Effects of inhibitors of the lipo-oxygenase family of enzymes on the store-operated calcium current I(CRAC) in rat basophilic leukaemia cells.
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In non-excitable cells, the major Ca2+ entry pathway is the store-operated pathway in which emptying of intracellular Ca2+ stores activates Ca2+ channels in the plasma membrane. In many cell types, store-operated influx gives rise to a Ca2+-selective current called I(CRAC) (Ca2+ release-activated Ca2+ current). Using both the whole-cell patch clamp technique to measure I(CRAC) directly and fluorescent Ca2+ imaging, we have examined the role of the lipo-oxygenase pathway in the activation of store-operated Ca2+ entry in the RBL-1 rat basophilic leukaemia cell-line. Pretreatment with a variety of structurally distinct lipo-oxygenase inhibitors all reduced the extent of I(CRAC), whereas inhibition of the cyclo-oxygenase enzymes was without effect. The inhibition was still seen in the presence of the broad protein kinase blocker staurosporine, or when Na+ was used as the charge carrier through CRAC channels. The lipo-oxygenase blockers released Ca2+ from intracellular stores but this was not associated with subsequent Ca2+ entry. Lipo-oxygenase blockers also reduced both the amount of Ca2+ that could subsequently be released by the combination of thapsigargin and ionomycin in Ca2+-free solution and the Ca2+ influx component that occurred when external Ca2+ was re-admitted. The inhibitors were much less effective if applied after I(CRAC) had been activated. This inhibition of I(CRAC) could not be rescued by dialysis with 5(S)-hydroxyperoxyeicosa-6E,8Z,11Z,14Z,tetraenoic acid (5-HPETE), the first product of the 5-lipo-oxygenase pathway. Our findings indicate that exposure to pharmacological tools that inhibit the lipo-oxygenase enzymes all decrease the extent of activation of the current. Our results raise the possibility that a lipo-oxygenase might be involved in the activation of I(CRAC). Alternative explanations are also discussed. (+info)
Caffeic acid phenethyl ester and curcumin: a novel class of heme oxygenase-1 inducers.
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Heme oxygenase-1 (HO-1) is a redox-sensitive inducible protein that provides efficient cytoprotection against oxidative stress. Curcumin, a polyphenolic natural compound that possesses anti-tumor and anti-inflammatory properties, has been reported recently to induce potently HO-1 expression in vascular endothelial cells (Free Rad Biol Med 28:1303-1312, 2000). Here, we extend our previous findings by showing that caffeic acid phenethyl ester (CAPE), another plant-derived phenolic agent, markedly increases heme oxygenase activity and HO-1 protein in astrocytes. The effect seems to be related to the peculiar chemical structures of curcumin and CAPE, because analogous antioxidants containing only portions of these two molecules were totally ineffective. At a final concentration of 30 microM, both curcumin and CAPE maximally up-regulated heme oxygenase activity while promoting marked cytotoxicity at higher concentrations (50-100 microM). Similar results were obtained with Curcumin-95, a mixture of curcuminoids commonly used as a dietary supplement. Incubation of astrocytes with curcumin or CAPE at concentrations that promoted maximal heme oxygenase activity resulted in an early increase in reduced glutathione followed by a significant elevation in oxidized glutathione contents. A curcumin-mediated increase in heme oxygenase activity was not affected by the glutathione precursor and thiol donor N-acetyl-L-cysteine. These data suggest that regulation of HO-1 expression by polyphenolic compounds is evoked by a distinctive mechanism which is not necessarily linked to changes in glutathione but might depend on redox signals sustained by specific and targeted sulfydryl groups. This study identifies a novel class of natural substances that could be used for therapeutic purposes as potent inducers of HO-1 in the protection of tissues against inflammatory and neurodegenerative conditions. (+info)
Chemiosmotic energy conservation with Na(+) as the coupling ion during hydrogen-dependent caffeate reduction by Acetobacterium woodii.
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Cell suspensions of Acetobacterium woodii prepared from cultures grown on fructose plus caffeate catalyzed caffeate reduction with electrons derived from molecular hydrogen. Hydrogen-dependent caffeate reduction was strictly Na(+) dependent with a K(m) for Na(+) of 0.38 mM; Li(+) could substitute for Na(+). The sodium ionophore ETH2120, but not protonophores, stimulated hydrogen-dependent caffeate reduction by 280%, indicating that caffeate reduction is coupled to the buildup of a membrane potential generated by primary Na(+) extrusion. Caffeate reduction was coupled to the synthesis of ATP, and again, ATP synthesis coupled to hydrogen-dependent caffeate reduction was strictly Na(+) dependent and abolished by ETH2120, but not by protonophores, indicating the involvement of a transmembrane Na(+) gradient in ATP synthesis. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) abolished ATP synthesis, and at the same time, hydrogen-dependent caffeate reduction was inhibited. This inhibition could be relieved by ETH2120. These experiments are fully compatible with a chemiosmotic mechanism of ATP synthesis with Na(+) as the coupling ion during hydrogen-dependent caffeate reduction by A. woodii. (+info)
Green coffee bean extract and its metabolites have a hypotensive effect in spontaneously hypertensive rats.
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The effects of a water-soluble green coffee bean extract (GCE) on blood pressure were investigated using spontaneously hypertensive rats (SHR). There was a dose-dependent reduction in blood pressure after a single ingestion (180 to 720 mg/kg, p.o.) or long-term ingestion (0.25 to 1% diet for 6 weeks) of GCE. A single oral ingestion (50 to 200 mg/kg) of 5-caffeoylquinic acid (5-CQA), the major component of GCE, dose-dependently decreased blood pressure, suggesting that 5-CQA is involved in the hypotensive effect of GCE in SHR. Because significant increases in caffeic acid (CA) or ferulic acid (FA) were detected in plasma after oral ingestion of 5-CQA in SHR, these acids (2.5, 5,10 micromol/kg) were intravenously injected into SHR under anesthesia and the carotid arterial pressure was measured. Of the two components, FA had a stronger depressor effect than CA. The depressor effect of FA (50 mg/kg, p.o.) was attenuated by the concurrent injection of atropine sulfate (5 mg/kg, s.c.), suggesting that the hypotensive effect of FA in SHR might be mediated via the muscarinic acetylcholine receptors. These findings indicate that oral ingestion of GCE or 5-CQA decreases blood pressure in SHR, and that FA, which is a metabolite of 5-CQA, is a candidate hypotensive component. (+info)
Plant-derived phenolic compounds prevent the DNA single-strand breakage and cytotoxicity induced by tert-butylhydroperoxide via an iron-chelating mechanism.
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The protective effects of selected members from a series of caffeic acid esters and flavonoids were tested in various toxicity paradigms using U937 cells, previously shown to be sensitive to either iron chelators or bona fide radical scavengers or to both classes of compounds. It was found that all the protective polyphenols were active at very low concentrations and that their effects were observed only under those conditions in which iron chelators also afforded protection. Consistently, active polyphenolic compounds, unlike the inactive ones, effectively chelated iron in an in vitro system. It follows that, at least under the experimental conditions utilized in the present study, the most prominent activity of these polyphenolic compounds resides in their ability to chelate iron. Further studies revealed that the protective effects afforded by the caffeic acid esters and flavonoids were largely mediated by the catechol moiety and that the relative biological potency of these compounds was a direct function of their lipophilicity. (+info)
Subulatin, an antioxidic caffeic acid derivative isolated from the in vitro cultured liverworts, Jungermannia subulata, Lophocolea heterophylla, and Scapania parvitexta.
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The new caffeic acid derivative, subulatin (1), was isolated from in vitro cultured liverworts, Jungermannia subulata, Lophocolea heterophylla, and Scapania parvitexta. The structure of 1 involved two caffeic acids, D-glucose, and 2-carboxy-6-(1,2-dihydroxy-ethyl)-4,5-dihydroxy-5,6-dihydro-4H-pyran. The connectivity of those and the absolute stereochemistry of 1 were elucidated on the basis of spectroscopic evidence. The antioxidative activity of 1 was comparable to that of alpha-tocopherol. (2'R)-Phaselic acid (2a) and (-)-9,2''-epiphylloyl-L-malic acid (4) were also isolated from J. subulata and L. heterophylla, respectively. A chiral HPLC analysis of the p-bromobenzoyl-malic acids derived from 2a showed that 2a from J. subulata was unusual (+)-trans-caffeoyl-D-malic acid. (+info)
Beneficial effects of caffeic acid phenethyl ester in a rat model of vascular injury.
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1. The aim of this study was to evaluate whether caffeic acid phenethyl ester (CAPE), an active component of propolis, was able to reduce neointimal formation in a model of endothelial injury of rat carotid artery (balloon angioplasty). Furthermore, we investigated the relationship between neointima formation and nuclear factor-kappaB (NF-kappaB) activation and we correlated NF-kappaB activation to the expression of inducible isoform of cyclo-oxygenase (COX-2) in injured carotids. 2. In control group a significant proliferation of neointima was observed 14 days after balloon angioplasty, which was correlated to an increase of NF-kappaB/DNA binding activity as well as p50/p65 nuclear levels compared to those observed in the carotids from sham-operated rats. Furthermore, NF-kappaB activation was correlated to increased COX-2, but not beta-actin, protein expression. 3. Treatment of rats for 14 days with CAPE (3, 10, 30 mg x kg(-1)) caused a significant inhibition of all the parameters assayed, except beta-actin protein expression. 4. These results indicate that treatment with CAPE may lead to a reduction of neointima formation by inhibiting NF-kappaB activation and suggest that this agent may have therapeutic relevance for the prevention of human restenosis. (+info)
Structural basis for the modulation of lignin monomer methylation by caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase.
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Caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) from alfalfa is an S-adenosyl-L-Met-dependent O-methyltransferase involved in lignin biosynthesis. COMT methylates caffeoyl- and 5-hydroxyferuloyl-containing acids, aldehydes, and alcohols in vitro while displaying a kinetic preference for the alcohols and aldehydes over the free acids. The 2.2-A crystal structure of COMT in complex with S-adenosyl-L-homocysteine (SAH) and ferulic acid (ferulate form), as well as the 2.4-A crystal structure of COMT in complex with SAH and 5-hydroxyconiferaldehyde, provide a structural understanding of the observed substrate preferences. These crystal structures identify residues lining the active site surface that contact the substrates. Structurally guided site-directed mutagenesis of active site residues was performed with the goal of altering the kinetic preferences for physiological substrates. The kinetic parameters of the COMT mutants versus wild-type enzyme are presented, and coupled with the high-resolution crystal structures, they will serve as a starting point for the in vivo manipulation of lignin monomers in transgenic plants. Ultimately, this structurally based approach to metabolic engineering will allow the further alteration of the lignin biosynthetic pathway in agronomically important plants. This approach will lead to a better understanding of the in vivo operation of the potential metabolic grid for monolignol biosynthesis. (+info)