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Changes in anterior pituitary hormone secretion and hypothalamic catecholamine metabolism during morphine withdrawal in the female rat
Brain Research, 346 (1985) 15-21 Elsevier
15
BRE 11083
Changes in Anterior Pituitary Hormone Secretion and Hypothalamic Catecholamine Metabolism During Morphine Withdrawal in the Female Rat STEVEN M. GABRIEL1, JAMES W. SIMPKINS 1and WILLIAM J. MILLARD2
1Department of Pharmacodynamics, Universityof Florida, Collegeof Pharmacy, Gainesville, FL and 2Department of Neurology, Massachusetts General Hospital Harvard Medical School Boston, MA (U. S.A.) (Accepted January 15th, 1985)
Key words: luteinizing hormone-- prolactin--fl-endorphin-- growth hormone-- endogenous opiate - - morphine withdrawal-catecholamine metabolism-- norepinephrine - - dopamine-- thyroid stimulating hormone
Studies were undertaken to evaluate the acute responses of hypothalamic noradrenergic and dopaminergic neurons and anterior pituitary hormones to naloxone (NAL)-precipitated morphine (MOR) withdrawal in the rat. Ovariectomized female rats were rendered MOR-dependent and injected with NAL (1 mg/kg b.w., s.c.). During precipitated MOR withdrawal, a decline in norepinephrine (NE) concentrations was preceded by an increase in the level of its metabolite normetanephrine (NME) in the medial basal hypothalamus (MBH) as well as the preoptic area-anterior hypothalamus (POA-AH). Both dopamine (DA) and its major acid metabolite, dihydroxyphenylacetic acid (DOPAC), showed increased concentrations in these two hypothalamic regions within 30 min of NAL administration. Elevated luteinizing hormone (LH) and fl-endorphin secretion was evident within 5 min of NAL injection to MOR-dependent rats, while serum prolaetin (PRL) increased 15 min into MOR withdrawal. Both growth hormone (GH) and thyroid-stimulating hormone (TSH) were depressed over the course of MOR withdrawal. Although a cause and effect relationship cannot be established, during NAL-precipitated MOR withdrawal, a heightened hypothalamic monoaminergic neuronal activity is accompanied by a differential response of anterior pituitary hormones. The observed responses, which are similar to those seen during acute stress, indicate that MOR withdrawal may activate the same mechanisms which mediate the neuroendocrine response to stress.
INTRODUCTION Morphine ( M O R ) and endogenous opioid peptides (EOP) have been shown to exert profound effects on brain monoaminergic neurons and on the secretion of the anterior pituitary hormones which they regulate. M O R and E O P inhibit hypothalamic norepinephrine (NE)I, 19 and tuberoinfundibular dopamine ( D A ) turnover20,51. Additionally, opiates stimulate prolactin (PRL)30, 35, fl-endorphin 27, and growth hormone (Gn)30, 35 secretion, and inhibit luteinizing h o r m o n e (LH) 7,30 and thyroid-stimulating hormone (TSH) 44 release. Through these effects on brain monoaminergic neurons and pituitary h o r m o n e secretion, endogenous and exogenous opiates can influence homeostatic processes in a variety of organs. A consequence of continued M O R treatment and the resulting opiate dependence is the abstinence
syndrome subsequent to narcotic antagonist exposure or the cessation of M O R treatment. Opiate withdrawal is throught to result in activation of brain adrenergic neuronsl,4,13, 40. The capacity of clonidine to relieve some of the symptoms of MOR-abstinence9,22, 26, presumably by reducing the withdrawalinduced acceleration in N E turnover 1, suggests that noradrenergic neurons are involved in the symptoms of opiate withdrawal. The role of D A neurons in the mechanism of opiate withdrawal is less certain however. The importance of elucidating the neuronal systems involved in opiate dependence and withdrawal is indicated by recent evidence for E O P dependence associated with chronic hyperprolactinemia 47, chronic stress6, 33, and the endogenous production of addictive alkaloids during chronic alcohol ingestion 11. The mechanisms involved in M O R dependence and with-
Correspondence: J. W. Simpkins, University of Florida, Department of Pharmacodynamics, College of Pharmacy, Box J-4, J.H.M.H.C., Gainesville, FL 32610, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
16 drawal may also be present in these conditons of EOP dependence. Thus, in the present study we evaluated the acute response of hypothalamic noradrenergic and dopaminergic neurons and anterior pituitary hormones to naloxone (NAL)-precipitated MOR withdrawal in the rat. MATERIALS AND METHODS
Animals Female Charles River rats (Sprague-Dawley, Willmington, MA) weighing 200-240 g were maintained in a light- (lights on 07.00-19.00 h) and temperature (26 + 1 °C)-controlled room. Purina Lab Chow and water were provided ad libitum. Rats were allowed to acclimate to the animal facilities for one week prior to experimentation, and all surgeries were performed under light ether anesthesia.
Experimental treatment M O R dependency was induced by subcutaneous implantation of pellets containing 75 mg M O R freebase. These tablets were prepared in our laboratory using the procedure originally described by Gibson and Tingstad 18. Animals received 2 additional pellets at 2 days after the first implant. This dosage regimen produces typical signs of M O R dependence and withdrawal4L To determine the effects of precipitated M O R withdrawal on hypothalamic catecholamine metabolism and anterior pituitary hormone secretion, female rats were ovariectomized and rendered MORdependent as described above. After 3 days of continuous M O R exposure, groups of rats (6 per group) were treated with NAL HC1 dissolved in saline (1 mg/kg b.w., s.c.) and killed by decapitation 5, 15, 30 or 60 rain later. A control group of MOR-dependent rats (t = 0) received saline rather than N A L and were killed 5 min later. Trunk blood was collected for later serum hormone analysis, brains were rapidly removed, placed on ice, and tissues containing the medial basal hypothalamus (MBH) and preoptic areaanterior hypothalamus ( P O A - A H ) were dissected as described previously 23. Tissues were immediately homogenized in 0.4 N perchloric acid (PCA) containing 10 rag% E D T A at a ratio of 1 mg tissue/10 pl PCA. The average weights for these tissues were 15.9 _+ 0.3 mg for MBH and 12.9 + 0.3 mg for POA-AH.
Analysis of amines and metabolites Concentrations of NE and DA were determined by the radioenzymatic assay procedure of Cuello et al. 10 using catecholamine-o-methyl transferase isolated from the rat liver by the method of Nikodjevic et al. 37. The sensitivities of this assay for NE and DA were 50 pg and 20 pg respectively. The NE metabolite, normetanephrine (NME), and the D A metabolite, dihydroxyphenylacetic acid (DOPAC) were assayed by amperometric methods following their separation by high-pressure liquid chromatography (HPLC). The separation of these metabolites was accomplished using reverse-phase chromatography with a mobile phase composed of 8% methanol, 0.2 mM octyl sodium sulfate, 0.1 M NaHzPO ~, and 0.1 mM E D T A at pH 3.1. The flow rate of this mobile phase was 0.7 ml/min across a Supelco LC-18 column (15 cm x 4.6 mm, 5/~m particle size). Detection of the metabolites was accomplished with an electrochemical detector (Bioanalytical Systems, Model LC-2A), with a potential difference between the working electrode and a reference A g Ag/CI 2 electrode set at + 0.9 V and currents generated at 5 nA/mV. To 100 pl of each M B H and P O A - A H sample was added 2 ng of dihydroxybenzoic acid ( D H B A ) as an internal standard. The concentrations of each of the metabolites was determined by the peak-height ratios of metabolite to D H B A in comparison to a standard curve of peak-height ratios. A sensitivity of less than 100 pg was found for both NME and DOPAC.
Radioimmunoassays Serum samples were assayed in dupficate or triplicate for PRL, GH, LH and TSH using the procedures described in the N I A D D K Kits provided. Data for these 4 hormone are expressed in terms of their respective reference preparations PRL-RP-S2, GHRP-1, LH-RP-1, or TSH-RP-1. The intraassay coefficient of variation for the PRL, GH, LH and TSH assays determined with t0 replicates of pooled serum which inhibited the binding of the labeled ligand 40-60%, was 6.7%, 7.8%, 6.8% and 6.2% respectively. All samples were assayed in a single run and therefore no interassay variability was determined. The sensitivity of these assays, defined as a 20% inhibition of binding of labeled ligand was 25 ng for TSH, 5 ng for GH, 0.9 ng for LH and 0.25 ng for PRL. Se-
17 rum concentrations of fl-endorphin were determined by the method described by Arnold et al.3. In this assay there is a 100% cross-reactivity of fl-endorphin and fl-lipotropin (on a molar basis); therefore the term immunoreactive fl-endorphin will be employed (IR-fl-ENDO). However, the antisera used does not cross-react with methionine-enkephalin, a-endorphin or 7-endorphin at concentrations as high as 5 ng/tube 3. In the present study, serum was assayed in duplicate and results are expressed in terms of the camel fl-endorphin standard (Biochem, Torrance, CA). The intraassay coefficient of variation of the flendorphin assay was 9.8% and the minimum sensitivity of the assay was 30 pg/ml serum. Analysis of variance and S t u d e n t - N e w m a n Keuls tests were used to evaluate the significance of differences among treatment groups. A significance level of P < 0.05 was chosen. Six to eight rats were used for each determination. RESULTS
hormone concentrations compared to time = 0 (controis). Serum hormone concentrations in MORtreated rats at 5 min after saline injection (control) were 11.27 + 3.15 ng/ml for LH, 100.4 _+ 12.9 ng/ml for IR-fl-ENDO, 12.5 + 3.03 mg/ml for PRL, 296.4 + 90.5 ng/ml for TSH, and 467.8 +_ 96.6 ng/ml for GH. N A L injection to MOR-dependent rats resulted in a marked secretion of LH and IR-fl-ENDO. Serum LH concentrations were increased 8-fold and serum IR-fl-ENDO concentrations were elevated 6-fold by 5 min after N A L injection (P < 0.05). Serum LH concentrations remained elevated and were significantly increased 1 h after NAL, while IR-fl-ENDO concentrations remained significantly elevated throughout the 60-min period following N A L administration. Serum PRL concentrations increased by 4-fold within 15 rain after N A L injection (P < 0.05). In contrast, two pituitary hormones, TSH and GH, were depressed during M O R withdrawal. Serum TSH concentrations were significantly decreased by greater than 60% at 30 and 60 min after NAL precipitated M O R withdrawal, while G H concentrations
Fig. 1 illustrates changes in serum LH, IR-flE N D O , PRL, TSH and G H during NAL-precipitated M O R withdrawal in the rat. To aid in the presentation, data were expressed as percent change in
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Fig. 1. Percent changes in serum immunoreactive fl-endorphin (IR-fl-ENDO), luteinizing hormone (LH), prolactin (PRL), thyroid-stimulating hormone (TSH), and growth hormone (GH) concentrations in MOR-dependent rats following the injection of NAL (1 mg/kg s.c.). Control animals (time = 0) received saline vehicle and were sacrificed 5 rain after injection. • P < 0.05 vs time = 0.
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Fig. 2. Changes in the concentrations of norepinephrine (NE)
and normetanephrine (NME) in the medial basal hypothalamus (MBH) and preoptic area-anterior hypothalamus (POA) following the injection of NAL (1 mg/kg s.c.) to MOR-dependent rats. Control rats (time = 0) received saline vehicle and were sacrificed 5 min after injection. * P < 0.05 vs time = 0.
18 were decreased 15 min after NAL injection (P < 0.05) and were undetectable in serum 60 min after NAL injection. During the course of NAL-induced withdrawal in MOR-dependent rats concentrations of NE in the MBH were reduced by 26% and 28% by 30 and 60 min respectively (P < 0.05, Fig. 2). Similarly, in the PAO-AH, a 29% and 40% decrease in NE concentrations occurred over the same time period. In both tissues, the decline in NE concentrations was preceded by a 2- to 3-fold increase in the concentrations of NME. Elevated NME levels persisted for 60 min in the MBH and 30 min in the P O A - A H (P < 0.05). In contrast, DA concentrations increased in the MBH, being significantly elevated by 21% and 40% at 30 and 60 min after NAL-induced MOR withdrawal (Fig. 3). In the P O A - A H , D A concentrations were increased 34% at 60 min after N A L injection, while concentrations of the D A metabolite, D O P A C , were increased in both hypothalamic regions at 30 and 60 min after NAL injection to MOR-dependent rats (P < 0.05).
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Fig. 3. Changes in the concentrations of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) in the medial basal hypothalamus (MBH) and preoptic area-anterior hypothalamus (POA) following the injection of NAL (1 mg/kg s.c.) to MORdependent rats. Control rats (time = 0) received saline vehicle and were sacrificed 5 min after injection. * P < 0.05 vs time = 0.
DISCUSSION
The present investigation shows that anterior pituitary hormones respond dramatically but differentially to acute M O R withdrawal, precipitated by the opiate antagonist, NAL. These hormonal alterations are accompanied by an apparent increase in neurotransmitter metabolism by noradrenergic and dopaminergic nerve terminals contained within the MBH and its closely associated POA-AH. In response to NAL, NE turnover was increased in both the MBH and P O A - A H , as shown by the rapid increase in NME concentrations and the subsequent decline in NE concentrations. NME is formed by the action of catecholamine-o-methyl transferase on released NE u. Thus, the dramatic increase in NME concentrations by 15 rain after N A L administration indicates a preceding release of NE in response to the narcotic antagonist. This rapid and sustained increase in NE release likely explains the decline in NE concentrations which occurs by 30-60 rain. The observations that NE release is increased in the thalamus 4 and locus coeruleus neuronal activity is increased 1 indicate that the activation of hypothalamic noradrenergic nerve terminals is part of a more generalized stimulation of brain noradrenergic systems during M O R withdrawal. The response of hypothalamic dopaminergic neurons to M O R withdrawal differed in two respects from that of noradrenergic neurons. First, levels of DOPAC, the major acid metabolite of DA 43, increased in both the MBH and P O A - A H , suggesting an increase in D A neurotransmission, while D A concentrations also increased. In view of the evidence for increased tyrosine hydroxylase and monoamine oxidase 40 activity in the caudate nucleus and tyrosine hydroxylase in the whole brain during M O R abstinence TM, our data suggest that D A turnover in the MBH and P O A - A H is increased and this is associated with an increase in de novo D A synthesis. Secondly, while NE turnover is increased by 15 min after NAL injection, the increase in D A turnover is delayed until 30 rain into withdrawal. This delay in activation of D A neurons may relate to the differential response of pituitary hormones to withdrawal, as discussed below. IR-fl-ENDO and LH showed a rapid secretory response to NAL-induced withdrawal while the re-
19 sponse of PRL was delayed. The increase in IR-flE N D O during withdrawal has been observed previously in both laboratory animals and human addicts in studies which evaluated a single time after N A L administration15, 22. The source of the IR-fl-ENDO which responds to withdrawal is likely to be the anterior pituitary gland, since circulating glucocorticoids increase during withdrawal and adrenocorticotropic hormone and fl-endorphin are released concomitantly from the anterior pituitary in response to many stimuli including stress 17,21,47. We and others, using less frequent sampling times, have observed marked increases in L H secretion following N A L administration to MOR-dependent ratsS, 45. In contrast, Morely et al. 34, did not observe changes in serum LH or PRL at 30 min after N A L administration to MOR-dependent male rats. Since for LH and PRL, the primary secretory response occurs within 15 min of NAL administration, it is likely that the apparent absence of a hormone secretory response in that earlier study was due to an inappropriate sampling time. We observed that both G H and TSH secretions decline from 15 to at least 60 min into withdrawal. These observations are consistent with previous reports of decreased G H and TSH levels at a single time point during M O R withdrawal32, 34. Since somatostatin can block the release of both TSH and GH, it is of interest that hypothalamic levels of somatostatin decline during M O R withdrawaP4, 50. If this rapid decline in hypothalamic somatostatin during M O R withdrawal represents increased release of the peptide, a reduction in both G H and TSH is a likely consequence. The brain noradrenergic system is stimulatory to LH38 and IR-fl-ENDO 46, while the role of NE in PRL secretion is unclear 47. It is not unreasonable, then, that associated with the initial increases in NE turnover was a marked increase in LH and IR-fl-ENDO secretion. By 30-60 min, when M B H and P O A - A H DA activity is increased, PRL secretion decreases but LH and IR-fl-ENDO levels remain elevated. While D A is clearly inhibitory to PRL secretion 31 and may account for the transient nature of the PRL response, the role of MBH and P O A - A H D A neurons in LH and IR-fl-ENDO secretion is less well defined 24,46. Thus, we are unable to draw firm conclu-
sions as to the role of the late acceleration in D A turnover on LH and IR-fl-ENDO secretion. Treatment of animals with selective antagonist of adrenergic or dopaminergic receptors, prior to N A L administration would help to resolve the role of these neuronal systems in the observed hormonal response. While NE is stimulatory to G H and TSH24, 46, D A appears to exert an inhibitory effect on TSH 23 and a stimulatory effect on GH24, 29. The inhibitory effects of withdrawal on TSH from 30 to 60 min can be explained by the acceleration in D A neuronal activity over this time period, but we cannot explain the decline in G H secretion after N A L administration in terms only of hypothalamic catecholamine neurons. The neuronal mechanism of this decline in G H secretion remains to be determined, but presumably involves enhanced release of somatostatin during withdrawap4, 50. Pretreatment of rats with somatostatin antagonists would resolve this issue. It should be noted that the neuronal and hormonal responses to NAL-induced M O R withdrawal are strikingly similar to those observed during acute stress. The acute response to stress includes increased hypothalamic NE turnover36,42,49,52, rapid increases in serum LH, PRL and IR-fl-ENDO2,14, 16,21,25,28,39,41, and diminished G H and TSH secretion14,25, although the response of hypothalamic D A neurons to stress is uncertain42, 52. Thus, M O R withdrawal may activate the same neuroendocrine mechanisms which mediate the response to stress. ACKNOWLEDGEMENTS The authors wish to thank Dupont Pharmaceuticals (Garden City, NY) for the generous gift of naloxone HC1 and the Pituitary Hormone Distribution Program of N I A D D K for the R I A Kits used. We also wish to recognize the technical assistance of Ms. Rebecca Thro and Ms. Sharon Layfield and the secretarial aid of Ms. Fran Paniello. This work was supported in part by N I H Grant AG02021 to J.W.S. and Grant HD17364 to W.J.M. A preliminary report of this work was presented at the 13th Annual Society for Neuroscience Meetings, Boston, MA, November 1983.
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15
BRE 11083
Changes in Anterior Pituitary Hormone Secretion and Hypothalamic Catecholamine Metabolism During Morphine Withdrawal in the Female Rat STEVEN M. GABRIEL1, JAMES W. SIMPKINS 1and WILLIAM J. MILLARD2
1Department of Pharmacodynamics, Universityof Florida, Collegeof Pharmacy, Gainesville, FL and 2Department of Neurology, Massachusetts General Hospital Harvard Medical School Boston, MA (U. S.A.) (Accepted January 15th, 1985)
Key words: luteinizing hormone-- prolactin--fl-endorphin-- growth hormone-- endogenous opiate - - morphine withdrawal-catecholamine metabolism-- norepinephrine - - dopamine-- thyroid stimulating hormone
Studies were undertaken to evaluate the acute responses of hypothalamic noradrenergic and dopaminergic neurons and anterior pituitary hormones to naloxone (NAL)-precipitated morphine (MOR) withdrawal in the rat. Ovariectomized female rats were rendered MOR-dependent and injected with NAL (1 mg/kg b.w., s.c.). During precipitated MOR withdrawal, a decline in norepinephrine (NE) concentrations was preceded by an increase in the level of its metabolite normetanephrine (NME) in the medial basal hypothalamus (MBH) as well as the preoptic area-anterior hypothalamus (POA-AH). Both dopamine (DA) and its major acid metabolite, dihydroxyphenylacetic acid (DOPAC), showed increased concentrations in these two hypothalamic regions within 30 min of NAL administration. Elevated luteinizing hormone (LH) and fl-endorphin secretion was evident within 5 min of NAL injection to MOR-dependent rats, while serum prolaetin (PRL) increased 15 min into MOR withdrawal. Both growth hormone (GH) and thyroid-stimulating hormone (TSH) were depressed over the course of MOR withdrawal. Although a cause and effect relationship cannot be established, during NAL-precipitated MOR withdrawal, a heightened hypothalamic monoaminergic neuronal activity is accompanied by a differential response of anterior pituitary hormones. The observed responses, which are similar to those seen during acute stress, indicate that MOR withdrawal may activate the same mechanisms which mediate the neuroendocrine response to stress.
INTRODUCTION Morphine ( M O R ) and endogenous opioid peptides (EOP) have been shown to exert profound effects on brain monoaminergic neurons and on the secretion of the anterior pituitary hormones which they regulate. M O R and E O P inhibit hypothalamic norepinephrine (NE)I, 19 and tuberoinfundibular dopamine ( D A ) turnover20,51. Additionally, opiates stimulate prolactin (PRL)30, 35, fl-endorphin 27, and growth hormone (Gn)30, 35 secretion, and inhibit luteinizing h o r m o n e (LH) 7,30 and thyroid-stimulating hormone (TSH) 44 release. Through these effects on brain monoaminergic neurons and pituitary h o r m o n e secretion, endogenous and exogenous opiates can influence homeostatic processes in a variety of organs. A consequence of continued M O R treatment and the resulting opiate dependence is the abstinence
syndrome subsequent to narcotic antagonist exposure or the cessation of M O R treatment. Opiate withdrawal is throught to result in activation of brain adrenergic neuronsl,4,13, 40. The capacity of clonidine to relieve some of the symptoms of MOR-abstinence9,22, 26, presumably by reducing the withdrawalinduced acceleration in N E turnover 1, suggests that noradrenergic neurons are involved in the symptoms of opiate withdrawal. The role of D A neurons in the mechanism of opiate withdrawal is less certain however. The importance of elucidating the neuronal systems involved in opiate dependence and withdrawal is indicated by recent evidence for E O P dependence associated with chronic hyperprolactinemia 47, chronic stress6, 33, and the endogenous production of addictive alkaloids during chronic alcohol ingestion 11. The mechanisms involved in M O R dependence and with-
Correspondence: J. W. Simpkins, University of Florida, Department of Pharmacodynamics, College of Pharmacy, Box J-4, J.H.M.H.C., Gainesville, FL 32610, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
16 drawal may also be present in these conditons of EOP dependence. Thus, in the present study we evaluated the acute response of hypothalamic noradrenergic and dopaminergic neurons and anterior pituitary hormones to naloxone (NAL)-precipitated MOR withdrawal in the rat. MATERIALS AND METHODS
Animals Female Charles River rats (Sprague-Dawley, Willmington, MA) weighing 200-240 g were maintained in a light- (lights on 07.00-19.00 h) and temperature (26 + 1 °C)-controlled room. Purina Lab Chow and water were provided ad libitum. Rats were allowed to acclimate to the animal facilities for one week prior to experimentation, and all surgeries were performed under light ether anesthesia.
Experimental treatment M O R dependency was induced by subcutaneous implantation of pellets containing 75 mg M O R freebase. These tablets were prepared in our laboratory using the procedure originally described by Gibson and Tingstad 18. Animals received 2 additional pellets at 2 days after the first implant. This dosage regimen produces typical signs of M O R dependence and withdrawal4L To determine the effects of precipitated M O R withdrawal on hypothalamic catecholamine metabolism and anterior pituitary hormone secretion, female rats were ovariectomized and rendered MORdependent as described above. After 3 days of continuous M O R exposure, groups of rats (6 per group) were treated with NAL HC1 dissolved in saline (1 mg/kg b.w., s.c.) and killed by decapitation 5, 15, 30 or 60 rain later. A control group of MOR-dependent rats (t = 0) received saline rather than N A L and were killed 5 min later. Trunk blood was collected for later serum hormone analysis, brains were rapidly removed, placed on ice, and tissues containing the medial basal hypothalamus (MBH) and preoptic areaanterior hypothalamus ( P O A - A H ) were dissected as described previously 23. Tissues were immediately homogenized in 0.4 N perchloric acid (PCA) containing 10 rag% E D T A at a ratio of 1 mg tissue/10 pl PCA. The average weights for these tissues were 15.9 _+ 0.3 mg for MBH and 12.9 + 0.3 mg for POA-AH.
Analysis of amines and metabolites Concentrations of NE and DA were determined by the radioenzymatic assay procedure of Cuello et al. 10 using catecholamine-o-methyl transferase isolated from the rat liver by the method of Nikodjevic et al. 37. The sensitivities of this assay for NE and DA were 50 pg and 20 pg respectively. The NE metabolite, normetanephrine (NME), and the D A metabolite, dihydroxyphenylacetic acid (DOPAC) were assayed by amperometric methods following their separation by high-pressure liquid chromatography (HPLC). The separation of these metabolites was accomplished using reverse-phase chromatography with a mobile phase composed of 8% methanol, 0.2 mM octyl sodium sulfate, 0.1 M NaHzPO ~, and 0.1 mM E D T A at pH 3.1. The flow rate of this mobile phase was 0.7 ml/min across a Supelco LC-18 column (15 cm x 4.6 mm, 5/~m particle size). Detection of the metabolites was accomplished with an electrochemical detector (Bioanalytical Systems, Model LC-2A), with a potential difference between the working electrode and a reference A g Ag/CI 2 electrode set at + 0.9 V and currents generated at 5 nA/mV. To 100 pl of each M B H and P O A - A H sample was added 2 ng of dihydroxybenzoic acid ( D H B A ) as an internal standard. The concentrations of each of the metabolites was determined by the peak-height ratios of metabolite to D H B A in comparison to a standard curve of peak-height ratios. A sensitivity of less than 100 pg was found for both NME and DOPAC.
Radioimmunoassays Serum samples were assayed in dupficate or triplicate for PRL, GH, LH and TSH using the procedures described in the N I A D D K Kits provided. Data for these 4 hormone are expressed in terms of their respective reference preparations PRL-RP-S2, GHRP-1, LH-RP-1, or TSH-RP-1. The intraassay coefficient of variation for the PRL, GH, LH and TSH assays determined with t0 replicates of pooled serum which inhibited the binding of the labeled ligand 40-60%, was 6.7%, 7.8%, 6.8% and 6.2% respectively. All samples were assayed in a single run and therefore no interassay variability was determined. The sensitivity of these assays, defined as a 20% inhibition of binding of labeled ligand was 25 ng for TSH, 5 ng for GH, 0.9 ng for LH and 0.25 ng for PRL. Se-
17 rum concentrations of fl-endorphin were determined by the method described by Arnold et al.3. In this assay there is a 100% cross-reactivity of fl-endorphin and fl-lipotropin (on a molar basis); therefore the term immunoreactive fl-endorphin will be employed (IR-fl-ENDO). However, the antisera used does not cross-react with methionine-enkephalin, a-endorphin or 7-endorphin at concentrations as high as 5 ng/tube 3. In the present study, serum was assayed in duplicate and results are expressed in terms of the camel fl-endorphin standard (Biochem, Torrance, CA). The intraassay coefficient of variation of the flendorphin assay was 9.8% and the minimum sensitivity of the assay was 30 pg/ml serum. Analysis of variance and S t u d e n t - N e w m a n Keuls tests were used to evaluate the significance of differences among treatment groups. A significance level of P < 0.05 was chosen. Six to eight rats were used for each determination. RESULTS
hormone concentrations compared to time = 0 (controis). Serum hormone concentrations in MORtreated rats at 5 min after saline injection (control) were 11.27 + 3.15 ng/ml for LH, 100.4 _+ 12.9 ng/ml for IR-fl-ENDO, 12.5 + 3.03 mg/ml for PRL, 296.4 + 90.5 ng/ml for TSH, and 467.8 +_ 96.6 ng/ml for GH. N A L injection to MOR-dependent rats resulted in a marked secretion of LH and IR-fl-ENDO. Serum LH concentrations were increased 8-fold and serum IR-fl-ENDO concentrations were elevated 6-fold by 5 min after N A L injection (P < 0.05). Serum LH concentrations remained elevated and were significantly increased 1 h after NAL, while IR-fl-ENDO concentrations remained significantly elevated throughout the 60-min period following N A L administration. Serum PRL concentrations increased by 4-fold within 15 rain after N A L injection (P < 0.05). In contrast, two pituitary hormones, TSH and GH, were depressed during M O R withdrawal. Serum TSH concentrations were significantly decreased by greater than 60% at 30 and 60 min after NAL precipitated M O R withdrawal, while G H concentrations
Fig. 1 illustrates changes in serum LH, IR-flE N D O , PRL, TSH and G H during NAL-precipitated M O R withdrawal in the rat. To aid in the presentation, data were expressed as percent change in
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Fig. 1. Percent changes in serum immunoreactive fl-endorphin (IR-fl-ENDO), luteinizing hormone (LH), prolactin (PRL), thyroid-stimulating hormone (TSH), and growth hormone (GH) concentrations in MOR-dependent rats following the injection of NAL (1 mg/kg s.c.). Control animals (time = 0) received saline vehicle and were sacrificed 5 rain after injection. • P < 0.05 vs time = 0.
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Fig. 2. Changes in the concentrations of norepinephrine (NE)
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18 were decreased 15 min after NAL injection (P < 0.05) and were undetectable in serum 60 min after NAL injection. During the course of NAL-induced withdrawal in MOR-dependent rats concentrations of NE in the MBH were reduced by 26% and 28% by 30 and 60 min respectively (P < 0.05, Fig. 2). Similarly, in the PAO-AH, a 29% and 40% decrease in NE concentrations occurred over the same time period. In both tissues, the decline in NE concentrations was preceded by a 2- to 3-fold increase in the concentrations of NME. Elevated NME levels persisted for 60 min in the MBH and 30 min in the P O A - A H (P < 0.05). In contrast, DA concentrations increased in the MBH, being significantly elevated by 21% and 40% at 30 and 60 min after NAL-induced MOR withdrawal (Fig. 3). In the P O A - A H , D A concentrations were increased 34% at 60 min after N A L injection, while concentrations of the D A metabolite, D O P A C , were increased in both hypothalamic regions at 30 and 60 min after NAL injection to MOR-dependent rats (P < 0.05).
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Fig. 3. Changes in the concentrations of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) in the medial basal hypothalamus (MBH) and preoptic area-anterior hypothalamus (POA) following the injection of NAL (1 mg/kg s.c.) to MORdependent rats. Control rats (time = 0) received saline vehicle and were sacrificed 5 min after injection. * P < 0.05 vs time = 0.
DISCUSSION
The present investigation shows that anterior pituitary hormones respond dramatically but differentially to acute M O R withdrawal, precipitated by the opiate antagonist, NAL. These hormonal alterations are accompanied by an apparent increase in neurotransmitter metabolism by noradrenergic and dopaminergic nerve terminals contained within the MBH and its closely associated POA-AH. In response to NAL, NE turnover was increased in both the MBH and P O A - A H , as shown by the rapid increase in NME concentrations and the subsequent decline in NE concentrations. NME is formed by the action of catecholamine-o-methyl transferase on released NE u. Thus, the dramatic increase in NME concentrations by 15 rain after N A L administration indicates a preceding release of NE in response to the narcotic antagonist. This rapid and sustained increase in NE release likely explains the decline in NE concentrations which occurs by 30-60 rain. The observations that NE release is increased in the thalamus 4 and locus coeruleus neuronal activity is increased 1 indicate that the activation of hypothalamic noradrenergic nerve terminals is part of a more generalized stimulation of brain noradrenergic systems during M O R withdrawal. The response of hypothalamic dopaminergic neurons to M O R withdrawal differed in two respects from that of noradrenergic neurons. First, levels of DOPAC, the major acid metabolite of DA 43, increased in both the MBH and P O A - A H , suggesting an increase in D A neurotransmission, while D A concentrations also increased. In view of the evidence for increased tyrosine hydroxylase and monoamine oxidase 40 activity in the caudate nucleus and tyrosine hydroxylase in the whole brain during M O R abstinence TM, our data suggest that D A turnover in the MBH and P O A - A H is increased and this is associated with an increase in de novo D A synthesis. Secondly, while NE turnover is increased by 15 min after NAL injection, the increase in D A turnover is delayed until 30 rain into withdrawal. This delay in activation of D A neurons may relate to the differential response of pituitary hormones to withdrawal, as discussed below. IR-fl-ENDO and LH showed a rapid secretory response to NAL-induced withdrawal while the re-
19 sponse of PRL was delayed. The increase in IR-flE N D O during withdrawal has been observed previously in both laboratory animals and human addicts in studies which evaluated a single time after N A L administration15, 22. The source of the IR-fl-ENDO which responds to withdrawal is likely to be the anterior pituitary gland, since circulating glucocorticoids increase during withdrawal and adrenocorticotropic hormone and fl-endorphin are released concomitantly from the anterior pituitary in response to many stimuli including stress 17,21,47. We and others, using less frequent sampling times, have observed marked increases in L H secretion following N A L administration to MOR-dependent ratsS, 45. In contrast, Morely et al. 34, did not observe changes in serum LH or PRL at 30 min after N A L administration to MOR-dependent male rats. Since for LH and PRL, the primary secretory response occurs within 15 min of NAL administration, it is likely that the apparent absence of a hormone secretory response in that earlier study was due to an inappropriate sampling time. We observed that both G H and TSH secretions decline from 15 to at least 60 min into withdrawal. These observations are consistent with previous reports of decreased G H and TSH levels at a single time point during M O R withdrawal32, 34. Since somatostatin can block the release of both TSH and GH, it is of interest that hypothalamic levels of somatostatin decline during M O R withdrawaP4, 50. If this rapid decline in hypothalamic somatostatin during M O R withdrawal represents increased release of the peptide, a reduction in both G H and TSH is a likely consequence. The brain noradrenergic system is stimulatory to LH38 and IR-fl-ENDO 46, while the role of NE in PRL secretion is unclear 47. It is not unreasonable, then, that associated with the initial increases in NE turnover was a marked increase in LH and IR-fl-ENDO secretion. By 30-60 min, when M B H and P O A - A H DA activity is increased, PRL secretion decreases but LH and IR-fl-ENDO levels remain elevated. While D A is clearly inhibitory to PRL secretion 31 and may account for the transient nature of the PRL response, the role of MBH and P O A - A H D A neurons in LH and IR-fl-ENDO secretion is less well defined 24,46. Thus, we are unable to draw firm conclu-
sions as to the role of the late acceleration in D A turnover on LH and IR-fl-ENDO secretion. Treatment of animals with selective antagonist of adrenergic or dopaminergic receptors, prior to N A L administration would help to resolve the role of these neuronal systems in the observed hormonal response. While NE is stimulatory to G H and TSH24, 46, D A appears to exert an inhibitory effect on TSH 23 and a stimulatory effect on GH24, 29. The inhibitory effects of withdrawal on TSH from 30 to 60 min can be explained by the acceleration in D A neuronal activity over this time period, but we cannot explain the decline in G H secretion after N A L administration in terms only of hypothalamic catecholamine neurons. The neuronal mechanism of this decline in G H secretion remains to be determined, but presumably involves enhanced release of somatostatin during withdrawap4, 50. Pretreatment of rats with somatostatin antagonists would resolve this issue. It should be noted that the neuronal and hormonal responses to NAL-induced M O R withdrawal are strikingly similar to those observed during acute stress. The acute response to stress includes increased hypothalamic NE turnover36,42,49,52, rapid increases in serum LH, PRL and IR-fl-ENDO2,14, 16,21,25,28,39,41, and diminished G H and TSH secretion14,25, although the response of hypothalamic D A neurons to stress is uncertain42, 52. Thus, M O R withdrawal may activate the same neuroendocrine mechanisms which mediate the response to stress. ACKNOWLEDGEMENTS The authors wish to thank Dupont Pharmaceuticals (Garden City, NY) for the generous gift of naloxone HC1 and the Pituitary Hormone Distribution Program of N I A D D K for the R I A Kits used. We also wish to recognize the technical assistance of Ms. Rebecca Thro and Ms. Sharon Layfield and the secretarial aid of Ms. Fran Paniello. This work was supported in part by N I H Grant AG02021 to J.W.S. and Grant HD17364 to W.J.M. A preliminary report of this work was presented at the 13th Annual Society for Neuroscience Meetings, Boston, MA, November 1983.
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