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BackgroundAbolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions.Methodology/Principal FindingsWe found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem −/− mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection.Conclusion/SignificanceCollectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.

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Selective adenosine A(3) agonists have potential utility for the prevention of perioperative myocardial ischemic injury. Herein, we report on the discovery and synthesis of compound 7. This amino nucleoside agonist possesses unprecedented levels of selectivity for the human adenosine A(3) receptor.

ABSTRACT:Clinical use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with an incidence of idiosyncratic hepatoxicity. The formation of reactive metabolites of DF in vivo has been proposed to be responsible for such toxicity. One type of reactive metabolite, a benzoquinone imine of DF formed through oxidation by cytochromes P450, can be trapped by glutathione in vitro in liver microsomes to form glutathione (GS) adducts. Three GS adducts from DF were reported in the literature, namely, 5-hydroxy (OH)-4-glutathione-DF, 4-OH-3-glutathione-DF and 5-OH-6-glutathione-DF, and they all have the same molecular weight of 616. Recently, we developed a sensitive and high throughput method for the detection of GS adducts from liver microsome incubation. This method uses a constant neutral loss scan of m/z 129, a "structurecharacteristic" fragment for GS adduct, on an automated chipbased nanoelectrospray (Advion NanoMate 100) attached to a tandem mass spectrometer (Sciex API 3000). The analysis of GS adducts from human liver microsome incubation with DF by the NanoMate 100-API 3000 method unambiguously revealed a new adduct ion with m/z 583 (MH ؉ ), in addition to the known adduct peak with m/z 617 (MH ؉ ). This new adduct was further confirmed to be 4-OH-2-glutathion-deschloro-diclofenac by liquid chromatography (LC) tandem mass spectrometry (MS), LC/MS-NMR, and comparison to a synthetic standard.Diclofenac (DF) is a nonsteroidal anti-inflammatory drug that is widely used for the treatment of osteoarthritis and rheumatoid arthritis (Small, 1989). Chemically reactive metabolites of DF have been proposed to explain the idiosyncratic hepatoxicity associated with the clinical use of the drug (Banks et al., 1995;Miyamoto et al., 1997;Shen et al., 1999;Tang et al., 1999b;Tang, 2003). One type of reactive metabolite associated with the formation of protein adducts (Maggs et al., 1995) is a benzoquinone imine intermediate generated via oxidation by cytochromes P450 (Shen et al., 1999;Poon et al., 2001). Metabolism of DF has been studied extensively (Corcoran et al., 2000), and it was proposed that P450-catalyzed oxidation of DF resulted in the formation of DF-2,5-quinone imine and DF-1Ј,4Ј-quinone imine intermediates through initial formation of 5-OH-DF and 4Ј-OH-DF, respectively (Tang et al., 1999a). These reactive intermediates can be trapped by glutathione (GSH) to form GS adducts (Baillie and Slatter, 1991) in vitro (in liver microsomes or hepatocytes), as well as in vivo (in bile and urine; often detected as degradation products, N-acetylcysteine, of the initially formed GS adduct). Three GS adducts from DF were reported in the literature, namely, 5-OH-4-GS-DF, 4Ј-OH-3Ј-GS-DF, and 5-OH-6-GS-DF (Tang et al., 1999a), and they all have the same molecular weight of 616.A neutral loss (NL) scan of m/z 129, a "structure-characteristic" fragment (loss of glutamate) for glutathione adducts, by liquid chromatography-tandem mass spectrometry (LC/MS/MS) was developed to detect reactive metabolite formation (Baill...