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NEUROPHARMACOLOGY

Suppressed Prolactin Response to Dynorphin A_1–13 in Methadone-Maintained Versus Control Subjects

The Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, New York

Received February 20, 2003; accepted April 24, 2003.

	Abstract

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Dynorphin A_1–13, a shortened sequence of the natural peptide dynorphin A_1–17, is a primarily -opioid receptor-preferring peptide. Previously, we showed that dynorphin A_1–13 administered to normal volunteers causes a prompt dose-dependent elevation in serum prolactin that may reflect a reduction in tuberoinfundibular dopaminergic tone. This study was conducted to determine whether tuberoinfundibular dopaminergic tone is reduced in methadone-maintained patients. Eight former heroin addicts on stable-dose methadone maintenance with no ongoing drug or alcohol abuse or dependence and 15 normal volunteer controls with no history of drug or alcohol dependence received dynorphin A_1–13 intravenously at doses of 120 µg/kg and 500 µg/kg. Studies began one hour before methadone dosing to avoid the expected increase in prolactin that coincides with peak plasma levels of methadone. After intravenous dynorphin A_1–13, a dose-response increase in serum prolactin, which peaked within 20 min, was observed in both groups. There was no difference in prolactin between the two groups at baseline or following a placebo. The prolactin response to each dose of dynorphin A_1–13 was significantly lower in the methadone-maintained volunteers compared with the controls. These results suggest that tuberoinfundibular dopaminergic tone is altered in methadone-maintained subjects. It is unknown whether altered dopaminergic tone existed before opiate addiction, is a result of heroin addiction, or is reflective of methadone maintenance. Whether methadone-maintained subjects also have decreased dopaminergic response to dynorphin and other -opioid receptor ligands in mesolimbic-mesocortical and nigrostriatal dopaminergic systems cannot be determined from this study.

Messenger RNA for prodynorphin, the precursor of the endogenous dynorphin peptides (dynorphin A and dynorphin B), is expressed throughout the mammalian brain with wide distribution within the limbic system (Hurd, 1996). Similarly, -opioid receptor mRNA is widely expressed within the limbic system of the human brain (Mathieu-Kia et al., 2001). The large presence of mRNA for these two proteins in the limbic region implicates the -opioid system in the modulation of affective states and the addictive diseases. -Receptor expression is increased in the nucleus accumbens and the caudate putamen of rats after chronic binge-pattern cocaine and, in human cocaine overdose victims, -receptor expression is increased in the nucleus accumbens and the amygdala (Unterwald et al., 1994; Staley et al., 1997). Similar drug-induced changes have also been reported for µ-opioid receptor expression (Unterwald et al., 1994; Zubieta et al., 1996).

µ- and -Receptor expression has been further localized to dopaminergic neurons within the mesolimbic-mesocortical, nigrostriatal, and tuberoinfundibular dopamine (TIDA) systems. Dynorphin A_1–17 infused directly into the nucleus accumbens of awake, freely moving rats decreases, whereas -endorphin, the principal endogenous µ-opioid receptor ligand, administered intracerebroventricularly increases basal levels of extracellular dopamine in the nucleus accumbens (Spanagel et al., 1991; Claye et al., 1997). In humans, dopamine is the final common mediator of prolactin release (Freeman et al., 2000). More specifically, TIDA neurons inhibit prolactin release through activity at dopamine D2 receptors in the anterior pituitary (Freeman et al., 2000). In this study, changes in serum prolactin after dynorphin A_1–13 administration are, therefore, interpreted as reflecting TIDA tonal response to dynorphin A_1–13 administration.

We have previously shown that dynorphin A_1–13 produces a dose-related increase in serum prolactin after intravenous administration to healthy human volunteers (Kreek et al., 1999). This effect is attenuated by pretreatment with either naloxone, a µ-preferring opioid antagonist with modest affinity at -receptors, or, to a greater extent by nalmefene, an opioid antagonist with high affinity at both µ- and -receptors (Kreek et al., 1999). These results suggest that dynorphin A_1–13 lowers TIDA through action at - and µ-receptors. Although dynorphin A_1–13 has been administered to human heroin addicts and patients on chronic opioid therapy for pain, none of these studies used a control group that would have allowed evaluation of possible dopaminergic modulation after long-term opiate exposure (Wen and Ho, 1982; Specker et al., 1998; Portenoy et al., 1999).

Stable-dose methadone therapy allows normalization in responsivity of several physiological systems (e.g., hypothalamic-pituitary-adrenal, hypothalamic-pituitary-gonadal, and immune) disrupted by heroin addiction (Kreek et al., 2002). Serum prolactin levels, however, increase 2 to 4 h after methadone administration (peak values coincide with peak plasma levels of methadone), a finding apparent during induction onto methadone therapy that persists during long-term stable dose methadone maintenance (Cushman and Kreek, 1974). This persistent methadone-induced increase in levels of serum prolactin may indicate that tolerance to the dopaminergic effect of methadone does not develop (Cushman and Kreek, 1974; Kreek, 1978). Serum prolactin response after dynorphin A_1–13 during methadone maintenance remains unknown. This study was conducted to determine whether the prolactin-elevating effect of intravenously administered dynorphin A_1–13 is modulated by chronic stable-dose methadone treatment. It is the first controlled study in stable-dose methadone-maintained patients to suggest that these subjects have an alteration in tuberoinfundibular dopaminergic tone.

	Materials and Methods

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Subjects. The study was approved by the Institutional Review Board of The Rockefeller University Hospital General Clinical Research Center (National Institutes of Health-General Clinical Research Center) and performed in accordance with the Helsinki Declaration of 1975. Written informed consent was obtained before participation; confidentiality was strictly maintained. Dynorphin A_1–13 was prepared for human use by Bachem (Torrance, CA) and its administration was conducted under Investigational New Drug status (held by M. J. Kreek, The Rockefeller University, Manhattan, NY) approved by the United States Food and Drug Administration. Normal volunteers were recruited from the local community by word of mouth and by advertisement. Stable methadone-maintained subjects (former opiate addicts without current drug or alcohol comorbidities) were recruited from local methadone maintenance clinics. All subjects were evaluated by laboratory personnel, consisting of registered nurses, internists, and psychiatrists trained in addiction medicine. Subjects were initially screened for the study by phone contact with a registered nurse. Evaluation for medical and psychiatric inclusion and exclusion criteria were made by an internist or psychiatrist using clinical interview, physical exam, and review of laboratory and corroborative data. Evaluations included general medical, psychiatric, and substance abuse histories; physical examination; EKG; and laboratory testing, including complete blood cell count, serum electrolytes, creatinine, blood urea nitrogen, liver function tests, thyroid function tests, hepatitis A, B, and C serologies, venereal disease research laboratory test, and urinalysis. Subjects recruited from methadone clinics gave consent for the investigators to speak with their counselors so that histories could be verified. Subjects were counseled about, and gave informed consent for, human immunodeficiency virus testing. Urine was collected on each of the outpatient visits and on a 24-h basis during inpatient studies. Aliquots of urine were tested daily, both during the screening process and during the inpatient stay, for the presence of mixed opioids, methadone, cocaine, cannabinoids, or benzodiazepines. Subjects were enrolled in the study and allowed to remain in the study only if outpatient and inpatient urine toxicology results were negative for all drugs (except methadone in the methadone-maintained group).

Females are more responsive to the prolactin effects of dynorphin A_1–13 (Kreek et al., 1999); therefore, to minimize any possible variance in serum prolactin levels due to gender, only male subjects were included in the current study. The study population consisted of two groups: stable-dose methadone-maintained former heroin addicts without ongoing drug or alcohol abuse or dependence and with no DSM-IV Axis I diagnosis other than opioid dependence (n = 8), ages 18 to 51 years (mean, 33.9 years; S.D., 13.8), including five smokers (one pack/day for three subjects and 0.5 pack/day for two subjects) and three nonsmokers; and normal healthy volunteer controls without DSM-IV Axis I diagnoses (n = 15), ages 25 to 53 (mean, 34.5 years; S.D., 7.4), including two smokers (1 and 0.5 pack/day, respectively) and 13 nonsmokers. Illicit drug use or alcohol use did not reach DSM-IV criteria for abuse or dependence in any of the controls. Subjects were free of significant active medical problems, including human immunodeficiency virus seropositivity, were not taking any prescription medications (other than methadone where appropriate) and were not regularly using over-the-counter medications or herbal preparations. All former heroin addicts were in a methadone maintenance treatment program for heroin dependence for a minimum of 6 months, and stabilized on the same dose of methadone for at least 1 month before the study (mean methadone dose 76.3 mg/day; S.D., 32.9; range, 20–120 mg/day). All subjects were medication-free (except methadone where appropriate) for at least 7 days before the study.

Procedures. Subjects were admitted to the inpatient unit one evening before the testing days. In most cases, testing was completed on three consecutive days. In some cases, testing was carried out during separate, closely scheduled admissions to accommodate time constraints of subjects. Subjects fasted at least 9 hours before the beginning of a testing day and were allowed to eat only after the first 2 h of testing had elapsed. Subjects who smoked were not permitted to do so from 60 min before and until 4 h after dynorphin A_1–13 or placebo infusion. Dynorphin A_1–13 was administered through, and blood samples were withdrawn from, a single indwelling intravenous catheter inserted at least an hour before the beginning of testing. In some cases, a functioning catheter from the previous day was used. Total blood volume sampled, including that taken during screening and testing, did not exceed 550 ml.

At 10:30 AM on each of the three testing days, a normal saline placebo (dynorphin A_1–13 0 µg/kg), low-dose dynorphin A_1–13 (120 µg/kg), or high-dose dynorphin A_1–13 (500 µg/kg) was administered in a single blind manner. To minimize risk, a dose run-up schedule was used at the request of the Food and Drug Administration. To further minimize risk and the dysphoria associated with rapid infusions of high-dose dynorphin A_1–13, the 500-µg/kg dose was infused over 8 min, whereas the placebo and low-dose dynorphin A_1–13 were infused over 2 min. Each subject received a placebo, the low dose of dynorphin A_1–13, and the high dose of dynorphin A_1–13, on separate days. Each subject served as his own control.

Methadone-maintained subjects received their daily dose of methadone at 11:30 AM, that is, 60 min after the test dose was administered. Peak plasma levels of methadone occur 2 to 4 h after methadone administration, and no increase in plasma levels of methadone occurs until 30 to 60 min after orally administered methadone (Inturrisi and Verebely, 1972; Kreek, 1973). Plateau levels are achieved approximately 6 h after dosing and are sustained at 22 to 26 h after methadone administration (Kling et al., 2000). Therefore, the study was designed to complete the observation of dynorphin A_1–13 effects before any methadone-induced effects on prolactin occur (i.e., to study the effects of dynorphin A_1–13 during plateau methadone levels).

Prolactin Assay. Serum prolactin levels were determined in blood samples drawn at sequential time points. Time points started 10 min before test substance administration, and then at time 0 (immediately before test substance administration), and then at 10, 20, 30, 40, 50, 60, 75, 90, 120, 150, 180, and 240 min after dynorphin A_1–13 or placebo administration. Blood was drawn into plain vacutainers, and immediately placed on ice. Samples were stored on ice for up to 40 min until centrifuged at 4°C at 1500g for 5 min. Serum was then removed, aliquoted, and stored at –40°C until assayed. Serum prolactin levels were determined by immunoradiometric assay procedures, with slight modifications (ICN Pharmaceuticals, Inc., Orangeburg, NY). Prolactin intra- and interassay coefficients of variation were 8.6 and 14.3%, respectively.

Data Analysis. Area under the curve (AUC) from 0 to 90 min after each dose of dynorphin A_1–13 (0, 120, and 500 µg/kg) was calculated for each subject. The dose-response effect of dynorphin A_1–13 on serum prolactin levels was examined in each subject group by one-way analysis of variance (ANOVA) with repeated measures, followed by Newman-Keuls post hoc tests. Then, to determine whether there were differences between groups in serum prolactin response to dynorphin A_1–13 administration, a two-way ANOVA of AUCs, group x dose, with repeated measure on the second factor, was used, followed by planned comparisons between groups at the 120- and 500-µg/kg doses. The complete time course of measured prolactin is shown for each group at each dose expressed as mean ± S.E.M.

A preliminary analysis of prolactin response, group (methadone-maintained, normal volunteers) x smoking status (yes or no) x dose (0, 120, and 500 µg/kg) showed no significant main effect of smoking status, nor any significant smoking status interaction effect, so the smoking status factor was not used in subsequent analyses.

	Results

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Prolactin Dose-Response Effect of Dynorphin A_1–13. AUCs of serum prolactin from 0 to 90 min after administration of dynorphin A_1–13 are shown in Fig. 1 with the responses of the normal volunteer (NV) control group in Fig. 1A and of methadone-maintained (MM) subjects in Fig. 1B. One-way ANOVA with repeated measures followed by Newman-Keuls post hoc tests showed that in the normal volunteers, both the 120- and the 500-µg/kg doses of dynorphin A_1–13 significantly increased prolactin AUCs (p < 0.0002) and that the 500-µg/kg dose caused a greater increase in prolactin AUC than did the 120-µg/kg dose (p < 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Dose response of serum prolactin measured as AUC from 0 to 90 min after infusion of dynorphin A1–13 is shown for the NV control group in A and for the MM group in B. Values shown are mean + S.E.M. One-way repeated measures ANOVA showed a significant main effect of dynorphin A1–13 dose in the normal volunteers, F(2,28) = 40.01, p < 0.000001. Newman-Keuls post hoc tests showed significant increases after both 120- and 500-µg/kg doses, ***, p < 0.0002, and that there was a greater response at the higher dose, , p < 0.05. In the methadone-maintained subjects also, there was a significant main effect of dynorphin dose, F(1,14) = 19.67, ***, p < 0.0001. Newman-Keuls post hoc tests showed that the 120- and the 500-µg/kg dose each significantly raised prolactin AUCs, **, p < 0.02 and ***, p < 0.0005, respectively, and that the higher dose caused a greater response, , p < 0.005.

In the methadone-maintained subjects also, there was a significant effect of dose, and both the 120- and the 500-µg/kg doses increased mean AUC over placebo (p < 0.02 and p < 0.0005, respectively). In these subjects also, the 500-µg/kg dose increased prolactin levels more than the 120-µg/kg dose (p < 0.005).

Difference between Methadone-Maintained and Control Subjects. The differences between subject groups in serum prolactin response patterns to administration of dynorphin A_1–13 are shown in Fig. 2. It is important to note that although there was no difference between subject groups in prolactin AUC from 0 to 90 min after the 0-µg/kg dose placebo administration, there was a significant difference between subject groups overall (p < 0.01). There was a significant effect of dose (p < 0.005), and groups differed in their response pattern across doses (p < 0.005). After both the 120- and 500-µg/kg doses, the mean AUC levels of the methadone-maintained subjects were lower than those of the normal volunteers at each dose (p < 0.005 and p < 0.02, respectively).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Differences in serum prolactin response to dynorphin A1–13 between subjects groups are shown at each dose. A two-way ANOVA, subject group x dose, with repeated measures on the second factor, showed a significant main effect of subject group, F(1,21) = 8.09, p < 0.01, a significant main effect of dose, F(2,42) = 39.55, p < 0.005, and a significant group x dose interaction, F(2,42) = 6.06, p < 0.005. Although there was no difference after the 0 dose placebo infusion, at both the 120- and 500-µg/kg dose, the prolactin response was significantly lower in the MM than in the NV group.

Secondary Prolactin Rise in Methadone-Maintained Subjects. Two to four hours after daily ingestion of methadone, there is a rise in serum levels of prolactin that can be seen in Fig. 3B, which shows all mean (± S.E.M.) prolactin measurements from 0 min before to 240 min after each dynorphin A_1–13 dose (0, 120, and 500 µg/kg). An arrow shows the time the daily methadone dose was taken on these study days. Thus, the time points of prolactin levels are from 60 min before to 180 min after the oral dose of methadone. As seen in Fig. 3B, the prolactin-elevating effect of dynorphin A_1–13 dissipates by the expected methadone-induced increase in prolactin. The serum prolactin values of the normal volunteers (without the methadone-induced secondary rise) are shown above in Fig. 3A. It is intriguing to note that in the methadone-maintained subjects, the relative peaks at 180 min are in inverse order to the magnitude of the dose of dynorphin A_1–13 they had received on that day. Due to limitations of total blood drawn, not all subjects had blood drawn at the 180-min time point in each condition, so statistical analyses could not be performed.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Mean (± S.E.M.) level of serum prolactin of the normal volunteers at each time point from 0 to 240 min after each dose of dynorphin A1–13 infusion are shown in A, and the serum prolactin levels of the methadone-maintained group in B. The time at which the methadone-maintained subjects took their daily dose of methadone is shown with an arrow. All subjects in each group had specimens drawn for measurements from 0 to 90 min, but due to limitations in blood volume over the 3 days of study, on each study day the number of samples from 120 to 240 min varied from a total of zero to 15 in the normal volunteers and from four to eight in the methadone-maintained subjects.

Lack of Relationship between Daily Methadone Dose and Prolactin Levels. Fig. 4 shows the prolactin AUCs from 0 to 90 min after the placebo dose plotted against the daily dose of methadone for each of the eight methadone-maintained subjects (Fig. 4A), and the increased AUC over placebo values after the 120- and 500-µg/kg dose of dynorphin A_1–13 (Fig. 4, B and C, respectively). It is clear from regression analysis that there is no significant correlation of methadone dose with prolactin AUC in the 0-µg/kg dose placebo condition nor is there a correlation in the increase over the 0-µg/kg dose of either the 120- or 500-µg/kg dose of dynorphin A_1–13.

View larger version (16K): [in this window] [in a new window]   Fig. 4. AUC of prolactin plotted against the daily dose of methadone after the 0-µg/kg dose of dynorphin A1–13 is shown in A with the regression line and 95% confidence intervals. In B and C are shown the increases in prolactin AUC over 0-µg/kg dose after the 120- and 500-µg/kg doses, respectively. (Note that the two lower data points overlap in B among the three subjects receiving the 90-mg dose of methadone.) In no case were the correlations significant (A: r = –0.2588; B: r = 0.1305; and C: r = 0.2750).

	Discussion

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Findings and Significance. Using levels of serum prolactin as a surrogate biomarker for TIDA tone, this study demonstrated that former heroin addicts on stable-dose methadone maintenance with no ongoing drug or alcohol abuse or dependence have an apparent reduction in TIDA responsivity to dynorphin A_1–13 administration. The effect was dose-dependent and significantly smaller than the dose-response increase in serum prolactin observed in the control group.

The observed decrease in TIDA responsivity to dynorphin A_1–13 in the methadone-maintained group may reflect a reduction in dopaminergic tone in these subjects. This may provide significant insight into an underlying substrate involved in the acquisition, persistence, and/or treatment of addictive diseases. Unfortunately, in human studies of the addictive diseases it is difficult to state when during the course of the addiction or its treatment such a reduction in TIDA tone may have taken place. Furthermore, it cannot be determined from this study whether these effects may be generalized to other, more behaviorally associated and characterized, dopaminergic pathways such as the mesolimbic-mesocortical dopamine systems.

Opiates and Prolactin. Prolactin, a hormone produced primarily in the anterior pituitary by dopamine D2-receptor containing lactotrophs, is a major regulator of mammary gland development and milk production. Its release seems to be mostly regulated through TIDA inhibition and no specific prolactin-releasing or -inhibiting factor has been identified in humans. In humans, there is circadian release of prolactin with an increase during the evening, although this is more an effect of sleep onset rather than true circadian regulation (Frantz, 1978).

Racemic methadone (that used in pharmacotherapy) has a plasma half-life of around 24 h (Inturrisi and Verebely, 1972; Kreek, 1973) and, during steady state, maintains approximately 30% µ-receptor occupancy, thereby allowing unoccupied µ-receptors to perform their usual physiological function (Kling et al., 2000). Although in vivo µ-receptor occupancy in the human brain during methadone maintenance has been determined with positron emission tomography (PET) imaging using the µ-selective antagonist [¹⁸F]cyclofoxy (Kling et al., 2000), no selective -receptor ligands are yet available for similar PET studies of -receptors in humans. Because methadone is a highly µ-receptor-selective full agonist and µ-receptors are available for normal physiological functions during methadone maintenance, there is no evidence that -receptor expression or function would become altered as a direct effect of methadone.

The effects of short- and long-acting opiates on serum prolactin have been extensively documented (for review, see Kreek et al., 2002). Active heroin addicts have increased prolactin levels compared with controls and drug-free medication-free former heroin addicts. During the initial period of methadone dose stabilization, serum prolactin levels rise 2 to 4 h after methadone dosing and may exceed normal levels as seen during active heroin addiction (Kreek, 1978). After methadone dose stabilization, however, serum prolactin levels return to within normal limits, although a daily opiate-induced elevation in prolactin coinciding with peak plasma levels of methadone persists (Kreek, 1978). Interestingly, although the level of prolactin and its expected evening, or sleep related, rise are normal in former heroin addicts on long-term methadone maintenance, tolerance to the methadone induced increase in serum prolactin does not develop (Kreek, 1978). This observation may indicate that, during chronic maintenance, methadone retains its ability to lower TIDA tone.

Opiates and Dopamine. The TIDA system is comprised of a small number of neurons with cell bodies in the periventricular and arcuate nuclei of the hypothalamus and axons projecting to the median eminence of the hypothalamus. Dopamine released from these axons enters the hypophysial portal capillary system and tonically inhibits prolactin release by binding to dopamine D2 receptors present on anterior pituitary lactotrophs. The TIDA system is tonically inhibited by dynorphinergic neurons (Manzanares et al., 1991). Peripherally administered dynorphin A_1–13, with its poor penetrance across the blood-brain barrier and opioid-specific actions, can be used as a pharmacological probe to evaluate the hypothalamic -opioid system (Chou et al., 1996; Gambus et al., 1998).

Administered peripherally, -endorphin and dynorphin A_1–13 each cause an increase in serum prolactin in normal human volunteers (Reid et al., 1981; Kreek et al., 1999). Studies in nonhuman primates and rats have confirmed that µ- and -opioid agonists suppress TIDA and elevate serum prolactin, changes that can be blocked by treatment with selective µ- and -opioid antagonists (Deyo et al., 1979; Durham et al., 1996; Butelman et al., 2002). Interestingly, although the selective µ-antagonist naloxone does not by itself affect serum prolactin in humans, we demonstrated previously in healthy volunteers that it attenuates dynorphin A_1–13-stimulated prolactin release, suggesting that dynorphin A_1–13 affects - and also µ-opioid systems (Kreek et al., 1999). In the same series of studies, doses of nalmefene (a µ-antagonist with modest -antagonism) with expected equipotence at µ-receptors as the naloxone resulted in greater attenuation of dynorphin A_1–13-induced prolactin release, reflecting action of dynorphin A_1–13 at - and also µ-receptors (Kreek et al., 1999).

The mesocortical and mesolimbic dopaminergic systems are central to the acquisition and maintenance of drug addiction and are inhibited by locally administered dynorphin A_1–17 and dynorphin A_1–13 and by systemically administered centrally penetrating -agonists (Spanagel et al., 1990; Claye et al., 1997). Di Chiara and Imperato (1988) have demonstrated µ-opioid agonist-induced elevations in dopamine release in the nucleus accumbens. An [¹¹C]raclopride PET study performed in 11 male active or former heroin addicts, nine of whom were on methadone maintenance, demonstrated decreased striatal uptake compared with controls, indicating reduced striatal dopamine D2 receptor availability and increased levels of striatal dopamine (Wang et al., 1997). These studies indicate that there are important interactions between the opioidergic and dopaminergic systems in the development of opiate addiction. There is, however, little evidence that the TIDA system is involved in these processes.

Dynorphin and Dopamine. Natural sequence dynorphin A_1–13 has high affinity at the -opioid receptor with approximately 5-fold less affinity at the µ-opioid receptor (Mansour et al., 1995). In vitro biotransformation studies of dynorphin A_1–13 and dynorphin A_1–17 in human and nonhuman primate blood have identified several peptides, including dynorphin A_1–6, which has greater affinity at µ- rather than -opioid receptors than dynorphin A_1–13 or dynorphin A_1–17 each of which has much greater affinity at - rather than µ-opioid receptors (Mansour et al., 1995; Chou et al., 1996; Yu et al., 1996). Although it is unknown whether dynorphin A_2–12, the major and nonopioid biotransformation product of dynorphin A_1–13, is pharmacologically active (as is the major dynorphin A_1–17 biotransformation product, the nonopioid dynorphin A_2–17), there is no indication that without the first tyrosine residue its action would be mediated through opioid receptors (Butelman et al., 1999). In fact, unlike dynorphin A_1–17, dynorphin A_2–17 does not stimulate prolactin release (Butelman et al., 1999). The findings of the present study were, therefore, likely mediated predominantly through the - (possibly also µ-) opioidergic effects of dynorphin A_1–13 and its major opioid biotransformation products. In addition, the size of dynorphin A_1–13, its rapid biotransformation into mostly nonopioid peptide fragments, and the physiological effects that persist beyond the time of biotransformation would predict that it has poor penetrance across the blood-brain barrier and that its prolactin elevating effects are - and possibly µ-mediated in the hypothalamic region lying outside of the blood-brain barrier. We have, however, reported that normal volunteer subjects experience negative subjective mood effects and positive drug effects after dynorphin A_1–13, indicating that there is some penetrance across the blood-brain barrier with mid-brain effects (King et al., 1999). In addition, dynorphin A_1–13 administration attenuated pain ratings in opiate maintained patients with chronic pain and lowered self-reported withdrawal symptoms in heroin addicts during acute withdrawal (Specker et al., 1998; Portenoy et al., 1999).

Although many of the effects opiates exert on dopaminergic pathways are indirect, mediated by opioid-responsive GABAergic interneurons (Svingos et al., 2001a), there are some direct interactions between the two systems. Ultra-structural studies of the nucleus accumbens have identified -opioid receptors on presynaptic dopaminergic terminals, possibly explaining the inhibitory effect of dynorphin on dopamine release in this region (Svingos et al., 2001b). In the TIDA system, neurons are primarily responsive to dopamine D2 receptor agonists (Durham et al., 1996). In the tuberoinfundibular and striatal dopaminergic systems, dopamine D2 receptor mediated activation may occur indirectly through inhibition of inhibitory dynorphinergic neurons. -Agonist-induced prolactin release in nonhuman primates can be blocked by a dopamine D2- but not by a D1-receptor agonist (Butelman and Kreek, 2001). Although this may provide a framework for the D2-receptor mediated effects on TIDA responsivity and how µ- and -receptor agonists each suppress TIDA responsivity, it does not completely explain why the methadone-maintained subjects in this study had a significantly smaller prolactin response to dynorphin A_1–13 than did the control group.

Summary and Conclusion. Compared with healthy controls, methadone-maintained subjects exhibited lowered tuberoinfundibular dopamine responsivity after dynorphin A_1–13 administration. The most likely explanation for the current findings is that there is an alteration in dopaminergic responsivity in the methadone-maintained subjects, in particular in D2 receptor-mediated dopaminergic responsivity, occurring through an unexplained mechanism. Whether this finding indicates altered dopaminergic responsivity that may have contributed to the acquisition of opiate addiction or results from opiate addiction and/or its treatment with methadone cannot be determined. Further studies of dopaminergic function using -opioid agonists and antagonists may provide a clearer understanding of the underlying neurobiological mechanisms of heroin and other addictions and lead to new therapeutic interventions.

	Acknowledgements

 
We gratefully acknowledge the assistance of Dr. Pauline McHugh, Wan-Xin Feng, David Fussell, Robert Gianotti, Lauren Hofmann, and Elizabeth Oosterhuis.

	Footnotes

 
This work was supported in part by National Institutes of Health (NIH)-National Institute on Drug Abuse (NIDA) Research Center Grant DA-P60-05130, NIH-NIDA Research Scientific Award Grant K05-DA00049, and General Clinical Research Center M01-RR00102.

DOI: 10.1124/jpet.103.050682.

ABBREVIATIONS: TIDA, tuberoinfundibular dopamine; DSM-IV, Diagnostic and Statistical Manual-Version IV; AUC, area under the curve; ANOVA, analysis of variance; NV, normal volunteer; MM, methadone-maintained; PET, positron emission tomography.

Address correspondence to: Gavin Bart, The Laboratory of the Biology of Addictive Diseases, The Rockefeller University, Box 171, 1230 York Ave., New York, NY 10021-6399. E-mail bartg{at}rockefeller.edu

	References

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Butelman ER, Ball JW, and Kreek MJ (2002) Comparison of the discriminative and neuroendocrine effects of centrally penetrating -opioid agonists in rhesus monkeys. Psychopharmacology 164: 115–120.[CrossRef][Medline]

Butelman ER, Harris TJ, Perez A, and Kreek MJ (1999) Effects of systemically administered dynorphin A(1–17) in rhesus monkeys. J Pharmacol Exp Ther 290: 678–686.[Abstract/Free Full Text]

Butelman ER and Kreek MJ (2001) -Opioid receptor agonist-induced prolactin release in primates is blocked by dopamine D₂-like receptor agonists. Eur J Pharmacol 423: 243–249.[CrossRef][Medline]

Chavkin C, James IF, and Goldstein A (1982) Dynorphin is a specific endogenous ligand of the opioid receptor. Science (Wash DC) 215: 413–415.[Abstract/Free Full Text]

Chou JZ, Chait BT, Wang R, and Kreek MJ (1996) Differential biotransformation of dynorphin A(1–17) and dynorphin A(1–13) peptides in human blood, ex vivo. Peptides 17: 983–990.[CrossRef][Medline]

Claye LH, Maisonneuve IM, Yu J, Ho A, and Kreek MJ (1997) Local perfusion of dynorphin A1–17 reduces extracellular dopamine levels in the nucleus accumbens. NIDA Res Monogr 174: 113.

Cushman P Jr and Kreek MJ (1974) Methadone-maintained patients. Effect of methadone on plasma testosterone, FSH, LH and prolactin. NY State J Med 74: 1970–1973.[Medline]

Deyo SN, Swift RM, and Miller RJ (1979) Morphine and endorphins modulate dopamine turnover in rat median eminence. Proc Natl Acad Sci USA 76: 3006–3009.[Abstract/Free Full Text]

Di Chiara G and Imperato A (1988) Opposite effects of µ and opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats. J Pharmacol Exp Ther 244: 1067–1080.[Abstract/Free Full Text]

Durham RA, Johnson JD, Moore KE, and Lookingland KJ (1996) Evidence that D2 receptor-mediated activation of hypothalamic tuberoinfundibular dopaminergic neurons in the male rat occurs via inhibition of tonically active afferent dynorphinergic neurons. Brain Res 732: 113–120.[CrossRef][Medline]

Frantz AG (1978) Prolactin. N Engl J Med 298: 201–207.[Medline]

Freeman ME, Kanyicska B, Lerant A, and Nagy G (2000) Prolactin: structure, function and regulation of secretion. Physiol Rev 80: 1523–1631.[Abstract/Free Full Text]

Gambus PL, Schnider TW, Minto CF, Youngs EJ, Billard V, Brose WG, Hochhaus G, and Shafer SL (1998) Pharmacokinetics of intravenous dynorphin A(1–13) in opioid-naive and opioid-treated human volunteers. Clin Pharmacol Ther 64: 27–38.[CrossRef][Medline]

Hurd YL (1996) Differential messenger RNA expression of prodynorphin and proenkephalin in the human brain. Neuroscience 72: 767–783.[CrossRef][Medline]

Inturrisi CE and Verebely K (1972) The levels of methadone in the plasma in methadone maintenance. Clin Pharmacol Ther 13: 633–637.[Medline]

King AC, Ho A, Schluger J, Borg L, and Kreek MJ (1999) Acute subjective effects of dynorphin A(1–13) infusion in normal healthy subjects. Drug Alcohol Depend 54: 87–90.[CrossRef][Medline]

Kling MA, Carson RE, Borg L, Zametkin A, Matochik JA, Schluger J, Herscovitch P, Rice KC, Ho A, Eckelman WC, et al. (2000) Opioid receptor imaging with positron emission tomography and [¹⁸F]cyclofoxy in long-term, methadone-treated former heroin addicts. J Pharmacol Exp Ther 295: 1070–1076.[Abstract/Free Full Text]

Kreek MJ (1973) Plasma and urine levels of methadone. Comparison following four medication forms used in chronic maintenance treatment. NY State J Med 73: 2773–2777.[Medline]

Kreek MJ (1978) Medical complications in methadone patients. Ann NY Acad Sci 311: 110–134.[Medline]

Kreek MJ, Borg L, Zhou Y, and Schluger J (2002) Relationships between endocrine functions and substance abuse syndromes: heroin and related short-acting opiates in addiction contrasted with methadone and other long-acting agonists used in pharmacotherapy of addiction, in Hormones, Brain and Behavior (Pfaff DW, Arnold AP, Etgen AM, Fahrbach SE, and Rubin RT eds) pp 781–830, Academic Press, New York.

Kreek MJ, Schluger J, Borg L, Gunduz M, and Ho A (1999) Dynorphin A1–13 causes elevation of serum levels of prolactin through an opioid receptor mechanism in humans: gender differences and implications for modulation of dopaminergic tone in the treatment of addictions. J Pharmacol Exp Ther 288: 260–269.[Abstract/Free Full Text]

Mansour A, Hoversten MT, Taylor LP, Watson SJ, and Akil H (1995) The cloned µ, and receptors and their endogenous ligands: evidence for two opioid peptide recognition cores. Brain Res 700: 89–98.[CrossRef][Medline]

Manzanares J, Lookingland KJ, and Moore KE (1991) Opioid receptor-mediated regulation of dopaminergic neurons in the rat brain. J Pharmacol Exp Ther 256: 500–505.[Abstract/Free Full Text]

Mathieu-Kia AM, Fan LQ, Kreek MJ, Simon EJ, and Hiller JM (2001) µ-, - and -opioid receptor populations are differentially altered in distinct areas of postmortem brains of Alzheimer's disease patients. Brain Res 893: 121–134.[CrossRef][Medline]

Moore MR and Black PM (1991) Neuropeptides. Neurosurg Rev 14: 97–110.[CrossRef][Medline]

Portenoy RK, Caraceni A, Cherny NI, Goldblum R, Ingham J, Inturrisi CE, Johnson JH, Lapin J, Tiseo PJ, and Kreek MJ (1999) Dynorphin A(1–13) analgesia in opioid-treated patients with chronic pain: a controlled pilot study. Clin Drug Investig 17: 33–42.

Reid RL, Hoff JD, Yen SS, and Li CH (1981) Effects of exogenous _h-endorphin on pituitary hormone secretion and its disappearance rate in normal human subjects. J Clin Endocrinol Metab 52: 1179–1184.[Abstract/Free Full Text]

Spanagel R, Herz A, Bals-Kubik R, and Shippenberg TS (1991) -Endorphin-induced locomotor stimulation and reinforcement are associated with an increase in dopamine release in the nucleus accumbens. Psychopharmacology 104: 51–56.[CrossRef][Medline]

Spanagel R, Herz A, and Shippenberg TS (1990) The effects of opioid peptides on dopamine release in the nucleus accumbens: an in vivo microdialysis study. J Neurochem 55: 1734–1740.[Medline]

Specker S, Wananukul W, Hatsukami D, Nolin K, Hooke L, Kreek MJ, and Pentel PR (1998) Effects of dynorphin A(1–13) on opiate withdrawal in humans. Psychopharmacology 137: 326–332.[CrossRef][Medline]

Staley JK, Rothman RB, Rice KC, Partilla J, and Mash DC (1997) 2 Opioid receptors in limbic areas of the human brain are upregulated by cocaine in fatal overdose victims. J Neurosci 17: 8225–8233.[Abstract/Free Full Text]

Svingos AL, Chavkin C, Colago EE, and Pickel VM (2001b) Major coexpression of -opioid receptors and the dopamine transporter in nucleus accumbens axonal profiles. Synapse 42: 185–192.[CrossRef][Medline]

Svingos AL, Garzon M, Colago EE, and Pickel VM (2001a) µ-Opioid receptors in the ventral tegmental area are targeted to presynaptically and directly modulate mesocortical projection neurons. Synapse 41: 221–229.[CrossRef][Medline]

Unterwald EM, Rubenfeld JM, and Kreek MJ (1994) Repeated cocaine administration upregulates and µ, but not , opioid receptors. Neuroreport 5: 1613–1616.[Medline]

Wang GJ, Volkow ND, Fowler JS, Logan J, Abumrad NN, Hitzemann RJ, Pappas NS, and Pascani K (1997) Dopamine D₂ receptor availability in opiate-dependent subjects before and after naloxone-precipitated withdrawal. Neuropsychopharmacology 16: 174–182.[CrossRef][Medline]

Wen HL and Ho WK (1982) Suppression of withdrawal symptoms by dynorphin in heroin addicts. Eur J Pharmacol 82: 183–186.[CrossRef][Medline]

Yu J, Butelman ER, Woods JH, Chait BT, and Kreek MJ (1996) In vitro biotransformation of dynorphin A (1–17) is similar in human and rhesus monkey blood as studied by matrix-assisted laser desorption/ionization mass spectrometry. J Pharmacol Exp Ther 279: 507–514.[Abstract/Free Full Text]

Zubieta JK, Gorelick DA, Stauffer R, Ravert HT, Dannals RF, and Frost JJ (1996) Increased µ opioid receptor binding detected by PET in cocaine-dependent men is associated with cocaine craving. Nat Med 2: 1225–1229.[CrossRef][Medline]

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