Acute Hypoxic Pulmonary Vasoconstriction Research Articles

Docosahexaenoic acid inhibits development of hypoxic pulmonary hypertension: In vitro and in vivo studies

Hypoxic pulmonary hypertension (HPH) is initiated by acute hypoxic pulmonary vasoconstriction followed by vascular remodeling and right ventricular failure. Numerous studies have shown that long-term supplementation with docosahexaenoic acid (DHA) may have a role in preventing cardiovascular disease. However, the effects of DHA on HPH remain unclear. Extracellular signal-regulated kinase 1/2 (ERK1/2) is known to be involved in the proliferation and migration of various cell types. In this study, we determined the role of DHA in the prevention of HPH via the ERK1/2 signal pathway. Rats were intragastrically administered with saline or DHA (100 mg) daily before exposure to room air or chronic hypoxia (O(2) content was maintained at 10%) for 8 h a day for 3 weeks. At the end of study, we compared the right ventricular systolic pressure (RVSP), the weight ratio of right ventricular (RV) free wall to the left ventricular (LV) including the septum (S) free wall, the percent wall thickness (wt.%) of small pulmonary arteries and the area of α-smooth muscle actin (α-SMA)-positive pulmonary artery smooth muscle cells (PASMCs) in the pulmonary arteries among the rats. In vitro, the proliferation and migration of PASMCs were through an MTT assay and a Boyden chamber, respectively. The phenotype marker expression and ERK1/2 activation in the PASMCs were through real-time PCR and western blot analysis, respectively. Under hypoxia, DHA treatment reduced the RVSP (20.41 mmHg ± 2.18 vs. 35.46 mmHg ± 3.21; P < 0.05), the weight ratio of RV free wall to left ventricular and septal free wall [RV/(LV + S)] (0.27 ± 0.03 vs. 0.38 ± 0.05; P < 0.05), wt.% (17.45 ± 1.58% vs. 59.65 ± 4.59%; P < 0.05) and the percentage of SMA-positive vessels (55.42 ± 5.18% vs. 84.71 ± 6.22%; P < 0.05) compared with those of the saline-treated rats. Hypoxia promoted the proliferation, migration and phenotype switching of PASMCs in vitro. However, DHA treatment suppressed the hypoxia-induced changes. Additionally, DHA attenuated the expression of hypoxia-activated ERK1/2 in the PASMCs. Our observations indicate that DHA may potentially prevent HPH.

Superoxide Generated at Mitochondrial Complex III Triggers Acute Responses to Hypoxia in the Pulmonary Circulation

The role of reactive oxygen species (ROS) signaling in the O(2) sensing mechanism underlying acute hypoxic pulmonary vasoconstriction (HPV) has been controversial. Although mitochondria are important sources of ROS, studies using chemical inhibitors have yielded conflicting results, whereas cellular models using genetic suppression have precluded in vivo confirmation. Hence, genetic animal models are required to test mechanistic hypotheses. We tested whether mitochondrial Complex III is required for the ROS signaling and vasoconstriction responses to acute hypoxia in pulmonary arteries (PA). A mouse permitting Cre-mediated conditional deletion of the Rieske iron-sulfur protein (RISP) of Complex III was generated. Adenoviral Cre recombinase was used to delete RISP from isolated PA vessels or smooth muscle cells (PASMC). In PASMC, RISP depletion abolished hypoxia-induced increases in ROS signaling in the mitochondrial intermembrane space and cytosol, and it abrogated hypoxia-induced increases in [Ca(2+)](i). In isolated PA vessels, RISP depletion abolished hypoxia-induced ROS signaling in the cytosol. Breeding the RISP mice with transgenic mice expressing tamoxifen-activated Cre in smooth muscle permitted the depletion of RISP in PASMC in vivo. Precision-cut lung slices from those mice revealed that RISP depletion abolished hypoxia-induced increases in [Ca(2+)](i) of the PA. In vivo RISP depletion in smooth muscle attenuated the acute hypoxia-induced increase in right ventricular systolic pressure in anesthetized mice. Acute hypoxia induces superoxide release from Complex III of smooth muscle cells. These oxidant signals diffuse into the cytosol and trigger increases in [Ca(2+)](i) that cause acute hypoxic pulmonary vasoconstriction.

99 TANSHINONE IIA ATTENUATED HYPOXIA-INDUCED PULMONARY HYPERTENSION VIA UPGRADING KV CHANNELS AND P27KIP1

Objectives: Pulmonary hypertension induced by hypoxia is the characterized by hypoxic pulmonary vasoconstriction (HPV) and pulmonary vascular structure remodeling (PVSR). Tanshinone IIA (TIIA) has numerous biological protective effects. Purpose of the paper is to investigated whether KV channels and P27kip1 were responsible for the protective effect of TIIA on HPV and PVSR. Basic procedures and methods: Effects of TIIA on intrapulmonary arteriole (IPA) rings and the IKV currents of pulmonary artery smooth muscle cells (PASMCs) isolated from rats in acute hypoxia experiments and chronic hypoxia experiments were determined. Main findings: results showed that TIIA attenuated acute hypoxia-induced IPA rings vasoconstriction which may be partially through recovering the down- regulation of IKV currents in PASMCs. TIIA markedly alleviated the increased weight of right ventricle/left ventricle plus septum ratio, medial width of pulmonary arterioles, decreased expression of Kv2.1 and Kv1.5 and the down-regulation of IKV currents induced by chronic hypoxia. In addition, TIIA increased the p27kip1 protein amount, but did not affect the p27kip1 mRNA amount. TIIA kept p27kip1 from being degraded through repressing the activity of Akt and decreasing the production of Skp-2. Conclusions: Principle conclusions: these data demonstrates that TIIA significantly attenuated acute and chronic hypoxia-induced HPV and PVSR, which were probably through up-regulation KV channels and p27kip1.

SGC stimulation totally reverses hypoxia‐induced pulmonary vasoconstriction alone and combined with dual endothelin‐receptor blockade in a porcine model

Stimulation of soluble guanylate cyclase (sGC) with BAY 41-8543 was hypothesized to attenuate acute hypoxic pulmonary vasoconstriction alone and combined with dual endothelin (ET)-receptor antagonist tezosentan. Measurements were taken in 18 anaesthetized pigs with a mean ± SEM weight of 31.1 ± 0.4 kg, in normoxia (FiO(2)~0.21) and hypoxia (FiO(2)~0.10) without (control protocol, n = 6), and with right atrial infusion of BAY 41-8543 at 1, 3, 6, 9 and 12 μg min(-1) per kg (protocol 2, n = 6) or tezosentan at 5 mg kg(-1) followed by BAY 41-8543 at 1, 3 and 6 μg min(-1) per kg (protocol 3, n = 6). Hypoxia (n = 18) increased (P < 0.001) mean pulmonary artery pressure (MPAP) and pulmonary vascular resistance (PVR) by 14.2 ± 0.6 mmHg and 2.8 ± 0.3 WU respectively. During sustained hypoxia without treatment, MPAP and PVR remained stable. BAY 41-8543 (n = 6) dose-dependently decreased (P < 0.001) MPAP and PVR by 15.0 ± 1.2 mmHg and 4.7 ± 0.7 WU respectively. Tezosentan (n = 6) decreased (P < 0.001) MPAP and PVR by 11.8 ± 1.2 mmHg and 2.0 ± 0.2 WU, respectively, whereafter BAY 41-8543 (n = 6) further decreased (P < 0.001) MPAP and PVR by 6.6 ± 0.9 mmHg and 1.9 ± 0.4 WU respectively. Both BAY 41-8543 and tezosentan decreased (P < 0.001) systemic arterial pressure and systemic vascular resistance. Blood-O(2) consumption remained unaltered (P = ns) during all interventions. BAY 41-8543 totally reverses the effects of acute hypoxia-induced pulmonary vasoconstriction, and enhances the attenuating effects of tezosentan, without affecting oxygenation. Thus, sGC stimulation, alone or combined with dual ET-receptor blockade, could offer a means to treat pulmonary hypertension related to hypoxia and potentially other causes.

Glc-6-PD and PKG contribute to hypoxia-induced decrease in smooth muscle cell contractile phenotype proteins in pulmonary artery

Persistent hypoxic pulmonary vasoconstriction (HPV) plays a significant role in the pathogenesis of pulmonary hypertension, which is an emerging clinical problem around the world. We recently showed that hypoxia-induced activation of glucose-6-phosphate dehydrogenase (Glc-6-PD) in pulmonary artery smooth muscle links metabolic changes within smooth muscle cells to HPV and that inhibition of Glc-6PD reduces acute HPV. Here, we demonstrate that exposing pulmonary arterial rings to hypoxia (20-30 Torr) for 12 h in vitro significantly (P < 0.05) reduces (by 30-50%) SM22α and smooth muscle myosin heavy chain expression and evokes HPV. Glc-6-PD activity was also elevated in hypoxic pulmonary arteries. Inhibition of Glc-6-PD activity prevented the hypoxia-induced reduction in SM22α expression and inhibited HPV by 80-90% (P < 0.05). Furthermore, Glc-6-PD and protein kinase G (PKG) formed a complex in pulmonary artery, and Glc-6-PD inhibition increased PKG-mediated phosphorylation of VASP (p-VASP). In turn, increasing PKG activity upregulated SM22α expression and attenuated HPV evoked by Glc-6-PD inhibition. Increasing passive tension (from 0.8 to 3.0 g) in hypoxic arteries for 12 h reduced Glc-6-PD, increased p-VASP and SM22α levels, and inhibited HPV. The present findings indicate that increases in Glc-6-PD activity influence PKG activity and smooth muscle cell phenotype proteins, all of which affect pulmonary artery contractility and remodeling.

Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction

BackgroundAcute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined.MethodWe employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability.ResultsIn hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W.ConclusionHypercapnia with and without acidosis increased HPV during conditions of sustained hypoxia. The increase of sustained HPV and endothelial permeability in hypoxic hypercapnia without acidosis was iNOS dependent.

Pulmonary Hypertension Associated with Acute Hypoxic Pulmonary Vasoconstriction in a Patient with Acute Myeloid Leukemia

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Precursors and inhibitors of hydrogen sulfide synthesis affect acute hypoxic pulmonary vasoconstriction in the intact lung

The effects of hydrogen sulfide (H(2)S) and acute hypoxia are similar in isolated pulmonary arteries from various species. However, the involvement of H(2)S in hypoxic pulmonary vasoconstriction (HPV) has not been studied in the intact lung. The present study used an intact, isolated, perfused rat lung preparation to examine whether adding compounds essential to H(2)S synthesis or to its inhibition would result in a corresponding increase or decrease in the magnitude of HPV. Western blots performed in lung tissue identified the presence of the H(2)S-synthesizing enzymes, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), but not cystathionine β-synthase (CBS). Adding three H(2)S synthesis precursors, cysteine and oxidized or reduced glutathione, to the perfusate significantly increased peak arterial pressure during hypoxia compared with control (P < 0.05). Adding α-ketoglutarate to enhance the 3-MST enzyme pathway also resulted in an increase (P < 0.05). Both aspartate, which inhibits the 3-MST synthesis pathway, and propargylglycine (PPG), which inhibits the CSE pathway, significantly reduced the increases in arterial pressure during hypoxia. Diethylmaleate (DEM), which conjugates sulfhydryls, also reduced the peak hypoxic arterial pressure at concentrations >2 mM. Finally, H(2)S concentrations as measured with a specially designed polarographic electrode decreased markedly in lung tissue homogenate and in small pulmonary arteries when air was added to the hypoxic environment of the measurement chamber. The results of this study provide evidence that the rate of H(2)S synthesis plays a role in the magnitude of acute HPV in the isolated perfused rat lung.

Hypoxia promotes rabbit pulmonary artery smooth muscle cells proliferation through a 15-LOX-2 product 15(S)-hydroxyeicosatetraenoic acid

The initial event of hypoxic pulmonary hypertension is acute hypoxic pulmonary vasoconstriction followed by remodeling of pulmonary arteries. Although 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] is found to be able to induce hypoxic pulmonary vasoconstriction, role of 15(S)-HETE in pulmonary artery smooth muscle cells (PASMCs) proliferation has been studied less. We sought evidence for a role of 15(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that hypoxia enhances 15-lipoxygenase-2 (15-LOX-2) expression and stimulates cultured rabbit PASMCs proliferation. 15(S)-HETE at concentration 0.1 μM stimulated proliferation of PASMCs and induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 15(S)-HETE-stimulated PASMC proliferation was blocked by the MEK inhibitors PD-98059. Hypoxia (3% O 2)-stimulated PASMC proliferation was blocked by U0126, a MEK inhibitor, as well as by NDGA and CDC, inhibitors of 15-LOX, but not by the p38 MAPK inhibitor SB-202190. We conclude that 15-LOX-2 and its product, 15(S)-HETE, are important intermediates in hypoxia-induced rabbit PASMC proliferation and may participate in hypoxia-induced pulmonary hypertension.

Neutral sphingomyelinase, NADPH oxidase and reactive oxygen species. Role in acute hypoxic pulmonary vasoconstriction

The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium-denuded PA segments and their inhibition prevented hypoxia-induced voltage-gated potassium channel (K(V) ) inhibition and pulmonary vasoconstriction. Consistently, H(2) O(2) , or its analogue t-BHP, decreased K(V) currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production, K(V) channel inhibition and vasoconstriction. Hypoxia induced p47(phox) phosphorylation and its interaction with caveolin-1. Inhibition of nSMase (GW4869) or PKCζ prevented p47(phox) phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase-PKCζ-NADPH oxidase as a necessary step required for ROS production and vasoconstriction.

Therapeutic efficacy of azaindole-1 in experimental pulmonary hypertension

An accumulating body of evidence incriminates Rho kinase (ROCK) in the pathogenesis of pulmonary hypertension (PH). The therapeutic efficacy of azaindole-1, a novel highly selective and orally active ROCK inhibitor, has not yet been investigated in PH. This study aimed to investigate the effects of azaindole-1 on 1) acute hypoxic pulmonary vasoconstriction (HPV), 2) proliferation of pulmonary arterial smooth muscle cells (PASMCs) and 3) animal models of PH. Azaindole-1 significantly inhibited HPV in isolated, ventilated and buffer-perfused murine lungs and proliferation of primary rat PASMCs in vitro. Azaindole-1 was administered orally from 21 to 35 days after monocrotaline (MCT) injection in rats and hypoxic exposure in mice. Azaindole-1 (10 and 30 mg per kg body weight per day in rats and mice, respectively) significantly improved haemodynamics and right ventricular hypertrophy. Moreover, the medial wall thickness and muscularisation of peripheral pulmonary arteries were ameliorated. Azaindole-1 treatment resulted in a decreased immunoreactivity for phospho-myosin phosphatase target subunit 1 and proliferating cell nuclear antigen in pulmonary vessels of MCT-injected rats, suggesting an impaired ROCK activity and reduced proliferating cells. Azaindole-1 provided therapeutic benefit in experimental PH, and this may be attributable to its potent vasorelaxant and antiproliferative effects. Azaindole-1 may offer a useful approach for treatment of PH.

Hypoxia Increases ROS Signaling and Cytosolic Ca2+ in Pulmonary Artery Smooth Muscle Cells of Mouse Lungs Slices

Precapillary arteries constrict during alveolar hypoxia in a response known as hypoxic pulmonary vasoconstriction (HPV). The mechanism by which pulmonary arterial smooth muscle cells (PASMCs) detect a decrease in Po(2) and trigger contraction is not fully understood. Previous studies in cultured PASMCs show that hypoxia induces an increase in reactive oxygen species (ROS) production, but these results may not reflect responses of PASMCs in their native tissue environment. We therefore assessed hypoxia-induced changes in cytosolic ROS in PASMCs of precision-cut mouse lung slices expressing the redox-sensitive protein, RoGFP. Superfusion of lung slices with hypoxic media (1.5% O(2)) resulted in a significant oxidation of RoGFP from normoxic baseline that was attenuated by overexpression of cytosolic catalase. Hypoxic superfusion also increased [Ca(2+)](i) above normoxic baseline; this response was significantly attenuated by cytosolic catalase overexpression or by the administration of EUK134, a synthetic SOD-catalase mimetic. The hypoxia-induced increase in [Ca(2+)](i) was abolished in the absence of extracellular Ca(2+), indicating that ROS signals trigger entry of extracellular calcium. Collectively, these results indicate that an increase in cytosolic ROS signaling is required for the increase in [Ca(2+)](i) in PASMCs in precision-cut mouse lung slices during the acute HPV response.

Changes in pulmonary blood flow distribution in monocrotaline compared with hypoxia-induced models of pulmonary hypertension: assessed using synchrotron radiation

We have previously described anatomical changes in pulmonary blood flow distribution in chronic hypoxic rats, associated with pulmonary arterial hypertension (PAH). In this study, we utilized synchrotron radiation microangiography to compare these changes in pulmonary blood flow with a PAH-model induced with monocrotaline (MCT), as the etiology for these two models of PAH is different. Three weeks after a subcutaneous injection of MCT (60 mg/kg) or vehicle (control), Sprague-Dawley rats were anesthetized, and microangiography was performed on the left lung to assess branching distribution of pulmonary blood flow and changes in vessel diameter during acute (8% O2 for 4 min) hypoxic pulmonary vasoconstriction--before and after sympathetic beta-adrenoceptor blockade (propranolol, 2 mg/kg, intravenous). Comparisons were made with chronic hypoxic rats using data previously published. We observed that adverse changes in pulmonary blood flow were comparable for both chronic hypoxia and MCT models of PAH. Specifically, the number of opaque third and fourth generation vessels was significantly and equally fewer than that of control rats. The acute hypoxic pulmonary vasoconstriction was not altered in the hypertensive lung, though sympathetic modulation of pulmonary vasoreactivity was enhanced by chronic hypoxia, but not MCT. In summary, we have demonstrated comparable adverse changes in pulmonary blood flow for chronic hypoxia and MCT models of PAH. In contrast, modulation of the hypoxic pulmonary vasoconstriction differs between the two PAH models, likely due to the impact that different pathological pathways have on the physiology of the whole organism. Such differences between models of PAH should be considered in future studies.

Heme Oxygenase-2 and Large-Conductance Ca2+-activated K+ Channels

Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. A complex of heme oxygenase-2 (HO-2) and the BK channel has been suggested as a universal oxygen sensor system. We investigated whether this complex serves as an oxygen sensor for the vascular effects of alveolar hypoxia in the lung. The investigations were performed in chronically hypoxic mice, in isolated perfused and ventilated lungs, and on the cellular level, including HO-2- and BK-channel deficient mice. Immunohistochemical analysis of mouse lungs identified HO-2 mainly in pulmonary arteries, the bronchial epithelium, and alveolar epithelial cells. BK channel alpha-subunit (BKalpha) immunoreactivity was found primarily in the bronchial and vascular smooth muscle layer. Immunofluorescence staining and coimmunoprecipitation suggested only a weak complexation of HO-2 and BKalpha in pulmonary arterial smooth muscle cells. The strength of acute and sustained HPV, determined in isolated perfused and ventilated lungs, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. Exposure of mice to 3 weeks of chronic hypoxia resulted in a slight down-regulation of HO-2 and no alteration in BKalpha expression. The degree of pulmonary hypertension that developed, quantified on the basis of right ventricular pressure, right-heart hypertrophy, and the degree of muscularization of precapillary pulmonary arteries, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. It is demonstrated that neither deletion of HO-2 nor BK channels affect acute, sustained, and chronic vascular responses to alveolar hypoxia in the lung.

Effect of Resveratrol in Acute Hypoxic Pulmonary Vasoconstriction in Isolated Lamb Pulmonary Arteries and Veins

Summary Resveratrol (RSV) is a natural phytoalexin with many biological effects, including antioxidant, anti- platelet, anti- atherogenic, anti- inflamatory, estrogenic properties, immunomodulation and chemoprevention. In the cardiovascular system, it has been shown to cause relaxation of vascular smooth muscle, and reports indicate that RSV induces vasodilatory effects in several vascular beds by acting in endothelium dependent and independent manners. The aim of the study was to determine the role of RSV on hypoxia induced vasoconstriction in precontracted isolated pulmonary arteries and veins. Isolated lamb pulmonary vessels were suspended in an organ bath filled with Krebs Heinseleit solution and isolated contractions were recorded continuously via an isometric transducer connected to a computerized polygraph system. The solution was aerated with room air or a gas mixture of 95% N2 + 5% CO2 (hypoxic). The oxygen concentration of the bathing medium was measured using an oxygen electrode. Serotonin (5-HT)(10 –5 M) for arteries and U46619, a thromboxane analog (TXA2) for veins, were used as pre contractile agents. Our results showed that in the presence of RSV (20 μM), hypoxia induced pulmonary vasoconstriction decreased in pulmonary veins pre contracted with U46619, but not in pulmonary arteries pre contracted with 5-HT.

EXOGENOUS ESTROGEN RAPIDLY ATTENUATES PULMONARY ARTERY VASOREACTIVITY AND ACUTE HYPOXIC PULMONARY VASOCONSTRICTION

Chronic estrogen exposure has been shown to affect pulmonary artery (PA) vasoreactivity. However, the immediate effects of exogenously administered 17beta-estradiol (E2) on vasopressor-induced PA vasoconstriction and acute hypoxic pulmonary vasoconstriction (HPV) have not yet been investigated. We hypothesized that exogenously administered E2 attenuates PA vasoreactivity and acute HPV through a rapid mechanism. Isometric force displacement was measured in isolated PA rings from proestrus female adult Sprague-Dawley rats, estrus, metestrus, or diestrus female adult Sprague-Dawley rats, and male adult Sprague-Dawley rats. The vasoconstrictor response in the absence of hypoxia (organ bath bubbled with 95% O2/5% CO2) was measured after stimulation with 1 microM of phenylephrine. Hypoxia was generated by changing the gas to 95% N2/5% CO2. The E2 was added to the organ bath in 0.1-nM, 0.5-nM, 1-microM, 500-microM, and 1-mM doses. The 1-mM dose caused an immediate decrease in force in PA rings from estrus, metestrus, or diestrus female adult Sprague-Dawley rats. In addition, 500 microM and 1 mM of E2 attenuated phenylephrine- and hypoxia-induced vasoconstriction and potentiated the vasodilatory phase of hypoxia. These effects were immediate and independent of sex or estrous cycle. Lower E2 doses did not mediate any significant effects. We conclude that high doses of exogenous E2 acutely attenuate PA vasoreactivity and acute HPV in a rapid and dose-dependent manner. A better understanding of how E2 modulates the pulmonary vasomotor response may allow for future therapeutic interventions in acute pulmonary hypertensive crises or in pulmonary arterial hypertension.

Activation of neutral sphingomyelinase is involved in acute hypoxic pulmonary vasoconstriction

AimsThe mechanisms involved in hypoxic pulmonary vasoconstriction (HPV) are not yet fully defined. The aim of the study was to determine the role of protein kinase C ζ (PKCζ) and neutral sphingomyelinase (nSMase) in HPV.Methods and resultsCeramide content was measured by immunocytochemistry and voltage-gated potassium channel (KV) currents were recorded by the patch clamp technique in isolated rat pulmonary artery smooth muscle cells (PASMC). Contractile responses were analysed in rat pulmonary arteries mounted in a wire myograph. Pulmonary pressure was recorded in anesthetized open-chest rats. Protein and mRNA expression were measured by western blot and RT–PCR, respectively. We found that hypoxia increased ceramide content in PASMC which was abrogated by inhibition of nSMase, but not acid sphingomyelinase (aSMase). The hypoxia-induced vasoconstrictor response in isolated pulmonary arteries and the inhibition of KV currents were strongly reduced by inhibition of PKCζ or nSMase but not aSMase. The nSMase inhibitor GW4869 prevented HPV in vivo. The vasoconstrictor response to hypoxia was mimicked by exogenous addition of bacterial Smase and ceramide. nSMase2 mRNA expression was ∼10-fold higher in pulmonary compared with mesenteric arteries. In mesenteric arteries, hypoxia failed to increase ceramide but exogenous SMase induced a contractile response.ConclusionnSMase-derived ceramide production and the activation of PKCζ are early and necessary events in the signalling cascade of acute HPV.

Abstract 2616: Role of the Soluble Epoxide Hydrolase in Hypoxic Pulmonary Vasoconstriction and Pulmonary Vascular Remodeling

The soluble epoxide hydrolase (sEH), which is expressed in pulmonary artery smooth muscle cells, metabolizes cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) to their less active diols. Preliminary findings indicate a role of the sEH on hypoxic pulmonary vasoconstriction (HPV) and a vasoconstrictor role of EETs in the pulmonary vasculature. Here we assessed the influence of hypoxia on the expression of the sEH, acute HPV and pulmonary vascular remodeling. In lungs from wild-type mice (WT), exposure to hypoxia (FiO 2 = 0.1) for 21 days decreased the expression of the sEH by 70% (RT-PCR), and increased the number of partially and fully muscularised resistance arteries (by 3-fold). In isolated lungs, pre-exposure to chronic hypoxia significantly increased baseline perfusion pressures (1.3-fold) and potentiated the acute HPV (1.5-fold). While an sEH inhibitor (1-adamantyl-3-cyclohexylurea; ACU) potentiated acute HPV in lungs from mice maintained in normoxic conditions, it had no effect on HPV in lungs from mice exposed to hypoxia. The EET antagonist, 14,15-EEZE, abolished the sEH inhibitor-dependent increase in acute HPV in normoxic lungs. Under normoxic conditions the muscularization of small pulmonary arteries was greater in lungs from sEH −/− mice than in lungs from WT mice and chronic hypoxia further increased the number of fully and partially muscularized arteries in these animals. sEH −/− mice also displayed an enhanced acute HPV (1.5-fold), compared to that observed in WT mice and chronic exposure to hypoxia did not further potentiate acute HPV. Taken together, these data indicate that the sEH is involved in hypoxia-induced pulmonary vascular remodeling and hypoxic pulmonary vasoconstriction.

Epoxyeicosatrienoic acids and the soluble epoxide hydrolase are determinants of pulmonary artery pressure and the acute hypoxic pulmonary vasoconstrictor response

Recent findings have indicated a role for cytochrome P-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in acute hypoxic pulmonary vasoconstriction (HPV). Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), we assessed the influence of the sEH and 11,12-EET on pulmonary artery pressure and HPV in the isolated mouse lung. In lungs from wild-type mice, HPV was significantly increased by sEH inhibition, an effect abolished by pretreatment with CYP epoxygenase inhibitors and the EET antagonist 14,15-EEZE. HPV and EET production were greater in lungs from sEH(-/-) mice than from wild-type mice and sEH inhibition had no further effect on HPV, while MSPPOH and 14,15-EEZE decreased the response. 11,12-EET increased pulmonary artery pressure in a concentration-dependent manner and enhanced HPV via a Rho-dependent mechanism. Both 11,12-EET and hypoxia elicited the membrane translocation of a transient receptor potential (TRP) C6-V5 fusion protein, the latter effect was sensitive to 14,15-EEZE. Moreover, while acute hypoxia and 11,12-EET increased pulmonary pressure in lungs from TRPC6(+/-) mice, lungs from TRPC6(-/-) mice did not respond to either stimuli. These data demonstrate that CYP-derived EETs are involved in HPV and that EET-induced pulmonary contraction under normoxic and hypoxic conditions involves a TRPC6-dependent pathway.

Hypoxia activates Calcium‐dependent Chloride Channels in Small Pulmonary Artery Smooth Muscle Cells

In small pulmonary artery (SPA) smooth muscle cells (SMC), Cl−/HCO3− exchange seemed to set the level of intracellular pH (pHi) and inhibiting the exchanger prevented the pHi increase during hypoxia. We hypothesized that Ca2+ dependent Cl− channels (ClCa) channels played a role in acute hypoxic pulmonary vasoconstriction (HPV). In patch‐clamp studies using the cell‐attached‐patch configuration we recorded single ClCa channels at different patch potentials in cultured SMC from bovine SPA. Hypoxia increased ClCa channel open times, as did 20 μM caffeine. The ClCa channel antagonist, niflumic acid (100 μM) reduced open times. Using the whole‐cell patch‐clamp configuration we recorded Cl− current amplitudes evoked with a given [Ca2+]i at different test potentials. We determined Cl− as the conducting ion species by comparing the set Cl− equilibrium potential (20 mV) with measured results (∼22 mV). These single channel and whole‐cell current studies show that SPA SMC possess ClCa currents and that they can be activated by hypoxia. Funding: Med Coll WI and VA Med Ctr.