Introduction

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Dynorphin A(1-17) is an endogenous agonist at -opioid receptors, although at high concentrations in vitro it can also produce agonist effects at µ- and -opioid receptors (e.g., Goldstein et al., 1981; Alt et al., 1998; Zhang et al., 1998). This peptide has relative binding selectivity for - over µ- and -receptors in rhesus monkey brain and in cloned rodent and human receptors (e.g., Raynor et al., 1994; Butelman et al., 1998; Zhang et al., 1998). Dynorphin A(1-17) also exhibits efficacy similar to that of arylacetamide -agonists such as U50,488 in cloned human -receptors, as measured by the accumulation of [³⁵S]GTPS (Zhu et al., 1997; Remmers et al., 1999).

The effects of a shorter fragment, dynorphin A(1-13), have been studied after systemic administration in humans and experimental animals (e.g., Gilbeau et al., 1986; Aceto and Bowman, 1992; Takemori et al., 1993; Specker et al., 1998). This research focus on the shorter dynorphin fragment is partly due to its early identification by sequencing, but dynorphin A(1-13) is not thought to be a major endogenous fragment in vivo (Goldstein et al., 1979, 1981; Chavkin et al., 1982). Several dynorphin fragments, including nonopioid des-Tyr¹ fragments, caused antinociceptive effects in mice after systemic administration; however, these effects were not mediated by opioid receptors, as shown by their insensitivity to naloxone (Hooke et al., 1995).

Full-length, natural sequence dynorphin A(1-17) has not been studied extensively after systemic administration in nonhuman primates or humans, and may be of further interest for several reasons. First, this endogenous opioid is more resistant to biotransformation in blood relative to dynorphin A(1-13), and second, it may give rise to different, possibly active biotransformation fragments (Chou et al., 1994; Muller and Hochhaus, 1995; Yu et al., 1996; Gambus et al., 1998). The main nonopioid biotransformation fragment, dynorphin A(2-17), is also studied here for comparison, because this fragment is active in behavioral assays after systemic administration in mice (Takemori et al., 1993; Hooke et al., 1995).

In the present studies, we characterized the effects of i.v. dynorphin A(1-17) and A(2-17) in rhesus monkeys, in assays of thermal antinociception and operant rate suppression, previously used to study nonpeptide -agonists (e.g., Dykstra et al., 1987; Negus et al., 1993; France et al., 1994). We also focused on the effects of these dynorphin peptides on the release of the anterior pituitary hormone prolactin, a neuroendocrine endpoint that is responsive to both - and µ-opioid agonists. The effects of -agonists on serum prolactin levels are probably mediated by hypothalamic opioid receptors, which modulate the dopaminergic tuberoinfundibular system, and are located in areas that may be outside the blood-brain barrier (e.g., Manzanares et al., 1991; Merchenthaler, 1991; Simpkins et al., 1991; Moore and Lookingland, 1995). The effects of the dynorphins were compared to those of the selective arylacetamide -agonist, U69,593, which is expected to cause agonist effects in all three endpoints (antinociception, operant, and neuroendocrine), as previously documented with selective -agonists in human and nonhuman primates (Negus et al., 1993; France et al., 1994; Ur et al., 1997). When possible, the effects of dynorphin A(1-17) were studied after pretreatment with the opioid antagonist, quadazocine (Dykstra et al., 1987), to determine whether the effects of this peptide were mediated by opioid receptors. The operant effects of dynorphin A(1-17) were also studied after pretreatment with the inhibitor of mast cell degranulation, cromolyn. These studies were carried out to determine whether behavioral effects of i.v. dynorphin A(1-17) may be a consequence of its ability to release inflammatory mediators (such as histamine; e.g., Sydbom and Terenius, 1985) from mast cells.

	Materials and Methods

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Subjects

Captive-bred, intact rhesus monkeys (age range: 4-7 years old; weight range: 3.8-6.8 kg), obtained from Biomedical Resources Foundation (Houston, TX), were used. They were housed singly in a room maintained at 20-22°C with controlled humidity, and a 12-h light/dark cycle (lights on at 7:00 AM). Monkeys used in serum prolactin (six females) and antinociception studies (three females, one male) were fed approximately 10 jumbo primate chow biscuits (Purina, Richmond, VA) daily, supplemented by fruit two times per week. Three of the female subjects were used both in the prolactin and the antinociception experiments. Monkeys in the food-reinforced responding studies (four females, one male) were fed appropriate amounts of chow to maintain body weight at levels approximately 90% of free-feeding levels. Water was freely available in home cages, via an automatic water spout.

Animals used in these studies were maintained in accordance with the Institutional Animal Care and Use Committee of Rockefeller University, and Guidelines of the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Health Council (Department of Health, Education and Welfare, Publication ISBN 0-309-05377-3, revised 1996).

Schedule-Controlled Food-Reinforced Responding

Apparatus and Procedure. The procedure used in the present study was similar to that described by Negus et al. (1993), but adapted to chair-trained rhesus monkeys. Chaired monkeys were trained and tested in melamine operant boxes (MED Associates, Georgia, VT). The boxes contained an operant panel with two levers within easy reach of the monkeys, on either side of a centrally located pellet dispenser (300-mg sucrose pellets were used; P.J. Noyes Co., Lancaster, NH). A 1-inch-diameter white light was placed 1 inch above each lever, and these lights were transilluminated with white light during response periods. The operant boxes were connected to a PC-compatible computer, via a MED Associates interface. The session contingencies and data collection were programmed with a menu-based desktop program (Schedule Manager, MED Associates).

Design and Data Presentation. Dynorphins A(1-17) and A(2-17) were studied in time course experiments (0.1-3.2 mg/kg i.v.). The data for dynorphin A(1-17) were analyzed in a two-way (dose × time) repeated measures ANOVA. Active doses of dynorphin A(1-17) were also studied after quadazocine (0.32 mg/kg) and cromolyn (10 mg/kg) pretreatment. The U69,593 cumulative s.c. dose-effect curve was also studied alone and after pretreatment with quadazocine and cromolyn. Test data on rate of responding were converted to individual % control values. Control was defined as the mean response rate for the training day immediately preceding the test. Individual ED₅₀ values were calculated by regression from the data points above and below 50% of control. Mean values (±95% CL) were calculated after log transformation of individual ED₅₀ values. An in vivo affinity estimate for quadazocine (apparent pK_B) was also calculated from the mean U69,593 ED₅₀ value, according to a modified equation (pK_B = log[B/DR  1]), where B was the dose of quadazocine in mol/kg and DR was the dose ratio (Negus et al., 1993). The same group of five subjects was used for the pharmacological comparisons in this assay; the 0.05  level was adopted for all of the studies presented here.

Warm Water Tail Withdrawal Assay (Antinociception)

Apparatus and Procedure. The procedure used in the present study has been described in detail previously (Dykstra and Woods, 1986). Monkeys were seated in primate restraint chairs, and the lower portion of the shaved tail (approximately 10 cm) was immersed in a polycarbonate flask containing water at either 40°, 50°, or 55°C. Monkeys were tested at the three water temperatures in varying order, with tests in the same monkey separated from each other by approximately 2 min. Tail withdrawal latencies were timed manually on a stopwatch. To prevent tissue damage, tails were removed from the water if they remained immersed for 20 s (cutoff latency). Sessions began with control determinations at each water temperature, presented in a varied order among the monkeys.

Data Analysis. Data for individual monkeys were converted to percent maximum possible effect (%MPE) by the following calculation: %MPE = [(test latency  control latency)/(cutoff latency  control latency)] × 100%. Individual ED₅₀ values were calculated from individual %MPE values by linear regression, and a mean ED₅₀ (±95% CL) was presented. Individual and mean quadazocine apparent pK_B values for U69,593 were also calculated (see above). The same group of four subjects was used for all the pharmacological comparisons in this assay.

Serum Prolactin Levels

Procedure. Chair-trained monkeys were tested after extensive habituation to the experimental conditions. Monkeys were chaired (in the same chairs as in the antinociception experiments) and brought into the experimental room between 0930 and 1000 on each test day. A single indwelling catheter (24 gauge; Angiocath, Becton Dickinson, Sandy, UT) was acutely placed in a superficial leg vein and secured with elastic tape. A multisample injection port (Terumo, Elkton, MD) was attached to the hub of the catheter; the port and catheter were flushed (0.3 ml of 50 U/ml heparinized saline) before use and after each blood sampling or i.v. injection. Approximately 15 min after catheter placement, two baseline blood samples were collected, 5 min apart from each other (defined as 10 and 5 min relative to the onset of dosing). At each sampling point, a 1.5-ml blood aliquot was placed in an EDTA Vacutainer (Becton Dickinson, Franklin Lakes, NJ; these samples were not analyzed in the present studies). This was followed by a second 1.5-ml blood sample, which was placed in a plain Vacutainer and kept at room temperature until the time of spinning (3000 rpm at 4°C) and serum separation. The serum samples (approximately 400 µl) were then kept at 40°C until the time of analysis. These samples were analyzed in duplicate with a standard human prolactin radioimmunoassay kit (44 samples/kit; Nichols Diagnostics Institute, San Juan Capistrano, CA), following manufacturer's instructions. The reported sensitivity limit of the assay was 0.14 ng/ml; the highest measurable concentration, using standard calibration curves, was 150 ng/ml; standard calibration curves were determined for each kit with human prolactin (3-150 ng/ml). The reported cross-reactivity of this assay was of largest magnitude for human growth hormone (0.07% cross-reactivity). The intra-assay coefficient of variation for samples tested with the present kits was 4.7%, whereas the interassay coefficient of variation was 10.1%.

Design and Data Presentation. Each experiment was carried out in four to five females in the follicular phase (days 2-12 of each cycle of approximately 28 days, as defined by the onset of visible bleeding). Consecutive experiments in the same subject were separated by at least 48 h (or 72 h after quadazocine administration). Dynorphin A(1-17) was studied under a single-dose time course design (0.00032-0.32 mg/kg). Selected dynorphin A(1-17) doses were also studied after 30-min pretreatment with quadazocine (0.32 mg/kg s.c.). Dynorphin A(2-17) (0.32 mg/kg i.v.) was studied in a time course procedure. An i.v. saline (0.1 ml/kg) time course was also studied for control purposes. U69,593 was studied under both time course (0.0032-0.032 mg/kg i.v.) and cumulative dose-effect curve designs (0.001-0.032 mg/kg i.v.); the U69,593 cumulative dose-effect curve was also redetermined after 30-min quadazocine (0.32 mg/kg s.c.) pretreatment.

Chemicals. Quadazocine methanesulfonate (Sanofi Winthrop, Malvern, PA) and cromolyn sodium (Sigma, St. Louis, MO) were dissolved in sterile water for s.c. injections in the midscapular region of the back (1-2 drops of lactic acid were added for quadazocine solutions). U69,593 (Pharmacia & Upjohn, Kalamazoo, MI) was dissolved in sterile water with one to two drops of lactic acid and injected s.c., as above, or i.v (for i.v. injections through the catheter port, the U69,593 stock solution was further diluted with saline). Dynorphins A(1-17) and A(2-17) (National Institution on Drug Abuse/Research Triangle Institute, Research Triangle Park, NC) were dissolved in saline approximately 5 min before use and injected i.v. Compounds were typically injected in volumes of 0.1 ml/kg. All doses are expressed as the above forms of the compounds.

	Results

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Schedule-Controlled Food-Reinforced Responding. Monkeys were shaped to lever-press and were gradually trained on the FR20, multiple-cycle session. After at least 4 weeks of training, animals reached a stable level of responding. The mean (n = 5) rate of responding for the 4 days before the onset of testing for each monkey was 1.89 responses/s (S.E.M. = 0.22). In a control experiment (n = 5), a s.c. saline injection (0.5 ml) was administered at the beginning of four consecutive response cycles. These saline injections did not affect rates of responding (mean rates of responding ranged from 104 to 110% of control). Likewise, a single i.v. saline bolus did not affect responding measured in a time course session (5-60 min after administration; mean rates of responding ranged from 96 to 108% of control).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Time course of the effects of i.v. dynorphin A(1-17) (top), and dynorphin A(2-17) and saline (bottom) on food-reinforced responding. Abscissae: time (min) from bolus injection; ordinates: rate of responding, expressed as a percentage of control (i.e., the mean rate in the previous day's training session). All points represent the mean ± S.E.M. (n = 4-5).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Dose dependence of the rate-decreasing effects of i.v. dynorphin (DYN) A(1-17) (top) and s.c. U69,593, and antagonist effects of quadazocine (0.32 mg/kg s.c.) or cromolyn (10 mg/kg s.c.) pretreatment. Abscissae: dose in mg/kg; all other details are as in Fig. 1.

Antinociception. After initial training, monkeys displayed a consistent pattern of tail withdrawal latencies in the different water temperatures. Monkeys typically left their tails in 40°C water until the cutoff (20 s), whereas they removed their tails from 50^o or 55^oC water rapidly (within 1-2 s). Saline (0.1 ml/kg), administered as an i.v. bolus, did not affect tail withdrawal latencies relative to control (n = 4; data not shown). Individual values in 50°C water after a saline i.v. bolus were all less than 5%MPE (tested 5-60 min after injection).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effects of bolus doses of s.c. U69,593 (U69) or i.v. dynorphin (DYN) A(1-17) or A(2-17) in the warm water tail withdrawal assay of thermal antinociception (top, 50°C; bottom, 55°C). Abscissae: dose in mg/kg; ordinates: %MPE (n = 4).

Serum Prolactin Levels. Preinjection prolactin values were reliably low in the subjects tested; the mean preinjection values in the experiments reported here were typically 5.5 ng/ml or less. The preinjection samples, obtained at 10 and 5 min relative to the onset of drug administration, did not differ from each other (see Fig. 4). Administration of a bolus saline i.v. injection (0.5 ml) did not elevate prolactin levels above preinjection values between 5 and 90 min after administration (see Fig. 4).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Time course of the effects of i.v. dynorphin (DYN) A(1-17) (top), dynorphin A(2-17) (middle), and U69,593 or saline (bottom) on serum prolactin levels in female subjects. Abscissae: time (min) from bolus injection (10 and 5 time points are preinjection baseline samples); ordinates: serum prolactin levels (n = 4; mean ± S.E.M.).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effects of quadazocine (0.32 mg/kg s.c.) antagonism on the time course of the peak dose of dynorphin (DYN) A(1-17) (0.0032 mg/kg) on serum prolactin levels, and surmountability with a larger dynorphin A(1-17) dose (0.32 mg/kg). All other details are as in Fig. 4 (n = 4).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Dose-effect curve for the effects of i.v. dynorphin (DYN) A(1-17) (top) and i.v. U69,593 (bottom) on serum prolactin levels, and quadazocine (QUAD) (0.32 mg/kg s.c.) antagonism. Abscissae: dose (mg/kg); all other details are as in Fig. 4 (n = 4-5).

	Discussion

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Food-Reinforced Responding. Dynorphin A(1-17) (0.1-3.2 mg/kg) decreased food-reinforced responding, but its effects were not mediated by - or µ-opioid receptors, as shown by a lack of sensitivity to the opioid antagonist quadazocine (0.32 mg/kg). This quadazocine dose antagonized the effects of both - and µ-agonists in this procedure (Negus et al., 1993; present studies). A -receptor-mediated effect of dynorphin A(1-17) could have been insensitive to this quadazocine dose (see Negus et al., 1993), but -opioid receptor mediation is unlikely, given the low affinity of dynorphin A(1-17) for -receptors in rhesus monkey brain (K_i = 129 nM; Butelman et al., 1998). However, future antagonism studies with the -selective antagonist naltrindole could directly test this possibility (e.g., Negus et al., 1994).

Antinociceptive Effects. Both dynorphin A(1-17) and A(2-17) produced partial antinociceptive effects in 50^oC water, and no effect in 55^oC water. By comparison, the selective -agonist (U69,593) was maximally effective in 50^oC water and partially effective in 55^oC water, up to the highest doses presently studied. Previous studies have demonstrated that some opioids [including partial agonists and dynorphin A(1-13)] may be effective in lower water temperatures (e.g., 48^o or 50^oC water), but not in higher water temperatures (55^oC) in this assay (e.g., Walker et al., 1993; Butelman et al., 1995). Possible reasons for the lack of more robust effects with dynorphin A(1-17) in this assay therefore include limited efficacy or affinity at -receptors. However, recent findings suggest that this peptide has high efficacy at cloned -opioid receptors in vitro, as measured by the accumulation of [³⁵S]GTPS (Zhu et al., 1997; Remmers et al., 1999), and has high affinity for [³H]U69,593 ("₁") sites in rhesus monkey brain (Butelman et al., 1998). Furthermore, dynorphin A(1-17) was approximately equipotent and equieffective with U69,593 in causing an opioid receptor-mediated increase in serum prolactin levels after i.v. administration (see below). Thus, the low potency and effectiveness of dynorphin A(1-17) relative to U69,593 in the antinociception assay may be due to factors in addition to the above pharmacodynamic variables. For example, it is currently unknown whether the partial antinociceptive effect of dynorphin A(1-17) was due to a nonopioid action of this peptide, such as that observed in the operant assay above.

Serum Prolactin Levels. In contrast to the low potency and/or low effectiveness of dynorphin A(1-17) in the above assays, dynorphin A(1-17) was equipotent and equieffective to U69,593 in increasing serum prolactin levels. Both compounds also produced sigmoidal dose-effect curves in this procedure. The dynorphin A(1-17) dose-effect curve was shifted to the right by quadazocine (0.32 mg/kg) pretreatment, but this shift was not parallel, probably due to variations in the magnitude of the shifts among the individual subjects. By contrast, quadazocine shifted the U69,593 dose-effect curve to the right in a parallel and surmountable manner; a quadazocine apparent pK_B value was therefore calculated for this effect. This value (7.2) was slightly higher than values observed for quadazocine against -opioid-mediated effects (e.g., Negus et al., 1993) but did not differ significantly from the apparent pK_B value obtained with U69,593 under identical dosing conditions in the antinociception assay. This finding is therefore consistent with a -opioid mediation of the prolactin-increasing effects of U69,593 in these studies. The nonparallel shift caused by quadazocine against dynorphin A(1-17) precludes the calculation of an apparent pK_B value, but the observed shift is consistent with an opioid mediation of this effect of dynorphin A(1-17) (see for comparison the lack of antagonism of the effects of dynorphin in the operant assay). One possible reason underlying the nonparallel shift in the dynorphin A(1-17) dose-effect curve could be a mediation of this effect by more than one opioid receptor type, for which quadazocine may have differential affinity (see Kenakin, 1993). Quadazocine has greater affinity for µ- than for -receptors in rhesus monkey brain (Negus et al., 1993), and both µ- and -opioid agonists can increase serum prolactin levels in mammals (see Moore and Lookingland, 1995; Ur et al., 1997). Dynorphin A(1-17) can also cause agonist effects at both - and µ-receptors in vitro, although its affinity for -receptors is higher than for µ-receptors in both human and nonhuman primates (Alt et al., 1998; Butelman et al., 1998; Zhang et al., 1998). Kappa- and µ-selective opioid antagonists may be used in the future to test whether both receptor types are involved in the prolactin-releasing effects of dynorphin A(1-17).

General Summary. The present studies are, to our knowledge, the first to compare the behavioral and neuroendocrine effects of i.v. dynorphin A(1-17) in primates. Dynorphin A(1-17) decreased food-reinforced responding, but its effect in this assay was of low potency (compared to U69,593) and was not mediated by - or µ-opioid receptors. This peptide was partially effective in the warm water (50°C) tail withdrawal assay of thermal antinociception, and its nonopioid biotransformation fragment, dynorphin A(2-17), was also partially effective, whereas U69,593 was maximally effective. By contrast, i.v. dynorphin A(1-17) was equieffective and equipotent to U69,593 in increasing serum prolactin levels, and the effects of both compounds were sensitive to opioid antagonist pretreatment, whereas dynorphin A(2-17) was inactive. The selectivity of i.v. dynorphin A(1-17) for this neuroendocrine effect versus operant or antinociceptive effects suggests that this peptide is more potent in stimulating the hypothalamic -opioid receptors thought to mediate prolactin release, relative to -opioid receptors that mediate these behavioral effects. One possible reason for this selectivity is that i.v. dynorphin A(1-17) may have greater access to the hypothalamic opioid receptors that mediate prolactin release, as these receptors may be functionally outside the blood-brain barrier (e.g., Merchenthaler, 1991). By contrast, the -receptors which presumably mediate the antinociceptive effect of U69,593 may be located in areas of the central nervous system that are less accessible to systemically administered dynorphin A(1-17).