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POINT-COUNTERPOINT COMMENTS
King's College London
Division of Allergy, Asthma and Lung Biology
School of Medicine
London, United Kingdom
The following letters are in response to the Point:Counterpoint series "Hypoxic pulmonary vasoconstriction is/is not mediated by increased production of reactive oxygen species" that appears in this issue.
To the Editor: In 1999, Archer and coworkers (1) demonstrated that mice with chronic granulomatous disease (CGD), which lack gp91phox, have HPV of the same amplitude as normal mice, indicating that NADPH oxidase is not involved in HPV-related O2 sensing. One particular result described in this paper, which seems to have been generally ignored but has always fascinated me, was that that lung ROS levels in the mice lacking gp91 were virtually nil.
It seems exceedingly unlikely that hypoxia could inhibit K+ channels and cause HPV by reducing an already nonexistent level of ROS production. If we accept these data, their clear implication is that it must be cell redox state, and not [ROS], that directly modulates K+ channels.
But hold on—cell redox state is mainly a function of the GSH/GSSG ratio, which is thought to be very high in most cells under normoxic conditions. Therefore the intracellular milieu is normally relatively reduced (3) and there is not much scope for hypoxia to cause further reduction. However, Michelakis et al. (2) later argued that the mitochondria of pulmonary artery smooth muscle cells basally produce huge amounts of ROS. Therefore, these cells may be in a relatively oxidized state, which hypoxia could then significantly reduce by lowering mitochondrial ROS production (see Ward et al., Ref. 4).
But now I am really confused. This cannot be what happened in the CGD mice, because their mitochondria apparently were not producing any ROS under normoxic conditions.
So how could they have normal HPV?!
REFERENCES
Institut für Physiologie
Universität Duisburg-Essen
Essen, Germany
To the Editor: O2 sensing is fundamental for all kinds of hypoxic response. Interestingly, the opponents refer to the HIF (hypoxia-inducible factor) system to underline their respective position (ROS up or down under hypoxia), but unfortunately a similar debate darkens the sky over HIF-dependent gene expression. Thus a common problem with ROS exists in all fields of oxygen sensing. One major problem is the insufficient methods for a reliable detection of ROS in vivo (5). Usual excitation of fluorescent indicators with blue/green laser light itself causes the production of ROS in cells even under hypoxic conditions (1). This can be avoided with the use of infrared lasers investigating dihydrorhodamine (DHR)-loaded cells when ROS went down under hypoxia (4). In addition, scavenging ROS with DHR decreased the activity of the HIF-prolyl-hydroxylases (PHDs) that target HIF-1a for degradation under normoxia (4). In contrast, JunD–/– cells that accumulated H2O2 showed reduced activity of PHDs and higher HIF-1 levels (3). Although the molecular details are not yet understood, the PHD oxygen sensors are susceptible to ROS. Whereas the opponents "most agree that mitochondria function as the oxygen sensor for HPV," the role of mitochondria in the field of the HIF-PHDs is far from being equivocally accepted. In fact, inhibitors of the mitochondrial electron transport chain may have a greater impact of the O2 availability than on ROS production (2). In conclusion, ROS may modulate oxygen sensing but appear secondary to O2-dependent PHD activity.
REFERENCES
Department of Physiology
New York Medical College
Valhalla, New York
To the Editor: Our perspective originates from identifying a peroxide-elicited cGMP-associated relaxing mechanism in bovine calf pulmonary arteries (1). This work evolved into evidence that BPA show a stable contraction to hypoxia associated with removal of this tonically active relaxing mechanism originating from cytosolic NADPH oxidase-derived peroxide (5). Hypoxia appears to also increase NADPH in pulmonary arteries (2). Bovine coronary arteries relax to hypoxia through a ROS-independent mechanism mediated through cytosolic NADPH oxidation, a process coordinating multiple mechanisms of lowering intracellular calcium (3). Although mitochondrial inhibitors have minimal effects on force and hypoxia-elicited responses in bovine arteries, they inhibit contractile function in rat arteries reported to show mitochondrial ROS-associated hypoxic responses. Our observations are similar to studies on small rat pulmonary arteries showing stable hypoxic contractions that support hypoxia decreasing mitochondrial ROS associated with cytosolic NADPH reduction and potassium channel closure (4). In contrast, studies supporting increased mitochondrial ROS use prolonged hypoxic exposures in larger rat pulmonary arteries and cultured smooth muscle cell preparations. The larger arteries do not maintain a stable contraction to hypoxia, and cellular studies lack evidence that increased mitochondrial ROS under hypoxia is specific for the pulmonary system. Thus the oxygen sensing mechanisms reported in these systems could be adapted processes lacking relevance to hypoxic pulmonary vasoconstriction. Therefore, our perspective is hypoxia initiates pulmonary artery contraction through decreasing ROS and increasing cytosolic NADPH, with Nox oxidases functioning as oxygen sensors, and altered cGMP and cytosolic NADPH redox may coordinate multiple force-regulating mechanisms mediating responses to hypoxia.
REFERENCES
Cellular and Integrative Physiology
Indiana University School of Medicine
Indianapolis, Indiana
California State University
Fullerton, California
To the Editor: Logistical issues must be reconciled when investigating potential mediators of hypoxic pulmonary vasoconstriction (HPV). While pulmonary vessels constrict, systemic vessels dilate in response to hypoxia. Why some vascular muscles contract whereas others relax when oxygen decreases must be considered. Hypoxic vasoconstriction is not unique to mammalian lung. Dorsal aortae of lower vertebrates such as lamprey and vessels of some animals phylogenetically lower than vertebrates such as the California blackworm constrict to hypoxia (2, 3). Hypoxic sensors may be universal or may vary between species and/or for hypoxic constrictor vs. dilator vessels. If the sensor is universal, then signaling pathways must deviate such that opposite responses can be the outcome of activation of a common sensor. Although hypoxia causes HPV and systemic vasodilation, the two circulatory systems do not typically respond oppositely to vasoactive reactive oxygen species (ROS). Vasoconstrictor oxidants such as hydrogen peroxide (H2O2) cause constriction in all vessels, large and small arteries, and veins in both pulmonary and systemic circulations (1). The current understanding of the signaling pathways of HPV and ROS-induced contraction does not support the idea that ROS are involved in mediating HPV. HPV is calcium and myosin light chain (MLC) phosphorylation dependent (5). H2O2-induced contraction is both calcium and MLC phosphorylation independent (1). The available data seem adequate to resolve the debate addressed by Ward vs. Weir and Archer (4). Because of the considerations addressed above and because of what is known about vasoconstrictor oxidants, it is difficult to accept that ROS mediate HPV.
REFERENCES
Department of Biomedical Sciences
School of Biology
University of St. Andrews
St. Andrews, Fife, United Kingdom
Dept. Bioquímica y Biología
Molecular y Fisiología
Facultad de Medicina
Universidad de Valladolid
Valladolid, Spain
To the Editor: Like Professor Ward, we have no qualms about going mano e mano with our friends in the South; we do care, honest!
Schumacker and coworkers developed an ingenious FRET-based ROS indicator that has provided support for a role for ROS in cell signaling by hypoxia. Unfortunately, the use of this tool is limited to cultured cells, which are less than representative of even acutely isolated cells. Supporting evidence has been provided by way of the fact that ROS scavengers attenuate HPV and that H2O2 induces pulmonary artery constriction (5). However, significant controversies remain.
1) The cellular mechanism of action of ROS scavengers is open to question.
2) Constriction by H2O2 is entirely independent of the endothelium, i.e., the precise characteristics of HPV are not reproduced (5).
3) The toxicity of H2O2 is inconsistent with the effects of hypoxia. Constriction by H2O2 is not reproducible, even at low concentrations (unpublished by many); does the magnitude of constriction decline markedly on the second exposure to H2O2 and fail on the third? At this point, is constriction by K+ abolished?
4) Hyperoxia, unlike hypoxia, fails to alter pulmonary vascular resistance or the distribution of blood flow in the lung, e.g., Ref. 3. However, hyperoxia increases ROS levels in the rat lung and in mitochondria thereof, e.g., Ref. 1 and the expression, as one might expect, of ROS scavengers (2).
5) In cultured cells, mitochondrial respiration seems not to be required as a permanent ROS source for physiological activities such as signaling, e.g., Ref. 4, in fact mitochondria are not required for such signaling mechanisms in a variety of cell systems.
REFERENCES
This article has been cited by other articles:
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  J. P. T. Ward Last Word: Point:Counterpoint authors respond to commentaries on "Hypoxic pulmonary vasoconstriction is/is not mediated by increased production of reactive oxygen species" J Appl Physiol, September 1, 2006; 101(3): 1004 - 1004. [Full Text] [PDF]
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