Incorporation of arachidonic and linoleic acid hydroperoxides into cultured human umbilical vein endothelial cells.

The current study assessed the differential incorporation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (12-HETE) and the linoleic acid (LA) oxidation products, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), into human umbilical vein endothelial cells (HUVEC). Approximately 80-90% of AA (10(-8)-10(-5)M) and 80% of LA (10(-8)-10(-5)M) were incorporated into HUVEC within 12h, while less than 50% of the hydroxy metabolites (12-HETE, 12-HPETE, 13-HODE, 13-HPODE) were incorporated into HUVEC over 48h. Further, treatment of HUVEC with either 12-HPETE or 13-HPODE (concentrations of 10(-5)M) had no effect on cell number at a 48h time point when compared with control. These results demonstrate that exogeneous hydroxy metabolites are incorporated into HUVEC to a lesser degree than were endogenous fatty acids. Further, we speculate that 12-HPETE and 13-HPODE are rapidly metabolized to substances without significant cytotoxic effects.

Respective role of lipoxygenase and nitric oxide-synthase pathways in plasma histamine-induced macromolecular leakage in conscious hamsters.

1. Intravital microscopy technique was used to determine the distribution of a fluorescent plasma marker (fluorescein-isothiocyanate-dextran, 150 kD; FD-150) into venular and interstitial compartments of dorsal skin fold preparations in conscious hamsters. 2. One mg kg(-1) histamine (i.v.) caused a biphasic decrease in venular fluorescence due to FD-150 extravasation in all organs (general extravasation). Immediately after injection, the venular fluorescence decreased and plateaued in 60 min. Ninety minutes after histamine injection, venular fluorescence further decreased until 180 min. Prior treatment with indomethacin (0.1 mg kg(-1), i.v.) did not modify the time-course of general extravasation but prevented histamine-induced venule dilatation. 3. Prior treatment with the 5-lipoxygenase activating protein (FLAP) inhibitor, 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-d imethyl-propanoic acid sodium (MK-886)(10 microg kg(-1), i.v.), the leukotriene receptor antagonist, benzenemethanol a-pentyl-3-(2-quinolinylmethoxy) (REV-5901)(1 mg kg(-1), i.v.), or the glutathione-S-transferase inhibitor, ethacrynic acid (1 mg kg(-1), i.v.), delayed by 60 min the onset of general extravasation caused by 1 mg kg(-1) histamine. 4. Prior treatment with lipoxygenase pathway inhibitors and N(G)-nitro-L-arginine-methylester (L-NAME)(100 mg kg(-1), i.v.) abolished the general extravasation and venule dilatation induced by 1 mg kg(-1) histamine. 5. Injection of 1 microg kg(-1) (i.v.), of leukotriene-C4 (LTC4) or -D4 (LTD4) induced immediate and sustained general extravasation and reduction in venule diameter, these effects being blocked by REV-5901. 6. Histamine (1 mg kg(-1), i.v.) induced biphasic decline in mean arterial blood pressure (MAP). An initial phase (from 0 to 60 min) was followed by a late phase beginning 90 min after histamine injection. L-NAME (100 mg kg(-1), i.v.) and aminoguanidine (1 mg kg(-1), i.v.) prevented the late phase of histamine-induced hypotension. 7. Thus, plasma histamine can trigger both an immediate cysteinyl-leukotriene (Cys-LT)-dependent and a late nitric oxide (NO)-mediated inflammatory cascade. Although the cyclo-oxygenase (COX) pathway might account for histamine-induced venule dilatation, it would not influence histamine-induced extravasation.

Evaluation of anti-inflammatory potential of fixed oil of Ocimum sanctum (Holybasil) and its possible mechanism of action.

The fixed oil of Ocimum sanctum (Labiatae) was found to possess significant anti-inflammatory activity against carrageenan- and different other mediator-induced paw edema in rats. Significant inhibitory effect was also observed in castor oil-induced diarrhoea in rats. It also inhibited arachidonic acid and leukotriene-induced paw edema. The results of anti-inflammatory activity of Ocimum sanctum support the dual inhibition of arachidonate metabolism as indicated by its activity in inflammation models that are insensitive to selective cyclooxygenase inhibitors. On the basis of the findings it may be inferred that Ocimum sanctum may be a useful anti-inflammatory agent which blocks both the pathways, i.e. cyclooxygenase and lipoxygenase, or arachidonic acid metabolism.

Overexpression of phospholipid hydroperoxide glutathione peroxidase suppressed cell death due to oxidative damage in rat basophile leukemia cells (RBL-2H3).

The role of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the cellular defense against oxidative stress was investigated in a novel cell model. We isolated stable transfectants of RBL-2H3 cells that overexpressed PHGPx. The activity of PHGPx in RBL2H3 cells that had been transfected with the 761bp cDNA for rat PHGPx (RPHGPx2) that we had cloned previously was 3.8 times higher than that in parent cells and in cells that had been mock-transfected with the vector without a cDNA insert. Cells that overexpressed PHGPx were three times more resistant than parent cells and mock-transfected cells to the cytotoxic effects of an radical initiator (AAPH) that induced the oxidative stress. This resistance to damage by AAPH of cells that overexpressed PHGPx was not observed after pretreatment with buthionine sulfoximine (BSO), an inhibitor of the synthesis of glutathione. Overexpression of PHGPx could suppress the peroxidation of membrane lipids and, in particular, the production of phosphatidylcholine hydroperoxide by AAPH in RBL2H3 cells. PHGPx was also able to prevent cell death in response to extracellular attack by a lipid peroxide. This is the first report to indicate directly that PHGPx can scavenge phosphatidylcholine hydroperoxide, which induces oxidative damage at the cellular level.

cAMP-specific phosphodiesterase inhibitor, rolipram, reduces eosinophil infiltration evoked by leukotrienes or by histamine in guinea pig conjunctiva.

The effect of rolipram, an isozyme IV-selective inhibitor of cAMP-specific phosphodiesterase, was evaluated in a guinea pig eye model of tissue eosinophilia. (R)-rolipram was administered by gavage to guinea pigs 1 h prior to topical ocular challenge with a mixture of leukotrienes (LTs) (10 ng LTB4 + 1000 ng LTD4/eye) or with histamine dihydrochloride (1 mg/eye). Conjunctivae were evaluated histologically 6 h after challenge. Eosinophil counts per millimeter of conjunctival epithelium in LT-challenged animals that received (R)-rolipram at dosages of 0.1, 0.3, 1, 3, or 10 mg/kg were reduced by 63, 63, 84, 81 and 90% respectively, compared to LT-challenged controls. Reduction was statistically significant (P < 0.05) at all dosages. Eosinophil counts per millimeter of epithelium in histamine-challenged animals that received 10 mg/kg (R)-rolipram were reduced by 79% compared to histamine-challenged controls (P < 0.01). The results indicate that (R)-rolipram inhibits the response to two distinct classes of mediator in this model of eosinophil infiltration, adding support to the contention that isozyme IV-selective cAMP phosphodiesterase inhibitors offer therapeutic potential for human asthma.

Prostacyclin does not play any cytoprotective role in endothelial cell injury induced by 15-hydroperoxy-eicosatetraenoic acid, an arachidonate lipoxygenase product.

Among various arachidonic acid metabolites examined, only 15(S)-hydroxperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), a lipoxygenase product, caused a time- and dose-dependent injury to bovine endothelial cells in culture. There also occurred a significant inhibition of endothelial prostacyclin (PGI2) production due to 15-HPETE. But there were obvious dissociations in time course and dose dependence between 15-HPETE-induced cellular injury and 15-HPETE-induced inhibition of PGI2 synthesis. In addition, the cytotoxicity of 15-HPETE was not aggravated even when the endothelial monolayers were pretreated with several inhibitors of PGI2 synthesis. Also, some stable analogues of PGI2 had no protective effect on the injury. These results suggest that the reduced production of PGI2 caused by 15-HPETE is not directly associated with the onset of cellular injury, and that PGI2 does not play any cytoprotective role in endothelial cell injury induced by at least such lipid peroxides as 15-HPETE.

Comparison of the damage-promoting effects of leukotrienes derived from eicosapentaenoic acid and arachidonic acid on the rat stomach.

The ability of leukotrienes derived from eicosapentaenoic acid were compared with counterpart leukotrienes derived from arachidonic acid in terms of their ability to affect susceptibility of the stomach to injury induced by a topical irritant and their ability to alter gastric blood flow. Intra-arterial infusion of leukotriene C4 (LTC4) and LTD4 (0.1-3 micrograms/kg/min for 5 min) produced dose-dependent increases in gastric mucosal damage induced by topically applied 20% ethanol, as assessed macroscopically, by changes in transmucosal potential difference and by measurement of efflux of protein into the gastric lumen. Similar doses of LTC5 or LTD5 did not produce significant changes in any of these three parameters, when compared with control rats receiving the vehicle. With a higher dose of LTC5 or LTD5 (5 micrograms/kg/min), significant damage was observed. LTC4 and LTD4 were also found to be more potent at reducing gastric blood flow than LTC5 and LTD5. These results demonstrate that the peptido-leukotrienes derived from eicosapentaenoic acid (LTC5 and LTD5) are on the order of five times less potent than the leukotrienes derived from arachidonic acid (LTC4 and LTD4), in terms of increasing the susceptibility of the gastric mucosa to damage and reducing gastric blood flow. These results may have important implications in terms of the hypothesis that fish oil diets may be protective or may accelerate healing in ulcerative diseases of the gastrointestinal tract.

Leukotriene B4 potentiates colonic ulceration in the rat.

The ability of various leukotrienes, platelet-activating factor and N-formyl-methyl-leucyl-phenylalanine to augment colonic damage induced by 30% ethanol was investigated in the rat. Each of the mediators was tested at a dose of 2 nmol, administered intracolonically in the ethanol vehicle. When colonic damage was assessed 72 hr later, only leukotriene B4 significantly augmented damage compared to the controls. The incidence of ulcers increased from 35% in the control group to 90% in the group receiving leukotriene B4. Leukotriene B4 administration also resulted in significant increases in colonic myeloperoxidase activity and colonic leukotriene B4 synthesis. To assess the possible contribution of infiltrating neutrophils to the increase in colonic leukotriene B4 synthesis that accompanies colonic inflammation, colitis was induced in normal and neutropenic rats by intracolonic administration of trinitrobenzene sulfonic acid. Neutropenia was achieved by treatment with an antineutrophil serum. In the neutropenic animals killed 4 hr after induction of colitis significant changes in leukotriene B4 synthesis were not observed, whereas a fourfold increase was observed in the controls. From these studies we conclude the following: (1) leukotriene B4, at a dose of 2 nmol, can significantly potentiate the colonic ulceration induced by 30% ethanol; (2) this action of leukotriene B4 is not shared by the same dose of the other inflammatory mediators tested; and (3) infiltrating neutrophils are the major source of colonic leukotriene B4 synthesis in a rat model of colitis.

The control of vascular endothelial cell injury.

The mechanism by which MCI-186 showed a potent cytoprotective effect on the in vitro endothelial cell injury due to 15-HPETE was studied. Stimulation of human leukocytes with various chemical mediators such as TPA, f-Met-Leu-Phe, LTB4, etc. elicited the production of active oxygens, which could be detected by luminol-dependent chemiluminescence. Among the chemical mediators tested, TPA elicited the chemiluminescence the most, and f-Met-Leu-Phe and LTB4 came next. When the leukocytes were directly placed on a monolayer of cultured endothelial cells, followed by stimulating the leukocytes with TPA, severe endothelial cell injury was observed. The effect of TPA was dose dependent. There was good correlation between the active oxygen releasing activity and the cytotoxic activity. When the leukocytes were placed on a filter which was set apart from the monolayer of endothelial cell in a culture dish, and stimulated the leukocytes with TPA, no cytotoxicity was observed. These data strongly suggest that the substance responsible for the cytotoxicity must be a very labile and short-lived substance, presumably active oxygens. On the other hand, MCI-186 was found to have a complete quenching activity to the chemiluminescence due to active oxygens in the TPA-leukocyte system. Taken together, these factors indicate that the potent cytoprotective effect of MCI-186 may be due to its specific radical scavenging activity.

Preventive effect of MCI-186 on 15-HPETE induced vascular endothelial cell injury in vitro.

Using cultured bovine aortic endothelial cells, the effects of MCI-186, a radical scavenger, were studied on arachidonic acid metabolism and on the cell injury caused by 15-HPETE. MCI-186 at 3 X 10(-5) M enhanced prostacyclin production in the intact endothelial cells without affecting phospholipase A2. When endothelial cell homogenates were used as an enzyme source, it was found that MCI-186 stimulated the conversion of arachidonic acid to prostacyclin like phenol, perhaps by trapping OH radicals produced in the process of the conversion of PGG2 to PGH2. On the other hand, MCI-186 was found to inhibit lipoxygenase metabolism of arachidonic acid in cell free homogenates of rat basophilic leukemia cells. The lipoxygenase inhibition caused by 3 X 10(-5) M MCI-186 was almost equivalent to that caused by 3 X 10(-6) M BW 755C. MCI-186 remarkably protected against endothelial cell damage caused by 15-HPETE. 3 X 10(-5) M of 15-HPETE caused endothelial cell death in about 60% of the population: however, pretreatment of the cells with 10(-5) M of MCI-186 or concomitant addition of 10(-5) M of MCI-186 with 15-HPETE to the cultures prevented the cell death completely. These results suggest that MCI-186 may become an unique anti-ischemic drug.