564 Biochimica et Biophysics Acia, 795 (;984) W-573 Ekvier BBA 51763 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA EFFECT OF K+ DEPOLARIZATION ON THE SVNTHESIS OF PROSTAGLANDINS AND HYDROXVEICOSATETRA(5,8,11,l4)ENOIC ACIDS (HETE) IN THE RAT zyxwvutsrqponmlkjihgf RETINA EVIDENCE FOR ESTERIFICATION OF 1ZHETE IN LIPIDS DALE L. BIRKLE and NICOLAS G. BAZAN * LSU Eye Center, Louisiana State University Medical Center School of Medicine, 136 South Roman Street, New Orleans LA 70112 (U.S.A.) (Received February 27th, 1984) Key words: Prostaglandin; Lipoxygenase; Hydroxyeicosatetraenoic acid; Glycerolipi4 Depolarization; (Rat retina) [ “C]Arachidonic acid is metabolized to prostaglandins and hydroxyeicosatetraenoic acids in the rat retina. After intravitreal injection of [‘4C]arachidonic acid, 25% of the injected radiolabel was recovered in the retinal lipids. Phosphatidylcholine and phosphatidylinositol were most actively labeled; however, all glycerolipids incorporated arachidonic acid. The synthesis of prostaglandins E,, F,,, D,, 6-keto-F,,, thromboxane & and hydroxyeicosatetraenoic acids was measured by high-performance liquid chromatogra- phy. The identity of 12HETE was confirmed by gas chromatography-mass spectrometry. Incubation of prelabeled retinas in vitro promoted the release of 1’4C]arachidonic acid from glycerolipids. A 12-fold increase in the synthesis of hydroxyeicosatetraenoic acids occurred with no change in the synthesis of prostaglandins. Incubation in a depolarizing medium (45 n&I K+) resulted in a selective increase in hydroxyeicosatetraenoic acids, an effect that was blocked by nordihydroguaiaretic acid (I @I) and eicosatetraynoic acid (10 CM). 12-[3H,]Hydroxyeicosatetraenoic acid, intravitreally injected, was iucorpo- rated into retinal lipids with a distribution similar to arachiionic acid. When retinas 1abeIed with 12-I 3H,]hy- droxyeicosatetraenoic acid were incubated, there was a large release of the incorporated radioactivity, aud metabolism to other products with the chromatographie properties of dihydroxyeicosatetraenoic acids. The release of 1Zhydroxyeicosatetraenoic acid was not affected by depolarizing conditions (45 mM K+); however, the conversion of 12-hydroxyeicosatetraenoic acid to dihydroxy isomers was stimulated by K +. These experiments demonstrate active pathways for the generation of eicosanoids in the rat retina that are sensitive to membrane depolarization and lipoxygenase inhibitors. Introduction The glycerolipids of the mammalian retina are enriched in polyunsaturated fatty acids, including * To whom correspondence should be addressed. Abbreviations: 20:4, arachidonic acid; HETE, hydroxyeicosa- tetraenoic acid; PI, phosphatidylinositol; PC, phosphatidylche line; PE, phosphatidylethanolamine; HPLC, high-performance liquid chromatography; NDGA, nordihydroguaiaretic acid; ETYA, eicosatetraynoic acid; PS, phosphatidylserine. OOW-2760/84/$03.00 0 1984 Elsevier Science Publishers B.V. arachidonic acid (20 : 4 (n-6)) [1,2]. The l-stearoyl- 2arachidonoyl species represents over SO% of the microsomal phosphatidylinositol (PI) from bovine retina. Phosphatidylcholine (PC), phosphatidyl- ethanolamine (PE) and triacylglycerols contain the largest amounts of arachidonic acid, inasmuch as they account for the largest proportion of retinal glycerolipids. Free 20 : 4 in the retina is increased by high Kf concentrations, dibutyryl CAMP and anoxia [3].