EGTA和La3+对几种逆境诱导辣椒样半萜环化酶基因表达的影响

用钙离子螯合剂EGTA及细胞膜钙离子通道拮抗剂La³⁺预处理辣椒叶片,以破坏辣椒叶片中的钙信使系统,再用紫外线、CuCl₂、HgCl₂处理辣椒叶片,研究表明EGTA和La³⁺预处理未能降低CuCl₂、HgCl₂、UV诱导辣椒倍半萜环化酶活化的作用,EGTA预处理反而对CuCl₂、HgCl₂、UV的诱导辣椒倍半萜环化酶活性作用有一定的促进效应.单独用EGTA处理也能诱导离体辣椒叶片表现出倍半萜环化酶活性.Northen Blot分析结果表明,EGTA能诱导辣椒倍半萜环化酶基因转录.研究表明,在辣椒倍半萜环化酶基因表达过程中,还存在钙信使系统以外的信号传递途径;非生物诱发因子对倍半萜环化酶基因表达诱导作用与生物Elicitor的诱导作用在信号传递上有差异.     

Some properties of, and the effect of temperature on the (Mg + Ca) ATPase from immature and adult rat brain

1. 1. (Mg²⁺ + Ca²⁺) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg²⁺ + Ca²⁺) ATPase seen during development was greatest in the synaptic membrane preparations.

2. 2. At 37°C both Na⁺ or K⁺ at concentrations higher than 30 mM inhibited the microsomal Mg²⁺ ATPase, but the (Mg²⁺ + Ca²⁺) ATPase was stimulated by both Na⁺ and K⁺. Synaptic membrane Mg²⁺ ATPase was inhibited by concentrations higher than 100 mM K⁺; Na⁺ however stimulated this enzyme at all concentrations. Much of this Na⁺ stimulated activity was ouabain sensitive. Synaptic membrane (Mg²⁺ + Ca²⁺) ATPase was stimulated by Na⁺ or K⁺, this stimulation follows approximate saturation kinetics with an apparent K_m of 18.8 mM Na⁺ or K⁺.

3. 3. Arrhenius plots of microsomal (Mg²⁺ + Ca²⁺) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole⁻¹ above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg²⁺ + Ca²⁺) ATPase from both immature and adult brain.

Calcium ion control of platelet thrombosthenin ATPase activity

This study demonstrates that Ca²⁺ regulates thrombosthenin ATPase activity, likening the control of platelet contraction to that of cardiac and skeletal muscle. Thrombosthenin, the platelet contractile protein, was isolated by repeated low ionic strength and isoelectric precipitation. Thrombosthenin superprecipitation and ATPase activity were measured in 10⁻⁴ M CaCl₂ (high ionized Ca²⁺) and 0.25 mM ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) (low ionized Ca²⁺). In both high and low Ca²⁺, superprecipitation, measured as an increase in turbidity, ocurred shortly after addition of ATP. ATP hydrolysis by thrombosthenin, which proceeded linearly for several hours, was greater in high Ca²⁺ (approx. 2.3 nmoles·mg⁻¹·min⁻¹) than in low Ca²⁺ (approx. 1.8 nmoles·mg⁻¹·min⁻¹). This difference, when analyzed by the Student's t-test for paired samples was highly significant (P < 0.001). Thrombosthenin ATPase activity was not significantly altered by azide, an inhibitor of mitochondrial ATPase, nor by ouabain, an inhibitor of (Na⁺ + K⁺)-activated ATPase. The dependence of thrombosthenin activation on ionized Ca²⁺, measured with the use of CaEGTA buffers, was studied. The Ca²⁺-dependent portion of thrombosthenin ATPase was half maximal at 4.5·10⁻⁷ M Ca²⁺. This corresponds to an apparent binding constant of 2.2·10⁶ M⁻¹, a value that is comparable to that of skeletal and cardiac muscle. These data suggest that a Ca²⁺ control mechanism similar to that of the troponin-tropomyosin complex of muscle exists in the platelet.     

Role of the calcium ion and the disulfide bond in the Burkholderia glumae lipase

The role of the Ca²⁺ ion that is present in the structure of Burkholderia glumae lipase was investigated. Previously, we demonstrated that the denatured lipase could be refolded in vitro into an active enzyme in the absence of calcium. Thus, an essential role for the ion in catalytic activity or in protein folding can be excluded. Therefore, a possible role of the Ca²⁺ ion in stabilizing the enzyme was considered. Chelation of the Ca²⁺ ion by EDTA severely reduced the enzyme activity and increased its protease sensitivity, however, only at elevated temperatures. Furthermore, EDTA induced unfolding of the lipase in the presence of urea. From these results, it appeared that the Ca²⁺ ion in B. glumae lipase fulfils a structural role by stabilizing the enzyme under denaturing conditions. In contrast, calcium appears to play an additional role in the Pseudomonas aeruginosa lipase, since, unlike B. glumae lipase, in vitro refolding of this enzyme was strictly dependent on calcium. Besides the role of the Ca²⁺ ion, also the role of the disulfide bond in B. glumae lipase was studied. Incubation of the native enzyme with dithiothreitol reduced the enzyme activity and increased its protease sensitivity at elevated temperatures. Therefore, the disulfide bond, like calcium, appears to stabilize the enzyme under detrimental conditions.     

Characterization of phospholipase A2 and acyltransferase activities in squid (Loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions.

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.

The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.

Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

The activation by Ca of the ATPase of extracted muscle fibrils with variation of ionic strength, pH and concentration of MgATP

1. The alteration of the Ca²⁺ requirements of the ATPase activity of fibrils from rabbits and crabs at varying ionic strength, pH and concentration of MgATP (i.e. MgATP²⁻ + MgHATP⁻) was investigated.

2. Under physiological conditions, it was found that the ATPase activity of rabbit and crab fibrils after an initial increase decreased steeply when the Ca²⁺ concentration is raised above 1×10⁻⁴ M. This is a primary effect of the over-optimal Ca²⁺ concentration and not a secondary one caused by the influence of accompanying ions.

3. The Ca²⁺ requirements for ATP splitting by rabbit fibrils remain constant at an ionic strength from 0.1 to 0.2 and for a MgATP concentration in the range from 0.5 to 10 mM. At I = 0.05 it is about 5 times smaller than at 0.1. When the pH is decreased from 8 to 7, the Ca²⁺ requirements are increased some 10 times but only 3 times when the pH is varied between 7 and 6.

4. In crab fibrils, there is no alteration of the Ca²⁺ requirements when the ionic strength is varied between 0.05 and 0.2, but a reduction of the pH from 8.0 to 6.0 raises the Ca²⁺ requirements for half activation and for threshold by a factor of 10. Changing the MgATP concentration increases the Ca²⁺ requirements only in the range from 1 to 5 mM, while the concentration required in 0.5 mM is identical with that at 1 mM, and 10 mM corresponds to 5 mM.

5. It can be deduced from the experimental results that at a pH above 6.0 maximal activation is always obtained if the Ca²⁺ concentration is 5×10⁻⁵ M. By contrast, relaxation is only achieved when the Ca²⁺ concentration is below 1×10⁻⁷ M for pH 7.0 and I > 0.1 or below 1×10⁻⁸ for pH > 7.0 or I < 0.1.

6. To achieve complete relaxation, an ethyleneglycoldiaminotetraacetate (EGTA) concentration of 1 mM is sufficient, even when there is a large degree of contamination by Ca²⁺ as long as the pH stays above 6.5.     

Ontogenesis of Ca++ and Mg++ Contents of Embryonic Chick Heart

The Ca⁺⁺ and Mg⁺⁺ contents of embryonic chick heart were studied by atomic absorption spectrophotometry during a period from 48 h of foetal development until 2-3 days post-hatching. The hearts were isolated and incubated for 40 min at 22°C in three different media aerated with 95% 0₂-5% C0₂. The media included: normal Ringer's; Ca⁺-free Ringer's with 3 mM EGTA; and Ca⁺⁺-free Ringer's with 3 mM EDTA. At 48 h, the tubular myocardium contained 7-3 mM Ca⁺⁺ per wet weight which decreased rapidly to 1-2 mM by 10 days of development and remained between 0-9 and 1-1 mM until hatching. The Ca⁺⁺ content paralleled the changes in Na⁺ content reported earlier. Treatment with excess chelators, EGTA or EDTA, resulted in removal of 65-75% of the Ca⁺⁺ content throughout development until the time of hatching, when 50% of the Ca⁺⁺ became firmly bound. In contrast to the results with Ca⁺⁺, myocardial Mg⁺⁺ content rose rapidly from an initial value of 3.2 mM at 48 h to 6.7 mM by the 5th day of development, and then gradually declined throughout the remaining foetal development to 4.8 mM 2-3 days post-hatching. The Mg⁺⁺ contents closely paralleled changes in K⁺ content during development, which were reported earlier. Treatment with EGTA and EDTA removed 13-22% and 19-28% of the myocardial Mg⁺⁺, respectively, during development until just prior to hatching, when only 10-12% could be removed by chelation.     

Inhibition of oxidative phosphorylation by Ca or Sr: A competition with Mg for the formation of adenine nucleotide complexes

Intramitochondrial Sr²⁺, similar to Ca²⁺, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca²⁺ and Sr²⁺ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca²⁺ or 5.0 mM Sr²⁺ when the concentration of Mg²⁺ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg²⁺ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca²⁺ or Sr²⁺ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg²⁺ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr²⁺ it can be concluded that intramitochondrial Ca²⁺ or Sr²⁺ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Ca-binding and Ca-sensitizing functions of cardiac native tropomyosin, troponin, and tropomyosin

Procedures are described by which troponin and tropomyosin can be isolated from cardiac muscle rapidly, with minimal damage by oxidation. Cardiac relaxing proteins inhibit actomyosin ATPase activity in the presence of ethyleneglycoltetraacetic acid (EGTA), and permit graded stimulation by Ca²⁺. This stimulation is independent of preexisting inhibition, and greater than that obtained with skeletal proteins. Characteristics of Scatchard plots for Ca²⁺ binding suggest that troponin contains one class of sites which interact at high fractional occupancy. Interaction appears to be enhanced by tropomyosin. Mean values for the estimated maximum affinity and capacity of six canine cardiac troponin preparations were: 4.92·10⁶ M⁻¹, and 21.58·10⁻⁶ moles·g⁻¹. Values for skeletal troponin were not significantly different. Native tropomyosin bound about half as much Ca²⁺ per g, with maximum affinity the same as troponin. Pure tropomyosin bound no Ca²⁺. Cardiac and skeletal proteins differ in that the former are much more labile, and more readily influenced by ions and drugs.     

Properties and function of clostridial membrane ATPase

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg²⁺ dependent; Co²⁺ and Mn²⁺ but not Ca²⁺ could replace Mg²⁺ to some extent; the activation by Mg²⁺ was slightly antagonized by Ca²⁺. Even in the presence of Mg²⁺, Na⁺ or K⁺ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]_{0.5 V} = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg²⁺. Solubilization, however, led to instability of the enzyme.

The clostridial solubilized and membrane-bound ATPase showed different properties similar to the “allotopic” properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10^-4 M, led to 80% inhibition of the membrane-bound enzyme; oligomycin, ouabain, or NaN₃ had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment.

Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H⁺ translocation. A H⁺-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prokaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H⁺-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

Inhibition of Neutrophil Superoxide Secretion By of Preservative, Methylhydroxybenzoate: Effects Mediated By Perturbation of Intracellular Ca2+?

The preservative, methylhydroxybenzoate inhibited O₂^- secretion from human neutrophils activated by both the chemotactic peptide fMet-Leu-Phe and phorbol myristate acetate (PMA): the low level of oxidant secretion activated by the ionophore A23187 was similarly reduced in preservative-treated suspensions. Oxidant secretion was similarly reduced in fMet-Leu-Phe and A23187 treated suspensions in which intracellular Ca²⁺ was buffered by loading with Quin-2, indicating that methylhydroxybenzoate may exert its effects by perturbation of intracellular Ca²⁺ -dependent processes. Methylhydroxybenzoate could mimic EGTA in preventing the Ca²⁺ dependent enhancement of trypsin activity and could also bind this cation in experiments using a Ca²⁺ electrode, although the preservative bound Ca²⁺ more slowly and had a lower affinity than EGTA. These data indicate that methylhydroxybenzoate may exert its effects on neutrophils by perturbation of Ca²⁺-dependent activation pathways and this phenomenon may also explain its other known pharmacological effects. Furthermore, these observations provide an insight into the mechanisms by which intracellular Ca²⁺ may regulate oxidant secretion.     

Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

The amylases produced by a Bacillus stearothermophilus were purified through a series of four steps. Two separable enzyme fractions having starch hydrolysing activity were eluted from a DEAE-cellulose column by NaCl gradient elution. The homogeneity of the purified enzymes was checked on polyacrylamide gel electrophoresis. The product formation studies indicated that fraction I was an -amylase whereas fraction II was a β-amylase. The molecular weights were determined to be 48 000 and 57 000 and the carbohydrate moiety was found to be 13.2 and 0.8% for - and β-amylase, respectively. The protein digest of these enzymes indicated a total number of 15 amino acids with aspartic and glutamic acid showing the highest value. The purified amylase showed maximal activity at 80°C and pH 6.9. Fe³⁺, Cd²⁺, Pb²⁺, Hg²⁺, Ni²⁺ and Ag¹⁺ were potent inhibitors whereas Zn²⁺, Mg²⁺, Mn²⁺ and Al³⁺ were mild inhibitors. Ca²⁺, Ba²⁺, Sr²⁺ and K⁺ stimulated amylase activity in the order of Ca²⁺ > Ba²⁺ > Sr²⁺ > K⁺. PCMB, EDTA and sodium iodoacetate were inhibitory whereas glutathione (GSH) and cysteine afforded protection of enzyme activity. EDTA showed dose-dependent noncompetitive inhibition of both - as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca²⁺ and PCMB inhibition by the addition of glutathione (reduced). The K_m for - and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.     

低氧应激下甘肃鼢鼠与SD 大鼠心脏抗氧化酶和ATP 酶活性的比较

为探讨低氧应激下甘肃鼢鼠心脏对抗氧化损伤和电生理紊乱的可能机制,对甘肃鼢鼠和SD 大鼠在4. 5%氧浓度下分别进行2 h、4 h、6 h、8 h、10 h、16 h 低氧应激,比较常氧和各时程低氧下二者心脏超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)和Ca^{2 +} - ATP 酶、Ca^{2 +} - Mg^{2 +} - ATP 酶、Na ⁺ - K ⁺ - ATP酶活性,以丙二醛(MDA)含量作为机体氧化损伤指标。结果显示,常氧组甘肃鼢鼠GR 活性比SD 大鼠高,SOD、CAT、Ca^{2 +}-ATP 酶、Ca^{2 +} - Mg^{2 +} - ATP 酶和Na ⁺ - K ⁺ - ATP 酶活性及MDA 含量与SD 大鼠相比均无显著性差
异;低氧组甘肃鼢鼠SOD、CAT、GR、Ca^{2 +} - ATP 酶、Ca^{2 +} - Mg^{2 +} - ATP 酶和Na ⁺ - K ⁺ - ATP 酶活性迅速升高,显著高于SD 大鼠,MDA 含量则显著低于SD 大鼠。说明甘肃鼢鼠心脏通过提高抗氧化酶活性清除低氧诱导产生的多余自由基,并通过提高ATP 酶活性保证心电活动正常、心率稳定,应对低氧应激。     

Peroxvnitrite Causes Aspartate Release from Dissociated Rat Cerebeliar Granule Neurones

Peroxynitrite (ONOO^-) is a powerful oxidant which is formed from the reaction between nitric oxide (NO) and superoxide anion. It has therefore been proposed to mediate the toxic actions caused by NO. Since ONOO^- may be formed in the central nervous system (CNS) in pathological conditions such as brain ischaemia, we decided to investigate whether this molecule induces the release of the endogenous excitatory amino acids glutamate and aspartate from neurones. We selected as biological model acutely dissociated rat cerebellar granule neurones in suspension to allow a direct interaction between ONOO^- and target cells. Peroxynitrite caused a concentration-dependent release of aspartate but not of glutamate from dissociated cerebellar granule neurones. Peroxyni-triteinduced aspartate release was inhibited by dithiothreitol, tetrodotoxin, and in Na⁺-deprived solutions and not affected by EGTA or pre-incubation with the cytosolic Ca²⁺ chelator BAPTA/ AM. Peroxynitrite also induced an increase in intracellular Ca²⁺ concentration which was not affected in the presence of EGTA. These data show that ONOO^- causes release of aspartate from cerebellar granule neurones and that this effect might arise from an alteration of Na⁺ membrane permeability leading subsequently to reversal of a Na⁺-dependent plasma membrane transporter of this excitatory amino acid. In addition, ONOO^- alters Ca²⁺ homeostasis likely due to Na⁺ overload. Taken together, these findings may help and elucidate some of the intimate mechanisms of NO-induced neuronal damage in pathological circumstances.     

Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O₂⁻) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O₂⁻ was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [¹⁴C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O₂⁻ in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O₂⁻ or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm². Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O₂⁻ (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O₂⁻, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ_m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca²⁺ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca²⁺ from internal stores. Thus, COM-induced mitochondrial O₂⁻ requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O₂⁻ production, which is probably regulated by Δψ_m.     

Mechanism of the activation of arachidonic acid release by oxysterols in NRK 49F cells: Role of calcium

We previously demonstrated that oxysterols added to the culture medium of NRK 49F cells labelled with [¹⁴C] arachidonic acid potentiated arachidonic acid (AA) release and prostaglandin (PG) E₂ biosynthesis induced by the activation of these cells with fetal calf serum (FCS). In the absence of FCS, oxysterols had no effect on AA release. As phospholipase (Plase) A₂ activity is Ca²⁺-dependent, we investigated whether oxysterol potentiating effect on AA release was related to an effect of these compounds on cell Ca²⁺ concentration. In this paper, we show that the intensity of potentiation by oxysterol varies with the external cell Ca²⁺ concentration; when external Ca²⁺ is chelated by EGTA, the oxysterol effect persists, though it is decreased. The Ca²⁺ channel inhibitor nifedipine does not decrease the potentiating effect of 25-OH cholesterol, indicating that, if oxysterol favours Ca²⁺ entry into the cell, the nifedipine inhibited channel is not involved. At the usual concentration (5 μm/ml), oxysterols are not able to increase, mimmediately or after a short time of contact (90 min) the concentration of intracellular free Ca²⁺ ([Ca²⁺])_i measured by fluorescence of Quinn-2; at very high concentration of oxysterol (25 μm/ml), [Ca²⁺]_i only slightly increases (+30%). The liberation of AA induced by cell activation with the Ca²⁺ ionophore ionomycin is also potentiated by 25-OH cholesterol. All these observations are not in favour of a proper effect o oxysterols on cell Ca²⁺ level.     

CATION MODULATION OF SYNAPTOSOMAL RESPIRATION

Abstract— Synaptosomes were prepared from the cerebral cortex of the adult rat by a rapid technique, involving the use of centrifugation in a Ficoll-sucrose discontinuous gradient. Adequate respiratory control ratios were obtained with glutamate and succinate plus rotenone. The addition of Na⁺ to the incubation medium stimulated synaptosomal, State-4 respiration, with a half-maximal response at 15 mM Na⁺. The stimulation by Na⁺ was inhibited by atractylate, oligomycin, ouabain or EDTA. A cooperative interaction between Na⁺ and low concentrations of Mg²⁺ was observed. A significant proportion (39 per cent) of the total Na-K ATPase (EC 3.6.1.4) activity in the discontinuous gradient was localized in the synaptosomal fraction. In the absence of exogenous Mg²⁺, Na⁺ induced a 64 per cent stimulation of the synaptosomal ATPase activity which was sensitive to ouabain. Such stimulation of ATP hydrolysis would account for the formation of increased amounts of ADP, with consequent recycling to ATP through adequately controlled oxidative phosphorylation. These observations demonstrate a significant role for transmembrane cationic gradients in the control of synaptosomal respiration and mitochondrial oxidative phosphorylation. The preparation exhibits moderate respiratory control and should prove useful in studies of integrated mitochondrial oxidative metabolism and neuronal membrane function.     

ATPase Activity in Isolated Flagella from Euglena gracilis

SYNOPSIS. The ATPase activity of isolated flagella was studied in Euglena gracilis strain Z in the presence of Mg⁺⁺ or Ca⁺⁺. With Mg⁺⁺, the optimum activity was at pH 7 and with Ca⁺⁺, at pH 9. The K _m values were respectively 6.6 × 10⁻⁴ and 3.6 × 10⁻⁴. Activity was influenced also by temperature and ionic strength. Results with inhibitors of membrane ATPase suggest the presence of a specific contractile system in the flagella. Our results are compatible with a multicomponent enzymic system containing 2 active ATPases.     

胞质Ca2+参与外源H2S促进盐碱胁迫下裸燕麦种子萌发

硫化氢（Hydrogen sulfide,H₂S）是植物新型气体信号分子,钙离子（Calcium,Ca²⁺）为重要的第二信使,两者在植物逆境响应中分别发挥着重要作用。为明确胞质Ca²⁺在外源H₂S促进盐碱胁迫下作物种子萌发中的作用,以裸燕麦（Avena nude）为材料,采用培养皿培养,以混合盐碱（NaCl、Na₂SO₄、Na₂CO₃、NaHCO₃的摩尔比为12：8：1：9）模拟甘肃裸燕麦种植地盐碱环境,蒸馏水为对照,测定了胞外Ca²⁺螯合剂乙二醇-双-（2-氨基乙醚）四乙酸（EGTA）、质膜Ca²⁺通道阻断剂氯化镧（LaCl₃）、液泡Ca²⁺释放抑制剂钌红（RR）和内质网钙泵阻断剂毒胡萝卜素（Thaps）分别与H₂S供体硫氢化钠（NaHS）共处理下种子的发芽势、发芽率、发芽指数、活力指数、平均发芽速率、胚根长和胚芽长7个发芽指标,利用隶属函数分析方法综合评价胞质Ca²⁺对H₂S缓解盐碱胁迫抑制种子萌发的影响。结果表明,随着盐碱胁迫浓度增大,裸燕麦种子的发芽势、发芽率、发芽指数、活力指数、平均发芽速率、胚根长和胚芽长显著下降。与对照相比,15~75 mmol·L^-1盐碱胁迫导致裸燕麦种子萌发的隶属函数综合评价值（D）显著降低,30 mmol·L^-1盐碱胁迫下D值下降了73.1%;100~1 000 μmol·L^-1 NaHS不同程度提高了裸燕麦种子萌发的D值,且100 μmol·L^-1 NaHS缓解30 mmol·L^-1盐碱胁迫下D值下降的作用最大;EGTA、LaCl₃和RR均显著逆转了100 μmol·L^-1 NaHS对30 mmol·L^-1盐碱胁迫下D值下降的缓解作用,而Thaps对NaHS的作用无显著影响。表明胞质Ca²⁺参与外源H₂S促进盐碱胁迫下裸燕麦种子萌发的信号传导过程,且胞质Ca²⁺主要来源于胞外Ca²⁺的内流和液泡中Ca²⁺的释放。     

Oxidative stress and Mrp2 internalization

Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Gö6850, a Ca²⁺ chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca²⁺-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 μM). Moreover, an increase in the intracellular Ca²⁺ level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca²⁺ elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.