铜、铅、镉、锌、汞和银离子复合污染对水螅的急性毒性效应

以水螅(Hydrasp)为例,通过单因子静态急性毒性试验方法和等毒性溶液法,分别研究Hg2 、Cu2 、Cd2 、Ag 、Zn2 和Pb2 对其单一和复合毒性效应。单一实验结果表明,它们对水螅毒性大小顺序为Hg2 >Cu2 >Cd2 >Ag >Zn2 >Pb2 。复合毒性实验表明,Zn2 与Cu2 、Hg2 、Pb2 、Ag ;Pb2 与Cu2 ;Hg2 与Ag ;Pb2 与Ag 这些组合对水螅联合急性毒性总体上表现出拮抗作用,Cd2 与Cu2 、Hg2 、Pb2 、Ag 组合总体上则是协同作用,Zn2 与Cd2 、Pb2与Hg2 、Cu2 与Hg2 ,Ag 在不同的浓度水平组合下明显表现出不同的毒性效应。     

Hg2+、Pd2+、Cd2+和Cu2+对沼泽红假单胞菌生长及净化氮、磷能力的影响

研究了在培养基中分别添加不同浓度的重金属离子Hg2+、Pd2+、Cd2+和Cu2+作为人工配制的污水对光合细菌沼泽红假单胞菌生长与净化PO4-P和NH4-N能力的影响.实验结果表明当培养基中Hg2+的浓度达到2×10-6mol@L-1,该菌生长趋势开始减慢;当增至4×10-6mol@L-1时,生长完全被抑制.Cu2+的浓度达到1×10-6mol@L-1,该菌生长趋势也开始减慢;当增至8×10-6mol@L-1时生长完全被抑制.Cd2+的浓度达4×10-5mol@L-1时,其延缓期大大延长;增至16×10-5mol@L-4时生长完全被抑制.培养基中Pd2+的浓度达到8×10-4mol@L-1时,对该菌生长并未产生影响.当培养基中重金属离子浓度未达到完全抑制其生长时,对其净化PO4-P和NH4-N能力的影响不显著.     

发状念珠蓝细菌细胞对Cu2+、Cr2+和Pb2+胁迫的响应

[目的] 对发状念珠蓝细菌细胞进行重金属离子Cu2+(CuSO4)、Cr2+(CrCl2)和Pb2+(PbCl2)胁迫,探讨发状念珠蓝细菌细胞对重金属离子胁迫的响应.[方法] 25℃,80 μmol/(m·s)光照下,BG11培养液培养发状念珠蓝细菌,利用不同浓度(0、0.1、1.0、10、100 mg/L) Cu2+、Cr2+和pb2+胁迫发状念珠蓝细菌细胞,测定其质膜透性、超氧化物歧化酶活性、丙二醛含量、脯氨酸含量、可溶性蛋白含量以及海藻糖含量,分析发状念珠蓝细菌细胞对重金属离子胁迫的响应.[结果] 在Cu2、Cr2和pb2胁迫下,发状念珠蓝细菌细胞的外渗率和丙二醛(malondialdehyde)含量随着重金属离子浓度的升高而升高,相对渗透率和膜脂过氧化水平的变化趋势一致.超氧化物歧化酶(superoxide dismutase)活性随重金属离子浓度的升高先升后降,脯氨酸含量随着重金属离子处理浓度的升高,呈先降后升的趋势,可溶性糖含量随浓度的增大而减少.[结论] 低浓度的重金属离子可以诱导发状念珠蓝细菌细胞产生结构和生理的应激响应,高浓度会导致发状念珠蓝细菌细胞膜结构和功能的严重损害.     

Relationships between Ca2+ release, Ca2+ cycling, and Ca2+-mediated permeability changes in mitochondria

Ruthenium red and/or EGTA prevent cyclic uptake and release of Ca2+ in mitochondria. These compounds inhibit but do not prevent the swelling of liver mitochondria induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. Ruthenium red and/or EGTA have complex effects on the release rate of Ca2+ and other cations induced by t-butyl hydroperoxide or N-ethylmaleimide. To determine the relationship between permeability changes and Ca2+ release in the absence of Ca2+ cycling, a novel method of data collection and analysis is developed which allows the relative time courses of Ca2+ release and Mg2+ release or swelling to be accurately and quantitatively compared. This method eliminates errors in time course comparisons which arise from the aging of mitochondrial preparations and allows data from different preparations to be directly contrasted. Using the method, it is shown that permeability changes caused by Ca2+-releasing agents are not secondary effects arising from Ca2+ cycling between uptake and release carriers. In the absence of Ca2+-cycling inhibitors, Ca2+ release induced by t-butyl hydroperoxide or N-ethylmaleimide is, in part, carrier-mediated. In the presence of EGTA and ruthenium red, Ca2+ release induced by either agent is mediated solely by the permeability pathway. No differences are apparent in the solute selectivity of the inner membrane permeability defect induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. A novel type of Ca2+ release from energized liver mitochondria is reported. This release is induced by EGTA, occurs in the absence of other releasing agents or nonspecific permeability changes, and is rapid (greater than or equal to 50 nmol/min/mg protein).     

Stability of Ca2+-, Cd2+-, Cu2+-[illite-humic] complexes and pH influence

Decomposition of fresh plant residues in soil is expected to produce humic fractions varying in molecular size. It was hypothesized that metal adsorption by soil, to some degree, will depend on humic acid content and molecular size. The latter is expected to vary in number and type of functional groups. In this study, illite-humic complexes were used to evaluate Ca²⁺, Cd²⁺, and Cu²⁺ adsorption and how this adsorption was affected by humic acids, differing in molecular size, under various pH values. Potentiometric titration using ion-selective electrodes with a stop-and-go procedure was employed to evaluate metal-[illite-humic] complex formation. The results showed that illite-humic complexes exhibited at least two types of metal-ion adsorption sites (low and high affinity) and molecular size of humic fractions had a large potential influence on total metal adsorption but a relatively smaller influence on metal-complex stability. Relative strength of metal-ion-[illite-humic] complexes followed the order of Cu²⁺>Cd²⁺>Ca²⁺ and were affected by pH, especially for low metal-ion affinity sites. Magnitude of metal-[illite-humic] stability constants, depending on molecular size of humic fraction and pH, varied on a log-scale from 3.52 to 4.21 for Ca²⁺, 4.38 to 5.18 for Cd²⁺and from 5.23 to 5.83 for Cu²⁺. There was an approximate 5-fold difference in these stability constants between the three different sizes of humic fractions. The larger the humic fraction, the lower the metal-[illite-humic] stability constant. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering,sore-operated Ca2+ entry,and Ca2+ store refilling

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.     

Translational suppression by Ca2+ ionophores: Reversibility and roles of Ca2+ mobilization, Ca2+ influx, and nucleotide depletion

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca²⁺ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50–300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca²⁺ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca²⁺ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH₃ pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca²⁺ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca²⁺. Ionomycin produced more Ca²⁺ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca²⁺ by ionomycin were readily reversed in GH₃ cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca²⁺ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca²⁺ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca²⁺-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca²⁺ accumulation. Increased Ca²⁺ contents in response to Ca²⁺ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.Abbreviations BSA bovine serum albumin - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - PKR double-stranded RNA-regulated protein kinase - ER endoplasmic reticulum - eIF eukaryotic initiation factor     

Presynaptic Ca2+ dynamics, Ca2+ buffers and synaptic efficacy

In synapses neurotransmitter release is triggered by elevation of Ca2+ concentration at a Ca2+ sensor of the release machinery. The Ca2+ concentration at the release site at the given time point is determined by Ca2+ dynamics within presynaptic terminal. It depends on a source of Ca2+ (usually voltage-gated Ca2+ channels), diffusional distance between the source of Ca2+ and the Ca2+ sensor and Ca2+ buffering by endogenous Ca2+ buffers. In many synapses transmitter release can be enhanced (facilitated) during repetitive activity of neurons. The main source of facilitation is activity-dependent increase of Ca2+ concentration at the release site. Several mechanisms of facilitation have been proposed, namely, accumulation of residual Ca2+, multi-site (X receptor) mechanism and partial Ca2+ buffer saturation mechanism. In this review we discuss theoretical and experimental evidence in favor of one or the other of proposed mechanisms.     

Ca2+ binding,ATP-dependent Ca2+ transport,and total tissue Ca2+ in embryonic and adult avian dystrophic pectoralis

Summary Avian muscular dystrophy is an autosomal recessive genetic disease characterized by early hypertrophy and loss of function of the pectoralis major. The disease is progressive, ultimately resulting in atrophy and heavy lipid deposition.Previous investigators have noted a decrease in the ability of the dystrophic sarcoplasmic reticulum to concentrate Ca²⁺. More recently, other investigators have shown an abnormal calcium uptake in avian dystrophic sarcoplasmic reticulum. They indicated, using freeze-fracture techniques, that a 90 Å particle of the vesicle membrane exhibited a decreased population and suggested that they might be the ATPase involved in calcium transport.Our studies confirm the earlier observations of a decreased rate of Ca²⁺ uptake and Ca²⁺ binding capacity of dystrophic fragmented sarcoplasmic reticulum vesicles which are isolated from both embryonic and adult pectoralis. These observations correlate in turn with a 75% drop in the Ca: ATP transport efficiency of the dystrophic sarcoplasmic reticulum determined by measuring the rate of³²P_i liberation from -ATP³² during active calcium transport by the isolated sarcoplasmic reticulum SR.In addition, we have found a quantitative deficiency in a 65,000 dalton component of the dystrophic fragmented SR at the time of myoblast fusion by measuring³⁵S-Methionine incorporation into the SR, coupled to high resolution polyacrylamide gel electrophoresis and radioautography. Analysis of total tissue calcium by atomic absorption spectroscopy revealed a decrease in the total calcium content of dystrophic muscle.     

Importance of K+-dependent Na+/Ca2+-exchanger 2, NCKX2, in motor learning and memory

Plasma membrane Na+/Ca2+-exchangers play a predominant role in Ca2+ extrusion in brain. Neurons express several different Na+/Ca2+-exchangers belonging to both the K+-independent NCX family and the K+-dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca2+ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K+ -dependent Na+/Ca2+-exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca2+ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca2+] that is essential for the development of synaptic plasticity associated with learning and memory.     

Effects of phalloidin on K+-dependent, Ca2+-independent neurotransmitter efflux and K+-facilitated, Ca2+-dependent neurotransmitter release.

Phalloidin tightly binds to actin and converts soluble actin into depolymerization-resistant actin filaments. Phalloidin promotes the potassium-dependent, calcium-independent efflux of γ-amino butyric acid and nore-pinephrine from synaptosomes but inhibits the potassium-facilitated, calcium-dependent release of these neurotransmitters. This suggests that an actomyosin system is involved in synaptic transmission.     

Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.     

Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

The luminal Ca2+ chelator,TPEN, inhibits NAADP-induced Ca2+ release

The regulation of Ca²⁺ release by luminal Ca²⁺ has been well studied for the ryanodine and IP₃ receptors but has been less clear for the NAADP-regulated channel. In view of conflicting reports, we have re-examined the issue by manipulating luminal Ca²⁺ with the membrane-permeant, low affinity Ca²⁺ buffer, TPEN, and monitoring NAADP-induced Ca²⁺ release in sea urchin egg homogenate. NAADP-induced Ca²⁺ release was almost entirely blocked by TPEN (IC₅₀ 17–25 μM) which suppressed the maximal extent of Ca²⁺ release without altering NAADP sensitivity. In contrast, Ca²⁺ release via IP₃ receptors was 3- to 30-fold less sensitive to TPEN whereas that evoked by ionomycin was essentially unaffected. The effect of TPEN on NAADP-induced Ca²⁺ release was not due to an increase in the luminal pH or chelation of trace metals since it could not be mimicked by NH₄Cl or phenanthroline. The fact that TPEN had no effect upon ionophore-induced Ca²⁺ release also argued against a substantial reduction in the driving force for Ca²⁺ efflux. We propose that, in the sea urchin egg, luminal Ca²⁺ is important for gating native NAADP-regulated two-pore channels.     

Differential Ca2+ influx, KCa channel activity, and Ca2+ clearance distinguish Th1 and Th2 lymphocytes

In Th1 and Th2 lymphocytes, activation begins with identical stimuli but results in the production of different cytokines. The expression of some cytokine genes is differentially induced according to the amplitude and pattern of Ca2+ signaling. Using fura- 2 Ca2+ imaging of murine Th1 and Th2 clones, we observed that the Ca2+ rise elicited following store depletion with thapsigargin is significantly lower in Th2 cells than in Th1 cells. Maximal Ca2+ influx rates and whole-cell Ca2+ currents showed that both Th1 and Th2 cells express indistinguishable Ca2+-release-activated Ca2+ channels. Therefore, we investigated other mechanisms controlling the concentration of intracellular Ca2+, including K+ channels and Ca2+ clearance from the cytosol. Whole-cell recording demonstrated that there is no distinction in the amplitudes of voltage-gated K+ currents in the two cell types. Ca2+-activated K+ (KCa) currents, however, were significantly smaller in Th2 cells than in Th1 cells. Pharmacological equalization of Ca2+-activated K+ currents in the two cell types reduced but did not completely eliminate the difference between Th1 and Th2 Ca2+ responses, suggesting divergence in an additional Ca2+ regulatory mechanism. Therefore, we analyzed Ca2+ clearance from the cytosol of both cell types and found that Th2 cells extrude Ca2+ more quickly than Th1 cells. The combination of a faster Ca2+ clearance mechanism and smaller Ca2+-activated K+ currents in Th2 cells accounts for the lower Ca2+ response of Th2 cells compared with Th1 cells.