doi: 10.1161/hh1201.093167
© 2001 American Heart Association, Inc.
Editorial |
Hypoxic Pulmonary Vasoconstriction
A Radical View
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 studies22 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
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