1 Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
2 Department of Neuroscience, Medical College of Ohio, 3036 Arlington Avenue, Toledo, OH 43614, USA
3 INSERM, EMI 0228, IFR118, Université des Sciences et Technologies de Lille 1, Bât. SN3, 59655 Villeneuve d'Ascq CEDEX, France
4 INSERM, U442, IFR46, Université Paris-Sud, Bât.443, 91405 Orsay CEDEX, France
* Author for correspondence (e-mail: thierry.capiod{at}univ-lille1.fr)
Accepted 25 August 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: CAI, 2-APB, CCE, Mitochondrial respiration, ARC
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The blocking effect of CAI on CCE would explain its role in cell proliferation, which mainly depends on the activation of this specific calcium-entry pathway. However, the actual mechanism by which it occurs may limit the therapeutic usefulness of this molecule.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calcium measurements
HEK-293 cells (5x106 cells/ml) were loaded with 4 µM Fura-2/AM in complete culture medium for 45 minutes at 37°C. Cells were treated with trypsin and washed once by centrifugation at 300 g for 1 minute in the same medium. Cell pellets were resuspended in 116 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 1 mM NaH2PO4, 5 mM NaHCO3, 0.1 mM EGTA, 20 mM HEPES, pH 7.3. Cell suspensions were transferred to a quartz cuvette and placed in the light beam of a Hitachi F2000 spectrofluorimeter, with continuous stirring, at 37°C. Changes in [Ca2+]i were recorded by measuring increases in the ratio of the readings obtained at excitation wavelengths of 340 and 380 nm.
Optical assessment of mitochondrial membrane potential (m)
HEK-293 cells were incubated in phosphate-buffered saline (PBS) (Gibco, Rockville, MD) supplemented with 2 µg/ml JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) for 30 minutes at room temperature in the dark. Cells were then centrifuged at 1200 g for 5 minutes and resuspended in dye-free physiological saline solution (PSS) containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.2 mM CaCl2, 10 mM HEPES, 10 mM Glucose, pH 7.2. The cells (100,000 cells/well) were then placed in 48-well culture plates (Corning plate 3548). A Fluostar Optima multi-well plate reader (BMG Labtechnologies, Durham, NC) was used to detect the fluorescence emission at 590 nm and 520 nm in response to alternating 530 nm and 485 nm excitation, respectively.
Mn2+ quenching
The entry of Ca2+ into individual intact cells was measured as the rate at which intracellular indo-1 was quenched by Mn2+, as previously described (Shuttleworth and Thompson, 1999).
Electrophysiological recordings
Macroscopic whole-cell currents were recorded using an Axopatch 1-C patch-clamp amplifier (Axon Instruments, Foster City, CA, USA) as previously described (Mignen and Shuttleworth, 2000).
Monitoring mitochondrial calcium dynamics
HEK-293 cells stably expressing the m3 muscarinic receptor (kind gift of Craig Logsdon, University of Michigan Medical School, Ann Arbor, MI) were grown in DMEM supplemented with 10% FCS, penicillin (200,000 U/ml) and streptomycin (100 µg/ml) (Gibco, Rockville, MD) on 25x25 mm clean glass coverslips, which formed the bottom of a perfusion chamber. Cells were loaded with 2 µM Rhod-2/AM, Rhod-FF/AM and Rhod-5N/AM mixture in PSS for 30 minutes at room temperature in the dark. Cells were then permeabilized for 3 minutes at 37°C in an intracellular saline solution (ISS) containing 10 µM digitonin but no added Ca2+. The ISS contained 130 mM KCl, 10 mM NaCl, 1 mM K3PO4, 1 mM ATP, 0.02 mM ADP, 2 mM succinate, 20 mM HEPES, 2 mM MgCl2 (adjusted to buffer Ca2+). Intracellular saline solutions with specific set calcium concentrations were obtained by adding HEDTA/Ca2+. EGTA (250 µM) was added to the `zero'-Ca2+ solution, pH 6.8. The Ca2+ challenge solutions (containing 3-3000 µM Ca2+) were exchanged using a pressure-driven perfusion system. Changes in [Ca2+]m were monitored by digital fluorescence imaging on a Nikon TE2000-S inverted fluorescence microscope (Nikon, Melville, NY) equipped with a monochrometer-based imaging system (TILL Photonics, Martinsried, Germany) and a Nikon 40x SuperFluor oil-immersion objective lens, NA 1.3. All fluorescent data were converted to F/F0=100[(F-F0)/F0], where F is the recorded fluorescence and F0 is the average of the first 15 frames of data. Full-frame images were collected at 1 second intervals for at least 400 seconds and changes are expressed as the percentage increase compared with F0.
TMRE plate reader analysis
HEK-293 cells were loaded with 1.5 µM TMRE (tetramethylrhodamine ethyl ester perchlorate) in PSS for 15 minutes at room temperature in the dark. Dye was then washed with fresh dye-free PSS and spun down at 300 g for 5 minutes. Cells were then resuspended in 24 ml PSS, loaded onto a 24-well plate at 1 ml per well, and allowed to rest for 30 minutes prior to monitoring by fluorescent plate reader. Cells were excited with 544 nm light and emission was measured at 590 nm. Wells were measured at 1 minute intervals for 10 minutes prior to treatment. Wells were then treated with 0 µM CAI (control), 10 µM CAI or 20 µM carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone (FCCP) and monitoring was resumed for an additional 30 minutes.
TMRE digital imaging analysis
HEK-293 cells were loaded on coverslips with 12.5 nM TMRE in PSS for 15 minutes at 37°C in the dark. Cells were then washed with PSS and kept at room temperature for 30 minutes before measuring to allow the dye to concentrate in the mitochondria. Following loading, coverslips were mounted in chambers and monitored by digital fluorescence imaging at 1 Hz. Solution changes were achieved by bath perifusion.
In some experiments [Ca2+]m was measured using a multi-well plate reader. In these experiments, HEK-293 cells were loaded with 200 nM MitoTracker Green FM and 1 µM Rhodamine mixture for 10 minutes, and permeabilized with digitonin. Cells were resuspended in ISS, and half of the cells were incubated with 10 µM CAI for 30 minutes. A Fluostar Optima multi-well fluorescence plate reader (BMG Labtechnologies) was used as described above. Following the addition of Ca2+ to each well, the 590/520 nm ratio was monitored at 5 minute intervals.
Confocal microscopy
Images of JC-1-labeled mitochondria were obtained using a Zeiss 510 Meta laser-scanning confocal microscope equipped with an Axiovert 200 MOT microscope with a 63x/NA 1.4 Plan-Apo oil-immersion objective. JC-1 dye was alternately excited with the 488 nm and 543 nm laser lines.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
After 5 minutes in the presence of CAI, half-maximal CCE inhibition was observed at 0.5 µM and maximal CCE inhibition at approximately 2 µM. However, the amplitude of the inhibitory effect of CAI on CCE depended on the duration of application of the drug. When added 10 seconds before Ca2+, 10 µM CAI reduced the CCE amplitude by 40%, whereas 10 µM CAI led to complete inhibition when added 5 minutes before Ca2+ (Fig. 2). The time-dependent CAI-evoked inhibitory effects on the CCE amplitude suggest that the calcium channels were inhibited by a complex mechanism. CAI had no effect on the [Ca2+]i increase observed in the absence of thapsigargin (data not shown), indicating that CAI affected CCE only. Owing to the potentially toxic effects of CAI (Enfissi et al., 2004), we did not use concentrations exceeding 10 µM.
|
|
CAI depolarizes the mitochondrial inner-membrane potential in a time- and concentration-dependent manner
Mitochondria play a crucial role in the regulation of CCE by buffering local increases in cytosolic Ca2+ thereby limiting the Ca2+-dependent inhibition of CCE channels (Gilabert and Parekh, 2000; Hoth et al., 1997
). Consistent with this, we showed that activation of CCE induced an increase in intramitochondrial Ca2+ concentration, which was blocked by 20 µM FCCP, as measured in intact HEK-293 cells loaded with the mitochondrial-selective Rhod-2 dyes (Fig. 4A). As the ability of mitochondria to take up Ca2+ is dependent on the maintenance of a highly negative intramitochondrial membrane potential, we performed an initial set of experiments to determine the effect of CAI on this potential (
m). The average change in
m was measured in a multi-well fluorescence plate reader using the mitochondrial-selective cationic dye, JC-1. The accumulation of JC-1 in mitochondria is driven by
m. At lower concentrations, JC-1 exists as a green fluorescent monomer, but at higher concentrations it aggregates as a red fluorescent form. Thus, the ratio of red-to-green fluorescence can be used to monitor
m without introducing errors due to mitochondrial swelling. HEK-293 cell suspensions were treated with 10 µM CAI to assess the time course and magnitude of the acute depolarization induced by CAI. The 590/520 nm ratios were calculated at 5 minute intervals for 35 minutes following the administration of vehicle (0.1% DMSO), 20 µM FCCP or 10 µM CAI (Fig. 4B). Drugs were added just before fluorescence acquisition. CAI significantly decreased
m within 5 minutes. At 15 minutes, the vehicle had not significantly altered
m (0.9±0.06; n=12). Treatment with 10 µM CAI or 20 µM FCCP, a protonophore that uncouples mitochondria and abolishes
m, significantly decreased the JC-1 ratio to 0.6±0.03 and 0.4±0.07, respectively, at 15 minutes (n=56 and 16; P<0.001 compared with levels in the controls). The depolarizing effect of CAI on HEK-293 cell mitochondria was validated by fluorescence imaging using confocal microscopy. JC-1-loaded control cells treated for 20 minutes with vehicle exhibited punctate red and green fluorescence, indicating a heterogeneous mitochondrial membrane potential (Fig. 4C). However, cultures that were treated for 20 minutes with 10 µM CAI or 20 µM FCCP displayed a largely diffuse green fluorescence, consistent with impairment of
m (Fig. 4C).
|
Because JC-1 dye provides only a threshold index, rather than a graded indication, of actual mitochondrial membrane potential, we performed a set of experiments to validate our observation that CAI reduced m. Treatment with TMRE at low concentration (12.5 nM) allowed both spontaneous changes in
m to be observed, as well as a reduction (or redistribution) of TMRE signal from mitochondria following treatment with 20 µM FCCP (Fig. 5A). (ROIs were placed on structures identified as mitochondria.) This method was adapted to acquire average
m changes in a multi-well plate reader (that lacked high resolution imaging) by using TMRE loaded at higher concentrations (Duchen et al., 2001
). In these experiments, the net cell fluorescence signal increases as dye redistributes to the cytosol and dequenches (Fig. 5B). Fig. 5C shows the averaged results of multi-well plate reader experiments where 0 or 10 µM CAI was applied to each well and the fluorescence intensity was monitored prior to and following treatment at 1 minute intervals. Similarly to JC-1 experiments, 10 µM CAI was found to significantly reduce
m compared with that in the control in a time-dependent manner. For example, 10 minutes after treatment, the control value was on average 2216±140 fluorescence units whereas the value in CAI-treated cells was 4173±418 fluorescence units (n=12 and 18, respectively; P=0.001). Moreover, the effect of 10 µM CAI was not significantly different from FCCP treatment (3157±162, n=13).
|
|
Next, we assessed the effects of CAI treatment on the relationship between the [Ca2+]m and the applied [Ca2+]. Cells were incubated with 10 µM CAI for 15 minutes prior to permeabilization and calcium was applied in the continued presence of 10 µM CAI. Representative traces of the
[Ca2+]m induced by addition of 30 µM Ca2+ in control conditions and following CAI treatment are shown in Fig. 6D. Line traces shown in the inset of Fig. 6D represent examples of the rate and magnitude of Ca2+ uptake in control mitochondria and in two other experiments where mitochondrial uptake was diminished or even decreased below resting fluorescence levels following CAI treatment. The rate and magnitude of
[Ca2+]m following CAI treatment were generally lower than those measured in control conditions. In some experiments, fluorescence was rapidly lost, suggesting that the permeability transition pore (PTP) had been activated. The PTP has been shown to be activated by elevated [Ca2+]m and by the generation of reactive oxygen species (ROS).
Effects of CAI and 2-APB on HEK-293 cell proliferation
Cell proliferation is tightly dependent on calcium influx (Munaron et al., 2004) and we recently showed that CAI blocks both proliferation and CCE in human hepatoma cells (Enfissi et al., 2004
). We assessed the proliferation of HEK-293 cells by measuring the incorporation of [3H]thymidine. Cells were first incubated for 24 hours in the presence of 10% FCS. The external medium was then replaced by fresh medium supplemented with various concentrations of CAI or 2-APB and the cells were incubated for a further 24 hours. CAI and 2-APB blocked the incorporation of [3H]thymidine into HEK-293 cells, with an IC50 of 1.6 and 50 µM, respectively (Fig. 7A,B), whereas the vehicle (0.1% DMSO) had no effect (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Functional mitochondria with the ability to take up calcium to buffer large increases in cytosolic calcium concentration (Jacobson and Duchen, 2004) are important in the overall control of CCE. Thus, as CCE is inactivated by high intracellular calcium concentrations, it is likely that those mitochondria that are located near to calcium channels (Varadi et al., 2004
), reduce this inactivation. Calcium hotspots as high as 200-300 µM measured in front of calcium channels (Llinas et al., 1992
) are in the range of concentrations detected by mitochondria. Furthermore, the slow rate of addition of calcium in our experiments probably leads to an overestimation of the measured value for mitochondrial calcium import. Consistent with this, inhibition of mitochondrial Ca2+ uptake by compounds that dissipate the mitochondrial membrane potential (such as oligomycin or CCCP) unmasks the Ca2+-dependent inactivation of CCE, resulting in a clear inhibition of calcium entry (Gilabert and Parekh, 2000
; Glitsch et al., 2002
; Hoth et al., 2000
; Hoth et al., 1997
). Mitochondrial respiration was first inhibited approximately 30 seconds after the addition of CAI at concentrations used to block CCE in Hep G2 cells (T.C., unpublished data). The time-dependent nature of the effects of CAI on mitochondria function in HEK-293 cells strongly suggest that CCE inhibition results from an alteration in the ability of mitochondria to buffer cell calcium. Thus, the CAI-induced block of CCE may be due to the direct blockage of calcium channels or to an indirect effect involving mitochondria and inactivation of the current by internal calcium. CAI does not block ISOC when applied at the peak of the current, ruling out a direct block of the channels. Furthermore, increasing the calcium buffering capacity of the cell has been shown to prevent CCE inactivation by mitochondria inhibitors (Glitsch et al., 2002
). We observed that 5 µM CAI reduced the SOC amplitude by about 50% whereas it totally blocked CCE in cell suspensions in which internal calcium is not buffered by EGTA. However, CCE was not sustained and declined to an intermediate plateau phase in HEK-293 cell suspensions. Hence, addition of CAI at the plateau has little effect on the amplitude of the calcium response. However, in human Huh-7 hepatoma cells, in which the plateau phase is often maintained at its maximal level, CAI reduces the amplitude of the CCE (Enfissi et al., 2004
). Alternatively, CAI may alter one of the mechanisms of SOC activation (Venkatachalam et al., 2002
), thus making the drug less effective when applied at the peak of the current activated by store depletion in whole-cell voltage-clamp experiments.
Our data clearly show that CAI does not inhibit Ca2+ entry via the ARC channels. It has also been shown that 2-APB has different effects on CCE and ARC channels (Mignen and Thompson et al., 2003). In Huh-7 cells, addition of 10% FCS evokes sustained increases in [Ca2+]i, which were sensitive to external calcium, 2-APB and CAI (Enfissi et al., 2004
). This suggests that CCE is the only calcium entry pathway activated at this serum concentration. Although CAI blocks cell proliferation induced by 10% FCS, we have no evidence that the effects of lower concentrations of serum would be sensitive to CAI. Hence, although our results clearly show that CCE is implied in cell proliferation, we cannot exclude the possibility that ARC or DAG-activated NCCE channels play a role in cell proliferation at lower serum concentrations.
Precisely how calcium influx via CCE influences cell proliferation is unclear. However, the progression through the cell cycle is regulated by several cyclins. CCE, as opposed to NCCE, activates calcineurin (Mignen et al., 2003), which in turn allows expression of cyclin A and E (Tomono et al., 1998
), and cyclin D2 induction is abolished in the presence of CCE blockers (Glassford et al., 2003
). Therefore, it is likely that calcium influx may block cell proliferation by preventing cyclin induction.
In summary, the reduced cell proliferation rate associated with CCE inhibition reflects the well-described antiproliferative and antimetastasic properties that have led to CAI being proposed as a potential drug for cancer treatment (Kohn and Liotta, 1995; Patton et al., 2003
). Moreover, the fact that CAI clearly inhibits CCE emphasizes the relationships between this specific calcium entry pathway and cell proliferation. However, our unexpected finding that the mitochondria represent a major target for CAI will probably limit its use in the treatment of cancers. Moreover, our results reinforce the crucial role that mitochondria play in the induction of cancer as membrane potential breakdown, in addition to the triggering of apoptosis (Henry-Mowatt et al., 2004
; Tirosh et al., 2003
), induces an inhibition of cell proliferation. The design of more specific CCE blockers holds much promise for the development of potential anticancer therapies, but this will almost certainly not be possible before the molecular identification of the specific calcium channels involved in this pathway.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alessandro, R., Masiero, L., Liotta, L. A. and Kohn, E. C. (1996). The role of calcium in the regulation of invasion and angiogenesis. In Vivo 10, 153-160.[Medline]
Bird, G. S., Aziz, O., Lievremont, J. P., Wedel, B. J., Trebak, M., Vazquez, G. and Putney, J. W., Jr (2004). Mechanisms of phospholipase C-regulated calcium entry. Curr. Mol. Med. 4, 291-301.[CrossRef][Medline]
Bruce, J. I., Giovannucci, D. R., Blinder, G., Shuttleworth, T. J. and Yule, D. I. (2004). Modulation of [Ca2+]i signaling dynamics and metabolism by perinuclear mitochondria in mouse parotid acinar cells. J. Biol. Chem. 279, 12909-12917.
Duchen, M. R., Jacobson, J., Keelan, J., Mojet, M. H. and Vergun, O. (2001). Functional imaging of mitochondrial within cells. In Methods in Cellular Imaging (ed. A. Periasamy), pp. 88-111. New York: Oxford University Press.
Enfissi, A., Prigent, S., Colosetti, P. and Capiod, T. (2004). The blocking of capacitative calcium entry by 2-aminoethyl diphenylborate (2-APB) and carboxyamidotriazole (CAI) inhibits proliferation in Hep G2 and Huh-7 human hepatoma cells. Cell Calcium 36, 459-467.[CrossRef][Medline]
Felder, C. C., Ma, A. L., Liotta, L. A. and Kohn, E. C. (1991). The antiproliferative and antimetastatic compound L651582 inhibits muscarinic acetylcholine receptor-stimulated calcium influx and arachidonic acid release. J. Pharmacol. Exp. Ther. 257, 967-971.[Abstract]
Gilabert, J. A. and Parekh, A. B. (2000). Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca(2+) current I(CRAC). EMBO J. 19, 6401-6407.
Glassford, J., Soeiro, I., Skarell, S. M., Banerji, L., Holman, M., Klaus, G. G., Kadowaki, T., Koyasu, S. and Lam, E. W. (2003). BCR targets cyclin D2 via Btk and the p85alpha subunit of PI3-K to induce cell cycle progression in primary mouse B cells. Oncogene 22, 2248-2259.[CrossRef][Medline]
Glitsch, M. D., Bakowski, D. and Parekh, A. B. (2002). Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake. EMBO J. 21, 6744-6754.
Golovina, V. A. (1999). Cell proliferation is associated with enhanced capacitative Ca(2+) entry in human arterial myocytes. Am. J. Physiol. 277, C343-C349.[Medline]
Golovina, V. A., Platoshyn, O., Bailey, C. L., Wang, J., Limsuwan, A., Sweeney, M., Rubin, L. J. and Yuan, J. X. (2001). Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation. Am. J. Physiol. Heart Circ. Physiol. 280, H746-H755.
Haverstick, D. M., Heady, T. N., Macdonald, T. L. and Gray, L. S. (2000). Inhibition of human prostate cancer proliferation in vitro and in a mouse model by a compound synthesized to block Ca2+ entry. Cancer Res. 60, 1002-1008.
Henry-Mowatt, J., Dive, C., Martinou, J. C. and James, D. (2004). Role of mitochondrial membrane permeabilization in apoptosis and cancer. Oncogene 23, 2850-2860.[CrossRef][Medline]
Hoth, M., Fanger, C. M. and Lewis, R. S. (1997). Mitochondrial regulation of store-operated calcium signaling in T lymphocytes. J. Cell Biol. 137, 633-648.
Hoth, M., Button, D. C. and Lewis, R. S. (2000). Mitochondrial control of calcium-channel gating: a mechanism for sustained signaling and transcriptional activation in T lymphocytes. Proc. Natl. Acad. Sci. USA 97, 10607-10612.
Hussain, M. M., Kotz, H., Minasian, L., Premkumar, A., Sarosy, G., Reed, E., Zhai, S., Steinberg, S. M., Raggio, M., Oliver, V. K. et al. (2003). Phase II trial of carboxyamidotriazole in patients with relapsed epithelial ovarian cancer. J. Clin. Oncol. 21, 4356-4363.
Jacobson, J. and Duchen, M. R. (2004). Interplay between mitochondria and cellular calcium signalling. Mol. Cell. Biochem. 256-257, 209-218.
Kohn, E. C. and Liotta, L. A. (1990). L651582: a novel antiproliferative and antimetastasis agent. J. Natl. Cancer Inst. 82, 54-60.[Abstract]
Kohn, E. C. and Liotta, L. A. (1995). Molecular insights into cancer invasion: strategies for prevention and intervention. Cancer Res. 55, 1856-1862.[Abstract]
Kohn, E. C., Figg, W. D., Sarosy, G. A., Bauer, K. S., Davis, P. A., Soltis, M. J., Thompkins, A., Liotta, L. A. and Reed, E. (1997). Phase I trial of micronized formulation carboxyamidotriazole in patients with refractory solid tumors: pharmacokinetics, clinical outcome, and comparison of formulations. J. Clin. Oncol. 15, 1985-1993.
Llinas, R., Sugimori, M. and Silver, R. B. (1992). Microdomains of high calcium concentration in a presynaptic terminal. Science 256, 677-679.[Medline]
Mignen, O. and Shuttleworth, T. J. (2000). I(ARC), a novel arachidonate-regulated, noncapacitative Ca(2+) entry channel. J. Biol. Chem. 275, 9114-9119.
Mignen, O., Thompson, J. L. and Shuttleworth, T. J. (2001). Reciprocal regulation of capacitative and arachidonate-regulated noncapacitative Ca2+ entry pathways. J. Biol. Chem. 276, 35676-35683.
Mignen, O., Thompson, J. L. and Shuttleworth, T. J. (2003). Calcineurin directs the reciprocal regulation of calcium entry pathways in nonexcitable cells. J. Biol. Chem. 278, 40088-40096.
Mignen, O., Thompson, J. L., Yule, D. I. and Shuttleworth, T. J. (2005a). Agonist activation of arachidonate-regulated Ca2+-selective (ARC) channels in murine parotid and pancreatic acinar cells. J. Physiol. 564, 791-801.
Mignen, O., Thompson, J. L., Yule, D. I. and Shuttleworth, T. J. (2005b). Agonist activation of ARC channels in parotid and pancreatic acinar cells. J. Physiol. 564, 791-801
Munaron, L., Antoniotti, S., Fiorio Pla, A. and Lovisolo, D. (2004). Blocking Ca2+entry: a way to control cell proliferation. Curr. Med. Chem. 11, 1533-1543.[Medline]
Parekh, A. B. and Putney, J. W., Jr (2005). Store-operated calcium channels. Physiol. Rev. 85, 757-810.
Patton, A. M., Kassis, J., Doong, H. and Kohn, E. C. (2003). Calcium as a molecular target in angiogenesis. Curr. Pharm. Des. 9, 543-551.[CrossRef][Medline]
Peppiatt, C. M., Collins, T. J., Mackenzie, L., Conway, S. J., Holmes, A. B., Bootman, M. D., Berridge, M. J., Seo, J. T. and Roderick, H. L. (2003). 2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels. Cell Calcium 34, 97-108.[Medline]
Shuttleworth, T. J. and Thompson, J. L. (1999). Discriminating between capacitative and arachidonate-activated Ca(2+) entry pathways in HEK293 cells. J. Biol. Chem. 274, 31174-31178.
Sweeney, M., Yu, Y., Platoshyn, O., Zhang, S., McDaniel, S. S. and Yuan, J. X. (2002). Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation. Am. J. Physiol. Lung Cell Mol. Physiol. 283, L144-L155.
Thebault, S., Roudbaraki, M., Sydorenko, V., Shuba, Y., Lemonnier, L., Slomianny, C., Dewailly, E., Bonnal, J. L., Mauroy, B., Skryma, R. et al. (2003). Alpha1-adrenergic receptors activate Ca(2+)-permeable cationic channels in prostate cancer epithelial cells. J. Clin. Invest. 111, 1691-1701.
Tirosh, O., Aronis, A. and Melendez, J. A. (2003). Mitochondrial state 3 to 4 respiration transition during Fas-mediated apoptosis controls cellular redox balance and rate of cell death. Biochem. Pharmacol. 66, 1331-1334.[CrossRef][Medline]
Tomono, M., Toyoshima, K., Ito, M., Amano, H. and Kiss, Z. (1998). Inhibitors of calcineurin block expression of cyclins A and E induced by fibroblast growth factor in Swiss 3T3 fibroblasts. Arch. Biochem. Biophys. 353, 374-378.[CrossRef][Medline]
Varadi, A., Cirulli, V. and Rutter, G. A. (2004). Mitochondrial localization as a determinant of capacitative Ca(2+) entry in HeLa cells. Cell Calcium 36, 499-508.[CrossRef][Medline]
Venkatachalam, K., van Rossum, D. B., Patterson, R. L., Ma, H. T. and Gill, D. L. (2002). The cellular and molecular basis of store-operated calcium entry. Nat. Cell Biol. 4, E263-E272.[CrossRef][Medline]
Yu, Y., Sweeney, M., Zhang, S., Platoshyn, O., Landsberg, J., Rothman, A. and Yuan, J. X. (2003). PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am. J. Physiol. Cell Physiol. 284, C316-C330.