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0022-3565/97/2801-0416$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics JPET 280:416 –421, 1997 Vol. 280, No. 1 Printed in U.S.A. Dynorphin A1–13 Stimulates Ovine Fetal Pituitary-Adrenal Function through a Novel Nonopioid Mechanism1 CINDY C. TAYLOR, DUNLI WU, YI SOONG, JENNY S. YEE and HAZEL H. SZETO Department of Pharmacology, Cornell University Medical College, New York, New York Accepted for publication September 13, 1996 Dynorphin A is a 17-amino acid peptide initially isolated from porcine pituitary tissue and postulated to be an endogenous k opioid receptor ligand (Goldstein et al., 1979; Goldstein et al., 1981; Huidoro-Toro et al., 1981; Chavkin et al., 1982). Dynorphin A1–13 is the N-terminal tridecapeptide of dynorphin A1–17 and has been found to be as potent as the natural peptide in opioid-binding assays and in the guinea pig ileum (Goldstein et al., 1979). The high density of k-opioid receptors in the hypothalamus (Mansour et al., 1994) and the high concentration of dynorphin peptides in both hypothalamus and pituitary (Hollt et al., 1980) suggest a possible involvement of dynorphin in the regulation of release of anterior pituitary hormones. In fact, dynorphin A1–13 significantly increased plasma prolactin levels when administered i.v. to adult male rhesus monkeys (Gilbeau et al., 1987), and both dynorphin and U50488H produced a significant increase in plasma levels of ir-ACTH and ir-corticosterone in adult rats (Pfeiffer et al., 1985; Iyenger et al., 1986). The sites and mechanisms by which dynorphin alters ACTH release have not been studied. The effects of opioids on Received for publication May 20, 1996. 1 This research was supported by Grant DA02475-16. C.C.T. is supported by training Grant from NIDA (DA07274-04). levels of 383.3 6 43.8 pg/ml and 32.8 6 9.0 ng/ml, respectively, at 15 min after administration. A similar increase in plasma immunoreactive-ACTH was seen after the same dose of dynorphin A1–17 (P 5 .02) but not dynorphin A2–17. This ACTH response to dynorphin A1–13 was shown to be insensitive to the opioid antagonist, naloxone (12 mg/hr), as well as antagonists of corticotropin releasing factor and arginine vasopressin. These data suggest that dynorphin A1–13 in the ovine fetus may be acting through a mechanism distinct from the k-opioid system and that the dynorphins may serve as secretagogues of ACTH directly at the anterior pituitary through nonopioid receptors. the pituitary-adrenal axis are thought to occur via a hypothalamic mechanism of action, most likely through the modulation of CRF or AVP, the two most potent secretagogues of ACTH (Pechnick, 1993). There is good evidence to support a role for CRF in the release of ACTH resulting from k-opioid agonists (Buckingham and Cooper, 1986; Nikolarakis et al., 1988). The prospect of opioid modulation of ACTH secretion via AVP has not previously been addressed. Studies investigating the interaction of the k-opioid system with AVP have been limited to the actions of k-opioid agonists on posterior pituitary function. It has been shown that administration of U50488H or dynorphin A1–13 causes diuresis via inhibiting AVP release most likely through a direct action of these agonists at posterior pituitary k receptors (Oiso et al., 1988). It is possible that dynorphin peptides may evoke ACTH release through a mechanism that is independent from AVP altogether and may be dependent solely on regulation of CRF. Alternatively, the combined hypothalamic secretion of AVP, CRF and dynorphin peptides (Roth et al., 1982; Watson et al., 1982) may implicate the dynorphin peptides as additional secretagogues of ACTH acting directly at the anterior pituitary independent of either CRF or AVP. Supporters of the view that dynorphin peptides act exclusively at opioid receptors would argue that the flaw in this hypothesis is that ABBREVIATIONS: HPA, hypothalamic-pituitary-adrenal; ACTH, adrenocorticotropin; AVP, arginine vasopressin; CRF, corticotropin releasing factor; ir-ACTH, immunoreactive adrenocorticotropin; ir-cortisol, immunoreactive cortisol; U50,488H, trans-(6)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide; icv, intracerebroventricular; ANOVA, analysis of variance. 416 Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017 ABSTRACT We previously reported that U50488H, a k-selective opioid agonist, stimulates the release of adrenocorticotropin (ACTH) in the ovine fetus via the release of hypothalamic arginine vasopressin and corticotropin releasing factor. In this study we examined the effects of the endogenous k-preferring opioid peptide, dynorphin A1–13, on fetal ACTH release using the unanesthetized, chronically catheterized fetal lamb model. Fetal plasma samples were collected at timed intervals after fetal administration of dynorphin A1–13 (0.5 mg/kg, i.v.) and subsequently analyzed by radioimmunoassay for immunoreactiveACTH and immunoreactive-cortisol. Dynorphin A1–13 produced a highly significant and rapid increase in immunoreactive-ACTH (P 5 .002) and immunoreactive-cortisol (P 5 .002) with peak 1997 Fetal HPA Activation by Dynorphin Materials and Methods Surgical procedure. Surgery was performed on 22 fetal sheep (gestational age 5 115–120 days) to insert chronic indwelling polyvinyl catheters as described in detail by Szeto et al. (1990). One catheter was inserted into the femoral artery and advanced to the distal aorta for blood sampling and another into the inferior vena cava via the femoral vein for drug infusion. An additional catheter was placed in the lateral cerebral ventricle for infusion of the CRF antagonist. Guidelines approved by the Institution for the Care and Use of Animals at Cornell University Medical College were followed for all surgical procedures and experimental protocols. Experimental protocol. Experiments were performed a minimum of 5 days after surgery to ensure complete recovery from surgical stress. The ewe was placed in a mobile cart with free access to food and water. All studies commenced at 0800 hr with a minimum of 2.5 hr to allow for acclimation of the animal to the study conditions. The study environment was free of external stressful stimuli to the best of our ability. As plasma hormone levels and responsiveness of the HPA axis varies diurnally, drugs were administered at approximately the same time for each study (1100 hr 6 30 min). A sample of fetal blood (2 ml) was collected for determination of basal arterial blood gases and pH (Radiometer ABL30, Cleveland, OH) as well as basal ir-ACTH and ir-cortisol levels 30 min before drug administration. An incomplete randomized cross-over design was used in the administration of the various drug treatments with each fetus receiving no more than three different treatments with a minimum of 2 days between studies. Gestational age on the day of the study ranged from 125 to 142 days. An estimation of fetal weight was made from physical observation at the time of surgery allowing for calculation of fetal weight on the day of the study factoring growth of 2% per day (Joubert, 1956). A more accurate estimation of the dose administered could then be determined after birth by measuring the actual birth weight and recalculating the study weight based on the 2% per day principle. Dynorphin A1–13 (Chiron Mimotopes Peptide Systems, San Diego, CA), dynorphin A1–17 (Multiple Peptide System, San Diego, CA), dynorphin A2–17 (Multiple Peptide System) or saline was administered to the fetus as an iv bolus (0.5 mg/kg) with n 5 5 in all groups except for dynorphin A1–17 (n 5 3). This dose of dynorphin peptides was chosen based on preliminary studies with doses ranging from 0.05 to 0.5 mg/kg. Blood samples (2 ml) were collected from the fetus 15, 30 and 60 min after drug administration. The opioid antagonist, naloxone (gift from Du Pont-Merck, Wilmington, DE; 12 mg/hr, i.v.), was administered to the fetus 1 hr before, during and 1 hr after the dynorphin A1–13 bolus (n 5 5) to test whether it is acting through classical opioid receptors. To determine whether dynorphin A1–13 is acting via the hypothalamic secretagogues, CRF or AVP, some fetuses were pretreated with antagonists of CRF or AVP before dynorphin A1–13 administration. a-Helical CRF [Sigma Chemical Co,, St. Louis, MO; 20 mg icv followed by 10 mg/hr icv infusion (Bennet et al., 1990)], was given 15 min before the dynorphin A1–13 bolus (n 5 3). The AVP antagonist [(b-mercapto-b, b-cyclopentamethylene-propionyl, O-Me2-Tyr, Arg8]vasopressin; Sigma)] was administered as an i.v. bolus (60 mg/kg) 10 min before dynorphin A1–13 (n 5 4) (Dunlap and Valego, 1989). It was necessary to administer a-helical CRF via the icv route because impractically large amounts would have had to be administered via the i.v. route (2.3 mg/kg) (Rivier et al., 1984). The dose of AVP antagonist was chosen because it has been shown to block an AVPinduced rise in ovine fetal ir-ACTH similar in magnitude to that which is produced by dynorphin A1–13 administration (Apostolakis et al., 1991). For the a-helical CRF, the dose used was shown to completely reverse the stimulation of ovine fetal breathing movements associated with CRF infusion (Bennet et al., 1990). Furthermore, previous studies in our laboratory have shown that these doses of antagonists resulted in a significant attenuation of a comparable increase in ovine fetal plasma ir-ACTH by U50488H (Taylor et al., 1996). In all antagonist studies, an additional control sample was collected after the administration of the antagonist and before the administration of dynorphin A1–13. Radioimmunoassay. Blood samples were centrifuged at 1500 rpm for 10 min at 4°C and subsequently frozen at -70°C until assayed. [125I]ACTH and [125I]cortisol radioimmunoassay kits (INCSTAR, Stillwater, MN) were used to measure fetal plasma ir-ACTH and ir-cortisol concentrations. These assays have been standardized in our laboratory using ovine fetal plasma (Taylor et al., 1996). Statistical analysis. All data are reported as mean 6 S.E.M. A single-factor ANOVA with repeated measures (factor 5 time) was used to determine drug effects on fetal plasma ir-ACTH and ircortisol levels. Friedmans repeated measures ANOVA on ranks was used if the normality test failed. The statistical significance of the change from control for each time point was assessed by Dunnett’s post hoc comparison. A multifactor ANOVA (factor 5 time, drug) was used to analyze the influence of antagonists on the dynorphin A1–13 response. To compare plasma levels of ir-ACTH after dynorphin A1–13 with different antagonists 15 min after drug administration, a single-factor ANOVA (factor 5 drug) followed by Dunnett’s post hoc test was used. Results Effects of dynorphin A1–13 on fetal plasma ir-ACTH and ir-cortisol. Basal levels of ir-ACTH and ir-cortisol were 46.2 6 7.2 pg/ml and 9.4 6 2.5 ng/ml, respectively. Fifteen minutes after the administration of dynorphin A1–13, there was a highly significant increase from control values in plasma ir-ACTH (x2 5 15.0, P 5 .002; see fig. 1) with peak ir-ACTH level of 383.3 6 43.8 pg/ml. Although ir-ACTH Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017 opioid receptors have not been shown to be present in the anterior pituitary (Mansour et al., 1995). However, the finding that dynorphin A1–17 is rapidly degraded to its nonopioid analog, dynorphin A2–17 (Muller and Hochhaus, 1995), and that many of the actions of dynorphin peptides are naloxoneinsensitive (Walker et al., 1982; Smith and Lee, 1988; Hooke et al., 1995), indicate that they are capable of acting via a nonopioid mechanism of action. Our study was an attempt to understand the regulation of the HPA axis by dynorphin peptides during fetal development. During fetal life the HPA axis plays a crucial role in proper development of organ systems, initiation of parturition and homeostasis. The development of an appropriate fetal stress response is essential to maintaining the wellbeing of the fetus and helps to ensure proper physiological controls through birth and into adulthood. Further, it may elucidate a mechanism by which exogenous administration of opioids during pregnancy can have detrimental effects on the fetus. We have administered dynorphin A1–13 and dynorphin A1–17 to the fetal lamb in utero and found a potent increase in fetal plasma ir-ACTH and ir-cortisol which was surprisingly naloxone-insensitive, not mimicked by dynorphin A2–17, and did not involve the release of AVP or CRF. This is in contrast to recent studies in our laboratory which have shown U50488H to stimulate fetal pituitary-adrenal function via a naloxone-sensitive mechanism involving hypothalamic AVP and CRF (Taylor et al., 1996). These data suggest that dynorphin A1–13 and dynorphin A1–17 in the fetus may be acting through a mechanism distinct from the k-opioid system and that the dynorphins may serve as secretagogues of ACTH directly at the anterior pituitary through nonopioid receptors. 417 418 Taylor et al. Vol. 280 Fig. 1. Effects of dynorphin A1–13 (f) alone and in the presence of naloxone (M) on fetal plasma (A) ir-ACTH and (B) ir-cortisol. Data are represented as absolute change from predrug control levels. Dynorphin A1–13 (0.5 mg/kg) was administered at time 0 min. Naloxone was given as a constant rate iv infusion (12 mg/h, iv) starting 60 min before, and continuing for 60 min after dynorphin A1–13 administration. The effects of naloxone alone (F) are also shown. *P , .05 as compared to predrug control levels. levels subsequently declined toward to control levels, plasma levels were still significantly elevated at 60 min after dynorphin A1–13 administration. There was a corresponding rise in ir-cortisol (F3,16 5 9.1, P 5 .002) at 15 and 30 min after dynorphin A1–13 administration. There was no effect of saline vehicle administration on plasma ir-ACTH or ir-cortisol (n 5 5) (see fig. 2). The effect of naloxone on the dynorphin A1–13 response. Administration of naloxone alone (n 5 6) had no effect on either ir-ACTH or ir-cortisol levels (fig. 1). Dynorphin A1–13 in the presence of naloxone (n 5 5) resulted in a significant rise in ir-ACTH levels (F3,16 5 20.1, P , .001) with plasma ir-ACTH reaching a peak of 291.9 6 42.7 pg/ml compared to 383.3 6 43.8 pg/ml with dynorphin A1–13 alone (see fig. 1). There was also a significant increase in ir-cortisol (F3,16 5 14.6, P , .001) with peak levels of 53.5 6 12.7 ng/ml compared to 32.8 6 9.0 ng/ml after dynorphin A1–13 alone. Neither the ir-ACTH nor the ir-cortisol response to dynorphin A1–13 was blocked by concurrent administration of naloxone (ir-ACTH: F1,32 5 1.5, P 5 .3, ir-cortisol: F1,32 5 1.1, P 5 .3). Effects of dynorphin A1–17 and dynorphin A2–17 on plasma ir-ACTH and ir-cortisol. Administration of the same dose of dynorphin A1–17 to the ovine fetus (n 5 3) also resulted in a significant increase in fetal plasma ir-ACTH (F 5 5.4, P 5 .02), although the magnitude of increase was significantly less than with dynorphin A1–13 (see fig. 2). In contrast, the same dose of dynorphin A2–17 had no effect on either plasma ir-ACTH (x2 5 7.1, P 5 .1) or ir-cortisol (x2 5 4.6, P 5 .2) (n 5 5; see fig. 2). The effects of AVP and CRF antagonists on the dynorphin A1–13 response. Administration of the AVP antagonist or CRF antagonist alone had no effect on plasma ir-ACTH or ir-cortisol. In the presence of the AVP antagonist, dynorphin A1–13 resulted in a highly significant increase in ir-ACTH (F3,12 5 80.0, P , .001) and ir-cortisol (F3,12 5 31.3, P , .001) (n 5 4). There was also a highly significant increase in ir-ACTH (F3,8 5 119.3, P , .001; n 5 3) and ir-cortisol (F3,8 5 12.6, P 5 .005; n 5 3) after dynorphin A1–13 in the presence of the CRF antagonist. Figure 3 illustrates the fetal plasma ir-ACTH levels after the administration of dynorphin A1–13 alone, and in the presence of the AVP antagonist or CRF antagonist. No significant difference was observed between administration of dynorphin A1–13 alone and in the presence of the AVP antagonist or CRF antagonist. Discussion Dynorphin-related peptides are known to have actions on the neuroendocrine system that are reversible by naloxone, including the inhibition of AVP and oxytocin release from the posterior pituitary (Pechnick, 1993). However, the role of dynorphin-related peptides in the anterior pituitary is not as well understood. Some evidence exists that the dynorphins can stimulate the release of ACTH, however, there has been conflicting data in regards to naloxone sensitivity. Iyengar et al. (1987) reported a significant rise in rat plasma ir-corticosterone after a 10-mg icv dose of dynorphin A1–13 that was blocked by naloxone (5 mg/kg, i.p.). However, Gunion et al. (1991) showed a similar rise in rat plasma ir-corticosterone after having administered icv dynorphin A1–17 (0.3–10 nmol/ rat) that was not blocked by naloxone (1 mg/kg, s.c.). Further, Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017 Fig. 2. Effects of saline (F), dynorphin A1–13 (f), dynorphin A1–17 (å) and dynorphin A2–17 (ç) on fetal plasma ir-ACTH. Data are represented as absolute change from predrug control levels. The dynorphin A1–13 data (f) are the same as shown in figure 1. All drugs were administered at time 0 min. *P , .05 as compared to predrug controls. 1997 the sustained actions of dynorphin A1–17 with mixed dynorphin-naloxone intraventricular injections confirmed the conclusion that the observed effects of dynorphin A1–17 were, in fact, not opioid mediated. Thus, a nonopioid component of HPA stimulation by the dynorphins is implied. In the present study, dynorphin A1–13 was capable of stimulating a highly significant and rapid rise in plasma ir-ACTH and ir-cortisol in the ovine fetus. The later peak in cortisol increase (30 min vs. 15 min) is consistent with the primary action of dynorphin A1–13 being at the level of the hypothalamus or pituitary rather than at the adrenal. This magnitude of ACTH release (7- to 10-fold) is comparable to that observed after 1 mg/kg of U50488H to the ovine fetus (Taylor et al., 1996), and is in the same range as those reported after high doses of CRF (Kerr et al., 1992) and AVP (Apostolakis et al., 1991), or a combination of CRF and AVP (Norman and Challis, 1987). Although higher doses of dynorphin A1–13 were not tested in this study, it is likely that the ACTH response is at near-maximal level. However, the time-action of dynorphin A1–13 on ir-ACTH was very different from that of U50488H (Taylor et al., 1996), with a much earlier peak and shorter duration of action. After an i.v. dose of U50488H, ir-ACTH increased gradually over a 60-min period, and plasma levels at 3 hr were still significantly elevated compared to control. The rapid increase in ir-ACTH after dynorphin A1–13 was unexpected given that the diffusion of a peptide drug should have been slower than that of the more lipid soluble benzeneacetamide. The magnitude of change in ir-ACTH was comparable for dynorphin A1–13 and U50488H even though the molar dose of dynorphin A1–13 was 7-fold lower than that of U50488H. The shorter duration of action of dynorphin A1–13 is consistent with the rapid degradation of dynorphin peptides in vivo (Muller and Hochhaus, 1995). The increase in ir-ACTH by dynorphin A1–13 was not antagonized by naloxone pretreatment, suggesting that it is not mediated by classical opioid receptors. The k-opioid antagonist, nor-binaltorphimine, was not practical for use in this 419 study because of its prolonged antagonism of 28 days (Horan et al., 1992). It is highly unlikely that the dose of naloxone administered was insufficient to block k-opioid receptors because it was three times the dose used to successfully block a comparable release of ir-ACTH by U50488H (Taylor et al., 1996). We therefore propose that dynorphin A1–13 is acting via a nonopioid mechanism to increase the release of ACTH from the ovine fetal pituitary. The apparent lack of action of dynorphin A1–13 on k-opioid receptors was surprising given that the affinity of dynorphin A1–13 for the recently cloned k receptor is actually 10-fold more than that of U50488H (Meng et al., 1993). This discrepancy may be explained by the poor distribution of dynorphin peptides across the bloodbrain barrier to the hypothalamus after i.v. administration. The N-terminal tyrosine of the endogenous opioid peptides is known to be required for binding and pharmacological activity at the opioid receptor (Walker et al., 1982) and the naloxone-insensitive actions of the dynorphins have been thought to be due to degradative fragments resulting from the loss of this crucial amino acid. Dynorphin A2–12 accounts for 15% of the dynorphin peptide fragments found in human blood within minutes of dynorphin A1–13 administration (Muller and Hochhaus, 1995). It has also been demonstrated that most of the naloxone-insensitive actions of dynorphin A1–17 can be mimicked by the des-[Tyr]1-dynorphin analogues (Shukla and Lemaire, 1994). Surprisingly, in this study dynorphin A2–17 had no effect on fetal plasma levels of ir-ACTH or ir-cortisol. It has been reported that b-endorphin can increase thyrotropin stimulating hormone secretion from superfused anterior pituitary tissue by a mechanism that is naloxone insensitive (Judd and Hedge, 1983). However, the nonopioid des-[Tyr]1-g-endorphin was unable to elicit a response. Thus, it appears as though the stimulatory effects of the dynorphins and other opioid peptides on the fetal anterior pituitary may be via a yet unreported or “novel” nonopioid mechanism in which the N-terminal tyrosine is necessary. The action of opioids on HPA function is generally thought to occur at the level of the hypothalamus via modulation of AVP and CRF (Pechnick, 1993). We recently reported that the effects of U504488H on ACTH release in the ovine fetus were naloxone-sensitive and significantly attenuated by both AVP and CRF antagonists (Taylor et al., 1996). In contrast, the stimulation of ACTH release by dynorphin A1–13 was found to be both nonopioid and independent of AVP or CRF modulation (see fig. 4). These results suggest a distinctly different mechanism, and possibly site of action, of dynorphin peptides and k-opioid agonists on ACTH release. Because dynorphin peptides can have both opioid and nonopioid actions, the possibility exists for dual regulation of ACTH release via k-opioid receptor-mediated release of AVP and CRF from the hypothalamus or directly at the anterior pituitary via nonopioid receptors. The possibility that dynorphin peptides may serve as additional ACTH secretagogues is supported by the finding that the dynorphins are known to be colocalized and coreleased with AVP and CRF (Roth et al., 1982; Watson et al., 1982). Further, it has been reported that fetal hypothalamic extracts contain a substantial ACTH-releasing capability that does not correspond with AVP or CRF (Currie and Brooks, 1992). In fact, we have found that there is a component of U50488H-induced ACTH release that is not blocked by either AVP or CRF antagonists (Taylor et al., Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017 Fig. 3. Effects of AVP antagonist (F) and CRF antagonist (å) on the stimulation of fetal plasma ir-ACTH by dynorphin A1–13. Data are represented as the change from predrug control levels. The dynorphin A1–13 data (F) are the same as shown in figure 1. The dynorphin A1–13 was administered at time 0 min with the AVP antagonist (60 mg/kg, iv) given 10 min before dynorphin A1–13, and the CRF antagonist (20 mg, icv 1 10 mg/hr infusion, icv) given 15 min before dynorphin A1–13. The change in ir-ACTH was significant (P , .05) for all three treatment groups at all time points. Fetal HPA Activation by Dynorphin 420 Taylor et al. Vol. 280 Although this hypothesis remains to be explored further, it is without question that the k-opioid and dynorphin systems play a prominent neuroendocrine role that may include maintenance of an appropriate fetal stress response and development of a functional HPA axis. References 1996) that perhaps may be accounted for by the concurrent release of dynorphin together with CRF and AVP. In our study, only the nonopioid actions of dynorphin A1–13 were apparent, probably due to the poor distribution of dynorphin peptides across the blood-brain barrier although the anterior pituitary essentially lies outside of the blood-brain barrier. Dynorphin peptides are not only coreleased with ACTH secretagogues, but their release from the hypothalamus has been shown to be modulated by CRF itself. There has been reported an increased release of dynorphin peptides from mouse spinal cords (Song and Takemori, 1992) or rat hypothalamic slices treated with CRF (Nikolarakis et al., 1986). Based on these studies, it is plausible that exogenously administered dynorphin peptides can bypass CRF-mediated control that may explain why the CRF antagonist was ineffective in blocking the effect of dynorphin A1–13. Thus, some of the secretory actions of CRF may be ascribed to the release of dynorphin peptides that could then stimulate ACTH release directly. In summary, it appears that in the ovine fetus, dynorphin A1–13 acts directly on the anterior pituitary to stimulate the release of ACTH. However, this stimulation is not mediated through classical opioid receptors. Several other actions of dynorphin have been found to be insensitive to naloxone, including a number of motor effects such as “barrel rolling” and flaccid paralysis (Walker et al., 1982; Shukla and Lemaire, 1994). The mechanisms behind these actions are not understood and the possibility exists that dynorphin-related peptides may be acting at other receptors as radioligand binding studies have shown that [3H]dynorphin A cannot be totally displaced by naloxone or other opioid ligands except dynorphin itself (Smith and Lee, 1988). A number of studies suggest that the NMDA receptor may play a role in the nonopioid actions of dynorphin A and related peptides (Shukla and Lemaire, 1994). 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