sq-23377 and 7-ketocholesterol

Recent studies have demonstrated that, unlike cholesterol, cholesterol oxidized at position 7 can reduce the maximal endothelium-dependent relaxation of isolated rabbit aortas (Circulation. 1997;95:723-731). The aim of the current study was to determine whether cholesterol oxides reduce the release of nitric oxide (NO) from human umbilical vein endothelial cells (HUVECs). The amount of NO released by histamine-stimulated HUVECs was determined by differential pulse amperometry using a nickel porphyrin- and Nafion-coated carbon microfiber electrode. The effects of cholesterol (preserved from oxidation by butylated hydroxytoluene), 7-ketocholesterol, 7beta-hydroxycholesterol, 5alpha,6alpha-epoxycholesterol, 19-hydroxycholesterol (60 microg/mL), and alpha-lysophosphatidylcholine (10 microg/mL) were compared. Pretreatment of HUVECs with cholesterol, 5alpha,6alpha-epoxycholesterol, or 19-hydroxycholesterol did not alter histamine-activated NO production. In contrast, pretreatment with 7-ketocholesterol or 7beta-hydroxycholesterol significantly decreased NO release. The inhibitory effect of 7-ketocholesterol was time and dose dependent and was maintained in the presence of L-arginine. In the absence of serum, lysophosphatidylcholine also reduced NO production. In ionomycin-stimulated cells, pretreatment with 7-ketocholesterol did not inhibit NO release. These results demonstrate that cholesterol derivatives oxidized at the 7 position, the main products of low density lipoprotein oxidation, reduce histamine-activated NO release in HUVECs. Such an inhibitory effect of cholesterol oxides may account, at least in part, for the ability of oxidized low density lipoprotein to reduce the endothelium-dependent relaxation of arteries.

Topics: Arginine; Cells, Cultured; Cholesterol; Endothelium, Vascular; Histamine; Humans; Hydroxycholesterols; Ionomycin; Ketocholesterols; Lysophosphatidylcholines; Nitric Oxide; Umbilical Veins

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca2+ level. Since both Ca2+ variations and protein kinase C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein kinase C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxysterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC50 (4 x 10(-9) M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE2 synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3 beta, 25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca2+ ion fluxes, therefore oxysterols could affect earlier events triggered by serum growth factor binding to their cell membrane receptors.

Topics: Animals; Arachidonic Acid; Blood; Calcitriol; Calcium; Cell Line; Dinoprostone; Hydroxycholesterols; Ionomycin; Ketocholesterols; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate