Chronic Treatment with Cholinesterase Inhibitors Increases T~2-Adrenoceptors in Rat Brain
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European Journal of Pharmacology, 153 (1988) 167-173 167 Elsevier EJP 50403 Chronic treatment with cholinesterase inhibitors increases t~2-adrenoceptors in rat brain Peggie J. Hollingsworth * Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI 48109, U.S.A. Received 4 February 1988, revised MS received 18 May 1988, accepted 31 May 1988 The specific binding of [3H]clonidine to a2-adrenoceptors on neural membranes isolated from various brain areas was determined with rats treated for 7-14 days with the cholinesterase inhihitors neostigmine, triorthocresyl phosphate (TOCP), diisopropylfluorophosphate (DFP) and paraoxon, or with vehicle. Treatment with all four inhibitors increased the number of clonidine binding sites in various brain areas. In those areas which demonstrated significant increases in [3H]clonidine binding, there was also a significant inhibition of acetylcholinesterase activity. The possibility is discussed that increases in brain a2-adrenoceptors are related to the alterations in mood seen in individuals chronically exposed to organophosphorus cholinesterase inhibitors. a2-Adrenoceptors; Acetylcholinesterase inhibition (chronic); [3H]Clonidine binding; Brain; (Rat) 1. Introduction has been postulated to exist in depression (Smith et al., 1981; Cohen et al., 1982). This hypothesis is More than 25 years ago Gershon and Shaw based in part upon observations which suggest (1961) reported that farm workers in Australia that antidepressant drugs and treatments cause a who were exposed for long periods of time to 'subsensitivity' of presynaptic a2-adrenoceptors organophosphorus insecticides develop a depres- (Smith and Hollingsworth, 1983; 1984). sion similar to melancholia (major depressive dis- The development of radioligand binding tech- order). Subsequently, a small body of evidence has niques to identify a2-adrenoceptors in the brain accumulated which supports the hypothesis that have stimulated an interest in the effects of psy- depression involves abnormahties of cholinergic chotropic drugs upon the number and/or affinity neuronal function. The 'amine theory' of depres- of these recepors (Bylund and U'Prichard, 1983; sion was generated almost simultaneously with U'Prichard, 1984). A number of studies have re- these early observations (Bunney and Davis, 1965; ported that the administration of antidepressant Schildkraut, 1965), and subsequent biological re- drugs decreases the specific binding of [3H]cloni- search on depression and upon the mechanisms of dine to neural membranes isolated from various action of antidepressant drugs has been dominated areas of the rat brain (Smith et al., 1981; Vetulani, by those who were exploring this theory. Recently, 1982; Smith and Hollingsworth, 1983). Such stud- an abnormality of prejunctional a2-adrenoceptors ies have strengthened the hypothesis that a 2- adrenoceptor function is altered in depression. Despite the postulated interaction between cholinergic and noradrenergic systems in depres- * To whom all correspondence should be addressed: Depart- ment of Pharmacology,0626, M6428 Medical Science I, The sion (Fernando et al., 1984; Potter et al., 1985; University of Michigan Medical School, Ann Arbor, MI Robinson et al., 1986), there has been no study of 48109, U.S.A. the possibility that the number and/or affinity of 0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division) 168 c%-adrenoceptors might be modified during long- homogenized in 5 ml of ice-cold Tris-sucrose buffer term exposure to cholinesterase inhibitors. The (Tris-HC1 5 mM; sucrose 250 mM; MgC12 1 mM; purpose of the present study was to determine pH 7.4). The homogenates were centrifuged at whether the chronic treatment of rats with various 1 100 × g for 10 min, and the supernatants were cholinesterase inhibitors might cause changes in saved. The supernatants were next centrifuged at the number and/or affinity of a2-adrenoceptors 40000 × g for 10 min, and the pellet saved. The in the rat brain. The cholinesterase inhibitors pellet was washed twice with 2 ml of fresh incuba- selected were TOCP, an organophosphorus com- tion buffer (Tris-HC1 40 mM; MgC12 10 mM; pH pound with unusual neurotoxicity (Davis and 7.5) and recentrifuged at 40000 × g for 10 min. Richardson, 1980); paraoxon, a metabolite of The final pellet was resuspended in an appropriate parathion, a commonly used insecticide, devoid of volume of Tris-incubation buffer. In equilibrium neurotoxicity (Johnson, 1975); DFP, a long-acting studies total [3H]clonidine binding was measured organophosphorus cholinesterase inhibitor; and in 1 ml aliquots of the fresh membranes which neostigmine, a non-organophosphorus were incubated in duplicate for 30 min at 25°C cholinesterase inhibitor. In the present study each with 0.5-64 nM [3H]clonidine. Non-specific bind- of the inhibitors were found to increase the num- ing was determined by adding unlabelled cloni- ber of binding sites for [3H]clonidine on mem- dine, 10 -5 M, as well as the radioligand to a branes isolated from certain areas of the rat brain. second pair of incubates. Specific binding was defined as the difference between total and non- specific binding. In drug competition studies, the 2. Materials and methods neural membranes were incubated with [3H]cloni- dine at a concentration of 4 nM in the presence of Groups of 6 male, Sprague-Dawley rats, 170-230 concentrations of the various cholinesterase in- g, were injected intraperitoneally (i.p.) with vehicle hibitors up to and including 100/~M. Incubations or with the various cholinesterase inhibitors for 7 were terminated by rapid filtration under vacuum or 14 days. The duration of treatment with any through Whatman GF/C glass fiber filters and by specific drug was determined by the ability of the washing with two 10 ml aliquots of Tris-incuba- animals to tolerate the drug. Twelve hours after tion buffer (25 o C). After termination of the in- the last injection, the rats were decapitated, their cubation, the glass fiber filters were air dried and brains removed rapidly and placed in ice-cold then placed in glass scintillation vials and counted Krebs physiological buffer solution and subse- for radioactivity as described by Smith et al. (1972). quently dissected according to a modification of A computerized program (McPherson, 1983) which the method described by Glowinski and Iversen utilizes Scatchard (Scatchard, 1949) and Hill (Hill, (1966). The Krebs buffer consisted of (mM): NaC1 1910) transformations was used to develop pre- 118; KC1 4.8; CaC12 1.3; KH2PO 4 1.2; MgSO 4 liminary estimates of the apparent dissociation 1.2; NaHCO 3 25; disodium EDTA 0.03; glucose constants (KD) and the maximum number of 10; ascorbic acid 0.06. The following regions were binding sites (Bmax) for the various radioligands. studied: hippocampus, amygdala, hypothalamus, The values obtained from this program were used parietal-occipital cortex, caudate nucleus and an as preliminary estimates which are required for area of the brainstem which contained the locus the non-linear regression analysis program devised coeruleus. by Munson and Rodbard (1980). This program, LIGAND, was used to develop the final values for 2.1. Measurement of specific binding of FH]cloni- K D and Bmax. dine to neural membranes in rat brain homogenates 2.2. Measurement of acetylcholinesterase activity The specific binding of [3H]clonidine to a 2- adrenoceptors in homogenates was measured as Acetylcholinesterase activity was assayed by a follows. Tissues pooled from the six rats were modification of the method described by Ellman 169 et al. (1961). Tissues were homogenized in ice-cold, 2.3. Drugs used 50 mM Tris-HC1/2 mM sodium edetate buffer (pH 8.0). The volume of buffer used was sufficient The following drugs were used: acetylthiocho- to give a final concentration of 20 mg of tissue line iodide (Sigma), clonidine hydrochloride (wet weight) per ml of buffer. To assay for (Boehringer-Ingelheim), [3H]clonidine hydrochlo- acetylcholinesterase activity, 0.25 ml of the homo- ride (New England Nuclear, specific activity 41.5 genate together with 2.75 ml of 0.1 mM tetraiso- mCi/mmol; radiochemical purity 98%), diisopro- propyl pyrophosphoramide (iso-OMPA), a specific pylfluorophosphate (Sigma); neostigmine methyl pseudocholinesterase inhibitor, were preincubated sulfate (Sigma); paraoxon (diethyl p-nitrophenyl under room air for 30 min at 37 ° C. The mixture phosphate, Aldrich); tetraisopropyl pyrophos- was allowed to cool to room temperature (22 ° C) phoramide (iso-OMPA, Sigma), triorthocresyl and placed in 3 ml quartz spectrophotometer cells. phosphate, (TOCP, Eastman). The paraoxon was To each sample was added in sequence: 0.1 ml of specially purified as described by Johnson (1977). color developer (10 mM 5,5'-dithio-bis(2-nitro- All drugs were dissolved in glass-distilled water benzoic acid), 0.1 M Na/K phosphate, pH 7.1, unless otherwise specified. Triorthocresyl phos- and 0.44 M NaHCO3) and 0.02 ml of substrate, phate was diluted with corn oil (Mazola). All drug acetylthiocholine iodide, 75 mM, and mixed. For doses are expressed in terms of their salts. blanks, the acetylthiocholine was replaced by 0.02 ml of distilled H20. The substrate and blank cells were placed in the sample and reference holders 3. Results respectively of a Beckman DU-40 spectropho- tometer, and the change in absorbance was re- corded at 412 nm. Protein determinations were 3.1. Binding of [3H]clonidine to neural membranes performed by the method of Lowry et al. (1951)