Regulation of human lung mast cell function by phosphatase inhibitors

Okadaic acid, a cell-permeant inhibitor of serine/threonine phosphatases (PP) was found to attenuate the IgE-mediated release of both performed (histamine) and newly formed (leukotriene C4) mediators from human lung mast cells (HLMC). Optimal inhibition (82 +/- 6%) of the IgE-triggered release of histamine was observed after a 2-h incubation of HLMC with okadaic acid (1 microM). Shorter (< 2 h) incubation times and lower (< 1 microM) concentrations of okadaic acid were less effective at inhibiting histamine release from HLMC. The extent to which okadaic acid prevented histamine release was highly dependent upon the strength of the stimulus with lower levels of stimulation more readily modulated. The efficacy of okadaic acid as an inhibitor was not dependent upon the nature of the stimulus because matched levels of histamine release (approximately 30%) induced by either IgE-mediated processes or the calcium ionophore A23187 were attenuated to comparable extents. A series of analogues of okadaic acid and a structurally-distinct PP inhibitor, calyculin, were also found to inhibit the IgE-induced release of mediators from HLMC with the following rank order of potency; calyculin (approximate IC50; 0.015 microM) > okadaic acid (0.2 microM) > okadaol (3.3 microM) > nor-okadaone (> 10 microM). These same PP inhibitors displayed a similar rank order of activity for the inhibition of mediator release induced by the ionophore A23187. Extracts of purified HLMC were found to liberate 32P from radiolabeled glycogen phosphorylase and this PP activity was inhibited by both low (2 nM) and high (5 microM) concentrations of okadaic acid suggesting that HLMC contain both PP type 2A (PP2A) and PP type 1 (PP1). The okadaic acid, analogues of okadaic acid, and calyculin attenuated mediator release at concentrations and with a rank order of activity that closely parallels their activities as inhibitors of PP suggests that PP are important in the regulation of HLMC function. This contention is further supported by the finding that PP activities are constitutively associated with HLMC and that these activities could be inhibited by okadaic acid.