Nitric Oxide–Mediated Zinc Release Contributes to Hypoxic Regulation of Pulmonary Vascular Tone
The metal binding protein metallothionein (MT) is a target for nitric oxide (NO), causing release of bound zinc that affects myogenic reflex in systemic resistance vessels. Here, we investigate a role for NO-induced zinc release in pulmonary vasoregulation. We show that acute hypoxia causes reversible constriction of intraacinar arteries (<50 μm/L) in isolated perfused mouse lung (IPL). We further demonstrate that isolated pulmonary (but not aortic) endothelial cells constrict in hypoxia. Hypoxia also causes NO-dependent increases in labile zinc in mouse lung endothelial cells and endothelium of IPL. The latter observation is dependent on MT because it is not apparent in IPL of MT−/− mice. Data from NO-sensitive fluorescence resonance energy transfer–based reporters support hypoxia-induced NO production in pulmonary endothelium. Furthermore, hypoxic constriction is blunted in IPL of MT−/− mice and in wild-type mice, or rats, treated with the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), suggesting a role for chelatable zinc in modulating HPV. Finally, the NO donor DETAnonoate causes further vasoconstriction in hypoxic IPL in which NO vasodilatory pathways are inhibited. Collectively, these data suggest that zinc thiolate signaling is a component of the effects of acute hypoxia-mediated NO biosynthesis and that this pathway may contribute to constriction in the pulmonary vasculature.
Acute hypoxic pulmonary vasoconstriction (HPV)1 is unique to the pulmonary vascular bed and is an important mechanism for matching blood flow to ventilation, thereby preventing arterial hypoxemia. Reductions in oxygen tension and associated changes in vascular resistance have been associated with increased endothelium-derived nitric oxide (NO).2 In the systemic circulation, this is believed to contribute to hypoxic vasodilation, whereas in the lung, NO biosynthesis will oppose hypoxic vasoconstrictor stimuli via activation of the soluble guanylyl cyclase (sGC)/cGMP pathway or by directly opening KCa2+ channels in pulmonary vascular smooth muscle.3,4
In addition to covalent modification of heme or nonheme iron, NO may exert significant biological activity via S-nitrosation of thiol groups. The zinc-thiolate moieties of the metal binding protein metallothionein (MT) are critical targets for NO,5,6 directly affecting intracellular zinc homeostasis.6,7 Although the physiological relevance of NO-induced changes in labile zinc is unknown, interactions between NO and MT facilitate myogenic reactivity in systemic resistance vessels.5 Indeed, whereas calcium has a well-documented critical role in pulmonary vasoregulation, little is known about the role of the other major divalent cation, zinc.
We used contemporary optical microscopy and fluorescent reporter molecules in live cells and isolated perfused lungs (IPLs) of rats and genetically modified mice to investigate the role of NO-induced changes in labile zinc on pulmonary vasoregulation. We hypothesized that hypoxia-induced acute increases in NO synthesis, in addition to opposing HPV via activation of sGC or direct activation of KCa2+ channels, contribute to vasoconstriction in pulmonary resistance vessels via S-nitrosation of the metal binding centers of MT and alterations in intracellular zinc homeostasis.
Materials and Methods
All studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh and following the guidelines of the American Physiological Society.
Cultures were grown at 37°C in an atmosphere with 5% CO2. Mouse lung endothelial cell8 and sheep pulmonary artery endothelial cell7 preparations are described elsewhere. Rat pulmonary microvascular endothelial cells (RPMECs) and rat aortic endothelial cells (RAECs) were purchased from VEC Technologies Inc (Rensselaer, NY) and grown in complete MCDB-131 media (VEC Technologies Inc).
Mouse and Rat Strains
Sprague–Dawley rats; Tie2-green fluorescent protein (GFP) mice (STOCK Tg(TIE2GFP)287Sato/J); MT−/− (129S7/SvEvBrd-Mt1tm1BriMt2tm1Bri) and wild-type controls (129S1/SvImJ, MT+/+) were purchased from The Jackson Laboratory (Bar Harbor, Me).
Isolated Perfused Mouse Lung
Mice were anesthetized, heparin was injected IV (50 U), and a thoracotomy was performed to expose heart and lungs. The trachea was cannulated, and heart and lungs were removed en bloc. Catheters were placed in the pulmonary artery and left atrium. Lungs were perfused via a peristaltic pump (0.8 mL/min) with a modified Krebs–Henseleit solution supplemented with 5.0 μmol/L meclofenamate and 5% dextran. Heart/lungs were then transferred to a glass-bottomed, humidified, temperature-controlled chamber. During image acquisition, ventilation was stopped and the lungs were held statically inflated. Perfusion pressure was monitored and recorded at constant flow (PowerLab, ADInstruments Inc, Colorado Springs, Colo). After establishing a baseline perfusion pressure with 21% O2, lungs were inflated with the hypoxic gas mixture (1.5% O2, 5% CO2, balance N2) for 10 minutes, followed by a return to 21% O2. The use of 1.5% O2 resulted in a drop in Po2 from 100 to 110 mm Hg to ≈30 to 35 mm Hg, as measured in the venous effluent using a Clarke electrode.
Isolated Perfused Rat Lung
Male rats (300 to 350 g) were anesthetized and injected IV with heparin, and a thoracotomy was performed to expose heart and lungs. Rats were ventilated (model 683, Harvard Apparatus, Holliston, Mass) at 55 breaths per minute with tidal volumes of <10 cm H2O. The pulmonary artery and left atrium were cannulated, and the ventilator was set to 2 cm H2O of positive end expiratory pressure. Lungs were perfused at 3 mL/min with warmed Krebs–Henseleit buffer supplemented with 3% Ficoll, 3.1 μmol/L meclofenamate, and 2.8 mmol/L CaCl2. The preparation equilibrated for 15 minutes, followed by priming with 100 ng of angiotensin II via bolus injection. The IPL was then exposed to 3 successive 5-minute episodes of alveolar hypoxia separated by 5 minutes of recovery. The perfusate was then switched to buffer containing 25 μmol/L N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), and the responses to 2 repeated hypoxic episodes were examined. Finally, the IPL was again exposed to 2 successive hypoxic episodes following removal of TPEN from the perfusate.
Analysis of Contractile Behavior in Isolated Cells
Matrices were prepared on 40-mm coverslips using rat tail (BD Bioscience, Bedford Mass), or bovine (PureCol, INAMED, Fremont, Calif) type 1 collagen. RPMECs and RAECs were seeded on the matrix 24 hours before imaging. Cells were imaged in a closed, thermocontrolled (37°C) stage insert (Bioptechs, Butler, Pa) under continuous flow of media at 0.3 mL/min, resulting in ≈8 dyn/cm2 of shear stress (KDS 100 syringe pump, KD Scientific, Holliston, Mass). Images were obtained using a Nikon TE2000E microscope equipped with a ×40 1.3NA oil immersion objective. After collection of baseline images, cells were exposed to perfusate that had been bubbled with anoxic gas (95% N2, 5% CO2), which acutely reduces oxygen tension to 13±2 mm Hg. Although changes in cell shape are easily quantified, they do not distinguish between active contractile events and passive changes caused by alterations in cellular anchoring. During active contraction, the cell exerts force on the surrounding matrix, allowing us to use differential interference contrast images of intrinsic collagen fiber structure, combined with online Deformation Quantification and Analysis (DQA) software (http://dqa.web.cmu.edu)9 to distinguish between active versus passive events by examining collagen displacement.
Imaging of Zinc-Sensitive Fluorophores in the IPL
FluoZin-3 (2.5 μmol/L) was added to the perfusate, and murine lungs were continuously perfused for 20 minutes, followed by a 20-minute washout period. The basal surface of the lung was placed in close proximity to a ×40 oil immersion objective (PlanNeoFluar, NA 1.3) for confocal imaging (510META, Carl Zeiss, Jena, Germany). FluoZin-3 was excited using the 488-nm line of the argon laser, and emissions were detected using a 505- to 550-nm bandpass filter. Sequential x, y, and z sections, which included the entire vessel, were collected during hypoxic exposure and the 3D anatomy of the vasculature was reconstructed using MetaMorph (Molecular Devices, Sunnyvale, Calif).
Fluorescence Resonance Energy Transfer
Details regarding the fluorescence resonance energy transfer (FRET) constructs, cygnet-2,10 and FRET · MT5,11 have been reported previously. FRET was detected in cell culture using spectral confocal microscopy (Zeiss 510META, Carl Zeiss).11 In brief, color separation of the donor (ECFP) and acceptor (EYFP) emission spectra were determined from the resolved image using a linear unmixing algorithm based on reference spectra obtained in cells expressing only ECFP or EYFP. Changes in the emissions ratio of the acceptor (EYFP, ≈525 nm) to the donor (ECFP, ≈480 nm) were monitored following exposure to hypoxia. In separate control experiments, FRET was confirmed by acceptor photobleaching.11
FRET in the IPL
We achieved expression of the FRET reporters in pulmonary endothelium of the mouse via tail vein injection of DOTAP:cholesterol liposomes,12 followed by 50 μg of cygnet-2 plasmid (at a 1:5−/+ charge ratio) or adenovirus containing cDNA for FRET · MT. Pulmonary adenoviral-mediated somatic gene transfer was shown to be significantly improved by preinjection of cationic liposomes.12 FRET was detected in real time using spectral confocal imaging of the intraacinar arteries of the IPL. Images were obtained with the ×40 oil immersion optic at 512×512 pixels. Acceptor photobleaching confirmed that the FRET · MT reporter was functional in the intact tissue (data not shown).
Results are given as means±SD. Data were analyzed using a 1-way ANOVA for multiple comparisons, with post hoc Tukey tests for pairwise comparisons. Significance was set at P<0.05.
Hypoxia Causes Active Constriction of Vessels in the Isolated Perfused Mouse Lung
The Tie2-GFP mouse expresses GFP under control of the endothelial-specific receptor tyrosine kinase (Tie2) promoter and hence defines the vascular bed. Confocal laser-scanning microscopy penetrates ≈100 μm into tissue, allowing visualization of intraacinar pulmonary arteries in murine IPL. Figure 1A (left) shows a 3D reconstruction of 46- and 41-μm segments of an intraacinar pulmonary artery. At 15 minutes after exposure to hypoxia, each segment decreased to a diameter of 39 μm (Figure 1A, middle) and returned to control values (48 and 42 μm; right) during recovery in normoxia. The reversibility is shown in a time lapse supplemental movie (Movie 1 in the online data supplement, available at http://circres.ahajournals.org). In repeat experiments (n=5, 3 to 5 vessels per experiment), there was a 9.2±1.1% (P<0.001) decrease in diameter in these small arteries (<50 μm) in response to hypoxia.
Isolated Pulmonary Microvascular Endothelial Cells Contract in Response to Hypoxia
Desmin staining (Figure I in the online data supplement) is consistent with data from other species, including rats, showing that the smooth muscle component of small pulmonary arteries (<100 μm) is either absent or is discontinuous in comparison to larger vessels,13,14 raising the possibility that endothelium contributes to the observed constriction in intraacinar arteries of the IPL. Indeed, isolated pulmonary endothelial cells do contract reversibly in response to hypoxia (supplemental Movie 2). Hypoxic exposure induced a 30±15.1% reduction in surface area in RPMECs, followed by a 25±18.9% recovery on return to normoxia (Figure 2A, n=26). Conversely, RAECs (n=8) constrict with thrombin but not hypoxia (Figure 2A and supplemental Figure II), illustrating that, like HPV, the hypoxia-induced contractile response is unique to endothelial cells derived from lung. To confirm that cells are actively contracting rather than undergoing passive shape changes, we examined collagen matrix deformation resulting from cell-applied forces (Figure 2B). The DQA software analyzes single-cell mechanics by tracking material displacement between time lapse images to create 2D density maps (Density Analysis) and continuous vector fields (Strain Analysis), as shown in Figure 2B. The resulting patterns were consistent in all RPMECs examined (n=26), demonstrating tension exerted by cells as they contract during hypoxia.
Acute Hypoxia Causes NO-Dependent Increases in Labile Zinc in the Mouse IPL and in Endothelial Cells
The effects of hypoxia on intracellular zinc were initially studied in primary cultures of mouse lung endothelial cells. Exposing cells to hypoxic media caused NO synthase (NOS)-dependent increases in labile zinc, as evidenced by increased fluorescence intensity of FluoZin-3 (supplemental Figure III). To establish the relevance of the hypoxia–NO–zinc signaling pathway in the intact organ, we used confocal microscopy to image FluoZin-3 in vasculature of IPL of MT+/+ mice. Detected fluorescence was significantly increased during hypoxia (Figure 3A, top) but prevented by the NOS inhibitor NG-nitro-l-arginine methyl ester (L-NAME) (1 mmol/L) (Figure 3A, middle). Furthermore, the IPL of MT-null mice showed no evident changes in fluorescence in response to hypoxia (Figure 3A, bottom), suggesting that changes in intracellular zinc were critically dependent on both NO production and MT (Figure 3B).
Zinc Chelation Attenuates HPV in the IPL
The isolated effect of altered zinc homeostasis on HPV was examined further using TPEN. Figure 4A shows representative pulmonary arterial pressure tracings from the IPL of a Tie2-GFP mouse. The addition of TPEN (25 μmol/L) to the perfusate attenuated the hypoxia-induced increase in pressure (Figure 4A, bottom). The response to hypoxia was restored following a 20-minute washout to remove TPEN (data not shown). Blunting of HPV by TPEN was both reproducible and significant (P<0.05, n=5) (Figure 4B). In contrast, the pressor response to 1 μmol/L U46619 (an increase of 3.1+0.8 cm H2O) was not affected by TPEN (an increase of 2.8±0.8 cm H2O).
We also examined the effects of zinc chelation on HPV in rat IPL (Figure 4C and 4D; n=6). As was the case in the mouse IPL, HPV was attenuated by TPEN (mean pressure change in hypoxia, 1.6±0.2 versus 0.7±0.3 cm H2O with TPEN, P<0.05), and this effect was reversed when TPEN was removed (mean pressure change on reexposure to hypoxia, 1.3±0.3 cm H2O). The hypoxic pressor responses in both the mouse and rat IPL were modest in our experience. Nonetheless, the effects of zinc chelation on HPV were apparent in the 2 species and were shown to be reversible, suggesting that the proposed NO-zinc signaling pathway is physiologically relevant.
Hypoxia Regulates Both cygnet-2 and FRET · MT Function Suggestive of Increased NO Production and Nitrosation of MT, Respectively
We previously described11 the use of genetically encoded FRET reporters to detect NO-related protein modifications including: (1) S-nitrosation, via the cysteine-rich protein MT (FRET · MT); and (2) nitrosyl-heme-Fe, via guanosine 3′,5′-cyclic monophosphate (cygnet-2). We used these approaches during hypoxic exposure in live endothelial cells. Hypoxia was associated with a significant (P<0.05) decrease in the FRET ratio for both reporter molecules (Figure 5) that was complete within 4 minutes. We have previously shown that FRET · MT is sensitive to NO donors as well as endothelial NOS (eNOS)-derived NO.5,7,11 The effects of hypoxia on FRET · MT were significantly blunted by NOS inhibition (Figure 5B), further demonstrating a role for NO in this response. The hypoxia-induced decrease in energy transfer observed with the cygnet-2 reporter (Figure 5C and 5D) was consistent with increases in cGMP, as previously reported in response to activation of sGC by NO donors.10,11 Furthermore, NOS inhibition (P<0.01) attenuated the changes (Figure 5D), indicating the importance of hypoxia-induced NO generation in mediating the responsiveness of cygnet-2.
We confirmed these NO-mediated events in the intact tissue using spectral confocal imaging of buffer perfused lungs expressing the FRET · MT or cygnet-2 reporters. Expression was confined to small intraacinar arteries and was predominantly endothelial as shown in Figure 6A (FRET · MT). In agreement with the cell culture data, hypoxia-induced decreases in energy transfer for both reporters (Figure 6D). These changes in FRET were evidenced by increases in the peak emission intensity of the donor and decreases in that of the acceptor (Figure 6B and 6C). The changes for FRET · MT were consistent with conformational changes and release of metals from the thiolate clusters of the core MT protein, as supported by hypoxia-induced increases in labile zinc (Figure 4).
HPV Was Attenuated in MT−/− Mice
We observed a blunting (P<0.05) of hypoxic induced increases in perfusion pressure in the IPLs of MT−/− versus MT+/+ mice (Figure 7; n=5). This effect was specific for hypoxia in that U46619-mediated increase in perfusion pressure was similar in MT−/− mice (3.1±0.8 cm H2O) and MT+/+ (3.5±0.8 cm H2O).
NO-Related Vasoconstriction in the Isolated Mouse Lung
We eliminated the potential vasodilatory limbs of NO-mediated effects on HPV via inhibition of sGC (ODQ, 10 μmol/L) and NO-sensitive large conductance Ca2+-activated potassium channels (BKCa2+, charybdotoxin [ChTx], 0.1 μmol/L) to confirm that NO could act as a vasoconstricting agent in buffer-perfused, isolated mouse lungs (Figure 8). In these experiments, hypoxia alone caused a 0.8±0.4 cm H2O increase (P<0.05) in perfusion pressure. When DETAnonoate was added in the presence of ODQ and ChTx, the NO donor caused a further increase in pressure during hypoxia (n=5, P<0.05). These effects were reversed by TPEN (25 μmol/L), suggesting that the vasoconstrictor effects of exogenous NO are mediated by changes in zinc. In separate sets of control experiments, ODQ alone caused a 2.4-fold increase in HPV (1.8±3.6 versus 0.8±2.8 cm H2O increase in perfusion pressure, P<0.05), whereas ChTx alone had no effect either on baseline pressure or HPV. The addition of DETAnonoate (100 μmol/L) alone to the perfusate decreased HPV from 2.6±0.8 cm H2O with hypoxia alone to 1.3±0.4 cm H2O in the presence of the NO donor (supplemental Figure IV).
We used a combination of optical imaging modalities and fluorescent reporter molecules to visualize the NO-MT-zinc signaling pathway in both pulmonary endothelial cells and intraacinar arteries of the isolated perfused mouse lung. Having confirmed that both intraacinar pulmonary arteries (Figure 1) and isolated pulmonary endothelial cells (Figure 2) actively constricted in response to hypoxia, we observed: i) hypoxia-induced changes in zinc homeostasis that were critically dependent on NO synthesis and MT in mouse lung endothelial cells (supplemental Figure III) and endothelium of the intact mouse IPL (Figure 3); and ii) hypoxia-induced production of NO in both cultured endothelial cells and endothelium of the IPL as revealed by FRET reporters for S-nitrosation of MT and activation of sGC (Figures 5 and 6⇑). Furthermore, following inhibition of the major NO-mediated effects on HPV (sGC and KCa2+ channels), the NO donor DETAnonoate was shown to enhance the hypoxic pressor response in the isolated mouse lung, and this effect was reversed by zinc chelation (Figure 8). Lastly, pharmacological (TPEN, Figure 4) and genetic (targeted ablation of zinc regulatory protein, MT; Figure 7) inhibition of hypoxic mediated elevations in zinc significantly blunted HPV. Collectively, these data suggest that hypoxia-induced increases in NO synthesis contribute to hypoxic vasoconstriction via formation of S-nitrosothiol in the metal binding center of MT and resultant changes in zinc homeostasis.
HPV and Nitric Oxide Production
Whereas exhaled NO decreases in perfused lungs in response to alveolar hypoxia, the effects on perfusate NOx levels appear to be both species and concentration dependent, requiring ≤1% inspired oxygen to reduce NOx in isolated rabbit lungs.15 In contrast, acute increases in pulmonary vascular resistance and HPV associated with pharmacological inhibition of NOS suggest that NO is generated during hypoxic exposure.3 In vitro data are similarly conflicting with reports of decreased eNOS activity in aortic endothelial cells16 but enhanced biosynthesis of NO in cultured pulmonary artery endothelial cells17 attributed to hypoxia-induced increases in calcium.18 Our data suggest that NO production is increased in the mouse IPL during hypoxia as the FRET efficiency of both the cygnet-2 and FRET · MT reporter molecules11 was decreased in a NOS-dependent manner following exposure to low pO2. Previously we noted that: (1) eNOS-derived NO, NO donors, and NO gas cause changes in FRET · MT5 and increases in labile zinc7; and (2) MT was the requisite target for NO resulting in the changes in zinc homeostasis.7 We also confirmed that FRET · MT was sensitive to DETAnonoate when expressed in the mouse IPL.19
NO and Acute HPV
Pharmacological inhibition of NO synthesis causes a 2-fold increase in HPV in the mouse IPL.20 Targeted disruption of individual NOS isoforms demonstrated that eNOS is the principal source of the NO-modulating acute responses to hypoxia.20,21 Thus, increases in NO would cause pulmonary vasodilatation, and attenuation of the hypoxic vasoconstrictor response, via stimulation of sGC and resultant increases in cGMP. Stable cGMP analogues decrease the strength of HPV and guanylate cyclase inhibition markedly amplifies the vasoconstrictor response to hypoxia in isolated rat lungs.22 Therefore, dissecting a potential vasoconstrictor response of NO is pharmacologically challenging. Nonetheless, when we inhibited the known vasodilatory limbs (sGC and BKCa2+) of NO-mediated effects on HPV, we observed a small but significant increase in pulmonary arterial pressure in response to DETAnonoate. Voelkel and colleagues23,24 described a paradoxical vasoconstrictive effect of normally vasodilatory stimuli, including NO and cGMP, in the pulmonary vasculature of hypoxic rat lungs that were perfused with red blood cell lysate. Although the mechanisms mediating NO-induced constriction remain uncertain, it appeared that second messenger function was altered by an undefined factor released during hemolysis. One possibility is that oxyhemoglobin (HbO2) from hemolyzed red blood cells acted to scavenge O, thus limiting the activation of sGC. However, HbO2 would not be expected to affect the NO-related species (nitrosonium) destined to participate in the S-nitrosation of MT.11 Although the present data were obtained in nonrecirculated, buffer perfused lungs, we cannot eliminate the possibility that there are trace amounts of hemolysate in the preparation that could affect the sGC pathway without altering NO-induced changes in labile zinc.
S-Nitrosation of MT in Hypoxia
Protein S-nitrosation has been observed following stimulation of all NOS isoforms.25 The favored in vitro reaction pathway for S-nitrosation involves NO and molecular oxygen to generate the nitrosonium donor N2O3 and therefore O2 is assumed to be necessary for NO-dependent protein S-nitrosation. However several mitochondrial proteins are nitrosated under anaerobic conditions, and it is possible that the oxidative requirements of this chemistry can be fulfilled in vivo by electron sinks other than molecular oxygen.26 Such issues highlight the importance of discerning between acute versus chronic and anoxic versus hypoxic effects on the signaling pathways of interest. Acute hypoxia has been associated with enhanced biosynthesis of NO in cultured pulmonary artery endothelial cells,17 whereas 4 to 24 hours of prolonged low oxygen decreases endothelial-derived NO production by disrupting the microenvironment of eNOS and l-arginine transport.27 S-Nitrosation of MT requires the presence of oxygen28 and will not occur in anoxia.29 Indeed anoxia is associated with pulmonary vasodilation.30 Accordingly, it is important to note that the gas mixtures in these studies were associated with Po2 measurements in the range of 10 to 15 mm Hg for cell culture and 30 to 35 mm Hg for IPL models.
Mouse Model of HPV
In contrast to other species, HPV in mice is relatively low (1 to 3 cm H2O).31 In addition, there are observed differences in hypoxic vascular reactivity between mouse strains.32 Regardless it is important to use genetically engineered animals to conclusively establish a role for MT in hypoxia-induced zinc release in the regulation of vascular tone. We document that HPV was reproducible over 2 hours of repeated 10 to 15 minutes hypoxic exposures (15 minutes recovery) and was increased by inhibition of both NOS (L-NAME) and sGC (ODQ) as shown in other animal models. Similar to the mouse data, zinc chelation (TPEN) reversibly blunted HPV in the IPL of rats. Although the hypoxic pressor responses in both the mouse and rat were modest in our experience, the effects of TPEN were apparent and reversible in the 2 species, suggesting that the modulating influence of hypoxia-induced zinc release on the HPV is of physiological relevance.
Although the precise mechanism underlying HPV remains unclear, it is recognized that unique intrinsic properties of pulmonary vascular smooth muscle (oxygen-sensing and coordination of ionic conductances leading to constriction) that are modulated by communication with endothelium (biosynthesis of vasoactive substances including NO) account for hypoxia-mediated vasoconstriction of pulmonary arteries. Nonetheless, previous studies using computer-enhanced videomicroscopy33 or x-ray microfocal angiographic images34 in perfused dog lungs, and our studies using scanning laser confocal microscopy of intraacinar pulmonary arteries of genetically modified mice reveal an important contribution of these small vessels (<50 μm in diameter) in HPV. Because this anatomic site is composed primarily of endothelial cells with solitary or discontinuous smooth muscle like cells (eg, pericytes) in their wall, the nature of contractile events within the microcirculation are likely to be distinct from vasoregulation of proximal pulmonary vessels. Pericytes have been shown to induce constriction by contraction of cell processes that partially envelop the capillary and could potentially contribute to the observed hypoxia-induced constriction of small pulmonary vessels. However, indirect evidence using vasoactive chemicals to induce reorganization of the endothelial microfilament system also suggests that endothelial cells play a role in capillary constriction in a number of vascular beds.35 Although our data show that isolated pulmonary (but not aortic) endothelial cells actively contract in response to hypoxia, the integrated subunit of intraacinar arteries under investigation contains both endothelium and a small component of discontinuous smooth muscle, and, as such, either or both cell types could contribute to vasomotor tone.
It is now apparent that: (1) S-nitrosation of zinc sulfur clusters is an important component of NO signaling; and (2) MT is a critical link between NO and intracellular zinc homeostasis.7 Our present data support the contention that zinc thiolate signaling is a component of acute hypoxia mediated NO biosynthesis and that this pathway may contribute to hypoxia-induced vasoconstriction within the pulmonary microcirculation. Although the precise mechanism by which increased labile zinc may cause vasoconstriction remains unclear, it is noteworthy that zinc-associated proteins account for a large part of mammalian proteome and that many of these candidate targets are components of signaling and effector pathways in cellular contraction. For example, the zinc-sensitive protein kinase C isoform, PKCε, is activated in response to hypoxia and has been shown to play a pivotal role in mediating acute hypoxic vasoconstriction in mice.36
Sources of Funding
This work was funded, in part, by NIH grants HL081421 (to C.M.S.); HL70807, HL65697, and GM53789 (to B.R.P.); and HL070807 and 1 U54 RR022241-01 (to S.C.W.); and by the American Heart Association (to C.M.S.).
Original received February 24, 2006; first resubmission received January 3, 2007; second resubmission received January 17, 2008; revised second resubmission received April 28, 2008; accepted May 6, 2008.
Archer SL, Huang JM, Hampl V, Nelson DP, Shultz PJ, Weir EK. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1994; 91: 7583–7587.
Pearce LL, Gandley RE, Han W, Wasserloos K, Stitt M, Kanai AJ, McLaughlin MK, Pitt BR, Levitan ES. Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc Natl Acad Sci U S A. 2000; 97: 477–482.
Kroncke KD, Fehsel K, Schmidt T, Zenke FT, Dasting I, Wesener JR, Bettermann H, Breunig KD, Kolb-Bachofen V. Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9. Biochem Biophys Res Commun. 1994; 200: 1105–1110.
St Croix CM, Wasserloos KJ, Dineley KE, Reynolds IJ, Levitan ES, Pitt BR. Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein. Am J Physiol Lung Cell Mol Physiol. 2002; 282: L185–L192.
Honda A, Adams SR, Sawyer CL, Lev-Ram V, Tsien RY, Dostmann WR. Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci U S A. 2001; 98: 2437–2442.
Le Cras TD, McMurtry IF. Nitric oxide production in the hypoxic lung. Am J Physiol Lung Cell Mol Physiol. 2001; 280: L575–L582.
Wei Z, Al-Mehdi AB, Fisher AB. Signaling pathway for nitric oxide generation with simulated ischemia in flow-adapted endothelial cells. Am J Physiol Heart Circ Physiol. 2001; 281: H2226–H2232.
Hampl V, Cornfield DN, Cowan NJ, Archer SL. Hypoxia potentiates nitric oxide synthesis and transiently increases cytosolic calcium levels in pulmonary artery endothelial cells. Eur Respir J. 1995; 8: 515–522.
St Croix CM, Pitt BR, Watkins SC. The use of contemporary fluorescent imaging technologies in biomedical research. Med Sci. 2005; 10: 16–29.
Archer SL, Rist K, Nelson DP, DeMaster EG, Cowan N, Weir EK. Comparison of the hemodynamic effects of nitric oxide and endothelium-dependent vasodilators in intact lungs. J Appl Physiol. 1990; 68: 735–747.
Voelkel N, Allard JD, Anderson SM, Burke TJ. cGMP and cAMP cause pulmonary vasoconstriction in the presence of hemolysate. J Appl Physiol. 1999; 86: 1715–1720.
Voelkel N, Lobel K, Westcott JY, Burke TJ. Nitric oxide-related vasoconstriction in lungs perfused with red cell lysate. FASEB J. 1995; 9: 379–386.
Gow AJ, Chen Q, Hess DT, Day BJ, Ischiropoulos H, Stamler JS. Basal and stimulated protein S-nitrosylation in multiple cell types and tissues. J Biol Chem. 2002; 277: 9637–9640.
Foster MW, Stamler JS. New insights into protein S-nitrosylation. Mitochondria as a model system. J Biol Chem. 2004; 279: 25891–25897.
Su Y, Block ER. Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol. 2000; 278: L1204–L1212.
Schwarz MA, Lazo JS, Yalowich JC, Allen WP, Whitmore M, Bergonia HA, Tzeng E, Billiar TR, Robbins PD, Lancaster JR Jr, Pitt BR. Metallothionein protects against the cytotoxic and DNA-damaging effects of nitric oxide. Proc Natl Acad Sci U S A. 1995; 92: 4452–4456.
Aravindakumar CT, Ceulemans J, De Ley M. Nitric oxide induces Zn2+ release from metallothionein by destroying zinc-sulphur clusters without concomitant formation of S-nitrosothiol. Biochem J. 1999; 344 (pt 1): 253–258.
Peake MD, Harabin AL, Brennan NJ, Sylvester JT. Steady-state vascular responses to graded hypoxia in isolated lungs of five species. J Appl Physiol. 1981; 51: 1214–1219.
Hillier SC, Graham JA, Hanger CC, Godbey PS, Glenny RW, Wagner WW Jr. Hypoxic vasoconstriction in pulmonary arterioles and venules. J Appl Physiol. 1997; 82: 1084–1090.
Clough AV, Haworth ST, Ma W, Dawson CA. Effects of hypoxia on pulmonary microvascular volume. Am J Physiol Heart Circ Physiol. 2000; 279: H1274–H1282.
Littler CM, Morris KG Jr, Fagan KA, McMurtry IF, Messing RO, Dempsey EC. Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction. Am J Physiol Heart Circ Physiol. 2003; 284: H1321–H1331.