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Long-term haloperidol treatment decreases somatostatin binding in rat brain
Neuropeptides (1989) 13,157-163 0 Longman Group UK Ltd 1989
Long-Term Haloperidol Treatment Decreases Sot6atostatin Binding in Rat Brain E. PEREZ-OSO,
B. COLAS, M.P. LOPEZ-RUIZ
Department of Biochemistry Henares, Madrid, Spain.
and Molecular
and E. ARILLA
Biology, Faculty of Medicine, University of Alcala de
Abstract-The effects of short and long-term haloperidol treatment on somatostatin concentration and specific binding in rat cerebral cortex and hippocampus were examined using the binding ligand 1251-Tyr’-somatostatin. Haloperidol treatment did not affect the concentration of somatostatin-like immunoreactivity in the two brain areas. Nevertheless, long-term, and not short-term, haloperidol treatment decreased the number of somatostatin receptors in the cerebral cortex and hippocampus. No significant differences in the apparent binding affinity values were seen after haloperidol treatment. When added at the time of the binding assay haloperidol 34.2bM produced a 42% and 27% decrease in cerebrocortical and hippocampal membrane somatostatin receptors respectively.
Introduction present not only in the hypothalamic tissue but also in other brain areas. Dense populations of cell bodies are present in many major areas including the neocortex, piriform cortex, hippocampus, amygdaloid complex as well as in some hypothalamic nuclei (1). The primary event mediating the effect of somatostatin in target tissues is interaction between the peptide and specific membrane receptors (2). The magnitude of somatostatin action can be modulated by its ambient concentration, the number of receptor sites and their somatostatin Somatostatin is a tetradecapeptide
Date received 3 August 1988 Date revised 1 November 1988 Date accepted 3 November 1988
affinity (3). It has been reported that somatostatin inhibits the extinction of active avoidance behaviour (4,5) while the behavioural effect of the peptide is said to be inhibited by the neuroleptic haloperidol (5). In addition, somatostatin has been reported to lengthen pentobarbital-induced sleeping time (6) while haloperidol shortens it (7). Bead and Martin (8) have found a dose-dependent reduction in striatal somatostatin-like immunoreactivity (SLI) following chronic haloperidol therapy. To date, the possible effect of haloperidol on brain somatostatin receptors is unknown. Therefore, we have investigated the effects of short and long-term treatment with haloperidol on the somatostatin receptors in rat cerebral cortex and hippocampus. The study also includes the determination of SLI concentrations at these sites. 157
158
NEUROPEPTIDES
addition of 1 ml dextran-coated charcoal (dextran: 0.2% w/v, Pharmacia T70, Uppsala, Sweden; The source for the chemicals used was the same as charcoal: Norit A, 2% w/v, Serva, Feinbiin (9). Synthetic Tyr”- somatostatin was radioochemica, Heidelberg, FRG). Dilution curves for iodinated as in (10) to a specific activity of about each brain area were parallel to the standard 360 cilg. curve. The intra-assay and inter-assay variation Male Sprague-Dawley rats (200-2208) were coefficients were respectively 6.5% and 8.1%. injected subcutaneously with haloperidol (Syntex Synaptosomal membranes were separated from Latin0 Laboratories, Barcelona, Spain) (OSmg/ cerebral cortex and hippocampus and prepared as Kg) daily for 3 or 21 days while controls received described by Reubi et al. (15). Cerebral cortex and saline injections, as previously described (11). hippocampus were homogenized in 10mM Rats were sacrificed by decapitation the day after HEPES-KOH pH 7.6 (low/v) with a Brinkmann their last haloperidol injection. The brains were polytron homogenizer (setting 5,15s). The homoremoved and the cerebral cortex and hippocampus genate was spun as 600g for 5 min at 4°C and the were rapidly dissected as per Glowinski and Iversupernatant centrifuged at 48000g for 30 min at sen (12). 4°C to produce a pellet which was then suspended For SLI measurement, cerebral cortex and in 10mM HEPES-KOH pH 7.6 pH 7.6 (low/v) hippocampus were rapidly homogenized using a and centrifuged as before. The resultant pellet was Breinkmann Polytron (setting 5, 3Os), in 1 ml 1 M resuspended in 50mM Tris-HCl buffer (pH 7.5). acetic acid. Extracts were boiled for 5 min in a Samples were stored at 70°C until assay. Protein water bath, chilled in ice, and aliquots (100~1) was determined as per Lowry et al. (13). were removed for protein determination (13). Specific somatostatin binding was measured Subsequently homogenates were centrifuged at according to the modified method of Czernik and 15000g for 15 min at 4”C, and the supernatant was Petrack (16). Brain membranes (about 1.5mg neutralized with 1M NaOH and stored at -70°C protein/ml) were incubated in 250 ~1 of a medium until assay. The rabbit antibody used in the containing 50mM Tris-HCl buffer (pH 7.5), 5 mM radioimmunoassay technique was raised in rabbits MgC12, 0.2% (w/v) bovine serum albumin and against somatostatin-14 conjugated to bovine 0.1 mg/ml bacitracin with 250pM ‘251-Tyr”-soserum albumin and is specific for somatostatin. matostatin either with or without O.Ol-10nM of However since the C-terminal portions of both unlabelled somatostatin. After 60 min incubation somatostatin-25 and somatostatin-28 are constiat 3O”C, the free radioligand was separated from tuted by somatostatin-14, the antiserum does not the bound radioligand by centrifugation at 12000g distinguish between these three forms. (Beckman microcentrifuge) for 1.5 min and the Somatostatin concentration was determined in resultant pellet was counted in a Beckman gamma counter. Nonspecific binding, i.e., binding occurtissue extracts by a modified radioimmunoassay method (14), with a sensitivity limit of lOpg/ml. ring in the presence of high concentration (lo-‘M) of unlabelled somatostatin, represented about Incubation tubes prepared in duplicate contained 20% of the binding observed in absence of native 100~1 samples of either unknown or standard peptide and was subtracted from the total bound solutions of 0-500pg cyclic somatostatin tetradecapeptide diluted in phosphate buffer (O.O5M, radioactivity in order to obtain the corresponding specific binding. ‘251-Tyr”-somatostatin inpH 7.2 containing 0.3% bovine serum albumin, activation after exposure to membranes in the 0.01 M EDTA), 200 l~,lappropriately diluted antisomatostatin serum, 100 t.~lfreshly prepared 1251- incubation medium was studied by observing the ability of the peptide to rebind to fresh membranes Tyr”-somatostatin diluted in buffer to give 60OOcpm (equivalent to 5-lOpg), and enough (17). To assess whether haloperidol exerts a direct buffer to give a final volume of 0.8ml. All reagents action on somatostatin receptors, haloperidol at as well as the assay tubes were kept chilled in ice, before 48 hours incubation at 4°C. Separation of concentrations ranging from 1mM to 1 nM was bound and free hormone was accomplished by included in the incubation medium at the time of Methods
LONG-TERM
HALOPERIDOL
Cerebral cwtex
TREATMENT
I
DECREASES
SOMATOSTATIN
Hippocampus
Fig 1 SLI concentrations in cerebral cortex and hippocampus in control (open bars) and haloperidol (closed bars) -treated rats. Values are expressed as the mean + SEM of five separated experiments. In each of the experiments, determination were made in duplicate. Differences were not statistically significant.
the binding assay using normal rat cerebrocortical and hippocampal membranes. Results were given in all cases as mean + S.E.M. The Students t test for unpaired variables was employed to assess differences between control and haloperidol-injected groups, as indicated in the figures. The numbers of receptors and affinity constant in Scatchard plots (18) were calculated by linear regression analysis with a Hewlett-Packard device.
159
BINDING IN RAT BRAIN
was determined (17). Membranes from different brain areas showed similar rate of peptide degradation, values being about 10% in control and in rats treated with haloperidol. Brain membrane preparations either from controls or from short-term haloperidol treated rats (3 days) exhibited an almost identical pattern of specific ‘251-Tyr’1-somatostatin binding both in the absence and presence of increasing concentrations (O.Ol-10nM) of unlabelled somatostatin (data not shown). In contrast, after 21 days of haloperidol treatment the receptor binding was decreased significantly throughout the whole range of peptide concentrations studied (Fig 2 and 3 left panel). binding Decreases in ‘251-Tyr”-somatostatin may be due to changes in the number of sites or in the dissociation constant (Kd). Scatchard analysis of ‘251-labelled ligand binding at a wide range of concentration after 21 days treatment indicates that haloperidol decreased the number of somatostatin receptors without changing the affinity constant (Table 1). The Kd values were in the nanomolar range, and Scatchard analysis indicated a single class of binding sites. To determine if haloperidol directly interferes with somatostatin receptors, ‘251-Tyr”-somatostatin binding to membrane receptors from cere1
Cerebmlcortex 5
105
t --------
Results The concentration of SLI in the cerebral cortex and hippocampus extracts are shown in Figure 1. Although administration of the haloperidol seemed to increase the SLI levels in both brain areas, the difference between groups was not significant (p > 0.05). Brain plasma membranes of both control and haloperidol-treated animals bound ‘251-pTyr”somatostatin in a time-dependent fashion, an apparent equilibrium being observed between 50-180 min at 30°C (data not shown). All subsequent binding studies were therefore conducted at 30°C for 60 min. In order to rule out the possibility of different somatostatin degrading activities in the membrane preparations that could affect interpretation of results, the degradation of the peptide
Bound,nM
Fig 2 Left panel: Competitive inhibition of specific *‘%Tyrl’somatostatin (1251-Tyr“-SS, 250pM) binding to membranes of the cerebral cortex by unlabelled somatostatin. Membranes (1Smg protein/ml) were incubated for 60 min at 30°C in the presence of 250pM “%Tyr”-somatostatin and increasing concentration of native peptide. Data correspond to control (@) and haloperidol (0) -treated rats for 21 days. Values are expressed as the mean + SEM of five replicate experiments. Right panel: Scatchard analysis of the same data.
160
NEUROPEPTIDES
These modificiations were seen after long-term (21 days) but not short-term (3 days) treatment with haloperidol. SLI levels were not modified in either brain area, as compared with control animals at any time studies. The levels in the cerebral cortex and hippocampus and the binding parameters of brain somatostatin receptors in control rats were similar to those previously reported by other authors (19, 20). It should be mentioned that the Scatchard analysis demonstrated the existence of only one type of somatostatin receptor. Although this Bound,nM rsq-IogM agrees with some studies (16, 19, 21), it differs Fig 3 Left panel: Competitive inhibition of specific 1z51-Tyr’1from other previously reported data (22, 23). It is somatostatin (250pM) binding to membranes of the hippo-
‘-:r
campus by unlabelled somatostatin. Membranes (1.5 mg protein/ml) were incubated for 60 min at 30°C in the presence of 250pM ‘2SI-Tyr11-somatostatin and increasing concentration of native peptide. Data correspond to control (0) and haloperidol (0) -treated rats for 21 days. Values are expressed as the mean f SEM of five replicate experiments. Right panel: Scatchard analysis of the same data.
v
1 Qo50
e”
.
a*
T
brocortical and hippocampal membranes was investigated in the absence and presence of added haloperidol (up to 10nM final concentration) (Figs 4 and 5). There was a significant decrease in binding capacity but not affinity at 34.2l.~M haloperidol in cerebrocortical and hippocampal membranes (Table 2).
0
i
J
0
Treatment
with Haloperidol
on Specific
Controls 3 days 21 days
0.495 f 0.13 0.395 + 0.09 0.469 + 0.14
a04
Somatostatin
Receptors
in
Hippocampus
Cerebral cortex Kd (nM)
002
Fig 4 Left panel: Direct effect of haloperidol on the competitive inhibition by unlabelled somatostatin of the specific binding of 1251-Tyr’1-somatostatin (‘*‘I-Tyr”-SS, 250pM) to normal rat cerebrocortical membranes (1.5mg protein/ml). Receptor binding was assessed in the absence (0) or presence (0) of 34.2t~,M haloperidol. Values are expressed as the mean f SEM of five replicate experiments. Right panel: Scatchard analysis of the binding data. The kinetic constants calculated by Scatchard analysis are given in Table 2.
The present study indicates that haloperidol treatment in the rat results in a decrease of somatostatin receptors in cerebral cortex and hippocampus.
of Short and Long-Term and hippocampus
I
hmd.nM
Discussion
Table 1 Effect Cerebral Cortex
0
I
Bmu.
194 * 14 247 + 41 112 + 11*
Kd (nM)
0.255 + 0.05 0.250 f 0.04 0.224 f 0.05
Bmar.
179 + 6 184 + 11 131 + 16*
Binding parameters were obtained by Scatchard (18) analysis of data from Figs 2-3, right panels. Bmax, Binding capacity (femtomoles of somatostatin bound per mg protein). Values represent the mean f SEM of five rats in each group. “p < 0.005 vs. control.
LONG-TERM
HALOPERIDOL
TREATMENT
DECREASES
SOMATOSTATIN
161
BINDING IN RAT BRAIN
radioimmunoassay. Other authors have shown that chronic haloperidol modify the synthesis of substance P (24, 25) and enkephalins (26). It is unlikely that changes in receptor binding are due to residual drug in the brain, since short-term injections of haloperidol fail to alter binding to somatostatin receptors. However, for the haloperidol to have an effect on the somatostatin receptors, a significant concentration of the drug must be present in the animal. -
11
10
9
tSS1,-logM
8
0
0030
aa5 Bwnd,nM
Fig 5 Left panel: Direct effect of haloperidol on the competitive inhibition by unlabelled somatostatin of the specific binding of ‘*‘I-Tyr” -somatostatin (rz51-Tyr”-SS, 250pM) to normal rat hippocampal membranes (1Smg protein/ml) Receptor binding was assessed in the absence (0) or presence (0) of 34.2 PM haloperidol. Values are expressed as the mean k SEM of five replicate experiments. Right panel: Scatchard analysis of the binding data. The kinetic constants calculated by Scatchard analysis are given in Table 2.
conceivable that differences in iz51-Tyr”-somatostatin specific activities could be the cause of this variation. The lack of modification in the concentration of SLI in the cerebral cortex and hippocampus after haloperidol treatment (3 or 21 days) agrees with Beal and Martin findings (8). It might be that even though the overall content of SLI in the cerebral cortex and hippocampus did not vary significantly, somatostatin synthesis and release rates may have changed. If this were the case, increased somatostatin release or turnover might lead to down-regulation of somatostatin receptors in these brain areas, even though we did not detect a significant increase in SLI content with
Since somatostatin activity is known to be presynaptically regulated by dopamine (27), the increase in somatostatin release could be a secondary reaction to haloperidol-induced (impairment of dopamine neurotransmision) block of dopamine receptors. The decrease of somatostatin binding to brain membranes from haloperidol treatment concurs well with the finding that this neuroleptic is a potent inhibitor of a variety of other neurotransmitter receptors. Thus, in addition to consistent antagonism of dopamine-2 receptors (28), the available haloperidol may also block, to a varying degree, (Y i-adrenergic (29), cholinergic (30), histamine (31) and serotonin receptors (32). However, binding to P-adrenergic and serotonin-2 receptors is unaffected by haloperidol (33). It is difficult to establish a direct relationship between both the in vitro and the in vivo actions of haloperidol upon somatostatin binding to rat brain membranes. Some of the pharmacological effects of haloperidol may well depend on decreases in the number of somatostatin receptors. For example, somatostatin administered intracerebroventricularly inhibits the extinction of active avoidance
Table 2 Equilibrium Parameters of Somatostatin Binding to Normal Rat Cerebrocortical Membranes in the Absence or Presence of 34.2 IAM Haloperidol in the Incubation Cerebral cortex Kd (nM)
Absence of haloperidol Presence of haloperidol
0.374 f 0.08 0.405 + 0.10
and Hippocampal
Hippocampus Bmax.
199 + 12 135 * 11*
Kd (nM)
Bmax.
0.322 _+0.06 0.368 f 0.08
202 t 12 107+9 *
Binding parameters were obtained by Scatchard (18) analysis of data from Figs 4-5, right panels. Bmax, Binding capacity (femtomoles of somatostatin bound per mg protein). Values represent the mean + SEM of five rats in each group. *p < 0.005 vs. absence of haloperidol.
162 behaviour (4,5) while that haloperidol inhibits the behavioural effects of the peptide (5). In addition, somatostatin has been reported to increase pentobarbital-induced sleeping time (6) and induces a frequent dissociation of the electroencephalogram (EEG) (34,35), while haloperidol has the opposite effect (7). Further experiments are required to explore our hypothesis however.
NEUROPEPTIDES 10. Greenwood,
11.
12.
13.
Acknowledgements This work was supported by a grant (88/0903) from the Fondo de Investigaciones Sanitarias de la Seguridad Social of Spain. The authors thank Carol F. Warren, from the Alcala de Henares University Institute of Education Sciences for her editorial help.
14.
15.
16.
References 1. Johansson, O., Hokfelt, T. and Elde, R. P. (1984). Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience 13: 265-339. 2. Schonbrunn, A. and Tashjian, A. H. Jr. (1978). Characterization of functional receptors for somatostatin in rat pituitary cells in culture. Journal of Biological Chemistry 253: 64736483. 3. Draznin, B. (1985). Somatostatin receptors in endocrine cells. In: P. M., Conn (Ed.) The receptors. Vol. II. Academic Press. Orlando, pp. 401-422. 4. Bollok, I., Vecsei, L. and Telegdy, G. (1983). The effect of interaction between propranolol and somatostatin on the active avoidance behaviour, open-field activity and electroconvulsive shock-induced amnesia of rats. Neuropeptides 3: 263-270. 5. Vecsei, L., Bollok, I. andTelegdy, G. (1983). The effect of linear somatostatin on the active avoidance behaviour and open-field activity on haloperidol, phenoxybenzamine and atropine pretreated rats. Acta Physiologica Hungarica 62: 205211. 6. Brown, M. and Vale, W. (1975). Central nervous system effects of hypothalamic peptides. Endocrinology 96: 13331336. 7. Fujii, T., Nakanishi, H., Morimoto, S. and Hara, N. (1987). Pharmacological assessment of the functional effects of the maternal exposure to drugs: transmission of the effects to the offspring of subsequent generations. In: Fujii, T. and Adams, P. M. (Eds.) Functional Teratogenesis. Teikyo University Press, Tokyo, pp. 159-173. 8. Beal, M. F. and Martin, J. B. (1984). Effectsof neuroleptic drugs on brain somatostatin-like immunoreactivity. Neuroscience Letters 47: 125-130. 9. Colas, B., Bodegas, G., Prieto, J. and Arilla, E. (1988). Partial enterectomy decreases somatostatin-binding sites in residual intestine of rabbits. Clinical Science 74: 499505.
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F. C., Hunter, W. M. and Glober. J.S. (1963). The preparation of ‘351-labelled human growth hormone of high specific radioactivity. Biochemical Journal 89: 114-123. Burt, D.R., Creese, I. and Snyder, S.H. (1977). Antischizophrenic drugs: chronic treatment elevates dopamine receptor binding in brain. Science 196: 326-328. Glowinski, J. and Iversen, L. L. (1966). Regional studies of catecholamines in the rat brain. Journal of Neurochemistry 13: 655-669. Lowry, 0. H., Rosebrough, N. J., Farr, A.L. and Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265-275. Pate& J. C. and Reichlin, S. (1978). Somatostatin in hypothalamus, extrahypothalamic brain and peripheral tissues of the rat. Endocrinology 102: 523-531. Reubi, J. C., Perrin, M. H., River, J. E. and Vale, V. (1981). High affinity binding sites for a somatostatin-28 analogue in rat brain. Life Sciences 28: 2191-2198. Czernik, A. J. and Petrack, B. (1983). Somatostatin receptor binding in rat cerebral cortex. Characterization using a nonreducible somatostatin analog. Journal of Biological Chemistry 258: 55255530. Aguilera, G., Parker, D. S. and Catt, K. J. (1982). Characterization of somatostatin receptors in the rat adrenal glomerulosa zone. Endocrinology 111: 1376-1384. Scatchard, G. (1949). The attraction of proteins for small molecules and ions. Annals of New York Academy of Sciences. 51: 660-671. Srikant, C. B. and Pate& Y. C. (1981). Somatostatin receptors: identification and characterization in rat brain membranes. Proceedings of the National Academy of Sciences of the United States of America 78: 3930-3934. Reubi, J. C. (1985). New specific radioligand for one subpopulation of brain somatostatin receptors. Life Sciences 36: 1829-1836. Epelbaum, J., Tapia Arancibia, L., Kordon, C. and Enjalbert, A. (1982). Characterization, regional distribution and subcellular distribution of ‘251-Tyr’-somatostatin binding sites in rat brain. Journal of Neurochemistry 38: 1515-1523. Reubi, J. C. (1984). Evidence for two somatostatin-14 receptor types in rat brain cortex. Neuroscience Letters 49: 259-263. Weightman, D. R., Whitford, C. A., Snell, C. R., Hirst, B. H., Brundish, D. E. and Kendall-Taylor, P. A. (1985). Rat brain membranes possess two high-affinity binding sites for (3H) somatostatin. Neuroscience Letters 55: 161-166. Radke, J. M., MacLennan, A. J., Vincent, S. R. and Fibiger, H. C. (1988). Comparison between short and long-term haloperidol administration on somatostatin and substance P concentrations in the rat brain. Brain Research 445: 55-60. Lindefors, N., Brodin, E. and Ungerstedt, U. (1986). Neuroleptic treatment induces region-specific changes in levels of neurokinin A and substance P in rat brain. Neuropeptides 7: 265-280. Hong, J. S., Yang, H. Y. T., Frata, W. and Costa, E. (1978). Rat striatal methionine-enkephalin content after
LONG-TERM
27.
28.
29.
30.
HALOPERIDOL
TREATMENT
DECREASES
SOMATOSTATIN
chronic treatment with cataleptogenic and noncataleptogenie antischizophrenic drugs. The Journal of Pharmacology and Experimental Therapeutics 205: 141-147. Lee. C. M. and Iversen, L. L. (1981). Release of somatostatin from extrahypothalamic rat brain slices: inhibition by dopamine and morphine. Brain Research 219: 355-361. Peroutka, S. J. and Snyder, S. H. (1980). Relationship of neuroleptic drug effects at brain dopamine, serotonin, -adrenergic, and histamine receptors to clinical potency. American Journal of Psychiatry 137: 1518-1522. Peroutka, S. J., U Pritchard, D. C. and Geenberg. D. A. (1977). Neuroleptic drug interactions with norepinephrines alpha-receptor binding sites in rat brain. Neuropharmacology 16: 549-556. Snyder, S. H., Banerjee, S. P., Yamamura, H. I. and Greenberg, D. A. (1974). Drugs, neuroleptics and schizophrenia. Science 184: 1243-1253.
BINDING
IN RAT BRAIN
163
31. Chang, R. S. L., Tran, V. T. and Snyder, S. H. (1979). Heterogeneity of histamine H receptors: species variations in H-mepyramine binding to brain membranes. Journal of Neurochemistry 32: 1653-1663. 32. Creese, I. and Snyder, S. H. (1978). H-spiroperidol labels serotonin receptors in rat cerebral cortex and hippocampus. European Journal of Pharmacology 49: 201-202. 33. Peroutka, S. J. and Snyder, S. H. (1980). Long-term antidepressant treatment decreases spiroperidol-labelled serotonin receptor binding. Science 210: 88-90. 34. Rezek, M., Havliceck, VG., Hughes, K. R. and Friesen, H. (1976). Central site of action of somatostatin (SRIF): role of hippocampus. Neuropharmacology 15: 499-504. 35. Enna, S. J. and Coyle, J. T. (1983). Neuroleptics. In: Coyle. 1. T. and Enna, S. J. (Eds.) Neuroleptics: Neurochemical, Behavioural and Clinical Perspectives. Raven Press, New York.
Long-Term Haloperidol Treatment Decreases Sot6atostatin Binding in Rat Brain E. PEREZ-OSO,
B. COLAS, M.P. LOPEZ-RUIZ
Department of Biochemistry Henares, Madrid, Spain.
and Molecular
and E. ARILLA
Biology, Faculty of Medicine, University of Alcala de
Abstract-The effects of short and long-term haloperidol treatment on somatostatin concentration and specific binding in rat cerebral cortex and hippocampus were examined using the binding ligand 1251-Tyr’-somatostatin. Haloperidol treatment did not affect the concentration of somatostatin-like immunoreactivity in the two brain areas. Nevertheless, long-term, and not short-term, haloperidol treatment decreased the number of somatostatin receptors in the cerebral cortex and hippocampus. No significant differences in the apparent binding affinity values were seen after haloperidol treatment. When added at the time of the binding assay haloperidol 34.2bM produced a 42% and 27% decrease in cerebrocortical and hippocampal membrane somatostatin receptors respectively.
Introduction present not only in the hypothalamic tissue but also in other brain areas. Dense populations of cell bodies are present in many major areas including the neocortex, piriform cortex, hippocampus, amygdaloid complex as well as in some hypothalamic nuclei (1). The primary event mediating the effect of somatostatin in target tissues is interaction between the peptide and specific membrane receptors (2). The magnitude of somatostatin action can be modulated by its ambient concentration, the number of receptor sites and their somatostatin Somatostatin is a tetradecapeptide
Date received 3 August 1988 Date revised 1 November 1988 Date accepted 3 November 1988
affinity (3). It has been reported that somatostatin inhibits the extinction of active avoidance behaviour (4,5) while the behavioural effect of the peptide is said to be inhibited by the neuroleptic haloperidol (5). In addition, somatostatin has been reported to lengthen pentobarbital-induced sleeping time (6) while haloperidol shortens it (7). Bead and Martin (8) have found a dose-dependent reduction in striatal somatostatin-like immunoreactivity (SLI) following chronic haloperidol therapy. To date, the possible effect of haloperidol on brain somatostatin receptors is unknown. Therefore, we have investigated the effects of short and long-term treatment with haloperidol on the somatostatin receptors in rat cerebral cortex and hippocampus. The study also includes the determination of SLI concentrations at these sites. 157
158
NEUROPEPTIDES
addition of 1 ml dextran-coated charcoal (dextran: 0.2% w/v, Pharmacia T70, Uppsala, Sweden; The source for the chemicals used was the same as charcoal: Norit A, 2% w/v, Serva, Feinbiin (9). Synthetic Tyr”- somatostatin was radioochemica, Heidelberg, FRG). Dilution curves for iodinated as in (10) to a specific activity of about each brain area were parallel to the standard 360 cilg. curve. The intra-assay and inter-assay variation Male Sprague-Dawley rats (200-2208) were coefficients were respectively 6.5% and 8.1%. injected subcutaneously with haloperidol (Syntex Synaptosomal membranes were separated from Latin0 Laboratories, Barcelona, Spain) (OSmg/ cerebral cortex and hippocampus and prepared as Kg) daily for 3 or 21 days while controls received described by Reubi et al. (15). Cerebral cortex and saline injections, as previously described (11). hippocampus were homogenized in 10mM Rats were sacrificed by decapitation the day after HEPES-KOH pH 7.6 (low/v) with a Brinkmann their last haloperidol injection. The brains were polytron homogenizer (setting 5,15s). The homoremoved and the cerebral cortex and hippocampus genate was spun as 600g for 5 min at 4°C and the were rapidly dissected as per Glowinski and Iversupernatant centrifuged at 48000g for 30 min at sen (12). 4°C to produce a pellet which was then suspended For SLI measurement, cerebral cortex and in 10mM HEPES-KOH pH 7.6 pH 7.6 (low/v) hippocampus were rapidly homogenized using a and centrifuged as before. The resultant pellet was Breinkmann Polytron (setting 5, 3Os), in 1 ml 1 M resuspended in 50mM Tris-HCl buffer (pH 7.5). acetic acid. Extracts were boiled for 5 min in a Samples were stored at 70°C until assay. Protein water bath, chilled in ice, and aliquots (100~1) was determined as per Lowry et al. (13). were removed for protein determination (13). Specific somatostatin binding was measured Subsequently homogenates were centrifuged at according to the modified method of Czernik and 15000g for 15 min at 4”C, and the supernatant was Petrack (16). Brain membranes (about 1.5mg neutralized with 1M NaOH and stored at -70°C protein/ml) were incubated in 250 ~1 of a medium until assay. The rabbit antibody used in the containing 50mM Tris-HCl buffer (pH 7.5), 5 mM radioimmunoassay technique was raised in rabbits MgC12, 0.2% (w/v) bovine serum albumin and against somatostatin-14 conjugated to bovine 0.1 mg/ml bacitracin with 250pM ‘251-Tyr”-soserum albumin and is specific for somatostatin. matostatin either with or without O.Ol-10nM of However since the C-terminal portions of both unlabelled somatostatin. After 60 min incubation somatostatin-25 and somatostatin-28 are constiat 3O”C, the free radioligand was separated from tuted by somatostatin-14, the antiserum does not the bound radioligand by centrifugation at 12000g distinguish between these three forms. (Beckman microcentrifuge) for 1.5 min and the Somatostatin concentration was determined in resultant pellet was counted in a Beckman gamma counter. Nonspecific binding, i.e., binding occurtissue extracts by a modified radioimmunoassay method (14), with a sensitivity limit of lOpg/ml. ring in the presence of high concentration (lo-‘M) of unlabelled somatostatin, represented about Incubation tubes prepared in duplicate contained 20% of the binding observed in absence of native 100~1 samples of either unknown or standard peptide and was subtracted from the total bound solutions of 0-500pg cyclic somatostatin tetradecapeptide diluted in phosphate buffer (O.O5M, radioactivity in order to obtain the corresponding specific binding. ‘251-Tyr”-somatostatin inpH 7.2 containing 0.3% bovine serum albumin, activation after exposure to membranes in the 0.01 M EDTA), 200 l~,lappropriately diluted antisomatostatin serum, 100 t.~lfreshly prepared 1251- incubation medium was studied by observing the ability of the peptide to rebind to fresh membranes Tyr”-somatostatin diluted in buffer to give 60OOcpm (equivalent to 5-lOpg), and enough (17). To assess whether haloperidol exerts a direct buffer to give a final volume of 0.8ml. All reagents action on somatostatin receptors, haloperidol at as well as the assay tubes were kept chilled in ice, before 48 hours incubation at 4°C. Separation of concentrations ranging from 1mM to 1 nM was bound and free hormone was accomplished by included in the incubation medium at the time of Methods
LONG-TERM
HALOPERIDOL
Cerebral cwtex
TREATMENT
I
DECREASES
SOMATOSTATIN
Hippocampus
Fig 1 SLI concentrations in cerebral cortex and hippocampus in control (open bars) and haloperidol (closed bars) -treated rats. Values are expressed as the mean + SEM of five separated experiments. In each of the experiments, determination were made in duplicate. Differences were not statistically significant.
the binding assay using normal rat cerebrocortical and hippocampal membranes. Results were given in all cases as mean + S.E.M. The Students t test for unpaired variables was employed to assess differences between control and haloperidol-injected groups, as indicated in the figures. The numbers of receptors and affinity constant in Scatchard plots (18) were calculated by linear regression analysis with a Hewlett-Packard device.
159
BINDING IN RAT BRAIN
was determined (17). Membranes from different brain areas showed similar rate of peptide degradation, values being about 10% in control and in rats treated with haloperidol. Brain membrane preparations either from controls or from short-term haloperidol treated rats (3 days) exhibited an almost identical pattern of specific ‘251-Tyr’1-somatostatin binding both in the absence and presence of increasing concentrations (O.Ol-10nM) of unlabelled somatostatin (data not shown). In contrast, after 21 days of haloperidol treatment the receptor binding was decreased significantly throughout the whole range of peptide concentrations studied (Fig 2 and 3 left panel). binding Decreases in ‘251-Tyr”-somatostatin may be due to changes in the number of sites or in the dissociation constant (Kd). Scatchard analysis of ‘251-labelled ligand binding at a wide range of concentration after 21 days treatment indicates that haloperidol decreased the number of somatostatin receptors without changing the affinity constant (Table 1). The Kd values were in the nanomolar range, and Scatchard analysis indicated a single class of binding sites. To determine if haloperidol directly interferes with somatostatin receptors, ‘251-Tyr”-somatostatin binding to membrane receptors from cere1
Cerebmlcortex 5
105
t --------
Results The concentration of SLI in the cerebral cortex and hippocampus extracts are shown in Figure 1. Although administration of the haloperidol seemed to increase the SLI levels in both brain areas, the difference between groups was not significant (p > 0.05). Brain plasma membranes of both control and haloperidol-treated animals bound ‘251-pTyr”somatostatin in a time-dependent fashion, an apparent equilibrium being observed between 50-180 min at 30°C (data not shown). All subsequent binding studies were therefore conducted at 30°C for 60 min. In order to rule out the possibility of different somatostatin degrading activities in the membrane preparations that could affect interpretation of results, the degradation of the peptide
Bound,nM
Fig 2 Left panel: Competitive inhibition of specific *‘%Tyrl’somatostatin (1251-Tyr“-SS, 250pM) binding to membranes of the cerebral cortex by unlabelled somatostatin. Membranes (1Smg protein/ml) were incubated for 60 min at 30°C in the presence of 250pM “%Tyr”-somatostatin and increasing concentration of native peptide. Data correspond to control (@) and haloperidol (0) -treated rats for 21 days. Values are expressed as the mean + SEM of five replicate experiments. Right panel: Scatchard analysis of the same data.
160
NEUROPEPTIDES
These modificiations were seen after long-term (21 days) but not short-term (3 days) treatment with haloperidol. SLI levels were not modified in either brain area, as compared with control animals at any time studies. The levels in the cerebral cortex and hippocampus and the binding parameters of brain somatostatin receptors in control rats were similar to those previously reported by other authors (19, 20). It should be mentioned that the Scatchard analysis demonstrated the existence of only one type of somatostatin receptor. Although this Bound,nM rsq-IogM agrees with some studies (16, 19, 21), it differs Fig 3 Left panel: Competitive inhibition of specific 1z51-Tyr’1from other previously reported data (22, 23). It is somatostatin (250pM) binding to membranes of the hippo-
‘-:r
campus by unlabelled somatostatin. Membranes (1.5 mg protein/ml) were incubated for 60 min at 30°C in the presence of 250pM ‘2SI-Tyr11-somatostatin and increasing concentration of native peptide. Data correspond to control (0) and haloperidol (0) -treated rats for 21 days. Values are expressed as the mean f SEM of five replicate experiments. Right panel: Scatchard analysis of the same data.
v
1 Qo50
e”
.
a*
T
brocortical and hippocampal membranes was investigated in the absence and presence of added haloperidol (up to 10nM final concentration) (Figs 4 and 5). There was a significant decrease in binding capacity but not affinity at 34.2l.~M haloperidol in cerebrocortical and hippocampal membranes (Table 2).
0
i
J
0
Treatment
with Haloperidol
on Specific
Controls 3 days 21 days
0.495 f 0.13 0.395 + 0.09 0.469 + 0.14
a04
Somatostatin
Receptors
in
Hippocampus
Cerebral cortex Kd (nM)
002
Fig 4 Left panel: Direct effect of haloperidol on the competitive inhibition by unlabelled somatostatin of the specific binding of 1251-Tyr’1-somatostatin (‘*‘I-Tyr”-SS, 250pM) to normal rat cerebrocortical membranes (1.5mg protein/ml). Receptor binding was assessed in the absence (0) or presence (0) of 34.2t~,M haloperidol. Values are expressed as the mean f SEM of five replicate experiments. Right panel: Scatchard analysis of the binding data. The kinetic constants calculated by Scatchard analysis are given in Table 2.
The present study indicates that haloperidol treatment in the rat results in a decrease of somatostatin receptors in cerebral cortex and hippocampus.
of Short and Long-Term and hippocampus
I
hmd.nM
Discussion
Table 1 Effect Cerebral Cortex
0
I
Bmu.
194 * 14 247 + 41 112 + 11*
Kd (nM)
0.255 + 0.05 0.250 f 0.04 0.224 f 0.05
Bmar.
179 + 6 184 + 11 131 + 16*
Binding parameters were obtained by Scatchard (18) analysis of data from Figs 2-3, right panels. Bmax, Binding capacity (femtomoles of somatostatin bound per mg protein). Values represent the mean f SEM of five rats in each group. “p < 0.005 vs. control.
LONG-TERM
HALOPERIDOL
TREATMENT
DECREASES
SOMATOSTATIN
161
BINDING IN RAT BRAIN
radioimmunoassay. Other authors have shown that chronic haloperidol modify the synthesis of substance P (24, 25) and enkephalins (26). It is unlikely that changes in receptor binding are due to residual drug in the brain, since short-term injections of haloperidol fail to alter binding to somatostatin receptors. However, for the haloperidol to have an effect on the somatostatin receptors, a significant concentration of the drug must be present in the animal. -
11
10
9
tSS1,-logM
8
0
0030
aa5 Bwnd,nM
Fig 5 Left panel: Direct effect of haloperidol on the competitive inhibition by unlabelled somatostatin of the specific binding of ‘*‘I-Tyr” -somatostatin (rz51-Tyr”-SS, 250pM) to normal rat hippocampal membranes (1Smg protein/ml) Receptor binding was assessed in the absence (0) or presence (0) of 34.2 PM haloperidol. Values are expressed as the mean k SEM of five replicate experiments. Right panel: Scatchard analysis of the binding data. The kinetic constants calculated by Scatchard analysis are given in Table 2.
conceivable that differences in iz51-Tyr”-somatostatin specific activities could be the cause of this variation. The lack of modification in the concentration of SLI in the cerebral cortex and hippocampus after haloperidol treatment (3 or 21 days) agrees with Beal and Martin findings (8). It might be that even though the overall content of SLI in the cerebral cortex and hippocampus did not vary significantly, somatostatin synthesis and release rates may have changed. If this were the case, increased somatostatin release or turnover might lead to down-regulation of somatostatin receptors in these brain areas, even though we did not detect a significant increase in SLI content with
Since somatostatin activity is known to be presynaptically regulated by dopamine (27), the increase in somatostatin release could be a secondary reaction to haloperidol-induced (impairment of dopamine neurotransmision) block of dopamine receptors. The decrease of somatostatin binding to brain membranes from haloperidol treatment concurs well with the finding that this neuroleptic is a potent inhibitor of a variety of other neurotransmitter receptors. Thus, in addition to consistent antagonism of dopamine-2 receptors (28), the available haloperidol may also block, to a varying degree, (Y i-adrenergic (29), cholinergic (30), histamine (31) and serotonin receptors (32). However, binding to P-adrenergic and serotonin-2 receptors is unaffected by haloperidol (33). It is difficult to establish a direct relationship between both the in vitro and the in vivo actions of haloperidol upon somatostatin binding to rat brain membranes. Some of the pharmacological effects of haloperidol may well depend on decreases in the number of somatostatin receptors. For example, somatostatin administered intracerebroventricularly inhibits the extinction of active avoidance
Table 2 Equilibrium Parameters of Somatostatin Binding to Normal Rat Cerebrocortical Membranes in the Absence or Presence of 34.2 IAM Haloperidol in the Incubation Cerebral cortex Kd (nM)
Absence of haloperidol Presence of haloperidol
0.374 f 0.08 0.405 + 0.10
and Hippocampal
Hippocampus Bmax.
199 + 12 135 * 11*
Kd (nM)
Bmax.
0.322 _+0.06 0.368 f 0.08
202 t 12 107+9 *
Binding parameters were obtained by Scatchard (18) analysis of data from Figs 4-5, right panels. Bmax, Binding capacity (femtomoles of somatostatin bound per mg protein). Values represent the mean + SEM of five rats in each group. *p < 0.005 vs. absence of haloperidol.
162 behaviour (4,5) while that haloperidol inhibits the behavioural effects of the peptide (5). In addition, somatostatin has been reported to increase pentobarbital-induced sleeping time (6) and induces a frequent dissociation of the electroencephalogram (EEG) (34,35), while haloperidol has the opposite effect (7). Further experiments are required to explore our hypothesis however.
NEUROPEPTIDES 10. Greenwood,
11.
12.
13.
Acknowledgements This work was supported by a grant (88/0903) from the Fondo de Investigaciones Sanitarias de la Seguridad Social of Spain. The authors thank Carol F. Warren, from the Alcala de Henares University Institute of Education Sciences for her editorial help.
14.
15.
16.
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