Diversity in Mitochondrial Function Explains Differences in Vascular Oxygen Sensing
Renal arteries (RAs) dilate in response to hypoxia, whereas the pulmonary arteries (PAs) constrict. In the PA, O2 tension is detected by an unidentified redox sensor, which controls K+ channel function and thus smooth muscle cell (SMC) membrane potential and cytosolic calcium. Mitochondria are important regulators of cellular redox status and are candidate vascular O2 sensors. Mitochondria-derived activated oxygen species (AOS), like H2O2, can diffuse to the cytoplasm and cause vasodilatation by activating sarcolemmal K+ channels. We hypothesize that mitochondrial diversity between vascular beds explains the opposing responses to hypoxia in PAs versus RAs. The effects of hypoxia and proximal electron transport chain (pETC) inhibitors (rotenone and antimycin A) were compared in rat isolated arteries, vascular SMCs, and perfused organs. Hypoxia and pETC inhibitors decrease production of AOS and outward K+ current and constrict PAs while increasing AOS production and outward K+ current and dilating RAs. At baseline, lung mitochondria have lower respiratory rates and higher rates of AOS and H2O2 production. Similarly, production of AOS and H2O2 is greater in PA versus RA rings. SMC mitochondrial membrane potential is more depolarized in PAs versus RAs. These differences relate in part to the lower expression of proximal ETC components and greater expression of mitochondrial manganese superoxide dismutase in PAs versus RAs. Differential regulation of a tonically produced, mitochondria-derived, vasodilating factor, possibly H2O2, can explain the opposing effects of hypoxia on the PAs versus RAs. We conclude that the PA and RA have different mitochondria.
The normoxic pulmonary circulation is vasodilated and accommodates the entire cardiac output at much lower pressures than the systemic circulation. During hypoxia, the pulmonary arteries (PAs) constrict (hypoxic pulmonary vasoconstriction, HPV), whereas systemic arteries, such as renal arteries (RAs), dilate. The mechanism of this opposing control of tone between the two vascular beds is unknown. Although the response of each bed to hypoxia is significantly modulated by the endothelium, the mechanism for the opposing responses to hypoxia appear to lie within the vascular smooth muscle cells (SMCs). Hypoxia increases intracellular Ca2+ ([Ca2+]i) and contracts PASMCs; in contrast, isolated SMCs from systemic arteries display decreased [Ca2+]i and relax in response to hypoxia.1
Both the control of tone and the response of O2-sensitive tissues to hypoxia involve redox-sensitive mechanisms (for review, see Wolin2). Activated oxygen species (AOS) are now recognized as important mediators in vascular cellular signaling. Several kinases and sarcolemmal potassium channels (K+ channels) are redox-sensitive and modulated by AOS. Cysteine-rich K+ channels are inhibited when reduced and activated when oxidized, and oxidants and AOS, including hydrogen peroxide (H2O2), are important K+ channel openers.3 K+ channels contribute to regulation of vascular tone through their control of SMC membrane potential (Em). Closing of K+ channels depolarizes Em, which causes an increase in the open probability of the voltage-gated L-type Ca2+ channels, Ca2+ influx, and vasoconstriction. In contrast, K+ channel openers cause hyperpolarization and vasodilatation.4
The major source of AOS and peroxides in SMCs are cytochrome-based oxidases, such as nicotinamide adenine dinucleotide phosphate (NADPH) and NADH, and the electron transport chain (ETC) in mitochondria.2 Both vascular oxidases and the ETC produce AOS in proportion to Po2, and thus have been proposed as candidates for vascular oxygen sensors.5,6⇓ Despite the low KM for O2 of the mitochondrial cytochromes, lung mitochondria make AOS in direct proportion to Po2 over the physiological range.7
Most O2-sensitive systems consist of a sensor that produces a mediator in response to changes in Po2, which in turn alters the function of an effector. O2- and redox-sensitive K+ channels have been shown to be the effectors in O2 sensing in several tissues including the PA, the ductus arteriosus, the carotid body, the neuroepithelial body, and the fetal adrenomedullary cells.4 Inhibition of these O2-sensitive K+ channels leads to depolarization, opening of L-type Ca2+ channels, Ca2+ influx, and vasoconstriction. The obligatory role of the L-type Ca2+ channel is suggested by the fact that blockers and agonists of this channel inhibit8 and enhance9 HPV, respectively. On the other hand there are reports suggesting that it is release of intracellular Ca2+, rather than influx of extracellular Ca2+, that initiates HPV.10 This controversy may relate to confusion regarding the pool of Ca2+ that initiates rather than sustains HPV and perhaps to the model used to study HPV (rings versus intact lungs).
There is growing consensus that the vascular O2 sensors are redox based. A variety of sensors have been proposed, including NADPH oxidase,11 NADH oxidase,12 and the ETC.6 It is likely that there is a diversity of sensors among different O2-sensitive tissues and species. The role of gp91phox-containing NADPH oxidase in O2 sensing was recently challenged, at least in the lung13 and the carotid body,14 although the role of novel oxidases (NOX) in HPV has not been assessed. The role of mitochondria as O2 sensors in the pulmonary circulation was proposed 15 years ago5 and is supported by several recent publications.6,15,16⇓⇓ Their role in O2 sensing appears to be widespread, because ETC inhibitors mimic hypoxia in several other O2-sensing organs, such as the carotid body17 and adrenal medullary cells.18 The acute hemodynamic effects of hypoxia and ETC inhibitors, unlike anoxia, do not relate to ATP depletion.19 Rather, we speculate that mitochondria alter vascular tone by regulating the production of diffusible redox mediators, such as AOS and peroxides. H2O2, with its long diffusion radius, can modify targets in the cytoplasm and membrane (guanylate cyclase and K+ channels), and thereby regulate tone.2 In addition, mitochondria are now recognized as important regulators of intracellular Ca2+.20
We hypothesized that the opposing responses of the pulmonary and systemic vascular beds to hypoxia are, at least in part, due to differences in mitochondria function. Although mitochondrial diversity has been shown among neurons21 and myocardial cells,22,23⇓ and between organs (pigeon heart versus rat liver mitochondria24) potential diversity of mitochondrial function and its link to tone has not been reported so far in the vasculature.
Materials and Methods
Perfused Lung-Kidney Model
In this model, the isolated lungs and kidneys are perfused in series with a shared, blood-free solution as previously described.25 The flow rate was kept constant in the lung (28 mL/min) and the kidney (10.5 mL/min) using separate perfusion pumps. Changes in PA and RA pressure were recorded in response to hypoxia or drugs.
Resistance PA and RA rings (4 to 5th order), denuded of endothelium, were studied as previously described.26
Whole-cell patch-clamp recordings were performed in freshly isolated PASMCS and RASMCs, as previously described.27
Lucigenin (10−5 mol/L) enhanced chemiluminescence, a measure of the production of AOS, was measured in vascular rings or isolated mitochondria using a Packard 1900CA Liquid Scintillation Analyzer as described.6 The amount of mitochondria studied was normalized for protein content. Both arteries and mitochondria were studied immediately after isolation.
H2O2 production in vascular rings and mitochondria at baseline and in response to hypoxia and ETC blockers was measured using the AmplexRed H2O2 assay kit (according to the manufacturer’s instructions) as well as DCFH-DA (2′,7′-dichlorofluorescin diacetate) assay, both from Molecular Probes. Background as well as drug autofluorescence was subtracted. Significant autofluorescence was seen with 10 μmol/L (but not 1 μmol/L) aqueous sodium cyanide (see online Figure 2, which can be found in the online data supplement available at http://www.circresaha.org).
Mitochondria isolation and respiration were studied using standard methodology.28,29⇓ Lung and Kidney mitochondria were kept at 4°C and studied immediately. A protein assay (BioRad) was performed on the isolated mitochondria, and the pellets were diluted accordingly to achieve equal mitochondrial protein loading. O2 consumption was measured with an O2 electrode using a MacLab D/A converter (AD Instruments). The maximum change in the rate of respiration in response to the proximal ETC complex substrates (glutamate 10 mmol/L for complex I and succinate 2.5 mmol/L for complex II) or substrate plus inhibitor (rotenone 5 μmol/L, antimycin A 50 μmol/L) was then calculated.
Mitochondrial Quality Control
To determine whether the quality of mitochondria was similar between the lung and kidney preparations (ie, to exclude possible differential damage of the one versus the other preparation during isolation), we used 2 standard quality control techniques: the NADH-supported respiration in intact versus lysed mitochondria and measurement of the respiratory control ration (RCR).28,29⇓
Immunoblotting of ETC proteins from isolated mitochondria and manganese superoxide dismutase (MnSOD) from isolated arteries was performed as previously described.27
Mitochondrial membrane potential (ΔΨm) was studied in first passage cultured PASMCs and RASM, using two different dyes that are widely used in studying ΔΨm, JC-1 and TMRM (tetramethylrhodamine methyl-ester perchlorate). Cells were loaded with either JC-1 (1 μmol/L) or TMRM (20 nmol/L) for 30 minutes (37°C). JC-1 is a cationic fluorescent dye that exhibits potential-dependent mitochondrial accumulation, in that it fluoresces green when uptaken in depolarized mitochondria, whereas it dimerizes and fluoresces red when uptaken in hyperpolarized mitochondria.30,31⇓ TMRM-loaded mitochondria exhibit stronger red fluorescence with hyperpolarization.32 ΔΨm was compared between PASMC and RASMC mitochondria at baseline and in response to hypoxia. All the imaging parameters were kept identical between the two preparations.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Rotenone Mimics Hypoxia Constricting the Pulmonary While Dilating the Renal Circulation
Rotenone (5 μmol/L) and hypoxia both constrict the pulmonary, whereas they dilate the renal vascular bed (Figures 1A and 1B). This difference is independent of nitric oxide and prostaglandins because the experiments were performed in the presence of inhibitors of the endothelial nitric oxide synthase, l-NG-nitroarginine methylester (L-NAME, 5×10−5 mol/L) and cyclooxygenase (meclofenamate, 1.7×10−5 mol/L). In contrast, both vascular beds constrict to angiotensin II (10−5 mol/L) and 4-aminopyridine (4-AP, 5 mmol/L), a Kv channel blocker. The order of hypoxia, rotenone, and 4-AP challenges varied.
Further evidence that the opposing response to hypoxia and rotenone is intrinsic to the SMCs and independent of the endothelium comes from tissue bath experiments. In endothelium-denuded vessels, rotenone (5 μmol/L) mimics hypoxia again, constricting PAs and dilating RAs, whereas 4-AP (5 mmol/L) constricts both RAs and PAs (Figure 1C).
Rotenone Mimics Hypoxia Inhibiting K+ Current in PASMCs While Activating it in RASMCs
Whole-cell patch clamping in freshly isolated SMCs from resistance PAs and RAs shows that rotenone inhibits outward K+ current (Ik) in PASMCs while it activates Ik in RASMCs (Figure 2). The effects of both hypoxia and rotenone were rapid and occurred within 5 minutes.
Production of AOS and H2O2 at Baseline and in Response to Hypoxia and ETC Inhibitors Is Differentially Regulated Between PAs and RAs
We measured baseline AOS production (during normoxia) as well as the effects of hypoxia and ETC inhibitors in isolated, denuded, resistance PA and RA rings using 3 independent methods. (1) Lucigenin-enhanced chemiluminescence, which preferentially detects superoxide anion levels,33 shows that baseline AOS levels are significantly higher in the PAs compared with the RAs, even after incubation with diphenyliodonium (DPI, 1 μmol/L), an inhibitor of NAD(P)H oxidase34 (Figure 3A). Rotenone’s effects on the production of AOS parallel those of hypoxia. They both decrease AOS production in isolated PA rings, confirming previous studies on whole lung preparations.6 In contrast, hypoxia and rotenone increased AOS production in the RAs (Figure 3A), confirming studies in coronary arteries.35 (2) The AmplexRed assay, which is specific for H2O2, shows that, concordant with the chemiluminescence data, the production of H2O2 is higher in the PAs versus the RAs at baseline (Figure 3B). Once again, a proximal ETC inhibitor (antimycin A, 50 μmol/L) mimics hypoxia, and they both decease the production of H2O2 in the PAs. However, neither hypoxia nor antimycin A alter the production of H2O2 in the RA (Figure 3B). (3) DCFH-DA fluorescence (Figure 3C), which like AmplexRed preferentially measures H2O2, once again decreased H2O2 in PAs but did not affect the RA H2O2 production. The proximal ETC (complex I) blocker rotenone once again mimicked hypoxia and decreased the H2O2 production in the PAs, although it did not have any effects on the RAs. The complex III blocker myxothiazol had a significant trend to inhibit H2O2 production in the PAs but did not reach statistical significance (P<0.057). Cyanide (1 μmol/L) did not affect H2O2 production in either artery. We observed a significant autofluorescence of the aqueous sodium cyanide solution itself at the dose of 10 μmol/L, which prevented us from studying it at this dose (see online Figure 1).
Lung Mitochondria Are Less Active and Produce More AOS and H2O2 Than Kidney Mitochondria
The fact that the PAs have higher AOS than the RAs, even in the presence of an NADPH oxidase inhibitor, suggests that mitochondria might be the source of this differentially regulated production of AOS. Metabolically active mitochondria are known to produce less AOS than less active mitochondria.20 We isolated lung and kidney mitochondria and studied respiration driven by substrates that enter the ETC proximally at the complexes I and II (glutamate and succinate; for results with glutamate alone versus succinate and glutamate, see the online data supplement, online Figure 1). We show that lung mitochondria have significantly lower rates of respiration compared with kidney mitochondria (Figure 4A). Rotenone and antimycin A inhibit respiration in both lung and kidney mitochondria, although the lung mitochondria are more sensitive to both inhibitors than are kidney mitochondria. The percent inhibition in respiration by rotenone for the lung and kidney respectively is −87±6 and −53±5 and for antimycin A is −96±3 and −68±15, respectively. The purity of our preparations is shown by electron microscopy, where mostly intact mitochondria and a few lysosomes are seen (Figure 4B).
The mitochondria isolation process could theoretically have resulted in preferential damage of one preparation versus the other, complicating their comparison. We therefore used 2 different assays to ensure that the quality of the mitochondria is similar in both preparations. Both preparations have similar RCRs (Figure 4C). These RCRs are lower that the RCRs from heart mitochondria (>10 in the literature29 and 11.4±0.4, n=3 in our hands), which is expected because blood vessels are metabolically less efficient than the myocardium. They show, however, that the quality of our preparations is similar. This was also confirmed by another assay, the ratio of NADH-driven respiration in sonicated versus intact mitochondria. Intact good quality mitochondria are impermeable and do not respond to NADH, whereas damaged “leaky” mitochondria (sonicated) respond to NADH with increased respiration.28 The ratio of NADH-supported respiration in sonicated/intact mitochondria is similar between the lung and kidney (Figure 4C).
We measured AOS and H2O2 production in lung and kidney mitochondria preparations with similar amounts of mitochondria, based on protein assays, and under identical conditions (Figures 5A and 5B). Lung mitochondria produce more AOS and H2O2 than the kidney mitochondria at baseline. The same proximal ETC substrate (glutamate+succinate) was used in both assays. The near complete elimination of the signal in the AmplexRed assay by catalase (10 000 U) confirms the specificity of the assay for H2O2 (Figure 5B). Antimycin A significantly inhibits the lung AOS production, but it does not alter the kidney mitochondria AOS production (Figure 5A). In contrast, the distal ETC inhibitor cyanide (10 μmol/L) does not alter AOS production in either preparation, confirming previous reports.6 These data are consistent with complexes I and III being the major sites of AOS production within the ETC.20
We then measured the expression of ETC complexes I and III (where most of the production of AOS occurs20) in PAs and RAs using immunoblotting. Although the ETC complexes consist of several subunits each, the commercially available subunits that we studied suggest that complexes I and III are expressed more in the RAs than the PAs (Figure 5C). Similar loading of the gels was confirmed by the Ponceau staining. In addition, using the same immunoblots (see online Figure 3B), we show that PAs express much more MnSOD, suggesting that the lower expression of proximal ETC complexes in this vessel is not a nonspecific finding or artifact of loading.
PASMC Mitochondria Are More Depolarized Than RASMC Mitochondria
We studied mitochondrial function within cultured PASMCs and RASMCs (first passage) loaded with 2 different dyes that are widely used in the measurement of ΔΨm, JC-1 and TMRM, using confocal microscopy (Figure 6).
With JC-1, mitochondria from RASMCs (n=44) have significantly higher ΔΨm than mitochondria from PASMCs (n=28) under identical loading and imaging conditions (Figure 6A). Extensive filamentous networks of mitochondria throughout the cytoplasm are shown in the representative pictures in Figure 6A. When the SMCs were superfused with a hypoxic solution, the PASMC mitochondria (n=30) hyperpolarized (decreased green/red ratio), whereas the RASMC mitochondria (n=40) depolarized.
In agreement with the JC-1 data, TMRM-loaded mitochondria from RASMCs (n=33) have significantly higher ΔΨm compared with mitochondria from PASMC, loaded and imaged under identical conditions (Figure 6B).
We report that physiologically significant mitochondrial diversity exists between the renal and the pulmonary circulations. This diversity exists both at baseline and in response to ETC inhibitors and hypoxia. We show that mitochondrial function is different not only between the PAs and RAs but between the lung and kidney as well, perhaps reflecting differences in the overall redox environment of the two organs. We propose a model by which this diversity might at least in part explain the differences of the two circulations in both their redox state and the response to hypoxia. Our data are supported by a multitude of techniques, including perfused organs, tissue baths, patch clamping, measurement of AOS by 3 different techniques, mitochondrial respiration, immunoblotting, and dynamic confocal imaging using 2 different ΔΨm-sensitive dyes.
Baseline Differences Between the PA/Lung and RA/Kidney Mitochondria
Under identical experimental conditions, lung mitochondria have slower respiratory rates (Figure 4A) than kidney mitochondria. Quality control studies, showed that there was no differential damage during the isolation procedure (Figures 4B and 4C). Direct imaging of vascular SMCs showed that PASMC are more depolarized than RASMC mitochondria (Figures 6A and 6B). In addition, and perhaps related to these differences, lung mitochondria produce more AOS and H2O2 than kidney mitochondria at baseline (Figure 4A and 4B). Although antimycin inhibited respiration in both preparations (Figure 4A), the AOS production in the lung is sensitive to antimycin, whereas the kidney AOS production is antimycin-insensitive (Figure 5A). Early work by Boveris and Chance24 showed similar differences in the antimycin sensitivity between the pigeon heart and rat liver mitochondria H2O2 production. This suggests that the regulation of AOS production within the pETC might be different among organs or vessels.
Redox Potential and the AOS Production Are Different in the PAs Compared With the RAs
We used 3 different techniques and showed that freshly isolated PAs have higher AOS and H2O2 production at baseline compared with the RAs (Figure 3). We also showed that hypoxia and proximal, but not distal ETC inhibitors, significantly decrease AOS production in the PA. Several groups have suggested that proximal ETC function is important in HPV.15,16⇓ However, these authors reported that AOS were increased by hypoxia and proximal ETC inhibition, whereas most groups find hypoxia decreases AOS.6,7,36,37⇓⇓⇓ A strength of our study is that AOS were measured in both mitochondria and in vessels using 3 different assays. The results of these assays were all concordant. In addition, we studied AOS production is freshly isolated arteries, which may be more physiological than the studies of AOS in cultured PASMCs, as performed by others.15 Even after inhibiting NADPH oxidase, there is residual AOS production, which is greater in the PA than the RA (Figure 3A), consistent with the concept that mitochondrial diversity exists between PAs and RAs, beyond any difference in NADPH oxidase that may exist.
Interestingly, although lucigenin-enhanced chemiluminescence was increased in RAs in response to hypoxia and proximal ETC inhibitors, the amount of H2O2 production was not altered. This might be perhaps due to lower levels of MnSOD in the RASMCs. MnSOD catalyzes the formation of H2O2 from superoxide, the major AOS detected by chemiluminescence. Indeed, we showed that both mRNA and expressed protein for MnSOD are significantly lower in the RAs (online data supplement results and online Figure 3).
The differences in AOS production in PAs versus RAs parallel the differences in AOS production in lung versus kidney mitochondria. This might be related to the fact that there might be a more generalized difference in the redox environment between the 2 organs that affects their vasculature as well. In vivo, the lungs and the resistance PAs are exposed to alveolar oxygen levels, which are higher than those in arterial blood perfusing the rest of the organs like the kidneys. This might have induced redox differences. In fact we show that the levels of reduced glutathione (GSH) in the lungs in normoxia are much higher than those in the kidney (online Figure 3). The findings that the lungs have a more oxidized redox potential than the kidneys and their importance are discussed in the online data supplement.
ETC Function Is Inversely Related to AOS Production: Lessons From Human and Animal Disease Models
The difference between PA and RA mitochondria is analogous to the comparison of mitochondria from healthy subjects versus patients with ETC complex I deficiency. Fibroblasts from complex I–deficient patients have increased mitochondrial-derived AOS production when compared with controls.38,39⇓ In addition, their fibroblasts have impaired mitochondrial function, as reflected by depolarized ΔΨm.38,39⇓ Furthermore, superoxide-induced MnSOD is upregulated in these patients.38 The normoxic PASMCs, like the fibroblasts from complex I deficiency patients, have (compared with the kidney) decreased levels of complex I (Figure 5C), decreased mitochondrial respiration (Figure 4A), and depolarized ΔΨm (Figure 6A). Lung mitochondria also make more superoxide and H2O2 than the kidney mitochondria (Figures 5A and 5B). The higher oxidative stress of PAs is associated with an apparently homeostatic induction of MnSOD expression and elevated GSH levels (online Figure 3). Furthermore, the fact that the RAs are relatively “deficient” in both GSH and MnSOD, both forms of oxidant defense, is in agreement with the finding that mice that genetically lack MnSOD also have 30% less GSH when compared with wild-type mice.40
How do these differences in ETC function and AOS production relate to the opposing response of the PA and RA to hypoxia? Important clues come from the striking similarities in the effects of ETC inhibitors and hypoxia discussed subsequently.
Proximal ETC Inhibitors Mimic the Opposing Effects of Hypoxia in PAs Versus RAs
We show that proximal ETC inhibitors and hypoxia inhibit PASMC Ik and constrict the PAs, whereas they activate RASMC Ik and dilate the renal circulation (Figures 1 to 2⇑). These inhibitors are the only class of drugs, to our knowledge, that mimic hypoxia in both circulations. Other important modulators of HPV do not mimic hypoxia in that they constrict both the pulmonary and systemic circulations. For example, endothelin-1 constricts both PAs41 and RAs42 and inhibits Kv currents depolarizing both the PASMCs43 and RASMCs.44 Similarly, the Kv blocker 4-AP constricts both vessels (Figure 1). This suggests that the mechanism of the opposing effects of hypoxia on the 2 circulations is intrinsic to the mitochondrial ETC, upstream from the Kv channels. Furthermore, because these opposing effects persist in the absence of endothelium (Figures 1A through 1C) and are present in isolated SMCs (Figures 2 and 6⇑), it suggests that they are intrinsic to the vascular SMCs and not the endothelium.
Vascular SMC Mitochondrial ETC Is an O2 Sensor
Our findings are in agreement with several recent studies that suggest a central role of mitochondria ETC and redox mediators in HPV,6,15,16⇓⇓ as previously proposed.5 Based on these studies, a potential mechanism for vascular O2 sensing includes (Figure 7) an O2 sensor (mitochondrial ETC or vascular oxidases) that regulates the production of a diffusible redox factor in proportion to O2 (oxygen radicals and peroxides, collectively known as AOS). Of the various species, H2O2 is an attractive candidate mediator because it is more stable than oxygen radicals, such as superoxide, it is freely diffusible, and it can affect several cellular mechanisms involved in tone control, including O2-sensitive K+ channels (effectors; for review, see Archer at al45).
Our study supports many of the known features of vascular O2 sensing: the high level of H2O2 production at baseline in the PAs could explain the relatively vasodilated (low-pressure) state of the pulmonary, compared with the systemic, circulation. Tonically produced AOS/H2O2 from the proximal ETC can diffuse to the cytoplasm and cause K+ channel activation, SCM hyperpolarization, decreased opening of the voltage-gated Ca2+ channels and decreased [Ca2+]i levels and vasodilatation (Figure 7). During acute hypoxia, the tonic production of these vasodilators (AOS/H2O2) decreases (Figure 3), thus promoting Ik inhibition (Figure 2) and vasoconstriction (Figure 1), ie, HPV. In contrast, a hypoxia-induced increase in AOS (Figure 3A) would increase Ik46 and promote RA vasodilatation.
We acknowledge that more than one O2-sensing mechanism might take place in one or both circulations at the same time. Thus, novel vascular oxidases could also contribute to redox signaling.
The concept of mitochondrial diversity in the vasculature is new and requires further study as it is likely relevant to other aspects of vascular function. In addition to its role in vascular O2 sensing, the diversity concept may have implications for apoptosis, vascular wall remodeling, or ischemia/reperfusion injury. It remains to be shown whether this mitochondrial diversity is due to a genetic difference intrinsic to the mitochondria or a result of the different redox environment that the PAs are exposed to, compared with the RAs.
Drs Michelakis and Archer are both supported by the Canadian Institutes for Health Research, The Heart and Stroke Foundation of Alberta, and the Alberta Heritage Foundation for Medical Research.
Original received October 30, 2000; resubmission received December 18, 2001; revised resubmission received May 23, 2002; accepted May 23, 2002.
- ↵Wolin MS. Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol. 2000; 20: 1430–1442.
- ↵Weir EK, Archer SL. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB. 1995; 9: 183–189.
- ↵Archer SL, Huang J, Henry T, Peterson D, Weir EK. A redox based oxygen sensor in rat pulmonary vasculature. Circ Res. 1993; 73: 1100–1112.
- ↵Freeman BA, Crapo JD. Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J Biol Chem. 1981; 256: 10986–10992.
- ↵McMurtry I, Davidson B, Reeves J, Grover R. Inhibition of hypoxic pulmonary vasoconstriction by calcium antagonists in isolated rat lungs. Circ Res. 1976; 38: 99–104.
- ↵Archer SL, Reeve HL, Michelakis EM, Puttagunta L, Waite R, Nelson DP, Dinauer MC, Weir EK. O2-sensing is preserved in mice lacking the 91 kD subunit of Nicotinamide Adenine Dinucleotide Phosphate Oxidase, NAD(P)H oxidase. Proc Natl Acad Sci U S A. 1999; 96: 7944–7949.
- ↵Waypa GB, Chandel NS, Schumacker PT. Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen sensing. Circ Res. 2001; 88: 1259–1266.
- ↵Mulligan E, Lahiri S, Storey BT. Carotid body O2 chemoreception and mitochondrial oxidative phosphorylation. J Appl Physiol. 1981; 51: 438–446.
- ↵Buescher P, Perse D, Pillai R, Litt M, Mitchell M, Sylvester JT. Energy state and vasomotor tone in hypoxic pig lungs. J Appl Physiol. 1991; 70: 1874–1881.
- ↵Palmer JW, Tandler B, Hoppel CL. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem. 1977; 252: 8731–8739.
- ↵Hampl V, Weir EK, Archer SL. Endothelium-derived nitric oxide is less important for basal tone regulation in the pulmonary than the renal circulation of the adult rat. J Vasc Med Biol. 1994; 5: 22–30.
- ↵Michelakis E, Weir E, Nelson D, Reeve H, Tolarova S, Archer S. Dexfenfluramine elevates systemic blood pressure by inhibiting potassium currents in vascular smooth muscle cells. J Phamacol Exper Ther. 1999; 291: 1143–1149.
- ↵Michelakis ED, Weir EK, Wu X, Nsair A, Waite R, Hashimoto K, Puttagunta L, Knaus HG, Archer SL. Potassium channels regulate tone in rat pulmonary veins. Am J Physiol Lung Cell Mol Physiol. 2001; 280: L1138–L1147.
- ↵Darley-Usmar VM, Rickwood D, Wolson MT. Mitochondria: A Practical Approach. Oxford, UK: IRL Press; 1987.
- ↵Li Y, Zhu H, Kuppusamy P, Roubaud V, Zweier J, Trush M. Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. J Biol Chem. 1998; 273: 2015–2023.
- ↵Cross A, Jones O. The effect of the inhibitor diphenylene iodonium on the superoxide generating system of neutrophils. Biochem J. 1986; 237: 111–116.
- ↵Paky A, Michael J, Burke-Wolin T, Wolin M. Endogenous production of superoxide by rabbit lungs: effects of hypoxia or metabolic inhibitors. J Appl Physiol. 1993; 74: 2868–2874.
- ↵Burke-Wolin T, Wolin MS. H2O2 and cGMP may function as an O2 sensor in the pulmonary artery. J Appl Physiol. 1989; 66: 167–170.
- ↵Barrientos A, Moraes C. Titrating the effects of mitochondrial complex I impairment in the cell physiology. J Biol Chem. 1999; 274: 16188–16197.
- ↵Williams MD, Van Remmen H, Conrad CC, Huang TT, Epstein CJ, Richardson A. Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice. J Biol Chem. 1998; 273: 28510–28515.