important role 1. Bergstrom, enzymatic conversion of a new R. Gryglews - PDF document

important role 1. Bergstrom, enzymatic c
important role 1. Bergstrom, enzymatic conversion of a new R. Gryglewski, from arteries transforms pros- to an substrate that hibits platelet 4. Roth, of prostaglandin synthase contracting substance guinea pig G. Herman, layers of thrombotic properties reacting substance polar substituents. 9. Palmblad, Malmsten, A. stimulator of neutrophil 10. Bryant, M. Bailey. 11. Borgeat, P., M. of arachidonic linolenic acid rabbit polymorphonuclear Selective feed-back inhibition of arachidonic Biochem. Biophys. poport. 1982. Positional specificity poxygenase: conversion arachidonic acid Bailey, A. L. biosynthesis in culture. Growth Cell. Biol. Protein measurement Biol. Chem. separation of Blood Coagulation, Haemostasis England. 662-663. 19. Bunting, substance (prostaglandin X) which of mesenteric inhibits plate- Marcus, A. A. Jaffe. human Biol. Chem. L. F. of histamine-mediated cultured human of endothelial 9 2 23. Whiting, J., M. Bailey. side effect synthesis in 24. Samuelsson, G. Crawford, of purified prostaglandin- inhibitor of platelet lipoxygenase. 27. Nakao, W. C. Murota. 1982. aortic smooth M. Bailey. 15-hydroxy-5,8,1 lt13-eicosatetraenoic Vanderhoek. 1982. of T-lymphocyte by guest, on January 4, 2021www.jlr.orgDownlo

aded from products released from smooth
aded from products released from smooth muscle cells thrombin before and after treatment. Confluent cultures cells were prelabeled prelabeled 1-"Clarachidonic acid. The washed, prelabeled cultures were superfused bovine thrombin units/ml) for and the products extracted and analyzed plates were radioautographed for 5 days. Upper thrombin plus aspirin, prelabeled cells were before addition and hydroxy eicosanoids also complete eicosanoid synthesis, not arachidonic the hydroxy eicosanoids are cyclooxygenase products. in which cant amounts PGE2, in this solvent system, gave quantitative conversion very little lipoxygenase activity cells which normal reaction cyclooxygenase involves identified as reaction (25). of this secondary muscle cells, large quantities of direct addition addition acid substrate since it (Fig. 7) mediator thrombin release of muscle cells have some has been lipoxygenase pathways. is ygenase (26). completely without affecting production. In particular interest that 12-HETE that 15-HETE inhibitor of 5-lipoxygenase pathway of leukotriene mast basophil cell line, however, that 15-HETE activate a has been inhibit certain agent leukotriene Thus, the muscle cells Cyclooxygenase pathway cultured rat.aorta

smooth by guest, on January 4, 2021ww
smooth by guest, on January 4, 2021www.jlr.orgDownloaded from TMS-methyl ester eicosanoid fraction shown spectra were taken at retention where the compounds overlapped the least. min) the presence the ions characteristic (229, 258, lower spectrum (173, 314, that the demonstrate that also synthesize thelial cell vascular tissues in thickness. muscle cell throughout the major potential total prostacyclin- vessel wall. muscle cells exposed directly prostacyclin synthesized have dif- cells may anti-platelet activ- muscle cells may as a muscle cell of interest, muscle cells releasing prostacyclin. release of prostacyclin cells in thrombin represents thrombotic properties of muscle cells in injurious conditions. clude degenerative conditions such as atherosclerosis, intimal layers considerable quantities monohydroxy fatty inhibition of by guest, on January 4, 2021www.jlr.orgDownloaded from ELUTION TIME GLC-MS profile of of monohydroxy-eicosanoids muscle cells. confluent cultures (IO-sq-cm) were containing arachidonic methylated with diazo the mono derivatives were analyzed on a 3-ft col- umn packed 3% SE-30 on at 10°/min to a ion, characteristic first major peak as a mixture 1-HETE and 15-HETE central large not a

n 11-HETE (229, 258, and of authentic 15
n 11-HETE (229, 258, and of authentic 15-HETE (1 14, and 343) Fig. 4 spectrum of compound eluting M/e ratios proportions of 1-HETE and mixture determined from the were approximately approximately I4C]arachidonic acid 5 min, as shown predominant compounds formed during lower doses of controls were hydroxy fatty acid release medium alone. Synthesis of 1-HETE and 15-HETE produc- rat smooth muscle cells. acid were isolated, in Fig. 4. During two trios of ions characteristic monitored. Note that for 15-HETE coeluted from the than those smooth muscle by guest, on January 4, 2021www.jlr.orgDownloaded from 100 150 200 250 authentic and Confluent cultures of formation, and GLC-MS as total ion profile spectra of the tetra retention time prostaglandin region of their KOH for treatment demonstrated quantitative conver- acteristics of conversion also TXB2 (which co-chro- muscle cells. compounds traveling hydroxy fatty arachidonic acid/ml) 25-sq-cm flasks, polyunsaturated mono- their corre- sponding saturated derivatives of 11-HETE and 15-HETE samples which to the the other profile as hydroxy fatty acid derivatives from the (the third total ion than that ticular ions and 343 for the 15-HETE monitoring of ions across profile o

f Fig. 5. 15-HETE, thus two compounds. s
f Fig. 5. 15-HETE, thus two compounds. spectra were by guest, on January 4, 2021www.jlr.orgDownloaded from exhaustion of [14C]arachidonic acid tabolites illustrated fusing medium. minor bands prostaglandin region ping bands were present hydroxy fatty acid region traveled immediately behind behind 14C]arachidonic acid band. The major product in the prostaglandin re- thentic sample of 6-keto-PGFI,. tentatively identified as PGE2, PGD2, Synthesis of prostaglandin region ment of indomethacin. Surprisingly, region of considered as probable and nonradioactive arachidonic acid this peak, to the same derivatization remaining com- ARACHIDONATE METABOLITES of products formed durin incubation incubation I-"Clarachidonic acid. Confluent cultures cultures 1j4C]arachidonic acid (0.75 pCi. 55 Ci/mol) in 1 ml of HNCTC-I35 for 5 min at 37OC. The products were extracted as and separated resulting chromatographic plate considerable quantities compounds corre- that chromatograph behind the by guest, on January 4, 2021www.jlr.orgDownloaded from platelet inhibitory aortic smooth arachidonic acid. Confluent cultures of rat aorta 37°C for medium containing pg of arachidonic acid. Aliquots incubated for platelet-rich plasma cuvett

es of a Chronolog Aggregometer stirring.
es of a Chronolog Aggregometer stirring. Aggregation of ethanol) min. Panel Upper curve, curve, preincubated with cell superfusate. Note two curves, medium water bath acid (ASA) incubation. Note that completely inhibits synthesis anti-platelet activity. Panel time periods before addition Note gradual decay of anti-platelet activity increasing time superfusates were tested for their inhibit platelet aggregation a standard aggre- gometer test rabbit platelet-rich Control platelets were arachidonic acid determine the optimum aggregation this optimum dose acid in min. Preincubation with cell aggregation (panel at 100°C for dropping the pH In addition, inhibit platelet aggregation Aliquots of fusate were held intervals before testing for antiaggregatory creasing time the per- plotted as arithmic function found to half-life of that reported half-life of cubation (1 Release of prostacyclin progressively increasing concentrations the range rapid, occurring self-limitingand does not terminate by guest, on January 4, 2021www.jlr.orgDownloaded from a Vanguard radioactivity scanner Autoscanner, scanner 1.3% butane approximately 20%. plates were in plastic Kodak X-ray Kodak Rapid min. After separation radioactive bands were

tentatively scraped from into 4 of Aqua
tentatively scraped from into 4 of Aquasol (New England Nuclear) converted to a quench Platelet aggregation studies muscle cells cm) were washed twice with (50 pg/ml) medium from effect on platelet to incubate for 21 min; for 30 the pH and returned to pH monolayers were Rabbit platelet-rich blood obtained from of 3-kg White rabbits described (1 PRP were incubated with 0.1-ml for 1 min. Aggregation stirring at 37°C. followed using to a chart prostacyclin and Confluent cultures were prelabeled incubation for containing from to 5 at 37°C. Derivatization procedures and acetate extracts metabolites were and the with diazomethane (1 for 5 carboxylic acid esters. For 6-keto PGFI, of PG12), at 70°C to convert keto groups to the (Pierce, 10 added to convert hydroxyl groups corresponding trimethylsilylethers. diately injected onto the For hydroxy fatty acids, materials were on a of Biosil to 2 acetate 95:5, 80:20, and ethyl acetate-methanol 90: acetate 80:20 fraction containing esters were methanol contain- platinum oxide (PtO) and for 5 resulting reaction then treated with Sylon- (Pierce, 10 convert them to their monotrimethylsilylether derivatives. studies were performed on a electron ion- voltage of 70 e.v. port tempe

rature 240°C for and the carrier Prosta
rature 240°C for and the carrier Prostaglandins were analyzed a 3 SP-2250 (Supelco) tained isothermally at 210°C. Hydroxy fatty acids, in- and 15-HETE on a maintained isothermally for then increased at 10°/min main- tained for Confluent monolayers muscle cells were superfused as described smooth muscle by guest, on January 4, 2021www.jlr.orgDownloaded from In addition cyclooxygenase derivatives, several lipoxygenase compounds of tance. A 5-lipoxygenase macrophages converts arachidonic intermediate, leukotriene to the to the derivatives, leuko- substance of anaphylaxis contractile agents particularly active acid (1 product together yeicosatriaenoic acids epoxy eicosatriaenoic acids fined. A 15-lipoxygenase certain T-lym- 1 2-lipoxygenase pathway and the 1). In activates a synthesis of vasoconstrictive report here a line of muscle cells synthesizes clooxygenase pathway stimuli such as AND METHODS rat aorta muscle cells obtained from were isolated from the of Wistar with col- in 25-sq-cm 75-sq-cm flasks of prostacyclin synthesis following in 10-sq-cm were grown medium buffered HEPES (Fisher) ferred to fetal bovine Bioproducts). Antibiotics for calcium-free (CMF) cells. Cell populations were using a Counter and p

rotein content a modification the proced
rotein content a modification the procedure al. (16). Serum-containing then added quoted into culture subculturing ratio became confluent time they were with [14C]arachidonic removed from washed twice with l-ml portions 25-sq-cm flasks flasks l-'4C]Arachidonic acid (0.75 pCi, 4 pg per 25-sq-cm flask, 10-sq-cm well, 2-sq-cm well. were incubated tubes containing pg each of 6-keto-PGF1,, PGFp,, and 15-HETE volumes of volumes of were collected water. When extracting and the layers were -20" until analyzed. were used hydroxy fatty acids, using of a of ethyl sample appli- were equilibrated water vapor and the phosphomolybdic acid 10°C for different procedures. plates were by guest, on January 4, 2021www.jlr.orgDownloaded from cyclooxygenase pathway in cultured muscle cells of Biochemistry, Washington University School of Medicine, Arachidonic acid that regulate vascular system. in vivo PGIp synthesized Arachidonic acid strictive components of SRSA. 15-HETE, a product rat aorta superfused briefly ['4C]arachidonic acid. were isolated thin-layer chromatography-radioautography, high mance liquid spectrometry. Prostacyclin identified as product both platelet ag- its tetra-trime- derivative. Minor quantities also synthesiz

ed. chromatographic properties acids wer
ed. chromatographic properties acids were also major amounts. indomethacin (10 hydroxy-eicosanoids were isolated scale in- compounds were trimethylsilylether derivatives chromatography-mass spectrometry. pounds were identified as 1-HETE), 15-hydroxy-5,8,1lI simultaneous ion of characteristic ions M/e ratios Rat smooth muscle cells, 24-hour incubation incubation '4C]arachidonic acid, released large enhanced amounts physiological levels to the boxane antagonist prostacyclin, muscle cells significant quantities leukotriene inhibitor 15- cyclooxygenase pathway stimuli such and 15-HETE muscle cells vascular homeostasis.-Bailey, Salata. Characterization 15-HETE, together prostacyclin, as major cyclooxygenase pathway arachidonic acid Arachidonic acid, released cellular phospholip- various stimuli, active compounds including endoperoxide precursor clooxygenase enzyme Thromboxane A*, sensitized lungs and other in response antigenic stimuli, has been substance released an inhibitor let aggregation a potent hydroxy eicosatetraenoic rabbit aorta contracting trihydroxy eicosatetraenoic acid; epoxy eicosatetraenoic acid; cells; ASA, BFT, boron platelet-rich plasma. 24, 1983 by guest, on January 4, 2021www.jlr.orgDownloaded f