CA2-ATPASE INHIBITORS AND PKC ACTIVATION SYNERGISTICALLY STIMULATE TNF-ALPHA PRODUCTION IN RBL-2H3 CELLS()

Objective and Design: To investigate the effect of Ca2+-ATPase inhibit ors on the production of TNF-alpha in rat basophilic leukemia (RBL-2H3 ) cells. Material: Two Ca2+-ATPase inhibitors, thapsigargin (TG) and c yclopiazonic acid (CPA), and three hydroquinone-antioxidants, 2,5-di-( tert-butyl)-1,4-hydroquinone (DTBHQ), 2,5-di-(tert/amyl)-1,4-hydroquin one (DTAHQ), 2-(tert-butyl)-1,4-hydroquinone (MTBHQ) were used. Treatm ent: Cells were treated with TG, CPA, DTBHQ, DTAHQ and MTBHQ for 3 h i n the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and relea sed TNF-alpha from the cells was measured (n > or = 4). Results: All C a2+-ATPase inhibitors (TG, CPA, DTBHQ and DTAHQ) induced TNF-alpha rel ease in a dose-dependent manner. TNF-alpha release was inhibited by tr eatment with protein kinase C inhibitors (staurosporine, Ro31-8220, ca lophostin C) (p < or = 0.05). In contrast, MTBHQ, which does not induc e increases in [Ca2+](i), did not induce the release of TNF-alpha. TNF -alpha release induced by DTBHQ and CPA was inhibited by treatment wit h actinomycin-D, the immunosuppressant FK506 and the glucocorticoid de xamethasone (p < or = 0.01). Conclusions: These results suggest 1) tha t [Ca2+](i) increase and subsequent activation of protein kinase C is necessary for the release of TNF-alpha, and they work synergistically, 2) that the TNF-alpha release induced by Ca2+-ATPase inhibitors can b e regulated at the transcriptional level.