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INFLAMMATION AND IMMUNOPHARMACOLOGY
Thoracic Medicine, National Heart and Lung Institute, Imperial College London, London, United Kingdom (K.K.M., M.C.C., L.M.C., P.J.B.); Department of Pharmacology, Rush University Medical Center, Chicago, Illinois (H.L.); Department of Biological Sciences, Biomedical Research Institute, University of Warwick, Coventry, United Kingdom (R.N.); and Department of Pharmacology and Therapeutics, Respiratory Research Group, University of Calgary, Calgary, Alberta, Canada (M.A.G.)
cAMP-elevating drugs are thought to mediate their biological effects by activating the cAMP/cAMP-dependent protein kinase (PKA) cascade. However, this hypothesis is difficult to confirm due to a lack of selective inhibitors. Here, we have probed the role of PKA in mediating inhibitory effects of several cAMP-elevating drugs in BEAS-2B epithelial cells using an adenovirus vector encoding a PKA inhibitor protein (PKI
) and have compared it to H-89, a commonly used small molecule PKA inhibitor. Initial studies established efficient gene transfer and confirmed functionality of PKI 48 h after virus infection. All cAMP-elevating drugs tested promoted the phosphorylation of cAMP response element-binding protein (CREB), activated a cAMP response element (CRE)-driven luciferase reporter gene, and suppressed both granulocyte/macrophage colony-stimulating factor (GM-CSF) generation and [3H]arachidonic acid (AA) release in response to interleukin-1
and monocyte chemotactic protein (MCP)-1, respectively. These effects were abolished by PKI. In contrast, H-89 behaved unpredictably under the same conditions. Thus, although CREB phosphorylation evoked by a range of cAMP-elevating drugs was abolished by H-89, neither activation of the CRE-dependent luciferase reporter gene construct nor the inhibition of GM-CSF generation was inhibited. Paradoxically, H-89 antagonized MCP-1-induced [3H]AA release and enhanced the inhibitory effect of submaximal concentrations of rolipram and 8-bromo-cAMP. We suggest that expression of PKI in susceptible cells provides a simple and unambiguous way to assess the role of PKA in cAMP signaling and to probe the mechanism of action of other drugs and cAMP-dependent responses where the participation of PKA is equivocal. Furthermore, these data suggest that H-89 is not a selective inhibitor of PKA and should be avoided.
Address correspondence to: Dr. Mark A. Giembycz, Department of Pharmacology and Therapeutics, Respiratory Research Group, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail: giembycz{at}ucalgary.ca
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