PRELIMINARY CHARACTERIZATION OF THE ROLE OF PROTEIN SERINE THREONINE PHOSPHATASES IN THE REGULATION OF HUMAN LUNG MAST-CELL FUNCTION/

1 Okadaic acid, a cell. permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-dependent release of mediators from human lung mast cells (HLMC). The concentration of okadaic acid required to inhibit by 50% (IC50) the IgE-dependent release of histami ne was 0.2 mu M. Okadaic acid also inhibited the IgE-mediated generati on of prostaglandin D-2 (PGD(2)) and sulphopeptidoleukotrienes (sLT) w ith IC50 values of 0.2 mu M and 0.6 mu M respectively. 2 The IgE-media ted generation of histamine: PGD(2) and sLT was inhibited by okadaic a cid and two analogues of okadaic acid, okadaol and okadaone, with the following rank order of activity; okadaic acid>okadaol>okadaone. This order of activity for the inhibition of mediator release parallels the activity of these compounds as inhibitors of isolated PPs. 3 Extracts of HLMC liberated P-32 from radiolabelled glycogen phosphorylase and this PP activity was inhibited by the PP inhibitors (all at 3 mu M), o kadaic acid (73+/-4% inhibition, P<0.0005), okadaol (26+/-7% inhibitio n, P<0.05) and okadaone (8+/-7% inhibition, P=0.52). The rank order of activity of okadaic acid>okadaol>okadaone parallels the activity of t hese compounds as inhibitors of isolated PPs. 4 Dephosphorylation of r adiolabelled glycogen phosphorylase by extracts of HLMC was inhibited by 15+/-3% (P<0.001) by a low (2 nM) concentration of okadaic acid and by 88+/-4% (P<0.0005) by a higher (5 mu M) concentration of okadaic a cid. Because 2 mM okadaic acid may act selectively to inhibit PP2A whe reas 5 mu M okadaic acid inhibits both PP1 and PP2A, these data sugges t that both PP1 and PP2A are present in HLMC. 5 Inhibitor 2, a PP1-sel ective inhibitor, attenuated (71+/-3% inhibition, P<0.05) PP activity in extracts of HLMC suggesting that HLMC contain PP1 and that it may c onstitute 71% of the phosphorylase PP activity in extracts of HLMC. 6 Radiolabelled casein, a PP2A-restricted substrate, was dephosphorylate d by extracts of purified HLMC and this activity was inhibited (81+/-8 % inhibition, P<0.005) by 2 nM okadaic acid suggesting that PP2A is re sident in HLMC. 7 Collectively, these data suggest that both PP1 and P P2A are resident in HLMC. However, although the data suggest that okad aic acid regulates responses in HLMC by interacting with PPs, it has n ot been possible to determine whether either PP1 or PP2A or both PPs a re involved in the okadaic acid-induced inhibition of mediator release from HLMC.