Elizabeth R. Hall

The effect of shear stress on the uptake and metabolism of arachidonic acid by human endothelial cells

The uptake and metabolism of arachidonic acid (AA) by human umbilical vein endothelial cells was studied for cells in stationary culture and for cells exposed to physiological levels of shear stress. For cells grown in stationary culture, the initial incorporation of arachidonic acid was primarily into diacylglycerol and phospholipids. Cells exposed to flow incorporated labeled arachidonic acid at a similar rate as cells maintained in stationary culture; however, the distribution of the label was altered by flow. The incorporation of arachidonic acid into diacylglycerol and phosphatidylinositol was increased in cells exposed to flow. The largest increase occurred for cells exposed to arterial levels of shear stress for the shortest time period studied, 0.5 h. Prostacyclin (PGI2) and PGF2 alpha were the principal arachidonic acid metabolites formed. Shear stress-stimulated cells preferentially produced PGI2 relative to other eicosanoid products. The initiation of flow caused a burst of AA metabolism which was highly specific for PGI2. This might represent an increase in the turnover of phosphatidylinositol-bound arachidonic acid which is specifically converted to PGI2 as a result of flow-induced membrane stresses.

The stimulation of arachidonic acid metabolism in human platelets by hydrodynamic stresses

Even though shear-induced platelet activation and aggregation have been studied for about 20 years, there remains some controversy concerning the arachidonic acid metabolites formed during stress activation and the role of thromboxane A2 in shear-induced platelet aggregation. In this study, platelets were labelled with [1-14C]arachidonic acid to follow the metabolism of arachidonic acid in stimulated platelets using HPLC and scintillation counting. Platelets activated by thrombin formed principally thromboxane A2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). In contrast, for platelets activated by shear--though arachidonic acid metabolism was stimulated--only 12-HETE was formed and essentially no cyclooxygenase metabolites were detected. This indicates that physical forces may initiate a different pathway for eicosanoid metabolism than most commonly used chemical stimuli and perhaps also implies that regulation of the cyclooxygenase activity may be a secondary level of regulation in eicosanoid metabolism.

The effect of fluid mechanical stress on cellular arachidonic acid metabolism.

The effect of sublytic levels of mechanical perturations of cells on cell metabolism were investigated by analyzing the products of arachidonic acid (used as a marker metabolite) in blood platelets, polymorphonuclear leucocytes, and cultured umbilical-vein endothelial cells after the suspensions of these cells were subjected to a shear stress in a modified viscometer. It is shown that the sublytic levels of mechanical stress stimulated the arachidonic acid metabolism in all these cell types. Possible biological implications of this stress-metabolism coupling are discussed.

Effect of Hemodynamic Shear on Arachidonic Acid Metabolism of Vascular Endothelium

Pathological conditions such as ischemia or postischemic recirculation stimulate the activity of cellular phospholipases, resulting in the release of arachidonic acid (AA) from membrane phospholipids [10, 13]. AA serves as the precursor for several biologically active substances, including prostaglandins and leukotrienes. The accumulation of these AA metabolites in cerebral tissue is believed to promote inflammation, edema formation, and decrease in blood flow.

Monoclonal antibody against TXB2: Its use in solid and liquid phase radirmmunoassays

Monoclonal antibodies to thromboxane B2 (TXB2) have been produced and characterized. Both liquid and solid phase radioimmunoassays have been developed using one of these monoclonal antibodies. The two assays gave similar results when used to quantitate TXB2 in 11 serum samples; however the solid phase assay was more sensitive than the liquid phase assay (i.e., 63 pg/ml vs 19 pg/ml) at a B/BO = 90%). Despite a difference in the sensitivity of the two assay systems, the cross-reactivity of the monoclonal antibody for PGD2, PGE2, PGF2 alpha and 6 keto-PGF1 alpha was the same.

Immobilization of catalytically active thromboxane synthase.

Thromboxane synthase has been immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme is catalytically active and has a slightly lower apparent Km for PGH2 than the detergent-solubilized enzyme. However, both imidazole- and pyridine-based inhibitors are equally effective in inhibiting the immobilized and solubilized enzyme preparations. Although the immobilized enzyme appears to be less stable than the solubilized enzyme it is sufficiently stable to be used as a model for studying the properties of the enzyme.

Substrate inactivation of lung thromboxane synthase preferentially decreases thromboxane A2 production

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The production of both products was inhibited by 1-benzylimidazole and furegrelate. Multiple additions of PGH2 dramatically reduced the ability of the enzyme to synthesize TXA2, but did not effect the synthesis of HHT. In addition, 1-benzylimidazole did not protect thromboxane synthase from inactivation with multiple additions of PGH2. When the enzyme was incubated with PGH2 in the presence of 1-benzylimidazole, the synthesis of TXA2 was inhibited. When the inhibitor was removed the enzyme had still been inactivated by PGH2 in the presence of 1-benzylimidazole. Thus the substrate inactivation of the enzyme does not require the production of TXA2. Our data suggests that the synthesis of TXA2 and HHT can be differentially inactivated and may occur at different sites on the enzyme.

Preparation of prostaglandin H2: extended purification/analysis scheme.

Publisher Summary The chapter presents a study on preparation of prostaglandin H 2 (PGH 2 ), discussing the extended purification/analysis scheme. PGH 2 is routinely prepared from ram seminal vesicles and isolated by gravity flow silicic acid chromatography. The chapter mentions the procedures that produce PGH 2 , which is contaminated with small amounts of HHT, HETEs, PGF 2α , PGE 2 , PGD 2 , and other unidentified nonarachidonic acid substances, and which has a purity that rarely exceeds 80%. The bonded-phase rechromatography methods described in the chapter provides homogeneous PGH 2 as assayed by high-performance liquid chromatography (HPLC) and ammonia, direct chemical ionization-mass spectrometry (DCI-MS). A fundamental limit to any technique used in the purification of PGH 2 resides in the reactivity of this molecule with protic solvents and silica-based adsorbents (presumably via the acidic Si-OH bond). A study evaluating various chromatography systems and solvents has shown that cyano-bonded, stationary-phase columns eluted with a hexane-2-propanol gradient is an effective means for further purification of PGH 2 .2-Propanol, used as the polar organic modifier in the mobile phase, appears to cause less PGH2 degradation than other alcohols that were tested. It is found that ammonia DCI-MS to be a particularly useful method for directly analyzing prostaglandin preparations, including those containing the labile PGH 2 molecule.

Generation of Monoclonal Antibodies to Prostaglandins and Their Potential Use in Detecting PGH Synthase Activity

Abstract Eicosanoids, both prostaglandins and leukotrienes, have been implicated as mediators in a number of physiological processes. Various tissues have been found to produce different types and quantities of eicosanoids. Tissues also differ in their eicosanoid profiles when the eicosanoids are produced under different conditions. The total amount of prostaglandins formed in response to cellular stimuli depends upon the release of arachidonic acid and its metabolism by PGH synthase (cyclooxygenase). Currently available assays for PGH synthase activity are too expensive, cumbersome, and insensitive to be used in screening a large number of samples for enzyme activity. The most sensitive assays available for prostaglandin detection are radioimmunoassays for specific prostaglandins. We discuss in this article the development of radioimmunoassays using monoclonal antibodies, both specific and pan-specific, for recognition of the prostaglandins.

Inactivation of thromboxane synthase with hydroperoxy-fatty acids

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The structure-activity relationship of several hydroperoxy fatty acids and their ability to inactivate thromboxane synthase was investigated. Millimolar quantities of hydrogen peroxide and tert-butylperoxide were required to inactivate the enzyme: whereas micromolar quantities of C18 and C20 hydroperoxy fatty acids inactivated the enzyme. Pretreatment of the enzyme with long chain hydroperoxy-fatty acids resulted in a decreased synthesis of both TXB2 and HHT.