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Effect of vitamin E on arachidonic acid-release in rat peritoneal macrophages
W Sakamoto1, K Fujie, J Nishihira
1Department of Biochemistry, School of Dentistry, Hokkaido University, Sapporo, Japan.
Abstract:
We have previously reported on suppression of the PGE2 production in PMA- and calcium ionophore A23187-stimulated macrophages isolated from vitamin E-treated rats. To further study the mechanism, we examined the effect of vitamin E on phospholipase A2 activity in both intact macrophages and cell-free homogenates measuring the release of [14C]arachidonic acid. In macrophages from vitamin E-treated rats, arachidonic acid release in intact cells as stimulated with PMA and calcium ionophore A23187 was hardly detected. In the cell-free homogenates, increase in phospholipase A2 activity of cytosol and particulate fractions by PMA and A23187 was partially suppressed. In unstimulated macrophages, most of phospholipase A2 was recovered in the cytosol fraction. The partially purified cytosolic phospholipase A2 showed a molecular mass 95 kDa on TSK gel G3000SW gel-filtration and on Western blot analysis using anti-rabbit platelet phospholipase A2 monoclonal antibody RHY-5. The activity of cytosolic 95 kDa phospholipase A2 was not inhibited in vitro by vitamin E. From these results, it was suggested that vitamin E needs intact macrophages to suppress arachidonic acid release.
Insights
Vitamin E suppresses arachidonic acid release in intact macrophages by inhibiting phospholipase A2 activity. This effect requires whole cells, as vitamin E did not inhibit the enzyme in cell-free preparations.
Area of Science:
- Biochemistry
- Immunology
- Cell Biology
Background:
- Vitamin E is known to suppress prostaglandin E2 (PGE2) production in stimulated macrophages.
- The precise mechanism by which vitamin E exerts this effect, particularly on phospholipase A2 (PLA2) activity, requires further investigation.
Purpose of the Study:
- To investigate the effect of vitamin E on phospholipase A2 activity in macrophages.
- To determine whether vitamin E requires intact cells or can act directly on the enzyme to suppress arachidonic acid release.
Main Methods:
- Macrophages from vitamin E-treated rats were stimulated with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187.
- Arachidonic acid release was measured using [14C]arachidonic acid.
- Phospholipase A2 activity was assessed in both intact cells and cell-free homogenates (cytosol and particulate fractions).
- Cytosolic PLA2 was partially purified and characterized by gel filtration and Western blot.
Main Results:
- Arachidonic acid release was significantly reduced in intact macrophages from vitamin E-treated rats.
- PMA and A23187-induced PLA2 activity in cell-free homogenates was only partially suppressed.
- The activity of the partially purified 95 kDa cytosolic PLA2 was not inhibited by vitamin E in vitro.
- Most unstimulated PLA2 activity was found in the cytosol fraction.
Conclusions:
- Vitamin E requires intact macrophages to suppress arachidonic acid release.
- The suppressive mechanism of vitamin E on PLA2 activity is likely dependent on cellular integrity rather than direct enzyme inhibition.