Abstract
The extent to which the prostacyclin (IP) receptor regulates the release of two proinflammatory chemokines from human airway epithelial cells was investigated using the novel and competitive IP-receptor antagonist 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452). In BEAS-2B human airway epithelial cells, taprostene, a selective IP-receptor agonist, suppressed interferon-γ-induced CXCL9 and CXCL10 release in a concentration-dependent manner. These effects were mimicked by 8-bromo-cAMP, and they were abolished in cells infected with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA). RO1138452 blocked the inhibitory effect of taprostene on chemokine output in a manner inconsistent with surmountable competitive antagonism. Comparable results were obtained using primary cultures of human airway epithelial cells. The basis of the antagonism imposed by RO1138452 was studied further using BEAS-2B cells stably transfected with a cAMP-response element (CRE) luciferase reporter. On this output, RO1138452 also behaved insurmountably. Mechanistically, this could not be attributed to covalent receptor inactivation, allosterism, or a state of hemiequilibrium. Other studies established that the degree by which RO1138452 antagonized taprostene-induced CRE-dependent transcription was not reversed over a 20-h “washout” period. This pharmacological profile is consistent with the behavior of a pseudo-irreversible antagonist where dissociation from its cognate receptor is so slow that re-equilibration is not achieved at the time the response is measured. Collectively, these data provide compelling evidence that human airway epithelial cells express inhibitory IP-receptors linked to the activation of PKA. Moreover, contrary to existing literature, RO1138452 behaved pseudoirreversibly, emphasizing the need, in drug discovery, to screen potential new medicines in the target tissue(s) of interest.
Footnotes
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M.A.G. is an Alberta Heritage Foundation for Medical Research Senior Scholar and is funded by the Canadian Institutes of Health Research (CIHR). D.P. is the recipient of a Canada Research Chair in Inflammatory Airway Diseases and is supported by the CIHR. S.L.T. acknowledges Nycomed Canada for postdoctoral fellowship support.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.129312.
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ABBREVIATIONS: PGI2, prostacyclin; GPCR, G protein-coupled receptor; IP, prostacyclin; RO1138452, 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxybenzyl)-phenyl]-amine; COPD, chronic obstructive pulmonary disease; CXCL9, monokine induced by interferon-γ; CXCL10, interferon-γ inducible protein of 10 kDa; CRE, cAMP-response element; KSFM, keratinocyte serum-free medium; HAEC, human primary airway epithelial cell; IFN, interferon; ELISA, enzyme-linked immunosorbent assay; MOI, multiplicity of infection; PKA, cAMP-dependent protein kinase; PKI, cAMP-dependent protein kinase inhibitor; CMV, cytomegalovirus; BWA 868C, 3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamino)hydantoin; L-161,982, [4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]; L-798,106, 5-bromo-2-methoxy-N-[3-(2-naphthalen-2-ylmethyl phenyl)acryloyl]-benzene sulfonamide; Br, bromo; iloprost, (5E)-5-[(3aS,4S,5R,6aS)-5-hydroxy-4-[(E,3S)-3-hydroxy-4-methyl-oct-1-en-6-ynyl]-3,3a,4,5,6,6a-hexahydro-1H-pentalen-2-ylidene]pentanoic acid; taprostene, 3-[(Z)-[(1S,3R,4S,5R)-4-[(E,3S)-3-cyclohexyl-3-hydroxy-prop-1-enyl]-3-hydroxy-8-oxabicyclo[3.3.0]oct-7-ylidene]methyl]benzoic acid; DP, PGD2 receptor; EP, PGE2 receptor; TP, thromboxane receptor; AFP-07, 18,19-didehydro-7,7-difluro-16S,20-dimethyl-PGI2; TEI-9063, 17,20-dimethylisocarbacyclin.
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. - Received July 27, 2007.
- Accepted October 24, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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