Generation of lipoxygenase metabolites of arachidonic acid by monolayer cultures of tracheal epithelial cells and intact tracheal segments from rabbits.
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.
Alpert
S E
SE
Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio.
Kramer
C M
CM
Brashler
J R
JR
Bach
M K
MK
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002470:Cell Survival; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008084:Lipoxygenase / Q000378:metabolism*; D011817:Rabbits; D011863:Radioimmunoassay; D014132:Trachea / Q000166:cytology / Q000378:metabolism*
D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D016718:Arachidonic Acid; D000001:Calcimycin; D008084:Lipoxygenase