REGULATION OF HUMAN BASOPHIL FUNCTION BY PHOSPHATASE INHIBITORS

1 Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-mediated release of the pre-for med mediator, histamine from human basophils in a time- and dose-depen dent manner. Optimal inhibition (77+/-4%, P<0.0001) of histamine relea se was observed following a 2 h incubation with 1 mu M okadaic acid. 2 Okadaic acid and two analogues of okadaic acid were also studied and were found to inhibit the IgE-dependent release of histamine. Concentr ations required to inhibit release by 50% (IC50) were 0.6 mu M for oka daic acid and 7.5 mu M for okadaol, whereas okadaone was inactive. 3 T he structurally-unrelated PP inhibitor, calyculin A, also inhibited Ig E-dependent histamine release from basophils dose-dependently and was approximately six fold more potent than okadaic acid. 4 The IgE-mediat ed generation of sulphopeptidoleukotrienes (sLT) from basophils was in hibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC50 0.3 mu M) > okadaol (3 mu M)> okadaone (inactive). 5 Okadaic acid, okadaol and okadaone (all at 3 mu M) inhibited the IgE-mediated generation of the cytokine interle ukin 4 (IL4) from human basophils by 67+/-9% (P<0.002), 48+/-14% (P<0. 05) and 8+/-7% (P=0.31), respectively. 6 Extracts of purified human ba sophils liberated P-32 from radiolabelled glycogen phosphorylase and t his PP activity was inhibited by 17+/-3% (P<0.0005) by a low (2 nM) co ncentration of okadaic acid and was inhibited by 96+/-1% (P<0.0001) by a higher (5 mu M) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a hig her (5 mu M) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils. 7 In total th ese data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.