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

Abstract

Production of superoxide (O(2)(·-)) 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 [Ca(2+)](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 Ca(2+) 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 Ca(2+)-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca(2+) measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca(2+)](i), while BAPTA-AM abolished the increase in [Ca(2+)](i). Furthermore, depletion of cellular Ca(2+) 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 O(2)(·-) and impaired buffering of cytosolic Ca(2+) by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.

Fig. 1.

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 gp91^phox 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 gp91^phox 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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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.

Fig. 6.

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.

Fig. 7.

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