共查询到20条相似文献,搜索用时 745 毫秒
1.
Activation of Na+ /H+ exchange on rat preadipocyte plasma membrane and its role in cell proliferation and differentiation 总被引:1,自引:0,他引:1
An in vitro cultured rat perirenal preadipocyte (PA) was established as a model system to investigate the role of the intracellular pH (pHi) and of the Na~ /H~ exchanger during PA proliferation and differentiation, pH sensitive probe, 2' ,7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), was employed to measure the pHi of PA and to determine the Na~ /H~ exchange activity. The results showed that there was Na~ /H~ exchange activity in the plasma membrane of PA, FCS stimulated DNA synthesis measured by ~3H-TdR incorporation, and the activation of Na~ /H~ exchanger resulted in phi increase (nearly 0.2 pH unit) within 2 min. Ethyl-isopropyl-amiloride (EIPA), a specific Na~ /H~ exchange inhibitor, inhibited Na~ /H~ exchange activity and DNA synthesis. In the absence of serum insulin did not stimulate DNA synthesis but did induce PA differentiation characterized by the appearance of adiposome in the cell and the enhancement of glycerol-3-phosphate dehydrogenase (G_3PDHase) activity. Meantime, insu 相似文献
2.
We have characterized a Na+/H+ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced
enzyme secretion. ZG Na+/H+ exchanger activity was estimated by measuring Na+ or Li+ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was
investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na+- or Li+-dependent ZG lysis was enhanced by increasing inward to outward directed H+ gradients. Na+-dependent ZG lysis was not prevented by an inside-positive K+ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na+ and H+ permeabilities and for coupled Na+/H+ exchange through an electroneutral carrier. Na+- and Li+-dependent ZG lysis was inhibited by EIPA (EC50∼25 μm) and benzamil (EC50∼100 μm), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into
the medium were obtained with Na+ and Li+ salts only, and were inhibited by EIPA, suggesting the presence of a Na+/H+ exchanger in the membrane. Na+ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-γ-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline
phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 μm), trifluoperazine (100 μm) and W-7 (500 μm), suggesting that the ZG Na+/H+ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na+ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic
acini and was higher in acini incubated in Na+ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 ± 0.4% of total amylase above basal) compared to buffer without Na+ (0 mm NaCl/135 mm KCl buffer; 4.7 ± 0.4% of total amylase above basal, P < 0.03). EIPA (50 μm) reduced CCK-OP-induced amylase secretion in Na+ containing buffer from 7.5 ± 0.6% to 4.1 ± 0.8% (P < 0.02). In the absence of Na+ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 μm EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na+/H+ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced
enzyme secretion from rat pancreatic acini.
Received: 7 December 1995/Revised: 2 April 1996 相似文献
3.
Intracellular myocardial Na+ overload during ischemia is an important cause of reperfusion injury via reversed Na+/Ca2+ exchange. Prevention of this Na+ overload can be accomplished by blocking the different Na+ influx routes. In this study the effect of ischemic inhibition of the Na+/H+ exchanger (NHE) on [Na+]i, pHi and post-ischemic contractile recovery was tested, using three different NHE-blockers: EIPA, cariporide and eniporide. pHi and [Na+]i were measured using simultaneous 31P and 23Na NMR spectroscopy, respectively, in paced (5 Hz) isolated, Langendorff perfused rat hearts while contractility was assessed by an intraventricular balloon. NHE-blockers (3 M) were administered during 5 min prior to 30 min of global ischemia followed by 30 min drug-free reperfusion. NHE blockade markedly reduced ischemic Na+ overload; after 30 min of ischemia [Na+]i had increased to 293 ± 26, 212 ± 6, 157 ± 5 and 146 ± 6% of baseline values in untreated and EIPA (p < 0.01 vs. untreated), cariporide (p < 0.01 vs. untreated) and eniporide (p < 0.01 vs. untreated) treated hearts, respectively. Ischemic acidosis did not differ significantly between groups. During reperfusion, however, recovery of pHi was significantly delayed in treated hearts. The rate pressure product recovered to 12.0 ± 1.9, 12.1 ± 2.1, 19.5 ± 2.8 and 20.4 ± 2.5 × 103 mmHg/min in untreated and EIPA, cariporide (p < 0.01 vs. untreated) and eniporide (p < 0.01 vs. untreated) treated hearts, respectively. In conclusion, blocking the NHE reduced ischemic Na+ overload and improved post-ischemic contractile recovery. EIPA, however, was less effective and exhibited more side effects than cariporide and eniporide in the concentrations used. 相似文献
4.
Danuta Krayer-Pawlowska Corinna Helmle-Kolb Marshall H. Montrose Reto Krapf Heini Murer 《The Journal of membrane biology》1991,120(2):173-183
Summary The present study describes a new perfusion technique—based on the use of a routine spectrofluorometer—which enables fluorometric evaluation of polarity, regulation and kinetics of Na+/H+ exchange at the level of an intact monolayer. Na+/ H+ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na+/H+ exchange activity was measured by monitoring changes in intracellular pH (pH
i
) after an acid load, using the pH-sensitive dye 27-bis (carboxyethyl) 5–6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pH
i
, recovery mechanism, which is Na+ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pH
i
=7.68; pH
o
=7.4) where Na+/H+ exchange is not detectable, transport rate increased as pH
i
decreased. A positive cooperativity characterized the interaction of internal H+ with the exchanger, and suggests multiple H+ binding sites. In contrast, extracellular [Na+] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na+ was 19mM at an intracellular pH of 7.1 and 60mM at an intracellular pH of 6.6. Inhibition of Na+/H+ exchange activity by EIPA was competitive with respect to extracellular [Na+] and theK
i
was 3.4 M. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pH
i
and processes associated with their polarized expression and regulation. 相似文献
5.
K. van Norren R. Gorissen F. Borgese J.M.P.M. Borggreven J.J.H.H.M. De Pont 《The Journal of membrane biology》1997,160(3):193-199
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
6.
Summary Rabbit erythrocytes are well known for possessing highly active Na+/Na+ and Na+/H+ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na+ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na+/Na+ exchange activity, determined as the Na
o
+
-dependent22Na efflux or Na
i
+
-induced22Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H
i
+
-induced Na+ entry sensitive to amiloride (Na+/H+ exchange activity). Similarly, EIPA, a strong inhibitor of the Na+/H+ exchanger, did not inhibit Na+/Na– countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na+/Na+ exchanger could be involved in Na+/H+ countertransport was suggested by studies in which the net Na+ transport sensitive to NEM was determined. As expected, net Na+ transport through this transport system was zero at different [Na+]
i
/[Na+]
o
ratios when intracellular pH was 7.2. However, at pH
i
=6.1, net Na+ influx occurred when [Na+]
i
was lower than 39mm. Valinomycin, which at low [K+]
o
was lower than 39mm. Valinomycin, which at low [K+]
o
clamps the membrane potential close to the K+ equilibrium potential, did not affect the net NEM-sensitive Na+ entry but markedly stimulated, the EIPA-and NEM-resistant Na+ uptake. This suggest that the net Na+ entry through the NEM-sensitive pathway at low pH
i
, is mediated by an electroneutral process possibly involving Na+/H+ exchange. In contrast, the EIPA-sensitive Na+/H+ exchanger is not involved in Na+/Na+ countertransport, because Na+ transport through this mechanism is not affected by an increase in cell Na– from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na+/Na+ and Na+/H+ exchangers, are mediated by distinct transport proteins. 相似文献
7.
Summary Suspensions of OK cells (a continuous renal epithelial cell line originating from the opossum kidney) were examined by flow cytometry. Three parameters were evaluated simultaneously; cell integrity as assayed by propidium iodide fluorescence, cell size as measured by time-of-flight, and intracellular pH as measured by fluorescence of 2,7-bis-(2-carboxyethyl)-5,6 carboxyfluorescein (BCECF). The suspension was shown to be composed of both intact singlets and doublets of cells, and no difference was noted in the behavior of these two populations with respect to the resting intracellular pH, or of the response of intracellular BCECF to changes in pH. Evidence suggests that using NH4 prepulses to create an acid load broadens the intracellular pH distribution. The population of OK cells demonstrates a recovery from this acid load which is very homogeneous with respect to its sensitivity to Na+ removal or EIPA (ethylisopropyl-amiloride), suggesting that virtually all cells utilize Na+/H+ exchange for this recovery. The data also suggest heterogeneity in the cellular pH recovery from an acid load with respect to the observed rates of Na+/H+ exchange. Despite this heterogeneity, the Na+/H+ exchanger is observed to focus the resting intracellular pH of the population to approximately pH 7.4–7.5. The response of the population to PTH suggests that the majority of cells respond to the hormone, and that the total Na+/H+ exchange in individual cells is only partially inhibited even in the presence of saturating PTH concentrations. 相似文献
8.
Liu L Schlesinger PH Slack NM Friedman PA Blair HC 《Journal of cellular physiology》2011,226(6):1702-1712
Osteoblasts synthesize bone in polarized groups of cells sealed by tight junctions. Large amounts of acid are produced as bone mineral is precipitated. We addressed the mechanism by which cells manage this acid load by measuring intracellular pH (pHi) in non‐transformed osteoblasts in response to weak acid or bicarbonate loading. Basal pHi in mineralizing osteoblasts was ~7.3 and decreased by ~1.4 units upon replacing extracellular Na+ with N‐methyl‐D ‐glucamine. Loading with 40 mM acetic or propionic acids, in normal extracellular Na+, caused only mild cytosolic acidification. In contrast, in Na+‐free solutions, weak acids reduced pHi dramatically. After Na+ reintroduction, pHi recovered rapidly, in keeping with Na+/H+ exchanger (NHE) activity. Sodium‐dependent pHi recovery from weak acid loading was inhibited by amiloride with the Ki consistent with NHEs. NHE1 and NHE6 were expressed strongly, and expression was upregulated highly, by mineralization, in human osteoblasts. Antibody labeling of mouse bone showed NHE1 on basolateral surfaces of all osteoblasts. NHE6 occurred on basolateral surfaces of osteoblasts mainly in areas of mineralization. Conversely, elevated HCO alkalinized osteoblasts, and pH recovered in medium containing Cl?, with or without Na+, in keeping with Na+‐independent Cl?/HCO exchange. The exchanger AE2 also occurred on the basolateral surface of osteoblasts, consistent with Cl?/HCO exchange for elimination of metabolic carbonate. Overexpression of NHE6 or knockdown of NHE1 in MG63 human osteosarcoma cells confirmed roles of NHE1 and NHE6 in maintaining pHi. We conclude that in mineralizing osteoblasts, slightly basic basal pHi is maintained, and external acid load is dissipated, by high‐capacity Na+/H+ exchange via NHE1 and NHE6. J. Cell. Physiol. 226: 1702–1712, 2011. © 2010 Wiley‐Liss, Inc. 相似文献
9.
N. C. Jørgensen 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1995,165(6):450-457
Simultaneous net uptake of Na+ and net extrusion of H+, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na+ and H+ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na+ and H+ with an exchange ratio of 1:1 Na+/H+ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO2/HCO
3
-
-buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na+.Abbreviations
DIDS
4,4-diisothiocyanatostilbene-2,2-disulphonate
-
DMSO
dimethylsulphoxide
-
IU
international unit
-
pH
e
extracellular pH
-
pH
i
intracellular pH
-
RBC
red blood cell 相似文献
10.
Summary Ehrlich ascites tumor cells undergoing regulatory volume decrease (RVD) exhibit cytoplasmic acidification as measured by an intracellular fluorescent pH indicator. The acidification results in an activation of the Na+/H+ exchanger. The intracellular pH set point for the activation is estimated to be around 7.0. The activation of the Na+/H+ exchanger leads to an incomplete RVD. In support of this conclusion, amiloride and Na+-free medium, known to limit the Na+/H+ exchange, indeed enhance the RVD response. Intracellular acidification and activation of Na+/H+ exchange may be a general response of cells undergoing RVD. 相似文献
11.
We used 31P NMR to investigate the temperature-dependence of intracellular pH (pH
i
) in isolated frog skeletal muscles. We found that ln[H+
i
] is a linear function of 1/T
abs paralleling those of neutral water (i.e., H+= OH−) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected
by inhibition of glycolysis and was not dependent upon the pH or [Na+] in the bathing solution. Insulin stimulation of Na+-H+ exchange shifted the intercept in the alkaline direction but had no effect on the slope. Acid loading followed by washout
resulted in an amiloride-sensitive return to the (temperature dependent) basal pH
i
.
These results show that the temperature dependence of activation of Na+-H+ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H+]/[OH−] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point
of the Na+-H+ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (ΔH°) under both control and insulin-stimulated
conditions that is similar to the ΔH° of the intracellular buffers and approximately half of the ΔH° for the dissociation
of water. Thus, the temperature-dependent component of the standard free-energy change (ΔF°) is unaffected by insulin stimulation,
suggesting that changes in Arrhenius activation energy (E
a
) may not be a part of the mechanism of hormone stimulation.
Received: 12 February 1997/Revised: 1 October 1997 相似文献
12.
Valeria Casavola Lorenzo Guerra Corinna Helmle-Kolb Stephan J. Reshkin Heini Murer 《The Journal of membrane biology》1992,130(2):105-114
Summary We have analyzed the mechanism of Na+-dependent pHi; recovery from an acid load in A6 cells (an amphibian distal nephron cell line) by using the intracellular pH indicator 27-bis(2-carboxyethyl)5, 6 carboxyfluorescein (BCECF) and single cell microspectrofluorometry. A6 cells were found to express Na+/H+-exchange activity only on the basolateral membrane: Na+/H+-exchange activity follows simple saturation kinetics with an apparent K
mfor Na+ of approximately 11 mm; it is inhibited in a competitive manner by ethylisopropylamiloride (EIPA). This Na+/H+-exchange activity is inhibited by pharmacological activation of protein kinase A (PKA) as well as of protein kinase C (PKC). Addition of arginine vasopressin (AVP) either at low (subnanomolar) or at high (micromolar) concentrations inhibits Na+/H+-exchange activity; AVP stimulates IP3 production at low concentrations, whereas much higher concentrations are required to stimualte cAMP formation. These findings suggest that in A6 cells (i) Na+/H+-exchange is located in the basolateral membrane and (ii) PKC activation (heralded by IP3 turnover) is likely to be the mediator of AVP action at low AVP concentrations.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation, and the Geigy Jubiläumsstiftung. Prof. Dr. V. Casavola and Dr. R. Guerra were supported by a research grant, No. 91.02470.CT14 of the Consiglio Nazionale della Ricerche (C.N.R.) We are grateful to Prof. Dr. B.C. Rossier of the Institute of Pharmacology of Lausanne (Switzerland) for the gift of the A6 cells, to H.P. Gaeggeler for the supply of the necessary culture media and to Jutka Forgo for her excellent help in the day-to-day culturing of the A6 cells. The secretarial assistance of D. Rossi is gratefully acknowledged. 相似文献
13.
S.F. Pedersen B. Kramhøft N.K. Jørgensen E.K. Hoffmann 《The Journal of membrane biology》1996,149(2):141-159
Amiloride-sensitive, Na+-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells.
This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel
increase in the amiloride-sensitive unidirectional Na+ influx is also observed. This indicates that hypertonic challenge activates a Na+/H+ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge,
and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes
in cellular pH
i
, but the overall picture confirms the results from cell suspensions.
Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells.
Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an
inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization.
Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of
intracellular free calcium with BAPTA.
At 25 mm HCO−
3, but not in nominally HCO−
3-free medium, Na+/H+ exchange contributes significantly to regulatory volume increase in Ehrlich cells.
Under isotonic conditions, the Na+/H+ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage.
Received: 2 March 1995/Revised: 29 September 1995 相似文献
14.
Transport of Na+ in isolated erythrocytes of the frog Rana ridibunda was studied using radioactive isotope 22
22Na. Treatment of erythrocytes with -adrenergic agonist isoproterenol (ISP) or with a combination of ISP and phosphodiesterase blocker 3-isobutyl-methyl-xanthine (IBMX) did not affect the Na+ transport into the cells. These data indicated that cAMP-dependent protein kinase A did not participate in regulation of the Na+ transport into the frog erythrocytes. Incubation of erythrocytes with protein kinase C activator phorbol ester (PMA, 0.15 µM) led to a pronounced increase of 22
22Na accumulation and intracellular Na+ concentration. These changes of the Na+ transport into the cells were completely blocked in the presence of 50 µM ethyl-isopropyl-amiloride (EIPA), a selective blocker of the NHE1-isoform of Na+/H+ exchanger. Hence, PMA produced activation of Na+/H+ exchange in frog erythrocytes. The unidirectional Na+ influx into erythrocytes amounted, on average, to 0.99 ± 0.12 and 147 ± 9 mmol/l cells/h for control and PMA-treated cells, respectively. The EIPA concentration producing a 50% inhibition of the PMA-induced Na+ influx (IC50) was 0.28 µM. A high sensitivity of the frog Na/H exchanger to EIPA indicates its similarity with the mammalian NHE1 isoform. The obtained data for the first time clearly indicate an important role of PKC in Na/H exchange regulation in the frog red blood cells. 相似文献
15.
Aporn Bualuang Kanteera Soontharapirakkul Aran Incharoensakdi 《Journal of applied phycology》2010,22(2):123-129
The activity of Na+/H+ exchanger to remove toxic Na+ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na+/H+ exchange activity since exogenously added Na+ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K
m (Na+) and V
max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H+ min−1 mg−1, respectively. For cells grown under salt-stress condition, the apparent K
m (Na+) and V
max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H+ min−1 mg−1, respectively. Three cations with decreasing efficiency namely Li+, Ca2+, and K+ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg2+. The exchange activity was strongly inhibited by Na+-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na+/H+ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na+/H+ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na+/H+ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na+/H+ exchange activity during 15 days of growth. 相似文献
16.
Nicolas Demaurex Robert R. Romanek John Orlowski Sergio Grinstein 《The Journal of general physiology》1997,109(2):117-128
We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using
the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na+-driven pHi recovery
from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably
transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP
perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of
the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange
even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also
known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger,
this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this
enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated
by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as
ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that
the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS
were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted
for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal
activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of
ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis
and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter. 相似文献
17.
Poliovirus-induced intracellular alkalinization involves a proton ATPase and protein phosphorylation
We reported previously that poliovirus infection induces alkalinization in HeLa cells and that an alkaline intracellular pH (pHi) promoted viral replication. Additional experiments were carried out to understand the underlying mechanism. Virus-infected or control monolayer cultures were incubated with nominally bicarbonate-free Eagle's minimal essential medium (MEM) buffered with N-2-hydroxyethylpiperazine-N-3-ethanesulfonic acid (HEPES), and immediately following preincubations, changes in pHi were monitored via benzoic acid uptake around 2 h postinfection. The absence of pH increase in cells infected with ultraviolet light-inactivated virus (UV-virus) indicated that viral gene expression was required for this effect. On the other hand, lack of effect of 3 mM guanidine, an inhibitor of poliovirus-specific RNA but not protein synthesis, suggested that translation of input viral genome RNA is sufficient for the pH increase. Activation of Na+/H+ exchange, Cl?HCO?3 exchange, or H+-ATPase was considered as possible mechanisms by which alkalinization occurs in virus-infected cells. Na+/H+ exchange was excluded because the pH effect occurred in a Na+/H+ exchange deficient HeLa cell mutant. Similarly, Cl?/HCO?3 exchange was excluded because virus-specific alkalinization was evident in the presence of Cl? or bicarbonate deficient medium and was not associated with an increase in HCO?3 uptake or a decrease in Cl? uptake. Lack of dependence on Na+, abrogation by 10 μM 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), and resistance to 1 mM vandate suggested that this effect was due to the activation of a vacuolar-type (V) proton ATPase. Studies using protein kinase inhibitors indicated that activation of the ATPase in virus-infected cells probably involved protein kinase C-mediated phosphorylation. © 1993 Wiley-Liss, Inc. 相似文献
18.
(Na+,K+)-ATPase is able to catalyze a continuous ATP?Pi exchange in the presence of Na+ and in the absence of a transmembrane ionic gradient. At pH 7.6 the Na+ concentration required for half-maximal activity is 85 mM and at pH 5.1 it is 340 mM. In the presence of optimal Na+ concentration, the rate of exchange is maximal at pH 6.0 and varies with ADP and Pi concentration in the assay medium. ATP?Pi exchange is inhibited by K+ and by ouabain. 相似文献
19.
D. Claveau I. Pellerin M. Leclerc M.G. Brunette 《The Journal of membrane biology》1998,165(3):265-274
In the rabbit as well as the rat, a Na+/H+ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform
derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal
membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined
the ethylpropylamiloride (EIPA)-sensitive 22Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 μm EIPA in the membrane suspension decreased the 15 sec Na+ uptake to 75.70 ± 4.70% and 50.30 ± 2.23% of the control values in vesicles from proximal and distal tubules, respectively.
The effect of EIPA on 35 mm Na+ uptake was concentration dependent, with a IC50 of 700 μm and 75 μm for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine
and clonidine up to a concentration of 2 mm, the 35 mm Na+ uptake by the distal membrane was strongly inhibited by cimetidine (IC50 700 μm) and modestly inhibited by clonidine (IC50 1.6 mm).
The incubation of proximal tubule suspensions with 1 mm (Bu2) cAMP decreased the 15-sec EIPA-sensitive Na+ uptake by the brush border membranes to 24.1 ± 2.38% of the control values. Unexpectedly, the same treatment of distal tubules
enhanced this uptake by 46.5 ± 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 ± 3.04% and 79.7 ± 3.21% of the control values
in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane
exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs
from that of the proximal tubule and probably corresponds to isoform 1.
Received: 6 March 1998/Revised: 6 July 1998 相似文献
20.
The present study is designed to investigate the role of Na+-H+ exchanger in the cardioprotective effect of ischaemic and angiotensin (Ang II) preconditioning. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Left ventricular developed pressure (LVDP) and dp/dt were recorded to evaluate myocardial contractility. Four episodes of ischaemic or Ang II preconditioning markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a Na+-H+ exchange inhibitor, produced no marked effect on ischaemic preconditioning and Ang II preconditioning induced cardioprotection. On the other hand, EIPA administration prior to global ischaemia produced a similar reduction in myocardial injury as was noted with ischaemic preconditioning or Ang II preconditioning. On the basis of these results, it may be concluded that inhibition of Na+-H+ exchanger protects against ischaemia-reperfusion induced myocardial injury whereas activation of Na+-H+ exchanger may not mediate the cardioprotective effect of ischaemic and Ang II preconditioning. 相似文献