Belén Fernández

Pathways for arachidonic acid mobilization in zymosan-stimulated mouse peritoneal macrophages

Resident peritoneal macrophages release arachidonic acid when challenged by zymosan, a phagocytosable particle. The present study was designed to investigate the pathways for arachidonic acid mobilization in zymosan-stimulated macrophages. Experiments were conducted with [3H]arachidonic acid-labeled macrophages to establish the relative contribution of acyltransferases, phospholipase A2, and diacylglycerol lipase to overall arachidonic acid release. Upon zymosan stimulation, [3H]arachidonic acid incorporation into phospholipids was significantly enhanced. Stimulus-induced activation of arachidonic acid incorporated was not observed immediately, but was found 5 min after cell challenge. On the other hand, the results indicated a rapid accumulation of intracellular free [3H]arachidonic acid that paralleled the appearance of both [3H]glycerol-labeled lysophosphatidylcholine and [3H]glycerol-labeled lysophosphatidylinositol, the by-products of phospholipase A2 action on phosphatidylcholine and phosphatidylinositol, respectively. A transient accumulation of [3H]arachidonate-carrying diacylglycerol was also observed. However, no appreciable alterations in the levels of [3H]monoacylglycerol were found. The phospholipase A2 inhibitor nordihydroguaiaretic acid substantially prevented the zymosan-induced arachidonic acid release. In contrast, RHC 80267, a diacylglycerol lipase inhibitor, though preventing diacylglycerol breakdown, did not have any effect on [3H]arachidonic acid release From these results, it is concluded that: (1) the phospholipase A2 pathway controls arachidonic acid release upon zymosan stimulation; (2) the diacylglycerol lipase pathway appears not to be involved in arachidonic acid release by stimulated cells; (3) the acyltransferases play a remarkable role in controlling free arachidonic acid levels, but they do not participate in the increase of free fatty acid levels observed upon cell stimulation.

Receptor-mediated activation of arachidonic acid release in mouse peritoneal macrophages is linked to extracellular calcium influx

The role of external calcium in platelet-activating factor- and zymosan-stimulated arachidonic acid release from mouse macrophages was investigated. Deprivation of external Ca2+ led to strong inhibition of receptor-mediated arachidonic acid release, which was completely restored when Ca2+ was added to the incubation medium. When arachidonic acid release was examined in Ca(2+)-depleted cells, the response took place only in presence of external Ca2+. Verapamil, a voltage-dependent Ca2+ channel blocker, nearly abolished arachidonic acid release in response to both platelet-activating factor and zymosan. These results suggest that extracellular Ca2+ influx is functionally linked to arachidonic acid release and hence to phospholipase A2 activation in mouse peritoneal macrophages.

Involvement of external calcium in the release of arachidonic acid by mouse peritoneal macrophages

The present investigation was undertaken to study the potential role of extracellular calcium on the release of arachidonic acid from mouse peritoneal macrophages. Both in phorbol ester‐treated and in Ca2+‐depleted cells, a rapid release of arachidonic acid was seen in direct response to added Ca2+. The response was directly dependent on the extracellular Ca2+concentration, with a Ca2+ threshold of 100 nM. These results support the notion that arachidonic acid release in macrophages is functionally coupled to influx of external calcium.

Acetylated low density lipoproteins promote the release and metabolism of arachidonic acid by murine macrophages

We studied the effect of acetyl-LDL on the synthesis of eicosanoids by primary cultures of mouse-resident peritoneal macrophages. A dosis of 150 micrograms of acetyl-LDL proteins promotes a maximal release of arachidonic acid metabolites into extracellular medium. This process is very rapid and reaches the maximal value about ten minutes after stimulation. The pattern of arachidonic acid metabolites released is different from that obtained when the cells are stimulated with a phagocytosable particle like zymosan or calcium ionophore A23187. The data show that the metabolism of arachidonic acid by cycloxigenase is diminished. In an unusual way the amounts of prostaglandin PGE2, 6-keto-prostaglandin PGF1 alpha and thromboxane TXB2 synthesized are similar.

Calcium- and G-protein-dependent activation of arachidonic acid release by concanavalin-A-stimulated mouse macrophages

In this work, signaling mechanisms put into function by concanavalin A in macrophages and its relationship to arachidonic acid release were investigated. After a lag period of approx. 3 min, concanavalin A induced the release of arachidonic acid from macrophages in a time- and dose-dependent manner. Removal of calcium from the extracellular medium led to a strong inhibition of the response. However, down-regulation of protein kinase C by prolonged treatment of macrophages with phorbol myristate acetate did not affect concanavalin-A-induced arachidonic acid release, suggesting that protein kinase C does not mediate the concanavalin A response. The role of G proteins in mediating the concanavalin A response was also investigated. Concanavalin-A-stimulated arachidonic acid release was inhibited by treatment with pertussis toxin but was enhanced by preincubation with cholera toxin. An increase of cAMP did not appear to mediate the stimulatory effect of cholera toxin since non-hydrolyzable cAMP derivatives or agents which raise cAMP levels, such as prostaglandin E2 and forskolin, were without effect on Con-A-stimulated arachidonate release. The direct G-protein activator fluoroaluminate was able to stimulate arachidonic acid release in a Ca(2+)-dependent manner. Combined treatment with fluoroaluminate and concanavalin A resulted in a greater than additive effect on arachidonic acid release. Altogether, these results suggest that concanavalin-A-induced arachidonic acid release in macrophages is co-ordinately regulated by Ca2+ and G proteins, but not by protein kinase C.