Comparative antioxidant effects of beta-adrenoceptor blockers, calcium antagonists and U-74500A against iron-dependent lipid peroxidation in murine ventricular microsomal membranes

Recently we have shown that ACE inhibitors and platelet activating factor antagonists inhibit iron-dependent lipid peroxidation in murine ventricular membranes and possess beneficial effects on ischemia and ischemia reperfusion-induced myocardial injury, which has been ascribed to their capacity to scavenge or impair oxygen free radical generation. In the present study we investigated the effects of beta-adrenoceptor blockers and calcium antagonists on iron-dependent lipid peroxidation (LPO) in murine ventricular membranes and compared them with the lazaroid U-74500A, a potent antioxidant. Fe(2+)-vitamin C induced LPO in a concentration- and time-dependent manner, measured as thiobarbituric acid reactive substances (TBARS) formation. Pretreatment of ventricular membranes with gallopamil, verapamil, propranolol and metaprolol at concentrations of 5 microM and higher inhibited Fe(2+)-vitamin C-induced LPO in a concentration-dependent manner with IC50 values of 192.8-208.3 microM; however, they were less potent than U-74500A (IC50 6.8 microM). In contrast, atenolol, timolol, diltiazem and nifedipine inhibited LPO at very high concentrations with IC50 values of 864.5-971.5 microM. Inhibition of LPO may not be due to the drugs' classical pharmacological actions, but rather to their characteristic chemical structures or physicochemical interactions with biological membranes. In view of the pathological importance of LPO in cardiac ischemic injury, inhibition of LPO by gallopamil, verapamil, propranolol and metaprolol may provide additional cardioprotective activity and thus reinforces their beneficial effects in the treatment of ischemic heart disease.     

Eugenol--an inhibitor of lipoxygenase-dependent lipid peroxidation

The effect of eugenol on enzymatic lipid peroxidation catalyzed by soybean lipoxygenase was studied in an in vitro system. Lipid peroxidation was inhibited by eugenol in a concentration-dependent manner. The half-maximal inhibition (IC50) was found to be 380 microM eugenol. Enzyme kinetic studies showed that eugenol non-competitively inhibited lipid peroxidation by altering the maximum velocity (Vmax) and without any change in Michaelis-Menten constant (Km) values. The inhibitory mechanism implies that eugenol does not inactivate the enzyme directly but may interfere with fatty acid radical intermediates due to its hydroxy radical scavenging ability and thus play a role in inhibiting the propagation of lipid peroxidation.     

A potent antioxidant, SB209995, inhibits oxygen-radical-mediated lipid peroxidation and cytotoxicity

The antioxidant activity of SB209995, a metabolite of carvedilol in human, was studied and compared with its parent compound, carvedilol. SB209995 or carvedilol inhibited iron-catalyzed lipid peroxidation assessed as thiobarbituric acid-reactive substance generated in brain homogenate with IC50s of 0.30 and 8.1 mumol/l, respectively. The oxidation of low-density lipoprotein (LDL) by macrophages or that initiated by Cu2+ ions was inhibited by SB209995 with IC50s of 59 nmol/l and 1.7 mumol/l, respectively. Under the same conditions, the IC50s of carvedilol were 3.8 and 17.1 mumol/l, respectively. Furthermore, SB209995 protected cultured endothelial cells against hydroxyl radical (OH.) or superoxide (O2-)-mediated lipid peroxidation and cytotoxicity, assessed as lactate dehydrogenase release and cell death. The results indicate that SB209995 is a more potent antioxidant than carvedilol and may contribute to the therapeutic effects of carvedilol.     

Antioxidant and free radical scavenging effects in extracts of the medicinal herb Achyrocline satureioides (Lam.) DC. ("marcela")

Achyrocline satureioides (Lam.) DC. (Compositae) is a medicinal herb used in Argentina, Uruguay, Brazil and Paraguay for its choleretic, antispasmodic and hepatoprotective properties. The presence of the flavonoid quercetin and its derivatives, and of different phenolic acids such as caffeic, chlorogenic and isochlorogenic acids in the aerial parts of this plant has led us to study the antioxidant activity of its extracts using different bioassays. The inhibition of luminol-enhanced chemiluminescence by the aqueous and methanolic extracts was used to show that their total reactive antioxidant potential index (TRAP; in microM Trolox equivalents) was 91.0 +/- 15.4 and 128.1 +/- 20.1 microM, respectively, while the total antioxidant reactivity index (TAR) was calculated to be 1537 +/- 148 and 1910 +/- 171 microM. Only the methanolic extract was capable of reducing iron (II)-dependent DNA damage. Lipid peroxidation was assessed by two different methods. The aqueous extract reduced hydroperoxide-initiated chemiluminescence in rat liver homogenates at all concentrations in a dose-dependent manner, with a calculated IC50 = 225 micrograms/ml, while the methanolic extract was only effective at higher concentrations (100 and 1000 micrograms/ml). Both aqueous and methanolic extracts were capable of reducing the production of thiobarbituric acid reactive substances (TBARS) in rat liver homogenates, with an IC50 > 1000 micrograms/ml. The results obtained suggest that the extracts of A. satureioides possess significant free radical scavenging and antioxidant activity in vitro, a fact that should encourage future in vivo studies.     

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.     

A novel antagonist, No. 7943, of the Na+/Ca2+ exchange current in guinea-pig cardiac ventricular cells

1. The effects of No. 7943 on the Na+/Ca2+ exchange current and on other membrane currents were investigated in single cardiac ventricular cells of guinea-pig with the whole-cell voltage-clamp technique. 2. No. 7943 at 0.1-10 microM suppressed the outward Na+/Ca2+ exchange current in a concentration-dependent manner. The suppression was reversible and the IC50 value was approximately 0.32 microM. 3. No. 7943 at 5-50 microM suppressed also the inward Na+/Ca2+ exchange current in a concentration-dependent manner but with a higher IC50 value of approximately 17 microM. 4. In a concentration-response curve, No. 7943 raised the K(m)Ca2+ value, but did not affect the Imax value, indicating that No. 7943 is a competitive antagonist with external Ca2+ for the outward Na+/ Ca2+ exchange current. 5. The voltage-gated Na+ current, Ca2+ current and the inward rectifier K+ current were also inhibited by No. 7943 with IC50S of approximately 14, 8 and 7 microM, respectively. 6. In contrast to No. 7943, 3', 4'-dichlorobenzamil (DCB) at 3-30 microM suppressed the inward Na+/Ca2+ exchange current with IC50 of 17 microM, but did not affect the outward exchange current at these concentrations. 7. We conclude that No. 7943 inhibits the outward Na+/Ca2+ exchange current more potently than any other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.     

Inhibition of microsomal lipid peroxidation and monooxygenase activities by eugenol

Previously we reported that eugenol (4-allyl-2-methoxyphenol) inhibits non-enzymatic peroxidation in liver mitochondria (E. Nagababu and N. Lakshmaiah, 1992, Biochemical Pharmacology. 43, 2393-2400). In the present study, we examined the effect of eugenol on microsomal mixed function oxidase mediated peroxidation using Fe+3-ADP-NADPH, carbon tetrachloride (CCL4)-NADPH and cumene hydroperoxide (CumOOH) systems. In the presence of eugenol the formation of thiobarbituric acid reactive substances (TBARS) was decreased in all the systems (IC50 values: 14 microM for Fe+3-ADP-NADPH, 4.0 microM for CCl4-NADPH and 15 microM for CumOOH). Oxygen uptake was also inhibited to a similar extent with Fe+3-ADP-NADPH and CumOOH systems. A comparative evaluation with other antioxidants showed that in Fe+3-ADP-NADPH and CumOOH systems, the antioxidant efficacy was in the order: butylated hydroxytoluene (BHT) > eugenol > alpha-tocopherol, while in CCl4-NADPH system the order was alpha-tocopherol > BHT > eugenol. Time course of inhibition by eugenol indicated interference in initiation as well as propagation of peroxidation. Eugenol did not inhibit cytochrome P-450 reductase activity but it inhibited P-450 - linked monooxygenase activities such as aminopyrine-N-demethylase, N-nitrosodimethylamine demethylase, benzo(a)pyrene hydroxylase and ethoxyresorufin-O-deethylase to different extents. However, CumOOH supported monooxygenases (aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase) required much higher concentrations of eugenol for inhibition. The concentration of eugenol required to inhibit monooxygenase activities was more than that required to inhibit peroxidation in all the systems. Eugenol elicited type 1 changes in the spectrum of microsomal cytochrome P-450. These results suggest that the inhibitory effect of eugenol on lipid peroxidation is predominantly due to its free radical quenching ability. Eugenol significantly protected against the degradation of cytochrome P-450 during lipid peroxidation with all the systems tested. These findings suggest that eugenol has the potential to be used as a therapeutic antioxidant. Further evaluation may throw more light on this aspect.     

U-101033E (2,4-diaminopyrrolopyrimidine), a potent inhibitor of membrane lipid peroxidation as assessed by the production of 4-hydroxynonenal, malondialdehyde, and 4-hydroxynonenal--protein adducts

4-Hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) are major lipid peroxidation products generated by free radical attack on membranes and appear to contribute to the cytotoxic effects of oxidative stress by a mechanism involving adduct formation with cellular proteins. In the present studies, we investigated the relationship between lipid peroxidation and eventual inactivation of plasma membrane proteins using a model system consisting of purified red blood cell membranes and Fe2+/EDTA. Using this system, we also analyzed the ability of a novel antioxidant, U-101033E (2,4-diaminopyrrolopyrimidine), to inhibit lipid peroxidation and associated protein damage. Our results demonstrated that significant levels of MDA and 4-HNE are generated in this model system, and that both aldehydes are capable of cross-linking membrane proteins. In addition, we used a monoclonal antibody to demonstrate the presence of 4-HNE-protein adducts in this system. The generation of 4-HNE-protein adducts closely paralleled the time course of lipid peroxidation and membrane protein cross-linking, suggesting that 4-HNE may contribute to membrane protein cross-linking. Analysis of U-101033E in this system showed that this antioxidant inhibited lipid peroxidation, prevented the appearance of 4-HNE-protein adducts, and strongly reduced membrane protein cross-linking, with an EC50 of 0.5 microM. We also show that these antioxidant effects were not due to the scavenging of superoxide anion. Thus, these studies demonstrate the potential usefulness of U-101033E for treating certain disease processes where lipid peroxidation plays a role in disease pathogenesis.     

Accumulation and metabolism of low density lipoprotein-derived cholesteryl linoleate hydroperoxide and hydroxide by macrophages

Cholesteryl linoleate hydroperoxide (CLOOH) and hydroxide (CLOH) are present in human atheroma. The intracellular metabolism of low density lipoprotein (LDL)-derived CLOOH and CLOH remain undefined because extensive free radical-mediated LDL oxidation, which modifies LDL apolipoprotein B sufficiently to allow endocytosis by the scavenger receptor (ScR), also degrades CLOOH and CLOH. This problem was approached by first acetylating LDL lysine residues (AcLDL) to achieve protein modification, then exposing AcLDL to the aqueous radical donor 2,2'-azobis(2-amidinopropane) HCl (AAPH), to generate mildly oxidized AcLDL (OxAcLDL). Murine peritoneal macrophages incubated with OxAcLDL accumulated large quantities of CE and small, non-toxic quantities of CLOOH and CLOH in a time- and concentration-dependent manner, and accumulation was inhibited by fucoidin. Inhibition of acyl CoA: cholesterol acyltransferase during loading did not inhibit the accumulation of either CLOOH or CLOH, whereas NH4Cl decreased intracellular clearance of accumulated CLOOH from 68.3 +/- 1.7% to 35.3 +/- 1.0% over 12 h, suggesting lysosomal or pre-lysosomal accumulation. Intracellular clearance of unoxidized lipoprotein-derived CE decreased from 84.0 +/- 5.9% to 43.1 +/- 2.3% over 12 h when cells were loaded with AcLDL or OxAcLDL, respectively. Aggregation of mildly oxidized LDL, even without acetylation, also promoted cellular accumulation of CLOOH and CLOH. We conclude that intracellular accumulation of cholesteryl linoleate hydroperoxide and cholesteryl linoleate hydroxide can follow charge modification or aggregation of mildly oxidized LDL, and that LDL-derived oxidation products may inhibit hydrolysis of LDL-derived CE in foam cell macrophages.     

A twin study of antisocial and neurotic symptoms in childhood

1. 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (ICl,Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2. PIT (0.1-100 microM) failed to stimulate P2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 microM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 microM), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 microM (pKB = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3. Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1-3 microM) potentiated ATP-responses in three experiments, while higher concentrations (3-100 microM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4. In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: alpha 1, alpha 2-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D1, D2, muscarinic, central benzodiazepine, H1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5. In guinea-pig isolated taenia caeci, PIT (12.5-50 microM) irreversibly antagonized relaxations to ATP (3-1000 microM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 microM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 microM) and guanethidine (17 microM), were not affected by PIT (50 microM, for 25-60 min). 6. These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.     

Effect of homocysteine on copper ion-catalyzed, azo compound-initiated, and mononuclear cell-mediated oxidative modification of low density lipoprotein

Homocysteine is an independent risk factor for cardiovascular diseases. The mechanisms by which elevated plasma concentrations of homocysteine are related to the pathogenesis of atherosclerosis are not fully understood. To examine whether homocysteine is implicated in atherogenesis through the modification of low density lipoprotein (LDL), the effect of homocysteine on the oxidation of LDL was studied by three different oxidation systems. Thus, LDL was subjected to Cu(2+)-catalyzed, azo compound-initiated, and peripheral blood mononuclear cell-mediated oxidative modification. The extent of modification was assessed by measuring the formation of conjugated dienes, lipid peroxides, thiobarbituric acid-reactive substances, and the relative electrophoretic mobility. Homocysteine at a normal plasma concentration (6 microM) showed no effect, whereas a concentration corresponding to moderate hyperhomocysteinemia (25 microM) or to concentrations seen in homocystinuria patients (100, 250, and 500 microM) protected LDL from modification of the lipid as well as of the protein moiety. One exception was observed: when the oxidation was initiated by copper ions, homocysteine at concentrations 6 and 25 microM stimulated the lipid peroxidation of LDL to a small, but statistically significant extent. High concentrations of homocysteine showed antioxidative properties as long as the thiol groups were intact, thereby delaying the onset of the oxidation. The 1,1-diphenyl-2-picrylhydracyl radical test demonstrated that homocysteine at concentrations > or = 50 microM possessed marked free radical scavenging capacity. Finally, LDL isolated from two patients with homozygous homocystinuria showed similar extent of Cu(2+)-catalyzed oxidation as LDL from a group of healthy control subjects. Taken together, our data suggest that low concentrations of homocysteine in the presence of copper ions may enhance the lipid peroxidation of LDL, whereas high concentrations of homocysteine may protect LDL against oxidative modification in the lipid as well as in the protein moiety. Thus, homocysteine-induced atherosclerosis may be explained by mechanisms other than oxidative modification of low density lipoprotein.     

Inhibition of oxidation of low density lipoprotein by troglitazone

The effect of a new oral hypoglycemic agent troglitazone, (+/-)-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl-methoxy)benz yl]-2,4-thiazolidinedione as an antioxidant against the free radical-mediated oxidation of low density lipoprotein (LDL) was studied. The oxidation of LDL gives cholesteryl ester hydroperoxide and phosphatidylcholine hydroperoxide as major primary products. Troglitazone incorporated exogenously into LDL inhibited the oxidations of LDL induced by either aqueous or lipophilic peroxyl radicals and suppressed the formation of lipid hydroperoxides efficiently. Ascorbic acid added into the aqueous phase spared both endogenous alpha-tocopherol and troglitazone in LDL. It was also found by absorption spectroscopic and electron spin resonance (ESR) studies that troglitazone reacted rapidly with a galvinoxyl radical to give a chromanoxyl radical which gives the same ESR spectrum as alpha-tocopherol. This ESR spectrum disappeared rapidly when ascorbic acid was added into the system. These results show that troglitazone acts as a potent antioxidant and protects LDL from oxidative modification.     

Lobeline and nicotine evoke [3H]overflow from rat striatal slices preloaded with [3H]dopamine: differential inhibition of synaptosomal and vesicular [3H]dopamine uptake

Lobeline is currently being developed as a substitution therapy for tobacco smoking cessation. Activation of CNS dopamine (DA) systems results in the reinforcing properties of nicotine. The present study compared the effects of lobeline and nicotine on rat striatum. Both lobeline and nicotine evoked [3H]overflow from striatal slices superfused in the presence of pargyline and nomifensine in the buffer. Marked DA depletion (42-67%) and a concomitant 2-fold increase in dihydroxyphenylacetic acid (DOPAC) in slices superfused with high concentrations (30-100 microM) of lobeline were observed. The effect of nicotine (10 microM) was inhibited in a concentration-dependent manner by mecamylamine (1-100 microM). However, lobeline (0.1-100 microM)-evoked [3H]overflow was calcium-independent, and was not antagonized by mecamylamine (1-100 microM), suggesting a mechanism of action other than stimulation of nicotinic receptors. Lobeline inhibited [3H]DA uptake into synaptosomes (IC50 = 80 +/- 12 microM) and vesicles (IC50 = 0.88 +/- 0.001 microM), whereas nicotine (< or =100 microM) did not inhibit synaptosomal or vesicular [3H]DA uptake. In the absence of pargyline and nomifensine in the buffer, endogenous DA was detected in superfusate only in those slices exposed to the highest concentration (100 microM) of lobeline. However, endogenous DOPAC concentration was increased in a concentration-dependent manner, indicating that lobeline exposure resulted in increased cytosolic DA which was rapidly metabolized to DOPAC. Under these conditions, lobeline (10-100 microM) also significantly depleted (66-85%) DA content; however, no change in DOPAC content was observed. The results suggest that, unlike nicotine, lobeline increases DA release by potent inhibition of DA uptake into synaptic vesicles, and a subsequent alteration in presynaptic DA storage.     

Inhibition of in vitro lipid peroxidation by stable steroidic nitroxyl radicals

4',4'-dimethylspiro (5 alpha-cholestane-3,2'-oxazolidin)-3'-yloxy (IK-1) and 7 alpha,12 alpha-dihydroxy-4',-4'-dimethylspiro (5 beta-cholan-24-oic-3,2'-oxazolidin)-3'-yloxy acid (IK-2), two stable steroidic nitroxyl radicals, were newly synthesized and tested as possible inhibitors of lipid peroxidation, induced by Fenton's reagent in both rat liver microsomes and egg phosphatidylcholine liposomes. The inhibitory activity, evaluated through the formation of thiobarbituric acid reactive substances (TBARS) and the conjugated diene, was compared with that of alpha-tocopherol and 2,2,6,6-tetramethylpiperidine-1-yloxy (TEMPO). In each model system IK-1 and IK-2 exhibited an IC50 of 8 microM and reduced the formation of TBARS and conjugated diene, showing IK-1 a potency comparable to alpha-tocopherol and higher than TEMPO. Moreover IK-1 and, to a lesser extent IK-2, reduced the lipid peroxidation induced in the microsomes by the water-soluble azo-initiator 2,2'-Azobis (2-methylpropionamidine) dihydrochloride (AMPH), indicating the IK-1 and IK-2 ability as chain-breaking antioxidants. The hydroxylamine 4',4'-dimethylspiro (5 alpha-cholestane-3,2'-oxazolidin)-3'-hydroxide (IK-3), obtained by chemical reduction of IK-1, was completely inactive as an inhibitor of lipid peroxidation in heat pre-treated microsomes and in liposomes. However in microsomes it was active since it was oxidized to the corresponding nitroxyl radical IK-1.     

Catecholamine regulation of the prefrontal cortex

In order to contribute to the understanding of the biological properties of nafazatrom, an antithrombotic agent (NAP), we studied its effects on peroxidation of low density lipoproteins (LDL), lipid liposomes, heart homopgenate, and its interaction with alpha-tocopherol radical. NAP decreased the FeSO4 and H202-induced peroxidation of phosphatidylcholine liposomes and heart homogenate, and it decreased peroxidation of LDL induced by CuSO4 or 2,2'-azobis(2-amidinopropane). The antioxidant effect of NAF was about 3 times less potent than that of alpha-tocopherol (alpha-TOC) in phosphatidylcholine liposomes, and NAF was about 2-4 times more efficient to decrease peroxidation of LDL than alpha-TOC. Possible interaction of NAF with alpha-tocopherol radical (alpha-TR) was studied by EPR spectroscopy. NAF decreased the concentration of alpha-TR, but it was about 100-times less efficient than vitamin C. This may indicate that NAF does not interfere with alpha-TR formation and/or reduction of alpha-TR in biological system. The obtained results may help the explanation of biological effects of NAF.     

Interventricular communication in the neonatal period]

Fe(II)-tetrakis-N,N,N',N'(2-pyridylmethyl) ethylenediamine (Fe-TPEN) catalyzes the dismutation of superoxide, and blocks the toxic effect of paraquat on Escherichia coli growth and survival. We examined antioxidative effects of Fe-TPEN on lipid peroxidation and t-butyl hydroperoxide induced cell damage. Fe-TPEN inhibited the FeSO4/H2O2 induced lipid peroxidation in the rat liver homogenates with an IC50 value of 30.2 microM, and protected Ac2F cell damage by t-butyl hydroperoxide in a dose-dependent manner (EC50 value is 2.6 microM). Also, hepatoprotective effect of Fe-TPEN (5 mg/kg, i.p.) was investigated using CCl4 induced liver injury in rats. This complex inhibited the elevation of serum alanine aminotransferase (AST) and aspartate aminotransferase (ALT) levels in CCl4 induced liver injuries, and improved submassive necrosis and fatty degeneration of the hepatocytes. Fe-TPEN also prevented the loss of total and nonprotein SH contents, glutathione peroxidase and glutathione-S-transferase activity in cytosol of rat liver. Although the exact mechanism of action is not clear, antioxidative properties as well as attenuation of hepatocellular defense systems by Fe-TPEN seem to be important on its potent hepatoprotective effect in CCl4-intoxicated rat.     

Effect of silibinin on oxidative damage of blood constituents

Silibinin (SDH) is a flavonoid with ascertained hepatoprotective effects, which have been partially attributed to its antioxidant properties. Oxidation of blood constituents could have a role in atherogenesis and interfere with the rheologic properties of the blood. In this study we investigated, whether SDH could protect some blood constituents against oxidative modification. In human plasma we measured TBARS and fluorescence generation as indicators of copper or azobis amidinopropane hydrochloride (AAPH) at 760 mm Hg PO2-induced lipid peroxidation. SDH at 50 microM inhibited copper-induced TBARS formation by 25% and fluorescence by 47%. SDH also inhibited AAPH-induced lipid peroxidation, but at 175 microM concentration only. Oxidative modification of albumine was evaluated by fluorescence generation. SDH at 50 microM inhibited copper/hydrogen peroxide fluorescence generation by 54% and at 2.5 microM it inhibited EDTA-Fe (II)/hydrogen peroxide fluorescence generation by 31%. The protection of albumin by SDH was confirmed by SDS-PAGE electrophoresis. Copper-induced red-cell lipid peroxidation was evaluated by TBARS formation. SDH at 250 microM inhibited copper-induced lipid peroxidation and hemolysis by 45% and 94%, respectively. SDH also inhibited hemolysis in red-cell suspensions exposed to hydrogen peroxide, but not lipid peroxidation. Our results show that SDH may protect blood constituents from oxidative damage.     

Gp120 can revert antagonism at the glycine site of NMDA receptors mediating GABA release from cultured hippocampal neurons

The effects of the human immunodeficiency virus type 1 envelope protein gp120 on the release of GABA elicited by N-methyl-D-aspartate (NMDA) from rat hippocampal neurons in primary culture has been investigated. NMDA (1-300 microM) increased in a concentration-dependent manner (EC50 =37.9+/-12 microM) the release of [3H]-GABA. The effect of 100 microM NMDA was prevented by 30 microM of the GABA transport inhibitor N-(4,4-diphenyl-3-butenyl)guvacine (SKF 100330A). Glycine (10 microM) or gp120 (0.01 microM) affected neither the basal nor the NMDA-evoked [3H]-GABA release. The NMDA (100 microM)-evoked release was prevented by 5,7-dichloro-kynurenic acid (5,7-DCKA), a selective antagonist at the glycine site of the NMDA receptor, in a concentration-dependent manner (IC50 approximately 0.3 microM). Glycine (3-10 microM) or gp120 (0.003-0.01 microM) produced reversal of the 5,7-DCKA antagonism in a way that suggested competition at a same site; gp120 was at least 3 orders of magnitude more potent than glycine. It is suggested that gp120 may mimic glycine at NMDA receptors.     

Characterization of the ocular antiallergic and antihistaminic effects of olopatadine (AL-4943A), a novel drug for treating ocular allergic diseases

Olopatadine (AL-4943A; KW-4679) [(Z)-11-[3-(dimethylamino)propylidene]-6, 11-dihydrodibenz[b,e]oxepine-2-acetic acid hydrochloride] is an antiallergic/antihistaminic drug under development for topical ocular use. The effects of the compound on release of proinflammatory mediators (histamine, tryptase and prostaglandin D2) from monodispersed human conjunctival mast cells were assessed. Histamine receptor subtype binding affinities and functional potencies were determined with ligand binding and phosphoinositide turnover assays, respectively. Olopatadine inhibited the release of histamine, tryptase and prostaglandin D2, in a concentration-dependent manner (IC50 = 559 microM). Evaluation of the interaction of olopatadine with histamine receptors revealed a relatively high affinity for the H1 receptor (Ki = 31.6 nM, pKi = 7.5 +/- 0.1, n = 7) but lower affinities for H2 receptors (Ki = 100 microM, pKi = 4.0 +/- 0.19, n = 7) and H3 receptors (Ki = 79.4 microM, pKi = 4.1 +/- 0.16, n = 7). The H1 selectivity of olopatadine was superior to that of other ocularly used antihistamines studied, such as ketotifen, levocabastine, antazoline and pheniramine. The profiling of olopatadine in 42 nonhistamine receptor binding assays revealed that olopatadine interacts with only two nonhistamine receptor/uptake sites to any significant degree (pIC50 < or = 5-6). Olopatadine inhibited histamine-induced phosphoinositide turnover in human conjunctival epithelial cells (IC50 = 10 nM, pIC50 = 8.0, n = 4) and in other human ocular cells (IC50 = 15.8-31.6 nM, pIC50 = 7.5-7.8) and exhibited apparent noncompetitive antagonist properties in these cells, with an estimated dissociation constant (Kb) of 19.9 nM (pKb = 7.7, n = 6). This combination of mast cell mediator release inhibition and selective H1 receptor antagonism suggests that olopatadine may be particularly useful in the treatment of ocular allergic diseases. Indeed, olopatadine has recently shown clinical efficacy in an allergic conjunctivitis model in human subjects.