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Effects of phencyclidine on 5-hydroxytryptophan- and suckling-induced prolactin release
204
Brain Research, 573 (1992) 204-208 (~ 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17465
Effects of phencyclidine on 5-hydroxytryptophan- and suckling-induced prolactin release James E Hyde Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, KY 40536-0084 (U.S.A.) (Accepted 8 October 1991)
Key words: Phencyclidine; Prolactin; Oxytocin; Dopamine; Serotonin; Lactation
The effects of acute and chronic exposure to phencyclidine (PCP) on the regulation of prolactin (PRL) secretion were examined. Female Sprague-Dawley rats were injected with PCP (10 mg/kg) or saline for 14 days, or just prior to the administration of the serotonin precursor, 5-hydroxytryptophan (5-HTP). A single injection of PCP had no effect on the 5-HTP-induced rise of plasma PRL levels. In contrast, chronic administration of PCP facilitated the release of PRL induced by 5-HTP. Peak plasma PRL levels were more than 3-fold higher after chronic PCP. The acute effect of PCP on suckling-induced PRL release was also examined. PCP delayed the rise of plasma PRL levels by suckling. The magnitude and profile of PRL, however, were similar to saline controls. The pups of PCP-treated dams failed to obtain milk during the suckling episode. Exogenous oxytocin restored the milk ejection reflex in PCP-treated dams. PCP had no effect on basal PRL release from anterior pituitary cells in vitro, and failed to alter the effects of TRH or dopamine. Conclusions: (1) chronic, but not acute, administration of PCP facilitates the 5-HTP-induced release of PRL, (2) acute exposure to PCP delays the suckling-induced rise in PRL and appears to inhibit oxytocin release. These data demonstrate that both acute and chronic PCP may alter the regulation of PRL release, likely through an indirect central mechanism. INTRODUCTION Phencyclidine (PCP) has been shown to have a wide spectrum of effects on neurochemical systems in the central nervous system ~2. F o r example, the release of dopamine and serotonin from some neurons appears to be a u g m e n t e d by PCP, and these effects are believed to be caused by an inhibition of neurotransmitter uptake. A s a result of its effects on hypothalamic neurons, PCP may also alter anterior pituitary h o r m o n e secretion. PCP inhibits prolactin ( P R L ) release in vivo 16'21'25, and this effect is likely due to the actions of PCP upon tuberoinfundibular dopaminergic neurons. I n d e e d , dopamine levels in hypophysial portal blood are increased after PCP administration 18. D o p a m i n e is the major physiological inhibitor of P R L release 1, and alterations in the activity of hypothalamic dopaminergic neurons are reflected by changes in plasma P R L levels. Serotonergic neurons also participate in the complex regulation of P R L secretion 2. A d m i n i s t r a t i o n of serotonin, its precursor, 5-hydroxytryptophan (5-HTP), or serotonin r e - u p t a k e blockers increase P R L secretion in the rat 4'15'17. D u e to its interactions with dopaminergic and serotonergic neurons, PCP may thus indirectly alter the
regulation of P R L secretion under certain physiological conditions. M o r e o v e r , PCP may exert a direct effect on P R L secretion, as PCP receptors have been identified within the anterior pituitary gland 28. The differential effects of acute and chronic exposure to PCP upon the regulation of P R L release have not been examined. The objectives of this study were to examine the effects of PCP on (1) drug-induced P R L release and (2) the intense physiological stimulus of suckling on P R L release. We are now reporting that chronic, but not acute, exposure to PCP facilitates the 5-HTP-induced release of PRL. M o r e o v e r , the acute administration of PCP delays the suckling-induced release of PRL. PCP d e m o n s t r a t e d no effect on thyrotropin-releasing h o r m o n e ( T R H ) - s t i m u l a t e d or dopamine-inhibited P R L secretion in vitro, suggesting that PCP alters the regulation of P R L release by an indirect, possibly hypothalamic, mechanism.
MATERIALS AND METHODS
Animals Female Sprague-Dawley rats (200-225 g; Harlan Industries, Indianapolis, IN) were used in this study. The rats were housed un-
Correspondence: J.E Hyde, Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky 40536-0084, U.S.A.
205 der controlled temperature and lighting conditions (lights on from 07.00-19.00 h). Food and water were available ad libitum.
800
POp
5-HTP experiments The acute and chronic effects of PCP on the 5-HTP-induced release of PRL were examined. In the acute study, rats were implanted with a jugular cannula under Brevital anesthesia (45 mg/kg b. wt., i.p.) one day prior to the experiment a. On the day of the experiment, rats were injected with PCP (10 mg/kg b. wt., s.c.; n = 6) or saline (0.1 ml/100 g b. wt., s.c.; n = 5) 90 min prior to the administration of 5-HTP (Sigma Chemical Co., St. Louis, MO). Blood samples (0.25 ml) were collected just prior to and 10, 20, 30, 60 and 90 min after 5-HTP administration (20 mg/kg b. wt., i.v.). In the chronic study, rats were injected with PCP (10 mg/kg b. wt./day, s.c.; n = 6) or saline (0.1 ml/100 g b. wt., s.c.; n = 5) for 13 days. On day 13, rats were implanted with a jugular cannula. On day 14, rats were injected with PCP (10 mg/kg b. wt.) or saline 90 min before 5-HTP administration. Blood samples (0.25 ml) were collected via the jugular cannula just prior to and 10, 20, 30, 60 and 90 min after 5-HTP administration (20 mg/kg b. wt., i.v.).
v
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< X 09 <
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0
20
40
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80
100
TIME (minutea)
F i g . 1. Stimulation of PRL secretion by 5-HTP administration. Adult female rats were injected with PCP (10 mg/kg b. wt., s.c.) or saline (0.1 ml/100 g b. wt.) 90 min prior to 5-HTP injection (20 mg/kg b. wt., i.v.). Each value is a mean _ S.E.M. (n = 5-6/data point).
Suckling experiments One day before the suckling experiment, dams (8-10 days postpartum; litter size adjusted to 10 pups on the day of birth) were implanted with a jugular cannula. The following day, dams were injected with PCP (10 mg/kg b. wt., s.c.; n = 6) or saline (s.c.; n = 7) and separated from their pups for 4 h. Blood samples (0.25 ml) were collected via the jugular vein cannula at various time intervals as previously described n. After a 20 rain control period without pups, 6 pups were weighed and reunited with their mothers. The 45 min suckling episode began when at least 4 pups were attached to nipples. The pups were then removed and weighed, and blood collection from the dam continued for an additional 90 min. In one experiment, dams (n = 5) were injected (i.v.) 3 times with oxytocin (5 m/IU) during the 45 min suckling episode.
Pituitary cell culture Monodispersed anterior pituitary cells were prepared as previously described 9'1°. Cells (50,000/well) were cultured in 96-well plates containing 0.3 ml Dulbecco's modified Eagles medium (MEM; Gibco, Grand Island, NY) supplemented with 10% horse serum, 2.5% fetal bovine serum, MEM non-essential amino acids, and antibiotics. After 4 days in culture, the cells were washed in medium 199 (Gibco) containing 0.1% bovine serum albumin (medium 199/BSA) 3 times (30 min each), and incubated in medium 199/BSA with or without test substances for either 15 min (TRH experiments) or 3 h (dopamine experiments). The medium was then frozen at -20°C until assayed for PRL content by radioimmunoassay. TRH (Peninsula Laboratories, Inc., Belmont, CA), dopamine (Sigma Chemical Co., St. Louis, MO) and PCP solutions were diluted immediately before use. Ascorbate (0.1 raM) was added to all solutions containing dopamine to inhibit oxidation. Ascorbate alone (56.4 _+ 4.2 ng PRL/well) had no effect on PRL release compared to controls with no ascorbate (59.3 + 5.1 ng PRL/well).
PRL radioimmunoassay and data analysis All blood samples were collected in tubes coated with heparird lithium, centrifuged immediately and plasma stored at -20°C until assayed for hormone content. Plasma and medium PRL levels were determined in triplicate with a radioimmunoassay kit provided by the National Hormone and Pituitary Program, NIDDK, University of Maryland School of Medicine, using rat PRL RP-3 as a reference preparation. The between- and within coefficients of variation were 8.7% and 6.3%, respectively. Data are expressed as the mean + S.E.M. Statistical analyses were performed by analysis of variance, followed by NewmanKeuls multiple range test where appropriate 29.
RESULTS
Effects of acute and chronic PCP on 5-HTP-induced P R L release
Acute injection of PCP lowered (P < 0.05) basal plasma PRL levels from 10.6 + 0.9 ng/ml to 5.3 + 0.6 ng/ml. However, the acute administration of PCP did not alter the 5-HTP-induced release of PRL (Fig. 1). The profiles of plasma PRL levels were similar in the saline and PCP-treated groups. In contrast, PCP failed to lower (P > 0.5) basal plasma PRL levels in rats treated with PCP for 2 weeks. However, rats treated with PCP for 2 weeks showed a greater (P < 0.01) response to 5-HTP than saline controls at all time points examined (Fig. 2). Peak plasma PRL levels were 3.4-fold higher in those animals exposed chronically to PCP.
800
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600
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400
200
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0 0
20
40
60
80
100
TIME (minutes)
Fig. 2. Stimulation of PRL secretion by 5-HTP administration. Adult female rats were treated with PCP (10 mg/kg b. wt., s.c.) or saline (0.1 ml/100 g b. wt.) for 14 days. PCP (10 mg/kg b. wt., s.c.) was administered 90 min prior to 5-HTP injection (20 mg/kg b. wt., i.v.). Each value is a mean + S.E.M. (n = 5-6/data point).
206 50
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20
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60
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Fig. 3. Delay of the suckling-induced rise in PRL by PCP. Three groups of rats were used: PCP alone (10 mg/kg b. wt., s.c.; n = 6), saline (0.1 ml/100 g b. wt.; n = 7), and PCP plus oxytocin (OT; 5 m/IU 3 times during suckling episode; n = 5). Pups were removed and PCP or saline injected 4 h before suckling. After a 20 min control period without pups, 6 pups were weighed and reunited with their mothers for 45 min. Pups were then removed, and blood collection continued for an additional 55 min. Each value is a mean ___ S.E.M. *Significantly greater than dams treated with PCP or PCP plus oxytocin (P < 0.05).
Effects of PCP on suckling-induced P R L release D a m s treated with PCP had lower ( P < 0.05) basal plasma P R L levels (7.4 + 0.5 ng/ml) than saline controls (16.3 + 1.9 ng/ml). A f t e r the initiation of suckling in dams treated with saline, plasma P R L levels were elevated at 15 min and reached p e a k levels at 30 min (Fig. 3). U p o n removal of the pups, plasma P R L levels declined and returned to basal levels by 100 min. In contrast, the increase in plasma P R L levels in dams treated with PCP was delayed after the initiation of suckling. P e a k plasma P R L levels were attained at 60 min, 15 min after the removal of the pups. P R L levels then declined and were significantly higher ( P < 0.05) than pre-suckling basal levels at 100 min. D a m s treated with PCP plus
~
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*
OT
4
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o
TRH
TRH ÷ POP
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TIME (minutes)
r---] CONTROL 1
PCP
100
*
-2
Fig. 4. Change in pups' weight as an index of milk intake during 45 min suckling episode. Each value is a mean + S.E.M. of 5-7 determinations. *Significantly lower than controls and oxytocin (OT)-treated groups (P < 0.05). For details of experimental groups see Fig. 3 legend.
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PCP
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Fig. 5. Effect of PCP on PRL release from anterior pituitary cells in vitro. Upper panel: pituitary cells were exposed to medium 199 alone (control), 1 /~M PCP, 25 nM TRH, or 25 nM TRH plus 1 /~M PCP (TRH + PCP) for 15 min. Each value represents a mean + S.E.M. of 5 experiments. *Significantly greater than control and PCP alone (P < 0.05). Lower panel: pituitary cells were exposed to medium 199 alone (control), 1/~M PCP, 50 nM dopamine (DA), or 50 nM DA plus 1/tM PCP (DA + PCP) for 3 h. Each value is a mean + S.E.M. of 5 experiments. *Significantly lower than control and PCP alone (P < 0.05).
intermittent injections of oxytocin during the suckling episode showed a similar profile of plasma P R L levels as those dams treated with PCP alone (P > 0.5). O t h e r modes of oxytocin administration (e.g. constant infusion) were not examined in this study. The weights of the pups before and after the suckling episode were d e t e r m i n e d as an index of milk intake. The pups of the saline-treated d a m gained weight during the 45 min of suckling (Fig. 4). In contrast, pups suckling the P C P - t r e a t e d d a m failed to attain milk ( P < 0.01). To d e t e r m i n e if PCP affects the milk ejection reflex, oxytocin was administered to P C P - t r e a t e d dams during the suckling episode. Pups exposed to dams treated with PCP plus oxytocin gained weight similar to those pups exposed to saline-treated dams (Fig. 4).
Effects of PCP on P R L release in vitro PCP (1 /~M) had no effect on basal P R L release in vitro at either 15 min or 3 h time intervals (Fig. 5). Furt h e r m o r e , PCP did not alter the stimulation of P R L release by 25 nM T R H or the inhibition of P R L secretion by 50 nM dopamine. Higher concentrations of PCP (10 /~M) also failed to alter P R L release (data not shown).
207 DISCUSSION This study demonstrates the differential abilities of acute and chronic PCP administration to affect PRL release. In addition to its effects on PRL, it appears that PCP also inhibits oxytocin secretion during suckling. In concert, the acute effects of PCP on PRL and oxytocin secretion during lactation are considerable. The ability of PCP to reduce basal PRL release may result in decreased milk production with the inhibition of the milk ejection reflex producing severe consequences. We have not examined the effects of chronic PCP ingestion during lactation on pup growth/weanling rate. The mechanism of 5-HTP-induced PRL release is not certain. It is presently believed that 5-HTP either reduces the activity of tuberoinfundibular dopaminergic neurons, or stimulates the release of a PRL-releasing factor. 5-HTP decreases dopamine synthesis in the median eminence 13. In addition, serotonin injections within the third ventricle reduce dopamine concentrations in hypophysial portal blood 22. Yet, serotonin is still capable of stimulating PRL release when dopamine is infused to maintain high levels22. Thus, a PRL-releasing factor has been postulated to mediate the effects of serotonin on PRL. Indeed, portal blood concentrations of vasoactive intestinal polypeptide (VIP), a putative PRL-releasing factor, are increased by serotonin, and passive immunization against VIP attenuates the 5-HTP-induced PRL release 14'27. Chronic administration of PCP may have altered one or several potential targets: dopamine neurons, serotonin neurons, or possibly PRL-releasing factor neurons. It is unlikely that hypothalamic dopamine neurons are dramatically altered because basal PRL levels were not markedly affected following chronic exposure to PCP. However, we cannot rule out the possibility that PCP may have altered the responsiveness of the dopaminergic neurons to specific stimuli. For example, the development of tolerance to the PCP-induced inhibition of plasma PRL levels after long-term (28 days) administration of PCP has been reported in male rats 16. The present study shows that PCP fails to lower PRL levels after 14 days in the female rat. The chronic effects of PCP on serotonergic neurons have not been thoroughly examined. Hypothalamic levels of serotonin in the mouse are increased after chronic (7 days), but not acute, PCP 19. In contrast, concentrations of serotonin in the mouse cortex are increased after acute, but not chronic, administration of PCP 19. Chronic PCP (14 days) has been reported to down-regulate the binding capacities of serotonergic ligands (5H T 1 and 5-HT 2 sites) in whole rat brain synaptic membranes 2°. Specific serotonin receptor changes in the
hypothalamus are presently unknown. The suckling-induced increase of plasma PRL levels is believed to be the result of several factors3. Dopamine levels in portal blood are reduced only transiently during simulated suckling in the lactating rat 24. Thus, a PRL-releasing factor has been postulated to play a major role in regulation of PRL release during suckling. Furthermore, the inhibition of dopamine release observed during the initial phases of suckling may alter the responsiveness of the lactotroph to secretagogues 7'23. Previous studies have shown that PCP (10 mg/kg) lowers plasma PRL levels for at least 4 h 25. The suckling experiments in the present study could not be conducted at an earlier time due to the profound behavioral effects of PCP and the interruption of maternal behavior. Due to the differences in time of exposure to PCP and the physiological states of the animals, comparisons between the 5-HTP and suckling experiments are difficult. However, similar to the rats in the 5-HTP study, basal PRL levels were lower in the lactating rats treated with PCP. The initial delay of the suckling-induced rise of PRL by acute injections of PCP may be the result of augmented dopamine release from tuberoinfundibular neurons. In the presence of an increased dopaminergic tone, the lactotrophs would be expected to be less susceptible to stimulation by subsequent PRL-releasing factors. Nevertheless, similar plasma PRL levels were attained in PCP-treated rats. The failure of the pups to gain weight during the suckling episode is likely due to the inhibition of oxytocin release from the neurohypophysis. The ability of exogenous oxytocin to restore the gain in pups' weight argues against the ability of PCP to inhibit oxytocin's actions within the mammary gland. The control of oxytocin release from the posterior pituitary is also complex6. Dopamine appears to inhibit oxytocin release s, and the facilitation of dopamine release from tuberohypophyseal dopamine neurons by PCP may account for the prolonged inhibition of oxytocin release. Oxytocin has also been postulated to be a PRL-releasing factor26. Thus, the effects of PCP on oxytocin release may, in part, play a role in the delay of the rise in PRL release. We are presently exploring the effects of PCP on dopamine and oxytocin release from the neurohypophysis. In summary, this study demonstrates that PCP may alter the regulation of PRL secretion in both acute and chronic manners. Although the mechanism(s) by which PCP mediates its actions on PRL release are not clear, the failure of PCP to alter PRL release in vitro argues in favor of a central mode of action. Dopaminergic and serotonergic neurons are potential targets, and future studies will be aimed to examine the effects of PCP on these neuronal systems in the hypothalamus and posterior pituitary.
208
Acknowledgements. This work was supported by Biomedical Research Support Grant RR05374 from the Biomedical Research Support Branch, Division of Research Facilities and Resources, NIH, and the University of Kentucky Medical Center Research Fund. We are indebted to the National Hormone and Pituitary Program,
NIDDK, University of Maryland School of Medicine for the PRL RIA kit, and the National Institute on Drug Abuse for providing the phencyclidine hydrochloride. Presented, in part, at the 20th Annual Society for Neuroscience Meeting, St. Louis, MO.
REFERENCES 1 Ben-Jonathan, N., Dopamine: a prolactin-inhibiting hormone, Endocrine Rev., 6 (1985) 564-589. 2 Ben-Jonathan, N., Arbogast, L.A. and Hyde, J.F., Neuroendocrine regulation of prolactin release, Prog. Neurobiol., 33 (1989) 399-447. 3 Ben-Jonathan, N., Hyde, J.F. and Murai, I., Suckling-induced rise in prolactin: mediation by prolactin-releasing factor from posterior pituitary, News Physiol. Sci., 3 (1988) 172-175. 4 Clemens, J.A., Sawyer B.D. and Cerimele, B., Further evidence that serotonin is a neurotransmitter involved in the control of prolactin secretion, Endocrinology, 100 (1977) 692-699. 5 Crowley, W.R., Shyr, S.W., Kacsoh, B. and Grosvenor, C.E., Evidence for stimulatory noradrenergic and inhibitory dopaminergic regulation of oxytocin release in the lactating rat, Endocrinology, 121 (1987) 14-20. 6 Grosvenor, C.E. and Mena, E, Regulating mechanisms for oxytocin and prolactin secretion during lactation. In E.E. Muller and R.M. MacLeod (Eds.), Neuroendocrine Perspectives, Vol. 1, Elsevier, Amsterdam, 1982, pp. 69-110. 7 Haisenleder, D.J., Moy, J.A., Gala, R.R. and Lawson, D.M., The effect of transient dopamine antagonism on thyrotropin-releasing hormone-induced prolactin release in ovariectomized rats treated with estradiol and/or progesterone, Endocrinology, 119 (1986) 1996-2003. 8 Harms, P.G. and Ojeda, S.R., A rapid and simple procedure for chronic cannulation of the rat jugular vein, J. Appl. Physiol., 36 (1974) 391-392. 9 Hyde, J.E and Keller, B.K., Galanin secretion from anterior pituitary cells in vitro is regulated by dopamine, somatostatin, and thyrotropin-releasing hormone, Endocrinology, (1991) 917922. 10 Hyde, J.F., Murai, I. and Ben-Jonathan, N., The rat posterior pituitary contains a potent prolactin-releasing factor: studies with perifused anterior pituitary ceils, Endocrinology, 121 (1987) 1531-1539. 11 Hyde, J.F., North, W.G. and Ben-Jonathan, N., The vasopressin-associated glycopeptide is not a prolactin-releasing factor: studies with lactating Brattleboro rats, Endocrinology, 125 (1989) 35-40. 12 Johnson Jr., K.M., Neurochemistry and neurophysiology of phencyclidine. In H.Y. Meltzer (Ed.), Psychopharmacology: The Third Generation in Progress, Raven, New York, 1987, pp. 1581-1588. 13 Johnston, C.A. and Negro-Vilar, A., Maturation of the prolactin and proopiomelanocortin-derived peptide responses to ether stress and morphine: neurochemical analysis, Endocrinology, 118 (1986) 797-804. 14 Kaji, H., Chihara, K., Abe, H., Kita, T., Kashio, Y., Okimura, Y. and Fu#ta, T., Effect of passive immunization with antisera to vasoactive intestinal polypeptide and peptide histidine iso-
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28 29
leucine amide on 5-hydroxy-l-tryptophan-induced prolactin release in rats, Endocrinology, 117 (1985) 1914-1919. Kamberi, I.A., Mical, R.S. and Porter, J.C., Effect of melatonin and serotonin on the release of FSH and prolactin, Endocrinology, 88 (1971) 1288-1293. Lozovsky, D., Sailer, C.E, Bayorh, M.A., Chiueh, C.C., Rice, K.C., Burke Jr., T.R. and Kopin, I.J., Effects of phencyclidine on rat prolactin, dopamine receptor and locomotor activity, Life Sci., 32 (1983) 2725-2731. Lu, K.H. and Meites, J., Effects of serotonin precursors and melatonin on serum prolactin release in rats, Endocrinology, 93 (1973) 152-155. Meltzer, H.Y., Simonovic, M. and Gudelsky, G.A., Phencyclidine-induced inhibition of rat prolactin secretion: increased portal blood dopamine, Eur. J. Pharmacol., 110 (1985) 143-146. Nabeshirna, T., Hiramatsu, M., Furukawa, H. and Kameyama, T., Effects of acute and chronic administrations of phencyelidine on the levels of serotonin and 5-hydroxyindoleacetic acid in discrete areas of mouse brain, Life Sci., 36 (1985) 939-946. Nabeshima, T., Noda, Y., Yamaguchi, K., Ishikawa, K., Furukawa, H. and Kametama, T., Acute and chronic phencyclidine administration changes serotonin receptors in rat brain, Eur. J. Pharmacol., 109 (1985) 129-130. Pechnick, R.N., George, R., Lee, R.J. and Poland, R.E., The effects of the acute administration of phencyclidine hydrochloride (PCP) on the release of corticosterone, growth hormone and prolactin in the rat, Life Sci., 38 (1986) 291-296. Pilotte, N.S. and Porter, J.C., Dopamine in hypophysial portal plasma and prolactin in systemic plasma of rats treated with 5-hydroxytryptamine, Endocrinology, 108 (1981) 2137-2141. Plotsky, P.M. and Neill, J.D., Interactions of dopamine and thyrotropin-releasing hormone in the regulation of prolactin release in lactating rats, Endocrinology, 111 (1982) 168-173. Plotsky, P.M. and Neill, J.D., The decrease in hypothalamic dopamine secretion induced by suckling: comparison of voltammetric and radioisotopic methods of measurement, Endocrinology, 110 (1982) 691-696. Sailer, C.F., Zerbe, R.L., Bayorh, M.A., Lozovsky, D. and Kopin, I.J., Phencyclidine suppresses plasma prolactin levels, Eur. J. Pharmacol., 83 (1982) 309-311. Samson, W.K., Lumpkin, M.D. and McCann, S.M., Evidence for a physiological role for oxytocin in the control of prolactin secretion, Endocrinology, 119 (1986) 554-560. Shimatsu, A., Kato, Y., Matsushita, N., Katakami, H., Yanaihara, N. and Imura, H., Stimulation by serotonin of vasoactive intestinal polypeptide release into rat hypophysial portal blood, Endocrinology, 111 (1982) 338-340. Stojilkovic, S.S., Dufau, M.L. and Catt, K.J., Receptors and secretory actions of o/phencyclidine agonists in anterior pituitary cells, Endocrinology, 121 (1987) 2044-2054. Zar, J.H., Biostatistical Analysis, Prentice-Hall, Englewood Cliffs, NJ, 1974, 151 pp.
Brain Research, 573 (1992) 204-208 (~ 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17465
Effects of phencyclidine on 5-hydroxytryptophan- and suckling-induced prolactin release James E Hyde Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, KY 40536-0084 (U.S.A.) (Accepted 8 October 1991)
Key words: Phencyclidine; Prolactin; Oxytocin; Dopamine; Serotonin; Lactation
The effects of acute and chronic exposure to phencyclidine (PCP) on the regulation of prolactin (PRL) secretion were examined. Female Sprague-Dawley rats were injected with PCP (10 mg/kg) or saline for 14 days, or just prior to the administration of the serotonin precursor, 5-hydroxytryptophan (5-HTP). A single injection of PCP had no effect on the 5-HTP-induced rise of plasma PRL levels. In contrast, chronic administration of PCP facilitated the release of PRL induced by 5-HTP. Peak plasma PRL levels were more than 3-fold higher after chronic PCP. The acute effect of PCP on suckling-induced PRL release was also examined. PCP delayed the rise of plasma PRL levels by suckling. The magnitude and profile of PRL, however, were similar to saline controls. The pups of PCP-treated dams failed to obtain milk during the suckling episode. Exogenous oxytocin restored the milk ejection reflex in PCP-treated dams. PCP had no effect on basal PRL release from anterior pituitary cells in vitro, and failed to alter the effects of TRH or dopamine. Conclusions: (1) chronic, but not acute, administration of PCP facilitates the 5-HTP-induced release of PRL, (2) acute exposure to PCP delays the suckling-induced rise in PRL and appears to inhibit oxytocin release. These data demonstrate that both acute and chronic PCP may alter the regulation of PRL release, likely through an indirect central mechanism. INTRODUCTION Phencyclidine (PCP) has been shown to have a wide spectrum of effects on neurochemical systems in the central nervous system ~2. F o r example, the release of dopamine and serotonin from some neurons appears to be a u g m e n t e d by PCP, and these effects are believed to be caused by an inhibition of neurotransmitter uptake. A s a result of its effects on hypothalamic neurons, PCP may also alter anterior pituitary h o r m o n e secretion. PCP inhibits prolactin ( P R L ) release in vivo 16'21'25, and this effect is likely due to the actions of PCP upon tuberoinfundibular dopaminergic neurons. I n d e e d , dopamine levels in hypophysial portal blood are increased after PCP administration 18. D o p a m i n e is the major physiological inhibitor of P R L release 1, and alterations in the activity of hypothalamic dopaminergic neurons are reflected by changes in plasma P R L levels. Serotonergic neurons also participate in the complex regulation of P R L secretion 2. A d m i n i s t r a t i o n of serotonin, its precursor, 5-hydroxytryptophan (5-HTP), or serotonin r e - u p t a k e blockers increase P R L secretion in the rat 4'15'17. D u e to its interactions with dopaminergic and serotonergic neurons, PCP may thus indirectly alter the
regulation of P R L secretion under certain physiological conditions. M o r e o v e r , PCP may exert a direct effect on P R L secretion, as PCP receptors have been identified within the anterior pituitary gland 28. The differential effects of acute and chronic exposure to PCP upon the regulation of P R L release have not been examined. The objectives of this study were to examine the effects of PCP on (1) drug-induced P R L release and (2) the intense physiological stimulus of suckling on P R L release. We are now reporting that chronic, but not acute, exposure to PCP facilitates the 5-HTP-induced release of PRL. M o r e o v e r , the acute administration of PCP delays the suckling-induced release of PRL. PCP d e m o n s t r a t e d no effect on thyrotropin-releasing h o r m o n e ( T R H ) - s t i m u l a t e d or dopamine-inhibited P R L secretion in vitro, suggesting that PCP alters the regulation of P R L release by an indirect, possibly hypothalamic, mechanism.
MATERIALS AND METHODS
Animals Female Sprague-Dawley rats (200-225 g; Harlan Industries, Indianapolis, IN) were used in this study. The rats were housed un-
Correspondence: J.E Hyde, Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky 40536-0084, U.S.A.
205 der controlled temperature and lighting conditions (lights on from 07.00-19.00 h). Food and water were available ad libitum.
800
POp
5-HTP experiments The acute and chronic effects of PCP on the 5-HTP-induced release of PRL were examined. In the acute study, rats were implanted with a jugular cannula under Brevital anesthesia (45 mg/kg b. wt., i.p.) one day prior to the experiment a. On the day of the experiment, rats were injected with PCP (10 mg/kg b. wt., s.c.; n = 6) or saline (0.1 ml/100 g b. wt., s.c.; n = 5) 90 min prior to the administration of 5-HTP (Sigma Chemical Co., St. Louis, MO). Blood samples (0.25 ml) were collected just prior to and 10, 20, 30, 60 and 90 min after 5-HTP administration (20 mg/kg b. wt., i.v.). In the chronic study, rats were injected with PCP (10 mg/kg b. wt./day, s.c.; n = 6) or saline (0.1 ml/100 g b. wt., s.c.; n = 5) for 13 days. On day 13, rats were implanted with a jugular cannula. On day 14, rats were injected with PCP (10 mg/kg b. wt.) or saline 90 min before 5-HTP administration. Blood samples (0.25 ml) were collected via the jugular cannula just prior to and 10, 20, 30, 60 and 90 min after 5-HTP administration (20 mg/kg b. wt., i.v.).
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F i g . 1. Stimulation of PRL secretion by 5-HTP administration. Adult female rats were injected with PCP (10 mg/kg b. wt., s.c.) or saline (0.1 ml/100 g b. wt.) 90 min prior to 5-HTP injection (20 mg/kg b. wt., i.v.). Each value is a mean _ S.E.M. (n = 5-6/data point).
Suckling experiments One day before the suckling experiment, dams (8-10 days postpartum; litter size adjusted to 10 pups on the day of birth) were implanted with a jugular cannula. The following day, dams were injected with PCP (10 mg/kg b. wt., s.c.; n = 6) or saline (s.c.; n = 7) and separated from their pups for 4 h. Blood samples (0.25 ml) were collected via the jugular vein cannula at various time intervals as previously described n. After a 20 rain control period without pups, 6 pups were weighed and reunited with their mothers. The 45 min suckling episode began when at least 4 pups were attached to nipples. The pups were then removed and weighed, and blood collection from the dam continued for an additional 90 min. In one experiment, dams (n = 5) were injected (i.v.) 3 times with oxytocin (5 m/IU) during the 45 min suckling episode.
Pituitary cell culture Monodispersed anterior pituitary cells were prepared as previously described 9'1°. Cells (50,000/well) were cultured in 96-well plates containing 0.3 ml Dulbecco's modified Eagles medium (MEM; Gibco, Grand Island, NY) supplemented with 10% horse serum, 2.5% fetal bovine serum, MEM non-essential amino acids, and antibiotics. After 4 days in culture, the cells were washed in medium 199 (Gibco) containing 0.1% bovine serum albumin (medium 199/BSA) 3 times (30 min each), and incubated in medium 199/BSA with or without test substances for either 15 min (TRH experiments) or 3 h (dopamine experiments). The medium was then frozen at -20°C until assayed for PRL content by radioimmunoassay. TRH (Peninsula Laboratories, Inc., Belmont, CA), dopamine (Sigma Chemical Co., St. Louis, MO) and PCP solutions were diluted immediately before use. Ascorbate (0.1 raM) was added to all solutions containing dopamine to inhibit oxidation. Ascorbate alone (56.4 _+ 4.2 ng PRL/well) had no effect on PRL release compared to controls with no ascorbate (59.3 + 5.1 ng PRL/well).
PRL radioimmunoassay and data analysis All blood samples were collected in tubes coated with heparird lithium, centrifuged immediately and plasma stored at -20°C until assayed for hormone content. Plasma and medium PRL levels were determined in triplicate with a radioimmunoassay kit provided by the National Hormone and Pituitary Program, NIDDK, University of Maryland School of Medicine, using rat PRL RP-3 as a reference preparation. The between- and within coefficients of variation were 8.7% and 6.3%, respectively. Data are expressed as the mean + S.E.M. Statistical analyses were performed by analysis of variance, followed by NewmanKeuls multiple range test where appropriate 29.
RESULTS
Effects of acute and chronic PCP on 5-HTP-induced P R L release
Acute injection of PCP lowered (P < 0.05) basal plasma PRL levels from 10.6 + 0.9 ng/ml to 5.3 + 0.6 ng/ml. However, the acute administration of PCP did not alter the 5-HTP-induced release of PRL (Fig. 1). The profiles of plasma PRL levels were similar in the saline and PCP-treated groups. In contrast, PCP failed to lower (P > 0.5) basal plasma PRL levels in rats treated with PCP for 2 weeks. However, rats treated with PCP for 2 weeks showed a greater (P < 0.01) response to 5-HTP than saline controls at all time points examined (Fig. 2). Peak plasma PRL levels were 3.4-fold higher in those animals exposed chronically to PCP.
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Fig. 2. Stimulation of PRL secretion by 5-HTP administration. Adult female rats were treated with PCP (10 mg/kg b. wt., s.c.) or saline (0.1 ml/100 g b. wt.) for 14 days. PCP (10 mg/kg b. wt., s.c.) was administered 90 min prior to 5-HTP injection (20 mg/kg b. wt., i.v.). Each value is a mean + S.E.M. (n = 5-6/data point).
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Fig. 3. Delay of the suckling-induced rise in PRL by PCP. Three groups of rats were used: PCP alone (10 mg/kg b. wt., s.c.; n = 6), saline (0.1 ml/100 g b. wt.; n = 7), and PCP plus oxytocin (OT; 5 m/IU 3 times during suckling episode; n = 5). Pups were removed and PCP or saline injected 4 h before suckling. After a 20 min control period without pups, 6 pups were weighed and reunited with their mothers for 45 min. Pups were then removed, and blood collection continued for an additional 55 min. Each value is a mean ___ S.E.M. *Significantly greater than dams treated with PCP or PCP plus oxytocin (P < 0.05).
Effects of PCP on suckling-induced P R L release D a m s treated with PCP had lower ( P < 0.05) basal plasma P R L levels (7.4 + 0.5 ng/ml) than saline controls (16.3 + 1.9 ng/ml). A f t e r the initiation of suckling in dams treated with saline, plasma P R L levels were elevated at 15 min and reached p e a k levels at 30 min (Fig. 3). U p o n removal of the pups, plasma P R L levels declined and returned to basal levels by 100 min. In contrast, the increase in plasma P R L levels in dams treated with PCP was delayed after the initiation of suckling. P e a k plasma P R L levels were attained at 60 min, 15 min after the removal of the pups. P R L levels then declined and were significantly higher ( P < 0.05) than pre-suckling basal levels at 100 min. D a m s treated with PCP plus
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Fig. 4. Change in pups' weight as an index of milk intake during 45 min suckling episode. Each value is a mean + S.E.M. of 5-7 determinations. *Significantly lower than controls and oxytocin (OT)-treated groups (P < 0.05). For details of experimental groups see Fig. 3 legend.
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Fig. 5. Effect of PCP on PRL release from anterior pituitary cells in vitro. Upper panel: pituitary cells were exposed to medium 199 alone (control), 1 /~M PCP, 25 nM TRH, or 25 nM TRH plus 1 /~M PCP (TRH + PCP) for 15 min. Each value represents a mean + S.E.M. of 5 experiments. *Significantly greater than control and PCP alone (P < 0.05). Lower panel: pituitary cells were exposed to medium 199 alone (control), 1/~M PCP, 50 nM dopamine (DA), or 50 nM DA plus 1/tM PCP (DA + PCP) for 3 h. Each value is a mean + S.E.M. of 5 experiments. *Significantly lower than control and PCP alone (P < 0.05).
intermittent injections of oxytocin during the suckling episode showed a similar profile of plasma P R L levels as those dams treated with PCP alone (P > 0.5). O t h e r modes of oxytocin administration (e.g. constant infusion) were not examined in this study. The weights of the pups before and after the suckling episode were d e t e r m i n e d as an index of milk intake. The pups of the saline-treated d a m gained weight during the 45 min of suckling (Fig. 4). In contrast, pups suckling the P C P - t r e a t e d d a m failed to attain milk ( P < 0.01). To d e t e r m i n e if PCP affects the milk ejection reflex, oxytocin was administered to P C P - t r e a t e d dams during the suckling episode. Pups exposed to dams treated with PCP plus oxytocin gained weight similar to those pups exposed to saline-treated dams (Fig. 4).
Effects of PCP on P R L release in vitro PCP (1 /~M) had no effect on basal P R L release in vitro at either 15 min or 3 h time intervals (Fig. 5). Furt h e r m o r e , PCP did not alter the stimulation of P R L release by 25 nM T R H or the inhibition of P R L secretion by 50 nM dopamine. Higher concentrations of PCP (10 /~M) also failed to alter P R L release (data not shown).
207 DISCUSSION This study demonstrates the differential abilities of acute and chronic PCP administration to affect PRL release. In addition to its effects on PRL, it appears that PCP also inhibits oxytocin secretion during suckling. In concert, the acute effects of PCP on PRL and oxytocin secretion during lactation are considerable. The ability of PCP to reduce basal PRL release may result in decreased milk production with the inhibition of the milk ejection reflex producing severe consequences. We have not examined the effects of chronic PCP ingestion during lactation on pup growth/weanling rate. The mechanism of 5-HTP-induced PRL release is not certain. It is presently believed that 5-HTP either reduces the activity of tuberoinfundibular dopaminergic neurons, or stimulates the release of a PRL-releasing factor. 5-HTP decreases dopamine synthesis in the median eminence 13. In addition, serotonin injections within the third ventricle reduce dopamine concentrations in hypophysial portal blood 22. Yet, serotonin is still capable of stimulating PRL release when dopamine is infused to maintain high levels22. Thus, a PRL-releasing factor has been postulated to mediate the effects of serotonin on PRL. Indeed, portal blood concentrations of vasoactive intestinal polypeptide (VIP), a putative PRL-releasing factor, are increased by serotonin, and passive immunization against VIP attenuates the 5-HTP-induced PRL release 14'27. Chronic administration of PCP may have altered one or several potential targets: dopamine neurons, serotonin neurons, or possibly PRL-releasing factor neurons. It is unlikely that hypothalamic dopamine neurons are dramatically altered because basal PRL levels were not markedly affected following chronic exposure to PCP. However, we cannot rule out the possibility that PCP may have altered the responsiveness of the dopaminergic neurons to specific stimuli. For example, the development of tolerance to the PCP-induced inhibition of plasma PRL levels after long-term (28 days) administration of PCP has been reported in male rats 16. The present study shows that PCP fails to lower PRL levels after 14 days in the female rat. The chronic effects of PCP on serotonergic neurons have not been thoroughly examined. Hypothalamic levels of serotonin in the mouse are increased after chronic (7 days), but not acute, PCP 19. In contrast, concentrations of serotonin in the mouse cortex are increased after acute, but not chronic, administration of PCP 19. Chronic PCP (14 days) has been reported to down-regulate the binding capacities of serotonergic ligands (5H T 1 and 5-HT 2 sites) in whole rat brain synaptic membranes 2°. Specific serotonin receptor changes in the
hypothalamus are presently unknown. The suckling-induced increase of plasma PRL levels is believed to be the result of several factors3. Dopamine levels in portal blood are reduced only transiently during simulated suckling in the lactating rat 24. Thus, a PRL-releasing factor has been postulated to play a major role in regulation of PRL release during suckling. Furthermore, the inhibition of dopamine release observed during the initial phases of suckling may alter the responsiveness of the lactotroph to secretagogues 7'23. Previous studies have shown that PCP (10 mg/kg) lowers plasma PRL levels for at least 4 h 25. The suckling experiments in the present study could not be conducted at an earlier time due to the profound behavioral effects of PCP and the interruption of maternal behavior. Due to the differences in time of exposure to PCP and the physiological states of the animals, comparisons between the 5-HTP and suckling experiments are difficult. However, similar to the rats in the 5-HTP study, basal PRL levels were lower in the lactating rats treated with PCP. The initial delay of the suckling-induced rise of PRL by acute injections of PCP may be the result of augmented dopamine release from tuberoinfundibular neurons. In the presence of an increased dopaminergic tone, the lactotrophs would be expected to be less susceptible to stimulation by subsequent PRL-releasing factors. Nevertheless, similar plasma PRL levels were attained in PCP-treated rats. The failure of the pups to gain weight during the suckling episode is likely due to the inhibition of oxytocin release from the neurohypophysis. The ability of exogenous oxytocin to restore the gain in pups' weight argues against the ability of PCP to inhibit oxytocin's actions within the mammary gland. The control of oxytocin release from the posterior pituitary is also complex6. Dopamine appears to inhibit oxytocin release s, and the facilitation of dopamine release from tuberohypophyseal dopamine neurons by PCP may account for the prolonged inhibition of oxytocin release. Oxytocin has also been postulated to be a PRL-releasing factor26. Thus, the effects of PCP on oxytocin release may, in part, play a role in the delay of the rise in PRL release. We are presently exploring the effects of PCP on dopamine and oxytocin release from the neurohypophysis. In summary, this study demonstrates that PCP may alter the regulation of PRL secretion in both acute and chronic manners. Although the mechanism(s) by which PCP mediates its actions on PRL release are not clear, the failure of PCP to alter PRL release in vitro argues in favor of a central mode of action. Dopaminergic and serotonergic neurons are potential targets, and future studies will be aimed to examine the effects of PCP on these neuronal systems in the hypothalamus and posterior pituitary.
208
Acknowledgements. This work was supported by Biomedical Research Support Grant RR05374 from the Biomedical Research Support Branch, Division of Research Facilities and Resources, NIH, and the University of Kentucky Medical Center Research Fund. We are indebted to the National Hormone and Pituitary Program,
NIDDK, University of Maryland School of Medicine for the PRL RIA kit, and the National Institute on Drug Abuse for providing the phencyclidine hydrochloride. Presented, in part, at the 20th Annual Society for Neuroscience Meeting, St. Louis, MO.
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