Related Experiment Videos
[Identification and characterization of two phospholipase A2 activities in resident mouse peritoneal macrophages]
Abstract:
Resident mouse peritoneal macrophages synthesize and release large amounts of prostaglandins in response to inflammatory stimuli. Release of prostaglandin E2 and 6-oxoprostaglandin F1 alpha occurs at a rate of 1 nmol/h per mg of cell protein. The mechanisms by which substrate arachidonic acid is released have yet to be established. We have therefore initiated studies to characterize those enzymes that can catalyse its release from phospholipid and may be of significance at the cellular level. We report initially the characterization of two phospholipase A2 activities in homogenates of mouse peritoneal macrophages. The first is active at pH 4.5 and is not dependent on Ca2+. The second is Ca2+-dependent and is optimally active at pH 8.5. Either phospholipase A2 activity is capable of hydrolysing [14C] arachidonic acid from [14C] arachidonic acid-labelled phospholipids in quantities sufficient to account for the amounts of prostaglandins by macrophages in culture. Phospholipid substrates are prepared from mouse LM fibroblasts in serum-free Higuchi medium containing radiolabelled phospholipid precursors. Single-labelled phospholipids bear the 14C label in the arachidonic acid moiety. Dual-labelled phospholipids bear a 14C label in the polar head group and a 3H label in the arachidonic acid moiety. Experiments with dual-labelled substrates establish that both phospholipase activities are of the A2 type as indicated by the equimolar recovery of [3H] arachidonic acid and [14C] lysophospholipid. Studies with aqueous sonicated dispersions of purified [14C] arachidonic acid-labelled phospholipid or mixed liposomal substrates formed from mixtures of cellular polar lipids reveal that the pH 4.5 activity hydrolyses phosphatidylethanolamine and phosphatidylcholine more efficiently when they are present in a mixture of other polar lipids. The pH 8.5 activity, however, hydrolyses the purified phospholipids more efficiently.
Insights
Mouse peritoneal macrophages release prostaglandins via two phospholipase A2 enzymes. These enzymes, active at pH 4.5 and pH 8.5, hydrolyze phospholipids to release arachidonic acid, a prostaglandin precursor.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Resident mouse peritoneal macrophages synthesize and release substantial quantities of prostaglandins upon inflammatory stimulation.
- Prostaglandin E2 and 6-oxoprostaglandin F1 alpha are released at a rate of 1 nmol/h per mg of cell protein.
- The precise mechanisms governing the release of the substrate arachidonic acid remain to be fully elucidated.
Purpose of the Study:
- To characterize enzymes responsible for releasing arachidonic acid from phospholipids in mouse peritoneal macrophages.
- To investigate the significance of these enzymes at the cellular level for prostaglandin synthesis.
Main Methods:
- Characterization of two distinct phospholipase A2 activities in macrophage homogenates.
- Assaying enzyme activity at different pH levels (4.5 and 8.5) and in the presence or absence of Ca2+.
- Utilizing radiolabeled phospholipids ([14C] arachidonic acid and dual-labeled [3H] arachidonic acid/ [14C] polar head group) to identify substrate hydrolysis and enzyme type.
Main Results:
- Two phospholipase A2 activities were identified: one Ca2+-independent at pH 4.5 and another Ca2+-dependent at pH 8.5.
- Both activities effectively hydrolyzed [14C] arachidonic acid from labeled phospholipids, providing sufficient substrate for prostaglandin synthesis.
- Dual-labeling experiments confirmed both activities as phospholipase A2, indicated by equimolar recovery of released [3H] arachidonic acid and [14C] lysophospholipid.
- The pH 4.5 activity preferentially hydrolyzed phosphatidylethanolamine and phosphatidylcholine within mixed lipid environments, while the pH 8.5 activity showed higher efficiency with purified phospholipids.
Conclusions:
- Mouse peritoneal macrophages possess at least two distinct phospholipase A2 enzymes crucial for releasing arachidonic acid.
- These enzymes play a significant role in providing the substrate necessary for prostaglandin production in macrophages.
- Understanding these phospholipase A2 activities offers insights into the regulation of inflammatory mediator synthesis.