Recent investigations on HPV have suggested that complex III ofthe electron transport chain may promote HPV. In short, it has been proposed that hypoxia inhibits oxidative phosphorylation, leading to a consequent increase in ROS at complex III in the electron transport chain, that is presumably driven by (3-NADH and FADH2 oxidation by complex I and complex II, respectively (52, 84, 85). More recently, however, it has been argued that the site of ROS production may be complex II and not complex III (67).
Irrespective of the exact site of ROS production, the possibility that a paradoxical increase in superoxide production by mitochondria may promote HPV, and therefore cADPR accumulation in pulmonary artery smooth muscle has been raised (52, 85). This proposal gains direct support from the finding that superoxide can increase cADPR synthesis by directly activating ADP-ribosyl cyclase (48, 62). Furthermore, Leach et al. (52, 53) suggest that it is an increase in superoxide and not an increase in 0-NADH that promotes cADPR accumulation. This is based on their observation that HPV may be blocked by inhibitors of complex I of the electron transport chain, and subsequently restored by addition of succinate, i.e., by facilitating FADH2 oxidation by complex II. Under each of these experimental conditions they report that the increase in NAD(P)H autofluorescence by hypoxia in intact arteries remained unaltered, a finding that they rightly cite as being inconsistent with the idea that an increase in promotes cADPR accumulation.
Clearly, superoxide can, under certain conditions, promote cADPR synthesis. However, when assessing the information before us, it is vitally important that we recognize the limits of the experimental evidence provided by a given study before we draw any conclusion as to the likely mechanisms involved. Herein lies the problem. Leach et al. (52) relied upon pre-constriction by prostaglandin F2a in order to elicit constriction of pulmonary arteries by hypoxia. Furthermore, under the conditions oftheir experiments they have been unable to record maintained cADPR-dependent constriction by hypoxia in arteries without endothelium (2). In short, they have yet to observe the component of HPV driven by cADPR-dependent SR Ca2+ release by hypoxia. In this respect, interpretation of the findings of Waypa et al. (84) in the rat lung is also problematic. In these studies too, pre-constriction was required to procure HPV. Given this fact, it is impossible to draw any conclusions as to the mechanisms by which hypoxia mediates, by a direct action on the smooth muscle, maintained smooth muscle constriction in pulmonary arteries. These studies could therefore be seen as providing strong evidence against the idea that hypoxia promotes cADPR accumulation by increasing superoxide production at complex II/III of the mitochondrial electron transport chain. In short, the proposed increase in superoxide and/or by hypoxia alone cannot explain cADPR accumulation by hypoxia in pulmonary artery smooth muscle. Support for this viewpoint may be derived from a recent investigation on carotid body glomus cells, which provided contrary findings with respect to the effects of mitochondrial inhibitors. In short, Ortega-Saenz et al. (65) conclude that the response of glomus cells to hypoxia is not linked to mitochondria in a simple way, and does not appear to be mediated by ROS production at complex II, or III of the ETC. This view is supported by the fact that mitochondrial inhibitors and ROS scavengers, respectively, do not alter the function of 02-sensing cells in a consistent, nor reproducible manner (32). These contrary findings underline the need to monitor the "metabolic setting" of02-sensing cells under all experimental conditions, and the need to strictly adhere to the physiological range of Po2 for a given cell type. This is highlighted by the Po2 window for cADPR accumulation by hypoxia. In short, a level of hypoxia too moderate to induce HPV in the absence of preconstriction may be insufficient to provide the metabolic stress (i.e., activation ofthe primary effector) required for significant cADPR accumulation in the smooth muscle. On the other hand, severe hypoxia could induce a marked increase in the P-NADH:P-NAD+ ratio without cADPR accumulation, due to a reduction in substrate (P-NAD+) availability. Either way it may be possible to observe increased superoxide and/or P-NADH formation, respectively, in pulmonary artery smooth muscle in the absence of cADPR-dependent, maintained constriction by hypoxia.
In addition to their studies on the whole lung, however, Waypa et al. (85) presented data from cultured pulmonary artery smooth muscle cells. These data are consistent with the idea that an increase in ROS production, measured by dichlorofluorescein fluorescence, at complex III in the ETC triggers an increase in cytoplasmic Ca2+ concentration. It is notable, however, that the increase in the Fura-2 fluorescence ratio reported is small and non-uniform in individual cells, and does not appear to be associated with cell contraction. Furthermore, one glance at the literature informs us that investigations from a variety of laboratories have provided contrary data (32). For example, in pulmonary arteries a decrease in ROS has been measured by lucigenin chemiluminescence (5, 6, 8), consistent with the classical view that a fell in Po2 results in a consequent fall in ROS (15). Not surprisingly, therefore, it has been stated that "the direct measurements of ROS are so demanding that they generate data supporting opposing views" (32, 77, 78). It is also important to note that cultured cells are known to lack many of the antioxidants normally present in cells in vivo, and are probably unrepresentative ofwild type cells (34). Therefore, studies on cultured cells may not be consistent with those carried out on acutely isolated cells, isolated arteries, or whole lungs, respectively. In this respect it should not be forgotten that the investigations of Leach et al. (52) and Waypa et al. (84) on the effects ofmitochondrial inhibitors on HPV in isolated pulmonary arteries and intact lungs, respectively, did not offer parallel measures of ROS levels. Furthermore, previous studies on pulmonary artery endothelial cells have shown a decrease in H202 production by hypoxia (91). This is significant, as the studies of Leach et al. (52) and Waypa et al. (84) may have been biased towards investigation of the effects of hypoxia on vasoconstrictor release from the pulmonary artery endothelium (see Chapter 6).
When considering the idea that cell signaling by hypoxia is mediated by ROS, however, I have most difficulty in explaining the fact that: a) Hyperoxia increases cellular ROS in a variety of preparations, including the rat lung and mitochondria derived from the lung (27, 41, 69, 76) and b) Hyperoxia but not hypoxia, increases the functional expression of antioxidants (18, 19, 40, 80). This is because short-term (hours) exposure to hyperoxia has little effect on resting tone of pulmonary arteries, and does not effect a change in pulmonary vascular resistance or the distribution of blood flow in the lung (36, 44). Allied to this, investigations on carotid body chemoreceptors have demonstrated that they are activated by hypoxia and inhibited by hyperoxia; the latter response being attributed to increased ROS (1,83). Furthermore, others have demonstrated that H202 decreases chemosensory discharge by the carotid body (66). Clearly, if increased cytoplasmic ROS acted as the primary effector of cell signaling by hypoxia and a common signaling pathway was present in all types of 02-sensing cell, then one would expect hypoxia and hyperoxia, respectively, to have similar effects. This is clearly not the case.
Consideration of ROS metabolism also questions a role for ROS in signaling by hypoxia. Superoxide dismutases rapidly convert superoxide to H202, which is subsequently reduced by glutathione peroxidase. Thus, H202 + 2GSH yields H20 + GSSG. GSH may then be regenerated by an NADPH glutathione reductase. Given these facts, an increase in cytoplasmic superoxide by hypoxia may be expected to elicit a consequent reduction in GSH and/or P-NADPH levels in pulmonary artery smooth muscle. In marked contrast, however, hypoxia has been shown to increase cellular GSH in pulmonary arteries and pulmonary artery endothelial cells (8, 21, 89), and hypoxia does not appear to alter P-NADPH levels in pulmonary artery smooth muscle (79). Furthermore, hyperoxia and not hypoxia increases glutathione uptake into endothelial cells (21). These findings are also inconsistent, therefore, with the idea that an increase in cytoplasmic superoxide and/or H202 act as primary mediators of HPV. There is also indirect evidence against a role for cytoplasmic ROS in increasing cADPR accumulation by hypoxia in pulmonary arteries. In short, superoxide has been shown to activate RyRs (46) and cADPR synthesis (48, 62), respectively, over a similar concentration range. One might expect SR Ca2+ release by hypoxia, therefore, to promote a degree of maintained pulmonary vasoconstriction in the presence of the cADPR antagonist 8-bromo-cADPR, because 8-bromo-cADPR does not block RyR activation per se (10, 22). In contrast, however, we have shown that 8-bromo-cADPR abolishes cADPR-dependent activation ofRyRs in pulmonary artery smooth muscle and HPV (10, 22).
14.1. Intramitochondrial Regulation of cADPR Synthesis in the Cytoplasm
One possible explanation for contrary findings with respect to the role of ROS in HPV could, however, lie in the site of production and effect, respectively. Previous investigations have demonstrated that ADP-ribosyl cyclase/CD38 activities may be present in the plasma membrane, SR membrane and mitochondrial membrane, respectively (56, 90). Furthermore, there is evidence to suggest that CD38 may be anchored to the mitochondrial outer membrane by proteins distinct from those that may anchor it to the plasma membrane (56). It is possible, therefore, that a buildup of mitochondrial superoxide may promote cADPR production in the cytoplasm. This is clear from the fact that enzyme activities of membrane associated CD38 are generally thought to be conferred by the extracellular/ extra-organellar domain, whilst the intra-organellar domain may offer a site for regulation (55). Thus, mitochondrial superoxide and/or H202 could directly modulate cADPR synthesis and thereby RyR activation due to the close association between mitochondria and the SR (17, 20, 33). To confirm this proposal, however, future investigations must demonstrate that cADPR synthesis occurs in the mitochondrial fraction of pulmonary artery smooth muscle homogenates free from any microsomal contamination. Furthermore, if cADPR synthesis does occur in a pure mitochondrial fraction, we must establish that the relatively small number of mitochondria in pulmonary artery smooth muscle is sufficient to support the observed level of cADPR accumulation by hypoxia. We must also account for the fact that hyperoxia and not hypoxia, increases the functional expression of antioxidants (18, 19, 40, 80).
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