Differential effect of silybin on the Fe2+-ADP and t-butyl hydroperoxide-induced microsomal lipid peroxidation

Summary We have observed a differential effect of silybin dihemisuccinate on rat liver microsonal oxygen consumption and on lipid peroxidation induced by NADPH-Fe²⁺-ADP and t-butyl hydroperoxide. These results are ascribed to the antioxidant properties of the flavonoid. The differences observed in the effect of the catalysts may be a consequence of the different capacity of silybin to act as a scavenger of free radicals formed by NADPH-Fe²⁺-ADP or t-butyl hydroperoxide.This research was supported in part by grant B-2019-8412 from Dirección de Investigación y Bibliotecas, Universidad de Chile.     

Inhibition of lipid peroxidation by diterpenoid fromPodocarpus nagi

A diterpenoid, totarol (1), fromPodocarpus nagi was evaluated as an antioxidant. This diterpenoid inhibited autoxidation of linoleic acid. Mitochondrial and microsomal lipid peroxidation induced by Fe(III)-ADP/NADH or Fe(III)-ADP/NADPH were also inhibited. Nagilactone E (2), a norditerpene lactone isolated from the same source, had no antioxidative activity. Furthermore, totarol protected red cells against oxidative hemolysis. This diterpene was shown to be effective in protecting biological systems against oxidative stresses.     

Effect of iron and hemoproteins on hydrogen peroxide-supported styrene oxidation to styrene oxide

Summary Fe²⁺, Fe³⁺ and their complexes with EDTA and hemin, methemalbumin and methemoglobin were active catalyzers of H₂O₂ supported styrene oxidation to styrene oxide. Methemoglobin was the most active compound; its peroxidative activity was comparable to that of cytochrome P-450 in liver microsomes of phenobarbital-treated rats. Cumene hydroperoxide supported styrene oxidation with methemoglobin and microsomal hemoproteins and was found to be more efficient than H₂O₂.This work was supported by C.N.R. (National Research Council) contract No. 79.03197.04.     

Activation of the carcinogen N-hydroxy-2-acetylaminofluorene by rat mammary peroxidase

A peroxidase preparation from rat mammary gland parenchymal cells, a target tissue of arylamine carcinogens, was shown to activate the carcinogen N-hydroxy-2-acetylaminofluorene via a nitroxyl free radical intermediate to the more active carcinogens nitrosofluorene and N-acetoxy-2-acetylaminofluorene. Hydrogen peroxide, cumene hydroperoxide, and linoleic acid hydroperoxide were effective as substrates. The antioxidants ascorbate, propyl gallate and reduced glutathione prevented the free radical activation route.     

Activation of the carcinogen N-hydroxy-2-acetylaminofluorene by rat mammary peroxidase

Summary A peroxidase preparation from rat mammary gland parenchymal cells, a target tissue of arylamine carcinogens, was shown to activate the carcinogen N-hydroxy-2-acetylaminofluorene via a nitroxyl free radical intermediate to the more active carcinogens nitrosofluorene and N-acetoxy-2-acetylaminofluorene. Hydrogen peroxide, cumene hydroperoxide, and linoleic acid hydroperoxide were effective as substrates. The antioxidants ascorbate, propyl gallate and reduced glutathione prevented the free radical activation route.     

Effects of barium on separately recorded fast and slow PIII responses in bullfrog retina

Investigation of Ba2+ effects on fast and slow PIII responses in isolated bullfrog retina revealed that Ba2+ suppressed slow PIII completely with little effect on fast PIII. A light-induced [K+]0 decrease in the photoreceptor layer was observed in spite of Ba2+ perfusion, indicating the suppressive action of Ba2+ on the K+ conductance of the Müller cell membrane.     

Peroxidase isoenzymes in cress seedlings (Lepidium sativum L.) and their inhibition by silybin, silydianin and silychristin

Seedlings of Lepidium sativum L. contain 4 peroxidase isoenzymes. Their de-novo-synthesis is significantly depressed in the presence of 3.10(-3) moles/1 silybin, silydianin and silychristin in the incubation medium respectively.     

Effect of free fatty acids and cholesterol in vitro on liver plasma membrane-bound enzymes

The effect of cholesterol and fatty acid treatment in vitro was tested on rat liver plasma membrane-bound enzymes and lipid fluidity. The observed alterations of membrane fluidity affect both (Na+-K+)-ATPase and Mg2+-ATPase activities but not 5'-nucleotidase; basal adenylate cyclase as well as its hormonal sensitivity were differentially affected by changes of membrane microenvironment.     

Evaluation of models for the mechanism of action of 4-hydroxyphenylpyruvate dioxygenase

Summary 4-Hydroxyphenylpyruvic acid was oxygenated with various complexes of oxygen with Fe²⁺, superoxide ion, hydroperoxide anion, triplet and singlet oxygen. Oxidation occurred giving varying amounts of the 4-hydroxy derivatives of benzaldehyde, benzoic acid, phenol, phenylacetic acid and 4-carboxymethylquinone, but no homogentisic acid. 4-Hydroxyphenylperacetic acid was prepared and shown not to undergo self-oxidation. Its ferrous salt decomposed to 4-hydroxybenzyl alcohol. It is concluded that thea-keto carboxylic function is the site of oxygenation in the enzyme process and that a trioxalanone rather than a peracid intermediate may be implicated.Acknowledgments. We thank a referee for drawing our attention to the work of Hawkinsin and we are indebted to the Swiss National Science Foundation for the support of this work (grant No 2.418.0.79).     

Effects of barium on separately recorded fast and slow PIII responses in bullfrog retina

Summary Investigation of Ba²⁺ effects on fast and slow PIII responses in isolated bullfrog retina revealed that Ba²⁺ suppressed slow PIII completely with little effect on fast PIII. A light-induced [K⁺]₀ decrease in the photoreceptor layer was observed in spite of Ba²⁺ perfusion, indicating the suppressive action of Ba²⁺ on the K⁺ conductance of the Müller cell membrane.Acknowledgment. This work was supported by research grant from the Ministry of Education, Science and Culture of Japan and Kinki University research grant 55–103. The author wishes to thank Prof. I. Hanawa at Kobe University for his valuable discussions.     

EP2 receptor stimulation promotes calcium responses in astrocytes via activation of the adenylyl cyclase pathway

Astrocytes are a heterogeneous population of cells that are endowed with a great variety of receptors for neurotransmitters and neuromodulators. Recently prostaglandin E₂ has attracted great interest since it is not only released by astrocytes but also activates receptors coupled to either phospholipase C or adenylyl cyclase. We report that EP₂ receptor stimulation triggers cAMP production but also causes release of Ca²⁺ from intracellular stores. This effect is shared by other receptors similarly coupled to adenylyl cyclase and elicited by direct stimulation of the enzyme or application of cAMP analogues. However, the stimulation of the Ca²⁺ response by cAMP is not mediated by protein kinase A, since a specific antagonist of this kinase had no effect. Such a cross-talk between cAMP and Ca²⁺ was not observed in all astrocytes. It might therefore reflect a specific resource of either a subpopulation or astrocytes in a specific functional state. Received 6 June 2006; received after revision 25 July 2006; accepted 31 August 2006     

Peroxidase-Isoenzyme in Kressekeimlingen (Lepidium sativum L.) und ihre Hemmung durch Silybin,Silydianin und Silychristin

Summary Seedlings ofLepidium sativum L. contain 4 peroxidase isoenzymes. Their de-novo-synthesis is significantly depressed in the presence of 3·10^–3 moles/l silybin, silydianin and silychristin in the incubation medium respectively.     

Zinc antagonizes the effect of botulinum type A toxin at the mouse neuromuscular junction

Zn2+ (10-100 microM) elevated the frequency of miniature end-plate potentials (MEPPs) in the mouse diaphragm. The effect did not depend on external Ca2+. Botulinum type A toxin (BTXA, 50 ng/ml) abolished MEPPs almost completely within 30 min. Zn2+ (100 microM) restored MEPPs and increased their frequency after they had been abolished by BTXA in Ca2+ -free solutions. The antagonistic effect of Zn2+ in the Ca2+ -free solution was reduced by exposing the diaphragm to the toxin in the Ca2+ -free solutions containing high K+. Thus, the action of BTXA is probably enhanced by depolarization of the motor nerve terminals.     

The action of the peptidoleukotriene LTD4 on intracellular calcium in rat mesangial cells

The dose-dependent effect of CGP 45715A on the LTD₄-induced Ca²⁺ response of glomerular mesangial cells has been studied. Our results demonstrate that the LTD₄-dependent increase in the cytosolic Ca²⁺ concentration primarily involves an InsP₃-mediated release of Ca²⁺ from intracellular storage sites and to a minor extent an enhanced influx of Ca²⁺ through receptor-operated Ca²⁺ channels located in the plasma membrane. The action of CGP 45715A on the Ca²⁺ response is an inhibitory one and is convincingly explained by a displacement of LTD₄ from its receptor site(s). The contractile effect of LTD₄ on pulmonary smooth muscle is proposed to be mainly caused by a receptor-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate.     

Inhibitory effects of phenolic compounds on CCl4-induced microsomal lipid peroxidation

The antiperoxidative effects of 35 phenolic compounds, most of them belonging to the flavonoid class, were investigated using CCl4-induced peroxidation of rat liver microsomes. This system was rather insensitive to gallic acid, methyl gallate and ellagic acid. Nevertheless it was inhibited by flavonoids and structure/activity relationships were established. The most potent compounds were gardenin D, luteolin, apigenin (flavones), datiscetin, morin, galangin (flavonols), eriodictyol (flavanone), amentoflavone (biflavone) and the reference compound, (+)-catechin. The natural polymethoxyflavone gardenin D has shown a potency comparable to that of (+)-catechin and higher than that of silybin. Thus, it may be considered as a new type of natural antioxidant with potential therapeutical applications.     

Tissue transglutaminase inhibits the TRPV5-dependent calcium transport in an N-glycosylation-dependent manner

Tissue transglutaminase (tTG) is a multifunctional Ca²⁺-dependent enzyme, catalyzing protein crosslinking. The transient receptor potential vanilloid (TRPV) family of cation channels was recently shown to contribute to the regulation of TG activities in keratinocytes and hence skin barrier formation. In kidney, where active transcellular Ca²⁺ transport via TRPV5 predominates, the potential effect of tTG remains unknown. A multitude of factors regulate TRPV5, many secreted into the pro-urine and acting from the extracellular side. We detected tTG in mouse urine and in the apical medium of polarized cultures of rabbit connecting tubule and cortical collecting duct (CNT/CCD) cells. Extracellular application of tTG significantly reduced TRPV5 activity in human embryonic kidney cells transiently expressing the channel. Similarly, a strong inhibition of transepithelial Ca²⁺ transport was observed after apical application of purified tTG to polarized rabbit CNT/CCD cells. Furthermore, tTG promoted the aggregation of the plasma membrane-associated fraction of TRPV5. Using patch clamp analysis, we observed a reduction in the pore diameter after tTG treatment, suggesting distinct structural changes in TRPV5 upon crosslinking by tTG. As N-linked glycosylation of TRPV5 is a key step in regulating channel function, we determined the effect of tTG in the N-glycosylation-deficient TRPV5 mutant. In the absence of N-linked glycosylation, TRPV5 was insensitive to tTG. Taken together, these observations imply that tTG is a novel extracellular enzyme inhibiting the activity of TRPV5. The inhibition of TRPV5 occurs in an N-glycosylation-dependent manner, signifying a common final pathway by which distinct extracellular factors regulate channel activity.     

Inhibitory effects of phenolic compounds on CCl4-induced microsomal lipid peroxidation

Summary The antiperoxidative effects of 35 phenolic compounds, most of them belonging to the flavonoid class, were investigated using CCl₄-induced peroxidation of rat liver microsomes. This system was rather insensitive to gallic acid, methyl gallate and ellagic acid. Nevertheless it was inhibited by flavonoids and structure/activity relationships were established. The most potent compounds were gardenin D, luteolin, apigenin (flavones), datiscetin, morin, galangin (flavonols), eriodictyol (flavanone), amentoflavone (biflavone) and the reference compound, (+)-catechin. The natural polymethoxyflavone gardenin D has shown a potency comparable to that of (+)-catechin and higher than that of silybin. Thus, it may be considered as a new type of natural antioxidant with potential therapeutical applications.     

Effects of increased potassium in scala tympani on auditory nerve sensitivity.

Raising the K+ concentration in scala tympani of the guinea-pig cochlea generally caused a substantial increase in the spontaneous firing rate of single auditory nerve fibres. This effect was not accompanied by any observed reduction in the threshold sensitivity of these fibres. These findings cast doubt on current theories of cochlear transduction.     

Mechanisms of lipid peroxidation: Iron catalyzed decomposition of fatty acid hydroperoxides as the basis of hydrocarbon evolution in vivo

Summary Ethane and pentane are evolved during peroxidation of tissue lipids in vivo and are believed to be derived from 3 and 6 fatty acids respectively. We present evidence supporting a scission mechanism as the means of forming these hydrocarbons during hydroperoxide decomposition. A fatty acid 6 hydroperoxide (methyl 13-hydroperoxy-6,9,11-octatrienoate), when incubated with a ferrous ion, yielded pentane as the major hydrocarbon (98%). Reaction with ferrous ion results in an alkoxy radical capable of undergoing scission to an aldehydic fatty acid and a hydrocarbon.We wish to thank Dr Ned Porter for both his generous donation of a supply of hydroperoxide and for his suggestions regarding possible reaction mechanisms. This research was supported by U.S.P.H.S. N.I.H. Grants ES00798, HL16264 and T32 GM07105.