Abstract
Arachidonic acid release is induced in macrophages with diverse agonists including calcium ionophores, phorbol myristate acetate (PMA), okadaic acid, and the phagocytic particle, zymosan, and correlates with activation of cytosolic phospholipase A2 (cPLA2). The role of calcium and phosphorylation of cPLA2 in regulating arachidonic acid release was investigated. Zymosan induced a rapid and transient increase in [Ca2+]i. This in itself is not sufficient to induce arachidonic acid release since ATP and platelet activating factor (PAF), agonists that induce transient calcium mobilization in macrophages, induced little arachidonic acid release. Unlike zymosan, which is a strong activator of mitogen-activated protein kinase (MAPK), ATP and PAF were weak MAPK activators and induced only a partial and transient increase in cPLA2 phosphorylation (gel shift). However, ATP or PAF together with colony stimulating factor-1 (CSF-1) synergistically stimulated arachidonic acid release. CSF-1 is a strong MAPK activator that induces a rapid and complete cPLA2 gel shift but not calcium mobilization or arachidonic acid release. Arachidonic acid release was more rapid in response to CSF-1 plus ATP or PAF than zymosan and correlated with the time course of the cPLA2 gel shift. Although low concentrations of ionomycin induced a lower magnitude of calcium mobilization than ATP, the response was more sustained resulting in arachidonic acid release. A23187 and ionomycin induced weak MAPK activation, and a partial and transient cPLA2 gel shift. The MAPK kinase inhibitor, PD 98059 suppressed A23187-induced MAPK activation and cPLA2 gel shift but had little effect on arachidonic acid release. These results indicate that in macrophages a transient increase in [Ca2+]i and sustained phosphorylation of cPLA2 can act together to promote arachidonic acid release but neither alone is sufficient. A sustained increase in calcium is sufficient for inducing arachidonic acid release. However, PMA and okadaic acid induce arachidonic acid release without increasing [Ca2+]i, although resting levels of calcium are required, suggesting alternative mechanisms of regulation.
Highlights
The production of the proinflammatory lipid mediators, the eicosanoids, is dependent on the availability of the precursor, free arachidonic acid
Stimulation of a variety of cell types with diverse agonists that induce arachidonic acid release has been shown to promote serine phosphorylation of cytosolic PLA2 (cPLA2) that is accompanied by an increase in cPLA2 activity and a decrease in electrophoretic mobility [1, 9, 10]. cPLA2 can be phosphorylated by protein kinase C, p42/p44 mitogen-activated protein kinases (MAPK), or protein kinase A in vitro but only phosphorylation by MAPK results in a significant increase in cPLA2 activity and induces a cPLA2 gel shift [10, 11]
We have previously reported that a variety of agonists that induce arachidonic acid release in peritoneal macrophages activate cPLA2 by serine phosphorylation through a MAPK-dependent mechanism [9, 24]
Summary
(Received for publication, May 22, 1997, and in revised form, November 13, 1997). Zhi-Hua Qiu‡, Miguel A. Zymosan induced a rapid and transient increase in [Ca2؉]i This in itself is not sufficient to induce arachidonic acid release since ATP and platelet activating factor (PAF), agonists that induce transient calcium mobilization in macrophages, induced little arachidonic acid release. The MAPK kinase inhibitor, PD 98059 suppressed A23187-induced MAPK activation and cPLA2 gel shift but had little effect on arachidonic acid release These results indicate that in macrophages a transient increase in [Ca2؉]i and sustained phosphorylation of cPLA2 can act together to promote arachidonic acid release but neither alone is sufficient. Stimulation of a variety of cell types with diverse agonists that induce arachidonic acid release has been shown to promote serine phosphorylation of cPLA2 that is accompanied by an increase in cPLA2 activity and a decrease in electrophoretic mobility (gel shift) [1, 9, 10]. In light of these observations, mouse peritoneal macrophages were used as a model to investigate the role of calcium and phosphorylation of cPLA2 in regulating arachidonic acid release
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