Role of cytosolic phospholipase A2 in arachidonic acid release of rat-liver macrophages: regulation by Ca2+ and phosphorylation

Abstract

In this study we have verified the existence of a cytosolic phospholipase A2 (cPLA2) in rat-liver macrophages. Stimulation of these cells with phorbol 12-myristate 13-acetate (PMA), zymosan and lipopolysaccharide (LPS), but not with the Ca(2+)-ionophore A23187, leads to phosphorylation of cPLA2 and activation of mitogen-activated protein (MAP) kinase, supporting the hypothesis that MAP kinase is involved in cPLA2 phosphorylation. We show furthermore, that the tyrosine kinase inhibitor genistein prevents the LPS- but not the PMA- or zymosan-induced phosphorylation of cPLA2 and activation of MAP kinase, indicating that tyrosine kinases participate in LPS- but not in PMA- and zymosan-induced cPLA2 phosphorylation and MAP kinase activation. Phosphorylation of cPLA2 does not strongly correlate with stimulation of the arachidonic acid (AA) cascade: (1) A23187, a potent stimulator of AA release, fails to induce cPLA2 phosphorylation; (2) withdrawal of extracellular Ca2+, which inhibits PMA-stimulated AA release (Dieter, Schulze-Specking and Decker (1988) Eur. J. Biochem. 177, 61-67), has no effect on PMA-induced phosphorylation of cPLA2; (3) LPS induces cPLA2 phosphorylation within minutes, whereas increased AA release upon treatment with LPS is detectable for the first time after 4 h; and (4) genistein, which prevents LPS-induced cPLA2 phosphorylation, does not inhibit AA release in response to LPS. From these data we suggest that a rise in intracellular Ca2+, but not phosphorylation of cPLA2, is essential for activation of the AA cascade in rat-liver macrophages.

Cytosolic Phospholipase A2 Is Required for Macrophage Arachidonic Acid Release by Agonists That Do and Do Not Mobilize Calcium

“…The role of cPLA 2 in regulating arachidonic acid release at basal calcium levels, however, has not been addressed. It has been proposed that arachidonic acid release in PMA-stimulated rat macrophages does not depend on a phospholipase A 2 but on the combined action of phospholipase C and diacylglycerol lipase (56). We now provide evidence that cPLA 2 is essential for arachidonic acid release and eicosanoid production induced by PMA and okadaic acid, which do not mobilize calcium in macrophages.…”

“…Arachidonic acid release under conditions of basal cytosolic calcium have also been reported in different mammalian systems, although the role of cPLA 2 has not been definitively established (21,25,61). In addition, cPLA 2 has been implicated in delayed arachidonic acid release, observed hours after stimulation, when calcium levels would not be expected to remain elevated (3,4,17,26,56). The ability of cPLA 2 to mediate arachidonic acid release without an increase in calcium levels cannot be explained by increased phosphorylation on Ser-505, which is not sufficient for cPLA 2 to mediate arachidonic acid release (17,25,31,36).…”

The Role of Calcium and Phosphorylation of Cytosolic Phospholipase A2 in Regulating Arachidonic Acid Release in Macrophages

“…In several models, arachidonic acid release has been linked to the influx of extracellular calcium (28 -31). From results using rat liver macrophages, it has been suggested that an increase in [Ca 2ϩ ] i , but not cPLA 2 phosphorylation, is necessary for arachidonic acid release (14). However, our results show that cPLA 2 phosphorylation on Ser-505 is required in macrophages when there is a transient increase in [Ca 2ϩ ] i but that it is not essential when there is a sustained increase in [Ca 2ϩ ] i from extracellular sources as induced by the calcium ionophores.…”

“…In macrophages, CSF-1 induces cPLA 2 phosphorylation but not arachidonic acid release although it can act synergistically with calcium mobilizing agonists (13). It has been suggested that an increase in intracellular calcium concentration [Ca 2ϩ ] i , but not phosphorylation of cPLA 2 , is essential for arachidonic acid release in rat liver macrophages (14). In platelets, thrombin-stimulated arachidonic acid release does not require phosphorylation of cPLA 2 on Ser-505 (15).…”

Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets

“…After this observation, many groups reported the concomitant activation of MAPK and phosphorylation of cPLA # in stimulated cells [12][13][14][15][16][17]. Several recent reports, however, dissociate cPLA # phosphorylation from MAPK activation.…”