Our laboratory has presented evidence that the lipoxygenase (LO) pathway of arachidonic acid (AA) metabolism plays a role in the regulation of vascular responses to pressor agonists such as angiotensin II (AngII). We now believe that in addition to stimulus-induced de novo generation of HETES, there may be another pathway of action for these bioactive products. Thus, HETEs, as well as other fatty acids, can remodel membrane phospholipids, affecting the subsequent actions of enzymes which act on these substrates. Several reports have shown that eicosanoids, such as HETEs, substitute for arachidonic acid at the sn-2 position of diacyglycerol. These changes occur through constant, ongoing acylation/deacylation reactions, are concentration and time dependent, and can result in the release of modified lipid messengers. We propose this modification may indirectly alter the kinetics of enzymes, such as phospholipases and protein kinase C, which act on these substrates. Using 3 unique 20 carbon fatty acids, we propose to investigate signaling control mechanisms in cultured rat VSMC. In order to test these hypotheses we will first survey the characteristics of the phospholipid remodeling, measuring the efficacy of uptake of the experimental fatty acids in our cell models and define the resulting changes in the membrane fatty acid profile. Next, we will expand on our historical data, quantitating the physiological consequences of these changes, using Ang II included calcium mobilization and protein kinase C activity as endpoints. Finally we will investigate the mechanism(s) of action(s) by measuring the kinetics of enzymes in vitro using synthetically produced phospholipids which have known fatty acid profiles.
Our Specific Aims are: 1) To profile using gas chromatography and mass spectroscopy, the changes in membrane fatty acid composition induced by 12(S)HETE, ETI, and EPA in cultured VSMC., 2) To quantitate the alterations in physiological response caused by the phospholipid remodeling induced by these fatty acids, and 3) To evaluate the effect of fatty acid substitution on phosphatidylcholine and phosphatidylinositol on phospholipase enzyme kinetics.
