Activation Of Store-operated Calcium Channels Research Articles

Molecular mechanism of tamoxifen‐induced calcium influx in human platelets

Tamoxifen, a non steroidal estrogen antagonist, is the most widely used drug for treatment and prevention of breast cancer. However, tamoxifen can cause thrombosis by an unknown mechanism. Using fura-2 to measure intracellular calcium in freshly isolated human platelets, we found that tamoxifen and its major metabolite 4-hydroxy tamoxifen at 5–10 uM promote calcium influx into platelets. Tamoxifen also potentiates action of platelet agonists (thrombin, vasopressin, ADP) to raise calcium. Tamoxifen effect was not attenuated by estrogen receptor antagonist ICI 182780 or COX inhibitor aspirin, but was blocked by the PLC inhibitor U-73122. Tamoxifen-induced calcium elevation was blocked by the lipid-soluble antioxidant vitamin E, but was unaffected by water-soluble vitamin C and quercetin. Tamoxifen effect was also blocked by the NADPH inhibitor apocynin and by wortmannin, an inhibitor of PI3 kinase, an upstream effector of NADPH oxidase. High molecular weight derivatives of 4-hydroxy tamoxifen conjugated to poly-methacrylic acid also promoted calcium elevation in platelets. An anti-estrogenic drug raloxifene, which is structurally related to tamoxifen, does not affect platelet calcium entry. Therefore tamoxifen may affect membrane-bound PLC, NADPH oxidase and PI3 kinase, leading to the activation of store-operated calcium channels, calcium entry into platelets, platelet activation and aggregation, which may be an underlying mechanism of tamoxifen-induced thrombosis. (Supported by grants from CHRB of Virginia and Cancer Research and Prevention Foundation to YD and Army Breast Cancer Research Program to RW).

Mechanisms of agonist-induced constriction in isolated human mesenteric arteries

We determined the calcium signalling pathways involved in the mechanisms of contraction of the vasoconstrictive agonists KCl, U46619 and PDBu in isolated human mesenteric arteries. The influence of gender, vessel diameter and age of the patients was also investigated. Human mesenteric arteries ( n = 76) were loaded in a wire myograph and maintained at a tension equivalent to the in vivo pressure of 100 mm Hg, bubbled with 95%O 2/5%CO 2 to maintain pH 7.4 in physiological saline solution (PSS). Cumulative concentration–response curves were obtained to KCl (100 μM–100 mM), U46619 (1 nM–1 μM) or PDBu (1 nM–1 μM), before or after a 30 min incubation with either the voltage-gated calcium channel (VGCC) blocker nifedipine (10 μM), the store-operated calcium channel (SOCC) blocker SK&F96365 (50 μM) or in calcium-free PSS (−Ca 2+PSS). The KCl response was abolished in −Ca 2+PSS and with nifedipine. The U46619 response was partially blocked in −Ca 2+PSS and with nifedipine and predominantly blocked by SK&F96365. Incubation in −Ca 2+PSS had no effect on the response to PDBu. Arteries from male patients responded significantly higher to KCl than arteries from female patients. This study demonstrates that KCl induces mesenteric vasoconstriction via activation of VGCCs, U46619 induces mesenteric vasoconstriction via activation of SOCCs, but also VGCCs and PDBu induce mesenteric vasoconstriction via a calcium-independent pathway.

Cellular uptake of 99mTcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation

A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ((99m)TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether (99m)TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. Uptake kinetics of (99m)TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of (99m)TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG2M cellular proliferation index. All calcium channel inhibitors significantly decreased the cellular uptake of (99m)TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of (99m)TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG2M. The uptake of (99m)TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that (99m)TcN-NOET might be a marker of cell proliferation.

Role of Ca 2+-independent phospholipase A 2 and cytochrome P-450 in store-operated calcium entry in 3T6 fibroblasts

Store-operated calcium (SOC) channels and capacitative Ca 2+ entry play a key role in cellular functions, but their mechanism of activation remains unclear. Here, we show that thapsigargin induces [ 3H] arachidonic acid (AA) release, 45Ca 2+ influx and a subsequent enhancement of intracellular calcium concentration ([Ca 2+] i. Thapsigargin-induced elevation of [Ca 2+] i was inhibited by cytochrome P-450 inhibitors and by cytochrome P-450 epoxygenase inhibitor and was reverted by 11,12 EET addition. However, cyclooxygenase and lipoxygenase inhibitors have no effect. Moreover, we observed that four EETs were able to induce 45Ca 2+ influx. Finally, we reported that the effect of 11,12 EET on 45Ca 2+ influx was sensible to receptor-operated Ca 2+ channel blockers (NiCl 2, LaCl 3) but not to voltage-dependent Ca 2+ channel blocker as verapamil. Thus, AA released by Ca 2+-independent phospholipase A 2 and AA metabolism through cytochrome P-450 pathway may be crucial molecular determinant in thapsigargin activation of SOC channels and store-operated Ca 2+ entry pathway in 3T6 fibroblasts. Moreover, EETs, the main cytochrome P-450 epoxygenase metabolites of AA, are involved in thapsigargin-stimulated Ca 2+ influx. In summary, our results suggest that EETs are components of calcium influx factor(s).

Store-operated calcium entry in differentiated C2C12 skeletal muscle cells

In this paper, we show further evidences for the existence of store-operated calcium entry in differentiated skeletal muscle C2C12 myotubes after Ca 2+ depletion in sarcoplasmic reticulum, using thapsigargin, a potent sarcoplasmic reticulum Ca 2+-ATPase inhibitor, caffeine as ryanodine receptor activator, and ATP which activates purinergic receptors. The quenching of fura 2 fluorescence emission by Mn 2+ also provided evidences for store-operated calcium entry because this quenching was accelerated when sarcoplasmic reticulum was depleted of Ca 2+. Ca 2+ entry was sensitive to Ni 2+, La 3+, Gd 3+ and 2-aminoethyl diphenyl borate but resistant to nifedipine, thus excluding L-type Ca 2+ channels in this type of calcium entry. Our data obtained using ATP for store depletion suggest that the level of Ca 2+ in internal stores could play a role in the regulation of store-operated calcium channel activity in this cell type.

2-Aminoethoxydiphenyl borate perturbs hormone-sensitive calcium stores and blocks store-operated calcium influx pathways independent of cytoskeletal disruption in human A549 lung cancer cells

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca 2+-signaling pathways, revealing differentially sensitive Ca 2+-influx pathways and long-term perturbation of hormone-sensitive Ca 2+ stores.

Functional role of lipid rafts in CD20 activity?

CD20 is a B-lymphocyte-specific integral membrane protein, implicated in the regulation of transmembrane calcium conductance, cell-cycle progression and B-lymphocyte proliferation. CD20 is proposed to function as a SOCC (store-operated calcium channel). SOCCs are activated by receptor-stimulated calcium depletion of intracellular stores. Sustained calcium conductivity across the plasma membrane mediated by SOCC activity is required for long-term calcium-dependent processes, such as transcriptional control and gene expression. Cross-linking of CD20 by antibodies (e.g. Rituxan) has been reported to induce a rapid redistribution of CD20 into specialized microdomains at the plasma membrane, known as lipid rafts. Recruitment of CD20 into lipid rafts and its homo-oligomerization are suggested to be crucial for CD20 activity and regulation. This review outlines recent biochemical studies characterizing the role of CD20 in calcium signalling in B-lymphocytes and evaluates an engagement of lipid rafts in the regulation of CD20-mediated calcium conductivity.

Inhibitory Mechanism of Store-operated Ca2+ Channels by Zinc

Capacitative calcium influx plays an important role in shaping the Ca(2+) response of various tissues and cell types. Inhibition by heavy metals is a hallmark of store-operated calcium channel (SOCC) activity. Paradoxically, although zinc is the only potentially physiological relevant ion, it is the least investigated in terms of inhibitory mechanism. In the present study, we characterize the inhibitory mechanism of the SOCC by Zn(2+) in the human salivary cell line, HSY, and rat salivary submandibular ducts and acini by monitoring SOCC activity using fluorescence imaging. Analysis of Zn(2+) inhibition indicated that Zn(2+) acts as a competitive inhibitor of Ca(2+) influx but does not permeate through the SOCC, suggesting that Zn(2+) interacts with an extracellular site of SOCC. Application of the reducing agents, dithiothreitol (DTT) and beta-mercaptoethanol, totally eliminated Zn(2+) and Cd(2+) inhibition of SOCC, suggesting that cysteines are part of the Zn(2+) and Cd(2+) binding site. Interestingly, reducing conditions failed to eliminate the inhibition of SOCC by La(3+) and Gd(3+), indicating that the Zn(2+) and lanthanides binding sites are distinct. Finally, we show that changes in redox potential and Zn(2+) are regulating, via SOCC activity, the agonist-induced Ca(2+) response in salivary ducts. The presence of a specific Zn(2+) site, responsive to physiological Zn(2+) and redox potential, may not only be instrumental for future structural studies of various SOCC candidates but may also reveal novel physiological aspects of the interaction between zinc, redox potential, and cellular Ca(2+) homeostasis.

Retrograde activation of store-operated calcium channel

Store-operated Ca 2+ entry represents an important mechanism for refilling of a depleted intracellular-reticulum Ca 2+ store following sustained activation of the IP 3 receptor or ryanodine receptor RyR/Ca 2+ release channel in the endoplasmic/sarcoplasmic reticulum (ER/SR). Recent studies have demonstrated the existence of store-operated Ca 2+ channel (SOC) in muscle cells, whose activation process appears to be coupled to conformational changes of the RyR. Regulation of the plasma membrane (PM)-resided SOC by the SR-located RyR requires an integrity of the junctional membrane structure between SR and PM. Proteins that interact with RyR or influence the Ca 2+ buffering capacity in the ER or SR lumen also participate in the activation process of SOC. Calsequestrin (CSQ) and calreticulin (CRT) are SR/ER-resident proteins, with highly negative charged regions at the carboxyl-terminal end that exhibit high buffering capacity for luminal Ca 2+. CSQ and CRT not only modulate the intracellular Ca 2+ release process but also might provide retrograde signals to regulate the function of SOC. The functional interplay between CSQ, RyR and SOC may serve essential roles of Ca 2+ signaling in muscle contraction and development. A tight link between the expression of CRT and operation of SOC exist in certain cancer cells, where the reduced sensitivity to apoptosis may correlate with the altered function of SOC.

Abnormal Calcium Homeostasis in Fibroblasts from Patients with Leigh Disease

Recently, we reported that in various cell lines under conditions of deenergization of the mitochondrial membrane, the release of Ca2+ from the endoplasmic reticulum (ER) does not produce the expected activation of store-operated calcium channels (SOCs) in the plasma membrane. In the present work, we examined the activation of SOCs in fibroblasts derived from three patients with Leigh disease (LS). We identified mutations in the SURF-1 gene in all these cells. Consequently, cytochrome oxidase (COX) deficiency was found in all these (LSCOX) cell lines and, thus, the main mitochondrial mechanism of generation of the electrochemical proton gradient on the mitochondrial membrane was naturally depressed. We demonstrated that, in untreated LSCOX fibroblasts, the rate of Ca2+-inflow through SOCs was low compared to the fibroblasts from healthy individuals even after thapsigargin-induced maximal release of Ca2+ from the ER. Moreover, the pretreatment of LSCOX fibroblasts with a protonophore did not modify this rate. Thus, in LSCOX fibroblasts, the activation of SOCs was naturally impaired. Our findings suggest that altered calcium metabolism, apart from severe energy production failure, may also contribute to developing pathological conditions in patients with COX-deficient Leigh disease related to SURF-1 gene mutation.

Mitochondrial uncoupling does not influence the stability of the intracellular signal activating plasma membrane calcium channels.

The effects of various concentrations of thapsigargin, a specific inhibitor of Ca2+-ATPase in the endoplasmic reticulum (ER) membrane, on calcium homeostasis in lymphoidal T cells (Jurkat) were investigated. Preincubation of these cells suspended in nominally calcium-free medium with 0.1 microM thapsigargin resulted in a complete release of Ca2+ from intracellular calcium stores. When the medium was supplemented with 3 mM CaCl2 the cells maintained constantly elevated level of cytosolic Ca2+. However, thapsigargin applied at lower concentration produced only a partial depletion of the stores. For example, in the cells pretreated with 1 nM thapsigargin and suspended in calcium-free medium approximately 75% of the calcium content was released from the intracellular stores. The addition of 3 mM CaCl2 to such cell suspension led to a transient increase in cytosolic calcium concentration, followed by a return to a lower steady-state. This phenomenon, related to the refilling of the ER by Ca2+, allowed to estimate the half-time for the process of cell recovery after activation of store-operated calcium channels. By this approach we have found that carbonyl cyanide m-chlorophenylhydrazone, which has been documented to inhibit calcium entry into Jurkat cells, does not influence the stability of the intracellular signal involved in the activation of store-operated calcium channels.

Mitochondria can modify the activity of store-operated calcium channels in the plasma membrane of Jurkat cells

Mitochondria can modify the activity of store-operated calcium channels in the plasma membrane of Jurkat cells

Effect of calmidazolium analogs on calcium influx in HL-60 cells

The structure–activity relationships of calmidazolium analogs with respect to intracellular calcium levels were investigated in HL-60 cells. Quaternized derivatives of miconazole and clotrimazole, known inhibitors of store-operated calcium (SOC) channels, were synthesized. The quaternary N-methyl derivatives of miconazole ( 3) and clotrimazole ( 6) had no effect on intracellular calcium levels, alone or after elevation of calcium induced by ATP. Calmidazolium alone induced a large increase in intracellular calcium levels in HL-60 cells ( ec 50 3 μM). Similar effects were observed for miconazole derivatives 1 ( ec 50 15 μM) and 2 ( ec 50 10 μM), wherein the diphenylmethyl group in calmidazolium was replaced by a 3,5-difluorobenzyl or cyclohexylmethyl group, respectively. The analogous clotrimazole derivatives 4 and 5 had no effect on intracellular calcium levels. The elevation of calcium levels by calmidazolium, 1, and 2 appears to be comprised of a calcium release component from inositol trisphosphate (IP 3)-sensitive stores followed by a large calcium influx component. Calcium influx was greater than that normally observed due to depletion of IP 3-sensitive calcium stores and activation of SOC channels. In addition, only a small component of the calmidazolium-elicited influx was inhibited by the SOC channel blocker miconazole. Thus, certain quaternized imidazoles substituted with large residues at both nitrogens of the imidazole ring caused both release and influx of calcium, the latter in part through SOC channels but mainly through an undefined cationic channel. Quaternized imidazoles, unlike the parent nonquaternary imidazole miconazole, did not block SOC channels. Inhibitory effects on calmodulin-activated phosphodiesterase did not correlate with effects on calcium release and influx.

Differential regulation of Ca2+ influx by fMLP and PAF in human neutrophils: possible involvement of store-operated Ca2+ channel.

Calcium (Ca2+) influx into human polymorphonuclear cells (PMNs) in response to N-formyl-Met-Leu-Phe (fMLP) and platelet-activating factor (PAF) stimulation was studied. Whole blood was taken by venous puncture from healthy human volunteers. PMNs were isolated, diluted, and incubated with 2 microM fura-2 AM. The cytosolic free calcium concentration, [Ca2+]i, in human neutrophils was determined by microfluorometry. We found that the net area under the fMLP- or PAF-induced [Ca2+]i rise curve in Ca2+-free medium decreased to 75% or 30% of the area under the curve in Ca2+ medium. Treatment of PMNs with phorbol myristate acetate (PMA), a protein kinase C activator, completely abolished the intracellular Ca2+ level stimulated by PAF, but not the intracellular Ca2+ level stimulated by fMLP. Treatment of PMNs with PAF did not abolish the intracellular Ca2+ level elevation stimulated by fMLP. In addition, treatment of PMNs with fMLP did not abolish intracellular Ca2+ level elevation stimulated by PAF. Loperamide, a positive modulator for store-operated calcium (SOC) channels, elicited an increase in intracellular calcium after the activation of SOC channels stimulated by fMLP or PAF. After the addition of guanosine 3',5'-cyclic monophosphate, N2,2'-O-Dibutyryl-, sodium salt (db-cGMP), the initial increase of PAF- or fMLP-induced PMNs intracellular Ca2+ fluorescences was well preserved, but the slope and the peak height of fluorescence curves declined compared with the curves without db-cGMP. In conclusion, we found that PAF and fMLP regulate the Ca2+ influx of PMNs in different ways. Most of the PAF-induced [Ca2+]i rise resulted from Ca2+ influx, and most of the fMLP-induced [Ca2+]i elevation resulted from intracellular stores release. The initial mobilization of intracellular Ca2+ stores in PAF-stimulated signals is mediated by protein kinase C (PKC) phosphorylation, but not in fMLP-stimulated route. SOC channels are present and important in the fMLP- or PAF-induced PMNs Ca2+ influx. There was no apparent cross-regulation between PAF- and fMLP-stimulated intracellular Ca2+ influx.

The role of mitochondria in the regulation of calcium influx into Jurkat cells.

In electrically nonexcitable cells the activity of the plasma membrane calcium channels is controlled by events occurring in mitochondria, as well as in the lumen of the endoplasmic reticulum. Thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, produces the release of calcium from the endoplasmic reticulum and thus, activation of store-operated calcium channels in the plasma membrane. However, thapsigargin failed to produce significant activation of the channels in Jurkat cells that had been pretreated with mitochondria-directed agents: an uncoupler (carbonyl cyanide m-chlorophenylhydrazone) and oligomycin. This is in spite of the fact that Jurkat cells pretreated with carbonyl cyanide m-chlorophenylhydrazone plus oligomycin are otherwise energetically competent, due to a high rate of glycolysis and the inhibition of mitochondrial F1Fo-ATPase by oligomycin. The pool of intracellular ATP was found not to be influenced by the pretreatments of cells with oligomycin or with oligomycin plus carbonyl cyanide m-chlorophenylhydrazone. In the control cells, we found that the ATP pool amounted to 23.2 +/- 1.9 nmoles per 107 cells (n = 4). In cells pretreated with oligomycin the level of ATP was 21.8 +/- 1.9 nmoles per 107 cells (n = 4), and in cells pretreated with both oligomycin and an uncoupler the level of ATP was 22.1 +/- 0.2 nmoles per 107 cells (n = 3). Moreover, in cells pretreated with oligomycin plus carbonyl cyanide m-chlorophenylhydrazone and suspended in a nominally calcium-free medium, thapsigargin produces transient increases in cytosolic calcium identical to those in the control cells. Thus, this pretreatment does not modify either the content of intracellular calcium stores and/or the activity of calcium ATPase in the plasma membrane. Similar results were obtained when Jurkat cells were challenged by myxothiazol, a potent inhibitor of mitochondrial cytochrome bc1 oxidoreductase. Thapsigargin, although producing calcium release from intracellular stores, was ineffective in triggering the activation of calcium channels in the plasma membrane in the case of cells pretreated with myxothiazol and oligomycin. Our results suggest that coupled mitochondria participate directly in the control of calcium channel activity in the plasma membrane of Jurkat cells. When the mitochondrial protonmotive force is collapsed, either by carbonyl cyanide m-chlorophenylhydrazone or myxothiazol, the channel remains inactive even under conditions of empty intracellular calcium stores.

Inhibitors of store-operated calcium channels: Imidazoles, phenothiazines, and other tricyclics

Imidazoles, such as SKF 96365, clotrimazole, and miconazole, have represented the major class of inhibitors of store-operated calcium (SOC) channels. In HL60 cells, ATP and thapsigargin cause depletion of intracellular calcium stores, resulting in activation of SOC channels. Tricyclic phenothiazines and other tricyclics, such as amitriptyline and clozapine, were found to inhibit SOC channels of HL60 cells, with trifluoperazine being of comparable potency to the imidazoles. The effects of the imidazoles and tricyclics on intracellular calcium levels in HL60 cells were compared. In addition to inhibiting SOC channels, imidazoles, in particular, miconazole, caused a marked release of intracellular calcium, apparently from the same intracellular pool depleted by ATP and thapsigargin. The phenothiazines and the other two tricyclics caused little or no release of intracellular calcium compared to that caused by miconazole. However, these compounds did cause a significant decrease in the release of intracellular calcium elicited by subsequent addition of ATP or thapsigargin. Loperamide, which appears to selectively and positively modulate activated SOC channels, augments the elevation of intracellular calcium elicited by the imidazoles and, to a lesser extent, the tricyclics. The augmentation of calcium levels by loperamide indicated that these agents had, through release of intracellular calcium, caused activation of SOC channels. Drug Dev. Res. 47:107–117, 1999. Published 1999 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Loperamide: A positive modulator for store-operated calcium channels?

The depletion of inositol trisphosphate-sensitive intracellular pools of calcium causes activation of store-operated calcium (SOC) channels. Loperamide at 10-30 microM has no effect on intracellular calcium levels alone, but augments calcium levels in cultured cells when SOC channels have been activated. In HL-60 leukemic cells, the apparent positive modulatory effect of loperamide on SOC channels occurs when these channels have been activated after ATP, thapsigargin, or ionomycin-elicited depletion of calcium from intracellular storage sites. Loperamide has no effect when levels of intracellular calcium are elevated through a mechanism not involving SOC channels by using sphingosine. Loperamide caused augmentation of intracellular calcium levels after activation of SOC channels in NIH 3T3 fibroblasts, astrocytoma 1321N cells, smooth muscle DDT-MF2 cells, RBL-2H3 mast cells, and pituitary GH4C1 cells. Only in astrocytoma cells did loperamide cause an elevation in intracellular calcium in the absence of activation of SOC channels. The augmentation of intracellular calcium elicited by loperamide in cultured cells was dependent on extracellular calcium and was somewhat resistant to agents (SKF 96365, miconazole, clotrimazole, nitrendipine, and trifluoperazine) that in the absence of loperamide effectively blocked SOC channels. It appears that loperamide augments influx of calcium through activated SOC channels.