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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Polyunsaturated fatty acids mobilize intracellular Ca²⁺ in NT2 human teratocarcinoma cells by causing release of Ca²⁺ from mitochondria

¹Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio; Departments of ²Medicine, ³Biochemistry, ⁴Dental Diagnostic Science, and ⁵Radiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas; and ⁶Department of Neurology, University of Virginia Health Sciences Center, Charlottesville, Virginia

Submitted 8 July 2005 ; accepted in final form 15 December 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
In a variety of disorders, overaccumulation of lipid in nonadipose tissues, including the heart, skeletal muscle, kidney, and liver, is associated with deterioration of normal organ function, and is accompanied by excessive plasma and cellular levels of free fatty acids (FA). Increased concentrations of FA may lead to defects in mitochondrial function found in diverse diseases. One of the most important regulators of mitochondrial function is mitochondrial Ca²⁺ ([Ca²⁺]_m), which fluctuates in coordination with intracellular Ca²⁺ ([Ca²⁺]_i). Polyunsaturated FA (PUFA) have been shown to cause [Ca²⁺]_i mobilization albeit by unknown mechanisms. We have found that PUFA but not monounsaturated or saturated FA cause [Ca²⁺]_i mobilization in NT2 human teratocarcinoma cells. Unlike the [Ca²⁺]_i response to the muscarinic G protein-coupled receptor agonist carbachol, PUFA-mediated [Ca²⁺]_i mobilization in NT2 cells is independent of phospholipase C and inositol-1,4,5-trisphospate (IP₃) receptor activation, as well as IP₃-sensitive internal Ca²⁺ stores. Furthermore, PUFA-mediated [Ca²⁺]_i mobilization is inhibited by the mitochondria uncoupler carboxyl cyanide m-chlorophenylhydrozone. Direct measurements of [Ca²⁺]_m with X-rhod-1 and ⁴⁵Ca²⁺ indicate that PUFA induce Ca²⁺ efflux from mitochondria. Further studies show that ruthenium red, an inhibitor of the mitochondrial Ca²⁺ uniporter, blocks PUFA-induced Ca²⁺ efflux from mitochondria, whereas inhibitors of the mitochondrial permeability transition pore cyclosporin A and bongkrekic acid have no effect. Thus PUFA-gated Ca²⁺ release from mitochondria, possibly via the Ca²⁺ uniporter, appears to be the underlying mechanism for PUFA-induced [Ca²⁺]_i mobilization in NT2 cells.

arachidonic acid; mitochondrial Ca²⁺ uniporter; G protein-coupled receptor; IP₃ receptor

At the cellular level, excessive FA can increase oxidative stress, influence ion channel activities, activate multiple kinase pathways, and induce apoptosis. All of these cellular effects of FA may contribute to the altered physiological functions of different tissues observed in association with lipid overload. Production of reactive oxygen species by polyunsaturated FA (PUFA) has been reported in HL-60 and HeLa cells (23, 24, 26, 59), neutrophils (11), and human breast carcinoma cells (6, 13, 70). PUFA-stimulated reactive oxygen species production in some cells has been shown to be mediated by NADPH oxidase activation (1).

The induction of apoptosis by FA has been reported in a variety of cells. In human retinoblastoma Y79 cells apoptosis is induced by arachidonic acid (AA) through the action of its oxidative metabolites (73). In contrast, AA-induced apoptosis in chronic myeloid leukemia cells does not require AA metabolism (55). AA is also partially responsible for the apoptotic effect of oxidized LDL in the macrophage cell line Chinese hamster ovary-K1 (48). Saturated FA, such as palmitic acid (PA) and stearic acid, induce apoptosis in human granulosa cells and cause downregulation of Bcl-2 and upregulation of Bax proteins (47). It has been shown in LLCPKc14 cells that AA causes apoptosis through increased production of ceramide (9). Docosahexaenoic acid (DHA) induces apoptosis in Jurkat cells by a protein phosphatase 1- and 2B-sensitive mechanism (67). In mitochondria isolated from rat liver, both AA and PA cause the opening of the mitochondrial permeability transition pore (MPT), a process leading to apoptosis (51, 64). These studies suggest that FA induce apoptosis by diverse mechanisms.

Growing evidence indicates that excessive concentrations of FA affect cell functions by altering the activities of various ion transporters and channels, including proton, K⁺, Na⁺, Cl^–, and Ca²⁺ currents, as well as nonselective cation channels and the cardiac Na⁺/Ca²⁺ exchanger (17, 25, 28, 36, 44, 63, 74). PUFA inhibit voltage-dependent Ca²⁺ channels in cardiac myocytes (17), retinal glial cells (4), and sympathetic neurons (39). The mechanisms by which PUFA modulate ion channels vary among different cell types and different channels. PUFA may directly bind and modulate the activity of some channels, whereas in other cases, PUFA regulate channel activities indirectly through metabolites and protein kinases (58). FA exert diverse effects on the transient receptor potential (TRP) or TRP-like families of Ca²⁺ channels (75). PUFA have been demonstrated to be reversible agonists for TRP and TRP-like channels (TRPC) in both Drosophila photoreceptors and Drosophila S2 cells (10). TRP channels in mammalian cells are composed of seven subfamilies, including TRPC, TRP vanilloid, and TRP melastatin (45). Some TRPC channels may be involved in the capacitative or store-operated Ca²⁺ entry during classic phospholipase C (PLC)-inositol-1,4,5-trisphosphate (IP₃)-mediated cytosolic, or intracellular, Ca²⁺ ([Ca²⁺]_i) signaling (75, 78). AA and other PUFA either inhibit or stimulate the store-operated Ca²⁺ influx during G protein-coupled receptor (GPCR)-mediated [Ca²⁺]_i mobilization depending on the cell type and the nature of the channel (58).

PUFA-induced [Ca²⁺]_i mobilization has also been observed in vascular endothelial and human embryonic kidney (HEK)-293 cells (33, 40, 65). In HEK-293 cells, [Ca²⁺]_i mobilization induced by AA and other PUFA involves activation of the AA-specific Ca²⁺ influx pathway I_avc (40, 65). In the presence of 10 µM Gd³⁺ AA- but not carbachol- or thapsigargin-mediated Ca²⁺ release was completely inhibited in HEK-293 cells, suggesting that AA-induced Ca²⁺ release in these cells may be mediated by a process distinct from the traditional PLC-IP₃ pathways (40). However, the mechanism of PUFA-induced [Ca²⁺]_i mobilization remains unclear.

In this study, experiments have been designed to clarify the role of the classic PLC-IP₃ pathway and other Ca²⁺ transport pathways in FA-mediated [Ca²⁺]_i mobilization. We have found that PUFA but not monounsaturated or saturated FA cause [Ca²⁺]_i mobilization in NT2 human teratocarcinoma cells. Unlike the [Ca²⁺]_i response to the muscarinic agonist carbachol, PUFA-mediated [Ca²⁺]_i mobilization in NT2 cells is independent of PLC and IP₃ receptor activation, as well as IP₃-sensitive internal Ca²⁺ stores. Furthermore, PUFA-mediated [Ca²⁺]_i mobilization is inhibited by the mitochondrial uncoupler carboxyl cyanide m-chlorophenylhydrozone (CCCP). Direct measurements of mitochondrial Ca²⁺ with X-rhod-1 and ⁴⁵Ca²⁺ indicate that PUFA induce Ca²⁺ efflux from mitochondria. These experiments suggest that PUFA-gated Ca²⁺ release from mitochondria is the underlying mechanism for PUFA-induced [Ca²⁺]_i mobilization in NT2 cells.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Materials. Fura-2 AM, Fluo-3 AM, BAPTA-AM, and X-rhod-1 AM were purchased from Molecular Probes (Eugene, OR). Thapsigargin was purchased from RBI (Natick, MA). Dulbecco's modified Eagle's medium, EGTA, trypsin-EDTA, Opti-MEM, and PBS [containing (in mM) 1 KH₂PO₄, 3 Na₂HPO₄, and 154 NaCl, pH 7.2] powders were obtained from Life Technologies (Gaithersburg, MD). Arachidonic acid and other fatty acids were purchased from Cayman (Ann Arbor, MI). The NT2 cell line and primary cultured human aortic endothelial cells (HAEC) were purchased from American Type Culture Collection (Manassas, VA) and Cambrex (Walkersville, MD), respectively. MDCB-131 medium, carbachol, CCCP, oligomycin, and other chemicals were from Sigma (St. Louis, MO).

Cell culture. NT2 cells were plated at a density of 10⁴ cells/cm² and cultured in 100 mm dishes in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum and penicillin (10 U/ml)/streptomycin (10 µg/ml), at 37°C in a humidified 5% CO₂-atmosphere incubator. Cells grown to near confluence were harvested with trypsin (0.05%)-EDTA (0.02%) and suspended in PBS solutions for use in experiments described below.

Human aortic endothelial cells (HAEC) were plated at a density of 6 x 10³/cm² in 75-ml flasks in MDCB-131, supplemented with 15 mM HEPES, 14 mM NaHCO₃, EGF (10 ng/ml), fibroblast growth factor (2.5 ng/ml), hydrocortisone (1 µg/ml), 10% fetal calf serum, and penicillin (10 U/ml)/streptomycin (10 µg/ml), and cultured at 37°C in a humidified 5% CO₂-atmosphere incubator. After 1 wk of culture, when the cells reached confluence, HAEC were harvested with trypsin (0.05%)-EDTA (0.02%) and labeled with fura-2 AM for measurement of [Ca²⁺]_i in cell suspensions or used to isolate mitochondria to measure [Ca²⁺]_m with X-rhod-1.

Measurement of [Ca²⁺]_i in suspensions of NT2 cells. NT2 cells suspended in a buffer containing (in mM) 140 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 1.2 KH₂PO₄, 10 glucose, and 10 HEPES, pH 7.4, were loaded with fura-2 AM (2 µM) at 37°C for 30 min with gentle shaking. Loaded cells were washed with 5 volumes of PBS supplemented with 1 mM CaCl₂ and 1 mM MgSO₄ (PBS1Ca). Alterations in [Ca²⁺]_i were measured by changes in fluorescence ratio with emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm. Fura-2-loaded cells in suspension were incubated at 37°C in a cuvette with a magnetic stirrer, and changes of fluorescence ratio were monitored in a fluorometer manufactured by Photon Technology International (PTI; Lawrenceville, NJ). For measurement of [Ca²⁺]_i in the absence of extracellular Ca²⁺, fura-2-loaded NT2 cells were washed with 5 volumes of PBS supplemented with 200 µM EGTA (pH 7.4) and 1 mM MgSO₄ (PBS0Ca) immediately before experiments; [Ca²⁺]_i measurements were performed in PBS0Ca solutions (76).

Measurement of mitochondrial Ca²⁺ in suspensions of NT2 cells. NT2 cells suspended in PBS were double labeled with X-rhod-1 AM (2 µM) and BAPTA-AM (50 µM), or labeled with X-rhod-1 AM alone, at 37°C for 30 min with gentle shaking. The loaded cells were washed with 5 volumes of PBS1Ca. Alterations in mitochondrial Ca²⁺ ([Ca²⁺]_m) were measured by changes in fluorescence intensity with emission wavelength of 602 nm and excitation wavelength of 578 nm. X-rhod-1-AM- and BAPTA-AM-loaded cells were incubated in PBS supplemented with 2 mM EGTA (pH 7.4) and 1 mM MgSO₄ at 37°C in a cuvette in the presence of constant magnetic stirring; cells loaded with X-rhod-1 alone were incubated in PBS supplemented with 2 mM EGTA/1 mM MgSO₄ or in PBS1Ca. Changes of X-rhod-1 fluorescence intensity were monitored in a PTI fluorometer.

Measurement of [Ca²⁺]_m in isolated mitochondria from NT2 cells and HAEC. Mitochondria from NT2 cells were isolated as described previously (30). Briefly, NT2 cells from seven to ten 100-mm dishes were suspended in PBS, incubated on ice, and centrifuged at 1,000 rpm for 15 min at 4°C in an Eppendorf 5804R centrifuge. Cells were resuspended in 2 ml ice-cold mitochondria isolation buffer containing 250 mM mannitol, 75 mM succinic acid, 100 µM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 7.5 (MB1). The cell suspensions were homogenized manually with 15–20 strokes in a glass-glass homogenizer. The homogenates were centrifuged at 1,000 rpm for 15 min at 4°C. The supernatants were collected and centrifuged at 10,000 g for 20 min at 4°C. The pellets were resuspended in 500 µl of MB1 and diluted to a final volume of 3 ml with PBS1Ca. The mitochondria suspensions were labeled with the mitochondrial Ca²⁺ fluorescent indicator X-rhod-1 AM (2 µM) at 37°C for 30 min, after which the mixtures were centrifuged at 10,000 g at room temperature in a tabletop high-speed centrifuge (Savant Instruments, Farmingdale, NY) for 10 min. The labeled mitochondria were resuspended in PBS1Ca and used for measurement of [Ca²⁺]_m. Alterations of [Ca²⁺]_m in suspensions of isolated mitochondria were measured by changes in fluorescence intensity with emission wavelength of 602 nm and excitation wavelength of 578 nm. X-rhod-1 loaded mitochondria were incubated in PBS0Ca at 37°C in a cuvette in the presence of continuous magnetic stirring, and changes of X-rhod-1 fluorescence intensity were monitored in a PTI fluorometer. The same procedure was adopted to isolate mitochondria and measure [Ca²⁺]_m from HAEC, except that the cells were grown in 75-ml flasks.

Measurement of PUFA-induced Ca²⁺ efflux in isolated mitochondria using ⁴⁵Ca²⁺. Loading of ⁴⁵Ca²⁺ into mitochondria was performed according to a previously reported procedure (8) with modification. Briefly, isolated mitochondria (0.4–1.2 mg protein) were resuspended in 500 µl of MB1, diluted to a final volume of 3 ml with PBS1Ca, and loaded with ⁴⁵Ca²⁺ at 37°C for 30 min with gentle agitation. The ⁴⁵Ca²⁺-loaded mitochondria were then diluted in 10 volumes of PBS0Ca-containing vehicle or 10 µg/ml PUFA or palmitic acid (PA). The mixtures were incubated at 37°C for 5 min and the ⁴⁵Ca²⁺ contents remaining in the mitochondria were counted in a liquid scintillation counter.

Data analysis. Individual figures shown in RESULTS are representative of at least three experiments. Statistical analysis of ⁴⁵Ca²⁺ measurements was performed using the Wilcoxon scores for variable assay and Monte Carlo estimate for significance.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Polyunsaturated but not saturated or monounsaturated FA induce [Ca²⁺]_i mobilization in NT2 cells. In suspensions of NT2 cells PUFA, including AA, DHA, eicosapentanoic acid, and linoleic acid (LA), increased [Ca²⁺]_i (Fig. 1, A–D). The initial rate of [Ca²⁺]_i increase and the amplitude of the [Ca²⁺]_i signal were dependent on the concentration of PUFA used, with half-maximal [Ca²⁺]_i response observed at 2.46 ± 0.05 µg/ml LA and AA. The monounsaturated FA oleic acid and the saturated FA PA did not induce [Ca²⁺]_i mobilization (Fig. 1E) even at a concentration (10 µg/ml) at which PUFA produced maximal [Ca²⁺]_i response. These results indicate that the PUFA-induced [Ca²⁺]_i responses in NT2 cells do not result from nonspecific detergent effects of FA on cell membranes.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Mobilization of intracellular Ca2+ concentration ([Ca2+]i) by polyunsaturated fatty acids (PUFA) in suspensions of NT2 cells. NT2 cells labeled with fura-2 (1.2 µM; incubated at 37°C for 30 min) in suspensions were treated with different FA as indicated, and changes in [Ca2+]i were measured as described in MATERIALS AND METHODS. Representative [Ca2+]i responses to increasing concentrations of arachidonic acid (AA; A), linoleic acid (LA; B), docosahexaenoic acid (DHA; C), and eicosapentanoic acid (EPA; D) are demonstrated by the traces. The arrows indicate the time of addition of different concentrations of FA as labeled. The y-axis is the fluorescence ratio of 340/380 in arbitrary units and the x-axis is time in seconds. E: traces are representative [Ca2+]i responses to the saturated FA palmitic acid (PA; 10 µg/ml) and the mono-unsaturated FA oleic acid (OA; 10 µg/ml).

View larger version (7K): [in this window] [in a new window]   Fig. 2. Eicosatetraynoic acid (ETYA)-mediated [Ca2+]i mobilization in NT2 cells. NT2 cells were treated with increasing concentrations of ETYA, and changes in [Ca2+]i were measured as described in MATERIALS AND METHODS.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effect of U73122 [GenBank] and U73343 [GenBank] on carbachol- and PUFA-mediated [Ca2+]i mobilization in NT2 cells. NT2 cells were either untreated (A and C) or treated with U73122 [GenBank] (10 µM) (B and D) or U73343 [GenBank] (10 µM) (E and F), followed by measurement of carbachol (100 µM)- and linoleic acid (LA; 10 µg/ml)-mediated [Ca2+]i mobilization, as described in MATERIALS AND METHODS.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effect of 2-APB on carbachol- and PUFA-mediated [Ca2+]i mobilization. NT2 cells in suspensions were either untreated (A and C) or treated with 2-APB (25 µM) for 15–30 min at room temperature (B and D). [Ca2+]i mobilization in response to carbachol (100 µM) and LA (10 µg/ml) was measured as described in MATERIALS AND METHODS.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Effect of thapsigargin (Tg) on carbachol- and PUFA-mediated [Ca2+]i mobilization in the absence of extracellular Ca2+. NT2 cells (2.5–5 x 105) were washed with 10 ml of PBS0Ca buffer immediately before [Ca2+]i measurements. A: LA-induced [Ca2+]i mobilization in the absence of extracellular Ca2+. B: [Ca2+]i responses of NT2 cells to sequential treatment with Tg, carbachol, and LA in the absence of extracellular Ca2+.

View larger version (5K): [in this window] [in a new window]   Fig. 6. Effect of carboxyl cyanide m-chlorophenylhydrazone (CCCP) on [Ca2+]i in NT2 cells. Fura-2-labeled NT2 cells in suspensions were treated with increasing concentrations of CCCP as indicated, and changes in [Ca2+]i were measured as described in MATERIALS AND METHODS.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Effect of CCCP on carbachol- and PUFA-mediated [Ca2+]i mobilization. NT2 cells in suspensions were either untreated (A, C, and E) or treated with CCCP (10 µM) for 15 min at room temperature (B, D, and F). After treatment, carbachol- (100 µM) and PUFA (AA and LA; 10 µg/ml)-mediated [Ca2+]i mobilization was measured as described in MATERIALS AND METHODS. The results of a typical experiment are presented in A–F.

The effects of CCCP observed in NT2 cells suggest that PUFA-induced [Ca²⁺]_i mobilization requires functional mitochondria. To determine how mitochondria are involved in the PUFA response, we compared the change of [Ca²⁺]_m during carbachol and PUFA treatment of NT2 cells. In these experiments, intact NT2 cells were labeled with the mitochondrial Ca²⁺ indicator X-rhod-1 and the fluctuation in cytosolic Ca²⁺ was buffered with BAPTA. In BAPTA and X-rhod-1 double-labeled cells, stimulation of the cells with carbachol had no effect on X-rhod-1 fluorescence in the absence of extracellular Ca²⁺ (Fig. 8A), indicating that activation of the traditional GPCR-PLC-IP₃ pathway under the experimental conditions used had no effect on [Ca²⁺]_m. In contrast, in the same cells in which carbachol had no effect on [Ca²⁺]_m subsequent treatment with AA (10 µg/ml) caused a reduction in X-rhod-1 fluorescence (Fig. 8B), suggesting a reduction in [Ca²⁺]_m in intact NT2 cells by AA. Under the same conditions, the saturated FA PA (10 µg/ml) showed no effect on X-rhod-1 fluorescence (Fig. 8C), whereas the PUFA LA also caused a reduction in X-rhod-1 fluorescence (Fig. 8D). These results suggest that PUFA but not carbachol or saturated FA cause Ca²⁺ release from mitochondria in intact NT2 cells.

View larger version (8K): [in this window] [in a new window]   Fig. 8. Effect of carbachol and PUFA on [Ca2+]m measured in intact NT2 cells. NT2 cells were loaded either sequentially with X-rhod-1 AM (2 µM) and BAPTA-AM (50 µM) (A–F) or with X-rhod-1 AM (2 µM) alone (G and H) at 37°C for 30 min. Loaded cells were resuspended in PBS supplemented with high EGTA (2 mM) (A–F, and H) or PBS supplemented with 1 mM CaCl2 and 1 mM MgSO4 (PBS1Ca) (G), and immediately used to measure carbachol-, PUFA-, or palmitic acid (PA)-induced changes in [Ca2+]m following the procedure described in MATERIALS AND METHODS. Mitochondrial blockers CCCP (10 µM) and rotenone (10 µM) were tested in panels E and F, respectively. Changes in [Ca2+]m following carbachol and FA treatment were indexed by alterations in X-rhod-1 fluorescence intensity (y-axis).

Additional experiments were conducted to compare the effects of carbachol and PUFA on X-rhod-1 signals in NT2 cells in the absence of BAPTA. In the presence of extracellular Ca²⁺, carbachol caused a transient increase in [Ca²⁺]_i, followed by an elevated plateau, indicating a typical GPCR response, whereas AA caused a more gradual increase of [Ca²⁺]_i (Fig. 8G). The AA response observed may represent the sum of the change in cytosolic and mitochondrial Ca²⁺. In Ca²⁺-free media, both carbachol and AA caused increases in [Ca²⁺]_i without BAPTA (Fig. 8H). However, after carbachol-induced [Ca²⁺]_i decayed to basal levels, addition of AA caused a reduction in X-rhod-1 signal (Fig. 8H). This observation is consistent with a decrease of [Ca²⁺]_m under conditions of enhanced removal of [Ca²⁺]_i by the endoplamic reticulum and plasma membrane Ca²⁺-ATPase, which has been reported to be activated during GPCR stimulation in other cell types (62, 77).

To confirm that PUFA may induce [Ca²⁺]_i mobilization by causing Ca²⁺ release from mitochondria, we examined the effect of PUFA in isolated mitochondria using both the mitochondrial Ca²⁺ indicator X-rhod-1 and ⁴⁵Ca²⁺. In these experiments, mitochondria were prepared from NT2 cells and labeled with X-rhod-1 or ⁴⁵Ca²⁺ in the presence of high concentration of Ca²⁺ (PBS1Ca buffer). The labeled mitochondria were then treated with PUFA and alterations in [Ca²⁺]_m were measured by X-rhod-1 fluorescence or by the remaining ⁴⁵Ca²⁺ content in the mitochondria. As demonstrated in Fig. 9A, DHA caused a concentration-dependent release of Ca²⁺ from mitochondria, as measured by X-rhod-1 fluorescence; LA and AA had similar effects (data not shown). Measurement of ⁴⁵Ca²⁺ indicated that AA and LA but not PA also caused a significant reduction in mitochondrial ⁴⁵Ca²⁺ content (Fig. 9B). Compared with control, AA and LA reduced the ⁴⁵Ca²⁺ content by 36.3 ± 12.4% (P = 0.0079, n = 5 measurements) and 35.9 ± 10.6% (P = 0.0039, n = 6), respectively, whereas PA had no significant effect on mitochondrial ⁴⁵Ca²⁺ content (P = 0.1256 vs. control, n = 5).

View larger version (7K): [in this window] [in a new window]   Fig. 9. Effect of PUFA and PA on [Ca2+]m in isolated mitochondria from NT2 cells. A: mitochondria were isolated and labeled with X-rhod-1 (2 µM) at 37°C for 30 min in PBS1Ca. Loaded mitochondria were resuspended in PBS supplemented with 200 µM EGTA and 1 mM MgSO4 (PBS0Ca) and Ca2+ release from mitochondria in response to increasing concentrations of DHA was measured immediately, as described in MATERIALS AND METHODS. B: isolated mitochondria were labeled with 45Ca2+ at 37°C for 30 min in PBS1Ca. 45Ca2+-loaded mitochondria were then transferred and incubated with PUFA (AA or LA; 10 µg/ml) or the saturated FA PA (10 µg/ml) in 10 vol of PBS0Ca at 37°C for 5 min. The mixtures were then filtered and washed three times with PBS0Ca and the 45Ca2+ content in mitochondria was counted. Values in the figure are means ± SE from 5–6 measurements. *P = 0.0079 vs. untreated (control); **P = 0.0039 vs. control.

View larger version (9K): [in this window] [in a new window]   Fig. 10. PUFA-induced [Ca2+]i mobilization in human aortic endothelial cells (HAEC) and mitochondrial Ca2+ efflux in isolated mitochondria of HAEC cells. A: confluent HAEC grown in 75-ml flasks were trypsinized and labeled with fura-2 AM (2 µM) at 37°C for 30 min. [Ca2+]i mobilization in response to AA (1–10 µg/ml as indicated) was measured in labeled cells, as described in MATERIALS AND METHODS. Typical AA concentration-dependent [Ca2+]i responses are shown. B: mitochondria were isolated from confluent HAEC and labeled with X-rhod-1, as described in MATERIALS AND METHODS. PUFA (AA or LA)-induced Ca2+ release from mitochondria was measured by the decrease in X-rhod-1 fluorescence intensity (y-axis).

View larger version (7K): [in this window] [in a new window]   Fig. 11. Effect of cyclosporin A (CsA), bongkrekic acid (BA), and ruthenium red on LA-induced Ca2+ efflux in isolated mitochondria from NT2 cells. Mitochondria were isolated and labeled with X-rhod-1 (2 µM) at 37°C for 30 min in PBS1Ca. Loaded mitochondria were resuspended in PBS0Ca and Ca2+ release from mitochondria in response to LA (10 µg/ml) was measured as described in MATERIALS AND METHODS. In experiments with CsA and BA (top), the reagents (each at 10 µM) were preincubated with loaded mitochondria for 15 min at room temperature before the addition of LA; ruthenium red (10 µM) was added immediately before LA (bottom). Changes in [Ca2+]m after LA treatment were indexed by alterations in X-rhod-1 fluorescence intensity (y-axis).

	DISCUSSION

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In several cell types, CCCP increases [Ca²⁺]_i by causing Ca²⁺ release from mitochondria (7, 12, 20, 29, 34, 49, 60, 67, 72). CCCP is a mitochondrial uncoupler that collapses the proton gradient across the mitochondrial inner membrane and thus eliminates the driving force for mitochondrial Ca²⁺ uptake. In addition, Ca²⁺ efflux from mitochondria requires the opening of a conducting pathway for Ca²⁺ exit. During GPCR-stimulated [Ca²⁺]_i mobilization CCCP prevents mitochondrial Ca²⁺ uptake, which leads to inhibition of the store-operated or capacitative Ca²⁺ influx. Consistently in NT2 cells CCCP eliminated the plateau phase of carbachol-mediated [Ca²⁺]_i mobilization, indicating the inhibition of store-operated Ca²⁺ influx (Fig. 7F). The effect of CCCP on [Ca²⁺]_i in resting cells has been found to vary among different cell types. For example, in studies (20) of pancreatic acinar cells, CCCP did not induce [Ca²⁺]_i signals in resting cells but did prevent mitochondrial Ca²⁺ uptake and cause [Ca²⁺]_i mobilization in agonist-stimulated cells. In chromaffin cells, CCCP triggered [Ca²⁺]_i mobilization and reduced [Ca²⁺]_m under resting conditions (46), indicating that in some cell types, energized mitochondria retain higher concentrations of ionic Ca²⁺ than the cytosol even in the resting state. Direct measurement of [Ca²⁺] in mitochondria with low-affinity aequorin yields a value of 5.8 µM (46), which is >50-fold higher than the normal resting [Ca²⁺]_i. Thus mitochondria may be an independent intracellular Ca²⁺ store. It is known that the two intracellular Ca²⁺ stores, i.e., the mitochondrial Ca²⁺ pool and the IP₃-sensitive endoplasmic reticulum pool, are dynamically linked by their structural proximity during [Ca²⁺]_i mobilization (57). Part of the Ca²⁺ released from the endoplasmic reticulum by the IP₃-gated channels is taken up by mitochondria (68), leading to increased [Ca²⁺]_m. The increase in [Ca²⁺]_m upregulates the activities of multiple enzymes involved in energy production, as indicated by changes in mitochondrial reduction-oxidation substrates (21, 68). On the other hand increased Ca²⁺ uptake into mitochondria and/or [Ca²⁺]_m also sensitize cells for the induction of apoptosis by proapoptotic factors (69). Mitochondrial Ca²⁺ uptake and subsequent efflux also modify the amplitude, duration, localization, and propagation of cytosolic Ca²⁺ transients (15, 19, 27, 71). In this study, we provide evidence that mitochondria may serve as an independent intracellular source for PUFA-responsive [Ca²⁺]_i mobilization in NT2 cells. CCCP-induced [Ca²⁺]_i mobilization was observed in resting NT2 cells (Fig. 6). The CCCP-mediated [Ca²⁺]_i signal in these cells may occur by elimination of the driving force for Ca²⁺ uptake, i.e., the proton gradient, and opening of a conducting pathway such as the mitochondrial Ca²⁺-induced Ca²⁺ release process (46) or Ca²⁺ leak along the Ca²⁺ gradient between mitochondria and cytosol. PUFA-induced mitochondrial Ca²⁺ release and [Ca²⁺]_i mobilization in NT2 cells may occur by depolarization of the mitochondrial proton gradient through an uncoupling protein-dependent mechanism (32) and/or opening of the MPT for Ca²⁺ exit (41, 50, 64). Pretreatment of NT2 cells with CCCP inhibits subsequent PUFA-induced [Ca²⁺]_i mobilization probably by causing Ca²⁺ efflux from mitochondria and depletion of the mitochondrial Ca²⁺ pool (Fig. 7).

In this study, we used U73122 [GenBank] and 2-APB to examine the involvement of the classic PLC-IP₃ signaling pathway in PUFA-induced [Ca²⁺]_i mobilization. U73122 [GenBank] is a synthetic aminosteroid PLC inhibitor. The specificity of the inhibitor toward PIP₂ specific PLCs has been validated by numerous studies (22, 38, 53, 54, 75) from different laboratories that had used various cell types. We observed that treatment of NT2 cells with U73122 [GenBank] completely blocked carbachol-induced [Ca²⁺]_i mobilization but had no effect on the PUFA response (Fig. 3). The results confirm that as in other cell types, U73122 [GenBank] specifically targets the GPCR-PLC-IP₃ signaling pathway in NT2 cells; [Ca²⁺]_i mobilization mediated by other mechanisms, such as PUFA-induced Ca²⁺ efflux from mitochondria, is unaffected (Fig. 3). Although 2-APB was originally reported as a cell permeable IP₃ receptor antagonist (43), recent studies (3, 37) demonstrated effects of this agent on other pathways that may or may not be related to PLC-IP₃-mediated [Ca²⁺]_i mobilization. Moreover, inhibition of IP₃ receptor-mediated Ca²⁺ release by 2-APB has been shown to be variable among different cell types (3, 37). The inhibitory effect of 2-APB may depend on the isoforms of IP₃ receptors expressed in the cell and the cytosolic concentrations of IP₃ during agonist stimulation (3). In NT2 cells 2-APB effectively blocked [Ca²⁺]_i mobilization in response to carbachol but not PUFA (Fig. 4). 2-APB was found to reduce the rate of the initial rise of PUFA-induced [Ca²⁺]_i mobilization by 80% (Fig. 4). This action of 2-APB could result from a nonspecific effect on mitochondria, insofar as 2-APB has been suggested to inhibit mitochondrial Ca²⁺ efflux in Jurkat T cells (52a).

Multiple mitochondrial pathways, including the Na⁺/Ca²⁺ and H⁺/Ca²⁺ exchangers, the Ca²⁺ uniporter, as well as the MPT, are capable of transporting Ca²⁺ out of mitochondria (14, 52, 56). Opening of MPT by PUFA and other FA has been reported in isolated mitochondria (2). The MPT is theoretically permeable to Ca²⁺ and other small molecules, which makes the MPT a possible candidate for mediating PUFA-induced Ca²⁺ efflux. In addition, the opening of MPT by PUFA may collapse the mitochondrial membrane potential and proton gradients and thus indirectly activate the reversal mode of the Ca²⁺ uniporter, or affect the activities of the Na⁺/Ca²⁺ and/or H⁺/Ca²⁺ exchangers, to release mitochondrial Ca²⁺. We have found that PUFA depolarize mitochondrial membrane potential in intact NT2 cells (data not shown). However, preincubation of NT2 mitochondria with CsA and BA had no effect on PUFA-induced Ca²⁺ efflux (Fig. 11), suggesting that the MPT and other pathways indirectly linked with MPT through mitochondrial membrane potential may not be involved in PUFA-mediated mitochondrial Ca²⁺ efflux. This does not exclude the possibility that PUFA might directly activate mitochondrial Ca²⁺ transporters to release Ca²⁺ in Ca²⁺-loaded mitochondria. Indeed, as we have demonstrated, the addition of ruthenium red to block the mitochondrial Ca²⁺ uniporter inhibited LA-induced mitochondrial Ca²⁺ efflux, implicating the involvement of the Ca²⁺ uniporter in PUFA-mediated mitochondrial Ca²⁺ efflux (Fig. 11). The Na⁺/Ca²⁺ exchanger blocker CGP37157 did not affect LA-induced [Ca²⁺]_i mobilization in NT2 cells (data not shown). PUFA and other FA may also depolarize the mitochondrial membrane potential by uncoupling protein 2 (UCP-2)-dependent mechanisms. However, the UCP-2 pathways are unlikely to be the underlying mechanism for PUFA-induced mitochondrial Ca²⁺ efflux because saturated and monounsaturated FA activate the UCP-2 pathway in other systems (35) but had no effect on [Ca²⁺]_i in NT2 cells and [Ca²⁺]_m in isolated mitochondria (Figs. 1 and 8). Additional studies are ongoing to define the role of the mitochondrial Ca²⁺ uniporter and possibly other transporters in PUFA-mediated mitochondrial Ca²⁺ efflux.

	GRANTS

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This work was partially supported by American Heart Association Grant 0235065N, National Heart, Lung, and Blood Institute Grant R01-HL-75011 (both to B.-X. Zhang), and by Department of Veterans Affairs grants (to B.-X. Zhang and M. S. Katz).

	ACKNOWLEDGMENTS

 
The authors thank Shuko Lee for help with data analysis and Drs. Paramita Ghosh and Amrita Kamat for helpful discussions and comments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: B.-X. Zhang, GRECC (182), South Texas Veterans Health Care System, Audie L. Murphy Division, 7400 Merton Minter Blvd., San Antonio, TX 78229 (e-mail: zhangb2{at}uthscsa.edu)

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.

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