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Peripheral benzodiazepine receptors on platelets in chronic and detoxified alcoholics
Neurophormocology Vol. 30, No. 6, pp. 665-669, 1991
0028-3908/91 $3.00 + 0.00 Pergamon Press plc
Printedin Great Britain
PERIPHERAL BENZODIAZEPINE RECEPTORS ON PLATELETS IN CHRONIC AND DETOXIFIED ALCOHOLICS* L. KARP,’ A. WEIZMAN,’ M. FLIMAN,* S. TYANO’ and M. GAVISH”~ ‘Geha Psychiatric Hospital, Beilinson Medical Center, Petah Tiqva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, ‘Israel Residential Center for Alcoholism, Ramat Gan and Talbieh Mental Health Center, affiliated with the Hebrew University-Hadassah Medical School, Jerusalem and ‘Department of Pharmacology, Faculty of Medicine and Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, P.O.B. 9649, 31096 Haifa, Israel (Accepted 22 January 1991) Summary-The
effect of chronic alcoholism and detoxification treatment with disulfiram on platelet peripheral benzodiaxepine receptors was studied in alcoholic males. Chronic consumption of alcohol did not alter the binding values for [‘H]PK 11195, as compared to non-alcoholics. Treatment for 3 weeks with disulfiram resulted in a significant increase in the density of peripheral benzodiazepine receptors, with no alteration in the affinity of these sites to the ligand. These results might be relevant to the cellular and metabolic effects of disulfiram. Key words-peripheral
benzodiaxepine receptor, PK 11195, alcoholism, disulfiram.
Specific high-affinity benzodiazepine receptors in the central nervous system (CNS) (Mohler and Okada,
1977; Squires and Braestrup, 1977) are coupled to the y-aminobutyric acid (GABA) receptor and chloride ion channel (Gavish and Snyder, 1981; Paul, Marangos and Skolnick, 1981). These central benzodiazepine receptors are involved in the anticonvulsant and anxiolytic effects of benzodiazepines (Mohler, Okada, Heitz and Ulrich, 1978; Tallman, Paul, Skolnick and Gallagher, 1980). In addition, peripheral-type benzodiazepine receptors have been identified in peripheral tissues, such as platelets, kidney, heart, adrenal and testis, as well as in the CNS (Wang, Taniguchi and Spector, 1980; Marangos, Patel, Boulenger and Clark-Rosenberg, 1982; Taniguchi, Wang and Spector, 1982; Schoemaker, Boles, Horst and Yamamura, 1983a; Anholt, De Souza, Kuhar and Snyder, 1985a; Anholt, De Souza, Oster-Geramite and Snyder, 1985b). They differ from the central receptors in their distribution in tissue, subcellular localization, pharmacological specificity and function, lack of coupling to GABA receptors and the chloride ion channel and specificity for ligand binding (Marangos et al., 1982; Anholt et al., 1985a, 1985b; Anholt, Aebi, Pedersen and Snyder, 1986a; Anholt, Pedersen, De Souza and Snyder, 1986b). Peripheral benzodiazepine receptors seem to to be under neural and hormonal control (Gavish, Okun, Weizman and Youdim, 1986a; Gavish, Weizman, Okun and Youdim, 1986~; Gavish, Weizman, Youdim and Okun, 1987; Basile, *Due to circumstances beyond the Publisher’s control, this article appears in print without author corrections.
Ostrowski and Skolnick, 1987; Fares, Bar-Ami, Haj-Yehia and Gavish, 1989). Ethanol, similary to benzodiazepines, possesses an anxiolytic and hypnotic effect. There is cross-tolerance and dependence between the two drugs, which suggests the possibility of a common mode of action (Goldstein, 1978). Chronic consumption of ethanol does not alter the binding capacity of [‘Hlflunitrazepam, [‘Hlmuscimol or [“Slbutylbicyclophosphorothionate to the brain of the rat (Greenberg, Cooper, Gordon and Diamond, 1984). Furthermore, the ability of GABA to enhance the binding of [‘Hlflunitrazepam is not affected by chronic treatment with ethanol or by withdrawal (Rastogi, Thyagarajan, Clothier and Ticku, 1986). It has recently been demonstrated that ethanol stimulates the GABA receptor-coupled transport of chloride ions in synaptoneurosomes from the brain of the rat (Suzdak, Schwartz, Skolnick and Paul, 1986). Chronic use of ethanol has been reported to induce a significant increase in the density of peripheral benzodiazepine receptors in the brain of mice and rats (Schoemaker, Thomas and Yamamura, 1983b; Tamborska and Marangos, 1986; Syapin and Alkana, 1988). A recent study in human8 demonstrated a significant reduction in the density of binding sites for [‘H]PK 11195 on platelets in chronic alcoholics, but not in alcoholics, abstinent for at least 4 months (SuranyiCadotte, Lafaille, Dongier, Dumas and Quirion, 1988). The present study was designed to reassess the effect of long-term consumption of alcohol and detoxification (using disulfiram as preventive treatment) on
665
L. KARP et al.
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peripheral benzodiazepine chronic alcoholics.
receptors on platelets in
METHODS
Materials
[)H] 1-( 2-chlorophenyl)-N-methyl-N-( 1 -methylpropyl)-3-isoquinoline carboxamide ([3H]PK 11195, 92.3 Ci/mmol) was purchased from New England Nuclear, Boston, Massachusetts. Unlabelled 7-chloro1,3-dihydro- 1-methyl-5-(p-chlorophenyl)-2H1,4benzodiazepine-Zone (Ro 5-4864) was kindly supplied by Drs H. Gutman and E. Kyburz, Hoffmann-La Roche, Basel, Switzerland. Lumax was purchased from Lumac, Schaesberg, The Netherlands. All other chemicals were purchased from commercial sources. Subjects
The population studied was comprised of three (currently drinking, groups: 9 alcohol-dependent with mean alcohol intake of 1923 f 494 g/week) male subjects, aged 30-52 years (mean f SD, 41.0 f 4.7 years); 10 detoxified male patients, aged 34-54 years (mean + SD, 42.7 f 6.4 years) and 9 non-alcoholic (with mean alcohol intake of 92 f 50 g/week) healthy volunteer male controls, aged 30-40 years (mean + SD, 38.7 f 1.7 years). All 19 patients met the DSM-III-R criteria for dependence on alcohol (American Psychiatric Association, 1987). These criteria include at least three of the following: (1) alcohol often taken in larger amounts or over a longer period than the person intended; (2) persistent desire or one or more unsuccessful efforts to cut down or control use of alcohol; (3) a great deal of time spent in activities necessary to get the substance, taking the substance or recovering from its effects; (4) frequent intoxication or withdrawal symptoms when expected to fulfil major role obligations at work, school or home, or when use of the substance is physically hazardous; (5) important social, occupational or recreational activities given up or reduced because of use of alcohol; (6) continued use of alcohol, despite knowledge of having persistent or recurrent social, psychological or physical problems that are caused or exacerbated by the use of the substance; (7) marked tolerance: need for markedly increased amounts of alcohol to achieve intoxication or desired effect or markedly diminished effect with continued use of the same amount; (8) characteristic withdrawal symptoms; and (9) alcohol often taken to relieve or avoid withdrawal symptoms. The alcoholics were all inpatients (in a closed department), participating in an alcohol-treatment program with no access to alcohol. The duration of abuse of alcohol was similar between the patients currently drinking and the detoxified alcoholics (mean + SD, 11.7 f 4.0 vs 9.6 f 3.0 years). On admission, each patient underwent a thorough physical, neurological and psychiatric examination and routine laboratory check-up. Exclusion criteria
were a past history or present symptoms of psychiatric or neurological disease, as well as concomitant abuse of drugs. None of the subjects had ever been treated with psychoactive drugs on a regular basis. All subjects were physically healthy. During the first 2 weeks of alcohol detoxification, the patients were treated with tapering doses of chlordiazepoxide (maximum dose, 30 mg/day per OS) to prevent symptoms of alcohol-withdrawal. Patients were treated thereafter with disulfiram (Antabuse) (250mg/day per OS) for 3 weeks. Membrane preparation
Blood samples (50 ml) for assessment of the binding of [3H]PK 11195 were withdrawn concomitantly from patients and controls, between 8:00 and 10:00 a.m., collected into plastic tubes, containing 8.8 ml of a mixture of 2.2% sodium citrate and 1.2% citric acid and spun at 180 g for 15 min at 23°C. Platelet-rich plasma was collected and spun at 1500 g for 15 min at 23°C. The platelet-containing pellet was frozen at -70°C until assay. The samples were thawed and prior to the binding assay, the pellet was homogenized in 20 ml of 50 mM Tris-HCl buffer, pH 7.4, at 4°C with a Brinkmann Polytron (setting 10) for 15 set and centrifuged at 49,000g for 15 min at 4°C. The procedure was immediately repeated. The pellet was homogenized in 20 ml of Tris-HCl buffer and used for binding studies. Binding assay for [IH]PK 11195
The binding of [3H]PK 11195 was conducted as previously described (Fares and Gavish, 1986; Gavish, Weizman, Karp, Tyano and Tanne, 1986b). The binding assay, in a final volume of 50 ~1, contained 400 ~1 platelet membranes (70-1OOpg protein) and 25 ~1 [3H]PK 11195 (final concentration 0.2-6 nM) in the absence (total binding) or presence (non-specific binding) of 10 p M unlabelled Ro 5-4864. After incubation for 60 min at 4”C, samples were filtered under vacuum over Whatman GF/B filters and washed three times with 3 ml of Tris-HCl buffer. Filters were placed in vials containing 5 ml of xylene-Lumax (3 : 1, v/v) and counted for radioactivity. Analysis of data
The equilibrium dissociation constant (K,) and maximum number of binding sites (B,,,,,) were determined by Scatchard analyses of saturation curves of 11195 binding of [3H]PK. The binding parameters were analyzed for each subject individually. Oneway analysis of variance (ANOVA) was used for intergroup comparisons. Results are expressed as mean f SEM. RESULTS
The platelet B,,, values for [)H]PK 11195 in the chronic alcoholics, detoxified alcoholics (after 5 weeks’ treatment: chlordiazepoxide for the first 2 weeks and
Benzodiazepine receptors in alcoholics l
4
chmnlcabholii
667 (n-9)
H detoxifkd akohoiia (n-10)
Fig. 1. Effect of chronic alcoholism and detoxification (under disulfiram maintenance treatment) on the maximum binding capacity of peripheral benzodiazepine receptors on platelets. Results are expressed as mean f SEM. *P < 0.05 compared to detoxified patients and controls [one-way ANOVA: F(2,25) = 4.131.
disulfiram maintenance for 3 weeks thereafter) and the control group are summarized in Fig. 1. No significant difference in the density of binding sites for [3H]PK 11195 in platelets was detected between chronic alcoholics and normal controls. However, the disulfiramtreated detoxified alcoholics revealed a significant increase in the number of peripheral benzodiazepine receptors, when compared to normal controls (32%; P < O.OS), as well as to chronic alcoholics (25%; P c 0.05). The KD values were similar in all three groups (chronic alcoholics, 3.1 + 0.2; detoxified alcoholics, 3.7 f 0.4; and controls, 3.3 f 0.3 nM; one-way ANOVA: F(2,25) = 1.03; not significant). DISCUSSION
These results demonstrated an unaltered density of peripheral benzodiazepine receptors on platelets in chronic alcoholics, while maintenance treatment with disulfiram resulted in up-regulation of this binding site. Previous studies have shown an increase in the binding of [3H]Ro 5-4864 to whole brain in mice (Schoemaker et al., 1983b; Syapin and Alkana, 1988) and to cerebral cortex, cerebellum and hippocampus in the brain of the rat (Tamborska and Marangos, 1986), after chronic (but not acute) exposure to ethanol. The changes in peripheral benzodiazepine receptors in rats did not extend to the olfactory bulb and kidney (Tamborska and Marangos, 1986). The elevation in the binding persisted for 3 days, after cessation of ethanol and thereafter returned to basal levels. In both studies, central benzodiazepine receptors remained unaltered (Schoemaker et al., 1983b; Tamborska and Marangos, 1986). Since administration of ethanol induced selective and bidirectional alterations in peripheral benzodiazepine receptors, it seems that these changes could not be attributed to non-specific alterations in membrane fluidity (Chin and Goldstein, 1977). One human study has shown a decreased density of peripheral benzodiazepine receptors on platelets
in currently drinking, but not in abstinent, alcoholics (Suranyi-Cadotte et al., 1988). This line of evidence suggests that peripheral benzodiazepine receptors are probably relevant to the neurobiological effects of ethanol consumption. However, no alteration could be detected in the density of peripheral benzodiazepine receptors on platelets in chronic alcoholics. The cause of the discrepancy between the two studies is unclear, but may be related to differences in the duration or quantity of consumption of alcohol (unfortunately, these details were not given in the study of SuranyiCadotte et al., 1988). The elevation in peripheral benzodiazepine receptors on platelets, observed in detoxified alcoholics, seemed to be related to the maintenance treatment with disulfiram. The up-regulation of these binding sites is unlikely to be attributable to administration of chlordiazepoxide, since the patients were free of this drug for 3 weeks. In a previous study, it was demonstrated that chronic treatment with diazepam in patients suffering from generalized anxiety disorder, induced an up-regulation of peripheral benzodiazepine receptors on platelets. However, 1 week after discontinuation of treatment with diazepam, the density of the binding sites was similar to that of untreated normal controls (Weizman, Tanne, Granek, Karp, Golomb, Tyano and Gavish, 1987). The enhancement in the binding of [‘H]PK 11195 was most probably not due to a psychotropic effect of disulfiram, since controlled studies did not demonstrate a sedative or anxiolytic effect of disulfiram in chronic alcoholics (Snyder and Keeler, 1981; Goyer, Brown, Minichiello and Major, 1984). It is not yet clear whether previous dependency on alcohol is a prerequisite for the up-regulatory effect of disulfiram on peripheral benzodiazepine receptors. Further studies, with non-dependent volunteers treated with disulfiram, can clarify this issue. In vitro competition experiments performed in this laboratory have shown that disulfiram is a competitive inhibitor (ICsO= 5 PM) at the peripheral benzodiazepine receptors in brain and kidney (unpublished
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data). Levels of disulfiram in plasma in alcoholic patients, maintained on preventive treatment with disulfiram reached a concentration of 1-2 PM (Kirvbakaran, Faiman, Liskow and Mayfield, 1986). Since the presence of disulfiram in vitro inhibited rather than stimulated the binding of [3H]PK 11195, it is suggested that the increase in the density of peripheral benzodiazepine receptors on platelets was attributable to the modulatory effect of chronic treatment with disulfiram. Furthermore, the procedure of preparation of membranes removed most of the disulfiram from the binding assay. Subcellular localization studies indicate that the peripheral benzodiazepine receptors are associated with the outer membrane of the mitochondria (Anholt et al., 1986a, 1986b; Basile and Skolnick, 1986). Drugs which are known to affect peripheral benzodiazepine receptors, produce stimulation or inhibition of cell proliferation (Matthew, Laskin, Zimmerman, Weinstein, Hsu and Engelhardt, 1981; Wang, Morgan and Spector, 1984). Disulfiram, on the other hand, produces hepatic hypertrophy and hyperplasia in rats (Fiala, Fiala and Keller, 1977). Disulfiram affects mitochondrial enzymes, such as NAD+-dependent aldehyde dehydrogenase (Vallari and Pietruszko, 1982) and monoamine oxidase (Schurr and Schoolar, 1978). The disulfiram-induced increase in peripheral benzodiazepine receptors on platelets might thus be relevant to the effects of this drug on cell growth. differentiation and metabolism.
Acknowledgements-This
work was supported in part by the Israel Institute for Psychobiology-the Charles E. Smith Family Foundation (Grant No. 26-90). We thank Mrs Zehava Tanne for technical assistance, Mrs Cila Aviram for clinical assistance and Miss Ruth Singer for typing and editing the manuscript.
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American Psychiatric Association, Washington, D.C. Anholt R. R. H., Aebi U., Pedersen P. L. and Snyder S. H. (1986a) Solubilization and ressembly of the mitochondrial benzodiazepine receptor. Biochemistry 25: 2120-2125. Anholt R. R. H., De Souza E. B., Kuhar M. J. and Snyder S. H. (1985a) Depletion of peripheral-type benzodiazepine receptors after hypophysectomy in rat adrenal gland and testis. Eur. J. Pharmaf. 110: 41-46. Anholt R. R. H.. De Souza E. B.. Oster-Geramite M. L. and Snyder S. H. (1985b) Peripheral-type benzodiazepine receptors: autoradiographic localization in whole-body sections of neonatal rats. J. Pharmac. exp. Ther. 223: 517-526. Anholt R. R. H., Pedersen P. L., De Souza E. B. and Snyder S. H. (1986b) The peripheral-type benzodiazepine receptor: localization io themitbchond;i-al outer membrane. J.biol. Chem. 261: 576-583. Basile A. S., Ostrowski N. L. and Skolnick P. (1987) Aldosterone-reversible decrease in the density of renal peripheral benzodiazepine receptors in the rat after adrenalectomy. J. Pharmac. exp. Ther. 240: 1006-1013.
BasiJeA. andSkoJ.nick P. (1986) Subcellular localization of “pheripheral tylpe” binding sites for benzodiazepines in ...““nl.nu -. A& -IV,--IVU. 1n4 ‘ZllP rat brain. J. NeU,VLIITlrl. Chin J. H. and Goldstein D. B. (1977) Drug tolerance in biomembranes: a snin label study of the effectsof ethanol. Science, Wash. 196: 684-685. Fares F., Bar-Ami S., Haj-Yehia Y. and Gavish M. (1989) Hormonal regulation of peripheral benzodiazepine binding sites in female rat adrenal gland and kidney. J. Receptor Res. 9: 1433157. Fares F. and Gavish M. (1986) Characterization of peripheral benzodiazepine binding sites in human term placenta. Biochem. Pharmac. 35: 227-230.
Fiala S., Fiala A. E. and Keller R. W. (1977) Activation of y-glutamyl transferase in rat liver by disulfiram and its effect on the first stage of azo-dye-induced carcinogenesis [Abstract]. Fed. Proc. 36: 349. Gavish M., Okun F., Weizman A. and Youdim M. B. H. (1986a) Modulation of peripheral benzodiazepine binding sites following chronic estradiol treatment. Eur. J. Pharmac. 127: 147-151.
Gavish M. and Snyder S. H. (1981) y-Aminobutyric acid benzodiazepine receptors: copurification and characterization. Proc. natn. Acad. Sci. U.S.A. 18: 1939-1942. Gavish M., Weizman A., Karp L., Tyano S. and Tanne Z. (1986b) Decreased peripheral benzodiazepine binding sites in platelets of neuroleptic-treated schizophrenics. Eur. J. Pharmac. 121: 275-279.
Gavish M., Weizman A., Okun F. and Youdim M. B. H. (1986~) Modulatory effects of thyroxine treatment on central and peripheral benzodiazepine receptor in the rat. J. Neurochem. 47: 1106611IO. Gavish M., Weizman A., Youdim M. B. H. and Okun F. (1987) Regulation of central and peripheral benzodiazepine receptors in progesterone-treated rats. Brain Res. 409: 3866390. Goldstein D. B. (1978) Alcohol-withdrawal reactions in mice: effects of drugs that modify neurotransmission. J. Pharmac. exp. Ther. 186: 1-9. Goyer P. F., Brown G. L., Minichiello M. P. and Major L. F. (1984) Mood-altering effects of disulfiram in alcoholics. J. Stud. Alcohol 45: 209-213. Greenberg D. A., Cooper E. C., Gordon I. and Diamond I. (1984) Ethanol and the GABA-benzodiazepine receptor complex. J. Neurochem. 42: 1062-1068. _ _ Kirvbakaran V., Faiman M. D., Liskow B. and Mayfield D. (1986) Plasma measurements of disulfiram and its metabolites in a case of severe disulfiram-ethanol reaction. Psychiar. J. Univ. Ottawa 11: 166-168. Marangos P. J., Pate1 J., Boulenger J. P. and ClarkRosenberg R. (1982) Characterization of peripheral-type benzodiazepine binding sites in brain using [jH]Ro 5-4864. Molec. Pharmac. 22: 26-32.
Matthew E., Laskin J. D., Zimmerman E. A., Weinstein I. B., Hsu K. C. and Engelhardt D. L. (1981) Benzodiapines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells. Proc. natn. Acad. Sci. U.S.A. 78: 3935-3939.
Mohler H. and Okada T. (1977) Benzodiazepine receptor: demonstration in the central nervous system. Science, Wash. 198: 849-851.
Mohler H., Okada T., Heitz P. H. and Ulrich J. (1978) Biochemical identification of the site of action of benzodiazepines in human brain. Life Sci. 22: 985-996. Paul S. M., Marangos P. J. and Skolnick P. (1981) The benzodiazepineGABA-chloride-ionophore receptor complex: site of minor tranquilizer action. Biol. Psychiaf. 16: 213-229.
Rastogi S. K., Thyagarajan R., Clothier J. and Ticku M. K. (1986) Effect of chronic treatment of ethanol on benzodiazepine and picrotoxin sites on the GABA receptor complex in regions of the brain of the rat. Neuropharmacology 25: 1179-I 184.
Benzodiazepine receptors in alcoholics Schoemaker H., Boles R. G., Horst W. D. and Yamamura H. I. (1983a) Specific high-affinity binding sites for [3H]Ro S-4864 in rat brain and kidney. J. Pharmac. exp. Ther. 225: 61-69. Schoemaker H., Thomas L. S. and Yamamura H. I. (1983b) Effect of chronic ethanol consumption on central- and peripheral-type benzodiazepine binding sites in the mouse brain. Brain Res. 253, 347-350. Schurr A., Ho B. T. and Schoolar J. C. (1978) The effects of disulfiram on rat liver mitochondrial monoamine oxidase. Life Sci. 22: 1979-1984. Snyder S. and Keeler M. (1981) Acute effects of disulfiram on anxiety levels of chronic alcoholics. Inz. Pharmacopsychiat.
16, 49-56.
Squires R. F. and Braestrup C. (1977) Benzodiazepine receptors in rat brain. Nature, Lond. 266: 732-734. Suranyi-Cadotte B., Lafaille F., Dongier M., Dumas M. and Quirion R. (1988) Decreased density of peripheral benzodiazepine binding sites on platelets of currently drinking but not abstinent alcoholics. Neuropharmacology 27: 443-44s.
Suzdak P. D., Schwartz R. D., Skolnick P. and Paul S. M. (1986) Ethanol stimulates GABA receptor-mediated chloride transport in rat brain synaptoneurosome. Proc. natn. Acad. Sci. U.S.A. 83: 4071-4075.
Syapin P. J. and Alkana R. L. (1988) Chronic ethanol
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exposure increases peripheral-type benzodiazepine receptors in brain. Eur. 2. ~harmac~‘147: 101-109.~ _ Tallman J. F.. Paul S. M.. Skolnick P. and Gallaaer D. W. (1986) Receptors for the age of anxiety: pharm&ology of the benzodiazepines. Science, Wash. 207: 274-28 1. Tamborska E. and Marangos P. J. (1986) Brain benzodiazepine binding sites in ethanol-dependent and withdrawal states. L.ife Sci. 38: 465-472. _ Tanieuchi T.. Wane J. K. T. and Soector S. (1982) [3H]Diazepam binding sites on rat heart and kidney. Biochem. Pharmac. 31, 589-590.
Vallari R. C. and Pietruszko R. (1982) Human aldehyde dehydrogenase: mechanism of inhibition by disulfiram. Science, Wash. 216: 637-639. Wang J. K. T., Morgan J. I. and Spector S. (1984) Differentiation of Friend erythroleukemia cells induced by benzodiazepines. Proc. natn. Acad. Sci. U.S.A. 81: 3770-3772. Wang J. K. T., Taniguchi T. and Spector S. (1986) Properties of [3H]diazepam binding on rat blood platelets. Life Sci. 27: 1881-1888. Weizman R., Tanne Z., Granek M., Karp L., Golomb S., Tyano S. and Gavish M. (1987) Peripheral benzodiazepine binding sites on platelet membranes are increased during diazepam treatment of anxious patients. Eur. J. Pharmac. 138: 289-292.
0028-3908/91 $3.00 + 0.00 Pergamon Press plc
Printedin Great Britain
PERIPHERAL BENZODIAZEPINE RECEPTORS ON PLATELETS IN CHRONIC AND DETOXIFIED ALCOHOLICS* L. KARP,’ A. WEIZMAN,’ M. FLIMAN,* S. TYANO’ and M. GAVISH”~ ‘Geha Psychiatric Hospital, Beilinson Medical Center, Petah Tiqva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, ‘Israel Residential Center for Alcoholism, Ramat Gan and Talbieh Mental Health Center, affiliated with the Hebrew University-Hadassah Medical School, Jerusalem and ‘Department of Pharmacology, Faculty of Medicine and Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, P.O.B. 9649, 31096 Haifa, Israel (Accepted 22 January 1991) Summary-The
effect of chronic alcoholism and detoxification treatment with disulfiram on platelet peripheral benzodiaxepine receptors was studied in alcoholic males. Chronic consumption of alcohol did not alter the binding values for [‘H]PK 11195, as compared to non-alcoholics. Treatment for 3 weeks with disulfiram resulted in a significant increase in the density of peripheral benzodiazepine receptors, with no alteration in the affinity of these sites to the ligand. These results might be relevant to the cellular and metabolic effects of disulfiram. Key words-peripheral
benzodiaxepine receptor, PK 11195, alcoholism, disulfiram.
Specific high-affinity benzodiazepine receptors in the central nervous system (CNS) (Mohler and Okada,
1977; Squires and Braestrup, 1977) are coupled to the y-aminobutyric acid (GABA) receptor and chloride ion channel (Gavish and Snyder, 1981; Paul, Marangos and Skolnick, 1981). These central benzodiazepine receptors are involved in the anticonvulsant and anxiolytic effects of benzodiazepines (Mohler, Okada, Heitz and Ulrich, 1978; Tallman, Paul, Skolnick and Gallagher, 1980). In addition, peripheral-type benzodiazepine receptors have been identified in peripheral tissues, such as platelets, kidney, heart, adrenal and testis, as well as in the CNS (Wang, Taniguchi and Spector, 1980; Marangos, Patel, Boulenger and Clark-Rosenberg, 1982; Taniguchi, Wang and Spector, 1982; Schoemaker, Boles, Horst and Yamamura, 1983a; Anholt, De Souza, Kuhar and Snyder, 1985a; Anholt, De Souza, Oster-Geramite and Snyder, 1985b). They differ from the central receptors in their distribution in tissue, subcellular localization, pharmacological specificity and function, lack of coupling to GABA receptors and the chloride ion channel and specificity for ligand binding (Marangos et al., 1982; Anholt et al., 1985a, 1985b; Anholt, Aebi, Pedersen and Snyder, 1986a; Anholt, Pedersen, De Souza and Snyder, 1986b). Peripheral benzodiazepine receptors seem to to be under neural and hormonal control (Gavish, Okun, Weizman and Youdim, 1986a; Gavish, Weizman, Okun and Youdim, 1986~; Gavish, Weizman, Youdim and Okun, 1987; Basile, *Due to circumstances beyond the Publisher’s control, this article appears in print without author corrections.
Ostrowski and Skolnick, 1987; Fares, Bar-Ami, Haj-Yehia and Gavish, 1989). Ethanol, similary to benzodiazepines, possesses an anxiolytic and hypnotic effect. There is cross-tolerance and dependence between the two drugs, which suggests the possibility of a common mode of action (Goldstein, 1978). Chronic consumption of ethanol does not alter the binding capacity of [‘Hlflunitrazepam, [‘Hlmuscimol or [“Slbutylbicyclophosphorothionate to the brain of the rat (Greenberg, Cooper, Gordon and Diamond, 1984). Furthermore, the ability of GABA to enhance the binding of [‘Hlflunitrazepam is not affected by chronic treatment with ethanol or by withdrawal (Rastogi, Thyagarajan, Clothier and Ticku, 1986). It has recently been demonstrated that ethanol stimulates the GABA receptor-coupled transport of chloride ions in synaptoneurosomes from the brain of the rat (Suzdak, Schwartz, Skolnick and Paul, 1986). Chronic use of ethanol has been reported to induce a significant increase in the density of peripheral benzodiazepine receptors in the brain of mice and rats (Schoemaker, Thomas and Yamamura, 1983b; Tamborska and Marangos, 1986; Syapin and Alkana, 1988). A recent study in human8 demonstrated a significant reduction in the density of binding sites for [‘H]PK 11195 on platelets in chronic alcoholics, but not in alcoholics, abstinent for at least 4 months (SuranyiCadotte, Lafaille, Dongier, Dumas and Quirion, 1988). The present study was designed to reassess the effect of long-term consumption of alcohol and detoxification (using disulfiram as preventive treatment) on
665
L. KARP et al.
666
peripheral benzodiazepine chronic alcoholics.
receptors on platelets in
METHODS
Materials
[)H] 1-( 2-chlorophenyl)-N-methyl-N-( 1 -methylpropyl)-3-isoquinoline carboxamide ([3H]PK 11195, 92.3 Ci/mmol) was purchased from New England Nuclear, Boston, Massachusetts. Unlabelled 7-chloro1,3-dihydro- 1-methyl-5-(p-chlorophenyl)-2H1,4benzodiazepine-Zone (Ro 5-4864) was kindly supplied by Drs H. Gutman and E. Kyburz, Hoffmann-La Roche, Basel, Switzerland. Lumax was purchased from Lumac, Schaesberg, The Netherlands. All other chemicals were purchased from commercial sources. Subjects
The population studied was comprised of three (currently drinking, groups: 9 alcohol-dependent with mean alcohol intake of 1923 f 494 g/week) male subjects, aged 30-52 years (mean f SD, 41.0 f 4.7 years); 10 detoxified male patients, aged 34-54 years (mean + SD, 42.7 f 6.4 years) and 9 non-alcoholic (with mean alcohol intake of 92 f 50 g/week) healthy volunteer male controls, aged 30-40 years (mean + SD, 38.7 f 1.7 years). All 19 patients met the DSM-III-R criteria for dependence on alcohol (American Psychiatric Association, 1987). These criteria include at least three of the following: (1) alcohol often taken in larger amounts or over a longer period than the person intended; (2) persistent desire or one or more unsuccessful efforts to cut down or control use of alcohol; (3) a great deal of time spent in activities necessary to get the substance, taking the substance or recovering from its effects; (4) frequent intoxication or withdrawal symptoms when expected to fulfil major role obligations at work, school or home, or when use of the substance is physically hazardous; (5) important social, occupational or recreational activities given up or reduced because of use of alcohol; (6) continued use of alcohol, despite knowledge of having persistent or recurrent social, psychological or physical problems that are caused or exacerbated by the use of the substance; (7) marked tolerance: need for markedly increased amounts of alcohol to achieve intoxication or desired effect or markedly diminished effect with continued use of the same amount; (8) characteristic withdrawal symptoms; and (9) alcohol often taken to relieve or avoid withdrawal symptoms. The alcoholics were all inpatients (in a closed department), participating in an alcohol-treatment program with no access to alcohol. The duration of abuse of alcohol was similar between the patients currently drinking and the detoxified alcoholics (mean + SD, 11.7 f 4.0 vs 9.6 f 3.0 years). On admission, each patient underwent a thorough physical, neurological and psychiatric examination and routine laboratory check-up. Exclusion criteria
were a past history or present symptoms of psychiatric or neurological disease, as well as concomitant abuse of drugs. None of the subjects had ever been treated with psychoactive drugs on a regular basis. All subjects were physically healthy. During the first 2 weeks of alcohol detoxification, the patients were treated with tapering doses of chlordiazepoxide (maximum dose, 30 mg/day per OS) to prevent symptoms of alcohol-withdrawal. Patients were treated thereafter with disulfiram (Antabuse) (250mg/day per OS) for 3 weeks. Membrane preparation
Blood samples (50 ml) for assessment of the binding of [3H]PK 11195 were withdrawn concomitantly from patients and controls, between 8:00 and 10:00 a.m., collected into plastic tubes, containing 8.8 ml of a mixture of 2.2% sodium citrate and 1.2% citric acid and spun at 180 g for 15 min at 23°C. Platelet-rich plasma was collected and spun at 1500 g for 15 min at 23°C. The platelet-containing pellet was frozen at -70°C until assay. The samples were thawed and prior to the binding assay, the pellet was homogenized in 20 ml of 50 mM Tris-HCl buffer, pH 7.4, at 4°C with a Brinkmann Polytron (setting 10) for 15 set and centrifuged at 49,000g for 15 min at 4°C. The procedure was immediately repeated. The pellet was homogenized in 20 ml of Tris-HCl buffer and used for binding studies. Binding assay for [IH]PK 11195
The binding of [3H]PK 11195 was conducted as previously described (Fares and Gavish, 1986; Gavish, Weizman, Karp, Tyano and Tanne, 1986b). The binding assay, in a final volume of 50 ~1, contained 400 ~1 platelet membranes (70-1OOpg protein) and 25 ~1 [3H]PK 11195 (final concentration 0.2-6 nM) in the absence (total binding) or presence (non-specific binding) of 10 p M unlabelled Ro 5-4864. After incubation for 60 min at 4”C, samples were filtered under vacuum over Whatman GF/B filters and washed three times with 3 ml of Tris-HCl buffer. Filters were placed in vials containing 5 ml of xylene-Lumax (3 : 1, v/v) and counted for radioactivity. Analysis of data
The equilibrium dissociation constant (K,) and maximum number of binding sites (B,,,,,) were determined by Scatchard analyses of saturation curves of 11195 binding of [3H]PK. The binding parameters were analyzed for each subject individually. Oneway analysis of variance (ANOVA) was used for intergroup comparisons. Results are expressed as mean f SEM. RESULTS
The platelet B,,, values for [)H]PK 11195 in the chronic alcoholics, detoxified alcoholics (after 5 weeks’ treatment: chlordiazepoxide for the first 2 weeks and
Benzodiazepine receptors in alcoholics l
4
chmnlcabholii
667 (n-9)
H detoxifkd akohoiia (n-10)
Fig. 1. Effect of chronic alcoholism and detoxification (under disulfiram maintenance treatment) on the maximum binding capacity of peripheral benzodiazepine receptors on platelets. Results are expressed as mean f SEM. *P < 0.05 compared to detoxified patients and controls [one-way ANOVA: F(2,25) = 4.131.
disulfiram maintenance for 3 weeks thereafter) and the control group are summarized in Fig. 1. No significant difference in the density of binding sites for [3H]PK 11195 in platelets was detected between chronic alcoholics and normal controls. However, the disulfiramtreated detoxified alcoholics revealed a significant increase in the number of peripheral benzodiazepine receptors, when compared to normal controls (32%; P < O.OS), as well as to chronic alcoholics (25%; P c 0.05). The KD values were similar in all three groups (chronic alcoholics, 3.1 + 0.2; detoxified alcoholics, 3.7 f 0.4; and controls, 3.3 f 0.3 nM; one-way ANOVA: F(2,25) = 1.03; not significant). DISCUSSION
These results demonstrated an unaltered density of peripheral benzodiazepine receptors on platelets in chronic alcoholics, while maintenance treatment with disulfiram resulted in up-regulation of this binding site. Previous studies have shown an increase in the binding of [3H]Ro 5-4864 to whole brain in mice (Schoemaker et al., 1983b; Syapin and Alkana, 1988) and to cerebral cortex, cerebellum and hippocampus in the brain of the rat (Tamborska and Marangos, 1986), after chronic (but not acute) exposure to ethanol. The changes in peripheral benzodiazepine receptors in rats did not extend to the olfactory bulb and kidney (Tamborska and Marangos, 1986). The elevation in the binding persisted for 3 days, after cessation of ethanol and thereafter returned to basal levels. In both studies, central benzodiazepine receptors remained unaltered (Schoemaker et al., 1983b; Tamborska and Marangos, 1986). Since administration of ethanol induced selective and bidirectional alterations in peripheral benzodiazepine receptors, it seems that these changes could not be attributed to non-specific alterations in membrane fluidity (Chin and Goldstein, 1977). One human study has shown a decreased density of peripheral benzodiazepine receptors on platelets
in currently drinking, but not in abstinent, alcoholics (Suranyi-Cadotte et al., 1988). This line of evidence suggests that peripheral benzodiazepine receptors are probably relevant to the neurobiological effects of ethanol consumption. However, no alteration could be detected in the density of peripheral benzodiazepine receptors on platelets in chronic alcoholics. The cause of the discrepancy between the two studies is unclear, but may be related to differences in the duration or quantity of consumption of alcohol (unfortunately, these details were not given in the study of SuranyiCadotte et al., 1988). The elevation in peripheral benzodiazepine receptors on platelets, observed in detoxified alcoholics, seemed to be related to the maintenance treatment with disulfiram. The up-regulation of these binding sites is unlikely to be attributable to administration of chlordiazepoxide, since the patients were free of this drug for 3 weeks. In a previous study, it was demonstrated that chronic treatment with diazepam in patients suffering from generalized anxiety disorder, induced an up-regulation of peripheral benzodiazepine receptors on platelets. However, 1 week after discontinuation of treatment with diazepam, the density of the binding sites was similar to that of untreated normal controls (Weizman, Tanne, Granek, Karp, Golomb, Tyano and Gavish, 1987). The enhancement in the binding of [‘H]PK 11195 was most probably not due to a psychotropic effect of disulfiram, since controlled studies did not demonstrate a sedative or anxiolytic effect of disulfiram in chronic alcoholics (Snyder and Keeler, 1981; Goyer, Brown, Minichiello and Major, 1984). It is not yet clear whether previous dependency on alcohol is a prerequisite for the up-regulatory effect of disulfiram on peripheral benzodiazepine receptors. Further studies, with non-dependent volunteers treated with disulfiram, can clarify this issue. In vitro competition experiments performed in this laboratory have shown that disulfiram is a competitive inhibitor (ICsO= 5 PM) at the peripheral benzodiazepine receptors in brain and kidney (unpublished
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data). Levels of disulfiram in plasma in alcoholic patients, maintained on preventive treatment with disulfiram reached a concentration of 1-2 PM (Kirvbakaran, Faiman, Liskow and Mayfield, 1986). Since the presence of disulfiram in vitro inhibited rather than stimulated the binding of [3H]PK 11195, it is suggested that the increase in the density of peripheral benzodiazepine receptors on platelets was attributable to the modulatory effect of chronic treatment with disulfiram. Furthermore, the procedure of preparation of membranes removed most of the disulfiram from the binding assay. Subcellular localization studies indicate that the peripheral benzodiazepine receptors are associated with the outer membrane of the mitochondria (Anholt et al., 1986a, 1986b; Basile and Skolnick, 1986). Drugs which are known to affect peripheral benzodiazepine receptors, produce stimulation or inhibition of cell proliferation (Matthew, Laskin, Zimmerman, Weinstein, Hsu and Engelhardt, 1981; Wang, Morgan and Spector, 1984). Disulfiram, on the other hand, produces hepatic hypertrophy and hyperplasia in rats (Fiala, Fiala and Keller, 1977). Disulfiram affects mitochondrial enzymes, such as NAD+-dependent aldehyde dehydrogenase (Vallari and Pietruszko, 1982) and monoamine oxidase (Schurr and Schoolar, 1978). The disulfiram-induced increase in peripheral benzodiazepine receptors on platelets might thus be relevant to the effects of this drug on cell growth. differentiation and metabolism.
Acknowledgements-This
work was supported in part by the Israel Institute for Psychobiology-the Charles E. Smith Family Foundation (Grant No. 26-90). We thank Mrs Zehava Tanne for technical assistance, Mrs Cila Aviram for clinical assistance and Miss Ruth Singer for typing and editing the manuscript.
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