Prostaglandins (PGs) now appear to have a central and complex role in inflammatory and proliferative processes, thus, they may be pertinent to the pathogenesis and treatment of cutaneous inflammatory and proliferative diseases. Despite the progress made in recent years, knowledge of the factors which regulate the release of the precursor fatty acids from membrane phospholipids, the biosynthesis and interconversions of the prostaglandins, and their mode of action in the skin is scanty. In order to gain a greater insight into the nature of these factors and into the mode of action of the prostaglandins in skin, we propose to direct our efforts towards the elucidation of the factors which regulate the release of precursor arachidonic acid (AA) from skin phospholipid, the biosynthesis and interconversions of the prostaglandins and the specific receptors for E- and F-prostaglandins in the skin. Specifically: (1) We shall determine the activity of phospholipase A2 in skin membrane preparations under a variety of skin conditions. The activity of this enzyme is determined by the hydrolysis of 1-acyl-2-(1-14C) arachidonyl-GPC as substrate. Relased 14C-AA is determined by thin layer chromatography (TLC). (2) We shall test and characterize possible endogenous inhibitors of PGE2 biosynthesis in skin by in vitro incubations with (1-14C) arachidonic acid. Radioactive PGs are identified after comparison with authentic standards on TLC-system; (3) We shall assay for the activity of the NADPH-dependent-PGE2-9-ketoreductase. This is determined by the incubation of (3H)PGE2 and soluble fractions of normal and proliferative skin specimens. The (3H)PGF2 formed from (3H)PGE2 is identified after TLC with reference standards; (4) Since PG- binding is an initial step in the biological action of PGs, we shall evaluate the binding capacities of (3H)PGE2 and (3H)PGF2 alpha for skin membrane receptors. Bound and unbound PGs are separated by column chromatography on Sephadex G50 (fine). Dissociation constants (Kds) are determined from the Scatchard plot analysis of the equilibrium binding data.
NIH ExPorter
NIH ExPorter