Cyclosporine A- and FK506-induced apoptosis in PC12 cells.

The purpose of this study was to examine, using glycogen synthase kinase (GSK) inhibitors, whether GSK-3 is involved in cyclosporine A (CsA)- and FK506-induced apoptosis in PC12 cells. CsA and FK506 increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation or fragmentation. Nerve growth factor (NGF) completely blocked cell death. Caspase-3 activation was accompanied by CsA- and FK506-induced cell death and inhibited by NGF. GSK-3 inhibitors such as alsterpaullone and SB216763 prevented CsA- and FK506-induced apoptosis. These results suggest that CsA and FK506 induce caspase-dependent apoptosis and that GSK-3 activation is involved in CsA- and FK506-induced apoptosis in PC12 cells.

A marked stimulation of Fe(3+)-dependent lipid peroxidation in phospholipid liposomes under acidic conditions.

Lipid peroxidation in phosphatidylcholine liposomes induced by Fe(3+) alone, assessed by thiobarbituric acid-reactive substances (TBARS) production, was markedly enhanced as the solution pH was lowered from 7.4 to 5.5. On the other hand, at physiological pH, TBARS production by Fe(3+) was almost negligible. Results of the radical scavenger experiments with superoxide dismutase, catalase and hydroxyl radical ((&z.rad;)OH) scavengers (sodium benzoate, mannitol and dimethylthiourea), deoxyribose degradation and ESR spectrometry suggest that the stimulation of Fe(3+)-dependent lipid peroxidation under acidic conditions is involved in generation of superoxide anion (O(2)(&z.rad;-)), hydrogen peroxide (H(2)O(2)) and (&z.rad;)OH during the reaction. The stimulation of Fe(3+)-dependent TBARS production by increasing the [H(+)] completely disappeared by triphenylphosphine (TPP) treatment of the liposomes, but the reaction was reversible with either incorporation of cumen hydroperoxide (CumOOH) into the TPP-treated liposomes or the addition of CumOOH to the treated liposomes. Incubation of the CumOOH-incorporated TPP-treated liposomes with Fe(3+) at pH 5.5 also resulted in (&z.rad;)OH generation. Based on these results, a possible mechanism of stimulatory effect of Fe(3+) on lipid peroxidation under acidic conditions is discussed.

Modification of ceftibuten transport by the addition of non-ionic surfactants.

The effects of non-ionic surfactants on the carrier-mediated transport of ceftibuten by rat intestinal brush-border membrane vesicles (BBMVs) were investigated. Ceftibuten uptake by BBMVs was measured by a rapid filtration technique. The concentration of surfactants for the uptake experiments was determined by a decrease in the turbidity of BBMV suspension and by the release of an impermeable probe, 2',7'-bis(carboxyethyl)-4(5)-carboxyfluorescein, from the vesicle inside. In fact, the surfactant concentration of 0. 03% (w/v) was selected to maintain the stability of BBMVs. The extent of ceftibuten uptake by BBMVs with various surfactants was correlated with their physicochemical properties, i.e. hydrophile-lipophile balance (HLB), critical micelle concentration (c.m.c.), average diameter of micelle colloid, and polydispersity determined by particle size distribution. The surfactants used were divided into two groups on the basis of polydispersity index (d(w)/d(n)), i.e. low polydispersity (d(w)/d(n) congruent with1) and high polydispersity d(w)/d(n)2). The ceftibuten uptake due to the addition of surfactants with low polydispersity increased with a decrease in the HLB number. These results indicate that the ceftibuten transport is modulated by the size distribution and hydrophobicity of surfactants. In addition, the effects of surfactants on the membrane lipid fluidity monitored by diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) were investigated. There was significant correlation between ceftibuten uptake and the fluorescence anisotropy of TMA-DPH-labeled membranes due to the addition of surfactants with low polydispersity (r=-0.81, P<0.0001). These results suggest that surfactants with low polydispersity, in part, increase or decrease the outer membrane leaflet, thereby enhancing or suppressing the ceftibuten transport by BBMVs, and that ceftibuten transport caused by surfactants with low polydispersity may be strongly dependent on the hydrophobic interaction.

Detection of superoxide anion radical in phospholipid liposomal membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran.

Utilization of a fluorescence dye, 1,3-diphenylisobenzofuran (DPBF) as a detector of superoxide anion radical (O2*-) was examined. The fluorescence intensity of DPBF incorporated in phospholipid liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) is effectively quenched by incubation with xanthine/xanthine oxidase system. On the other hand, xanthine or xanthine oxidase alone did not induce quenching of the DPBF fluorescence in the liposomes. Xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes was almost completely protected by the addition of superoxide dismutase (SOD, 1 U/ml), but not by heat-denatured SOD (10 min boiling) at the same concentration. On the other hand, catalase (1 U/ml), and hydroxyl radical and singlet oxygen scavengers (10 mM sodium benzoate, 300 mM mannitol, 1 mM tryptophan and 1 mM sodium azide) did not protect xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes. The concentration dependence profiles of xanthine oxidase on the DPBF fluorescence quenching and O2*- generation showed that there is a good correlation between these parameters. Under the present experimental conditions, approximately 7 microM H(2)O(2)/30 min were produced, but the addition of H(2)O(2) (1 mM) to DPBF-labeled liposomes did not quench the dye fluorescence in the liposomes. Temperature dependence profiles of the DPBF fluorescence quenching induced by xanthine/xanthine oxidase treatment and the excimer fluorescence formation of pyrene molecules embedded in the liposomal membrane suggested that the quenching efficiency of the DPBF fluorescence is largely dependent on their lipid dynamics. Based on these results, we proposed the possibility that DPBF fluorescence quenching method is able to be used as a simple method for detecting O2*- inside the membrane lipid layer and that DPBF fluorescence quenching by O2*- is controlled by the physical state of membrane lipids.

Apoptotic cell death and caspase-3 activation induced by N-methyl-D-aspartate receptor antagonists and their prevention by insulin-like growth factor I.

The effect of N-methyl-D-aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK-801 and 2-amino-5-phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK-801 (an NMDA antagonist) for 1-2 days induced apoptotic cell death in a dose-dependent manner (1 nM to 10 microM). NMDA (25 microM), however, inhibited the MK-801 (0.1 microM)-induced apoptotic cell death. MK-801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase-3 was accompanied by MK-801-induced cell death in a dose-dependent manner, and an inhibitor of caspase-3 reduced the cell death. Further, cycloheximide (0.2 microg/ml) completely protected the cells from MK-801-induced apoptotic cell death and caspase-3 activation. Insulin-like growth factor I completely attenuated MK-801-induced apoptotic cell death and caspase-3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.

A marked stimulation of Fe2+-initiated lipid peroxidation in phospholipid liposomes by a lipophilic aluminum complex, aluminum acetylacetonate.

In the present study, the efficacy of a lipophilic Al complex, aluminum acetylacetonate, as a stimulator of Fe2+-initiated lipid peroxidation in phospholipid liposomes was examined, and results were compared with those from the liposomes treated with AlCl3. The extent of lipid peroxidation was assessed by the formation of thiobarbituric acid-reactive substances (TBARS). The results indicated that the stimulatory effect of Al complex on Fe2+-initiated lipid peroxidation in phosphatidylcholine liposomes was more effective than that of AlCl3 under the same conditions. The concentration dependence of Al complex on TBARS production showed that the concentration of the complex required to induce half-maximal stimulation of TBARS production was 43 microM. In contrast, the stimulatory effect of AlCl3 was not observed until the AlCl3 concentration is increased above 300 microM. In addition, it was found that there is a linear relationship between the TBARS values and the residual amounts of Fe2+ at an earlier stage (within 2 min after the addition of Fe2+) of the lipid peroxidation in PC liposomes with different concentrations of Al complex, suggesting that Fe2+ oxidation process is closely related to the stimulatory effect of Al complex. The stimulatory effect of Al complex upon the lipid peroxidation completely disappeared by treatment of Al complex-treated liposomes with Triton X-100. The results of fluorescence anisotropy measurements using 12-(9-anthroyloxy)stearic acid-labeled liposomes suggested that treatment of the liposomes with Al complex caused a decrease in their lipid fluidity. Furthermore, it was found that there is a correlation between the extents of the fluorescence anisotropy and the Fe2+ oxidation parameters in the liposomes with different concentrations of Al complex. From these results, it is suggested that the Al effect on Fe2+-initiated lipid peroxidation in the phospholipid liposomes is markedly enhanced by incorporation of Al complex into the liposomal membranes and that an acceleration of Fe2+ oxidation due to a strengthened packing between the acyl chains in the lipid layer may be one possible mechanism for the occurrence of a marked stimulatory effect of Al complex on Fe2+ initiated lipid peroxidation.

Apoptotic cell death and CPP32-like activation induced by thapsigargin and their prevention by nerve growth factor in PC12 cells.

Thapsigargin, an endoplasmic reticular Ca2+-ATPase inhibitor, induced apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by the activation of CPP32-like protease, a member of the interleukin-1beta converting enzyme protease (ICE) family, but not the activation of ICE-like protease. Nerve growth factor (NGF) completely inhibited the cell death and CPP32-like activation induced by thapsigargin while Ac-Asp-Glu-Val-Asp-CHO, an inhibitor of CPP32-like protease, reduced the cell death. PD98059, a specific inhibitor of Map kinase kinase, did not reduce the protective effect of NGF on thapsigargin-induced cell death. These results suggest that calcium ion-induced apoptotic cell death was mediated by CPP32-like, but not ICE-like, protease and was regulated by a neurotrophic factor possibly, through the Map kinase cascade independent pathway.

Apoptotic cell death and caspase 3 (CPP32) activation induced by calcium ionophore at low concentrations and their prevention by nerve growth factor in PC12 cells.

A23187 (a calcium ionophore) at low concentration (0.1 microM) induced apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by the activation of caspase-3 (CPP32), a member of the interleukin-1beta-converting enzyme protease. On the other hand, A23187 at high concentration (2 microM) induced necrotic cell death not accompanied by the activation of CPP32. Nerve growth factor inhibited the cell death and CPP32 activation induced by 0.1 microM A23187, but not the cell death induced by 2 microM A23187. Acylaspartyl-glutamyl-valyl-aspartyl-aldehyde, an inhibitor of CPP32, reduced the cell death induced by 0.1 microM A23187. These results suggest that calcium-ion-induced apoptotic cell death was mediated by CPP32 activation in PC12 cells.

Effect of aluminum ion on Fe(2+)-induced lipid peroxidation in phospholipid liposomes under acidic conditions.

The effects of Al3+ on Fe(2+)-induced lipid peroxidation in phospholipid liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) were examined under acidic conditions. The stimulatory effect of Al3+ on Fe(2+)-induced lipid peroxidation in the liposomes showed a biphasic response against pH variation, and the maximum stimulation was observed around pH 6.0. In addition, it was found that the stimulatory effect of Al3+ on the lipid peroxidation was dependent on the proportion of PS in the liposomes. On the other hand, the lipid peroxidation in PC liposomes was not stimulated by the addition of Al3+. From these findings, it is suggested that the Al3+ effect on Fe(2+)-induced lipid peroxidation under acidic conditions is largely dependent on the phospholipid composition. Trivalent cations such as Tb3+ and Ga3+ also stimulated Fe(2+)-induced lipid peroxidation in PC/PS liposomes under acidic conditions, but divalent cations (Zn2+ and Mn2+) showed no stimulatory effect. The extents of Fe2+ disappearance and Fe3+ formation during the reaction were enhanced by the addition of Al3+ or Ga2+, but Tb3+ had no effect on Fe2+ disappearance. The results with 1,6-diphenyl-1,3,5-hexatriene (DPH) showed that the fluorescence anisotropy of DPH-labeled PC/PS liposomes under acidic conditions was increased by the addition of Al3+. Furthermore, there is a relation between the extents of the fluorescence anisotropy of the complex and TBARS production. In contrast, the fluorescence anisotropy of DPH molecules embedded in PC liposomes was not changed by the addition of Al3+. Based on these results, a possible mechanism of the stimulatory effect of Al3+ on Fe(2+)-induced lipid peroxidation under acidic conditions is discussed.

Contribution of peroxidation products to oxidative inactivation of rat liver microsomal glucose-6-phosphatase.

Exposure of rat liver microsomes to ascorbic acid/Fe(2+) caused decreases in the membrane-bound glucose-6-phosphate (G-6-Pase) activity and the protein thiols after a short lag period (4 min). Under the same conditions, the production of thiobarbituric acid-reactive substances and fluorescent products was also initiated from 4 min after the start of the treatment, although conjugated diene was formed immediately on incubation of the microsomes with ascorbic acid/Fe(2+). After centrifugation of the treated microsomes, the fluorescent products and the enzyme activity remained in the membrane fraction. The results of kinetic studies of the enzyme activity indicated that ascorbic acid/Fe(2+)-induced inhibition of the enzyme activity is mainly due to an increased Km value for the substrate. A decreased activity of the microsomal G-6-Pase was also observed when the microsomes were incubated with aldehydes such as malondialdehyde, n-heptaldehyde, acetaldehyde, and trans-2-nonenal. However, loss of protein thiols was detected only upon treatment of the microsomes with trans-2-nonenal. Glucose-6-phosphate (G-6-P)effectively prevented ascorbic acid/Fe(2+)- or trans-2-nonenal-induced inhibition of the enzyme activity, but the substrate failed to protect the protein thiols in both systems. The results of fluorescence anisotropy measurements of diphenylhexatriene-labeled microsomes suggested that changes in the lipid dynamics are not directly related to peroxidation- mediated inhibition of the enzyme activity. Based on these results, a possible reason for the inhibition of the microsomal G-6-Pase activity associated with ascorbic acid/Fe(2+) treatment is discussed.

In vitro influences of alcohols on mouse synaptosomes, and structure-activity relationships.

Little information is available on the structure-central nervous system membrane toxicity relationship of alcohols. The purpose of the present study was to study in vitro influence of alcohols (n = 20) on the activity of the toxic indicator Na+/K(+)-adenosine triphosphatase (Na+/K(+)-ATPase) and acetylcholinesterase (AchE), and membrane fluidity in mouse brain synaptosomes, in terms of the structure-activity relationship. The potency of inhibition for the enzymes (IC50) and the potency of increasing membrane fluidity (IC12.5) were determined experimentally, and n-octanol/water partition coefficient (P) and the steric constant Taft Es are cited from the literature. Regression analysis revealed that log 1/IC50 for Na+/K(+)-ATPase is a function of log P and Taft Es. The situation was true for AchE activity. The results indicate that the hydrophobicity expressed as log P and the steric effect of the alcohols play an important role in inhibiting both enzyme activities. A linear relationship between log 1/IC12.5 for membrane fluidity and log P is shown, indicating a significant effect of the alcohols on membrane fluidity. Based on these results, it is suggested that the alcohols inhibit the Na+/K(+)-ATPase and AchE activity through a direct action on the enzymes and/or through changing the membrane fluidity.

Physical-chemical-activity relationship of organic solvents: effects on Na(+)-K(+)-ATPase activity and membrane fluidity in mouse synaptosomes.

Physical-chemical-activity relationship of aromatic hydrocarbons (n = 10) and alkyl acetates (n = 16) with respect to their in vitro effects on synaptosomal membranes was studied. Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase) activity and membrane fluidity, which was determined using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene, were used as potential indicators of neuronal cell toxicity. The potency of inhibition for the enzyme (IC50), the potency of increasing membrane fluidity (IC12.5), and n-octanol/water partition coefficient (P) were all determined experimentally for 26 solvents. Correlation analyses were made on aromatic hydrocarbons and on alkyl acetates. There were linear relationships between log P and pIC50 (log1/IC50) values, and between log P and pIC12.5 (log1/IC12.5) values, indicating that the hydrophobicity of the solvents determines their toxic ability to affect membrane environment; the more hydrophobic the solvents are, the more toxic they are. A direct linear relationship between Na(+)-K(+)-ATPase activity pIC50 and membrane fluidity pIC12.5 values was also shown. This predictive correlation suggests a similar mechanism of membrane surface interaction govering both processes that are common to the test solvents. The present results confirm the importance of the lipid environment of neuronal membranes in maintaining the normal function of membrane-bound protein.

Oxygen radical-induced inhibition of alkaline phosphatase activity in reconstituted membranes.

The effects of lipid peroxidation on membrane-bound enzyme activity were examined using reconstituted membranes consisting of intestinal alkaline phosphatase (ALP) and phosphatidylcholine (PC) or dipalmitoylphosphatidylcholine (DPPC). When the PC-reconstituted membranes were incubated with ascorbic acid/Fe2+, the ALP activity decreased with increases in the thiobarbituric acid-reactive substances and conjugated diene values in a time-dependent manner. The kinetic studies on the ALP activity with varying the p-nitrophenyl phosphate or beta-glycerophosphate concentrations showed that the inhibition of the enzyme activity by treatment with these oxidizing agents is mainly due to a decrease in the Vmax value rather than a change in the Km value. The results with several antioxidants suggested that ascorbic acid/Fe(2+)-induced inhibition of the ALP activity is related to generation of .OH radicals. Modification of the reconstituted membranes with malondialdehyde, trans-2-nonenal, or n-heptaldehyde did not affect the ALP activity, suggesting that the secondary degraded products of lipid hydroperoxides had no influence on the enzyme activity. Increasing bityrosine production in the membrane constituents was observed by ascorbic acid/Fe2+ treatment, depending on the incubation time. This finding suggests the possibility that amino acid modifications in the protein molecule are induced by the treatment. Furthermore, the contribution of the lipid organization in ascorbic acid/Fe(2+)-induced inhibition of the ALP activity in the reconstituted membranes was examined by measurements of the fluorescence anisotropy of diphenylhexatriene-labeled membranes. In addition, it was found that the ALP activity in DPPC-reconstituted membranes was also inhibited by treatment with ascorbic acid/Fe2+, similar to the case in PC-reconstituted ones. On the basis of these results, a possible mechanism of ascorbic acid/Fe(2+)-induced inhibition of membrane-bound ALP activity is discussed.

Inhibition of Ca(2+)-induced aggregation of porcine intestinal brush-border membranes by lipid peroxidation.

The effects of lipid peroxidation on Ca(2+)-induced aggregation of porcine intestinal brush-border membranes were examined using a system consisting of ascorbic acid/Fe2+/tert-butyl hydroperoxide (t-BuOOH). Incubation of the membranes with ascorbic acid/Fe2+/t-BuOOH resulted in inhibition of Ca(2+)-induced aggregation of the membranes with the formation of thiobarbituric acid-reactive substances, depending on the hydroperoxide concentration and the incubation time. The inhibition of the membrane aggregation associated with ascorbic acid/Fe2+/t-BuOOH treatment was effectively prevented by the addition of an antioxidant, 3(2)-tert-butyl-4-hydroxyanisole, to the reaction mixture. Studies with 8-anilino-1-naphthalenesulfonate (ANS) revealed that there is a linear relationship between the apparent dissociation constants (Kd) of ANS-membrane complexes and the aggregating efficiencies of the membranes with different levels of lipid peroxidation This suggests that inhibition of the membrane aggregation by lipid peroxidation involves a change in the membrane surface charge density. Modification of the membranes with malondialdehyde also resulted in a decrease in the aggregating efficiency of the membranes with a decrease in the Kd value of ANS-membrane complex. In addition, the contribution of the lipid organization to membrane aggregation was examined by measuring the fluorescence anisotropy of diphenylhexatriene-labeled membranes in the presence of a lipid fluidizer, benzyl alcohol. The results are discussed in terms of peroxidation-induced inhibition of intramembrane interactions of the membranes.

Changes in SH reactivity of the protein in porcine intestinal brush-border membranes associated with lipid peroxidation.

The effects of lipid peroxidation on the SH reactivity of the proteins in porcine intestinal brush-border membranes were examined using a fluorogenic thiol reagent, N-[7-dimethyl-amino-4-methylcoumarinyl]maleimide (DACM) in relation to lipid organization. Changes in the lipid organization were assessed by measurement of the rate of incorporation of 1,6-diphenyl-1,3,5-hexatriene (DPH) into the membrane lipids and the fluorescence anisotropy of DPH-labeled membranes. Treatment of the membranes with an oxygen-radical-generating system, i.e., ascorbic acid/Fe2+/tert-butyl hydroperoxide (t-BuOOH), resulted in decrease in the rate of DACM incorporation into the SH groups of the membrane proteins (DACM-labeling) and the amount of DACM labeled to the SH groups with a decrease in the lipid fluidity, depending on the formation of thiobarbituric acid-reactive substances and conjugated diene. Pretreatment of the membranes with diphenylamine effectively prevented the ascorbic acid/Fe2+/t-BuOOH-induced decreases in the DACM-labeling and DPH incorporation rates, whereas neither superoxide dismutase, catalase, sodium benzoate, nor mannitol showed a protective effect. The contribution of the lipid fluidity to the SH reactivity to DACM of the proteins in the membranes with different levels of lipid peroxidation was further examined using a lipid fluidizer, benzyl alcohol. The results showed that the DPH incorporation rate increased in proportion to increasing concentrations of the alcohol regardless of the peroxidation level of the membranes, whereas the susceptibility of the SH reactivity of the membrane proteins as to benzyl alcohol transitionally changed as the membranes were peroxidized to levels greater than 400 nmol conjugated diene/mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of Fe(2+)-induced lipid peroxidation in phosphatidylcholine liposomes by aluminium ions at physiological pH.

The effects of aluminium ions on Fe(2+)-induced lipid peroxidation in egg yolk phosphatidylcholine liposomes were examined under various conditions. The degree of Fe(2+)-induced lipid peroxidation of the liposomes was dependent on pH of the reaction mixture: pH 5.0 > pH 7.4. However, Fe2+ did not induce lipid peroxidation in the liposomes at pH 9.0. The addition of AlCl3 to the liposomal suspension resulted in a marked stimulation of Fe(2+)-induced liposomal peroxidation at pH 7.4, depending on the concentration of AlCl3. On the other hand, Fe3+, Cu2+, Pb2+, Cd2+ and Cr6+ did not induce lipid peroxidation in the liposomes at pH 7.4 regardless of the presence and absence of AlCl3. Fe3+ enhanced Fe(2+)-induced liposomal peroxidation at pH 7.4 but is unrelated to the stimulatory effect of AlCl3. In the absence of AlCl3, Fe(2+)-induced liposomal peroxidation was observed after a lag phase of about 15 min. The lag phase of the reaction was shortened by the addition of AlCl3 in a dose-dependent fashion. The shortening of the lag phase was also observed by the decrease of Fe2+ concentration or by the co-presence of Fe3+ in the reaction mixture. In addition, it was found that AlCl3 stimulates Fe2+ disappearance and Fe3+ formation. The addition of AlCl3 to the liposomal suspension at pH 7.4 resulted in a marked increase of the turbidity of the suspension. On the other hand, the turbidity of the liposomal suspension at pH 5.0 did not change by the addition of AlCl3.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-toxicity relationship of monoketones: in vitro effects on beta-adrenergic receptor binding and Na(+)-K(+)-ATPase activity in mouse synaptosomes.

The structure-toxicity relationship of monoketones, a class of organic solvents widely used in industry, was investigated with respect to their in vitro effects on synaptosomal membrane proteins. The toxic parameters used were Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase), a well-known marker enzyme often used as a membrane toxicity model, and 3H-dihydroalprenolol (3H-DHA)-labeled beta-adrenergic receptor binding that has been shown to be vulnerable to solvent-induced changes in membrane fluidity. In vitro treatments with 12 kinds of monoketones (carbon chain length from 3-10) dose-dependently inhibited both 3H-DHA binding to mouse synaptosomes and Na(+)-K(+)-ATPase activity. The potency of inhibition (IC50) for both the two parameters was linearly related to n-octanol/water partition coefficient and synaptosome/buffer partition coefficient of the test compounds. Additions of monoketones did not significantly alter the number of 3H-DHA binding sites but markedly decreased their affinity. In each monoketone, the IC50 values for 3H-DHA binding and Na(+)-K(+)-ATPase activity were generally within the same range. The anisotropy of fluorescence probe 1,6-diphenyl-1,3,5-hexatriene-labeled synaptosomal membranes was dose-dependently decreased by the monoketones, implying increased membrane fluidity. These results indicate that increasing lipophilicity of monoketones results in increased solvent penetration of synaptic membrane preparations, leading to conformational changes in membrane structure and increased ability to inhibit both neuroreceptor binding and enzyme activity. The present data confirm the importance of the lipid micro-environment of membranes in maintaining the normal functions of membrane-bound proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of lipid peroxidation on surface charge density of the porcine intestinal brush-border membranes.

The effects of lipid peroxidation on the surface charge density of the porcine intestinal brush-border membranes were studied using an oxygen-radical-generating system consisting of ascorbic acid, ferrous ion and tert-butyl hydroperoxide (tert-BuOOH). Changes in the membrane surface charge density were monitored using a fluorescent dye, 8-anilino-1-naphthalenesulfonate (ANS). The incubation of the membranes with ascorbic acid/Fe2+/tert-BuOOH resulted in a decrease of the fluorescence intensity of the ANS-membrane complex with a red-shift in the emission maximum, depending on the hydroperoxide concentration and the incubation time. The kinetic studies on ANS-binding showed that the apparent dissociation constant of ANS-membrane complex decreased by treatment with ascorbic acid/Fe2+/tert-BuOOH. Similar results were also obtained by treatment of the membranes with other oxidizing systems, hematin/tert-BuOOH and dipyridyl/Fe2+/tert-BuOOH. These results proposed the possibility that lipid peroxidation of the membranes causes an increase of the positive charge on the membrane surface. The results of the dependence of the ionic strength with increasing KCl concentrations in the medium upon the ANS-binding affinity for the membranes further supported this interpretation.

A decrease of lipid fluidity of the porcine intestinal brush-border membranes by treatment with malondialdehyde.

The effect of treatment of the porcine intestinal brush-border membranes with malondialdehyde (MDA) on their lipid fluidity was examined using a fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). When the membranes were treated with MDA, the fluorescence anisotropy of DPH-labeled membranes increased and the amount of DPH molecules incorporated into the membranes decreased from 3.25 to 2.23 nmol/mg protein. In addition, the response of the fluorescence anisotropy of DPH-labeled membranes to benzyl alcohol, a well-known fluidizer, was markedly suppressed by treatment of the membranes with MDA. These results suggest that treatment of the membranes with MDA causes a decrease of the membrane lipid fluidity. This interpretation was further supported by the increase observed in the fluorescence anisotropy of DPH-labeled liposomes prepared from the extracted lipids of MDA-treated membranes. The results of SDS-polyacrylamide gel electrophoresis suggested that the formation of high-molecular-weight aggregates of the membrane proteins is not involved in the increase of the fluorescence anisotropy of DPH-labeled membranes by treatment with MDA. On the basis of these results, changes in the physical properties of the intestinal brush-border membranes by treatment with MDA are discussed.

Changes in surface charge density of lecithin liposomes by lipid peroxidation. A fluorescence study with 8-anilino-1-naphthalenesulfonate.

Treatment of lecithin liposomes with 100 microM ascorbic acid and 10 microM ferrous ion resulted in the formation of fluorescent products exhibiting an emission maximum at 430 nm and a decrease in the fluorescence intensity of 8-anilino-1-naphthalenesulfonate (ANS) bound to the liposomes without change in the emission maximum. The degree of ascorbic acid/Fe(2+)-induced decrease in the ANS fluorescence was dependent on the extent of fluorescent product formation. The results of kinetic studies on ANS-binding to the liposomes showed that treatment of the liposomes with ascorbic acid/Fe2+ causes an increase of the apparent dissociation constant (Kd) of ANS-liposome complex. This indicates that lipid peroxidation of the liposomes by treatment with ascorbic acid/Fe2+ decreases the binding affinity of ANS to the liposomes. In addition, it was also found that there is a good correlation between degrees of the Kd value and the formation of fluorescent products. The fluorescence properties, i.e. emission maximum and response of the fluorescence intensity for borohydride reduction, of the products formed by lipid peroxidation of the liposomes were similar to those derived from modification of the liposomes with monofunctional aldehydes such as acetaldehyde and heptaldehyde. From these results, it is suggested that the decrease of ANS-binding affinity to the liposomes by treatment with ascorbic acid/Fe2+ may be due to changes in the surface charge density of the liposomes relating to the formation of fluorescent products.