title: Phosphodiesterase-4 inhibition improves corticosteroid insensitivity in pulmonary endothelial cells under oxidative stress.
abstract: BACKGROUND
  Several clinical studies have shown that smoking in asthmatics and chronic obstructive pulmonary disease patients is closely associated with corticosteroid refractoriness. In this work, we have analyzed glucocorticoid insensitivity in human pulmonary artery endothelial cells (HPAECs) under cigarette smoke extract (CSE) exposure as well as the possible additive effects of the combination therapy with a phosphodiesterase (PDE)-4 inhibitor.
  
  
  METHODS
  Interleukin (IL)-8 was measured in cell supernatants by ELISA. Histone deacetylase (HDAC), histone acetylase (HAT), and intracellular cAMP levels were measured by colorimetric assays and enzyme immunoassay, respectively. PDE4 isotypes and glucocorticoid receptor (GR)-α and β expression were measured by real-time RT-PCR.
  
  
  RESULTS
  The PDE4 inhibitor rolipram dose dependently inhibited the IL-8 secretion induced by CSE 5%. In contrast, dexamethasone 1 μM did not show inhibitory effect on IL-8 secretion. Combination of subeffective rolipram concentrations at 10 nM increased the inhibitory effect of dexamethasone to ~45% of inhibition. Cigarette smoke extract 5% inhibited HDAC activity and increased HAT activity generating glucocorticoid insensitivity. Rolipram did not modify the HDAC activity, however partially inhibited the increase in HAT activity at 1 μM. PDE4 isotypes were up-regulated by CSE 5% with the consequent cAMP down-regulation. Dexamethasone reduced all PDE4 isotypes expression and showed additive effects with rolipram enhancing cAMP levels. Furthermore, rolipram enhanced GR-α expression and inhibited the increase in GR-β induced by CSE.
  
  
  CONCLUSIONS
  Combination of rolipram and dexamethasone shows additive properties in HPAECs under glucocorticoid insensitive conditions. These results may be of potential value in future anti-inflammatory therapies using combination of PDE4 inhibitors and glucocorticoids.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain.
authors.forename: J L
authors.initials: JL
authors.lastname: Ortiz
authors.forename: J
authors.initials: J
authors.lastname: Milara
authors.forename: J
authors.initials: J
authors.lastname: Lluch
authors.forename: A
authors.initials: A
authors.lastname: De Diego
authors.forename: C
authors.initials: C
authors.lastname: Sanz
authors.forename: J
authors.initials: J
authors.lastname: Cortijo
mesh_terms: D000305:Adrenal Cortex Hormones / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D051548:Histone Acetyltransferases / Q000378:metabolism; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D008168:Lung / Q000187:drug effects* / Q000378:metabolism*; D018384:Oxidative Stress; D058988:Phosphodiesterase 4 Inhibitors / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D020889:Rolipram / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D000305:Adrenal Cortex Hormones; D016209:Interleukin-8; D058988:Phosphodiesterase 4 Inhibitors; D017382:Reactive Oxygen Species; D011965:Receptors, Glucocorticoid; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone; D000242:Cyclic AMP; D051548:Histone Acetyltransferases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D006655:Histone Deacetylases; D020889:Rolipram