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(Circulation Research. 2002;91:649.)
© 2002 American Heart Association, Inc.

Editorials

Hypoxic Pulmonary Vasoconstriction

Ups and Downs of Reactive Oxygen Species

James S.K. Sham

From the Division of Pulmonary and Critical Care Medicine, Johns Hopkins Medical Institutions, Baltimore, Md.

Correspondence to James S.K. Sham, Division of Pulmonary and Critical Care Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21224. E-mail jsks{at}welchlink.welch.jhu.edu

Key Words: hydrogen peroxide • calcium • ryanodine receptor • hypoxia

Reduction in alveolar oxygen tension causes reversible hypoxic pulmonary vasoconstriction (HPV), which is thought to serve as an adaptive mechanism for diverting blood flow from poorly ventilated to better-ventilated regions of the lung to improve ventilation-perfusion matching. Since first described by Von Euler and Liljestrand¹ 50 years ago, significant advances have been made in determining the O₂ dependence, temporal characteristics, loci of response, physiological conditions, and neurohumoral factors that influence HPV in intact animal, isolated lung, and pulmonary arterial rings. Recent studies using isolated pulmonary arterial smooth muscle cells (PASMCs) indicate that hypoxia directly causes membrane depolarization, increase in intracellular [Ca²⁺], and cell-shortening; thus, the essential elements of HPV are contained in PASMCs, even though one or more endothelially derived mediators, such as ET-1 and/or an unknown factor,^2,3 are required for the full expression of hypoxic response. However, despite this progress, the precise cellular mechanism of O₂ sensing for HPV remains controversial.

There are several proposed mechanisms for O₂ sensing in PASMCs, most of which are related to reactive oxygen species (ROS).⁴ The first hypothesis proposed that a smooth muscle microsomal NADH oxidoreductase, which generates H₂O₂ via a PO₂-dependent production of superoxide, acts as the O₂ sensor; H₂O₂ interacts with catalase to activate soluble guanylate cyclase and increase cGMP to provide tonic vasorelaxation during normoxia.^5,6 Under hypoxic conditions, production of H₂O₂ from NADH oxidase would be reduced, leading to the removal of the vasorelaxant influence, and pulmonary vasoconstriction.

The second hypothesis proposed that hypoxia activates a sarcolemmal NADPH oxidase, similar to that found in neutrophils, to paradoxically increase superoxide production to signal HPV. This notion is supported by the evidence that NADPH oxidase was detected in pulmonary arteries by Western blot analysis and immunostaining of gp91^phox subunit, and by spectrometric analysis of cytochrome b_-245. Hypoxia was found to increase superoxide anion production, which was blocked by the inhibitor of NADPH oxidase, diphenyleneiodium (DPI), in cultured PASMCs.⁷ Moreover, DPI blocked hypoxic responses in isolated lung, isolated arteries, and PASMCs.^8,9 However, the supporting evidence is weakened by the fact that DPI is a nonspecific blocker for many flavoprotein-dependent enzymes including complex I of the mitochondrial electron transport chain (ETC) and NO synthase (NOS), and other proteins such as K⁺ and Ca²⁺ channels, and by the unaltered HPV in the phagocyte NADPH oxidase gp91^phox subunit knockout mice.^10,11

The third hypothesis has received the most attention. It proposed a redox-based regulation of K_V channel as the O₂ sensing mechanism for HPV.¹² According to this hypothesis, hypoxia reduces mitochondrial electron transport, decreases ROS generation, and shifts the ratio of redox couples (ie, GSSG:GSH and NAD⁺:NADH) toward a more reduced state, leading to inhibition of K_V channels, membrane depolarization, Ca²⁺ influx via L-type Ca²⁺ channels, increase in cytosolic [Ca²⁺], and vasoconstriction. Consistent with this hypothesis, hypoxia or mitochondrial ETC inhibitors, rotenone and antimycin A, caused inhibition of K_V current in PASMCs, vasoconstriction, and decreased luminol-enhanced chemiluminescence in isolated lungs, suggesting a decrease in ROS production.^12,13 Agents that promoted cytosolic oxidation were reported to activate, whereas agents that promote cytosolic reduction caused inhibition of K_V currents.¹⁴ However, the role of K_V channel in initiating HPV has been questioned because an antagonist of K_V channels failed to inhibit HPV,^2,15 and a functional sarcoplasmic reticulum (SR) was found to be required for HPV.¹⁶ Hence, it is proposed alternatively that K_V channel inhibition during hypoxia is secondary to Ca²⁺ release from SR. Beside K_V channels, ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase may also act as redox sensors for HPV.¹⁷ A reduction of ß-NAD⁺:ß-NADH ratio during hypoxia was reported to stimulate ADP-ribosyl cyclase and inhibit cyclic ADP-ribose hydrolase, leading to accumulation of cyclic ADP-ribose, an endogenous activator of ryanodine receptors, to activate Ca²⁺ release from SR.

Recently, Waypa et al¹⁸ proposed the fourth hypothesis of O₂ sensing. Using parallel experiments in isolated perfused lungs and PASMCs, they found that HPV was blocked by inhibitors acting at ETC sites proximal, but not distal, to superoxide production; mutant PASMCs lacking mitochondrial ETC failed to contract to hypoxia, whereas contraction to U46619 was unaffected. Moreover, 2',7'-dichlorofluorescin diacetate (DCF) in PASMCs detected an increase in cytosolic ROS, which was blocked by myxothiazol, during hypoxia. Antioxidants and a Cu,Zn superoxide dismutase (SOD) inhibitor also blocked HPV. Based on these results, they proposed that mitochondria indeed function as O₂ sensors, but it is the increase instead of decrease in mitochondrial ROS generation that triggers HPV, and H₂O₂ is probably the signal molecule. Even though many questions have yet to be answered (see previous editorial¹⁹), this hypothesis gained support from Leach et al²⁰ who found similarly in isolated intrapulmonary arteries that inhibition of complex I and III did not cause vasoconstriction but abolished HPV, whereas inhibition of cytochrome oxidase potentiated hypoxic constriction. More interestingly, succinate, a substrate for complex II, reversed the effects of complex I but not complex III inhibition on HPV. In this issue of Circulation Research, Waypa et al²¹ push their case further by demonstrating that the proximal ROS-generating sites of ETC are required for the hypoxia-induced increase in cytoplasmic [Ca²⁺], and verified the link between H₂O₂ and the increase in cytoplasmic [Ca²⁺] by showing exogenous application of a low concentration of H₂O₂ (50 µmol/L) elicited a Ca²⁺ response and overexpression of catalase in PASMCs attenuated the hypoxia and H₂O₂, but not the angiotensin II, induced Ca²⁺ responses.

The major controversy in the above mentioned hypotheses of O₂ sensing is whether ROS go up or down in PASMCs during hypoxia. Such discrepancy is likely related to differences in preparations and detection methods, each of which has its own severe specific limitations.²² To date, three reported measurements in cultured PASMCs with either lucigenin chemiluminescence or DCF fluorescence all showed increases in ROS release during hypoxia.^7,18,23 Similar observations were also made in other cell systems. These results provide a strong case for an augmented ROS release in PASMCs during hypoxia. In contrast, observations of a reduction in ROS during hypoxia were made mainly by detection of lucigenin or luminol enhanced chemiluminescence in isolated perfused lungs.^6,12 Despite the known problem of redox cycling of lucigenin, the chemiluminescence signal of such preparations reflected the global superoxide anion generated by all pulmonary cells (including airway epithelial and smooth muscle, endothelial, and vascular smooth muscle cells) in the lung surface. Hence, it did not provide a direct measurement of ROS in PASMCs. However, Michelakis et al¹³ recently measured ROS in endothelium-denuded resistance PA rings using three different detection methods (lucigenin-enhanced chemiluminescence, AmplexRed H₂O₂ assay, and DCF fluorescence), and all showed a decrease in ROS release during hypoxia. This motivates consideration of other factors that could lead to different results in ROS detection in pulmonary arteries/PASMCs during hypoxia. ROS are produced from a variety of biosynthetic sources, including mitochondrial ETC, NADPH and NADH oxidases, cyclooxygenase, lipoxygenase, xanthine oxidase, and nitric oxide synthase, and each of these sources may respond differently to hypoxia. For example, hypoxia may cause a decrease in ROS production from NADH oxidase^5,6 but an increase in ROS release from mitochondrial ETC. In this case, it may be possible that depending on subcellular locations of these ROS sources, and different compartmentation of the detection probes (cytosolic versus intraorganelle; intra- versus extracellular compartments), the net signals are different in arterial tissues and in cultured cells. Moreover, if one considers the possibility of local ROS signaling due to spatial confinement, similar to the differential signaling of sarcolemmal NOS-3 to L-type Ca²⁺ channels and sarcoplasmic NOS-1 to ryanodine receptors reported in cardiac myocytes,²⁴ one can even speculate about oxidation of effector molecules by ROS generated from a spatially-confined source in a globally-reduced environment. However, detection of subcellular ROS or redox transient in PASMCs has not yet been made.

Extending their previous experiments on antioxidants and inhibitors of SOD,¹⁸ Waypa et al,²¹ using catalase overexpressed PASMCs, have provided further evidence suggesting that H₂O₂ (or hydroxyl radical) is the signaling molecule for HPV. It is tempting to speculate that ROS release from mitochondrial ETC activates Ca²⁺ release via ryanodine receptors to initiate HPV. This idea is especially compelling because H₂O₂ can activate ryanodine receptors directly by oxidation of sulfhydryls of the channel,²⁵ mitochondria and sarcoplasmic reticulum are functionally coupled in vascular smooth muscle cell,^26,27 local Ca²⁺ release from ryanodine receptors (Ca²⁺ sparks) causes membrane depolarization, instead of hyperpolarization, in PASMCs,²⁸ and a functional SR is required for HPV.¹⁶ ROS may also stimulate cyclic ADP-ribose synthesis to activate ryanodine receptors in a calmodulin-dependent manner.²⁹ The notion of mitochondria/SR interaction fits nicely with previous findings to form a coherent hypothesis that hypoxia enhances ROS (probably H₂O₂) generation from mitochondrial ETC, activates ryanodine receptors to release SR Ca²⁺, which then leads to store-operated calcium influx, altered activity of sarcolemmal (K_V or other) ion channels, membrane depolarization, and calcium influx through L-type Ca²⁺ channels.

However, the picture is complicated by the fact that H₂O₂ can cause Ca²⁺ release from mitochondria.³⁰ Exogenous application of H₂O₂ to unstimulated pulmonary arteries was found to cause an endothelium-independent contraction, but it was unaltered by Ca²⁺-free external solution or inhibiting Ca²⁺ release by ryanodine and thapsigargin,^31,32 an obvious deviation from HPV. Moreover, H₂O₂ can cause a variety of effects on different Ca²⁺ pathways, including activation of L-type Ca²⁺ channel and nonselective cation channel, uncoupling Na⁺,K⁺-ATPase, and altered activities of SR Ca²⁺-ATPase and Na⁺-Ca²⁺ exchanger. Hence, the Ca²⁺ response elicited by H₂O₂ could be rather complex. Because the H₂O₂-induced Ca²⁺ response in this study had not been characterized, it is unclear whether it resembles the Ca²⁺ response associated with HPV, ie, blocked by free Ca²⁺ external solution, L-type Ca²⁺ channel antagonist, ryanodine, and thapsigargin. It is also unfortunate, probably due to technical limitations, that kinetic data of ROS production and Ca²⁺ response elicited during hypoxia are not available for comparison.

In conclusion, Waypa et al²¹ have made a further step toward validating their hypothesis. But until precise characterizations of the temporal and spatial aspects of ROS generation and thorough comparison of ETC ROS- and hypoxia-induced Ca²⁺ responses are available, ROS will continue their ups and downs on the stage of HPV.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

References

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This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sham, J. S.K. Search for Related Content PubMed PubMed Citation Articles by Sham, J. S.K. Related Collections Pulmonary biology and circulation Pulmonary circulation and disease Oxidant stress Other Vascular biology