A new metabolite of arachidonic acid, formed during interaction between thrombin-or collagenstimulated platelets and unstimulated neutrophils, has been demonstrated by both thin-layer radiochromatography and high-performance liquid chromatography. Production of the 3H-labeled metabolite in combined suspensions containing[3H]arachidonate-labeled platelets and unlabeled neutrophils from aspirin-treated donors suggested that platelet 3H-labeled 12S-hydroxy-5,8-cis,10-trans,14-cis-icosatetraenoic acid (12-HETE) was the precursor. This was confirmed by identification of the same product when purified 12-[3H]HETE was added directly to unstimulated neutrophils. Hydrogenation and oxidation of the isolated product, followed by gas chromatography-mass spectrometry showed the structure to be 12S,20-dihydroxyicosatetraenoic acid. These experiments further show that platelet stimuli known to occur in vivo may initiate metabolic interactions between different cell types via the arachidonic acid pathway.

Endogenous carcinogens arising from normal metabolism are known to cause modifications to DNA by reacting with bases to form adducts. Malondialdehyde is a well characterized endogenous carcinogen that forms an adduct with 2'-deoxyguanosine. This adduct has been detected in rat and human liver DNA by gas cbromatographylelectron capture negative-ion chemical ionization mass spectrometry. A method has been developed based on liquid chromatography/electrospray ionization tandem mass spectrometry for the characterization of endogenous DNA adducts. Using this method, it was unequivocally establisbed that the malondialdebyde adduct of 2'-deoxyguanosine was a constituent of human liver DNA. The methodology that has been developed should be applicable to the cbaracterization of other endogenous DNA adducts. There are few structural or biological data available to assess the importance of endogenous DNA adducts as premutagenic lesions responsible for the development of cancer. The availability of methodology based on liquid chromatograpby/electrrospray ionization tandem mass spectrometry for the characterization of DNA adducts should greatly facilitate future studies in this area.

Accumulating experimental and clinical evidence indicates that a time for reappraisal of therapeutic modalities designed to inhibit the eicosanoid pathway as it may affect vascular disease may be approaching. Pharmacologic agents originally used were chosen because they were capable of suppressing platelet functions such as aggregation, release, and adhesion. The goals of clinical trials were to evaluate medications that would prevent or reduce platelet accumulation in critically located blood vessels of the heart, brain, and extremities and on vascular prostheses. Evaluation of results of therapeutic trials has been difficult and this is superimposed on less-than-complete knowledge of the basic pharmacology of the drugs that have been used. Participation of neutrophils and possibly macrophages in the thrombotic process is now well recognized on morphologic grounds. Because different cell types such as platelets, neutrophils, and endothelial cells have been shown to interact biochemically by sharing precursors and intermediates of the eicosanoid pathway, the pharmacologic approach to inhibition of vascular disease may require reevaluation. Neutrophils appear to lack a cyclooxygenase pathway but serve as a source of the lipoxygenase product leukotriene B4 (LTB4). Actions of LTB4 include neutrophil aggregation, adhesion of neutrophils to endothelial cells, chemotaxis, chemokinesis, and plasma exudation. We have demonstrated in vitro that released free arachidonic acid from aspirin-treated platelets can serve as a source of neutrophil LTB4. Leukotrienes C4, D4, and E4 are agonists for various functions of vascular endothelium and smooth muscle. Most pharmacologic agents used in the treatment of vascular diseases inhibit the cyclooxygenase pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

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Incubation of cultured human umbilical vein endothelial cells with -4C]-arachidonic acid, followed by RP-HPLC analysis, resulted in the appearance of two principal radioactive products besides 6-keto-PGF,. The first peak was HHT, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by GC/MS and was shown to be the lipoxygenase product 1 5-HETE. Stimulation of endothelial cells with thrombin enhanced 15-HETE synthesis from arachidonate. Subsequent experiments showed that 5-HETE and 12-HETE were also synthesized by endothelial cells, but no evidence of leukotriene synthesis was found. Incubation of the 15-HETE precursor 1 5-HPETE with endothelial cells resulted in the formation of four distinct ultraviolet light-absorbing peaks. Ultraviolet and GC/MS analysis showed these peaks to be 8, 15-diHETEs that differed only in their hydroxyl configuration and cis-trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14,15-LTA4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggests that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-LTA4. Circulation 72, No. 4, 708-712, 1985. PG12* is the principal prostaglandin biosynthesized by cultured human umbilical vein endothelial cells in response to thrombin, histamine, A-23 187, trypsin, and ATP. LTC4 and LTD4 were shown to stimulate PGI2 biosynthesis in human endothelial cells, and agonist-specific desensitization to LTC4 was also observed.6. 7 Although leukotrienes are known to stimulate prostaglandin biosynthesis in endothelial cells, no physical evidence of arachidonate lipoxygenation by endothelial cells has appeared. In this report we present evidence that human umbilical vein endothelial cells do contain a 15-lipoxygenase.