Characteristics of arachidonic acid metabolism of human endothelial cells in culture

The subject of this work is to examine the hypothesis that some sublytic levels of mechanical perturbation of cells can stimulate cell metabolism. As a marker metabolite, we have chosen arachidonic acid. Principal metabolites for platelets include the cyclooxygenase product thromboxane A2(TXA2) and the lipoxygenase product 12-hydroperoxy-eicosatetraenoic acid (12-HPETE). Polymorphonuclear leukocytes (PMNLs) initally produce principally 5-HPETE, somtimes leading to the formation leukotrienes, though many other metabolites of arachidonic acid have been isolated from activated neutrophils. Human umbilical vein endothelial cells utilize arachidonic acid to produce mainly prostaglandin I2(PGI2). All of these metabolites are biologically active and modulate cell function - sometimes in quite contrasting ways. We will show that levels of sublytic mechanical stress exposure can stimulate arachidonic acid metabolism in all three of the cell types mentioned above. The biological implications of this stress/metabolism coupling may be quite far reaching.Human platelets, leukocytes and endothelial cells all appear to be sensitive to mechanical stress induced activation of arachidonic acid metabolism. Sheared PRP exhibited greatly increased synthesis of 12-HETE and surprisingly little thromboxane B2 production. This indicates that shear stress stimulation of platelets may produce quite different arachidonic acid metabolism than that seen with many direct chemical stimuli, such as thrombin or collagen.Our data demonstrate that a substance derived from shear induced platelet activation may activate the C-5 lipoxygenase of human PMNL under stress, leading to the production of LTB4. We hypothesize that this substance maybe 12-HPETE. LTB4 is known to be a very potent chemotactic factor and to induce PMNL aggregation and degranulation. Our studies provide further evidence that lipoxygenase products of one cell type can modulate production of lipoxygenase products in a second cell type, and that shear stress can initiate cell activation. This kind of coupling could have far reaching implications in terms of our understanding of cell/cell interaction in flowing systems, such as acute inflammation, artificial organ implantation and tumor metastasis.The data on PGI2 production by endothelial cells demonstrate that physiological levels of shear stress can dramatically increase arachidonic acid metabolism. Step increases in shear stress lead to a burst in production of PGI2 which decayed to a steady state value in several minutes. This longer term stimulation of prostacyclin production rate increased linearly with shear stress over the range of 0-24 dynes/cm2. In addition, pulsatile flow of physiological frequency and amplitude caused approximately 2.4 times the PGI2 production rate as steady flow with the same mean stress. Although only PGI2 was measured, it is likely that other arachidonic acid metabolites of endothelial cells are also affected by shear stress.The ability of cells to respond to external stimuli involves the transduction of a signal across the plasma membrane. One such external stimulus appears to be fluid shear stress. Steady shear flow induces cell rotation in suspended cells, leading to a periodic membrane loading, with the peak stress proportional to the bulk shear stress. On anchorage-dependent cells, such as endothelial cells, steady shear stress may act by amplifying the natural thermal or Brownian fluttering or rippling of the membrane. There are several possible mechanisms by which shear stress induced membrane perturbation could mimic a hormone/receptor interaction, leading to increased intracellular metabolism. Shear stress may induce increased phospholipase C activity, caused by translocation of the enzyme, increased substrate (arachidonic acid) pool availability to phospholipase C (particularly from that stored in phosphoinositols) due to shear-induced membrane movements or changes in membrane fluidity, direct activation of calcium - activated phospholipase A2 by increased membrane calcium ion permeability, or most probably by a combination of these mechanisms.

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Regulation of synthesis of prostacyclin and HETEs in human endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1989.256.6.c1168 ◽

1989 ◽

Vol 256 (6) ◽

pp. C1168-C1175 ◽

Cited By ~ 37

Author(s):

B. O. Ibe ◽

J. R. Falck ◽

A. R. Johnson ◽

W. B. Campbell

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Caffeic Acid ◽

Inhibitory Concentration ◽

Nordihydroguaiaretic Acid ◽

Hydroxyeicosatetraenoic Acid ◽

Human Endothelial Cells ◽

Lipoxygenase Pathway ◽

Cells In Culture ◽

Regulation Of Synthesis

Human umbilical endothelial cells in culture synthesize prostacyclin (PGI2), 15-hydroxyeicosatetraenoic acid (15-HETE), and 12-hydroxyeicosatetraenoic acid (12-HETE). The synthesis of these eicosanoids was measured by specific radioimmunoassays after stimulation by arachidonic acid, A23187, bradykinin, melittin, or histamine. Under all conditions, the synthesis of PGI2 paralleled and exceeded the synthesis of 15-HETE and 12-HETE. Indomethacin inhibited arachidonic acid-stimulated PGI2 and 15-HETE synthesis but enhanced 12-HETE synthesis. Meclofenamate gave similar qualitative results. Drugs that act as inhibitors of lipoxygenase in some tissues, such as nordihydroguaiaretic acid (NDGA), caffeic acid, esculin, diethylcarbamazine, quercetin, and 5,8,11,14-eicosatetrayenoic acid (ETYA) were nonspecific in their inhibition of PGI2, 12-HETE, and 15-HETE synthesis. For example, NDGA inhibited arachidonic acid-stimulated release with a 50% inhibitory concentration (IC50) of 0.39 microM for PGI2, 0.25 microM for 15-HETE, and 0.10 microM for 12-HETE. These results show that endothelial cells metabolize both endogenous and exogenous arachidonic acid to PGI2, 15-HETE, and 12-HETE. These data also suggest, based on results with inhibitors, that PGI2 and 15-HETE are products of cyclooxygenase, whereas 12-HETE is produced via a different enzymatic pathway, most likely a lipoxygenase pathway.

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