The Effect of Hypoxia on Cellular Redox Status 21 Sources of ROS Production

Cellular oxygen sensing is a highly conserved mechanism in evolution, most likely developed by primitive organisms with the first appearance of oxygen approximately 3 billion years ago (21, 50). It has long been recognized that ROS such as superoxide anion (02'~), hydrogen peroxide (H202) and hydroxyl radical are produced in aerobic cells as by-products of oxidative metabolism, either during mitochondrial electron transport or by several oxidoreductases and the metal-catalyzed oxidation of metabolites.

Stimulated production of ROS by a multicomponent nicotinamide adenine dinucleotide phosphate reduced (NAD(P)H) oxidase was first described in phagocytic cells like neutrophils and macrophages due to the transient consumption of 02 ("the respiratory burst"). During the last decade, production of ROS by NAD(P)H oxidase or by the NADH isoforms has also been demonstrated in a variety of cells in 02-sensitive tissues, including neuroepithelial body (NEB) cells (78), PASMCs (42, 46, 47), endothelial cells (85), and carotid bodies (38). A central role for an NAD(P)H oxidase in 02 sensing in lung NEB cells was recently examined by Fu et al. (30) who found that K+ channels of NEB cells in lung slices recorded from a knockout mouse, lacking the gp91pll0X subunit, were insensitive to acute hypoxia. They concluded that during normoxia NAD(P)H oxidase usually generates ROS that keep the K+ channels open. During hypoxia the decrease in ROS would allow the channels to close. In bovine pulmonary arteries the changes in 02 tension (Po2) may regulate vascular relaxation through changes in ROS (77). Wolin et al. paper reported that H202 production increased in normoxia, relative to hypoxia, and caused PA SMC relaxation through an increase in cGMP. Diphenylene iodonium (DPI), the non-selective NAD(P)H oxidase inhibitor, would be expected to reduce ROS and thus resemble hypoxia. In isolated and cultured NEB cells DPI does in fact inhibit K+ current as would be predicted if H202 produced by NAD(P)H oxidase promotes the K+ current seen in normoxia (69). DPI did not mimic the hypoxia response when given during normoxia, although this could be due to inhibition of Ca2+ channels. In the study of Archer et al. (7), the gp91phox knockout mouse has normal HPV and hypoxia still inhibits K+ channels in their PASMCs, suggesting that NAD(P)H oxidase containing gp91phox, is not required for HPV (Fig. 1). Consequently, the role of NAD(P)H oxidase in oxygen sensing is still uncertain.

Mitochondria were proposed over 30 years ago as potential 02 sensors in the carotid body (45). Under hypoxic conditions, depletion of high-energy phosphates, a shift toward the reduced form of redox couples, or cytochromes with an unusually low affinity for Po2 could act as a sensor. There are several studies providing further support for involvement of mitochondria in 02-sensing in Hep 3B cells (15, 16, 17), cardiomyocytes (12, 13, 17) and in human ductus arteriosus (44). Complementary to these findings, Waypa et al. have recently shown that proximal but not distal inhibitors, acting at electron transport chain (ETC) in mitochondria, suppressed HPV without affecting the response to other vasoconstrictors. They proposed that mitochondrial complex III is important in HPV, increasing ROS production under hypoxia and H202 is probably the signal molecule (70, 71).

Figure 1. HPV is preserved in mice lacking the gp91 subunit of NAD(P)H oxidase (knock-out mouse) but there is virtually no oxygen radical production at the lung surface, measured by luminol-chemiluminescence (upper panels) (From Ref. 72).

However, after more than two decades of investigation in this field it is still disputed whether ROS go up or down during acute exposure of the lung to hypoxia at physiological levels. Several studies have shown that acute hypoxia decreases production and tissue levels of ROS. In a small cell lung carcinoma cell line (H-146), hypoxia has been shown to reduce ROS production measured by DCF fluorescence (51). In addition, a decrease in superoxide anion production in neutrophils was reported as measured by cytochrome c reduction (31). Similar observations were made using chemiluminescence techniques, with luminol or lucigenin enhancement, during hypoxia in the rat lung (6), mouse lung (7), rabbit lung (53) and rabbit ductus arteriosus (60). Finally, Michelakis et al. recently provided supportive evidence for decreased ROS release in denuded resistance pulmonary artery rings during hypoxia using three different detection methods, DCF fluorescence, lucigenin-enhanced chemiluminescence and AmplexRed H202 assay (43). Similarly, an increase in P02 increased ROS production in the human ductus arteriosus, as demonstrated by the same group (44). In contrast, measurements with either DCF fluorescence or lucigenin-enhanced chemiluminescence in cultured PASMCs all showed increases in ROS production during hypoxia (37, 42, 70). This discrepancy may relate to differences in preparation methods or experimental techniques used. In addition, questions arise concerning the precise subcellular locations of ROS production.

2.2. Cytosolic Redox Status

Regardless of the direction of the change in the level of ROS, ROS signaling can be divided into two general mechanisms of action: a) direct effect of ROS and b) alterations in intracellular redox state.

2.2.1. Direct Effect of ROS

ROS can alter protein structure and function by modifying critical amino acid residues by removing or donating electrons. The reduction or oxidation of the sulfhydryl groups of amino acids, such as cysteine, have emerged as a molecular mechanism behind many cellular processes, including DNA transcription, ion channel modification, and regulation of cytosolic free Ca2+ concentration ([Ca2+]cyt). Oxidative modifications of critical amino acids within the functional domain of proteins (notably voltage-gated K+ channels) may occur in several ways. The best described of such modifications involves cysteine residues. The sulfhydryl group (-SH) of a single cysteine residue may be oxidized to form sulfenic (-SOH), sulfinic (-S02H), sulfonic (-S03H), or S-glutathionylated (-SSG) derivatives. Such alterations caused by ROS may alter the activity of K+ channels, changing channel gating or open probability and link changes in Po2 to vascular tone.

2.2.2. Alterations in Intracellular Redox State

The effects of ROS in signalling have often been attributed to a "shift" in the redox potential of the cells. In comparison with the extracellular environment, the cytosol is normally maintained under strong "reducing" conditions. Schafer and Buettner (63) suggested recently that the glutathione disulfide (GSSG)/glutathione (GSG) ratio can serve as a good indicator of the cellular redox state. This ratio results primarily from a combination of the rates of H202 removal by GSH (reduced glutathione) peroxidase and GSSG reduction by GSH reductase, regulating the GSH concentration. Hypoxia increases the ratios of the reduced to the oxidized forms of the cytosolic redox pairs, such as glutathione

(GSH/GSSG) and/or NADH/NAD, NADPH/NADP (4, 18, 64) and thus shifts the cells to a more reduced state. The redox couples within the cytosol could also affect and modify channel activity, as will be extensively reviewed in the following part of the chapter.

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