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1.
Koh-ichi Enomoto Kishio Furuya Shunichi Yamagishi Takami Oka Takashi Maeno 《Cell biochemistry and function》1995,13(4):279-286
The relationship between the increase of intracellular Ca2+ and the release of arachidonic acid by bradykinin and pyrophosphonucleotides was studied in cultured mammary tumour cells, MMT060562. Bradykinin, ATP, UTP and UDP induced an increase of intracellular Ca2+ and the release of arachidonic acid from phospholipids into the extracellular fluid. Release of arachidonic acid was also induced by the application of the Ca2+ ionophore, A23187. Liberation of arachidonic acid by bradykinin and ATP was reduced by mepacrine, a blocker of phospholipase A2 and W-7, a calmodulin antagonist. It is suggested that the increase in cytosolic Ca2+-induced release of arachidonic acid occurs through activation of calmodulin-dependent phospholipase A2. 相似文献
2.
We examined the effect of tricyclic antidepressants on intracellular Ca2+ signalling in cultured cells of neuronal and glial origin. High concentrations of amitriptyline and desipramine increased the intracellular Ca2+ in PC-12 and U-87 MG cells. In PC-12 cells amitriptyline induced a biphasic rise in intracellular Ca2+. A rapid and transient increase due to release of Ca2+ from intracellular pools was followed by sustained elevation of [Ca2+]i due to influx from the extracellular medium. Desipramine evoked the Ca2+ release from intracellular pools but the influx of Ca2+ was not elicited. In U-87 MG cells both the drugs induced Ca2+ release from intracellular pools, however amitriptyline also induced a transient influx of Ca2+. To delineate the mechanisms involved in mobilization of Ca2+ by the drugs pharmacological agents that inhibit IP3 formation in cells and Ca2+ channel blockers were used and changes in [Ca2+]i and membrane potential were monitored. The results show that both the drugs release Ca2+ from IP3 sensitive pools by activation of phospholipase C and amitriptyline in addition activates a non specific cation channel in the plasma membrane of cells. Paradoxically at relatively lower concentrations (< 50 M) amitriptyline and desipramine inhibited the Ca2+ signal induced by adenosine triphosphate in both the cell types. Our data demonstrate that tricyclic antidepressants at different doses may have inhibitory or stimulatory effects on cellular Ca2+ signalling. 相似文献
3.
In order to examine the role of phospholipids in the activation of membrane bound Ca2+/Mg2+ ATPase, the activities of Ca2+ ATPase and Mg2+ ATPase were studied in heart sarcolemma after treatments with phospholipases A, C and D. The Mg2+ ATPase activity was decreased upon treating the sarcolemmal membranes with phospholipases, A, C and D; phospholipase A produced the most dramatic effect. The reduction in Mg2, ATPase activity by each phospholipase treatment was associated with a decrease in the Vmax value without any changes in the Ka value. The depression of Mg2+ ATPase in the phospholipase treated preparations was not found to be due to release of fatty acids in the medium and was not restored upon reconstitution of these membranes by the addition of synthetic phospholipids such as lecithin, lysolecithin or phosphatidic acid. In contrast to the Mg2+ ATPase, the sarcolemmal Ca2+ ATPase was affected only slightly by phospholipase treatments. The greater sensitivity of Mg- ATPase to phospholipase treatments was also apparent when deoxycholate-treated preparations were employed. These results indicate that glycerophospholipids are required for the sarcolemmal Mg2+ ATPase activity to a greater extent in comparison to that for the Ca2+ ATPase activity and the phospholipids associated with Mg2+ ATPase are predominantly exposed at the outer surface of the membrane. 相似文献
4.
Summary Calcium signaling systems in nonexcitable cells involve activation of Ca2+ entry across the plasma membrane and release from intracellular stores as well as activation of Ca2+ pumps and inhibition of passive Ca2+ pathways to ensure exact regulation of free cytosolic Ca2+ concentration ([Ca2+]
i
). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca2+ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca2+ entry and release while bradykinin appeared only to release Ca2+ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca2+]
i
response to these agonists was examined by four methods. Low concentrations of TPA (2×10–10
m) had no effect on Ca2+ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca2+]
i
to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca2+]
i
changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca2+ entry, fura-2 loaded cells were suspended in Mn2+ rather than Ca2+ buffers. Addition of EGF or TGF- resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10–8
m) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca2+ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca2+]
i
, showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca2+]
i
transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca2+ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca2+]
i
signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca2+ for EGF-mediated Ca2+ release which also respond differently to TPA. 相似文献
5.
Yorimasa Ogata Naomi Niisato Tetsuo Negishi Takeshi Sakurai Shunsuke Furuyama Hiroshi Sugiya 《Cell biology international》1995,19(8):637-646
In fura-2-loaded human periodontal ligament (HPDL) cells, bradykinin induced a rapidly transient increase and subsequently sustained increase in cytosolic Ca2+ ([Ca2+]i). When external Ca2+ was chelated by EGTA, the transient peak of [Ca2+]i was reduced and the sustained level was abolished, implying the Ca2+ mobilization consists of intracellular Ca2+ release and Ca2+ influx. Thapsigargin, a specific Ca2+-ATPase inhibitor for inositol 1,4,5-trisphosphate (1,4,5-1P3)-sensitive Ca2+ pool, induced an increase in [Ca2+]i in the absence of external Ca2+. After depletion of the intracellular Ca2+ pool by thapsigargin, the increase in [Ca2+]i induced by bradykinin was obviously reduced. Bradykinin also stimulated formation of inositol polyphosphates including 1,4,5-IP3. These results suggest that bradykinin stimulates intracellular Ca2+ release from the 1,4,5-1P3-sensitive Ca2+ pool. Bradykinin stimulated prostaglandin E2 (PGE2) release in the presence of external Ca2+, but not in the absence of external Ca2+. Ca2+ ionophore A23187 and thapsigargin evoked the release of PGE2 in the presence of external Ca2+ despite no activation of bradykinin receptors. These results indicate that bradykinin induces Ca2+ mobilization via activation of phospholipase C and PGE2 release caused by the Ca2+ influx in HPDL cells. 相似文献
6.
Albrecht Schwab John Geibel Wenhui Wang Hans Oberleithner Gerhard Giebisch 《The Journal of membrane biology》1993,132(2):125-136
Summary We studied the mechanism of K++ channel activation by minoxidil-sulfate (MxSO4) in fused Madin-Darby canine kidney (MDCK) cells. Patch-clamp techniques were used to assess single channel activity, and fluorescent dye techniques to monitor cell calcium. A Ca+2+-dependent inward-rectifying K++ channel with slope conductances of 53±3 (negative potential range) and 20±3 pS (positive potential range) was identified. Channel activity is minimal in cell-attached patches. MxSO4 initiated both transient channel activation and an increase of intracellular Ca+2+ (from 94.2±9.1 to 475±12.6 nmol/liter). The observation that K++ channel activity of excised inside-out patches was detected only at Ca+2+ concentrations in excess of 10 mol/liter suggests the involvement of additional mechanisms during channel activation by MxSO4.Transient K++ channel activity was also induced in cell-attached patches by 10 mol/liter of the protein kinase C activator 1-oleoyl-2-acetyl-glycerol (OAG). OAG (10 mol/liter in the presence of 1.6 mmol/liter ATP) increased the Ca+2 sensitivity of the K+ channel in inside-out patches significantly by lowering the K
mfor Ca+2 from 100 mol/liter to 100 nmol/liter. The channel activation by OAG was reversed by the protein kinase inhibitor H8. Staurosporine, a PKC inhibitor, blocked the effect of MxSO4 on K+ channel activation. We conclude that MxSO4-induced K+ channel activity is mediated by the synergistic effects of an increase in intracellular Ca+2 and a PKC-mediated enhancement of the K+ channel's sensitivity to Ca+2.A. Schwab was recipient of a Feodor-Lynen-Fellowship from the Alexander von Humboldt-Stiftung. This work was supported by NIH grant DK 17433. The authors thank Nikon Instruments Partners in Research Program for their support and generous use of equipment during the course of this study. Minoxidil-sulfate was kindly provided by Upjohn, Kalamazoo, MI. 相似文献
7.
Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group 总被引:1,自引:0,他引:1
The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca2+–Mg2+)-ATPase in the plasma membranes of rat liver was investigated. (Ca2+–Mg2+)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5–5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25–1.0 M) significantly increased (Ca2+-Mg2+)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca2+–Mg2+)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 M) increasing (Ca2+–Mg2+)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca2+–Mg2+)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme. 相似文献
8.
Bradykinin‐mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c‐Kit+ progenitor cells
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Gang Li Hui Che Wei‐Yin Wu Ling‐Jun Jie Guo‐Sheng Xiao Yan Wang Gui‐Rong Li 《Journal of cellular and molecular medicine》2018,22(10):4688-4699
Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c‐Kit+ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin‐mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c‐Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ () by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store‐operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin‐induced cell cycle progression and migration were not observed in cells with siRNA‐silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin‐induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin‐mediated increase in free via ER‐IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c‐Kit+ progenitor cells. 相似文献
9.
Toshitaka Ikehara Hirotaka Nishisako Yuki Minami Hiromi Ichinose Tairo Shiraishi Mitsuo Kitamura Masayuki Shono Hitoshi Houchi Kazuyoshi Kawazoe Kazuo Minakuchi Kazuo Yoshizaki Yohsuke Kinouchi Hiroshi Miyamoto 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
It has been reported that exposure to electromagnetic fields influences intracellular signal transduction. We studied the effects of exposure to a time-varying 1.5 T magnetic field on membrane properties, membrane cation transport and intracellular Ca2+ mobilization in relation to signals. We also studied the mechanism of the effect of exposure to the magnetic field on intracellular Ca2+ release from Ca2+ stores in adrenal chromaffin cells.Methods
We measured the physiological functions of ER, actin protein, and mitochondria with respect to a neurotransmitter-induced increase in Ca2+ in chromaffin cells exposed to the time-varying 1.5 T magnetic field for 2 h.Results
Exposure to the magnetic field significantly reduced the increase in [Ca2+]i. The exposure depolarized the mitochondria membrane and lowered oxygen uptake, but did not reduce the intracellular ATP content. Magnetic field-exposure caused a morphological change in intracellular F-actin. F-actin in exposed cells seemed to be less dense than in control cells, but the decrease was smaller than that in cytochalasin D-treated cells. The increase in G-actin (i.e., the decrease in F-actin) due to exposure was recovered by jasplakinolide, but inhibition of Ca2+ release by the exposure was unaffected.Conclusions and general significance
These results suggest that the magnetic field-exposure influenced both the ER and mitochondria, but the inhibition of Ca2+ release from ER was not due to mitochondria inhibition. The effect of eddy currents induced in the culture medium may indirectly influence intracellular actin and suppress the transient increase in [Ca2+]i. 相似文献10.
G. Bkaily D. Econornos L. Potvin J. -L. Ardilouze C. Marriott J. Corcos D. Bonneau Chin Nok Fong 《Molecular and cellular biochemistry》1992,117(1):93-106
The effect of high K– concentration, insulin and the L-type Ca2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca2+]i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca2+-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K+]o induced a rapid transient increase of [Ca2+]i that was followed by a sustained component. The early transient increase of [Ca2+]i by high [+]o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca2+ blocker was found to decrease both the early transient and the sustained increase of [Ca2+]i induced by depolarization of the cell membrane with high [K+]o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca2+]i that was blocked by PN 200-110 or by lowering the extracellular Ca2+ concentration with EGTA. The sustained increase of [Ca2+], induced by high [K+]o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na+ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca2+ current and the delayed outward K+ current. These results suggest that the early increase of (Ca2+]i during depolarization of the cell membrane of heart and VSM cells with high [K+]o is due to the opening and decay of an L-type Ca 2+ channel. However, the sustained increase of [Ca2+]i during a sustained depolarization is due to the activation of a resting (R) Ca 2+ channel that is insensitive to lowering [ATP]i and sensitive to insulin. 相似文献
11.
Margaret Colden-Stanfield William P. Schilling Lourival D. Possani Diana L. Kunze 《The Journal of membrane biology》1990,116(3):227-238
Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca2+ concentration, [Ca2+]
i
, accompanied by an increase in surface membrane K+ permeability. In this study, electrophysiological measurement of K+ current was combined with86Rb+ efflux measurements to characterize the K+ flux pathway in BAECs. Bradykinin- and Ca2+-activated K+ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca2+ activation is in the range of 10 to 100nm [Ca2+]
i
. The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K+ current present in control conditions, an inwardly rectifying K+ current, was blocked by Ba2+ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of86Rb– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,86Rb+ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca2+-activated membrane currents. The results confirm that bradykinin-stimulated86Rb+ efflux occurs via Ca2+-activated K+ channels. The blocking agents identified may provide a means for interpreting the role of the Ca2+-activated K+ current in the response of BAECs to bradykinin. 相似文献
12.
Enhancement of plasma membrane (Ca2+-Mg2+)-ATPase activity in regenerating rat liver: Involvement of endogenous activating protein regucalcin 总被引:1,自引:0,他引:1
The alteration of (Ca2+-Mg2+)-ATPase activity in the plasma membranes of regenerating rat liver after a partial hepatectomy was investigated. Liver was surgically removed about two thirds of that of sham-operated rats. The reduced liver weight by partial hepatectomy was restored about 50% at 24 h after the surgery, and it was completely restored at 72 h. Regenerating liver significantly increased calcium content and plasma membrane (Ca2+-Mg2+)-ATPase activity between 12–48 h after hepatectomy. Those increases were maximum at 24 h after the surgery. The regenerating liver-induced increase in hepatic plasma membrane (Ca2+-Mg2+)-ATPase activity was completely abolished by the presence of anti-regucalcin IgG (1.0–4.0 g/ml). The regenerating liver-induced increase in hepatic plasma membrane (Ca2+-Mg2+)-ATPase activity was clearly inhibited by N-ethylmaleimide (2.5 and 5.0 mM) addition into the enzyme reaction mixture. This NEM effect was also seen for the activatory effect with regucalcin (0.25 M) addition on the enzyme activity in the plasma membranes from normal rat liver. The endogenous regucalcin may play a cell physiological role in the activation of the plasma membrane (Ca2+-Mg2+)-ATPase to maintain the intracellular calcium level in regenerating rat liver. 相似文献
13.
Richard H. Foster John S. Davis Robert V. Farese 《Molecular and cellular biochemistry》1990,95(2):157-166
Previous studies have shown that external calcium (Ca2+) is required for the effects of angiotensin II (AII) on aldosterone secretion in adrenal glomerulosa zone. Using bovine adrenal glomerulosa cells prepared by collagenase dispersion, we examined whether external Ca2+ is required for the activation of phospholipase C by AII. Adrenal glomerulosa cells were exposed to Ca-EGTA buffered media to provide accurate estimates of external free Ca2+ concentrations. Phospholipase C activation was evaluated by measurement of inositol phosphates production. At 0.1 M Ca2+ and less, sustained AII effects on inositol monophosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were markedly inhibited. Increasing the Ca2+ concentration to 50kM or greater fully restored All-induced inositol phosphates production. AII-induced increases in cytosolic Ca2+ measured by Quin-2 fluorescence, were diminished at lower external Ca2+ concentrations. Treating adrenal glomerulosa cells with Chelex-100, a strong Ca2+ binding resin, blocked early activation of phospholipase C by AII. Inhibition of IP3 production was also observed when inhibitors of Ca2+ movement across the plasma membrane were used, viz., La2+, TMB-8 and nifedipine. The requirement for Ca2+ during AII-induced activation of phospholipase C may be explained, at least partly by a requirement for Ca2+ at a site between the AII receptor and Phospholipase C. 相似文献
14.
This work investigated the role of Ca2+ mobilization and heterotrimeric G protein activation in mediating angiotensin II-dependent tyrosine phosphorylation signaling patterns. We demonstrate that the predominant, angiotensin II-dependent, tyrosine phosphorylation signaling patterns seen in vascular smooth muscle cells are blocked by the intracellular Ca2+ chelator BAPTA-AM, but not by the Ca2+ channel blocker verapamil. Activation of heterotrimeric G proteins with NaF resulted in a divergent signaling effect; NaF treatment was sufficient to increase tyrosine phosphorylation levels of some proteins independent of angiotensin II treatment. In the same cells, NaF alone had no effect on other cellular proteins, but greatly potentiated the ability of angiotensin II to increase the tyrosine phosphorylation levels of these proteins. Two proteins identified in these studies were paxillin and Jak2. We found that NaF treatment alone, independent of angiotensin II stimulation, was sufficient to increase the tyrosine phosphorylation levels of paxillin. Furthermore, the ability of either NaF and/or angiotensin II to increase tyrosine phosphorylation levels of paxillin is critically dependent on intracellular Ca2+. In contrast, angiotensin II-mediated Jak2 tyrosine phosphorylation was independent of intracellular Ca2+ mobilization and extracellular Ca2+ entry. Thus, our data suggest that angiotensin II-dependent tyrosine phosphorylation signaling cascades are mediated through a diverse set of signaling pathways that are partially dependent on Ca2+ mobilization and heterotrimeric G protein activation. 相似文献
15.
The regulatory role of Ca2+-stimulated adenosine 5-triphosphatase (Ca2+-ATPase) in Ca2+ transport system of rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca2+-ATPase activity was calculated by subtracting Mg2+-ATPase activity from (Ca2+–Mg2+)-ATPase activity. The release of Ca2+ from the Ca2+-loaded nuclei was evoked progressively after Ca2+ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca2+ from the Ca2+-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca2+ uptake and induced a promotion of Ca2+ release from the Ca2+-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca2+-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca2+ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca2+ channels, did not prevent the NAD+ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca2+ release, suggesting that Ca2+ channels do not involve on Ca2+ release from the nuclei. These results indicates that an inhibition of nuclear Ca2+-ATPase activity causes the decrease in nuclear Ca2+ uptake and the release of Ca2+ from the Ca2+-loaded nuclei. The present finding suggests that Ca2+-ATPase plays a critical role in the regulatory mechanism of Ca2+ uptake and release in rat liver nuclei. 相似文献
16.
Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca2+ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca2+ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 μM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2′,7′-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N′-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca2+ concentration, which was completely prevented by the antioxidants. An intracellular Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca2+ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca2+ increase and induction of apoptosis were significantly inhibited by an extracellular Ca2+ chelator or Na+/Ca2+ exchanger blockers (bepridil and benzamil), whereas neither Ca2+ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca2+ through the activation of reverse mode of Na+/Ca2+ exchanger. However, tBOOH decreased intracellular Na+ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca2+ concentration by direct activation of the reverse mode of Na+/Ca>2+ exchanger, rather than indirect elevation of intracellular Na+ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca2+ may mediate this action of tBOOH. These results further suggest that Na+/Ca2+ exchanger may be a target for the management of oxidative hepatotoxicity. 相似文献
17.
M. V. Kustov V. V. Tsvilovskii A. V. Zholos M. F. Shuba T. B. Bolton 《Neurophysiology》2006,38(1):1-8
Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca2+]i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications
of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 μM carbachol, CCh, as well as 10.0 and
100.0 μM ATP) induced a rise in the [Ca2+]i. Carbachol evoked an increase in the [Ca2+]i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise
in the [Ca2+]i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly
differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally
Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca2+]i in myocytes. The absence of the slow component in the [Ca2+]i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is
not related to activation of the entry of Ca2+ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP
induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca2+]i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted
only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 μM), completely blocked rises
in the [Ca2+]i induced by both CCh and ATP; therefore, the release of Ca2+ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the
difference in the release of Ca2+ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and
complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional
effects of the above-mentioned neurotransmitters on smooth muscles.
Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 3–10, January–February, 2006. 相似文献
18.
The effects of lead on Ca2+ homeostasis in nerve terminals was studied. Incubation with leadin vitro stimulated the activity of calmodulin and the maximum effect was observed at 30 M lead, higher concentrations had an inhibitory effect.In vivo exposure to lead increased the activity of calmodulin by 45%. Lead had an inhibitory effect on Ca2+ ATPase activity in both calmodulin-rich and calmodulin-depleted synaptic plasma membranes, the IC50 values for inhibition being 13.34 and 16.69 M respectively. Exogenous addition of calmodulin (5 g) and glutathione (1 mM) to calmodulin rich synaptic plasma membranes reversed the inhibition by IC50 concentration of lead.In vivo exposure of lead also significantly reduced the Ca2+ ATPase activity, resulting in an increase in intrasynaptosomal calcium. Concomitant with the increase in intrasynaptosomal calcium, lipid peroxidation values also increased significantly in lead-treated animals. In addition lead also had an inhibitory effect on depolarization induced Ca2+ uptake and the inhibition was found to be a competitive one. The results sugest that lead exerts its toxic effects by modifications of the intracellular calcium messenger system which would have serious consequences on neuronal functioning. 相似文献
19.
The phospholipid requirement for Ca2+-stimulated, Mg2+-dependent ATP hydrolysis (Ca2+/Mg2+-ATPase) and Mg2+-stimulated ATP hydrolysis (Mg2+-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidation of the membranes with phospholipase A2 (Hog pancreas), phospholipase C (Bacillus cereus) and phospholipase D (cabbage). Treatment with phospholipase A2 caused an increase in the activities of both Ca2+/Mg2+-ATPase and Mg2+-ATPase whereas with phospholipase C treatment both the enzyme activities were inhibited. Phospholipase D treatment had no effect on Ca2+/Mg2+-ATPase but Mg2+-ATPase activity was inhibited. Inhibition of Mg2+-ATPase activity after phospholipase C treatment was relieved with the addition of phosphatidylinositol-4,5-bisphosphate (PIP2) and to a lesser extent with phosphatidylinositol-4-phosphate (PIP) and phosphatidylcholine (PC). Phosphatidylserine (PS), phosphatidic acid (PA), PIP and PIP2 brought about the reactivation of Ca2+/Mg2+-ATPase. Phosphatidylinositol (PI) and PA inhibited Mg2+-ATPase activity.K
ms for Ca2+ (0.47 M) and Mg2+ (60 M) of the enzyme were found to be unaffected after treatment with the phospholipases. 相似文献
20.
Sculptoreanu A Abramovici H Abdullah AA Bibikova A Panet-Raymond V Frankel D Schipper HM Pinsky L Trifiro MA 《Molecular and cellular biochemistry》2000,203(1-2):23-31
We have analyzed Ca2+ currents in two neuroblastoma-motor neuron hybrid cell lines that expressed normal or glutamine-expanded human androgen receptors (polyGln-expanded AR) either transiently or stably. The cell lines express a unique, low-threshold, transient type of Ca2+ current that is not affected by L-type Ca2+ channel blocker (PN 200-110), N-type Ca2+ channel blocker (-conotoxin GVIA) or P-type Ca2+ channel blocker (Agatoxin IVA) but is blocked by either Cd2+ or Ni2+. This pharmacological profile most closely resembles that of T-type Ca2+ channels [1-3]. Exposure to androgen had no effect on control cell lines or cells transfected with normal AR but significantly changed the steady-state activation in cells transfected with expanded AR. The observed negative shift in steady-state activation results in a large increase in the T-type Ca2+ channel window current. We suggest that Ca2+ overload due to abnormal voltage-dependence of transient Ca2+ channel activation may contribute to motor neuron toxicity in spinobulbar muscular atrophy (SBMA). This hypothesis is supported by the additional finding that, at concentrations that selectively block T-type Ca2+ channel currents, Ni2+ significantly reduced cell death in cell lines transfected with polyGln-expanded AR. 相似文献