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1.
2.
Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H+ gradient (pHin=6.0, pHout=8.0)22Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pHin=pHout=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K 1 =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K+-stimulatedp-nitrophenyl phosphatase. In addition22Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H+ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na+/H+ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.  相似文献   

3.
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.
Basolateral membrane vesicles from rat jejunal enterocytes, especially purified of brush-border contamination, were used for Na+ uptake. The basolateral membrane vesicles are osmotically active and under our experimental conditions Na+ binding is much lower than transport. An outwardly directed proton gradient stimulates Na+ uptake at both 5 microM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH4Cl (NH3 diffusion). Membrane potential effects can be excluded by having valinomycin plus K+ on both sides of the vesicles. These results suggest that there is an Na+/H+ exchanger in the basolateral membrane of rat enterocytes.  相似文献   

5.
Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.  相似文献   

6.
Summary Studies were performed on purified brush-border membranes from the kidney of the rabbit to examine the relation between protein kinase C and the Na+/H+ exchanger in these membranes. The brush-border membranes were transiently opened by exposure to hypotonic media and the membrane proteins phosphorylated by exposure to ATP and phorbol esters or partially purified protein kinase C. The membranes were resealed and the intravesicular space acidified by incubation in a sodium-free isotonic solution (pH 5.5). The rate of uptake of 1mm 22Na+ (pH 7.5), with and without amiloride (1mm), was assayed and the proton gradient-stimulated, amiloride-inhibitable component of22Na+ taken as a measure of the activity of the Na+/H+ exchanger. 12-0-tetradecanoyl phorbol-13-acetate (TPA) increased the amiloride-sensitive component of22Na+ uptake TPA did not affect the amiloride-insensitive component of22Na+ uptake or the equilibrium concentration of sodium. TPA also did not affect the rate of dissipation of the proton gradient in the absence of sodium or the rate of sodium-dependent or-independent uptake ofd-glucose. Other active phorbol esters stimulated the rate of Na+/H+ exchange, but phorbol esters of the 4 configuration did not. Incubation of the opened membranes in partially purified protein kinase C increased the rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake. The stimulatory effect of TPA and protein kinase C was not additive. In the absence of ATP, neither TPA nor protein kinase C affected Na+/H+ exchange transport. To determine the membrane-bound protein substrates, parallel experiments were conducted with -[32P] ATP in the phosphorylating solutions. The reaction was stopped by SDS and the phosphoproteins resolved by PAGE and autoradiography. TPA stimulation of protein kinase C resulted in phosphorylation of approximately 13 membrane-bound proteins ranging in apparent molecule from 15,000 to 140,000 daltons. These studies indicate that activation of endogenous renal brush-border protein kinase C by phorbol esters or exposure of these membranes to exogenous protein kinase C increases the rate of proton gradient-stimulated, amiloride-inhibitable sodium transport. Protein kinase C activation also results in phosphorylation of a finite number of membrane-bound proteins.  相似文献   

7.
Endothelin stimulates Na+/H+ exchange in vascular smooth muscle cells   总被引:2,自引:0,他引:2  
The effect of endothelin (ET) on the intracellular pH (pHi) of vascular smooth muscle cells (VSMC), was investigated using a fluorescent pH indicator 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). ET at concentrations of over 10(-9) M caused dose-dependent transient acidification followed by Na(+)-dependent and amiloride-sensitive alkalization of the cells due to stimulation of Na+/H+ exchange. The alkalization induced by ET was Ca2(+)-dependent and was inhibited by a calcium channel blocker, nicardipine. Pretreatment with H-7, an inhibitor of protein kinase C, also inhibited the ET-induced cell alkalization. These results indicate that ET stimulates Na+/H+ exchange, resulting in alkalization of VSMC and that this ET-induced cell-alkalization is probably linked to Ca2+ influx and activation of protein kinase C.  相似文献   

8.
Summary We have studied the kinetic properties of rabbit red cell (RRBC) Na+/Na+ and Na+/H+ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na+ and H+ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Na i and H l were prepared by nystatin and DIDS treatment of acid-loaded cells. Na+/Na+ EXC was measured as Na o -stimulated Na+ efflux and Na+/H+ EXC as Na o -stimulated H+ efflux and pH o -stimulated Na+ influx into acid-loaded cells.The activation of Na+/Na+ EXC by Na o at pH i 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (K m 2.2 mM) and low affinity (K m 108 mM) sites for a single- or two-carrier system. The activation of Na+/H+ EXC by Na o (pH i 6.6, Na i <1 mM) also showed high (K m 11 mM) and low (K m 248 mM) affinity sites. External H+ competitively inhibited Na+/Na+ EXC at the low affinity Na o site (K H 52 nM) while internally H+ were competitive inhibitors (pK 6.7) at low Na i and allosteric activators (pK 7.0) at high Na i .Na+/H+ EXC was also inhibited by acid pH o and allosterically activated by H i (pK 6.4). We also established the presence of a Na i regulatory site which activates Na+/H+ and Na+/Na+ EXC modifying the affinity for Na o of both pathways. At low Na i , Na+/Na+ EXC was inhibited by acid pH i and Na+/H+ stimulated but at high Na i , Na+/Na+ EXC was stimulated and Na+/H+ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Na o sites,cis-inhibited by external H o , allosterically modified by the binding of H+ to a H i regulatory site and regulated by Na i . These findings are consistent with Na+/Na+ EXC being a mode of operation of the Na+/H+ exchanger.Na+/H+ EXC was partially inhibited (80–100%) by dimethyl-amiloride (DMA) but basal or pH i -stimulated Na+/Na+ EXC (pH i 6.5, Na i 80 mM) was completely insensitive indicating that Na+/Na+ EXC is an amiloride-insensitive component of Na+/H+ EXC. However, Na+ and H+ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA: this also indicates that the Na+/H+ EXC might operate in reverse or uncoupled modes in the absence of Na+/Na+ EXC.In summary, the observed kinetic properties can be explained by a model of Na+/H+ EXC with several conformational states, H i and Na i regulatory sites and loaded/unloaded internal and external transport sites at which Na+ and H+ can compete. The occupancy of the H+ regulatory site induces a conformational change and the occupancy of the Na i regulatory site modulates the flow through both pathways so that it will conduct Na+/H+ and/or Na+/Na+ EXC depending on the ratio of internal Na+:H+.  相似文献   

9.
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   

10.
Superoxide (O2-) increases Na+ reabsorption in the thick ascending limb (THAL) by enhancing Na/K/2Cl cotransport. However, the effects of O2- on other THAL transporters, such as Na(+)/H+ exchangers, are unknown. We hypothesized that O2- stimulates Na(+)/H+ exchange in the THAL. We assessed total Na(+)/H+ exchange activity by measuring recovery of intracellular pH (pH(i)) after acid loading in isolated perfused THALs before and after adding xanthine oxidase (XO) and hypoxanthine (HX). We found that XO and HX decreased total pH(i) recovery rate from 0.26 +/- 0.05 to 0.21 +/- 0.04 pH units/min (P < 0.05), and this net inhibition decreased steady-state pH(i) from 7.52 to 7.37. Because THALs have different Na(+)/H+ exchanger isoforms on the luminal and basolateral membrane, we tested the effects of xanthine oxidase and hypoxanthine on luminal and basolateral Na(+)/H+ exchange by adding dimethylamiloride to either the bath or lumen. Xanthine oxidase and hypoxanthine increased luminal Na(+)/H+ exchange from 3.5 +/- 0.8 to 6.7 +/- 1.4 pmol.min(-1).mm(-1) (P < 0.01) but decreased basolateral Na(+)/H+ exchange from 10.8 +/- 1.8 to 6.8 +/- 1.1 pmol.min(-1).mm(-1) (P < 0.007). To ascertain whether these effects were caused by O2- or H2O2, we examined the ability of tempol, a superoxide dismutase mimetic, to block these effects. In the presence of tempol, xanthine oxidase and hypoxanthine had no effect on luminal or basolateral Na(+)/H+ exchange. We conclude that O2- inhibits basolateral and stimulates luminal Na(+)/H+ exchangers, perhaps because different isoforms are expressed on each membrane. Inhibition of basolateral Na(+)/H+ exchange may enhance stimulation of luminal Na(+)/H+ exchange by providing additional protons to be extruded across the luminal membrane. Together, the effects of O2- on Na(+)/H+ exchange may increase net HCO3- reabsorption by the THAL.  相似文献   

11.
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   

12.
Summary The present studies were designed to test our previous suggestion that Na+/H+ exchange was activated by muscarinic stimulation of rat parotid acinar cells. Consistent with this hypothesis, we demonstrate here that intact rat parotid acini stimulated with the muscarinic agonist carbachol in HCO 3 -free medium show an enhanced recovery from an acute acid load as compared to similarly challenged untreated preparations. Amiloride-sensitive22Na uptake, due to Na+/H+ exchange, was also studied in plasma membrane vesicles prepared from rat parotid acini pretreated with carbachol. This uptake was stimulated twofold relative to that observed in vesicles from control (untreated) acini. This stimulation was time dependent, requiring 15 min of acinar incubation with carbachol to reach completion, and ws blocked by the presence of the muscarinic antagonist atropine (2×10–5 m) in the pretreatment medium. The effect of carbachol was dose dependent withK 0.53×10–6 m. Stimulation of the exchanger was also seen in vesicles prepared from acini pretreated with the -adrenergic agonist epinephrine, but not with the -adrenergic agonist isoproterenol, or with substance P. Kinetic analysis indicated that the stimulation induced by carbachol was due to an alkaline shift in the pH responsiveness of the exchanger in addition to an increasedapparent transport capacity. Taken together with previous results from this and other laboratories, these results strongly suggest that the Na+/H+ exchanger and its regulation are intimately involved in the fluidsecretory response of the rat parotid.  相似文献   

13.
The effects of elevating intracellular cAMP levels on Na+ transport across the apical membrane of Necturus gallbladder epithelium were studied by intracellular and extracellular microelectrode techniques. Intracellular cAMP was raised by serosal addition of the phosphodiesterase inhibitor theophylline (3 mM) or mucosal addition of either 8-Br-cAMP (1 mM) or the adenylate cyclase activator forskolin (10 microM). During elevation of intracellular cAMP, intracellular Na+ activity (alpha Nai) and intracellular pH (pHi) decreased significantly. In addition, acidification of the mucosal solution, which contained either 100 or 10 mM Na+, was inhibited by approximately 50%. The inhibition was independent of the presence of Cl- in the bathing media. The rates of change of alpha Nai upon rapid alterations of mucosal [Na+] from 100 to 10 mM and from 10 to 100 mM were both decreased, and the rate of pHi recovery upon acid loading was also reduced by elevated cAMP levels. Inhibition was approximately 50% for all of these processes. These results indicate that cAMP inhibits apical membrane Na+/H+ exchange. The results of measurements of pHi recovery at 10 and 100 mM mucosal [Na+] and a kinetic analysis of recovery as a function of pHi suggest that the main or sole mechanism of the inhibitory effect of cAMP is a reduction in the maximal rate of acid extrusion. In conjunction with the increase in apical membrane electrodiffusional Cl- permeability, produced by cAMP, which causes a decrease in net Cl- entry (Petersen, K.-U., and L. Reuss, 1983, J. Gen. Physiol., 81:705), inhibition of Na+/H+ exchange contributes to the reduction of fluid absorption elicited by this agent. Similar mechanisms may account for the effects of cAMP in other epithelia with similar transport properties. It is also possible that inhibition of Na+/H+ exchange by cAMP plays a role in the regulation of pHi in other cell types.  相似文献   

14.
The effects of thyrotropin-releasing hormone (TRH) and 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytosolic pH (pHi) were studied on GH4C1 pituitary cells loaded with the fluorescent pH indicator bis(carboxyethyl)carboxyfluorescein (BCECF) and the fluorescent Ca2+ indicator quin2. TRH, which was minimally effective at around 10(-9) M, and TPA, 100 nM, produced very small elevations in pHi of about 0.05 pH units from the normal basal resting pHi of GH4C1 cells of around 7.05. The effects were more marked after acid-loading the cells using 1 micrograms of nigericin/ml. Preincubation with amiloride or replacing the extracellular Na+ with choline+ completely blocked the elevations stimulated by TRH or TPA, consistent with an activation of the Na+/H+ antiport mechanism. The effects were completely independent of the cytoplasmic free calcium concentration ([Ca2+]i). The calcium ionophore ionomycin produced an elevation in [Ca2+]i with no concomitant effect on pHi, and amiloride, although completely inhibiting the pH change stimulated by TRH, failed to affect the initial stimulated [Ca2+]i transient. Although the data are consistent with an elevation in pHi by TRH which is caused by stimulation of a protein kinase C and subsequent activation of the antiporter, the rapidity of the onset of the pHi response to TRH could not be mimicked by a combination of TPA and ionomycin. These results, together with previous findings which show that secretion can be mimicked by TPA and ionomycin, suggest that TRH-stimulated Na+/H+ exchange plays no part in the acute stimulation of secretion, but that TRH increases the pH-sensitivity of the antiport system during increased synthesis of prolactin and growth hormone.  相似文献   

15.
16.
Nerve growth factor (NGF) inhibits transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). To investigate the mechanism of this inhibition, MTALs were perfused in vitro in Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pHi recovery after lumen or bath Na+ addition. NGF (0.7 nM in the bath) had no effect on apical Na+/H+ exchange activity, but inhibited basolateral Na+/H+ exchange activity by 50%. Inhibition of basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) secondarily reduces apical Na+/H+ exchange activity and HCO3- absorption in the MTAL (Good, D. W., George, T., and Watts, B. A., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12525-12529). To determine whether a similar mechanism could explain inhibition of HCO3- absorption by NGF, apical Na+/H+ exchange activity was assessed in physiological solutions (146 mM Na+) by measurement of the initial rate of cell acidification after lumen EIPA addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF inhibited apical Na+/H+ exchange activity. Inhibition of HCO3- absorption by NGF was eliminated in the presence of bath EIPA or in the absence of bath Na+. Also, NGF blocked inhibition of HCO3- absorption by bath EIPA. We conclude that NGF inhibits basolateral Na+/H+ exchange activity in the MTAL, an effect opposite from the stimulation of Na+/H+ exchange by growth factors in other systems. NGF inhibits transepithelial HCO3- absorption through inhibition of basolateral Na+/H+ exchange, most likely as the result of functional coupling in which primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal tubule HCO3- absorption.  相似文献   

17.
Endocytic uptake of [3H]sucrose and lucifer yellow, markers for fluid-phase endocytosis, was studied in cultures of the renal epithelial cell lines LLC-PK1 and OK. Endocytosis in LLC-PK1 cells was inhibited when the cells were grown in the presence of gentamicin (1 mg/ml) for 4 days or when the cells were treated with concanavalin A (1 mg/ml) for 5 h. These changes occurred without perturbation of intracellular Na+ and K+ content, indicating that the cells maintained normal ion gradients. The inhibition of endocytosis was accompanied by marked increases in the apparent Vmax for Na+-dependent cell uptake of solutes such as Pi and L-alanine. The apparent Km was unchanged. In contrast, treatment of OK cells with concanavalin A produced marked stimulation of endocytosis and inhibition of the Na+-dependent uptake of Pi and L-glutamate. These changes occurred in the absence of changes in intracellular Na+ and K+ content. Neither gentamicin nor concanavalin A had a direct effect on Na+/solute cotransport in these cell lines. The changes in Na+/Pi cotransport induced by concanavalin A in both LLC-PK1 and OK cells were blocked by keeping the cells at 4 degrees C during exposure to the lectin, suggesting that endocytosis may be part of the mechanism which mediates the changes in solute uptake. The reciprocal relationship between the changes in endocytosis and the changes in Na+/solute cotransport is consistent with the possibility that the number of Na+/solute cotransporters present in the plasma membrane may be altered by an increase or decrease in the rate of membrane internalization by endocytosis. The Vmax changes in Na+/solute cotransport provide indirect support for this conclusion.  相似文献   

18.
Electrogenic 2 Na+/1 H+ exchange in crustanceans   总被引:4,自引:0,他引:4  
Summary Hepatopancreatic brush border membrane vesicles of the freshwater prawn,Macrobrachium rosenbergii and the marine lobster,Homarus americanus exhibited22Na uptake which was Cl-independent, amiloride sensitive, and stimulated by a transmembrane H gradient (H i >H o ). Sodium influx by vesicles of both species were sigmoidal functions of [Na] o , yielding Hill coefficients that were not significantly different (P>0.5) than 2.0. Estimations of half-saturation constants (K Na) were 82.2mm (prawn) and 280.1mm (lobster), suggesting a possible adaptation of this transporter to environmental salinity.Trans-stimulation andcis-inhibition experiments involving variable [H] suggested that the exchangers in both species possessed single internal cation binding sites (pK 6.5–6.7) and two external cation binding sites (prawn, pK 4.0 and 5.7; lobster pK 3.5 and 6.1). Similarcis inhibition studies using amiloride as a competitive inhibitor of Na uptake supported the occurrence of dual external sites (prawn,K i 50 and 1520 m; lobsterK i 9 and 340 m). Electrogenic Na/H exchange by vesicles from both crustaceans was demonstrated using equilibrium shift experiments where a transmembrane potential was used as the only driving force for the transport event. Transport stoichiometries of the antiporters were determined using Static Head analysis where driving forces for cation transfer were balanced using a 101 Na gradient, a 1001 H gradient, and a stoichiometry of 2.0. These electrogenic 2 Na/1 H exchangers appear thermodynamically capable of generating sufficient gastric acidification for organismic digestive activities.  相似文献   

19.
Na+/H+ exchange in the cyanobacterium Synechococcus 6311   总被引:9,自引:0,他引:9  
The cyanobacterium Synechococcus 6311 adapts to grow in 0.6 M NaCl by developing an efficient system for sodium extrusion. In the present investigation cells loaded with NaC1 were subjected to a large dilution. Changes in fluorescence quenching of acridine orange as a function of transmembrane Na+ gradients provide evidence that Na+/H+ exchange activity greatly enhanced in salt-adapted cells.  相似文献   

20.
Summary LLC-PK1 cells (a continuous epithelioid cell line with renal characteristics) are examined by microspectrofluorometry as single cells, in order to determine the mechanism of intracellular pH (pH i ) recovery from an acid load imposed by ammonium preincubation and removal (NH4 prepulse). Initial experiments evaluate the intracellular K+ levels through a null point analysis of total cellular K+ with flame photometry. The response of BCECF (a pH-sensitive fluorescent dye) is then calibrated, using saturating concentrations of nigericin to cause defined changes in pH i . For experiments with the microspectrofluorometer, LLC-PK1 cells were grown on either glass coverslips or filters (the latter attached to plastic coverslips with a hole under the filter). The cells on glass coverslips demonstrate a Na+-dependent recovery from an (NH4 prepulse) acid load which is sensitive to 1 M ethylisopropylamiloride. They also demonstrate a set point of activation of Na+/H+ exchange. When examined for changes in pH i due to changes in membrane potential, plasma membrane proton conductance could not be detected at resting pH i . Cells grown on filters also demonstrate a pH i recovery from an acid load which is Na+ dependent and ethylisopropylamiloride sensitive, but in this configuration, the majority of cells (22/23 preparations) require Na+ at the basolateral membrane for rapid pH i recovery. The morphology and polarity of the cells grown on permeable supports appears normal at the electron-microscopic level. The results are not affected by changes in cell seeding density or collagen treatment of the filters.  相似文献   

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