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(Circulation Research. 2002;90:1307.)
© 2002 American Heart Association, Inc.

Integrative Physiology

Diversity in Mitochondrial Function Explains Differences in Vascular Oxygen Sensing

Evangelos D. Michelakis, Vaclav Hampl, Ali Nsair, XiCheng Wu, Gwyneth Harry, Al Haromy, Rachita Gurtu, Stephen L. Archer

From the Department of Medicine (Cardiology) and the Vascular Biology Group (E.D.M., A.N., X.W., G.H., A.L., R.G., S.L.A.), University of Alberta, Edmonton, Alberta, Canada; and Charles University, Physiology (V.H.), Prague, Czech Republic.

Correspondence to Evangelos D. Michelakis, MD, Cardiology, University of Alberta, 2C2.36 Walter Mackenzie Health Sciences Centre, Edmonton, AB, Canada T6G 2B7. E-mail emichela{at}cha.ab.ca

	Abstract

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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.

Key Words: rotenone • K⁺ channels • redox • pulmonary circulation • oxygen sensor

	Introduction

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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 PO_2, and thus have been proposed as candidates for vascular oxygen sensors.^5,6 Despite the low K_M 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

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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.

Tissue Baths
Resistance PA and RA rings (4 to 5th order), denuded of endothelium, were studied as previously described.²⁶

Patch Clamping
Whole-cell patch-clamp recordings were performed in freshly isolated PASMCS and RASMCs, as previously described.²⁷

Chemiluminescence
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 Measurement
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).

View larger version (29K): [in this window] [in a new window]   Figure 2. Opposing effects of rotenone and hypoxia on PASMC vs RASMC Ik. Both hypoxia and rotenone inhibit whole cell PASMC Ik, whereas they both increase RASMC Ik.

Mitochondria Respiration
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
Immunoblotting of ETC proteins from isolated mitochondria and manganese superoxide dismutase (MnSOD) from isolated arteries was performed as previously described.²⁷

Confocal Microscopy
Mitochondrial membrane potential (m) was studied in first passage cultured PASMCs and RASM, using two different dyes that are widely used in studying m, 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 m 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.

	Results

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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-N^G-nitroarginine methylester (L-NAME, 5x10^-5 mol/L) and cyclooxygenase (meclofenamate, 1.7x10^-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.

View larger version (28K): [in this window] [in a new window]   Figure 1. Opposing actions of rotenone and hypoxia on the renal vs the pulmonary circulation. A and B, Both rotenone (5 µmol/L) and hypoxia (PO2 40 mm Hg) constrict the pulmonary and dilate the renal circulation in the presence of L-NAME and meclofenamate. 4-AP (5 mmol/L) and angiotensin II (AII, 0.1 µg bolus, total perfusate 50 mL) constrict both vascular beds. C, Rotenone mimics hypoxia, constricting PA rings while dilating RA rings (both denuded of endothelium), whereas 4-AP and phenylephrine (PE) constrict both vessels.

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 (I_k) in PASMCs while it activates I_k 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).

View larger version (35K): [in this window] [in a new window]   Figure 3. Differences between the PA and RA production of AOS. A, Lucigenin-enhanced chemiluminescence at normoxic baseline is much higher in denuded PA rings than in denuded RA rings (P<0.001), even in the presence of the NAD(P)H oxidase inhibitor DPI (*P<0.05). Both rotenone and hypoxia (PO2 40 mm Hg) decrease AOS in the PA (P<0.01) and increase AOS production in the RA (**P<0.05). B, PAs produce more H2O2 than RAs (P<0.01). Both hypoxia and antimycin A decrease H2O2 production in the PA (P<0.05) but not the RA. C, PAs produce more H2O2 than RAs. Both rotenone and hypoxia decrease H2O2 production in the PA but not in the RA. Myxothiazol and cyanide do not alter H2O2 production in either vessel (*P<0.05 compared with normoxic PAs; P<0.01 compared with normoxic PAs).

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).

View larger version (46K): [in this window] [in a new window]   Figure 4. PA mitochondria are less active than RA mitochondria. A, Baseline respiration is greater in isolated kidney vs lung mitochondria. Rotenone and antimycin A inhibit respiration significantly in both organs (P<0.01, *P<0.05). Substrates=glutamate (10 mmol/L)+succinate (2.5 mmol/L). B, Mitochondrial preparations are >90% pure as seen by electron microscopy. A few lysosomes are seen. D, Quality assessment by 2 assays (respiratory control ratio and the ratio of NADH-supported respiration in sonicated/intact mitochondria) shows that the condition of mitochondria from the lung and kidney is similar.

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

View larger version (29K): [in this window] [in a new window]   Figure 5. Lung mitochondria produce more AOS than kidney mitochondria, perhaps due to differences in the expression of ETC complexes I and III. A, Lucigenin-enhanced chemiluminescence is higher at baseline in the lung vs kidney mitochondria (*P<0.05). Antimycin, but not cyanide, decrease AOS production in the lung but not in the kidney (P<0.05). B, Catalase-sensitive production of H2O2 is higher in the lung vs kidney mitochondria at baseline (*P<0.05). Glutamate+succinate were used as substrates in both A and B. C, Expression of complex I and III (immunoblotting) is less in the PA.

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 m, JC-1 and TMRM, using confocal microscopy (Figure 6).

View larger version (45K): [in this window] [in a new window]   Figure 6. m is more negative in RASMC mitochondria vs PASMC. A, JC-1–loaded PASMCs and RASMCs. Representative confocal images are shown in the left (x40). Mean data for baseline m, expressed as the ratio of the relative fluorescent units in the green and red channels, are shown on the right. PASMC mitochondria are more depolarized than the RASMC mitochondria. Hypoxia hyperpolarizes PASMC mitochondria, whereas it depolarizes RASMC mitochondria. B, TMRM-loaded RASMC mitochondria exhibit stronger red fluorescence, suggesting they are more hyperpolarized than PASMC mitochondria loaded and imaged under identical conditions (*P<0.05, **P<0.0001).

With JC-1, mitochondria from RASMCs (n=44) have significantly higher m 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 m compared with mitochondria from PASMC, loaded and imaged under identical conditions (Figure 6B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 m-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 m.^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 m (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 I_k and constrict the PAs, whereas they activate RASMC I_k 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).

View larger version (58K): [in this window] [in a new window]   Figure 7. Proposed sensors, mediators, and effectors in vascular O2 sensing.

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 I_k inhibition (Figure 2) and vasoconstriction (Figure 1), ie, HPV. In contrast, a hypoxia-induced increase in AOS (Figure 3A) would increase I_k⁴⁶ 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.

	Acknowledgments

 
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.

Received October 30, 2000; revision received December 18, 2001; accepted May 23, 2002.

	References

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