Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin.
FcepsilonRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 and 4 nM, respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these 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 active A:B complexes. We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcineurin A subunit, which binds the calcineurin B subunit with high affinity but does not possess catalytic activity (B subunit knock-out, BKO). In these transfected cells, the dose-response curve for the inhibition of FcepsilonRI-mediated exocytosis by FK506 was shifted to the left, indicating an increased drug sensitivity of BKO-transfected cells. We conclude that FK506 inhibition of FcepsilonRI-mediated exocytosis in mast cells specifically targets calcineurin activity.
Hultsch
T
T
Department of Dermatology, University of Mainz, Germany. HULTSCH@goofy.ZDV.UNI-MAINZ.DE
Brand
P
P
Lohmann
S
S
Saloga
J
J
Kincaid
R L
RL
Knop
J
J
D000818:Animals; D019703:Calcineurin / Q000378:metabolism / Q000502:physiology*; D015550:Cell Degranulation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D018274:Electroporation; D005089:Exocytosis / Q000187:drug effects*; D007166:Immunosuppressive Agents / Q000008:administration & dosage / Q000494:pharmacology*; D015471:Leukemia, Basophilic, Acute / Q000473:pathology; D008407:Mast Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017455:Receptors, IgE / Q000502:physiology*; D012680:Sensitivity and Specificity; D016559:Tacrolimus / Q000008:administration & dosage / Q000494:pharmacology*; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000502:physiology
D007166:Immunosuppressive Agents; D017455:Receptors, IgE; D019703:Calcineurin; D016559:Tacrolimus