DIRECT EVIDENCE THAT FK506 INHIBITION OF FC-EPSILON-RI-MEDIATED EXOCYTOSIS FROM RBL MAST-CELLS INVOLVES CALCINEURIN

Fc epsilon RI-mediated exocytosis of preformed mediators from mast cel ls and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is s ensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 a nd 4 nM, respectively) but not rapamycin, The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of ino sitol phosphates or calcium flux, Here we report experiments using a m olecular approach to assess the role of calcineurin, a serine/threonin e phosphatase thought to be the primary pharmacological target of thes e drugs, Calcineurin's activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit, We hypothesized that calcineurin-sensitive signalling events should be affected by the depletion of calcineurin B subunits, thereby reducing the number of a ctive A:B complexes, We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcine urin A subunit, which binds the calcineurin B subunit with high affini ty but does not possess catalytic activity (B subunit knock-out, BKO), In these transfected cells, the dose-response curve for the inhibitio n of FceRI-mediated exocytosis by FK506 was shifted to the left, indic ating an increased drug sensitivity of BKO-transfected cells, We concl ude that FK506 inhibition of FceRI-mediated exocytosis in mast cells s pecifically targets calcineurin activity.