The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the release of arachidonic acid (AA) from mouse keratinocytes. A distinct difference was observed between the fatty acid release profile elicited by TPA and other stimuli. These findings led to the investigation of keratinocyte phospholipase A 2 (PLA 2 ), which catalyzes the release of sn -2 fatty acids from membrane phospholipids and regulates the production of eicosanoids. We characterized and identified several forms of PLA 2 in mouse keratinocytes, a cytosolic or cPLA 2 and two secretory or sPLA 2 s in the membrane. The PLA 2 in keratinocyte cytosol is sensitive to heating and acid treatment, while resistant to reducing reagent. The PLA 2 in keratinocyte membrane is resistant to heating and acid treatment, while sensitive to reducing reagent. These characteristics suggested the presence of a cPLA 2 and at least one type of sPLA 2 . Inhibitor data further confirmed the identities of these PLA 2 s. The cPLA 2 was activated by TPA, and appeared to be responsible for the majority of the specific release of AA observed in mouse keratinocytes treated with TPA. The calcium ionophore A23187, and 4 ␣ -TPA did not elicit the selectivity towards AA observed with TPA. The release of linoleic acid (LA) and oleic acid (OA) from A23187and 4 ␣ -TPA-treated keratinocytes suggests activation of sPLA 2 . These activities may be due to the existence of both type I and type II sPLA 2 , as both were identified by polymerase chain reactions.In conclusion, keratinocytes express several forms of phospholipase A 2 that differ in their substrate specificities and mechanisms of activation, resulting in distinct agonist-specific fatty acid release profiles.-Li-Stiles, B., H-H. Lo, and S. M. Fischer. Identification and characterization of several forms of phospholipase A 2 in mouse epidermal keratinocytes.

Male Syrian hamsters and male CD-1 mice were fed diets supplemented with ethyl esters of oleic, linoleic, arachidonic, and eicosapentaenoic acids (1.1-5%, w/w) for 3-4 wk. Plasma and serum triglycerides were significantly higher in the arachidonic acid-supplemented animals compared to those in the other supplementation groups. Changes in serum insulin and glucose levels did not appear to be related to the changes in circulating triglycerides observed in the arachidonic acid-supplemented group. These data indicate that dietary arachidonic acid elevates circulating triglyceride levels compared to other unsaturated fatty acids in hamsters and mice by unknown mechanisms.

The differential activation of different members of the phospholipase A 2 (PLA 2 ) superfamily and their regulation are important as one or more of them regulates the production of eicosanoids and others may contribute to the formation of other lipid mediators. We previously reported the existence of two forms of secretory or sPLA 2 in mouse keratinocytes, namely type I and type II sPLA 2 . We show here that mouse keratinocyte sPLA 2 s were potently activated by protease treatment and inhibited by protease inhibitors. We also observed that G protein effectors induced substantial release of oleic acid (OA) from prelabeled mouse keratinocytes. A G i /G 0 protein activator significantly enhanced the hydrolysis of OA and this increase was not responsive to either pertussis toxin or cholera toxin treatment. Although there was a significant negative correlation between intracellular cAMP levels and OA hydrolysis, experimentally increasing cAMP with forskolin treatment had no effect on sPLA 2 activity. Arachidonic acid but not its metabolites was also shown to marginally activate keratinocyte sPLA 2 by 1.5-fold. These results lead to the conclusion that mouse keratinocyte sPLA 2 s can be regulated primarily by proteolytic activation and a G protein pathway. -Li-Stiles, B., and S. M. Fischer. Mechanism(s) of activation of secretory phospholipase A 2 s in mouse keratinocytes. J.

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