(Chest. 2005;128:556S-558S.)
© 2005
American College of Chest Physicians
Redox Activation of Intracellular Calcium Release Channels (Ryanodine Receptors) in the Sustained Phase of Hypoxia-Induced Pulmonary Vasoconstriction*
Wanglei Du, MS;
Melissa Frazier, BA;
Timothy J. McMahon, MD PhD and
Jerry P. Eu, MD
* From the Division of Pulmonary, Allergy and Critical Care Medicine, Duke University Medical Center, Durham, NC.
Correspondence to: Jerry Eu, MD, Division of Pulmonary, Allergy and Critical Care Medicine, PO Box 3168, Duke University Medical Center, Durham, NC 27710; e-mail: eu000001{at}duke.edu
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Abstract
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Hypoxia-induced pulmonary vasoconstriction (HPV) is an important adaptive process that remains incompletely understood. In preconstricted rat pulmonary arteries (inner diameter, 250 to 400 µm), hypoxia (pO2 approximately 10 mm Hg) induces an initial transient phase and a more slowly developing sustained phase of vasoconstriction. Since the release of calcium ions (Ca2+) from intracellular stores by redox-sensitive intracellular Ca2+ release channels known as ryanodine receptors (RyRs) in pulmonary arterial smooth-muscle cells (PASMCs) may play a role in HPV, and considerable evidence now supports that levels of reactive oxygen species (ROS) are paradoxically increased in PASMC under hypoxia, we investigated whether redox activation of RyRs by ROS may transduce HPV. By reverse transcriptase-polymerase chain reaction, we found that all three RyR isoforms are expressed in rat pulmonary arteries and in PASMCs. The sustained phase, but not the transient phase, of HPV can be prevented by pretreating pulmonary arteries with RyR inhibitors ryanodine (200 µmol/L) or dantrolene (50 µmol/L). The addition of dantrolene, ryanodine or the thiol-reducing agent dithiothreitol (1 mmol/L) during the sustained phase of HPV reversed the hypoxic vasoconstriction. In contrast, the superoxide scavenger nitroblue tetrazolium (500 nmol/L) prevented further hypoxic pulmonary vasoconstriction during the sustained phase of HPV but did not reverse it. Taken together, our data suggest that redox activation of RyRs by ROS has an important role in transducing the sustained contraction of pulmonary arteries under hypoxia.
Key Words: hypoxia-induced pulmonary vasoconstriction • redox regulation • ryanodine receptors
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Introduction
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Hypoxia-induced pulmonary vasoconstriction (HPV) is an important adaptive process that diverts blood flow away from poorly oxygenated lung regions to well-oxygenated regions, thereby optimizing oxygenation/gas exchange in patients with significant respiratory diseases. Persistent hypoxic pulmonary vasoconstriction, however, can lead to the remodeling of pulmonary vasculature, debilitating pulmonary hypertension, and other untoward clinical sequelae.1 Previous studies2 have shown that HPV is in part triggered by the inactivation of voltage-dependent potassium channels in pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, resulting in the depolarization of cell membrane and extracellular Ca2+ entry via voltage-dependent Ca2+ channels.
However, the full manifestation of HPV is likely mediated by more than one cellular mechanism in the pulmonary vasculature working synergistically and/or sequentially. In ex vivo studies1 of isolated small pulmonary arteries (small vessel myography studies), hypoxia typically induces an initial transient phase and a more slowly developing sustained phase of vasoconstriction (Fig 1
, bottom left, C). Moreover, in addition to the contribution of L-type Ca2+ channels in HPV, previous studies13 have shown that Ca2+ release from intracellular stores of PASMCs by intracellular Ca2+ release channels known as ryanodine receptors (RyRs) may also be important in HPV. Since we and others45 have found that all three isoforms of RyRs contain regulatory thiols that are sensitive to redox modifications, and considerable evidence now supports that levels of reactive oxygen species (ROS; ie, superoxide anion, hydrogen peroxide, and their metabolites) are paradoxically increased in PASMCs under hypoxia,67 we therefore examined pulmonary arteries from rats to test the hypothesis that redox activation of RyRs by ROS may transduce HPV.
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Figure 1. Top left, A: RT-PCR of rat pulmonary arteries (PA) [inner diameter, 250 to 400 µm] and PASMCs using isoform-specific primers (labeled below lanes) for three isoforms of RyR. All three isoforms of RyR were detected in rat pulmonary arteries and PASMCs. RT-PCR of positive control tissues (skeletal muscle [Sk], heart [Ht], and brain [Br] for RyR-1, RyR-2 and RyR-3 isoforms, respectively) showed the expected amplified products.9 Top right, B: Immunofluorescence studies using a nonisoform specific anti-RyR monoclonal antibody (Calbiochem; La Jolla, CA) show the distribution of RyR within PASMCs (original x 63). Another nonisoform specific anti-RyR mAb (Affinity BioReagents; Golden, CO) also showed the same cellular distribution and the secondary antibody alone was negative (data not shown). Bottom left, C: Representative small-vessel myography recordings demonstrate the role of RyRs in the HPV response of rat pulmonary arteries. A control pulmonary artery had an initial transient phase and a more slowly developing sustained phase of vasoconstriction in response to hypoxia. Pulmonary arteries pretreated with RyR inhibitors ryanodine (200 µmol/L) or dantrolene (50 µmol/L) had a relatively preserved transient phase of HPV but lacked the sustained phase. Bottom right, D: The sustained phase of HPV can be reversed by adding an inhibitor of RyRs such as ryanodine. Adding a superoxide scavenger nitroblue tetrazolium (NBT) [500 nmol/L] during this phase prevented further hypoxic contraction of the pulmonary artery, thus suggesting that superoxide and/or its downstream metabolites may play a significant role in this phase of HPV. A thiol-reducing agent (DTT, 1 mmol/L) completely reversed the sustained phase of HPV. Similar experiments were repeated three times, and identical results were obtained each time. Taken together, these data suggest that RyRs are activated by ROS during the sustained phase of HPV.
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Materials and Methods
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Endothelium-intact intrapulmonary arteries (inner diameter, 250 to 400 µm) were isolated from pentobarbital-euthanized male Wistar rats (weight approximately 250 g). These vessels were placed in Krebs buffer, dissected to remove connective tissues, and processed immediately for PASMC isolation,8 reverse transcriptase-polymerase chain reaction (RT-PCR) analysis,9 or small-vessel myography.10 In certain experiments, primary PASMCs were cultured for 3 to 4 days in Dulbecco modified Eagle medium plus 10% fetal bovine serum prior to RT-PCR analysis or immunofluorescence studies.8
In small-vessel myography studies,10 each isolated rat pulmonary arterial ring (approximately 3 to 5 mm in length) was mounted horizontally on two tungsten triangles and suspended between a stainless-steel hook connected to a force transducer (Grass FT-03; Grass; West Warwick, RI) and a glass hook in the organ bath. These vessel rings were equilibrated in Krebs buffer at 37°C for 30 min with constant bubbling of a premixed gas consisting of 20% O2, 5% CO2, and balance N2. A resting tension of 0.5 g was applied to each ring. After a pre-equilibration period, these pulmonary arterial rings were first constricted with KCl (50 mmol/L) to establish references for comparison. Prior to introducing hypoxia (1% O2, 5% CO2, and balance N2, pO2 approximately 10 mm Hg) to the organ baths, these pulmonary arteries were preconstricted with 1 µmol/L of phenylephrine.10 Changes in isometric tensions were amplified (Grass DC pre/amplifier model 7DAF; Grass) and recorded digitally (Polyview Software; Grass-Telefactor; West Warwick, RI).
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Results
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All three RyR isoforms are expressed in rat pulmonary arteries and in isolated PASMCs (> 99% positive for smooth-muscle cell-specific 
-actin) by RT-PCR analysis (Fig 1, top left, A). The distribution of RyRs in PASMCs as assessed by an immunofluorescence method8 using monoclonal, nonisoform specific anti-RyR antibodies appears to be diffuse throughout the cytoplasm (Fig 1, top right, B).
In preconstricted rat pulmonary arteries, the sustained phase of HPV can be prevented by pretreating these vessels with the RyR inhibitors ryanodine (200 µmol/L) or dantrolene (50 µmol/L) [Fig 1, bottom left, C]. The changes in tension under hypoxia measured at 30 min after the induction of hypoxia (corresponding to the sustained phase of HPV), were – 1.8 ± 4.4% and – 16.6 ± 6.7% of KCl-induced contraction (mean ± SEM) in ryanodine-pretreated (n = 7) and dantrolene-pretreated (n = 11) groups, respectively. In contrast, a vigorous sustained contraction was typically observed in the control group (+ 29.3 ± 3.6% of KCl-induced contraction, n = 15) [p < 0.001 vs either RyR inhibitor group]. Inhibitors of RyRs, however, also have a modest suppressive effect on the transient phase of HPV. Peak transient contraction of pulmonary arteries pretreated with ryanodine or dantrolene was 20.5 ± 1.8% and 23.2 ± 2.9% of the KCl response, respectively, vs 34.4 ± 3.1% in the control group (p < 0.01 vs either RyR inhibitor group).
In separate experiments, the addition of dantrolene, ryanodine, or a thiol-reducing agent, dithiothreitol (DTT) [1 mmol/L] during the sustained phase of HPV reproducibly reversed the hypoxic vasoconstriction (Fig 1, bottom right, D), thus implicating the oxidation of RyR regulatory thiol groups during the sustained contraction of pulmonary arteries under hypoxia. Furthermore, addition of the superoxide scavenger nitroblue tetrazolium (500 nmol/L) during the sustained phase of HPV prevented further hypoxic pulmonary vasoconstriction (but did not reverse it), thus suggesting superoxide and/or its downstream metabolites are endogenous activators of RyRs in PASMCs during this phase of HPV.
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Discussion
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ROS have been found to be paradoxically increased in PASMCs under hypoxia,67 possibly due to the uncoupling of the mitochondrial electron transport chain or other mechanisms. Moreover, the generation of these ROS has been found to be essential for HPV in isolated lung preparations,11 and scavenging superoxide greatly attenuates the sustained phase of HPV.12 The cellular targets of ROS in PASMCs have not been identified. Our studies45 suggest that regulatory thiol groups of RyRs could be the downstream targets of ROS in the signaling pathway of HPV, especially during the sustained phase of HPV. Our data thus suggest a link between previous independent observations that ROS76 and RyRs31 have important roles in the signaling pathway of HPV.
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Footnotes
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Abbreviations: DTT = dithiothreitol; HPV = hypoxia-induced pulmonary vasoconstriction; PASMC = pulmonary arterial smooth muscle cell; ROS = reactive oxygen species; RT-PCR = reverse transcriptase-polymerase chain reaction; RyR = ryanodine receptor
This work was supported by research grants from the American Lung Association (RG-191-N) and the American Heart Association (Beginning Grant-in-Aid, Mid-Atlantic Affiliate).
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References
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Abstract
Introduction
