0013-7227/79/1054-0923$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 4
Printed in U.S.A.
The Effects of Metergoline and Other Serotonin Receptor Antagonists on Serum Corticosterone in Rats RAY W. FULLER* AND HAROLD D. SNODDY The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206
heptadine and spiperone, which gave significant but incomplete antagonism at 1 mg/kg. Methysergide at 3 mg/kg did not alter the effect of quipazine. Metergoline did not antagonize the elevation of serum corticosterone by other agents throught to act via serotoninergic mechanisms, namely fluoxetine, fenfluramine, L-5-hydroxytryptophan, N,iV-dimethyl-5-methoxytryptamine, and l-(m-trifluoromethylphenyl)piperazine. Thus, the interactions between metergoline and quipazine may have occurred at receptors that are not serotonin receptors or that represent a subset of serotonin receptors not mediating the actions of serotoninergic agents other than quipazine. (Endocrinology 105: 923, 1979)
ABSTRACT. Metergoline antagonized the elevation of serum corticosterone by quipazine in rats. The ED50 of metergoline was less than 0.1 mg/kg, ip, and the effects of a 3 mg/kg dose persisted for more than 24 h. Metergoline did not antagonize the elevation of serum corticosterone by theophylline or ketamine {i.e. did not prevent corticosterone release nonspecifically) and did not affect the concentration of quipazineMn the brain. Since quipazine is a serotonin receptor agonist, the antagonistic effects of metergoline may have been due to competition with quipazine at serotonin receptor sites in the brain. Some other agents capable of blocking serotonin receptors also antagonized the elevation of serum corticosterone by quipazine. These included LY53857, which gave complete blockade at 3 mg/kg, and cypro-
R
groups of five in hanging wire cages in an air-conditioned (24 C) and light-controlled room for at least 1 week after delivery. Lights were on from 0700-1900 h daily. Food and water were available ad libitum. Drugs received as gifts from pharmaceutical companies were metergoline (Farmitalia, Milan, Italy), cyproheptadine (Merck, Rahway, NJ), methysergide (Sandoz, Hanover, NJ), spiperone (Janssen, Beerse, Belgium), fenfluramine hydrochloride (A. H. Robins, Richmond, VA), and ketamine hydrochloride (Bristol, Syracuse, NY). Purchased drugs were L-5-hydroxytryptophan (Sigma, St. Louis, MO), N,iV-dimethyl-5-methoxytryptamine (Aldrich Chemical Co., Milwaukee, WI), and l-(ra-trifluoromethylphenyl)piperazine (Aldrich). The remaining drugs were synthesized at Eli Lilly and Co. (Indianapolis, IN; quipazine maleate, theophylline, fluoxetine hydrochloride, and LY53857). LY53857 is 4-isopropyl-7-methyl-9-(2-hydroxy-l-ethylpropoxycarbonyl)4,6,6A,7,8,9,10,10A-octahydroindolo[4,3-FG)quinoline maleate. All drugs were administered ip at an injection volume of 1 ml/kg in aqueous solution (except metergoline and theophylline, which were given in acacia suspension). Pyrogen-free glass-distilled water was used in preparing the solutions or suspensions. Control rats received the same volume of the injection vehicle. Rats were killed by decapitation, and trunk blood was collected and allowed to clot. Serum was obtained by centrifugation and assayed immediately or stored frozen at -15 C before assay. Corticosterone concentration was assayed spectrofluorometrically by the method of Solem and Brinck-Johnsen (9). Statistical analysis of the data by Student's t test was done on an Olivetti desktop computer. Unless otherwise indicated, all data are expressed as the mean ± SE for five rats per group.
ECENTLY, we (1) reported that quipazine, a serotonin receptor agonist, increased serum corticosterone concentration in rats. This observation extended earlier findings that serotonin precursors (2, 3) and serotonin uptake inhibition (4) increased serum corticosterone and thus added to the evidence that serotonin neurons in the brain have a positive role (5-7) in the control of corticotropin-releasing factor from the hypothalamus. The fact that the effect of quipazine was completely prevented by pretreatment with metergoline, a known serotonin receptor antagonist (8), was taken as evidence that quipazine was acting via serotonin receptors to elevate serum corticosterone (1). We have now studied the effects of metergoline more thoroughly and report here that metergoline is a potent and long lasting antagonist of quipazine-induced elevation of serum corticosterone but does not antagonize the elevation of serum corticosterone by agents acting through nonserotoninergic mechanisms or by agents other than quipazine thought to act by serotoninergic mechanisms. Effects of other serotonin receptor antagonists, especially LY53857, are also reported. The structural relationships between LY53857 and metergoline are shown in Fig. 1. Materials and Methods Male Wistar rats, weighing about 150 g, were obtained from Harlan Industries (Cumberland, IN). They were housed in Received January 15, 1979. * To whom requests for reprints should be addressed. 923
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FULLER AND SNODDY
924
Endo • 1979 Vol 105 • No 4
TABLE 1. Duration of effect of metergoline at two dose levels in antagonizing serum corticosterone elevation by quipazine Metergoline pretreatment interval
I-CH3
Metergoline at 3 mg/kg No metergoline lh
16 h 24 h Metergoline at 1 mg/kg No metergoline lh
16 h 24 h 48 h
Serum corticosterone (/ig/100 ml) Control
Quipazine
6.3 ± 9.0 ± 6.5 ± 5.3 ±
0.4 2.2 0.5 0.3
43.5 ± 4.6" 12.5 ± 3.5* 7.0 ± 0.3* 8.8 ± 1.8*
6.5 ± 6.5 ± 7.7 ± 6.5 ± 8.4 ±
0.6 0.3 1.7 0.6 1.3
42.0 ± 4.2" 12.0 ± 3.4* 23.3 ± 5.1C 21.1 ± 4.5C 46.1 ± 3.4"
Serum corticosterone concentration was measured 1 h after the injection of quipazine maleate (10 mg/kg, ip). Metergoline (1 or 3 mg/ kg, ip) was injected at 1, 16, 24, or 48 h before quipazine. " P< 0.001 vs. control. *NS. ' P < 0.025 vs. control.
R} = C H 3 C H -
- 50
1*1 .
CH 3 O
.003.01.03.1 .3 Mitiriilin list
II
R 2 =COCHCHCH 3 CH3OH Metergoline
rii
LY53857
FIG. 1. Structural relationship between metergoline and LY53857.
Results Antagonism of quipazine by metergoline Table 1 shows the ability of metergoline pretreatment to prevent the elevation of serum corticosterone by quipazine and the duration of metergoline's effect. The 3 mg/kg dose of metergoline prevented the elevation of serum corticosterone by quipazine when given 1 h before quipazine, thus confirming our earlier findings; this dose also completely prevented the corticosterone increase when given 16 or 24 h before quipazine. A lower dose (1 mg/kg) was also effective after 1 h of pretreatment and partially antagonized the quipazine effect at 16 and 24 h. By 48 h after metergoline pretreatment, quipazine caused a normal elevation of serum corticosterone. The effect of various doses of metergoline given 1 h before quipazine is shown in Fig. 2. A dose of 0.3 mg/kg completely prevented the effect of quipazine. Lower doses caused less or no inhibition. Plotting the percentage of antagonism of the effect of quipazine vs. the dose of metergoline (Fig. 2, top right inset) indicated an ED50
0 0
0 .003 .01
. 0 3 .1 .3 Metergoline dose «
0 10 10 10 10 10 10 Quipazine dose
FIG. 2. Dose dependence of the antagonism by metergoline of serum corticosterone elevation by quipazine. Quipazine (10 mg/kg) was injected ip 1 h before the rats were killed and 1 h after metergoline was injected ip at various doses. *, Groups that differed significantly (P < 0.05) from the vehicle-treated control group (left). Groups treated with 0.1 and 0.3 mg/kg doses of metergoline in addition to quipazine differed significantly (P < 0.05) from the group receiving quipazine alone. Inset (upper right), Percentage antagonism plotted against metergoline dose on a logarithmic scale; from this graph, the ED«> value for metergoline was estimated to be approximately 0.03 mg/kg.
value for metergoline of approximately 0.03 mg/kg in antagonizing serum corticosterone elevation by quipazine. Metergoline did not alter brain levels of quipazine under conditions in which the elevation of corticosterone had been completely suppressed. One hour after injection of quipazine maleate (10 mg/kg, ip), quipazine concentrations in whole brains were 9.18 ± 3.46 jiig/g in control rats and 10.14 ± 2.23 jtig/g in rats pretreated with metergoline (1 mg/kg, ip) 1 h before quipazine.
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SEROTONIN AND SERUM CORTICOSTERONE
Effect of metergoline on corticosterone elevation by other agents To obtain evidence that this ability of metergoline to prevent serum corticosterone elevation was not nonspecific, we determined its effect on serum corticosterone elevation by other agents not acting via serotoninergic mechanisms (Table 2). The 3 mg/kg dose of metergoline antagonized the elevation of serum corticosterone by quipazine in this experiment but had no effect on the elevation of serum corticosterone by theophylline or ketamine. The effect of metergoline on agents other than quipazine that are thought to act via serotoninergic mechanisms is shown in Table 3. Metergoline completely prevented the elevation of serum corticosterone by quipazine in this experiment but did not significantly alter the elevation of serum corticosterone by iV,N-dimethyl-5TABLE 2. Effect of metergoline on corticosterone elevation by quipazine and by agents acting through nonserotoninergic mechanisms Serum corticosterone (fig/100 ml)
Treatment
Saline Quipazine Theophylline Ketamine
Vehicle-pretreated 11.2 ± 0.9 53.7 ± 4.0 46.0 ± 3.0
49.5 ± 8.2
Metergolinepretreated 14.4 ± 2.0 24.2 ± 5.4 49.2 ± 5.7 59.0 ±1.6
Effect of metergoline (P value)
NS NS
TABLE 3. Effect of metergoline on corticosterone elevation by various agents thought to act via serotoninergic mechanisms
Saline Quipazine Fluoxetine Fenfluramine L-5-Hydroxytryptophan iV,iV-Dimethyl-5-methoxytryptamine l-(/n-Trifluoromethylphenyl) piperazine
TABLE 4. Effect of other serotonin antagonists on serum corticosterone elevation by quipazine
Antagonist None LY53857 Cyproheptadine Spiperone Methysergide
Serum corticosterone (fig/100 ml) Control
Quipazine
4.6 ± 5.7 ± 5.2 ± 4.8 ± 4.0 ±
43.1 ± 2.3 8.3 ± 2.0" 21.4 ± 7.4" 21.3 ± 6.3" 46.8 ± 2.2
0.5 1.1 1.1 0.5 1.0
Effect of quipazine (P value)
Vol. 105, No. 4
Printed in U.S.A.
The Effects of Metergoline and Other Serotonin Receptor Antagonists on Serum Corticosterone in Rats RAY W. FULLER* AND HAROLD D. SNODDY The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206
heptadine and spiperone, which gave significant but incomplete antagonism at 1 mg/kg. Methysergide at 3 mg/kg did not alter the effect of quipazine. Metergoline did not antagonize the elevation of serum corticosterone by other agents throught to act via serotoninergic mechanisms, namely fluoxetine, fenfluramine, L-5-hydroxytryptophan, N,iV-dimethyl-5-methoxytryptamine, and l-(m-trifluoromethylphenyl)piperazine. Thus, the interactions between metergoline and quipazine may have occurred at receptors that are not serotonin receptors or that represent a subset of serotonin receptors not mediating the actions of serotoninergic agents other than quipazine. (Endocrinology 105: 923, 1979)
ABSTRACT. Metergoline antagonized the elevation of serum corticosterone by quipazine in rats. The ED50 of metergoline was less than 0.1 mg/kg, ip, and the effects of a 3 mg/kg dose persisted for more than 24 h. Metergoline did not antagonize the elevation of serum corticosterone by theophylline or ketamine {i.e. did not prevent corticosterone release nonspecifically) and did not affect the concentration of quipazineMn the brain. Since quipazine is a serotonin receptor agonist, the antagonistic effects of metergoline may have been due to competition with quipazine at serotonin receptor sites in the brain. Some other agents capable of blocking serotonin receptors also antagonized the elevation of serum corticosterone by quipazine. These included LY53857, which gave complete blockade at 3 mg/kg, and cypro-
R
groups of five in hanging wire cages in an air-conditioned (24 C) and light-controlled room for at least 1 week after delivery. Lights were on from 0700-1900 h daily. Food and water were available ad libitum. Drugs received as gifts from pharmaceutical companies were metergoline (Farmitalia, Milan, Italy), cyproheptadine (Merck, Rahway, NJ), methysergide (Sandoz, Hanover, NJ), spiperone (Janssen, Beerse, Belgium), fenfluramine hydrochloride (A. H. Robins, Richmond, VA), and ketamine hydrochloride (Bristol, Syracuse, NY). Purchased drugs were L-5-hydroxytryptophan (Sigma, St. Louis, MO), N,iV-dimethyl-5-methoxytryptamine (Aldrich Chemical Co., Milwaukee, WI), and l-(ra-trifluoromethylphenyl)piperazine (Aldrich). The remaining drugs were synthesized at Eli Lilly and Co. (Indianapolis, IN; quipazine maleate, theophylline, fluoxetine hydrochloride, and LY53857). LY53857 is 4-isopropyl-7-methyl-9-(2-hydroxy-l-ethylpropoxycarbonyl)4,6,6A,7,8,9,10,10A-octahydroindolo[4,3-FG)quinoline maleate. All drugs were administered ip at an injection volume of 1 ml/kg in aqueous solution (except metergoline and theophylline, which were given in acacia suspension). Pyrogen-free glass-distilled water was used in preparing the solutions or suspensions. Control rats received the same volume of the injection vehicle. Rats were killed by decapitation, and trunk blood was collected and allowed to clot. Serum was obtained by centrifugation and assayed immediately or stored frozen at -15 C before assay. Corticosterone concentration was assayed spectrofluorometrically by the method of Solem and Brinck-Johnsen (9). Statistical analysis of the data by Student's t test was done on an Olivetti desktop computer. Unless otherwise indicated, all data are expressed as the mean ± SE for five rats per group.
ECENTLY, we (1) reported that quipazine, a serotonin receptor agonist, increased serum corticosterone concentration in rats. This observation extended earlier findings that serotonin precursors (2, 3) and serotonin uptake inhibition (4) increased serum corticosterone and thus added to the evidence that serotonin neurons in the brain have a positive role (5-7) in the control of corticotropin-releasing factor from the hypothalamus. The fact that the effect of quipazine was completely prevented by pretreatment with metergoline, a known serotonin receptor antagonist (8), was taken as evidence that quipazine was acting via serotonin receptors to elevate serum corticosterone (1). We have now studied the effects of metergoline more thoroughly and report here that metergoline is a potent and long lasting antagonist of quipazine-induced elevation of serum corticosterone but does not antagonize the elevation of serum corticosterone by agents acting through nonserotoninergic mechanisms or by agents other than quipazine thought to act by serotoninergic mechanisms. Effects of other serotonin receptor antagonists, especially LY53857, are also reported. The structural relationships between LY53857 and metergoline are shown in Fig. 1. Materials and Methods Male Wistar rats, weighing about 150 g, were obtained from Harlan Industries (Cumberland, IN). They were housed in Received January 15, 1979. * To whom requests for reprints should be addressed. 923
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 23:24 For personal use only. No other uses without permission. . All rights reserved.
FULLER AND SNODDY
924
Endo • 1979 Vol 105 • No 4
TABLE 1. Duration of effect of metergoline at two dose levels in antagonizing serum corticosterone elevation by quipazine Metergoline pretreatment interval
I-CH3
Metergoline at 3 mg/kg No metergoline lh
16 h 24 h Metergoline at 1 mg/kg No metergoline lh
16 h 24 h 48 h
Serum corticosterone (/ig/100 ml) Control
Quipazine
6.3 ± 9.0 ± 6.5 ± 5.3 ±
0.4 2.2 0.5 0.3
43.5 ± 4.6" 12.5 ± 3.5* 7.0 ± 0.3* 8.8 ± 1.8*
6.5 ± 6.5 ± 7.7 ± 6.5 ± 8.4 ±
0.6 0.3 1.7 0.6 1.3
42.0 ± 4.2" 12.0 ± 3.4* 23.3 ± 5.1C 21.1 ± 4.5C 46.1 ± 3.4"
Serum corticosterone concentration was measured 1 h after the injection of quipazine maleate (10 mg/kg, ip). Metergoline (1 or 3 mg/ kg, ip) was injected at 1, 16, 24, or 48 h before quipazine. " P< 0.001 vs. control. *NS. ' P < 0.025 vs. control.
R} = C H 3 C H -
- 50
1*1 .
CH 3 O
.003.01.03.1 .3 Mitiriilin list
II
R 2 =COCHCHCH 3 CH3OH Metergoline
rii
LY53857
FIG. 1. Structural relationship between metergoline and LY53857.
Results Antagonism of quipazine by metergoline Table 1 shows the ability of metergoline pretreatment to prevent the elevation of serum corticosterone by quipazine and the duration of metergoline's effect. The 3 mg/kg dose of metergoline prevented the elevation of serum corticosterone by quipazine when given 1 h before quipazine, thus confirming our earlier findings; this dose also completely prevented the corticosterone increase when given 16 or 24 h before quipazine. A lower dose (1 mg/kg) was also effective after 1 h of pretreatment and partially antagonized the quipazine effect at 16 and 24 h. By 48 h after metergoline pretreatment, quipazine caused a normal elevation of serum corticosterone. The effect of various doses of metergoline given 1 h before quipazine is shown in Fig. 2. A dose of 0.3 mg/kg completely prevented the effect of quipazine. Lower doses caused less or no inhibition. Plotting the percentage of antagonism of the effect of quipazine vs. the dose of metergoline (Fig. 2, top right inset) indicated an ED50
0 0
0 .003 .01
. 0 3 .1 .3 Metergoline dose «
0 10 10 10 10 10 10 Quipazine dose
FIG. 2. Dose dependence of the antagonism by metergoline of serum corticosterone elevation by quipazine. Quipazine (10 mg/kg) was injected ip 1 h before the rats were killed and 1 h after metergoline was injected ip at various doses. *, Groups that differed significantly (P < 0.05) from the vehicle-treated control group (left). Groups treated with 0.1 and 0.3 mg/kg doses of metergoline in addition to quipazine differed significantly (P < 0.05) from the group receiving quipazine alone. Inset (upper right), Percentage antagonism plotted against metergoline dose on a logarithmic scale; from this graph, the ED«> value for metergoline was estimated to be approximately 0.03 mg/kg.
value for metergoline of approximately 0.03 mg/kg in antagonizing serum corticosterone elevation by quipazine. Metergoline did not alter brain levels of quipazine under conditions in which the elevation of corticosterone had been completely suppressed. One hour after injection of quipazine maleate (10 mg/kg, ip), quipazine concentrations in whole brains were 9.18 ± 3.46 jiig/g in control rats and 10.14 ± 2.23 jtig/g in rats pretreated with metergoline (1 mg/kg, ip) 1 h before quipazine.
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SEROTONIN AND SERUM CORTICOSTERONE
Effect of metergoline on corticosterone elevation by other agents To obtain evidence that this ability of metergoline to prevent serum corticosterone elevation was not nonspecific, we determined its effect on serum corticosterone elevation by other agents not acting via serotoninergic mechanisms (Table 2). The 3 mg/kg dose of metergoline antagonized the elevation of serum corticosterone by quipazine in this experiment but had no effect on the elevation of serum corticosterone by theophylline or ketamine. The effect of metergoline on agents other than quipazine that are thought to act via serotoninergic mechanisms is shown in Table 3. Metergoline completely prevented the elevation of serum corticosterone by quipazine in this experiment but did not significantly alter the elevation of serum corticosterone by iV,N-dimethyl-5TABLE 2. Effect of metergoline on corticosterone elevation by quipazine and by agents acting through nonserotoninergic mechanisms Serum corticosterone (fig/100 ml)
Treatment
Saline Quipazine Theophylline Ketamine
Vehicle-pretreated 11.2 ± 0.9 53.7 ± 4.0 46.0 ± 3.0
49.5 ± 8.2
Metergolinepretreated 14.4 ± 2.0 24.2 ± 5.4 49.2 ± 5.7 59.0 ±1.6
Effect of metergoline (P value)
NS NS
TABLE 3. Effect of metergoline on corticosterone elevation by various agents thought to act via serotoninergic mechanisms
Saline Quipazine Fluoxetine Fenfluramine L-5-Hydroxytryptophan iV,iV-Dimethyl-5-methoxytryptamine l-(/n-Trifluoromethylphenyl) piperazine
TABLE 4. Effect of other serotonin antagonists on serum corticosterone elevation by quipazine
Antagonist None LY53857 Cyproheptadine Spiperone Methysergide
Serum corticosterone (fig/100 ml) Control
Quipazine
4.6 ± 5.7 ± 5.2 ± 4.8 ± 4.0 ±
43.1 ± 2.3 8.3 ± 2.0" 21.4 ± 7.4" 21.3 ± 6.3" 46.8 ± 2.2
0.5 1.1 1.1 0.5 1.0
Effect of quipazine (P value)
