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) and eicosapentaenoic acid (20:5n-3) in membrane phospholipids on receptor-mediated phospholipase C beta (PLC-beta) activity in cultured rat ventricular myocytes was investigated. For this purpose, cells were grown 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 basal- 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 18:2n-6-treated cells, the incorporation of this 18:2n-6 in the phospholipids 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 acid (22:5n-3) in the phospholipids increased from 0.5 and 2.7 mol % in control cells to 23.2 and 9.7 mol %, respectively. When 20:5n-3-treated cells were stimulated with phenylephrine or endothelin-1, the inositolphosphate production decreased by 33.2% and increased by 43.4%, respectively, 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 stimulated with 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 with GTP gamma S, in the presence of Ca2+, was inhibited 19.3%. This suggests that the 20:5n-3 effect on intact cardiomyocytes could be exerted either on the level of agonist-receptor, receptor-GTP-binding-protein coupling or GTP-binding-protein-PLC-beta interaction. Investigation of the time course of saponin-induced permeabilization of the cardiomyocytes, measured by the release of lactate dehydrogenase, unmasked a slight decrease in the rate of permeabilization by 20:5n-3 pretreatment, indicating a protective effect. This led the authors to measure the cholesterol/phospholipid molar ratio, the double bond index of membrane phospholipids, and the membrane fluidity; the latter by using a diphenylhexatriene probe. In 20:5n-3-pretreated cells, a strong increase in the cholesterol/phospholipid molar ratio (from 0.23 to 0.39), a marked increase in the double bond index (from 1.76 to 2.33), and a slight decrease in fluidity (steady-state anisotropy rss of the diphenylhexatriene probe increased from 0.196 to 0.217) were observed. Thus, treatment of cardiomyocytes for 4 days with 20:5n-3, but not with 18:2n-6, causes alterations of receptor-mediated phospholipase C beta activity. A causal relationship may exist between the 20:5n-3 causes alterations of the physicochemical properties in the bilayer and of the agonist-stimulated phosphatidylinositol cycle activity.
de Jonge
H W
HW
Department of Biochemistry, COEUR, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, The Netherlands.
Dekkers
D H
DH
Bastiaanse
E M
EM
Bezstarosti
K
K
van der Laarse
A
A
Lamers
J M
JM
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D015118:Eicosapentaenoic Acid / Q000737:chemistry / Q000378:metabolism*; D019332:Endothelin-1 / Q000494:pharmacology; D006352:Heart Ventricles / Q000648:ultrastructure; D008560:Membrane Fluidity / Q000187:drug effects; D009200:Myocardial Contraction / Q000187:drug effects; D010656:Phenylephrine / Q000494:pharmacology; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011942:Receptors, Adrenergic, alpha / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D010738:Type C Phospholipases / Q000502:physiology*; D016276:Ventricular Function
D000316:Adrenergic alpha-Agonists; D019332:Endothelin-1; D010743:Phospholipids; D011942:Receptors, Adrenergic, alpha; D010656:Phenylephrine; D015118:Eicosapentaenoic Acid; D010738:Type C Phospholipases