Protein kinase C‐αmediates TNF release process in RBL‐2H3 mast cells

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Abstract
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. 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. 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. 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 (α,βI andβII) and transfected GFP‐tagged cPKC, respectively. Both methods clearly demonstrated that 1 μMazelastine selectively inhibits ionomycin‐triggered translocation ofαPKC without acting onβI orβIIPKC. In antigen‐stimulated cells, such a low concentration of azelastine did not affect eitherαPKC translocation or TNF release, suggesting a functional link betweenαPKC and the TNF‐releasing step. These results suggest thatαPKC mediates the TNF release process and azelastine inhibits TNF release by selectively interfering with the recruitment ofαPKC in the pathway activated by ionomycin in RBL‐2H3 cells. British Journal of Pharmacology(2005)145, 415–423. doi:10.1038/sj.bjp.0706207