The effects of heroin on prolactin levels in male rhesus monkeys: use of cumulative-dosing procedures

Psychoneuroendocrinology 27 (2002) 319–336 www.elsevier.com/locate/psyneuen

The effects of heroin on prolactin levels in male rhesus monkeys: use of cumulative-dosing procedures夽 Carrie A. Bowen *, S. Stevens Negus, Maureen Kelly, Nancy K. Mello Alcohol and Drug Abuse Research Center, McLean Hospital-Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA Received 16 February 2001; received in revised form 12 June 2001; accepted 13 June 2001

Abstract There is considerable evidence that mu opioid receptors are involved in the regulation of anterior pituitary function. For example, in nonhuman primates and humans, mu agonists generally increase prolactin (PRL) levels. In contrast, mu antagonists decrease or have no effect on PRL levels. The goal of this study was to assess the potential utility of cumulative-dosing procedures to evaluate the endocrine effects of mu opioid receptor ligands. The effects of single and multiple, cumulative doses of the mu agonist heroin and the mu-selective antagonist quadazocine on PRL levels were investigated in four male rhesus monkeys. Cumulative dose-response curves were determined by infusing increasing drug doses at 60 min intervals over 290 min. Blood samples for PRL analysis were collected at 25 and 50 min after each cumulative infusion. Samples were collected at similar time points following single drug dose administration. Heroin (0.01–0.32 mg/kg, IV) administration dose-dependently increased PRL levels. Maximum levels of heroininduced PRL levels were equivalent after single and cumulative doses. Quadazocine alone (0.032–1.0 mg/kg, IM) did not alter PRL levels significantly. However, quadazocine (0.1 mg/kg, IM) antagonized heroin-stimulated increases in PRL levels and produced a significant rightward shift in the heroin dose-effect curve. These data suggest that a cumulative-dosing procedure similar to that used in behavioral pharmacology may be useful to study the endocrine pharmacology of mu opioids in rhesus monkeys.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Prolactin; Heroin; Quadazocine; Rhesus monkey; Cumulative dose; Single dose

* Corresponding author. Tel.: +1-617-855-2478; fax: +1-617-855-2519. E-mail address: [email protected] (C.A. Bowen). 夽 Preliminary data were presented at the 2000 annual meeting of the college on problems of drug dependence in San Juan, Puerto Rico. 0306-4530/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 3 0 ( 0 1 ) 0 0 0 5 3 - 1

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1. Introduction Prolactin (PRL) is synthesized and released by lactotrophs of the anterior pituitary gland. This hormone is regulated by multiple neurotransmitter systems in the central nervous system. For example, dopamine plays a major role in the inhibition of PRL secretion (Ben-Jonathan, 1985; Yen and Jaffe, 1999). Hypothalamic dopaminergic cells, called tuberoinfundibular dopamine (TIDA) neurons, are located in the arcuate nucleus and send axonal projections to the median eminence. Dopamine is secreted by these neurons into the portal circulation, and the secreted dopamine binds to D2 dopamine receptors in the plasma membranes of lactotrophs. D2 receptor stimulation by dopamine inhibits PRL synthesis and release (Maurer, 1980). TIDA neurons are unusual among dopaminergic cells because they do not contain autoreceptors and do not regulate dopamine release via local negative feedback mechanisms (Demarest and Moore, 1979; Yen and Jaffe, 1999). There is considerable evidence that endogenous opioid systems also regulate PRL secretion, possibly by inhibiting TIDA neurons and disinhibiting lactotrophs. Opioids produce their effects by acting at three main types of opioid receptors, the mu, kappa and delta receptors (Kieffer, 1995), and the mu and kappa receptors are thought to mediate the majority of opioid effects on PRL secretion (Gilbeau et al., 1985; Leadem and Yagenova, 1987; Soaje and Deis, 1999; Butelman et al., 1999a,b,c). For example, mu and kappa opioid receptors and neurons containing opioid peptides are present both in the arcuate nucleus of the hypothalamus, where TIDA neuron cell bodies are located, and in regions that innervate the arcuate nucleus (Khachaturian et al., 1985; Mansour et al. 1988, 1995; Peckys and Landwehrmeyer, 1999). In fact, opioid-containing terminals have been reported to contact directly TIDA neuron cell bodies (Fitzimmons et al., 1992). Opioid peptides are believed to reduce dopamine turnover and release from hypothalamic TIDA neurons (Annunziato, 1979; Van Loon et al., 1980; Wilkes and Yen, 1980; Demarest and Moore, 1981; Wehrenberg et al., 1981). Consistent with reduced dopamine turnover and release, both mu and kappa opioid agonists increase PRL levels in nonhuman primates and humans (Ellingboe et al., 1980; Gilbeau et al., 1985; Ur et al., 1997; Butelman et al., 1999a,b,c). In contrast, opioid antagonists decrease or have no effect on PRL levels in nonhuman primates and humans (Gold et al., 1979; Rubin et al., 1979; Volavka et al., 1979; Ellingboe et al., 1980; Gilbeau et al., 1985; Mello et al. 1989, 2000). The inconsistent effects of opioid antagonists may reflect procedural differences and/or fluctuations in endogenous opioid peptide levels and activity (Kerdelhue et al., 1983; Lim and Funder, 1983; Almeida et al., 1988). Diurnal variations in endocrine responses to mu opioid agonists and antagonists have been reported in humans, with lower sensitivity in the morning (Frecska et al., 1988; Martin del Campo et al., 1994). Thus, the inconsistent effects of opioid antagonists may be related to the time of day (e.g., morning versus afternoon or evening) in which the hormone samples were collected. Most studies of opioid effects on PRL secretion have used a pharmacological approach that involves the administration of a single dose of drug followed by measurements of the dependent variable(s) at specific times after drug administration. One limitation of this approach is that several studies are necessary to characterize

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the complete drug dose-response relationships that are necessary for a comprehensive pharmacological evaluation of a drug’s endocrine effects. In contrast, in behavioral pharmacology, cumulative-dosing procedures often are used to complement singledose studies and to assess complete dose-effect curves in a single test session (Boren, 1966). Cumulative dosing involves the administration of multiple, increasing doses of drug at regular intervals and measurement of the dependent variable(s) at one or more time points following each cumulative drug dose. This procedure has been used to study a range of drug-induced behavioral effects in nonhuman primates, including drug discrimination (Bertalmio et al., 1982; Lamas et al., 1995; Brandt et al., 1999), drug-induced antinociception (Dykstra et al., 1987; Negus and Mello, 1999) and schedule-controlled behavior (Bergman et al., 1985; Spealman et al., 1989; Negus et al., 1993). Cumulative-dosing procedures also have been used in physiological studies of drug-induced respiratory effects (Howell et al. 1988, 1990; Butelman et al., 1993). Recently, these procedures also have been employed in endocrine pharmacology to investigate the effects of kappa opioid receptor ligands on PRL levels in rhesus monkeys (Butelman et al., 1999a,b,c). The purpose of the present study was to evaluate the utility of cumulative dosing procedures in studies of mu opioid-induced endocrine effects and, in particular, to apply these procedures to a pharmacological analysis of the effects of heroin on PRL release in rhesus monkeys. Heroin is a widely abused opioid agonist that stimulates PRL secretion in humans (Ellingboe et al., 1980), but the pharmacological mechanisms of action that underlie heroin’s endocrine effects have not been elucidated fully. The effects of heroin are thought to be mediated by its metabolites, 6-monoacetyl morphine and morphine, which act primarily at mu opioid receptors (Way et al., 1960; Inturrisi et al., 1983; Selley et al., 2001). However, heroin and 6-monoacetylmorphine may also act directly or indirectly at delta opioid receptors (Rady et al. 1991, 1994; Martin et al., 2000). Moreover, although delta receptors appear to play a relatively minor role in the regulation of PRL release (Leadem and Yagenova, 1987; Walsh and Clarke, 1996; Soaje and Deis, 1999), delta agonists may stimulate PRL release under some conditions and may contribute to the effects of endogenous opioid peptides (Xu, 1992; Kehoe et al., 1993). To test the hypothesis that heroin’s effects on PRL release are mu-receptor mediated, the effects of heroin were determined both alone and after pretreatment with the opioid antagonist quadazocine (Ward et al., 1983). Quadazocine is selective for mu receptors, and it has been used extensively to characterize the behavioral effects of opioid agonists in rhesus monkeys (e.g., Negus et al., 1993). Initially, the effects of heroin and quadazocine were tested alone using both single-dosing and cumulative-dosing procedures to permit a comparison of results using the two dosing regimens. Subsequently, quadazocine was administered as a pretreatment both to a single heroin dose and to a cumulative series of heroin doses. Single-dosing studies assessed the ability of quadazocine to block the effects of a maximally-effective dose of heroin. Cumulative-dosing studies permitted rapid determination of complete heroin dose-effect curves in the absence and presence of the antagonist. These latter studies examined the degree to which heroin could surmount the antagonist effects of quadazocine and permitted the application of in vivo apparent pKB analysis to

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draw inferences regarding the receptor population mediating heroin’s endocrine effects.

2. Methods 2.1. Subjects These studies were conducted in four adult male rhesus monkeys (Macaca mulatta) that had been adapted to the laboratory and to primate restraint chairs on several occasions before the experiments began. The monkeys were maintained at ad lib weight (8.0–14.5 kg) and were fed monkey chow (Lab Diet Jumbo Monkey biscuits, PMI Foods, Inc., St. Louis, MO), fresh fruit, primate treats and multiple vitamins each day. Water was available continuously in the home cage via an automated watering system. The animal housing room was maintained at constant temperature and humidity. A 12-hour light/dark cycle was in effect (lights on at 0700 h). Monkeys also had access to toys, foraging trays and visual stimulation (other monkeys in the room and televised nature programs) for environmental enrichment. The experimental histories of the animals were varied and included studies of the endocrine effects of cocaine (monkeys 179F, 90B115 and 91D242), the endocrine effects of nalmefene (monkey 179F), the reinforcing effects of cocaine (monkey 90B115), and the analgesic effects of opioids (monkey 89B084). A repeated measures design was employed in which each monkey received all treatments and served as its own control. The experiments always were conducted at the same time of day, and successive studies were separated by at least two weeks. Animal maintenance and research were conducted in accordance with guidelines provided by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, NIH. This protocol was approved by the McLean Hospital Institutional Animal Care and Use Committee. The facility is licensed by the US Department of Agriculture. A veterinarian qualified in primate medicine oversaw the general health of the animals. 2.2. Acute saphenous vein catheterization On a study day, the morning feeding was postponed to prevent aspiration and vomiting during anesthesia. The monkey was sedated with ketamine (5–10 mg/kg, IM) prior to implantation of a saphenous vein catheter in each leg. Ketamine was used because it does not interfere significantly with the activity of the hypothalamic– pituitary–adrenal axis (Ferin et al., 1976). Using aseptic procedures, a 22 gauge Deseret radiopaque intracath (Deseret Medical, Parke–Davis Co., Sandy, UT) was inserted into a saphenous vein. The needle and internal stylet were removed, and the catheter was joined to a length of sterile silicon tubing. The saphenous vein in the opposite leg was implanted with an identical catheter using the same procedure. One catheter was used for IV drug administration and fluid replacement, and the other catheter was used for blood sample collection.

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Following catheterization, the monkey was placed in a standard primate restraint chair. To reduce any possible effects of stress or ketamine administration, blood sample collection did not begin until at least 90 min after catheterization. During this period, the animal was given limited food treats to minimize any possible effects of 18 h of food restriction on hormone levels (see Cameron and Nosbisch, 1991, for review). Each study began with the collection of a blood sample at time ⫺10 min, before administration of the treatment drug or control solution, to determine baseline levels of PRL. In single-dose experiments, the effects of the mu agonist heroin (0.032 and 0.32 mg/kg; IV), the mu-selective antagonist quadazocine (0.1 and 1.0 mg/kg, IM), and saline (IV) were examined. To study the time course of acute mu ligand effects, a single drug dose was administered as a bolus over 10–30 s starting at time 0 min and immediately followed by a saline flush. Blood samples were collected at 25, 50, 85, 110, 145, 170, 205, 230, 265 and 290 min after acute administration of drug or saline. Identical sample collection intervals were used during cumulative drug or saline administration (see below). In cumulative-dose experiments, an entire dose-effect function was determined for heroin (0.01–3.2 mg/kg; IV) or quadazocine (0.032–1.0 mg/kg, IM) within a single experimental session. For these studies, saline and a series of four drug doses were administered at 60 min intervals, and each dose increased the total, cumulative dose by 0.5 log unit. Blood samples were collected at 25 and 50 min after each injection. Following collection of the final blood sample, the animal was sedated with ketamine, the catheters were removed from the saphenous veins and the legs were bandaged. An iron injection (200 mg, IM; Iron–Gard 200, Fermenta Animal Health Co., Kansas City, MO) was administered to the animal to reduce the risk of anemia. Then, the monkey was returned to its home cage. Pretreatment tests also were conducted to determine if quadazocine antagonized the endocrine effects of heroin and if the antagonism was surmountable. In the singledose paradigm, a mu-selective dose of quadazocine (0.1 mg/kg; IM) was administered 30 min prior to heroin administration (0.32 mg/kg, IV) at time 0 min. In the cumulative-dose paradigm, quadazocine (0.1 mg/kg; IM) was administered at time ⫺1 min, followed by the administration of saline (at time 0 min) and cumulative heroin doses (at times 60, 120, 180 and 240 min). Blood samples were collected in a manner identical to that described above. The selection of this quadazocine dose and pretreatment time was based on data from behavioral experiments that demonstrated antagonism of the effects of mu agonists in rhesus monkeys (Negus et al., 1993). 2.3. Measurement of PRL concentrations Plasma PRL concentrations initially were measured in duplicate using a direct, double-antibody radioimmunoassay kit purchased from Pantex, Santa Monica, CA. Due to the discontinuation of this PRL kit, a different PRL kit was used for some experiments. The new kit purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA), measured serum PRL concentrations in duplicate using a solid-phase immuno-

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radiometric assay. Results were expressed in nanograms per milliliter (ng/ml). For the Pantex kit, the assay sensitivity was 1.7 ng/ml and the intra- and interassay coefficients of variation were 9.3 and 11.8%, respectively. For the ICN kit, the assay sensitivity was 0.10 ng/ml and the intra- and interassay coefficients of variation were 4.8 and 5.8%, respectively. 2.4. Drugs Heroin hydrochloride was obtained from the National Institute on Drug Abuse (Rockville, MD). Quadazocine methanesulfonate was supplied by Sanofi Pharmaceuticals (Malvern, PA). Drugs were dissolved in sterile water at a concentration of 50 mg/ml. Stock solutions were filter-sterilized using a 0.22 micron filter and stored in sterile pyrogen-free vials. Dilutions of stock solutions with sterile saline permitted injection of the appropriate drug doses in the appropriate volumes (0.1–1.5 ml). Drug doses were calculated on the basis of the salt form of the drugs and each monkey’s body weight. 2.5. Data analysis To control for differences in baseline hormone levels between animals, the PRL data were transformed to percent of baseline levels prior to analysis. The level of significance was P⬍0.05 for all analyses. For single-dose studies, the data were analyzed as percent change from baseline PRL values over time. The effects of each dose of heroin and quadazocine were analyzed by a two-factor repeated measures analysis of variance (ANOVA; SuperAnova, Abacus Concepts, Inc., Berkeley, CA) with dose and time as the two factors. If a factor showed significance, post-hoc comparisons were carried out using Sheffe’s F test. Since only three of the four monkeys were tested with the lower heroin dose (0.032 mg/kg, IV), the statistical analyses were performed with and without the inclusion of the low heroin dose data. A significant finding is reported if a factor showed a significant difference in both analyses. For cumulative-dose studies, PRL concentrations were measured at 25 and 50 min after each injection. Because the two values usually were similar for a given monkey, the data were pooled and plotted as a single average value for each cumulative drug dose. Differences in PRL concentrations between baseline and drug dose administration were analyzed by a one-factor repeated measures ANOVA with dose as the single factor. In addition, an ED250 value was calculated for heroin in the presence and absence of the antagonist. The ED250 value was defined as the dose of heroin that produced a 250% increase in PRL concentration from the baseline value. A 250% change was chosen for this analysis because it was the greatest change observed in all four monkeys. Individual ED250 values were derived mathematically using the least-squares method by log–linear interpolation with at least three points on the linear portion of the dose-effect curve. Mean ED250 values were compared using a paired t-test. Paired t-tests also were used to compare the average maximal

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PRL stimulation following single- versus cumulative-dose administration of 0.032 and 0.32 mg/kg heroin. The mean in vivo apparent pKB value for quadazocine in combination with heroin was calculated using the equation pKB=⫺log(B/[DR⫺1]) (Negus et al., 1993). In this equation, pKB is the ⫺log dose of the antagonist that produces a two-fold rightward shift in the agonist dose-effect curve, B is the dose of antagonist in mol/kg, and DR is the ratio (dose ratio) of the ED250 for heroin in the presence of the antagonist to the ED250 for heroin alone. In vivo apparent pKB analysis is a well-established method for quantifying the potency of an antagonist in blocking the effects of an agonist, and it can be used to draw inferences regarding the receptor population that mediates the agonist’s effects. For example, quadazocine binds to all three opioid receptor types, but it has highest affinity for mu opioid receptors, lower affinity for kappa receptors and even lower affinity for delta opioid receptors (Negus et al., 1993). In vivo, quadazocine antagonizes the effects of mu, kappa and delta opioid agonists; however, it is most potent in blocking the effects of mu agonists, less potent in blocking the effects of kappa agonists, and least potent in blocking the effects of delta agonists (Negus et al., 1993). As a result, pKB values for quadazocine antagonism of mu agonists are higher than pKB values for antagonism of kappa or delta agonists (e.g., Negus et al., 1993). In the present study, a pKB value for quadazocine antagonism of heroin was determined from cumulative dose-effect curves as described above and compared to the pKB values determined in other studies for quadazocine antagonism of highly selective mu, kappa and delta agonists. Our hypothesis predicted that the pKB value for quadazocine antagonism of heroin’s effect on PRL levels would be similar to pKB values for quadazocine antagonism of mu opioid receptor-mediated effects determined in other in vivo assays (Negus et al., 1993).

3. Results 3.1. Single-dose studies Mean untransformed baseline PRL levels ranged from 4.1–9.7 ng/ml using the plasma-based assay kit (Pantex) and 9.3–15.7 ng/ml using the serum-based assay kit (ICN Pharmaceuticals, Inc). Administration of saline and a low dose of heroin (0.032 mg/kg, IV) did not alter PRL concentrations significantly (data not shown). Fig. 1 shows the time course of changes in PRL concentrations after acute administration of 0.32 mg/kg heroin, 0.1 mg/kg quadazocine, and a combination of quadazocine (0.1 mg/kg) and heroin (0.32 mg/kg). Administration of 0.32 mg/kg heroin significantly increased PRL levels to an average (±1 SEM) of 931±214% of baseline (i.e., 6.6±1.8 ng/ml to 69.1±28.2 ng/ml) within 25 min after drug injection (P⬍0.05). PRL levels remained significantly above baseline for 230 min post injection. In contrast, 0.1 mg/kg quadazocine did not significantly alter PRL levels. Administration of a higher dose of quadazocine (1.0 mg/kg) also had no effect on PRL levels (data not shown). However, pretreatment with 0.1 mg/kg quadazocine significantly antagonized the

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Fig. 1. The acute effects of 0.32 mg/kg heroin, 0.1 mg/kg quadazocine and a combination of these drug doses on PRL levels. Data are expressed as the average (±1 SEM) percent of baseline response over time. Vertical lines indicate times of drug administration. Note that when heroin or quadazocine was administered alone, the drug was administered at time 0 min. When the drug combination was administered, quadazocine was administered as a pretreatment (time ⫺30 min) to heroin (time 0 min). Each data point is the average of four monkeys. An asterisk indicates that the PRL level was significantly different from baseline levels (P⬍0.05).

0.32 mg/kg heroin-induced increase in PRL levels. Following administration of the drug combination, PRL levels were similar to those following quadazocine alone. 3.2. Cumulative-dose studies Fig. 2 shows the effects of saline and cumulative doses of heroin and quadazocine on PRL levels. Saline was administered as five consecutive control infusions (Panel 1) or as the initial dose (0 mg/kg) in the cumulative-dose series (Panels 2 and 3). Saline administration did not significantly alter PRL levels. Cumulative heroin administration resulted in dose-related increases in PRL concentrations. PRL increases were significantly different from saline following administration of 0.1 and 0.32 mg/kg cumulative heroin (P⬍0.05). The highest dose of heroin, 0.32 mg/kg, produced an average PRL increase of 852±365% (i.e., 7.6±2.7 ng/ml to 46.6±17.0 ng/ml). PRL levels were not altered significantly by cumulative administration of quadazocine. The effect of the antagonist quadazocine on the cumulative heroin dose-effect function is shown in Fig. 3. Pretreatment with a mu-selective dose of quadazocine (0.1 mg/kg) surmountably antagonized heroin-induced PRL secretion and produced a rightward shift of the dose-effect function. There was a significant difference between the heroin ED250 values determined in the presence and absence of the

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Fig. 2. The effects of successive doses of saline and cumulative doses of heroin and quadazocine on PRL levels. Data are expressed as the average (±1 SEM) percent of baseline response as a function of the number of saline injections or the cumulative heroin and quadazocine dose. Blood samples were collected 25 and 50 min after each saline or drug injection. Data from the two time points were pooled and averaged across monkeys to yield a single value for each cumulative drug dose. Each data point is the average of four monkeys. An asterisk indicates that the PRL level was significantly different from the 0 mg/kg heroin condition (P⬍0.05).

Fig. 3. The effects of saline and 0.1 mg/kg quadazocine pretreatments on the cumulative heroin doseeffect function. Data are expressed as the average (±1 SEM) percent of baseline response as a function of cumulative heroin dose. Blood samples were collected 25 and 50 min after each drug injection. Data from the two time points were pooled and averaged across monkeys to yield a single value for each cumulative drug dose. Each data point is the average of four monkeys. The vertical line represents the time of quadazocine administration. B indicates baseline PRL levels prior to administration of quadazocine or heroin. An asterisk indicates that the PRL level was significantly different from baseline and 0 mg/kg heroin conditions (P⬍0.05). † Indicates heroin ED250 significantly different from saline pretreatment condition (P⬍0.05).

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antagonist (i.e., 0.72±0.14 vs. 0.04±0.02 mg/kg, respectively, P⬍0.05). The mean (±1 SEM) in vivo apparent pKB value for quadazocine in combination with heroin was 8.0 (±0.18). Fig. 4 summarizes the maximal PRL responses in individual monkeys and the group 25–50 min following single- and cumulative-dose administration of heroin. The top panel shows PRL levels following 0.032 mg/kg heroin. The magnitudes of the PRL responses to 0.032 mg/kg heroin showed some variability across monkeys and across administration conditions (i.e., range of 161–344% of baseline). However, the group data indicate that the average (±1 SEM) PRL stimulation did not differ significantly between the single- and cumulative-dose administration conditions (i.e., 229±58% and 257±32% of baseline, respectively; P=0.91). The bottom panel of Fig. 4 shows PRL levels following 0.32 mg/kg heroin. Similar to the 0.032 mg/kg heroin data, the magnitudes of the PRL responses to 0.32 mg/kg heroin showed variability across monkeys and across administration conditions (i.e., range of 280–1881% of baseline). However, the group data indicate that the average PRL stimulation did not differ significantly between the single- and cumulative-dose administration conditions (i.e., 922±126% and 852±365% of baseline, respectively; P=0.82).

4. Discussion 4.1. Opioid effects on prolactin levels The present experiment was designed to further characterize mu opioid receptorrelated endocrine effects in nonhuman primates by examining the effects of a mu agonist, heroin, and a mu-selective antagonist, quadazocine, on PRL levels. Each mu opioid ligand was administered under both single- and cumulative-dosing conditions. The data obtained suggest that a cumulative-dosing procedure may be an effective way to examine the dose-related endocrine effects of opioids. Most information about the effects of heroin on PRL secretion has come from studies in human drug abusers (Chan et al., 1979; Ellingboe et al., 1980; Spagnolli et al., 1987; Ragni et al., 1988). To our knowledge, this is the first study of the effect of heroin on PRL levels in rhesus monkeys. The overall findings are consistent with previous reports of the effects of other mu opioid agonists on PRL levels. As with heroin in the present study, other mu agonists (e.g., morphine) have been shown to rapidly increase PRL levels in rats (Shaar and Clemens, 1980), normal human subjects (Delitala et al., 1983; Pende et al., 1986) and Macaque monkeys (Gold et al., 1979; Wehrenberg et al., 1981; Belchetz et al., 1982; Gilbeau et al., 1985; Van Vugt et al., 1989). In macaque monkeys, mu agonists appear to stimulate PRL levels via dopaminergic mechanisms (Wehrenberg et al., 1981). In the present study, the PRL-stimulating effect of heroin was surmountably antagonized by the mu opioid antagonist, quadazocine. Other investigators have reported antagonism of morphine-induced PRL secretion by the opioid antagonists naloxone and nalmefene in monkeys, but these studies did not examine the surmountability of the effect (Gold et al., 1979; Gilbeau et al., 1985; Van Vugt et al., 1989).

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Fig. 4. The effects of single and cumulative administration of 0.032 and 0.32 mg/kg heroin on PRL levels in individual monkeys and the group. Data are expressed as the percent of baseline response as a function of the subject number or group. Blood samples were collected at 25 and 50 min after heroin administration. Data from the two time points were averaged for each monkey, and pooled and averaged across monkeys, to yield a single PRL value for each acute and cumulative heroin dose. The abbreviation “nt” indicates that the single dose of 0.032 mg/kg heroin was not tested in monkey 90B115. Therefore, group data for the 0.032 mg/kg heroin conditions represent averages of three (single-dose administration) and four (cumulative-dose administration) monkeys. Group data for the 0.32 mg/kg heroin conditions represent averages of four monkeys. The dashed vertical line indicates the separation between individual and group data.

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In addition, quadazocine alone did not affect PRL release significantly. This finding is consistent with a study of the effects of oral naltrexone in men with a history of heroin addiction (Ellingboe et al., 1980) and with a study of rhesus monkeys treated with the opioid antagonist nalmefene, IV (Mello et al., 2000). However, administration of the opioid antagonists naloxone and naltrexone, IV, has been reported to decrease basal PRL levels in monkeys (Gold et al., 1979; Gilbeau et al., 1985; Mello et al., 1989). As noted in the Introduction, one explanation for the reported differences in the effects of opioid antagonists may be fluctuations in endogenous opioid peptide levels and activity (Kerdelhue et al., 1983; Lim and Funder, 1983; Almeida et al., 1988). In rats, there is evidence for diurnal changes in hypothalamic and pituitary opioid peptide levels (Kerdelhue et al., 1983; Lim and Funder, 1983). Specifically, β-endorphin levels peaked late in the day, around 1600 h in the medial basal hypothalamus and around 2000 h in the anterior lobe of the pituitary (Kerdelhue et al., 1983). Circadian rhythms also have been reported in the number of opioid receptor binding sites in the rat hypothalamus, with maximal binding observed late in the day, around 2200 h (Naber et al., 1981; Giardino et al., 1989). Thus, during the later part of the day when hypothalamic opioid tone and corresponding PRL levels are sufficiently high, the administration of an opioid antagonist may block the effect of endogenous opioids and decrease PRL secretion. Conversely, during the earlier part of the day when endogenous hypothalamic opioid tone and corresponding PRL levels are low, the administration of an opioid antagonist may not significantly affect PRL levels. Diurnal variations in endocrine responses to naloxone and the mu agonist fentanyl have been reported in humans, with significantly greater responses observed in the afternoon and evening (Frecska et al., 1988; Martin del Campo et al., 1994). Alternatively, a floor effect may explain the inconsistent effects of opioid antagonists on PRL levels. Because basal PRL concentrations are low (i.e., usually below 10 ng/ml), significant antagonist-induced decreases may be difficult to observe. This explanation is consistent with the fact that nonhuman primate studies that reported lower baseline PRL levels (below 20 ng/ml) were unable to detect a significant effect of an opioid antagonist on PRL levels (present study; Butelman et al., 1999a,b,c; Mello et al., 2000); whereas, a study reporting higher baseline PRL levels (20–27 ng/ml) detected a significant reduction of PRL levels following opioid antagonist treatment (Mello et al., 1989). The reasons for these differences in basal PRL measurements are unclear, but they may include differences in the time of day of the experiments, differences in the initial stress levels of the animals and/or differences in the PRL assays. 4.2. Cumulative-dosing: advantages and limitations Often, the effects of a single dose of a drug on an endocrine endpoint are studied, and several studies are necessary to characterize a complete drug dose-effect curve. In contrast, the cumulative-dosing procedure involves the administration of multiple, increasing drug doses over the course of an experimental session, permitting the determination of a complete drug dose-effect curve within a single study day. Thus,

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the cumulative-dosing procedure may be used to rapidly characterize a drug’s potency and efficacy in an individual animal. Cumulative-dosing procedures have been used successfully in behavioral pharmacology research to study the effects of multiple drug doses within a single test session. Cumulative-dosing procedures have been employed in studies of drug discrimination (Bertalmio et al., 1982; Lamas et al., 1995; Brandt et al., 1999), drug-induced antinociception (Dykstra et al., 1987; Negus and Mello, 1999) and schedule-controlled behavior (Bergman et al., 1985; Spealman et al., 1989; Negus et al., 1993). In an early report, Bertalmio et al. (1982) noted the similarity of results between single- and cumulative-dosing procedures in rhesus monkeys trained in a drug discrimination paradigm. Single- and cumulative-dosing procedures also have yielded similar results in other drug discrimination studies in rats, rhesus monkeys and humans (Lamas et al., 1995; Schechter, 1997; Smith and Bickel, 1999). In the present study, the physiologic validity of the cumulative-dosing procedure was demonstrated by the fact that single- and cumulative-dose heroin administration resulted in average maximal increases in PRL levels that were not statistically different. These results demonstrate that cumulative administration of heroin produces dose-related stimulation of PRL levels and that single and cumulative doses of heroin produce similar increases in PRL levels. Such findings suggest that cumulative-dosing procedures may have utility in endocrine pharmacology as well as behavioral pharmacology research. One example of the utility of cumulative-dosing procedures is in the conduct of antagonism studies to investigate receptor mechanisms that mediate drug-induced endocrine effects. To our knowledge, Butelman et al. (1999a,b,c) were the first to employ a cumulative-dosing paradigm to measure endocrine endpoints. Specifically, dose-dependent increases in PRL levels following cumulative administration of opioid agonists with different efficacies at the kappa receptor were reported (Butelman et al., 1999a,b,c). Cumulative administration of the long-acting opioid antagonist nalmefene did not increase PRL levels, but nalmefene (0.1 mg/kg, SC) attenuated the release of PRL induced by cumulative kappa agonist administration (Butelman et al., 1999a). In addition, quadazocine (0.32 and 1.0 mg/kg, SC) produced a surmountable antagonism of cumulative kappa agonist-induced PRL release (Butelman et al., 1999b,c). In one study, quadazocine (0.32 mg/kg, SC) pretreatment produced a 10-fold rightward shift in the dose-effect curve for the kappa agonist U69,593, and the in vivo apparent pKB of quadazocine against the effects of U69,593 on PRL levels was 7.2 [95% CL=6.7–7.6] (Butelman et al., 1999c). In contrast, in the present study, quadazocine (0.1 mg/kg, IM) produced an 18-fold rightward shift of the heroin dose-effect curve, and the in vivo apparent pKB of quadazocine was 8.0 [95% CL=7.7–8.3]. This in vivo apparent pKB value is similar to those reported for quadazocine’s antagonism of other mu opioid receptor-mediated effects (Negus et al., 1993). Together, these findings are consistent with the conclusion that the effects of heroin on PRL levels are mediated by mu opioid receptors. In addition, there is approximately a 10-fold difference in the ability of quadazocine to antagonize kappa and mu opioid receptor-mediated effects on PRL levels. Overall, these findings

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indicate that a cumulative-dosing procedure can be used to conduct comprehensive pharmacological analyses in endocrine research. One limitation of the cumulative-dosing procedure is that drug doses must be administered in ascending order to reduce the possibility that the effect of the previous dose will alter the response to the subsequent dose. Also, the determination of an appropriate inter-injection interval requires prior knowledge of the time course of a drug-stimulated effect. If a drug has active metabolites, the results may be difficult to attribute to any specific compound without further study. Although a wide dose range can be studied using a cumulative-dosing regimen, doses must be selected carefully to avoid toxic cumulative effects. Lastly, the present findings and those of Butelman et al. (1999a,b,c) indicate the utility of the cumulative-dosing procedure to study the PRL-stimulating effects of both mu and kappa opioid agonists, but additional research will be necessary to determine if the results generalize to the study of other hormones and other drug classes. 4.3. Conclusions The most significant finding of this study was that the same opioid drugs produced similar effects on PRL levels under both single- and cumulative-dose conditions. Although this was not the first study to use cumulative-dosing procedures in endocrine research, the strength of this report lies in the fact that it directly compared single- and cumulative-dose administration and demonstrated the reliability of cumulative-dosing procedures. In addition, cumulative-dosing procedures permit thorough investigations of dose-related effects in a fraction of the time that it would take to study dose-effect relationships using traditional single-dose procedures. Overall, we conclude that cumulative-dosing procedures facilitate the study of dose-related endocrine effects of opioids in a systematic, reliable and efficient manner. Moreover, the present results confirm that heroin dose-dependently stimulates PRL levels, and antagonism studies with quadazocine indicate that heroin’s effects are mediated by mu opioid receptors.

Acknowledgements This research was supported by grants T32-DA07252, P50-DA04059 and K05DA00101 from the National Institutes of Health, National Institute on Drug Abuse. The technical assistance of Rebecca Callahan, Nicholas Diaz–Migoyo, Ashton Koo, Kendall Szeliga, David Zuzga, and Glen Matlock is appreciated greatly. We thank Beth A. Moseley, D.V.M, for veterinary care and Bruce J. Stephen for assistance with data analysis. We also wish to thank Dr. Johannes Veldhuis for thoughtful discussions on this topic.

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