The effect of tryptophan, 5-hydroxytryptophan, serotonin, histidine and histamine on the peroxidation of lipids in hepatic mitochondrial membranes]

The method of recording the superweak chemifluorescence was applied to the study of the effect of tryptophan, 5-oxytryptophan, sertonin, histidine and histamine on the peroxication of lipids in the membranes of mitochondria of rat liver in the presence of Fe++ ions inducing this process. 5-oxytryptophan and serotonin inhibited this process in a concentration of 10(-5)-10(-4) M (protein content -1.8 mg per 1 ml of mitochondrial suspension). On the basis of studying the kinetics of the initial portion of the ascending branch of the "slow flare up" constants of the antioxidatinve activity (constituting 2.2-10(3) and 9.8-10(3) M-1 for 5-oxytryptophan and serotonin, respectively) was calculated. The antioxidative action is associated with the presence of the phenol group in the molecule of the compound under study. It is supposed that the action of 5-oxytryptophan and serotonin on peroxidation of lipids in the membranes was of significance for the regulation of permeability of the biological membranes, along with their effect on the other membrane processes.     

Antioxidative activity of new N-oxides of tertiary amines: membrane model and chromogen studies

Potential antioxidative activities of three series of newly synthesized N-oxides were studied. Individual components in each of the series differed in the lipophilicities and number of free radical scavenging groups. Various methods were used to determine their antioxidative efficiencies: Prevention of erythrocyte membrane lipid oxidation induced by UV irradiation and chromogen experiments in which antioxidative efficiencies of compounds were compared to that of the standard antioxidant Trolox (a water-soluble vitamin E analogue). Additionally, some hemolytic (pig erythrocytes) and differential scanning calorimetry (DSC) measurements were performed to determine a mechanism of the interaction between membranes and N-oxides. It was found that N-oxides, especially those of long alkyl chains (> C12H25), readily interacted with both, erythrocyte and liposomal membranes. No marked differences were found in their protection of erythrocytes against oxidation. In most cases inhibition of oxidation changed between 15% and 25%. Still, it was far better than in chromogen experiments where suppression of free radicals reached 20% in the best case. It may be concluded that antioxidative capabilities of N-oxides are moderate. Studies on the interaction mechanism showed that incorporation of particular compounds into model membranes varied. Hemolysing activities of compounds increased with the elongation of the alkyl chain but differed for corresponding compounds of particular series indicating that lipophilicity of compounds is not the only factor determing their interaction with erythrocyte membranes. DSC experiments showed that N-oxides, upon incorporation into 1,2-dipalmitoyl-3-sn-phosphatidylcholine liposomes, shifted the subtransition (Tp) and the main transition (Tm). The shifts observed depended on the alkyl chain length. The effects differed for each series. It seems that in the case of long alkyl chain compounds the domain formation may take place. Generally, the decrease of Tm was greatest for the same compounds that exhibited the best hemolytic efficacy. The same conclusion concerns the decrease of cooperativity of the main transition and the observed changes suggest an increase in membrane fluidity. Both, erythrocyte and DSC experiments seem to indicate that compounds of particular series incorporate in a somewhat different way into membranes.     

Inhibition of lipid peroxidation and protein oxidation in rat liver mitochondria by curcumin and its analogues

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, 1) is a yellow ingredient isolated from turmeric (curcumin longa). It has been shown to exhibit a variety of biological activities including antioxidative activity. In order to find more active antioxidants with 1 as the lead compound we synthesized curcumin analogues, i.e., 1-(3,4-dihydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (2), 1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione (3), 1,7-bis-(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione (4), 1-(3,4-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (5), 1,7-bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5-dione (6), and 1,7-diphenyl-1,6-heptadiene-3,5-dione (7), and evaluated their antioxidative activity. The in vitro oxidative damage to both lipids and proteins in rat liver mitochondria was used as a model to study the free radical-induced oxidative damage of biological lipids as well as proteins and the protective effects of these curcumin analogues. It was found that these compounds, except 6 and 7, could effectively inhibit the free radical induced lipid peroxidation and protein oxidative damage of rat liver mitochondria by H-atom abstraction from the phenolic groups. Compound 2 which bear ortho-diphenoxyl functionality exhibited remarkably higher antioxidative activity for lipids and proteins than curcumin and other analogues, and the 4-hydroxy-3-methoxyphenyl group also play an important role in the antioxidative activity.     

Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells.

The binding of secretory component (SC) to epithelial cells and its role in the specific uptake of immunoglobulin A (IgA) dimer has been studied in rabbit mammary gland and liver. SC, Mr approximately 80,000, secreted by epithelial cells of the mammary gland was found associated with the cell surface of mammary cells in intact tissue. Dispersed mammary cells and plasma membrane-enriched fractions obtained from mammary glands of midpregnant rabbits bound 125I-labeled SC in a saturable time- and temperature-dependent process. The association rate followed a second order reversible reaction (k+1 approximately equal to 2.7 x 10(6) M-1 min-1 at 4 degrees C) and equilibrium was reached in about 4 h at 4 degrees C. The dissociation rate for membranes was first order (k-1 approximately equal to 1.7 x 10(-2) min-1 at 4 degrees C), whereas displacement from cells was incomplete. The apparent affinity constant was similar for membranes and cells (Ka approximately equal to 5 x 10(8) M-1) with one class of binding sites. The number of binding sites varied from one animal to another (260 to 7,000 sites/mammary cell) in relation to endogenous occupancy by SC, which was assessed by immunocytochemistry and complement-mediated cytotoxicity. Rabbit liver and heart membranes did not bind SC, and serum proteins present in rabbit milk failed to interact with mammary cells or membranes. Mammary membranes or cells and liver membranes bound 125I-labeled IgA dimer in a saturable, reversible time- and temperature-dependent process. Association and dissociation rate constants at 4 degrees C (k+1 approximately equal to 5 x 10(6) M-1 min-1 and k-1 approximately equal to 5 x 10(-3) min-1, respectively) and the apparent affinity constant (Ka approximately equal to 10(9) M-1) were similar for liver and mammary membranes; these parameters differed, however, from those reported for free SC-IgA dimer interaction. The binding capacity of membranes for IgA dimer was directly related to the amount of free SC bound to membranes. Interaction of IgA dimer with mammary or liver membranes or cells was abrogated by excess of free SC and was prevented by preincubation of membranes or cells with Fab antibody fragments directed against SC. These data indicate that the first step in the translocation process of polymeric immunoglobulins across epithelia consists of binding of SC to the surface of epithelial cells which in turn acts as a receptor for the specific uptake of IgA dimer.     

Chelating and antiradical effect of rutin during peroxidation of lipids from microsomes and liposomes

The antioxidative effect of rutin (vitamin P) on Fe2+-induced lipid peroxidation (LPO) in bovine heart microsomes and lecithin liposomes was studied. It was shown that the LPO-induced inhibition of microsomes and liposomes in the presence of rutin occurs via two mechanisms, i.e., association of Fe2+ ions to form an inactive complex and a direct interaction between rutin and free radicals. The contribution of these mechanisms depends on the composition of the reaction mixture. In bovine heart microsomes and liposomes, ascorbic acid has a dual activity towards LPO. At high concentrations of Fe2+ necessary for LPO induction (approximately 1 x 10(-3) M), ascorbic acid blocks LPO, whereas at low Fe2+ concentrations (less than 1 x 10(-4) M) it has a prooxidative effect. A combined use of ascorbic acid and rutin results in an additive antioxidative effect at high Fe2+ concentrations (approximately 1.10(-3) M). However, at low Fe2+ concentrations rutin acts as an antagonist of the prooxidative effect of ascorbic acid.     

Effect of phytyl side chain of vitamin E on its antioxidant activity

Inhibition of the oxidation of methyl linoleate and soybean phosphatidylcholine in homogeneous solution and in aqueous dispersion by four chain-breaking antioxidants, vitamin E (alpha-tocopherol), 2,2,5,7,8-pentamethyl-6-chromanol, 2,6-di-tert-butyl-4-methylphenol, and stearyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate, was studied to examine the effect of the phytyl side chain of vitamin E on its antioxidant activity. These four antioxidants exerted similar antioxidative activities. They were also effective as antioxidants in protecting the oxidation of soybean phosphatidylcholine liposomes in water dispersion. However, when they were incorporated into dimyristoyl phosphatidylcholine liposomes, only 2,2,5,7,8-pentamethyl-6-chromanol and 2,6-di-tert-butyl-4-methylphenol could suppress the oxidation of soybean phosphatidylcholine liposomes dispersed in the same aqueous system. It was concluded that the antioxidative properties of vitamin E and its model without the phytyl side chain are quite similar within micelles and liposomes as well as in homogeneous solution but that the phytyl side chain enhances the retainment of vitamin E in liposomes and suppresses the transfer of vitamin E between liposomal membranes.     

Indole-3-propionic acid, a melatonin-related molecule, protects hepatic microsomal membranes from iron-induced oxidative damage: relevance to cancer reduction

Excessive free iron and the associated oxidative damage are commonly related to carcinogenesis. Among the antioxidants known to protect against iron-induced oxidative abuse and carcinogenesis, melatonin and other indole compounds recently have received considerable attention. Indole-3-propionic acid (IPA), a deamination product of tryptophan, with a structure similar to that of melatonin, is present in biological fluids and is an effective free radical scavenger. The aim of the study was to examine the effect of IPA on experimentally induced oxidative changes in rat hepatic microsomal membranes. Microsomes were preincubated in presence of IPA (10, 3, 2, 1, 0.3, 0.1, 0.01 or 0.001 mM) and, then, incubated with FeCl(3) (0.2 mM), ADP (1.7 mM) and NADPH (0.2 mM) to induce oxidative damage. Alterations in membrane fluidity (the inverse of membrane rigidity) were estimated by fluorescence spectroscopy and lipid peroxidation by measuring concentrations of malondialdehyde+4-hydroxyalkenals (MDA+4-HDA). IPA, when used in concentrations of 10, 3 or 2 mM, increased membrane fluidity, although at these concentrations it did not influence lipid peroxidation significantly. The decrease in membrane fluidity due to Fe(3+) was completely prevented by preincubation in the presence of IPA at concentrations of 10, 3, 2 or 1 mM. The enhanced lipid peroxidation due to Fe(3+) was prevented by IPA only at the highest concentration (10 mM). It is concluded that Fe(3+)-induced rigidity and, to a lesser extent, lipid peroxidation in microsomal membranes may be reduced by IPA. However, IPA in high concentrations increase membrane fluidity. Besides melatonin, IPA may be used as a pharmacological agent to protect against iron-induced oxidative damage to membranes and, potentially, against carcinogenesis.     

Adrenergic versus VIPergic control of cyclic AMP in human colonic crypts

The actions of catecholamines on VIP-induced cyclic AMP is studied in human colon. We show that: (1) Epinephrine in the 10(-7)-10(-3) M concentration range (ED50 = 11.10(-6) M) inhibits VIP-induced cyclic AMP rise in isolated colonic epithelial cells; the maximal inhibition reaches 30% of VIP effect; epinephrine alters the efficacy of the peptide and does not modify its potency; epinephrine also reduces the basal cyclic AMP level. (2) The inhibition is found with other alpha adrenergic agonists with the order of potencies epinephrine greater than norepinephrine greater than phenylephrine. Clonidine has a poor intrinsic activity but antagonizes the action of epinephrine. (3) The inhibition of VIP action by epinephrine is reversed by the alpha antagonists dihydroergotamine, phentolamine and the alpha 2 antagonist yohimbine, while unaffected by the beta antagonist propranolol and the alpha 1 antagonist prazosin, (4) Epinephrine inhibits VIP-stimulated adenylate cyclase activity in preparations of colonic plasma membranes. Thus catecholamines exert through an alpha 2 adrenoreceptor a negative control on basal and VIP-stimulated cyclic AMP formation in human colon. We suggest that colonic cyclic AMP metabolism undergoes a dual control: VIPergic, activator and adrenergic, inhibitor.     

Effects of coenzyme Q(10) administration on its tissue concentrations,mitochondrial oxidant generation,and oxidative stress in the rat

Coenzyme Q (CoQ(10)) is a component of the mitochondrial electron transport chain and also a constituent of various cellular membranes. It acts as an important in vivo antioxidant, but is also a primary source of O(2)(-*)/H(2)O(2) generation in cells. CoQ has been widely advocated to be a beneficial dietary adjuvant. However, it remains controversial whether oral administration of CoQ can significantly enhance its tissue levels and/or can modulate the level of oxidative stress in vivo. The objective of this study was to determine the effect of dietary CoQ supplementation on its content in various tissues and their mitochondria, and the resultant effect on the in vivo level of oxidative stress. Rats were administered CoQ(10) (150 mg/kg/d) in their diets for 4 and 13 weeks; thereafter, the amounts of CoQ(10) and CoQ(9) were determined by HPLC in the plasma, homogenates of the liver, kidney, heart, skeletal muscle, brain, and mitochondria of these tissues. Administration of CoQ(10) increased plasma and mitochondria levels of CoQ(10) as well as its predominant homologue CoQ(9). Generally, the magnitude of the increases was greater after 13 weeks than 4 weeks. The level of antioxidative defense enzymes in liver and skeletal muscle homogenates and the rate of hydrogen peroxide generation in heart, brain, and skeletal muscle mitochondria were not affected by CoQ supplementation. However, a reductive shift in plasma aminothiol status and a decrease in skeletal muscle mitochondrial protein carbonyls were apparent after 13 weeks of supplementation. Thus, CoQ supplementation resulted in an elevation of CoQ homologues in tissues and their mitochondria, a selective decrease in protein oxidative damage, and an increase in antioxidative potential in the rat.     

Antioxidant action of ubiquinol homologues with different isoprenoid chain length in biomembranes

Ubiquinones (CoQn) are intrinsic lipid components of many membranes. Besides their role in electron-transfer reactions they may act as free radical scavengers, yet their antioxidant function has received relatively little study. The efficiency of ubiquinols of varying isoprenoid chain length (from Q0 to Q10) in preventing (Fe2+ + ascorbate)-dependent or (Fe2+ + NADPH)-dependent lipid peroxidation was investigated in rat liver microsomes and brain synaptosomes and mitochondria. Ubiquinols, the reduced forms of CoQn, possess much greater antioxidant activity than the oxidized ubiquinone forms. In homogenous solution the radical scavenging activity of ubiquinol homologues does not depend on the length of their isoprenoid chain. However in membranes ubiquinols with short isoprenoid chains (Q1-Q4) are much more potent inhibitors of lipid peroxidation than the longer chain homologues (Q5-Q10). It is found that: i) the inhibitory action, that is, antioxidant efficiency of short-chain ubiquinols decreases in order Q1 greater than Q2 greater than Q3 greater than Q4; ii) the antioxidant efficiency of long-chain ubiquinols is only slightly dependent on their concentrations in the order Q5 greater than Q6 greater than Q7 greater than Q8 greater than Q9 greater than Q10 and iii) the antioxidant efficiency of Q0 is markedly less than that of other homologues. Interaction of ubiquinols with oxygen radicals was followed by their effects on luminol-activated chemiluminescence. Ubiquinols Q1-Q4 at 0.1 mM completely inhibit the luminol-activated NADPH-dependent chemiluminescent response of microsomes, while homologues Q6-Q10 exert no effect. In contrast to ubiquinol Q10 (ubiquinone Q10) ubiquinone Q1 synergistically enhances NADPH-dependent regeneration of endogenous vitamin E in microsomes thus providing for higher antioxidant protection against lipid peroxidation. The differences in the antioxidant potency of ubiquinols in membranes are suggested to result from differences in partitioning into membranes, intramembrane mobility and non-uniform distribution of ubiquinols resulting in differing efficiency of interaction with oxygen and lipid radicals as well as different efficiency of ubiquinols in regeneration of endogenous vitamin E.     

Calcium-Dependent Calmodulin Binding to Chromaffin Granule Membranes: Presence of a 65-Kilodalton Calmodulin-Binding Protein

The presence of calmodulin-binding sites on chromaffin granule membranes has been investigated. Saturable, high-affinity 125I-calmodulin-binding sites (KD = 9.8 nM; Bmax = 25 pmol/mg protein) were observed in the presence of 10(-4) M free calcium. A second, nonsaturable, calmodulin-binding activity could also be detected at 10(-7) M free calcium. No binding occurred at lower calcium levels. When chromaffin granule membranes were delipidated by solvent extraction, calmodulin binding was observed at 10(-4) M free calcium. However no binding was detected at lower calcium concentrations. Thus it appears that a calcium concentration of 10(-7) M promotes the binding of calmodulin to some solvent-soluble components of the chromaffin granule membrane. Calmodulin-binding proteins associated with the granule membrane identified by photoaffinity cross-linking. A calmodulin-binding protein complex, of molecular weight 82K, was formed in the presence of 10(-4) M free calcium. This cross-linked product was specific because it was not detected either in the absence of calcium, in the presence of nonlabeled calmodulin, or in the absence of cross-linker activation. When solvent-treated membranes were used, a second, specific, calmodulin-binding protein complex (70K) was formed. Since the apparent molecular weight of calmodulin in our electrophoresis system was 17K, these experiments suggested the presence of two calmodulin-binding proteins, of molecular weights 65K and 53K, in the chromaffin granule membrane. This result was confirmed by the use of calmodulin-affinity chromatography. When detergent-solubilized membranes were applied on the column in the presence of calcium, two polypeptides of apparent molecular weights of 65K and 53K were specifically eluted by EGTA buffers. Since detergent treatments or solvent extractions are necessary to detect the 53K calmodulin-binding protein, it is concluded that only the 65K calmodulin-binding polypeptide may play a role in the interaction between calmodulin and secretory granules in chromaffin cells.     

The binding of ribosomal subunits to endoplasmic reticulum membranes

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60-80% of added 60S subunits and 10-15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane-60S subunit-40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.     

Mechanism of desensitization of adenylate cyclase in lutropin. GTP-dependent uncoupling of the receptor

Lutropin-sensitive adenylate cyclase ((EC 4.6.1.1) ATP pyrophosphate-lyase (cyclizing)) in purified rat ovarian plasma membranes is stimulated by lutropin 2- to 3-fold in the absence, but 15- to 20-fold in the presence of GTP or p(NH)ppG. Following 10 to 15 min of incubation at 30 degrees C in the presence of lutropin, enzyme activity declined (50%) in the presence of GTP but not in the presence of p(NH)ppG. This desensitizing process induced by lutropin and GTP is not seen if NaF is also included in the incubation medium. The desensitized state of the enzyme persists at 4 degrees C in membranes washed free of the incubation medium. In this state the enzyme is characterized by: (i) a reduced response to lutropin even in the presence of p(NH)ppG; (ii) its response to NaF is not different from that of untreated enzyme; (iii) it reconverts to a fully responsive state following incubation (10 min, 30 degrees C) in GTP-free medium, a process accelerated by p(NH)ppG; (iv) the receptor content as well as the stability of the receptor.hormone complex does not differ from that of untreated fully responsive enzyme. It is proposed that desensitization results from a GTP-dependent, hormone-stimulated reaction that leads to impaired coupling of the enzyme system. The desensitized state induced is transient and may revert to a responsive one under specified conditions.     

Positive inotropic effect of ethylephrine on the isolated rat atria

Ethylephrine, a sympathomimetic amine which belongs to the phenolamine group, was assayed on the driven left rat atrium. The frequency response curve was performed for norepinephrine and ethylephrine. The maxima was attained for both compounds at 1 Hz. The agonist under study has an inotropic action less potent than the classical catecholamines. Propranolol (10(-8) and 10(-7) M) produced a parallel shift to the right in the log dose-response curves of ethylephrine with no decrease in the maximal response, indicating that the antagonism was competitive. In the presence of cocaine or with reserpine-pretreatment the sensitivity of the preparation to the amine did not vary. The alpha-blocker, phentolamine (10(-8) to 3.10(-5) M) did not possess an inotropic effect per se. In contrast, phentolamine, delivered to the bath beforehand, did not block the agonist. However at 10(-8) and 10(-7) M increase the maximal response both in normal and reserpinized preparations. It is suggested that ethylephrine is a direct inotropic preparation. It is suggested that ethylephrine is a direct inotropic agent on the driven left rat atrium and its effects are mediated by beta-receptors. The results also indicate the lack of evidence that ethylephrine has any action on the alpha-receptors.     

5-methoxytryptophol preserves hepatic microsomal membrane fluidity during oxidative stress

Lipid peroxidation is a degenerative chain reaction in biological membranes that may be initiated by exposure to free radicals. This process is associated with changes in the membrane fluidity and loss of several cell membrane-dependent functions. 5-methoxytryptophol (ML) is an indole isolated from the mammalian pineal gland. The purpose of this study was to investigate the effects of ML (0. 01mM-10mM) on membrane fluidity modulated by lipid peroxidation. Hepatic microsomes obtained from rats were incubated with or without ML (0.01-10 mM). Then lipid peroxidation was induced by FeCl(3), ADP, and NADPH. Membrane fluidity was determined using fluorescence spectroscopy. Malonaldehyde (MDA) +4-hydroxyalkenals (4-HDA) concentrations were estimated as an indicator of the degree of lipid peroxidation. With oxidative stress, membrane fluidity decreased and MDA+4-HDA levels increased. ML (0.01-3 mM) reduced membrane rigidity and the rise in MDA+4-HDA formation in a concentration-dependent manner. 10 mM ML protected against lipid peroxidation but failed to prevent the membrane rigidity. In the absence of oxidative reagents, ML (0.3-10 mM) decreased membrane fluidity whereas MDA+4-HDA levels remained unchanged. This indicates that ML may interact with membrane lipids. The results presented here suggest that ML may be another pineal indoleamine (in addition to melatonin) that resists membrane rigidity due to lipid peroxidation.     

Pharmacological control of muscular activity in the sea urchin larva--IV. Effects of monoamines and adenosine.

1. The muscular activity of the sea urchin pluteus is affected by catecholamines. alpha-Agonists in high concentrations bring about a strong, temporary, stimulation. 2. The stimulation by beta-agonists tends to be masked by different mechanisms. 3. Serotonin brings about a strong stimulation of long duration, even at 10(-7) M. It reactivates larvae where the activity has declined after exposure to catecholamines. A dopamine-effect at 10(-5)-5 x 10(-6) M is similar to that of serotonin. The effect of adenosine is similar to that of dc-c-AMP. 4. The action of the alpha- and beta-agonists and adenosine appears to involve an increased respectively excessive Ca2(+)-influx, directly or indirectly mediated by c-AMP. 5. It is suggested that a strong Ca2(+)-influx induces an outflux of K+ leading to hyperpolarization. Serotonin may decrease the K(+)-outflux.     

Action of DCFH and BODIPY as a probe for radical oxidation in hydrophilic and lipophilic domain

Fluorogenic probes such as 2',7'-dichlorofluorescin (DCFH) have been extensively used to detect oxidative events and to measure antioxidant capacity. At the same time, however, the inherent drawbacks of these probes such as non-specificity towards oxidizing species have been pointed out. The present study was carried out to analyze the action and dynamics of 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY) and DCFH as a fluorescent probe in the free radical-mediated lipid peroxidation in homogeneous solution, aqueous suspensions of liposomal membranes and LDL and plasma. The rate constant for the reaction of BODIPY with peroxyl radicals was estimated as 6.0 x 10(3) M(-1) s(-1), which makes BODIPY kinetically an inefficient probe especially in the presence of potent radical-scavenging antioxidants such as tocopherols, but a convenient probe for lipid peroxidation. On the other hand, the reactivity of DCFH toward peroxyl radicals was as high as Trolox, a water-soluble analogue of alpha-tocopherol. Thus, DCFH is kinetically more favored probe than BODIPY and could scavenge the radicals within lipophilic domain as well as in aqueous phase. The partition coefficients for BODIPY and DCFH were obtained as 4.57 and 2.62, respectively. These results suggest that BODIPY may be used as an efficient probe for the free radical-mediated oxidation taking place in the lipophilic domain, especially after depletion of alpha-tocopherol, while it may not be an efficient probe for detection of aqueous radicals.     

Investigations on the binding and antioxidant properties of the plant flavonoid fisetin in model biomembranes

Plant flavonoids are emerging as potent therapeutic drugs for free radical mediated diseases, for which cell membranes generally serve as targets for lipid peroxidation and related deleterious effects. Screening and characterization of these ubiquitous, therapeutically potent polyphenolic compounds, require a clear understanding regarding their incorporation and possible location in membranes, as well as quantitative estimates of their antioxidative and radical scavenging capacities. Here, we demonstrate the novel use of the intrinsic fluorescence characteristics of the plant flavonoid fisetin (3,3^′,4^′,7-OH flavone) to explore its binding and site(s) of solubilisation in egg lecithin liposomal membranes. Spectrophotometric assays have been used to obtain quantitative estimates of its antioxidative capacity. Furthermore, our quantum mechanical semi-empirical calculations provide a quantitative measure for the free radical scavenging activity of fisetin from the OH (at 3, 3^′, 4^′, 7 positions of the molecule)-bond dissociation enthalpies. Implications of these findings are discussed.     

Activity and phosphorylation state of glucose transporters in plasma membranes from insulin-, isoproterenol-, and phorbol ester-treated rat adipose cells

The counterregulatory action of catecholamines on insulin-stimulated glucose transport and its relation to glucose transporter phosphorylation were studied in isolated rat adipose cells. Plasma membranes exhibiting reduced glucose transport activity were prepared as described previously (Joost, H. G., Weber, T. M., Cushman, S. W., and Simpson, I. A. (1986) J. Biol. Chem. 261, 10033-10036) from cells treated with insulin, and subsequently with isoproterenol and adenosine deaminase. In these membranes, transporter affinity for cytochalasin B binding was significantly reduced (KD = 133.5 +/- 14 versus 89.8 +/- 11 nM, means +/- S.E.) with no change in number of sites or immunoreactivity of the transporter on Western blots. Reconstituted plasma membrane transport was significantly lower with isoproterenol treatment (0.50 +/- 0.12 versus 0.97 +/- 0.27 nmol/mg protein/10 s). In contrast, transport activity reconstituted from corresponding intracellular transporters (from low density microsomes) was unchanged (5.4 +/- 2.2 versus 6.9 +/- 1.2 nmol/mg protein/10 s). Thus, the intrinsic activity change of the transporter produced by catecholamines appears to reflect a structural modification that is confined to the plasma membrane and not recycled into the intracellular compartment. In cells equilibrated with [32P]phosphate, neither insulin nor isoproterenol induced [32P]phosphate incorporation into the glucose transporter immunoprecipitated from plasma membranes. Conversely, phorbol 12-myristate 13-acetate stimulated significant incorporation of [32P]phosphate into the glucose transporter in insulin-stimulated cells without any change in plasma membrane transport activity or transporter concentration. Thus, the phosphorylation state of the glucose transporter does not seem to be involved in either signaling transporter translocation or triggering changes in transporter intrinsic activity.     

Calcium inhibition of cardiac adenylyl cyclase. Evidence for two distinct sites of inhibition

Increasing the free calcium concentration from 10(-8) M to 10(-4) M inhibited cardiac sarcolemmal adenylyl cyclase activated by the addition of 5 X 10(-4) M forskolin or 1 X 10(-4) M GTP or Gpp(NH)p. The calcium inhibition curve in the presence of all three activators was shallow and best fit by a two site model of high affinity (less than 1.0 microM) and low affinity (greater than 0.1 mM). Gpp(NH)p appeared to decrease the sensitivity of adenylyl cyclase to inhibition by calcium at the high affinity site. Similar inhibition constants were obtained with each of the activators. Calmodulin content of native freeze-thaw vesicles was 76.2 +/- 14.2 ng/mg. Treatment of the vesicles with 1 mM EGTA to remove calmodulin significantly reduced calmodulin content to 19.7 +/- 1.35 ng/mg. This treatment had no significant effect on the calcium inhibition profile. Increasing free calcium to 3 X 10(-6) M was shown to have no effect on the EC50 estimated for either Gpp(NH)p or forskolin but did slightly increase the EC50 estimated for Mg2+ in the presence of maximal concentrations of either activator. Nevertheless, maximally stimulating concentrations of Mg2+ were unable to overcome calcium inhibition. Pretreatment of sarcolemmal membranes with pertussis toxin was shown to have no significant effect on calcium inhibition of adenylyl cyclase. The results suggest that the overall inhibitory action of calcium was most likely calmodulin independent and involved a direct interaction with the catalytic subunit at two distinct sites of high and low affinity. At the low affinity site calcium most likely competes with Mg2+ for an allosteric divalent cation binding site.(ABSTRACT TRUNCATED AT 250 WORDS)