Endomorphins, endogenous opioid peptides, provide antioxidant defense in the brain against free radical-induced damage

Oxidative stress has been considered to be a major cause of cellular injuries in a variety of chronic health problems, such as carcinogenesis and neurodegenerative disorders. The brain appears to be more susceptible to oxidative damage than other organs. Therefore, the existence of antioxidants may be essential in brain protective systems. The antioxidative and free radical scavenging effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, have been investigated in vitro. The oxidative damage was initiated by a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrocholoride) (AAPH) and hydrogen peroxide (H2O2). The linoleic acid peroxidation, DNA and protein damage were monitored by formation of hydroperoxides, by plasmid pBR 322 DNA nicking assay and single-cell alkaline electrophoresis, and by SDS-polyacrylamide gel electrophoresis. Endomorphins can inhibit lipid peroxidation, DNA strand breakage, and protein fragmentation induced by free radical. Endomorphins also reacted with galvinoxyl radicals in homogeneous solution, and the pseudo-first-order rate constants were determined spectrophotometrically by following the disappearance of galvinoxyl radicals. In all assay systems, EM1 was more potent than EM2 and GSH, a major intracellular water-soluble antioxidant. We propose that endomorphins are one of the protective systems against free radical-induced damage in the brain.     

Endomorphins and morphine limit anoxia-reoxygenation-induced brain mitochondrial dysfunction in the mouse

The protection of brain mitochondria from oxidative stress is an important therapeutic strategy against ischemia-reperfusion injury and neurodegenerative disorders. Isolated brain mitochondria subjected to a 5 min period of anoxia followed by 5 min reoxygenation mirrored the effect of oxidative stress in the brain. The present study attempts to evaluate the protective effects of endomorphin 1 (EM1), endomorphin 2 (EM2), and morphine (Mor) in an in vitro mouse brain mitochondria anoxia-reoxygenation model. Endomorphins (EM1/2) and Mor were added to mitochondria prior to anoxia or reoxygenation. EM1/2 and Mor markedly improved mitochondrial respiratory activity with a decrease in state 4 and increases in state 3, respiratory control ratio (RCR) and the oxidative phosphorylation efficiency (ADP/O ratio), suggesting that they may play a protective role in mitochondria. These drugs inhibited alterations in mitochondrial membrane fluidity, lipoperoxidation, and cardiolipin (CL) release, which indicates protection of the mitochondrial membranes from oxidative damage. The protective effects of these drugs were concentration-dependent. Furthermore, these drugs blocked the enhanced release of cytochrome c (Cyt c), and consequently inhibited the cell apoptosis induced by the release of Cyt c. Our results suggest that EM1/2 and Mor effectively protect brain mitochondria against oxidative stresses induced by in vitro anoxia-reoxygenation and may play an important role in the prevention of deleterious effects during brain ischemia-reperfusion and neurodegenerative diseases.     

Endomorphins, endogenous opioid peptides, induce apoptosis in human leukemia HL-60 cells

Opioids play a role in the apoptosis machinery. We studied the induction of apoptosis in endomorphin 1 (EM1) and endomorphin 2 (EM2), 2 newly isolated endogenous mu-opioid receptor agonists. These endomorphins were able to reduce the viability of cultured HL-60 cells. The antiproliferative properties of endomorphins appeared to be attributable to their induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 indicated down-regulation, but the Bax, Fas, and FasL expression showed up-regulation as compared with the untreated control cells. These data support the idea that endomorphins induce apoptosis in HL-60 cells through the activation of the Bcl-2-Bax and the Fas-FasL pathway. We suggest that endomorphins may play an important role in the regulation of tumor cell death.     

C-terminal amide to alcohol conversion changes the cardiovascular effects of endomorphins in anesthetized rats

Endomorphin1-ol (Tyr-Pro-Trp-Phe-ol, EM1-ol) and endomorphin2-ol (Tyr-Pro-Phe-Phe-ol, EM2-ol), with C-terminal alcohol (-ol) containing, have been shown to exhibit higher affinity and lower intrinsic efficacy in vitro than endomorphins. In the present study, in order to investigate the alterations of systemic hemodynamic effects induced by C-terminal amide to alcohol conversion, responses to intravenous (i.v.) or intracerebroventricular (i.c.v.) injection of EM1-ol, EM2-ol and their parents were compared in the system arterial pressure (SAP) and heart rate (HR) of anesthetized rats. Both EM1-ol and EM2-ol induced dose-related decrease in SAP and HR when injected in doses of 3-100 nmol/kg, i.v. In terms of relative vasodepressor activity, it is interesting to note that EM2-ol was more potent than endomorphin2 [the dose of 25% decrease in SAP (DD25) = 6.01+/-3.19 and 13.99+/-1.56 nmol/kg, i.v., respectively] at a time when responses to EM1-ol were less potent than endomorphin1. Moreover, decreases in SAP in response to EM1-ol and EM2-ol were reduced by naloxone, atropine sulfate, L-NAME and bilateral vagotomy. It indicated that the vasodepressor responses were possibly mediated by a naloxone-sensitive, nitric oxide release, vagus-activated mechanism. It is noteworthy that i.c.v. injections of -ol derivatives produced dose-related decreases in SAP and HR, which were significantly less potent than endomorphins and were attenuated by naloxone and atropine sulfate. In summary, the results of the present study indicated that the C-terminal amide to alcohol conversion produced different effects on the vasodepressor activity of endomorphin1 and endomorphin2 and endowed EM2-ol distinctive hypotension characters in peripheral (i.v.) and central (i.c.v.) tissues. Moreover, these results provided indirect evidence that amidated C-terminus might play an important role in the regulation of the cardiovascular system.     

Kinetics of lipid peroxidation in mixtures of HDL and LDL, mutual effects.

In view of the proposed central role of LDL oxidation in atherogenesis and the established role of HDL in reducing the risk of atherosclerosis, several studies were undertaken to investigate the possible effect of HDL on LDL peroxidation. Since these investigations yielded contradictory results, we have conducted systematic kinetic studies on the oxidation in mixtures of HDL and LDL induced by different concentrations of copper, 2, 2'-azo bis (2-amidinopropane) hydrochloride (AAPH) and myeloperoxidase (MPO). These studies revealed that oxidation of LDL induced either by AAPH or MPO is inhibited by HDL under all the studied conditions, whereas copper-induced oxidation of LDL is inhibited by HDL at low copper/lipoprotein ratio but accelerated by HDL at high copper/lipoprotein ratio. The antioxidative effects of HDL are only partially due to HDL-associated enzymes, as indicated by the finding that reconstituted HDL, containing no such enzymes, inhibits peroxidation induced by low copper concentration. Reduction of the binding of copper to LDL by competitive binding to the HDL also contributes to the antioxidative effect of HDL. The acceleration of copper-induced oxidation of LDL by HDL may be attributed to the hydroperoxides formed in the "more oxidizable" HDL, which migrate to the "less oxidizable" LDL and enhance the oxidation of the LDL lipids induced by bound copper. This hypothesis is supported by the results of experiments in which native LDL was added to oxidizing lipoprotein at different time points. When the native LDL was added prior to decomposition of the hydroperoxides in the oxidizing lipoprotein, the lag preceding oxidation of the LDL was much shorter than the lag observed when the native LDL was added at latter stages, after the level of hydroperoxides became reduced due to their copper-catalyzed decomposition. The observed dependence of the interrelationship between the oxidation of HDL and LDL on the oxidative stress should be considered in future investigations regarding the oxidation of lipoprotein mixtures.     

Oxidized low density lipoprotein suppresses lipopolysaccharide-induced inflammatory responses in microglia: oxidative stress acts through control of inflammation

Low density lipoprotein (LDL) is readily oxidized under certain conditions, resulting in the formation of oxidized LDL (oxLDL). Despite numerous in vitro reports that reveal the pathogenic role of oxidative stress, anti-oxidative strategies have underperformed in the clinic. In this study, we examine the role of oxLDL in brain inflammatory responses using cultured rat brain microglia. We demonstrate that oxLDL inhibits lipopolysaccharide (LPS)-induced inflammatory responses in these cells. It also decreases LPS-induced expression of inducible nitric oxide synthase and production of nitric oxide, and reduces LPS-induced secretion of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Oxysterols, known components of oxLDL and endogenous agonists of liver X receptor, can simulate the inhibitory effects of oxLDL in LPS-activated microglia. In addition, their inhibitory effects were mimicked by liver X receptor (LXR) agonists and potentiated by a retinoid X receptor agonist, suggesting these molecules heterodimerize to function as oxysterol receptors. Taken together, our results demonstrate that oxLDL inhibits LPS-induced inflammatory responses in brain microglia and that these inhibitory effects are mediated by oxysterols and, at least in part, by the nuclear receptor LXR. Our results suggest an additional mechanism of action for oxidative stress that acts indirectly via modulation of inflammatory responses. Although further studies are needed, these results answer in part the question of why anti-oxidative strategies have not been successful in clinical situations. Moreover, as brain inflammation participates in the initiation and progression of several neurodegenerative disorders, the present data provide information that should prove a useful guide for designing therapeutic strategies to combat oxidative brain diseases.     

Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants

Human serum paraoxonase (PON1) can protect low density lipoprotein (LDL) from oxidation induced by either copper ion or by the free radical generator azo bis amidinopropane hydrochloride (AAPH). During LDL oxidation in both of these systems, a time-dependent inactivation of PON arylesterase activity was observed. Oxidized LDL (Ox-LDL) produced by lipoprotein incubation with either copper ion or with AAPH, indeed inactivated PON arylesterase activity by up to 47% or 58%, respectively. Three possible mechanisms for PON inactivation during LDL oxidation were considered and investigated: copper ion binding to PON, free radical attack on PON, and/or the effect of lipoprotein-associated peroxides on the enzyme. As both residual copper ion and AAPH are present in the Ox-LDL preparations and could independently inactivate the enzyme, the effect of minimally oxidized (Ox-LDL produced by LDL storage in the air) on PON activity was also examined. Oxidized LDL, as well as oxidized palmitoyl arachidonoyl phosphatidylcholine (PAPC), lysophosphatidylcholine (LPC, which is produced during LDL oxidation by phospholipase A2-like activity), and oxidized cholesteryl arachidonate (Ox-CA), were all potent inactivators of PON arylesterase activity (PON activity was inhibited by 35%-61%). PON treatment with Ox-LDL (but not with native LDL), or with oxidized lipids, inhibited its arylesterase activity and also reduced the ability of the enzyme to protect LDL against oxidation. PON Arylesterase activity however was not inhibited when PON was pretreated with the sulfhydryl blocking agent, p-hydroxymercurybenzoate (PHMB). Similarly, on using recombinant PON in which the enzyme's only free sulfhydryl group at the position of cysteine-284 was mutated, no inactivation of the enzyme arylesterase activity by Ox-LDL could be shown. These results suggest that Ox-LDL inactivation of PON involves the interaction of oxidized lipids in Ox-LDL with the PON's free sulfhydryl group. Antioxidants such as the flavonoids glabridin or quercetin, when present during LDL oxidation in the presence of PON, reduced the amount of lipoprotein-associated lipid peroxides and preserved PON activities, including its ability to hydrolyze Ox-LDL cholesteryl linoleate hydroperoxides. We conclude that PON's ability to protect LDL against oxidation is accompanied by inactivation of the enzyme. PON inactivation results from an interaction between the enzyme free sulfhydryl group and oxidized lipids such as oxidized phospholipids, oxidized cholesteryl ester or lysophosphatidylcholine, which are formed during LDL oxidation. The action of antioxidants and PON on LDL during its oxidation can be of special benefit against atherosclerosis since these agents reduce the accumulation of Ox-LDL by a dual effect: i.e. prevention of its formation, and removal of Ox-LDL associated oxidized lipids which are generated during LDL oxidation.     

Differential antioxidant properties of red wine in water soluble and lipid soluble peroxyl radical generating systems

Red wine and its components have been shown to possess cardioprotective and anti-atherogenic effects. Additionally, red wine and many of its components like catechin, epicatechin, rutin, transresveratrol and quercetin possess antioxidant properties. Oxidized low density lipoprotein (LDL) is involved in the development of an atherosclerotic lesion. Red wine, therefore, may be anti-atherogenic because of its antioxidant effects on LDL modification. This study examined the antioxidant effects of catechin, epicatechin, rutin, transresveratrol, quercetin and Merlot wines on LDL oxidation. Merlot was chosen because although other red wines have been tested, limited information exists for this variety. Oxidation was carried out with AAPH (2,2-Azo-bis(2-amidinopropane) dihydrochloride) and AMVN (2,2-Azo-bis(2,4-dimethylvaleronitrile)), as water and lipid soluble peroxyl radical generating systems (FRGS), respectively. This allowed us to determine the lipophilic antioxidant characteristics of the wine and its components. Conjugated diene assays were used to measure LDL oxidation over 6 hrs. In an AAPH system, all polyphenolic compounds except transresveratrol displayed an antioxidant effect. LDL oxidation by AAPH was also inhibited by aliquots of Merlot wine. No antioxidant effects were observed in an AMVN environment except for a mild antioxidant effect by quercetin. Surprisingly, incubation of LDL with Merlot wine strongly protected against oxidation by AMVN. In summary, the five phenolic compounds displayed antioxidant effects in a water soluble free radical generating system, but only quercetin showed this in a lipid soluble one. However, red wine inhibited LDL oxidation by both water and lipid soluble free radical generating systems. Our data suggest, therefore, that red wines contain unidentified antioxidants that provide protection against LDL oxidation within a lipid soluble environment. (Mol Cell Biochem 263: 211–215, 2004)     

Cyclosporin A does not increase the oxidative susceptibility of low density lipoprotein in vitro

Accelerated atherosclerosis is the leading cause of morbidity in renal transplant recipients. The pathogenic mechanisms responsible for the progression of atherosclerosis in renal transplant recipients have not been elucidated. Cyclosporin A (CsA) is an immunosuppressive agent used post-transplant and may contribute to increased oxidative susceptibility of low density lipoprotein (LDL). There is a paucity of data testing the effect of CsA on LDL oxidation. Hence, the aim of this study was to test the effect of in vitro enrichment of LDL with CsA on LDL oxidation. LDL oxidation in presence of different concentrations of CsA was tested using metal-dependent (copper), metal-independent (AAPH) and cell-mediated (macrophages) oxidation systems. In all 3 systems, CsA had no significant effect on LDL oxidation. Also, pre-incubation of LDL with CsA did not affect LDL oxidation and LDL alpha tocopherol levels. Thus, the results of our studies with CsA indicate that it is not a direct pro-oxidant.     

In vivo protection of synaptosomes from oxidative stress mediated by Fe2+/H2O2 or 2,2-azobis-(2-amidinopropane) dihydrochloride by the glutathione mimetic tricyclodecan-9-yl-xanthogenate

D609 (tricyclodecan-9-yl-xanthogenate) is a phosphatidylcholine-specific phospholipase C inhibitor that also has been reported to protect rodents against oxidative damage caused by lethal doses of ionizing radiation. We previously showed that D609 mimics glutathione. D609 has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies from our laboratory have also shown that D609 reduces the Alzheimer amyloid beta-peptide (1-42)-induced oxidative stress and cytotoxicity in neuronal cell culture. The present study was undertaken to test the hypothesis that D609 would provide neuroprotection against free radical oxidative stress in vivo. Synaptosomes isolated from gerbils, previously injected intraperitoneally (ip) with D609, were treated with the oxidants Fe2+/H2O2 or 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH), which produce free radicals. Synaptosomes isolated from the gerbils ip injected with D609 and treated with Fe2+/H2O2 or AAPH showed significant reduction in reactive oxygen species, levels of protein carbonyl, protein-bound hydroxynonenal (a lipid peroxidation product), and 3-nitrotyrosine (another marker of protein oxidation formed by reaction of tyrosine residues with peroxynitrite) compared to oxidative stress in synaptosomes isolated from gerbils that were injected with saline, but treated with Fe2+/H2O2 or AAPH. These results are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders.     

Free and peptide-bound DOPA can inhibit initiation of low density lipoprotein oxidation

Hydroxyl radicals have been shown to convert free tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA) which has reducing properties. During protein or peptide oxidation such reducing species are also formed from tyrosine residues. Free DOPA or peptide-bound DOPA (PB-DOPA) is able to promote radical-generating events, facilitating the damage of biomolecules such as nucleic acids. Radical induced lipid oxidation in low density lipoprotein (LDL) transforms the lipoprotein into an atherogenic particle. As PB-DOPA has been found in atherosclerotic plaques, we tested the ability of free and PB-DOPA to influence LDL oxidation. Free DOPA, in contrast to tyrosine had strong inhibitory action on both, the copper-ion initiated and metal ion independent (AAPH-induced) lipid oxidation. Free DOPA also inhibited LDL oxidation induced by the copper transport protein ceruloplasmin. To test if PB-DOPA was also able to inhibit LDL oxidation, DOPA residues were generated enzymatically in the model peptides insulin and tyr-tyr-tyr, respectively. PB-DOPA formation substantially increased the ability of both molecules to inhibit LDL oxidation by copper or AAPH. We hypothesize that DOPA-peptides and -proteins may have the potential to act as efficacious antioxidants in the atherosclerotic plaque.     

Free radical-mediated chain oxidation of low density lipoprotein and its synergistic inhibition by vitamin E and vitamin C

The oxidation of human low density lipoprotein (LDL) initiated by free radical initiator and its inhibition by vitamin E and water-soluble antioxidants have been studied. It was found that the kinetic chain length was considerably larger than 1, suggesting that LDL was oxidized by a free radical chain mechanism. Vitamin E acted as a lipophilic chain-breaking antioxidant. Water-soluble chain-breaking antioxidants such as ascorbic acid and uric acid suppressed the oxidation of LDL initiated by aqueous radicals but they could not scavenge lipophilic radicals within LDL to break the chain propagation. Ascorbic acid acted as a synergistic antioxidant in conjunction with vitamin E.     

Protein hydroperoxides are a major product of low density lipoprotein oxidation during copper, peroxyl radical and macrophage-mediated oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble alpha-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

In vivo protection of synaptosomes by ferulic acid ethyl ester (FAEE) from oxidative stress mediated by 2,2-azobis(2-amidino-propane)dihydrochloride (AAPH) or Fe(2+)/H(2)O(2): insight into mechanisms of neuroprotection and relevance to oxidative stress-related neurodegenerative disorders

Ferulic acid ethyl ester (FAEE) is an ester derivative of ferulic acid, the latter known for its anti-inflammatory and antioxidant properties. Previous studies from our laboratory have shown that ferulic acid protects synaptosomal membrane system and neuronal cell culture systems against hydroxyl and peroxyl radical oxidation. FAEE is lipophilic and is able to penetrate lipid bilayer. Previous studies reported that FAEE reduces Alzheimer's amyloid beta peptide Abeta(1-42)-induced oxidative stress and cytotoxicity in neuronal cell culture by direct radical scavenging and by inducing certain antioxidant proteins. In the present study we tested the hypothesis that FAEE would provide neuroprotection against free radical oxidative stress in vivo. Synaptosomes were isolated from the gerbils that were previously injected intraperitoneally (i.p.) with FAEE or DMSO and were treated with oxidants, Fe(2+)/H(2)O(2) or 2,2-azobis(2-amidino-propane)dihydrochloride (AAPH). Synaptosomes isolated from the gerbil previously injected i.p. with FAEE and treated with Fe(2+)/H(2)O(2) and AAPH showed significant reduction in reactive oxygen species (ROS), levels of protein carbonyl, protein bound 4-hydroxynonenal (HNE, a lipid peroxidation product), and 3-nitrotyrosine (3-NT, another marker of protein oxidation formed by reaction of tyrosine residues with peroxynitrite) compared to Fe(2+)/H(2)O(2) or AAPH induced oxidative stress in synapotosomes isolated from the brain of gerbils that were previously injected with DMSO. The synaptosomes isolated from gerbil pre-injected with FAEE and subsequently treated with AAPH or Fe(2+)/H(2)O(2) showed induction of heme oxygenase (HO-1) and heat shock protein 70 (HSP-70) but reduced inducible nitric oxide synthase (iNOS) levels. These results are discussed with reference to potential use of this lipophilic antioxidant phenolic compound in the treatment of oxidative stress-related neurodegenerative disorders.     

Antioxidative effects of green tea polyphenols on free radical initiated and photosensitized peroxidation of human low density lipoprotein

Antioxidative effects of the main polyphenolic components extracted from green tea leaves, i.e. (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and gallic acid (GA), against free radical initiated peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either thermally by a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), or photochemically by a triplet sensitizer benzophenone (BP). The reaction kinetics was monitored by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) presented in the native LDL. Kinetic analysis of the antioxidation process demonstrates that these green tea polyphenols are effective antioxidants against both AAPH-initiated and BP-photosensitized LDL peroxidation. The antioxidative action of the green tea polyphenols includes trapping the initiating and/or propagating peroxyl radicals with the activity sequence EC>EGCG>ECG>EGC>GA for the AAPH initiated peroxidation, and reducing the alpha-tocopheroxyl radical to regenerate alpha-tocopherol with the activity sequence of ECG>EC>EGCG>EGC>GA and ECG>EGCG>GA>EC>EGC for the AAPH-initiated and BP-photosensitized peroxidations respectively.     

Endomorphins interact with tachykinin receptors

Soon after the discovery of endomorphins several studies indicated differences between pharmacological effects of endomorphins and other MOR selective ligands, as well as differences between the effects of endomorphin I and endomorphin II. We now propose that these differences are the result of an additional non-opioid property of endomorphins, namely, their weak antagonist properties with respect to tachykinin NK1 and NK1 receptors.     

Inhibition of human low density lipoprotein oxidation by flavonols and their glycosides

Antioxidative effects of the flavonols and their glycosides, i.e., quercetin (Q), quercetin galactopyranoside (QG), quercetin rhamnolpyranoside (QR), rutin (R), morin (MO), myrecetin (MY), kaempferol (K) and kaempferol glucoside (KG), against free radical initiated peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either by a water-soluble initiator 2,2'-azobis(2-amidino propane hydrochloride) (AAPH), or by cupric ion (Cu2+). The reaction kinetics were monitored either by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) presented in the native LDL, or by the formation of thiobarbituric acid reactive substances (TBARS). Kinetic analysis of the antioxidation process demonstrates that these flavonols and their glycosides are effective antioxidants against AAPH- and Cu(2+)-initiated LDL peroxidation, the flavonols bearing ortho-dihydroxyl groups possess significantly higher antioxidant activity than those bearing no such functionalities, and the glycosides are less active than their parent aglycones.     

Protein Hydroperoxides are a Major Product of Low Density Lipoprotein Oxidation During Copper,Peroxyl Radical and Macrophage-mediated Oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble α-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

Strictinin as an efficient antioxidant in lipid peroxidation

The antioxidant effect of strictinin (SOH), which was extracted from green tea leaves, against the peroxidation of linoleic acid in sodium dodecyl sulfate (SDS) and cetyl trimethylammonium (CTAB) micelles, against the peroxidation of low-density lipoprotein (LDL) and against oxidative hemolysis of human red blood cells (RBCs), has been studied. The peroxidation of linoleic acid and LDL, and oxidative hemolysis of RBCs were initiated thermally by a water-soluble azo initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), and the reaction kinetics in micelles and LDL were monitored by uptake of oxygen. The synergistic antioxidant effect of SOH with alpha-tocopherol (Vitamin E) was also studied by following the decay kinetics of alpha-tocopherol. Kinetic analysis of the antioxidation process demonstrates that SOH, used either alone or in combination with alpha-tocopherol, is an effective antioxidant against lipid peroxidation, but its effects significantly depend on the reaction medium.     

Abnormal modulation of cholinergic neurotransmission by endomorphin 1 and endomorphin 2 in isolated bronchus of type 1 diabetic rats

To assess whether diabetes alters the regulatory effects of mu-opioid receptor (MOR) agonists on the cholinergic bronchoconstriction, we investigated the inhibitory effects of endomorphins (EMs) on the electrical field stimulation (EFS)-induced cholinergic bronchoconstriction in type 1 diabetic rats. At 4 weeks after the onset of diabetes, both the EFS- and exogenous acetylcholine (ACh)-induced bronchoconstriction in diabetes in vitro were greater than those in non-diabetes rats. Furthermore, endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited the response to EFS in diabetic rat isolated bronchus in a concentration- and frequency-dependent manner, which is in agreement with that in non-diabetes. However, the inhibitory effects of EMs on the EFS-induced bronchoconstriction in diabetes were significantly weaker than those in non-diabetes. Both EM1 and EM2 (1 microM) had no effect on the contractile response to exogenous ACh, indicating a prejunctional effect. Furthermore, the inhibitory effect on the EFS-induced bronchoconstriction was blocked by naloxone (10 microM). Eight weeks after the induction of diabetes, both the EFS- and exogenous ACh-induced bronchoconstrictions in diabetes were further enhanced compared to those in short-time (4 weeks) diabetic rats. Moreover, the inhibitory effects of EMs on the EFS-induced bronchoconstriction were further attenuated. These results suggest that dysfunction of presynaptic inhibitory modulation through opioid receptor by EMs may take place in the bronchus of diabetic rats.