Abstract
Abstract In asthmatic epithelium, the TH2 cytokine milieu results in production of phospholipid-esterified 15-HETE and elevated mucus secretion. A high-throughput 15-HETE binding assay was developed using 15-HETE esterified to biotin and Streptavidin-AlexaFluor488 to form the ligand (15HETE-Bt:SA-AlF). 15HETE-Bt:SA-AlF binding to Beas-2B cells and PBEC grown under air-liquid interface were assessed from 2- 60 min. Specificity of binding was monitored with unlabeled fatty acids [0.05-10 μM], biotin-hydrazide and SA-ALF. CyclicAMP (cAMP) was quantified using a competitive immunoassay kit. Intracellular calcium [Ca2+]i was measured (Fluo-3 / Fura-red) by flow cytometry. Beas-2B (n=8) and PBEC (n=3 donors) demonstrated time-dependent binding of 15HETE-Bt:SA-AlF, reaching equilibrium at 30 min., 4 deg.C. Binding of 15HETE-Bt:SA-AlF was dose-dependently displaced on Beas2B with 15-HETE>12-HETE, 5-HETE > 5,15diHETE,>15-KETE >> LTB4 (p< .05, 15-HETE vs. other fatty acids) whereas biotin-hydrazide:SA-ALF or SA-ALF were ineffective. In Beas2B cells, 15-HETE-Bt [0.1 - 10 μM] induced pM [cAMP] (n =4) whereas cholera toxin pretreatment (1 μg/ml, 5 hr.) inhibited 55-100% of cAMP production (n=2). A transient increase in [Ca2+]i was detected with 3-10 μM 15-HETE-Bt or 15-HETE in Beas2B (n=2). Bronchial epithelial cells exhibit specific, reversible binding of esterified 15-HETE suggesting a unique Gs- GPCR for esterified 15-HETE that may regulate airway inflammation in asthma.
Published Version
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