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Pathways for arachidonic acid mobilization in zymosan-stimulated mouse peritoneal macrophages
J Balsinde1, B Fernández, J A Solís-Herruzo
1Centro de Investigación del Hospital Universitario Doce de Octubre, Madrid, Spain.
Abstract:
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.
Insights
Resident peritoneal macrophages release arachidonic acid via the phospholipase A2 pathway when stimulated by zymosan. Diacylglycerol lipase is not involved in this process, while acyltransferases regulate free arachidonic acid levels.
Area of Science:
- Cellular biology
- Immunology
- Biochemistry
Background:
- Resident peritoneal macrophages are key immune cells.
- Zymosan is a phagocytosable particle used to stimulate macrophages.
- Arachidonic acid release is a critical signaling event in macrophages.
Purpose of the Study:
- To investigate the pathways responsible for arachidonic acid mobilization in zymosan-stimulated macrophages.
- To determine the relative contributions of acyltransferases, phospholipase A2, and diacylglycerol lipase.
- To elucidate the mechanism of arachidonic acid release.
Main Methods:
- Macrophages were labeled with [3H]arachidonic acid.
- Experiments utilized specific inhibitors: nordihydroguaiaretic acid (phospholipase A2 inhibitor) and RHC 80267 (diacylglycerol lipase inhibitor).
- Levels of labeled arachidonic acid, lysophospholipids, and diacylglycerol were measured.
Main Results:
- Zymosan stimulation enhanced [3H]arachidonic acid incorporation into phospholipids.
- A rapid accumulation of intracellular free [3H]arachidonic acid was observed, paralleling lysophosphatidylcholine and lysophosphatidylinositol formation.
- Phospholipase A2 inhibition significantly reduced arachidonic acid release, while diacylglycerol lipase inhibition had no effect.
Conclusions:
- The phospholipase A2 pathway is the primary controller of arachidonic acid release in zymosan-stimulated macrophages.
- The diacylglycerol lipase pathway is not involved in zymosan-induced arachidonic acid release.
- Acyltransferases regulate free arachidonic acid levels but do not mediate the stimulated increase.