Characterization of high-affinity 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on normal human keratinocytes.

Abstract

Eicosanoids are thought to play an important role in the pathogenesis of inflammatory skin diseases. The object of the present study was the detection and characterization of putative 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites in normal human keratinocytes. Keratinocytes were obtained from foreskin and dermatome-shaved normal human skin. Radioligand binding assays were performed with 12(S)-[3H]HETE on cultured cells. Analysis of saturation curves suggested a one-site model for 12(S)-HETE binding with a KD of 3.84 +/- 0.18 nM and receptor number Bmax of 2.32 +/- 0.12 x 10(5) per cell. Ligand binding was reversible. The rank order of potency in competition for 12(S)-[3H]HETE was 12(S)-HETE > 12(R)- HETE > or = leukotriene B4. Preincubation of cells with 12(S)-HETE (2 x 10(-6) M) resulted in down-regulation of the binding site by approximately 50%. The identification and characterization of specific 12(S)-HETE binding sites on normal human keratinocytes should enable further elucidation of the role of 12-HETE in cutaneous biology and in the pathophysiology of psoriasis and other inflammatory and hyperproliferative dermatoses.