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1.
Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na+ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na+ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K+ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I50) of Na+/H+ exchange and K+ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I50 for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na+/H+ exchange by these drugs. 相似文献
2.
The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive 86Rb uptake and amiloride-sensitive 24Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na+/H+ and Na+/K+ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na+/H+ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na+/H+ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na+ pump, Na+/H+ exchange) without eliciting corresponding regulatory mechanisms (Na+ stat, pH stat). 相似文献
3.
H+ extrusion by the isolated skins of two amphibia, Rana ridibunda and Bufo bufo was studied in order to test for the presence of exchange mechanisms of the type Na+/H+ and Cl?/HCO3?, which have been described in several epithelial structures. The preparations were mounted in chambers of the Ussing type, so that the short-circuit current could be used as a function of Na+ transport and the pH-stat technique was utilized to determine the rates of H+ extrusion under different experimental conditions. These conditions were either the withdrawal of the ions intervening in the mentioned exchanges (Cl- or Na+, or the addition of drugs with well-known effects on Na+ uptake and transport (antidiuretic hormone and amiloride).In the frog skin, H+ excretion was detected in solutions containing either Cl? or SO42?, with identical rates. Again, Na+ substitution by Mg2+ had no effect on H+ excretion rates, neither did the suppression of Na+ influx by amiloride or its stimulation by antidiuretic hormone. These experiments were repeated with similar results in gland-free preparations of the epidermis of frog skin separated from the corion by the action of collagenase.Experiments in toad skin showed that H+ excretion could not be detected when Cl? was present in the outer medium, but became apparent if an impermeant anion, SO42?, was used. This observation is compatible with the existence of an exchange mechanism of the type Cl?/HCO3?. Secondly, in these preparations H+ extrusion increased after stimulation with antidiuretic hormone and decreased when amiloride was used or when Na+ was substituted by Mg2+, suggesting that at least a fraction of the total H+ efflux is linked to Na+ influx. In the isolated frog skin this mechanism does not seem to be operative. 相似文献
4.
Na+ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na+ uptake was assayed by the rapid filtration technique in the presence and the absence of an uphill pH gradient. Amiloride strongly decreased Na+ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na+ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na+ uptake was lower but still sensitive to amiloride. The Lineweaver-Burk plot of Na+ uptake consistently showed a single kinetics. Increasing the pH gradient decreased Km values of the amiloride-sensitive Na+ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive Na+ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na+. Since ethylisopropylamiloride, the specific Na/H exchanger inhibitor mimicked the effects of amiloride, decreasing by 98% the 10 mM Na+ uptake, whereas benzamil, the Na+ channel blocker, had no effect, it was concluded that the amiloride sensitive Na+ uptake was predominantly or exclusively due to a Na+-H+ exchanger activity. K+ in trans-position significantly decreased the amiloride sensitive uptake. In contrast, the presence of the cation in cis-position had no effect. The amiloride resistant Na+ transport was neither influenced by pH, nor saturable. Incubation of the placental tissue with 100 μM or 1 mM dibutyryl cAMP, 0.1 or 1 μM phorbol myristate acetate, 10−7 M insulin, 10−10 M angiotensin II, or 10−8 M human parathyroid hormone (PTH) did not influence Na+ transport by subsequently prepared brush border membranes. Finally, we failed to demonstrate any Na+-H+ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of co-substrates such as phosphate and aminoacids, the Na+-H+ exchange is probably the unique mechanism of Na+ transport by the placental brush border membrane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the isoform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane. © 1996 Wiley-Liss, Inc. 相似文献
5.
T. I. Ivanova A. O. Sherstobitov G. P. Gusev 《Journal of Evolutionary Biochemistry and Physiology》2001,37(5):528-534
To study H+ transport, the lamprey red blood cells were acidified to pH 6.0 by a pretreatment with an ionophore, nigericin. Incubation of the acidified cells in NaCl-medium at pH 8.0 was accompanied by a rapid H+ efflux from the erythrocytes. There was a tenfold decrease of the H+ efflux rate on addition to NaCl-medium of dimethylamiloride or on replacing Na+ in the medium (KCl-medium, pH 8.0). A high rate of Na+ influx into the acidified erythrocytes occurred only in the presence of H+ gradient (pH medium 8.0), but not in its absence (pH medium 6.0). The Na+-dependent H+ efflux from the cells and H+-dependent Na+ influx into the cells were quantitatively similar (about 700 mmol/l cells/h). A rapid elevation of the intracellular Na+ concentration as measured by flame photometry was also observed during incubation of the acidified cells in NaCl-medium (pH 8.0). The H+-dependent Na+ influx and an increase of the Na+ content in the acidified cells were significantly inhibited by amiloride. The data obtained for the first time prove with certainty the presence of the Na+/H+ exchanger in erythrocytes of the river lamprey. 相似文献
6.
M.Z. Abedin D.I.N. Giurgiu Z.R. Abedin E.A. Peck X. Su P.R. Smith 《The Journal of membrane biology》2001,182(2):123-134
Gallbladder Na+ absorption is linked to gallstone formation in prairie dogs. Na+/H+ exchange (NHE) is one of the major Na+ absorptive pathways in gallbladder. In this study, we measured gallbladder Na+/H+ exchange and characterized the NHE isoforms expressed in prairie dogs. Na+/H+ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na+ flux and apical 22Na+ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J
Na
ms was higher than J
Na
sm with J
Na
net absorption. Mucosal DMA inhibited transepithelial Na+ flux in a dose-dependent fashion, causing J
Na
ms equal to J
Na
sm and blocking J
Na
net absorption at 100 μm. Basal 22Na+ uptake rate was 10.9 ± 1.0 μmol · cm−2· hr−1 which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression
of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na+/H+ exchange accounts for a substantial fraction of gallbladder apical Na+ entry and most of net Na+ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na+ uptake and transepithelial Na+ absorption.
Received: 9 February 2001/Revised: 11 April 2001 相似文献
7.
Summary Exposure of Ehrlich cells to isotonic Na+-propionate medium induces a rapid cell swelling. This treatment is likely to impose an acid load on the cells. Cell swelling is absent in K+-propionate medium but may be induced by the ionophore nigericin, which mediates K+/H+ exchange. Cell swelling in Na+-propionate medium is blocked by amiloride, but an alternative pathway is introduced by addition of the ionophore monensin, which mediates Na+/H+ exchange. Consequently, swelling of Ehrlich cells in Na+-propionate medium is due to the operation of an amiloride-sensitive, Na+-specific mechanism. It is concluded that this mechanism is a Na+/H+ exchange system, activated by cytoplasmic acidification. We have previously demonstrated that the heavy metal salt CuSO4 in micromolar concentrations inhibits regulatory volume decrease (RVD) of Ehrlich cells following hypotonic swelling. The present work shows that CuSO4 inhibits RVD as a result of a net uptake of sodium, of which the major part is sensitive to amiloride. Measurements of intracellular pH show that CuSO4 causes significant cytoplasmic alkalinization, which is abolished by amiloride. Concomitantly, CuSO4 causes an amiloride-sensitive net proton efflux from the cells. The combined results confirm that a Na+/H+ exchange system exists in Ehrlich cells and demonstrate that the heavy metal salt CuSO4 activates this Na+/H+ exchange system. 相似文献
8.
The cytoplasmic pH undergoes a biphasic change when neutrophils are activated. The role of Ca2+ in initiating these changes was investigated. No correlation was found between the increased cytosolic [Ca2+] and the stimulation of the Na+/H+ antiport. Similarly, the cytoplasmic acidification elicited by activation in Na+-free media was found to be unrelated to [Ca2+]. Reversal of Na+/H+ exchange was also ruled out as the source of the acidification. Data using a variety of soluble activators indicate that metabolic acid generation is largely responsible for the observed drop in cytoplasmic pH. 相似文献
9.
Summary We have investigated the kinetic properties of the human red blood cell Na+/H+ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H+ (H
i
) and external Na+ (Na
o
) in red blood cells of normal subjects.Red blood cells with different cell Na+ (Na
i
) and pH (pH
i
) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na+ influx were measured by varying pH
i
(from 5.7 to 7.4), external pH (pH
o
), Na
i
and Na
o
and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na+ influx (Na
i
<2.0 mmol/liter cell, Na
o
= 150mm) increased sigmoidally (Hill coefficient 2.5) when pH
i
fell below 7.0 and the external pH
o
was 8.0, but increased linearly at pH
o
6.0. The net Na+ influx driven by an outward H+ gradient was estimated from the difference of Na+ influx at the two pH
o
levels (pH
o
8 and pH
o
6). The H+-driven Na+ influx reached saturation between pH
i
5.9 and 6.1. TheV
max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK
m
for H
i
to activate H+-driven Na+ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na+/H+ exchange. The stoichiometric ratio between H+-driven Na+ influx and Na+-driven H+ efflux was 11. The dependence of Na+ influx from Na
o
was studied at pH
i
6.0, and Na
i
lower than 2 mmol/liter cell at pH
o
6.0 and 8.0. The meanK
m
for Na
o
of the H+-gradient-driven Na+ influx was 55±7mm.An increase in Na
i
from 2 to 20 mmol/liter cell did not change significantly H+-driven net Na+ influx as estimated from the difference between unidirectional22Na influx and efflux. Na+/Na+ exchange was negligible in acid-loaded, DIDS-treated cells. Na+ and H+ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na+ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na+/H+ exchanger.It is concluded that human red cell Na+/H+ exchange performs 11 exchange of external Na+ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H+ and external Na+ is similar to other cells, but it displays a larger variability in theV
max between individuals. 相似文献
10.
Ken-ichi Yano Toru Maruyama Naoki Makino Hirosuke Matsui Takashi Yanaga 《Molecular and cellular biochemistry》1993,121(1):75-83
Although many causal factors have been proposed for the ischemia-reperfusion injury, the exact mechanisms for interdependent derangements of mechanical, electrical and metabolic events remains unclear. For this purpose, the Langendorff-perfused rat hearts were subjected to regional brief ischemia followed by reperfusion to study the protective effects of amiloride, an inhibitor of Na+–H+ exchange. Amiloride (0.1 mM) attenuated the rise in tissue Na+ and Ca2+, both duration and incidence of arrhythmias (p<0.05 vs. control), sarcolemmal injury (assessed by Na–K ATPase) and lipid peroxidation (assessed by malonedialdehyde formation) during reperfusion. Treatment of hearts with monensin, a sodium inophore, reversed the protective effects of amiloride. Reduction in transsarcolemmal Na+ and pH gradients during ischemia exhibited protective effects similar to those seen with amiloride. These results suggest that cardiac dysfunction, sarcolemmal injury and triggered arrhythmias during ischemia-reperfusion are due to the occurrence of intracellular Ca2+ overload caused by the activation of Na+–H+ exchange and Na+–Ca2+ exchange systems in the myocardium. 相似文献
11.
The Na+ uptake into neuroblastoma x glioma hybrid cells was measured in Hepes-buffered EMEM containing 10% calf serum and 5 mM ouabain in the presence and absence of amiloride (1.0 mM). Amiloride was found to markedly inhibit net Na+ influx (by approximately 50%). Examination of the effect of amiloride on net Na+ influx in the absence of calf serum revealed that a significant amiloride-sensitive Na+ influx remains even under serum-deprived conditions, although the degree of amiloride inhibition (35%) is substantially lower than that found in the presence of serum. The amiloride-insensitive portion of Na+ influx was found to be independent of serum effects. Estimation of resting membrane potential was made by measurement of the steady state distribution of the lipophilic cation, TPP+, in the presence and absence of amiloride. A large, immediate increase in TPP+ uptake, indicative of a membrane hyperpolarization, was seen upon addition of amiloride. Determination of the effect of amiloride on resting membrane potential of serum-deprived cells showed that cells are hyperpolarized to a greater extent in the presence than in the absense of amiloride, and that serum exerts a depolarizing effect on the cells. Thus, serum-stimulation of Na+ influx results in a depolarization of resting membrane potential, while amiloride inhibition of Na+ influx causes a hyperpolarization. These data strongly suggest that NG108-15 cells possess an electrogenic Na+ influx pathway that is sensitive to amiloride inhibition and enhanced by serum. 相似文献
12.
The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na+/H+ or Li+/H+ exchange was demonstrated by diluting Na2SO4 or Li2SO4 loaded vesicles into Na+- or Li+-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K+ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na+ conductance smaller than electroneutral Na+/H+ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na+ conductance greater than the H+ conductance was suggested. K+ gradients also induced changes of pH, at about 10% of the Na+ or Li+ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K+ was a low affinity substrate for the Na+/H+ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K+ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K+ conductance. Inward Cl? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl? conductance greater than the H+ conductance and a Cl?/OH? exchange were present. The rate of Na+/H+ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent Km for Na+ or Li+ of about 10 mM and an EA of 14.3 kcal per mol. A 61-fold Na2SO4 gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H+ and Cl? pathways could alter the effects of the Na+/H+ exchange. 相似文献
13.
We have studied the movements of H+ from the in vitro frog skin into the outside solution because it has been suggested that the movement of sodium from the outside solution into the skin may result from the forced exchange of Na+ by H+.Our main observations can be summarized as follows: (a) Hydrogen moves from the skin into the outside solution at a rate of 0.04 μequiv · cm?2 · h?1 while Na+ influx had a value of 0.49 μequiv · cm?2 · h?1. (b) The rate of H+ secretion is not significantly affected by substituting the Na+ in the outside solution by K+ nor by inhibiting Na+ influx with amiloride (5 · 10?5 M). (c) Acetazolamide (5 · 10?3 M) blocked H+ secretion without altering the potential difference across the skin. (d) The rate of H+ production is not underestimated because it may have been neutralized by HCO3? secreted into the outside solution in exchange for Cl?. Substituting all the Cl? by SO42? in the outside solutions does not result in an increase in the rate of H+ production. (e) The steady-state rate of H+ secretion is not affected by large changes in electrochemical potential gradients for H+. Neither abolishing the potential difference across the skin nor a 10-fold change in H+ concentration in the outside solution affected significantly the steady-state rate of H+ secretion. (f) The H+ secretion was abolished by the metabolic inhibitors dinitrophenol (1 · 10?4 M) and Antimycin A (1.5 · 10?6 M) which also markedly reduced the potential difference across the skin.Observations (a), (b), and (c) suggest that H+ and Na+ movements across the outer border of the isolated frog skin are not coupled. The ratio of Na+ to H+ movements is very different from unity and Na+ movements can be abolished without any effects on H+ secretion and conversely H+ movements can be abolished without interruption of Na+ uptake.A second conclusion suggested by these results is that the H+ secretion does not result from movement of H+ following its electrochemical potential gradient since that rate of secretion is not affected by marked changes in either potential or [H+]. Furthermore, the effects of metabolic inhibitors suggest that H+ secretion requires the expenditure of energy by the cell. 相似文献
14.
Maria Ereciska David Nelson Fiorenzo Dagani Judith Deas† Ian A. Silver† 《Journal of neurochemistry》1993,61(4):1356-1368
Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na+]i., [K+]i, and [Ca2+]i, and pH under conditions when [H+]i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K+ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca2+], and [Na+]., The increase in [Na+], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K+ and subsequent depolarization lead to an increase in Na+ influx and stimulation of the Na+/K+ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H+] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K+], depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia. 相似文献
15.
Based on sequence analysis, the salt overly sensitive (SOS1) gene has been suggested to function as a Na+/H+ antiporter located at the plasma membrane of plant cells, being expressed mostly in the meristem zone of the root and in the parenchyma cells surrounding the vascular tissue of the stem. In this study, we compared net H+ and Ca2+ fluxes and intracellular pH and [Ca2+]cyt in the root meristem zone of Arabidopsis wild‐type (WT) and sos mutants before and after salt stress. In addition, we studied the effect of pretreatment with amiloride (an inhibitor of Na+/H+ antiporters) on net ion fluxes, intracellular pH and intracellular Ca2+ activity ([Ca2+]cyt) in WT plants and sos1 mutants before and after salt stress. Net ion fluxes were measured using microelectrode ion flux estimation (MIFE) and intracellular pH and [Ca2+]cyt using fluorescence lifetime imaging microscopy (FLIM) techniques. During the first 15 min after NaCl application, sos1 mutants showed net H+ efflux and intracellular alkalinization in the meristem zone, whereas sos2 and sos3 mutants and WT showed net H+ influx and slight intracellular acidification in the meristem zone. Treatment with amiloride led to intracellular acidification and lower net H+ flux in WT plants and to a decrease in intracellular Ca2+ in WT and sos1 plants. WT plants pretreated with amiloride did not show positive net H+ flux and intracellular acidification. After NaCl application, internal pH shifted to higher values in WT and sos1 plants. However, absolute values of H+ fluxes were higher and internal pH values were lower in WT plants pretreated with amiloride compared with sos1 mutants. Therefore, the SOS1 transporter is involved in H+ influx into the meristem zone of Arabidopsis roots, or it may function as a Na+/H+ antiporter. Amiloride affects SOS1 and other Na+/H+ antiporters in plant cells because of its ability to decrease the H+ gradient across the plasma membrane. 相似文献
16.
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
17.
Erythrocytes of lamprey Lampetra fluviatilis were incubated in standard isotonic medium at 20°C with 22Na to determine the unidirectional Na+ influx. Cell incubation in the presence of various protein phosphatase inhibitors (NaF, cantharidin, calyculin A) led to a considerable increase of Na+ transport into erythrocytes. The stimulation of Na+ influx into erythrocytes rose with increase of concentration of calyculin A within the range of 10–100 nM. The calyculin A concentration producing a 50% activation of Na+ transport amounted to 41.5 nM. Under optimal experimental conditions, the Na+ influx increased from control level of 5–8 to 20–40 mmol/l cells/h under effect of protein phosphatase blockers. The Na+ transport induced by these inhibitors was completely suppressed on addition of amiloride to the incubation medium. The treatment of lamprey erythrocytes with protein phosphatase inhibitors was accompanied by a small (~12%), but statistically significant decrease of intracellular Na+ content. A small decrease of intracellular K+ content in erythrocyte was observed only under the effect of NaF. The obtained data allow making the conclusion that protein phosphatases of the PP1 and PP2A types play a significant role in regulation of Na+ transport across the lamprey erythrocyte membrane in both directions. 相似文献
18.
A. Bosia D. Ghigo F. Turrini E. Nissani G. P. Pescarmona H. Ginsburg 《Journal of cellular physiology》1993,154(3):527-534
Intraerythrocytic malaria parasites produce vast amounts of lactic acid through glycolysis. While the egress of lactate is very rapid, the mode of extrusion of H+ is not known. The possible involvement of a Na+/H+ antiport in the extrusion of protons across the plasma membrane of Plasmodium falciparum has been investigated by using the fluorescent pH probe 6-carboxyfluorescein. The resting cytosolic pH was 7.27 ± 0.1 in ring stage parasites and 7.31 ± 0.12 in trophozoites. Spontaneous acidification of parasite cytosol was observed in Na+-medium and realkalinization occurred upon addition of Na+ to the medium in a concentration-dependent manner, with no apparent saturation. The rate of H+-at the ring stage was higher than that at the trophozoite stage due to the larger surface/volume ratio of the young parasite stage. Na+-H+-was: 1) inhibited by the Na+/H+ inhibitors amiloride and 5-(N-ethyl-isopropyl) amiloride (EIPA), though at relatively high concentrations; 2) augmented with rising pH6 (pHi = 6.2 [Na+]o = 30 mM); and 3) decreased with increasing pHi (pHo = 7.4; [Na+]o = 30 mM). The pHi and the pHo dependencies of H+-were almost identical at all parasite stages. Only at pHi > 7.6 efflux was totally obliterated. The target of this inhibitory effect is probably other than the antiport. Results indicate that H+-is mediated by a Na+/H+ antiport which is regulated by host and parasite pH and by the host cytosol sodium concentration. The proton transport capacity of the antiport can easily cope with all the protons of lactic acid produced by parasite's glycolysis. © 1993 Wiley-Liss, Inc. 相似文献
19.
The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na+/H+ antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na+/H+ exchange in a competitive manner with a Ki of 2.5 and 5.9 micromolar for ΔpH-dependent 22Na+ influx in tonoplast vesicles and Na+-dependent H+ efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [3H]MIA to tonoplast membranes revealed a high affinity binding component with a Kd of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na+/H+ antiport. Photolabeling of the tonoplast with [3H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog. 相似文献
20.
Summary Suspensions of LLC-PK1 cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na+ and K+ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05±0.01,n=5) of cells which have recovered in (pH 7.4) Na+-containing medium is not affected over several minutes by addition of 100 M amiloride or removal of extracellular Na+ (Na
o
+
/H
i
+
and Na
i
+
/H
o
+
exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na
o
+
/H
i
+
exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H+ efflux or net Na+ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a set point of Na+/H+ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05. 相似文献