SUBSTANCE P-INDUCED MAST CELL MEDIATOR RELEASE IS ASSOCIATED WITH STEM CELL FACTOR-DEPENDENT UPREGULATION OF Gαi-2 AND Gαi-3 PROTEINS AND DOWNSTREAM SIGNALING THROUGH A PROTEIN KINASE C-DEPENDENT PATHWAY

Abstract

51 Aims: Substance P (SP)-induced mast cell activation has been implicated in the pathogenesis of gastrointestinal inflammation, but the precise nature of mast cell/SP interactions has not been completely defined. We previously reported that stem cell factor (SCF) conferred reactivity to mast cells stimulated by substance P through a signaling pathway involving G-proteins (Gastroenterology 1996; 110: A911). We therefore assessed whether SCF influenced the expression of G-proteins in mast cells and examined the role of protein kinase C (PKC) in SP-induced mast cell activation. Methods: Bone marrow cultured mast cells (BMCMCs) were maintained in either SCF (50 ng/ml) or 20% Concanavalin A-stimulated spleen conditioned medium (CM) for 4-6 weeks. Western blot analysis of G-protein expression was then performed on BMCMCs in SCF or CM. BMCMCs in SCF or CM were incubated with3 H-5-hydroxytryptamine (5-HT) in the presence or absence of the PKC inhibitors calphostin C (CC; 1 μM) or bisindolylmaleimide I (BIM; 10μM), and then challenged with either SP (10-4 M) or medium alone. Radioactive counts in the supernatants and cell pellets were measured and specific 5-HT release calculated. Results: SP induced significant 5-HT release from BMCMCs in SCF compared with medium alone (12.5 ± 1.5% vs. 1.6 ± 0.2%, respectively, p < 0.05), while no 5-HT release occurred from BMCMCs in CM. Western blot analysis demonstrated a marked increase in the expression of the G-proteins Gαi-2 (by 5 fold) and Gαi-3 (by 20 fold) in BMCMCs in SCF compared with BMCMCs in CM. Furthermore, the PKC inhibitors CC or BIM inhibited SP-induced 5-HT release from BMCMCs in SCF (by 74% and 100%, respectively). Conclusions: We found that mast cells maintained in SCF acquire the ability to release serotonin upon stimulation with SP and express increased levels of Gαi-2 and Gαi-3 proteins. Furthermore, our data demonstrate that SP-induced mast cell pre-formed mediator release involves a protein kinase C-dependent pathway.