Effect of the calcium-binding protein regucalcin on the Ca2+ transport system in rat liver microsomes: the protein stimulates Ca2+ release

The effect of the calcium-binding protein regucalcin on the Ca2+ transport system in the liver microsomes from fed rats was investigated. Ca2+ transport was assayed by the method of Millipore filtration to estimate microsomal 45Ca2+ accumulation following addition of 10 mM adenosine triphosphate (ATP). 45Ca2+ uptake was retarded by the presence of regucalcin (1.0-4.0 microM). This retardation was remarkable at 1 min after regucalcin addition, while appreciable retardation was no longer seen at 5 min. Regucalcin (2.0 microM)-induced retardation of 45Ca2+ uptake was prevented by the presence of calmodulin (5 micrograms/ml). Calmodulin alone (1 and 5 micrograms/ml) caused a significant increase in 45Ca2+ uptake at 3 min after the start of incubation. Also, regucalcin (2.0 microM)-induced retardation of 45Ca2+ uptake was completely blocked by the presence of a Ca2(+)-trapping agent, oxalate (3 mM). On the other hand, 45Ca2+, which accumulated in microsomes during 5 min after ATP addition, was markedly released by the addition of regucalcin. This release was dose-dependent (0.5-4.0 microM). Guanosine triphosphate (GTP; 10-100 microM) caused a significant release of 45Ca2+ from the microsomes. The presence of regucalcin (2.0 microM) further enhanced the GTP effect. Regucalcin (2.0 microM)-induced release of 45Ca2+ was not blocked by the presence of the protein thiol-protecting agent dithiothreitol (0.1 mM). The presence of oxalate (3 mM) completely blocked the effect of regucalcin on 45Ca2+ release from the microsomes. These results indicate that regucalcin stimulates Ca2+ release from liver microsomes, and that the protein retards the microsomal Ca2+ uptake. The present study suggests that regucalcin can regulate the Ca2+ transport system in rat liver microsomes.     

Regucalcin-induced Ca2+ release from rat liver microsomes: the effect is inhibited by heparin

The effect of heparin on the calcium-binding protein regucalcin-stimulated Ca2+ release from rat liver microsomes was investigated. Ca2+ release was assayed by the method of Millipore filtration to estimate microsomal 45Ca2+ accumulation following the addition of 10 mM adenosine triphosphate. The addition of regucalcin (1.0 microM) or inositol 1,4,5-trisphosphate [Ins(1,4,5)P3; 1.0 microM] stimulated 45Ca2+ release from rat liver microsomes. These effects were completely inhibited by the presence of heparin (10.0 micrograms/ml). Regucalcin did not enhance the effect of Ins(1,4,5)P3. These results suggest that regucalcin affects 45Ca2+ release involved in Ins(1,4,5)P3 action in rat liver microsomes.     

Activation of hepatic microsomal Ca2+-adenosine triphosphatase by calcium-binding protein regucalcin

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca2+-adenosine triphosphatase (ATPase) activity in hepatic microsomes was investigated. Mg2+-ATPase activity was clearly increased by the presence of 50 microM Ca2+. Regucalcin (1.0-4.0 microM) caused a remarkable elevation (about 3-fold) of Ca2+-ATPase activity. Also, Mg2+-ATPase activity was increased (about 1.6-fold) by the presence of regucalcin (2.0 and 4.0 microM). Guanosine-5'-O-(3-thiotriphosphate) (GTPrs; 10(-5) and 10(-4) M) and nicotinamide adenine dinucleotide phosphate oxidized form (NADP+; 10(-5) to 10(-3) M) or reduced form (NADPH; 10(-4) and 10(-3) M) significantly increased Ca2+-ATPase activity. These increases were not enhanced by the presence of regucalcin (2.0 microM). Of various metal ions, a comparatively low concentration of V5+ (10(-5) M) or Cd2+ (10(-6) M) significantly increased Ca2+-ATPase activity, while Hg2+, Zn2+, Cu2+ and Mn2+ did not have such an effect. Regucalcin (2.0 microM) did not enhance the effect of V5+ and Cd2+ on Ca2+-ATPase activity. The present finding, that regucalcin activates hepatic microsomal Ca2+-ATPase, suggests a cell physiological role of regucalcin as an activator in the microsomal Ca2+-pump activity. This action of regucalcin may not be influenced by other regulators.     

Effects of Ca2+ and V5+ on glucose-6-phosphatase activity in rat liver microsomes: the Ca2+ effect is reversed by regucalcin

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on glucose-6-phosphatase in the microsomes of rat liver was investigated. Addition of Ca2+ up to 2.5 microM to the enzyme reaction mixture caused a significant increase of glucose-6-phosphatase activity in hepatic microsomes, while Ni2+, Zn2+, Cd2+, Cu2+, Mn2+ and Co2+ (20 microM) did not have an appreciable effect. Vanadate (V5+) markedly inhibited the enzyme activity; a significant inhibitory effect was seen at 10 microM V5+. The Ca2+-induced increase of glucose-6-phosphatase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 microM of the protein. Regucalcium had no effect on the basal activity of the enzyme. Meanwhile, the inhibitory effect of V5+ (10-100 microM) on glucose-6-phosphatase was not appreciably blocked by the presence of regucalcin (up to 2.0 microM). The present data suggest that hepatic microsomal glucose-6-phosphatase is uniquely regulated by Ca2+ and V5+, of various metals, and that the Ca2+ effect is reversed by regucalcin. The present study supports the view that regucalcin plays an important role as a regulatory protein in liver cell function related to Ca2+.     

Hepatic calcium-binding protein regucalcin decreases Ca2+/calmodulin-dependent protein kinase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on cytosolic Ca2+/calmodulin-dependent protein kinase activity was investigated. The increase in cytosolic Ca2+/calmodulin-dependent protein kinase activity with passage of incubation time was clearly prevented by the presence of regucalcin (1.0 microM). An appreciable effect of regucalcin was seen at 0.5 microM. The cytosolic Ca2+/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca2+ (0.25-1.0 mM). This increase was clearly blocked by the presence of regucalcin (1.0 microM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2.5-15 micrograms). In the presence of regucalcin (1.0 microM), trifluoperazine (50 microM), an antagonist of calmodulin, significantly decreased the cytosolic Ca2+/calmodulin-dependent protein kinase activity. These results suggest that regucalcin can regulate the Ca2(+)-calmodulin effect in liver cytosol.     

Effects of Ca2+, Zn2+ and Cd2+ on uridine diphosphate-glucuronyltransferase and beta-glucuronidase activities in rat liver microsomes

The effect of various metals on uridine diphosphate (UDP)-glucuronyltransferase and beta-glucuronidase activities in rat liver microsomes was investigated. The presence of Mn2+, Cd2+, Zn2+, V5+, Ni2+, Co2+, Cu+ or Ca2+ (20 microM) in the enzyme reaction mixture did not cause a significant alteration of UDP-glucuronyltransferase activity in hepatic microsomes. Of these metals, Zn2+ and Cd2+ (20 microM) caused a remarkable increase in hepatic microsomal beta-glucuronidase activity. Appreciable effects of Zn2+ and Cd2+ on beta-glucuronidase activity were seen at 5.0 microM, and the effects were saturated at 50 microM. Ca2+ (5.0-50 microM) and/or the Ca2(+)-binding protein regucalcin (2.0 microM) did not have an appreciable effect on UDP-glucuronyltransferase and beta-glucuronidase activities in hepatic microsomes. Thus, Zn2+ and Cd2+ uniquely increased beta-glucuronidase activity. The Zn2(+)- and Cd2(+)-induced increase in beta-glucuronidase activity was completely reversed by the presence of an SH group-protecting reagent (dithiothreitol). The response of the microsomal enzyme to Zn2+ and Cd2+ (20 microM) was no longer seen after treatment with 0.2% Triton X-100 [polyoxyethylene(10)octylphenyl ether], indicating that the stimulation by these metals is dependent on membrane association. The present study suggests that, of various metals tested, Zn2+ and Cd2+ can uniquely increase hepatic microsomal beta-glucuronidase activity and that their effect is based on binding to membranous SH groups, beside the enzyme protein.     

Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry

The mitochondrial membrane potential (DeltaPsi(m)) is an important indicator of the energetic state of both the mitochondria and the cells. To develop a sensitive, convenient, and rapid method for the measurement of DeltaPsi(m), we carried out cell fluorescence assays using the Agilent 2100 bioanalyzer system which, unlike the conventional flow cytometry, is based on microfluidic technology employing fluorescence detection with a 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)) fluorescent probe. The use of DiOC(6)(3) in the fluorometer was shown to be feasible for monitoring variations in DeltaPsi(m) in the mitochondria isolated from rat liver and treated with rotenone, succinate, ADP, and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Flow cytometry analysis showed severe reduction of fluorescence intensity in Jurkat cells after treatment with 1.0 and 10 microM FCCP. However, fluorescence microscopy demonstrated obvious accumulation of fluorescence in the mitochondria and induction of diffuse cytoplasmic fluorescence not localized to the mitochondria in these cells. The dose response range of DiOC(6)(3) in the Agilent 2100 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm-2.0 microM. Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 microM DiOC(6)(3) was observed after treatment with 10 microM FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring DeltaPsi(m) in cell assays.     

Interaction of Cu+ with mitochondria.

The uptake of Cu+ by rat liver mitochondria is rapid and extensive. Respiration is stimulated by 10 microM Cu+ then inhibited and the inhibition could not be relieved with uncoupling agents. Collapse of the membrane potential is induced by 5-10 microM Cu+. These effects are partially inhibited by radical scavengers indicating the involvement of radical production in these events. Reduction of the GSH content and production of peroxidation products by higher amounts of Cu+ was also demonstrated. Swelling of non-respiring rat liver and heart mitochondria in sodium or lithium acetate was used to study effects of Cu+ on the Na+/H+ exchanger. Swelling is stimulated by 5-100 microM Cu+. In the presence of a radical scavenger the swelling is reduced. In sodium nitrate media diltiazem-sensitive stimulated swelling is observed. Amiloride was found to inhibit Cu(+)-induced efflux of Ca2+. At high concentrations of Cu+, a general increase in permeability was the dominant feature.     

Haematoporphyrin derivative (Photofrin II) photosensitization of isolated mitochondria: inhibition of ADP/ATP translocator

To gain further insight into the mechanism by which irradiation of mitochondria in the presence of haematoporphyrin derivative (Photofrin II) (PF II) causes impairment of mitochondrial oxidative phosphorylation, the rate of ADP/ATP exchange via the ADP/ATP translocator was measured fluorometrically is isolated rat liver mitochondria. In accord with noncompetitive inhibition, PF II photosensitization decreases the maximum rate of exchange Vmax (20.8 and 9.6 nmol ATP effluxed min-1 x mg protein in the control and after 2 min irradiation, respectively) without changing the ADP affinity for the carrier (Km = 5 microM in both cases). Comparison of the rate of oxygen uptake by mitochondria stimulated by either ADP or by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) confirms that the adenine nucleotide carrier is a major target of photodynamic action which causes oxidative phosphorylation impairment.     

Photosensitization of isolated mitochondria by hematoporphyrin derivative (Photofrin): effects on bioenergetics.

Isolated rat liver mitochondria were incubated in the presence of 6 micrograms/ml of Photofrin and irradiated at the wavelength of 365 nm. After 45 s irradiation (30 W/m2), coupling defined as stimulation of respiration by externally added adenosine 5'-diphosphate (ADP) is totally lost. In contrast, membrane potential created by addition of succinate or adenosine 5'-triphosphate (ATP) is only slightly affected. Similarly, the ADP/O ratio is not modified after 20 s irradiation. These data suggest that modification of the mitochondrial membrane potential is not a primary event after irradiation.     

Effect of the ionic strength and prostaglandin E2 on the free Ca2+ concentration and the Ca2+ influx in human red blood cells

Human red blood cells (RBCs) were loaded with the Ca(2+)-sensitive fluorescent dye fura-2 to investigate the effects of media ionic strength and prostaglandin E2 (PGE2) on the intracellular free Ca2+ concentration ([Ca2+]i). [Ca2+]i of intact RBCs in a Ca(2+)-containing physiological (high) ionic strength (HIS) solution was 75.1 +/- 8.3 nM after 5 min incubation, increasing to 114.9 +/- 9.6 nM after 1 h. In Ca(2+)-containing low ionic strength (LIS) solutions, [Ca2+]i was significantly lower than in the Ca(2+)-containing HIS solution (p = 0.041 or 0.0385 for LIS solutions containing 200 or 250 mM sucrose, respectively), but, as in HIS solution, an increase of [Ca2+]i was seen after 1 h. In Ca(2+)-free (0 Ca2+ plus 15 microM EGTA) media, [Ca2+]i decreased (ranging from 15 to 21 nM), but were not significantly different in HIS or LIS, and did not change following 1 h incubation. The effect of the ionic strength and PGE2 on passive Ca2+ influx was investigated on ATP-depleted RBCs. Ca2+ influx was faster during the initial 10 min in comparison with the subsequent time period (10-45 min), both in HIS and LIS media, decreasing from 20.3 +/- 1.9 to 12.9 +/- 1.3 micromol/(lcells x h) in HIS, and from 36.7 +/- 5.3 to 8.6 +/- 1.2 micromol/(lcells x h) in LIS. Prostaglandin E2 (PGE2; 10(-7)-10(-11) M), dissolved in deionised water or in ethanol, did not affect [Ca2+]i in either normal or in ATP-depleted RBCs suspended in Ca(2+)-containing HIS medium. Finally, the addition of carbachol (100 microM) did not affect [Ca2+]i. The present findings suggest that stimulation of the Ca(2+)-activated K+ channel by PGE2, reported in [J. Biol. Chem. 271 (1996) 18651], cannot be mediated via increased [Ca2+]i.     

Non-respiring rat liver mitochondria do not have a Ca2+/2H+ antiporter

Liver mitochondria take up Ca2+ by the Ca2+ uniporter, whereas at steady state efflux is believed to occur mainly by means of a ruthenium red-insensitive Ca2+/2H+ antiporter. The latter activity was studied in respiration-inhibited mitochondria in the presence of ruthenium red and was measured as Ca2+ uptake following acidification of the matrix by addition of nigericin, which catalyzes K+/H+ exchange. Ca2+ uptake was stimulated by protonophorous uncoupling agents and inhibited by increasing the concentration of ruthenium red. However, the rates were always smaller than those obtained by addition of valinomycin instead of nigericin. This indicates that under these conditions, Ca2+ fluxes are not mediated by a Ca2+/2H+ antiporter but by residual uniporter activity.     

The effects of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose on rat liver mitochondrial respiration

The inhibitory effects of pure galloylglucose (1,2,3,4,6-penta-O-galloyl-beta-D-glucose) on the respiratory chain of rat liver mitochondria were investigated. The respiratory control ratio (RCR) decreased by 50% on addition of 20 microM pentagalloylglucose to highly coupled mitochondria, but the adenosine-5'-diphosphate/oxygen (ADP/O) ratio decreased only slightly. The RCR disappeared and the ADP/O ratio could not be measured at concentrations of pentagalloylglucose above 30 microM. On the other hand, the uncoupler-induced oxygen consumption was also inhibited. These findings suggest that pentagalloylglucose at low concentrations inhibits the electron transport system to decrease the RCR, but scarcely impairs the membrane, practically retaining the coupled reaction, while at high concentrations it impairs the structural integrity of the mitochondrial membrane. Pentagalloylglucose competitively inhibited succinate dehydrogenase activity, and noncompetitively inhibited reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase and ubiquinol-1 oxidase activities of submitochondrial particles (SMP). However, it did not show significant inhibition of the cytochrome c oxidase activity of SMP. It is thus concluded that pentagalloylglucose, which is the lowest-molecular-weight component of tannic acid, exerts its effect on mitochondrial respiration and oxidative phosphorylation through action on the membrane and on succinate dehydrogenase, NADH dehydrogenase and cytochrome bc1 complex of mitochondria.     

Inhibition of superoxide generation and of increase in intracellular Ca2+ concentration by zinc in rat neutrophils stimulated with zymosan

The effect of Zn2+ on the O2- generation and change in intracellular Ca2+ concentration ([Ca2+]i) of rat peritoneal neutrophils was studied. Zymosan (serum-treated zymosan (STZ))-induced O2- generation was inhibited by Zn2+ at concentrations as low as 10 microM. A large amount of the inhibition was observed in the absence of extracellular Ca2+ but the inhibition could not be restored by increasing the extracellular Ca2+ concentration, indicating that Zn2+ does not necessarily inhibit the O2- generation competitively with extracellular Ca2+. In the absence of extracellular Ca2+, Zn2+ inhibited STZ-induced transient increase in [Ca2+]i in the concentration range that evoked a marked inhibition in the O2- generation. On the other hand, Zn2+ did not inhibit significantly STZ-induced uptake of 45Ca2+ from extracellular medium by the cells. From these results, it is suggested that Zn2+ inhibits STZ-induced release of Ca2+ from intracellular storage sites, resulting in the suppression of the activation mechanism of neutrophils.     

An HPLC assay for rat liver ferrochelatase activity

A rapid, reliable, sensitive and reproducible HPLC method was developed for the assay of ferrochelatase activity in rat liver. The assay was carried out aerobically with Zn2+ and mesoporphyrin or protoporphyrin IX as substrates. Zn-porphyrins formed were extracted with dimethyl sulphoxide/methanol (30:70, v/v) containing Zn-deuteroporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for mesoporphyrin was 5.9 microM, for protoporphyrin IX 8.8 microM and for zinc 6.0 microM. The specific activities were 33.1 +/- 5.0 nmol Zn-mesoporphyrin or 13.4 +/- 2.0 nmol Zn-protoporphyrin formed per hour per mg of protein for mitochondria and 12.3 +/- 2.2 nmol Zn-mesoporphyrin or 4.6 +/- 0.9 nmol Zn-protoporphyrin per hour per mg of protein for liver homogenate.     

EFFECTS OF DEMETHYLCHLORTETRACYCLINE PHOTOTOXICITY ON THE STRUCTURE and FUNCTIONS OF RAT LIVER MITOCHONDRIA

The phototoxic effects of demethylchlortetracycline (DMCT) with UVA radiation on isolated rat liver mitochondria were studied. DMCT at concentration of 21.5 microM with 5.53 x 10(-2) J cm-3 of UVA was found to be a potent uncoupler of oxidative phosphorylation. DMCT alone also uncoupled mitochondria but at higher concentrations (105 microM). ATPase activity was remarkably induced in mitochondria exposed to DMCT (21.5 microM) plus UVA (120% of DNP-stimulated ATPase activity). Content of ATP in such mitochondria when measured after addition of ADP was much smaller than that in control mitochondria. Ultrastructurally, mitochondria treated either with DMCT (21.5 microM) or with UVA alone stayed in the condensed configuration. On the other hand, uncoupled mitochondria treated with DMCT plus UVA became swollen and were changed into the orthodox configuration.     

Effect of light on calcium transport in bull sperm cells.

The effect of light on calcium transport was studied. Bull sperm cells were irradiated with an He-Ne (630 mm) laser and a 780 nm diode laser at various energy doses, and 45Ca2+ uptake was measured by the filtration technique. It was found that there is an accelerated Ca2+ transport in the irradiated cells, which means that laser light can stimulate Ca2+ exchange through the cell membrane. This may cause transient changes in the cytoplasmic Ca2+ concentration which, in spermatozoa, has a regulatory role in control of motility and acrosome reaction, and in other cells can trigger mitosis.     

CHEMILUMINESCENT DIPHENYLACETALDEHYDE OXIDATION BY MITOCHONDRIA IS PROMOTED BY CYTOCHROMES and LEADS TO OXIDATIVE INJURY OF THE ORGANELLE

Plant and animal mitochondria promote the aerobic oxidation of diphenylacetaldehyde (DPAA). This process is accompanied by chemiluminescence and rotenone-insensitive oxygen uptake. Tn rat liver and potato tubers, mitochondrial swelling is concurrently detected. Light emission and oxygen consumption decreased (about 50%) in cytochrome c-depleted mitochondria. A model system–cytochrome c or b5/dihexadecylphosphate liposomes–was also able to oxidize DPAA with parallel reduction of the cytochrome. Reduction of respiratory complex I or I plus II by addition of rotenone or antimycin A, respectively, did not prevent DPAA oxidation. However, when all cytochrome was reduced by addition of cyanide, aldehyde oxidation was completely suppressed. Altogether these data indicate that respiratory cytochromes are responsible for DPAA oxidation with production of excited species and consequent mitochondrial permeabilization.     

不同条件下离体大鼠肝脏线粒体能量代谢微量热分析

为了探究线粒体的能量代谢过程,本文以离体大鼠肝脏线粒体为模型,利用多通道、高灵敏度的热活性检测仪TAM Ⅲ,实时监测了不同线粒体浓度、不同底物、不同缓冲液、几种呼吸抑制剂以及Ca²⁺和线粒体渗透转换孔抑制剂CsA存在时线粒体的能量代谢,获得了完整的热功率―时间曲线,并通过计算得到了线粒体能量代谢的热动力学参数。通过分析发现：（1）线粒体浓度越大,代谢越快;（2）直接底物琥珀酸钠使线粒体代谢更快;（3）高浓度Ca²⁺能够刺激线粒体快速产热,且在长期代谢进程中,线粒体渗透转换孔抑制剂CsA并不能改变Ca²⁺造成的影响;（4）不同缓冲液对线粒体代谢的影响基于其组分的不同,缓冲液中含有呼吸底物;（5）呼吸抑制剂都能抑制线粒体的能量代谢,尤其是复合物IV的抑制剂NaN₃,高浓度下使代谢停止。     

重稀碱金属Cs~+跨人红细胞膜行为的研究(英文)

采用原子吸收光谱法检测体外人红细胞摄取Cs+的含量,系统讨论了胞外Cs+浓度,温育时间、温育温度、介质pH值对人红细胞摄取Cs+过程的影响。选用不同离子通道或离子载体的特异性抑制剂进一步探讨Cs+的跨膜途径和机理。结果显示,各实验参数对人红细胞摄取Cs+均有一定的促进作用。Cs+主要借助Na+/K+-泵的主动运输方式跨膜;少量的Cs+能"漏入"细胞,微量的Cs+可以模拟Na+/Li+-反向协同运输的方式跨膜;在允许HCO3-存在的pH环境下,少量Cs+以Cl-/CsCO3-交换的形式通过膜上带3蛋白进入人红细胞;Ca2+通道对Cs+没有通透作用。