Generation and Metabolism of Lipoxygenase Products in Normal and Membrane-Damaged Cultured Human Keratinocytes

• Published in: Journal of investigative dermatology Vol. 93; no. 4; pp. 486 - 491
• Main Author: Green, Floyd A
• Format: Journal Article
• Language: English
• Published: Danvers, MA Elsevier Inc 01-10-1989; Nature Publishing
• Subjects: Adult; Analytical, structural and metabolic biochemistry; Arachidonate Lipoxygenases - biosynthesis; Arachidonate Lipoxygenases - metabolism; Biological and medical sciences; Cell Membrane - enzymology; Cell Membrane - pathology; Cells, Cultured; Eicosanoic Acids - metabolism; Enzymes and enzyme inhibitors; Epidermal Cells; Epidermis - enzymology; Freezing; Fundamental and applied biological sciences. Psychology; Humans; Hydroxyeicosatetraenoic Acids - metabolism; Infant, Newborn; Keratins - metabolism; Oxidoreductases; Reference Values; Time Factors; Freeze thaw cycle; Human; Enzyme; Arachidonic acid derivatives; HPLC chromatography; Keratinocyte; Lipoxygenase; Metabolism; HETE
• ISSN: 0022-202X; 1523-1747
• DOI: 10.1111/1523-1747.ep12284046

The production and metabolism of lipoxygenase eicosanoids were studied in cultured human keratinocytes. The identity of these eicosanoid structures was established by a variety of chromatographic and analytical techniques. Normal cultured keratinocytes did not produce lipoxygenase eicosanoids either spontaneously or when given arachidonic acid in the presence of permeabilizing concentrations of ethanol or dimethyl sulfoxide. Freeze-thawing of human neonatal and adult keratinocytes resulted in a rapid release of linoleic and arachidonic acids over time. Activation of a latent 15-lipoxygenase was demonstrated by the synthesis of 15-hydroryeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid, and both these products were greatly increased in amount when the corresponding fatty acid precursor was added. Eicosanoid production by cells of newborn and adult origin was indistinguishable. Rapid metabolism of exogenous 15-HETE by normal keratinocytes was observed. Measurable quantities of esterified 15-HETE were found after 1 min, but by 18–20 h all the esterified 15-HETE was degraded to the extent that 80% of the recovered radioactivity was found in water-soluble form. In contrast, when labeled or unlabeled 5-HETE was used a much larger fraction was esterified intact (30% as opposed to 10%) and at the end of 18–20 hours a substantial peak of esterified 5-HETE remained. Intact esterified [3H] HETE were recovered only in the triacylglycerol fraction. The key findings that ω-6 lipoxygenase products are generated but not esterified by membrane-damaged keratinocytes, whereas these products are esterified but not generated by normal keratinocytes, may be of importance in transcellular metabolic control.