sq-23377 and azelastine

1 To clarify the mechanism of mast cell TNF secretion, especially its release process after being produced, we utilized an antiallergic drug, azelastine (4-(p-chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepin-4-yl)-1-(2H)- phthalazinone), which has been reported to inhibit TNF release without affecting its production in ionomycin-stimulated RBL-2H3 cells. 2 Such inhibition was associated with the suppression of an ionomycin-induced increase in membrane-associated PKC activity rather than the suppression of Ca2+ influx, suggesting that PKC might be involved in TNF release process. 3 To see whether conventional PKC family (cPKCs) are involved, we investigated the effects of a selective cPKC inhibitor (Gö6976) and an activator (thymeleatoxin) on TNF release by adding them 1 h after cell stimulation. By this time, TNF mRNA expression had reached its maximum. Gö6976 markedly inhibited TNF release, whereas thymeleatoxin enhanced it, showing a key role of cPKC in TNF post-transcriptional process, possibly its releasing step. 4 To determine which subtype of cPKCs could be affected by azelastine, Western blotting and live imaging by confocal microscopy were conducted to detect the translocation of endogenous cPKC (alpha, betaI and betaII) and transfected GFP-tagged cPKC, respectively. Both methods clearly demonstrated that 1 microM azelastine selectively inhibits ionomycin-triggered translocation of (alpha)PKC without acting on betaI or betaIIPKC. 5 In antigen-stimulated cells, such a low concentration of azelastine did not affect either (alpha)PKC translocation or TNF release, suggesting a functional link between (alpha)PKC and the TNF-releasing step. 6 These results suggest that (alpha)PKC mediates the TNF release process and azelastine inhibits TNF release by selectively interfering with the recruitment of (alpha)PKC in the pathway activated by ionomycin in RBL-2H3 cells.

Topics: Animals; Blotting, Western; Carbazoles; Cell Line, Tumor; Dinitrophenols; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression; Green Fluorescent Proteins; Indoles; Ionomycin; Mast Cells; Microscopy, Confocal; Phthalazines; Protein Kinase C; Protein Kinase C-alpha; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serum Albumin, Bovine; Transfection; Tumor Necrosis Factor-alpha

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.

Topics: Animals; Antigens; Calcium; Calcium Channel Blockers; Cell Degranulation; Imidazoles; Ionomycin; Ionophores; Lipoxygenase Inhibitors; Mast Cells; Phthalazines; Protein Kinase C; Rats; RNA, Messenger; Signal Transduction; Tumor Necrosis Factor-alpha