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21.
The kinetic properties and inhibitor sensitivity of the Na+-H+ exchange activity present in the inner membrane of rat heart and liver mitochondria were studied. (1) Na+-induced H+ efflux from mitochondria followed Michaelis-Menten kinetics. In heart mitochondria, the Km for Na+ was 24 +/- 4 mM and the Vmax was 4.5 +/- 1.4 nmol H+/mg protein per s (n = 6). Basically similar values were obtained in liver mitochondria (Km = 31 +/- 2 mM, Vmax = 5.3 +/- 0.2 nmol H+/mg protein per s, n = 4). (2) Li+ proved to be a substrate (Km = 5.9 mM, Vmax = 2.3 nmol H+/mg protein per s) and a potent competitive inhibitor with respect to Na+ (Ki approximately 0.7 mM). (3) External H+ inhibited the mitochondrial Na+-H+ exchange competitively. (4) Two benzamil derivatives of amiloride, 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil and 3',5'-bis(trifluoromethyl)benzamil were effective inhibitors of the mitochondrial Na+-H+ exchange (50% inhibition was attained by approx. 60 microM in the presence of 15 mM Na+). (5) Three 5-amino analogues of amiloride, which are very strong Na+-H+ exchange blockers on the plasma membrane, exerted only weak inhibitory activity on the mitochondrial Na+-H+ exchange. (6) The results indicate that the mitochondrial and the plasma membrane antiporters represent distinct molecular entities. 相似文献
22.
Intracellular pH changes of cultured bovine aortic endothelial cells in response to ATP addition 总被引:3,自引:0,他引:3
T Kitazono K Takeshige E J Cragoe S Minakami 《Biochemical and biophysical research communications》1988,152(3):1304-1309
The changes of the intracellular pH (pHi) of cultured bovine aortic endothelial cells were fluorometrically monitored using 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). A biphasic pHi change was observed by addition of ATP: an initial acidification followed by an alkalinization of about 0.2 pH unit above the resting level of pHi 7.23. The alkalinization was dependent on [Na+]o and [H+]o, and was inhibited by 5-(N,N-hexamethylene)amiloride, indicating that the alkalinization is mediated by the Na+/H+ exchanger. The 50% effective concentration of ATP was about 1.4 microM. ADP similarly induced pHi changes, whereas AMP and adenosine were inactive. The pHi changes induced by ATP were dependent on the extracellular Ca2+, and the addition of calcium ionophore A23187 induced similar pHi changes. The results indicate that ATP activates the Na+/H+ exchanger in cultured bovine aortic endothelial cells and the activation is mediated by the P2-purinergic receptor and is dependent on the extracellular Ca2+. 相似文献
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The intracellular pH of murine splenic B lymphocytes was measured using the pH-sensitive fluorescent dye, bis(carboxyethyl)carboxyfluorescein (BCECF). After stimulation of B lymphocytes with anti-immunoglobulin antibody plus cytochalasin D, two agents that act in synergy to promote S-phase entry, a late increase in pH was detected that occurred prior to the onset of DNA synthesis. The degree of alkalinization observed was comparable to that produced by two additional mitogenic regimens. Cytoplasmic alkalinization was not blocked by dimethylamiloride. Cytoplasmic alkalinization represents a sign of, and may play a role in, stimulation of B lymphocytes to enter S phase. 相似文献
26.
The influence of calcium, sodium, and the Na+/Ca2+ antiport on susceptibility to cytolysin/perforin-mediated cytolysis 总被引:1,自引:0,他引:1
R P Kraut D Bose E J Cragoe A H Greenberg 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(9):3498-3505
In NK cell-mediated cytolysis, the degranulation of the NK cell, and the binding and polymerization of its effector molecule cytolysin/perforin, are Ca2+ dependent. To determine if increases in the target cell free intracellular Ca2+ concentration ([Ca2+]i) are also important for cytotoxicity, we examined the effects of Ca2+ substitutes, promotors of Ca2+ influx; and inhibitors of Ca2+ efflux on the cytolysis mediated by purified cytolysin. Ca2+ promoted cytolysin-mediated cytolysis but could also inactivate cytolysin on preincubation in the absence of target cells. Ba2+ and Sr2+ could not alone promote cytolysin-mediated cytolysis but did enhance Ca2(+)-driven cytolysis. Preincubation with Ba2+ and Sr2+ did not inactivate cytolysin. One interpretation which these results suggested was that neither Ba2+ nor Sr2+ were involved in cytolysin polymerization and pore formation, but could act intracellularly as Ca2+ analogs once pore formation had occurred. The synergistic interaction of cytolysin and the Ca2+ ionophore A23187 further supported a role for increased [Ca2+]i in cytolysis. Inhibitors of Na+/Ca2+ exchange, namely low Na+ medium, ouabain, and the amiloride analogs 2',4'-dimethylbenzamil, 5-(N-4-chlorobenzyl)-2',4'dimethylbenzamil, and alpha-phenylbenzamil all markedly enhanced cytolysin-mediated cytolysis. These results support the hypothesis that the Ca2+ dependency of NK cell- and cytolysin-mediated cytolysis is related to increases in target cell [Ca2+]i. Furthermore, they indicate that Na+/Ca2+ exchange is an important counterlytic mechanism. 相似文献
27.
G B Mills E J Cragoe E W Gelfand S Grinstein 《The Journal of biological chemistry》1985,260(23):12500-12507
In several cell types, proliferation initiated by growth factors is associated with a rapid increase in cytoplasmic pH (pHi). This cytoplasmic alkalinization is due to the activation of an amiloride-sensitive Na+/H+ antiport. It is unclear whether growth factor-induced activation of the antiport or the resultant increase in pHi is the trigger for proliferation, an obligatory requirement for proliferation, or simply an associated phenomenon. Interleukin 2 (IL 2) acts as a growth factor for mitogen or antigen-stimulated thymus-derived (T) lymphocytes. In this study, we established that IL 2 produces an increase in pHi and determined whether this increase in pHi plays a role in the proliferative response to IL 2. Monitoring pHi with an intracellularly trapped, pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, we demonstrated that IL 2 rapidly (less than 90 s) initiates an increase in pHi in IL 2-sensitive human and murine T cells. Because intracellular alkalinization requires extracellular Na+ and is amiloride-sensitive, it likely occurs through activation of the Na+/H+ antiport. Using partitioning of a weak acid, 5,5-dimethyl-2,4-oxazolidinedione, we confirmed that the IL 2-dependent increase in pHi is sustained for several hours and returns to near base-line levels by 18 h. We also investigated the consequence of preventing Na+/H+ exchange on the proliferative response induced by IL 2. IL 2-driven proliferation occurred in nominally bicarbonate-free medium in the presence of concentrations of amiloride analogs sufficient to inhibit the Na+/H+ antiport and prevent intracellular alkalinization. These data suggest that although the antiport is activated by binding of IL 2 to its receptor, intracellular alkalinization is not essential for IL 2-dependent proliferation. It seems unlikely that either cytoplasmic alkalinization or activation of the Na+/H+ antiport are triggers for T cell proliferation. 相似文献
28.
Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol
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S P Soltoff M K McMillian E J Cragoe L C Cantley B R Talamo 《The Journal of general physiology》1990,95(2):319-346
The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
29.
B Vanhaesebroeck E J Cragoe J Pouysségur R Beyaert F Van Roy W Fiers 《FEBS letters》1990,260(2):319-322
Cytotoxicity of tumor necrosis factor (TNF) on L929s cells was efficiently blocked by several amiloride analogs but not by amiloride itself. This protection did not require RNA or protein synthesis. Na+/H+ antiporter-negative L-M(TK-) cells (LAP) could be killed by TNF, showing that the Na+/H+ exchanger is not required for TNF-cytotoxicity. Similar protection against TNF-mediated cell lysis by amiloride derivatives was found for LAP and L929s cells, excluding a blockade of the Na+/H+ antiporter as the cause of the protection against TNF by these agents. 相似文献
30.
A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils 总被引:2,自引:0,他引:2
A Na+/Ca2+ exchange mechanism has been recently described in human neutrophils that constitutes the principal pathway for Ca2+ influx into resting cells. The potential role of this system in regulating the respiratory burst in response to activation by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine was explored. In the presence of 1 mM Ca2+, a variety of di- and trivalent cations suppressed the generation of O(-2) radicals in a series of decreasing efficacy: La3+ approximately Zn2+ much greater than Sr2+ approximately Cd2+ greater than Ba2+ greater than Co2+ greater than Ni2+ approximately Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na+/Ca2+ counter-transport. Benzamil, phenamil, and 2',4'-dichlorobenzamil, analogues of amiloride which selectively block Na+/Ca2+ exchange in neutrophils, likewise suppressed the release of O(-2) with apparent Ki values of approximately 30 microM. The effect of the cations was competitive with Ca2+, while the interaction between the benzamil derivatives and Ca2+ appeared to be noncompetitive in nature. Both the divalent cations and benzamil also inhibited the rise in cytoplasmic Ca2+ as monitored by fura-2 fluorescence: these agents reduced peak cytosolic Ca2+ levels after N-formyl-methionyl-leucyl-phenylalanine stimulation to values seen in the absence of extracellular Ca2+. These results are compatible with the hypothesis that the influx of Ca2+ via Na+/Ca2+ exchange contributes to the transient elevation in intracellular free Ca2+. The polyvalent cations block the entry of critical Ca2+ ions by competing with Ca2+ for binding to the translocation site on the exchange carrier, while benzamil acts by lowering the maximal transport rate. These studies emphasize that Na+/Ca2+ exchange through its effects on cytoplasmic Ca2+ plays a major regulatory role in activation of the respiratory burst in chemotactic factor-stimulated neutrophils. 相似文献