Regulation of Leukocyte Degranulation by cGMP-Dependent Protein Kinase and Phosphoinositide 3-Kinase: Potential Roles in Phosphorylation of Target Membrane SNARE Complex Proteins in Rat Mast Cells

Abstract

We examined the roles of cGMP-dependent protein kinase (PKG) and PI3K in degranulation induced by fMLF and by FcεRI 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 β-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.

An Important Role of the Src Family Kinase Lyn in Stimulating Platelet Granule Secretion

“…These results are consistent with the report that insulin stimulates secretion of ATP from dense granule via the NOcGMP-PKG pathway (49) and with the findings that the NOcGMP-PKG pathway stimulates exocytosis in leukocytes and neuronal cells (21)(22)(23). The cGMP-dependent granule secretion is associated with the PKG-dependent association between syntaxin 2 VAMP3 (vesicle-associated membrane protein 3) (49) in platelets and phosphorylation of soluble SNAP (N-ethylmaleimide-sensitive factor-attached protein)-23, and syntaxins 2 and 4 in leukocytes (21). However, the stimulatory effect cGMP in platelet activation was not repeated by some studies (50,51).…”

“…We have recently discovered that platelet granule secretion can be stimulated via the phosphoinositide 3-kinase (PI3K)-Akt-nitric oxide (NO)-cGMP pathway (16 -20). It has also been shown that granule secretion in leukocytes and neuronal cells can also be stimulated via the NO-cGMP pathway (21)(22)(23).…”

Role of G protein-coupled receptors in inflammation

“…In fact, exogenous expression of FPR1 or C5aR in a rat basophilic leukemia cell line renders these cells capable of releasing β-hexosaminidase upon stimulation, demonstrates the sufficiency of these receptors to trigger degranulation [25] . Simultaneous activation of several signaling pathways is required for degranulation [26] . As with other secretory cells, fusion of intracellular granules or vesicles in phagocytes requires calcium influx, which is triggered by GPCR signaling ( Figure 1).…”

Akt isoforms differentially regulate neutrophil functions

“…23 Antibody detection of phosphorylated p47 phox was conducted as detailed in a recent publication. 28 The Western blot result was quantified with the use of ImageJ software (National Institutes of Health).…”

“…Taking advantage of this feature, Servant et al 22 used the PH domain of Akt as a probe to detect chemoattractant signaling in polarized neutrophils. The PI3K/Akt signaling pathway is one of the key components for the induced release of neutrophil granule contents, 23 although the exact mechanisms remain unknown. Akt is one of the protein kinases that phosphorylate p47 phox , resulting in its membrane translocation and activation of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.…”