Regulation of human basophil function by phosphatase inhibitors.

Abstract

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-mediated release of the pre-formed mediator, histamine from human basophils in a time- and dose-dependent manner. Optimal inhibition (77 +/- 4%, P < 0.0001) of histamine release was observed following a 2 h incubation with 1 microM 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. Concentrations required to inhibit release by 50% (IC50) were 0.6 microM for okadaic acid and 7.5 microM for okadaol, whereas okadaone was inactive. 3. The structurally-unrelated PP inhibitor, calyculin A, also inhibited IgE-dependent histamine release from basophils dose-dependently and was approximately six fold more potent than okadaic acid. 4. The IgE-mediated generation of sulphopeptidoleukotrienes (sLT) from basophils was inhibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC50 0.3 microM) > okadaol (3 microM) > okadaone (inactive). 5. Okadaic acid, okadaol and okadaone (all at 3 microM) inhibited the IgE-mediated generation of the cytokine interleukin 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 basophils liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by 17 +/- 3% (P < 0.0005) by a low (2 nM) concentration of okadaic acid and was inhibited by 96 +/- 1% (P < 0.0001) by a higher (5 microM) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a higher (5 microM) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils. 7. In total these data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.