Doi:10.1016/j.pnpbp.2007.01.023

Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 848 – 857 Effects of haloperidol and its pyridinium metabolite on plasma membrane permeability and fluidity in the rat brain Tetsuhito Murata a,⁎, Nobuyuki Maruoka a,b, Naoto Omata a, Yasuhiro Takashima a, Kazuo Igarashi c, Fumiyo Kasuya d, Yasuhisa Fujibayashi b, Yuji Wada a a Department of Neuropsychiatry, University of Fukui, Fukui 910-1193, Japan b Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193, Japan c Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0212, USA d Faculty of Pharmaceutical Sciences, Kobe-gakuin University, Nishi-ku, Kobe 651-21, Japan Received 26 August 2006; received in revised form 10 January 2007; accepted 23 January 2007 Available online 3 February 2007 The use of antipsychotic drugs is limited by their tendency to produce extrapyramidal movement disorders such as tardive dyskinesia and parkinsonism. In previous reports it was speculated that extrapyramidal side effects associated with the butyrophenone neuroleptic agenthaloperidol (HP) could be caused in part by the neurotoxic effect of its pyridinium metabolite (HPP+). Although both HPP+ and HP have beenshown to induce neurotoxic effects such as loss of cell membrane integrity, no information exists about the difference in the neurotoxic potency,especially in the potency to induce plasma membrane damage, between these two agents. In the present study, we compared the potency of theinteraction of HPP+ and HP with the plasma membrane integrity in the rat brain. Membrane permeabilization (assessed as [18F]2-fluoro-2-deoxy- D-glucose-6-phosphate release from brain slices) and fluidization (assessed as the reduction in the plasma membrane anisotropy of 1,6-diphenyl1,3,5-hexatriene) were induced by HPP+ loading (at ≥ 100 μM and ≥ 10 μM, respectively), while comparable changes were induced only at ahigher concentration of HP (= 1 mM). These results suggest that HPP+ has a higher potency to induce plasma membrane damage than HP, andthese actions of HPP+ may partly underlie the pathogenesis of HP-induced extrapyramidal side effects.
2007 Elsevier Inc. All rights reserved.
Keywords: Haloperidol pyridinium; Membrane fluidity; Membrane permeability; Neurotoxicity; Parkinsonism; Tardive dyskinesia (TD) () and parkinsonism ( Antipsychotic drugs have been widely used to treat TD is characterized psychiatric disorders, including schizophrenia, but the use of by involuntary movements of facial, buccal, and masticatory these drugs is limited by their tendency to produce a range of muscles. Parkinsonism is characterized by motor deficits such extrapyramidal movement disorders such as tardive dyskinesia as bradykinesia, rigidity, resting tremor and impairment ofpostural reflexes. The underlying pathophysiological mechan-isms of the development of these movement disorders as side Abbreviations: 2DG, 2-deoxy-D-glucose; DHAA, dehydroabietic acid; effects of antipsychotic therapy still remain to be elucidated.
DMSO, dimethyl sulfoxide; DPH, 1,6-diphenyl-1,3,5-hexatriene; [18F]FDG, The butyrophenone neuroleptic agent haloperidol {4-(4- nol, HP} was introduced into therapy over 40 years ago, and is still one of the most widely used antipsychotic drugs dine; PSL, photostimulated luminescence; TD, tardive dyskinesia; TLC, thin- HP shares some structural Corresponding author. Tel.: +81 776 61 8363; fax: +81 776 61 8136.
E-mail address: (T. Murata).
0278-5846/$ - see front matter 2007 Elsevier Inc. All rights reserved.
T. Murata et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 848–857 MPTP is a contaminant in a synthetic heroin substitute and has To develop a sensitive assay for detecting cell membrane been identified as the toxic agent responsible for an irreversible permeability changes, several laboratories developed a proce- neurodegenerative condition very similar to Parkinson's disease dure which utilizes the leakage of radio-labeled 2-deoxy-D- glucose-6-phosphate (2DG-6-phosphate) from cells generally accepted that the toxic effects of MPTP are mediated by 1-methyl-4-phenylpyridine (MPP+), the pyridinium metab- Advantages of this method include: (1) It uses a biochemically olite of MPTP HP is extensively well-characterized probe, 2DG. (2) 2DG is rapidly taken up into metabolized in the liver, with only about 1% of the administered cells via a glucose transporter (GLUT) and phosphorylated to a dose excreted unchanged in the urine metabolically inert compound, 2DG-6-phosphate. (3) The The major biotransformation pathways of HP in humans method is highly sensitive because of the high level of have been extensively characterized ( accumulated intracellular radioactivity and the comparatively HP undergoes dehydration to the tetrahydropyridine derivative, small size of 2DG.
"Membrane fluidity" is the mobility of the membrane tetrahydropyridine (HPTP), a compound with structural features components such as lipids and proteins, and is assessed by similar to those of MPTP. Both HP and HPTP undergo oxidation measuring the Brownian motion of fluorescent probes incorpo- to the pyridinium metabolite, 4-(4-chlorophenyl)-1-[4-(4-fluor- rated into the membrane bilayer ). 1,6- ophenyl)-4-oxobutyl]-pyridinium (HPP+). HPP+ shares some diphenyl-1,3,5-hexatriene (DPH) is the most frequently structural similarity and toxic actions with MPP+, which employed fluorescent probe which distributes throughout the suggests that HPP+ might induce extrapyramidal side effects hydrophobic core of the membrane bilayer ( (). HPP+ has been shown to be present in ), and is widely used to quantify membrane significant quantities in urine plasma and post-mortem When DPH is immobilized in a rigid matrix and brain samples ) in schizophrenic patients excited by polarized light, it emits fluorescence that is polarized treated with HP. In patients undergoing treatment with HP, a parallel to the exciting light. To the extent that DPH moves significantly linear relationship was found between the HP dose during the lifetime of the excited state, the polarization of its and the serum concentration of HPP+, as well as between the fluorescent emission is reduced. Thus, a decrease in fluores- serum concentrations of HP and HPP+ (It has cence polarization reflects increased mobility of the probe in the also been shown that in psychiatric patients who were treated membrane, i.e., an increase in the membrane fluidity. "Aniso- with HP, the severity of TD and parkinsonism was associated tropy" is a term often used in the fluorescence polarization field, with an increased blood level of HPP+ and is also inversely related to fluidity.
These reports led us to speculate that In the present study, to investigate the effects of HPP+ and extrapyramidal side effects associated with HP could be caused HP on plasma membrane permeability, [18F]2-fluoro-2-deoxy- in part by neurotoxic effects of HPP+.
D-glucose ([18F]FDG) uptake in fresh rat brain slices was Both HPP+ and HP have been reported to induce neuronal serially and two-dimensionally measured using a dynamic positron autoradiography technique ( The results were compared with the leakage of cytoplasmic lactate dehydrogenase (LDH) from the slices into Although several laboratories have previously the medium, which is generally accepted as an indicator of reported differences in the neurotoxic properties between HPP+ plasma membrane damage and cell injury ). Also, to investigate the effects of these agents on plasma membrane fluidity, plasma membrane anisotropy in the rat brain there is not yet a consensus of opinion.
was measured spectrofluorometrically using DPH as a fluores- Generally, it is well known that antipsychotic drugs act as cent probe. Additional studies were conducted to measure antagonists against various neurotransmitter receptors on the TBARS levels after the addition of HPP+ and HP to evaluate plasma membrane. However, the targets of these drugs are so whether oxidative stress is involved in the mechanisms of diverse that none of the specific interactions of the drugs is membrane alteration induced by these agents.
likely to be the sole mechanism of action. Also, since thesedrugs have low molecular weight and high lipophilicity, it seems unlikely that they would remain exclusively at the cellsurface and react only with specific receptors. Rather, it is 2.1. Dynamic positron autoradiography technique natural to assume that they would become widely distributedthroughout the cell and interact directly with the plasma All animal procedures were approved by the Animal Care membrane. Indeed, both HPP+ () and HP and Use Committee of University of Fukui and conducted in have been shown to induce loss of cell accordance with the Guide for the Care and Use of Laboratory membrane integrity in vitro. However, no information exists Animals as adopted and promulgated by the National Institutes about the difference in the potency to induce plasma membrane of Health. Male Wistar rats (250–300 g) were decapitated and damage between these two agents.
their brains were removed. Sagittal brain slices (300 μm in

T. Murata et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 848–857 tivity of [18F]FDG was 1–2 Ci/mmol at the end of the synthesis,and the total concentration (labeled plus unlabeled) used in theexperiment was 0.45–1.16 μg/ml (2.5–6.4 μM). After 1 h ofpre-incubation, the slices were incubated in Krebs–Ringersolution containing [18F]FDG diluted to 150 kBq/ml. The sliceswere then incubated with various concentrations of HP orHPP+, and the effects of these agents on [18F]FDG uptake wereevaluated. We additionally examined whether the [18F]FDGuptake during the loading of either HPP+ or HP was influencedby addition of a GLUT inhibitor cytochalasin B (300 μM) Fig. 1. Schematic view of the apparatus for the incubation of brain slices and the (Sigma Chemical Co., St. Louis, MO, USA), in order to detection of the radioactivity signal on a radioluminography plate. a, brain slice evaluate whether the [18F]FDG uptake was mediated via (300 μm thick); b, 300-μm-thick bathing solution layer; c, radioluminography GLUTs. HP and cytochalasin B were dissolved in dimethyl plate; d, stainless steel ring; e, outer chamber; f, inner chamber; g, hole on the side sulfoxide (DMSO). HPP+ was dissolved in methanol. The final wall of inner chamber; h, nylon net (80 μm thick); i, polyvinylidene chloride film(10 μm thick); j, polytetrafluoroethylene catheter for bubbling; k, Krebs–Ringer concentration of the vehicle (DMSO or methanol) in the solution containing [18F]FDG. RI and BM are defined as the radioactivity signal incubation medium was 0–1%, depending on the solubility of [photostimulated luminescence (PSL)/mm2] on the radioluminography plate the test compounds. DMSO and methanol at these concentra- detected beneath the brain region of interest and the bathing medium solution, tions had no effect on [18F]FDG uptake.
respectively. Images were obtained in a dark environment at 36 °C.
The exposed radioluminography plates (BAS-MP 2040S, Fuji Photo Film Co., Tokyo, Japan) were scanned using a BAS- thickness) were prepared with a microslicer (DTK-2000, 1500 (Fuji Photo Film Co.). The pixel size was 100 μm. The Dosaka EM, Kyoto, Japan), and incubated as previously regions of the brain slices were identified by referring to a brain described (). The system map of the rat ). The image data is shown schematically in The outer chamber was filled obtained were quantitatively analyzed as follows. The radioac- with Krebs–Ringer solution and the inner chamber was tivity of 18F decreases with the same time course (half- immersed in it. The bottom of the inner chamber was made of life = 109.7 min) in both the brain slices and the surrounding a nylon net, and the bottom of the outer chamber was a 10-μm- bathing medium, and it is not necessary to compensate for the thick polyvinylidene chloride film that was penetrable to the radioactive decay when the radioactivity pixel value of a region beta and gamma rays of 18F. The prepared slices were placed in of interest is divided by that of the bathing medium. Thus, the the inner chamber and covered with a 300-μm-thick stainless relative increment of [18F]FDG uptake in the region of interest steel ring whose upper side was covered by a nylon net. The can be expressed in decay-corrected form by the following ratio: incubation volume was 80 ml. During the incubation, theKrebs–Ringer solution was bubbled with a mixture of 95% O2 Relative Uptake Ratio ¼ ðRI−BMÞ=BM and 5% CO2. 18F was produced by 18O (p,n) 18F nuclearreactions, and [18F]FDG was produced by the method of where RI is defined as the radioactivity signal [photostimulated using an automated [18F]FDG synthesis luminescence (PSL)/mm2] on the radioluminography plate system (NKK Co. Ltd., Tokyo, Japan). The specific radioac- detected beneath the region of interest, and BM as the average Fig. 2. Time-resolved pseudocolor images of [18F]FDG uptake in sagittally sectioned rat brain slices. Time zero is when HPP+ was added to the incubation medium.
Two typical slices under the control condition with its diagram (A) and before and after the loading of 100 μM HPP+ (B) for three representative time periods (−10–0 min, 150–160 min, and 430–440 min) are shown. The filled regions in the diagram represent the five brain regions examined in the present study (frontal cortex,caudate putamen, thalamus, hippocampus, and cerebellum). For decay correction, the color-coding was based on the relative uptake ratio (see text for furtherexplanation).

T. Murata et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 848–857 membranes were diluted in 50 mM Tris–HCl buffer, pH 7.4,and mixed with the fluorescent probe DPH (0.8 mg protein/ml,3.3 μM DPH). The concentration of DPH used was based onprevious reports and the DPH stock solution (1 mM)was prepared in tetrahydrofuran. The mixture was incubated at25 °C for 10 min, and then the reaction was stopped by theaddition of a large volume of the above buffer, and the mixturewas centrifuged at 15,000 ×g and 4 °C for 20 min. The resultantpellet was washed twice with the buffer and resuspended in the Fig. 3. Effect of treatment with various concentrations of HPP+ on the time– same buffer. Various concentrations of HP or HPP+ were added course of [18F]FDG uptake in the striatum. Ordinate: relative uptake ratio of18F-radioactivity (see text for further explanation). Abscissa: time in minutes.
to DPH-labeled membrane solutions (0.07 mg protein/ml) and The point at which the drug was applied (= time zero) is indicated by the incubated at 36 °C for 10 min. The 10-min incubation period arrow. Values are the means obtained for six slices (SD is omitted).
was based on a pilot experiment in which the maximum effectof HP or HPP+ on the membrane anisotropy described below radioactivity signal (PSL/mm2) on the radioluminography plate was reached after 10 min of incubation, which is in accord with detected beneath the bathing medium solution surrounding each the findings for alcohols () and other organic brain slice.
solvents The finalconcentration of the vehicle (DMSO or methanol) in the 2.2. [18F]FDG metabolite analysis reaction mixture was 0–1%, depending on the solubility of thetest compound. DMSO or methanol alone at these concentra- In order to measure the release of the [18F]FDG metabolite tions had no effect on membrane fluidity. Fluorescence from the brain slices induced by the administration of HP or measurements were carried out at 36 °C with a Beacon 2000 HPP+, metabolite analysis was performed by thin-layer fluorescence polarization system (Invitrogen Corporation, San chromatography (TLC) on Whatman LK6DF silica gel plates Diego, CA, USA). The excitation and emission wavelengths (Clifton, NJ, USA) with a solvent system of acetonitrile/water were 330 and 420 nm, respectively. The steady-state fluores- (95:5). The metabolites in the incubation medium were sampled cence polarization (P) was expressed using the formula after the administration of HP or HPP+ and separated by TLC.
The TLC plates were exposed to a radioluminography plate.
P ¼ ðIt−I8Þ=ðIt þ I8Þ 2.3. Assessment of plasma membrane damage and cell injury where I‖ and I⊥ are the emission intensities parallel and Leakage of LDH from the slices into the incubation medium perpendicular, respectively, to the plane of the excitation light.
was measured using an LDH Cytotoxicity Detection Kit (Takara The fluorescence anisotropy (A) was calculated from the Bio Inc., Shiga, Japan) according to the manufacturer's fluorescence polarization value using the formula ( instructions. Results were expressed as percentage of control, which was considered as 100%.
A ¼ 2P=ð3−PÞ 2.4. Fluorescence anisotropy measurement The amount of protein was determined with a Bio-Rad In this study, the P2 fraction of was protein assay kit (Hercules, CA, USA) using bovine serum prepared as a crude synaptosomal membrane fraction. Although albumin as the standard.
the P2 fraction is enriched in synaptosomes but also containsrelatively large amounts of myelin fragments and free mitochon-dria, it can be obtained in 70–80 min and the long preparationtimes of the original method () can beavoided. Briefly, the rat brain tissue was homogenized in 10 vol of0.32 M sucrose using an Ultrasonic Disrupter (UR-20P, TomySeiko Co. Ltd., Tokyo, Japan). Each homogenate was centrifugedat 1000 ×g and 4 °C for 10 min. The supernatant was removed andcentrifuged at 12,000 ×g and 4 °C for 20 min. The resultant pelletwas suspended in 20 vol of 50 mM Tris–HCl buffer, pH 7.4, andwashed twice by centrifugation at 12,000 ×g and 4 °C for 20 min.
The final pellet was resuspended in the same buffer and used for Fig. 4. Effect of treatment with various concentrations of HP on the time–course of [18F]FDG uptake in the striatum. Ordinate: relative uptake ratio of the measurement of membrane anisotropy.
18F-radioactivity (see text for further explanation). Abscissa: time in minutes.
Membrane anisotropy was measured by the method of The point at which the drug was applied (= time zero) is indicated by the arrow. Values are the means obtained for six slices (SD is omitted).

T. Murata et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 848–857 2.5. Lipid peroxidation assay Brain slices were treated with either 1 mM HPP+ or 1 mM HP for 60 min. The slices were then homogenized and aliquots wereused to determine the levels of thiobarbituric acid-reactivesubstances (TBARS) using Lipid Peroxidation Test Wako(Wako Pure Chemical Industries Ltd, Osaka, Japan) accordingto the manufacturer's instructions. In this assay, lipid peroxidationis quantified by measuring malondialdehyde (MDA), an endproduct of oxidative lipid degradation. MDA forms a 1:2 adductwith thiobarbituric acid (TBA) and produces a TBA pigmentwhich can be measured by fluorometry or spectrophotometry Fig. 6. Effect of treatment with 1 mM HPP+ and 1 mM HP on LDH leakage from (The interaction of 50 μM Fe2+ and 3 mM brain slices into the bathing medium 480 min after the administration of the hydrogen peroxide was used as a positive control. The quantities drugs. Samples treated with 1% Triton X-100 were defined as positive controls.
of TBARS were expressed as nanomoles per milligram of protein, Results are expressed as percentage of control, which was considered as 100%, and presented as a percentage of control values.
and represent the means ± SD obtained in six slices. The Mann–Whitney U-testwas used to evaluate the significance of differences. ⁎P b 0.05 compared withcontrol values.
HPP+ was synthesized as previously described before and after the loading of 100 μM HPP+ (B) for three ). The purity of the HPP+ was 97–98%. HP, representative time periods (−10–0 min, 150–160 min, and DPH and cytochalasin B were purchased from Sigma Chemical 430–440 min). Time zero is defined as the time when HPP+ or Co. (St. Louis, MO, USA). The LDH Cytotoxicity Detection HP was introduced into the bathing medium containing brain Kit was purchased from Takara Bio Inc. (Shiga, Japan). The slices. Rat brain slices from five representative brain regions Bio-Rad protein assay kit was obtained from Bio-Rad (frontal cortex, striatum, thalamus, hippocampus, and cerebel- Laboratories Inc. (Hercules, CA, USA). The Lipid Peroxidation lum) were analyzed.
Test Wako was obtained from Wako Pure Chemical Industries To depict dynamic changes of [18F]FDG uptake, the relative Ltd. (Osaka, Japan). All other chemicals were from Nacalai uptake ratio at 10-min intervals in the striatum before and after Tesque Inc. (Kyoto, Japan).
loading various concentrations (10, 30, 100 μM and 1 mM) ofHPP+ or HP was plotted against time [the results for the control 2.7. Statistical analysis condition and for the HPP+ loading (10, 30, 100 μM and 1 mM)at 20-min intervals are shown in The slope of the graph The presented values are shown as the means ± SD. The indicates the rate of [18F]FDG uptake. The slope of the graph Mann–Whitney U-test was used to evaluate the significance of for 10–30 μM HPP+ was definitely increased during the entire differences. P b 0.05 was considered statistically significant.
time course. However, when slices were loaded with buffercontaining ≥100 μM HPP+, the slope of the graph initially increased, then gradually decreased and finally became negative(this finding may reflect the outflow of [18F]FDG metabolites shows time-resolved images of [18F]FDG uptake of from the brain slices). Similar results were obtained in each of two typical brain slices under the control condition (A) and the brain regions examined (data not shown).
Fig. 5. Effect of treatment with 100 μM HPP+ on the time–course of [18F]FDGuptake in the striatum. HPP+ was administered alone or co-administered with Fig. 7. Effects of treatment with various concentrations of HPP+ or HP on the a GLUT inhibitor cytochalasin B (300 μM). Ordinate: relative uptake ratio of DPH fluorescence anisotropy as an index of membrane fluidity. Data represent 18F-radioactivity (see text for further explanation). Abscissa: time in minutes.
the means ± SD obtained for six samples. The Mann–Whitney U-test was used The point at which each agent was applied is indicated by an arrow. Values are to evaluate the significance of differences. ⁎P b 0.05 compared with control the means obtained for six slices (SD is omitted).
T. Murata et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 848–857 In contrast, the slope of the graph for 10–100 μM HP was Treatment with either 1 mM HPP+ or 1 mM HP did not similar to that of the control. Significant changes in the slope of significantly alter TBARS levels in comparison to those in the the graph (i.e., initial increase and subsequent gradual decrease in the slope of the graph) were induced by HP loading only atthe concentration of 1 mM [the results for the control condition and for the HP loading (100 μM and 1 mM) at 20-min intervalsare shown in Similar results were obtained in each of the Previous studies have shown differences in the neurotoxic regions examined (data not shown).
properties between HPP+ and HP. Sagittal mouse brain slices The enhancement of the [18F]FDG uptake induced by both incubated in vitro with HP (10 nM) showed time- and HPP+ (≥10 μM) and HP (1 mM) is inhibited by addition of concentration-dependent inhibition of complex I 300 μM cytochalasin B in each of the brain regions examined ). However, similar concentrations of HPP+ failed to (the results for the 100 μM HPP+ loading, and cytochalasin B inhibit complex I activity, and comparable inhibition was loading at 140 min after the administration of HPP+ in the obtained only at a 10,000-fold higher concentration of HPP+ striatum are shown in ). Therefore, it is suggested that the (100 μM) (). On the other hand, because enhancement of the [18F]FDG uptake was mediated via GLUTs.
the neuronal accumulation of HP metabolites via monoamine The TLC data suggested that the major [18F]FDG metabolite transporters could facilitate subsequent toxic events in the released from the slices as a result of the administration of HPP+ (≥100 μM) or HP (1 mM) was [18F]FDG-6-phosphate, not laboratories examined the interactions of HPP+ and HP with the [18F]FDG (data not shown). Because [18F]FDG-6-phosphate monoamine transporters and demonstrated that HPP+ was more cannot be transported via GLUTs, the efflux of [18F]FDG-6- active for inhibiting monoamine uptake into cells phosphate was probably not mediated by GLUTs; rather, it is likely that the increased plasma membrane permeability of the ). Thus, no consensus has cells allowed intracellular [18F]FDG-6-phosphate to leak from yet been reached as to the difference in the potency of inducing neurotoxicity between HPP+ and HP.
The LDH assay revealed that in the case of 1 mM HPP+ and The present study is the first to investigate the difference in 1 mM HP exposure, which began to induce the release of [18F] the potency of inducing plasma membrane damage between FDG-6-phosphate around 200–300 min after the administration HPP+ and HP by using [18F]FDG-6-phosphate leakage as an of the drug ), no significant increase in the release of index of membrane permeabilization and DPH fluorescence LDH was observed even at 480 min after the administration of anisotropy as an index of membrane fluidization. In this study, the drug (). These results indicate that the leakage of LDH increases in membrane permeability and fluidity were induced is clearly slower than that of [18F]FDG-6-phosphate.
by HPP+ loading (≥100 μM and ≥10 μM, respectively) At concentrations ≥ 10 μM, HPP+ induced a dose-dependent ). Similar concentrations of HP failed to affect decrease in anisotropy (i.e., an increase in membrane fluidity), membrane permeability or fluidity, and comparable changes while 10–100 μM HP induced no significant changes in were obtained only at a higher concentration of HP (1 mM) anisotropy; a significant decrease in anisotropy was obtained These results suggest that HPP+ has a higher by HP loading only at the concentration of 1 mM [the results for potency for inducing plasma membrane damage than HP.
the control condition, for the HPP+ loading (10 and 100 μM) and We found that when the release of [18F]FDG-6-phosphate for the HP loading (100 μM and 1 mM) are shown in ].
was already induced at 480 min after the administration of bothHPP+ and HP, no significant increase in the release of LDH wasobserved, which suggests that the leakage of LDH is clearlyslower than that of [18F]FDG-6-phosphate Apossible explanation for this is that HPP+ and HP could initiallyinduce small membrane holes that became enlarged with time.
The holes were not initially large enough to allow the leakage oflarger molecules such as LDH, but a small increase in hole sizewith time eventually enabled the leakage of LDH. The lagbetween the leakage of [18F]FDG-6-phosphate and LDH couldreflect the time needed for the hole to become large enough forLDH leakage to be possible.
Amphiphilic molecules have both hydrophobic and hydro- philic moieties and accumulate in the membrane bilayer and can Fig. 8. Effect of treatment with 1 mM HPP+ and 1 mM HP on the formation of consequently affect the membrane fluidity. For example, the thiobarbituric acid-reactive substances (TBARS) in rat brain slices. Data amphiphilic peptide gramicidin-S penetrates and accumulates in represent the means ± SD obtained in six slices. Samples treated with 50 μM the membrane bilayer. The penetration of gramicidin-S creates a FeSO4 and 3 mM H2O2 were examined as positive controls. The quantities of space in the interior of the membrane, resulting in an increase in TBARS were expressed as percentage of the control values. The Mann– the movement of the acyl chains of phospholipid molecules and Whitney U-test was used to evaluate the significance of differences. ⁎P b 0.05compared with control value.
thus in the membrane fluidization ). The T. Murata et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 848–857 accumulation of amphiphilic molecules destabilizes the mem- evaluate whether the [18F]FDG uptake was mediated via GLUTs.
brane structure, resulting in the release of membrane components The enhancement of the [18F]FDG uptake induced by both HPP+ and a concomitant enhancement of permeability (≥10 μM) and HP (≥1 mM) was prevented by addition of a ). Antipsychotic drugs such as HP and chlorpromazine GLUT inhibitor cytochalasin B (). Therefore it is suggested are known to have an amphiphilic nature that the enhancement of the [18F]FDG uptake was mediated via ). Although HPP+ possesses a fixed GLUTs, rather than the GLUT-independent [18F]FDG influx positive charge, which renders the molecule hydrophilic, it can (passive diffusion) due to membrane damage. Nevertheless, we still be considered to be lipophilic because of its complex alkyl could not exclude the possibility that the membrane permeability aromatic structure (). Therefore, it seems is underestimated in dynamic positron autoradiography since the reasonable to categorize HPP+ as an amphiphilic agent. In the summation of radioactivities derived from [18F]FDG taken up present study, increased lipophilicity of HP and HPP+ did not into the tissue and [18F]FDG-6-phosphate that leaked out from the necessarily correlate with potency to interact with the membrane, tissue was measured in this method.
since HPP+ [partition coefficient (log P) = 1.23], which is less It has been reported that haloperidol induces the generation lipophilic than HP (log P = 3.85) was of reactive oxygen species in rat neurons ( more potent as a membrane destabilizer than HP. Accordingly, it is Treatment with haloperidol led to increased lipid peroxidation suggested that the ability to interact with the membrane is not due and concomitant reductions in brain levels of glutathione in rats to the mere increase of lipophilicity of HP and HPP+, but mainly to the amphiphilic nature of HP and HPP+. Our results are also and humans ). Free radicals have been consistent with a previous report which showed that when reported to increase plasma membrane permeability ( erythrocytes were transformed into stomatocytes by the addition Several laboratories have of cationic amphiphilic agents, membrane fluidization was always reported that oxidative stress and lipid peroxidation increase observed, thus indicating a close correlation between stomatocyte membrane fluidity formation and an increase in membrane fluidity ).
These reports prompted us to speculate that HPP+ and HP may Our data showed that HPP+ (≥ 10 μM) and HP (1 mM) induce oxidative stress and lipid peroxidation, and consequently enhanced the [18F]FDG uptake in the initial phase after the drug increase membrane permeabilization and fluidization. Based on administration. A possible explanation for the mechanism by this hypothesis, TBARS levels after the addition of HPP+ and which HPP+ or HP induced enhancement of the [18F]FDG HP were measured. Contrary to our hypothesis, treatment with uptake is the drug-induced inhibition of mitochondrial activity.
either 1 mM HPP+ or 1 mM HP did not alter the TBARS levels It has been shown that both HPP+ ( significantly (suggesting that HPP+ and HP did not induce lipid peroxidation. Therefore, it is unlikely that oxidative inhibit complex I in the mitochondrial stress is involved in the mechanisms of membrane alteration respiratory chain. It has also been demonstrated that HPP+ is a induced by HPP+ and HP.
stronger inhibitor of mitochondrial respiration than MPP+ HPP+ is mainly produced by the microsomal-catalyzed () which is well known to be a oxidation of HP with cytochrome P450, which is distributed mitochondrial complex I inhibitor (). In principally in the liver ( the present study, the enhancement of glycolytic activity may have served to compensate for the decrease in aerobic ). Enzyme activity responsible for the conversion of metabolism when oxidative phosphorylation was inhibited by HP to HPP+ was not found in rat brain mitochondria ( HPP+ or HP at the mitochondrial respiratory chain level.
Therefore, it seems unlikely that HPP+ was gen- Dehydroabietic acid (DHAA), which is known to be an erated during the incubation of the brain slices. This, however, amphiphilic compound, was reported to cause a dose-dependent may not be the case, since HP and HPTP are biotransformed by reduction in the cellular ATP content and a concomitant mouse brain preparations to HPP+ ).
enhancement of the glycolytic activity of rainbow trout Consequently, it is also possible that HPP+ is formed from hepatocytes, suggesting that ATP depletion is likely due to HP in the brain slices, which may have partly affected the increased consumption of cellular ATP caused by amphiphilic membrane alteration induced by HP in the present study.
action of DHAA in the cell membrane ().
The therapeutic use of antipsychotic drugs is limited by their Therefore, in the present study, through their amphiphilic tendency to produce a range of extrapyramidal movement properties, HP and HPP+ could induce ATP depletion and disorders such as TD enhancement of glycolytic activity. It has been suggested that and parkinsonism these mechanisms cause further ATP depletion, and depletion of ATP to a critically low level increases membrane permeability Although the pathophysiological basis of TD remains unclear, TD has been attributed to the supersensitivity of dopamine It could be argued that GLUT-independent [18F]FDG influx (passive diffusion) would be induced if a membrane structure is suggested that antipsychotic drugs fluidize damaged. Therefore, we additionally examined whether the membranes, thus promoting fusion between the neurovesicle enhancement of the [18F]FDG uptake induced by either HPP+ or membrane and the presynaptic membrane. This membrane- HP was prevented by addition of a GLUT inhibitor in order to fluidizing action would thus lead to an increased spontaneous T. Murata et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 848–857 secretion of dopamine, and this may underlie TD. Although drug-induced parkinsonism is usually attributed to the anti-dopaminergic properties of antipsychotic drugs, a wide variety For the first time, we investigated the effect of HP and its of processes have been suggested to be involved in the pyridinium metabolite HPP+ on plasma membrane perme- pathogenesis of Parkinson's disease and parkinsonism. Recent- ability in relation to the effect of these agents on plasma ly, Volles et al. proposed that membrane permeabilization by membrane fluidity in the central nervous system. Membrane protein α-synuclein is a pathogenetic mechanism involved in permeabilization and fluidization were induced by HPP+ loading Parkinson's disease; that is, inappropriate membrane permea- (≥ 100 μM and ≥ 10 μM, respectively). Similar concentrations of bilization by α-synuclein could cause the degeneration and HP failed to change membrane permeability and fluidity in this death of neurons in several ways: unregulated calcium flux into model; indeed, comparable changes were obtained only at a the cytosol, depolarization of the mitochondrial membrane, or higher concentration of HP (1 mM). These results suggested that leakage of dopamine into the cytoplasm ( HPP+ had a higher potency to induce plasma membrane dam- These reports together with our age than HP, which may partly underlie the pathogenesis of findings prompt us to speculate that the induction of membrane HP-induced extrapyramidal side effects such as TD and permeabilization and fluidization by HPP+ may play a role, at least partially, in the pathogenesis of HP-induced extrapyrami-dal side effects such as parkinsonism and TD. Further studies will be needed to clarify the relationship between membraneactions of HPP+ and the pathogenesis of HP-induced extrapy- This work was in part supported by 21st Century COE ramidal side effects.
program "Biomedical Imaging Technology Integration Program" One of the limitations of our findings is that the concentra- from the Japan Society for the Promotion of Science (JSPS).
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