LIPOCORTIN-1 AND THE CONTROL OF ARACHIDONIC-ACID RELEASE IN CELL SIGNALING - GLUCORTICOIDS INHIBIT G-PROTEIN-DEPENDENT ACTIVATION OF CPLA(2) ACTIVITY

In pre labelled A549 cells epidermal growth factor (EGF) (10 nM) stimulates the release of [5,6,8,9,11,12,14,15-H-3(N)]-arachidonic acid (H-3-AA) by approximately 70%. Increasing Ca2+ with thapsigargin (50 nM) stimulates 3H-AA release by approximately 120%. However, the combined use of these two agents results in a synergistic stimulation of H-3-AA release by over 700%. The EGF stimulated release is sensitive to pertussis toxin (10ng/mL) and guanosine 5'-0-(2-thiodiphosphate) suggesting a G protein-mediated event. This is supported by the fact that the G protein activators AIF(4)(-) and guanosine 5'-O-(2-thiotriphosphate) both stimulate 3H-AA release. The stimulation of (3)HAA release by both EGF or direct G protein activation is completely blocked following pre-treatment for 3 hr with 1 nM dexamethasone. This effect is reversed with a neutralizing antibody to lipocortin-l (1 mu g/mL) suggesting that this protein mediates the inhibitory effects of glucocorticoids on agonist activated H-3-AA release. Thapsigargin stimulation of 3H-AA release is insensitive to dexamethasone treatment. A peptide fragment from the N-terminus of lipocortin-1-Lc13-25 (20-200 ug/mL) mimics the effect of glucocorticoid in suppressing both EGF and G protein activated 3H-AA release. A peptide with Me-Tyr substituting Tyr(21) is much reduced in activity suggesting that the presence of this residue is essential. As peptide Lc13-25 is not derived from the Ca2+/phospholipid binding domain of the native protein then sequestration of phospholipid substrate for PLA, remains an unlikely mechanism of action for this peptide.