Adp-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor. a primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction

Hypoxic pulmonary vasoconstriction is unique to pulmonary arteries and serves to match lung perfusion to ventilation. However, in disease states this process can promote hypoxic pulmonary hypertension. Hypoxic pulmonary vasoconstriction is associated with increased NADH levels in pulmonary artery smooth muscle and with intracellular Ca(2+) release from ryanodine-sensitive stores. Because cyclic ADP-ribose (cADPR) regulates ryanodine receptors and is synthesized from beta-NAD(+), we investigated the regulation by beta-NADH of cADPR synthesis and metabolism and the role of cADPR in hypoxic pulmonary vasoconstriction. Significantly higher rates of cADPR synthesis occurred in smooth muscle homogenates of pulmonary arteries, compared with homogenates of systemic arteries. When the beta-NAD(+):beta-NADH ratio was reduced, the net amount of cADPR accumulated increased. This was due, at least in part, to the inhibition of cADPR hydrolase by beta-NADH. Furthermore, hypoxia induced a 10-fold increase in cADPR levels in pulmonary artery smooth muscle, and a membrane-permeant cADPR antagonist, 8-bromo-cADPR, abolished hypoxic pulmonary vasoconstriction in pulmonary artery rings. We propose that the cellular redox state may be coupled via an increase in beta-NADH levels to enhanced cADPR synthesis, activation of ryanodine receptors, and sarcoplasmic reticulum Ca(2+) release. This redox-sensing pathway may offer new therapeutic targets for hypoxic pulmonary hypertension.     

Hypoxic pulmonary vasoconstriction: role of ion channels.

Acute hypoxia induces pulmonary vasoconstriction and chronic hypoxia causes structural changes of the pulmonary vasculature including arterial medial hypertrophy. Electro- and pharmacomechanical mechanisms are involved in regulating pulmonary vasomotor tone, whereas intracellular Ca(2+) serves as an important signal in regulating contraction and proliferation of pulmonary artery smooth muscle cells. Herein, we provide a basic overview of the cellular mechanisms involved in the development of hypoxic pulmonary vasoconstriction. Our discussion focuses on the roles of ion channels permeable to K(+) and Ca(2+), membrane potential, and cytoplasmic Ca(2+) in the development of acute hypoxic pulmonary vasoconstriction and chronic hypoxia-mediated pulmonary vascular remodeling.     

Mitochondrial ROS-PKCepsilon signaling axis is uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle cells

The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCepsilon activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCepsilon protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression prevented hypoxia-induced increases in total PKC and PKCepsilon activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCepsilon translocation peptide inhibitor or PKCepsilon gene deletion decreased hypoxic increase in [Ca(2+)](i) in PASMCs, whereas the conventional PKC inhibitor GO6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCepsilon, contributing to hypoxia-induced increase in [Ca(2+)](i) and contraction in PASMCs.     

CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin

Pulmonary intralobar arteries express heme oxygenase (HO)-1 and -2 and release carbon monoxide (CO) during incubation in Krebs buffer. Acute hypoxia elicits isometric tension development (0.77 +/- 0.06 mN/mm) in pulmonary vascular rings treated with 15 micromol/l chromium mesoporphyrin (CrMP), an inhibitor of HO-dependent CO synthesis, but has no effect in untreated vessels. Acute hypoxia also induces contraction of pulmonary vessels taken from rats injected with HO-2 antisense oligodeoxynucleotides (ODN), which decrease pulmonary HO-2 vascular expression and CO release. Hypoxia-induced contraction of vessels treated with CrMP is attenuated (P < 0.05) by endothelium removal, by CO (1-100 micromol/l) in the bathing buffer, and by endothelin-1 (ET-1) receptor blockade with L-754142 (10 micromol/l). CrMP increases ET-1 levels in pulmonary intralobar arteries, particularly during incubation in hypooxygenated media. CrMP also causes a leftward shift in the concentration-response curve to ET-1, which is offset by exogenous CO. In anesthetized rats, pretreatment with CrMP (40 micromol/kg iv) intensifies the elevation of pulmonary artery pressure elicited by breathing a hypoxic gas mixture. However, acute hypoxia does not elicit augmentation of pulmonary arterial pressure in rats pretreated concurrently with CrMP and the ET-1 receptor antagonist L-745142 (15 mg/kg iv). These data suggest that a product of HO activity, most likely CO, inhibits hypoxia-induced pulmonary vasoconstriction by reducing ET-1 vascular levels and sensitivity.     

Kv3.4通道参与15-羟二十碳四烯酸诱导的大鼠肺动脉收缩

通过组织浴槽血管环方法观察Kv3．4通道特异阻断剂BDS-Ⅰ对15-羟二十碳四烯酸（15-hydroxyeicosatetraenoic acid,15-FETE）收缩肺动脉血管的影响;通过酶法分离、培养Wistar大鼠肺动脉血管平滑肌细胞（pulmonary artery smooth musclecells,PASMCs）,RT-PCR和Western blot技术观察15-HETE对大鼠PASMCs上Kv3．4通道表达的影响,以探讨Kv3．4通道在15-HETE收缩肺动脉过程中的作用。结果如下：（1）15-HETE以浓度依赖方式使肺动脉环张力增加,对缺氧组大鼠肺动脉环张力作用更为明显,与正常对照组相比差异显著;（2）除去肺动脉内皮后,15-HETE引起血管收缩的强度较内皮完整时增强,呈剂量依赖性收缩反应;（3）阻断Kv3．4通道可抑制15-HETE收缩肺动脉;（4）15-HETE下调PASMCs膜上Kv3．4通道mRNA及蛋白质表达。上述观察结果提示Kv3．4通道参与由15-HETE引起的缺氧肺动脉血管收缩（hypoxic pulmonary vasoconstriction,HPV）。     

Constitutive and inducible thymic stromal lymphopoietin expression in human airway smooth muscle cells: role in chronic obstructive pulmonary disease

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that triggers dendritic cell-mediated T helper (Th)-2 inflammatory responses. Previous studies have demonstrated that human airway smooth muscle cells (HASMC) play a critical role in initiating or perpetuating airway inflammation by producing chemokines and cytokines. In this study, we first evaluated the expression of TSLP in primary HASMC and investigated how proinflammatory cytokines (TNF-alpha and IL-1beta) and Th-2 cytokines (IL-4, IL-9) regulate TSLP production from HASMC. TSLP mRNA and protein were assessed by real-time RT-PCR, ELISA, and immunofluorescence from primary HASMC cultures. Primary HASMC express constitutive level of TSLP. Incubation of HASMC with IL-1 or TNF-alpha resulted in a significant increase of TSLP mRNA and protein release from HASMC. Furthermore, combination of IL-1beta and TNF-alpha has an additive effect on TSLP release by HASMC. Primary HASMC pretreated with inhibitors of p38 or p42/p44 ERK MAPK, but not phosphatidylinositol 3-kinase, showed a significant decrease in TSLP release on IL-1beta and TNF-alpha treatment. Furthermore, TSLP immunoreactivity was present in ASM bundle from chronic obstructive pulmonary disease (COPD) and to lesser degree in normal subjects. Taken together, our data provide the first evidence of IL-1beta- and TNF-alpha-induced TSLP expression in HASMC via (p38, p42/p44) MAPK signaling pathways. Our results raise the possibility that HASMC may play a role in COPD airway inflammation via TSLP-dependent pathway.     

尼氟灭酸在大鼠低氧高二氧化碳性肺血管收缩中的作用

目的：探讨氯离子通道阻断剂一尼氟灭酸（NFA）在大鼠低氧高二氧化碳性肺血管收缩（HHPV）中的作用。方法：采用大鼠HHPV模型,二、三级动脉环分别随机分4组（n=8）：常氧组（N组）、低氧高二氧化碳组（H组）、DMSO组（HD组）、尼氟灭酸组（NFA组）。在急性低氧高二氧化碳介质中,采用NFA分别孵育肺二、三级肺动脉环,按照低氧高二氧化碳反应性测定的方法测定其二、三级肺动脉血管环张力的变化,并观察NFA对HHPV的影响。结果：①H组二、三级肺动脉均呈现双向性收缩变化（I期快速收缩、快速舒张;II期持续性收缩）与N组相比有显著性差异（P〈0．05,P〈0．01）;②NFA组二、三级肺动脉环的低氧高二氧化碳性血管收缩作用明显减弱,尤其是Ⅱ期的持续收缩,与HD组相比有显著性差异（P〈0．05,P〈0．01）。结论：氯离子通道阻断剂一尼氟灭酸可减轻大鼠二、三级肺动脉环的张力变化率（尤其是Ⅱ期的持续性收缩）,从而发挥拮抗HHPV的作用。     

Effects of hypoxia on vertebrate blood vessels

Hypoxia contracts mammalian respiratory vessels and increases vascular resistance in respiratory tissues of many vertebrates. In systemic vessels these responses vary, hypoxia relaxes mammalian vessels and contracts systemic arteries from cyclostomes. It has been proposed that hypoxic vasoconstriction in cyclostome systemic arteries is the antecedent to mammalian hypoxic pulmonary vasoconstriction, however, phylogenetic characterization of hypoxic responses is lacking. In this study, we characterized the hypoxic response of isolated systemic and respiratory vessels from a variety of vertebrates using standard myography. Pre-gill/respiratory (ventral aorta, afferent branchial artery, pulmonary artery) and post-gill/systemic (dorsal and thoracic aortas, efferent branchial artery) from lamprey (Petromyzon marinus), sandbar shark (Carcharhinus plumbeus), yellowfin tuna (Thunnus albacares), American bullfrog (Rana catesbeiana), American alligator (Alligator mississippiensis), Pekin duck (Anas platyrhynchos domesticus), chicken (Gallus domesticus) and rat (Rattus norvegicus) were exposed to hypoxia at rest or during pre-stimulation (elevated extracellular potassium, epinephrine or norepinephrine). Hypoxia produced a relaxation or transient contraction followed by relaxation in all pre-gill vessels, except for contraction in lamprey, and vasoconstriction or tri-phasic constriction-dilation-constriction in all pulmonary vessels. Hypoxia contracted systemic vessels from all animals except shark and rat and in pre-contracted rat aortas it produced a transient contraction followed by relaxation. These results show that while the classic "systemic hypoxic vasodilation and pulmonary hypoxic vasoconstriction" may occur in the microcirculation, the hypoxic response of the vertebrate macrocirculation is quite variable. These findings also suggest that hypoxic vasoconstriction is a phylogenetically ancient response.     

Hypoxic pulmonary vasoconstriction is enhanced by inhibition of the synthesis of an endothelium derived relaxing factor

Inhibition of the synthesis of endothelium derived relaxing factor by NG-monomethyl-L-arginine, a competitive inhibitor of the synthesis of nitric oxide from L-arginine, enhances hypoxic pulmonary vasoconstriction in pulmonary artery rings and isolated, Krebs albumin perfused rat lungs. L-arginine rapidly reduces hypoxic vasoconstriction, particularly in lungs treated with NG-monomethyl-L-arginine. Following administration of NG-monomethyl-L-arginine, bradykinin-induced vasodilatation is inhibited (p less than 0.01) and a bradykinin-induced vasoconstriction develops (p less than 0.001). NG-monomethyl-L-arginine does not significantly diminish acetylcholine-induced vasodilatation in the isolated lung. NG-monomethyl-L-arginine causes an endothelium-dependent vasoconstriction in pulmonary artery rings.     

Angiotensin II stimulates nitric oxide production in pulmonary artery endothelium via the type 2 receptor

We previously reported that angiotensin II stimulates an increase in nitric oxide production in pulmonary artery endothelial cells. The aims of this study were to determine which receptor subtype mediates the angiotensin II-dependent increase in nitric oxide production and to investigate the roles of the angiotensin type 1 and type 2 receptors in modulating angiotensin II-dependent vasoconstriction in pulmonary arteries. Pulmonary artery endothelial cells express both angiotensin II type 1 and type 2 receptors as assessed by RT-PCR, Western blot analysis, and flow cytometry. Treatment of the endothelial cells with PD-123319, a type 2 receptor antagonist, prevented the angiotensin II-dependent increase in nitric oxide synthase mRNA, protein levels, and nitric oxide production. In contrast, the type 1 receptor antagonist losartan enhanced nitric oxide synthase mRNA levels, protein expression, and nitric oxide production. Pretreatment of the endothelial cells with either PD-123319 or an anti-angiotensin II antibody prevented this losartan enhancement of nitric oxide production. Angiotensin II-dependent enhanced hypoxic contractions in pulmonary arteries were blocked by the type 1 receptor antagonist candesartan; however, PD-123319 enhanced hypoxic contractions in angiotensin II-treated endothelium-intact vessels. These data demonstrate that angiotensin II stimulates an increase in nitric oxide synthase mRNA, protein expression, and nitric oxide production via the type 2 receptor, whereas signaling via the type 1 receptor negatively regulates nitric oxide production in the pulmonary endothelium. This endothelial, type 2 receptor-dependent increase in nitric oxide may serve to counterbalance the angiotensin II-dependent vasoconstriction in smooth muscle cells, ultimately regulating pulmonary vascular tone.     

Hypoxia-induced activation in small isolated pulmonary arteries from the cat

Effects of hypoxia on force development and membrane potential were studied in isolated small (less than 300 microns diam) and large (greater than 500 microns diam) pulmonary arteries from cats. There was a consistent and reproducible hypoxic constrictor response in small pulmonary arteries that began at PO2 values between 350 and 300 Torr and reached a maximum at PO2 between 50 and 30 Torr. In the small artery smooth muscle cell the membrane potential, which was -51 +/- 1.4 mV at a PO2 of 400 Torr, was depolarized to -37 +/- 2 mV at a PO2 of 50 Torr. In contrast, larger arteries did not exhibit significant hypoxic constriction or depolarization upon exposure to low PO2. Constriction in small arteries was not blocked by phentolamine. Treatment with a low dose of indomethacin (10(-9) M) augmented the response; however, a larger dose of indomethacin (10(-3) M) blocked the constriction to hypoxia but not to 30 mM KCl. Depolarization during hypoxia was not blocked by ouabain. Results of this study suggest that the hypoxic response of these isolated small pulmonary vessels may be like that seen in the intact lung. Furthermore, these data suggest that hypoxic vasoconstriction may be mediated by electrical events occurring at the pulmonary arterial muscle cell membrane either directly or via mediators released from the vessel wall.     

Energy state, pH, and vasomotor tone during hypoxia in precontracted pulmonary and femoral arteries

To assess effects of smooth muscle energy state and intracellular pH (pH(i)) on pulmonary arterial tone during hypoxia, we measured ATP, phosphocreatine, P(i), and pH(i) by (31)P-NMR spectroscopy and isometric tension in phenylephrine-contracted rings of porcine proximal intrapulmonary arteries. Hypoxia caused early transient contraction followed by relaxation and late sustained contraction. Energy state and pH(i) decreased during relaxation and recovered toward control values during late contraction. Femoral arterial rings had higher energy state and lower pH(i) under baseline conditions and did not exhibit late contraction or recovery of energy state and pH(i) during hypoxia. In pulmonary arteries, glucose-free conditions abolished late hypoxic contraction and recovery of energy state and pH(i), but endothelial denudation abolished only late hypoxic contraction. NaCN had little effect at 0. 1 and 1.0 mM but caused marked vasorelaxation and decreases in energy state and pH(i) at 10 mM. These results suggest that 1) regulation of tone, energy state, and pH(i) differed markedly in pulmonary and femoral arterial smooth muscle, 2) hypoxic relaxation was mediated by decreased energy state or pH(i) due to hypoxic inhibition of oxidative phosphorylation, 3) recovery of energy state and pH(i) in hypoxic pulmonary arteries was due to accelerated glycolysis mediated by mechanisms intrinsic to smooth muscle, and 4) late hypoxic contraction in pulmonary arteries was mediated by endothelial factors that required hypoxic recovery of energy state and pH(i) for transduction in smooth muscle or extracellular glucose for production and release by endothelium.     

Hypoxia activates NADPH oxidase to increase [ROS]i and [Ca2+]i through the mitochondrial ROS-PKCepsilon signaling axis in pulmonary artery smooth muscle cells

The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells, including pulmonary artery smooth muscle cells (PASMCs), remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22(phox), p47(phox), and p67(phox) were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47(phox) protein to the plasma membrane in pulmonary, but not mesenteric, arteries. The Nox inhibitor apocynin and p47(phox) gene deletion attenuated the hypoxic increase in intracellular concentrations of reactive oxygen species and Ca(2+) ([ROS](i) and [Ca(2+)](i)), as well as contractions in mouse PASMCs, and abolished the hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKCepsilon translocation peptide inhibitor, and PKCepsilon gene deletion, but not the conventional PKC inhibitor GO6976, prevented the hypoxic increase in Nox activity in pulmonary arteries and [ROS](i) in PASMCs. The PKC activator phorbol 12-myristate 13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H(2)O(2) removal significantly inhibited the hypoxic activation of Nox, whereas Gpx1 gene deletion had the opposite effect. Exogenous H(2)O(2) increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase [ROS](i) through the mitochondrial ROS-PKCepsilon signaling axis, providing a positive feedback mechanism to contribute to the hypoxic increase in [ROS](i) and [Ca(2+)](i) as well as contraction in PASMCs.     

Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.     

Intestinal inflammation downregulates smooth muscle CPI-17 through induction of TNF-alpha and causes motility disorders

Motility disorders are frequently observed in intestinal inflammation. We previously reported that in vitro treatment of intestinal smooth muscle tissue with IL-1beta decreases the expression of CPI-17, an endogenous inhibitory protein of smooth muscle serine/threonine protein phosphatase, thereby inhibiting contraction. The present study was performed to examine the pathophysiological importance of CPI-17 expression in the motility disorders by using an in vivo model of intestinal inflammation and to define the regulatory mechanism of CPI-17 expression by proinflammatory cytokines. After the induction of acute ileitis with 2,4,6,-trinitrobenzensulfonic acid, CPI-17 expression declined in a time-dependent manner. This decrease in CPI-17 expression was parallel with the reduction of cholinergic agonist-induced contraction of smooth muscle strips and sensitivity of permeabilized smooth muscle fibers to Ca(2+). Among the various proinflammatory cytokines tested, TNF-alpha and IL-1beta were observed to directly inhibit CPI-17 expression and contraction in cultured rat intestinal tissue. Moreover, both TNF-alpha and IL-1beta inhibited CPI-17 expression and contraction of smooth muscle tissue isolated from wild-type and IL-1alpha/beta double-knockout mice. However, IL-1beta treatment failed to inhibit CPI-17 expression and contraction in TNF-alpha knockout mice. In beta-escin-permeabilized ileal tissues, pretreatment with anti-phosphorylated CPI-17 antibody inhibited the carbachol-induced Ca(2+) sensitization in the presence of GTP. These findings suggest that CPI-17 was downregulated during intestinal inflammation and that TNF-alpha plays a central role in this process. Downregulation of CPI-17 may play a role in motility impairments in inflammation.     

Expression and self-regulatory function of cardiac interleukin-6 during endotoxemia

Interleukin (IL)-6 reportedly has negative inotropic and hypertrophic effects on the heart. Here, we describe endotoxin-induced IL-6 in the heart that has not previously been well characterized. An intraperitoneal injection of a bacterial lipopolysaccharide into C57BL/6 mice induced IL-6 mRNA in the heart more strongly than in any other tissue examined. Induction of mRNA for two proinflammatory cytokines, IL-1beta and tumor necrosis factor (TNF)-alpha, occurred rapidly before the induction of IL-6 mRNA and protein. Although stimulation of isolated rat neonatal myocardial cells with IL-1beta or TNF-alpha induced IL-6 mRNA in vitro, nonmyocardial heart cells produced higher levels of IL-6 mRNA upon stimulation with IL-1beta. In situ hybridization and immunohistochemical analyses localized the IL-6 expression primarily in nonmyocardial cells in vivo. Endotoxin-induced expression of cardiac IL-1beta, TNF-alpha, and intercellular adhesion molecule 1 was augmented in IL-6-deficient mice compared with control mice. Thus cardiac IL-6, expressed mainly by nonmyocardial cells via IL-1beta action during endotoxemia, is likely to suppress expression of proinflammatory mediators and to regulate itself via a negative feedback mechanism.     

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.     

Hypoxia-induced reactive oxygen species downregulate ETB receptor-mediated contraction of rat pulmonary arteries

Production of reactive oxygen species (ROS) may be increased during hypoxia in pulmonary arteries. In this study, the role of ROS in the effect of hypoxia on endothelin (ET) type B (ETB) receptor-mediated vasocontraction in lungs was determined. In rat intrapulmonary (approximately 0.63 mm ID) arteries, contraction induced by IRL-1620 (a selective ETB receptor agonist) was significantly attenuated after 4 h of hypoxia (30 mmHg Po2) compared with normoxic control (140 mmHg Po2). The effect was abolished by tiron, a scavenger of superoxide anions, but not by polyethylene glycol (PEG)-conjugated catalase, which scavenges H2O2. The hypoxic effect on ETB receptor-mediated vasoconstriction was also abolished by endothelium denudation but not by nitro-L-arginine and indomethacin. Exposure for 4 h to exogenous superoxide anions, but not H2O2, attenuated the vasoconstriction induced by IRL-1620. Confocal study showed that hypoxia increased ROS production in pulmonary arteries that were scavenged by PEG-conjugated SOD. In endothelium-intact pulmonary arteries, the ETB receptor protein was reduced after 4 h of exposure to hypoxia, exogenous superoxide anions, or ET-1. BQ-788, a selective ETB receptor antagonist, prevented these effects. ET-1 production was stimulated in endothelium-intact arteries after 4 h of exposure to hypoxia or exogenous superoxide anions. This effect was blunted by PEG-conjugated SOD. These results demonstrate that exposure to hypoxia attenuates ETB receptor-mediated contraction of rat pulmonary arteries. A hypoxia-induced production of superoxide anions may increase ET-1 release from the endothelium and result in downregulation of ETB receptors on smooth muscle.     

Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.