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Antagonism of serotonin receptor mediated neuroendocrine and temperature responses by atypical neuroleptics in the rat
European Journal of Pharmacology, Elsevier
463
151 (1988) 463-469
ELJP 50335
Antagonism of serotonin receptor mediated neuroendocrine and temperature responses by atypical neuroleptics in the rat J. Frank Nash *, Herbert Case Western Reserve Uniuersity, School
Y. Meltzer
ofMedicine,Department
and Gary A. Gudelsky
of Psychiatry and Pharmocologv,
Cleveland, Ohro 44106, U.S.A.
Received 12 April 1988, accepted 19 April 1988
The ability of atypical and typical antipsychotics to antagonize serotonin (5HT) receptor-mediated temperature and neuroendocrine responses was tested in rats. Clozapine, melperone and setoperone, three atypical neuroleptics, blocked in a dose-dependent manner, the hyperthermic response to the S-HT agonist, MK-212, whereas chlorpromazine and haloperidol were ineffective. The hypothermic response to the 5-HT,, a g onist, 8-OH-DPAT, was unaltered by any of the atypical neuroleptics tested. Similarly, MK-212-induced corticosterone secretion was blocked in a dose-related manner by clozapine, melperone and setoperone but was relatively unaffected by either haloperidol or chlorpromazine. The increase in corticosterone secretion observed following 8-OH-DPAT administration was not attenuated by pretreatment with the atypical or typical antipsychotics tested. These data indicate that atypical haloperidol neuroleptics are effective 5-HT, but not 5-HT,, antagonists in vivo. Conversely, the typical neuroleptics, and chlorpromazine do not block the 5-HT receptors involved in activation of the hypothalamic-pituitary-adrenal axis or thermoregulation. Atypical
neuroleptics;
Antipsychotics;
Corticosterone;
1. Introduction The ability of antipsychotic drugs to interact with serotonergic, as well as dopaminergic, binding sites was demonstrated by Leysen et al. (1978). These authors suggested that both serotonergic and dopaminergic receptors were involved in the mechanism of action of antipsychotic drugs. Recently, the treatment of chronic schizophrenia with antipsychotics which possess significant 5-HT, antagonist properties has been reported to result in the amelioration of negative symptoms (flat affect, withdrawal, loss of motivation) as well as in a low incidence of extrapyramidal side effects (Bjerkenstedt et al., 1979; Ceulemans et al., 1985; Gelders et al., 1986). These data are suggestive
* To whom all correspondence should be addressed. 0014-2999/88/$03.50
Body temperature;
Serotonin
antagonism
that the ratio of affinities for 5-HT, and dopamine (D-2) receptors may, in part, lead to the atypical nature of some antipsychotic drugs. Indeed, Altar et al. (1986) reported that several atypical neuroleptics (e.g. clozapine, fluperlapine, melperone, setoperone and RMI 81582) differed from typical neuroleptics (e.g. haloperidol, metoclopramide and pimozide) in their relative affinities for 5-HT, and D-2 binding sites. Specifically, the atypical neuroleptics displayed a greater affinity for 5-HT, binding sites in the frontal cortex and a lower affinity for D-2 binding sites in the caudate-putamen as compared to the typical neuroleptics. Several studies have been undertaken in which the antiserotonergic effects of antipsychotic agents have been examined. Sulpizio et al. (1978) found that clozapine blocked fenfluramine-induced hyperthermia in a dose-dependent manner. In con-
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
464
trast, typical antipsychotics were reported not to block this response. Moreover, Fink et al. (1984) demonstrated that clozapine, but not haloperidol, blocked the ability of LSD to potentiate apomorphine-induced hypermotility and concluded that clozapine had antiserotonergic properties. Furthermore, clozapine has been shown to block the discriminative stimulus properties of quipazine, a 5-HT agonist (Friedman et al., 1985). Fuller and Mason (1986) reported that clozapine, but not fluphenazine, attenuated quipazine-induced corticosterone secretion. Collectively, these data are consistent with the data from binding studies (Altar et al., 1986) and are suggestive that clozapine possesses significant antiserotonergic properties. The data are not as convincing (nor consistent) regarding the antiserotonergic properties of typical neuroleptics. Although typical neuroleptics were found to lack significant antiserotonergic effects in the aforementioned studies, Peroutka et al. (1981) reported that numerous typical neuroleptics (e.g. chlorpromazine, haloperidol) antagonized 5-hydroxytryptophan (5-HTP)-induced head twitches in mice. Additionally, Ortmann et al. (1982) demonstrated that typical neuroleptics prevented the development of the serotonin syndrome elicited by 5-HTP. The effects of 5-HT agonists on body temperature and corticosterone secretion in the rat have been ascribed to the activation of specific 5-HT receptor subtypes. For example, stimulation of 5-HT, sites with quipazine or 6-chloro-2-(1piperazinyl)-pyrazine (MK-212) in heat-adapted rats elicits a hyperthermic response (Gudelsky et al., 1986; Pawlowski, 1984). Conversely, activation of 5-HT1, receptors with 8-hydroxy-2-(di-n-propyl-amino) tetralin (8-OH-DPAT) produces hypothermia in rats (Hjorth, 1985; Goodwin and Green, 1985; Gudelsky et al., 1986). Interestingly, activation of either 5-HT, sites by MK-212 or 5-HTr, sites by 8-OH-DPAT results in an elevation of serum corticosterone concentrations (Koenig et al., 1987). In the present study we have assessed the ability of the atypical antipsychotics clozapine, melperone and setoperone and the typical antipsychotics, haloperidol and chlorpromazine to at-
tenuate 5-HT, and 5-HT,, changes in body temperature secretion.
receptor-mediated and corticosterone
2. Materials and methods 2.1. Animals Male, Sprague-Dawley rats weighing 200-250 g (Zivic Miller Laboratory, Hillson Park, PA), were used in all experiments. Animals were housed 6 per cage in a temperature-controlled room (2224’ C) with a light/dark cycle of 12/12 h (lights on at 06:OO). Food (Wayne Lab Blox) and tap water were available ad libitum. 2.2. Drugs Chlorpromazine and 8-OH-DPAT were purchased from Sigma Chemical, Co. (St. Louis, MO) and Research Biochemicals, Inc. (Wayland, MA), respectively. The other drugs were generously provided by the manufacturers: clozapine (Sandoz Pharmaceuticals, E. Hanover, NJ), haloperidol (McNeil Pharmaceuticals, Springhouse, PA), MK212 (Merck, Sharp 62 Dohme, Co., Westpoint, PA), melperone (Leo, Malmo, Sweden) and setoperone (Janssen Pharmaceutics, Beerse, Belgium). All drugs except 8-OH-DPAT were dissolved in 0.1 N HCl and injected i.p. 8-OH-DPAT was dissolved in sodium metabisulfite and administered S.C. 2.3. Experimental
design
On the day prior to an experiment in which serum corticosterone concentrations were determined, the animals were housed six per cage and transferred to the experimental room. On the day of the experiment, MK-212 (2.5 mg/kg i.p.) was injected 60 min before the rats were decapitated and 60 min following the administration of a neuroleptic. 8-OH-DPAT (0.1 mg/kg s.c.) was injected 30 min prior to sacrifice and 60 min following neuroleptic pretreatment. The doses of MK-212 and 8-OH-DPAT used in these experiments were the approximate ED,, values obtained
465
in previously reported dose-response studies (Koenig et al., 1987). For each experiment, the vehicle used to dissolve the drugs was injected. Following decapitation, trunk blood was collected and allowed to clot. Serum was obtained following centrifugation and stored at -2O’C until the time of assay. On the day of an experiment in which body temperatures were to be determined, rats were transferred to a temperature-controlled room. The rats were acclimated for l-2 h at either 29 o C for the study of antagonism of MK-21Zinduced hyperthermia, or 23 o C for the study of antagonism of 8-OH-DPAT-induced hypothermia. Body temperatures were recorded 30 min, 15 min and immediately prior to the administration of the antipsychotics. With the exception of clozapine, the neuroleptics were injected 60 min prior to the administration of MK-212 (1 mg/kg i.p.) or 8OH-DPAT (0.1 mg/kg s.c.). Clozapine was injected 30 min prior to the administration of MK212 or 8-OH-DPAT. The doses of MK-212 and 8-OH-DPAT used in these temperature experiments were chosen on the basis of previously reported dose-response studies (Gudelsky et al., 1986). A final measurement of body temperature was performed 30 min after the injection of MK212 or 8-OH-DPAT. Rectal temperature measurements were made using a telethermometer (Model 44TA, Yellow Springs, Inst. Co.) and a thermistor probe. Each rat was slightly restrained in a large sheet following which the thermistor probe, lubricated with a small amount of petroleum jelly, was inserted 5 cm into the rectum until a stable temperature was obtained.
TABLE
1
The effect of MK-212 and 8-OH-DPAT on serum corticosterone (pg/dl) concentration alone or following pretreatment with the atypical antipsychotics clozapine, melperone or setoperone. Drugs were administered according to the procedure described in Methods. Each value is the mean f S.E.M. of six rats. Pretreatment
(mg/-
Treatment
kg)
Vehicle
Vehicle Clozapine
(2.5) (5.0)
Melperone
(2.5) (5.0)
Setoperone
(1 .O) (2.5)
MK-212
8-OH-DPAT
6.8 f 1.6
34.1+ 2.8 a
19.1+ 1.7 a
6.7k1.5 4.5 f 1.6
25.3 + 3.7 a*b N.D. 14.5k2.9 a,b 23.8k3.3
3.0+1.2’ 21.3f1.4 1.6*0.5 = 11.3k2.9 10.3 + 2.1 16.4&2.8d
a.b N.D. a.b 16.9k2.0
’
’
18.7 f 2.9 axb N.D. 20.5kl.S b 31.7 + 3.9 a
a Significantly greater than the values for rats pretreated with the vehicle or antipsychotic, P < 0.05. b Significantly less than the value for vehicle pretreated rats administered MK-212, P < 0.05. ’ Significantly less than the value for rats given only the vehicle injections, P < 0.05. d Significantly greater than the value for rats given only the vehicle injections, P < 0.05.
2.5. Statistical
analysis
The data obtained from studies of the interaction between antipsychotics and 5-HT agonists on serum corticosterone were analysed with a two-way analysis of variance. The data obtained in the temperature experiments were analysed with oneway analysis of variance. Differences between treatment groups were evaluated using the Student-Newman-Keuls test. In each test, statistical significance was set at a level of P < 0.05.
2.4. Assays 3. Results Serum concentrations of corticosterone were determined by radioimmunoassay (RIA) following the procedure described by Abraham (1977). [3H]Corticosterone was purchased from New England Nuclear (Boston, MA), and the antiserum was purchased from Radioassay Systems Laboratories, Inc. (Carson, CA). The unlabeled corticosterone used in preparing the RIA standard was purchased from Steraloids, Inc. (Wilton, NH). The assay sensitivity was 25 pg/ml.
The effects of clozapine, melperone and setoperone on MK-212 or 8-OH-DPAT-induced corticosterone secretion are presented in table 1. Clozapine and melperone (2.5 and 5 mg/kg) antagonized the MK-212-induced elevation of serum corticosterone concentrations in a doserelated manner but did not antagonize the increase in serum corticosterone concentrations produced by 8-OH-DPAT. The administration of
466 TABLE 2 Effect of haloperidol of chlorpromazine on MK-212- and S-OH-DPAT-induced elevations of serum corticosterone (pg/dl) concentrations. Drugs were administered according to the procedure described in Methods. Each value is the mean (pg/dl) f S.E.M. of six rats. Pretreatment Vehicle Haloperidol Chlorpromazine
(mg/-
Treatment
kg)
Vehicle
MK-212
(1.0)
5.5k1.9 8.7kl.7
33.1f2.0 28.7+3.2
a a
(10.0)
12.9k2.3
23.1+3.0
a.b 21.1k4.6
&OH-DPAT 26.8 + 2.5 a 22.1*5.1 ’ ’
a Significantly greater than the values for vehicle or antipsychotic pretreated rats given the vehicle, P < 0.05. b Significantly less than the value for the vehicle pretreated rats administered MK-212, P < 0.05.
clozapine did not significantly alter serum corticosterone concentrations whereas melperone significantly decreased basal corticosterone concentrations. The administration of setoperone (2.5 mg/kg) alone significantly increased serum corticosterone concentrations (table 1). Nevertheless, setoperone (1.0 and 2.5 mg/kg) also significantly attenuated the MK-212-induced increase in serum costicosterone concentrations. The 8-OH-DPAT-induced increase in serum corticosterone concentrations was not antagonized by setoperone. In fact, the stimulatory effects of setoperone and S-OHDPAT on corticosterone secretion were additive. The effects of haloperidol and chlorpromazine on the 5-HT agonist-induced secretion of corticosterone are presented in table 2. Haloperidol (1.0 mg/kg) T alone, had no effect on corticosterone secretion nor did it antagonize the elevation of corticosterone concentrations produced by either MK-212 or S-OH-DPAT. The administration of chlorpromazine (10 mg/kg) alone resulted in a slight but non-significant elevation of serum corticosterone concentrations. The effect of MK-212 on corticosterone secretion was partially antagonized by chlorpromazine, whereas the stimulatory effect of 8-OH-DPAT was unaltered. Previous studies have shown that MK-212-induced hyperthermia is antagonized not only by ketanserin, but also by the 5-HT, antagonist pirenperone and other 5-HT antagonists with a
high affinity for the 5-HT, binding site (Gudelsky et al., 1986). To confirm and extend these previous findings, the highly selective 5-HT, antagonist, ritanserin (0.5 and 1.0 mg/kg) was administered, i.p. to heat-adapted rats (N = 6/group) 60 min prior to the administration of MK-212 (1.0 mg/kg). Body temperature was recorded 30 min following MK-212 administration. In rats pretreated with vehicle, MK-212 significantly increased body temperature (1.4 + 0.1” C); both the 0.5 and 1.0 mg/kg doses of ritanserin significantly (P < 0.05) attenuated MK-212-induced hyperthermia to a similar extent (0.6 k 0.1 and 0.5 + O.l” C, respectively). The dose-dependent inhibition of MK-212-induced hyperthermia by clozapine, melperone and setoperone is shown in fig. 1. The effect of MK-212 (1.0 mg/kg) was significantly (P < 0.05) inhibited by approximately 80% in rats treated with 10 mg/kg of any one of these atypical antipsychotics. The least effective dose of clozapine, melperone or
Inhibition
of MK-212
-
Induced
Hyperthermia
a
0
-N
0
1
*
1 Dose
3 (mg/kg)
I
10
Fig. 1. Antagonism of hyperthermia induced by MK-212. Melperone (MP) setoperone (ST) or vehicle (0 dose) was injected i.p. 1 h prior to MK-212 (1 mg/kg i.p.). Clozapine (CZ) was injected i.p. 30 min before MK-212 (1 mg/kg i.p.). Symbols represent the meanf S.E.M. of six rats. Statistically significant (P -z 0.05) antagonism of MK-212~induced hyperthermia was achieved at a dose of 1 mg/kg for each of the atypical neuroleptics tested.
467
setoperone which produced a significant (P < 0.05) antagonism of the MK-21Zinduced hyperthermia was 1.0 mg/kg. In contrast, neither haloperidol (1 mg/kg i.p.) nor chlorpromazine (10 mg/kg i.p.) significantly affected MK-212-induced hyperthermia. The increases in body temperature produced by MK-212 in vehicle-, haloperidoland chlorpromazinetreated rats were 1.2 k 0.2, 1.2 f 0.3 and 1.5 f 0.1 o C, respectively. A slight hyperthermia of approximately 0.4” C was produced by all of the neuroleptic agents at an ambient temperature of 29 o C, whereas a hypothermic response of approximately 1 o C was produced at an ambient temperature of 23” C. Although the atypical antispsychotics effectively antagonized MK-21Zinduced hyperthermia and the typical neuroleptics did not, there was no correlation for the entire group of antipsychotic agents between their effects on basal body temperature and antagonism of MK-212-induced hyperthermia. At the highest dose tested, 10 mg/kg, the atypical neuroleptics clozapine, melperone and setoperone had no effect on 8-OH-DPAT (0.1 mg/kg s.c.)-induced hypothermia in rats tested at an ambient temperature of 23 o C. The decreases in body temperature produced by 8-OH-DPAT in rats pretreated with vehicle, clozapine, melperone and setoperone were - 1.3 f 0.2, -0.9 * 0.2, - 1.2 k 0.1 and - 1.0 f 0.2, respectively. Thus, although the atypical neuroleptics decreased body temperature alone compared to the effect of vehicle treatment, 8-OH-DPAT produced a similar net decrease in body temperature in all of the treatment groups.
4. Discussion The ability of clozapine, melperone and setoperone to block MK-212-induced corticosterone secretion and hyperthermia is consistent with the view that these atypical neuroleptics possess significant 5-HT antagonist properties. Although MK-212 is not a selective 5-HT, agonist (Engel et al., 1986; Lorens and Van de Kar, 1987),
the increase in serum corticosterone secretion and hyperthermia elicited by MK-212 is thought to be mediated by 5-HT, receptors, since these responses are blocked in a dose-dependent manner by the 5-HT, antagonists ketanserin and pirenperone (Gudelsky et al., 1986; Koenig et al., 1987) as well as by ritanserin. Therefore, the results of the present study are supportive of the view that the atypical neuroleptics are antagonists at receptors of the 5-HT, subtype. These data are in good agreement with the data obtained from radioligand binding studies (Altar et al., 1986) in which clozapine, melperone and setoperone were reported to have a relatively high affinity for 5-HT, receptors. The atypical antipsychotics did not alter the corticosterone response or the hypothermic response following activation of 5-HT,, receptors with 8-OH-DPAT. Hence, these atypical antipsychotics appear to be relatively selective in their interaction with 5-HT, receptor systems. However, interactions of these agents with other 5-HT receptor subtypes has not been determined, to our knowledge. The effects of chlorpromazine, haloperidol as well as clozapine on body temperature have been extensively studied (for review see: Clark and Lipton, 1985). In general, the administration of these antipsychotics results in a slight hypothermia (- 1.0-1.5 “C) in rats maintained at room temperature and a slight hyperthermia or no effect in heat-adapted (29OC) rats. Similar results were obtained in the present studies. More importantly, there was no correlation between the effects of the antipsychotics on basal body temperature when administered alone and their ability to antagonize MK-212- or 8-OH-DPAT-induced hyper- and hypothermia, respectively. Thus, in contrast to the marked inhibition of 5-HT, receptor-mediated corticosterone secretion and hyperthermia achieved by clozapine, melperone and setoperone, these responses were essentially unaffected by the typical neuroleptics, haloperidol and chlorpromazine. However, chlorpromazine did produce a partial antagonism of MK-212-induced corticosterone secretion. These results contrast with those reported by Ortman et al. (1982) in which chlorpromazine and haloperi-
468
do1 were both more potent than clozapine in blocking the L-SHTP-induced 5-HT syndrome. Additionally, Peroutka et al. (1981) found that clozapine, haloperidol and chlorpromazine were nearly equipotent in their ability to inhibit 5HTP-induced head twitches and displace [ 3H]spiperone from cortical membrane binding sites. Differences between these studies and the results reported herein could possibly be attributed to the non-specific effects of administration of high doses of 5-HTP or to the sedative effects of neuroleptics. Alternatively, the different populations of 5-HT, receptors that are involved in corticosterone secretion, hyperthermia and the 5HTP syndrome may have differential sensitivities to the effects of typical neuroleptics. The potency with which the atypical neuroleptics antagonized MK-212-induced corticosterone secretion and hyperthermia does not appear to correlate well with the reported affinities of the agents for 5-HT, receptors. Altar et al. (1986) reported the apparent IC,, values obtained in radioligand competition studies for a number of antipsychotics. From these data, setoperone was 30 times more potent than melperone or clozapine at 5-HT, sites. However, these differences in affinity for 5-HT, binding sites were not reflected in the potency to inhibit MK-21Zinduced hyperthermia. Clozapine, melperone and setoperone were essentially equipotent in this measure of 5-HT, antagonism. A number of explanations could account for the differences between the potency of these agents in vitro and the failure to reflect this potency in vivo. For example, pharmacokinetic differences between these agents could affect the concentration of drug at the site of action. In addition, the complex effects on several neurotransmitter systems ultimately determines the pharmacological characteristics of the atypical antipsychotics in vivo. In summary, the atypical antipsychotics, clozapine, melperone and setoperone exhibited significant 5-HT, antagonist properties, whereas chlorpromazine and haloperidol did not. Although a limited number of antipsychotics were tested, it appears that this pharmacological property may differentiate atypical from typical antipsychotics.
Acknowledgements This work was supported, in part, by USPHS MH 41684, MH 41683, MH 41594 and grants from the Cleveland and Sawyer Foundations. JFN is the recipient of a NARSAD fellowship extension award. HYM is the recipient of a USPHS Research Career Scientist Award MH 47808. The authors thank Mrs. Diane Mack for her secretarial assistance in preparing this manuscript.
References Abraham, G.E., 1977, Handbook of Radioimmunoassay, Vol. 5 (Marcel Dekker, New York), p 591. Altar, C.A., A.M. Wasley, R.F. Neale and G.A. Stone, 1986, Typical and atypical antipsychotic occupancy of D, and S, receptors: An autoradiographic analysis in rat brain, Brain Res. Bull. 16, 517. Bjerkenstedt, L., B. Gullberg, C. H&rnryd and G. Sedvall, 1979, Relationship between clinical and biochemical effects of melperone and thiotbixene in psychotic women, Arch. Psychiat. Nerve&r. 227, 181. Ceulemans, D.L.S., Y.G. Gelders, M.-L.J.A. Hoppenbrouwers, A.J.M. Reyntjens and P.A.J. Janssen, 1985, Effect of serotonin antagonism in schizophrenia: A pilot study with setoperone, Psychopharmacology 85, 329. Clark, W.G. and J.M. Lipton, 1985, Changes in body temperature after administration of amino acids, peptides, dopamine neuropeptides, dopamine neuroleptics and related agents: II, Neurosci. Biobehav. Rev. 9, 299. Engel, G., M. Gothert, D. Hoyer, E. S&licker and K. Hilenbrand, 1986, Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT,, binding sites, Naunyn-Schmiedeb. Arch. Pharmacol. 332, 1. Fink, H., R. Morgenstem and W. Oelssner, 1984, Clozapine a serotonin antagonist?, Pharmacol. B&hem. Behav. 20, 513. Friedman, R.L., E. Sanders-Bush and R.L. Barrett, 1985, Clozapine blocks disruptive and discriminative stimulus effects of quipazine, European J. Pharmacol. 106, 191. Fuller, R.W. and N.R. Mason, 1986, Flumerapine, an antagonist of central dopamine and serotonin receptors, Res. Commun. Chem. Pathol. Pharmacol. 54, 23. Gelders, V., G. Vanden Bussche, A. Reyntjens and P. Janssen, 1986, Serotonin-S, receptor blockers in the treatment of chronic schizophrenia, Clin. Neuropharmacol. 9, 325. Goodwin, G.M. and A.D. Green, 1985, A behavioral and biochemical study in mice and rats of putative selective agonists and antagonists for 5-HT, and 5-HT, receptors, Br. J. Pharmacol. 84, 743. Gudelsky, G.A., J.I. Koenig and H.Y. Meltzer, 1986, Thermoregulatory responses to serotonin (5-HT) receptor stimulation in the rat. Evidence for opposing roles of 5-HT, and 5HTi, receptors, Nemopharmacology 25, 1307.
469 Hjorth, S., 1985, Hypothermia in the rat induced by the potent serotonergic agent I-OH-DPAT, J. Neural Transm. 61,131. Koenig, J.I., G.A. Gudelsky and H.Y. Meltzer, 1987, Stimulation of corticosterone and /I-endorphin secretion in the rat by selective 5-HT receptor subtype activation, European J. Pharmacol. 137, 1. Leysen, L.E., C.J.E. Niemegeers, J.P. Tollenaere and P.M. Laduron, 1978, Serotonergic component of neuroleptic receptors, Nature 272, 168. Lorens, S.A. and L.D. Van de Kar, 1987, Differential effects of serotonin (5HT,, and S-HT,) agonists and antagonists on renin and corticosterone secretion, Neuroendocrinology 45, 305. Ortmann, R., S. Bischoff, E. Radeke, 0. Buech and A. Delini-
Stula, 1982, Correlations between different measures of antiserotonin activity of drugs: Study with neuroleptics and serotonin receptor blockers, Naunyn-Schmiedeb. Arch. Phannacol. 321, 265. Pawlowski, L., 1984, Amitriptyline and femoxetine, but not clomipramine or citalopram, antagonize hyperthermia induced by directly acting 5-hydoxytryptamine-like drugs in heat adapted rats, J. Pharm. Pharmacol. 36, 197. Peroutka, S.J, R.M. Lebovitz and S.H. Snyder, 1981, Two distinct central serotonin receptors with different physiological functions, Science 212, 827. Sulpizio, A., P.J. Fowler and E. Macko, 1978, Antagonism of fenfluramine-induced hyperthennia: A measure of central serotonin inhibition, Life Sci. 22, 1439.
463
151 (1988) 463-469
ELJP 50335
Antagonism of serotonin receptor mediated neuroendocrine and temperature responses by atypical neuroleptics in the rat J. Frank Nash *, Herbert Case Western Reserve Uniuersity, School
Y. Meltzer
ofMedicine,Department
and Gary A. Gudelsky
of Psychiatry and Pharmocologv,
Cleveland, Ohro 44106, U.S.A.
Received 12 April 1988, accepted 19 April 1988
The ability of atypical and typical antipsychotics to antagonize serotonin (5HT) receptor-mediated temperature and neuroendocrine responses was tested in rats. Clozapine, melperone and setoperone, three atypical neuroleptics, blocked in a dose-dependent manner, the hyperthermic response to the S-HT agonist, MK-212, whereas chlorpromazine and haloperidol were ineffective. The hypothermic response to the 5-HT,, a g onist, 8-OH-DPAT, was unaltered by any of the atypical neuroleptics tested. Similarly, MK-212-induced corticosterone secretion was blocked in a dose-related manner by clozapine, melperone and setoperone but was relatively unaffected by either haloperidol or chlorpromazine. The increase in corticosterone secretion observed following 8-OH-DPAT administration was not attenuated by pretreatment with the atypical or typical antipsychotics tested. These data indicate that atypical haloperidol neuroleptics are effective 5-HT, but not 5-HT,, antagonists in vivo. Conversely, the typical neuroleptics, and chlorpromazine do not block the 5-HT receptors involved in activation of the hypothalamic-pituitary-adrenal axis or thermoregulation. Atypical
neuroleptics;
Antipsychotics;
Corticosterone;
1. Introduction The ability of antipsychotic drugs to interact with serotonergic, as well as dopaminergic, binding sites was demonstrated by Leysen et al. (1978). These authors suggested that both serotonergic and dopaminergic receptors were involved in the mechanism of action of antipsychotic drugs. Recently, the treatment of chronic schizophrenia with antipsychotics which possess significant 5-HT, antagonist properties has been reported to result in the amelioration of negative symptoms (flat affect, withdrawal, loss of motivation) as well as in a low incidence of extrapyramidal side effects (Bjerkenstedt et al., 1979; Ceulemans et al., 1985; Gelders et al., 1986). These data are suggestive
* To whom all correspondence should be addressed. 0014-2999/88/$03.50
Body temperature;
Serotonin
antagonism
that the ratio of affinities for 5-HT, and dopamine (D-2) receptors may, in part, lead to the atypical nature of some antipsychotic drugs. Indeed, Altar et al. (1986) reported that several atypical neuroleptics (e.g. clozapine, fluperlapine, melperone, setoperone and RMI 81582) differed from typical neuroleptics (e.g. haloperidol, metoclopramide and pimozide) in their relative affinities for 5-HT, and D-2 binding sites. Specifically, the atypical neuroleptics displayed a greater affinity for 5-HT, binding sites in the frontal cortex and a lower affinity for D-2 binding sites in the caudate-putamen as compared to the typical neuroleptics. Several studies have been undertaken in which the antiserotonergic effects of antipsychotic agents have been examined. Sulpizio et al. (1978) found that clozapine blocked fenfluramine-induced hyperthermia in a dose-dependent manner. In con-
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
464
trast, typical antipsychotics were reported not to block this response. Moreover, Fink et al. (1984) demonstrated that clozapine, but not haloperidol, blocked the ability of LSD to potentiate apomorphine-induced hypermotility and concluded that clozapine had antiserotonergic properties. Furthermore, clozapine has been shown to block the discriminative stimulus properties of quipazine, a 5-HT agonist (Friedman et al., 1985). Fuller and Mason (1986) reported that clozapine, but not fluphenazine, attenuated quipazine-induced corticosterone secretion. Collectively, these data are consistent with the data from binding studies (Altar et al., 1986) and are suggestive that clozapine possesses significant antiserotonergic properties. The data are not as convincing (nor consistent) regarding the antiserotonergic properties of typical neuroleptics. Although typical neuroleptics were found to lack significant antiserotonergic effects in the aforementioned studies, Peroutka et al. (1981) reported that numerous typical neuroleptics (e.g. chlorpromazine, haloperidol) antagonized 5-hydroxytryptophan (5-HTP)-induced head twitches in mice. Additionally, Ortmann et al. (1982) demonstrated that typical neuroleptics prevented the development of the serotonin syndrome elicited by 5-HTP. The effects of 5-HT agonists on body temperature and corticosterone secretion in the rat have been ascribed to the activation of specific 5-HT receptor subtypes. For example, stimulation of 5-HT, sites with quipazine or 6-chloro-2-(1piperazinyl)-pyrazine (MK-212) in heat-adapted rats elicits a hyperthermic response (Gudelsky et al., 1986; Pawlowski, 1984). Conversely, activation of 5-HT1, receptors with 8-hydroxy-2-(di-n-propyl-amino) tetralin (8-OH-DPAT) produces hypothermia in rats (Hjorth, 1985; Goodwin and Green, 1985; Gudelsky et al., 1986). Interestingly, activation of either 5-HT, sites by MK-212 or 5-HTr, sites by 8-OH-DPAT results in an elevation of serum corticosterone concentrations (Koenig et al., 1987). In the present study we have assessed the ability of the atypical antipsychotics clozapine, melperone and setoperone and the typical antipsychotics, haloperidol and chlorpromazine to at-
tenuate 5-HT, and 5-HT,, changes in body temperature secretion.
receptor-mediated and corticosterone
2. Materials and methods 2.1. Animals Male, Sprague-Dawley rats weighing 200-250 g (Zivic Miller Laboratory, Hillson Park, PA), were used in all experiments. Animals were housed 6 per cage in a temperature-controlled room (2224’ C) with a light/dark cycle of 12/12 h (lights on at 06:OO). Food (Wayne Lab Blox) and tap water were available ad libitum. 2.2. Drugs Chlorpromazine and 8-OH-DPAT were purchased from Sigma Chemical, Co. (St. Louis, MO) and Research Biochemicals, Inc. (Wayland, MA), respectively. The other drugs were generously provided by the manufacturers: clozapine (Sandoz Pharmaceuticals, E. Hanover, NJ), haloperidol (McNeil Pharmaceuticals, Springhouse, PA), MK212 (Merck, Sharp 62 Dohme, Co., Westpoint, PA), melperone (Leo, Malmo, Sweden) and setoperone (Janssen Pharmaceutics, Beerse, Belgium). All drugs except 8-OH-DPAT were dissolved in 0.1 N HCl and injected i.p. 8-OH-DPAT was dissolved in sodium metabisulfite and administered S.C. 2.3. Experimental
design
On the day prior to an experiment in which serum corticosterone concentrations were determined, the animals were housed six per cage and transferred to the experimental room. On the day of the experiment, MK-212 (2.5 mg/kg i.p.) was injected 60 min before the rats were decapitated and 60 min following the administration of a neuroleptic. 8-OH-DPAT (0.1 mg/kg s.c.) was injected 30 min prior to sacrifice and 60 min following neuroleptic pretreatment. The doses of MK-212 and 8-OH-DPAT used in these experiments were the approximate ED,, values obtained
465
in previously reported dose-response studies (Koenig et al., 1987). For each experiment, the vehicle used to dissolve the drugs was injected. Following decapitation, trunk blood was collected and allowed to clot. Serum was obtained following centrifugation and stored at -2O’C until the time of assay. On the day of an experiment in which body temperatures were to be determined, rats were transferred to a temperature-controlled room. The rats were acclimated for l-2 h at either 29 o C for the study of antagonism of MK-21Zinduced hyperthermia, or 23 o C for the study of antagonism of 8-OH-DPAT-induced hypothermia. Body temperatures were recorded 30 min, 15 min and immediately prior to the administration of the antipsychotics. With the exception of clozapine, the neuroleptics were injected 60 min prior to the administration of MK-212 (1 mg/kg i.p.) or 8OH-DPAT (0.1 mg/kg s.c.). Clozapine was injected 30 min prior to the administration of MK212 or 8-OH-DPAT. The doses of MK-212 and 8-OH-DPAT used in these temperature experiments were chosen on the basis of previously reported dose-response studies (Gudelsky et al., 1986). A final measurement of body temperature was performed 30 min after the injection of MK212 or 8-OH-DPAT. Rectal temperature measurements were made using a telethermometer (Model 44TA, Yellow Springs, Inst. Co.) and a thermistor probe. Each rat was slightly restrained in a large sheet following which the thermistor probe, lubricated with a small amount of petroleum jelly, was inserted 5 cm into the rectum until a stable temperature was obtained.
TABLE
1
The effect of MK-212 and 8-OH-DPAT on serum corticosterone (pg/dl) concentration alone or following pretreatment with the atypical antipsychotics clozapine, melperone or setoperone. Drugs were administered according to the procedure described in Methods. Each value is the mean f S.E.M. of six rats. Pretreatment
(mg/-
Treatment
kg)
Vehicle
Vehicle Clozapine
(2.5) (5.0)
Melperone
(2.5) (5.0)
Setoperone
(1 .O) (2.5)
MK-212
8-OH-DPAT
6.8 f 1.6
34.1+ 2.8 a
19.1+ 1.7 a
6.7k1.5 4.5 f 1.6
25.3 + 3.7 a*b N.D. 14.5k2.9 a,b 23.8k3.3
3.0+1.2’ 21.3f1.4 1.6*0.5 = 11.3k2.9 10.3 + 2.1 16.4&2.8d
a.b N.D. a.b 16.9k2.0
’
’
18.7 f 2.9 axb N.D. 20.5kl.S b 31.7 + 3.9 a
a Significantly greater than the values for rats pretreated with the vehicle or antipsychotic, P < 0.05. b Significantly less than the value for vehicle pretreated rats administered MK-212, P < 0.05. ’ Significantly less than the value for rats given only the vehicle injections, P < 0.05. d Significantly greater than the value for rats given only the vehicle injections, P < 0.05.
2.5. Statistical
analysis
The data obtained from studies of the interaction between antipsychotics and 5-HT agonists on serum corticosterone were analysed with a two-way analysis of variance. The data obtained in the temperature experiments were analysed with oneway analysis of variance. Differences between treatment groups were evaluated using the Student-Newman-Keuls test. In each test, statistical significance was set at a level of P < 0.05.
2.4. Assays 3. Results Serum concentrations of corticosterone were determined by radioimmunoassay (RIA) following the procedure described by Abraham (1977). [3H]Corticosterone was purchased from New England Nuclear (Boston, MA), and the antiserum was purchased from Radioassay Systems Laboratories, Inc. (Carson, CA). The unlabeled corticosterone used in preparing the RIA standard was purchased from Steraloids, Inc. (Wilton, NH). The assay sensitivity was 25 pg/ml.
The effects of clozapine, melperone and setoperone on MK-212 or 8-OH-DPAT-induced corticosterone secretion are presented in table 1. Clozapine and melperone (2.5 and 5 mg/kg) antagonized the MK-212-induced elevation of serum corticosterone concentrations in a doserelated manner but did not antagonize the increase in serum corticosterone concentrations produced by 8-OH-DPAT. The administration of
466 TABLE 2 Effect of haloperidol of chlorpromazine on MK-212- and S-OH-DPAT-induced elevations of serum corticosterone (pg/dl) concentrations. Drugs were administered according to the procedure described in Methods. Each value is the mean (pg/dl) f S.E.M. of six rats. Pretreatment Vehicle Haloperidol Chlorpromazine
(mg/-
Treatment
kg)
Vehicle
MK-212
(1.0)
5.5k1.9 8.7kl.7
33.1f2.0 28.7+3.2
a a
(10.0)
12.9k2.3
23.1+3.0
a.b 21.1k4.6
&OH-DPAT 26.8 + 2.5 a 22.1*5.1 ’ ’
a Significantly greater than the values for vehicle or antipsychotic pretreated rats given the vehicle, P < 0.05. b Significantly less than the value for the vehicle pretreated rats administered MK-212, P < 0.05.
clozapine did not significantly alter serum corticosterone concentrations whereas melperone significantly decreased basal corticosterone concentrations. The administration of setoperone (2.5 mg/kg) alone significantly increased serum corticosterone concentrations (table 1). Nevertheless, setoperone (1.0 and 2.5 mg/kg) also significantly attenuated the MK-212-induced increase in serum costicosterone concentrations. The 8-OH-DPAT-induced increase in serum corticosterone concentrations was not antagonized by setoperone. In fact, the stimulatory effects of setoperone and S-OHDPAT on corticosterone secretion were additive. The effects of haloperidol and chlorpromazine on the 5-HT agonist-induced secretion of corticosterone are presented in table 2. Haloperidol (1.0 mg/kg) T alone, had no effect on corticosterone secretion nor did it antagonize the elevation of corticosterone concentrations produced by either MK-212 or S-OH-DPAT. The administration of chlorpromazine (10 mg/kg) alone resulted in a slight but non-significant elevation of serum corticosterone concentrations. The effect of MK-212 on corticosterone secretion was partially antagonized by chlorpromazine, whereas the stimulatory effect of 8-OH-DPAT was unaltered. Previous studies have shown that MK-212-induced hyperthermia is antagonized not only by ketanserin, but also by the 5-HT, antagonist pirenperone and other 5-HT antagonists with a
high affinity for the 5-HT, binding site (Gudelsky et al., 1986). To confirm and extend these previous findings, the highly selective 5-HT, antagonist, ritanserin (0.5 and 1.0 mg/kg) was administered, i.p. to heat-adapted rats (N = 6/group) 60 min prior to the administration of MK-212 (1.0 mg/kg). Body temperature was recorded 30 min following MK-212 administration. In rats pretreated with vehicle, MK-212 significantly increased body temperature (1.4 + 0.1” C); both the 0.5 and 1.0 mg/kg doses of ritanserin significantly (P < 0.05) attenuated MK-212-induced hyperthermia to a similar extent (0.6 k 0.1 and 0.5 + O.l” C, respectively). The dose-dependent inhibition of MK-212-induced hyperthermia by clozapine, melperone and setoperone is shown in fig. 1. The effect of MK-212 (1.0 mg/kg) was significantly (P < 0.05) inhibited by approximately 80% in rats treated with 10 mg/kg of any one of these atypical antipsychotics. The least effective dose of clozapine, melperone or
Inhibition
of MK-212
-
Induced
Hyperthermia
a
0
-N
0
1
*
1 Dose
3 (mg/kg)
I
10
Fig. 1. Antagonism of hyperthermia induced by MK-212. Melperone (MP) setoperone (ST) or vehicle (0 dose) was injected i.p. 1 h prior to MK-212 (1 mg/kg i.p.). Clozapine (CZ) was injected i.p. 30 min before MK-212 (1 mg/kg i.p.). Symbols represent the meanf S.E.M. of six rats. Statistically significant (P -z 0.05) antagonism of MK-212~induced hyperthermia was achieved at a dose of 1 mg/kg for each of the atypical neuroleptics tested.
467
setoperone which produced a significant (P < 0.05) antagonism of the MK-21Zinduced hyperthermia was 1.0 mg/kg. In contrast, neither haloperidol (1 mg/kg i.p.) nor chlorpromazine (10 mg/kg i.p.) significantly affected MK-212-induced hyperthermia. The increases in body temperature produced by MK-212 in vehicle-, haloperidoland chlorpromazinetreated rats were 1.2 k 0.2, 1.2 f 0.3 and 1.5 f 0.1 o C, respectively. A slight hyperthermia of approximately 0.4” C was produced by all of the neuroleptic agents at an ambient temperature of 29 o C, whereas a hypothermic response of approximately 1 o C was produced at an ambient temperature of 23” C. Although the atypical antispsychotics effectively antagonized MK-21Zinduced hyperthermia and the typical neuroleptics did not, there was no correlation for the entire group of antipsychotic agents between their effects on basal body temperature and antagonism of MK-212-induced hyperthermia. At the highest dose tested, 10 mg/kg, the atypical neuroleptics clozapine, melperone and setoperone had no effect on 8-OH-DPAT (0.1 mg/kg s.c.)-induced hypothermia in rats tested at an ambient temperature of 23 o C. The decreases in body temperature produced by 8-OH-DPAT in rats pretreated with vehicle, clozapine, melperone and setoperone were - 1.3 f 0.2, -0.9 * 0.2, - 1.2 k 0.1 and - 1.0 f 0.2, respectively. Thus, although the atypical neuroleptics decreased body temperature alone compared to the effect of vehicle treatment, 8-OH-DPAT produced a similar net decrease in body temperature in all of the treatment groups.
4. Discussion The ability of clozapine, melperone and setoperone to block MK-212-induced corticosterone secretion and hyperthermia is consistent with the view that these atypical neuroleptics possess significant 5-HT antagonist properties. Although MK-212 is not a selective 5-HT, agonist (Engel et al., 1986; Lorens and Van de Kar, 1987),
the increase in serum corticosterone secretion and hyperthermia elicited by MK-212 is thought to be mediated by 5-HT, receptors, since these responses are blocked in a dose-dependent manner by the 5-HT, antagonists ketanserin and pirenperone (Gudelsky et al., 1986; Koenig et al., 1987) as well as by ritanserin. Therefore, the results of the present study are supportive of the view that the atypical neuroleptics are antagonists at receptors of the 5-HT, subtype. These data are in good agreement with the data obtained from radioligand binding studies (Altar et al., 1986) in which clozapine, melperone and setoperone were reported to have a relatively high affinity for 5-HT, receptors. The atypical antipsychotics did not alter the corticosterone response or the hypothermic response following activation of 5-HT,, receptors with 8-OH-DPAT. Hence, these atypical antipsychotics appear to be relatively selective in their interaction with 5-HT, receptor systems. However, interactions of these agents with other 5-HT receptor subtypes has not been determined, to our knowledge. The effects of chlorpromazine, haloperidol as well as clozapine on body temperature have been extensively studied (for review see: Clark and Lipton, 1985). In general, the administration of these antipsychotics results in a slight hypothermia (- 1.0-1.5 “C) in rats maintained at room temperature and a slight hyperthermia or no effect in heat-adapted (29OC) rats. Similar results were obtained in the present studies. More importantly, there was no correlation between the effects of the antipsychotics on basal body temperature when administered alone and their ability to antagonize MK-212- or 8-OH-DPAT-induced hyper- and hypothermia, respectively. Thus, in contrast to the marked inhibition of 5-HT, receptor-mediated corticosterone secretion and hyperthermia achieved by clozapine, melperone and setoperone, these responses were essentially unaffected by the typical neuroleptics, haloperidol and chlorpromazine. However, chlorpromazine did produce a partial antagonism of MK-212-induced corticosterone secretion. These results contrast with those reported by Ortman et al. (1982) in which chlorpromazine and haloperi-
468
do1 were both more potent than clozapine in blocking the L-SHTP-induced 5-HT syndrome. Additionally, Peroutka et al. (1981) found that clozapine, haloperidol and chlorpromazine were nearly equipotent in their ability to inhibit 5HTP-induced head twitches and displace [ 3H]spiperone from cortical membrane binding sites. Differences between these studies and the results reported herein could possibly be attributed to the non-specific effects of administration of high doses of 5-HTP or to the sedative effects of neuroleptics. Alternatively, the different populations of 5-HT, receptors that are involved in corticosterone secretion, hyperthermia and the 5HTP syndrome may have differential sensitivities to the effects of typical neuroleptics. The potency with which the atypical neuroleptics antagonized MK-212-induced corticosterone secretion and hyperthermia does not appear to correlate well with the reported affinities of the agents for 5-HT, receptors. Altar et al. (1986) reported the apparent IC,, values obtained in radioligand competition studies for a number of antipsychotics. From these data, setoperone was 30 times more potent than melperone or clozapine at 5-HT, sites. However, these differences in affinity for 5-HT, binding sites were not reflected in the potency to inhibit MK-21Zinduced hyperthermia. Clozapine, melperone and setoperone were essentially equipotent in this measure of 5-HT, antagonism. A number of explanations could account for the differences between the potency of these agents in vitro and the failure to reflect this potency in vivo. For example, pharmacokinetic differences between these agents could affect the concentration of drug at the site of action. In addition, the complex effects on several neurotransmitter systems ultimately determines the pharmacological characteristics of the atypical antipsychotics in vivo. In summary, the atypical antipsychotics, clozapine, melperone and setoperone exhibited significant 5-HT, antagonist properties, whereas chlorpromazine and haloperidol did not. Although a limited number of antipsychotics were tested, it appears that this pharmacological property may differentiate atypical from typical antipsychotics.
Acknowledgements This work was supported, in part, by USPHS MH 41684, MH 41683, MH 41594 and grants from the Cleveland and Sawyer Foundations. JFN is the recipient of a NARSAD fellowship extension award. HYM is the recipient of a USPHS Research Career Scientist Award MH 47808. The authors thank Mrs. Diane Mack for her secretarial assistance in preparing this manuscript.
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