Effects of antioxidants apocynin and the natural water-soluble antioxidant from spinach on cellular damage induced by lipopolysaccaride in the rat.
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Oxidative damage plays a key role in septic shock induced by the endotoxin lipopolysaccaride (LPS) by enhancing the formation of reactive oxygen species such as superoxide anion radicals, peroxides, and their secondary product, malondialdehyde, especially in the liver. In this study, histopathologic changes in several organs were compared among groups of male Wistar rats that had been injected with LPS following prophylactic pretreatment with either of 2 antioxidants, a group that had been injected with LPS without pretreatment with antioxidants, an untreated control group, and groups that had been injected with either of the 2 antioxidants only. The antioxidants used were a water-soluble natural antioxidant from spinach (NAO) and the NADPH oxidase inhibitor apocynin. Hematoxylin-and-eosin-stained slides were prepared, and lesions were semiquantitatively scored. Exposure to LPS alone was associated with multifocal hepatocellular necrosis and acute inflammation, thymic and splenic lymphoid necrosis, ocular retinal hemorrhage and acute endophthalmitis, adrenal medullary vacuolation and necrosis and acute inflammation, and decreased adrenal cortical cytoplasmic vacuolation (consistent with depletion of steroidal hormone contents). Results indicated that pretreatment with both antioxidants for 8 days reduced, in some organs, the necrotic and inflammatory changes associated with the LPS challenge. These findings suggest a potential therapeutic application for these antioxidants in clinical sepsis. (+info)
The prophylactic effects of natural water-soluble antioxidant from spinach and apocynin in a rabbit model of lipopolysaccharide-induced endotoxemia.
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Radical-scavenging antioxidants, as part of the cellular defense system, function to inhibit the formation and propagation of free radicals and active oxygen species formation. In previous studies we demonstrated that endotoxin lipopolysaccharide (LPS) promotes oxidative stress and associated pathological changes in a rat model and that use of selected antioxidants was effective in reducing LPS-related lipid peroxidation product formation in the liver, as well as LPS-related pathological changes in different organs. In this study, several toxicological parameters (ie, clinical signs, blood chemistry, and histopathological changes) were compared among groups of male New Zealand rabbits injected with LPS following prophylactic pretreatment with either of 2 antioxidants, a group injected with LPS without pretreatment with antioxidants, groups injected with either of the 2 antioxidants only, and an untreated control group. The antioxidants used were a water-soluble natural antioxidant (NAO) from spinach and the NADPH oxidase inhibitor, apocynin. Exposure to LPS alone was associated clinically with depression, tachypnea, outer ear vasodilation, and iris congestion; biochemically with a significant increase in blood total bilirubin, transaminase activity, and glucose, total cholesterol, and triglyceride levels; macroscopically with multiple whitish areas in the liver; and histologically with hepatocellular focal necrosis and acute inflammation, thymic and splenic lymphoid necrosis and depletion, acute uveitis and hemorrhages in the ciliary processes, and decreased adrenal cortical cytoplasmic vacuolation considered consistent with depletion of steroidal hormone contents. The NAO had more effective prophylactic capacities than the apocynin. The protective effects were obvious in all investigated parameters. The results indicate the possible therapeutic efficacy of NAO in the treatment of clinical endotoxemia associated with gram-negative bacterial sepsis that is known to be associated with oxidative stress. (+info)
Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells.
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To characterize the volume-sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [(3)H]-taurine efflux from acutely isolated SON. Taurine release induced by hypotonic stimulus (250 mosmol l(-1)) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter. The osmodependent release of taurine was blocked by a variety of Cl(-) channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4-bromophenacyl bromide, mibefradil) known to block volume-activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. Although the molecular identity of volume-sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC(3), phospholemman and VDAC(1), and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC(2). In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume-sensitive anion channel, which is likely to be formed by yet unidentified proteins. (+info)
Inhibitors of prostaglandin synthesis inhibit human prostate tumor cell invasiveness and reduce the release of matrix metalloproteinases.
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Eicosanoids modulate the interaction of tumor cells with various host components in cancer metastasis. Their synthesis involves the release of arachidonic acid (AA) from cellular phospholipids by phospholipase A2 (PLA2), followed by metabolism by cyclooxygenases (COXs) and lipooxygenases (LOXs). This study aimed to identify the pathway(s) of AA metabolism that are required for the invasion of prostate tumor cells. DU-145 and PC-3 human prostate cancer cell lines were used to test the effect of inhibitors of PLA2, COX, or LOX on the invasion of prostate tumor cells through Matrigel in vitro using the Boyden chamber assay and fibroblast-conditioned medium as the chemoattractant. We used nontoxic doses that did not inhibit simple cell motility and did not decrease clonogenic survival. All of the inhibitors caused a significant reduction in AA release from treated cells compared with control cells, which indicated that the treatments were biochemically active. Invasion through Matrigel was inhibited by the PLA2 inhibitor 4-bromophenacyl bromide (4-BPB), the general COX inhibitor ibuprofen (IB), and the highly selective COX-2 inhibitor NS398. Inhibition of cell invasiveness by 4-BPB (1.0 microM), IB (10.0 microM), and NS398 (10.0 microM) was reversed by the addition of prostaglandin E2 (PGE2). PGE2 alone, however, did not stimulate invasiveness, which suggests that its production is necessary for rendering the cells invasive-permissive but not sufficient for inducing invasiveness. In contrast, we found no significant inhibition of invasion of prostate tumor cells treated with esculetin (1.0 microM) or nordihydroguiaretic acid (1.0 microM), which are specific inhibitors of LOX. We also tested the effect of 4-BPB, IB, NS398, and esculetin on the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), as key enzymes in the proteolysis of Matrigel during invasion, using gelatin zymograms and Western blots. Cells that received 4-BPB, IB, or NS398, but not esculetin showed a significant reduction in the levels of proMMP-2, MMP-9, and proMMP-9 in the culture medium. DU-145 cells did not secrete TIMP-1, and the drugs did not alter the secretion of TIMP-2. This work highlights the role played by COX in disturbing the balance between MMPs and TIMPs in prostate cancer cells, and it points to the potential use of COX inibitors, especially COX-2 selective inhibitors, in the prevention and therapy of prostate cancer invasion. (+info)
Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus.
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Our studies were conducted to explore the role of hepatic fatty acid-binding protein (L-FABP) in fatty acid transport to the nucleus. Purified rat L-FABP facilitated the specific interaction of [(3)H]oleic acid with the nuclei. L-FABP complexed with unlabeled oleic acid decreased the nuclear association of [(3)H]oleic acid:L-FABP; however, oleic acid-saturated bovine serum albumin (BSA) or fatty acid-free L-FABP did not. The peroxisome-proliferating agents LY171883, bezafibrate, and WY-14,643 were also effective competitors when complexed to L-FABP. Nuclease treatment did not affect the nuclear association of [(3)H]oleic acid:L-FABP; however, proteinase treatment of the nuclei abolished the binding. Nuclei incubated with fluorescein-conjugated L-FABP in the presence of oleic acid were highly fluorescent whereas no fluorescence was observed in reactions lacking oleic acid, suggesting that L-FABP itself was binding to the nuclei. The nuclear binding of FABP was concentration dependent, saturable, and competitive. LY189585, a ligand for L-FABP, also facilitated the nuclear binding of fluorescein-conjugated L-FABP, although it was less potent than oleic acid. A structural analog that does not bind L-FABP, LY163443, was relatively inactive in stimulating the nuclear binding. Potential interactions between L-FABP and nuclear proteins were analyzed by Far-Western blotting and identified a 33-kDa protein in the 500 mm NaCl extract of rat hepatocyte nuclei that bound strongly to biotinylated L-FABP. Oleic acid enhanced the interaction of L-FABP with the 33-kDa protein as well as other nuclear proteins. We propose that L-FABP is involved in communicating the state of fatty acid metabolism from the cytosol to the nucleus through an interaction with lipid mediators that are involved in nuclear signal transduction. (+info)
Specificity and mechanism of action of some commonly used protein kinase inhibitors.
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The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays. (+info)
Permissive role of protein kinase C alpha but not protein kinase C delta in sphingosine 1-phosphate-induced Rho A activation in C2C12 myoblasts.
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Rho GTPases participate in various important signaling pathways and have been implicated in myogenic differentiation. Here the first evidence is provided that in C2C12 myoblasts sphingosine 1-phosphate (SPP) rapidly and transiently induced membrane association of Rho A in a pertussis toxin-insensitive manner. The bioactive lipid preferentially relocalized the GTPase to Golgi-enriched membrane. Translocation of Rho A was abolished by inhibition or down-regulation of protein kinase C (PKC). Notably, treatment with Go6976, an inhibitor of conventional PKCs, which selectively blocked PKC alpha in these cells, prevented SPP-induced Rho A translocation. Conversely rottlerin, a selective inhibitor of PKC delta, was without effect, demonstrating that SPP signaling to Rho A involves PKC alpha but not PKC delta activation. This novel functional relationship between the two proteins may have a role in SPP-mediated regulation of downstream effectors. (+info)
Peroxisome proliferator-activated receptor alpha activates transcription of the brown fat uncoupling protein-1 gene. A link between regulation of the thermogenic and lipid oxidation pathways in the brown fat cell.
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High expression of the peroxisome proliferator-activated receptor alpha (PPARalpha) differentiates brown fat from white, and is related to its high capacity of lipid oxidation. We analyzed the effects of PPARalpha activation on expression of the brown fat-specific uncoupling protein-1 (ucp-1) gene. Activators of PPARalpha increased UCP-1 mRNA levels severalfold both in primary brown adipocytes and in brown fat in vivo. Transient transfection assays indicated that the (-4551)UCP1-CAT construct, containing the 5'-regulatory region of the rat ucp-1 gene, was activated by PPARalpha co-transfection in a dose-dependent manner and this activation was potentiated by Wy 14,643 and retinoid X receptor alpha. The coactivators CBP and PPARgamma-coactivator-1 (PGC-1), which is highly expressed in brown fat, also enhanced the PPARalpha-dependent regulation of the ucp-1 gene. Deletion and point-mutation mapping analysis indicated that the PPARalpha-responsive element was located in the upstream enhancer region of the ucp-1 gene. This -2485/-2458 element bound PPARalpha and PPARgamma from brown fat nuclei. Moreover, this element behaved as a promiscuous responsive site to either PPARalpha or PPARgamma activation, and we propose that it mediates ucp-1 gene up-regulation associated with adipogenic differentiation (via PPARgamma) or in coordination with gene expression for the fatty acid oxidation machinery required for active thermogenesis (via PPARalpha). (+info)