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1.
In previous studies cadmium chloride (CdCl 2) nonlethally inhibited Y-1 mouse adrenal tumor cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. In addition, dibutyryl cAMP-stimulated 20DHP secretion was unaffected by CdCl 2, while the site of the unstimulated effect was indirectly shown to involve steps between endogenous cholesterol utilization and 20-hydroxycholesterol association with mitochondrial cytochrome P450 side-chain cleavage enzyme. In the present study we determined CdCl 2 effects on plasma membrane sites preceding pre-dbcAMP-stimulation of 20DHP secretion. Y-1 cells were incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited adrenocorticotropin- (ACTH)-stimulated steroid secretion by 50%) together with exogenously added maximally stimulating concentrations of ACTH, cholera toxin, forskolin, or adenosine triphosphate Cholera toxin, forskolin and ATP bypass specific plasma membrane sites involved in the synthesis of intracellular cAMP and activate the steroid hormone biosynthetic pathway. Cadmium effects on ACTH-stimulated endogenous cAMP secretion were also examined. CdCl 2 significantly reduced Y-1 cell 20DHP secretion following exposure to ACTH, cholera toxin, forskolin, and ATP; it also significantly decreased endogenous cAMP secretion into culture medium. These data may be interpreted to suggest that CdCl 2 altered Y-1 cell regulation of adenyl cyclase activity, which reduced cAMP-activated cholesterol uptake by mitochondria as a consequence.Abbreviations ACTH
adrenocorticotropin
- ATP
adenosine triphosphate
- ANOVA
analysis of variance
- CdCl 2 or Cd 2+
cadmium chloride
- cAMP
cyclic 3,5-adenosine monophosphate
- CTX
cholera toxin
- dbcAMP
dibutyryl cAMP, N, O-dibutyryl-3,5-adenosine monophosphate
- EGTA
ethylene glycol bis tetraacetic acid
- FMEM
serum-free Eagle's Minimum Essential Medium with all other supplements
- FSK
forskolin
- Hepes
N-2-hydroxyethylpiperazine- N-1,2-ethanesulfonic acid
- IC 50'
concentration inhibiting stimulated steroid secretion by 50%
- IU
international unit
- MEM
Eagle's Minimum Essential Medium
- P450scc
cytochrome P450 side-chain cleavage enzyme
- PREG
pregnenolone
- PROG
progesterone
- SEM
standard error of the mean
- SMEM
serum-containing Eagle's Minimum Essential Medium with supplements
- 20DHP
20--hydroxy-4-pregnen-3-one 相似文献
2.
In vitro and in vivo cadmium toxicity studies focus almost exclusively on CdCl 2 effects. Only a few studies have used adrenocortical cells and tissue to determine cadmium salt effects during stress of adrenocorticotropin stimulation. Because several biologically relevant water-soluble cadmium salts exist, this study extended work with CdCl 2 to evaluate the acute adrenocortical cell steroid secretory responses to non-lethal cadmium acetate (CdAc 2) and CdSO 4
4 concentrations. Control or ACTH-stimulated cultured Y-1 mouse adrenal tumor cells (ATCC) which secrete 20-dihydroprogesterone (20-DHP) were incubated for 0.5 h in serum-free medium (FMEM) with or without 0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 500.0 and 1000.0 µg CdAc 2 or CdSO 4/ml FMEM (1.9, 3.8, 19.0, 38.0, 190.0, 380.0 and 1900.0 µmol/L, respectively). For each salt, cell viability was measured at the end of the incubation using live cell trypan blue exclusion. In addition, cumulative CdAc 2 effects during 4 h incubations and effect reversibility were determined for control and stimulated cells. After each experimental incubation, the 20-DHP secreted into the medium was determined by radioimmunoassay. Over 80% of all control or ACTH-stimulated cells were viable after incubation in the presence or absence of various CdAc 2 or CdSO 4 concentrations. Cadmium acetate and sulfate inhibited basal and ACTH-stimulated steroid secretion in a dose-dependent manner. For basal steroid secretion the CdAc 2 concentration that first significantly inhibited was 0.5 µg/ml medium (1.9 µmol/L); stimulated secretion was significantly inhibited beginning at 5.0 µg/ml (19.0 µmol/L) and the concentration reducing stimulated 20-DHP secretion by 50% (IC 50) was 5.6 µg/ml (21.3 µmol/L). Similarly, the first CdSO 4 concentration to significantly inhibit basal and ACTH-stimulated steroid secretion was 10.0 µg/ml medium (39.0 µmol/L); the IC 50 was 7.8 µg/ml (29.8 µmol/L). Except that basally secreting Cd 2+
2+-treated cells almost doubled 20-DHP secretion after Cd 2+ removal and subsequent incubation with ACTH, all basal and ACTH-stimulated steroid secretion was irreversibly inhibited by every CdAc 2 concentration. All CdAc 2 concentrations initiated and maintained cumulative inhibitory effects on basal and ACTH-stimulated steroid secretion over a 4 h period. Reversibility and cumulative CdSO 4 treatment studies were not conducted. Based on the results from the present studies, both CdAc 2 and CdSO 4 appeared to incrementally inhibit control and ACTH-stimulated steroidogenesis without affecting cell viability and to be more potent inhibitors of adrenocortical cell steroid secretion than CdCl 2. Finally, CdAc 2 effects on control and stimulated cells were cumulative and irreversible. 相似文献
3.
In previous studies cadmium chloride (CdCl 2) nonlethally inhibited Y-1 adrenal mouse adrenal tumour cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. We studied CdCl 2 effects on unstimulated steroidogenesis using Y-1 cells incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited ACTH-stimulated steroid secretion by 50%). Exogenously added 20-hydroxycholesterol (20OHC), 22( R)-hydroxycholesterol (22OHC), 25-hydroxycholesterol (25OHC), pregnenolone (PREG), or progesterone (PROG) were used to bypass any rate-limited steroidogenic pathway sites that CdCl 2 might inhibit. 25OHC is a biologically active nonpathway steroid, while 20OHC, 22OHC, PREG, and PROG are pathway steroids; each increased unstimulated 20DHP secretion nearly 10-fold. Although CdCl 2 could not reduce dibutyryl cyclic AMP- (dbcAMP)-stimulated 20DHP secretion significantly, it did significantly reduce basal and 25OHC-induced 20DHP secretion 25% below untreated levels. When 20OHC, 22OHC, PREG, or PROG were incubated with unstimulated Y-1 cells, their synthesis into 20DHP was unaffected by cadmium. dbcAMP bypasses the plasma membrane enzyme complex that synthesizes intracellular cAMP during exogenous ACTH stimulation; dbcAMP was not inhibited by CdCl 2. The rate-limited step accelerated by cAMP involves plasma membrane and/or cytoplasmic cholesterol transport to and through outer and inner mitochondrial membranes before the cholesterol is synthesized into pregnenolone by side-chain cleavage enzymes on the inner membrane matrix face. Little is known regarding the mechanisms controlling unstimulated steroidogenesis. Under unstimulated conditions the 25-, 20- and 22( R)-monohydroxyls of cholesterol facilitate plasma membrane, cytoplasm and inner and outer mitochondrial solubility, diffusion and/or transport to bypass rate-limited steps and augment unstimulated steroid synthesis. Since conversion of endogenous mitochondrial cholesterol and 25OHC, but not dbcAMP-mobilized cytoplasmic cholesterol, 20OHC or 22OHC conversion, to 20DHP is inhibited by CdCl 2, this suggests that (a) control of mitochondrial cholesterol supplies is independent of the cAMP-regulated mitochondrial steps in the 20DHP steroid synthetic pathway, (b) CdCl 2 specifically inhibited endogenous mitochondrial cholesterol and 25OHC utilization, (c) CdCl 2 toxicity may affect adrenal, testicular, ovarian, and placental basal steroidogenic functions, and (d) 25OHC may be a useful compound to examine unstimulated steroid synthesisAbbreviations ACTH
adrenocorticotropin
- ANOVA
analysis of variance
- CdCl 2
cadmium chloride
- cAMP
cyclic 3,5-adenosine monophosphate
- DMSO
dimethylsulfoxide
- DNA
deoxyribonucleic acid
- FMEM
serum-free Eagle's Minimum Essential Medium
- Hepes
N-2-hydroxyethyl-piperazine- N-1,2-ethanesulfonic acid
- 20OHC
20-hydroxycholesterol
- 22OHC
22( R)-hydroxycholesterol
- 25OHC
25-hydroxycholesterol
- IC 50'
concentration inhibiting stimulated steroid secretion by 50%
- IU
international unit
- MEM
Eagle's Minimum Essential Medium
- P450scc
cytochrome P450 side-chain cleavage enzyme
- PREG
pregnenolone
- PROG
progesterone
- RNA
ribonucleic acid
- SEM
standard error of the mean
- SMEM
serum-containing Eagle's Minimum Essential Medium
- 20DHP
20-hydroxy-4-pregnen-3-one 相似文献
4.
Cultured Y-1 mouse adrenal tumor cells, which secrete 20--hydroxy-4-pregnen-3-one (20-DHP), were used to investigate the acute nonlethal effects of incremental cadmium chloride (CdCl 2) concentrations on basal and maximally stimulated steroid secretion. In addition, cumulative CdCl 2 effects during 4-hr incubations, effect reversibility, and viability were determined. Cells were incubated in 1 ml serum-free Eagle's Minimal Essential Medium (FMEM) with or without 0.5 IU (ca. 1.5 M) adrenocorticotropin (ACTH) in the presence or absence of CdCl 2. Following incubation, cell viability was quantitated using trypan blue exclusion. The 20-DHP secreted into the experimental incubation medium was measured by radioimmunoassay. CdCl 2 levels of 10.0 g/ml or greater significantly inhibited basal 30 min steroid secretion in a dose-dependent manner; ACTH-stimulated steroid secretion was significantly inhibited by levels 5.0 g/ml or greater. At least 80% of all control and stimulated cells in the presence or absence of cadmium ions excluded trypan blue. The reduction in ACTH-stimulated steroid secretion was greater than the reduction in basal steroid secretion at any cadmium concentration level. The CdCl 2 concentration that reduced stimulated steroid hormone secretion by 50% (IC 50) was 45.0 g/ml. Exposing Y-1 cells to either 5.0, 10.0, 45.0 or 500.0 g CdCl 2/ml FMEM for periods ranging from 0.5 to 4 hr inhibited ACTH-stimulated steroid secretion in a time-dependent manner. After 30 min exposure to 10.0, 45.0 or 500.0 g CdCl 2/ml FMEM with or without ACTH, cadmium inhibition was irreversible. When 5.0 g CdCl 2/ml was used, basal and stimulated inhibition was reversible by reincubating in medium containing ACTH alone. The relatively greater cadmium effects on ACTH stimulated steroidogenesis might suggest that cadmium modulated the rate-limited transducing system between the ACTH plasma membrane receptor complex and cholesterol side-chain cleaving mitochondrial enzymes. However, cadmium influences on basal secretion indicated effects on the non-rate-limited steroidogenic pathway.Abbreviations ACTH
adrenocorticotropin
- ANOVA
analysis of variance
- CdCl 2
cadmium chloride
- Ci
Curie
- DNA
deoxyribonucleic acid
- FMEM
serum-free Eagle's Minimum Essential Medium
- HEPES
N-2-hydroxyethylpiperazine- N-1,2-ethanesulfonic acid
- IC 50
concentration inhibiting stimulated steroid secretion by 50%
- IU
international unit
- MEM
Eagle's Minimum Essential Medium
- RIA
radioimmunoassay
- RNA
ribonucleic acid
- SEM
standard error of the mean
- SMEM
serum-containing Eagle's Minimum Essential Medium
- 20-DHP
20--hydroxy-4-pregnen-3-one 相似文献
5.
Although cadmium-induced apoptosis of lymphocytes is one of common features in the immunotoxicity of cadmium, the membrane
pathway for intracellular cadmium accumulation is not fully elucidated. To characterize membrane Cd 2+ transport of rat thymocytes, the change in intracellular Cd 2+ concentration under various conditions was examined by the use of Fluo-3, a fluorescent probe for monitoring the change in
intracellular concentration of divalent metal cations. The membrane Cd 2+ transport was estimated by the augmentation of Fluo-3 fluorescence induced by bath application of CdCl 2. Lowering temperature strongly suppressed the augmentation of Fluo-3 fluorescence by CdCl 2, suggesting that the metabolic process can be involved in membrane Cd 2+ transport. External acidification (decreasing pH) and membrane depolarization by adding KCl attenuated the augmentation,
indicating the requirement of electrochemical driving force for membrane Cd 2+ transport into the cells. Bath application of CaCl 2 and ZnCl 2 equally decreased the augmentation, suggesting their competition with Cd 2+ at the membrane transport. The augmentation by CdCl 2 was lesser in the cells treated with N-ethylmaleimide inducing chemical depletion of cellular thiols. The result suggests
the contribution of sulfhydryl groups to membrane Cd 2+ transport. Taken together, it is suggested that the cells possess a temperature-sensitive membrane Cd 2+ pathway, driven by electrochemical gradient of Cd 2+ and transmembrane potential, with competitive binding site. Based on the characteristics described above, it is unlikely
that the membrane Cd 2+ transport in rat thymocytes is attributed to a single transport system although it has characteristics that are similar to
those of divalent cation transporter 1. 相似文献
6.
Treatment of Catharanthus roseus hairy roots with antagonists, like verapamil and CdCl 2, that block the Ca 2+ flux across the plasma membrane enhanced the total alkaloid content by 25% and their secretion 10 times. The specific Ca 2+ chelator, EGTA, stimulated 90% of the total alkaloid secretion. Treatment with inhibitors of intracellular Ca 2+ movement, like TMB-8 and trapsigargin, enhanced the total alkaloid content by 74% and their secretion into the culture media by 4- to 6-fold. The results suggest that an inhibition of external and internal Ca 2+ fluxes induces an increase in the indole alkaloid accumulation and secretion in C. roseus hairy roots. 相似文献
7.
Prolactin (PRL) release and intracellular free calcium concentration [Ca 2+] i were measured in two populations of normal rat lactotrophs (light and heavy fractions) in culture. Spontaneous PRL release of heavy fraction cells was more sensitive to dihydropyridines (DHPs; Bay K 8644 and nifedipine) when compared to the light fraction lactotrophs. The stimulatory effect of thyrotropin-releasing hormone (TRH) on PRL release from heavy fraction cells was inhibited by Cd 2+ and mimicked by Bay K 8644. Indo-1 experiments revealed that TRH-increased [Ca 2+] i was reversibly inhibited by Cd 2+. In a Ca 2+-free EGTA-containing medium, TRH did not modify [Ca 2+] i.Abbreviations [Ca 2+] i
intracellular free calcium concentration
- DA
dopamine
- DHP
dihydropyridine(s)
- DMEM
Dulbecco's Modified Eagle's Medium
- Ins(1,4,5)P 3
inositol 1,4,5-trisphosphate
- PRL
prolactin
- RIA
radioimmunoassay
- TRH
thyrotropin-releasing hormone
- VGCC
voltage-gated calcium channel 相似文献
8.
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
9.
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
10.
We have studied Cd 2+-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd 2+] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 M, Cd 2+ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 M, Cd 2+ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO 3 or NH 4NO 3 medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd 2+ effects on mitochondria treated in sequence with 100 M of Ca 2+, Sr 2+, Mn 2+ or Ba 2+(Me 2+) and 7.5 M RR, as well as the alterations in Cd 2+ action on the uptake of 137Cs + by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca 2+ or RR. The evidence obtained indicate that Ca 2+ exhibits a synergestic action on all Cd 2+ effects examined, whereas Sr 2+ and Mn 2+, conversely, are antagonistic. In the presence of RR, the Cd 2+ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd 2+, conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd 2+-stimulated swelling and 137Cs + (analog of K +) uptake in the acetate media. For the first time, we have shown that Cd 2+-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition. 相似文献
11.
Intact cells of Synechococcus elongatus were treated with different concentrations (0.1 and 1.0 mM = Cd 0.1, Cd 1.0) of CdCl 2 for 24 h. Cd 0.1 treatment stimulated growth of the cell culture and chlorophyll (Chl) a concentration in the culture. Cd 1.0 inhibited both the above mentioned parameters. The oxygen evolving activity of intact cells (H 2O BQ) as well as of isolated thylakoid membranes, TM (H 2O DCPIP; H 2O PBQ + FeCy) decreased after 24 h of Cd 1.0 cultivation to 7 %. Photosystem 1 (PS1) activity was less sensitive to the effect of Cd 2+ than PS2 activity. CdCl 2 concentration in cultivation media after 24 h of cultivation proved that the cyanobacterium cells take up these ions to a large extent from the cultivation medium. After 24 h of the Cd 1.0 treatment only 12 % of the amount of Cd 2+ originally added to the cultivation medium was found. The ratio of external-antenna pigments, phycocyanin, and allophycocyanin to Chl increased approximately twofold with growing Cd 2+ concentration in the cultivation medium. This ratio was found in both TM and dodecylmaltoside extracts. 相似文献
12.
Evidence for a primary role for intracellular Ca 2+ in the stimulation of pancreatic enzyme secretion is reviewed. Measurements of cytoplasmic free Ca 2+ concentration have allowed direct demonstration of its importance in triggering enzyme secretion and defined the concentration range over which membrane Ca 2+ pumps must work to regulate intracellular Ca 2+. Current evidence suggests a key role for the Ca 2+ Mg-ATPase of rough endoplasmic reticulum in regulating intracellular Ca 2+ and accumulating a Ca 2+ store which is released by the action of inositol-l,4,5 trisphosphate following stimulation of secretion.Abbreviations Used EGTA
(ethylene dioxy) diethylene-dinitrilotetraacetic acid
- BAPTA
1,2-bis (2-aminophenoxy) ethane NNN,N-tetracetic acid
- InsP 3
inositol trisphosphate
- Ins-1,4,5P 3 and Ins-1,3,4P 3
isomers of inositol trisphosphate with the position of phosphate groups assigned
- Ins-1,3,4,5P 4
inositol tetrakisphosphate 相似文献
13.
The transport of Cd 2+ and the effects of this ion on secretory activity and metabolism were investigated in β cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 μmol/kg dry wt. After 60 min of incubation in a Ca 2+-deficient medium containing 2.5 μM Cd 2+ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca 2+. The incorporation of Cd 2+ was stimulated either by raising the concentration of glucose to 20 mM or K + to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K +. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd 2+-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd 2+ efflux into a Ca 2+-deficient medium. Concentrations of Cd 2+ up to 2.5 μM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd 2+ concentration was above 10 μM. Basal insulin release was stimulated by 5 μM Cd 2+. At a concentration of 160 μM, Cd 2+ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the β cell uptake of Cd 2+ is facilitated by the activation of voltage-dependent Ca 2+ channels. Apparently, the accumulation of Cd 2+ mimics that of Ca 2+ also involving a component of intracellular sequestration promoted by glucose. 相似文献
14.
Summary The inhibition of Ca 2–-ATPase, (Na ++K +)-ATPase and Na +/Ca 2+ exchange by Cd 2+ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven 45Ca 2+ uptake into inside-out membrane vesicles displayed a K
m
for Ca 2+ of 88±17 nm, and was extremely sensitive to Cd 2+ with an IC 50 of 8.2±3.0 pM Cd 2+, indicating an inhibition via the Ca 2+ site. (Na ++K +)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd 2+, displaying an IC 50 of 2.6±0.6 m Cd 2+. Cd 2+ ions apparently compete for the Mg 2+ site of the (Na – +K +)-ATPase. The Na +/Ca 2+ exchanger was inhibited by Cd 2+ with an IC 50 of 73±11 n m. Cd 2+ is a competitive inhibitor of the exchanger via an interaction with the Ca 2+ site (K
i
= 11 n m). Bepridil, a Na + site specific inhibitor of Na +/Ca 2+ exchange, induced an additional inhibition, but did not change the K
i
of Cd 2+. Also, Cd 2+ is exchanged against Ca 2+, albeit to a lesser extent than Ca 2+. The exchanger is only partly blocked by the binding of Cd 2+. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na +/Ca 2+ exchanger. We conclude that intracellular Cd 2+ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.We would like to thank Dr. D. Fackre (University of Alberta, Canada) for stimulating discussions and Mr. F.A.T. Spanings (University of Nijmegen, The Netherlands) for excellent fish husbandry. The fura-2 measurements of intracellular Ca 2+ concentrations in tilapia enterocytes were carried out in the Department of Physiology, School of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Th.J.M. Schoenmakers and G. Flik were supported by travel grants from the Foundation for Fundamental Biological Research (BION) and the Netherlands Organization for Scientific Research (NWO). 相似文献
15.
Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca 2+ transport have been examined in intact human red blood cells. Active Ca 2+ efflux was determined from the initial rate of 45Ca 2+ loss after CoCl 2 was added to block Ca 2+ loading via the ionophore A23187. Ca 2+-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca 2+ in the presence of A23187. The apparent Ca affinity of active Ca 2+ efflux ( K
0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca 2+-ATPase assay ( K
0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca 2+ efflux and Ca 2+-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg 2+-depleted cells, and completely restored by reintroduction of intracellular Mg 2+. Active Ca 2+ efflux was inhibited almost completely by raising external CaCl 2 (but not MgCl 2) to 20 mm, probably by interaction of Ca 2+ at the externally oriented E 2P conformation of the pump. Cd 2+ was more potent than Ca 2+ in this inhibition, while Mn 2+ was less potent and 10 mm Ba 2+ was without effect. A Ca 2+: proton exchange mechanism for active Ca 2+ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca 2+ efflux at least twofold above the efflux rate at pH 7.8 Ca 2+ transport was not affected by decreasing the membrane potential across the red cell. 相似文献
16.
Acute effects of heavy metal ions on shrimp have been an area of intense study worldwide. However, the molecular mechanism by which cadmium-induced injury occurs remains largely unclear, and methods for mitigating toxicity in vivo have rarely been reported. In this study, the changes in respiratory burst and intracellular free calcium in haemocytes of pacific white shrimp, Litopenaeus vannamei, after exposure to Cd 2+ (CdCl 2) were examined using flow cytometry. Meanwhile, DNA damage and repair in haemocytes and hepatopancreas cells were studied using the comet assay. Respiratory burst generation, intracellular Ca 2+ concentration ([Ca 2+]i) and DNA damage in haemocytes and hepatopancreas cells all exhibited a dose-dependent increase and a time-dependent change after treatment with Cd 2+ compared with controls. These results indicate that Cd can induce oxidative stress and DNA damage in the shrimp L. vannamei. Moreover, the results also demonstrate that these parameters can be used as sensitive indicators of exposure to this genotoxicant. 相似文献
17.
Abstract: Carbachol or elevated K + stimulated 45Ca 2+ uptake into chromaffin cells two- to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca 2+ uptake and catecholamine secretion induced by the mixed nicotinic-muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca 2+ uptake occurred within 15 s of stimulation by carbachol or elevated K + at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K + was similar to that of Ca 2+ uptake. There was a close correlation between Ca 2+ uptake and catecholamine secretion at various concentrations of Ca 2+. The concentration dependencies for inhibition of both processes by Mg 2+ or Cd 2+ were similar. Ca 2+ uptake saturated with increasing Ca 2+ concentrations, with an apparent Km for both carbachol-induced and elevated K +-induced Ca 2+ uptake of approximately 2 m M. The Ca 2+ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca 2+ entry and a presumed increase in cytosolic Ca 2+ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca 2+- dependent processes associated with catecholamine secretion. Ca 2+ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage-sensitive Ca 2+ channels. 相似文献
18.
The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca 2 + concentration ([Ca 2 +] i) and proliferation was examined by using the Ca 2 +-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca 2 +] i in a concentration-dependent manner. Celecoxib-induced [Ca 2 +] i increase was partly reduced by removal of extracellular Ca 2 +. Celecoxib-induced Ca 2 + influx was independently suggested by Mn 2 + influx-induced fura-2 fluorescence quench. In Ca 2 +-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2 +-ATPase, caused a monophasic [Ca 2 +] i increase, after which celecoxib only induced a tiny [Ca 2 +] iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca 2 +] i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca 2 +] i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca 2 +] i increase in renal tubular cells by stimulating both extracellular Ca 2 + influx and intracellular Ca 2 + release and is highly toxic to renal tubular cells in vitro. 相似文献
19.
Intracellular Ca 2+ (Ca i) signaling following the binding of surface receptors activates a Ca 2+ permeable plasma membrane conductance which has been shown to be associated with store depletion in a number of cell types. We examined the activation of this conductance in human monocyte-derived macrophages (HMDMs) using whole-cell voltage-clamp techniques coupled with fura-2 microfluorimetry and characterized the importance of external pH (pH o) as a modulator of current amplitude. Current activation was observed following experimental maneuvers designed to deplete intracellular Ca 2+-stores including: (i) dialysis of the cell with 100 m inositol 1,4,5-triphosphate (IP 3), (ii) intracellular dialysis with high concentrations of the Ca 2+ buffers EGTA and BAPTA, or (iii) exposure of the cell to the Ca 2+-ATPase inhibitor thapsigargin (1 m). Currents associated with store depletion were inwardly rectifying with kinetics, inactivation, and selectivity that appeared similar irrespective of the mode of activation. Currents were Ca 2+ selective with a selectivity sequence of Ca 2+ > Sr 2+ Mg 2+ = Mn 2+ = Ni 2+. The Ca 2+ influx current was modulated by changes in pH o; modulation was not produced as a consequence of changes in internal pH (pH i). External acidification led to a reversible reduction in current amplitude with a pKa at pH 8.2. Changes in pH o alone failed to induce current activation. These observations are consistent with a scheme by which changes in pH o, as would be encountered by macrophages at sites of inflammation, could change the time course and magnitude of the Ca i transient associated with receptor activation by regulating the influx of Ca 2+ ions.The authors wish to gratefully acknowledge the expert technical assistance of Weiwen Xie without whom the study could not have been completed. This work was supported by National Institutes of Health GM36823. 相似文献
20.
Summary 1. Real-time monitoring of dopamine (DA) release from rat striatal slices demonstrated that endothelin (ET)-3 (0.1–10 M) produced a biphasic DA release consisting of transient and sustained components. When extracellular Ca 2+ was removed, the sustained but not transient response remarkably decreased.2. ET-3 (1–10 M) stimulated an increase in the intracellular Ca 2+ concentration ([Ca 2+] i), which also consisted of two components. The external Ca 2+ depletion inhibited primarily the sustained component of the Ca 2+ response to ET-3.3. ET-3 increased inositol 1,4,5-trisphosphate (IP3) concentrations in striatal slices. This response peaked at 10 to 20 sec and returned to the basal level 2 min after stimulation, an event which was in good accord with a prompt and transient phase of both cytosolic Ca 2+ activity and DA release evoked by ET-3.4. Thus, ET-3 produces a transient and a sustained release of DA from striatal slices by stimulating intracellular Ca 2+ mobilization via IP3 formation and extracellular Ca 2+ influx, respectively. 相似文献
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