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

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‐1 H ‐azepin‐4‐yl)‐1‐(2 H )‐ 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 Ca 2+ 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 ( α , β I and β II) and transfected GFP‐tagged cPKC, respectively. Both methods clearly demonstrated that 1  μ M azelastine selectively inhibits ionomycin‐triggered translocation of α PKC without acting on β I or β IIPKC. 5 In antigen‐stimulated cells, such a low concentration of azelastine did not affect either α PKC translocation or TNF release, suggesting a functional link between α PKC and the TNF‐releasing step. 6 These results suggest that α PKC mediates the TNF release process and azelastine inhibits TNF release by selectively interfering with the recruitment of α PKC in the pathway activated by ionomycin in RBL‐2H3 cells.British Journal of Pharmacology (2005) 145 , 415–423. doi: 10.1038/sj.bjp.0706207