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 produces Ca2+-mobilizing second messengers such as inositol 1,4,5-triphosphate or sphingosine-1-phosphate, which induce Ca2+ release from internal stores. The resulting depletion of Ca2+ stores activates Ca2+ channels in plasma membranes designated as Ca2+ release-activated Ca2+ (CRAC) channels. Ionomycin appears to cause activation of CRAC channels by depleting intracellular Ca2+ stores rather than by acting as an ionophore. We compared the effects of azelastine, an anti-allergic drug, on TNF-alpha secretion, on Ca2+ signal, and on degranulation in an antigen- or ionomycin-stimulated rat mast RBL-2H3 cell line. Azelastine inhibited TNF-alpha release at concentrations lower than those needed for the inhibition of degranulation. In antigen-stimulated cells, azelastine also inhibited equipotently TNF-alpha mRNA expression/protein synthesis, TNF-alpha release and Ca2+ influx. In ionomycin-stimulated cells, however, azelastine inhibited TNF-alpha release to a greater extent than TNF-alpha mRNA expression/protein synthesis and Ca2+ influx, indicating that azelastine inhibits the release process more potently than transcription or production of TNF-alpha by interfering with a signal other than Ca2+. Pretreatment with 1 microM azelastine inhibited ionomycin-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 involving activation of protein kinase C.