共查询到20条相似文献,搜索用时 31 毫秒
1.
S-Adenosyl-L-methionine (AdoMet) has been found to bind specifically to the plasma membrane of promyelocytic leukemia cells, HL-60. The Kd for AdoMet is 4.2.10(-6) M and the Bmax is 4.0.10(-12) mol/10(7) HL-60 cells. The binding is not related to the adenosine receptor since neither adenosine, ADP, nor ATP affect the ligand-receptor reaction. When HL-60 cells were incubated with physiological concentrations of [methyl-3H]AdoMet (20 microM) at 36 degrees C, AdoMet did not equilibrate with the intracellular pool, nor were any [3H]methyl groups incorporated into nucleic acids or proteins. In contrast, significant amounts of [3H]methyl groups were incorporated into membrane phospholipids. When cells were incubated with 20 microM [methyl-3H]AdoMet, [3H]methyl groups were transferred to phosphatidylethanolamine, -monomethylethanolamine, and -dimethylethanolamine yielding phosphatidylcholine. However, the rate of methyl transfer with AdoMet was only 22% of that observed when cells were incubated with a comparable amount of [methyl-3H]methionine. Both the binding of AdoMet and the methylation of phospholipids were inhibited by exogenous S-adenosyl-L-homocysteine. Therefore, the binding may be linked to a phospholipid methyltransferase. 相似文献
2.
Three forms of AdoMet synthetase were separated from sheep liver. The apparent molecular weights of the native isozymes were 122,000, 62,400 and 70,800 for the alpha-, beta 1- and beta 2-form, respectively and beta 1 was the predominant form. The alpha-form exhibited negative cooperativity with [S] 0.5 values of 31 microM for methionine and 62 microM for ATP; while the two beta-forms exhibited positive cooperativity with [S]0.5 values for methionine of 82 microM and 70 microM and those for ATP of 572 microM and 505 microM for the beta 1- and beta 2-form, respectively. Dimethylsulfoxide markedly stimulated the activities of the two beta-forms at low methionine concentrations. However, at high methionine levels, it inhibited the activity of the beta 2-form but not that of the beta 1-form. The effect of dimethylsulfoxide on the alpha-form was not significant. AdoMet was inhibitory at high concentrations. However, it had a slight stimulatory effect on the two beta-forms at low concentrations when methionine level was also low. These results suggest that AdoMet synthetase is a regulatory enzyme and the reaction rate in vivo can be directly influenced by substrate and product concentrations. 相似文献
3.
Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2 总被引:11,自引:0,他引:11
P C Johnson J A Ware P B Cliveden M Smith A M Dvorak E W Salzman 《The Journal of biological chemistry》1985,260(4):2069-2076
The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone. 相似文献
4.
T Shimizu 《Cryobiology》1988,25(1):38-43
Echinocytes appeared in thawed-washed erythrocytes previously frozen with glycerol in response to Ca2+ plus divalent cation ionophore A23187. The occurrence of echinocytes depended on the concentrations of Ca2+. When the incidence of echinocytes was plotted against the log dose of Ca2+, a sigmoidal curve was obtained and fitted the probit plots well. ED50 values for frozen-thawed and fresh erythrocytes were 38 +/- 10 and 40 +/- 9 microM (mean +/- SD) CaCl2, respectively. The effects of divalent cations, such as Co2+, Mn2+, Ba2+, and Mg2+, on echinocyte formation were examined. Increasing Mg2+ concentrations only shifted the dose-response curve parallel to the right side. However, in cryopreserved erythrocytes in the medium with high Mg2+ content, the degree of the shift was less than that of fresh erythrocytes. ED50 values in 2 mM Mg2+ medium for frozen-thawed and fresh erythrocytes were 62 +/- 15 and 130 +/- 12 microM CaCl2, respectively. Under this condition, the lowest adenosine triphosphate (ATP) levels of about 60% of the control level were established at Ca2+ concentrations in cryopreserved erythrocytes lower than those in fresh ones. Then the utilization of cellular ATP decreased with echinocyte formation. These results indicate that futile hydrolysis of ATP in cryopreserved erythrocytes in high Mg2+ media may reduce Ca2+ excretion by activation of the Ca2+ pump in plasma membranes, resulting in echinocyte formation in low Ca2+ concentration. 相似文献
5.
Epidermal growth factor-stimulated DNA synthesis requires an influx of extracellular calcium 总被引:2,自引:0,他引:2
The dependency of normal cell proliferation on adequate extracellular Ca2+ levels was further investigated by determining the role of Ca2+ influx in epidermal growth factor (EGF)-induced rat liver epithelial (T51B) cell DNA synthesis. Fura-2-loaded T51B cells responded with an increase in [Ca2+]i to EGF (5-50 ng/ml) that was blocked by low (25 microM) extracellular Ca2+ or by pretreatment with 50 microM La3+ to inhibit plasma membrane Ca2+ flux. Confluent T51B cells treated for 24 h with EGF (0.1-50 ng/ml) dose-dependently incorporated [3H]-thymidine into cell nuclei. Low extracellular Ca2+ or addition of La3+ prevented the EGF-stimulated rise in labeled nuclei, indicating that a movement of Ca2+ into the cell was required for DNA synthesis. This was supported by our findings that bradykinin, which induced a rise in [Ca2+]i by opening plasma membrane Ca2+ channels in T51B cells (but not A23187, thrombin or ATP, which raise [Ca2+]i primary through mobilization of intracellular Ca2+ stores), potentiated DNA synthesis stimulated by submaximal doses of EGF. Potentiation of the action of EGF by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), indicates that activation of protein kinase C and an influx of Ca2+ share a common mechanism for initiating DNA synthesis. 相似文献
6.
Interactions between endothelin-1 (ET)-induced phosphoinositide (PI) hydrolysis and agents that increase Ca2+ influx (i.e. A23187 and ionomycin) or induce depolarization (i.e. KCl) were investigated using C6 glioma. A23187 dose-dependently potentiated ET (30 nM)- and ATP (100 microM)-induced [3H]inositol phosphate (IP) accumulation. This potentiation was associated with an increase in the maximal stimulation elicited by both ET and ATP but their EC50 values were unchanged. This effect of A23187 occurred at concentrations that did not affect basal PI turnover; i.e. 10 nM-3 microM. Ionomycin within the range of 1 nM-1 microM also significantly enhanced ET-induced PI breakdown and this effect was associated with an increase of [Ca2+]i. KCl in a concentration-dependent manner (14.7-54.7 mM) markedly inhibited PI breakdown elicited by ET and ATP, but had much less inhibition on basal activity and no effect on A23187- and ionomycin-induced responses. In parallel, KCl added before or after ET, sharply attenuated the increase of ET-induced [Ca2+]i but did not affect basal level or ionomycin-induced [Ca2+]i response. Neither the potentiation by A23187 nor the inhibition by KCl of ET-induced PI turnover was observed in cultured cerebellar astrocytes. Our results suggest that the cell type-specific regulation by Ca2+ ionophores and KCl on ET-induced PI metabolism is closely related to perturbation of [Ca2+]i. 相似文献
7.
S C Chow G E Kass M J McCabe S Orrenius 《Archives of biochemistry and biophysics》1992,298(1):143-149
The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry. 相似文献
8.
Free intracellular Mg2+ concentration ([Mg2+]i) was measured in cold-stored human erythrocytes by the method of null-point titration with ionophore A23187. [Mg2+]i was 311 +/- 41 microM (mean +/- S.D.) for cells stored 0-10 days, increasing to 458 +/- 64 microM for cells stored 22-48 days. The values for stored cells were higher than those previously determined by a 31P-NMR method (Bock et al. (1985) Blood 65, 1526-1530); however, the null-point method requires extensive washing of the cells, which we have found to increase NMR-measured [Mg2+]i. The null-point values still represent a small fraction of total cell Mg2+, and confirm that binding of Mg2+ to ligands other than ATP and 2,3-bisphosphoglycerate must increase during storage. As an initial test of whether this may imply suboptimal availability of Mg2+ for cell preservation, we used A23187 to prepare erythrocytes with altered Mg2+ content, then removed ionophore and stored the cells in plasma-free medium for up to 2 weeks. Higher Mg2+ content had a very significant positive correlation (P less than 0.0001) with higher cell ATP concentrations. Storage did not significantly affect basal or Na+-stimulated efflux of Mg2+ from Mg2+-loaded red cells. 相似文献
9.
1, 5-Dihydroxy-2, 3-dimethoxy-xanthone (HM-5) is one of the naturally-occurring xanthones of a Tibetan medicinal herb Halenia elliptica. Recently, it has been shown that HM-5 is one of the phase I metabolites of 1-hydroxy-2, 3, 5-trimethoxy-xanthone (HM-1), the major active component of H. elliptica with potent vasorelaxant actions. This study investigated the vasorelaxant effect of HM-5 and its mechanism(s). HM-5 (0.35-21.9 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 4.40+/-1.08 microM. Unlike HM-1, the effect of HM-5 was endothelial-independent such that removal of the endothelium did not affect its vasodilator potency. Nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME, 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM) did not affect the vasodilatory effects of HM-5, thus confirming the non-involvement of endothelium related mechanisms. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-5 was inhibited by a potassium channel blocker, TEA (10 mM), and 4-aminopyridine (4-AP, a K(v) blocker; 1 mM) but not by other K+ channel blockers such as iberiotoxin (100 nM), barium chloride (100 microM) and glibenclamide (10 microM). The involvement of Ca2+ channel was studied in artery rings pre-incubated with Ca2+-free buffer (intact endothelium or endothelium-denuded) and primed with 1 microM 5-HT or 60 mM KCl prior to the addition of CaCl2 to elicit contraction. In the 5-HT-primed preparations, HM-5 (34.7 microM) significantly inhibited the CaCl(2)-induced vasoconstriction (89.9% inhibition in intact endothelium artery rings; 83.3% inhibition in endothelium-denuded rings). In the KCl-primed preparations, HM-5 (34.7 microM) produced a 34% inhibition in endothelium-denuded rings. The same concentration of HM-5 inhibited (by 62.3%) the contractile response to 10 microM phorbol 12, 13-diacetate (PDA), a protein kinase C activator, in Ca2+-free solutions. Taken together, this study showed that the mechanisms of the vasorelaxant effects of HM-5 were distinctly different from those of its parent drug HM-1. The vasorelaxant effect of HM-5 was mediated through opening of potassium channel (4-AP) and altering intracellular calcium by partial inhibition of Ca2+ influx through L-type voltage-operated Ca2+ channels and intracellular Ca2+ stores. 相似文献
10.
Previous studies have shown that hypertonic mannitol or NaCl increases the release of [3H]arachidonate and immunoreactive prostaglandin E in inner medullary slices incubated in Ca2+-free media containing EGTA. By contrast, the stimulation of these parameters by ionophore A23187 and by arginine-vasopressin are abolished in Ca2+-free media plus EGTA. In the present study, the effects of Ca2+ deprivation and the intracellular Ca2+ antagonist TMB-8 [8-N,N-diethylamino)octyl-3,4,5 -trimethoxybenzoate-HCl) were further examined to assess the Ca2+ dependence of the actions of different stimuli of prostaglandin E synthesis in rat renal inner medulla. Ca2+-free media without EGTA abolished increases in [3H]arachidonate and immunoreactive prostaglandin E release induced by ionophore A23187, but not those induced by arginine-vasopressin, suggesting that different pools of Ca2+ subserve expression of the actions of these two stimuli. At low concentrations, TMB-8 (10-25 microM) inhibited increases in [3H]arachidonate and immunoreactive prostaglandin E release induced by arginine-vasopressin, but did not influence effects of Ca2+ plus ionophore A23187 or hypertonicity on these parameters. At higher concentrations (100-500 microM), TMB-8 suppressed effects of ionophore A23187, hyperosmolar NaCl and mannitol on immunoreactive prostaglandin E and [3H]arachidonate release from slices. The effects of a sub-optimal inhibitory concentration of TMB-8 on ionophore A23187 actions were overcome by increasing Ca2+ in the media from 1.5 to 5 mM. Ca2+ deprivation, or concentrations of EGTA or TMB-8, that were effective in suppressing increases in immunoreactive prostaglandin E induced by ionophore A23187, arginine-vasopressin or hypertonicity, did not modify increases in immunoreactive prostaglandin E induced by exogenous arachidonate. Moreover, in microsomal fractions of inner medulla, TMB-8 suppressed Ca2+-dependent increases in phospholipase A2 and C activities, an effect which was competitive with Ca2+. Thus, Ca2+ deprivation and TMB-8 act at a step in the immunoreactive prostaglandin E synthetic pathway proximal to cyclooxygenase activity, and probably at the level of Ca2+-dependent acyl hydrolase activity. The results with TMB-8 indicate that an intracellular pool of Ca2+ is involved in expression of the actions of hypertonicity to increase [3H]arachidonate release and immunoreactive prostaglandin E in inner medulla. 相似文献
11.
External ATP induces [3H] dopamine [( 3H]DA) release in rat pheochromocytoma cells (PC-12 cells). The ATP-induced release is a saturable process with half-effective concentration of EC50 = 80 microM. ADP is a poor secretagogue of [3H]DA (one-sixth of ATP) and AMP is devoid of secretory capabilities. Adenosine and the non-hydrolyzable analogues of ATP, AppNHp and AppCp are ineffective as inducers of [3H]DA, release, or as inhibitors of the ATP-induced [3H]DA release. The most potent antagonist of ATP-induced release is Coomassie Blue (IC50 = 25 microM), compared to ADP beta S (IC50 = 500 microM). The overall rank order of potency is ATP greater than ADP much greater than AMP greater than adenosine, which is characteristic of the P2-purinergic receptor. ATP-induced secretion is absolutely Ca2+ dependent, indicating an exocytotic process and is independent of Mg2+ (up to 2 mM) suggesting that the active species is not ATP4-. (a) The ATP-induced 45Ca2+ influx into the cells is in good correlation to ATP induction of release (IC50 = 80 and 90 microM, respectively) and is carried over to ADP which has a diminished ability to induce both release and 45Ca2+ influx. (b) Divalent cations (Ba2+ greater than Sr2+ greater than Ln3+ greater than Mn2+) replace Ca2+ and support ATP-induced release similar to their effectiveness in supporting bradykinin- and K+ (50 mM)-induced release in PC-12 cells (Weiss, C., Sela, D., and Atlas, D. (1990) Neurosci. Lett. 119, 241-245). Combined together the absolute requirement of [Ca2+]ex for release, inhibition of release by Gd3+ (IC50 = 100 microM), Ni2+, and Co2+ (IC50 = 1 mM), and support of release by Ba2+, Sr2+, and Mn2+, we suggest that ATP induces Ca2+ entry via ligand-operated Ca2+ channels as previously suggested for ATP in smooth muscle cells (Benham, C.D., Bolton, T.B., Byren, N.G., and Large, W.A. (1987) J. Physiol. (Lond.) 387, 473-488). No significant inhibition by 1 microM verapamil, 10 microM nifedipine, or 2 mM Cd2+ argues against ATP activation of voltage-dependent Ca2+ channels as similarly shown for ATP-induced [3H]noradrenaline release (Inoue, K., Nakazawa, K., Fujimoro, K., and Takanaka, A. (1989) Neurosci. Lett. 106, 294-299). Thus, the widely distributed ATP receptor might play an essential role in Ca2+ homeostasis of the cell by introducing Ca2+ into the cell via specific ligand-gated Ca2+ channels. 相似文献
12.
In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca(2+)-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed. Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca(2+)-free washings, did not have degranulated ER. Quiescent cells incubated with 0.7 mM adenosine 5'-triphosphate (ATP) in Ca(2+)-free HEPES-buffered saline produced very similar ER degranulations. Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment. In the absence of extracellular Ca2+, transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores. Substituting ATP with 1 microM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation. In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation. ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca(2+)-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER. It therefore seems possible for Ca2+ depletion, or at least a reduction, to be causally related to ER degranulation. If that were the case, ER granularity would appear to be a facultative rather than a constitutive state. 相似文献
13.
In rat erythrocytes, the regulation of Na+/Mg2+ antiport by protein kinases (PKs), protein phosphatases (PPs), intracellular Mg2+, ATP and Cl- was investigated. In untreated erythrocytes, Na+/Mg2+ antiport was slightly inhibited by the PK inhibitor staurosporine, slightly stimulated by the PP inhibitor calyculin A and strongly stimulated by vanadate. PMA stimulated Na+/Mg2+ antiport. This effect was completely inhibited by staurosporine and partially inhibited by the PKC inhibitors Ro-31-8425 and BIM I. Participation of other PKs such as PKA, the MAPK cascade, PTK, CK I, CK II, CAM II-K, PI 3-K, and MLCK was excluded by use of inhibitors. Na+/Mg2+ antiport in rat erythrocytes can thus be stimulated by PKCalpha. In non-Mg2+ -loaded erythrocytes, ATP depletion reduced Mg2+ efflux and PMA stimulation in NaCl medium. A drastic activation of Na+/Mg2+ antiport was induced by Mg2+ loading which was not further stimulated by PMA. Staurosporine, Ro-31-8425, BIM I and calyculin A did not inhibit Na+/Mg2+ antiport of Mg2+ -loaded cells. Obviously, at high [Mg2+]i Na+/Mg2+ antiport is maximally stimulated. PKCalpha or PPs are not involved in stimulation by intracellular Mg2+. ATP depletion of Mg2+ -loaded erythrocytes reduced Mg2+ efflux and the affinity of Mg2+ binding sites of the Na+/Mg2+ antiporter to Mg2+. In non-Mg2+ -loaded erythrocytes Na+/Mg2+ antiport essentially depends on Cl-. Mg2+ -loaded erythrocytes were less sensitive to the activation of Na+/Mg2+ antiport by [Cl-]i. 相似文献
14.
15.
Phosphatidylinositol (PtdIns) is the key precursor of phosphoinositide-derived intracellular mediators. The effects of changing the rate of PtdIns synthesis on mitogenic activity of human amnion-derived WISH cells were investigated. Incubation of the cells with [3H]inositol caused a time- and dose-dependent PtdIns labeling. Exogenous Ca2+ inhibited [3H]inositol incorporation in a dose-dependent fashion; half-maximal inhibition occurred with 0.3-1.0 mM Ca2+. In contrast, removal of cytosolic Ca2+ by ionophore A23187 and 1 mM EGTA induced enhancement of the PtdIns labeling as a function of A23187 concentration, perhaps through release of inhibitory effects of endogenous Ca2+. The A23187-stimulated PtdIns labeling with [3H]inositol was not abolished by additional unlabeled inositol, suggesting that [3H]inositol labeling of PtdIns occurred mainly through de novo synthesis catalyzed by PtdIns synthase (EC 2.7.8.11). In cells with PtdIns synthase activity decreased by exogenous Ca2+, [3H]thymidine incorporation was also inhibited, while A23187 caused dose-dependent enhancement of thymidine incorporation. The changes in PtdIns synthase activity occurred in parallel with changes in mitogenic activity caused by increasing the dose of exogenous Ca2+ or A23187. A similar lowering of mitogenic activity was observed upon suppression of PtdIns synthase by pemirolast potassium (9-methyl-3-1H-tetrazol-5yl-4H-pyrido[1,2-a]pyridin-4-one potassium) via a Ca(2+)-independent mechanism. These data demonstrate that changes in PtdIns synthase activity by some agents acting via different mechanisms are associated with parallel changes in thymidine incorporation, and suggest that PtdIns production is tightly coupled to cell proliferation in human amnion cells. 相似文献
16.
Calcium Enhances In Vitro Free Radical-Induced Damage to Brain Synaptosomes, Mitochondria, and Cultured Spinal Cord Neurons 总被引:2,自引:1,他引:1
Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal gamma-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 microM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+. 相似文献
17.
Requirement of ATP in bacterial chemotaxis 总被引:13,自引:0,他引:13
J I Shioi R J Galloway M Niwano R E Chinnock B L Taylor 《The Journal of biological chemistry》1982,257(14):7969-7975
Evidence is presented that chemotaxis requires ATP or a closely related metabolite, in addition to its known requirements of ATP for synthesis of S-adenosylmethionine (AdoMet) and maintenance of the proton motive force. Previous studies demonstrated a loss of tumbling and chemotaxis, and depletion of ATP when hisF auxotrophs of Salmonella typhimurium are starved for histidine (Galloway, R. J., and Taylor, B. L. (1980) J. Bacteriol. 144, 1068-1075). In the present study, intracellular [AdoMet], membrane potential, and [ATP] were measured in a hisF mutant of S. typhimurium. Membrane potential, determined from partitioning of [3H]tetraphenylphosphonium ion between the inside and the outside of the cell, was about -150 mV at pH 7.6, and did not decrease in histidine starvation but was slightly increased. The concentration of AdoMet decreased from 0.4 mM to 0.3 mM during starvation but when cycloleucine, an inhibitor of AdoMet synthetase, was used to decrease [AdoMet] by a similar amount in histidine-fed cells there was little change in tumbling frequency. Intracellular [ATP] was reduced from 4.5 mM to less than 0.2 mM by histidine starvation. About 0.2 mM ATP was necessary for spontaneous tumbling. A similar [ATP] was required for tumbling in arsenate-treated cells. Adenine at concentrations as low as 20 nM caused a transient increase in both tumbling frequency and [ATP] in histidine-starved cells. Thus, out of three parameters tested, only the intracellular [ATP] correlated with changes in tumbling frequency in the histidine-starved cells. 相似文献
18.
We previously showed that A23187 in high ionophore/protein ratios almost completely inhibits the sarcoplasmic reticulum Ca(2+)-ATPase [Hara, H. & Kanazawa, T. (1986) J. Biol. Chem. 261, 16584-16590]. In an attempt to obtain information on the mechanism of this inhibition, the effects of A23187 on conformational changes involved in the Ca(2+)-induced activation of the enzyme were investigated. The purified enzyme from sarcoplasmic reticulum of rabbit skeletal muscle as well as the purified enzyme labeled with fluorescein 5-isothiocyanate (FITC) were preincubated with A23187 in the absence of Ca2+ at pH 7.0 and 0 degrees C for 45 min. The activation of the enzyme following addition of CaCl2 was assessed by determining the capacity for rapid formation of phosphoenzyme from ATP. This activation was strongly inhibited by the preincubation with A23187. This indicates that the previously observed inhibition of the Ca(2+)-ATPase is mostly due to hindrance of the Ca(2+)-induced activation of the enzyme. In the control, in which the FITC-labeled enzyme was preincubated without A23187, the fluorescence intensity of the bound FITC decreased in a biphasic manner upon addition of CaCl2. The first rapid phase of this fluorescence drop was unaffected by A23187, whereas its second slow phase was almost completely inhibited by this drug. These results show that the Ca(2+)-dependent conformational change is biphasic and that the second slow phase (but not the first rapid phase) of this conformational change is inhibited by A23187. This suggests that the observed inhibition of Ca2+ activation is attributed to hindrance of the second slow phase of the Ca(2+)-dependent conformational change. 相似文献
19.
Y. H. Ehrlich† R. M. Snider‡ E. Kornecki M. G. Garfield§ R. H. Lenox 《Journal of neurochemistry》1988,50(1):295-301
The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
A M Brown 《Biochimica et biophysica acta》1979,554(1):195-203
Intact dog erythrocytes, whose Ca2+ permeability had been increased with A23187 still maintained intracellular Ca2+ below electrochemical equilibrium indicating that they could extrude Ca2+. This extrusion required no Na+ gradient but apparently depended on intracellular ATP and Mg2+ suggesting that it was mediated by an ATP-fuelled Ca2+ pump. 相似文献