EICOSAPENTAENOIC ACID INCORPORATION IN MEMBRANE PHOSPHOLIPIDS MODULATES RECEPTOR-MEDIATED PHOSPHOLIPASE-C AND MEMBRANE FLUIDITY IN RAT VENTRICULAR MYOCYTES IN CULTURE

The influence of increased incorporation of linoleic acid (18:2n-6) an d eicosapentaenoic acid (20:5n-3) in membrane phospholipids on recepto r-mediated phospholipase C beta (PLC-beta) activity in cultured rat ve ntricular myocytes was investigated. For this purpose, cells were grow n for 4 days in control, stearic acid (18:0)/oleic acid (18:1n-9), 18: 2n-6 and 20:5n-3 enriched media, and subsequently assayed for the basa l- and phenylephrine- or endothelin-1-induced total inositol phosphate formation. The various fatty acid treatments resulted in the expected alterations of fatty acid composition of membrane phospholipids. In 1 8:2n-6-treated cells, the incorporation of this 18:2n-6 in the phospho lipids increased from 17.1 mol % in control cells to 38.9 mol %. In 20 :5n-3-treated cells, incorporation of 20:5n-3 and docosapentaenoic aci d (22:5n-3) in the phospholipids increased from 0.5 and 2.7 mol % in c ontrol cells to 23.2 and 9.7 mol %, respectively. When 20:5n-3-treated cells were stimulated with phenylephrine or endothelin-1, the inosito lphosphate production decreased by 33.2% and increased by 43.4%, respe ctively, as compared to cells grown in control medium. No effects were seen in 18:2n-6-treated cells. When 18:0/18:1n-9-treated cells were s timulated endothelin-1, inositolphosphate formation increased by 26.4% , whereas phenylephrine-stimulated inositolphosphate formation was not affected. In saponin-permeabilized cells, that were pre-treated with 20:5n-3, the formation of total inositolphosphates after stimulation w ith GTP gamma S, in the presence of Ca2+, was inhibited 19.3%. This su ggests that the 20:5n-3 effect on intact cardiomyocytes could be exert ed either on the level of agonist-receptor. receptor-GTP-binding-prote in coupling or GTP-binding-protein-PLC-beta interaction. Investigation of the time course of saponin-induced permeabilization of the cardiom yocytes, measured by the release of lactate dehydrogenase, unmasked a slight decrease in the rate of permeabilization by 20:5n-3 pretreatmen t, indicating a protective effect. This led the authors to measure the cholesterol/phospholipid molar ratio, the double bond index of membra ne phospholipids, and the membrane fluidity; the latter by using a dip henylhexatriene probe. In 20: 5n-3-pretreated cells, a strong increase in the cholesterol/ phospholipid molar ratio (from 0.23 to 0.39), a m arked increase in the double bond index (from 1.76 to 2.33), and a sli ght decrease in fluidity (steady-state anisotropy r(ss) of the dipheny lhexatriene probe increased from 0.196 to 0.217) were observed. Thus, treatment of cardiomyocytes for 4 days with 20:5n-3, but not with 18:2 n-6, causes alterations of receptor-mediated phospholipase C beta acti vity. A causal relationship may exist between the 20:5n-3-induced alte rations of the physicochemical properties in the bilayer and of the ag onist-stimulated phosphatidylinositol cycle activity. (C) 1996 Academi c Press Limited