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1.
The mechanism underlying the generation of cytosolic free Ca 2+ ([Ca 2+i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca 2+] i oscillations with variable patterns. The bombesin-induced [Ca 2+] i oscillations could last more than 1 h and glucose was required for maintaining these [Ca 2+ fluctuations. Bombesin-evoked [Ca 2+] i oscillations were dependent on extracellular Ca 2+ entry and were attenuated by membrane hype rpolarization or by L-type Ca 2+ channel blockers. These [Ca 2+] i oscillations were apparently not associated with fluctuations in plasma membrane Ca 2+ permeability as monitored by the Mn 2+ quenching technique. 2,5-di-( tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro- m-cresol, which interfere with intracellular Ca 2+ stores, respectively, by inhibiting Ca 2+-ATPase of endoplasmic reticulum and by affecting Ca 2+-induced Ca 2+ release, disrupted bombesin-induced [Ca 2+] i oscillations. 4-chloro- m-resol raised [Ca 2+] i by mobilizing an intracellular Ca 2+ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca 2+] i It raised [Ca 2+] i by promoting Ca 2+ entry while inhibiting bombesin-elicited [Ca 2+] i oscillations. Our results suggest that in bombesin-elicited [Ca 2+] i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca 2+ stores; and (ii) the Ca 2+ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca 2+] i oscillations. The interplay between intracellular Ca 2+ stores and voltage-sensitive and voltage-insensitive extracellular Ca 2+ entry determines the [Ca 2+] i oscillations evoked by bombesin. 相似文献
2.
By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20–50 μM acylcarnitines increased calcium concentration in cytoplasm ([Ca 2+] i) and caused cell death after a lag-period of 4–8 min. This effect was independent of extracellular calcium level and Ca 2+ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300–500 μM had little effect on [Ca 2+] i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these conditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of [Ca 2+] i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine. 相似文献
3.
It is well established that intracellular calcium ([Ca 2+] i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca 2+ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSC Ca) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSC Ca that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca 2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca 2+ oscillations followed by an overall increase in [Ca 2+] i. The latter occurs also in HL-1 cells in Ca 2+-free solution and after depletion of sarcoplasmic reticulum Ca 2+ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130–150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca 2+ oscillations followed by a compensatory increase in [Ca 2+] i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion. 相似文献
4.
In the resting state, the Ca 2+ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca 2+, which is mediated by Ca 2+-ATPase (SERCA), and passive leakage of Ca 2+. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments,
we examined possible pathways of passive leakage of Ca 2+ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements
of the concentration of Ca 2+ in the ER ([Ca 2+] ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with
the help of β-escin (40 μg/ml); the Ca 2+ concentration in the cytoplasm ([Ca 2+]
i
) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca 2+ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin,
resulted in complete inhibition of the uptake of Ca 2+ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding
solutions and was accompanied by the leakage of Ca 2+ from the ER; this is confirmed by a gradual drop in the [Ca 2+] ER. Such a leakage of Ca 2+ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol
1,4,5-trisphosphate-sensitive receptors (InsP 3R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides
from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca 2+ from the ER. This effect did not depend on the functioning of SERCA, InsP 3R, or RyR. Therefore, passive leakage of Ca 2+ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the
ER membrane.
Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 339–346, July–August, 2005. 相似文献
5.
Using a Ca 2+-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K +]
e
up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca 2+]
i
, whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA 2+-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca 2+]
i
and a decrease in the K +-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca 2+]
i
. Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca 2+ influx and Ca 2+-induced Ca 2+ release from the endoplasmic reticulum; the initial level of [Ca 2+]
i
was restored at the joint involvement of Ca 2+-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria. 相似文献
6.
Rationale: Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored. Objective: To identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action. Methods and Results: Freshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free Ca2+ concentration ([Ca2+]i) and contractility was performed in individual CMs paced at 0.3 Hz. Our findings demonstrate that TRPA1 stimulation with AITC results in a dose-dependent increase in peak [Ca2+]i and a concomitant increase in CM fractional shortening. Further analysis revealed a dose-dependent acceleration in time to peak [Ca2+]i and velocity of shortening as well as an acceleration in [Ca2+]i decay and velocity of relengthening. These effects of TRPA1 stimulation were not observed in CMs pre-treated with the TRPA1 antagonist, HC-030031 (10 µmol/L) nor in CMs obtained from TRPA1?/? mice. Moreover, we observed no significant increase in cAMP levels or PKA activity in response to TRPA1 stimulation and the PKA inhibitor peptide (PKI 14–22; 100 nmol/L) failed to have any effect on the TRPA1-mediated increase in CM contractile function. However, TRPA1 stimulation resulted in a rapid phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII) (1–5 min) that correlated with increases in CM [Ca2+]i and contractile function. Finally, all aspects of TRPA1-dependent increases in CM [Ca2+]i, contractile function and CaMKII phosphorylation were virtually abolished by the CaMKII inhibitors, KN-93 (10 µmol/L) and autocamtide-2-related peptide (AIP; 20 µmol/L). Conclusions: These novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca2+ availability and handling leading to increases in CM contractile function. Cardiac TRPA1 ion channels may represent a novel therapeutic target for increasing the inotropic and lusitropic state of the heart. 相似文献
7.
The effect of the natural product diindolylmethane on cytosolic Ca 2+ concentrations ([Ca 2+] i) and viability in PC3 human prostate cancer cells was explored. The Ca 2+-sensitive fluorescent dye fura-2 was applied to measure [Ca 2+] i. Diindolylmethane at concentrations of 20–50 µM induced [Ca 2+] i rise in a concentration-dependent manner. The response was reduced partly by removing Ca 2+. Diindolylmethane-evoked Ca 2+ entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca 2+, incubation with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca 2+] i rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca 2+] i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca 2+] i rise. At concentrations of 50–100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca 2+ with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca 2+] i rise in PC3 cells by evoking phospholipase C-dependent Ca 2+ release from the endoplasmic reticulum and Ca 2+ entry via phospholipase A 2-sensitive store-operated Ca 2+ channels. Diindolylmethane caused cell death in which apoptosis may participate. 相似文献
8.
Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly
elevated the intracellular calcium level ([Ca 2+] i) in a dose-dependent manner and activated the L-type Ca 2+ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca 2+] i elevation was abolished in the presence of the ET A receptor blocker BQ123, but was not affected by the ET B receptor blocker BQ788. ET-1-induced an increase in [Ca 2+] i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine
receptor. The ET-1-induced [Ca 2+] i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor
(AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca 2+ channel current and an increase of open-state probability (NPo) of an L-type single Ca 2+ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated
that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca 2+ channel activation and Ca 2+-induced Ca 2+ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway.
Supported by the National Natural Science Foundation of China (Grant No. 200830870910). 相似文献
10.
Glucagon induces intracellular Ca 2+ ([Ca 2+] i) elevation by stimulating glucagon receptor (GCGR). Such [Ca 2+] i signaling plays important physiological roles, including glycogenolysis and glycolysis in liver cells and the survival of β-cells. Previous studies indicated that phospholipase C (PLC) might be involved in glucagon-mediated [Ca 2+] i response. Other studies also debated whether cAMP accumulation mediated by GCGR/Gα s coupling contributes to [Ca 2+] i elevation. But the exact mechanisms remain uncertain. In the present study, we found that glucagon induces [Ca 2+] i elevation in HEK293 cells expressing GCGR. Removing extracellular Ca 2+ did not affect glucagon-stimulated [Ca 2+] i response. But depleting the intracellular Ca 2+ store by thapsigargin completely inhibited glucagon-induced [Ca 2+] i response. Experiments with forskolin and adenylyl cyclase inhibtor revealed that cAMP is not the cause of [Ca 2+] i response. Further studies with Gα q/11 RNAi and pertussis toxin (PTX) indicated that both Gα q/11 and Gα i/o are involved. Combination of Gα q/11 RNAi and Gα i/o inhibition almost completely abolished glucagon-induced [Ca 2+] i signaling. 相似文献
11.
The Ins(1,4,5) P3 receptor acts as a central hub for Ca 2+ signaling by integrating multiple signaling modalities into Ca 2+ release from intracellular stores downstream of G-protein and tyrosine kinase-coupled receptor stimulation. As such, the Ins(1,4,5) P3 receptor plays fundamental roles in cellular physiology. The regulation of the Ins(1,4,5) P3 receptor is complex and involves protein-protein interactions, post-translational modifications, allosteric modulation, and regulation of its sub-cellular distribution. Phosphorylation has been implicated in the sensitization of Ins(1,4,5) P3-dependent Ca 2+ release observed during oocyte maturation. Here we investigate the role of phosphorylation at T-930, a residue phosphorylated specifically during meiosis. We show that a phosphomimetic mutation at T-930 of the rat Ins(1,4,5) P3 receptor results in decreased Ins(1,4,5) P3-dependent Ca 2+ release and lowers the Ins(1,4,5) P3 binding affinity of the receptor. These data, coupled to the sensitization of Ins(1,4,5) P3-dependent Ca 2+ release during meiosis, argue that phosphorylation within the coupling domain of the Ins(1,4,5) P3 receptor acts in a combinatorial fashion to regulate Ins(1,4,5) P3 receptor function. 相似文献
12.
Striated muscles (skeletal and cardiac) are major physiological targets of insulin and this hormone triggers complex signaling pathways regulating cell growth and energy metabolism. Insulin increases glucose uptake into muscle cells by stimulating glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface. The canonical insulin-triggered signaling cascade controlling this process is constituted by well-mapped tyrosine, lipid and serine/threonine phosphorylation reactions. In parallel to these signals, recent findings reveal insulin-dependent Ca 2+ mobilization in skeletal muscle cells and cardiomyocytes. Specifically, insulin activates the sarco-endoplasmic reticulum (SER) channels that release Ca 2+ into the cytosol i.e., the Ryanodine Receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP 3R). In skeletal muscle cells, a rapid, insulin-triggered Ca 2+ release occurs through RyR, that is brought about upon S-glutathionylation of cysteine residues in the channel by reactive oxygen species (ROS) produced by the early activation of the NADPH oxidase (NOX2). In cardiomyocytes insulin induces a fast and transient increase in cytoplasmic [Ca 2+] i trough L-type Ca 2+ channels activation. In both cell types, a relatively slower Ca 2+ release also occurs through IP 3R activation, and is required for GLUT4 translocation and glucose uptake. The insulin-dependent Ca 2+ released from IP 3R of skeletal muscle also promotes mitochondrial Ca 2+ uptake. We review here these actions of insulin on intracellular Ca 2+ channel activation and their impact on GLUT4 traffic in muscle cells, as well as other implications of insulin-dependent Ca 2+ release from the SER. 相似文献
13.
We investigated the contribution of L-, N- and P/Q-type Ca 2+ channels to the [Ca 2+] i changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca 2+ imaging. Selective Ca 2+ channel blockers, namely nitrendipine, ω-Conotoxin GVIA (ω-GVIA) and ω-Agatoxin IVA (ω-AgaIVA) were used. The [Ca 2+] i changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but
the N-methyl- d-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca 2+ channel blocker caused differential inhibitory effects on [Ca 2+] i responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses
below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above
90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by ω-GVIA or ω-AgaIVA. Thus,
in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case
of ω-GVIA or ω-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what
concerns their L-, N- and P/Q- type Ca 2+ channels activated by stimulation of the AMPA/kainate receptors.
Special issue article in honor of Dr. Ricardo Tapia. 相似文献
14.
We used MCF-7 human breast cancer cells that endogenously express Cav3.1 and Cav3.2 T-type Ca 2+ channels toward a mechanistic study on the effect of EGCG on [Ca 2+] i. Confocal Ca 2+ imaging showed that EGCG induces a [Ca 2+] i spike which is due to extracellular Ca 2+ entry and is sensitive to catalase and to low-specificity (mibefradil) and high-specificity (Z944) T-type Ca 2+channel blockers. siRNA knockdown of T-type Ca 2+ channels indicated the involvement of Cav3.2 but not Cav3.1. Application of EGCG to HEK cells expressing either Cav3.2 or Cav3.1 induced enhancement of Cav3.2 and inhibition of Cav3.1 channel activity. Measurements of K + currents in MCF-7 cells showed a reversible, catalase-sensitive inhibitory effect of EGCG, while siRNA for the Kv1.1 K + channel induced a reduction of the EGCG [Ca 2+] i spike. siRNA for Cav3.2 reduced EGCG cytotoxicity to MCF-7 cells, as measured by calcein viability assay. Together, data suggest that EGCG promotes the activation of Cav3.2 channels through K + current inhibition leading to membrane depolarization, and in addition increases Cav3.2 currents. Cav3.2 channels are in part responsible for EGCG inhibition of MCF-7 viability, suggesting that deregulation of [Ca 2+] i by EGCG may be relevant in breast cancer treatment. 相似文献
15.
Summary Intracellular calcium [Ca 2+]
i
measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca 2+]
i
transient followed by a steady-state increase as the characteristic [Ca 2+]
i
response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca 2+]
i
signal in single cells. The distribution of [Ca 2+]
i
in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca 2+]
i
present in the subplasmalemmal space and in one cell pole. [Ca 2+]
i
gradients within these regions were not constant but showed temporal changes in the form of [Ca 2+]
i
oscillations and spatial changes in the form of [Ca 2+]
i
waves. [Ca 2+]
i
oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca 2+] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca 2+], was 171 ± 13 n m and the mean oscillation amplitude was 194 ± 12 n m. Generation of [Ca 2+]
i
waves was also independent of influx of extracellular Ca 2+. [Ca 2+]
i
waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca 2+]
i
induced by different agonists were encoded into changes of baseline [Ca 2+]
i
and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca 2+]
i
regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca 2+]
i
regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca 2+]
i
oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar 9,Met(O 2) 11]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI. 相似文献
16.
BackgroundWe have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca 2+ [Ca 2+] i. We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca 2+] i. MethodsHSV were investigated in vitro either unfrozen within 12 h after removal or after storage at −196 °C in a cryomedium containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose as cryoprotectant additives. ResultsCryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration-response curves for CaCl 2 on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (−)-( R) enantiomer of SDZ 202-791 whereas the Ca 2+ channel activating (+)-( S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl 2 when tested after cryostorage. These functional effects were reflected by changes in [Ca 2+] i as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-( S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 μM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage. ConclusionsData suggested that cryopreservation modified [Ca 2+] i of venous smooth muscle cells (1) through depolarization-induced changes in Ca 2+ influx and (2) through activation of Rho kinase signaling pathways. 相似文献
17.
Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca 2+] i levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca 2+-sensitive fluorescent probe. Thapsigargin at concentrations between 10?nM and 10 µM increased [Ca 2+] i in a concentration-dependent fashion. The Ca 2+ signal was reduced partly by removing extracellular Ca 2+ indicating that Ca 2+ entry and release both contributed to the [Ca 2+] i rise. This Ca 2+ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca 2+ channels or by modulation of protein kinase C activity. In Ca 2+-free medium, pretreatment with the endoplasmic reticulum Ca 2+ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca 2+ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca 2+] i rise, suggesting that thapsigargin released Ca 2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca 2+] i rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca 2+ with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca 2+] i rises by causing phospholipase C-independent Ca 2+ release from the endoplasmic reticulum and Ca 2+ influx via phospholipase A2-sensitive Ca 2+ channels. Thapsigargin also induced cell death via Ca 2+-dependent pathways and Ca 2+-independent apoptotic pathways. 相似文献
18.
The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca 2+ concentrations ([Ca 2+] i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca 2+] i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 µM increased [Ca 2+] i in a concentration-dependent manner. The Ca 2+ signal was reduced partly by removing extracellular Ca 2+. The econazole-induced Ca(2+) influx was insensitive to L-type Ca 2+ channel blockers and protein kinase C modulators. In Ca 2+-free medium, after pretreatment with 20 µM econazole, [Ca 2+] i rises induced by 1 µM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca 2+] i rises. Inhibition of phospholipase C with 2 µM U73122 did not change econazole-induced [Ca 2+] i rises. At concentrations between 10 and 80 µM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 µM econazole was not reversed by prechelating cytosolic Ca 2+ with BAPTA. This shows that in SIRC cells econazole induces [Ca 2+] i rises by causing Ca 2+ release from the endoplasmic reticulum and Ca 2+ influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca 2+] i rise. 相似文献
19.
Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca 2+ concentration ([Ca 2+] d) and diastolic Na + concentration ([Na +] d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd 3+)-sensitive Ca 2+ entry and inositol triphosphate (IP 3) signaling pathways in abnormal [Ca 2+] d and [Na +] d were investigated. Our results showed an age-dependent increase in both [Ca 2+] d and [Na +] d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd 3+ treatment significantly reduced both [Ca 2+] d and [Na +] d at all ages. In addition, blockade of the IP 3-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd 3+ normalized both [Ca 2+] d and [Na +] d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca 2+ and Na + overload mediated at least in part by enhanced Ca 2+ entry through Gd 3+ sensitive transient receptor potential channels (TRPC), and by IP 3 receptors. 相似文献
20.
AbstractResveratrol is a natural compound that affects cellular Ca 2+ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca 2+ concentrations ([Ca 2+] i) and viability in PC3 human prostate cancer cells. The Ca 2+-sensitive fluorescent dye fura-2 was used to measure [Ca 2+] i and WST-1 was used to measure viability. Resveratrol-evoked [Ca 2+] i rises concentration-dependently. The response was reduced by removing extracellular Ca 2+. Resveratrol-evoked Ca 2+ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca 2+-free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca 2+] i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca 2+] i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca 2+] i. Previous studies showed that resveratrol between 10 and 100?µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10?μM) increased cell viability, which was abolished by chelating cytosolic Ca 2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10?µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca 2+] i by evoking phospholipase C-independent Ca 2+ release from the endoplasmic reticulum and Ca 2+ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10?µM also caused Ca 2+-dependent cell proliferation. 相似文献
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