Hypoxia Augments Mitochondrial Reactive Oxygen Species

During mitochondrial respiration under normal oxygen conditions, 02 is chemically reduced to water by the transfer of four electrons at cytochrome oxidase. The resulting free energy change is conserved in the form of ATP synthesis. It has been estimated that 2-3% ofthe 02 consumed by mitochondria is incompletely reduced, yielding ROS (4). Univalent electron transfer to 02 generates superoxide, a modestly stable free radical anion. Superoxide can potentially be generated at a number of different sites, including complex I, the ubisemiquinone site of complex III, and other electron transfer proteins (31). ROS do not appear to be generated by cytochrome oxidase itself, due to the high-affinity kinetic trapping of 02 at thebinuclear center, which occurs while the four electrons are sequentially transferred. The superoxide generated during the Q-cycle within the mitochondrial electron complex III (bc1 complex) is considered the main site of ROS generation within the electron transport chain (Figure 1). The Q-cycle involves the transfer of two electrons to ubiquinone from complex I or complex II resulting in the reduction of ubiquinone to ubiquinol. Subsequently, ubiquinol oxidation requires donation of two electrons: the first electron transfer is to the iron-sulfur centers and cytochrome c1 resulting in the oxidation of ubiquinol to ubisemiquinone. This reaction is inhibited by myxothiazol. The second electron is transferred to cytochrome b and results in the oxidation of ubisemiquinone to ubiquinone. This step is inhibited by antimycin A. The oxidation of ubisemiquinone to ubiquinone is the main site of ROS generation during the Q-cycle. The availability of 02, the reduction state of the electron carriers, and the mitochondrial membrane potential determine the ability of electron transport chain to generate superoxide.

Recent data suggest that hypoxia increases the generation of ROS within mitochondria. Using fluorescent dyes to detect an oxidative signal, we initially studied the oxidation of 2', 7'-dichlorofluorescin (DCFH) in cardiomyocytes and Hep3B cells under controlled 02 conditions (5, 7). The cells were studied in a flow-through chamber maintained at 37°C on an inverted microscope. The perfusate was bubbled with different 02 concentrations in a water-jacketed equilibration column mounted above the microscope stage and was delivered to the chamber via a short length of tubing. The reduced diacetate form ofthe dye DCFH was continually present in the medium (5 |iM). Oxidation of the dye within the cell yields the fluorescent compound 2',7'-dichlorofluorescein (DCF), which was detected with a 12-bit digital cooled charge-coupled device camera. In the absence of dye, no fluorescence can be detected. After addition of dye to the medium, evidence of cell fluorescence is detected within a few minutes. Dye oxidized outside of the cell, or dye oxidized within the cell that leaks out, is carried away in the perfusate flow. Under steady-state conditions, cellular fluorescence reflects a balance between the rate of oxidation of DCFH in the cell and the rate at which oxidized dye escapes and is carried away. Paradoxically, oxidation of DCFH increased as the 02 concentration was decreased from normoxic levels (16%02), with minimal effect seen at 8% and a maximal effects observed at 1% 02. Within minutes of decreasing the 02 concentration, intracellular fluorescence increased, reflecting an increase in the rate of dye oxidation. On return to normoxia, fluorescence decreased as the rate of escape of oxidized dye exceeded the rate of dye oxidation in the cell. The increase in DCFH oxidation can be prevented by ebselen, a synthetic glutathione peroxidase, and by diethyldithiocarbamate(DDC), a cytosolic inhibitor of Cu,Zn superoxide dismutase (SOD). Ebselen and DDC prevent the cytosolic formation of hydrogen peroxide (H202) indicating that the increase in DCFH oxidation during hypoxia is primarily occurring by H202 production.

Figure 1. In the electron transportchain, ubisemiquinone appears to be a major site of superoxide generation because of its predisposition for univalent electron transfer to 02. Electron transport inhibition with rotenone (complex I) or myxothiazol limits the generation of superoxide by attenuating the formation of ubisemiquinone whereas antimycin A, an inhibitor of complex III, augments superoxide generation by increasing the lifetime of ubisemiquinone.

The potential role of mitochondria as a source of ROS generation has been examined by oxidation of DCFH in the presence of electron transport chain inhibitors (Fig. 1). Both rotenone or myxothiazol prevent the increase in oxidation of DCFH during hypoxia. Rotenone inhibits complex I while myxothiazol blocks electron transfer to the Rieske iron-sulfur center within complex III. In contrast, antimycin A, which inhibits the oxidation of cytochrome b562 at complex III, augmented the increase in the oxidation of DCFH. Thus, inhibitors that block electron transfer upstream from that site (rotenone, myxothiazol) tend to prevent the formation of ubisemiquinone and thereby diminish ROS generation. Mitochondrial inhibitors that act at more downstream sites (antimycin, cyanide) tend to augment ROS generation by increasing the generation of ubisemiquinone. Further evidence of mitochondria as a source of oxidant production during hypoxia comes from the observation

Mitochondria

Ubisemiquinone

Figure 1. In the electron transportchain, ubisemiquinone appears to be a major site of superoxide generation because of its predisposition for univalent electron transfer to 02. Electron transport inhibition with rotenone (complex I) or myxothiazol limits the generation of superoxide by attenuating the formation of ubisemiquinone whereas antimycin A, an inhibitor of complex III, augments superoxide generation by increasing the lifetime of ubisemiquinone.

Mitochondria that 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) abolishes the oxidation of DCFH during hypoxia. DIDS blocks anion channels in both inner and outer mitochondrial membranes, thereby preventing the leakage of superoxide from mitochondria into the cytosol.

The pharmacological inhibition of the electron transport chain has been confirmed using p° cells, a functional mitochondria-deficient cell line. Rapidly-dividing cells can be mutated into a state by incubation with ethidium bromide, which inhibits the replication ofmitochondrial DNA that is required for critical subunits of certain mitochondrial electron transport complexes and for part of the F0F| ATP synthase. The p° cells are therefore incapable of supporting mitochondrial respiration or oxidative phosphorylation. Survival and growth of p° cells requires glycolytically-derived ATP. Because p° cells lack mitochondrial electron transport, they cannot generate mitochondrial ROS. In p° HepSB cells, hypoxia failed to stimulate oxidative signaling, as evidenced by an absence of DCFH oxidation. Based on studies using both mitochondrial inhibitors and p° cells, we conclude that hypoxia stimulates superoxide production at the ubisemiquinone site. Subsequently, superoxide enters the cytosol by anion channels where it is converted into H202 by cytosolic Cu,Zn superoxide dismutase.

After our initial observations in HEP3B cells and cardiac myocytes on DCFH oxidation, Gillespie and colleagues demonstrated that primary cultures of rat main PASMCs increased DCFH oxidation during hypoxia (19). Hypoxia-induced DCF fluorescence was attenuated by the addition of the antioxidants dimethylthiourea and catalase. Recently, we also demonstrated that rat PASMCs acutely exposed to hypoxia exhibit a marked increase in intracellular DCF fluorescence suggestive of an increase in ROS (34). Myxothiazol attenuated the increase in DCFH oxidation during hypoxia, implicating the mitochondrion as a major source of oxidant production in PASMCs under hypoxic conditions. Thus, a variety of mammalian cells including PASMCs exhibit an increase in ROS during hypoxia.

Was this article helpful?

0 0
Reducing Blood Pressure Naturally

Reducing Blood Pressure Naturally

Do You Suffer From High Blood Pressure? Do You Feel Like This Silent Killer Might Be Stalking You? Have you been diagnosed or pre-hypertension and hypertension? Then JOIN THE CROWD Nearly 1 in 3 adults in the United States suffer from High Blood Pressure and only 1 in 3 adults are actually aware that they have it.

Get My Free Ebook


Post a comment