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American Journal of Physiology - Regulatory, Integrative and Comparative Physiology logoLink to American Journal of Physiology - Regulatory, Integrative and Comparative Physiology
. 2012 Mar 21;302(10):R1134–R1142. doi: 10.1152/ajpregu.00842.2010

Mitochondrial reactive oxygen species and calcium uptake regulate activation of phagocytic NADPH oxidase

Sergey I Dikalov 1,✉, Wei Li 2, Abdulrahman K Doughan 2, Raul R Blanco 2, A Maziar Zafari 2,3
PMCID: PMC3362147  PMID: 22442197

Abstract

Production of superoxide (O2·−) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca2+]i was measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca2+ uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca2+-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca2+ measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca2+]i, while BAPTA-AM abolished the increase in [Ca2+]i. Furthermore, depletion of cellular Ca2+ with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O2·− and impaired buffering of cytosolic Ca2+ by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.

Keywords: mitochondria, NADPH oxidase, calcium, superoxide, electron spin resonance

age is the major risk factor for cardiovascular diseases (20). Heart disease and stroke incidence rise steeply after age 65, accounting for more than 40% of all deaths among people aged 65 to 74 and almost 60% at age 85 and above (21). People, aged 65 and older are much more likely than younger people to suffer a heart attack, to have a stroke, or to develop coronary heart disease and high blood pressure leading to heart failure (5). These age-related conditions are well correlated with a decline in mitochondrial function (4); however, the precise interactions of mitochondrial dysfunction and cardiovascular diseases are not clear.

Production of superoxide (O2·−) by NADPH oxidases significantly contributes to the development of heart failure, hypertension, and atherosclerosis (18, 30). However, factors responsible for activation of NADPH oxidases are not completely understood (22). Interestingly, mitochondria are the second largest source of vascular O2·− (3), and mitochondrial dysfunction may be an initiating event in cardiovascular diseases (28). We, therefore, hypothesized that mitochondrial dysfunction can be involved in the regulation of vascular NADPH oxidases and, thereby, promote vascular disease.

We have recently demonstrated an important role for the Nox2 isoform of NADPH oxidases in cardiovascular disease (23). Nox2, previously known as gp91phox, is present in endothelial and phagocytic cells and contributes to blood pressure regulation, inflammation, and cell growth (14, 19, 37, 40). Nox2 function requires several cytoplasmic factors, such as rac1, p22phox, p67phox, and p47phox (17). The latter factor p47phox is of particular importance since its translocation from the cytoplasm and assembly into the Nox2 catalytic complex leads to hundred-fold increase of Nox2 catalytic activity and results in a phenomenon called “oxidative burst” due to robust increase in O2·− production by phagocytic cells. Under normal unstimulated conditions, basal production of O2·− by Nox2 is very slow, but treatment of cells with PKC activator PMA results in rapid phosphorylation of p47phox and assembly of the activated Nox2 complex (41). PMA stimulation, therefore, represents a convenient model for PKC-dependent Nox2 activation.

Cultured human lymphoblasts are commonly used to study phagocytic NADPH oxidase, since they predominantly express Nox2, but not the Nox1 isoform, and maintain functional mitochondria (31). Age-associated increased mitochondrial uncoupling and reduced ATP production (4) can be mimicked by mitochondrial inhibitors, such as complex I inhibitor rotenone, mitochondrial proton ionophore CCCP, inhibitors of mitochondrial complex III stigmatellin, or antimycin A (25). In this work, we studied the effect of mitochondrial inhibitors and an inhibitor of mitochondrial Ca2+ uniporter (12) on PMA-dependent activation of phagocytic NADPH oxidase in human lymphoblasts and human blood (Fig. 1). Production of O2·− by phagocytic NADPH oxidase was measured by the extracellular spin probe 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium chloride (CAT1H) (10) and electron spin resonance (ESR) (8). Our data show that decreased calcium uptake by mitochondria and production of mitochondrial reactive oxygen species (ROS) significantly increase phagocytic NADPH oxidase activity.

Fig. 1.

Fig. 1.

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Measurements of phagocytic NADPH oxidase activity in mouse peripheral blood mononuclear cell (PBMC) and human lymphoblasts by analysis of extracellular O2·− using cell-impermeable spin probe 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium chloride (CAT1H). A: typical electron spin resonance (ESR) spectrum of CAT1-nitroxide. Arrow indicates the low field component of ESR spectrum, which was used to monitor accumulation of CAT1-nitroxide during reaction of CAT1H with extracellular O2·−. B: measurements of O2·− production by PBMC (2.5 × 105/ml) isolated from C57/B6 wild-type and gp91phox KO mice. PBMC O2·− production was measured in unstimulated and PMA (10 μM)-stimulated cells. Extracellular O2·− detection was confirmed by inhibition of ESR signal with Cu,Zn-SOD (50 units/ml). As expected, PMA did not stimulate O2·− production in PBMC isolated from gp91phox KO mice. C: measurements of O2·− in human lymphoblasts (10 × 106/ml) by nitroxide production using extracellular CAT1H probe and mitochondria-targeted 1-hydroxy-4-[2-(triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine (mTH) probe. Lymphoblasts were treated with NADPH oxidase activator PMA or stimulator of mitochondrial O2·− antimycin A (AA; 10 μM). PMA-stimulated nitroxide accumulation was 5 times higher in the presence of CAT1H compared with mTH. Stimulation of mitochondrial O2·− by antimycin A was detected by mTH, while nitroxide accumulation with CAT1H probe was not affected by antimycin A. D: regulation of NADPH oxidase activity and general scheme showing detection of extracellular O2·− by CAT1H and mitochondrial O2·− by mTH with only minor contribution of extracellular O2·−. These data confirmed that CAT1H specifically measures activity of phagocytic NADPH oxidase, and it is not detected in mitochondrial O2·−. *P < 0.001 vs. control. **P <0.001 vs. WT. §P < 0.001 vs. control.

METHODS

Reagents.

Spin probe CAT1H and mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine (mTH) were purchased from Enzo Life Sciences (Plymouth Meeting, PA). Fura-2 AM was obtained from Invitrogen (Carlsbad, CA). SOD and phorbol 12-myristate 13-acetate (PMA) were acquired from Sigma-Aldrich (St. Louis, MO). All other reagents were obtained from Sigma-Aldrich.

Immortalized cell lines.

In collaboration with the Emory University General Clinical Research Center, human immortalized lymphoblast cell lines were developed from peripheral blood mononuclear cells of human subjects, as described previously (8). The study was approved by the Institutional Review Board of Emory University and the Atlanta Veterans Affairs Medical Center's Research and Development Committee. All subjects provided informed consent.

To initiate B lymphocyte cultures, lymphocytes were infected with the B95–8 strain of Epstein-Barr Virus (EBV) (33). After EBV-transformation, B lymphocytes were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine,100 U/ml penicillin, and 100 mg/ml streptomycin, at 37°C in a humid atmosphere saturated with 5% CO2. The medium was changed twice weekly. Cell counts and viability of >95% (trypan blue stain) were monitored on a daily basis for 2 wk until they were harvested for ESR experiments.

Superoxide measurement with ESR.

Superoxide radical was measured by ESR using a Bruker EMX spectrometer (Bruker Biospin) by extracellular CAT1H (1 mM) or mitochondria-targeted mTH (50 μM) spin probes (7). Briefly, cells were washed with PBS and resuspended in Krebs-HEPES buffer (KHB). Subsequently, 1 × 106 human lymphoblasts were placed in 0.1 ml KHB with 0.1 mM diethylenetriaminepentaacetic acid and supplemented with mitochondrial inhibitors prior to treatment with PMA. NADPH oxidase activity was determined at baseline and upon PKC-dependent stimulation with PMA following accumulation of nitroxide by monitoring the low-field component of ESR spectrum (Fig. 1A). Detection of O2·− was confirmed by inhibition of CAT1 accumulation with 50 units/ml SOD. SOD inhibited extracellular accumulation of nitroxide. Background oxidation of spin probes was subtracted from nitroxide accumulation in cellular samples. Results were expressed as picomoles of O2·− per 106 cells per minute.

Measurements of intracellular calcium [Ca2+]i.

[Ca2+]i was measured spectrofluorometrically as previously described (36). Briefly, cells were incubated with fura-2 AM (2 μM), and the cell dispersion agent Pluronic F-127 (0.04%) at 37°C in the dark for 30 min. Values of [Ca2+]i were obtained by determining maximum fura-2 fluorescence ratio (Rmax) and minimum (Rmin) using 10 μM digitonin followed by 30 mM EGTA, as described previously (16).

Statistics.

Experiments were analyzed using the Student-Neuman-Keuls post hoc test and ANOVA. P levels <0.05 were considered significant.

RESULTS

Measurement of phagocytic NADPH oxidase activity by ESR and hydroxylamine spin probes.

We have previously reported that human lymphoblasts produce predominantly extracellular O2·− because supplementation with extracellular SOD almost completely blocked O2·− detection by the cell-permeable probe (8). In this work, we used the cell-impermeable spin probe CAT1H for the detection of extracellular O2·− as a measure of phagocytic NADPH oxidase activity (Fig. 1, scheme). CAT1H rapidly react with O2·−, producing stable CAT1-nitroxide; therefore, O2·− production by NADPH oxidase can be measured by accumulation of CAT1 (Fig. 1A). To validate detection of phagocytic NADPH oxidase activity, we have investigated O2·− production by mouse peripheral blood mononuclear cell (PBMC) isolated from C57/B6 or gp91phox KO mice (Fig. 1B). It was found that PMA stimulation resulted in 10-fold increase in CAT1 accumulation, which was inhibited by supplementation of extracellular SOD. As expected, PMA stimulation did not affect basal O2·− production by PBMC isolated from gp91phox KO mice. It was important that supplementation of extracellular SOD inhibited both basal and PMA-stimulated O2·− production by wild-type PBMC. These data indicate that PMA-stimulated extracellular O2·− production reflects activation of phagocytic NADPH oxidase, and it can be measured by extracellular spin probe CAT1H (7).

To determine potential contribution of mitochondrial O2·− in extracellular O2·−, we have measurements of O2·− in human lymphoblasts by nitroxide production using extracellular CAT1H probe and mitochondria-targeted mTH probe (Fig. 1C). Lymphoblasts were acutely treated with NADPH oxidase activator PMA or stimulator of mitochondrial O2·− antimycin A prior to supplementation of the spin probe. Antimycin A inhibits electron transfer through the mitochondrial respiratory chain, which results in collapse of the mitochondrial membrane potential, overreduction of complex III, and increased O2·− production on complex III (24), while PMA stimulates PKC-dependent activation of NADPH oxidase. It was found that PMA-stimulated nitroxide accumulation was 5 times higher in the presence of CAT1H compared with mTH. Furthermore, stimulation of mitochondrial O2·− by antimycin A was detected by mTH, while nitroxide accumulation with CAT1H probe was not affected by antimycin A. These data show that CAT1H detects only extracellular O2·− and mitochondrial O2·− does not contribute to extracellular O2·−, which makes CAT1H a specific probe for O2·− production by phagocytic NADPH oxidase (Fig. 1D). Meanwhile, mTH was not effective in the detection of extracellular O2·− due to lower concentration of mTH (50 μM) compared with CAT1H (1 mM). However, mitochondria-targeted mTH (7) did show significant increase in mitochondrial O2− in antimycin A-treated cells. It is very important to note that supplementation of SOD did not inhibit nitroxide accumulation in mTH and antimycin A-supplemented cells, which support the intracellular detection of mitochondrial O2·− by mTH.

Lymphoblast and PBMC data confirmed that CAT1H specifically measures activity of phagocytic NADPH oxidase, and it does not detect mitochondrial O2·−, while mTH predominantly detects mitochondrial O2·− with only minor contribution of extracellular O2·−.

Stimulation of phagocytic NADPH oxidase by PMA in the presence of mitochondrial inhibitors.

In this work, we sought to determine the role of mitochondria in Ca2+-dependent regulation of phagocytic NADPH oxidase and investigate potential contribution of mitochondrial ROS in redox-dependent activation of NADPH oxidase (Fig. 1D). For this aim, we have investigated the effect of mitochondrial inhibitors, such as complex III inhibitor antimycin A on PMA-stimulated NADPH oxidase activity (Fig. 2). Supplementation with antimycin A did not significantly affect basal extracellular O2·− production (Fig. 2A). However, combination of antimycin A and PMA synergistically increased O2·− production. CAT1H did not detect antimycin A-stimulated increase in mitochondrial O2·− without PMA; therefore, the increase of PMA-stimulated O2·− production in antimycin A-treated cells may represent additional activation of the phagocytic NADPH, and it is not contaminated by mitochondrial O2·−. These results may suggest a potential role of mitochondrial ROS in activation of phagocytic NADPH oxidase.

Fig. 2.

Fig. 2.

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ESR detection of cellular O2·− using mitochondria-targeted mTH or cell-impermeable CAT1H probes. A: measurements of extracellular O2·− in lymphoblasts in the presence of CAT1H and stimulated with PMA (10 μM) and/or antimycin A (AA; 10 μM). Cu,Zn-SOD abolished CAT1 accumulation both in resting and PMA-stimulated cells. AA and PMA synergistically increased activity of phagocytic NADPH oxidase. B: measurements of cellular and mitochondrial O2·− in lymphoblasts in the presence of mTH and stimulated with PMA (10 μM) and/or AA (10 μM). Cu,Zn-SOD did not completely inhibited mT-nitroxide accumulation due to detection of mitochondrial O2·−. Both antimycin A and PMA increased O2·− production; however, combined treatment synergistically enhanced activity of phagocytic NADPH oxidase. Experiments were performed in Ca2+-free PBS prepared by Chelex-100 treatment. Results are expressed as means ± SE; n = 3–5. *P < 0.001 vs. Control. **P <0.05 vs. Control. §P < 0.01 vs. Control. #P < 0.05 vs. AA. ¶P < 0.001 vs. AA + PMA.

To investigate the potential role of mitochondrial ROS in regulation of phagocytic NADPH oxidase, we have compared production of extracellular O2·− and generation of cellular and mitochondrial O2·−, which was measured by cell-permeable and mitochondria-targeted spin probe mTH (7). In contrast to CAT1H detection of O2·− in cells supplemented with mTH has been only partially attenuated by Cu,Zn-SOD because extracellular SOD does inhibit detection of mitochondrial O2·−. The addition of SOD to PMA-stimulated cells inhibited O2·− detection by mTH to the level of unstimulated cells (Fig. 2B, SOD). As expected, antimycin A significantly increased O2·− detection by mTH, but the addition of SOD to antimycin A-treated cells led to only minor decrease in O2·− measurement. These data confirm that antimycin A treatment causes specific increase in intracellular ROS without significant elevation of extracellular O2·− or NADPH oxidase activity. Of note, CCCP did not significantly increase EPR signal in mTH-supplemented cells. Interestingly, supplementation of both antimycin A and PMA synergistically increased O2·− production, which was substantially inhibited by SOD to the level of antimycin A-treated cells. The fact that extracellular O2·− was generated mainly by phagocytic NADPH oxidase was supported by 1) the lack of extracellular O2·− in response to antimycin A in unstimulated cells measured by CAT1H (Fig. 2A: AA); and 2) the inhibition of PMA-induced O2·− production by gp91ds-tat, a chimeric peptide that attenuates the association of p47phox with Nox2 to the same extent (35%, data not shown), as was previously reported (29).

These data suggest that antimycin A-induced mitochondrial uncoupling and production of mitochondrial ROS may stimulate activity of phagocytic NADPH oxidase. To further elucidate the role of mitochondria on stimulation of phagocytic NADPH oxidase, we studied O2·− production in cells treated with the complex I inhibitor rotenone, mitochondrial proton ionophore CCCP, inhibitors of mitochondrial complex III stigmatellin, or antimycin A (Fig. 3) (25). We observed that in PMA-treated samples, rotenone significantly increased the activity of phagocytic NADPH oxidase similar to the proton ionophore CCCP. These data suggest that the decrease in mitochondrial membrane potential in the presence of rotenone or CCCP may result in increased activation of phagocytic NADPH oxidase. Interestingly, inhibition of complex III with stigmatellin or antimycin A further increased O2·− production compared with PMA+CCCP-treated cells. It is important to note that in contrast to CCCP, treatment with stigmatellin and antimycin A should result in increased production of mitochondrial O2·− and H2O2 (24). The difference in the extent of inhibition of NADPH oxidase activity by CCCP compared with complex III inhibitors may implicate mitochondrial ROS in PKC-dependent stimulation of the phagocytic NADPH oxidase.

Fig. 3.

Fig. 3.

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Stimulation of phagocytic NADPH oxidase by PMA in the presence of mitochondrial inhibitors. Superoxide production was measured in unstimulated or PMA-stimulated lymphoblasts in the presence of ethanol as a vehicle, inhibitor of mitochondrial complex I rotenone (10 μM), mitochondrial proton ionophore CCCP (10 μM), inhibitors of mitochondrial complex III stigmatellin (10 μM), or antimycin A (10 μM). Rotenone significantly increased activity of phagocytic NADPH oxidase similar to CCCP, while inhibitors of complex III stigmatellin and antimycin A further increased O2− production compared with PMA + CCCP-treated cells. Results are expressed as means ± SE; n = 3–6. *P < 0.001 vs. Control. **P < 0.05 vs. PMA. ***P < 0.01 vs. PMA. +P < 0.01 vs. PMA+CCCP. #P <0.001 vs. PMA + rotenone.

It has been previously shown that PKC-mediated activation of NADPH oxidase depends on the level of intracellular calcium [Ca2+]i (39). Intracellular calcium is known to be regulated by endoplasmic reticulum, but the role of mitochondria is unknown. Of note, the findings reported above have maintained when experiments were performed in KHB or calcium-free phosphate buffer prepared with chelating resin Chelex-100. Therefore, we tested the hypothesis that inhibition of mitochondrial uptake of calcium would enhance the activity of the phagocytic NADPH oxidase via increase in intracellular calcium [Ca2+]i.

Effect of mitochondrial Ca2+ uptake on activation of phagocytic NADPH oxidase.

Mitochondria absorb Ca2+ using the mitochondrial calcium uniporter, which can be inhibited by decreased mitochondrial membrane potential or ruthenium red (15). We studied the role of the calcium uniporter in the activation of the phagocytic NADPH oxidase using lymphoblasts treated with ruthenium red, chelator of intracellular Ca2+ BAPTA-AM, mitochondrial proton ionophore CCCP, and complex III inhibitor antimycin A. We found that ruthenium red did not significantly change the basal O2·− production in unstimulated cells. Supplementation of PMA-stimulated cells with ruthenium red, however, resulted in robust increase in NADPH oxidase activity compared with cells treated with PMA alone (Fig. 4). Interestingly, inhibition of the calcium uniporter with CCCP increased NADPH oxidase activity in PMA-stimulated cells similar to ruthenium red-treated cells. Supplementation with antimycin A resulted in higher NADPH oxidase activity compared with ruthenium red-treated cells, which is in line with a possible role for mitochondrial ROS in NADPH oxidase activation (9). Cotreatment with antimycin A and ruthenium red was not more effective than antimycin A alone, suggesting the inhibition of the calcium uniporter in antimycin A-treated cells. Furthermore, treatment of cells with the chelator of intracellular Ca2+ BAPTA-AM inhibited PMA-induced activation of NADPH oxidase and attenuated the effect of antimycin A on NADPH oxidase activity (Fig. 4). These data show that inhibition of Ca2+ uptake by mitochondria significantly increased activity of phagocytic NADPH oxidase, which was attenuated by chelation of intracellular Ca2+ with BAPTA-AM.

Fig. 4.

Fig. 4.

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Effect of mitochondrial Ca2+ uptake on activation of phagocytic NADPH oxidase in human B lymphoblasts. Stimulation of phagocytic NADPH oxidase by PMA in the presence of mitochondrial inhibitors. Superoxide production was measured in unstimulated or PMA-stimulated lymphoblasts in the presence of ethanol as a vehicle (Control), inhibitor of mitochondrial complex III AA (10 μM), inhibitor of mitochondrial Ca2+ uniporter ruthenium red (RR; 5 μM), chelator of intracellular Ca2+ BAPTA-AM (BAPTA, 0.1 mM), or mitochondrial proton ionophore CCCP (10 μM). Inhibition of Ca2+ uptake by mitochondria with RR significantly increased activity of phagocytic NADPH oxidase similar to CCCP, while chelation of intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ with thapsigargin (TG; 1 μM) reduced O2·− production in PMA-treated cells. Results are expressed as mean ± SE; n = 4–6. *P < 0.001 vs. Control. **P <0.05 vs. PMA. +P < 0.01 vs. PMA. #P < 0.001 vs. PMA. $P < 0.05 vs. AA + PMA. ¶P < 0.001 vs. PMA. §P < 0.01 vs. CCCP + PMA.

The role of intracellular Ca in mitochondria-mediated stimulation of NADPH oxidase was further analyzed in Ca2+-depleted cells. Lymphoblasts were treated with an inhibitor of sarcoplasmic reticulum Ca2+ ATPase, thapsigargin (27), to deplete Ca2+ stores (35), and then supplemented with antimycin A, CCCP, and PMA. It was found that depletion of Ca2+ stores attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly (Fig. 4,TG).

The above data indicate that inhibition of Ca2+ uptake by mitochondria would increase intracellular Ca2+. We, therefore, investigated the effects of mitochondrial inhibitors on intracellular Ca2+ levels in human lymphoblasts. Lymphoblasts were loaded with the probe for intracellular Ca2+ fura-2 AM and then treated with mitochondrial inhibitors or a chelator of intracellular Ca2+ BAPTA-AM prior to addition of PMA. We found that treatment of lymphoblasts with antimycin A or CCCP increased the level of intracellular Ca2+ similar to ruthenium red (Fig. 5); PMA supplementation further increased intracellular Ca2+. As expected treatment with the chelator of intracellular Ca2+ BAPTA-AM resulted in significant decrease of [Ca2+]i (Fig. 5F). These data strongly support our hypothesis that inhibition of mitochondrial uptake of Ca2+ stimulates activity of phagocytic NADPH oxidase via an increase in the intracellular Ca2+ level.

Fig. 5.

Fig. 5.

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Effects of mitochondrial inhibitors and the intracellular Ca-chelator BAPTA-AM on PMA stimulation of and intracellular Ca2+ levels in human lymphoblasts. Lymphoblasts were loaded with fura-2 AM (10 μM, 20 min) and then treated with mitochondrial inhibitors, BAPTA-AM, and PMA. Intracellular Ca2+ levels were measured spectrofluorometrically using fura-2 AM by the ratio of fluorescence, as previously described (36). Graphs show typical measurements of intracellular Ca2+ in cells treated with PMA (A), CCCP + PMA (B), antimycin A + PMA (C), RR + PMA (D) and BAPTA-AM + PMA (E). F: measurements of cytosolic Ca2+. Results are expressed as means ± SE; n = 3–5. *P < 0.001 vs. Control. **P <0.05 vs. PMA. +P < 0.001 vs. PMA.

The above studies show the role for mitochondrial uptake of Ca2+ in activation of the NADPH oxidase in lymphoblasts but do not provide insight into whether the role of mitochondria could be extended to other phagocytic cells. Therefore, we investigated the effect of inhibition of the mitochondrial calcium uniporter on O2·− production in whole blood.

Mitochondrial function and O2·− production in whole blood.

To confirm the role of the mitochondrial calcium uniporter on activation of phagocytic NADPH oxidase in whole blood, we measured O2·− production in samples with and without PMA stimulation and supplemented with calcium uniporter inhibitor ruthenium red, chelator of intracellular Ca2+ BAPTA-AM, or inhibitor of redox-sensitive K+ATP channel 5-hydroxydecanoate, which may improve mitochondrial function and increase mitochondrial potential (6, 11). We found that supplementation of ruthenium red resulted in the robust increase of O2·− production in samples without PMA, while BAPTA-AM inhibited basal O2·− production (Fig. 6A). As expected, supplementation of blood with ruthenium red significantly increased PMA-stimulated O2·− production, while BAPTA-AM attenuated PMA-induced O2·− production (Fig. 6B). Interestingly, supplementation with 5-hydroxydecanoate significantly inhibited PMA-stimulated O2·− production similar to BAPTA-AM, which can be associated with increased activity of the calcium uniporter due to the enhanced mitochondrial membrane potential in 5-hydroxydecanoate-treated cells (11). These data support the role of the mitochondrial calcium uniporter in the regulation of the phagocytic NADPH oxidase.

Fig. 6.

Fig. 6.

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Effect of mitochondrial inhibitors on phagocytic NADPH oxidase activity in whole human blood. A: typical time scans of O2·− production in the whole blood in the presence of DMSO as a vehicle (Control), inhibitor of K+ATP channel 5-hydroxydecanoate (5HD; 10 μM), chelator of intracellular Ca2+ BAPTA-AM (BAPTA, 0.1 mM), or inhibitor of mitochondrial Ca2+ uniporter RR (5 μM). B: typical time scans of O2·− production in the whole blood in the presence of PMA (2 μM) and supplemented with DMSO (Control), 5-hydtoxydecanoate (5HD), BAPTA-AM (BAPTA), or RR. Figure shows typical ESR time scans observed in at least three independent experiments. *P < 0.02 vs. Control. **P <0.01 vs. Control + PMA.

DISCUSSION

The present study provides evidence for a mitochondrial role in stimulation of the phagocytic NADPH oxidase. We observed that inhibition of the calcium uniporter with ruthenium red or with complex I or complex III inhibitors resulted in significant increase of intracellular calcium and increased NADPH oxidase activity. The role of mitochondria in stimulation of the phagocytic NADPH oxidase can be summarized in two pathways: 1) uptake of cytoplasmic Ca2+ by the calcium uniporter, and 2) production of mitochondrial ROS (Fig. 7). Indeed, effects of mitochondrial inhibitors were attenuated with the chelator of intracellular Ca2+ BAPTA-AM both in cultured human lymphoblasts and in whole blood. Furthermore, complex III inhibitors stigmatellin and antimycin A increased production of mitochondrial ROS (24) and inhibited calcium uniporter function, which resulted in more potent stimulation of PMA-induced O2·− production compared with the inhibition of the calcium uniporter alone by ruthenium red or CCCP. Further analysis confirmed that mitochondria regulate activation of phagocytic NADPH oxidase also in whole blood.

Fig. 7.

Fig. 7.

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Possible role of mitochondria in activation of the phagocytic NADPH oxidase.

It has been previously reported that activation of phagocytic NADPH oxidase is Ca2+ and H2O2 dependent (13, 34). Intracellular Ca2+ may stimulate PKC-dependent p47phox phosphorylation and cause Ca2+-dependent phosphorylation of Nox2 (13). Interestingly, activation of NADPH oxidase in astrocytes resulted in transient increases in intracellular Ca2+ and caused loss of mitochondrial potential (1, 2). The mitochondrial response, which consisted of Ca2+-dependent transient depolarization, was prevented by antioxidants and NADPH oxidase inhibitors, suggesting an upstream role of Nox2-mediated ROS in Ca2+ increase and mitochondrial dysfunction. Indeed, increase of intracellular ROS may lead to elevated intracellular Ca2+, which may cause mitochondrial Ca2+ overload followed by collapse of mitochondrial potential and apoptosis (25). Previous works, however, did not study the role of mitochondrial ROS or mitochondrial Ca2+ uptake in the regulation of NADPH oxidase activity. In our paper, we were focused on the physiological role of mitochondrial Ca2+ uptake, which attenuates NADPH oxidase activation without mitochondrial depolarization or cell apoptosis.

It has been previously shown that phagocytic NADPH oxidase of human lymphoblasts produces mainly extracellular superoxide (8). Treatment of these cells with mitochondrial inhibitors did not significantly change the amount of extracellular superoxide in nonstimulated cells (Figs. 2, 3, and 4). However, inhibition of the calcium uniporter with ruthenium red or treatment with complex I or complex III inhibitors resulted in increased NADPH oxidase activity in the presence of PMA. Antimycin A and stigmatellin may stimulate production of intracellular superoxide and H2O2 (24, 26). It is, therefore, conceivable that these mitochondrial inhibitors can affect redox regulation of the phagocytic NADPH oxidase. Indeed, we have previously reported that the H2O2 scavenger catalase attenuated PMA-induced activation of the phagocytic NADPH oxidase (8). Meanwhile, CCCP, which is known to inhibit production of mitochondrial ROS (25), actually stimulated NADPH oxidase activity. Furthermore, specific inhibition of the Ca2+ uniporter also stimulated NADPH oxidase activity. Similar effects of antimycin A, CCCP, and ruthenium red suggest that output of these mitochondrial inhibitors is primarily due to a decrease in the mitochondrial membrane potential, which is the driving force of the Ca2+ uniporter. Indeed, treatment of cells with antimycin A, CCCP, or ruthenium red resulted in similar increase of intracellular Ca2+ (Fig. 5). These data imply that mitochondria play a crucial role in the control of Ca2+-dependent stimulation of the phagocytic NADPH oxidase. Notably, antimycin A provided significantly higher stimulation of NADPH oxidase compared with CCCP or ruthenium red (Figs. 3 and 4), which implicates mitochondrial ROS in stimulation of the phagocytic NADPH oxidase. Therefore, we suggest that mitochondria affect phagocytic NADPH oxidase activity via both Ca2+-dependent and ROS-dependent mechanisms (Fig. 7).

We suggest that relevance of our findings is not limited to phagocytic cells and phagocytic NADPH oxidase. Indeed, it has been recently shown that overexpression of mitochondrial SOD2 attenuates ANG II-mediated activation of NADPH oxidase in endothelial cells (9). ANG II mainly activates Nox2 in these cells, which is an analog of phagocytic NADPH oxidase expressed in the vasculature. Interestingly, depletion of SOD2 resulted in overstimulation of NADPH oxidase, and treatment with mitochondria-targeted SOD mimetic mitoTEMPO has reversed the effect of SOD depletion and attenuated activation of NADPH oxidase in endothelial cells. Furthermore, in vivo experiments showed that SOD2 overexpression or mitoTEMPO supplementation has reduced NADPH oxidase activity in vasculature and inhibited vascular O2·− production. These data imply that mitochondrial ROS stimulate activation of Nox2 and possibly other Nox isoforms, such as Nox1. Interestingly, mitoTEMPO has improved mitochondrial respiration (9), which may be associated with increased mitochondrial membrane potential. Therefore, additional studies are required to identify the specific role of mitochondrial Ca2+ uptake and ROS in regulation of vascular NADPH oxidase activity.

A growing body of evidence has emerged linking inflammation and aging with the development and progression of cardiovascular diseases (38). The molecular mechanisms underlying chronic inflammatory conditions, such as hypertension and atherosclerosis during cellular senescence, are not clearly defined. Cellular damage by ROS is a primary driving force for aging and increased activation of redox-regulated transcription factors, such as NF-κB, that regulate the expression of proinflammatory genes, has been reported (32).

It has been previously observed that mitochondrial function declines with age or is altered due to side effects of pharmacological treatments (4). Our data suggest that mitochondrial impairment can promote activation of phagocytic NADPH oxidase, which may enhance inflammation and ultimately contribute to the development and progression of cardiovascular diseases. Thus, it is conceivable that healthy or younger mitochondria represent an important barrier in stimulation of phagocytic NADPH oxidase, while age-impaired mitochondria may promote NADPH oxidase activation, resulting in enhanced oxidative stress, which would increase inflammation and impair mitochondrial function. Therefore, age-associated mitochondrial dysfunction may play a significant role in the vicious cycle of oxidative stress and inflammation, contributing to the development and progression of cardiovascular diseases.

Perspectives and Significance

It was known that production of superoxide (O2·−) by NADPH oxidases may significantly alter mitochondrial function. In this work, we report that mitochondria provide feedforward stimulation of PKC-dependent phagocytic NADPH oxidase. Increased production of mitochondrial ROS and impaired mitochondrial buffering of cytosolic calcium may result in enhanced NADPH oxidase activity and may contribute to the development and progression of cardiovascular diseases.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: S.I.D. and A.M.Z. conception and design of research; S.I.D., W.L., A.K.D., and R.R.B. performed experiments; S.I.D., W.L., A.K.D., and R.R.B. analyzed data; S.I.D. interpreted results of experiments; S.I.D. prepared figures; S.I.D. and A.M.Z. drafted manuscript; S.I.D. and A.M.Z. edited and revised manuscript; S.I.D. and A.M.Z. approved final version of manuscript.

ACKNOWLEDGMENTS

This research was supported by National Institutes of Health grants PO-1 HL-058000, PO-1 HL-075209, R01HL-094469, and by a National Institutes of Health cardiovascular training grant (T32 HL-07745) to R. R. Blanco.

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