CALCIUM SIGNALING AND PROTEIN-KINASE-C FOR TNF-ALPHA SECRETION IN A RAT MAST-CELL LINE

In mast cells, like other nonexcitable cells, receptor activation prod uces Ca2+-mobilizing second messengers such as inositol 1,4,5-triphosp hate or sphingosine-1-phosphate, which induce Ca2+ release from intern al stores. The resulting depletion of Ca2+ stores activates Ca2+ chann els in plasma membranes designated as Ca2+ release-activated Ca2+ (CRA C) channels. Ionomycin appears to cause activation of CRAC channels by depleting intracellular Ca2+ stores rather than by acting as an ionop hore. We compared the effects of azelastine, an anti-allergic drug, on TNF-alpha secretion, on Ca2+ signal, and on degranulation in an antig en- or ionomycin-stimulated rat mast RBL-2H3 cell line. Azelastine inh ibited TNF-alpha release at concentrations lower than those needed for the inhibition of degranulation. In antigen-stimulated cells, azelast ine also inhibited equipotently TNF-alpha mRNA expression/protein synt hesis, TNF-alpha release and Ca2+ influx. In ionomycin-stimulated cell s, however, azelastine inhibited TNF-alpha release to a greater extent than TNF-alpha mRNA. expression/protein synthesis and Ca2+ influx, in dicating that azelastine inhibits the release process more potently th an transcription or production of TNF-alpha by interfering with a sign al other than Ca2+. Pretreatment with 1 mu M azelastine inhibited iono mycin-induced, but not antigen-induced, protein kinase C translocation to the membranes. These results suggest that TNF-alpha transcription/ production is mainly regulated by Ca2+ influx, but the release process of TNF-alpha is regulated by additional mechanism(s) possibly involvi ng activation of protein kinase C.