共查询到20条相似文献,搜索用时 15 毫秒
1.
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
2.
Stewart AK Kurschat CE Burns D Banger N Vaughan-Jones RD Alper SL 《American journal of physiology. Cell physiology》2007,292(2):C909-C918
Activity of the AE2/SLC4A2 anion exchanger is modulated acutely by pH, influencing the transporter's role in regulation of intracellular pH (pHi) and epithelial solute transport. In Xenopus oocytes, heterologous AE2-mediated Cl/Cl and Cl/HCO3 exchange are inhibited by acid pHi or extracellular pH (pHo). We have investigated the importance to pH sensitivity of the eight histidine (His) residues within the AE2 COOH-terminal transmembrane domain (TMD). Wild-type mouse AE2-mediated Cl/Cl exchange, measured as DIDS-sensitive 36Cl efflux from Xenopus oocytes, was experimentally altered by varying pHi at constant pHo or varying pHo. Pretreatment of oocytes with the His modifier diethylpyrocarbonate (DEPC) reduced basal 36Cl efflux at pHo 7.4 and acid shifted the pHo vs. activity profile of wild-type AE2, suggesting that His residues might be involved in pH sensing. Single His mutants of AE2 were generated and expressed in oocytes. Although mutation of H1029 to Ala severely reduced transport and surface expression, other individual His mutants exhibited wild-type or near-wild-type levels of Cl transport activity with retention of pHo sensitivity. In contrast to the effects of DEPC on wild-type AE2, pHo sensitivity was significantly alkaline shifted for mutants H1144Y and H1145A and the triple mutants H846/H849/H1145A and H846/H849/H1160A. Although all functional mutants retained sensitivity to pHi, pHi sensitivity was enhanced for AE2 H1145A. The simultaneous mutation of five or more His residues, however, greatly decreased basal AE2 activity, consistent with the inhibitory effects of DEPC modification. The results show that multiple TMD His residues contribute to basal AE2 activity and its sensitivity to pHi and pHo. pH regulation; histidine residues; Cl/HCO3 exchange 相似文献
3.
Bevensee Mark O.; Bashi Esther; Schlue Wolf-Rudiger; Boyarsky Gregory; Boron Walter F. 《American journal of physiology. Cell physiology》1999,276(3):C674
Using thepH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF),we examined the effect of hyperosmolar solutions, which presumablycaused cell shrinkage, on intracellular pH(pHi) regulation in mesangialcells (single cells or populations) cultured from the rat kidney. Thecalibration of BCECF is identical in shrunken and unshrunken mesangialcells if the extracellular K+concentration ([K+])is adjusted to match the predicted intracellular[K+]. ForpHi values between ~6.7 and~7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (~300mosmol/kgH2O). In the nominalabsence ofCO2/HCO3,exposing cell populations to a HEPES-buffered solution supplementedwith ~300 mM mannitol (600 mosmol/kgH2O) causes steady-statepHi to increase by ~0.4. The pHi increase is due to activationofNa+/H+exchange because, in single cells, it is blocked in the absence ofexternal Na+ or in the presence of50 µM ethylisopropylamiloride (EIPA). Preincubating cells in aCl-free solution for atleast 14 min inhibits the shrinkage-induced pHi increase by 80%. Wecalculated the pHi dependence oftheNa+/H+exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) thepHi dependence of the totalacid-extrusion rate and 2) thepHi dependence of theEIPA-insensitive acid-loading rate. Shrinkage alkali shifts thepHi dependence ofNa+/H+exchange by ~0.7 pH units. 相似文献
4.
Bouzinova EV Praetorius J Virkki LV Nielsen S Boron WF Aalkjaer C 《American journal of physiology. Cell physiology》2005,289(6):C1448-C1456
Several studies suggest the involvement of Na+ and HCO3 transport in the formation of cerebrospinal fluid. Two Na+-dependent HCO3 transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pHi) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH2-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pHi in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO2/HCO3 into the bath solution. This increase was Na+ dependent and inhibited by the Cl and HCO3 transport inhibitor DIDS (200 µM). This suggests the presence of Na+-dependent, partially DIDS-sensitive HCO3 uptake. The pHi recovery after acid loading revealed an initial Na+ and HCO3-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO2/HCO3 was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na+- and HCO3-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na+-driven Cl bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse 相似文献
5.
Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes 总被引:18,自引:0,他引:18
McLean LA Roscoe J Jorgensen NK Gorin FA Cala PM 《American journal of physiology. Cell physiology》2000,278(4):C676-C688
Malignantgliomas exhibit alkaline intracellular pH (pHi) and acidicextracellular pH (pHe) compared with nontransformedastrocytes, despite increased metabolic H+ production. Theacidic pHe limits the availability ofHCO3, thereby reducing both passiveand dynamic HCO3-dependent buffering.This implies that gliomas are dependent upon dynamic HCO3-independent H+buffering pathways such as the type 1 Na+/H+exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state pHi(pHi = 7.31-7.48) than normal astrocytes(pHi = 6.98), and increased rates of recovery fromacidification, under nominallyCO2/HCO3-free conditions.Inhibition of NHE1 in the absence ofCO2/HCO3 resulted inpronounced acidification of gliomas, whereas normal astrocytes wereunaffected. When suspended inCO2/HCO3 medium astrocytepHi increased, yet glioma pHi unexpectedlyacidified, suggesting the presence of anHCO3-dependent acid loadingpathway. Nucleotide sequencing of NHE1 cDNA from the gliomasdemonstrated that genetic alterations were not responsible for thisaltered NHE1 function. The data suggest that NHE1 activity issignificantly elevated in gliomas and may provide a useful target forthe development of tumor-selective therapies. 相似文献
6.
Mulkey DK Henderson RA Ritucci NA Putnam RW Dean JB 《American journal of physiology. Cell physiology》2004,286(4):C940-C951
Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pHi). pHi was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pHi in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H2O2 decreased pHi by 0.19 ± 0.007, 0.20 ± 0.015, 0.15 ± 0.013, and 0.08 ± 0.002 pH unit, respectively. Hypercapnia decreased pHi by 0.26 ± 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pHi, causing a 0.42 ± 0.03 (n = 21) pH unit acidification. CT slowed pHi recovery mediated by Na+/H+ exchange (NHE) from NH4Cl-induced acidification by 53% (n = 20) in -buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pHi. These results indicate that oxidative stress 1) decreases pHi in some SC neurons, 2) together with hypercapnia has an additive effect on pHi, 3) partially inhibits NHE, and 4) directly affects excitability of CO2/H+-chemosensitive SC neurons independently of pHi changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction. hyperoxic hyperventilation; O2 toxicity; pH regulation; brain stem; reactive oxygen species 相似文献
7.
Cowley EA Sellers MC Illsley NP 《American journal of physiology. Cell physiology》2005,288(4):C891-C898
Resting or basal intracellular pH (pHi) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO3) or 7.24 ± 0.03 (with HCO3). Ion substitution and inhibitor experiments were performed to determine whether common H+-transporting species were operating to maintain basal pHi. Removal of extracellular Na+ or Cl or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) had no effect. Acidification with the K+/H+ exchanger nigericin reduced pHi to 6.25 ± 0.15 (without HCO3) or 6.53 ± 0.10 (with HCO3). In the presence of extracellular Na+, recovery to basal pHi was prompt and occurred at similar rates in the absence and presence of HCO3. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pHi after acidification. Recovery was inhibited by removal of Na+ or addition of amiloride, whereas removal of Cl and addition of H2DIDS were ineffective. Addition of the Na+/H+ exchanger monensin to cells that had returned to basal pHi elicited a further increase in pHi to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pHi approached the basal level, despite the continued presence of a driving force for H+ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pHi is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na+/H+ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein 相似文献
8.
Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH(2)-terminal cytoplasmic domain 总被引:3,自引:0,他引:3
Stewart AK Chernova MN Kunes YZ Alper SL 《American journal of physiology. Cell physiology》2001,281(4):C1344-C1354
The role ofintracellular pH (pHi) in regulation of AE2 function inXenopus oocytes remains unclear. We therefore compared AE2-mediated 36Cl efflux fromXenopus oocytes during imposed variation of extracellular pH(pHo) or variation of pHi at constantpHo. Wild-type AE2-mediated 36Clefflux displayed a steep pHo vs. activity curve, withpHo(50) = 6.91 ± 0.04. SequentialNH2-terminal deletion of amino acid residues in tworegions, between amino acids 328 and 347 or between amino acids 391 and510, shifted pHo(50) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocytepHi at constant pHo and were shown to beneither substrates nor inhibitors of AE2-mediated Cltransport. At constant pHo, AE2 was inhibited byintracellular acidification and activated by intracellularalkalinization. Our data define structure-function relationships withinthe AE2 NH2-terminal cytoplasmic domain, which demonstratesdistinct structural requirements for AE2 regulation by intracellularand extracellular protons. 相似文献
9.
Choi Inyeong; Romero Michael F.; Khandoudi Nassirah; Bril Antoine; Boron Walter F. 《American journal of physiology. Cell physiology》1999,276(3):C576
Our group recentlycloned the electrogenicNa+-HCO3cotransporter (NBC) from salamander kidney and later from mammaliankidney. Here we report cloning an NBC isoform (hhNBC) from a humanheart cDNA library. hhNBC is identical to human renal NBC (hkNBC),except for the amino terminus, where the first 85 amino acids in hhNBCreplace the first 41 amino acids of hkNBC. About 50% of the amino acidresidues in this unique amino terminus are charged, compared with~22% for the corresponding 41 residues in hkNBC. Northern blotanalysis, with the use of the unique 5' fragment of hhNBC as aprobe, shows strong expression in pancreas and expression in heart andbrain, although at much lower levels. InXenopus oocytes expressing hhNBC,adding 1.5% CO2/10 mMHCO3 hyperpolarizes the membrane andcauses a rapid fall in intracellular pH(pHi), followed by apHi recovery. Subsequent removalof Na+ causes a depolarization anda reduced rate of pHi recovery.Removal of Cl from the bathdoes not affect the pHi recovery.The stilbene derivative DIDS (200 µM) greatly reduces thehyperpolarization caused by addingCO2/HCO3.In oocytes expressing hkNBC, the effects of addingCO2/HCO3and then removing Na+ were similarto those observed in oocytes expressing hhNBC. We conclude that hhNBCis an electrogenicNa+-HCO3cotransporter and that hkNBC is also electrogenic. 相似文献
10.
Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA 总被引:1,自引:0,他引:1
Loiselle FB Morgan PE Alvarez BV Casey JR 《American journal of physiology. Cell physiology》2004,286(6):C1423-C1433
Human NBC3 is an electroneutral Na+/HCO3 cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO3 metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO2 + H2O HCO3 + H+ in many tissues. We investigated whether NBC3, like some Cl/HCO3 exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with Kd = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 ± 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 µM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pHi) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pHi recovery rate by 69 ± 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [-32P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO3 transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct. pH regulation; bicarbonate transport; metabolon 相似文献
11.
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
12.
Wenzel Uwe; Diehl Daniela; Herget Martina; Daniel Hannelore 《American journal of physiology. Cell physiology》1998,275(6):C1573
The reabsorption of filtered di- andtripeptides as well as certain peptide mimetics from the tubular lumeninto renal epithelial cells is mediated by anH+-coupledhigh-affinity transport process. Here we demonstrate for the first timeH+-coupled uptake of dipeptidesinto the renal proximal tubule cell lineLLC-PK1. Transport was assessed1) by uptake studies using theradiolabeled dipeptideD-[3H]Phe-L-Ala,2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and3) by measurement of intracellularpH (pHi) changes as aconsequence of H+-coupleddipeptide transport. Uptake ofD-Phe-L-Alaincreased linearly over 11 days postconfluency and showed all thecharacteristics of the kidney cortex high-affinity peptide transporter,e.g., a pH optimum for transport ofD-Phe-L-Alaof 6.0, an apparent Km value forinflux of 25.8 ± 3.6 µM, and affinities of differently chargeddipeptides or the -lactam antibiotic cefadroxil to the binding sitein the range of 20-80 µM.pHi measurements established thepeptide transporter to induce pronounced intracellular acidification inLLC-PK1 cells and confirm itspostulated role as a cellular acid loader. 相似文献
13.
Miyata Y Muto S Yanagiba S Asano Y 《American journal of physiology. Cell physiology》2000,278(6):C1218-C1229
To examine theeffect of hyperosmolality on Na+/H+ exchanger(NHE) activity in mesangial cells (MCs), we used apH-sensitive dye,2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pHi) in a single MC from ratglomeruli. All the experiments were performed inCO2/HCO3-free HEPESsolutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH2O) treated with mannitol caused cellalkalinization. The hyperosmolality-induced cell alkalinization wasinhibited by 100 µM ethylisopropylamiloride, a specific NHEinhibitor, and was dependent on extracellular Na+. Thehyperosmolality shifted the Na+-dependent acid extrusionrate vs. pHi by 0.15-0.3 pH units in thealkaline direction. Removal of extracellular Cl byreplacement with gluconate completely abolished the rate of cellalkalinization induced by hyperosmolality and inhibited the Na+-dependent acid extrusion rate, whereas, under isosmoticconditions, it caused no effect on Na+-dependentpHi recovery rate or Na+-dependent acidextrusion rate. The Cl-dependent cell alkalinizationrate under hyperosmotic conditions was partially inhibited bypretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS,and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acidextrusion rate via NHE is greater under hyperosmotic conditions thanunder isosmotic conditions at a wide range of pHi,3) the NHE activation under hyperosmotic conditions, but notunder isosmotic conditions, requires extracellularCl, and 4) theCl-dependent NHE activation under hyperosmoticconditions partly occurs via Cl channel andmicrotubule-dependent processes. 相似文献
14.
Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0 总被引:1,自引:0,他引:1
During maturation of oocytes,Cl conductance (GCl) oscillatesand intracellular pH (pHi) increases. ElevatingpHi permits the protein synthesis essential to maturation.To examine whether changes in GCl andpHi are coupled, the Cl channel ClC-0 washeterologously expressed. Overexpressing ClC-0 elevatespHi, decreases intracellular Cl concentration([Cl]i), and reduces volume. Acuteacidification with butyrate does not activate acid extrusion inClC-0-expressing or control oocytes. The ClC-0-induced pHichange increases after overnight incubation at extracellular pH 8.5 butis unaltered after incubation at extracellular pH 6.5. Membranedepolarization did not change pHi. In contrast, hyperpolarization elevates pHi. Thus neither membranedepolarization nor acute activation of acid extrusion accounts for theClC-0-dependent alkalinization. Overnight incubation in lowextracellular Cl concentration increases pHiand decreases [Cl]i in control and ClC-0expressing oocytes, with the effect greater in the latter. Incubationin hypotonic, low extracellular Cl solutions preventedpHi elevation, although the decrease in[Cl]i persisted. Taken together, ourobservations suggest that KCl loss leads to oocyte shrinkage, whichtransiently activates acid extrusion. In conclusion, expressing ClC-0in oocytes increases pHi and decreases[Cl]i. These parameters are coupled viashrinkage activation of proton extrusion. Normal, cyclical changes ofoocyte GCl may exert an effect onpHi via shrinkage, thus inducing meiotic maturation. 相似文献
15.
Fraire-Zamora JJ González-Martínez MT 《American journal of physiology. Cell physiology》2004,287(6):C1688-C1696
Human sperm are endowed with putative voltage-dependent calcium channels (VDCC) that produce measurable increases in intracellular calcium concentration ([Ca2+]i) in response to membrane depolarization with potassium. These channels are blocked by nickel, inactivate in 12 min in calcium-deprived medium, and are remarkably stimulated by NH4Cl, suggesting a role for intracellular pH (pHi). In a previous work, we showed that calcium permeability through these channels increases approximately onefold during in vitro "capacitation," a calcium-dependent process that sperm require to fertilize eggs. In this work, we have determined the pHi dependence of sperm VDCC. Simultaneous depolarization and pHi alkalinization with NH4Cl induced an [Ca2+]i increase that depended on the amount of NH4Cl added. VDCC stimulation as a function of pHi showed a sigmoid curve in the 6.67.2 pHi range, with a half-maximum stimulation at pH 7.00. At higher pHi (7.3), a further stimulation occurred. Calcium release from internal stores did not contribute to the stimulating effect of pHi because the [Ca2+]i increase induced by progesterone, which opens a calcium permeability pathway that does not involve gating of VDCC, was unaffected by ammonium. The ratio of pHi-stimulated-to-nonstimulated calcium influx was nearly constant at different test depolarization values. Likewise, depolarization-induced calcium influx in pHi-stimulated and nonstimulated cells was equally blocked by nickel. In our capacitating conditions pHi increased 0.11 pH units, suggesting that the calcium influx stimulation observed during sperm capacitation might be partially caused by pHi alkalinization. Additionally, a calcium permeability pathway triggered exclusively by pHi alkalinization was detected. mammalian sperm; capacitation; intracellular calcium 相似文献
16.
Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport 总被引:1,自引:0,他引:1
Zeiske Wolfgang; Smets Ilse; Ameloot Marcel; Steels Paul; van Driessche Willy 《American journal of physiology. Cell physiology》1999,277(3):C469
We report, for the epithelialNa+ channel (ENaC) in A6 cells,the modulation by cell pH (pHc)of the transepithelial Na+ current(INa), thecurrent through the individual Na+channel (i), the openNa+ channel density(No), and thekinetic parameters of the relationship betweenINa and theapical Na+ concentration. Thei andNo were evaluatedfrom the Lorentzian INa noise inducedby the apical Na+ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pHc shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH4Cl pulses orbasolateral arrest of theNa+/H+exchanger (Na+ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pHc were positivelycorrelated to changes inINa and theapically dominated transepithelial conductance. The sole pHc-sensitive parameter underlyingINa wasNo. Only thesaturation value of theINa kinetics wassubject to changes in pHc.pHc-dependent changes inNo may be causedby influencingPo, the ENaC openprobability, or/and the total channel number,NT = No/Po. 相似文献
17.
Pathways for HCO3− transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl−---HCO3− exchange was assessed directly by 36Cl− tracer flux measurements and indirectly by determinants of acridine orange absorbance changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3− concentration gradient (pHo 6/pHi 7.5) stimulated Cl− uptake compared to Cl− uptake under 100% N2 in the presence of a pH gradient alone. Mediated exchange of Cl− for HCO3 was suggested by the HCO3− gradient-induced concentrative accumulation of intravesicular Cl−. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO3− gradient-driven Cl− uptake further suggesting chemical as opposed to electrical Cl−HCO3− exchange coupling. Although basolateral membrane vesicle Cl− uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl conductive pathway served to distinguish this mode of Cl− translocation from HCO3 gradient-driven Cl− uptake. No evidence for
cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pHo 7.5/pHi 6), a HCO3− dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl− concentration gradient. The basolateral membrane vesicle origin of the observed Cl−−HCO3− exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl− on HCO3− gradient-driven Na+ uptake suggesting a basolateral membrane Na+−HCO3− for Cl− exchange mechanism, no effect of Na+ on Cl−HCO3− exchange was observed in the present study. 相似文献
18.
Anderson SE Kirkland DM Beyschau A Cala PM 《American journal of physiology. Cell physiology》2005,288(1):C57-C64
Evidence suggests that 1) ischemia-reperfusion injury is due largely to cytosolic Ca2+ accumulation resulting from functional coupling of Na+/Ca2+ exchange (NCE) with stimulated Na+/H+ exchange (NHE1) and 2) 17-estradiol (E2) stimulates release of NO, which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na+ and therefore Ca2+ accumulation, thereby limiting ischemia-reperfusion injury. NMR was used to measure cytosolic pH (pHi), Na+ (Na), and calcium concentration ([Ca2+]i) in Krebs-Henseleit (KH)-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia-reperfusion was 20 min of baseline perfusion, 40 min of global ischemia, and 40 min of reperfusion. The E2 protocol was identical, except that 1 nM E2 was included in the perfusate before ischemia and during reperfusion. E2 significantly limited the changes in pHi, Na and [Ca2+]i during ischemia (P < 0.05). In control OVX vs. OVX+E2, pHi fell from 6.93 ± 0.03 to 5.98 ± 0.04 vs. 6.96 ± 0.04 to 6.68 ± 0.07; Na rose from 25 ± 6 to 109 ± 14 meq/kg dry wt vs. 25 ± 1 to 76 ± 3; [Ca2+]i changed from 365 ± 69 to 1,248 ± 180 nM vs. 293 ± 66 to 202 ± 64 nM. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 µM N-nitro-L-arginine methyl ester. Thus the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca2+]i is greater than can be accounted for by the thermodynamic effect of reduced Na accumulation on NCE. myocardial ischemia; Na+/H+ exchange; Na+/Ca2+ exchange; nuclear magnetic resonance; ischemic biology; ion channels/membrane transport; transplantation 相似文献
19.
Maglova Lilia M.; Crowe William E.; Altamirano Anibal A.; Russell John M. 《American journal of physiology. Cell physiology》1998,275(2):C515
The effects ofhuman cytomegalovirus (HCMV) infection onCl/HCO3exchanger activity in human lung fibroblasts (MRC-5 cells) were studiedusing fluorescent, ion-sensitive dyes. The intracellular pH(pHi) of mock- and HCMV-infectedcells bathed in a solution containing 5%CO2-25 mMHCO3 were nearly the same. However,replacement of external Clwith gluconate caused anH2DIDS-inhibitable (100 µM)increase in the pHi ofHCMV-infected cells but not in mock-infected cells. Continuous exposureto hyperosmotic external media containing CO2/HCO3caused the pHi of both cell typesto increase. The pHi remainedelevated in mock-infected cells. However, in HCMV-infected cells, thepHi peaked and then recoveredtoward control values. This pHirecovery phase was completely blocked by 100 µMH2DIDS. In the presence ofCO2/HCO3, there was an H2DIDS-sensitivecomponent of net Cl efflux(external Cl wassubstituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl (in the nominal absenceofCO2/HCO3),the H2DIDS-sensitive netCl efflux was much greaterfrom HCMV- than from mock-infected cells. In mock-infected cells,H2DIDS-sensitive, netCl efflux decreased aspHi increased, whereas forHCMV-infected cells, efflux increased aspHi increased. All these resultsare consistent with an HCMV-induced enhancement ofCl/HCO3exchanger activity. 相似文献
20.
Aqueous humorsecretion is in part linked to transport by nonpigmented ciliary epithelium (NPE) cells. During thisprocess, the cells must maintain stable cytoplasmic pH(pHi). Because a recent reportsuggests that NPE cells have a plasma membrane-localized vacuolarH+-ATPase, the present study wasconducted to examine whether vacuolar H+-ATPase contributes topHi regulation in a rabbit NPEcell line. Western blot confirmed vacuolarH+-ATPase expression as judged byH+-ATPase 31-kDa immunoreactivepolypeptide in both cultured NPE and native ciliary epithelium.pHi was measured using2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF).Exposing cultured NPE to K+-richsolution caused a pHi increase weinterpret as depolarization-induced alkalinization. Alkalinization wasalso caused by ouabain or BaCl2. Bafilomycin A1 (0.1 µM; aninhibitor of vacuolar H+-ATPase)inhibited the pHi increase causedby high K+. ThepHi increase was also inhibited byangiotensin II and the metabolic uncoupler carbonyl cyanidem-chlorophenylhydazone but not by ZnCl2,4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid(SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), omeprazole, low-Clmedium, -free medium, orNa+-free medium. BafilomycinA1 slowed thepHi increase after an NH4Cl (10 mM) prepulse. However,no detectable pHi change was observed in cells exposed to bafilomycinA1 under control conditions. Thesestudies suggest that vacuolarH+-ATPase is activated bycytoplasmic acidification and by reduction of the protonelectrochemical gradient across the plasma membrane. We speculate thatthe mechanism might contribute to maintenance of acid-base balance inNPE. 相似文献