Recent reports indicate that products of the lipoxygenation of arachidonic acid termed hydroxy-eicosatetraeinoic acids (HETE's) are potent mediators of more fundamental importance than the cyclooxygenase products (prostaglandins, thromboxanes and prostacyclins). The biosynthesis of these substances in the skin are presently unknown. The present proposal will again pioneer: (i) to determine whether or not and how much of these potent lipoxygenase products: monohydroxy-ETE (5-HETE), dihydroxy-ETE (5,12-HETE) as well as the glutathionyl derivatives termed leukotrienes (LTC4 and LED4) are formed in normal and abnormal epidermal preparations. We will determine whether or not they potentiate or block the actions of the cyclooxygenase products such as PGE2 and PGF2 in the skin. Novel synthesis of inhibitors of the lipoxygenase pathway will be carried out and their effects on skin will be investigated. (ii) An exciting revelation from our preliminary studies is that UVB-irradiation of skin does generate local photolytic products from 7-dehydrocholesterol, a precursor of Vit D3 in the skin. We shall isolate, purify and test the effects of each product on the release of arachidonic acid from keratinocytes pre-labeled with 14C-arachidonic acid (AA) and directly on the hydrolysis of AA from 1-acyl-2-[1-14C] arachydonyl-syn-glycero-3-phosphocholine by phospholipase A2 prepared from skin. (iii) An understanding of prostaglandin action in the skin must include knowledge of receptors for these substances in the tissue. We will establish a correlation between PG-receptor activity and cAMP or cGMP accumulation in epidermal and keratinocyte preparations, then determine the effects of PG-antagonists on known actions of PGS and cyclic nucleotides. Source materials for these studies will be obtained from normal and hyperproliferating epidermal tissues from EFA-deficient, 20:3,n9-treated skin and mouse keratinocytes in culture. Techniques of column chromatography, TLC, GLC and HPLC will be used. We envisage that these studies will reveal an immense information on the nature of endogenous modulators of phospholipid and arachidonic acid turnover in the epidermal tissue and keratinocyte. These informations will form the basis for future investigations into human skin hyperproliferative disorders and their control.
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