Epithelial Strips: An Alternative Technique for Examining Arachidonate Metabolism in Equine Tracheal Epithelium

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Abstract
We have developed an alternative method for examining equine tracheal epithelial arachidonic acid (AA) metabolism that utilizes strips of pseudostratified columnar epithelium attached to a layer of elastic tissue 80 to 130 microns thick. We compared the responses of this preparation with those of enzymatically dispersed suspensions of tracheal epithelium obtained from the same animal. Strips incubated with [3H]AA incorporated 40.8 +/- 3.6% of added radioactivity and released 2.55 +/- 0.23% of incorporated radioactivity when stimulated with 5 microM A23187. Values for the cell suspension were 59.6 +/- 1.6% and 1.90 +/- 0.08%, respectively. Stimulation with 50 microM histamine or bradykinin resulted in significant release of free [3H]AA only from the strips. High-performance liquid chromatography radioactivity profiles of eicosanoids released following stimulation with 5 microM A23187 demonstrated peaks that coeluted with free AA, prostaglandin (PG) E2, and PGF2 alpha for the strips, and free AA, leukotriene B4, and 5-HETE for the cell suspensions. The absence of PGE2 production by cell suspensions was confirmed by assaying immunoreactive PGE2 in supernatants from unlabeled strips and suspensions stimulated with 5 microM A23187. Epithelial strips produced 10.3 +/- 1.3 ng PGE2/ml supernatant, whereas 5 x 10(6) cells in suspension produced less than 100 pg/ml. Despite the lack of PG production by the cell suspensions, immunocytochemical staining with an anti-PGH synthase antibody demonstrated the presence of PGH synthase in epithelial cells of both preparations. These data indicate that, in contrast to epithelial cell suspensions, epithelial strips synthesize cyclooxygenase metabolites and respond to peptide agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

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