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benzodiazepine receptor complex
Regulatory Peptides, 27 (1990) 355-365
355
Elsevier REGPEP 00884
Thymopentin antagonizes stress-induced changes of GABA/benzodiazepine receptor complex Vija Klusa 2, Raul-AUan Kiivet 1, Ruta Muceniece 2, Inga Liepa 2, Jaanus Harro i, Simon Svirskis 2, Aldis Andermanis 2 and Lembit Rago 1 ~Department of Pharmacology, Tartu University, Tartu, Estonia and 2Institute of Organic Synthesis, Latvian SSR Academy of Sciences, Riga, Latvia (U.S.S.R.) (Received 9 December 1988; revised version received and accepted 13 November 1989)
Key words: Thymopentin; Anxiety model; Benzodiazepine receptor
Summary Intraperitoneal administration of thymopentin, a thymopoetin II-derived pentapeptide, had no stable and evident effect in the two anxiety models (elevated plus-maze and licking-conflict test) studied. However, in the elevated plus-maze test thymopentin antagonized the behavioral effects of DMCM, a ]~-carboline derivative with anxiogenic properties. Further, it was demonstrated that the licking-conflict test procedure itself produced a significant elevation of plasma corticosterone levels, increased the number of [3H]flunitrazepam and decreased the number of [3H]muscimol binding sites in rat hippocampus. The forced-swimming stress similarly to the licking-conflict test also caused an increase in hippocampal [ 3H]flunitrazepam binding sites. Although ineffective behaviorally in the tests for anxiety, thymopentin pretreatment effectively reversed the changes in corticosterone levels caused by the licking-conflict test. Moreover, it normalized the changed number of benzodiazepine and GABA receptors after stressful stimuli. It is well known that not all anxiolytic drugs (i.e. buspirone) are equally active in behavioral tests for anxiety. According to our data we propose that thymopentin has stress-protective activity. As in vivo and in vitro thymopentin did not change [3H]flunitrazepam and [3H]muscimol binding, the direct effect of this peptide on the GABA-benzodiazepine-C1- ionophore receptor complex is unlikely. The action of this peptide on GABA release and/or metabolism can be suggested.
Correspondence: L. Rago, Department of Pharmacology, Tartu University, Ulikooli 18, 202 400 Tartu, Estonia, U.S.S.R. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
356 Introduction
In 1975 Schlesinger and Goldstein purified two homologous peptides from calf thymus [ 1]. These peptides were able to inhibit the nerve-muscle transmission in mice and they were named thymopoetin I and II (TP I and TP II). Later it was demonstrated that thymopoetins, in a much higher doses than that necessary to inhibit nerve-muscle transmission, have immunoregulatory properties. Both these peptides induce early differentiation of T-cells. They facilitate the transformation of prothymocytes into thymocytes that possess characteristics for the T-lymphocytes' morphologic and functional features. Thymopoetins also inhibit the differentiation of/~-lymphocytes and act on the functions of mature lymphocytes [2]. Several fragments and analogues of thymopoetin fragments have been synthesized to study the relations between the structure and mechanism of action and to localize the active center. Thymopentin is a biologically active pentapeptide fragment (32-36 TP II) of 49 amino acids containing thymopoetin II [3]. The immunological activity and imrounopharmacological profile of thymopentin resembles that of TP I and TP II and is described comprehensively elsewhere [4-6]. It has been suggested that the CNS, immune- and endocrine systems are closely interacting. Indeed, it is well known that many neuropeptides (endorphines, enkephalines, somatostatin, substance P, neurotensin etc.) have immunoregulatory properties [7-9]. On the other hand, immune system cells (i.e., lymphocytes) are able to produce many neuropeptides [9,10]. The possible psychotropic action of immunoregulatory peptides from the thymus has not been studied thoroughly yet. In the present study, an attempt was made to clarify this problem, and the potential anxiolytic and stress-protective activity of thymopentin was examined using behavioral methods, benzodiazepine and GABA receptor binding assays.
Materials and Methods
Animals and drugs Adult male albino laboratory rats (220-250 g) and mice (22-30 g), both from Rappolovo farm (Leningrad) were housed at constant conditions of temperature and photo-period (light 06.00-18.00 h) and had free access to food and water. DMCM (methyl-6,7-dimethoxy-4-ethyl-/~-carboline-3-carboxylate) (Schering, Berlin) was dissolved using a drop of Tween-80. Thymopentin was synthesized at the Institute of Organic Synthesis of Latvian Academy of Sciences (Riga) and was dissolved in saline immediately before the experiment. For binding studies, flunitrazepam (Hoffman-La Roche, Basel, Switzerland) and muscimol (Research Biochemicals, MA, U.S.A.) were used as displacing agents. Exploratory activity of mice in an elevated plus-maze The method used was described by Fellow and File [11] with the exception that originally it was used for rats. The plus-maze consisted of two open arms, 22 x 5 cm, and two enclosed arms, 22 x 5 x 15 cm with an open roof, arranged such that the two
357 arms of the same kind were opposite to each other. The central compartment of the plus-maze was an open square, 5 x 5 cm. The maze was elevated to the height of 25 cm. During a 4-min test period the following observations were made: (a) the latency period of the In'st open-arm entry, (b) the number of open-arm entries and, (c) the total time spent in open arms. To begin the experiment mice were placed at the center of the plus-maze. Licking-conflict test in rats The licking-conflict test (Vogel-type) procedure [ 12] was used to test thymopentin activity. On the third day, water-deprived (48 h) rats were pretreated with saline (1 ml/kg i.p.) and 30 min later placed alone in a perspex cage (20 x 24 x 22 cm) with a metal grid floor. The rat was allowed 10 min to f'md a metal nozzle, of the type used in their home cages, and drink 40 licks. Licking was counted automatically with a capacity sensor attached to the water nozzle. On the fourth day, during a 10 min observation period, a footshock (350 #A, 300 ms) was delivered via a scrambler connected to the bars of the grid floor. After the licking conflict-test the rats were immediately decapitated and the brains rapidly removed on ice. Forced-swimming stress The forced-swimming stress was carried out as described previously [ 16] with minor modifications. In short, the stress was produced by forcing the rats to swimm in a water basin (50 x 40 x 25 cm) at 20 + 1 °C for 3 min. After termination of the forced swimming the animals were immediately killed by decapitation and the brains were rapidly removed and kept on ice for further binding studies. Assay of corticosterone Trunk blood (500 #1) from each animal was collected in heparinized test-tubes, thoroughly mixed with a vortex mixer and spun for 7 min. Plasma was stored at - 20 ° C until assayed. Corticosterone levels in plasma were assayed by a modification of the sulfuric acid-induced fluoresence method [ 13]. In short, to a sample of plasma (100 #1) iso-octane (300/~1) was added in 1.5 ml polypropylene test-tubes. After thorough mixing, the tubes were centrifuged shortly. The iso-octane layer was removed by aspiration, and chloroform (600 #1) was added to the tubes. After mixing and centrifugation the top layer was removed by aspiration, and 500 #1 of the chloroform was placed into glass test-tubes. 1 ml of a sulfuric acid/ethanol (65 : 35, v/v) mixture was added to test-tubes. The contents were mixed, centrifuged, and 30 min later the fluoresence was measured. In vitro binding studies The hippocampi were rapidly dissected and homogenized in 30 vol. of ice-cold Tris-HCl (pH 7.4) using a Potter-S glass-Teflon homogenizer (1000 rpm, 10 passings). For [ 3H ]flunitrazepam binding, the membranes were washed twice in Tris-HC1 buffer by centrifugation (40,000 g for 20 min) and resuspended. Half of these membranes were stored overnight at - 20 ° C, and after melting they were additionally washed four times in the same buffer to use them for [ 3H ]muscimol binding. Binding of [ 3H ]flunitrazepam (spec. act. 81 Ci/mmol, Amersham Radiochemicals) was carried out in the presence of
358 0.125-8 n M of the tritiated ligand using a total incubation volume of 500 #1, 10 # M of flunitrazepam was used to determine nonspecific binding. After 60 min incubation on ice, the reaction was stopped by rapid filtration over W h a t m a n G F / B filters. The filters were washed with 4 x 3 ml of ice-cold Tris-HC1 buffer. [3H]muscimol (spec. act. 18 Ci/mmol, A m e r s h a m Radiochemicals) binding was carried out in the presence of 2 - 6 0 n M of the labeled ligand using a total incubation volume of 500 #1. Nonspecific binding was detected in the presence of 10 # M o f unlabeled muscimol. After 15 min of incubation on ice, the reaction was stopped by centrifugation (10,000 g for 5 min). The resultant pellets were superficially washed gently twice with 1 ml of ice-cold Tris-HC1. Specific binding was calculated by subtracting the nonspecific from total binding at each given radioactivity concentration. Protein content was measured by the Lowry et al. [ 14] method.
Statistics Results are expressed as a mean + S.E.M. D a t a were statistically analysed using Student's t-test.
Results
The effect of thymopentin on the exploratory behavior of control and DMCM-treated mice in an elevated plus-maze Recently it was demonstrated that an elevated plus-maze is a simple and reliable method to test various anxiolytic or anxiogenic c o m p o u n d s not only in rats but also in mice [ 15]. In the current study it was shown that thymopentin (0.02-0.5 mg/kg) in an elevated plus-maze did not change any o f the parameters studied. D M C M (1 mg/kg), a fl-carboline derivative with anxiogenic properties, increased the latency o f the tra'st TABLE I The effect of thymopentin and DMCM on the exploratory behavior of mice in an elevated plus-maze Thymopentin and DMCM were administered i.p. 40 and 15 min prior to the experiment respectively. Results are expressed as a mean + S.E.M. Treatment (mg/kg)
No. of animals
Latency of 1st open arm entry (s)
No. of open arm entries
Total time spent in open arm (s)
Saline Thymopentin 0.02 0.10 0.50 DMCM 1.00 DMCM 1.00 + Thymopentin 0.50
14
31+ 5
21+3
46_+ 7
8 10 10 10
34+ 39+ 24+ 68 +
25+4 18+6 24+5 8 + 3*
48+ 67+ 53+ 24 +
12
51 _+ 16
18 + 4
36 + 7
* P < 0.05, as compared to saline-treated animals.
7 8 8 19
9 12 10 4*
359 open-arm entry and significantly reduced the total number of open-arm entries along with the time spent in the open arms. The concomitant administration of thymopentin (0.5 mg/kg) with D M C M eliminated the behavioral effects of the latter (Table I). Smaller doses of thymopentin (0.02 and 0.1 mg/kg) were almost ineffective against D M C M (data not shown).
The effect of thymopentin on licking conflict in rats The action of thymopentin (0.02-0.5 mg/kg) on punished licking responses was examined in several experiments. Although in one experiment out of four experiments thymopentin markedly increased punished licking responses, we have to conclude that thymopentin does not have a statistically significant effect in this test (data not shown).
The effect of thymopentin on changes ofplasma corticosterone levels which were due to licking conflict In our previous experiments we noted that the licking-conflict procedure itself causes a significant rise in plasma corticosterone levels (from + 30 up to + 85% of that of the control animals). This effect of conflict testing procedure on corticosterone levels was effectively counteracted by pretreatment of animals with diazepam. Although, contrary to diazepam, thymopentin did not have any clear-cut anxiolytic effect in Vogel-test it lowered the corticosterone levels which were elevated due to the conflict testing procedure (Table II).
The effect of thymopentin on [3H]flunitrazepam and [~H]muscimol binding The in vivo administration of several doses of thymopentin (0.1-1.0 mg/kg i.p.) on [ 3H]flunitrazepam and [ 3H]muscimol binding was without significant effects in hippocampus (Tables III and IV). After pretreatment with thymopentin, [ 3H]flunitrazepam binding remained unchanged also in cerebral cortex, striatum and cerebellum. In spite of the absence or presence of protease inhibitors (bacitracin, P M S F and leupeptin) TABLE II The effectofin vivopretreatmentwith thymopentinon changesofcorticosteronelevelsin rat plasma caused by conflicttesting (Vogel-type)procedure Saline,thymopentinand diazepamwere administeredi.p.40 rain beforethe experiment.The data expressed are means of eight individual animals for each group + S.E.M. Experimental group (mg/kg)
Plasma corticosterone (#g/dl)
Saline Vogel + saline Vogel + diazepam 2.0 Vogel + thymopentin 0.1 0.5 1.0
25 + 3 43 + 6* 24 + 2** 34 + 7 29 _+4 22 + 4**
* P < 0.05, as compared to saline-treated controls. ** P < 0.05, as compared to Vogel + saline group.
360 TABLE III The effect of in vivo administration of thymopentin on baseline (experiment I) and changes caused by conflict-testing (Vogel-type) procedure (experiment II) of [aH]flunitrazepam binding in rat hippocampus Thymopentin was administered i.p. 40 min before the conflict test. The pooled tissue of eight animals was used for the binding assay. The data expressed are mean + S.E.M. of at least four experiments, each carried out in triplicate. Experimental group (mg/kg)
[3H]flunitrazepam binding Bmax (fmol/mg of protein)
K D (nM)
Experiment I Saline Thymopentin 0.1 0.5 1.0
713 738 808 709
+ 56 + 43 _+ 61 + 29
1.74 1.85 1.98 1.67
+ 0.12 + 0.24 + 0.31 +_ 0.19
Experiment II Saline Vogel + saline Vogel + thymopentin 0A 0.5
632 881 698 609
+ + + +
1.85 2.20 1.85 2.05
+ + + +
48 51" 18 45**
0.14 0.12 0.36 0.46
* P < 0.05, as compared to saline controls. ** P < 0.05, as compared to Vogel + saline group.
TABLE IV The effect of in vivo administration of thymopentin on baseline (exl~riment I) and changes caused by conflict-testing (Vogel-type) procedure (experiment II) of [3H]museimol binding in rat hippocampus Thymopentin was administered i.p. 40 rain before the conflict test. The pooled tissue of eight animals per group was used for the binding assay. The data expressed are mean + S.E.M. of at least four experiments, each carried out in triplicate. Experimental group (mg/kg)
[3H]muscimol binding Bm~ (fmol/mg of protein)
K D (nM)
Experiment I Saline Thymopentin 0.1 0.5
1108 _+ 112 834 __. 98 956 + 127
16.8 + 2.1 18.1 + 3.2 14.9 + 3.2
Experiment II Saline Vogel + saline Vogel + thymopentin 0.1 0.5
1281 995 1183 1203
15.3 19.6 17.8 20.6
* P < 0.05, as compared to saline controls.
+ 89 + 53* + 97 __+.115
+ + + +
3.8 5.8 4.3 6.4
361 thymopentin was unable to change [3H]flunitrazepam binding in vitro (data not presented).
The effect of thymopentin on changes of [ 3H]flunitrazepam and [3H]muscimol binding in rat hippocampus which were due to licking conflict Recently, it has been shown that several models of stress can change benzodiazepine binding sites [16,17]. Therefore, we decided to study the effect of the licking-conflict procedure on the benzodiazepine binding sites in hippocampus. It was found that the licking-conflict procedure enhanced significantly the number of [3H]flunitrazepam binding sites, without reliable changes in the affinity. The pretreatment of rats with thymopentin dose-dependently eliminated the changes in the benzodiazepine receptor caused by the Vogel-test (Table III). In contrast to benzodiazepine binding, the licking-conflict procedure caused a statistically significant decrease in the number of [3H]muscimol binding sites. This effect of the Vogel-test was counteracted also by pretreatment with thymopentin (Table IV). When GABA/benzodiazepine receptor density ratios were calculated for the Vogeltest groups in which GABA and benzodiazepine receptors were studied in paralell (see Tables III and IV), it was found that the licking conflict caused a marked decrease of this ratio. Pretreatment of rats with thymopentin dose-dependently eliminated the decline, and 0.5 mg/kg of thymopentin almost completely restored the GABA/ benzodiazepine receptor ratio (Table V).
The effect of thymopentin on changes of [3H]flunitrazepam binding in rat hippocampus due to swimming stress A short-time forced-swimming stress in rats resulted in an increased number of [3H]flunitrazepam binding sites in hippocampus (Table VI). The affinity for the ligand did not change significantly although the same tendency as in the licking conflict rats was observed. In both cases the alTmity for the ligand was somewhat lower than in control animals (Tables III and VI). Pretreatment of rats with 0.5 mg/kg ofthymopentin prior to swimming stress eliminated the increase of benzodiazepine binding sites caused by this test procedure (Table VI). TABLE V The effectofin vivoadministrationofchangescausedbythymopentinconflict-testing(Vogel-type)procedure in GABA/benzodiazepinereceptor ratio in rat hippocampus The bound maximumvalues of [3H]muscimoland [3H]flunitrazepambinding are derivedfrom tables III and II, respectively. Experimentalgroup (mg/kg)
GABA/benzodiazepine receptor densityratio
% of change
Saline Vogel + saline Vogel + thymopentin O.1 0.5
2.03 1.13 1.69 1.97
- 44 - 17 -3
362 TABLE VI The effect ofthymopentin on changes in [3H]flunitrazepam binding in rat hippocampus, caused by swimming stress Thymopentin was administered i.p. 40 min prior to swimming stress. The pooled tissue of six animals was used for this study. The data expressed are mean _+ S.E.M. of at least four experiments, each carried out in triplicate. Experimental group (mg/kg)
[3H]flunitrazepam binding Bma x
Saline Swimming stress + saline Swimming stress + thymopentin 0.5
(fmol/mg of protein)
K D (nM)
752 + 54
1.64 + 0.15
1084 + 76*
1.86 + 0.12
897 + 68
1.58 + 0.18
* P < 0.05, as compared to saline group.
Discussion
Thymopentin (0.02-0.5 mg/kg) was inactive in the elevated plus-maze model of anxiety and in most experiments where Vogel-type licking-conflict procedure was used. The inactiveness in behavioral tests for anxiety does not allow to conclude that a compound is completely devoid of anxiolytic activity. It should be mentioned that the majority of animal models of anxiety have been introduced using classical benzodiazepine (BD) tranquilizers and the models meet the needs of this group best. For example buspirone, a clinically effective non-benzodiazepine tranquilizer, has no anxiolytic activity in the elevated plus-maze [11] and is far less efficacious than BD anxiolytics in traditional punishment producers like Vogel-type licking-conflict test [ 18 ]. Although itself inactive in the elevated plus-maze test, in a higher dose (0.5 mg/kg) thymopentin counteracted the behavioral effects of DMCM, an anxiogenic fl-carboline. As thymopentin is devoid of any activity on BD receptors in vitro as well as in vivo, the behavioral antagonism with DMCM, a BD receptor inverse agonist, is difficult to explain. In a separate series of experiments it was demonstrated that Vogel-test procedure itself caused a marked rise in corticosterone levels. It is of interest to mention that anxiogenic BD receptor ligands, like DMCM and similarly to various stressful stimuli, also cause increase in plasma corticosterone in otherwise unstressed animals [ 19]. The effect of the licking-conflict procedure on corticosterone levels was dose-dependently reduced by pretreatment with thymopentin. Taking into consideration the fact that stressful stimuli are able to change BD receptors [ 16,17], we decided to study the effect of the licking-conflict procedure on [3H]flunitrazepam binding characteristics in the hippocampus. So far, it has been difficult to ascribe the induction or attenuation of emotional tension and excessive psychomotor reactivity to certain neurochemical
363 changes in a single brain structure. Nevertheless, it is well known that brain structures belonging to the limbic system are of special importance in the regulation of emotional behavior. For the current study, hippocampus, a structure belonging to the limbic system and containing a large number of neurotransmitters (like GABA, glutamate, aspartate, acetylcholine, NE and 5-HT) whose distribution and synaptic role is known to a considerable extent, was chosen. It was found that the Vogel-test caused a marked increase in [3H]flunitrazepam binding sites in hippocampus. This was in correlation with our recent finding that another stressful situation, the forced-swimming stress, also increased the number of BD binding sites [20]. Moreover, pretreatment with thymopentin dose-dependently prevented the action of the Vogel-test procedure on BD receptors. Similarly to licking-conflict testing, the forced-swimming stress caused an increase in the number of benzodiazepine binding sites, which was counteracted by pretreatment with thymopentin. As in vivo administration ofthymopentin had no effect on BD receptors, the question arises why this pentapeptide normalizes BD receptor binding changes caused by stressful situations. To answer this question, GABA A receptors labeled with [ 3H]muscimol were also studied after the Vogel-test procedure. It was demonstrated that, contrary to the BD binding sites, the density of [ 3H ]muscimol binding sites was diminished. These data are in correlation with the earlier findings where it has been demonstrated that stress reduces the number of GABA A receptors [21,22]. Although in control animals thymopentin was without clear-cut effect on hippocampal [ 3n]muscimol binding sites, the effect of the Vogel-test on GABA receptors was again counteracted by in vivo pretreatment with thymopentin. When the GABA/benzodiazepine receptor density ratios were calculated, it was found that the licking-conflict procedure lowered the ratio, and pretreatment with thymopentin restored it close to control values. Apparently, the GABA/benzodiazepine receptor density ratio reflects in some aspects the functional capacity of the GABA- benzodiazepine-C1- ionophore receptor complex. The protective action of thymopentin on the level of B D and GABA receptors during various kinds of stressful stimuli is difficult to explain. Thymopentin neither antagonized nor potentiated picrotoxin induced convulsions (Dr. R. Muceniece, unpublished data). Therefore it seems unlikely that thymopentin has any direct action on the GABA-benzodiazepine-Cl- ionophore receptoral complex subunits. One must consider also the very short half-life of thymopentine in vivo and in vitro, about 30 and 60 s respectively [23,24]. Nevertheless, we cannot exclude some indirect action on GABA release and/or metabolism. Recently, it was found that thymopentin enchances GABA release in vitro (personal communication from Dr. A. P. Dolshenko) and normalizes the GABA content reduced by stress in rat hippocampus and cerebral cortex [25]. Concerning the relatively high doses of thymopentin used in this study, the very low toxicity of thymopentin should be considered: in mice, the LDso is more than 250 mg/kg [4]. In conclusion, we would like to emphasize that in spite of the ineffectiveness in the two behavioral anxiety models, thymopentin has a unique stress-protective action at the level of the GABA-benzodiazepine-C1- ionophore complex. As the direct action of this peptide on benzodiazepine and GABA receptors is unlikely, the involvement of other mechanisms like GABA release and/or metabolism can be suggested.
364
References 1 Schlesinger, D.H. and Goldstein, G., The amino acid sequence of thymopentin II, Cell, 5 (1975), 361-365. 2 Basch, R.S. and Goldstein, G., Induction of T-cell differentiation in vitro by thymin, a purified polypeptide hormone of the thymus, Proc. Nat. Acad. Sci. USA, 71 (1974), 1474-1478. 3 Goldstein, G., Scheid, M.P. and Boyse, E. A., A synthetic pentapeptide with biological activity characteristic of the thymic hormone thymopentin, Science, 204 (1979), 1309-1310. 4 Goldstein, G. and Lau, C.Y., Immunoregulation by thymopentin, J. Supramolecular Struct., 14 (1980), 225-261. 5 Lau, C.Y., Freestone, J.A. and Goldstein, G., Effect of thymopentin pentapeptide (TP5) on autoimmunity. I. TP5 supression of induced erytrocyte auto antibodies in C3H mice, J. Immunol., 125 (1980), 1634-1638. 6 Verhaegen, H., De Cock, W., De Cre, J. and Goldstein, G., Restoration of the impaired lymphocyte stimulation in old people by thymopentin pentapeptide, J. Clin. Lab., 6 (1981), 103-105. 7 Wybran, J., Enkephalins and endorphins as modifiers of the immune system: present and future, Fed. Proc., 44 (1985) 92-94. 8 Payan, D.G. and Goitze, E.J., Modulation of lymphocyte function by sensory neuropeptides, J. Immunol., 135 (1985) 738-786. 9 Lygran, I., Revhang, A., Burhol, P. G., G-iereksky, K. C. and Jenssen, T. G., Vasoactive intestinal peptide and somatostatin in leukocyte, Scand. J. Lab. Invest., 44 (1984) 347-351. 10 Smith, E.M., Phan, M., Kruger, T.E., Coppenhaver, D.H. and Blalock, J.E., Human lymphocyte production of immunoreactive thymotropin, Proc. Natl. Acad. Sci. USA, 80 (1983) 6010-6013. 11 Pellow, S. and File, S.E., Anxiolytic and anxiogenic drug effect on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat, Pharmacol. Biochem. Behav., 24 (1986) 525-529. 12 Vogel, R.A., Beer, B. and Clody, D. E., A simply and reliable conflict procedure for testing anti-anxiety agents, Psychopharmacology (Berlin), 21 (1971) 1-7. 13 Glick, D., von Redlich, D. and Levine, S., Fluorometric determination of corticosterone and cortisol in 0.02-0.05 millilitres of plasma or submilligram samples of adrenal tissue, Endocrinology, 74 (1964) 653-655. 14 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193 (1951) 265-275. 15 Rligo, L.K., Kiivet, R.-A., Harro, J. and P~ld, M., Behavioral differences in an elevated plus-maze: correlation between anxiety and decreased number of GABA and bemzodiazepine receptors in mouse cerebral cortex, Naunyn Schmiedeberg's Arch. Pharmacol, 337 (1988) 675-678. 16 Medina, J.H., Nowas, M.L. and Wolfman, C., Levi de Stein, M. and De Robertis, E., Benzodiazepine receptors in rat cerebral cortex and hippoeampus undergo rapid and reversible changes after acute stress, Neuroseience, 9 (1983) 331-335. 17 Soubrie, P., Thiebot, M.H., Jobert, A., Montrastruc, S.L., Henry, F. and Hemon, M., Decreased convulsant potency of picrotoxin and enchanced [3H]flunitrazepam cortical binding following stressful manipulations in rats, Brain Res., 189 (1980) 505-517. 18 Weissman, B.A., Barrett, J. E., Brady, L. S., Witkin, J.M., Mendeison, W. B., Paul, S.M. and Skoinick, P., Behavioral and neurochemieal studies on the anticonfliet actions of buspirone, Drug Dev. Res., 4 (1984) 83-89. 19 Stephens, D.N., Schneider, H.H., Kehr, W., Jensen, L.H., Petersen, E. and Honore, T., Modulation of anxiety by ~-carbolines and other benzodiazepine receptor ligands: Relationship of pharmacological to biochemical measures of efficacy, Brain Res. Bull., 19 (1987) 309-318. 20 R~go, L.K., Kiivet, R.-A., Harro, J. and POld, M., Central- and peripheral-type benzodiazepine receptors: similar regulation by stress and GABA receptor agonists, Pharmacol. Bioehem. Behav., 32 (1989) 879-883. 21 Concas, A., Corda, M.G. and Biggio, G., Involvement of benzodiazepine recognition sites in the foot shock-induced decrease of low affinity GABA receptors in the rat cerebral cortex, Brain Res., 341 (1985) 50-56.
365 22 Corda, M. G., and Biggio, G., Stress and GABA-ergic transmission: biochemical and behavioral studies. In G. Biggio and E. Costa (Eds.), GABA-ergic transmission and anxiety, Raven Press, New York, 1986, pp. 121-136. 23 Tischio, J. P., Patrick, J.E., Neintraub, H.S., Chasin, M., and Goldstein, G., Short in vitro half-life Of thymopoietin 32-36 pentapeptide in human plasma, Int. J. Pept. Protein Res., 14 (1979) 479-484. 24 Heavner, G. A., Kroon, D.J., Andhya, T., Goldstein, G., Biologically active analogs ofthymopentin with enchanced enzymatic stability, Peptides, 7 (1986) 1015-1019. 25 Klusa, V., Behavioral and neurochemical effects of thymopentin an immunoprotein fragment, Abstracts, World Meeting of the International Society for Research on Agression, Multidisciplinary Studies On Agression, Swansea, July 2-6, 1988, p. 62:
355
Elsevier REGPEP 00884
Thymopentin antagonizes stress-induced changes of GABA/benzodiazepine receptor complex Vija Klusa 2, Raul-AUan Kiivet 1, Ruta Muceniece 2, Inga Liepa 2, Jaanus Harro i, Simon Svirskis 2, Aldis Andermanis 2 and Lembit Rago 1 ~Department of Pharmacology, Tartu University, Tartu, Estonia and 2Institute of Organic Synthesis, Latvian SSR Academy of Sciences, Riga, Latvia (U.S.S.R.) (Received 9 December 1988; revised version received and accepted 13 November 1989)
Key words: Thymopentin; Anxiety model; Benzodiazepine receptor
Summary Intraperitoneal administration of thymopentin, a thymopoetin II-derived pentapeptide, had no stable and evident effect in the two anxiety models (elevated plus-maze and licking-conflict test) studied. However, in the elevated plus-maze test thymopentin antagonized the behavioral effects of DMCM, a ]~-carboline derivative with anxiogenic properties. Further, it was demonstrated that the licking-conflict test procedure itself produced a significant elevation of plasma corticosterone levels, increased the number of [3H]flunitrazepam and decreased the number of [3H]muscimol binding sites in rat hippocampus. The forced-swimming stress similarly to the licking-conflict test also caused an increase in hippocampal [ 3H]flunitrazepam binding sites. Although ineffective behaviorally in the tests for anxiety, thymopentin pretreatment effectively reversed the changes in corticosterone levels caused by the licking-conflict test. Moreover, it normalized the changed number of benzodiazepine and GABA receptors after stressful stimuli. It is well known that not all anxiolytic drugs (i.e. buspirone) are equally active in behavioral tests for anxiety. According to our data we propose that thymopentin has stress-protective activity. As in vivo and in vitro thymopentin did not change [3H]flunitrazepam and [3H]muscimol binding, the direct effect of this peptide on the GABA-benzodiazepine-C1- ionophore receptor complex is unlikely. The action of this peptide on GABA release and/or metabolism can be suggested.
Correspondence: L. Rago, Department of Pharmacology, Tartu University, Ulikooli 18, 202 400 Tartu, Estonia, U.S.S.R. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
356 Introduction
In 1975 Schlesinger and Goldstein purified two homologous peptides from calf thymus [ 1]. These peptides were able to inhibit the nerve-muscle transmission in mice and they were named thymopoetin I and II (TP I and TP II). Later it was demonstrated that thymopoetins, in a much higher doses than that necessary to inhibit nerve-muscle transmission, have immunoregulatory properties. Both these peptides induce early differentiation of T-cells. They facilitate the transformation of prothymocytes into thymocytes that possess characteristics for the T-lymphocytes' morphologic and functional features. Thymopoetins also inhibit the differentiation of/~-lymphocytes and act on the functions of mature lymphocytes [2]. Several fragments and analogues of thymopoetin fragments have been synthesized to study the relations between the structure and mechanism of action and to localize the active center. Thymopentin is a biologically active pentapeptide fragment (32-36 TP II) of 49 amino acids containing thymopoetin II [3]. The immunological activity and imrounopharmacological profile of thymopentin resembles that of TP I and TP II and is described comprehensively elsewhere [4-6]. It has been suggested that the CNS, immune- and endocrine systems are closely interacting. Indeed, it is well known that many neuropeptides (endorphines, enkephalines, somatostatin, substance P, neurotensin etc.) have immunoregulatory properties [7-9]. On the other hand, immune system cells (i.e., lymphocytes) are able to produce many neuropeptides [9,10]. The possible psychotropic action of immunoregulatory peptides from the thymus has not been studied thoroughly yet. In the present study, an attempt was made to clarify this problem, and the potential anxiolytic and stress-protective activity of thymopentin was examined using behavioral methods, benzodiazepine and GABA receptor binding assays.
Materials and Methods
Animals and drugs Adult male albino laboratory rats (220-250 g) and mice (22-30 g), both from Rappolovo farm (Leningrad) were housed at constant conditions of temperature and photo-period (light 06.00-18.00 h) and had free access to food and water. DMCM (methyl-6,7-dimethoxy-4-ethyl-/~-carboline-3-carboxylate) (Schering, Berlin) was dissolved using a drop of Tween-80. Thymopentin was synthesized at the Institute of Organic Synthesis of Latvian Academy of Sciences (Riga) and was dissolved in saline immediately before the experiment. For binding studies, flunitrazepam (Hoffman-La Roche, Basel, Switzerland) and muscimol (Research Biochemicals, MA, U.S.A.) were used as displacing agents. Exploratory activity of mice in an elevated plus-maze The method used was described by Fellow and File [11] with the exception that originally it was used for rats. The plus-maze consisted of two open arms, 22 x 5 cm, and two enclosed arms, 22 x 5 x 15 cm with an open roof, arranged such that the two
357 arms of the same kind were opposite to each other. The central compartment of the plus-maze was an open square, 5 x 5 cm. The maze was elevated to the height of 25 cm. During a 4-min test period the following observations were made: (a) the latency period of the In'st open-arm entry, (b) the number of open-arm entries and, (c) the total time spent in open arms. To begin the experiment mice were placed at the center of the plus-maze. Licking-conflict test in rats The licking-conflict test (Vogel-type) procedure [ 12] was used to test thymopentin activity. On the third day, water-deprived (48 h) rats were pretreated with saline (1 ml/kg i.p.) and 30 min later placed alone in a perspex cage (20 x 24 x 22 cm) with a metal grid floor. The rat was allowed 10 min to f'md a metal nozzle, of the type used in their home cages, and drink 40 licks. Licking was counted automatically with a capacity sensor attached to the water nozzle. On the fourth day, during a 10 min observation period, a footshock (350 #A, 300 ms) was delivered via a scrambler connected to the bars of the grid floor. After the licking conflict-test the rats were immediately decapitated and the brains rapidly removed on ice. Forced-swimming stress The forced-swimming stress was carried out as described previously [ 16] with minor modifications. In short, the stress was produced by forcing the rats to swimm in a water basin (50 x 40 x 25 cm) at 20 + 1 °C for 3 min. After termination of the forced swimming the animals were immediately killed by decapitation and the brains were rapidly removed and kept on ice for further binding studies. Assay of corticosterone Trunk blood (500 #1) from each animal was collected in heparinized test-tubes, thoroughly mixed with a vortex mixer and spun for 7 min. Plasma was stored at - 20 ° C until assayed. Corticosterone levels in plasma were assayed by a modification of the sulfuric acid-induced fluoresence method [ 13]. In short, to a sample of plasma (100 #1) iso-octane (300/~1) was added in 1.5 ml polypropylene test-tubes. After thorough mixing, the tubes were centrifuged shortly. The iso-octane layer was removed by aspiration, and chloroform (600 #1) was added to the tubes. After mixing and centrifugation the top layer was removed by aspiration, and 500 #1 of the chloroform was placed into glass test-tubes. 1 ml of a sulfuric acid/ethanol (65 : 35, v/v) mixture was added to test-tubes. The contents were mixed, centrifuged, and 30 min later the fluoresence was measured. In vitro binding studies The hippocampi were rapidly dissected and homogenized in 30 vol. of ice-cold Tris-HCl (pH 7.4) using a Potter-S glass-Teflon homogenizer (1000 rpm, 10 passings). For [ 3H ]flunitrazepam binding, the membranes were washed twice in Tris-HC1 buffer by centrifugation (40,000 g for 20 min) and resuspended. Half of these membranes were stored overnight at - 20 ° C, and after melting they were additionally washed four times in the same buffer to use them for [ 3H ]muscimol binding. Binding of [ 3H ]flunitrazepam (spec. act. 81 Ci/mmol, Amersham Radiochemicals) was carried out in the presence of
358 0.125-8 n M of the tritiated ligand using a total incubation volume of 500 #1, 10 # M of flunitrazepam was used to determine nonspecific binding. After 60 min incubation on ice, the reaction was stopped by rapid filtration over W h a t m a n G F / B filters. The filters were washed with 4 x 3 ml of ice-cold Tris-HC1 buffer. [3H]muscimol (spec. act. 18 Ci/mmol, A m e r s h a m Radiochemicals) binding was carried out in the presence of 2 - 6 0 n M of the labeled ligand using a total incubation volume of 500 #1. Nonspecific binding was detected in the presence of 10 # M o f unlabeled muscimol. After 15 min of incubation on ice, the reaction was stopped by centrifugation (10,000 g for 5 min). The resultant pellets were superficially washed gently twice with 1 ml of ice-cold Tris-HC1. Specific binding was calculated by subtracting the nonspecific from total binding at each given radioactivity concentration. Protein content was measured by the Lowry et al. [ 14] method.
Statistics Results are expressed as a mean + S.E.M. D a t a were statistically analysed using Student's t-test.
Results
The effect of thymopentin on the exploratory behavior of control and DMCM-treated mice in an elevated plus-maze Recently it was demonstrated that an elevated plus-maze is a simple and reliable method to test various anxiolytic or anxiogenic c o m p o u n d s not only in rats but also in mice [ 15]. In the current study it was shown that thymopentin (0.02-0.5 mg/kg) in an elevated plus-maze did not change any o f the parameters studied. D M C M (1 mg/kg), a fl-carboline derivative with anxiogenic properties, increased the latency o f the tra'st TABLE I The effect of thymopentin and DMCM on the exploratory behavior of mice in an elevated plus-maze Thymopentin and DMCM were administered i.p. 40 and 15 min prior to the experiment respectively. Results are expressed as a mean + S.E.M. Treatment (mg/kg)
No. of animals
Latency of 1st open arm entry (s)
No. of open arm entries
Total time spent in open arm (s)
Saline Thymopentin 0.02 0.10 0.50 DMCM 1.00 DMCM 1.00 + Thymopentin 0.50
14
31+ 5
21+3
46_+ 7
8 10 10 10
34+ 39+ 24+ 68 +
25+4 18+6 24+5 8 + 3*
48+ 67+ 53+ 24 +
12
51 _+ 16
18 + 4
36 + 7
* P < 0.05, as compared to saline-treated animals.
7 8 8 19
9 12 10 4*
359 open-arm entry and significantly reduced the total number of open-arm entries along with the time spent in the open arms. The concomitant administration of thymopentin (0.5 mg/kg) with D M C M eliminated the behavioral effects of the latter (Table I). Smaller doses of thymopentin (0.02 and 0.1 mg/kg) were almost ineffective against D M C M (data not shown).
The effect of thymopentin on licking conflict in rats The action of thymopentin (0.02-0.5 mg/kg) on punished licking responses was examined in several experiments. Although in one experiment out of four experiments thymopentin markedly increased punished licking responses, we have to conclude that thymopentin does not have a statistically significant effect in this test (data not shown).
The effect of thymopentin on changes ofplasma corticosterone levels which were due to licking conflict In our previous experiments we noted that the licking-conflict procedure itself causes a significant rise in plasma corticosterone levels (from + 30 up to + 85% of that of the control animals). This effect of conflict testing procedure on corticosterone levels was effectively counteracted by pretreatment of animals with diazepam. Although, contrary to diazepam, thymopentin did not have any clear-cut anxiolytic effect in Vogel-test it lowered the corticosterone levels which were elevated due to the conflict testing procedure (Table II).
The effect of thymopentin on [3H]flunitrazepam and [~H]muscimol binding The in vivo administration of several doses of thymopentin (0.1-1.0 mg/kg i.p.) on [ 3H]flunitrazepam and [ 3H]muscimol binding was without significant effects in hippocampus (Tables III and IV). After pretreatment with thymopentin, [ 3H]flunitrazepam binding remained unchanged also in cerebral cortex, striatum and cerebellum. In spite of the absence or presence of protease inhibitors (bacitracin, P M S F and leupeptin) TABLE II The effectofin vivopretreatmentwith thymopentinon changesofcorticosteronelevelsin rat plasma caused by conflicttesting (Vogel-type)procedure Saline,thymopentinand diazepamwere administeredi.p.40 rain beforethe experiment.The data expressed are means of eight individual animals for each group + S.E.M. Experimental group (mg/kg)
Plasma corticosterone (#g/dl)
Saline Vogel + saline Vogel + diazepam 2.0 Vogel + thymopentin 0.1 0.5 1.0
25 + 3 43 + 6* 24 + 2** 34 + 7 29 _+4 22 + 4**
* P < 0.05, as compared to saline-treated controls. ** P < 0.05, as compared to Vogel + saline group.
360 TABLE III The effect of in vivo administration of thymopentin on baseline (experiment I) and changes caused by conflict-testing (Vogel-type) procedure (experiment II) of [aH]flunitrazepam binding in rat hippocampus Thymopentin was administered i.p. 40 min before the conflict test. The pooled tissue of eight animals was used for the binding assay. The data expressed are mean + S.E.M. of at least four experiments, each carried out in triplicate. Experimental group (mg/kg)
[3H]flunitrazepam binding Bmax (fmol/mg of protein)
K D (nM)
Experiment I Saline Thymopentin 0.1 0.5 1.0
713 738 808 709
+ 56 + 43 _+ 61 + 29
1.74 1.85 1.98 1.67
+ 0.12 + 0.24 + 0.31 +_ 0.19
Experiment II Saline Vogel + saline Vogel + thymopentin 0A 0.5
632 881 698 609
+ + + +
1.85 2.20 1.85 2.05
+ + + +
48 51" 18 45**
0.14 0.12 0.36 0.46
* P < 0.05, as compared to saline controls. ** P < 0.05, as compared to Vogel + saline group.
TABLE IV The effect of in vivo administration of thymopentin on baseline (exl~riment I) and changes caused by conflict-testing (Vogel-type) procedure (experiment II) of [3H]museimol binding in rat hippocampus Thymopentin was administered i.p. 40 rain before the conflict test. The pooled tissue of eight animals per group was used for the binding assay. The data expressed are mean + S.E.M. of at least four experiments, each carried out in triplicate. Experimental group (mg/kg)
[3H]muscimol binding Bm~ (fmol/mg of protein)
K D (nM)
Experiment I Saline Thymopentin 0.1 0.5
1108 _+ 112 834 __. 98 956 + 127
16.8 + 2.1 18.1 + 3.2 14.9 + 3.2
Experiment II Saline Vogel + saline Vogel + thymopentin 0.1 0.5
1281 995 1183 1203
15.3 19.6 17.8 20.6
* P < 0.05, as compared to saline controls.
+ 89 + 53* + 97 __+.115
+ + + +
3.8 5.8 4.3 6.4
361 thymopentin was unable to change [3H]flunitrazepam binding in vitro (data not presented).
The effect of thymopentin on changes of [ 3H]flunitrazepam and [3H]muscimol binding in rat hippocampus which were due to licking conflict Recently, it has been shown that several models of stress can change benzodiazepine binding sites [16,17]. Therefore, we decided to study the effect of the licking-conflict procedure on the benzodiazepine binding sites in hippocampus. It was found that the licking-conflict procedure enhanced significantly the number of [3H]flunitrazepam binding sites, without reliable changes in the affinity. The pretreatment of rats with thymopentin dose-dependently eliminated the changes in the benzodiazepine receptor caused by the Vogel-test (Table III). In contrast to benzodiazepine binding, the licking-conflict procedure caused a statistically significant decrease in the number of [3H]muscimol binding sites. This effect of the Vogel-test was counteracted also by pretreatment with thymopentin (Table IV). When GABA/benzodiazepine receptor density ratios were calculated for the Vogeltest groups in which GABA and benzodiazepine receptors were studied in paralell (see Tables III and IV), it was found that the licking conflict caused a marked decrease of this ratio. Pretreatment of rats with thymopentin dose-dependently eliminated the decline, and 0.5 mg/kg of thymopentin almost completely restored the GABA/ benzodiazepine receptor ratio (Table V).
The effect of thymopentin on changes of [3H]flunitrazepam binding in rat hippocampus due to swimming stress A short-time forced-swimming stress in rats resulted in an increased number of [3H]flunitrazepam binding sites in hippocampus (Table VI). The affinity for the ligand did not change significantly although the same tendency as in the licking conflict rats was observed. In both cases the alTmity for the ligand was somewhat lower than in control animals (Tables III and VI). Pretreatment of rats with 0.5 mg/kg ofthymopentin prior to swimming stress eliminated the increase of benzodiazepine binding sites caused by this test procedure (Table VI). TABLE V The effectofin vivoadministrationofchangescausedbythymopentinconflict-testing(Vogel-type)procedure in GABA/benzodiazepinereceptor ratio in rat hippocampus The bound maximumvalues of [3H]muscimoland [3H]flunitrazepambinding are derivedfrom tables III and II, respectively. Experimentalgroup (mg/kg)
GABA/benzodiazepine receptor densityratio
% of change
Saline Vogel + saline Vogel + thymopentin O.1 0.5
2.03 1.13 1.69 1.97
- 44 - 17 -3
362 TABLE VI The effect ofthymopentin on changes in [3H]flunitrazepam binding in rat hippocampus, caused by swimming stress Thymopentin was administered i.p. 40 min prior to swimming stress. The pooled tissue of six animals was used for this study. The data expressed are mean _+ S.E.M. of at least four experiments, each carried out in triplicate. Experimental group (mg/kg)
[3H]flunitrazepam binding Bma x
Saline Swimming stress + saline Swimming stress + thymopentin 0.5
(fmol/mg of protein)
K D (nM)
752 + 54
1.64 + 0.15
1084 + 76*
1.86 + 0.12
897 + 68
1.58 + 0.18
* P < 0.05, as compared to saline group.
Discussion
Thymopentin (0.02-0.5 mg/kg) was inactive in the elevated plus-maze model of anxiety and in most experiments where Vogel-type licking-conflict procedure was used. The inactiveness in behavioral tests for anxiety does not allow to conclude that a compound is completely devoid of anxiolytic activity. It should be mentioned that the majority of animal models of anxiety have been introduced using classical benzodiazepine (BD) tranquilizers and the models meet the needs of this group best. For example buspirone, a clinically effective non-benzodiazepine tranquilizer, has no anxiolytic activity in the elevated plus-maze [11] and is far less efficacious than BD anxiolytics in traditional punishment producers like Vogel-type licking-conflict test [ 18 ]. Although itself inactive in the elevated plus-maze test, in a higher dose (0.5 mg/kg) thymopentin counteracted the behavioral effects of DMCM, an anxiogenic fl-carboline. As thymopentin is devoid of any activity on BD receptors in vitro as well as in vivo, the behavioral antagonism with DMCM, a BD receptor inverse agonist, is difficult to explain. In a separate series of experiments it was demonstrated that Vogel-test procedure itself caused a marked rise in corticosterone levels. It is of interest to mention that anxiogenic BD receptor ligands, like DMCM and similarly to various stressful stimuli, also cause increase in plasma corticosterone in otherwise unstressed animals [ 19]. The effect of the licking-conflict procedure on corticosterone levels was dose-dependently reduced by pretreatment with thymopentin. Taking into consideration the fact that stressful stimuli are able to change BD receptors [ 16,17], we decided to study the effect of the licking-conflict procedure on [3H]flunitrazepam binding characteristics in the hippocampus. So far, it has been difficult to ascribe the induction or attenuation of emotional tension and excessive psychomotor reactivity to certain neurochemical
363 changes in a single brain structure. Nevertheless, it is well known that brain structures belonging to the limbic system are of special importance in the regulation of emotional behavior. For the current study, hippocampus, a structure belonging to the limbic system and containing a large number of neurotransmitters (like GABA, glutamate, aspartate, acetylcholine, NE and 5-HT) whose distribution and synaptic role is known to a considerable extent, was chosen. It was found that the Vogel-test caused a marked increase in [3H]flunitrazepam binding sites in hippocampus. This was in correlation with our recent finding that another stressful situation, the forced-swimming stress, also increased the number of BD binding sites [20]. Moreover, pretreatment with thymopentin dose-dependently prevented the action of the Vogel-test procedure on BD receptors. Similarly to licking-conflict testing, the forced-swimming stress caused an increase in the number of benzodiazepine binding sites, which was counteracted by pretreatment with thymopentin. As in vivo administration ofthymopentin had no effect on BD receptors, the question arises why this pentapeptide normalizes BD receptor binding changes caused by stressful situations. To answer this question, GABA A receptors labeled with [ 3H]muscimol were also studied after the Vogel-test procedure. It was demonstrated that, contrary to the BD binding sites, the density of [ 3H ]muscimol binding sites was diminished. These data are in correlation with the earlier findings where it has been demonstrated that stress reduces the number of GABA A receptors [21,22]. Although in control animals thymopentin was without clear-cut effect on hippocampal [ 3n]muscimol binding sites, the effect of the Vogel-test on GABA receptors was again counteracted by in vivo pretreatment with thymopentin. When the GABA/benzodiazepine receptor density ratios were calculated, it was found that the licking-conflict procedure lowered the ratio, and pretreatment with thymopentin restored it close to control values. Apparently, the GABA/benzodiazepine receptor density ratio reflects in some aspects the functional capacity of the GABA- benzodiazepine-C1- ionophore receptor complex. The protective action of thymopentin on the level of B D and GABA receptors during various kinds of stressful stimuli is difficult to explain. Thymopentin neither antagonized nor potentiated picrotoxin induced convulsions (Dr. R. Muceniece, unpublished data). Therefore it seems unlikely that thymopentin has any direct action on the GABA-benzodiazepine-Cl- ionophore receptoral complex subunits. One must consider also the very short half-life of thymopentine in vivo and in vitro, about 30 and 60 s respectively [23,24]. Nevertheless, we cannot exclude some indirect action on GABA release and/or metabolism. Recently, it was found that thymopentin enchances GABA release in vitro (personal communication from Dr. A. P. Dolshenko) and normalizes the GABA content reduced by stress in rat hippocampus and cerebral cortex [25]. Concerning the relatively high doses of thymopentin used in this study, the very low toxicity of thymopentin should be considered: in mice, the LDso is more than 250 mg/kg [4]. In conclusion, we would like to emphasize that in spite of the ineffectiveness in the two behavioral anxiety models, thymopentin has a unique stress-protective action at the level of the GABA-benzodiazepine-C1- ionophore complex. As the direct action of this peptide on benzodiazepine and GABA receptors is unlikely, the involvement of other mechanisms like GABA release and/or metabolism can be suggested.
364
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