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1.
In previous studies, nonlethal CdCl 2 concentrations apparently inhibited basal Y-1 mouse adrenal tumor cell endogenous mitochondrial cholesterol conversion to pregnenolone. In addition, CdCl 2 inhibited all agents stimulating both plasma membrane-dependent cAMP synthesis and 20-hydroxy-4-pregnen-3-one (20DHP) secretion. Bypassing the plasma membrane using dibutyryl-cAMP (dbcAMP) stimulated cytoplasmic cholesterol metabolism and 20DHP secretion in the presence of CdCl 2. Since CdCl 2 competed at metabolic steps requiring Ca 2+ in other tissues, experiments were designed to examine Cd 2+ competition with Ca 2+ during steroidogenesis. Sets of cells incubated with either medium or adrenocorticotropin (ACTH) with or without CdCl 2 were also treated with 0, 1.0, 5.0 or 10.0 mmol/L CaCl 2 in the presence or absence of EGTA, a relatively specific Ca 2+, but not Cd 2+, chelating agent. Another experimental cell set incubated with either medium or ACTH, with or without CdCl 2, was treated with or without 1 mmol/L A23187, an ionophore specifically facilitating extracellular Ca 2+ transfer across plasma membranes. Besides determining Ca 2+ involvement in steroidogenesis using steroid secretion as an endpoint, we directly measured Ca 2+ concentrations using intracellular fura-2 fluorescence. Following loading with 2 mol/L fura-2, cells remained untreated or medium was infused with CdCl 2, ACTH, ACTH/CdCl 2 or ACTH followed after 50 s by CdCl 2. Using Ca 2+-supplemented media, we observed that Cd 2+ inhibition of ACTH-stimulated 20DHP secretion was completely reversed. Standard Ca 2+-containing medium supplemented with Ca 2+ also enhanced maximally stimulated 20DHP secretion by ACTH. 20DHP secretion by ACTH-treated and ACTH/Cd 2+-treated cells was only reduced by EGTA, when Ca 2+ was not supplemented. The ionophore A23187 increased basal and ACTH-stimulated 20DHP secretion by Cd 2+-treated cells, suggesting that extracellular Ca 2+ resources may compete against Cd 2+ effects on plasma membrane cAMP synthesis and on basal cholesterol metabolism by mitochondria. No time-dependent change in Ca 2+ concentrations occurred within untreated cell suspensions. ACTH stimulation caused a 25 s burst in Ca 2+ concentrations before returning to basal, steady-state levels. Cd 2+ also stimulated intracellular fura-2 fluorescence. Untreated cell suspensions infused with Cd 2+ exhibited a continuous rise in intracellular fluorescence. ACTH/CdCl 2-treated cells exhibited a hyperbolic rise in intracellular fluorescence over the 300 s study period. Cells treated with Cd 2+ 50 s after ACTH treatment initially exhibited the 25 s fluorescence burst followed by a Cd 2+-induced hyperbolic rise in intracellular Cd 2+. These fluorescence measurements suggested that cytoplasmic Ca 2+ changes do not appear to be necessary for basal 20DHP synthesis and secretion; only a 25 s burst in intracellular Ca 2+ is necessary to a slightly higher plateau level for stimulated 20DHP synthesis and secretion. Cd 2+ freely enters the cell under basal conditions and Cd 2+ entry is accelerated by ACTH stimulation. Data were consistent with Ca 2+ being required for optimal stimulated steroid production and Cd 2+ probably competing with Ca 2+ during basal mitochondrial cholesterol metabolism and plasma membrane ACTH-stimulated cAMP generation. 相似文献
2.
Summary Evaluation of the potential adverse human health effects of low-level chronic exposure to heavy metals is dependent on the
basic knowledge of the cellular and molecular toxicology of these metals. The use of various cell culture systems has greatly
facilitated our knowledge of the cellular effects. Inasmuch as most of the acute and chronic toxic effects of metals occur
primarily on the renal proximal tubules, the development of a rat kidney epithelial cell culture has provided a unique system
to study the uptake and mechanism of toxicity of metals and their intracellular binding ligands. In the presence of d-valine, fibroblast growth was retarded and a primary epithelial monolayer culture was selectively grown from rat kidney cells.
A distinct difference in the uptake of chemically similar divalent metals, such as Pb 2+, Hg 2+, Cd 2+, and Zn 2+, was observed in these cells. Both Pb 2+ and Hg 2+ were more avidly taken up by kidney cells than Cd 2+ and Zn 2+ salts and they also showed increased toxicity. On the other hand, the cellular uptake of Cd from cadmium-metallothionein
(CdMT) was much less than from CdCl 2, but CdMT was about seven times more toxic than CdCl 2 when added to the renal cell culture. The cytotoxicity of CdCl 2 was decreased significantly with pretreatment of the cells with CdCl 2, although this had no effect on the toxicity of CdMT. The cellular toxicity of CdMT occurred probably during the process
of its transport across the plasma membrane whereas that of CdCl 2 occurred after it had entered the cell. Thus rat kidney epithelial cells may be a useful tool to study the mechanism of renal
toxicity of environmental chemicals and drugs.
This work was funded by grants-in-aid of research from the Kidney Foundation of Canada. 相似文献
3.
Cd 2+ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd 2+-resistant due to possession of cadA-coded Cd 2+ efflux system, recognized here as P-type Cd 2+-ATPase. This Cd 2+ pump utilizing cellular energy—ATP, ?μ H + (electrochemical proton potential) and respiratory protons, extrudes Cd 2+ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd 2+ extrusion remains unknown. Here we propose that two Cd 2+ taken up by strain 17810R via Mn 2+ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd 2+-ATPase, forming SCdS. This stops Cd 2+ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd 2+, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd 2+/H + exchange. In 1 mM phosphate buffer (low P iB), external Cd 2+ competing with decreased number of P i-dependent protons, binds to ψ s of Cd 2+-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd 2+/Cd 2+ exchange and blocks dithiols in ODHC. However, Mg 2+ pretreatment preventing external Cd 2+ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd 2+ via Cd 2+/H + exchange, despite low P iB. 相似文献
4.
To identify some of the mechanisms involved in the high resistance to Cd 2+ in the protist Euglena gracilis, we studied the effect of Cd 2+ exposure on its energy and oxidative stress metabolism as well as on essential heavy metals homeostasis. In E. gracilis heterotrophic cells, as in other organisms, CdCl 2 (50 μM) induced diminution in cell growth, severe oxidative stress accompanied by increased antioxidant enzyme activity and
strong perturbation of the heavy metal homeostasis. However, Cd 2+ exposure did not substantially modify the cellular respiratory rate or ATP intracellular level, although the activities of
respiratory complexes III and IV were strongly decreased. In contrast, an enhanced capacity of the alternative oxidase (AOX)
in both intact cells and isolated mitochondria was determined under Cd 2+ stress; in fact, AOX activity accounted for 69-91% of total respiration. Western blotting also revealed an increased AOX
content in mitochondria from Cd 2+-exposed cells. Moreover, AOX was more resistant to Cd 2+ inhibition than cytochrome c oxidase in mitochondria from control and Cd 2+-exposed cells. Therefore, an enhanced AOX seems to be a relevant component of the resistance mechanism developed by E. gracilis against Cd 2+-stress, in addition to the usual increased antioxidant enzyme activity, that enabled cells to maintain a relatively unaltered
the energy status. 相似文献
5.
To determine the onset of the Cd 2+-hyperaccumulating phenotype in Euglena gracilis, induced by Hg 2+ pretreatment (Avilés et al. in Arch Microbiol 180:1–10, 2003), the changes in cellular growth, Cd 2+ uptake, and intracellular contents of sulfide, cysteine, γ-glutamylcysteine, glutathione and phytochelatins during the progress
of the culture were analyzed. In cells exposed to 0.2 mM CdCl 2, the Cd 2+-hyperaccumulating phenotype was apparent only after 48 h of culture, as indicated by the significant increase in cell growth
and higher internal contents of sulfide and thiol-compounds, along with a higher γ-glutamylcysteine synthetase activity. However,
the stiochiometry of thiol-compounds/Cd 2+ accumulated was similar for both control and Hg 2+-pretreated cells. Moreover, the value for this ratio was 2.1 or lower after 48-h culture, which does not suffice to fully
inactivate Cd 2+. It is concluded that, although the glutathione and phytochelatin synthesis pathway is involved in the development of the
Cd 2+-hyperaccumulating phenotype in E. gracilis, apparently other pathways and sub-cellular mechanisms are also involved. These may be an increase in other Cd 2+ chelating molecules such as di- and tricarboxylic acids, phosphate and polyphosphates, as well as Cd 2+ compartmentation into organelles.
César Avilés: In memoriam. 相似文献
6.
Loading of Ca 2+-sensitive fluorescent probes into plant cells is an essential step to measuring activities of cytoplasmic free Ca 2+ ions with a fluorescent imaging technique. A major barrier to the loading of the fluorescent probes into plant cells using the acetoxymethyl (AM) esters of the Ca 2+-sensitive dyes is the presence of cell-wall associated esterases. These esterases hydrolyse the esterified form of the fluorescent probes, rendering the probes membrane-impermeable. A novel non-invasive loading protocol was described in this paper to load the Ca 2+-sensitive fluorescent probe Fluo-3/AM ester into apical cells of intact wheat roots by incubating the roots in Fluo-3/AM ester solution at 4°C for 2 h followed by 2-h incubation in the dye-free solution at 20°C. The incubation at low temperature inhibited extracellular hydrolysis of Fluo-3/AM ester but had less effect on diffusion of membrane-permeable Fluo-3/AM ester across the plasma membrane, because hydrolysis of Fluo-3/AM ester by extracellular esterases is a chemical process (high Q 10), while diffusion of Fluo-3/AM across the plasma membrane is a physical process (low Q 10). The Fluo-3/AM ester, accumulated in the root cells during the low temperature incubation, was then cleaved by intracellular esterases during the incubation at 20°C, releasing the membrane-impermeable Ca 2+-sensitive Fluo-3 in the cytoplasm. The root cells loaded with Fluo-3 showed strong intracellular fluorescence under confocal microscopy. The fluorescence from the root cells was sensitive to the Ca 2+ ionophore and hydrogen peroxide, indicating that the intracellular fluorescence was due to intracellular Ca 2+ ions. 相似文献
7.
The mechanism for cadmium (Cd 2+) uptake into the cytosol of protoplasts from 5- to 7-day-old wheat seedlings ( Triticum aestivum L. cv. Kadett) was investigated by a new method, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. Cadmium fluorescence gradually increased in the cytosol of shoot and root protoplasts upon repeated additions of CdCl 2 to the external medium, reflecting an uptake of Cd 2+. The uptake was inhibited by calcium and potassium chloride, as well as by Verapamil and tetraethylammonium (TEA), inhibitors of calcium and potassium channels, respectively. Calcium competitively inhibited the cadmium uptake. The metabolic inhibitors vanadate and dinitrophenol partly inhibited the uptake, suggesting it was dependent on membrane potential. The results indicate that cadmium is taken up by channels permeable to both calcium and potassium. The total uptake of cadmium into the protoplasts was also detected by unidirectional flux analyses using 109Cd 2+, and showed approximately the same maximal concentration of Cd 2+ as the fluorescence measurements. By combining the two methods it is possible to detect both uptake into the cytosol and into the vacuole.Abbreviations BTC-5N, AM Acetoxymethyl ester of 5-nitrobenzothiazole coumarin - DNP 2,4-Dinitrophenol - TEA Tetraethylammonium 相似文献
8.
Cadmium (Cd 2+) is a nonessential divalent metal ion that causes toxicity in multiple organs in humans. In order for toxicity to occur Cd 2+ must first enter cells by utilizing transport pathways for essential metals. This review focuses on studies in which Cd 2+ transport was directly demonstrated by electrophysiological, radiotracer or Cd 2+-sensitive fluorescent dye techniques. The chemistry of Cd 2+ and metal ions in general is addressed in the context of properties relevant for transport through membrane proteins, such
as hydration energy. Apart from transport by the ZIP transporters SLC39A8 and SLC39A14, which is not topic of the review,
uptake of free Cd 2+ has been demonstrated for the Fe 2+/H + cotransporter divalent metal transporter 1. Moreover, the multiligand endocytic receptors megalin and cubilin take up cadmium-metallothionein
complexes via receptor-mediated endocytosis. The role of ATP binding cassette transporters in Cd 2+ efflux from cells is also discussed. Both the multidrug resistance-associated protein 1 and cystic fibrosis transmembrane
conductance regulator are likely to transport cadmium–glutathione complexes out of cells, whereas transport of free Cd 2+ by the multidrug resistance P-glycoprotein remains controversial. Finally, arguments for and against Cd 2+ transport by Ca 2+ channels are presented. Most N- and L-type Ca 2+ channels are closed at resting membrane potential (with the exception of CaV1.3 channels) and therefore unlikely to allow
significant Cd 2+ influx under physiological conditions. CaV3.1 and CaV3.2 T-type calcium channels are permeated by divalent metal ions, such
as Fe 2+ and Mn 2+ because of considerable “window” currents close to resting membrane potential and could be responsible for tonic Cd 2+ entry. TRPM7 and the mitochondrial Ca 2+ uniporter are other likely candidates for Cd 2+ transporters, whereas the role of Orai proteins, the store-operated calcium channels carrying Ca 2+ release-activated Ca 2+ current, in Cd 2+ influx remains to be investigated. 相似文献
9.
Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca 2+ entry into the cells via L-type voltage-gated Ca 2+ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond
pulse-induced effects on intracellular Ca 2+ level ([Ca 2+] i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca 2+ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude
of the rise in [Ca 2+] i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and
P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca 2+] i response of the cells, suggesting Ca 2+ influx occurs only via VGCCs. Lowering extracellular K + concentration from 5 to 2 mM or pulsing cells in Na +-free medium suppressed the pulse-induced rise in [Ca 2+] i in the majority of cells. Thus, both membrane potential and Na + entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca 2+ influx. However, activation of voltage-gated Na + channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca 2+] i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple
types of VGCCs in chromaffin cells in a manner involving Na + transport across the plasma membrane. Whether Na + transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined. 相似文献
10.
Cadmium is a highly toxic metal entering cells by a variety of mechanisms. Its toxic action is far from being completely understood, although specific interaction with the cellular calcium metabolism has been indicated. Metal ions that influence intracellular Ca 2+ concentrations or compete with Ca 2+ for protein binding sites may exert an effect on actin filaments, whose assembly and disassembly are both regulated by a number of calcium-dependent factors. Cadmium is such a metal. Much evidence demonstrates that cadmium interferes with the dynamics of actin filaments in various types of cells. Here we show that, at high (0.8–1.0 mM) concentrations, CdCl 2 causes actin denaturation. At such Cd 2+ concentrations, actin precipitates (really actin, as shown by SDS-PAGE, see Fig. 1B) in the form of irregular, disordered clots, clearly appreciable by electron microscopy. Denaturation seems to be reversible since, after Cd 2+ removal by dialysis, the polymerizability of sedimented actin is restored almost completely. On the other hand, at concentrations ranging from 0.25 to 0.6 mM, CdCl 2 is more effective as an actin polymerizing agent than both MgCl 2 and CaCl 2. The Cd-related increase in the actin assembly rate is ascribable to an enhanced nucleation rather than to an increased monomer addition to filament growing ends. The latter, in contrast, appears quite slow. Critical concentration measurements revealed that the extent of polymerization of both Mg- and Cd-assembled actin are very close ( Cc ranges from 0.25 to 0.5 μM), while Ca-polymerized actin shows a polymerization extent markedly lower ( Cc=4.0 μM). By both the fluorescent Ca 2+ chelator Quin-2 assay and limited proteolysis of actin by trypsin and α-chymotrypsin, the real substitution of G-actin-bound Ca 2+ by Cd 2+ has been appreciated. The increase in Quin-2 fluorescence after addition of excess CdCl 2 indicates that, in our experimental conditions, Ca 2+ tightly-bound to actin is partially (60–70%) replaced by Cd 2+, forming Cd-actin. Electrophoretic patterns after limited proteolysis reveal that the trypsin cleavage sites in the segment 61–69 of the actin polypeptide chain are less accessible in Cd-actin than in Ca-actin, although the cation-dependent effect is less pronounced in Cd-actin than in Mg-actin. Our results are consistent with some of the consequences on microfilament organization observed in Cd 2+-treated cells; however, considering the positive effect of Cd 2+ on actin polymerization in solution we have noticed that this was never observed in vivo. A different indirect effect of Cd 2+ on some cellular event(s) influencing cytoplasmic actin polymerization appears to be reasonable. © 1997 Elsevier Science B.V. All rights reserved. 相似文献
11.
Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd 2+) and lead (Pb 2+). Leadmium Green has been used in various paradigms, such as tracking Cd 2+ sequestration in plant cells, heavy metal export in protozoa, and Pb 2+ absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd 2+, Pb 2+, and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer’s claims, Leadmium Green was sensitive to Cd 2+ (K D ~600 nM) and also Pb 2+ (K D ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca 2+, Co 2+, Mn 2+ and Ni 2+. Leadmium Green also responded to Zn 2+ with a K D of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd 2+ or Zn 2+ in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn 2+ was much less than that produced by Cd 2+. When tested in a model of oxidant-induced liberation of endogenous Zn 2+, Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd 2+, particularly in models where Cd 2+ accumulates rapidly, and when concomitant fluctuations of intracellular Zn 2+ are minimal. 相似文献
12.
We investigated the effect of moderate Cu 2+ and Cd 2+ stress by applying chlorophyll (Chl) fluorescence and P 700 absorbance measurements to monitor the photosynthetic electron transport activity of 3-week-old Pisum sativum L. cv. Petit Proven?al plants grown in a modified Hoagland solution containing 50 ??M CuSO 4 or 5 ??M CdCl 2. Both heavy metals caused a slight inhibition in PSII photochemistry as indicated by the decrease in the effective quantum efficiency of PSII (?? PSII), the maximum electron transport capacity (ETR max), and the maximum quantum yield for electron transport (??). PSI photochemistry was also affected by these heavy metals. Cu 2+ and Cd 2+ decreased the quantum efficiency of PSI (?? PSI) as well as the number of electrons in the intersystem chain, and the Cu 2+ treatment significantly reduced the number of electrons from stromal donors available for PSI. These results indicate that PSII and PSI photochemistry of pea plants are both sensitive to moderate Cu 2+ and Cd 2+ stress, which in turn is easily detected and monitored by Chl fluorescence and P 700 absorbance measurements. Therefore, monitoring the photochemistry of pea plants with these noninvasive, yet sensitive techniques offers a promising strategy to study heavy metal toxicity in the environment. 相似文献
13.
Nitric oxide (NO) is a bioactive gas and functions as a signaling molecule in plants exposed to diverse biotic and abiotic
stresses including cadmium (Cd 2+). Cd 2+ is a non-essential and toxic heavy metal, which has been reported to induce programmed cell death (PCD) in plants. Here,
we investigated the role of NO in Cd 2+-induced PCD in tobacco BY-2 cells ( Nicotiana tabacum L. cv. Bright Yellow 2). In this work, BY-2 cells exposed to 150 μM CdCl 2 underwent PCD with TUNEL-positive nuclei, significant chromatin condensation and the increasing expression of a PCD-related
gene Hsr203J. Accompanied with the occurring of PCD, the production of NO increased significantly. The supplement of NO by sodium nitroprusside
(SNP) had accelerated the PCD, whereas the NO synthase inhibitor Nω-nitro- l-arginine methyl ester hydrochloride ( l-NAME) and NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) alleviated this
toxicity. To investigate the mechanism by which NO exerted its function, Cd 2+ concentration was measured subsequently. SNP led more Cd 2+ content than Cd 2+ treatment alone. By contrast, the prevention of NO by l-NAME decreased Cd 2+ accumulation. Using the scanning ion-selective electrode technique, we analyzed the pattern and rate of Cd 2+ fluxes. This analysis revealed the promotion of Cd 2+ influxes into cells by application of SNP, while l-NAME and cPTIO reduced the rate of Cd 2+ uptake or even resulted in net Cd 2+ efflux. Based on these founding, we concluded that NO played a positive role in CdCl 2-induced PCD by modulating Cd 2+ uptake and thus promoting Cd 2+ accumulation in BY-2 cells. 相似文献
14.
The inotropic Cd 2+ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd 2+ at concentrations of 1, 10, and 20 mM is established to decrease dose dependently (21.3, 50.3, and 72.0%, respectively) the
muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Na 2+-channels of the L-type (Ca v 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd 2+ at concentrations of 15 and 25 mM produces swelling of non-energized and energized mitochondria in isotonic (with KNO 2 and NH 2NO 3) and hypoosmotic (with 25 mM CH 3COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 mM Cd 2+ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the
medium with Cd 2+ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition
into the medium of DNP—uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate.
Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd 2+ is due not only to its direct blocking action on Ca 2+-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability
of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K + transport into the matrix of mitochondria, and inhibition of their respiratory chain. 相似文献
15.
Extracellular ATP triggers changes in intracellular Ca 2+, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors
might mediate the Ca 2+ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are
expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence
imaging of Ca 2+ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology
of the Ca 2+ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity
of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed
mRNA for P2Y 2, P2Y 6, and P2Y 12 metabotropic receptors and P2X 1, P2X 2, P2X 3, P2X 4, P2X 5, and P2X 7 ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca 2+, while 100 μ M suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca 2+ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely
with 1 μ M PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells,
and that membrane trafficking is regulated by a P2 receptor showing unusual properties. 相似文献
16.
The toxic metal ion cadmium (Cd 2+) induces pleiotropic effects on cell death and survival, in part through effects on cell signaling mechanisms and cytoskeletal dynamics. Linking these phenomena appears to be calmodulin‐dependent activation of the Ca 2+/calmodulin‐dependent protein kinase II (CaMK‐II). Here we show that interference with the dynamics of the filamentous actin cytoskeleton, either by stabilization or destabilization, results in disruption of focal adhesions at the ends of organized actin structures, and in particular the loss of vinculin and focal adhesion kinase (FAK) from the contacts is a result. Low‐level exposure of renal mesangial cells to CdCl 2 disrupts the actin cytoskeleton and recapitulates the effects of manipulation of cytoskeletal dynamics with biological agents. Specifically, Cd 2+ treatment causes loss of vinculin and FAK from focal contacts, concomitant with cytoskeletal disruption, and preservation of cytoskeletal integrity with either a calmodulin antagonist or a CaMK‐II inhibitor abrogates these effects of Cd 2+. Notably, inhibition of CaMK‐II decreases the migration of FAK‐phosphoTyr925 to a membrane‐associated compartment where it is otherwise sequestered from focal adhesions in a Cd 2+‐dependent manner. These results add further insight into the mechanism of the CaMK‐II‐dependent effects of Cd 2+ on cellular function. J. Cell. Biochem. 114: 1832–1842, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
17.
The effect of cadmium on the photosynthetic activity of Synechocystis PCC 6803 was monitored in this study. The oxygen evolving capacity of Synechocystis treated with 40 μM CdCl 2 was depressed to 10% of the maximum in 15 min, indicating that Cd 2+ penetrated rapidly into the cells and blocked the photosynthetic activity. However, neither photosystem II (PSII) nor photosystem I (PSI) activity showed a significant short-term decrease which would explain this fast decrease in the whole-chain electron transport. Thermoluminescence measurements have shown that the charge separation and stabilization in PSII remains essentially unchanged during the first few hours following the Cd 2+ treatment. The electron flow through PSI was monitored by following the redox changes of the P700 reaction centers of PSI. Alterations in the oxidation kinetics of P700 in the Cd 2+-treated cells indicated that Cd 2+ treatment might affect the available electron acceptor pool of P700, including the CO 2 reduction and accumulation in the cells. Perturbed angular correlation of γ-rays (PAC) using the radioactive 111mCd isotope was used to follow the Cd 2+ uptake at a molecular level. The most plausible interpretation of the PAC data is that Cd 2+ is taken up by one or more Zn proteins replacing Zn 2+ in Synechocystis PCC 6803. Using the radioactive 109Cd isotope, a protein of approximately 30 kDa that binds Cd 2+ could be observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results indicate that Cd 2+ might inactivate different metal-containing enzymes, including carbonic anhydrase, by replacing the zinc ion, which would explain the rapid and almost full inhibition of the photosynthetic activity in cyanobacteria. 相似文献
19.
Abscisic acid (ABA), a widely known phytohormone involved in the plant response to abiotic stress, plays a vital role in mitigating Cd2+ toxicity in herbaceous species. However, the role of ABA in ameliorating Cd2+ toxicity in woody species is largely unknown. In the present study, we investigated ABA restriction on Cd2+ uptake and the relevance to Cd2+ stress alleviation in Cd2+-hypersensitive Populus euphratica. ABA (5 μM) markedly improved cell viability and growth but reduced membrane permeability in CdCl2 (100 μM)-stressed P. euphratica cells. Moreover, ABA significantly increased the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), contributing to the scavenging of Cd2+-elicited H2O2 within P. euphratica cells during the period of CdCl2 exposure (100 μM, 24–72 h). ABA alleviation of Cd2+ toxicity was mainly the result of ABA restriction of Cd2+ uptake under Cd2+ stress. Steady-state and transient flux recordings showed that ABA inhibited Cd2+ entry into Cd2+-shocked (100 μM, 30 min) and short-term-stressed P. euphratica cells (100 μM, 24–72 h). Non-invasive micro-test technique data showed that H2O2 (3 mM) stimulated the Cd2+-elicited Cd2+ influx but that the plasma membrane (PM) Ca2+ channel inhibitor LaCl3 blocked it, suggesting that the Cd2+ influx was through PM Ca2+-permeable channels. These results suggested that ABA up-regulated antioxidant enzyme activity in Cd2+-stressed P. euphratica and that these enzymes scavenged the Cd2+-elicited H2O2 within cells. The entry of Cd2+ through the H2O2-mediated Ca2+-permeable channels was subsequently restricted; thus, Cd2+ buildup and toxicity were reduced in the Cd2+-hypersensitive species, P. euphratica. 相似文献
20.
Despite intense research, the mechanism of Cd 2+ toxicity on photosynthesis is still elusive because of the multiplicity of the inhibitory effects and different barriers in plants. The quick Cd 2+ uptake in Synechocystis PCC 6803 permits the direct interaction of cadmium with the photosynthetic machinery and allows the distinction between primary and secondary effects. We show that the CO 2‐dependent electron transport is rapidly inhibited upon exposing the cells to 40 µm Cd 2+ (50% inhibition in ~15 min). However, during this time we observe only symptoms of photosystem I acceptor side limitation and a build of an excitation pressure on the reaction centres, as indicated by light‐induced P700 redox transients, O 2 polarography and changes in chlorophyll a fluorescence parameters. Inhibitory effects on photosystem II electron transport and the degradation of the reaction centre protein D1 can only be observed after several hours, and only in the light, as revealed by chlorophyll a fluorescence transients, thermoluminescence and immunoblotting. Despite the marked differences in the manifestations of these short‐ and long‐term effects, they exhibit virtually the same Cd 2+ concentration dependence. These data strongly suggest a cascade mechanism of the toxic effect, with a primary effect in the dark reactions. 相似文献
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