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J. Biol. Chem., Vol. 275, Issue 26, 19978-19984, June 30, 2000

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Depletion of Intracellular Ca²⁺ by Caffeine and Ryanodine Induces Apoptosis of Chinese Hamster Ovary Cells Transfected with Ryanodine Receptor^*

Zui Pan, Derek Damron^§, Anna-Liisa Nieminen^¶, Manjunatha B. Bhat, and Jianjie Ma

From the Departments of  Physiology & Biophysics and ^¶ Anatomy, Case Western Reserve University School of Medicine and the ^§ Center for Anesthesiology Research, Cleveland Clinic Foundation, Cleveland, Ohio 44106

Received for publication, October 12, 1999, and in revised form, March 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recent studies have suggested a central role for Ca²⁺ in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca²⁺ homeostasis. Here we report that depletion of Ca²⁺ from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ovary cells. Stable expression of ryanodine receptor (RyR) in these cells enables rapid and reversible changes of both cytosolic Ca²⁺ and ER Ca²⁺ content via activation of the RyR/Ca²⁺ release channel by caffeine and ryanodine. Sustained depletion of the ER Ca²⁺ store leads to apoptosis in Chinese hamster ovary cells, whereas co-expression of Bcl-xL and RyR in these cells prevents apoptotic cell death but not necrotic cell death. The anti-apoptotic effect of Bcl-xL does not correlate with changes in either the Ca²⁺ release process from the ER or the capacitative Ca²⁺ entry through the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a stage downstream of ER Ca²⁺ release and capacitative Ca²⁺ entry.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apoptosis, an evolutionarily conserved programmed cell death process, plays a central role in both development and homeostasis of tissues. Malfunctions in this process contributes to many diseased states, such as Alzheimer's disease, cancer, and AIDS (1). Characteristic features of apoptosis include DNA fragmentation, condensation of nuclear chromatin, cell membrane blebbing, and activation of a number of specific biochemical pathways (2). Although the sequence of events from the initial apoptotic trigger leading up to DNA fragmentation and eventual cell death is not completely understood, several genes that play essential roles in the regulation of apoptosis have been identified. Among them are the ones encoding a family of Bcl-2-related proteins that either inhibit (e.g. Bcl-2 and Bcl-xL) or facilitate apoptosis (e.g. Bax and Bad) (3, 4). Several studies have suggested a central role for Ca²⁺ in the initiation of apoptosis. Many apoptotic stimuli, including growth factor withdrawal (5) and activation of surface antigen receptors (6), are known to alter the concentration of Ca²⁺ in the cytosol and the storage of Ca²⁺ in the intracellular organelles (7). Compounds that directly affect the intracellular Ca²⁺ homeostasis, such as Ca²⁺ ionophores and thapsigargin (TG),¹ have been shown to induce apoptosis in a variety of cells (8-11). However, the elevated cytosolic Ca²⁺ does not necessarily correlate with apoptosis (12, 13). It is not clear whether the elevation of cytosolic Ca²⁺ per se, or the depletion of intracellular Ca²⁺ store serves as the primary trigger for apoptosis. The anti-apoptotic effect of Bcl-2 has been implicated with changes in intracellular Ca²⁺ (5, 14), but the cellular mechanism underlying the correlation between Bcl-2 and Ca²⁺ remains largely unknown.

Ca²⁺ release from the ER can occur through inositol 1,4,5-trisphosphate receptor (IP₃R) and/or ryanodine receptor (RyR), both of which function as Ca²⁺ release channels in the ER membrane. The level of IP₃R has been shown to increase in lymphocytes induced to undergo apoptosis, and lymphocytes deficient in IP₃R are resistant to glucocorticoid-induced apoptosis (15, 16). Furthermore, dantrolene, an agent that blocks Ca²⁺ release from the ER could prevent apoptosis in cultured PC-12 cells (17). Transforming growth factor-1, one of the potent inducers of apoptosis, has been shown to up-regulate the expression of a form of RyR in pulmonary epithelial cells (18, 19). Thus, Ca²⁺ release channels in the ER appear to play important roles in the signal transduction pathway of apoptosis.

In the present study, we examined the possibility that depletion of the ER Ca²⁺ stores by activation of RyR/Ca²⁺ release channel can directly induce apoptosis in cultured Chinese hamster ovary (CHO) cells. We have stably transfected CHO cells with the RyR and Bcl-xL genes, and measured the changes in cytosolic Ca²⁺ as well as ER Ca²⁺ content through activation of the Ca²⁺ release channel with caffeine and ryanodine. The changes in morphology and chromatin structure of cells undergoing apoptosis or necrosis were characterized with confocal microscopic imaging and DNA laddering assays. Our data show that depletion of ER Ca²⁺ store can serve as a trigger for apoptosis in CHO cells, and the anti-apoptotic effect of Bcl-xL likely occurs at a stage downstream of ER Ca²⁺ release.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids-- The full-length cDNA (~15.3 kilobases) of rabbit skeletal muscle RyR (RyR1) was cloned into the expression vector pRRS11 with transcription occurring under the control of SV40 promoter (20). The E4032A mutant of RyR1 was generated using the Altered Sites II mutagenesis kit purchased from Promega (Madison, WI). A 4.25-kilobase cDNA fragment (nucleotide 10982-15230) encoding the carboxyl-terminal portion of RyR was subcloned into the pAlter vector, and a designed oligonucleotide was used to change the glutamate residue at amino acid position 4032 (Glu⁴⁰³²) into alanine (Ala). The fragment containing the E4032A mutation was subcloned back into its original position in pRRS11, to generate the E4032A-RyR1 mutant. The mutagenesis was confirmed by restriction enzyme digestion and sequencing. The full-length cDNA encoding the human Bcl-xL protein was amplified by polymerase chain reaction using pProex-1 (Bcl-xL) as the template (21), and subcloned into NheI and XhoI sites of the pCEP4 eukaryotic expression vector (Invitrogen, Carlsbad, CA). The pRRS11 vector contains the neomycin (G418) resistance gene and the pCEP4 vector contains the hygromycin resistance gene, which allow selection of cells co-transfected with the RyR and Bcl-xL cDNAs, using G418 and hygromycin, separately.

Cell Culture and Gene Transfection-- CHO cells were grown at 37 °C and 5% CO₂ in Ham's F-12 medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The expression plasmids were introduced into the cells (60-70% confluent) using LipofectAMINE reagent (Life Technologies Inc., Gaithersburg, MD) following the manufacturer's instructions. 48 h after transfection, the cells were selected with G418 (0.5 mg/ml, for pRRS11 based plasmids), hygromycin (0.26 mg/ml, for pCEP4 based plasmids), or both (for co-expression). Resistant colonies were isolated and characterized for RyR1 or Bcl-xL expressing using Western blot analysis.

Western Blot Assay-- Control and transfected CHO cells were harvested and washed twice with ice-cold phosphate-buffered saline, and lysed with modified RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 1 mM EGTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate) in the presence of protease inhibitors (0.1 mM phenlymethylsulfonyl fluoride, 1.0 µM pepstatin, 1 mM benzamidine, 10 µM leupeptin, 1 µg/ml aprotinin). The whole cell lysate was mixed with a 2× sample buffer (200 mM Tris-Cl, pH 6.7, 9% SDS, 6% -mercaptoethanol, 15% glycerol, 0.01% bromphenol blue) and separated on a 3-12% linear gradient SDS-polyacrylamide gel after heating the samples at 60 °C for 15 min. The proteins were transferred to a polyvinylidene difluoride membrane and probed with anti-RyR1 (34C, mouse monoclonal) (22) or anti-Bcl-xL (H-62, rabbit polyclonal, Santa Cruz Biotechnology Inc., Santa Cruz, CA) antibodies. The protein-antibody complexes were then blotted with a horseradish peroxidase-linked secondary antibody and the signal detected on Kodak films using chemiluminescent kit (Pierce, Rockford, IL).

Confocal Microscopic Images-- The ~70% confluent CHO cells were treated with 10 mM caffeine and 10 µM ryanodine for 18 h. Hoechst 33342 (10 µM, Sigma) was added to the culture medium for 15 min at 37 °C in a dark chamber. The cell death process was monitored with a Zeiss laser scanning confocal microscope using a 63× oil immersion objective, with the excitation wavelength set at 351 nm and the emission filter set at 417-482 nm (23).

Intracellular Ca²⁺ Measurement in Single Cell-- CHO cells were grown in TC3 dishes (Bioptechs, Inc., Butler, PA) and loaded with 2 µM Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min at 37 °C in a balanced salt solution (BSS) (140 mM NaCl, 2.8 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, pH 7.2). The cells were then left for a further 30-min period in the bathing solution at room temperature to allow ester hydrolysis to go to completion. Using a dual-wavelength spectrofluorometer, with excitation wavelengths at 340 and 380 nm and emission at 510 nm, fluorescence measurements were performed at 37 °C in a temperature-regulated chamber, mounted on the stage of an inverted fluorescence microscope (Olympus IX-70). Single-cell fluorescence spectra were continuously monitored at a sampling frequency of 50 Hz and collected with a PTI spectrofluorometer (Photon Technology International, Monmouth Junction, NJ) (24). The release of intracellular Ca²⁺ in individual cells was measured following exposure to caffeine, ryanodine, ATP, or thapsigargin in a Ca²⁺-free BSS solution (plus 0.5 mM EGTA) by rapid solution exchange.

DNA Fragmentation Assays-- Cells were seeded onto 100-mm tissue culture dishes at exponential growth phase and allowed to reach 70% confluence. Caffeine (10 mM), ryanodine (10 µM), or EGTA (0.5 mM) were added into the medium directly. 18 h later, both floating and attached cells were collected and washed with ice-cold PBS. The cells were pelleted and lysed in 500 µl of lysis buffer (10 mM EDTA, 150 mM NaCl, 0.15 mM sodium citrate). After the cell suspension was well dispersed, 50 µl of 10% N-laurosarcosine and 50 µl of proteinase K (1 mg/ml) were added separately. DNA was extracted and analyzed for fragmentation following the procedures of McGahon et al. (25).

Quantification of Cell Viability and Apoptosis-- Differential uptakes of fluorescent DNA binding dyes acridine orange (4 µg/ml) and ethidium bromide (8 µg/ml) were used to determine viable and nonviable cells in a given population (25). A viable cell will have a red cytoplasm with bright green nucleus due to intercalation of acridine orange into the DNA. Ethidium bromide is only taken up by nonviable cell after disruption of plasma membrane, and this dye also intercalates into DNA, making it appear orange. Thus a dying cell will have a bright nucleus (ethidium overwhelms acridine) and its cytoplasm will appear dark red. Early apoptotic cells whose membranes are still intact will have green nuclei, but the initiation of DNA fragmentation will result in patched labeling of the nuclei by acridine orange. As the cells progress through the apoptotic pathway and membrane blebbing starts to occur, ethidium bromide enters the cell resulting in orange staining of the nucleus. Cells entering the late apoptotic stage will have bright orange areas of condensed chromatin that will distinguish them from necrotic ones, which have a uniform orange color. For each measurement, a minimum of 200 total cells were counted and the percentage of total apoptotic cells was obtained by adding early and late apoptotic cells. The percentage of necrotic cells was recorded by counting cells with bright orange chromatin with organized structure. Different extracellular agonists, i.e. 10 mM caffeine, 10 µM ryanodine, 0.5 mM EGTA, 0.5 mM ATP or AMP-PNP (Sigma), were added to the medium directly. To suppress the elevation of cytosolic Ca²⁺, cells were pretreated with 10 µM BAPTA-AM (Molecular Probes) added to the culture medium at 37 °C for 90 min.

Statistical Analysis-- Values are mean ± S.E. Significance was determined by Student's t test. A value of p < 0.01 was used as criterion for statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stable Expression of RyR and Bcl-xL in CHO Cells-- The cDNAs encoding the wild type or E4032A-RyR1 were introduced into CHO cells using the LipofectAMINE reagent. Individual clones of CHO cells stably expressing RyR were obtained after selection with G418. As shown in the Western blot (Fig. 1A), the parental CHO cells do not contain detectable amounts of endogenous RyR protein (lane 1, CHO-WT), whereas an ample amount RyR proteins can be detected in the cells transfected with wt-RyR1 (lane 2, CHO-RyR) and E4032A-RyR1 (lane 3, CHO-E4032A), respectively. Stable clones of CHO cells co-expressing Bcl-xL and wt-RyR1 were generated by transfection of Bcl-xL cDNA into CHO-RyR cells, as revealed by the distinct band of 30-kDa protein recognized by the anti-Bcl-xL antibody, and ~560-kDa protein corresponding to the wt-RyR1 (lane 4, RyR-xL). The level of Bcl-xL protein in parental CHO cells was not detectable with the anti-Bcl-xL antibody. As controls, we have also transfected the mock pCEP4 vector in the CHO-wt and CHO-RyR, and upon selection with hygromycin, the following clones were generated, CHO-C4 and RyR-C4, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Caffeine- and ryanodine-mediated movement of intracellular Ca2+ in CHO cells transfected with ryanodine receptor. A, Western blot of RyR and Bcl-xL stably expressed in CHO cells. Proteins from the whole cell lysate were separated on a 3-12% linear SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The top portion of the membrane was blotted with the 34C antibody (against RyR1) and the bottom portion of the membrane was blotted with the H-62 antibody (against Bcl-xL). Lane 1, parental CHO cells; lane 2, CHO-RyR; lane 3, CHO-E4032A; lane 4, RyR-xL. B, caffeine-induced release of intracellular Ca2+. a, CHO-E4032A; b, CHO-RyR; c, RyR-C4; d, RyR-xL. 10 mM caffeine (in Ca2+-free BSS) stimulated Ca2+ release from ER in CHO-RyR, RyR-C4, or RyR-xL, but not in CHO-E4032A. The traces are representative of 5-68 cells in 3-24 independent experiments. C, depletion of ER Ca2+ stores by caffeine and ryanodine. e, CHO-E4032A; f, CHO-RyR; g, RyR-xL. The cells were pretreated with 10 µM ryanodine for 3 min, and 10 mM caffeine in Ca2+-free BSS solution was applied to the cells. Following the caffeine-induced release of Ca2+ from the ER membrane, the cells were re-loaded with 2 mM Ca2+-BSS for 3-6 min. The bath solution was then changed to a Ca2+-free solution (0.5 mM EGTA) followed by quick addition of 10 µM TG. Inhibition of the Ca2+-ATPase by TG resulted in gradual increase of cytosolic Ca2+ in CHO-E4032A, but not in CHO-RyR1 or RyR-xL. Each trace represents 4-12 cells in four independent experiments.

The levels of protein expression in these cells were maintained in over 30 passages, suggesting that the CHO cells can take up well these exogenous RyR1 or Bcl-xL proteins. Morphologically, CHO cells expressing wt-RyR1 appear to be slightly larger than the parental cells, but otherwise they exhibit normal growth and proliferation.

Depletion of ER Ca²⁺ Stores via Activation of RyR-- Our previous studies have shown that the expressed RyR1 proteins are localized to the ER membrane of CHO cells, and they maintain functional Ca²⁺ release channel activity in response to stimulation with caffeine (Ref. 20, and see Fig. 1B). The glutamate residue at amino acid position 4032 (Glu⁴⁰³²) is believed to play a critical role in the Ca²⁺-dependent activation of the RyR1/Ca²⁺ release channel, as mutation of the corresponding residue in RyR3 (E3872A), a brain isoform of RyR, produces a mutant Ca²⁺ release channel with reduced Ca²⁺ activation profile (26). As shown in Fig. 1B, addition of 10 mM caffeine to CHO-E4032A cells did not cause release of Ca²⁺ from the intracellular stores. These cells maintain an intact ER Ca²⁺ pool, since addition of thapsigargin caused release of Ca²⁺ from the ER (see Fig. 1C). Moreover, selection with hygromycin did not affect the intracellular Ca²⁺ movement, as reflected by the similar response to caffeine in CHO-RyR and RyR-C4 cells (Fig. 1B). The inability of E4032A-RyR to release Ca²⁺ in response to caffeine and ryanodine serves as important controls for our subsequent experiments (see below).

Interestingly, the RyR-xL cells respond to caffeine with intracellular Ca²⁺ release that is not very different from the CHO-RyR cells (Fig. 1B, lower trace). On average, the peak of caffeine-induced Ca²⁺ release had a ratio of F₃₄₀/F₃₈₀ = 4.5 ± 0.6 (n = 15) and 4.3 ± 0.4 (n = 6) for CHO-RyR and RyR-xL cells, respectively. In addition, the resting cytosolic [Ca²⁺] did not show significant difference between CHO-RyR (F₃₄₀/F₃₈₀ = 1.0 ± 0.4) (n = 32) and RyR-xL cells (F₃₄₀/F₃₈₀ = 1.2 ± 0.3) (n = 11). The data suggest that Bcl-xL does not interfere with the caffeine- and ryanodine-induced Ca²⁺ release from the ER membrane.

Ryanodine, as a specific ligand for RyR, induces a global conformational change in the RyR protein, and locks the Ca²⁺ release channel in a permanent open state at micromolar concentrations (27). It binds only to the open state of the Ca²⁺ release channel, such that no significant release of intracellular Ca²⁺ was observed when 10 µM ryanodine alone was applied to cells at the resting state prior to stimulation with caffeine (Fig. 1C). But the combined application of ryanodine and caffeine resulted in fast elevation of cytosolic Ca²⁺ in CHO-RyR and RyR-xL cells but not in CHO-E4032A cells, as shown in Fig. 1C. After ryanodine/caffeine-induced Ca²⁺ release, although extracellular solution was changed back to BSS-Ca²⁺ to allow cells to reload with Ca²⁺, there was no detectable amount of thapsigargin-induced Ca²⁺ release from the ER in CHO-RyR and RyR-xL cells. Thus, with sustained opening of the Ca²⁺ release channel by ryanodine, the Ca²⁺ content in the ER had been completely depleted. Notice that the capacitative Ca²⁺ entry (CCE) becomes significantly larger in cells pretreated with ryanodine. The result is consistent with the suggestion that sustained depletion of ER Ca²⁺ is required for full activation of the CCE pathway (28, 29).

Apoptosis of CHO Cells Induced by Activation of RyR/Ca²⁺ Channels-- Perturbation of ER Ca²⁺ homeostasis with caffeine and ryanodine has significant impact on the propagation and proliferation of CHO cells. Typically, within 16-24 h after treatment with caffeine and ryanodine, less than 10% of the CHO-RyR cells remained viable. To monitor the cell death process, confocal microscopic imaging of individual CHO cells stained with Hoechst 33342 was used. The Hoechst dye is a fluorescent indicator that binds specifically to the nuclear chromatin, which is traditionally used to monitor the apoptotic status of cells. As shown in Fig. 2A, 18 h after treatment with 10 mM caffeine and 10 µM ryanodine, the majority of the adhering CHO-RyR cells had a condensed nucleus with fragmented chromatin structure, which is the hallmark of cells undergoing apoptosis. In contrast, most of the CHO-E4032A cells remained healthy with normal nuclear structure following identical treatment with caffeine and ryanodine. Interestingly, the RyR-xL cells had a unique pattern of Hoechst dye staining that is different from those of CHO-RyR and CHO-E4032A cells, with the same treatment with caffeine and ryanodine. Although the nuclei of RyR-xL cells exhibited extended shape, they remained intact with no apparent chromatin fragmentation.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   ER Ca2+-depletion induced apoptosis in CHO cells. A, confocal images of CHO cell after staining with Hoechst 33342. The pictures were taken 18 h after treatment with 10 µM ryanodine and 10 mM caffeine. Most of CHO-RyR exhibited plasma membrane blebbing, chromatin condensation, and fragmentation, which are typical features of apoptosis (middle). In contrast, these apoptotic features were absent in CHO-E4032A (left), or RyR-xL (right). B, DNA laddering assays. CHO-E4032A, CHO-RyR1, or RyR-xL cells were treated for 18 h in standard medium with caffeine (10 mM), ryanodine (10 µM), and EGTA (0.5 mM). Shown are genomic DNAs separated on a 1.5% agarose gel. Depletion of intracellular Ca2+ by caffeine and ryanodine induced DNA fragmentation in CHO-RyR (lanes 3 and 4), but not in CHO-E4032A (lane 1), where the mutant Ca2+ channel could not be activated by caffeine. Prolonged exposure of extracellular EGTA leads to depletion of intracellular Ca2+ stores, and thus caused DNA fragmentation in both CHO-RyR (lanes 5 and 6) and CHO-E4032A (lane 2). The characteristic DNA laddering pattern was absent in RyR-xL cells in all conditions tested (lanes 8-11).

One of the common features of cells undergoing apoptosis is the fragmentation of genomic DNA into laddering patterns of oligonucleosome-sized fragments. Fig. 2B is a representative agarose gel of extracted genomic DNA from CHO cells following 18 h treatment with 10 mM caffeine, 10 µM ryanodine, or 0.5 mM EGTA. In CHO-RyR cells, DNA laddering was clearly visible after treatment with caffeine, especially with caffeine plus ryanodine (Fig. 2B, lane 3, 4). However, the CHO-E4032A cells showed no detectable DNA fragmentation following treatment with caffeine and ryanodine (lane 1). Prolonged incubation of cells with 0.5 mM EGTA (with nominal Ca²⁺ present in the culture medium) caused DNA laddering in both CHO-RyR and CHO-E4032A cells (lane 2, 5, and 6). In contrast, no DNA fragmentation was observed in RyR-xL, whether treated with caffeine, caffeine plus ryanodine, or combination of caffeine, ryanodine, and EGTA (lane 8, 9, and 10).

Based on the morphological changes and the DNA laddering patterns of cells treated with caffeine, ryanodine, and EGTA, the data indicate that depletion of ER Ca²⁺ stores via activation of the RyR/Ca²⁺ release channel can induce apoptosis in CHO cells. Furthermore, Bcl-xL as an anti-apoptotic regulator can prevent the ER-Ca²⁺ depletion induced apoptosis in CHO cells.

Time-dependent Effect of Caffeine, Ryanodine, and EGTA on CHO Cell Viability-- To further characterize the time-dependent changes in cell viability following perturbation of ER Ca²⁺ homeostasis, we did the following studies. Apoptotic and necrotic cells were recorded by using DNA binding dyes acridine orange and ethidium bromide following the protocol of McGahon et al. (25). As shown in Fig. 3, combined application of caffeine and ryanodine, by virtue of their ability to deplete ER Ca²⁺ stores, induced substantial loss of viability in CHO-RyR cells (Fig. 3B). By 12 h, about 50% of the CHO-RyR cells had entered the apoptotic process, and by 24 h, less than 3% of cells remained viable. In contrast, 82% of the CHO-E4032A cells remained viable by 24 h and the majority of cell death was by necrosis, likely due to the pleiotropic effects of caffeine (Fig. 3A).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Time-dependent changes in cell viability following treatment with caffeine, ryanodine, and EGTA. Quantification of apoptotic (rectangle), necrotic (circle) CHO cells, and cell viability (triangle) was carried out as described under "Experimental Procedures." CHO-E4032A (A), CHO-RyR (B), or RyR-xL (C) were treated with 10 µM ryanodine and 10 mM caffeine without (left panels) or with 0.5 mM EGTA (right panels). Each point represents the mean ± S.E. of four determinations. For each determination, 200-400 cells were counted for staining with acridine orange and ethidium bromide.

As shown in Fig. 1, depletion of ER Ca²⁺ stores induced a large and sustained increase in cytosolic Ca²⁺ due to the CCE across the plasma membrane (Fig. 1C). To determine if the sustained high concentration of Ca²⁺ or the entry of Ca²⁺ from the extracellular space is important in the induction of apoptosis, we studied the ability of caffeine and ryanodine to induce apoptosis in a Ca²⁺-free medium (with 0.5 mM EGTA). This Ca²⁺-free medium significantly augmented apoptosis instead of increasing the viability of CHO-RyR cells (compare left versus right panels of Fig. 3B). By 9 h, only 16% cells remained viable and most of the dying cells had entered the apoptosis stages. Moreover, pronounced apoptosis could be observed in CHO-E4032A cells, 12 h after the addition of EGTA (Fig. 3A, right), which is also evident in the DNA laddering patterns (Fig. 2B, lane 2). The results are consistent with the observation that prolonged incubation of cells with EGTA lead to eventual depletion of the intracellular Ca²⁺ stores (30). Together, our data suggest that it is the depletion of the ER Ca²⁺ pool, rather than the elevation of cytosolic Ca²⁺, that is responsible for the apoptotic cell death.

Consistent with the anti-apoptotic effect of Bcl-xL, the RyR-xL cells had less than 5% apoptotic death even after 24 h treatment with caffeine and ryanodine. Although Bcl-xL had a dramatic effect on cell viability, it is interesting to note that a significant portion of the RyR-xL cells died via necrosis, rather than apoptosis, following depletion of the ER Ca²⁺ stores. This is clearly seen when 0.5 mM EGTA was added to the extracellular medium (Fig. 3C, right). Thus, co-expression of Bcl-xL and wt-RyR1 in CHO cells prevented the apoptotic cell death pathway, but not the necrotic cell death process.

Effect of Extracellular ATP on Intracellular Ca²⁺ Release and Cell Viability-- CHO cells contain purinergic receptors on the surface membrane, and binding of extracellular ATP to this receptor leads to generation of IP₃ in the cytosol which, in turn, activates the IP₃R channel in the ER membrane. Studies from other investigators have shown that application of ATP could induce apoptosis in cultured cells (31, 32). To compare the effect of ATP with those of caffeine and ryanodine on the apoptosis of CHO cells, the following assays were used. First, the ability of extracellular ATP to release Ca²⁺ from intracellular stores was compared in CHO-C4 and RyR-C4 cells. As shown in Fig. 4A, addition of 0.5 mM ATP to the extracellular medium caused fast release of Ca²⁺ from ER with a magnitude that is comparable between CHO-C4 and RyR-C4 cells. After ATP-induced Ca²⁺ release, addition of 10 mM caffeine failed to cause further Ca²⁺ release from intracellular stores; and pretreatment of the RyR-C4 cells with caffeine and ryanodine also abolished the ATP-induced calcium release (Fig. 4B). The results indicate that the ATP-induced Ca²⁺ release share the same pool with the caffeine- and ryanodine-induced Ca²⁺ release, and overexpression of RyR in the ER membrane does not affect the function of the IP₃ receptor.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   ATP-induced Ca2+ release and apoptosis in CHO cells. A, traces represent the ratio of Fura-2 fluorescence (F340/F380) measured in a single cell of CHO-C4 (a), RyR-C4 (b), or RyR-xL (c). Application of 0.5 mM ATP () produced intracellular Ca2+ release, which is similar to that generated by caffeine. All the measurements were done at 37 °C in Ca2+-free BSS (containing 0.5 mM EGTA). B, RyR-C4 cells were used. ATP, 0.5 mM; caffeine, 10 mM; ryanodine, 10 µM. The traces are representative of 6 cells in two independent experiments. C, time dependent changes in cell viability following treatment with 0.5 mM ATP are shown. a, CHO-E4032A; b, CHO-RyR; c, RyR-xL.

Next, we compared the effect of extracellular ATP on the viability of CHO-E4032A, CHO-RyR, and RyR-xL cells. As shown in Fig. 4C, addition of 0.5 mM ATP produced progressive cell death in all three types of cell lines. But the on-set of ATP-induced cell death is significantly slower than that generated by caffeine and ryanodine. 24 h after the addition of ATP, only about 12% of CHO-RyR cells had entered the apoptotic process (Fig. 4C, middle), which is in contrast to the near complete apoptosis observed after exposure to caffeine and ryanodine (Fig. 3B). Similar ATP-induced apoptosis processes were also observed in CHO-E4032A (Fig. 4C, top). The slow ATP-induced apoptosis process is unlikely due to the hydrolysis of extracellular ATP by the ecto-ATPase, since AMP-PNP, a poorly hydrolyzable analog of ATP, produced similar effect (not shown). Thus, overexpression of RyR in CHO cells does not affect the intrinsic ability of cells to undergo apoptosis triggered by extracellular ATP. The difference between the ATP- and caffeine-induced cell death could reflect functional differences between RyR and IP₃R. The IP₃ receptor is known to enter a desensitized state with sustained stimulation with IP₃, and the IP₃R channel has a narrower window of intracellular Ca²⁺ dependence compared with the RyR channel, i.e. the Ca²⁺-dependent activation and inactivation phases of the IP₃R channel exhibit significant overlap (33, 34). Thus, transient activation of IP₃R is unlikely to cause sustained depletion of the ER Ca²⁺ store, which may be sufficient to maintain certain viability of the cells.

Effect of Bcl-xL on Capacitative Ca²⁺ Entry and Cytosolic [Ca²⁺]-- Studies from other investigators have suggested that part of the anti-apoptotic effect of Bcl-2 family proteins could be correlated with the altered CCE pathway across the plasma membrane (5, 14). This does not seem to be the case with CHO cells, based on the following two observations. First, the amount of Ca²⁺ stored in ER membrane did not change with or without the presence of Bcl-xL, as reflected by the magnitude of caffeine-induced Ca²⁺ release in CHO-RyR and RyR-xL cells (see Fig. 1B). Second, the amount of CCE following ER Ca²⁺ depletion remained essentially the same, whether or not Bcl-xL is present in the cells. As shown in Fig. 5A, there is no significant difference in either the basal level of cytosolic Ca²⁺ ([Ca²⁺]_min) or the peak of Ca²⁺ entry ([Ca²⁺]_max) following the addition of Ca²⁺ (2 mM) to the extracellular solution. However, significant differences between the CHO-RyR and RyR-xL cells was observed in the cytosolic [Ca²⁺] following sustained treatment with caffeine and ryanodine. 2-8 h after the depletion of ER Ca²⁺, the cytosolic [Ca²⁺] in RyR-xL cells appear to be significantly higher than that in CHO-RyR cells (Fig. 5B).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Capacitative Ca2+ entry and sustained cytosolic Ca2+ in CHO cells following depletion of the ER Ca2+ stores. A, capacitative Ca2+ entry was defined as the changes of fluorescence ratio 340 nm/380 nm in 0.5 mM EGTA-BSS ([Ca2+]min) and in 2 mM Ca2+-BSS ([Ca2+]max). CHO-RyR cells and RyR-xL were treated with 10 mM caffeine and 10 µM ryanodine in 0.5 mM EGTA-BSS, then bath solutions were changed to 2 mM Ca2+-BSS. Neither [Ca2+]min nor the peak Ca2+ entry [Ca2+]max shows significant differences between the two cell types. B, at 2 and 8 h after addition of caffeine and ryanodine in culture medium, fluorescence ratio 340/380 in CHO-RyR and RyR-xL cells were measured to represent the concentration of cytosolic Ca2+ ([Ca2+]2 h, [Ca2+]8 h). The concentration of cytosolic Ca2+ at both time points in RyR-xL cells (hatched bar) was significantly higher than that in CHO-RyR cells (blank bar).

Effect of BAPTA-AM on ER Ca²⁺ Release and Cell Viability-- One of the observations in Fig. 3 was that chelation of extracellular Ca²⁺ with EGTA appeared to accelerate the cell death process in CHO cells. A potential caveat of using EGTA is that it might introduce an oxidative stress to the mitochondria, such as glutathione efflux from mitochondria, which may affect the apoptosis process (35). And to some extent, prolonged exposure to low Ca²⁺ may cause cells to lose adhesion, which may also lead to apoptosis. As an alternative way of buffering cytosolic Ca²⁺, CHO cells were treated with a membrane-permeable Ca²⁺ chelator, BAPTA-AM (10 µM). As shown in Fig. 6A, in cells pretreated with BAPTA-AM, the ATP-induced cytosolic Ca²⁺ elevation, as well as the caffeine-induced cytosolic Ca²⁺ elevation, were dramatically reduced (trace b comparing with trace a). And BAPTA appeared to be stably remained in the cytosol since its effects on ATP-induced or caffeine-induced cytosolic Ca²⁺ elevation in CHO-RyR cells after another 2 h were similar with the effects produced immediately after BAPTA-AM treatment (trace c comparing with b). The effects of BAPTA pretreatment on cell viability at 24 h after addition of 10 mM caffeine and 10 µM ryanodine are summarized in Fig. 6B. In 4 paired experiments with control (+dimethyl sulfoxide) or 10 µM BAPTA-AM-treated cells, there were no significant differences in the percentage of cells undergoing apoptosis, studied in all three types of cell lines, CHO-E4032A, CHO-RyR, and RyR-xL. But, the percentage of cells undergoing necrosis was dramatically reduced in RyR-xL cells. This is likely due to the buffering capacity of the cytosolic Ca²⁺ by BAPTA (see Fig. 5C). With the measurement of cytosolic Ca²⁺ in individual RyR-xL cells, we found that pretreatment with BAPTA lead to significant reduction of the sustained cytosolic [Ca²⁺], 2 h after stimulation by caffeine and ryanodine: the ratio of F₃₄₀/F₃₈₀ decreased from 1.7 ± 0.3 (+dimethyl sulfoxide) to 1.2 ± 0.1 (+BAPTA-AM).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   BAPTA-AM reduces elevation of cytosolic Ca2+ and inhibits necrosis in CHO cells after caffeine and ryanodine treatment. Cells were incubated with 10 µM BAPTA-AM (dissolved in dimethyl sulfoxide) in extracellular medium at 37 °C. Control cells were pretreated with equal volumes of dimethyl sulfoxide without BAPTA-AM. After 90 min, the cells were changed back to standard medium. A, representative traces of ATP-induced Ca2+ release and caffeine-induced Ca2+ release were plotted in control (a), immediately (b, black lines), or 2 h (c, gray lines) after pretreatment with BAPTA-AM in CHO-RyR cells. B, the effect on BAPTA on apoptosis (top) and necrosis (bottom) was measured 24 h after addition of 10 caffeine (10 mM) and ryanodine (10 µM). The number of necrotic cells in RyR-xL has significant difference (*).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stable expression of RyR enables rapid and reversible changes of Ca²⁺ release across the ER membrane, thus providing a convenient means for studying the role of Ca²⁺ signaling in apoptosis. Our data demonstrate that depletion of ER Ca²⁺ stores is capable of inducing apoptosis in CHO cells, which is in agreement with previous studies showing that inhibition of the ER Ca²⁺-ATPase by thapsigargin leads to apoptotic cell death (8-11). Moreover, the anti-apoptotic effect of Bcl-xL in our system appears to be independent of changes in ER Ca²⁺ release and capacitative Ca²⁺ entry. This suggests that Bcl-xL prevents apoptosis at a step that is downstream of ER Ca²⁺ homeostasis.

The presence of RyR in the ER membrane of CHO-RyR cells has several advantages over the endogenous IP₃R present in the parental CHO cells. First, it allows control of Ca²⁺ release in a reversible manner with caffeine, as well as control of the ER Ca²⁺ content with ryanodine. With the combined application of caffeine and ryanodine, it is possible to achieve a status of sustained depletion of the ER Ca²⁺ store, and such status offers important tests to the cellular mechanism of Ca²⁺ signaling in apoptosis. In contrast, the IP₃R exhibits desensitization upon binding of IP₃, and opening of the IP₃R channel shows substantial inactivation when the cytosolic Ca²⁺ reaches low micromolar concentrations (34). These intrinsic properties of the IP₃R channel help to maintain the ER Ca²⁺ homeostasis, but they also prevent broader manipulations of Ca²⁺ movement across the ER membrane. Another advantage with the heterogonous expression of RyR is the possibility of producing mutant Ca²⁺ release channels with altered responses to caffeine or regulation by cytosolic Ca²⁺ (36). For example, the E4032A-RyR1 mutant, which differs from the wt-RyR1 in only a single amino acid substitution, shows no response to caffeine and ryanodine. Through comparison between CHO-WT and CHO-E4032A cells, we show that overexpression of RyR does not affect the proliferation and propagation properties of the CHO cells.

Inhibition of the ER Ca²⁺-ATPase by TG has been shown to induce apoptosis in a variety of cell types (8-11). But, TG-induced Ca²⁺ release from ER is a relatively slow process compared with the Ca²⁺ spikes generated by activation of IP₃R or RyR/Ca²⁺ channels. Studies from other investigators have demonstrated that, in addition to changes in the global cytosolic [Ca²⁺], the spatial and temporal patterns of Ca²⁺ signals also play important roles in cellular responses (37, 38). Based on morphological characterization (Hoechst dye staining of the nuclear chromatin) and DNA laddering assays, our data clearly show that sustained activation of the RyR channel by caffeine and ryanodine could lead to apoptosis in CHO-RyR but not in CHO-E4032A cells (Fig. 2). Compared with the caffeine- and ryanodine-induced apoptosis in CHO cells, we found the Ca²⁺ signals generated by extracellular ATP through IP₃ pathway did not induce significant apoptosis, i.e. the extent of cell death was smaller and the time-dependent changes in cell viability was significantly delayed. Considering that IP₃R only provides transient Ca²⁺ spikes which may not be sufficient to trigger apoptosis, our data are in agreement with the work by Szalai et al. (39) showing that IP₃-linked mitochondrial Ca²⁺ signals induced apoptosis only in cells that were pretreated with apoptotic stimuli.

Sustained depletion of the ER Ca²⁺ store by caffeine and ryanodine results in activation of the capacitative Ca²⁺ entry pathway, and thus causing sustained elevation of cytosolic Ca²⁺ (29, 40). This elevated cytosolic Ca²⁺ could have a number of effects on the cellular functions. To distinguish the effect of elevation in cytosolic Ca²⁺ from that of depletion of intracellular Ca²⁺ stores on the apoptosis of CHO cells, we have added EGTA to the extracellular medium and pretreated the cells with membrane permeable Ca²⁺ chelator, BAPTA-AM. Both of the methods, by which the sustained elevation of cytosolic Ca²⁺ can be prevented, failed to inhibit apoptosis in CHO-RyR cells after caffeine and ryanodine treatment. The presence of BAPTA in the cytosol adds to a buffering capacity of the cell to cytosolic Ca²⁺ and itself alone did not have a significant effect on cell viability (at 10 µM concentration). Since the number of cells undergoing necrosis in RyR-xL cells following stimulation by caffeine and ryanodine was significantly reduced by BAPTA, the elevation of cytosolic Ca²⁺ might be important in the execution phase of the necrotic death. Together, our data suggest that depletion of the ER Ca²⁺ stores rather than elevation of cytosolic Ca²⁺ lead to apoptosis.

Overexpressing Bcl-xL protein prevented RyR-xL cells from apoptosis. Moreover, the anti-apoptotic effect of Bcl-xL in our system appears to be independent of changes in ER Ca²⁺ release and capacitative Ca²⁺ entry. This suggests that Bcl-xL prevents apoptosis at a step that is downstream of ER Ca²⁺ homeostasis. Immunocytochemistry studies reveal that Bcl-xL and other Bcl-2 family proteins are primarily localized to the ER, the nucleus, and, for the most part, the outer membrane of mitochondria (41, 42). Recent studies by Rizzuto et al. (43, 44) showed that mitochondria and ER form a close network, providing the possibility that the active site of ER Ca²⁺ release may be directly coupled with the mitochondria Ca²⁺ uptake (45). The protective effect of Bcl-xL on apoptosis could in part reflect its effect on the mitochondria's Ca²⁺ handling capacity. However, it remains controversial whether Bcl-2-related proteins enhance mitochondria's Ca²⁺ loading capacity or it prevents intracellular Ca²⁺ stores from overloading (46-48). Further studies to investigate the relationship between ER Ca²⁺ release and mitochondrial Ca²⁺ uptake, and to understand the effect of Bcl-xL on the communication between ER and mitochondria should provide new insights into the cellular and molecular mechanism of Ca²⁺ signaling in the initial phase of apoptosis.

	ACKNOWLEDGEMENTS

We thank Drs. X. Xu and S. M. Hayek for suggestions in experiments and helpful discussions in preparing the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant RO1-AG15556, the Charlotte Geyer Foundation, and an Established Investigatorship from the American Heart Association (to J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 216-368-2684; Fax: 216-368-1693; E-mail: jxm63@po.cwru.edu.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M908329199

	ABBREVIATIONS

The abbreviations used are: TG, thapsigargin; ER, endoplasmic reticulum; RyR, ryanodine receptor; IP₃R, inositol 1,4,5-trisphosphate receptor; CHO, Chinese hamster ovary; BSS, balanced salt solution; CCE, capacitative Ca²⁺ entry; BAPTA-AM, (acetoxymethyl)-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; AMP-PNP, adenosine 5'-(,-iminotriphosphate).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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