Alpert S E, Kramer C M, Brashler J R, Bach M K
Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio.
Exp Lung Res. 1990 May-Jun;16(3):211-33. doi: 10.3109/01902149009108841.
We compared the profile of lipoxygenase metabolites of arachidonic acid (AA) generated by cultured rabbit tracheal epithelial (TE) cells with that produced by intact rabbit tracheal segments at baseline and following addition of exogenous AA or calcium ionophore A23187. Lipoxygenase metabolites in effluent media were resolved by high-pressure liquid chromatography and quantitated by radioimmunoassay for monohydroxyeicosanoid (HETE) and leukotriene (LT) metabolites [5-, 12-, and 15-HETE; LTB4, LTC4, LTD4]. Following incubation with exogenous AA (10 micrograms/ml), cultured TE cells generated immunoreactive products that coeluted with authentic 5-, 12-, and 15-HETE standards. 12-HETE was the predominant metabolite. Whereas the generation of HETEs by TE monolayers was dependent on addition of exogenous AA, intact tracheal segments demonstrated a baseline production of 12-HETE and lesser amounts of 5- and 15-HETE as well as unidentified metabolites with UV absorbance at 280 nm. Incubation of tracheal segments with AA resulted in augmented metabolite production. In cultured TE cells, small quantities of HETEs were present intracellularly esterified to membrane phospholipids or free in the cytosol, and significant increases in free cytosolic 12- and 15-HETE were detected postincubation with AA. Calcium ionophore (5 microM) did not induce significant increases in HETE production in either cultured TE cells or tracheal segments. Minimal or no immunoreactive LTs B4, C4, and D4 were produced by TE monolayers or tracheal segments at baseline or following addition of AA or ionophore. Production of HETEs by cultured TE cells was not associated with decreased viability, release of intracellular lactic dehydrogenase, or loss of cells from the monolayers. Preincubation of monolayer cultures or tracheal segments with 5,8,11,14-eicosatetraynoic acid prior to addition of exogenous AA inhibition metabolite production. Our observations provide further documentation for the generation of lipoxygenase metabolites by TE cells and suggest that the array of metabolites generated by cultured TE cells may not be representative of the entire spectrum of AA metabolites produced by intact native epithelium.
我们比较了培养的兔气管上皮（TE）细胞产生的花生四烯酸（AA）脂氧合酶代谢产物谱与完整兔气管段在基线时以及添加外源性AA或钙离子载体A23187后产生的代谢产物谱。流出培养基中的脂氧合酶代谢产物通过高压液相色谱分离，并用放射免疫分析法对单羟基二十碳烯酸（HETE）和白三烯（LT）代谢产物[5-、12-和15-HETE；LTB4、LTC4、LTD4]进行定量。用外源性AA（10微克/毫升）孵育后，培养的TE细胞产生了与 authentic 5-、12-和15-HETE标准品共洗脱的免疫反应性产物。12-HETE是主要代谢产物。虽然TE单层细胞产生HETEs依赖于添加外源性AA，但完整的气管段显示出基线时产生12-HETE以及少量的5-和15-HETE，以及在280nm处有紫外吸收的未鉴定代谢产物。气管段与AA孵育导致代谢产物产生增加。在培养的TE细胞中，少量的HETEs以细胞内酯化为膜磷脂的形式存在或游离于细胞质中，与AA孵育后检测到游离细胞质中12-和15-HETE显著增加。钙离子载体（5微摩尔）在培养的TE细胞或气管段中均未诱导HETE产生显著增加。TE单层细胞或气管段在基线时或添加AA或离子载体后产生的免疫反应性LTs B4、C4和D4极少或没有。培养的TE细胞产生HETEs与细胞活力降低、细胞内乳酸脱氢酶释放或单层细胞丢失无关。在添加外源性AA抑制代谢产物产生之前，用5,8,11,14-二十碳四烯酸对单层培养物或气管段进行预孵育。我们的观察结果为TE细胞产生脂氧合酶代谢产物提供了进一步的证据，并表明培养的TE细胞产生的代谢产物阵列可能不代表完整天然上皮产生的AA代谢产物的整个谱。
