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(Circulation Research. 2001;88:1259.)
© 2001 American Heart Association, Inc.

Cellular Biology

Model for Hypoxic Pulmonary Vasoconstriction Involving Mitochondrial Oxygen Sensing

Gregory B. Waypa, Navdeep S. Chandel, Paul T. Schumacker

From the Department of Medicine, The University of Chicago, Chicago, Ill.

Correspondence to Paul T. Schumacker, PhD, Department of Medicine, MC6026, The University of Chicago, 5841 S Maryland Ave, Chicago, IL 60637. E-mail pschumac{at}medicine.bsd.uchicago.edu

Abstract

Abstract—We tested whether mitochondria function as the O₂ sensor underlying hypoxic pulmonary vasoconstriction (HPV). In buffer-perfused rat lungs, rotenone, myxothiazol, and diphenyleneiodonium, which inhibit mitochondria in the proximal region of the electron transport chain (ETC), abolished HPV without attenuating the response to U46619. Cyanide and antimycin A inhibit electron transfer in the distal region of the ETC, but they did not abolish HPV. Cultured pulmonary artery (PA) myocytes contract in response to hypoxia or to U46619. The hypoxic response was abolished while the response to U46619 was maintained in mutant (⁰) PA myocytes lacking a mitochondrial ETC. To test whether reactive oxygen species (ROS) derived from mitochondria act as signaling agents in HPV, the antioxidants pyrrolidinedithiocarbamate and ebselen and the Cu,Zn superoxide dismutase inhibitor diethyldithiocarbamate were used. These abolished HPV without affecting contraction to U46619, suggesting that ROS act as second messengers. In cultured PA myocytes, oxidation of intracellular 2',7'-dichlorofluorescin diacetate (DCFH) dye increased under 2% O₂, indicating that myocytes increase their generation of H₂O₂ during hypoxia. This was attenuated by myxothiazol, implicating mitochondria as the source of increased ROS during HPV. These results indicate that mitochondrial ATP is not required for HPV, that mitochondria function as O₂ sensors during hypoxia, and that ROS generated in the proximal region of the ETC act as second messengers in the response.

Key Words: reactive oxygen species • hypoxia • redox signaling • pulmonary circulation • oxidants

Hypoxic pulmonary vasoconstriction (HPV) diverts blood flow away from the lung during fetal development and optimizes lung gas exchange after birth by enhancing the matching of blood flow and ventilation. Excised lungs retain the HPV response.^{1 2 3 4 5 6} Rings of pulmonary artery (PA) constrict under low O₂ conditions^{7 8} even if denuded of endothelium.^{9 10} Even isolated PA myocytes contract during hypoxia,¹¹ indicating that the O₂ sensor is intrinsic to those cells.

Although HPV has been well characterized, the underlying mechanism of O₂ sensing is not established. Among the putative O₂ sensors that have been proposed, mitochondria have been discounted because the K_m of cytochrome oxidase for O₂ is too low to permit detection of physiological hypoxia.¹² Moreover, inhibition of cytochrome oxidase with cyanide failed to abolish HPV.² However, our studies have implicated mitochondria in the O₂ sensing underlying functional and transcriptional responses to hypoxia in other cells.^{13 14 15} Those data suggest that mitochondria generate reactive oxygen species (ROS) in response to low PO₂, which constitutes an O₂-dependent signal.^{14 15} Oxidant signaling during hypoxia appears to originate at complex III, which could continue to function despite inhibition of complex IV with cyanide. The present study sought to determine whether mitochondria also function as the O₂ sensor during HPV and whether ROS generated by mitochondria function as second messengers in that response.

Materials and Methods

Isolated Perfused Lung
Lungs from Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, Ind) were isolated as described previously.¹⁶ Lungs and heart were removed en bloc and the PA and left atrium were cannulated and perfused (8 mL/min) with a buffered salt solution containing BSA (0.5% wt/vol) and indomethacin (10 mg/L). Perfusate was maintained at 38°C, pH 7.4, and bubbled with 5% O₂, 5% CO₂, and 90% N₂. Lungs were ventilated with a humidified mixture of 21% O₂, 5% CO₂, and 74% N₂ (normoxia) at 54 breaths/min, tidal volume of 2 to 3 mL, and end-expiratory pressure of 3 cm H₂O. Left atrial and PA pressures were continuously recorded. All animals were housed and cared for under National Research Council guidelines for care and use of laboratory animals.

HPV in Isolated Lungs
Angiotensin II (10 nmol/L) was added to the perfusate, and HPV was induced by switching from normoxia to hypoxia (2% O₂, 5% CO₂, 93% N₂). Hypoxia-induced changes in pulmonary vascular impedance are represented as the change in PA pressure during constant flow, compared with normoxia, in cm H₂O. Two hypoxic challenges were averaged to define the baseline response before experimental intervention. The experimental agents were added to the reservoir and recirculated, after which two more hypoxic challenges were administered and averaged. In the continued presence of the agents, the stable thromboxane A₂ analogue U46619 (5 ng/mL) was added to the reservoir to determine the vasoconstrictor response to this receptor-mediated agonist.

PA Myocytes
PA microvessel myocytes were isolated after the method of Marshall et al.⁷ Myocytes were plated on collagen-coated coverslips and grown until 70% or 15% confluent, for 2',7'-dichlorofluorescin diacetate (DCFH) or contraction studies, respectively. Mutant (⁰) PA myocytes were generated from wild-type cells by incubation in ethidium bromide (25 ng/mL) for 2 weeks.¹⁷ This inhibits replication of mitochondrial DNA, which encodes critical subunits of the ETC.¹⁸ The absence of cytochrome oxidase subunit II was confirmed by polymerase chain reaction. Myocytes on coverslips were placed in a flow-through chamber on an inverted microscope and studied under controlled [O₂] at 37° using Hoffman-modulation optics. Cell contraction was assessed from changes in cell length after 30 minutes and was expressed as percent decrease from the original length at t=0, {[(original length)-(length at 30 minutes)/(original length)]x100}.¹¹

Measurement of ROS
ROS generation in PA myocytes was assessed using DCFH-DA (5 µmol/L, Molecular Probes). In the presence of H₂O₂, this probe is oxidized to 2',7'-dichlorofluorescein (DCF), which was quantified using fluorescence imaging (excitation: 488 nm, emission: 535 nm) and reported as percent of initial values, after subtracting background (Universal Imaging).

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

Results

HPV in Isolated Perfused Lung
Figure 1A shows a representative PA pressure tracing for an isolated rat lung during alveolar hypoxia or in response to U46619. Hypoxia (2% O₂, 5% CO₂, 93% N₂) increased PA pressure by 8.3±0.6 cm H₂O (Hypoxia, Figure 1B). Administration of drug vehicle (0.1% DMSO) had no effect on the HPV response (7.2±0.6 cm H₂O) compared with hypoxia. Addition of U46619 (5 ng/mL) increased PA pressure by 17.8±2.5 cm H₂O, confirming the ability to respond to a receptor-mediated vasoconstrictor. Washout and replacement with fresh perfusate containing angiotensin II had no effect on HPV (8.3±0.5 cm H₂O). These results confirm the suitability of this model for studies of HPV.

View larger version (46K): [in this window] [in a new window]   Figure 1. Changes in PA pressure during hypoxia in an isolated perfused lung. A, Typical tracing of a control lung during hypoxia. B, Average change in PA pressure during hypoxia, determined by subtracting baseline pressure from the maximal value. Drug Vehicle+Hypoxia denotes hypoxia-induced response in the presence of DMSO (0.1%). U46619 denotes the response to U46619 (5 ng/mL). Hypoxia Post-wash denotes the hypoxia-induced increase in pressure after washout with fresh perfusate. Data are mean±SE; n=4.

Mitochondria as O₂ Sensors During HPV
To determine the requirement for electron transport in HPV, the flavoprotein inhibitor diphenyleneiodonium (DPI) was added to the isolated lung perfusate before hypoxia. DPI (10 µmol/L) blunted HPV compared with controls (6.6±0.6 cm H₂O) without affecting the response to U46619 (11±3.8 cm H₂O) (Figure 2A). In cultured PA myocytes, hypoxia elicited contraction compared with normoxia; this response was attenuated in the presence of DPI (Table 1).

View larger version (13K): [in this window] [in a new window]   Figure 2. Changes in PA pressure during hypoxia in an isolated perfused lung. A, Hypoxia-induced response in the presence of DPI (10 µmol/L). B, Hypoxia-induced response in the presence of rotenone (5 µg/mL). C, Hypoxia-induced response in the presence of rotenone (50 ng/mL). D, Hypoxia-induced response in the presence of rotenone (5 ng/mL). Data are mean±SE; n=4. *P<0.05 compared with Hypoxia.

View this table: [in this window] [in a new window]   Table 1. PA Myocyte Contraction Studies

To further clarify the mitochondrial ETC requirement for HPV, the inhibitor rotenone was added to the perfusate before hypoxia. Rotenone at 5 µg/mL inhibited HPV compared with controls (6.2±0.6 cm H₂O) (Figure 2B), but it also abolished the response to U46619 (1.2±0.2 cm H₂O). Rotenone at 50 ng/mL inhibited the rate of O₂ uptake by isolated pulmonary cells by 78%, and it abolished HPV (8.0±1.0 cm H₂O) (Figure 2C) without attenuating the response to U46619 (13.2±1.5 cm H₂O). At 5 ng/mL, rotenone had no effect on HPV or the response to U46619 (Figure 2D). In cultured PA myocytes, rotenone at 50 ng/mL abolished the contractile response to hypoxia without affecting the response to U46619 (Table 1).

Myxothiazol inhibits complex III by blocking electron transfer to the Rieske iron-sulfur center in the bc₁ complex.¹⁹ Myxothiazol at 50 ng/mL inhibited lung mitochondrial O₂ uptake by 99%. It also attenuated HPV (1.6±0.2 cm H₂O) compared with controls (12.2±0.8 cm H₂O), without affecting the response to U46619 (17±0.6 cm H₂O) (Figure 3A). These effects were reversed after washout with fresh perfusate (11.6±1.9 cm H₂O). In cultured PA myocytes, myxothiazol attenuated the contractile response to hypoxia without affecting the response to U46619 (Table 1).

View larger version (15K): [in this window] [in a new window]   Figure 3. Changes in PA pressure during hypoxia in an isolated perfused lung. A, Hypoxia-induced response in the presence of myxothiazol (50 ng/mL). B, Hypoxia-induced response in the presence of antimycin A (1 ng/mL). C, Hypoxia-induced response in the presence of cyanide (10 µmol/L). D, Hypoxia-induced response in the presence of DIDS (200 µmol/L). Data are mean±SE; n=4. *P<0.05 compared with Hypoxia.

Antimycin A inhibits the oxidation of cytochrome b₅₆₂ at complex III.¹⁹ Antimycin A (10 ng/mL) inhibited HPV but also attenuated the response to U46619 (not shown). At 1 ng/mL, antimycin A attenuated mitochondrial O₂ uptake by 97% without inhibiting HPV (8.9±3.7 cm H₂O) compared with controls (7.4±1.4 cm H₂O) and without affecting the response to U46619 (13.8±2.5 cm H₂O) (Figure 3B). In PA myocytes, antimycin A elicited contraction during normoxia (Table 1). Cyanide inhibits complex IV of mitochondria. Cyanide at 10 µmol/L inhibited mitochondrial O₂ uptake by 89% and augmented HPV (10.2±0.8 cm H₂O) compared with controls (7.6±0.5 cm H₂O) without affecting the response to U46619 (14.8±1.2 cm H₂O) (Figure 3C).

To clarify the requirement for mitochondria in HPV, mutant ⁰ cells were generated from wild-type rat PA myocytes. Contraction of ⁰ cells under hypoxia (2% O₂) was attenuated compared with controls, while the response to U46619 was preserved (Table 1).

Increased ROS Signaling in HPV
To determine whether increases in ROS are required for HPV, the thiol reductant pyrrloidinedithiocarbamate (PDTC) was added before hypoxic challenge of isolated lungs. As an antioxidant,^{15 20} PDTC appears to enhance H₂O₂ clearance by reducing oxidized glutathione. PDTC (5 µmol/L) attenuated HPV to 2.6±0.7 cm H₂O compared with controls (7.3±0.8 cm H₂O) (Figure 4A), without affecting the response to U46619 (6.8±1.3 cm H₂O). The HPV response was restored (6.2±0.7 cm H₂O) after the PDTC was washed out and perfusate was replaced. At 10 µmol/L, PDTC also blocked HPV (2.1±0.5 cm H₂O) compared with controls (7.4±1.1 cm H₂O), without affecting the response to U46619 (9.2±1.2 cm H₂O) (Figure 4B). However, HPV was not restored (2.4±0.5 cm H₂O) after washout of this higher concentration. In cultured PA myocytes, PDTC inhibited the contractile response to hypoxia (Table 1).

View larger version (15K): [in this window] [in a new window]   Figure 4. Effects of antioxidants on the change in PA pressure during hypoxia in an isolated perfused rat lung model. A, Hypoxia-induced response in the presence of PDTC (5 µmol/L). B, Hypoxia-induced response in the presence of PDTC (10 µmol/L). C, Hypoxia-induced response in the presence of ebselen (50 µmol/L). D, Hypoxia-induced response in the presence of DDC (1 mmol/L). Data are mean±SE; n=4. *P<0.05 compared with hypoxia.

Ebselen, a synthetic glutathione peroxidase, was added to the perfusate before hypoxic challenge to clarify the role of H₂O₂ in HPV. Ebselen (50 µmol/L) attenuated HPV (1.0±0.1 cm H₂O) compared with controls (6.4±0.4 cm H₂O) (Figure 4C), without altering the response to U46619 (8.1±1.1 cm H₂O). The HPV response was not restored after washout (1.4 cm H₂O). In cultured PA myocytes, ebselen also attenuated contraction during hypoxia (Table 1).

To clarify the relative importance of superoxide versus H₂O₂ in HPV, a cytosolic Cu,Zn superoxide dismutase (SOD) inhibitor, diethyldithiocarbamate (DDC), was added to the perfusate before hypoxic challenge. By inhibiting the formation of H₂O₂ in the cytosol, this should attenuate HPV if H₂O₂ is required for signaling, but could enhance the response if superoxide itself was involved. DDC (1 mmol/L) blunted HPV (2.1±0.5 cm H₂O) compared with controls (6.5±0.8 cm H₂O) (Figure 4D) without inhibiting the response to U46619 (18.3±0.8 cm H₂O). Inhibition of HPV by DDC was reversed after washout (6.2±0.5 cm H₂O). To test whether peroxide is sufficient to cause vasoconstriction, H₂O₂ (100 µmol/L) was added during normoxia. This increased PA pressure by 4.1±0.4 cm H₂O (Table 2). When administered to normoxic cultured PA myocytes, H₂O₂ also elicited contraction (Table 1).

View this table: [in this window] [in a new window]   Table 2. Transient Changes in PA Pressure

Some mitochondrial inhibitors induce pulmonary vasoconstriction during normoxia.^{1 2} In our study, DPI (10 µmol/L), rotenone (50 and 5 µg/mL), antimycin A (10 ng/mL), and cyanide (10 µmol/L) caused transient (<10 minutes) increases in PA pressure during normoxia (Table 2). However, myxothiazol (50 ng/mL) had no effect. The transient increase in PA pressure induced by cyanide was attenuated by pretreatment with wither ebselen or myxothiazol. Neither PDTC, ebselen, DDC, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), nor apocynin altered PA pressure during normoxia.

Mitochondrial Generation of ROS During Hypoxia
To determine the source of ROS during hypoxia, cultured PA myocytes were studied using DCFH dye at 37°C in a flow-through chamber. When the [O₂] bubbling the media was switched from 16% to 2% O₂, DCF fluorescence increased (Figure 5A). Return to 16% O₂ was associated with a return toward baseline in each case, which likely reflects leakage of oxidized dye from the cells. The response to hypoxia was repeated in the same field of cells before and after addition of myxothiazol. At 100 ng/mL, myxothiazol attenuated the increase in DCFH oxidation during hypoxia (Figure 5B).

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of hypoxia on DCFH oxidation in PA myocytes. A, DCF fluorescence intensity when [O2] was decreased from 16% to 2%. Representative tracing; values are percent, relative to baseline. B, DCF fluorescence during 2% O2 before and after addition of myxothiazol (100 ng/mL). Data are mean±SE; n=5, *P<0.005.

Superoxide generated in the mitochondria would require an anion channel to reach the cytosol. If so, then an inhibitor of anion channels should attenuate cytosolic ROS signaling during hypoxia. To test this, we evaluated the anion channel inhibitor DIDS in perfused lungs. DIDS (200 µmol/L) inhibited HPV (2.8±0.7 cm H₂O) compared with hypoxia alone (9.4±0.7 cm H₂O) (Figure 3D), without affecting the response to U46619 (13.4±2.4 cm H₂O). HPV was restored after washout (8.4±1.1 cm H₂O). In PA myocytes, DIDS blunted contraction during hypoxia without affecting the response to U46619. DIDS also attenuated the contractile response to antimycin A seen during normoxia (Table 1).

Alternative Sources of ROS
To test the involvement of NADPH oxidase in HPV,^{4 7} the inhibitor apocynin was added before hypoxia. Apocynin (3 mmol/L) abolished HPV compared with controls (6.3±0.7 cm H₂O), but it also abolished the response to U46619 (0.6±0.2 cm H₂O). At a lower concentration (300 µmol/L) that suppresses the respiratory burst of alveolar macrophages,²¹ apocynin failed to inhibit HPV (6.6±0.6 cm H₂O) compared with controls (9.5±1.0 cm H₂O), but did not inhibit the response to U46619 (12.7±2.3 cm H₂O).

Discussion

Role of Mitochondria as the O₂ Sensor in HPV
We used parallel studies in intact lungs and in cultured PA myocytes to test whether mitochondria function as the O₂ sensor underlying HPV. In perfused lungs, inhibitors of the proximal region of the mitochondrial ETC including DPI, rotenone, and myxothiazol abrogated the hypoxia-induced increase in PA pressure without affecting the response to U46619. In PA myocytes, these inhibitors also abolished contraction during hypoxia. By contrast, more distal inhibitors of the ETC failed to abolish HPV. In this regard, cyanide augmented the HPV response, and antimycin A caused constriction of PA myocytes during normoxia. Thus, the response to hypoxia requires electron transport but does not require mitochondrial ATP because all of the inhibitors block oxidative phosphorylation, yet only the proximal inhibitors selectively abolish HPV.

To use a nonpharmacological approach, we generated mutant ⁰ cells from wild-type PA myocytes. These cells lack mitochondrial DNA, which encodes a number of essential subunits in the ETC complexes.¹⁷ These cells do not respire and depend on glycolytic ATP, yet they are morphologically indistinguishable from wild-type cells. We observed that ⁰ PA myocytes retain the contractile response to U46619 but fail to respond to hypoxia. These findings support the conclusion that mitochondria function as the O₂ sensor underlying HPV. Interestingly, we previously found that ⁰ Hep3B selectively lose the ability to activate the transcription factor HIF-1 during hypoxia, yet they retained the ability to respond to other stimuli such as cobalt chloride.¹⁵ Collectively, these observations suggest that a similar mitochondrial O₂ sensing mechanism may be responsible for HPV and for HIF-1–mediated transcriptional activation during hypoxia.

ROS as Second Messengers in HPV
Our results suggest that the mitochondrial ETC acts as an O₂ sensor during hypoxia by releasing ROS that function as signaling messengers (Figure 6). Superoxide generation is known to occur at the ubisemiquinone site of complex III via univalent electron transfer to O₂.²² The antioxidants PDTC, ebselen, and DDC blocked the response to hypoxia without affecting the U46619 response, suggesting that ROS and, in particular, H₂O₂ act as second messengers. The predicted increase in oxidant signaling during hypoxia was confirmed using the intracellular probe DCFH in cultured PA myocytes during hypoxia. Myxothiazol attenuated the increase in fluorescence during hypoxia, consistent with its expected inhibition of ubisemiquinone generation at complex III. These results further implicate ROS generated by the mitochondrial ETC in the signaling process.

View larger version (33K): [in this window] [in a new window]   Figure 6. Model describing the mechanism of O2 sensing by mitochondria underlying the HPV response. Boxes show sites of inhibition.

Previous studies also implicate increased ROS generation in the response to hypoxia. Monaco et al²³ showed that HPV was augmented when catalase was inhibited with aminotriazole. Weissmann et al⁵ observed that SOD and 4,5-dihydroxy-1,3-benzenedisulfonic acid (to accelerate H₂O₂ generation from superoxide) did not affect HPV, suggesting that H₂O₂, rather than superoxide, is involved. By contrast, nitrobluetetrazolium, which traps superoxide and prevents H₂O₂ formation, attenuated HPV. Finally, our data and previous studies show that H₂O₂ constricts the pulmonary circulation during normoxia.²⁴ These findings are consistent with a role for increased H₂O₂ as a signaling molecule involved in HPV.

Mechanism of ROS Generation During Hypoxia
Our previous studies demonstrated that hypoxia affects cytochrome oxidase, causing it to cycle at a more reduced state.²⁵ We had suggested that the increase in reduction state of that complex should cause a similar redox change at more proximal ETC sites, which might explain the increase in ROS generation at complex III during hypoxia.¹⁴ However, we later observed that hypoxia still increased ROS generation when electron transport at the distal end of complex III was inhibited by antimycin A.¹⁵ In the presence of an inhibitor, more proximal ETC complexes become fully reduced while those at more distal locations become oxidized. The observations that hypoxia augmented ROS signaling during antimycin A, and that antimycin A and cyanide failed to abolish the hypoxic constriction, suggest that the O₂-sensing site must be located upstream from the antimycin A inhibition site. Moreover, the O₂ sensor must still be able to function if the ETC chain is fully reduced.

During normoxia, ETC inhibitors acting at sites distal to ubisemiquinone (eg, cyanide, azide, or antimycin A) tend to augment ROS generation by increasing the reduction state of the ubiquinone pool.^{14 15 19 26} During hypoxia, our model suggests that the biophysical process for ROS generation from that site is amplified, even when the complex is fully reduced. By contrast, if the complex becomes fully oxidized by ETC inhibition at a more proximal site (eg, rotenone, DPI, or myxothiazol) then ROS generation and O₂ sensing are abolished by the lack of electrons. In accordance with this model (Figure 6), distal inhibitors of the ETC such as cyanide induced constriction during normoxia, through a mechanism that could be inhibited by ebselen (an antioxidant) or myxothiazol (a more proximal ETC inhibitor). Similarly, Rounds and McMurtry¹ previously found that antimycin A elicits vasoconstriction in normoxic lungs, a response we reproduced (Table 2). Also, Archer et al² found that cyanide induced vasoconstriction during normoxia and augmented HPV without affecting the response to angiotensin II or KCl. Collectively, these observations are consistent with our proposed model, but the mechanism by which hypoxia amplifies ROS generation at complex III is not yet known.

An alternative explanation for the increase in ROS signaling during hypoxia involves the regulation of superoxide egress from mitochondria to cytosol. Superoxide would presumably require an anion channel to escape from the matrix. The inner membrane anion channel (IMAC) could conceivably function as that pathway. If the IMAC were an O₂-sensitive channel that increased its conductance during hypoxia, the egress of superoxide from the matrix could increase even if the rate of mitochondrial superoxide generation were to decrease during hypoxia. This could explain why ROS signaling in the cytosol is increased during moderate hypoxia, and why DIDS, which inhibits mitochondrial IMAC,^{27 28} attenuated HPV and abolished PA myocyte contraction during hypoxia. It should be noted that DIDS is also a thiol-reactive compound; however, pretreatment with DIDS had no effect on H₂O₂-induced vasoconstriction in the lung (see online data supplement available at http://www.circresaha.org). In either case, it appears that superoxide enters the cytosol where it is dismuted to H₂O₂ by cytosolic Cu,Zn SOD. Inhibition of SOD by DDC abrogated the hypoxic responses, indicating that superoxide conversion to H₂O₂ is required for the response.

Downstream Signaling in HPV
Smooth muscle contraction during HPV requires an increase in cytosolic [Ca²⁺]. Although H₂O₂ appears to act as a signaling messenger in the sequence leading to calcium activation, the details of that pathway are not fully understood. One possibility is that potassium channels become inhibited through redox-mediated signaling, resulting in membrane depolarization and the opening of voltage-dependent calcium channels.^{2 9 10 11 29 30 31} However, other studies show that PA myocytes demonstrate increases in cytosolic [Ca²⁺] and cell contraction in the presence of 4-aminopyridine, a pharmacological inhibitor of these Kv channels.³² Their results indicate that HPV can be elicited by a mechanism that does not require closure of Kv channels. One possibility is that H₂O₂ might cause oxidation of mitochondrial pyridine nucleotides, resulting in mitochondrial Ca²⁺ release.³³ However, further studies are required to address this theory.

Drug-Induced Contraction During Normoxia
In previous studies, the mitochondrial inhibitor rotenone attenuated HPV but also produced a transient increase in PA pressure immediately after administration.¹ Rounds and McMurtry¹ suggested that this was due to an inhibition of ATP production, whereas Archer et al² suggested that a shift in the cytosolic redox status elicits contraction. We also observed a small transient increase in PA pressure on addition of rotenone, cyanide, or DPI. However, myxothiazol had no such effect, so not all ETC inhibitors elicit vasoconstriction. We suggest that the transient responses to some compounds reflect nonspecific effects on the pulmonary circulation. By contrast, their effects on the subsequent response to hypoxia reflect their influence on HPV.

Alternative ROS Sources
Marshall et al⁷ suggested that hypoxia accelerates ROS generation by a membrane-bound NADPH oxidase, based on their observation that DPI inhibited the oxidant signal and the contractile response to hypoxia. DPI inhibits a wide range of flavoproteins including NADPH oxidase, mitochondrial complex I,³⁴ glutathione reductase, nitric oxide synthase, and prostaglandin synthetase.^{34 35} Therefore, the site of inhibition responsible for the attenuation of HPV is not known. It is conceivable that DPI abolished HPV by inhibiting mitochondrial complex I and abolished their chemiluminescence signal through a separate effect on NAD(P)H oxidase. Grimminger et al⁴ also found that DPI attenuated HPV without affecting the vascular response to U46619, consistent with our findings. To address the possible involvement of NADPH oxidase in HPV, we used apocynin, a selective inhibitor of the neutrophil form of this enzyme.²¹ At concentrations that preserved the response to U46619, apocynin failed to attenuate either HPV or PA myocyte contraction during hypoxia. In homozygous knockout animals lacking the gp91^phox subunit of the NADPH oxidase complex, Archer et al³⁶ found that the response to hypoxia was preserved, further suggesting a lack of involvement of that system in HPV.

In summary, our results demonstrate that HPV requires mitochondrial electron transport proximal to the ubisemiquinone site but does not require the entire mitochondrial ETC to be functional. ROS generated by mitochondria appear to function as second messengers during hypoxia and contribute to the signal transduction process leading to smooth muscle cell contraction in HPV.

Acknowledgments

This research was supported by NIH Grants HL32646, HL35440, HL66315, and HL10405. The authors gratefully acknowledge the technical assistance of Carol Mathieu, Dr Ningfang Chen, and Dr Matthew Mack in these studies.

Footnotes

Original received December 27, 1999; resubmission received December 21, 2000; revised resubmission received April 23, 2001; accepted April 24, 2001.

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