Cardiovascular Research

Cardiovascular Research 2006 71(4):620-629; doi:10.1016/j.cardiores.2006.04.009

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Copyright © 2006, European Society of Cardiology

Oxygen sensors in hypoxic pulmonary vasoconstriction

Norbert Weissmann^*, Natascha Sommer, Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, Werner Seeger and Friedrich Grimminger

University of Giessen Lung Center (UGLC), Medical Clinic II, Justus-Liebig-University Giessen, Klinikstrasse 36, 35392 Giessen, Germany

^* Corresponding author. Tel.: +49 641 99 42351; fax: +49 641 99 42419. Email address: Norbert.Weissmann{at}UGLC.de Norbert.Weissmann{at}innere.med.uni-giessen.de

Received 31 January 2006; revised 16 March 2006; accepted 9 April 2006

	Abstract

Top Abstract 1. Introduction 2. Characteristics of hypoxic... 3. The mitochondria as... 4. NAD(P)H-oxidase as a... 5. The role of... 6. Cytochrome P450 enzymes... 7. Concluding remarks References

 
Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism adapting lung perfusion to regional ventilation. Perturbations to HPV, such as those occurring in pneumonia, acute respiratory distress syndrome and liver failure, can result in arterial hypoxemia. Under conditions of general hypoxia, HPV increases pulmonary vascular resistance and thus causes acute pulmonary hypertension. Despite intensive research, the underlying mechanisms of HPV have not been fully elucidated. Deciphering signalling pathways that result in HPV could suggest novel approaches to address a failure of HPV, as well as for the treatment of pulmonary hypertension associated with HPV. Within this context, this review focuses on current concepts in the oxygen sensing mechanisms that underlie HPV.

KEYWORDS Hypoxic pulmonary vasoconstriction; Oxygen; Oxygen sensing; Lung; Hypoxia

	1. Introduction

Top Abstract 1. Introduction 2. Characteristics of hypoxic... 3. The mitochondria as... 4. NAD(P)H-oxidase as a... 5. The role of... 6. Cytochrome P450 enzymes... 7. Concluding remarks References

 
Hypoxic pulmonary vasoconstriction is a physiological response of the lung to alveolar hypoxia, which redistributes pulmonary blood flow from areas of low oxygen partial pressure to areas of high oxygen availability. This mechanism thus optimises gas exchange and helps to prevent arterial hypoxemia [1,2].

Impairment of HPV under pathophysiological conditions, including acute respiratory distress syndrome [3] or hepato-pulmonary syndrome [4], or during anaesthesia [5], may result in poor arterial blood oxygenation. Alternatively, global, prolonged alveolar hypoxia that occurs at high altitude, or during impairment of respiratory functions (for example, as occurs in chronic obstructive pulmonary disease (COPD), pneumonia, fibrosis, or neurological diseases) may result in pulmonary hypertension. Both persistent HPV and hypoxia-altered gene regulation may contribute to hypoxia-induced pulmonary hypertension.

Due to the opposing functions of lung and systemic vessels–one taking up, the other delivering oxygen–different responses to hypoxia have emerged. While systemic vessels of adults dilate during hypoxia, pulmonary vessels constrict. From an ontogenetic point of view, hypoxic pulmonary vasoconstriction may better be termed "normoxic pulmonary vasodilation". In utero, persistent vasoconstriction of pulmonary vessels helps prevent perfusion of non-inflated lungs. After birth, inflation of the alveoli and the concomitant increase in alveolar oxygen partial pressure leads to vasodilation and perfusion of the lung vasculature. Although the importance of HPV for pulmonary gas exchange was recognised early [6], the underlying oxygen sensing and signal transduction processes have not been clarified. Elucidation of the oxygen sensing and signal transduction mechanisms of HPV could serve as the basis for the development of new therapeutic approaches to treat diseases associated with a disturbance in HPV or acute pulmonary hypertension associated with global HPV. Within this context, this review focuses on current concepts of oxygen sensing mechanisms that underlie HPV (Fig. 1).

View larger version (60K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Current concepts of the oxygen sensing of hypoxic pulmonary vasoconstriction. Possible oxygen sensors are shown in green, mediators of hypoxic pulmonary vasoconstriction (HPV) in yellow. For details see text. pO2, oxygen partial pressure; , mitochondrial membrane potential; SR, sarcoplasmic reticulum; HETE, hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; Em, cellular membrane potential; BK, large conductance Ca2+- and voltage-gated potassium channel; HO-2, hemoxygenase-2. (A) Includes those concepts that comprise a decrease in reactive oxygen species (ROS) as a trigger for HPV (red lines), (B) those that comprise an increase in ROS as a trigger for HPV (blue lines).

 

	2. Characteristics of hypoxic pulmonary vasoconstriction and location of the oxygen sensor

Top Abstract 1. Introduction 2. Characteristics of hypoxic... 3. The mitochondria as... 4. NAD(P)H-oxidase as a... 5. The role of... 6. Cytochrome P450 enzymes... 7. Concluding remarks References

 
Pulmonary artery pressure is increased during hypoxic ventilation [7,8], leading von Euler and Liljestrand to suggest that ventilation-perfusion matching was the purpose of this increase in pulmonary artery pressure [6]. It was believed that a self-regulatory mechanism intrinsic to the lung controls HPV, since HPV still occurs after denervation of the lung and in explanted lungs, thus excluding neural or humoral effects [9,10]. Furthermore, there were no histological or pharmacological hints of a contribution by neural mechanisms [11,12]. Along these lines, foetal pulmonary arterioles co-transplanted with neonatal lung tissue into the hamster cheek pouch demonstrated HPV before innervation [13].

The strength of the HPV response depends on species, age, gender, pCO₂, pH, and methodology employed [11,14–16], although its effector mechanism is independent from these factors. HPV is a highly conserved process in mammals [17–19], birds [20], reptiles [21], and even fish [22]. HPV is triggered by mild hypoxia (alveolar pO₂<100 mm Hg) [18]. The precapillary smooth muscle layer of the resistance vessels, located at the acinus entrance, has been identified as the effector cell type [23–26]. Since isolated pulmonary artery smooth muscle cells (PASMC) respond to hypoxia by contraction and an elevation in intracellular Ca²⁺ levels, these cells represent both the sensor and effector cell type [27–31] in the context of acute hypoxia. For sustained (>30 min) hypoxia, a contribution of endothelial cells must also be considered [32,33].

The kinetics of sustained HPV have not been fully resolved [34–36]. There is no doubt that HPV occurs, and that HPV can be rapidly switched off, since HPV has to adapt perfusion to ventilation immediately upon changes in the alveolar oxygen partial pressure [18]. For sustained hypoxia a temporary vasodilation has been described, followed by a secondary vasoconstrictor response. Sustained HPV may be of major relevance for continuous ventilation-perfusion matching and under conditions of generalised hypoxia, which results in pulmonary hypertension.

Pathways leading to contraction of precapillary PASMC rely on an increase in cytosolic calcium, including an influx from the extracellular space as well as from intracellular stores, and membrane depolarisation attributed to closure of potassium channels [37]. The role of potassium channels in HPV is reviewed elsewhere in this Review Focus. For sustained HPV, a Ca²⁺ sensitisation, in addition to an increase in cytosolic calcium, possibly via activation of Rho-kinase, has been suggested [38–40]. However, it is not yet clear, if Rho-kinase and/or other protein kinases only play a modulating or an indispensible role in HPV [41].

	3. The mitochondria as possible oxygen sensors of HPV

Top Abstract 1. Introduction 2. Characteristics of hypoxic... 3. The mitochondria as... 4. NAD(P)H-oxidase as a... 5. The role of... 6. Cytochrome P450 enzymes... 7. Concluding remarks References

 
Apart from being the main site of oxygen consumption, two arguments are in favour of a role for mitochondria as primary oxygen sensors. First, inhibitors of the mitochondrial electron transport chain (ETC) specifically inhibit HPV [31,42,43], and second, PASMCs without a functional respiratory chain do not show hypoxia-specific responses [29].

In general, mitochondria have diverse functions in the cell related to energy conservation, apoptosis, calcium regulation and intracellular signalling [44]. Mitochondria generate a proton gradient across the mitochondrial membrane, thereby providing energy for ATP synthesis. The electrons are transferred along a redox gradient, and finally to molecular oxygen. The ETC consists of several complexes: complexes I and II, which provide the electrons; complex III including an electron cycle; and complex IV, the final centre for the reduction of oxygen.

Electrons from the ETC may be "accidentally" transferred to molecular oxygen, resulting in the generation of superoxide radicals. After conversion by superoxide dismutase (SOD), the resulting H₂O₂ can readily diffuse through the membrane into the cytosol. Alternatively, superoxide can pass through an anion channel from the intermembrane space into the extramitochondrial environment [43,45,46]. The putative role of mitochondria during HPV is depicted in Fig. 1, .

Two main theories exist concerning a role for mitochondria and reactive oxygen species (ROS) in HPV. (1) The original redox hypothesis, proposed by Weir, Archer and colleagues, assumed a decrease in mitochondrial ROS, shifting the cellular redox state towards a more reduced state, resulting in the inhibition of K_v channels (Fig. 1A, a). This closure of potassium channels is mediated by the redox pairs GSH/GSSG and NADH/NAD [36,47]. (2) In contrast, Schumacker, Chandel and co-workers suggested that an increase in ROS production during hypoxia triggers intracellular calcium release and thus HPV [43,48] (Fig. 1B, a). The latter theory assumes that mitochondrial complex III is the ROS producing site.

3.1. Mitochondria-dependent decrease of ROS in HPV
Early investigations found that the mitochondrial inhibitors rotenone, antimycin A, azide, and cyanide, as well as dinitrophenol, increased vascular pressure under normoxic conditions and subsequently inhibited HPV in isolated blood-perfused rat lungs [49]. Later, Archer, Weir and colleagues demonstrated that hypoxia and the proximal ETC inhibitors, rotenone and antimycin A, decreased lung ROS release (detected by chemiluminescence), whereas distal inhibition with cyanide increased ROS release during normoxia. Under normoxic conditions, rotenone and antimycin A increased pulmonary artery pressure and inhibited HPV, while cyanide increased vascular pressure but did not decrease HPV [50]. These findings are in line with the observation that rotenone and antimycin A mimicked HPV in isolated pulmonary arteries and in PASMCs, and decreased ROS production, concomitant with inhibition of potassium channels in PASMCs [31]. In contrast, these agents had opposing effects in renal tissue. These differences were attributed to a higher basal ROS production in the lung pulmonary arteries, compared to renal arteries, due to the lower respiration rates in lung mitochondria, the reduced complexes I and III content, and a lower membrane potential in pulmonary compared with renal arteries [31]. Higher ROS production has also been observed in patients with complex I deficiency [51], perhaps explaining the high ROS production by mitochondria of PASMCs under normoxic conditions, compared to studies that provide evidence that there is low or no ROS release by intact mitochondria at all [52,53]. Archer and colleagues concluded from these pharmacological interventions that ETC-inhibition proximal to the site of mitochondrial ROS release at complex I or III attenuated the reduction potential of this complex, reduced ROS production, and increased pulmonary arterial pressure. Blockade of the electron mitochondrial respiratory chain distal to complex III had no effect on pulmonary vascular tone [54]. Thus, the decreased ROS release under hypoxic conditions triggers HPV via K⁺-channels as explained above (Fig. 1A, a).

3.2. Mitochondria-dependent increase of ROS in HPV
Although a decrease in ROS production during hypoxia could be explained by decreased availability of oxygen, a prerequisite for increased mitochondria-derived ROS during hypoxia indicate an alteration in the properties of the ETC, which could be achieved by hypoxia-induced inhibition of cytochrome c oxidase and electron flow as initially suggested after investigations in hepatocytes [55]. Paul Schumacker's group later provided evidence that increased ROS release from the semiubiquinone binding site in mitochondrial complex III occurs under hypoxia because (1) in isolated rat lungs the proximal ETC-inhibitors rotenone, DPI and myxothiazol inhibited HPV, while the distal inhibitors antimycin A and cyanide did not [43]; (2) proximal inhibitors also attenuated hypoxia-induced contraction and increase in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) of PASMCs [29]; (3) catalase overexpression inhibited the hypoxia-induced [Ca²⁺]_i increase, as well as the hypoxia-induced increase in ROS [29,43]; and (4) myxothiazol attenuated hypoxia-induced increase in ROS, abolished HPV, and blocked hypoxia-induced [Ca²⁺]_i increase; but (5) antimycin A had no specific effects on the hypoxia-induced responses in isolated lungs or PASMCs [29,43]. In line with these observations, inhibition of the hypoxia-induced elevation in [Ca²⁺]_i by rotenone could be reversed by succinate in isolated pulmonary arteries of the rat [56].

Data from our laboratory obtained in isolated perfused rabbit lungs are consistent with the effect of the proximal electron chain inhibitors (inhibiting HPV without being hypoxia mimics) and the complex III inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) (mimicking HPV), but disagree with respect to the effects of antimycin A, inhibiting HPV without being a hypoxia mimic and cyanide, inhibiting HPV [42,57]. Complex II of the ETC has also been suggested to be a source of ROS release in HPV in a study in murine lung sections [58]. While ROS production under normoxic conditions required complexes I and III in this investigation, ROS generation under hypoxic conditions also required complex II. Inhibition of the reversed enzymatic reaction of the succinate dehydrogenase, i.e., fumarate reductase, by application of succinate, specifically abolished ROS generation under hypoxic, but not normoxic, conditions [58].

While these studies substantially relied on the use of inhibitors, PASMCs that lack a functional ETC were incapable of generating ROS under hypoxic conditions, and lost their response to hypoxia, although they still responded to the thromboxane mimetic U46619 [GenBank] [43,59].

It has been suggested that ROS can escape from the mitochondria through an anion channel, and subsequently induce vasoconstriction by downstream effects on Ca²⁺ metabolism and sensitivity (Fig. 1B, a). Alternatively, these channels are dependent on mitochondrial membrane potential [46]. This may in turn be affected by respiration, calcium metabolism and mitochondrial ATP-sensitive potassium channels, that may also affect mitochondrial ROS release and thus again interact with HPV [60], suggesting an even more complex role in HPV than simple ROS trafficking. The current data do not support a conclusive role for mitochondria in HPV, particularly concerning their contribution to ROS release. The diverging effects of different mitochondrial inhibitors in various investigations may be explained by the different experimental settings, species, tissues and concentrations of the inhibitors used, as well as the methods used for quantification of ROS. They may also be attributed to properties of these agents exceeding simple variation of ROS production, for example, also affecting calcium homeostasis and ATP production [45,61].

3.3. Mitochondria and calcium homeostasis
Mitochondria play a role in cytosolic calcium homeostasis through a calcium uniporter that is driven by the mitochondrial membrane potential, and the concentration of cytosolic calcium [45]. A decrease in membrane potential due to impaired respiration induces mitochondrial calcium release, and the intracellular calcium profile can be shaped by mitochondrial buffering of changes in [Ca₂₊]_i [44].

In carotid body cells, hypoxia <60 mm Hg leads to mitochondrial membrane depolarisation [62], which could reduce mitochondrial Ca²⁺ uptake, resulting in an increase in cytosolic calcium (Fig. 1, b). Cyanide, rotenone and uncouplers like carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) increase [Ca²⁺]_i in PASMCs during calcium release from the sarcoplasmic reticulum [63–65]. It was therefore suggested that inhibition of mitochondrial calcium uptake by FCCP or hypoxia augmented intracellular calcium increase [66]. Furthermore, an increase in hypoxic tone and HPV in isolated lungs has been described using mitochondrial uncouplers [18]. Thus, mitochondria may play an as-yet-unproven role in HPV related to Ca²⁺-homeostasis independent of or in addition to mitochondrial ROS release.

3.4. Mitochondrial ATP production, energy state, and cADP-ribose
The role of ATP as a second messenger for HPV has been suggested since oxidative phosphorylation is the main oxygen consumption site, and inhibitors of the ETC and of glycolysis caused vasoconstriction [67,68]. However, the role of ATP was questioned because (1) changes in the ATP content or deterioration of energy state during acute hypoxia could not be detected [69–71], (2) the K_m for cytochrome c oxidase seemed to be too low to decrease ATP under conditions of mild hypoxia [72], and (3) inhibitors of the cytochrome c oxidase induced vasoconstriction under normoxic conditions, but did not abolish HPV in some investigations [43]. In contrast oxidative ATP generation may be impaired and the cellular energy state is maintained by upregulation of glycolysis during sustained HPV [56,69,70]. For example, in isolated ferret lungs, Wiener and colleagues showed that high glucose levels prevented vasodilation following acute HPV, while pyruvate did not [73,74], suggesting that glucose metabolism beyond pyruvate is responsible for the inhibition of sustained HPV. Similarly, low glucose levels suppressed sustained HPV in isolated rat small pulmonary arteries. Since pyruvate did not reverse suppression of sustained HPV in this study, glucose may facilitate sustained HPV by a mechanism independent from glucose metabolism downstream of pyruvate [56]. However, the glucose concentration applied was much lower than that used by Wiener and colleagues, perhaps explaining these different results [74].

A recent new concept assumes that mild hypoxia leads to inhibition of the respiratory chain and a small decrease in ATP production, which does not affect energy state, but rather acts as a second messenger (Fig. 1, c). Thus, an increase in the AMP/ATP ratio activates AMP-activated protein kinase 1 (AMPK) and increases cyclic ADP-ribose (cADPR) that releases calcium through ryanodine-sensitive calcium stores as a first step in the HPV signalling mechanism [75]. This is an extension of the oxygen sensing mechanism proposed some years ago, assuming that during sustained HPV, hypoxia increases β-NADH levels, which then increase the net amount of cADPR synthesised from β-NAD+ by ADP-ribosyl cyclase, and simultaneously inhibit cADPR degradation by cADP-ribosyl hydrolase [76]. A decrease in ATP levels under mild hypoxia may be assisted by a low oxygen affinity cytochrome c oxidase in PASMCs, as has been proposed for carotid body cells [77,78]. Alternatively, the cADP-ribose system may also be regulated by interference with ROS, since low levels of superoxide stimulated calcium release via cADPR [79]. That this possible link to ROS plays a role in HPV, however, remains to be proven.

	4. NAD(P)H-oxidase as a possible oxygen sensor of HPV

Top Abstract 1. Introduction 2. Characteristics of hypoxic... 3. The mitochondria as... 4. NAD(P)H-oxidase as a... 5. The role of... 6. Cytochrome P450 enzymes... 7. Concluding remarks References

 
NAD(P)H-oxidases are superoxide-generating enzymes. Classical leukocyte NADPH-oxidase is a multiprotein complex, consisting of membrane-bound gp91^phox (now also termed NOX2) and p22, which comprise the cytochrome b558; and cytosolic p47^phox, p67^phox, and p40^phox. Superoxide production by NADPH-oxidase is induced by assembly of these two sets of subunits. Activation can be induced by at least a phosphorylation of p47^phox and Rac GTPase activation [80]. A variety of NADPH-oxidase isoforms have been identified that can substitute for NOX2 (e.g. NOX1, NOX3, NOX4, and DUOX). These isoforms have unique features, including the release of superoxide into the intracellular milieu, rather than extracellularly, and produce lower amounts of superoxide than the phagocytic type [81,82]. Two isoforms of p47^phox and p67^phox, termed NOXO1 and NOXA1 interact with NOX1 to generate high amounts of superoxide without activation by protein kinase C-dependent phosphorylation [83,84]. Regulation of NADPH-oxidase activity may involve phospholipase A2 and protein kinase C [85,86]. NAD(P)H-oxidases might also be activated by depolarisation of the cell [87] or lead to depolarisation itself [88].

The concept of NAD(P)H-oxidases as oxygen sensors for HPV (Fig. 1, ) emerged against (1) the background that they are oxygen sensing candidates in other oxygen sensor systems [89] and (2) the study of Thomas et al. [90] which demonstrated that the NADPH-oxidase inhibitor DPI inhibited HPV. We have confirmed these data, and excluded interference with NO as a second target of DPI [91]. The NAD(P)H-oxidase concept got a second impetus after the investigations of Marshall et al. [92] and Wolin et al. [93]. The former group suggested an NADPH-oxidase related increase in superoxide as the mechanism underlying HPV, while the latter group suggested that an NADH oxidoreductase-related decrease in superoxide and H₂O₂, through stimulation of the soluble guanylate cyclase, could decrease vascular tone under normoxic conditions. This hypothesis thus suggested a "loss of normoxic vasodilation" during HPV triggered by this pathway.

Thus, two diverging concepts regarding the contribution of NAD(P)H-oxidase-derived superoxide currently exist: one proposing an upregulation (Fig. 1B, ) and the second a downregulation of superoxide (Fig. 1A, ).

4.1. NAD(P)H-oxidase-dependent increase in ROS in HPV
In an elegant study, Marshall and colleagues described an NAD(P)H-oxidase in pulmonary arteries with an unusually low redox potential. Isolated smooth muscle cells from small pulmonary arteries demonstrated an increase in superoxide production that was derived from an NAD(P)H-oxidase with an unusually low redox potential [92]. An upregulation of superoxide, and subsequently H₂O₂, as the underlying pathway of HPV has also been suggested by data from our laboratory [94]. However, the major drawback of studies suggesting an NAD(P)H-oxidase as a pulmonary oxygen sensor was that they relied primarily on one NAD(P)H-oxidase inhibitor, DPI, which also inhibits other FAD-dependent enzymes, the mitochondrial ETC, as well as potassium channels [95,96]. Therefore, different NAD(P)H oxidase inhibitors were investigated: apocynin, which, however, interfered with vascular tone in general in isolated rabbit lung studies [91], and 4-(2-aminoethyl)benzenesulfonyl fluoride, which selectively inhibited HPV in isolated rabbit lungs, but not vasoconstriction induced by other mechanisms [97]. This study also suggested an NAD(P)H-oxidase-derived increase in ROS as the underlying mechanism of HPV [97,98]. In line with these studies, protein kinase C (PKC), a possible activator of the NADPH-oxidase, has been suggested to regulate HPV via NADPH-oxidases [86] (although PKC may also affect HPV without interaction with an NADPH-oxidase [41]), and a phospholipase A₂ knockout mouse exhibited reduced HPV that may also interfere with the NADPH-oxidase pathway [85,99]. This theory was confounded by the observation that gp91^phox-deficient mice fully responded to acute hypoxia [100]. Nevertheless, these experiments cannot rule out an NAD(P)H oxidase isoform being active as an oxygen sensor. In line with this hypothesis, we recently demonstrated that mice deficient in the cytosolic NADPH-oxidase subunit p47 exhibited 25% reduced acute, but unchanged sustained HPV. This supported the concept that isoforms of the leukocyte NADPH-oxidase may, at least in part, function as oxygen sensors in HPV. That NADPH-oxidases in principle are involved in hypoxic signalling pathways was shown in studies on neuroepithelial bodies (NEB) [89].

4.2. NAD(P)H-oxidase dependent decrease of ROS in HPV
The group of Wolin and Burke-Wolin suggested that an NADH oxidase-mediated decrease in superoxide, and subsequently H₂O₂, under hypoxic conditions may lead to decreased GMP levels, through reduced stimulation of the soluble guanylate cyclase (sGC), and thus vasoconstriction [93,101]. In addition, NO may act synergistically in this pathway with respect to HPV. In principle, cGMP release from sGC can be triggered by H₂O₂, CO and NO. The release of NO in the lung is dependent on oxygen, as it is reduced under hypoxic conditions [102,103]. However, the H₂O₂-sGC concept was challenged by data that demonstrated that only NO-triggered sGC stimulation interferes specifically with HPV [104]. Recently Wolin et al. have put forward an interesting new version of their oxygen-sensing concept. According to this hypothesis, the concentration of NADPH, and therefore ROS production, is higher in pulmonary vessel cells (compared to coronary smooth muscle cells) due to higher levels of glucose-6-phosphate-dehydrogenase, the rate-limiting enzyme of pentose phosphate metabolism by the pentose phosphate pathway (PPP) [105]. Under hypoxic conditions, high levels of glucose-6-phosphate-dehydrogenase compete with glycolysis, maintain high NADPH levels, and therefore–in combination with hypoxic inhibition of NAD(P)H-oxidases–maintain high reduction levels in pulmonary cells. In contrast, in coronary smooth muscle cells NADPH is oxidised because of an inhibited PPP [101]. Thus, inhibition of PPP decreases HPV via activation of the cGMP pathway [106]. While the decrease in ROS formation by NAD(P)H-oxidases can be explained by a lack of the substrate oxygen, it remains unclear how NAD(P)H-oxidases may increase superoxide release under hypoxic conditions. The concept of an NAD(P)H-oxidase-derived increase in ROS has to assume that oxygen is not the rate limiting factor in ROS production by NAD(P)H-oxidases, but rather that the activity of the enzyme is regulated by other mechanisms, for example, an increased electron flux through the oxidase under hypoxic conditions. We have recently published data that suggest that NADPH-oxidase-derived lung superoxide release can be increased during hypoxia [107]. However, molecular proof of such mechanism is still lacking.

	5. The role of reactive oxygen species (ROS) in HPV

Top Abstract 1. Introduction 2. Characteristics of hypoxic... 3. The mitochondria as... 4. NAD(P)H-oxidase as a... 5. The role of... 6. Cytochrome P450 enzymes... 7. Concluding remarks References

 
ROS play a key role in HPV signalling, however, there is no consensus regarding the question of whether ROS are increased or decreased under hypoxic conditions (for review see [108,109]) (Fig. 1A and B). This disagreement has implications for the interpretation of the mitochondrial and NAD(P)H-oxidase concepts of oxygen sensing, therefore, we will briefly summarise here the main investigations that have focused on oxygen-dependent ROS release in the pulmonary system.

Direct measurement of ROS in isolated rat lungs revealed a decrease in superoxide under hypoxic conditions using luminol and lucigenin enhanced chemiluminescence [50,110]. These results are consistent with (a) decreased intravascular superoxide release in [107], and (b) decreased H₂O₂ levels exhaled from [111] isolated rabbit lungs undergoing hypoxic ventilation. Furthermore, ROS detection with three different dyes (amplex red, 2',7'-dichlorofluorescin diacetate (DCFH), and lucigenin) suggested decreased ROS levels in rat pulmonary arteries maintained under hypoxic conditions [31]. Intravascular superoxide release in isolated rabbit lungs quantified with a spin probe in electron spin resonance spectroscopy (ESR) indicated a decrease in superoxide release under hypoxic conditions, however, with a tendency towards a smaller decrease in severe hypoxia [107]. In contrast, measurements in isolated porcine pulmonary arteries suggested a hypoxia-mediated increase in ROS using lucigenin chemiluminescence and DCFH, and these observations are supported by ESR spectroscopy measurements indicating release of hydroxyl and alkyl radicals during hypoxia [30]. Along these lines, cellular measurement of ROS release suggested a hypoxia-mediated increase in calf [92], rat [43,112] and rabbit PASMCs [58]. These results were recently confirmed by a new, elegant technique, quantifying ROS with a fluorescence resonance energy transfer (FRET) sensor technique, supporting the concept of increased ROS generation [113]. We have also recently demonstrated an increase in intravascular superoxide release during hypoxic ventilation in p47^phox-deficient mice, suggesting a non-phagocytic source of increased ROS release during HPV, that is camouflaged by an overall decreased phagocytic ROS release under hypoxic conditions [57].

Although it has been suggested that limitations in the methods used for ROS detection are responsible for the discordant effects observed, it also remains possible that alternative explanations may exist. While fluorescent probes have been criticised for yielding for false-positive results, for example, as a consequence of redox cycling, the ESR techniques may also have some limitation, since they indirectly quantify ROS generation. Convincing data have been generated with the new FRET sensor technique [113]. Nevertheless this investigation could also not explain discrepancies observed by Michelakis et al. [31], using three different methods that compared the pulmonary and the renal system, which were by themselves conclusive.

Apart from artefacts related to the ROS detection methodology, the experimental models (isolated lungs, vessels, or cells) employed and the kinetics and timeframe of the measurements must also be taken into account as potential sources of error. Similarly, the question as to the location of the ROS must also be considered. For example, is it intravascular or exhaled ROS release that is representative of, or participates in, the signalling that underlies HPV, which is suggested to occur within the vascular smooth muscle cell? Therefore, the apparently conflicting conclusions concerning ROS in HPV may be explained by the hypothesis that a local, subcellular and compartmentalised regulation of ROS triggers HPV, and that this signal is obscured in the background of a general decrease in ROS production in the remainder of the cell that is not linked to HPV.

	6. Cytochrome P450 enzymes and heme oxygenase-2 as possible oxygen sensors of HPV

Top Abstract 1. Introduction 2. Characteristics of hypoxic... 3. The mitochondria as... 4. NAD(P)H-oxidase as a... 5. The role of... 6. Cytochrome P450 enzymes... 7. Concluding remarks References

 
Arachidonic acid (AA)-associated pathways activated by cyclooxygenase and lipoxygenase are well known potent modulators of vessel tone, but have not been shown to mediate any HPV-specific reaction [14,114]. However, the third group of metabolites derived from AA by cytochrome P450 monooxygenases, namely hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), are suggested to be involved in HPV [115–117], since oxygen serves as a substrate for these enzymes (Fig. 1, ). Cytochrome P450 enzymes are implicated in a new oxygen-sensing concept: in addition to heme proteins, hemoxygenase-2 (HO-2) may play a role in oxygen-sensing via cytochrome P450-dependent release of CO [118]. A large conductance calcium and voltage dependent potassium channel (BK (Ca)) is tightly associated with HO-2 and is activated by HO-2-derived CO under normoxic conditions [119,120] (Fig. 1, ). Carotid body cells demonstrated an HO-2-dependent hypoxic BK channel inhibition, which indicated a possible role of HO-2 as an oxygen sensor that controls channel activity during oxygen deprivation. However, this elegant and new concept of oxygen sensing still needs to be proven in the pulmonary system.

	7. Concluding remarks

Top Abstract 1. Introduction 2. Characteristics of hypoxic... 3. The mitochondria as... 4. NAD(P)H-oxidase as a... 5. The role of... 6. Cytochrome P450 enzymes... 7. Concluding remarks References

 
Although intensive research concerning the mechanism of HPV started 60 years ago with the recognition of its importance for pulmonary gas exchange, the current review demonstrates that we are far from being able to draw a conclusive picture of its regulation, even at the initial step: the oxygen sensing process. Currently, more than five different concepts are discussed, some of them suggesting completely opposing mechanisms. One major aspect of this discussion is the fact that some investigations found an increase, while others a decrease in ROS generation under hypoxic conditions, which are proposed to be involved in the downstream signalling of the oxygen sensing process. While this was initially attributed to the different techniques used for ROS detection, and criticised as not being reliable, recent new techniques like ESR spectroscopy and FRET sensor technologies may help to overcome these problems, although these techniques are also unlikely to be free of methodological problems. These conflicting results may also reflect the different models employed (e.g. cellular, vessel, intact organ, intact animal investigations), and the duration of the hypoxic treatment applied. Furthermore, a localised subcellular increase in ROS may trigger HPV, but this localised effect may be covered by a decrease in ROS in the remainder of the cell, which may not be involved in the HPV pathway. Also, it has to be taken into account that the different sensors suggested may affect each other, for example, proper mitochondrial function is a prerequisite for NAD(P)H-oxidase systems to be operative.

More provocatively, one may also hypothesise that changes in ROS levels occur as a consequence of the alteration of the cellular oxygen partial pressure but are not directly linked to HPV [121], although no convincing evidence for such a hypothesis has been provided.

Although not discussed in detail in this review, different signal transduction processes, and possibly oxygen sensing processes, are involved in the regulation of the very acute phase of HPV (occurring within seconds) and the sustained phase (several hours) of hypoxia. The latter is proposed to result in hypoxia-induced pulmonary hypertension. In this regard it was shown that ATP, oxidative phosphorylation, Ca²⁺ metabolism, NAD(P)H-oxidase and mitochondria play different roles in acute and sustained HPV. Bearing this in mind, we have to take into account that HPV is a multifactorial process [35]. In this regard, we have recently suggested that both a mitochondrial and an NAD(P)H-oxidase mechanism contribute to the regulation of acute HPV [57].

Identification of the pulmonary oxygen sensing processes underlying HPV remains a tremendous challenge, even 60 years after von Euler and Liljestrand's initial observations. However, elucidation of the molecular mechanism(s) that regulate this process would be a key step in the development of novel approaches to address an impaired HPV response, and for the treatment of HPV-related diseases. To reach this goal, new molecular tools and subcellular approaches will have to be developed and refined.

	Acknowledgements

 
The authors thank Dr. Rory Morty for linguistic editing of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft SFB 547, project B7.

	Notes

 
Time for primary review 22 days

	References

Top Abstract 1. Introduction 2. Characteristics of hypoxic... 3. The mitochondria as... 4. NAD(P)H-oxidase as a... 5. The role of... 6. Cytochrome P450 enzymes... 7. Concluding remarks References

 

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