Shawn D. Harmon

20-Hydroxyeicosatetraenoic Acid (20-HETE) Metabolism in Coronary Endothelial Cells

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 ω-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[3H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[3H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca2+-dependent cytosolic phospholipase A2 released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[3H]HETE from the PCEC, a finding that also is consistent with a Ca2+-dependent mobilization process. PCEC also converted 20-[3H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon β-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.

Effect of the Δ6-desaturase inhibitor SC-26196 on PUFA metabolism in human cellsinhibitor SC-26196 on PUFA metabolism in human cells

The objective of this study was to determine the effect of 2,2-diphenyl-5-(4-{[(1E)-pyridin-3-yl-methylidene]-amino}piperazin-1-yl)pentanenitrile (SC-26196), a Δ6-desaturase inhibitor, on PUFA metabolism in human cells. SC-26196 inhibited the desaturation of 2 μM [1-14C] 18∶2n−6 by 87–95% in cultured human skin fibroblasts, coronary artery smooth muscle cells, and astrocytes. By contrast, SC-26196 did not affect the conversion of [1-14C]20∶3n−6 to 20∶4 in the fibroblasts, demonstrating that it is selective for Δ6-desaturase. The IC50 values for inhibition of the desaturation of 2 μM [1-14C] 18∶3n−3 and [3-14C]24∶5n−3 in the fibroblasts, 0.2–0.4 μM, were similar to those for the inhibition of [1-14C] 18∶2n−6 desaturation, and the rates of recovery of [1-14C] 18∶2n−6 and [3-14C] 24∶5n−3 desaturation after removal of SC-26196 from the culture medium also were similar. SC-26196 reduced the conversion of [3-14C] 22∶5n−3 and [3-14C] 24∶5n−3 to DHA by 75 and 84%, respectively, but it had no effect on the retroconversion of [3-14C] 24∶6n−3 to DHA. These results demonstrate that SC-26196 effectively inhibits the desaturation of 18- and 24-carbon PUFA and, therefore, decreases the synthesis of arachidonic acid, EPA, and DHA in human cells. Furthermore, they provide additional evidence that the conversion of 22∶5n−3 to DHA involves Δ6-desaturation.