Pregnancy-Specific Modulatory Role of Mitochondria on Adenosine 5'-Triphosphate-Induced Cytosolic [Ca2+] Signaling in Uterine Artery Endothe
http://www.100md.com
《内分泌学杂志》
Perinatal Research Laboratories, Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, Wisconsin 53715
Abstract
Vascular endothelial cells respond to extracellular ATP by inositol 1,4,5-trisphosphate-mediated Ca2+ release from the endoplasmic reticulum followed by Ca2+ influx and subsequent synthesis of vasodilators. In this study, the contribution of mitochondria in shaping the ATP-induced Ca2+ increase was examined in ovine uterine artery endothelial cells from nonpregnant and pregnant (late gestation) ewes (NP- and P-UAEC, passage 4). The mitochondrial protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced a rapid mitochondrial depolarization. CCCP also slowly increased cytosolic [Ca2+] ([Ca2+]c), which then gradually declined to 10–20 nM above resting level. Pretreatment with CCCP for 30 min significantly inhibited both ATP and thapsigargin-induced [Ca2+]c, with inhibition in NP-UAEC more effective than in P-UAEC. Pretreatment of mitochondrial permeability transition pore inhibitor cyclosporine A did not affect CCCP-induced mitochondrial depolarization, but delayed CCCP-induced [Ca2+]c for about 12–15 min (we termed this the "window of time"). During the cyclosporine A-delayed window of time of CCCP-induced [Ca2+]c, ATP induced a normal Ca2+ response, but after this window of time, ATP-induced [Ca2+]c was significantly inhibited. Pretreatment of oligomycin B to prevent intracellular ATP depletion by F0F1-ATPase did not reduce the inhibition of ATP-induced [Ca2+]c by CCCP. Ruthenium red, a mitochondrial Ca2+ uptake blocker, did not mimic the inhibition of Ca2+ signaling by CCCP. In conclusion, our data show that mitochondrial Ca2+ depletion after dissipation of mitochondrial membrane potential with CCCP inhibits ATP-induced [Ca2+]c, mediated at the level of Ca2+ release from the endoplasmic reticulum. Moreover, our data revealed that P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c than NP-UAEC.
Introduction
PREGNANCY IS ASSOCIATED with dramatic increases in uterine blood flow to meet the continually increasing needs of the growing fetus. The importance of this adaptive response is shown by the observation that direct impairment of uterine arterial flow results in intrauterine growth retardation and low birth weight, which in turn correlates with neonatal morbidity (1, 2, 3). The endothelium-derived vasodilator molecules nitric oxide (NO) and prostaglandin I2 (PGI2) are critically involved in the dramatic increase in uterine blood flow that occurs during pregnancy (4). Pregnancy-specific changes in uterine artery (UA) endothelial function are not only dependent on tonic stimuli (such as estrogen), but also are both programmed at the level of cell signaling and are retained in culture (4, 5, 6, 7). Thus, we have developed a unique cell model that retains functional differences between UA endothelial cells (UAEC) from healthy nonpregnant (NP) and pregnant (P) late-term ewes (8). After culture to passage 4, the P-UAEC still show increased NO and PGI2 production over the NP-UAEC in response to agonists (8, 9), which is associated with an increased activation of signaling kinases, and a more sustained and oscillatory nature of the Ca2+ response (7, 9, 10). Because activation of both endothelial NO synthase and cytosolic phospholipase A2 is dependent at least in part on cytosolic [Ca2+] ([Ca2+]c) (11), such changes in [Ca2+]c may well underlie in part the pregnancy-specific increase in NO and PGI2 production.
Many studies have shown that shear stress can release ATP from endothelial cells (12, 13, 14). Extracellular ATP binds to and activates P2Y receptors. P2Y purinoceptors, in turn, are coupled to G proteins, the majority of which activate phospholipase C, leading to the production of inositol 1,4,5-trisphosphate (InsP3) and the subsequent release of Ca2+ from endoplasmic reticulum (ER) (15). The 20- to 50-fold increase in uterine blood flow during pregnancy results in a dramatic rise in shear stress (4), so released ATP may be all the more important as a physiological vasodilator during pregnancy.
The main intracellular Ca2+ store is the ER, but mitochondria also take up and release Ca2+ very efficiently and are often strategically located close to Ca2+ sources (16, 17, 18, 19). Although it has been known for many years that mitochondria can transport Ca2+, recent evidence has suggested that these organelles are important physiological modulators for intracellular Ca2+ signaling (20). Apart from shaping global Ca2+ signals by acting as simple passive-uptake slow-release buffers in the bulk cytoplasm (18), the intimate connection with ER allows mitochondria to shape Ca2+ signals (21) by modulating the release of Ca2+ from the ER (22) and the influx of Ca2+ across the plasma membrane (23), or by providing a local source of Ca2+ for ER refilling (24). Dysregulation of mitochondrial Ca2+ homeostasis is now recognized to play a key role in several pathologies. For example, Ca2+ overload mitochondrial matrix can lead to enhanced generation of reactive oxygen species, triggering of the permeability transition pore, and cytochrome c release, leading to apoptosis (25).
In view of this and our former data on Ca2+ signaling adaptation in UAEC from P vs. NP ewes, we investigated the role of the mitochondria in shaping the Ca2+ response in UAEC and whether the role changes in pregnancy. We found that indeed mitochondria do play a role and, further, this role is altered in pregnancy. The details of these findings and the implications to both normal pregnancy and disease are discussed.
Materials and Methods
Materials
ATP (disodium salt), carbonyl cyanide m-chlorophenylhydrazone (CCCP), cyclosporine A (CsA), and oligomycin B were purchased from Sigma (St. Louis, MO). BAPTA-AM, LY294002, CaCl2, and thapsigargin were purchased from Calbiochem (San Diego, CA). Fura 2-AM and rodamine 123 were obtained from Molecular Probes (Eugene, OR). Unless noted otherwise, MEM D-Val, and all other cell culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY). Thirty-five-millimeter dishes with glass coverslip windows for [Ca2+]c imaging studies were purchased from MatTek (Ashland, MA).
Isolation of UAECs
Uterine arteries were obtained from Polypay and mixed Western breed NP sheep (n = 4) and P ewes at 120–130 d of gestation (n = 6) during nonsurvival surgery, as described previously (7, 8). Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison Research Animal Care Committees of both the Medical School and the College of Agriculture and Life Sciences and follow the recommended guidelines of The American Veterinary Medical Association for humane treatment and euthanasia of laboratory farm animals. Briefly, primary uterine arteries were flushed free of blood and digested with collagenase. Freshly isolated endothelial cells (passage 0) were plated to 35-mm dishes in MEM. Cells were then grown and passaged to approximately 70% confluence in T75 flasks, at which point they were passaged once more (passage 3) to medium containing 10% dimethylsulfoxide and frozen in liquid nitrogen for long-term storage. Cells at passage 3 from four separate animals each were thawed and grown to passage 4, combined, and then split 1:8 before freezing with 10% dimethylsulfoxide (Sigma) for later plating at lower density as required. Note in test studies, the data from these cells were indistinguishable from our previously published data on prepassage 4 cells (8).
Fura 2 Ca2+ imaging studies
UAEC were plated to 35-mm dishes with glass coverslip windows and grown to 60–70% confluence. Immediately before use, cells were loaded with 5 μM Fura 2-AM (Molecular Probes) in Krebs buffer (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM KH2PO4, 6 mM glucose, 25 mM HEPES, 2 mM CaCl2, pH 7.4) for 45 min. The cells were then rinsed three times and covered with 2 ml prewarmed (37 C) Krebs buffer and incubated a further 30 min to complete ester hydrolysis. Cells were washed once more and covered with 1 ml Krebs buffer, and Fura 2 loading was verified by viewing at 380 nm UV excitation on a Nikon inverted microscope (Nikon, Melville, NY). Approximately 20 cells were then set in the field of view and recordings commenced, using alternate excitation at 340 and 380 nm at 50-msec intervals, and measuring emitted light using a photomultiplier. From the ratio of emission at 510 nm detected at the two excitation wavelengths, and by comparison to a standard curve established for the same settings using buffers of known free [Ca2+], the [Ca2+]c was then calculated in real time using the InCyt Pm2 software (Intracellular Imaging, Inc., Cincinnati, OH).
Determination of mitochondrial membrane potential ()
The cells (passage 4) grown on 35-mm dishes with glass coverslip windows were loaded with 2.5 μg/ml rhodamine 123 (Rh123; Molecular Probes) for 30 min at 37 C. Rh123 is taken up selectively by mitochondria, and its uptake is dependent of (27). The lipophilic dye Rh123 is cationic and is accumulated in the negatively charged mitochondrial matrix where it is partially quenched (28, 29). When the mitochondria depolarize, the dye is dequenched. This is observed as an increase in the fluorescence of the cell under microfluoroscopic analysis (28, 29).
Rh123 fluorescence is excited at 485 nm, and emission at 535 nm was measured using a photomultiplier connected to a fluorescence microscope (Diaphot 150; Nikon). The fluorescent intensities are quantified using InCyt Pm1 software (Intracellular Imaging, Inc.). In isolated mitochondria, the relationship between Rh123 fluorescence and is linear at a range of 55–220 mV (30). The Rh123 fluorescence, which is quenched at resting , increases with mitochondrial membrane depolarization.
Statistical analysis
For each experiment, fluorescent signal was averaged from approximately 20 cells. Data are typically from 6–10 separate dishes/independent experiments and are presented as means ± SE. Data were analyzed by Student’s t test and ANOVA, as appropriate. A value of P < 0.05 was considered statistically significant.
Results
CCCP-induced depolarization of mitochondria in UAEC
The mitochondrial matrix normally is negative with respect to the cell cytoplasm, with the across the inner membrane ranging from –90 to –160 mV (31, 32). To confirm that the protonophore CCCP depolarized mitochondria in UAEC, cells were loaded with Rh123 and examined using fluorescence microscope.
As shown in Fig. 1A, the addition of CCCP (2 μM) caused an immediate increase in Rh123 fluorescence in P-UAEC, which was gradually decreased to a level that is still much higher than basal level, and then gradually returned to the peak level. Pretreatment of CsA, a potent inhibitor of the mitochondrial permeability transition pore (MPTP) (33), for 5 min did not prevent the CCCP-induced loss of (Fig. 1B). Similar results were obtained with NP-UAEC (not shown). CCCP did not lead to any detectable changes in cell shape or loss of cell viability for up to several hours (not shown).
CCCP-induced release of Ca2+ from mitochondria
Mitochondrial Ca2+ uptake is driven by the negative mitochondrial potential, which is provided by the transmembrane electrochemical proton gradient (34). Depolarized mitochondria lose the potential gradient necessary for storing Ca2+, and any sequestered Ca2+ is then released (35). CCCP dissipates both the proton and electrical potential gradients and thus releases Ca2+ from mitochondria, preventing further uptake of the ion by the organelle. As shown in Fig. 2, upon addition of CCCP to the bath solution, [Ca2+]c increased gradually in 2 min to a level about 40–50 nM higher than resting level, and then decreased gradually in 20 min to a basal level that is about 10–20 nM higher than resting level.
Pretreatment of CsA for 5 min delayed the CCCP-induced increase in [Ca2+]c for about 12–15 min, confirming that the rise of [Ca2+]c in response to CCCP was initially due to release from MPTP. These data indicate that Ca2+ release from mitochondria by depolarizing is primarily through the opening of MPTP. Because the pore was blocked with CsA, other mechanisms (putative Ca2+/H+ antiport, for example) may contribute to the delayed release of Ca2+ from mitochondria.
Inhibitory effect of CCCP on ATP-induced [Ca2+]c in NP- and P-UAEC
Cells loaded with the Ca2+-sensitive fluorescent dye Fura 2 were exposed to the InsP3-generating agonist ATP (100 μM). Figure 3A shows representative recordings of ATP-induced [Ca2+]c in the P-UAEC. ATP induced a rapid increase in [Ca2+]c, which peaked within 20 sec and was followed by a sustained phase. After ATP washout for 20 min, ATP-induced [Ca2+]c could be totally recovered, which makes it possible to challenge the cell with ATP for a second time. Pretreatment of 2 μM CCCP for 30 min significantly inhibited ATP-induced [Ca2+]c in P-UAEC (Fig. 3A). As summarized in Fig. 3B, there are no significant differences between the ATP-induced maximal increase in [Ca2+]c in P- and NP-UAEC, and CCCP inhibited ATP-induced [Ca2+]c by 57% in P-UAEC but by 83% in NP-UAEC, respectively. These results suggest that, although the initial Ca2+ release is similar in P-UAEC and NP-UAEC, P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c.
Inhibitory effect of CCCP on thapsigargin-induced [Ca2+]c in NP- and P-UAEC
We next used thapsigargin, an inhibitor of the ER Ca2+-ATPase (36), to release Ca2+ from ER. Figure 4A shows representative recordings of thapsigargin-induced [Ca2+]c in P-UAEC. Pretreatment with 2 μM CCCP inhibited the thapsigargin-induced [Ca2+]c in P-UAEC. As shown in Fig. 4B, there are no significant differences between thapsigargin-induced maximal increase in [Ca2+]c in P- and NP-UAEC, and CCCP inhibited thapsigargin-induced [Ca2+]c by 51% in P-UAEC but by 69% in NP-UAEC, respectively. These results suggest that CCCP inhibited thapsigargin-induced [Ca2+]c in both P- and NP-UAEC; the extent in NP-UAEC is again more significant than that in P-UAEC.
Effects of CsA on CCCP-inhibited [Ca2+]c in response to ATP in P-UAEC
We investigated the potential mechanisms that underlie the inhibition of Ca2+ signaling after dissipation of with CCCP. The possibility that release of Ca2+ from mitochondria contributes to a CCCP-induced inhibitory effect on Ca2+ was examined. As shown in Fig. 5B, ATP was added to the bath solution 200 sec after CCCP (at this time CCCP-increased [Ca2+]c was still high); ATP-induced [Ca2+]c was significantly inhibited. Because the MPTP inhibitor, CsA, delayed the CCCP-induced Ca2+ release from mitochondria (Fig. 2), ATP was added during or after this window of time. As shown in Fig. 5C, P-UAEC were pretreated with CsA for 5 min before CCCP to block MTMP, and ATP was added to bath solution 200 sec after CCCP. Under this condition, ATP still induced a [Ca2+]c response similar to that under control condition (summarized in Fig. 6). However, when ATP was added after the delayed [Ca2+]c, ATP-induced [Ca2+]c was significantly inhibited (Fig. 6).
No direct involvement of ATP depletion in CCCP-inhibited [Ca2+]c response
F0F1-ATPase is the "universal" enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. F0F1-ATPase can also reverse itself and hydrolyze ATP to pump protons against an electrochemical gradient after dissipation of the proton gradient (37). Thus, dissipation of with CCCP should lead to local depletion of ATP. Therefore, one possible consequence of mitochondrial depolarization is reduction of intracellular ATP concentration ([ATP]i). Because ATP is known to enhance InsP3R activation (38), and ER Ca2+-ATPases are regulated by the ATP to ADP ratio, such a decrease in [ATP]i in the local area around ER could have contributed to the inhibition of Ca2+ signaling in cells after mitochondrial depolarization. To ensure that CCCP did not inhibit Ca2+ signaling, by reduction of local ATP levels, cells were pretreated with oligomycin B (5 μM), to block the mitochondrial F0F1-ATPase, before addition of CCCP. As shown in Fig. 6, pretreatment of oligomycin B did not reduce the ability of CCCP to inhibit the ATP-evoked Ca2+. Moreover, as shown in Figs. 1, 2, and 5, CsA did not prevent the CCCP-induced loss of , but delayed CCCP-induced Ca2+ release. In other words, CsA did not prevent CCCP-induced ATP depletion, but prevented CCCP-induced inhibition of [Ca2+]c in response to ATP during this window of time. Theoretically, prolonged incubation with CCCP will progressively decrease the local ATP level, so if the inhibition of Ca2+ signaling by CCCP is due to depletion of ATP, prolonged incubation with CCCP will result in an increased degree of inhibition of Ca2+. However, when cells were incubated with CCCP for 60 min before ATP application, the cells responded in a similar manner to those treated with CCCP for 30 min (data not shown). As shown in Fig. 5B, even 200 sec of pretreatment of CCCP resulted in significant inhibition of ATP-induced Ca2+ response. Collectively, these results indicate that intracellular ATP depletion did not underlie CCCP-induced inhibition of Ca2+ signaling.
Effects of mitochondrial Ca2+ uptake blocker on ATP-induced [Ca2+]c
Because mitochondrial potential is the driving force for mitochondrial Ca2+ uptake, CCCP should prevent mitochondrial Ca2+ uptake. It is possible that blocking mitochondrial Ca2+ uptake may mediate CCCP-induced inhibition of Ca2+ signaling. To test this hypothesis, we established the experimental conditions that allow a selective blockade of mitochondrial Ca2+ uptake in UAEC. For this we used ruthenium red, a blocker of the mitochondrial uniporter (22). Fig. 7A illustrates that 5 μM ruthenium red did not inhibit ATP-induced [Ca2+]c in P-UAEC; these results are summarized in Fig. 7B. These data indicate that the blockade of mitochondrial Ca2+ uptake is not involved in CCCP-induced inhibition of Ca2+ signaling.
Discussion
To control Ca2+-dependent intracellular processes, cells have developed a variety of mechanisms that regulate [Ca2+]c. Mitochondria are clearly important in the regulation of [Ca2+]c in many cells, not only in pathological but also physiological conditions (39), and the specific nature of this regulation is cell-specific. For example, mitochondrial depolarization accelerates InsP3-mediated Ca2+ waves in cortical astrocytes (20), but blocks InsP3-evoked Ca2+ release in colonic smooth muscle cells (40).
Endothelial cells play an important role in modulating vascular resistance and blood flow through their abilities to produce vasodilators such as NO and PGI2. This is most apparent during late pregnancy, when enhanced elevation of [Ca2+]c in UA endothelium in response to agonists in part leads to enhanced production of both NO and PGI2. Herein we address the possibility that an altered agonist (ATP)-induced [Ca2+]c response in UA endothelium between the NP- and P- state may in fact be mediated through changes at the level of the mitochondria.
Protonophores, such as CCCP, which collapse , are widely used to inhibit mitochondrial Ca2+ handling. In the present study, we observed that mitochondrial depolarization with CCCP significantly reduced the ATP-evoked [Ca2+]c in UAEC. There was no significant difference between ATP-induced peak increase in [Ca2+]c in P- and NP-UAEC, but CCCP was more efficient to inhibit ATP-induced [Ca2+]c in NP-UAEC than P-UAEC, confirming an altered role for mitochondria in pregnancy. To investigate the effects of CCCP at the level of ER, we examined the response of cells to thapsigargin, a potent and selective inhibitor of the ER Ca2+-ATPase that releases Ca2+ from ER by preventing Ca2+ uptake back to ER (36). The ability of CCCP to significantly inhibit thapsigargin-induced [Ca2+]c indicates that the inhibition of ATP-induced Ca2+ signaling by CCCP mainly occurred at the level of the ER by preventing Ca2+ release. Similar to the experiments with ATP, thapsigargin-induced [Ca2+]c was inhibited by CCCP pretreatment to a much greater degree in NP-UAEC than P-UAEC. This suggests that pregnancy-induced adaptation in Ca2+ signaling includes changes at the level of the mitochondria, and that P-UAEC has a greater capacity to resist the inhibitory effect of CCCP on agonist-stimulated [Ca2+]c.
Investigating the protective mechanisms in the normal P-UA may allow us to understand the pathophysiological etiology of preeclampsia, a condition in part associated with impaired Ca2+ signaling at least in hand vein endothelial cells (5). It is not yet clear whether the lack of such pregnancy-specific changes in endothelial [Ca2+]c signaling is due to failed adaptation at the level of the mitochondria. Nonetheless, two recent clinical reports directly implicate abnormal mitochondrial function with the preeclamptic condition (41, 42). For this reason, it is important to first know more about the molecular basis for the normal pregnancy-specific change in endothelial cell mitochondrial function before proceeding to investigate pathological pregnancy states. To that end, we used P-UAEC to further investigate the potential mechanisms underlying the inhibition of Ca2+ signaling after mitochondrial dysfunction as a result of CCCP pretreatment. Because pretreatment with CCCP could result in mitochondrial depolarization, opening of MPTP, release of Ca2+ from MPTP, depletion of local [ATP]i, change in cytoplasmic pH, and/or the blockade of mitochondria Ca2+ uptake, all were examined in the present study.
Mitochondrial Ca2+ uptake is driven by the negative mitochondrial potential, which is provided by the transmembrane electrochemical proton gradient (34). Depolarized mitochondria lose the potential gradient necessary for storing Ca2+, and any sequestered Ca2+ is then released (35). Thus, we examined the role of depletion of mitochondria Ca2+ store on ATP-induced [Ca2+]c. Indeed, CCCP application resulted in a increase in [Ca2+]c. This increase in [Ca2+]c by CCCP could be completely inhibited within 12–15 min by pretreatment of MPTP inhibitor CsA, indicating that MPTP opening was the primary mechanism of Ca2+ release from mitochondria. Because the pore was blocked with CsA, other mechanisms (putative Ca2+/H+ antiport, for example) may contribute to the delayed release of Ca2+ from mitochondria. During the CsA-created window of time, ATP could still induce a [Ca2+]c response similar to that under control conditions. However, when ATP was added after the delayed [Ca2+]c, ATP-induced [Ca2+]c was significantly inhibited. Collectively, these results indicate that depletion of Ca2+ from mitochondria results in inhibition of ATP-induced [Ca2+]c, and that the basal level of mitochondrial Ca2+ is necessary for normal ER function. Unlike Ca2+, some other mitochondrial factors, such as cytochrome c, can be only released through MPTP (25). Because MPTP was inhibited by CsA, this minimized the possibility that these factors also mediated CCCP-inhibited Ca2+ signaling.
The most direct effect of CCCP is mitochondrial depolarization. In the present study, ATP responded normally during the CsA-created window of time, whereas CCCP-induced mitochondrial membrane depolarization was not affected by CsA. These results suggest that mitochondrial depolarization itself does not directly inhibit ATP-induced [Ca2+]c, which is in contrast with reports showing that mitochondrial depolarization directly inhibits InsP3-induced Ca2+ release in HeLa cells (43).
Because CCCP is a protonophore, another obvious effect of CCCP application would be a change in cytoplasmic pH, which is known to modulate InsP3-induced Ca2+ release in some cells (44). However, such a change in cytoplasmic pH did not underlie the inhibition of Ca2+ release in UAEC, because CsA did not prevent the CCCP-induced loss of , but delayed CCCP-induced Ca2+ release from mitochondria.
Dissipation of with CCCP should lead to the inhibition of mitochondrial ATP synthesis due to the lack of a proton gradient. In addition, during CCCP treatment, the F0F1-ATPase could run in the reverse direction and rapidly consume ATP (37). Therefore, one possible consequence of mitochondrial depolarization is a reduction of local [ATP]i level around the ER. Because ATP is known to enhance InsP3R activation (38), such a decrease in [ATP]i in the vicinity of ER could have contributed to the inhibition of Ca2+ signaling in cells after CCCP treatment. However, 1) pretreatment of F0F1-ATPase inhibitor oligomycin B (5 μM) did not reduce the inhibition of ATP-induce [Ca2+]c by CCCP; 2) CsA did not prevent the CCCP-induced loss of , but delayed CCCP-induced Ca2+ release, and extracellular ATP responded normally during this window of time; 3) prolonged incubation with CCCP did not result in progressively increased degree of inhibition of Ca2+. These results indicate that the inhibition of Ca2+ signaling by depletion of mitochondrial Ca2+ with CCCP in UAEC is unlikely to be explained simply by changes in intracellular ATP levels. Our results are consistent with the studies with smooth muscle cell (40) and HeLa cell (43).
Mitochondria rapidly take up Ca2+ from the cytosol via a ruthenium red-sensitive uniporter spanning the inner mitochondrial membrane. This electrogenic passive uptake is driven by the enormous negative potential across the inner mitochondrial membrane, a consequence of proton extrusion along the electron transport chain. Thus mitochondria may shape intracellular Ca2+ signals, both spatially and temporally, as a direct consequence of their rapid Ca2+ buffering capacity (26). Mitochondrial depolarization leads to blockade of Ca2+ uptake, so the mitochondria are no longer available to act as a sink for ER-released Ca2+, which reflect the positive feedback effects of Ca2+ on the InsP3R (22). However, the observation that blockade of the Ca2+ uniporter by ruthenium red did not mimic CCCP-induced inhibition of Ca2+ signaling excluded the possibility that blockage of Ca2+ uptake mediate the inhibition of Ca2+ by CCCP.
In summary, we found that mitochondrial Ca2+ depletion after dissipation of with CCCP inhibits ATP-induced [Ca2+]c, and this inhibition occurred at ER level by inhibiting Ca2+ release from ER. Change in cytoplasmic pH, depletion of local ATP, and blockade of mitochondria Ca2+ uptake did not underlie the inhibition of ATP-induced Ca2+ release in UAEC. Moreover, our data revealed that P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c than NP-UAEC. Further studies are needed to explore the mechanism of pregnancy-specific resistance to mitochondrial dysfunction and its associated adverse effect on Ca2+ signaling.
Footnotes
This work was supported by National Institutes of Health Grants HL64601 (to I.M.B.), HD 38843 (Project 1 to I.M.B.), and HD050578 (to F.-X.Y.).
Abbreviations: [ATP]I, Intracellular ATP concentration; [Ca2+]c, cytosolic [Ca2+]; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CsA, cyclosporine A; , mitochondrial membrane potential; ER, endoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; MPTP, mitochondrial permeability transition pore; NO, nitric oxide; NP, nonpregnant; P, pregnant; PGI2, prostaglandin I2; Rh123, rhodamine 123; UA, uterine artery; UAEC, UA endothelial cell.
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Abstract
Vascular endothelial cells respond to extracellular ATP by inositol 1,4,5-trisphosphate-mediated Ca2+ release from the endoplasmic reticulum followed by Ca2+ influx and subsequent synthesis of vasodilators. In this study, the contribution of mitochondria in shaping the ATP-induced Ca2+ increase was examined in ovine uterine artery endothelial cells from nonpregnant and pregnant (late gestation) ewes (NP- and P-UAEC, passage 4). The mitochondrial protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced a rapid mitochondrial depolarization. CCCP also slowly increased cytosolic [Ca2+] ([Ca2+]c), which then gradually declined to 10–20 nM above resting level. Pretreatment with CCCP for 30 min significantly inhibited both ATP and thapsigargin-induced [Ca2+]c, with inhibition in NP-UAEC more effective than in P-UAEC. Pretreatment of mitochondrial permeability transition pore inhibitor cyclosporine A did not affect CCCP-induced mitochondrial depolarization, but delayed CCCP-induced [Ca2+]c for about 12–15 min (we termed this the "window of time"). During the cyclosporine A-delayed window of time of CCCP-induced [Ca2+]c, ATP induced a normal Ca2+ response, but after this window of time, ATP-induced [Ca2+]c was significantly inhibited. Pretreatment of oligomycin B to prevent intracellular ATP depletion by F0F1-ATPase did not reduce the inhibition of ATP-induced [Ca2+]c by CCCP. Ruthenium red, a mitochondrial Ca2+ uptake blocker, did not mimic the inhibition of Ca2+ signaling by CCCP. In conclusion, our data show that mitochondrial Ca2+ depletion after dissipation of mitochondrial membrane potential with CCCP inhibits ATP-induced [Ca2+]c, mediated at the level of Ca2+ release from the endoplasmic reticulum. Moreover, our data revealed that P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c than NP-UAEC.
Introduction
PREGNANCY IS ASSOCIATED with dramatic increases in uterine blood flow to meet the continually increasing needs of the growing fetus. The importance of this adaptive response is shown by the observation that direct impairment of uterine arterial flow results in intrauterine growth retardation and low birth weight, which in turn correlates with neonatal morbidity (1, 2, 3). The endothelium-derived vasodilator molecules nitric oxide (NO) and prostaglandin I2 (PGI2) are critically involved in the dramatic increase in uterine blood flow that occurs during pregnancy (4). Pregnancy-specific changes in uterine artery (UA) endothelial function are not only dependent on tonic stimuli (such as estrogen), but also are both programmed at the level of cell signaling and are retained in culture (4, 5, 6, 7). Thus, we have developed a unique cell model that retains functional differences between UA endothelial cells (UAEC) from healthy nonpregnant (NP) and pregnant (P) late-term ewes (8). After culture to passage 4, the P-UAEC still show increased NO and PGI2 production over the NP-UAEC in response to agonists (8, 9), which is associated with an increased activation of signaling kinases, and a more sustained and oscillatory nature of the Ca2+ response (7, 9, 10). Because activation of both endothelial NO synthase and cytosolic phospholipase A2 is dependent at least in part on cytosolic [Ca2+] ([Ca2+]c) (11), such changes in [Ca2+]c may well underlie in part the pregnancy-specific increase in NO and PGI2 production.
Many studies have shown that shear stress can release ATP from endothelial cells (12, 13, 14). Extracellular ATP binds to and activates P2Y receptors. P2Y purinoceptors, in turn, are coupled to G proteins, the majority of which activate phospholipase C, leading to the production of inositol 1,4,5-trisphosphate (InsP3) and the subsequent release of Ca2+ from endoplasmic reticulum (ER) (15). The 20- to 50-fold increase in uterine blood flow during pregnancy results in a dramatic rise in shear stress (4), so released ATP may be all the more important as a physiological vasodilator during pregnancy.
The main intracellular Ca2+ store is the ER, but mitochondria also take up and release Ca2+ very efficiently and are often strategically located close to Ca2+ sources (16, 17, 18, 19). Although it has been known for many years that mitochondria can transport Ca2+, recent evidence has suggested that these organelles are important physiological modulators for intracellular Ca2+ signaling (20). Apart from shaping global Ca2+ signals by acting as simple passive-uptake slow-release buffers in the bulk cytoplasm (18), the intimate connection with ER allows mitochondria to shape Ca2+ signals (21) by modulating the release of Ca2+ from the ER (22) and the influx of Ca2+ across the plasma membrane (23), or by providing a local source of Ca2+ for ER refilling (24). Dysregulation of mitochondrial Ca2+ homeostasis is now recognized to play a key role in several pathologies. For example, Ca2+ overload mitochondrial matrix can lead to enhanced generation of reactive oxygen species, triggering of the permeability transition pore, and cytochrome c release, leading to apoptosis (25).
In view of this and our former data on Ca2+ signaling adaptation in UAEC from P vs. NP ewes, we investigated the role of the mitochondria in shaping the Ca2+ response in UAEC and whether the role changes in pregnancy. We found that indeed mitochondria do play a role and, further, this role is altered in pregnancy. The details of these findings and the implications to both normal pregnancy and disease are discussed.
Materials and Methods
Materials
ATP (disodium salt), carbonyl cyanide m-chlorophenylhydrazone (CCCP), cyclosporine A (CsA), and oligomycin B were purchased from Sigma (St. Louis, MO). BAPTA-AM, LY294002, CaCl2, and thapsigargin were purchased from Calbiochem (San Diego, CA). Fura 2-AM and rodamine 123 were obtained from Molecular Probes (Eugene, OR). Unless noted otherwise, MEM D-Val, and all other cell culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY). Thirty-five-millimeter dishes with glass coverslip windows for [Ca2+]c imaging studies were purchased from MatTek (Ashland, MA).
Isolation of UAECs
Uterine arteries were obtained from Polypay and mixed Western breed NP sheep (n = 4) and P ewes at 120–130 d of gestation (n = 6) during nonsurvival surgery, as described previously (7, 8). Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison Research Animal Care Committees of both the Medical School and the College of Agriculture and Life Sciences and follow the recommended guidelines of The American Veterinary Medical Association for humane treatment and euthanasia of laboratory farm animals. Briefly, primary uterine arteries were flushed free of blood and digested with collagenase. Freshly isolated endothelial cells (passage 0) were plated to 35-mm dishes in MEM. Cells were then grown and passaged to approximately 70% confluence in T75 flasks, at which point they were passaged once more (passage 3) to medium containing 10% dimethylsulfoxide and frozen in liquid nitrogen for long-term storage. Cells at passage 3 from four separate animals each were thawed and grown to passage 4, combined, and then split 1:8 before freezing with 10% dimethylsulfoxide (Sigma) for later plating at lower density as required. Note in test studies, the data from these cells were indistinguishable from our previously published data on prepassage 4 cells (8).
Fura 2 Ca2+ imaging studies
UAEC were plated to 35-mm dishes with glass coverslip windows and grown to 60–70% confluence. Immediately before use, cells were loaded with 5 μM Fura 2-AM (Molecular Probes) in Krebs buffer (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM KH2PO4, 6 mM glucose, 25 mM HEPES, 2 mM CaCl2, pH 7.4) for 45 min. The cells were then rinsed three times and covered with 2 ml prewarmed (37 C) Krebs buffer and incubated a further 30 min to complete ester hydrolysis. Cells were washed once more and covered with 1 ml Krebs buffer, and Fura 2 loading was verified by viewing at 380 nm UV excitation on a Nikon inverted microscope (Nikon, Melville, NY). Approximately 20 cells were then set in the field of view and recordings commenced, using alternate excitation at 340 and 380 nm at 50-msec intervals, and measuring emitted light using a photomultiplier. From the ratio of emission at 510 nm detected at the two excitation wavelengths, and by comparison to a standard curve established for the same settings using buffers of known free [Ca2+], the [Ca2+]c was then calculated in real time using the InCyt Pm2 software (Intracellular Imaging, Inc., Cincinnati, OH).
Determination of mitochondrial membrane potential ()
The cells (passage 4) grown on 35-mm dishes with glass coverslip windows were loaded with 2.5 μg/ml rhodamine 123 (Rh123; Molecular Probes) for 30 min at 37 C. Rh123 is taken up selectively by mitochondria, and its uptake is dependent of (27). The lipophilic dye Rh123 is cationic and is accumulated in the negatively charged mitochondrial matrix where it is partially quenched (28, 29). When the mitochondria depolarize, the dye is dequenched. This is observed as an increase in the fluorescence of the cell under microfluoroscopic analysis (28, 29).
Rh123 fluorescence is excited at 485 nm, and emission at 535 nm was measured using a photomultiplier connected to a fluorescence microscope (Diaphot 150; Nikon). The fluorescent intensities are quantified using InCyt Pm1 software (Intracellular Imaging, Inc.). In isolated mitochondria, the relationship between Rh123 fluorescence and is linear at a range of 55–220 mV (30). The Rh123 fluorescence, which is quenched at resting , increases with mitochondrial membrane depolarization.
Statistical analysis
For each experiment, fluorescent signal was averaged from approximately 20 cells. Data are typically from 6–10 separate dishes/independent experiments and are presented as means ± SE. Data were analyzed by Student’s t test and ANOVA, as appropriate. A value of P < 0.05 was considered statistically significant.
Results
CCCP-induced depolarization of mitochondria in UAEC
The mitochondrial matrix normally is negative with respect to the cell cytoplasm, with the across the inner membrane ranging from –90 to –160 mV (31, 32). To confirm that the protonophore CCCP depolarized mitochondria in UAEC, cells were loaded with Rh123 and examined using fluorescence microscope.
As shown in Fig. 1A, the addition of CCCP (2 μM) caused an immediate increase in Rh123 fluorescence in P-UAEC, which was gradually decreased to a level that is still much higher than basal level, and then gradually returned to the peak level. Pretreatment of CsA, a potent inhibitor of the mitochondrial permeability transition pore (MPTP) (33), for 5 min did not prevent the CCCP-induced loss of (Fig. 1B). Similar results were obtained with NP-UAEC (not shown). CCCP did not lead to any detectable changes in cell shape or loss of cell viability for up to several hours (not shown).
CCCP-induced release of Ca2+ from mitochondria
Mitochondrial Ca2+ uptake is driven by the negative mitochondrial potential, which is provided by the transmembrane electrochemical proton gradient (34). Depolarized mitochondria lose the potential gradient necessary for storing Ca2+, and any sequestered Ca2+ is then released (35). CCCP dissipates both the proton and electrical potential gradients and thus releases Ca2+ from mitochondria, preventing further uptake of the ion by the organelle. As shown in Fig. 2, upon addition of CCCP to the bath solution, [Ca2+]c increased gradually in 2 min to a level about 40–50 nM higher than resting level, and then decreased gradually in 20 min to a basal level that is about 10–20 nM higher than resting level.
Pretreatment of CsA for 5 min delayed the CCCP-induced increase in [Ca2+]c for about 12–15 min, confirming that the rise of [Ca2+]c in response to CCCP was initially due to release from MPTP. These data indicate that Ca2+ release from mitochondria by depolarizing is primarily through the opening of MPTP. Because the pore was blocked with CsA, other mechanisms (putative Ca2+/H+ antiport, for example) may contribute to the delayed release of Ca2+ from mitochondria.
Inhibitory effect of CCCP on ATP-induced [Ca2+]c in NP- and P-UAEC
Cells loaded with the Ca2+-sensitive fluorescent dye Fura 2 were exposed to the InsP3-generating agonist ATP (100 μM). Figure 3A shows representative recordings of ATP-induced [Ca2+]c in the P-UAEC. ATP induced a rapid increase in [Ca2+]c, which peaked within 20 sec and was followed by a sustained phase. After ATP washout for 20 min, ATP-induced [Ca2+]c could be totally recovered, which makes it possible to challenge the cell with ATP for a second time. Pretreatment of 2 μM CCCP for 30 min significantly inhibited ATP-induced [Ca2+]c in P-UAEC (Fig. 3A). As summarized in Fig. 3B, there are no significant differences between the ATP-induced maximal increase in [Ca2+]c in P- and NP-UAEC, and CCCP inhibited ATP-induced [Ca2+]c by 57% in P-UAEC but by 83% in NP-UAEC, respectively. These results suggest that, although the initial Ca2+ release is similar in P-UAEC and NP-UAEC, P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c.
Inhibitory effect of CCCP on thapsigargin-induced [Ca2+]c in NP- and P-UAEC
We next used thapsigargin, an inhibitor of the ER Ca2+-ATPase (36), to release Ca2+ from ER. Figure 4A shows representative recordings of thapsigargin-induced [Ca2+]c in P-UAEC. Pretreatment with 2 μM CCCP inhibited the thapsigargin-induced [Ca2+]c in P-UAEC. As shown in Fig. 4B, there are no significant differences between thapsigargin-induced maximal increase in [Ca2+]c in P- and NP-UAEC, and CCCP inhibited thapsigargin-induced [Ca2+]c by 51% in P-UAEC but by 69% in NP-UAEC, respectively. These results suggest that CCCP inhibited thapsigargin-induced [Ca2+]c in both P- and NP-UAEC; the extent in NP-UAEC is again more significant than that in P-UAEC.
Effects of CsA on CCCP-inhibited [Ca2+]c in response to ATP in P-UAEC
We investigated the potential mechanisms that underlie the inhibition of Ca2+ signaling after dissipation of with CCCP. The possibility that release of Ca2+ from mitochondria contributes to a CCCP-induced inhibitory effect on Ca2+ was examined. As shown in Fig. 5B, ATP was added to the bath solution 200 sec after CCCP (at this time CCCP-increased [Ca2+]c was still high); ATP-induced [Ca2+]c was significantly inhibited. Because the MPTP inhibitor, CsA, delayed the CCCP-induced Ca2+ release from mitochondria (Fig. 2), ATP was added during or after this window of time. As shown in Fig. 5C, P-UAEC were pretreated with CsA for 5 min before CCCP to block MTMP, and ATP was added to bath solution 200 sec after CCCP. Under this condition, ATP still induced a [Ca2+]c response similar to that under control condition (summarized in Fig. 6). However, when ATP was added after the delayed [Ca2+]c, ATP-induced [Ca2+]c was significantly inhibited (Fig. 6).
No direct involvement of ATP depletion in CCCP-inhibited [Ca2+]c response
F0F1-ATPase is the "universal" enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. F0F1-ATPase can also reverse itself and hydrolyze ATP to pump protons against an electrochemical gradient after dissipation of the proton gradient (37). Thus, dissipation of with CCCP should lead to local depletion of ATP. Therefore, one possible consequence of mitochondrial depolarization is reduction of intracellular ATP concentration ([ATP]i). Because ATP is known to enhance InsP3R activation (38), and ER Ca2+-ATPases are regulated by the ATP to ADP ratio, such a decrease in [ATP]i in the local area around ER could have contributed to the inhibition of Ca2+ signaling in cells after mitochondrial depolarization. To ensure that CCCP did not inhibit Ca2+ signaling, by reduction of local ATP levels, cells were pretreated with oligomycin B (5 μM), to block the mitochondrial F0F1-ATPase, before addition of CCCP. As shown in Fig. 6, pretreatment of oligomycin B did not reduce the ability of CCCP to inhibit the ATP-evoked Ca2+. Moreover, as shown in Figs. 1, 2, and 5, CsA did not prevent the CCCP-induced loss of , but delayed CCCP-induced Ca2+ release. In other words, CsA did not prevent CCCP-induced ATP depletion, but prevented CCCP-induced inhibition of [Ca2+]c in response to ATP during this window of time. Theoretically, prolonged incubation with CCCP will progressively decrease the local ATP level, so if the inhibition of Ca2+ signaling by CCCP is due to depletion of ATP, prolonged incubation with CCCP will result in an increased degree of inhibition of Ca2+. However, when cells were incubated with CCCP for 60 min before ATP application, the cells responded in a similar manner to those treated with CCCP for 30 min (data not shown). As shown in Fig. 5B, even 200 sec of pretreatment of CCCP resulted in significant inhibition of ATP-induced Ca2+ response. Collectively, these results indicate that intracellular ATP depletion did not underlie CCCP-induced inhibition of Ca2+ signaling.
Effects of mitochondrial Ca2+ uptake blocker on ATP-induced [Ca2+]c
Because mitochondrial potential is the driving force for mitochondrial Ca2+ uptake, CCCP should prevent mitochondrial Ca2+ uptake. It is possible that blocking mitochondrial Ca2+ uptake may mediate CCCP-induced inhibition of Ca2+ signaling. To test this hypothesis, we established the experimental conditions that allow a selective blockade of mitochondrial Ca2+ uptake in UAEC. For this we used ruthenium red, a blocker of the mitochondrial uniporter (22). Fig. 7A illustrates that 5 μM ruthenium red did not inhibit ATP-induced [Ca2+]c in P-UAEC; these results are summarized in Fig. 7B. These data indicate that the blockade of mitochondrial Ca2+ uptake is not involved in CCCP-induced inhibition of Ca2+ signaling.
Discussion
To control Ca2+-dependent intracellular processes, cells have developed a variety of mechanisms that regulate [Ca2+]c. Mitochondria are clearly important in the regulation of [Ca2+]c in many cells, not only in pathological but also physiological conditions (39), and the specific nature of this regulation is cell-specific. For example, mitochondrial depolarization accelerates InsP3-mediated Ca2+ waves in cortical astrocytes (20), but blocks InsP3-evoked Ca2+ release in colonic smooth muscle cells (40).
Endothelial cells play an important role in modulating vascular resistance and blood flow through their abilities to produce vasodilators such as NO and PGI2. This is most apparent during late pregnancy, when enhanced elevation of [Ca2+]c in UA endothelium in response to agonists in part leads to enhanced production of both NO and PGI2. Herein we address the possibility that an altered agonist (ATP)-induced [Ca2+]c response in UA endothelium between the NP- and P- state may in fact be mediated through changes at the level of the mitochondria.
Protonophores, such as CCCP, which collapse , are widely used to inhibit mitochondrial Ca2+ handling. In the present study, we observed that mitochondrial depolarization with CCCP significantly reduced the ATP-evoked [Ca2+]c in UAEC. There was no significant difference between ATP-induced peak increase in [Ca2+]c in P- and NP-UAEC, but CCCP was more efficient to inhibit ATP-induced [Ca2+]c in NP-UAEC than P-UAEC, confirming an altered role for mitochondria in pregnancy. To investigate the effects of CCCP at the level of ER, we examined the response of cells to thapsigargin, a potent and selective inhibitor of the ER Ca2+-ATPase that releases Ca2+ from ER by preventing Ca2+ uptake back to ER (36). The ability of CCCP to significantly inhibit thapsigargin-induced [Ca2+]c indicates that the inhibition of ATP-induced Ca2+ signaling by CCCP mainly occurred at the level of the ER by preventing Ca2+ release. Similar to the experiments with ATP, thapsigargin-induced [Ca2+]c was inhibited by CCCP pretreatment to a much greater degree in NP-UAEC than P-UAEC. This suggests that pregnancy-induced adaptation in Ca2+ signaling includes changes at the level of the mitochondria, and that P-UAEC has a greater capacity to resist the inhibitory effect of CCCP on agonist-stimulated [Ca2+]c.
Investigating the protective mechanisms in the normal P-UA may allow us to understand the pathophysiological etiology of preeclampsia, a condition in part associated with impaired Ca2+ signaling at least in hand vein endothelial cells (5). It is not yet clear whether the lack of such pregnancy-specific changes in endothelial [Ca2+]c signaling is due to failed adaptation at the level of the mitochondria. Nonetheless, two recent clinical reports directly implicate abnormal mitochondrial function with the preeclamptic condition (41, 42). For this reason, it is important to first know more about the molecular basis for the normal pregnancy-specific change in endothelial cell mitochondrial function before proceeding to investigate pathological pregnancy states. To that end, we used P-UAEC to further investigate the potential mechanisms underlying the inhibition of Ca2+ signaling after mitochondrial dysfunction as a result of CCCP pretreatment. Because pretreatment with CCCP could result in mitochondrial depolarization, opening of MPTP, release of Ca2+ from MPTP, depletion of local [ATP]i, change in cytoplasmic pH, and/or the blockade of mitochondria Ca2+ uptake, all were examined in the present study.
Mitochondrial Ca2+ uptake is driven by the negative mitochondrial potential, which is provided by the transmembrane electrochemical proton gradient (34). Depolarized mitochondria lose the potential gradient necessary for storing Ca2+, and any sequestered Ca2+ is then released (35). Thus, we examined the role of depletion of mitochondria Ca2+ store on ATP-induced [Ca2+]c. Indeed, CCCP application resulted in a increase in [Ca2+]c. This increase in [Ca2+]c by CCCP could be completely inhibited within 12–15 min by pretreatment of MPTP inhibitor CsA, indicating that MPTP opening was the primary mechanism of Ca2+ release from mitochondria. Because the pore was blocked with CsA, other mechanisms (putative Ca2+/H+ antiport, for example) may contribute to the delayed release of Ca2+ from mitochondria. During the CsA-created window of time, ATP could still induce a [Ca2+]c response similar to that under control conditions. However, when ATP was added after the delayed [Ca2+]c, ATP-induced [Ca2+]c was significantly inhibited. Collectively, these results indicate that depletion of Ca2+ from mitochondria results in inhibition of ATP-induced [Ca2+]c, and that the basal level of mitochondrial Ca2+ is necessary for normal ER function. Unlike Ca2+, some other mitochondrial factors, such as cytochrome c, can be only released through MPTP (25). Because MPTP was inhibited by CsA, this minimized the possibility that these factors also mediated CCCP-inhibited Ca2+ signaling.
The most direct effect of CCCP is mitochondrial depolarization. In the present study, ATP responded normally during the CsA-created window of time, whereas CCCP-induced mitochondrial membrane depolarization was not affected by CsA. These results suggest that mitochondrial depolarization itself does not directly inhibit ATP-induced [Ca2+]c, which is in contrast with reports showing that mitochondrial depolarization directly inhibits InsP3-induced Ca2+ release in HeLa cells (43).
Because CCCP is a protonophore, another obvious effect of CCCP application would be a change in cytoplasmic pH, which is known to modulate InsP3-induced Ca2+ release in some cells (44). However, such a change in cytoplasmic pH did not underlie the inhibition of Ca2+ release in UAEC, because CsA did not prevent the CCCP-induced loss of , but delayed CCCP-induced Ca2+ release from mitochondria.
Dissipation of with CCCP should lead to the inhibition of mitochondrial ATP synthesis due to the lack of a proton gradient. In addition, during CCCP treatment, the F0F1-ATPase could run in the reverse direction and rapidly consume ATP (37). Therefore, one possible consequence of mitochondrial depolarization is a reduction of local [ATP]i level around the ER. Because ATP is known to enhance InsP3R activation (38), such a decrease in [ATP]i in the vicinity of ER could have contributed to the inhibition of Ca2+ signaling in cells after CCCP treatment. However, 1) pretreatment of F0F1-ATPase inhibitor oligomycin B (5 μM) did not reduce the inhibition of ATP-induce [Ca2+]c by CCCP; 2) CsA did not prevent the CCCP-induced loss of , but delayed CCCP-induced Ca2+ release, and extracellular ATP responded normally during this window of time; 3) prolonged incubation with CCCP did not result in progressively increased degree of inhibition of Ca2+. These results indicate that the inhibition of Ca2+ signaling by depletion of mitochondrial Ca2+ with CCCP in UAEC is unlikely to be explained simply by changes in intracellular ATP levels. Our results are consistent with the studies with smooth muscle cell (40) and HeLa cell (43).
Mitochondria rapidly take up Ca2+ from the cytosol via a ruthenium red-sensitive uniporter spanning the inner mitochondrial membrane. This electrogenic passive uptake is driven by the enormous negative potential across the inner mitochondrial membrane, a consequence of proton extrusion along the electron transport chain. Thus mitochondria may shape intracellular Ca2+ signals, both spatially and temporally, as a direct consequence of their rapid Ca2+ buffering capacity (26). Mitochondrial depolarization leads to blockade of Ca2+ uptake, so the mitochondria are no longer available to act as a sink for ER-released Ca2+, which reflect the positive feedback effects of Ca2+ on the InsP3R (22). However, the observation that blockade of the Ca2+ uniporter by ruthenium red did not mimic CCCP-induced inhibition of Ca2+ signaling excluded the possibility that blockage of Ca2+ uptake mediate the inhibition of Ca2+ by CCCP.
In summary, we found that mitochondrial Ca2+ depletion after dissipation of with CCCP inhibits ATP-induced [Ca2+]c, and this inhibition occurred at ER level by inhibiting Ca2+ release from ER. Change in cytoplasmic pH, depletion of local ATP, and blockade of mitochondria Ca2+ uptake did not underlie the inhibition of ATP-induced Ca2+ release in UAEC. Moreover, our data revealed that P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c than NP-UAEC. Further studies are needed to explore the mechanism of pregnancy-specific resistance to mitochondrial dysfunction and its associated adverse effect on Ca2+ signaling.
Footnotes
This work was supported by National Institutes of Health Grants HL64601 (to I.M.B.), HD 38843 (Project 1 to I.M.B.), and HD050578 (to F.-X.Y.).
Abbreviations: [ATP]I, Intracellular ATP concentration; [Ca2+]c, cytosolic [Ca2+]; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CsA, cyclosporine A; , mitochondrial membrane potential; ER, endoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; MPTP, mitochondrial permeability transition pore; NO, nitric oxide; NP, nonpregnant; P, pregnant; PGI2, prostaglandin I2; Rh123, rhodamine 123; UA, uterine artery; UAEC, UA endothelial cell.
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