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1.
The effect of Zn2+ on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by measuring the changes in the fluorescence of the Ca2+-sensitive dye fura-2. Zn2+ significantly increased cytoplasmic free Ca2+ levels ([Ca2+]i) at concentrations of 2-100 microM. The maximum response was obtained at concentrations of 25-100 microM. The [Ca2+]i rise induced by 100 microM Zn2+ consisted of a gradual rise and a plateau phase, and was primarily mediated by La3+-sensitive extracellular Ca2+ influx because the [Ca2+]i rise was abolished by pretreatment with 100 microM La3+ or removal of extracellular Ca2+, and that Zn2+ induced Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength which was prevented by pretreatment with 100 microM La3+. Pretreatment with 100 microM Zn2+ for 220 s did not reduce the [Ca2+]i rise induced by the endoplasmic reticulum (ER) Ca2+ pump inhibitor, thapsigargin, suggesting that Ca2+ release from the ER played a minor role in the Zn2+-induced [Ca2+]i rise. Zn2+ (100 microM) nearly abolished the capacitative Ca2+ entry induced by ATP (100 microM). We also investigated the effect of Zn2+ pretreatment on the [Ca2+]i rise induced by ATP. Zn2+ (100 microM) affected ATP-induced [Ca2+]i rise by abolishing capacitative Ca2+ entry and increasing [Ca2+]i on its own without altering Ca2+ release from intracellular sources. The effect of Zn2+ on [Ca2+]i was dissociated from changes in membrane potential.  相似文献   

2.
The effect of the antidepressant fluoxetine on Ca2+ signaling in cultured cells was largely unknown. The effect of various concentrations of fluoxetine on [Ca 2+] i in populations of bladder female transitional cancer (BFTC) cells was evaluated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca 2+] i concentration dependently (20-100 microM) with an EC50 value of 30 microM. The response was inhibited by 50-60% on extracellular Ca2+ removal. In Ca2+ -free medium, pretreatment with 1 microM thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) abolished 50 microM fluoxetine-induced Ca2+ release; whereas pretreatment with fluoxetine did not alter the thapsigargin-induced Ca2+ response. Addition of 3 mM Ca2+ increased [Ca 2+] i after pretreatment with 50 microM fluoxetine in Ca2+ -free medium, suggestive of fluoxetine-induced capacitative Ca2+ entry. Suppression of inositol 1,4,5-trisphosphate formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 50 microM fluoxetine-induced Ca2+ release. Collectively, this study shows that fluoxetine increased [Ca 2+] i in bladder cancer cells in a concentration-dependent fashion, by releasing Ca2+ from thapsigargin-sensitive Ca2+ stores in an IP3-independent manner, and by inducing Ca2+ influx from extracellular medium.  相似文献   

3.
Econazole is an antifungal drug with different in vitro effects. However, econazole's effect on osteoblast-like cells is unknown. In human MG63 osteosarcoma cells, the effect of econazole on intracellular Ca2+ concentrations ([Ca2+]i) was explored by using fura-2. At a concentration of 0.1 microM, econazole started to cause a rise in [Ca2+]i in a concentration-dependent manner. Econazole-induced [Ca2+]i rise was reduced by 74% by removal of extracellular Ca2+. The econazole-induced Ca2+ influx was mediated via a nimodipine-sensitive pathway. In Ca2+ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca+ -ATPase, caused a [Ca2+]i rise, after which the increasing effect of econazole on [Ca2+]i was abolished. Pretreatment of cells with econazole to deplete Ca2+ stores totally prevented thapsigargin from releasing Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not econazole-induced, [Ca2+]i rise. Econazole inhibited 76% of thapsigargin-induced store-operated Ca2+ entry. These findings suggest that in MG63 osteosarcoma cells, econazole increases [Ca2+]i by stimulating Ca2+ influx and Ca2+ release from the endoplasmic reticulum via a phospholipase C-independent manner. In contrast, econazole acts as a potent blocker of store-operated Ca2+ entry.  相似文献   

4.
Carvedilol is a useful cardiovascular drug for treating heart failure, however, the in vitro effect on many cell types is unclear. In human MG63 osteosarcoma cells, the effect of carvedilol on intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Carvedilol at concentrations greater than 1 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50=15 microM). Carvedilol-induced [Ca2+]i rise was reduced by 60% by removal of extracellular Ca2+. Carvedilol-induced Mn2+-associated quench of intracellular fura-2 fluorescence also suggests that carvedilol induced extracellular Ca2+ influx. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of carvedilol on [Ca2+]i was inhibited by 50%. Conversely, pretreatment with carvedilol to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not carvedilol-induced, [Ca2+]i rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, did not alter carvedilol-induced [Ca2+]i rise. Separately, overnight treatment with 0.1-30 microM carvedilol inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, carvedilol increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum and other stores via a phospholipase C-independent manner. Carvedilol may be cytotoxic to osteoblasts.  相似文献   

5.
The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca2+ dye. Histamine at concentrations between 0.1 and 50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1 microM. The [Ca2+]i response comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In the absence of extracellular Ca2+, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 10 microM histamine did not increase [Ca2+]i. After pretreatment with 10 microM histamine in a Ca2+-free medium for several minutes, addition of 3 mM Ca2+ induced [Ca2+]i increases. Histamine (10 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 microM pyrilamine but was not altered by 50 microM cimetidine. Collectively, the present study shows that histamine induced [Ca2+]i transients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca2+ release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca2+ entry.  相似文献   

6.
Huang CC  Cheng HH  Lin KL  Cheng JS  Tsai JY  Liao WC  Fang YC  Jan CR 《Toxicology》2009,255(1-2):58-64
The effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in corneal epithelial cells. This study examined whether tamoxifen altered [Ca2+]i and viability in SIRC corneal epithelial cells. Tamoxifen at concentrations > or = 1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 6 microM. The Ca2+ signal was reduced substantially by removing extracellular Ca2+. Tamoxifen induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to Ca2+ entry inhibitors and protein kinase C modulators. After pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were abolished; conversely, tamoxifen pretreatment abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not change the [Ca2+]i rises. At concentrations of 5-30 microM, tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 15 microM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5-30 microM tamoxifen. Tamoxifen (30 microM did not induce production of reactive oxygen species (ROS). Collectively, in SIRC cells, tamoxifen induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via unknown pathways. Tamoxifen-caused cytotoxicity was partly mediated by a Ca2+-independent apoptotic pathway.  相似文献   

7.
The effects of the environmental toxicant, triethyltin, on Ca2+ mobilization in Madin-Darby canine kidney (MDCK) cells have been examined. Triethyltin induced an increase in cytosolic free Ca2+ levels ([Ca2+]i) at concentrations larger than 2 microM in a concentration-dependent manner. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was partly reduced by removing extracellular Ca2+. In Ca(2+)-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, reduced 50 microM triethyltin-induced [Ca2+]i increase by 80%. Conversely, pretreatment with triethyltin abolished thapsigargin-induced Ca2+ release. Pretreatment with U73122 (2 microM) to inhibit phospholipase C-coupled inositol 1,4,5-trisphosphate formations failed to alter 50 microM triethyltin-induced Ca2+ release. Incubation with triethyltin at a concentration (1 microM) that did not increase basal [Ca2+]i for 3 min did not alter ATP (10 microM)- and bradykinin (1 microM)-induced [Ca2+]i increases. Collectively, this study shows that triethyltin altered Ca2+ movement in renal tubular cells by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing Ca2+ influx.  相似文献   

8.
The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca2+ levels ([Ca2+]i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. AM404 induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, Ni2+, nifedipine and verapamil. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), AM404-induced [Ca2+]i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca2+]i rise. At concentrations between 10 and 200 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM AM404 was partly reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.  相似文献   

9.
The effect of calmidazolium on Ca(2+) signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca(2+) probe. Calmidazolium at 2-5 microM increased [Ca(2+)](i) concentration dependently. The [Ca(2+)](i) rise induced by 2-5 microM calmidazolium comprised an immediate rise and a slow decay. External Ca(2+) removal partly inhibited the Ca(2+) signals, suggesting that calmidazolium activated external Ca(2+) influx and internal Ca(2+) release. In Ca(2+)-free medium, pretreatment with 3 microM calmidazolium abolished the Ca(2+) release induced by 1 microM thapsigargin, an endoplasmic reticulum Ca(2+) pump inhibitor, and, vice versa, pretreatment with thapsigargin inhibited calmidazolium-induced Ca(2+) release. This indicates that thapsigargin-sensitive Ca(2+) store was the source of calmidazolium-induced Ca(2+) release. Calmidazolium (3 microM) induced Mn(2+) quench of fura-2 fluorescence at 360 nm excitation wavelength, which was suppressed by 0.1 mM La(3+). Addition of 3 mM Ca(2+) increased [Ca(2+)](i) after pretreatment with 3-5 microM calmidazolium in Ca(2+)-free medium. This implies that calmidazolium activated concentration-dependent capacitative Ca(2+) entry. Calmidazolium (3 microM) augmented the capacitative Ca(2+) entry induced by 1 microM thapsigargin or 0.1 mM ATP by 38%. Calmidazolium (3 microM)-induced Ca(2+) release was blocked by pretreatment with 40 microM aristolochic acid and 2 microM U73122 (2 microM) to inhibit phospholipase A(2) and phospholipase, respectively, but pretreatment with 0.1 mM propranolol to inhibit phospholipase D had no effect. This suggests that calmidazolium induced internal Ca(2+) release in a manner dependent on phospholipases C- and A(2)-coupled events.  相似文献   

10.
The effect of thimerosal, a reactive oxidant, on cytoplasmic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored by using the Ca2+-sensitive dye fura-2. Thimerosal acted in a concentration-dependent manner with an EC50 of 0.5 microM. The Ca2+ signal comprised a gradual rise and a sustained elevation. Removal of extracellular Ca2+ reduced 80% of the signal. In Ca2+-free medium, the [Ca2+]i rise induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) was completely inhibited by pretreatment with 5 microM thimerosal. The thimerosal (5 microM)-induced Ca2+ release was not changed by inhibition of phospholipase C with 2 microM U73122. Collectively, this study shows that thimerosal induced [Ca2+]i rises in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity.  相似文献   

11.
This study examined the effect of tamoxifen, an anti-breast cancer drug, on Ca2+ handling in bladder female transitional cancer cells. Changes in cytosolic free Ca2+ levels were recorded by using the Ca2+-sensitive dye fura-2. In a dose-dependent manner, tamoxifen induced intracellular free Ca2+ concentrations ([Ca2+]i) increases between 5 and 20 microM with an EC50 of 10 microM. External Ca2+ removal reduced the response by 60+/-6%. Addition of 3 mM Ca2+ caused a [Ca2+]i increase after pretreatment with 10 microM tamoxifen in Ca2+-free medium. In Ca2+-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca2+]i increase induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 1 microM thapsigargin prevented tamoxifen from releasing more Ca2+. Inhibition of phospholipase C-dependent inositol 1,4,5-tris-phosphate formation with 2 microM U73122 did not alter 10 microM tamoxifen-induced Ca2+ release. The [Ca2+]i increase induced by 5 microM tamoxifen was not altered by 10 microM La3+, nifedipine, verapamil, and diltiazem. Collectively, it was found that tamoxifen increased [Ca2+]i in bladder cancer cells by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from external medium.  相似文献   

12.
1. We investigated the characteristics of endothelin (ET)-induced contraction and changes in intracellular Ca2+ concentration ([Ca2+]i) using the fura-2-loaded and non-loaded rabbit iris dilator. ET-1 and ET-2 (3-100 nM) and ET-3 (30-100 nM) caused contraction in a concentration-dependent fashion. 2. The selective ETB-receptor agonists, IRL1620 and sarafotoxin S6c produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of potencies for the contraction (pD2 value) was ET-1 = ET-2 > ET-3 >> sarafotoxin S6c = IRL1620. 3. The contractile response to ET-3 was antagonized by pretreatment with BQ-123 (10 nM), a selective ETA receptor antagonist. The contractile responses to ET-1 and ET-2 were antagonized by pretreatment with BQ-123 (10 microM), but not at a concentration of 10 nM. 4. ETs increased [Ca2+]i and sustained muscle contraction. ET-1 (100 nM), ET-2 (100 nM), and ET-3 (1 microM) induced an elevation of [Ca2+]i consisting of two components: first a rapid and transient elevation to reach a peak, followed by a second, sustained elevation; a sustained contraction was produced without a transient contraction. The ETB receptor-selective agonist, IRL1620 (1 microM) and sarafotoxin S6c (1 microM) also induced a rapid and transient elevation of [Ca2+]i to reach a peak and a sustained elevation, together with only a small contraction or no contraction. 5. ET-1 (100 nM) induced a transient increase in [Ca2+]i in a Ca(2+)-free, 2 mM EGTA-containing physiological saline solution (Ca(2+)-free PSS), and a small sustained contraction which was significantly different from that induced by ET-1 (100 nM) in normal PSS. The ET-1-induced increase in [Ca2+]i and sustained contraction were not affected by the voltage-dependent Ca2+ channel blocker, nicardipine (10 microM). The ET-1-induced transient increase in [Ca2+]i was significantly reduced by the sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor, cyclopiazonic acid (30 microM); however, the ET-1-induced sustained contraction was not affected by this agent. 6. The selective ETA receptor antagonist, BQ-123 (100 nM) reduced the ET-3 (100 nM)-induced contraction, but did not affect the transient increase or elevation of the second phase of [Ca2+]i. However, this antagonist at 1 microM did not affect the ET-1 (100 nM)- and ET-2 (100 nM)-induced elevation of [Ca2+]i and contractile response, or the IRL1620-induced elevation of [Ca2+]i. 7. The selective ETB receptor antagonist, BQ-788 (1 microM) reduced the transient increase in [Ca2+]i induced by ET-1 (30 nM), ET-2 (30 nM), ET-3 (100 nM) and IRL1620 (1 microM), but did not affect the sustained elevation of [Ca2+]i and contractile responses produced by ET-1, ET-2 and ET-3. 8. Pretreatment with IRL1620 (1 microM) reduced the increase in [Ca2+]i induced by IRL1620 (1 microM) and sarafotoxin S6c (1 microM), as well as the ET-1 (100 nM)-, ET-2 (100 nM)- and ET-3 (1 microM)-induced elevation of [Ca2+]i, whereas in the presence of IRL1620, ET-1-, ET-2- and ET-3-induced contractions were unaltered. 9. These results suggest that ETA and ETB receptor subtypes exist in the rabbit iris dilator muscle, and that the ETA receptor is divided into: (1) BQ-123-sensitive ETA subtypes activated by ET-1, ET-2 and ET-3, and (2) BQ-123-insensitive ETA subtypes activated by ET-1 and ET-2, which cause the sustained increase of [Ca2+]i and contraction; in contrast, ETB receptor subtypes are activated by ET-1, ET-2, ET-3, IRL1620 and sarafotoxin S6c and cause the transient and sustained increase in [Ca2+]i which is not able to contract the smooth muscle.  相似文献   

13.
The effect of N-palmitoyl-L-serine phosphoric acid (L-NASPA), which has been used as an inhibitor of lysophosphatidic acid receptors, on intracellular Ca2+ concentration ([Ca2+]i) in human osteosarcoma MG63 cells was measured by using fura-2. L-NASPA (0.1-10 microM) caused a rapid and transient plateau [Ca2+]i rise in a concentration-dependent manner (EC50=0.5 microM). The L-NASPA-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+ but was not altered by L-type voltage-gated Ca2+ channel blockers. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, induced a [Ca2+]i rise, after which the increasing effect of L-NASPA on [Ca2+]i was completely inhibited; also, pretreatment with L-NASPA partly reduced thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phospholipase C, abolished histamine (but not L-NASPA)-induced [Ca2+]i rise. Overnight incubation with 1 microM L-NASPA did not affect cell proliferation, but 10-20 microM L-NASPA exerted 4% and 15% inhibition, respectively. Collectively, L-NASPA rapidly increased [Ca2+]i in MG63 cells by evoking both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic at higher concentrations.  相似文献   

14.
1. The effects of the specific protein kinase C (PKC) inhibitor, GF109203X, were measured on the cytoplasmic Ca2+ concentration ([Ca2+]i), and on histamine H1 receptor- and thapsigargin-mediated increases in [Ca2+]i in DDT1 MF-2 smooth muscle cells. 2. After pretreatment of cells with GF109203X (5 microM, 45 min), the histamine (100 microM)-induced initial rise in [Ca2+]i, representing Ca2+ mobilization from internal stores, was inhibited (by 59 +/- 7%). The slowly declining phase of the histamine induced Ca2+ response, reflecting Ca2+ entry, was enhanced (83 +/- 26%) in the presence of the PKC inhibitor. 3. The histamine induced release of Ca2+ from internal stores, measured after blocking Ca2+ entry with LaCl3 was inhibited by GF109203X in a concentration-dependent manner (IC50: 3.1 +/- 1.1 microM). 4. Histamine-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was not changed in the presence of GF109203X. 5. The PKC activating phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM), strongly reduced histamine-induced Ins(1,4,5)P3 formation (58 +/- 16%). This effect was reversed by GF109203X (5 microM). Furthermore, PMA diminished histamine evoked Ca2+ release (50 +/- 6%) and blocked Ca2+ entry completely. 6. The rise in [Ca2+]i caused by blocking endoplasmic reticulum Ca2(+)-ATPase with thapsigargin (1 microM), was strongly reduced (57 +/- 3%) after pretreatment of cells with GF109203X. Downregulation of PKC by long-term pretreatment of cells with PMA (1 microM, 48 h) did not abolish this effect of GF109203X (48 +/- 3% inhibition). 7. In permeabilized DDT, MF-2 cells preloaded with 45Ca2+ in the presence of GF109203X, the amount of 45Ca2+ released by Ins(1,4,5)P3 (10 microM) was markedly reduced (42 +/- 9%). GF109203X did not release Ca2+ itself and did not impair Ins(1,4,5)P3 receptor function. 8. Uptake of 45Ca2+ by intact cells, representing Ca2+ entry, was enhanced by GF109203X (65 +/- 11%), by histamine (24 +/- 6%) and also by thapsigargin (121 +/- 10%). The GF109203X- and the thapsigargin-induced uptake of 45Ca2+ were not additive. 9. These data suggest that GF109203X reduces the filling-state of intracellular Ins(1,4,5)P3 sensitive Ca2+ stores by inhibiting the Ca2+ uptake into these stores, thereby promoting store-dependent (capacitive) Ca2+ entry.  相似文献   

15.
This study investigated the effect of the anti-anginal drug, fendiline, on intracellular free Ca2+ levels ([Ca2+]i) in HA/ 22 human hepatoma cells by using fura-2 as a fluorescent Ca2+ dye. Fendiline (1-100 microM) increased [Ca2+]i with an EC50 of 25 microM. Removal of extracellular Ca2+ reduced the [Ca2+]i signals by 51 +/- 5%. Fendiline (10 microM)-induced Ca2+ release was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not alter 10 microM fendiline-induced Ca2+ release. Several other calmodulin antagonists, such as phenoxybenzamine (100-200 microM), trifluoperazine (5-50 microM), and fluphenazine-N-chloroethane (2-100 microM), had no effect on [Ca2+]i. Together, it was found that fendiline increased [Ca2+]i in human hepatoma cells by discharging Ca2+ from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ entry. This effect of fendiline does not appear to be via antagonism of calmodulin.  相似文献   

16.
Azelastine (1-300 microM) inhibited contractions of isolated porcine trachea induced by high K+, carbachol and endothelin-1 (ET-1) with a decrease in [Ca2+]cyt (as measured by fura-2-fluorescence). Verapamil (0.1-10 microM) also inhibited the high K(+)-induced increases in [Ca2+]cyt and contraction, although it only partially inhibited the responses evoked by carbachol or ET-1. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), carbachol induced a transient increase in [Ca2+]cyt and force by releasing Ca2+ from cellular stores. Azelastine (100 microns) completely inhibited these contransient changes. In the absence of extracellular Ca2+, carbachol and 12-deoxyphorbol 13-isobutyrate (DPB) induced small sustained contractions without increasing [Ca2+]cyt. Azelastine inhibited these contractions. In muscle permeabilized with alpha-toxin, Ca2+ (0.3-3 microM) induced contraction in a concentration-dependent manner. DPB (without GTP) and carbachol or ET-1 (with GTP) enhanced the Ca(2+)-induced contraction. Azelastine partially inhibited the contraction induced by 0.3 microM Ca2+ but not the contraction induced by 3 microM Ca2+, and strongly inhibited the potentiating effects of DPB, carbachol and ET-1. Azelastine had no effect on the content of cyclic AMP or cyclic GMP. These results suggest that azelastine inhibits smooth muscle contraction by (i) decreasing [Ca2+]cyt, by inhibition of Ca2+ channels, (ii) decreasing agonist-induced Ca2+ release, and (iii) direct inhibition of contractile elements.  相似文献   

17.
Riluzole is an effective neuroprotective drug. Its effect on intracellular free Ca2+ levels ([Ca2+]i) has not been explored. This study examined the effect of riluzole on [Ca2+]i in IMR32 neuroblastoma cells using fura-2 as a Ca2+ probe. Riluzole 0.1-1 mM increased [Ca2+]i in a concentration-dependent manner. Removal of extracellular Ca2+ inhibited the response by 52 +/- 5%. The [Ca2+]i increase induced by 0.2 mM riluzole was unaltered by 0.1 mM La3+ or 10 microM verapamil, but was inhibited by 51 +/- 4% by 10 microM nifedipine. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) reduced the 0.2 mM riluzole-induced Ca2+ release by 44 +/- 3%; this reduction was augmented to 66 +/- 5% by additionally depleting the Ca2+ stores in the Golgi complex with 50 microM brefeldin A. Inhibition of inositol 1,4,5-trisphosphate formation by 2 microM U73122, a phospholipase C inhibitor, did not affect Ca2+ release induced by 0.2 microM riluzole. It was concluded that the neuroprotective agent riluzole increased [Ca2+]i in IMR32 neuroblastoma cells concentration-dependently by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner and also by inducing nifedipine-sensitive Ca2+ influx.  相似文献   

18.
The effect of 5,8,11-eicosatriynoic acid, a widely used lipoxygenase inhibitor, on Ca2+ fate in Madin Darby canine kidney cells was examined by using fura-2 as a Ca2+ probe. At concentrations between 2-100 microM 5,8,11-eicosatriynoic acid increased [Ca2+]i concentration-dependently with an EC50 of 20 microM . Extracellular Ca2+ removal decreased the Ca2+ signals, indicating that 5,8,11-eicosatriynoic acid triggered Ca2+ release and Ca2+ influx. 5,8,11 -Eicosatriynoic acid (30 microM) induced a [Ca2+]i increase in Ca2+-free medium after pretreatment with carbonylcyanide m-chlorophenylhydrazone (2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor for 20 min. Conversely, 5,8,11-eicosatriynoic acid pretreatment almost abolished the Ca2+ release induced by carbonylcyanide m-chlorophenylhydrazone and thapsigargin. These results suggest that 30 microM 5,8,11-eicosatriynoic acid released Ca2+ from the endoplasmic reticulum, mitochondria and other stores. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 2-50 microM 5,8,11-eicosatriynoic acid for 10 min. in Ca2+-free medium concentration-dependently. Pretreatment with 10 microM La3+ abolished 30 microM 5,8,11-eicosatriynoic acid -induced [Ca2+]i increases, but adding La3+ during the decay phase had no effect. 5,8,11-Eicosatriynoic acid-induced Ca2+ release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), but was decreased by 60% by 40 microM aristolochic acid. Several other lipoxygenase inhibitors such as baicalein (50 microM), 5.8.11.14-eicosatetraynoic acid (ETYA; 0.1-0.2 mM), caffeic acid (5-50 microM), esculetin (5-50 microM), alpha-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV-5901; 0.1-0.2 mM) and alpha-pentyl-4-(2-quinolinylmethoxy)-benzenemethanol (L-655238; 80-100 microM) had no effect on [Ca2+]i. Collectively, the data suggest that the lipoxygenase inhibitor 5,8,11-eicosatriynoic acid induced a [Ca2+]i increase in renal tubular cells concentration-dependently, by releasing intracellular Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing extracellular Ca2+ influx in a La3+-sensitive manner.  相似文献   

19.
I investigated the role of protein kinase C (PKC) in regulation of the capacitative Ca2+ entry and steroidogenesis in bovine adrenocortical (BA) cells. Thapsigargin (TG)-treatment depleted intracellular Ca2+ stores followed by induction of Ca2+ influx from the extracellular pool and also increasing of Mn2+ influx as an indicator of divalent cation influx in BA cells. Calphostin C, a PKC inhibitor, inhibited the TG-induced [Ca2+]i elevation dose-dependently (0.1-1 microM) and attenuated Mn2+ entry. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, potentiated the elevation of [Ca2+]i and enhanced Mn2+ entry by TG treatment. These results suggest that PKC may modulate capacitative Ca2+ entry in BA cells. In the presence of extracellular Ca2+, TG enhanced cortisol production in BA cells. Calphostin C attenuated the TG-induced steroidogenesis dose-dependently (0.25-1 microM). PMA enhanced the steroidogenesis dose-dependently (1-100 nM). These results suggested that PKC may have a modulatory effect on the capacitative Ca2+ entry that links to steroidogenesis in BA cells.  相似文献   

20.
The effect of the antidepressant nortriptyline, on bone cells is unknown. In human osteosarcoma MG63 cells, the effect of nortriptyline on intracellular Ca2+ concentration ([Ca2+]i) and proliferation was measured by using fura-2 and tetrazolium, respectively. Nortriptyline (> or = 10 microM) caused a [Ca2+]i rise in a concentration-dependent manner (EC50 = 200 microM). Nortriptyline-induced [Ca2+]i rise was prevented by 60% by removal of extracellular Ca2+ but was not altered by voltage-gated Ca2+ channel blockers. In Ca2+ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ -ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of nortriptyline on [Ca2+]i was abolished; also, pretreatment with nortriptyline abolished thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, did not affect nortriptyline-induced [Ca2+]i rise; however, activation of protein kinase C decrease nortriptyline-induced [Ca2+]i rise by 32%. Overnight incubation with 50 and 100 microM nortriptyline killed 78% and 97% of cells, respectively; while 10 microM nortriptyline had no effect. These data suggest that nortriptyline rapidly increases [Ca2+]i in human osteosarcoma cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic at high concentrations.  相似文献   

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