Novel role of phospholipase C-
1: regulation of liver mitochondrial Ca2+ uptake
Clayton D. Knox,1
Andrey E. Belous,1
Janene M. Pierce,1
Aya Wakata,1
Ian B. Nicoud,2
Christopher D. Anderson,1
C. Wright Pinson,1 and
Ravi S. Chari1,2
Departments of 1Surgery and 2Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-4753
Submitted 29 January 2004
; accepted in final form 20 April 2004
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ABSTRACT
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Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-
1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37°C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-
1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.
calcium; uniporter; inositol trisphosphate; cat; 2-APB
MITOCHONDRIAL CA2+ (mCa2+) handling plays a central role in the regulation of hepatocyte cell function. In addition to its control of signal transduction, cell cycle, and rate of cellular respiration, mCa2+ is an important determinant of apoptosis (49). When excessive Ca2+ enters the mitochondria and mCa2+ overload occurs, mitochondrial transmembrane potential (m
) is dissipated and permeability transition pore formation occurs. This releases cytochrome c (22), apoptosis-inducing factor (23), and a host of other proapoptotic factors into the cytoplasm, in which they activate caspases and initiate a cascade of cellular events leading to apoptosis (reviewed in Ref. 49).
Cytosolic Ca2+ enters the mitochondria almost exclusively through the mCa2+ uniporter, a Ca2+ channel located on the inner mitochondrial membrane (25, 29). The opened uniporter allows Ca2+ to passively flow down its electrochemical gradient into the negatively charged mitochondrial matrix without the exchange of another ion or molecule (39). There are two modes of mCa2+ uptake, the normal and rapid modes. Both are thought to occur through the uniporter and are sensitive to ruthenium red (42). The rate of mCa2+ uptake is dependent on two variables: cytosolic Ca2+ concentration (cytosolic [Ca2+]) and m
(37, 46). Under physiological conditions, when the cytosolic [Ca2+] is
100 nm and m
is 150 to 180 mV (internally negative), the uniporter is relatively inactive, and negligible Ca2+ transport occurs (51). However, when cytosolic Ca2+ levels increase and the uniporter opens, mitochondria accumulate Ca2+. Although the exact structure and mechanism of the uniporter are unknown, spermine (21, 28) and Ca2+ (21) stimulate the uniporter, and ruthenium red is known to be a selective inhibitor (35). Enzymatic regulation of uniporter activity has not been thoroughly investigated.
Phospholipase C (PLC) is a signaling enzyme that hydrolyzes phosphatidylinositol-4,5-biphosphate (PIP2) to produce inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). I(1,4,5)P3 directly stimulates increases in cytosolic [Ca2+] through interactions with I(1,4,5)P3 receptors on the endoplasmic reticulum, whereas DAG activates protein kinase C, another critical component in cellular Ca2+ maintenance (reviewed in Ref. 33). Whereas the close relationship between PLC and Ca2+ within the cell is clear, the presence of PLC has not been described in mitochondria. However, mitochondrial PIP2 was recently identified (48). Due to the direct role of PLC in cellular Ca2+ maintenance, we hypothesized that PLC may exist in mitochondria and play a role in mCa2+ handling. We now demonstrate that PLC-
1 exists within the mitochondria and plays an essential role in mCa2+ uptake through the mCa2+ uniporter.
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EXPERIMENTAL PROCEDURES
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Reagents.
Unless otherwise noted, all reagents were obtained from Sigma (St. Louis, MO).
Preparation of cellular fractions.
The Vanderbilt University Institutional Animal Care and Use Committee approved the protocol for animal use in this study. Livers were isolated from male Sprague-Dawley rats (300 g) as previously described (18). Whole mitochondria were separated from rat liver as previously described by Belous et al. (3). Briefly, the liver was minced and homogenized in liver homogenization buffer (LHB; 0.2 M mannitol, 50 mM sucrose, 10 mM KCl, and 1 mM Na2EDTA, pH = 7.4, KOH). The homogenate was filtered through gauze and centrifuged two times for 10 min at 1,000 g. The supernatant was collected and centrifuged for 10 min at 3,000 g. The pellet was washed twice and resuspended in Ca2+-free LHB without EDTA to a concentration of 2 mg/ml. All steps were performed at 4°C.
Mitochondrial membranes were prepared as described by Hagopian (12). Purified mitochondria in suspension were sonicated twice for 60 s and then centrifuged twice for 10 min at 7,000 g. The supernatant was collected and centrifuged for 1 h at 150,000 g. The resulting pellet contained purified mitochondrial membranes.
In separate animals, plasma membranes were isolated essentially as previously described (27a). Livers were perfused with 4°C PBS via the portal vein, harvested, and minced in a solution containing sucrose, Tris, and MgCl2 (STM; 0.25 M sucrose, 10 mM Tris, 1.0 mM MgCl2, protease inhibitors, pH = 7.4, KOH). The mixture was homogenized and filtered through gauze. STM (0.25 M) was added to the homogenate and centrifuged twice for 5 min at 260 g. The supernatant was centrifuged for 10 min at 1,500 g, and the resulting pellet was resuspended in a mixture of 0.25 and 2.0 M STM to achieve a final density of 1.18 g/cm3. Ultracentrifuge tubes (Kendro/Sorval, Asheville, NC) were filled to 90% of maximum volume with this mixture and 0.25 M STM was layered on top. Tubes were then centrifuged for 60 min at 113,000 g. Plasma membranes, which accumulated at the interface of the two STM layers, were mixed with water and then centrifuged for 10 min at 1,500 g. The pellet contained purified plasma membranes.
Western blot analysis.
Purity of the whole mitochondria preparation was confirmed by using Western blot technique as previously reported (3). Mitochondrial membrane purity was further analyzed by using additional Western blots probed for alkaline phosphatase, Na+-K+- ATPase, calreticulin, and prohibitin. Sixty-microgram samples of mitochondrial membranes and the respective antibody controls were electrophoresed on 12% acrylamide gels and transferred to polyvinylidene difluoride membranes. Rabbit anti-alkaline phosphatase (RDI, Flanders, NJ) and rabbit anti-Na+-K+-ATPase antibodies (Novus Biologicals, Littleton, CO) were used to detect heavy and light plasma membrane fraction contamination, respectively, with rat liver plasma membranes serving as a positive control. An anticalreticulin antibody (Novus Biologicals) was used to determine endoplasmic reticulum contamination, with rat brain whole cell lysate as a positive control. Antiprohibitin antibodies were used to confirm the presence of mitochondria, with rat liver whole cell lysate used as a positive control. Blots were probed by using antibodies directed against different isoforms of PLC. Mouse anti-PLC-
1 (BD Transduction Labs, Lexington, KY), rabbit anti-PLC-
2 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-PLC-
1 (BD Transduction Labs), rabbit anti-PLC-
2 and -
3 (Santa Cruz Biotechnology), mouse anti-PLC-
1 (BD Transduction Labs), and goat anti-PLC-
(Santa Cruz Biotechnology) antibodies were tested.
Measurement of mCa2+ uptake.
All mCa2+ uptake studies were performed in incubation buffer (100 mM KCl, 20 mM HEPES, 5 mM MgCl2, pH = 7.4, KOH) as previously described by Belous et al. (3). A combination of metabolic substrates pyruvic acid and malic acid (final concentration: 1 mM each) was used as a source of mitochondrial energy. Radioactive 45Ca2+ (New England Nuclear Life Science Products, Boston, MA) was added to the incubation buffer (final concentration range: 0.12.0 µM). The experiments were initiated by the addition of mitochondria to the incubation buffer (final concentration: 0.2 mg/ml). Mitochondria were incubated in a 37°C shaking water bath for 30 min, filtered, and washed. Accumulated mitochondrial 45Ca2+ was then measured by using liquid scintillation counting in a Beckman LS6000IC beta counter. Mitochondria in incubation buffer without pyruvic acid and malic acid served as background controls. Ruthenium red (10 µM), a selective uniporter antagonist, was tested in all conditions to determine inhibitory capacity on observed mCa2+ uptake and to ensure that all observed Ca2+ uptake was through the uniporter.
Mechanisms of mCa2+ uptake.
Metabolic substrates pyruvic acid and malic acid (1 mM each) were added to reaction tubes containing incubation buffer and 0.2 µM 45Ca2+. The selective, membrane-permeable PLC inhibitor U-73122 (Calbiochem, San Diego, CA) was added at various concentrations from 0.1 to 15 µM to determine the effect of PLC inhibition on mCa2+ uptake. Parallel experiments used identical concentrations of its inactive analog U-73343 (Calbiochem) or DMSO as a vehicle control. Spermine, a polyamine, was used to stimulate uniporter activity at a final concentration of 5 µM. Additionally, U-73122 was added together with spermine to determine how PLC inhibition would affect the stimulated uniporter.
To investigate the possible role of I(1,4,5)P3 and DAG in mCa2+ uptake, the following I(1,4,5)P3- and DAG-signaling agonists and antagonists were employed: I(1,4,5)P3, adenophostin A, 2-aminoethoxydiphenylborate (2-APB), xestospongin C (XeC), 1-oleoyl-2-acetyl-sn-glycerol (OAG), and DAG-lactone. I(1,4,5)P3 was added to mCa2+ uptake reaction tubes to a final concentration of 50 µM, the concentration previously reported to fully stimulate endoplasmic reticulum I(1,4,5)P3 receptors in rat hepatocytes (7). Adenophostin A, a potent I(1,4,5)P3 receptor agonist resistant to degradation (44), was also tested. 2-APB is a membrane-permeable, selective I(1,4,5)P3 receptor inhibitor that blocks I(1,4,5)P3 signaling without disrupting the binding of I(1,4,5)P3 to the I(1,4,5)P3 receptor (4, 24, 26, 31, 41). 2-APB was added to mCa2+ uptake reaction tubes at various concentrations in the range of the IC50 values reported for its inhibition of I(1,4,5)P3 receptors (24). XeC is another highly specific and membrane-permeable inhibitor of I(1,4,5)P3 receptors and was used to confirm the effects of 2-APB (10). In parallel experiments, two membrane-permeable analogs of DAG, OAG and DAG-lactone, were tested to determine if DAG affects mCa2+ uptake.
Statistics.
Each experiment was performed on at least 3 separate animals. All measurements in each condition and at each [Ca2+] were performed in triplicate. Data are presented as means ± SE. Statistical significance was assessed by using two-tailed Students t-test with unequal variance, and P < 0.05 was taken as significant.
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RESULTS
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PLC-
1 exists in mitochondria.
The purity of the mitochondrial membrane preparations was confirmed by Western blot. Negligible alkaline phosphatase, Na+-K+-ATPase, and calreticulin were visualized on the mitochondrial membrane preparations, which were highly enriched with prohibitin compared with liver whole cell lysate (Fig. 1). To confirm this, densitometric analysis was performed. There was no detectable activity above background in mitochondrial membrane fractions at the band size for alkaline phosphatase, Na+-K+-ATPase, and calreticulin (n = 3).

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Fig. 1. Western blot analyses demonstrated that isolated mitochondrial membranes (MM) were free of heavy and light plasma membrane fraction, as well as microsomal contamination. Prohibitin, a mitochondrial protein, was enriched in mitochondrial membranes. Positive controls (pos.) for each protein were as follows: Na+-K+-ATPase and alkaline phosphatase, liver plasma membranes; calreticulin, brain whole cell; prohibitin, liver whole cell.
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Western blot analysis revealed a single band at the appropriate molecular weight of
88 kDa for PLC-
1 in the isolated mitochondrial membranes and plasma membranes (Fig. 2). These data suggest that within both the cell and the mitochondria, PLC-
1 is found in association with membranes.
PLC-
1 and -
2 were also observed in the mitochondrial membrane preparations, although they showed only a faint signal (Fig. 2). Blots were also probed for PLC-
1, -
2, -
3, and -
, but PLC-
4 was excluded, because it is not present in rat liver (36). PLC-
1, -
3, and -
were enriched in the liver plasma membranes compared with whole cell lysate, whereas PLC-
2 was not found in association with plasma membranes. None of these four isoforms were detected in mitochondrial membranes (Fig. 2).
PLC inhibition blocks mCa2+ uptake.
After confirming the presence of PLC-
1 in the mitochondria, we characterized its influence on the regulation of mCa2+ handling by using a number of experimental conditions. Mitochondria were incubated in incubation buffer with 0.2 µM 45Ca2+ for varying time points from 160 min, and maximal 45Ca2+ uptake was found to occur after 30 min (data not shown), which was calculated to be 12 pm·mg mitochondrial protein1·min1. This value was taken as baseline. In background controls that contained no metabolic substrates, uptake was <10% of baseline. Ruthenium red completely blocked mCa2+ uptake at a concentration of 10 µM, confirming that the 45Ca2+ was entering the mitochondria exclusively through the mCa2+ uniporter (data not shown).
The aminosteroid U-73122 is a potent PLC inhibitor with few nonspecific effects (5, 6). It has no effect on other molecules involved in Ca2+ signaling, such as cAMP, protein kinase C, and phospholipase A2 (47). U-73343, which differs from U-73122 by a sole double bond, does not inhibit PLC and was used as a negative control. Concentrations of 5 µM or greater of U-73122 inhibited mCa2+ uptake by 90100% in the presence of 0.2 µM extramitochondrial 45Ca2+ with an IC50 of 2.03.0 µM U-73122 (Fig. 3A). This value is consistent with the 1.02.1 µM IC50 reported for PLC inhibition in intact cells (5). When tested over the same concentration range, U-73343 had no effect on mCa2+ uptake (Fig. 3B).

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Fig. 3. Mitochondrial Ca2+ uptake after 30-min incubation of isolated mitochondria in 37°C incubation buffer containing 0.2 µM 45Ca2+. Uptake in the presence of 0.14% DMSO (vehicle) was taken as baseline. A: PLC inhibitor U-73122 dose-dependently inhibited mitochondrial Ca2+ uptake when added to the incubation buffer at 0.5- to 7.5-µM concentrations. B: U-73343, the inactive analog of U-73122, did not significantly affect mitochondrial Ca2+ uptake when tested by using the same concentration range (n = 3).
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To test the effects of increasing [Ca2+] on uniporter activity, varying 45Ca2+ concentrations were used in the incubation buffer. As the concentration of extramitochondrial 45Ca2+ was increased from 0.1 µM to 1.0 µM, mCa2+ uptake increased up to 12-fold. At all concentrations of extramitochondrial 45Ca2+, 10 µM ruthenium red completely blocked mCa2+ uptake, indicating that all observed mCa2+ uptake was occurring through the uniporter. To determine whether PLC inhibition would be able to inhibit this increased mCa2+ uptake in a similar manner, mitochondria were incubated in incubation buffer containing 5 µM U-73122 with the same range of Ca2+ concentrations. At all tested concentrations of extramitochondrial Ca2+, 5 µM U-73122 blocked mCa2+ uptake (Fig. 4).
Spermine stimulation of uniporter activity is inhibited by PLC antagonism.
Once the relationship between PLC activity and mCa2+ uptake was established, we sought to determine whether or not U-73122 would affect mCa2+ uptake during spermine stimulation of the uniporter. At a concentration of 5 µM, spermine stimulated mCa2+ uptake by 34 ± 13%. This effect was completely inhibited by the addition of 10 µM ruthenium red. When added to mitochondria in the presence of spermine, 2 µM U-73122 blocked mCa2+ by 50 ± 10% and 5 µM U-73122 blocked mCa2+ uptake by 99 ± 0.4% (Fig. 5).

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Fig. 5. Mitochondrial Ca2+ uptake after 30-min incubation of isolated mitochondria in 37°C incubation buffer containing 0.2 µM 45Ca2+. Bar 1 represents baseline mitochondrial Ca2+ uptake in the presence of substrates alone and in the absence of agonists/antagonists. The effect of the following agents was determined: bar 2, 4 µM U-73343; bar 3, 5 µM spermine; bar 4, 5 µM spermine plus 2 µM U-73122; bar 5, 5 µM spermine plus 5 µM U-73122; bar 6, 5 µM spermine plus 10 µM ruthenium red. Spermine significantly increased mitochondrial Ca2+ uptake, whereas U-73122 dose-dependently blocked Ca2+ uptake in the presence of spermine. Inhibition with ruthenium red confirmed that Ca2+ uptake was uniporter dependent (n = 3, *P < .05).
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2-APB and XeC block mCa2+ uptake.
PLC-
1 is known to affect cellular Ca2+ through hydrolysis of PIP2 to yield the second messengers I(1,4,5)P3 and DAG. Because PIP2 has recently been identified in the mitochondria (48), we considered that I(1,4,5)P3 and DAG could be involved in mCa2+ uptake. I(1,4,5)P3 generated at the cell membrane promotes increased cytosolic [Ca2+] through its interaction with I(1,4,5)P3 receptors on the endoplasmic reticulum. This pathway is inhibited by 2-APB (31) and XeC (10). In our experiments, 2-APB blocked mCa2+ uptake and exhibited a dose-response curve consistent with that reported in the literature for I(1,4,5)P3 receptor inhibition. Fifty percent inhibition of mCa2+ uptake was obtained with 5075 µM 2-APB (Fig. 6A), and an IC50 of 42 µM has been reported in intact cells (24). To confirm these effects, XeC was also tested. It reduced mCa2+ uptake by 77 ± 6% at 10 µM (Fig. 6C).

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Fig. 6. Mitochondrial Ca2+ uptake after 30-min incubation of isolated mitochondria in 37°C incubation buffer containing 0.2 µM 45Ca2+. A: 2-aminoethoxydiphenylborate (2-APB) dose-dependently inhibited mitochondrial Ca2+ uptake. Ca2+ uptake in the absence of 2-APB was taken as baseline. B: addition of 50 µM inositol-1,4,5-trisphosphate [I(1,4,5)P3] or 1 µM adenophostin A (Ad A), a selective and stable I(1,4,5)P3 receptor agonist, had no significant effect on mitochondrial Ca2+ uptake. Baseline Ca2+ uptake was established in the absence of agonists/antagonists. C: baseline Ca2+ uptake was established in the presence of 0.1% DMSO (vehicle). Ten micrograms xestospongin C (XeC), a permeant I(1,4,5)P3 receptor antagonist, and 10 µM 1,2-diacylglycerol-lactone (DAG-L), a permeant DAG analog, were tested. Both compounds significantly decreased mitochondrial Ca2+ uptake. D: membrane-permeable DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG), an inhibitor of PLC activity, dose-dependently inhibited mitochondrial Ca2+ uptake. Ca2+ uptake in the presence of 1.0% DMSO (vehicle) was taken as baseline (n = 3, *P < 0.01).
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Extramitochondrial I(1,4,5)P3 and adenophostin A do not affect mCa2+ uptake.
I(1,4,5)P3 or the I(1,4,5)P3 receptor agonist adenophostin A were added to mCa2+ uptake reaction tubes. I(1,4,5)P3 was added at concentrations ranging from 5 to 50 µM but had no effect on mCa2+ uptake. Previous reports have shown that 50 µM I(1,4,5)P3 is sufficient for potent I(1,4,5)P3 receptor stimulation in hepatocytes. In parallel experiments, adenophostin A [IC50 = 9.2 nM in rat hepatocytes (8)] was added at concentrations up to 1 µM and also had no effect on mCa2+ uptake (Fig. 6B). Because neither compound is membrane permeable, we conclude that there is no uniporter-coupled I(1,4,5)P3 binding site on the outer mitochondrial membrane that is accessible from the cytosol.
DAG analogs inhibit mCa2+ uptake.
When added at concentrations 125 µM, the membrane-permeable DAG analog OAG dose-dependently blocked mCa2+ uptake in our experiments. The IC50 for this inhibition was
5 µM, and complete inhibition was observed at 25 µM (Fig. 6D). To confirm these effects, a second DAG analog, DAG-L, was tested. At 10 µM, DAG-L blocked mCa2+ uptake by 76 ± 3% (Fig. 6C).
Mito-Tracker Red fluorescence.
Mitochondrial viability was assessed by using Mito-Tracker Red (Molecular Probes, Eugene, OR) fluorescence microscopy to ensure that mitochondria maintained m
, indicating viability. Staining with Mito-Tracker Red (final concentration: 100 nM) confirmed that isolated mitochondria were sufficiently energized to maintain m
and take up extramitochondrial Ca2+ in the presence of incubation buffer and the metabolic substrates pyruvic and malic acid. When exposed individually to all the experimental agonists and antagonists at concentration ranges used above, mitochondria remained structurally intact and viable. Representative images for viable mitochondria, nonviable mitochondria, and mitochondria incubated with U-73122 and U-73343 are shown in Fig. 7.

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Fig. 7. Grayscale images (magnification, x400) from fluorescent microscopic imaging of isolated, whole rat liver mitochondria after incubation with Mito Tracker Red fluorescent dye (final concentration: 100 nM), a positively charged probe that, when sequestered by mitochondria, is indicative of mitochondrial membrane potential and viability. Mitochondria were incubated for 15 min at 37°C in the presence of incubation buffer and 0.2 µM Ca2+, plus metabolic substrates pyruvate and malate (final concentration: 1 mM each) (A); no metabolic substrates (B); metabolic substrates plus U-73122 (final concentration: 5 µM) (C); and metabolic substrates plus U-73343 (final concentration: 5 µM) (D). A, C, and D show intact and viable mitochondria that have sequestered Mito Tracker dye; B shows mitochondria that, in the absence of metabolic substrates, were unable to maintain membrane potential and thus were not viable. U-73122 and U-7343 did not affect mitochondrial viability (C and D).
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DISCUSSION
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Through the uptake and release of cytosolic Ca2+, mitochondria are able to affect cellular [Ca2+] in a number of ways. They may sequester cytosolic Ca2+ as concentration spikes occur, acting as a temporary buffer and maintaining cytosolic [Ca2+] (34). Mitochondria are also capable of propagating agonist-induced Ca2+ signals originating at cell membrane receptors and controlling the amplitude of these Ca2+ oscillations, thus serving as cellular transduction modulators (32, 38). Through their management of the Ca2+ release-activated Ca2+ channels on the cell membrane and the rate and magnitude of Ca2+ influx, mitochondria are also important regulators of cell cycle and malignant transformation (15, 16, 52). The balance between cytosolic [Ca2+] and mitochondrial [Ca2+] is crucial to cell survival, because mCa2+ overload during ischemia-reperfusion can lead to mitochondrial permeability transition and apoptosis (17).
The mechanisms controlling mCa2+ handling are poorly understood. Our experiments confirmed the findings of others regarding the increased uniporter activity in the presence of rising extramitochondrial [Ca2+] (13). An increase in extra-mitochondrial [Ca2+] from 0.2 µM to 1.0 µM caused a 12-fold increase in mCa2+ uptake in our experiments, demonstrating the important relationship between intra- and extramitochondrial [Ca2+]. Kirichok et al. (19) recently reported a number of novel findings regarding uniporter physiology. Their studies using patch-clamped mitoplasts demonstrated that the uniporter is an inwardly rectifying Ca2+ channel with high selectivity and that there does not appear to be a direct mechanism for Ca2+-dependent inhibition of the Ca2+ current generated by the uniporter. Thus Ca2+ current through the uniporter can increase until it becomes saturated at extra-mitochondrial Ca2+ concentrations of 100120 mM. Furthermore, although the density of the uniporter channels in the inner mitochondrial membrane appeared to be less than that of voltage-gated Ca2+ channels in the cell membrane, their studies showed that the Ca2+ currents generated by the uniporter at micromolar concentrations of cytosolic Ca2+ are comparable with those generated by voltage-gated Ca2+ channels at millimolar Ca2+ concentrations. This high conductivity is dependent on m
, whereas the selectivity appears to be mediated by a high-affinity Ca2+ binding site on the uniporter.
Attenuation of mCa2+ uptake in the face of increased cytosolic Ca2+ decreases apoptosis (20), and inhibition of mCa2+ uptake with ruthenium red in intact hepatocytes significantly reduces cytochrome c release after ischemia/reperfusion (14). In our experiments, U-73122 dose-dependently blocked mCa2+ uptake at extramitochondrial Ca2+ concentrations up to 20 times higher than physiological levels. This suggests that PLC-
1 is essential for mCa2+ uptake not only under physiological conditions, but also during potent uniporter stimulation by potentially toxic levels of cytosolic Ca2+. Although PLC-
1 is the only isoform we were able to clearly identify in mitochondria, we acknowledge that U-73122 is not specific to PLC-
1 and can effectively bind and inactivate all isoforms of PLC (6, 9, 30, 40). The fact that U-73122 is membrane permeable was important to this study, because permeabilizing the mitochondria to deliver an inhibitor would have destroyed m
.
Spermine has been widely used as a uniporter agonist (28). Stimulation of the uniporter by spermine was also sensitive to PLC inhibition in our experimental conditions, implicating a direct involvement of PLC-
1 in uniporter activity. Interestingly, spermine is also known to stimulate PLC-
1 activity. Because the mechanism of spermines uniporter stimulation is unknown, this raises the possibility that spermine increases mCa2+ uptake by stimulating PLC-
1 activity rather than by directly stimulating uniporter activity.
We found mitochondrial PLC-
1 to be associated with mitochondrial membranes, which is consistent with the fact that the ligand and signal transduction interactions of this enzyme, specifically I(1,4,5)P3 formation, take place at a membrane surface (2, 45, 50). The I(1,4,5)P3 receptor antagonists 2-APB and XeC both inhibit mCa2+ uptake, suggesting a possible role for I(1,4,5)P3 or an I(1,4,5)P3-like receptor in the mechanism controlling mCa2+ uptake. In the cell, 2-APB and XeC disrupt the signaling cascade responsible for I(1,4,5)P3-induced Ca2+ release from the endoplasmic reticulum. I(1,4,5)P3 receptors on the endoplasmic reticulum are ligand-gated Ca2+ channels, and XeC and 2-APB do not interfere with the ability of I(1,4,5)P3 to bind to the I(1,4,5)P3 receptor (10, 31). Their mechanism of action may therefore be allosteric I(1,4,5)P3 receptor inhibition or physical blockade of the calcium pore formed by the I(1,4,5)P3 receptor (10). Thus the effects of 2-APB and XeC on mCa2+ uptake can be interpreted in two different ways. The uniporters ability to function as a Ca2+ channel could be regulated by I(1,4,5)P3 via an I(1,4,5)P3 receptor-like site on the uniporter. This would require that the uniporter contain sequences similar to 2-APBs and XeCs allosteric inactivation sites on the I(1,4,5)P3 receptors in the endoplasmic reticulum, if such sites and mechanisms exist. Conversely, Gafni et al. (10) provide evidence that on the endoplasmic reticulum, XeC most likely acts by binding to and blocking the Ca2+ pore formed by the activated I(1,4,5)P3 receptor. If this is the case, the Ca2+ pore formed by the activated uniporter could be blocked in the same manner. Because both the uniporter and I(1,4,5)P3 receptor are Ca2+ pore-forming molecules, the latter explanation seems more likely.
Mitochondria sense and respond to changes in cytosolic Ca2+ that are frequently controlled by PLC-mediated I(1,4,5)P3 production. Because 2-APB and XeC prevented mCa2+ uptake, it is interesting to consider an I(1,4,5)P3 binding site on the outer mitochondrial membrane or on the exterior surface of the inner mitochondrial membrane (either on the uniporter itself or some regulatory protein), allowing mitochondria to respond directly to cytosolic I(1,4,5)P3. I(1,4,5)P3 and adenophostin A are membrane impermeable and are thus unable to bind to any site inside the mitochondrial matrix or on the interior surface of the inner mitochondrial membrane. They may or may not be able to penetrate the outer mitochondrial membrane. Neither I(1,4,5)P3 nor adenophostin A had any effect on mCa2+ uptake, suggesting that if a role exists for I(1,4,5)P3 signaling in mCa2+ uptake, then I(1,4,5)P3 must be generated through machinery located inside the mitochondria. The necessary precursor molecule PIP2 is also associated with both the inner and outer mitochondrial membrane (48).
The effects of 2-APB and XeC suggest that PLCs hydrolytic activity is involved in mCa2+ uptake. As PLC hydrolyzes PIP2 to I(1,4,5)P3 and DAG, the increasing concentration of DAG slows the rate of PIP2 hydrolysis, thus decreasing I(1,4,5)P3 production (11, 43). OAG is analogous to DAG in its inhibition of PLC hydrolytic activity (11, 43). The inhibitory effects of OAG and DAG-lactone on mCa2+ in our experiments further support a role for PLC activity in mCa2+ uptake. It is also possible that our observations using DAG analogs were due to activation of protein kinase C, although such pathways have only been described in intact cells (27).
In summary, PLC-
1 exists in liver mitochondria and regulates ruthenium red-sensitive mCa2+ uptake. Basal levels of PLC-
1 activity are required for uniporter activity under physiological conditions, highlighting the central role of PLC-
1 in normal mCa2+ uptake. I(1,4,5)P3 receptor antagonists 2-APB and XeC block mCa2+ uptake, suggesting either a role for internal mitochondrial I(1,4,5)P3 signaling or a biophysical similarity between the uniporter and the I(1,4,5)P3 receptors on the endoplasmic reticulum. In the presence of potentially toxic concentrations of extramitochondrial Ca2+ that normally cause permeability transition, inhibition of mitochondrial PLC-
1 completely blocks mCa2+ uptake. Mitochondrial PLC-
1 therefore represents a novel target in the study of the relationship between mitochondrial and cellular physiology in the liver.
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GRANTS
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This work was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-593901 (to R. S. Chari) and National Cancer Institute Grant CA-6848507 (to R. S. Chari).
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ACKNOWLEDGMENTS
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This work received the Research Award from the American Association for the Study of Liver Disease (AASLD) and was presented, in part, at the AASLD meeting in Boston, MA, October 2428, 2003.
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FOOTNOTES
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Address for reprint requests and other correspondence: R. S. Chari, Dept. of Surgery, Vanderbilt Univ. Medical Center, Nashville, TN 37232-4753 (E-mail: ravi.chari{at}vanderbilt.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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