Abstract
alpha-Tocopherol and three derivatives in which the phytol chain is modified or deleted were examined for their effect on cultured keratinocyte arachidonic acid metabolism. 2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC), in which the phytol chain is replaced by a methyl group, inhibited basal, bradykinin (BK)- and A23187-stimulated prostaglandin E2 (PGE2) synthesis with an apparent Ki of 1.3 microM. The Ki of the analogue with six carbon atoms in the side chain (C6) was 5 microM while that of the C11 analogue was 10 microM. No effect of alpha-tocopherol was observed. The mechanism of inhibition was studied using PMC. The effect of PMC on phospholipase and cyclooxygenase activity was assayed using stable isotope mass measurements of PGE2 formation, which assesses arachidonate release and cyclooxygenase metabolism simultaneously. BK-stimulated formation of PGE2, derived from endogenous phospholipid, was decreased 60% by 5 microM PMC and eliminated by 50 microM PMC, compared with controls. No difference in PGE2 formed from exogenous arachidonic acid was observed, indicating no effect of PMC on cyclooxygenase activity. In contrast, no effect of 5 microM PMC was observed on BK-stimulated [3H]arachidonic acid release from prelabeled cultures. The capacity of PMC to inhibit phospholipase activity in vitro was also assessed. PMC inhibited hydrolysis of phospholipid substrate by up to 60%. These results suggest that alpha-tocopherol analogues with alterations in the phytol chain inhibit eicosanoid synthesis by preferential inhibition of phospholipase.
Highlights
Sayed using stableisotope mass measurementsof PGE2 Potentially, the effects of tocopherols on eicosanoid synformation,which assesses arachidonate release and thesis maybe limited by their poor solubility in aqueous cyclooxygenase metabolism simultaneously
No which the phytol side chain is modified were exdifferencein PGE2 formedfromexogenous arachi- amined for their capacity to modify cellular arachidonic acid donic acid was observed, indicating no effect of PMC metabolism
Two additional analogues, designed C6 and C11, contain 6 or 11carbons in the phytol side chain (Fig. 1).a-Tocopherol or its analogues were added to confluent cultures in concentrations between 0.25 and 25 p ~an,d thecumulative PGE2 synthesisover the 24 h was determined by immunoassay
Summary
Two additional analogues, designed C6 and C11, contain 6 or 11carbons in the phytol side chain (Fig. 1).a-Tocopherol or its analogues were added to confluent cultures in concentrations between 0.25 and 25 p ~an,d thecumulative PGE2 synthesisover the 24 h was determined by immunoassay. Time of Pretreatment in Hours glandin synthesisstimulated by bradykinin.Prostaglandin synthesis was inhibited by 5 p~ PMC a t all concentrations of with 10 p~ of the calcium ionophore A23187 at intervals after bradykinin tested (Fig. 6). In PMC-treated thesis-The effect of PMC treatment on cell viability was cultures, the inhibition of ionophore-induced prostaglandin examined.
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