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 histamine was 0.2 microM. Okadaic acid also inhibited the IgE-mediated generation of prostaglandin D2 (PGD2) and sulphopeptidoleukotrienes (sLT) with IC50 values of 0.2 microM and 0.6 microM respectively. 2. The IgE-mediated generation of histamine, PGD2 and sLT was inhibited by okadaic acid 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 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by the PP inhibitors (all at 3 microM), okadaic acid (73 +/- 4% inhibition, P < 0.0005), okadaol (26 +/- 7% inhibition, 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 these compounds as inhibitors of isolated PPs. 4. Dephosphorylation of radiolabelled 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 microM) concentration of okadaic acid. Because 2 nM okadaic acid may act selectively to inhibit PP2A whereas 5 microM okadaic acid inhibits both PP1 and PP2A, these data suggest that both PP1 and PP2A are present in HLMC. 5. Inhibitor 2, a PP1-selective inhibitor, attenuated (71 +/- 3% inhibition, P < 0.05) PP activity in extracts of HLMC suggesting that HLMC contain PP1 and that it may constitute 71% of the phosphorylase PP activity in extracts of HLMC. 6. Radiolabelled casein, a PP2A-restricted substrate, was dephosphorylated 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 resident in HLMC. 7. Collectively, these data suggest that both PP1 and PP2A are resident in HLMC. However, although the data suggest that okadaic acid regulates responses in HLMC by interacting with PPs, it has not been possible to determine whether either PP1 or PP2A or both PPs are involved in the okadaic acid-induced inhibition of mediator release from HLMC.
Peirce
M J
MJ
Department of Medicine & Pharmacology, University of Sheffield, Royal Hallamshire Hospital.
Cox
S E
SE
Munday
M R
MR
Peachell
P T
PT
D006636:Histamine Release / Q000187:drug effects*; D006801:Humans; D008168:Lung / Q000502:physiology*; D008407:Mast Cells / Q000502:physiology*; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010766:Phosphorylation
D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases