This Article Extract 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 Sylvester, J. T. Search for Related Content PubMed PubMed Citation Articles by Sylvester, J. T. Related Collections Cell signalling/signal transduction Pulmonary biology and circulation

(Circulation Research. 2001;88:1228.)
© 2001 American Heart Association, Inc.

Editorial

Hypoxic Pulmonary Vasoconstriction

A Radical View

J. T. Sylvester

From the Division of Pulmonary and Critical Care Medicine, The Johns Hopkins School of Medicine, Baltimore, Md.

Correspondence to J.T. Sylvester, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail jsylv{at}welchlink.welch.jhu.edu

Key Words: KeyWords • hypoxia • vasoconstriction • vascular smooth muscle • reactive oxygen species • mitochondria

In 1894, when Bradford and Dean¹ reported that asphyxia caused pulmonary hypertension, no one paid much attention. But ever since 1946, when von Euler and Liljestrand² reported that acute hypoxia increased pulmonary arterial pressure attributable to pulmonary vasoconstriction, investigators have been hard at work to determine the underlying mechanisms. They have kept at it for more than half a century because of the important roles hypoxic pulmonary vasoconstriction (HPV) plays in health and disease.

Early work on HPV was performed almost exclusively in intact animals or isolated lungs. These preparations provided reproducible relevant responses, but their complexity placed limits on mechanistic investigation. The last decade has seen accelerated use of more reduced preparations, such as isolated vessels and vascular cells. These preparations provide more investigative precision, but relevance is sometimes uncertain, and special conditions are often necessary to achieve adequate reproducibility. Because of these problems, the mechanisms of HPV remain unknown, and a rapidly growing mass of inconsistent data has generated confusion and frustration, leading one investigator to title his symposium on HPV, "Can everyone be right?" and another investigator to title his review, "Can anyone be right?" Nevertheless, areas of tentative consensus are emerging.³

The primary mechanisms of HPV are contained entirely within pulmonary vascular tissue. The main locus of the response is small distal pulmonary arteries. The smooth muscle effector pathway depends on an increase in cytoplasmic calcium concentration ([Ca²⁺]_c) caused by influx of calcium from extracellular fluid. Voltage-gated calcium channels provide a major influx pathway; however, release of calcium from sarcoplasmic reticulum (SR) seems to be essential, and influx also occurs through other pathways, such as channels dependent on internal calcium stores.³ One hypothesis resolves this complexity by proposing that hypoxia first causes SR release of calcium, which then leads to store-dependent calcium influx, altered activity of sarcolemmal ion channels, membrane depolarization, and calcium influx through voltage-gated channels.⁴ The resulting increase in [Ca²⁺]_c triggers calmodulin-mediated activation of myosin light chain kinase, actin-myosin interaction, and contraction.

Hypoxia depolarizes both pulmonary arteries and pulmonary arterial myocytes.³ The identity of the ion channels responsible for hypoxic depolarization is under active investigation. Voltage-dependent potassium (Kv) channels, known regulators of membrane potential in vascular smooth muscle, are inhibited by hypoxia⁵ ; however, HPV was not prevented by 4-aminopyridine (4-AP), a Kv channel blocker, raising doubts that these channels play an exclusive role.^{6 7} Other possibilities include calcium-dependent chloride channels and a newly described Kv channel subtype insensitive to 4-AP.³ Recently, Robertson et al⁸ reported that hypoxia increased [Ca²⁺]_c and caused constriction in pulmonary arteries exposed to high external [K⁺] and inhibitors of voltage-dependent calcium channels. These provocative observations question the necessity for membrane depolarization and the role of sarcolemmal ion channels in HPV.

For the effector pathway to be fully expressed as HPV, previous activation (priming) of smooth muscle seems to be required. In distal pulmonary arteries, vigorous HPV was abolished by endothelial denudation or BQ123 (an ET-1 receptor antagonist) and restored after denudation by exposure to a threshold ET-1 concentration.⁹ In myocytes from the same vessels, ET-1 priming potentiated a small hypoxic contraction 8-fold while leaving a small increase in [Ca²⁺]_c unaffected.⁷ Thus, pulmonary arterial myocytes contain both sensor and effector pathways for HPV, but full expression of these pathways requires priming by basal release of ET-1 from endothelium.³ Mechanisms of priming may include increased myofilament calcium sensitivity and partial depolarization of resting membrane potential. Although the degree and mediators of priming may vary with species, preparations, and conditions, consistent inhibition of HPV by ET-1 receptor antagonists in intact animals suggests that ET-1 plays an important physiological role.³

The mechanism of oxygen sensing is the most important, controversial, and studied but least understood aspect of HPV. The search for the sensor has proceeded from the premise that O₂ must be rate limiting to a system of biochemical reactions, which alters the contractile state of pulmonary vascular smooth muscle. Accordingly, mitochondria have been a recurrent focus of attention. In isolated lungs and intact animals, inhibitors of electron transport and uncouplers of oxidative phosphorylation caused pulmonary vasoconstriction during normoxia, suggesting that mitochondria might signal HPV through decreases in energy state.¹⁰ However, energy state did not decrease during HPV in isolated lungs¹¹ or sustained hypoxic contraction in isolated pulmonary arteries.¹² Thus, changes in energy state seem more likely to modulate or permit HPV than to trigger the response.

If sufficiently severe, hypoxia slows mitochondrial electron transport, leading to accumulation of reducing equivalents, decreased reactive oxygen species (ROS) production, and a shift of cytosolic redox state toward reduction. Such changes have been demonstrated in isolated lungs and pulmonary arteries and could act as signals for HPV.^{13 14} Indeed, it has been proposed that HPV is triggered by redox-dependent gating of Kv channels in pulmonary arterial myocytes.⁵ Consistent with this proposal, reductants caused depolarization, Kv channel inhibition, and constriction in pulmonary vascular tissue, whereas oxidants had opposite effects.¹⁵ However, as noted above, enthusiasm for this hypothesis has diminished because antagonists of Kv channels did not prevent HPV.^{6 7} Of course, O₂-dependent changes in redox state could act in other ways. In rat pulmonary arteries, decreased smooth muscle NAD⁺/NADH attributable to hypoxia seemed to augment cADP-ribose concentration, which caused constriction by activating calcium release from SR ryanodine receptors.¹⁶

In this issue of Circulation Research, Waypa et al¹⁷ turn these notions on their head and present a compelling case that mitochondria function as O₂ sensors by increasing release of ROS during hypoxia. In carefully performed, well-controlled, complementary experiments in isolated lungs and pulmonary arterial myocytes, HPV was (1) blocked by agents that inhibit mitochondrial electron transport upstream from sites of superoxide (·O₂^-) generation but not by inhibitors acting downstream from these sites; (2) absent in mutant pulmonary arterial myocytes without mitochondria; and (3) inhibited by antioxidants and an antagonist of Cu,Zn superoxide dismutase (SOD). Furthermore, hypoxia oxidized intracellular dichlorofluorescin (DCF), indicating increased H₂O₂ production, and this oxidation was blocked by myxothiazol, a proximal inhibitor of electron transport. These results suggest that HPV occurred independently of mitochondrial ATP production and was triggered by increased release of ROS from proximal portions of the mitochondrial electron transport chain.

As always, there are reasons to be cautious. Reactants other than H₂O₂ may have oxidized DCF. DCF distribution and kinetics in pulmonary arterial smooth muscle are unknown. Proximal inhibitors of electron transport caused vasoconstriction during normoxia. Inconsistencies with previous results need to be explained. Toxic effects of higher inhibitor concentrations may explain why cyanide and antimycin A previously blocked HPV.¹⁰ Preparations that obscured or altered release of ROS from myocytes may explain why hypoxia previously decreased ROS production.^{14 18 19} On the other hand, why did antioxidants previously cause pulmonary vasoconstriction rather than vasodilation?¹⁵ Why did inhibitors of SOD potentiate rather than prevent HPV?²⁰ Why did myxothiazol have no effect on hypoxic enhancement of ROS production?²¹

Despite these uncertainties, it is clear that Waypa et al¹⁷ have pointed out a new and unexpected direction for HPV research. One of the most intriguing questions raised by their work is how hypoxia increases release of ROS in pulmonary arterial smooth muscle, an observation now reported by three other laboratories.^{21 22 23} In the simplest conceptualization, electrons accumulating during hypoxia upstream from cytochrome oxidase would react nonenzymatically with O₂ to form ·O₂^-; however, under these conditions it is difficult to understand how ·O₂^- production could increase, because increased availability of electrons would be offset by decreased availability of oxygen. Previous work from the authors’ laboratory suggests a more complicated explanation.²⁴ In Hep3B cells, hypoxia increased mitochondrial production of ROS even when the proximal portion of the electron transport chain was fully reduced by antimycin A. Thus, hypoxic enhancement of ROS production seems to occur at sites upstream from ubisemiquinone (where antimycin A acts) because of factors other than electron availability. As the authors suggest, these factors could include O₂-dependent facilitation of electron transfer to O₂ or enhanced egress of ·O₂^- through O₂-dependent anion channels in the inner mitochondrial membrane. Consistent with the latter possibility, they found that DIDS (an anion channel blocker) prevented HPV.¹⁷

Where is the ROS signal received? Although the results of Waypa et al¹⁷ suggest an intracellular site, previous studies^{22 23 25} demonstrated that exogenous SOD or catalase blocked both hypoxic enhancement of ROS production and HPV. If these large proteins did not enter cells, then ROS released during hypoxia must have either exited myocytes to act at the external sarcolemmal surface or traversed the extracellular space before reentry to act at intracellular loci. Alternatively, SOD and catalase may have entered cells by endocytosis, which occurs in smooth muscle²⁶ and may be enhanced by ROS.²⁷

As previous work from the laboratory of Waypa et al¹⁷ makes clear, hypoxic enhancement of mitochondrial ROS production is not limited to pulmonary arterial myocytes and Hep3B cells but also occurs in cardiomyocytes, endothelial cells, and macrophages.^{24 28 29 30} This ubiquity suggests that unique hypoxic responses characteristic of different tissues, such as hypoxic constriction in pulmonary vessels and ischemic preconditioning in cardiomyocytes, derive from differences in downstream transduction pathways rather than O₂ sensing. Although evidence is rapidly accumulating that ROS play important roles in a wide variety of physiological and pathophysiologic processes, the transduction pathways activated by ROS during HPV are unknown.

Could HPV be signaled by a mitochondrial property linked to, but different from, ROS production? Mitochondria store and release calcium and may actively participate in calcium signaling.³¹ Moreover, increased mitochondrial metabolism of H₂O₂ by glutathione peroxidase may increase NAD⁺, leading to increased ADP-ribose production, monoADP-ribosylation of mitochondrial proteins, and calcium release.³² Thus, the role of mitochondrial calcium signaling in HPV deserves full evaluation.

Until these and many other questions have been answered, the significance of this work will not be known. For the moment, however, Waypa et al¹⁷ have added a new and interesting spice to the HPV stew. Now it is time to stir.

Footnotes

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

References

1. Bradford J, Dean H. The pulmonary circulation. J Physiol (Lond). 1894;16:34–96.
2. von Euler U, Liljestrand G. Observations on the pulmonary arterial blood pressure of the cat. Acta Physiol Scand. 1946;12:301–320.
3. Sylvester JT, Sham JSK, Shimoda LA, Liu Q. Cellular mechanisms of acute hypoxic pulmonary vasoconstriction. In: Scharf SM, Pinsky MR, Magder S, eds. Respiratory-Circulatory Interactions in Health and Disease. New York, NY: Marcel Dekker; 2001:351–359.
4. Post JM, Gelband CH, Hume JR. [Ca²⁺]_i inhibition of K⁺ channels in canine pulmonary artery: novel mechanism for hypoxia-induced membrane depolarization. Circ Res. 1995;77:131–139.[Abstract/Free Full Text]
5. Archer SL, Weir EK, Reeve HL, Michelakis E. Molecular identification of O₂ sensors and O₂-sensitive potassium channels in the pulmonary circulation. Adv Exp Med Biol. 2000;475:219–240.[Medline] [Order article via Infotrieve]
6. Hasunuma K, Rodman DM, McMurtry IF. Effects of K⁺ channel blockers on vascular tone in the perfused rat lung. Am Rev Respir Dis. 1991;144:884–887.[Medline] [Order article via Infotrieve]
7. Sham JS, Crenshaw BR, Jr, Deng LH, Shimoda LA, Sylvester JT. Effects of hypoxia in porcine pulmonary arterial myocytes: roles of Kv channel and endothelin-1. Am J Physiol Lung Cell Mol Physiol. 2000;279:L262–L272.[Abstract/Free Full Text]
8. Robertson TP, Hague D, Aaronson PI, Ward JP. Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat. J Physiol. 2000;525:669–680.[Abstract/Free Full Text]
9. Liu Q, Sham JS, Shimoda LA, Sylvester JT. Hypoxic constriction of porcine distal pulmonary arteries: endothelium and endothelin dependence. Am J Physiol Lung Cell Mol Physiol. 2001;280:L856–L865.[Abstract/Free Full Text]
10. Rounds S, McMurtry IF. Inhibitors of oxidative ATP production cause transient vasoconstriction and block subsequent pressor responses in rat lungs. Circ Res. 1981;48:393–400.[Abstract/Free Full Text]
11. Buescher PC, Pearse DB, Pillai RP, Litt MC, Mitchell MC, Sylvester JT. Energy state and vasomotor tone in hypoxic pig lungs. J Appl Physiol. 1991;70:1874–1881.[Abstract/Free Full Text]
12. Leach RM, Sheehan DW, Chacko VP, Sylvester JT. Energy state, pH, and vasomotor tone during hypoxia in precontracted pulmonary and femoral arteries. Am J Physiol Lung Cell Mol Physiol. 2000;278:L294–L304.[Abstract/Free Full Text]
13. Shigemori K, Ishizaki T, Matsukawa S, Sakai A, Nakai T, Miyabo S. Adenine nucleotides via activation of ATP-sensitive K⁺ channels modulate hypoxic response in rat pulmonary artery. Am J Physiol. 1996;270:L803–L809.[Abstract/Free Full Text]
14. Archer SL, Huang J, Henry T, Peterson D, Weir EK. A redox-based O₂ sensor in rat pulmonary vasculature. Circ Res. 1993;73:1100–1112.[Abstract/Free Full Text]
15. Reeve HL, Weir EK, Nelson DP, Peterson DA, Archer SL. Opposing effects of oxidants and antioxidants on K⁺ channel activity and tone in rat vascular tissue. Exp Physiol. 1995;80:825–834.[Abstract]
16. Wilson HL, Dipp M, Thomas JM, Lad C, Galione A, Evans AM. ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor. a primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction. J Biol Chem. 2001;276:11180–11188.[Abstract/Free Full Text]
17. Waypa GB, Chandel NS, Schumacker PT. Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen sensing. Circ Res. 2001;88:1259-1266.[Abstract/Free Full Text]
18. Mohazzab KM, Fayngersh RP, Kaminski PM, Wolin MS. Potential role of NADH oxidoreductase-derived reactive O₂ species in calf pulmonary arterial PO₂-elicited responses. Am J Physiol. 1995;269:L637–L644.[Abstract/Free Full Text]
19. Mohazzab KM, Wolin MS. Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle. Am J Physiol. 1994;267:L815–L822.[Abstract/Free Full Text]
20. Abdalla S, Will JA. Potentiation of the hypoxic contraction of guinea-pig isolated pulmonary arteries by two inhibitors of superoxide dismutase. Gen Pharmacol. 1995;26:785–792.[Medline] [Order article via Infotrieve]
21. Marshall C, Mamary AJ, Verhoeven AJ, Marshall BE. Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction. Am J Respir Cell Mol Biol. 1996;15:633–644.[Abstract]
22. Killilea DW, Hester R, Balczon R, Babal P, Gillespie MN. Free radical production in hypoxic pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2000;279:L408–L412.[Abstract/Free Full Text]
23. Liu Q, Kuppusamy P, Sham JS, Shimoda LA, Zweier JL, Sylvester JT. Increased production of reactive oxygen species (ROS) by pulmonary arterial smooth muscle is required for hypoxic pulmonary vasoconstriction (HPV). Am J Respir Crit Care Med. 2001;163:A395. Abstract.
24. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci U S A. 1998;95:11715–11720.[Abstract/Free Full Text]
25. Becker LB, vanden Hoek TL, Shao ZH, Li CQ, Schumacker PT. Generation of superoxide in cardiomyocytes during ischemia before reperfusion. Am J Physiol. 1999;277:H2240–H2246.[Abstract/Free Full Text]
26. Muir EM, Bowyer DE. Dependence of fluid-phase pinocytosis in arterial smooth-muscle cells on temperature, cellular ATP concentration and the cytoskeletal system. Biochem J. 1983;216:467–473.[Medline] [Order article via Infotrieve]
27. Sundqvist T, Liu SM. Hydrogen peroxide stimulates endocytosis in cultured bovine aortic endothelial cells. Acta Physiol Scand. 1993;149:127–131.[Medline] [Order article via Infotrieve]
28. Duranteau J, Chandel NS, Kulisz A, Shao Z, Schumacker PT. Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes. J Biol Chem. 1998;273:11619–11624.[Abstract/Free Full Text]
29. Ali MH, Schlidt SA, Chandel NS, Hynes KL, Schumacker PT, Gewertz BL. Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction. Am J Physiol. 1999;277:L1057–L1065.[Abstract/Free Full Text]
30. Chandel NS, Trzyna WC, McClintock DS, Schumacker PT. Role of oxidants in NF-B activation and TNF- gene transcription induced by hypoxia and endotoxin. J Immunol. 2000;165:1013–1021.[Abstract/Free Full Text]
31. Duchen MR. Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death. J Physiol (Lond). 1999;516:1–17.[Abstract/Free Full Text]
32. Richter C, Gogvadze V, Laffranchi R, Schlapbach R, Schweizer M, Suter M, Walter P, Yaffee M. Oxidants in mitochondria: from physiology to diseases. Biochim Biophys Acta. 1995;1271:67–74.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				N. Sommer, A. Dietrich, R. T. Schermuly, H. A. Ghofrani, T. Gudermann, R. Schulz, W. Seeger, F. Grimminger, and N. Weissmann Regulation of hypoxic pulmonary vasoconstriction: basic mechanisms Eur. Respir. J., December 1, 2008; 32(6): 1639 - 1651. [Abstract] [Full Text] [PDF]

				M. Mittal, M. Roth, P. Konig, S. Hofmann, E. Dony, P. Goyal, A.-C. Selbitz, R. T. Schermuly, H. A. Ghofrani, G. Kwapiszewska, et al. Hypoxia-Dependent Regulation of Nonphagocytic NADPH Oxidase Subunit NOX4 in the Pulmonary Vasculature Circ. Res., August 3, 2007; 101(3): 258 - 267. [Abstract] [Full Text] [PDF]

				C. Tekinbas, H. Ulusoy, E. Yulug, M. Muharrem Erol, A. Alver, E. Yenilmez, S. Geze, and M. Topbas One-lung ventilation: For how long? J. Thorac. Cardiovasc. Surg., August 1, 2007; 134(2): 405 - 410. [Abstract] [Full Text] [PDF]

				N. Weissmann, N. Sommer, R. T. Schermuly, H. A. Ghofrani, W. Seeger, and F. Grimminger Oxygen sensors in hypoxic pulmonary vasoconstriction Cardiovasc Res, September 1, 2006; 71(4): 620 - 629. [Abstract] [Full Text] [PDF]

				J. P. T. Ward, T. P. Robertson, and P. I. Aaronson Capacitative calcium entry: a central role in hypoxic pulmonary vasoconstriction? Am J Physiol Lung Cell Mol Physiol, July 1, 2005; 289(1): L2 - L4. [Full Text] [PDF]

				L. C. Ng, S. M Wilson, and J. R Hume Mobilization of sarcoplasmic reticulum stores by hypoxia leads to consequent activation of capacitative Ca2+ entry in isolated canine pulmonary arterial smooth muscle cells J. Physiol., March 1, 2005; 563(2): 409 - 419. [Abstract] [Full Text] [PDF]

				G. B. Waypa and P. T. Schumacker Hypoxic pulmonary vasoconstriction: redox events in oxygen sensing J Appl Physiol, January 1, 2005; 98(1): 404 - 414. [Abstract] [Full Text] [PDF]

				N. Weissmann, N. Ebert, M. Ahrens, H. A. Ghofrani, R. T. Schermuly, J. Hanze, L. Fink, F. Rose, J. Conzen, W. Seeger, et al. Effects of Mitochondrial Inhibitors and Uncouplers on Hypoxic Vasoconstriction in Rabbit Lungs Am. J. Respir. Cell Mol. Biol., December 1, 2003; 29(6): 721 - 732. [Abstract] [Full Text] [PDF]

				V. Sauzeau, M. Rolli-Derkinderen, S. Lehoux, G. Loirand, and P. Pacaud Sildenafil Prevents Change in RhoA Expression Induced by Chronic Hypoxia in Rat Pulmonary Artery Circ. Res., October 3, 2003; 93(7): 630 - 637. [Abstract] [Full Text] [PDF]

				S. Bonnet, E. Dumas-de-La-Roque, H. Begueret, R. Marthan, M. Fayon, P. Dos Santos, J.-P. Savineau, and E.-E. Baulieu Dehydroepiandrosterone (DHEA) prevents and reverses chronic hypoxic pulmonary hypertension PNAS, August 5, 2003; 100(16): 9488 - 9493. [Abstract] [Full Text] [PDF]

				Y.-X. Wang, Y.-M. Zheng, I. Abdullaev, and M. I. Kotlikoff Metabolic inhibition with cyanide induces calcium release in pulmonary artery myocytes and Xenopus oocytes Am J Physiol Cell Physiol, February 1, 2003; 284(2): C378 - C388. [Abstract] [Full Text] [PDF]

				J. S.K. Sham Hypoxic Pulmonary Vasoconstriction: Ups and Downs of Reactive Oxygen Species Circ. Res., October 18, 2002; 91(8): 649 - 651. [Full Text] [PDF]

				Y. Morio and I. F. McMurtry Ca2+ release from ryanodine-sensitive store contributes to mechanism of hypoxic vasoconstriction in rat lungs J Appl Physiol, February 1, 2002; 92(2): 527 - 534. [Abstract] [Full Text] [PDF]

This Article Extract 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 Sylvester, J. T. Search for Related Content PubMed PubMed Citation Articles by Sylvester, J. T. Related Collections Cell signalling/signal transduction Pulmonary biology and circulation