinositol 3-kinase orprotein kinase C did not affect NT-3-induced inhibition, but inhibitionby NT-3 was eliminated by genistein, consistent with involvement of areceptor tyrosine kinase. These results demonstrate that NT-3 inhibitsHCO absorption via a cAMP- and PKA-dependentpathway. NT-3 and NGF regulate MTAL ion transport through differentsignal transduction mechanisms. These studies establish a direct rolefor NT-3 in regulation of renal tubule transport and identify the MTALas an important target for neurotrophins, which may be involved in thecontrol of renal acid excretion. 相似文献
10.
Alvarez de la Rosa D Canessa CM 《American journal of physiology. Cell physiology》2003,284(2):C404-C414
The purpose of this study was to examinethe role of the serum- and glucocorticoid-induced kinase (SGK) in theactivation of the epithelial sodium channel (ENaC) by aldosterone,arginine vasopressin (AVP), and insulin. We used atetracycline-inducible system to control the expression of wild-type(SGK ), constitutively active (S425Dmutation; SGK ), or inactive (K130Mmutation; SGK ) SGK in A6 cellsindependently of hormonal stimulation. The effect of SGK expression onENaC activity was monitored by measuring transepithelialamiloride-sensitive short-circuit current (Isc) of transfected A6 cell lines. Expression ofSGK orSGK and aldosterone stimulation haveadditive effects on Isc. Although SGK could playsome role in the aldosterone response, our results suggest that othermechanisms take place. SGK abrogatesthe responses to AVP and insulin; hence, in the signaling pathways ofthese hormones there is a shared step that is stimulated by SGK.Because AVP and insulin induce fusion of vesicles to the apicalmembrane, our results support the notion that SGK promotes incorporation of channels in the apical membrane. 相似文献
11.
Role of glutamate transporters in the regulation of glutathione levels in human macrophages 总被引:3,自引:0,他引:3
Rimaniol Anne-Cecile; Mialocq Patricia; Clayette Pascal; Dormont Dominique; Gras Gabriel 《American journal of physiology. Cell physiology》2001,281(6):C1964
Cysteine is the limiting precursor forglutathione synthesis. Because of its low bioavailability, cysteine isgenerally produced from cystine, which may be taken up through twodifferent transporters. The cystine/glutamate antiporter(x system) transports extracellular cystine inexchange for intracellular glutamate. The XAG transportsystem takes up extracellular cystine, glutamate, and aspartate. Bothare sensitive to competition between cystine and glutamate, and excessextracellular glutamate thus inhibits glutathione synthesis, anonexcitotoxic mechanism for glutamate toxicity. We demonstratedpreviously that human macrophages express the glutamate transportersexcitatory amino acid transporter (EAAT)1 and EAAT2 (which do nottransport cystine, X system) and overcomecompetition for the use of cystine transporters. We now showthat macrophages take up cystine through the x andnot the XAG system. We also found that glutamate, although competing with cystine uptake, dose-dependently increases glutathione synthesis. We used inhibitors to demonstrate that this increase ismediated by EAATs. EAAT expression in macrophages thus leads toglutamate-dependent enhancement of glutathione synthesis by providingintracellular glutamate for direct insertion in glutathione and alsofor fueling the intracellular pool of glutamate andtrans-stimulating the cystine/glutamate antiporter. 相似文献
12.
Gap junctions and fluid flow response in MC3T3-E1 cells 总被引:2,自引:0,他引:2
Saunders M. M.; You J.; Trosko J. E.; Yamasaki H.; Li Z.; Donahue H. J.; Jacobs C. R. 《American journal of physiology. Cell physiology》2001,281(6):C1917
In thecurrent study, we examined the role of gap junctions in oscillatoryfluid flow-induced changes in intracellular Ca2+concentration and prostaglandin release in osteoblastic cells. Thiswork was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Ourresults demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E2 (PGE2) release, whereas cellswith diminished gap junctional communication do not. Furthermore, wefound that cytosolic Ca2+ (Ca ) responsewas unaltered by the disruption in gap junctional communication and wasnot significantly different among the cell lines. Thus our resultssuggest that gap junctions contribute to the PGE2 but notto the Ca response to oscillatory fluid flow. Thesefindings implicate gap junctional intercellular communication (GJIC) inbone cell ensemble responsiveness to oscillatory fluid flow and suggestthat gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone. 相似文献
13.
Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains 总被引:6,自引:0,他引:6
Lohi H Lamprecht G Markovich D Heil A Kujala M Seidler U Kere J 《American journal of physiology. Cell physiology》2003,284(3):C769-C779
The solute carrier gene family SLC26consists of tissue-specific anion exchanger genes, three of themassociated with distinct human recessive disorders. By a genome-drivenapproach, several new SLC26 family members have been identified,including a kidney- and pancreas-specific gene, SLC26A6. We report thefunctional characterization of SLC26A6 and two new alternativelyspliced variants, named SLC26A6c and SLC26A6d. Immunofluorescencestudies on transiently transfected cells indicated membranelocalization and indicated that both NH2- and COOH-terminaltails of the SLC26A6 variants are located intracellularly, suggesting atopology with an even number of transmembrane domains. Functionalexpression of the three proteins in Xenopus oocytesdemonstrated Cl and SO transportactivity. In addition, the transport of SO andCl was inhibited by DIDS and HCO . We demonstrated also that the COOH terminus of SLC26A6 binds to the firstand second PDZ domains of the Na+/H+ exchanger(NHE)3 kinase A regulatory protein (E3KARP) and NHE3 regulatory factor(NHERF) proteins in vitro. Truncation of the last three amino acids(TRL) of SLC26A6 abrogated the interaction but did not affect transportfunction. These results demonstrate that SLC26A6 and its two splicevariants can function as anion transporters linked to PDZ-interactionpathways. Our results support the general concept of microdomainorganization for ion transport and suggest a mechanism for cysticfibrosis transmembrane regulator (CFTR)-mediated SLC26A6 upregulationin pancreatic duct cells. 相似文献
14.
Sciortino Christopher M.; Shrode Lamara D.; Fletcher Bonnie R.; Harte Peter J.; Romero Michael F. 《American journal of physiology. Cell physiology》2001,281(2):C449
Na+-dependent Cl/HCO exchange activity helps maintain intracellular pH (pHi)homeostasis in many invertebrate and vertebrate cell types. Ourlaboratory cloned and characterized a Na+-dependentCl/HCO exchanger (NDAE1) fromDrosophila melanogaster (Romero MF, Henry D, Nelson S, HartePJ, and Sciortino CM. J Biol Chem 275:24552-24559, 2000). In the present study we usedimmunohistochemical and Western blot techniques to characterize thedevelopmental expression, subcellular localization, and tissue distribution of NDAE1 protein in D. melanogaster. We haveshown that a polyclonal antibody raised against the NH2terminus of NDAE1 (CWR57) recognizes NDAE1 electrophysiologicallycharacterized in Xenopus oocytes. Moreover, our resultsbegin to delineate the NDAE1 topology, i.e., both the NH2and COOH termini are intracellular. NDAE1 is expressed throughoutDrosophila development in the central and peripheral nervoussystems, sensilla, and the alimentary tract (Malpighian tubules, gut,and salivary glands). Coimmunolabeling of larval tissues with NDAE1antibody and a monoclonal antibody to theNa+-K+-ATPase -subunit revealed that themajority of NDAE1 is located at the basolateral membranes of Malpighiantubule cells. These results suggest that NDAE1 may be a keypHi regulatory protein and may contribute to basolateralion transport in epithelia and nervous system of Drosophila. 相似文献
15.
Flow-induced calcium oscillations in rat osteoblasts are age, loading frequency, and shear stress dependent 总被引:5,自引:0,他引:5
Donahue SW Jacobs CR Donahue HJ 《American journal of physiology. Cell physiology》2001,281(5):C1635-C1641
Bone adaptation tomechanical loading is dependent on age and the frequency and magnitudeof loading. It is believed that load-induced fluid flow in the porousspaces of bone is an important signal that influences bone cellmetabolism and bone adaptation. We used fluid flow-induced shear stressas a mechanical stimulus to study intracellular calcium(Ca ) signaling in rat osteoblastic cells (ROB)isolated from young, mature, and old animals. Fluid flow producedhigher magnitude and more abundant [Ca2+]ioscillations than spontaneous oscillations, suggesting that flow-induced Ca signaling encodes a differentcellular message than spontaneous oscillations. ROB from old ratsshowed less basal [Ca2+]i activity and wereless responsive to fluid flow. Cells were more responsive to 0.2 Hzthan to 1 or 2 Hz and to 2 Pa than to 1 Pa. These data suggest that thefrequency and magnitude of mechanical loading may be encoded by thepercentage of cells displaying [Ca2+]ioscillations but that the ability to transduce this information may bealtered with age. 相似文献
16.
Satoh H Moriyama N Hara C Yamada H Horita S Kunimi M Tsukamoto K Iso-O N Inatomi J Kawakami H Kudo A Endou H Igarashi T Goto A Fujita T Seki G 《American journal of physiology. Cell physiology》2003,284(3):C729-C737
Mutations inNa+-HCO cotransporter (NBC-1) causeproximal renal tubular acidosis (pRTA) associated with ocularabnormalities. One pRTA patient had increased serum amylase, suggestingpossible evidence of pancreatitis. To further delineate a link betweenNBC-1 inactivation and pancreatic dysfunction, immunohistochemicalanalysis was performed on rat and human pancreas using antibodiesagainst kidney-type (kNBC-1) and pancreatic-type (pNBC-1) transporters.In rat pancreas, the anti-pNBC-1 antibody labeled acinar cells and bothapical and basolateral membranes of medium and large duct cells. Inhuman pancreas, on the other hand, the anti-pNBC-1 antibody did notlabel acinar cells, although it did label the basolateral membranes ofthe entire duct system. The labeling by anti-kNBC-1 antibody wasdetected in only a limited number of rat pancreatic duct cells. Toexamine the effects of pRTA-related mutations, R342S and R554H, onpNBC-1 function, we performed functional analysis and found that bothmutants had reduced transport activities compared with the wild-typepNBC-1. These results indicate that pNBC-1 is the predominant variant that mediates basolateral HCO uptake into duct cellsin both rat and human pancreas. The loss of pNBC-1 function ispredicted to have significant impact on overall ductal HCO secretion, which could potentially lead topancreatic dysfunction. 相似文献
17.
Yao H Gu XQ Douglas RM Haddad GG 《American journal of physiology. Cell physiology》2001,281(4):C1205-C1210
To determine the role ofmembrane transporters in intracellular pH (pHi) regulationunder conditions of low microenvironmental O2, we monitoredpHi in isolated single CA1 neurons using the fluorescentindicator carboxyseminaphthorhodafluor-1 and confocal microscopy. Aftertotal O2 deprivation or anoxia (PO2 0 Torr), a large increase in pHi was seen in CA1neurons in HEPES buffer, but a drop in pHi, albeit small,was observed in the presence of HCO . Ionicsubstitution and pharmacological experiments showed that the largeanoxia-induced pHi increase in HEPES buffer was totallyNa+ dependent and was blocked by HOE-694, stronglysuggesting the activation of the Na+/H+exchanger (NHE). Also, this pHi increase in HEPES bufferwas significantly smaller in Na+/H+ exchangerisoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons,demonstrating that NHE1 is responsible for part of the pHiincrease following anoxia. Both chelerythrine and H-89 partly blocked,and H-7 totally eliminated, this anoxia-induced pHiincrease in the absence of HCO . We conclude that1) O2 deprivation activatesNa+/H+ exchange by enhancing protein kinaseactivity and 2) membrane proteins, such as NHE, activelyparticipate in regulating pHi during low-O2states in neurons. 相似文献
18.
CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts 总被引:4,自引:0,他引:4
Barrière H Poujeol C Tauc M Blasi JM Counillon L Poujeol P 《American journal of physiology. Cell physiology》2001,281(3):C810-C824
To study the potentialinfluence of cystic fibrosis conductance regulator (CFTR) onintracellular pH regulation during apoptosis induction, we usedPS120 Chinese hamster lung fibroblasts devoid of theNa+/H+ exchanger (NHE1 isoform) transfectedwith constructs, allowing the expression of CFTR and/or NHE1. Kineticsof lovastatin-induced apoptosis were measured by orceinstaining, double staining with Hoechst-33258, propidium iodide, DNAfragmentation, and annexin V labeling. In PS120 control cells, thepercentage of apoptotic cells after 40 h of lovastatintreatment was 23 ± 3%, whereas in PS120 CFTR-transfected cells,this percentage was 40 ± 4%. In PS120 NHE1 cells, thetransfection with CFTR did not modify the percentage of apoptoticcells after 40 h (control: 19 ± 3%, n = 8;CFTR: 17 ± 1%, n = 8), indicating that blockingintracellular acidification by overexpressing theNa+/H+ exchanger inhibited the enhancement ofapoptosis induced by CFTR. In all cell lines, the initial pHvalues were identical (pH = 7.46 ± 0.04, n = 9), and treatment with lovastatin led to intracellular acidification.However, the pH value after 40 h was lower in PS120 CFTR-transfected cells (pH = 6.85 ± 0.02, n = 10) than in PS120 cells (pH = 7.15 ± 0.03, n = 10). To further investigate the origin of thisincreased intracellular acidification observed in CFTR-transfected cells, the activity of the DIDS-inhibitableCl/HCO exchanger was studied.8-Bromoadenosine 3',5'-cyclic monophosphate incubation resulted inCl/HCO exchanger activation in PS120 CFTR-transfected cells but had no effect on PS120 cells. Together, ourresults suggest that CFTR can enhance apoptosis in Chinese hamster lung fibroblasts, probably due to the modulation of the Cl/HCO exchanger, resulting in a more efficient intracellular acidification. 相似文献
19.
Cougnon M Benammou S Brouillard F Hulin P Planelles G 《American journal of physiology. Cell physiology》2002,282(6):C1445-C1453
To investigate theeffects of reactive oxygen species (ROS) on NH permeation in Xenopus laevis oocytes, we used intracellulardouble-barreled microelectrodes to monitor the changes in membranepotential (Vm) and intracellular pH(pHi) induced by a 20 mM NH4Cl-containingsolution. Under control conditions, NH4Cl exposure induceda large membrane depolarization (to Vm = 4.0 ± 1.5 mV; n = 21) and intracellularacidification [reaching a change in pHi(pHi) of 0.59 ± 0.06 pH units in 12 min]; theinitial rate of cell acidification (dpHi/dt) was0.06 ± 0.01 pH units/min. Incubation of the oocytes in thepresence of H2O2 or -amyloid protein had nomarked effect on the NH4Cl-induced pHi. Bycontrast, in the presence of photoactivated rose bengal (RB),tert-butyl-hydroxyperoxide (t-BHP), orxanthine/xanthine oxidase (X/XO), the same experimental maneuverinduced significantly greater pHi anddpHi/dt. These increases in pHiand dpHi/dt were prevented by the ROS scavengershistidine and desferrioxamine, suggesting involvement of the reactivespecies 1gO2 and ·OH. Using thevoltage-clamp technique to identify the mechanism underlying theROS-measured effects, we found that RB induced a large increase in theoocyte membrane conductance (Gm). ThisRB-induced Gm increase was prevented by 1 mMdiphenylamine-2-carboxylate (DPC) and by a low Na+concentration in the bath. We conclude that RB, t-BHP, andX/XO enhance NH influx into the oocyte via activationof a DPC-sensitive nonselective cation conductance pathway. 相似文献
20.
Virkki Leila V.; Wilson Darren A.; Vaughan-Jones Richard D.; Boron Walter F. 《American journal of physiology. Cell physiology》2002,282(6):C1278
We havefunctionally characterized Na+-driven bicarbonatetransporter (NBC)4, originally cloned from human heart by Pushkin etal. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the fourNBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no.AF293337) in Xenopus laevis oocytes and assayed membrane potential (Vm) and pH regulatory function withmicroelectrodes. Exposing an NBC4c-expressing oocyte to a solutioncontaining 5% CO2 and 33 mM HCO elicited a large hyperpolarization, indicating that the transporter iselectrogenic. The initial CO2-induced decrease inintracellular pH (pHi) was followed by a slow recovery thatwas reversed by removing external Na+. Two-electrodevoltage clamp of NBC4c-expressing oocytes revealed largeHCO - and Na+-dependent currents. When wevoltage clamped Vm far from NBC4c's estimatedreversal potential (Erev), the pHirecovery rate increased substantially. Both the currents andpHi recovery were blocked by 200 µM4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimatedthe transporter's HCO :Na+ stoichiometryby measuring Erev at different extracellularNa+ concentration ([Na+]o)values. A plot of Erev againstlog[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well asthe absolute Erev values, are consistent with a2:1 stoichiometry. In conclusion, the behavior of NBC4c, which wepropose to call NBCe2-c, is similar to that of NBCe1, the firstelectrogenic NBC. 相似文献