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Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612
We examined the roles of cGMP-dependent protein kinase (PKG) and PI3K in degranulation induced by fMLF and by Fc
RI cross-linking. In rat basophilic leukemia-2H3 cells expressing formyl peptide receptor, the PKG inhibitors KT5823 and Rp-8-Br-PET-cGMP, as well as the PI3K inhibitor LY294002, reduced agonist-stimulated 
-hexosaminidase release in a dose-dependent manner. These inhibitors also abolished vesicular fusion with the plasma membrane, as evidenced by diminished annexin V staining. Agonist-induced degranulation was completely blocked when LY294002 was applied together with one of the PKG inhibitors, suggesting an additive and possibly synergistic effect. In contrast, the PKG inhibitors did not affect fMLF-induced intracellular calcium mobilization and Akt phosphorylation. Likewise, LY294002 did not alter fMLF-induced elevation of intracellular cGMP concentration, and the inhibitory effect of LY294002 was not reversed by a cell-permeable analog of cGMP. Treatment with fMLF induced phosphorylation of soluble N-ethylmaleimide-sensitive factor-attachment protein (SNAP)-23, syntaxins 2, 4, and 6, and Monc18-3. The induced phosphorylation of SNAP-23 and syntaxins 2 and 4 was blocked by Rp-8-Br-PET-cGMP and LY294002. However, LY294002 was less effective in inhibiting Munc18-3 phosphorylation. The induced phosphorylation of syntaxin 6 was not effectively blocked by either Rp-8-Br-PET-cGMP or LY294002. Treatment of human neutrophils with the PKG inhibitors and LY294002 reduced enzyme release from primary, secondary, and tertiary granules. These results suggest that PKG and PI3K are involved in degranulation, possibly through phosphorylation of target membrane SNAP receptor proteins and their binding proteins.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants AI033503 and HL068819 from the National Institutes of Health (NIH). M.N. was supported by a National Research Service Award/NIH Institutional T32 Training Grant, "Training Program in Signal Transduction and Cellular Endocrinology" (T32 DK07739) and is currently a recipient of the American Heart Association Midwest Affiliate Predoctoral Fellowship.
2 Address correspondence and reprint requests to Dr. Richard D. Ye, Department of Pharmacology, University of Illinois, 835 South Wolcott Avenue, Chicago, IL 60612. E-mail address: yer{at}uic.edu
3 Abbreviations used in this paper: SNAP, soluble N-ethylmaleimide-sensitive factor-attachment protein; SNARE, SNAP receptor protein; t-SNARE, target membrane SNARE; PLC, phospholipase C; PKC, protein kinase C; PKG, cGMP-dependent protein kinase; sGC, soluble guanylyl cyclase; RBL, rat basophilic leukemia; FPR, formyl peptide receptor; DN, dominant negative; LTF, lactoferrin; MMP, matrix metalloproteinase; IBMX, 3-isobutyl-1-methylxanthine; NOS, NO synthase; eNOS, endothelial NOS; fMLF, formyl-methionyl-leucyl-phenylalanine.
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