AOS as a Mediator

AOS from mitochondria and other cellular sources have been traditionally regarded as toxic by-products of metabolism with the potential to cause damage to lipids, proteins, and DNA. To protect against the potentially damaging effects of AOS, cells possess several isoforms of the antioxidant enzyme as superoxide dismutase (SOD; which reduces 02"to H202), as well as catalase (which reduces H202 to HjO and 02). Thus, oxidative stress may be broadly defined as an imbalance between oxidant production and the antioxidant capacity of the cell to prevent oxidative injury. Oxidative stress has been implicated in a large number of human diseases including atherosclerosis, pulmonary fibrosis, cancer, neurodegenerative diseases and aging (29). Yet the relationship between oxidative stress and pathobiology of these diseases is not clear, largely due to a lack of understanding of the mechanisms by which AOS function in both physiological and disease states.

Accumulating evidence suggests that AOS are not always injurious byproducts of cellular metabolism, but can also be important participants in cell signaling. The evidence can be found in bacteria as they use AOS to oxidize cysteine residues in oxidative stress-responsive protein (OxyR) to activate transcription (136). Similarly studies in E. coli showed that AOS was instrumental in oxidizing SoxR, triggering events which culminated in transcription of various factors including SOD (31). While bacteria use AOS as a physiological second messenger molecule, sea urchin uses H202 to form a protective envelope around the freshly fertilized oocyte thus enabling procreation in animals (110). In mammals, the role of AOS has been studied extensively and has been shown to be an integral second messenger molecule in events critical for immunomodulation, regeneration of tissues, maintaining vasoreactivity of blood vessels and neural conduction (119).

One of the primary AOS is 02". Superoxide itself can be toxic, especially through inactivation of proteins that contain Fe-S centers such as aconitase, succinate dehydrogenase, and mitochondrial NADH-ubiquinone oxidoreductase (34). Fortunately, 02" in aqueous solution is short-lived. This instability in aqueous solution is based on rapid dismutation of 02'~ to H202, a reaction facilitated by higher concentrations of the protonated form of 02" (H02*) in more acidic pH conditions (34). The stability is further reduced by enzymes that have evolved with the task of detoxifying oxygen free radicals, collectively named the superoxide dismutases. There are three of them in mammalian systems: a cytosolic CuZn superoxide (SOD1; CuZnSOD), an intramitochondrial manganese superoxide dismutase (SOD2; MnSOD) and extracellular CuZn superoxide dismutase (SODS). Collectively, they catalyze the following reaction:

The dismutation reaction has an overall rate constant of 5 x 105 M'1 s'1 atpH

7.0. SOD speeds up this reaction almost 104 fold (Kd = 1.6 x 109 M'1 s1) (34). Thus, in physiological systems, superoxides are converted to the stable H202. Because of its stability, H202 is used as a primary signaling molecule (24).

AOS have been implicated in HPV. Although, the precise mechanism behind AOS mediated HPV is not fully elucidated yet, the implication of AOS in HPV has been widely recognized. Though AOS has been an accepted signal transduction molecule of HPV, two schools of thoughts (outlined below) prevail on how the AOS levels and subsequent HPV is regulated in physiological system.

5.1. The Mitochondrial Redox Hypothesis

The redox theory of HPV proposes that there is a tonic, basal production of AOS in the PASMCs, most likely generated by mitochondria (7). The "normal" rate of generation of AOS causes physiological oxidation of K+ channels in PASMCs, thus maintaining "normoxic vasodilation", thereby contributing to the low basal tone of pulmonary arteries. Under conditions of alveolar hypoxia, production of AOS falls in proportion to the inspired 02 concentration (Fig. 7 A). The result is the inhibition of normal AOS mediated dilatation and hence constriction (4). In support, our laboratory showed that a decrease in whole lung AOS production, as measured by chemiluminescence, precedes HPV (by seconds) (14). Non-redox dependent PA constrictors, such as angiotensin II, do not acutely alter AOS production. Furthermore, when antimycin A and rotenone were used to inhibit mitochondrial ETC, a similar decrease in measured chemiluminescence and increased PA pressure was observed (Fig. 7B) (10). Paky et al. also noticed inhibition ofAOS production by hypoxia and antimycin A (82). However, these studies generated skepticism in some quarters because ofconcerns that lucigenin may itself trigger the redox cycle and purportedly has a propensity to measure cellular reductase activity independent from superoxide radicals. Furthermore, while the strength of these studies was the online measurement of AOS and its correlation with vascular tone, the chemiluminescence signal reflected the global superoxide anion generation by all pulmonary cells near the lung surface (potentially including neutrophils, airway epithelium and smooth muscle, endothelium, and vascular SMCs) (109). To address these concerns, we utilized three different radical detection methods (lucigenin-enhanced chemiluminescence, AmplexRed H202 assay, and 2'-7'-dichlorofluorescein (DCF)) for AOS measurement (72). We showed that in endothelium-denuded resistance PA rings, AOS levels were decreased during hypoxia as measured by these three different techniques. Furthermore, this decrease in AOS production was also noted in PA rings treated with proximal ETC inhibitors (72). This has been also supported by studies in other tissues and experimental models showing a similar decrease in AOS production during HPV (Fig. 7) (82, 97).

Figure 7. A: AOS production decreases with hypoxia, just prior to the onset of HPV. PA pressure and AOS production (luminol-enhanced chemiluminescence) were measured simultaneously in isolated rat lungs. The decrease in AOS production precedes the increase in PA pressure by seconds and occurs in a 02 "dose dependent" manner. B: Chronic hypoxia reveals similarities between acute hypoxia and rotenone. Simultaneous luminol chemiluminescence and PA pressure measurements in a normoxic isolated rat lung show that rotenone constricts the PA and decreases AOS production in a manner similar to hypoxia. In lungs from a rat exposed to chronic hypoxia (0.45 atmospheres for -3 weeks), HPV is suppressed while angiotensin II (All) constriction is unchanged (Reproduced from Refs. 14,97).

Figure 7. A: AOS production decreases with hypoxia, just prior to the onset of HPV. PA pressure and AOS production (luminol-enhanced chemiluminescence) were measured simultaneously in isolated rat lungs. The decrease in AOS production precedes the increase in PA pressure by seconds and occurs in a 02 "dose dependent" manner. B: Chronic hypoxia reveals similarities between acute hypoxia and rotenone. Simultaneous luminol chemiluminescence and PA pressure measurements in a normoxic isolated rat lung show that rotenone constricts the PA and decreases AOS production in a manner similar to hypoxia. In lungs from a rat exposed to chronic hypoxia (0.45 atmospheres for -3 weeks), HPV is suppressed while angiotensin II (All) constriction is unchanged (Reproduced from Refs. 14,97).

In the classical AOS response, ischemia-reperfusion, AOS are universally agreed to be reduced in the ischemic phase (analogous to hypoxia), and only increase with reperfusion (analogous to the termination of hypoxia) (42). Thus, it has been consistently shown that hypoxia or conditions mimicking hypoxia (proximal ETC inhibitors) decrease AOS production. In fact this hypothesis has been extended to another 02-sensing tissue, i.e., the ductus arteriosus (DA) (74). Studies in our laboratory have shown that DASMCs responded in accordance to the mitochondrial redox hypothesis. Inhuman DASMCs, 02 causes constriction. We showed that, in response to increased ambient production was increased in DASMCs. This increase caused constriction of DASMCs, which was associated with an inhibition ofplasmalemmal K+ channels. Thus it appears that the response ofthe K+ channelsto H202 accounts for the opposing response of the PA and the DA to As in the PA, rotenone and antimycin mimic the effects of hypoxia on the DA (i.e., cause vasodilation) (74). Hence, it appears that during hypoxia, when ambient Po2 is low, AOS production is decreased which subsequently translates into pulmonary vasoconstriction and dilatation of the ductus arteriosus.

5.2. Increased Mitochondrial AOS Production Hypothesis

In opposition to the theory that hypoxia results in a shift towards reduced state or decreased AOS levels, other laboratories have suggested a paradoxical increase in AOS generation during hypoxia. Marshall et al. found an increase in chemiluminescence during hypoxia in isolated PASMCs (66). A similar increase in oxidant signaling has also been reported in various hypoxic models. Recently, in parallel experiments in isolated perfused lungs and PASMCs (124), Waypa et al. showed that intracellular DCF was increased during hypoxic treatment, a response blocked by myxothiazol, a proximal ETC inhibitor. Based on these results the authors postulated that mitochondria indeed function as 02 sensor, but that increased rather than decreased mitochondrial AOS production triggers HPV (124). They agreed with the proposed role of H202 as a probable signaling molecule. However, the use ofDCF to measure AOS is controversial as previous studies suggest DCF as a suboptimal fluorescent probe to measure 02'~ based radicals. DCF readily detects nitric oxide (in fact even more efficiently than it detects AOS) (95). In addition, DCF itself has been shown to be a source of H202 generation, further complicating and questioning the use ofDCF as a fluorescent probe to detect 02 radicals (102). Rota et al. evaluated the sensitivity and specificity of DCF and found that, depending on the chemical environment, DCF is quite effective in generating AOS and therefore should not be used as a reliable probe (102). Furthermore, previously published studies have shown a decrease in AOS production, as detected using DCF as a fluorescent marker (28). Collectively, these studies show that DCF may not be an optimal marker for measuring AOS levels. Teleologically, an increase in AOS levels in the face of hypoxiaseems unlikely. Electrons accumulating during hypoxiainmitochondria would react with 02 to produce 02'~. However, under hypoxic conditions, it is difficult to conceptualize that 02" production would increase as increased availability of electrons cannot be offset by decreased 02 availability. Furthermore, even in the myocardium where ischemia-reperfusion studies have long been conducted, AOS production decreases during ischemia (58) and increased AOS is only seen during reperfusion (58, 137). It would seem that the AOS status in the lung during hypoxia is more analogous to that of the heart during ischemia-not the reperfusion or reoxygenation phase. With cessation of hypoxia AOS levels in the lung rise, as expected and as is consistent with the termination of ischemia in the heart (albeit without much of an overshoot). Therefore, the studies cumulatively suggest that, under hypoxic conditions, withdrawal of AOS is the mediator of vasoconstriction in pulmonary vascular beds.

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