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The metal binding protein, metallothionein (MT) is a target for 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 intra-acinar arteries (<40 μM) 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 upon MT as it is not apparent in IPL of MT−/− mice. Data from NO-sensitive fluorescence resonance energy transfer (FRET)-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 this pathway may contribute to constriction in the pulmonary vasculature.
Acute hypoxic pulmonary vasoconstriction (HPV)1 is uniqueto 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-derivednitric oxide (NO)2. In the systemic circulation, this is believed to contribute to hypoxic vasodilation, wheres in the lung, NO biosynthesis will oppose hypoxic vasoconstrictor stimuli via activation of the soluble guanylyl cyclase (sGC)/3′,5′-cyclic guanosine monophosphate (cGMP) pathway or by directly opening KCa2+ channels in pulmonary vascular smooth muscle3,4.
In addition to covalent modification of heme or non-heme 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 NO5,6, directly affecting intracellular zinc homeostasis6,7. While the physiological relevance of NO-induced changes in labile zinc is unknown, interactions between NO and MT facilitate myogenic reactivity in systemic resistance vessels5. Indeed, while 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 (IPL) 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, contributes to vasoconstriction in pulmonary resistance vessels via S-nitrosation of the metal binding centers of MT and alterations in intracellular zinc homeostasis.
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 cells (MLEC)8 and sheep pulmonary artery endothelial cells (SPAEC)7 preparations are described elsewhere. Rat pulmonary microvascular endothelial cells (RPMEC) and rat aortic endothelial cells (RAEC) were purchased from VEC Technologies Inc. (Resselaer, NY) and grown in complete MDCB-131 media (VEC Technologies Inc).
Sprague Dawley rats; Tie2-Green Fluorescent Protein(GFP) mice (STOCK Tg(TIE2GFP)287Sato/J); MT −/− (129S7/SvEvBrd-Mt1tm1BriMt2tm1Bri)and WT controls (129S1/SvImJ, MT +/+) were purchased fromJackson Laboratories (Bar Harbor, ME).
Mice were anesthetized, heparin was injected i.v. (50 U) and a thoracotomy performed to expose heart and lungs. The trachea was cannulated and heart and lungs 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 μM 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 held statically inflated. Perfusion pressure was monitoredand recorded at constant flow (PowerLab, ADInstruments, Inc., Colorado Springs, CO). After establishing a baseline perfusion pressure with21% O2, lungs were inflated with the hypoxic gas mixture (1.5% O2, 5% CO2, balance N2) for 10 min followed by a return to 21% O2. The use of 1.5% O2 resulted in a drop in PO2 from 100–110 mmHg to ~30–35 mmHg as measured in the venous effluent using a Clarke electrode.
Male rats (300–350g) were anesthetized, injected i.v. with heparin and a thoracotomy performed to expose heart and lungs. Rats were ventilated (model 683, Harvard Apparatus, Holliston, MA) at 55 breaths/min with tidal volumes less than 10 cm H2O. The pulmonary artery and left atrium were cannulated, and the ventilator set to 2 cmH2O of positive end expiratory pressure (PEEP). Lungs were perfused at 3ml/min with warmed Krebs-Henseleit Buffer supplemented with 3% Ficoll, 3.1 μM meclofenamate and 2.8 mM CaCl2. The preparation equilibrated for 15 min followed by priming with 100 ng angotensin II via bolus injection. The IPL was then exposed to three successive 5 min episodes of alveolar hypoxia separated by 5 min recovery. The perfusate was then switched to buffer containing 25 μM TPEN and the responses to two repeated hypoxic episodes were examined. Finally, the IPL was again exposed to two successive hypoxic episodes following removal of TPEN from the perfusate.
Matrices were prepared on 40 mm coverslips using rat tail (BD Bioscience, Bedford MA), or bovine (PureCol, INAMED, Fremont, CA) type 1 collagen. RPMEC and RAEC were seeded on the matrix 24 hours prior to imaging. Cells were imaged in a closed, thermocontrolled (37oC) stage insert (Bioptechs, Butler, PA) under continuous flow of media at 0.3ml/min, resulting in approximately 8dyn/cm2 of shear stress (KDS 100 syringe pump, KD Scientific, Holliston, MA). Images were obtained using a Nikon TE2000E microscope equipped with a 40X 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 mmHg,. Although changes in cell shape are easily quantified, they do not distinguish between active contractile events and passive changes due to alterations in cellular anchoring. During active contraction, the cell exerts force on the surrounding matrix allowing us to use differential interference contrast (DIC) 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 vs. passive events by examining collagen displacement.
FluoZin-3 (2.5 μM) was added to the perfusate and murine lungs continuously perfused for 20 minutes, followed by a 20 min washout period. The basal surface of the lung was placed in close proximity to a 40X 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 detected using a 505- to 550-nm bandpass filter. Sequential XYZ-sections which included the entire vessel were collected during hypoxic exposure and the three-dimensional anatomy of the vasculature was reconstructed using MetaMorph (Molecular Devices, Sunnyvale, CA).
Details regarding the FRET constructs, cygnet-210 and FRET.MT511 were reported previously. FRET was detected in cell culture using spectral confocal microscopy (Zeiss 510META, Carl Zeiss, Jena, Germany)11. In brief, color separation of the donor (ECFP) and acceptor (EYFP) emission spectra was 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 photo-bleaching11.
We achieved expression of the FRET reporters in pulmonary endothelium of the mouse via tail vein injection of DOTAP:cholesterol liposomes12 followed by 50 μg 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 pre-injection of cationic liposomes12. FRET was detected in real time, using spectral confocal imaging of the intra-acinar arteries of the IPL. Images were obtained with the 40X oil immersion optic at 512 × 512 pixels. Acceptor photo-bleaching confirmed that the FRET.MT reporter was functional in the intact tissue (data not shown).
Results are given as mean ±SD. Data were analyzed using a one-way analysis of variance for multiple comparisons with post-hoc Tukey tests for pairwise comparisons. Significance was set at P < 0.05.
The Tie2-GFP mouse expresses GFP under control of endothelial-specific receptor tyrosine kinase (Tie2) promoter and hence defines the vascular bed. Confocal laser scanning microscopy penetrates about 100 μm into tissue allowing visualization of intra-acinar pulmonary arteries in murine IPL. Figure 1A (left) shows a three dimensional reconstruction of 46 and 41 μm segments of an intra-acinar pulmonary artery. At 15 min 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 repeat experiments (n=5, 3–5 vessels per experiment), there was a 9.2 ± 1.1% (P < 0.001) decrease in diameter in these small arteries (≤40 μm) in response to hypoxia.
Desmin staining (Supplemental Data, Figure 1) is consistent with data from other species, including rats, showing that the smooth muscle component of small pulmonary arteries (< 100μm) is either absent, or is discontinuous in comparison to larger vessels13,14 raising the possibility that endothelium contributes to the observed constriction in intra-acinar 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 RPMEC followed by a 25% ± 18.9% recovery on return to normoxia (Figure 2A, n=26). Conversely, RAEC (n=8) constrict with thrombin but not hypoxia (Figure 2A and Supplemental Figure 2), 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 fromcell-applied forces (Figure 2B). The DQA software analyses 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 RPMEC examined (n=26) demonstrating tension exerted by cells as they contract during hypoxia.
The effects of hypoxia on intracellular zinc were initially studied in primary cultures of MLEC. Exposing cells to hypoxic media caused NOS-dependent increases in labile zinc evidenced by increased fluorescence intensity of FluoZin-3(Supplemental Figure 3). 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, upper panel) but prevented by the NOS inhibior L-NAME, 1mM (Figure 3A, middle panel). Furthermore, the IPL of MT-null mice showed no evident changes in fluorescence in response to hypoxia (Figure 3A, lower panel) suggesting changes in intracellular zinc were critically dependent upon both NO production and metallothionein. (Figure 3B).
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. Addition of TPEN (25 μM) to the perfusate attenuated the hypoxia-induced increase in pressure (Figure 4A, lower panel). 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 μM 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 D, n=6). As was the case in the mouse IPL, HPV was attenuated by TPEN (mean pressure change in hypoxia 1.6 ± 0.2 vs. 0.7 ± 0.3 cm H2O with TPEN, P < 0.05), and this effect was reversed when TPEN was removed (mean pressure change upon re-exposure 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 two species and were shown to be reversible suggesting that the proposed NO-zinc signaling pathway is physiologically relevant.
We previously described11 the use of genetically-encoded FRET reporters to detect NO-related protein modifications including: (a) S-nitrosation, via the cysteine-rich protein metallothionein (FRET.MT); and (b) 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 min. We have previously shown that FRET.MT is sensitive to NO donors as well as endothelial NO synthase (eNOS)-derived NO5,7,11. 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 D) was consistent with increases in cGMP, as previously reported in response to activation of sGC by NO donors10,11. 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 intra-acinar 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 C). The changes for FRET.MT were consistent with conformational changes and release of metals from the thiolate clusters of the core metallothionein protein as supported by hypoxia-induced increases in labile zinc (Figure 4).
We observed a blunting (P<0.05) of hypoxic induced increases in perfusion pressure in the IPLs of MT−/− vs. 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 cmH2O) and MT+/+ (3.5 ± 0.8 cmH2O).
We eliminated the potential vasodilatory limbs of NO-mediated effects on HPV via inhibition of sGC (ODQ, 10 μM) and NO-sensitive large conductance Ca2+-activated potassium channels (BKCa2+, charybdotoxin, ChTx, 0.1 μM) in order 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 cmH2O 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 μM), 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 vs. 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 μM) 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 4).
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 intra-acinar arteries of the isolated perfused mouse lung. Having confirmed that both intra-acinar 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 metallothionein in mouse lung endothelial cells (Supplemental Figure 3) 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 and6).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, pharmacologic (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.
While exhaled NO decreases in perfused lungs in response to alveolar hypoxia, the effects on perfusate NO−x levels appear to be both species and concentration dependent, requiring ≤1% inspired oxygen to reduce NO−x 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 exposure3. In vitro data is similarly conflicting with reports of decreased eNOS activity in aortic endothelial cells16 but enhanced biosynthesis of NO in cultured pulmonary artery endothelial cells 17 attributed to hypoxia-induced increases in calcium18. 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 homeostasis7. We also confirmed that FRET.MT was sensitive to DETAnonate when expressed in the mouse IPL19.
Pharmacological inhibition of NO synthesis causes a 2-fold increase in HPV in the mouse IPL20. Targeteddisruption of individual NOS isoforms demonstrated that eNOS is the principal source of the NO modulating acute responsesto hypoxia20, 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 markedlyamplifies the vasoconstrictor response to hypoxia in isolated rat lungs22. 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 (RBC) lysate. Though the mechanisms mediating NO-induced constriction remain uncertain, it appeared that second messenger function was altered by an undefined factor releasedduring hemolysis. One possibility is that oxyhemoglobin (HbO2) from hemolyzed red blood cells acted to scavenge •NO 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 metallothionein11. While the present data were obtained in non-recirculated, 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.
Protein S-nitrosation has been observed following stimulation of all NOS isoforms25. 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 vs. chronic and anoxic vs. hypoxic effects on the signaling pathways of interest. Acute hypoxia has been associated with enhanced biosynthesis of NO in cultured pulmonary artery endothelial cells17 whereas 4–24 hrs of prolonged low oxygen decreases endothelial derived NO production by disrupting the microenvironment of eNOS and L-arginine transport27. S-nitrosation of MT requires the presence of oxygen28 and will not occur in anoxia29. Indeed anoxia is associated with pulmonary vasodilation30. Accordingly, it is important to note that the gas mixtures in these studies were associated with pO2 measurements in the range of 10–15 mmHg for cell culture and 30–35 mmHg for IPL models.
In contrast to other species, HPV in mice is relatively low (1–3 cmH2O)31. In addition, there are observed differences in hypoxic vascular reactivity between mouse strains32. 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 two hours of repeated 10–15 min hypoxic exposures (15 min 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 two 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, current dogma suggests 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 hypoxic 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 intra-acinar pulmonary arteries of genetically modified mice reveal an important contribution of these small vessels (≤40 μm diameter) in HPV. Since this anatomic site is composed primarily of endothelial cells with solitary or discontinuous smooth muscle like cells (e.g. 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 which 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 beds35. While our data show that isolated pulmonary (but not aortic) endothelial cells actively contract in response to hypoxia, the integrated subunit of intra-acinar 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: a) S-nitrosation of zinc sulfur clusters is an important component of NO signaling; and b) MT is a critical link between NO and intracellular zinc homeostasis7. 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 hypoxic 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 many of these candidate targets are components of signaling and effector pathways in cellular contraction. For example, the zinc sensitive protein kinase C isoform, PKC epsilon, is activated in response to hypoxia and has been shown to play a pivotal role in mediating acute hypoxic vasoconstriction in mice36.
SOURCES OF FUNDING
Funded in part by NIH HL081421 (CMS); HL70807, HL65697, and GM53789 (BRP); HL070807 and 1 U54 RR022241-01 (SCW); and the American Heart Association (CMS).