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CCK-33 antagonizes apomorphine-induced growth hormone secretion and increases basal prolactin levels in man
Neuropeptides
4: 281-291, 1984
CCK-33 ANTAGONIZES APOHORPHINE-INDUCED GROWTH HORMONE INCREASES BASAL PROLACTIN LEVELS IN MAN.
SECRETION AND
N.P.V. Nair', S. Lall", J.X. Thavundayil', P.L. Wood', P. Etienne', and H. Guyda3. 'Douglas Hospital Research Center, 6875 Lasalle Boulevard, Verdun, Quebec, Canada H4H lR3, 'Department of Psychiatry, Montreal General Hospital, Montreal, P.Q., Canada, 3Protein and Polypeptide Laboratory, Montreal, P.Q., Canada (reprint requests to NN). ABSTRACT Cholecystokinin (CCK-33) (225 Ivy Dog Units intravenously) had no effect on basal growth hopone (GH) secretion but antagonized the GH response to the dopamine receptor agonist, apomorphine HCl (0.5 mg SC) (N = 7), and induced a transient increase in basal prolactin (PRL) secretion (N = 8) in normal men. These findings are similar to those described with neuroleptics and are compatible with an inhibitory effect of CCK-33, or fragments, on dopamine function in man, at least in the hypothalamic-pituitary axis. However, an inhibitory effect of CCK-33 on the release of GH and a stress-induced increase in PRL secretion cannot be excluded. INTRODUCTION Cholecystokinin (CCK-33) and related peptides have been identified in animal and human brain (l-5) and shown to bind to distinct receptors (6,7). The function of these peptides in the central nervous system is unclear. The observation that CCK-like immunoreactivity may be contained within dopamine (DA) neurons suggests that CCK-33 or fragments may modulate DA function (2, 8-10). Whereas some evidence for such an effect has been found in animal studies (11-13) whether CCK-peptides affect DA function in man is unknown. Alterations in DA function are believed to play a role in the pathophysiology of schizophrenia (14). Neuroleptics block DA receptors and it is this property which is believed to subserve their antischizophrenic activity (14, 15) and their ability to increase basal prolactin (PRL) secretion (16) and antagonize the growth hormone (GH) response to the DA receptor agonist, apomorphine (APO) (17, 18). Recently there have been reports that CCK-33 (19) and ceruletide (ZO), a decapeptide chemically related to CCK-8, exert a therapeutic effect in schizophrenic patients on neuroleptic treatment. This may indicate an inhibitory effect of these peptides on DA function in man. In previous work we found that CCK-33 281
had no effect on PRL or APO-induced GH secretion but this lack of effect may have been due to use of an insufficient dose of the peptide (21). Accordingly, in the present study we have investigated the effect of a much larger dose of CCK-33 on DA function by studying the effect of this peptide on basal PRL secretion and APO-induced GH secretion in normal subjects. SUBJECTS AND METHODS Eight physically healthy, non-obese male volunteers without psychiatric history, aged 21-38 years, on no medication and who gave their informed consent served as subjects. Each subject was investigated on four separate occasions, namely, with CCK-33 (Kabi Diagnostica, Sweden; 225 Ivy Dog Units in 10 ml physiological saline intravenously) plus Apo HCl (0.5 mg subcutaneously), CCK-33 plus Apo placebo (physiological saline), CCK-33 placebo (10 ml physiological saline) plus Apo, and CCK-33 placebo plus Apo placebo. The interval between tests was at least 48 hours. The dose of CCK-33 used in the present study is about three times the dose used in radiological investigation of gallbladder contraction. In previous work one Ivy Dog Unit/kg CCK-33 (approximately 75 Ivy Dog Units per subject) was insufficient to affect APO-induced GH secretion or basal PRL secretion (21). Accordingly, in the present study it was decided to use 225 Ivy Dog Units of CCK-33. Following intravenous CCK-33 gallbladder contraction occurs within l-3 minutes and lasts over 2 hours (Kabi Diagnostica, Sweden). The dose of Apo HCl was chosen on the basis of previous studies which show that 0.5 mg increases GH secretion (22-25). This dose is insufficient to decrease basal PRL levels (22-25). To decrease basal PRL much larger doses of Apo are required (26) which cause distressing side effects and so were not used in the present study. After an overnight fast at 08:OO hours a 19-gauge scalp vein needle was inserted into an arm vein and kept open with heparin-saline. Samples of blood (3 ml) were drawn at -30, -15, 0, 15, 30, 45, 60, 75, 90 and 120 minutes. CCK-33 (or placebo) was injected over a 'lo-minute period commencing at -10 minutes. Apo (or placebo) was administered at time 0 minutes. Samples were centrifuged and serum stored at -2O'C until assayed by radioimmunoassay for GH (27) and PRL (28). To avoid inter-assay variability, samples were run in one assay. Data were analyzed by analysis of variance, repeated measures model, followed by tests for simple effects where appropriate. The mean individual peak GH concentrations were analyzed by the paired 't' test. Data are presented as the mean f standard error of the mean. RESULTS Following CCK-33, on both occasions each of the 8 subjects experienced abdominal cramps and nausea within 2-5 minutes of commencing the injection; the symptoms lasted 5-15 minutes. After Apo alone 7 of the subjects experienced drowsiness and yawning, 6 transient nausea occurring lo-15 minutes after Apo injection and lasting 5 minutes or less and 6 of the When CCK-33 was subjects experienced spontaneous penile erections. administered prior to Apo none of the subjects experienced nausea following the Apo injection. Post-apomorphine drowsiness, yawning and erections occurred in 4, 3 and 1 subject, respectively, following CCK-33 pretreatment. None of the subjects experienced side effects after placebo alone.
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Apo increased GH secretion compared with placebo (interaction: One of the 8 subjects, however, showed no F(6, 84) = 12.93; p < 0.001). increase in GH after Apo alone (peak 2.5 mg/ml) or after CCK-33 plus Apo (peak 1.7 ng/ml) and was excluded from analysis of data evaluating the effect of CCK-33 on APO-induced GH secretion. In the remaining 7 subjects Apo alone increased GH from a mean baseline of 2.3 ? 0.6 ng/ml to a mean individual peak of 18.3 + 2.3 ng/ml (range: 12.5 - 27 ng/ml). CCK-33 significantly inhibited the GH response to Apo (interaction: F(6, 72) = 3.84; p < 0.01) (Fig. 1). The APO-induced GH concentrations were significantly inhibited at 45 (p < 0.001) and 60 mins (p < 0.005) (Fig. 1)
-30
0
30
60
90
120
Time (min)
Fig. 1.
Effect of CCK-33 on apomorphine-induced growth hormone secretion. CCK-33 (225 Ivy Dog Units) (or placebo) was administered to 7 men intravenously over a lo-minute period commencing at -10 minutes and apomorphine hydrochloride (APO) (0.5 mg subcutaneously) (or placebo) at 0 minutes. I----m = CCK-33 plus placebo; O----O = CCK-33 plus Apo; O---C = placebo plus Apo; IJ--0 = placebo plus placebo. "p < 0.005, **p < 0.001 compared with CCK-33 plus Apo.
The mean individual peak GH concentrations after CCK-33 plus Apo (8.9 ? 2.6 ng/ml, range: 2.1 - 20 ng/ml) was significantly decreased compared with Apo alone (18.3 ? 2.3 ng/ml) (p < 0.02). CCK-33 alone or placebo alone had no significant effect on basal GH levels (F (9, 63) < 1 in both cases). After CCK-33 alone one of the 8 subjects showed an increase in GH concentration from a baseline of
283
1.0 ng/ml to a peak of 24 ng/ml. In the remaining 7 subjects the peak concentrations after CCK-33 varied from 1.0 - 2.4 ng/ml. There was no significant difference in GH concentration between CCK-33 alone and placebo alone (interaction: F (7, 98) = 1.12; p = NS). The mean individual peak after CCK-33 alone was 4.6 i 2.8 ng/ml and after placebo alone 2.6 + 0.4 ng/ml (range: 1.3 - 4.5 ng/ml) (p z NS). CCK-33 alone significantly increased PRL concentrations compared with placebo alone (interaction: F (7, 98) = 4.23; p < 0.01) (Fig. 2); PRL
18 -
l
*
WI14-
6I -30
Fig. 2.
I 60
30
I 90
1 120
Time (min)
Effect of CCK-33 on basal prolactin secretion. CCK-33 (225 Ivy Dog Units) (or placebo) was administered to 8 men intravenously over a lo-minute period commencing at -10 minutes and placebo (physiological saline) administered subcutaneously at time 0 minutes. a----m = CCK-33 plus placebo; a---- u= placebo plus placebo. *p < 0.05; **p < 0.001.
concentrations were significantly higher at the 15 minute (p < 0.001) and 30 minute (p < 0.05) periods (Fig. 2). The mean individual peak PRL concentration was significantly higher after CCK-33 alone (15.0 + 2.7 ng/ ml) compared with placebo alone (9.8 + 0.8 ng/ml) (p < 0.05). There was no significant difference between Apo alone and placebo alone on PRL concentration (interaction: F (6, 84) = 1.95; p = NS). PRL was significantly increased after CCK-33 plus Apo compared with placebo alone (interaction: F (6, 84) = 18.67; p < 0.001) (Fig. 3); significantly higher values were noted at the 15 (p < 0.001) and 30 (p < 0.005) minute time periods. Also, prolactin concentrations were significantly higher after CCK-33 plus Apo compared with Apo alone (interaction: F (6, 84) = 14.35; p < 0.001); significantly higher values were present at the 15 (p < O.OOl), 30 (p < 0,001) and 45 min (p < 0.005) periods. The mean
284
I 0
I 30
I
60
I 90
I
120
Time (min) Fig. 3.
Effect of CCK-33 plus apomorphine on basal prolactin secretion. CCK-33 (225 Ivy Dog Units) (or placebo) was administered to 8 men intravenously and apomorphine HCl (APO) (0.5 mg subO----O = CCK-33 cutaneously) (or placebo) at time 0 minutes. Cl = placebo plus plus APO;.H = placebo plus Apo; Uplacebo. :p < 0.005, ?tp < 0.001 compared with placebo plus Apo. "p < 0.005, **p < 0.001 compared with placebo plus placebo.
individual peak after CCK-33 plus Apo (16.6 ? 2.3 ng/ml) was significantly higher than after placebo alone (9.8 + 0.8 ng/ml) (p < 0.01) and after Apo alone (7.9 + 0.7 ng/ml> (p < 0.01). DISCUSSION Apo, a DA receptor agonist in animals (29) and man (30), stimulates GH secretion in humans (31) by an effect on postsynaptic DA receptors (32) within the hypothalamic-pituitary axis which lie outside the blood brain barrier and which are not linked to adenylate cyclase (24,25). PRL secretion is modulated by an inhibitory DA mechanism in man (33). These observations have led to the clinical evaluation of the effect of psychotropic agents on DA function by investigating the effect of various drugs on APO-induced GH secretion and on basal PRL secretion (34-36). Neuroleptics, including the atypical major tranquillizer, clozapine, antagonize the GH response to Apo (26, 37-39). Most neuroleptics also increase basal PRL secretion (16, 18). In the present study, CCK-33 antagonized Apoinduced GH secretion and caused a modest though transient elevation in basal PRL secretion. These observations are compatible with an inhibitory effect of CCK-33 on postsynaptic DA function in man, at least in the 285
hypothalamic-pituitary axis. The relatively small increase in PRL in contrast to a more pronounced antagonism of APO-induced GH secretion is similar to that observed with the atypical neuroleptic, clozapine (18, 38). In previous work (21) we used only about one-third of the dose used in the present study; this probably accounts for the lack of effect noted in our earlier investigation with CCK-33. CCK-33 and CCK-8 have been identified in human brain (2, 5) and receptors for these peptides have been identified in the hypothalamus (6, 7) so that whether the presently observed hormonal effects are due to CCK-33 or mediated by CCK-8 or other fragments cleaved from the parent molecular is unclear. In this regard it is known that CCK-33 is cleaved by peptidases in blood (40, 41) and CCK-converting enzymes in brain (42). Approximately, 32% of CCK-33 is destroyed (in rat plasma) after 45 minutes (40) and the half life of CCK-8 in human plasma is about 50 minutes (40). In contrast to our findings in man in whom CCK-33 had no effect on basal GH secretion, in animal studies CCK peptides appear to exert a stimulatory effect. Thus, in incubated rat anterior pituitary CCK-8 and CCK-39 stimulate GH secretion (43). CCK-8 also induces GH secretion from cultured GH3 pituitary cells and reverses the inhibitory effects of somatostatin on GH secretion (43). Vijayan et al. (44) found that CCK-8 only induced GH secretion after intraventricular administration but not after intravenous use or in incubated rat pituitary. In keeping with our present findings, some studies in animals also point to a stimulatory effect of CCK peptides on PRL secretion. Vijayan et al. (44) found that CCK-8 increased PRL secretion after intraventricular or intravenous administration; no effect was observed in the pituitary in vitro. Malarkey et al. (45) noted an increase in PRL secretion in rat pituitary monolayer cultures following CCK-8 or CCK-33 but no effect of CCK-8 on human monolayer cultures obtained from a single normal pituitary and a single prolactinoma. Though our data support an inhibitory effect of CCK-33 on DA function Thus, CCK-33 may antagonize Apoalternative explanations are possible. induced GH secretion by inhibiting the release of GH either by a direct effect on the pituitary somatotrophe or by inhibiting the release of Against this view is the failure of growth hormone releasing hormone. CCK-33 to decrease basal GH levels; in fact in one subject a brisk increase in GH followed CCK-33 administration. Also, in animal studies a stimulatory or absent effect of CCK-peptides on GH secretion have been However, noted. All subjects experienced abdominal cramps and nausea. stress cannot explain the inhibitory effect of CCK-33 on APO-induced GH secretion as stress is believed to increase GH secretion in man (46). Stress may increase PRL secretion in humans (47) so that the transient elevation of basal PRL concentrations by CCK-33 may be a nonspecific stress response rather than the manifestation of an antagonistic effect Incidental observations during the on DA inhibition of PRL secretion. present study reveal that CCK-33 antagonized other effects of Apo that are believed to be mediated by DA mechanisms, namely, drowsiness (48, 49), penile erections (37, 50) and nausea (29). These findings favour an inhibitory effect of CCK-33 on DA function in man. Further, several
286
studies in animals also support an inhibitory effect of CCK peptides on DA function. Thus, CCK peptides decrease the number of DA binding sites in certain brain regions (51, 52), suppress the behavioural effects of L-dopa (11) and methamphetamine (12), antagonize Apo (13) and methylphenidate-induced stereotyped behaviour (53) and inhibit tail pinchinduced eating (54). CCK-8 antagonizes conditioned-avoidance behaviour in the rat (13) and CCK-8 and ceruletide induce catalepsy in mice (53, 55). These behavioural observations in animals and our present endocrine findings are characteristic of neuroleptics. Kowever, some studies have questioned whether CCK-8 interacts with DA function (56). Because of stress effects noted in the present study it is possible that the effect of CCK-33 on Apo may be mediated by an indirect action on DA function. In summary, CCK-33 antagonized the GH response to Apo and increased basal PRL secretion. These findings are compatible with an inhibitory effect of CCK-33 or fragments on DA function in man though other interpretations are possible. Studies on the effect of CCK-33 on other stimuli of GH secretion may help to determine whether this peptide antagonizes DA function or whether it inhibits the final common pathway involved in GH release. ACKNOWLEDGEMENTS We thank J. Parodo for technical assistance, Andrg Achim, Ph.D. and George Schwartz for statistical analysis of data, Yvonne Hall, R.N. and staff of the research ward for clinical assistance and Nadia Zajac for secretarial assistance. We also thank the NIAMDD, NIH (USA) for the supply of growth hormone radioimmunoassay reagents. This work was supported in part by the Medical Research Council (Canada). REFERENCES 1.
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Accepted
1913184
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and antistereotypic Neuropharmacology 20,
4: 281-291, 1984
CCK-33 ANTAGONIZES APOHORPHINE-INDUCED GROWTH HORMONE INCREASES BASAL PROLACTIN LEVELS IN MAN.
SECRETION AND
N.P.V. Nair', S. Lall", J.X. Thavundayil', P.L. Wood', P. Etienne', and H. Guyda3. 'Douglas Hospital Research Center, 6875 Lasalle Boulevard, Verdun, Quebec, Canada H4H lR3, 'Department of Psychiatry, Montreal General Hospital, Montreal, P.Q., Canada, 3Protein and Polypeptide Laboratory, Montreal, P.Q., Canada (reprint requests to NN). ABSTRACT Cholecystokinin (CCK-33) (225 Ivy Dog Units intravenously) had no effect on basal growth hopone (GH) secretion but antagonized the GH response to the dopamine receptor agonist, apomorphine HCl (0.5 mg SC) (N = 7), and induced a transient increase in basal prolactin (PRL) secretion (N = 8) in normal men. These findings are similar to those described with neuroleptics and are compatible with an inhibitory effect of CCK-33, or fragments, on dopamine function in man, at least in the hypothalamic-pituitary axis. However, an inhibitory effect of CCK-33 on the release of GH and a stress-induced increase in PRL secretion cannot be excluded. INTRODUCTION Cholecystokinin (CCK-33) and related peptides have been identified in animal and human brain (l-5) and shown to bind to distinct receptors (6,7). The function of these peptides in the central nervous system is unclear. The observation that CCK-like immunoreactivity may be contained within dopamine (DA) neurons suggests that CCK-33 or fragments may modulate DA function (2, 8-10). Whereas some evidence for such an effect has been found in animal studies (11-13) whether CCK-peptides affect DA function in man is unknown. Alterations in DA function are believed to play a role in the pathophysiology of schizophrenia (14). Neuroleptics block DA receptors and it is this property which is believed to subserve their antischizophrenic activity (14, 15) and their ability to increase basal prolactin (PRL) secretion (16) and antagonize the growth hormone (GH) response to the DA receptor agonist, apomorphine (APO) (17, 18). Recently there have been reports that CCK-33 (19) and ceruletide (ZO), a decapeptide chemically related to CCK-8, exert a therapeutic effect in schizophrenic patients on neuroleptic treatment. This may indicate an inhibitory effect of these peptides on DA function in man. In previous work we found that CCK-33 281
had no effect on PRL or APO-induced GH secretion but this lack of effect may have been due to use of an insufficient dose of the peptide (21). Accordingly, in the present study we have investigated the effect of a much larger dose of CCK-33 on DA function by studying the effect of this peptide on basal PRL secretion and APO-induced GH secretion in normal subjects. SUBJECTS AND METHODS Eight physically healthy, non-obese male volunteers without psychiatric history, aged 21-38 years, on no medication and who gave their informed consent served as subjects. Each subject was investigated on four separate occasions, namely, with CCK-33 (Kabi Diagnostica, Sweden; 225 Ivy Dog Units in 10 ml physiological saline intravenously) plus Apo HCl (0.5 mg subcutaneously), CCK-33 plus Apo placebo (physiological saline), CCK-33 placebo (10 ml physiological saline) plus Apo, and CCK-33 placebo plus Apo placebo. The interval between tests was at least 48 hours. The dose of CCK-33 used in the present study is about three times the dose used in radiological investigation of gallbladder contraction. In previous work one Ivy Dog Unit/kg CCK-33 (approximately 75 Ivy Dog Units per subject) was insufficient to affect APO-induced GH secretion or basal PRL secretion (21). Accordingly, in the present study it was decided to use 225 Ivy Dog Units of CCK-33. Following intravenous CCK-33 gallbladder contraction occurs within l-3 minutes and lasts over 2 hours (Kabi Diagnostica, Sweden). The dose of Apo HCl was chosen on the basis of previous studies which show that 0.5 mg increases GH secretion (22-25). This dose is insufficient to decrease basal PRL levels (22-25). To decrease basal PRL much larger doses of Apo are required (26) which cause distressing side effects and so were not used in the present study. After an overnight fast at 08:OO hours a 19-gauge scalp vein needle was inserted into an arm vein and kept open with heparin-saline. Samples of blood (3 ml) were drawn at -30, -15, 0, 15, 30, 45, 60, 75, 90 and 120 minutes. CCK-33 (or placebo) was injected over a 'lo-minute period commencing at -10 minutes. Apo (or placebo) was administered at time 0 minutes. Samples were centrifuged and serum stored at -2O'C until assayed by radioimmunoassay for GH (27) and PRL (28). To avoid inter-assay variability, samples were run in one assay. Data were analyzed by analysis of variance, repeated measures model, followed by tests for simple effects where appropriate. The mean individual peak GH concentrations were analyzed by the paired 't' test. Data are presented as the mean f standard error of the mean. RESULTS Following CCK-33, on both occasions each of the 8 subjects experienced abdominal cramps and nausea within 2-5 minutes of commencing the injection; the symptoms lasted 5-15 minutes. After Apo alone 7 of the subjects experienced drowsiness and yawning, 6 transient nausea occurring lo-15 minutes after Apo injection and lasting 5 minutes or less and 6 of the When CCK-33 was subjects experienced spontaneous penile erections. administered prior to Apo none of the subjects experienced nausea following the Apo injection. Post-apomorphine drowsiness, yawning and erections occurred in 4, 3 and 1 subject, respectively, following CCK-33 pretreatment. None of the subjects experienced side effects after placebo alone.
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Apo increased GH secretion compared with placebo (interaction: One of the 8 subjects, however, showed no F(6, 84) = 12.93; p < 0.001). increase in GH after Apo alone (peak 2.5 mg/ml) or after CCK-33 plus Apo (peak 1.7 ng/ml) and was excluded from analysis of data evaluating the effect of CCK-33 on APO-induced GH secretion. In the remaining 7 subjects Apo alone increased GH from a mean baseline of 2.3 ? 0.6 ng/ml to a mean individual peak of 18.3 + 2.3 ng/ml (range: 12.5 - 27 ng/ml). CCK-33 significantly inhibited the GH response to Apo (interaction: F(6, 72) = 3.84; p < 0.01) (Fig. 1). The APO-induced GH concentrations were significantly inhibited at 45 (p < 0.001) and 60 mins (p < 0.005) (Fig. 1)
-30
0
30
60
90
120
Time (min)
Fig. 1.
Effect of CCK-33 on apomorphine-induced growth hormone secretion. CCK-33 (225 Ivy Dog Units) (or placebo) was administered to 7 men intravenously over a lo-minute period commencing at -10 minutes and apomorphine hydrochloride (APO) (0.5 mg subcutaneously) (or placebo) at 0 minutes. I----m = CCK-33 plus placebo; O----O = CCK-33 plus Apo; O---C = placebo plus Apo; IJ--0 = placebo plus placebo. "p < 0.005, **p < 0.001 compared with CCK-33 plus Apo.
The mean individual peak GH concentrations after CCK-33 plus Apo (8.9 ? 2.6 ng/ml, range: 2.1 - 20 ng/ml) was significantly decreased compared with Apo alone (18.3 ? 2.3 ng/ml) (p < 0.02). CCK-33 alone or placebo alone had no significant effect on basal GH levels (F (9, 63) < 1 in both cases). After CCK-33 alone one of the 8 subjects showed an increase in GH concentration from a baseline of
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1.0 ng/ml to a peak of 24 ng/ml. In the remaining 7 subjects the peak concentrations after CCK-33 varied from 1.0 - 2.4 ng/ml. There was no significant difference in GH concentration between CCK-33 alone and placebo alone (interaction: F (7, 98) = 1.12; p = NS). The mean individual peak after CCK-33 alone was 4.6 i 2.8 ng/ml and after placebo alone 2.6 + 0.4 ng/ml (range: 1.3 - 4.5 ng/ml) (p z NS). CCK-33 alone significantly increased PRL concentrations compared with placebo alone (interaction: F (7, 98) = 4.23; p < 0.01) (Fig. 2); PRL
18 -
l
*
WI14-
6I -30
Fig. 2.
I 60
30
I 90
1 120
Time (min)
Effect of CCK-33 on basal prolactin secretion. CCK-33 (225 Ivy Dog Units) (or placebo) was administered to 8 men intravenously over a lo-minute period commencing at -10 minutes and placebo (physiological saline) administered subcutaneously at time 0 minutes. a----m = CCK-33 plus placebo; a---- u= placebo plus placebo. *p < 0.05; **p < 0.001.
concentrations were significantly higher at the 15 minute (p < 0.001) and 30 minute (p < 0.05) periods (Fig. 2). The mean individual peak PRL concentration was significantly higher after CCK-33 alone (15.0 + 2.7 ng/ ml) compared with placebo alone (9.8 + 0.8 ng/ml) (p < 0.05). There was no significant difference between Apo alone and placebo alone on PRL concentration (interaction: F (6, 84) = 1.95; p = NS). PRL was significantly increased after CCK-33 plus Apo compared with placebo alone (interaction: F (6, 84) = 18.67; p < 0.001) (Fig. 3); significantly higher values were noted at the 15 (p < 0.001) and 30 (p < 0.005) minute time periods. Also, prolactin concentrations were significantly higher after CCK-33 plus Apo compared with Apo alone (interaction: F (6, 84) = 14.35; p < 0.001); significantly higher values were present at the 15 (p < O.OOl), 30 (p < 0,001) and 45 min (p < 0.005) periods. The mean
284
I 0
I 30
I
60
I 90
I
120
Time (min) Fig. 3.
Effect of CCK-33 plus apomorphine on basal prolactin secretion. CCK-33 (225 Ivy Dog Units) (or placebo) was administered to 8 men intravenously and apomorphine HCl (APO) (0.5 mg subO----O = CCK-33 cutaneously) (or placebo) at time 0 minutes. Cl = placebo plus plus APO;.H = placebo plus Apo; Uplacebo. :p < 0.005, ?tp < 0.001 compared with placebo plus Apo. "p < 0.005, **p < 0.001 compared with placebo plus placebo.
individual peak after CCK-33 plus Apo (16.6 ? 2.3 ng/ml) was significantly higher than after placebo alone (9.8 + 0.8 ng/ml) (p < 0.01) and after Apo alone (7.9 + 0.7 ng/ml> (p < 0.01). DISCUSSION Apo, a DA receptor agonist in animals (29) and man (30), stimulates GH secretion in humans (31) by an effect on postsynaptic DA receptors (32) within the hypothalamic-pituitary axis which lie outside the blood brain barrier and which are not linked to adenylate cyclase (24,25). PRL secretion is modulated by an inhibitory DA mechanism in man (33). These observations have led to the clinical evaluation of the effect of psychotropic agents on DA function by investigating the effect of various drugs on APO-induced GH secretion and on basal PRL secretion (34-36). Neuroleptics, including the atypical major tranquillizer, clozapine, antagonize the GH response to Apo (26, 37-39). Most neuroleptics also increase basal PRL secretion (16, 18). In the present study, CCK-33 antagonized Apoinduced GH secretion and caused a modest though transient elevation in basal PRL secretion. These observations are compatible with an inhibitory effect of CCK-33 on postsynaptic DA function in man, at least in the 285
hypothalamic-pituitary axis. The relatively small increase in PRL in contrast to a more pronounced antagonism of APO-induced GH secretion is similar to that observed with the atypical neuroleptic, clozapine (18, 38). In previous work (21) we used only about one-third of the dose used in the present study; this probably accounts for the lack of effect noted in our earlier investigation with CCK-33. CCK-33 and CCK-8 have been identified in human brain (2, 5) and receptors for these peptides have been identified in the hypothalamus (6, 7) so that whether the presently observed hormonal effects are due to CCK-33 or mediated by CCK-8 or other fragments cleaved from the parent molecular is unclear. In this regard it is known that CCK-33 is cleaved by peptidases in blood (40, 41) and CCK-converting enzymes in brain (42). Approximately, 32% of CCK-33 is destroyed (in rat plasma) after 45 minutes (40) and the half life of CCK-8 in human plasma is about 50 minutes (40). In contrast to our findings in man in whom CCK-33 had no effect on basal GH secretion, in animal studies CCK peptides appear to exert a stimulatory effect. Thus, in incubated rat anterior pituitary CCK-8 and CCK-39 stimulate GH secretion (43). CCK-8 also induces GH secretion from cultured GH3 pituitary cells and reverses the inhibitory effects of somatostatin on GH secretion (43). Vijayan et al. (44) found that CCK-8 only induced GH secretion after intraventricular administration but not after intravenous use or in incubated rat pituitary. In keeping with our present findings, some studies in animals also point to a stimulatory effect of CCK peptides on PRL secretion. Vijayan et al. (44) found that CCK-8 increased PRL secretion after intraventricular or intravenous administration; no effect was observed in the pituitary in vitro. Malarkey et al. (45) noted an increase in PRL secretion in rat pituitary monolayer cultures following CCK-8 or CCK-33 but no effect of CCK-8 on human monolayer cultures obtained from a single normal pituitary and a single prolactinoma. Though our data support an inhibitory effect of CCK-33 on DA function Thus, CCK-33 may antagonize Apoalternative explanations are possible. induced GH secretion by inhibiting the release of GH either by a direct effect on the pituitary somatotrophe or by inhibiting the release of Against this view is the failure of growth hormone releasing hormone. CCK-33 to decrease basal GH levels; in fact in one subject a brisk increase in GH followed CCK-33 administration. Also, in animal studies a stimulatory or absent effect of CCK-peptides on GH secretion have been However, noted. All subjects experienced abdominal cramps and nausea. stress cannot explain the inhibitory effect of CCK-33 on APO-induced GH secretion as stress is believed to increase GH secretion in man (46). Stress may increase PRL secretion in humans (47) so that the transient elevation of basal PRL concentrations by CCK-33 may be a nonspecific stress response rather than the manifestation of an antagonistic effect Incidental observations during the on DA inhibition of PRL secretion. present study reveal that CCK-33 antagonized other effects of Apo that are believed to be mediated by DA mechanisms, namely, drowsiness (48, 49), penile erections (37, 50) and nausea (29). These findings favour an inhibitory effect of CCK-33 on DA function in man. Further, several
286
studies in animals also support an inhibitory effect of CCK peptides on DA function. Thus, CCK peptides decrease the number of DA binding sites in certain brain regions (51, 52), suppress the behavioural effects of L-dopa (11) and methamphetamine (12), antagonize Apo (13) and methylphenidate-induced stereotyped behaviour (53) and inhibit tail pinchinduced eating (54). CCK-8 antagonizes conditioned-avoidance behaviour in the rat (13) and CCK-8 and ceruletide induce catalepsy in mice (53, 55). These behavioural observations in animals and our present endocrine findings are characteristic of neuroleptics. Kowever, some studies have questioned whether CCK-8 interacts with DA function (56). Because of stress effects noted in the present study it is possible that the effect of CCK-33 on Apo may be mediated by an indirect action on DA function. In summary, CCK-33 antagonized the GH response to Apo and increased basal PRL secretion. These findings are compatible with an inhibitory effect of CCK-33 or fragments on DA function in man though other interpretations are possible. Studies on the effect of CCK-33 on other stimuli of GH secretion may help to determine whether this peptide antagonizes DA function or whether it inhibits the final common pathway involved in GH release. ACKNOWLEDGEMENTS We thank J. Parodo for technical assistance, Andrg Achim, Ph.D. and George Schwartz for statistical analysis of data, Yvonne Hall, R.N. and staff of the research ward for clinical assistance and Nadia Zajac for secretarial assistance. We also thank the NIAMDD, NIH (USA) for the supply of growth hormone radioimmunoassay reagents. This work was supported in part by the Medical Research Council (Canada). REFERENCES 1.
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Accepted
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and antistereotypic Neuropharmacology 20,