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Accumulating evidence indicates that the endogenous opioid peptides dynorphinA-(1-17) and dynorphinA-(1-13) interact not only with opioid but also with yet poorly characterized non-opioid receptors. The latter have been implicated in a number of the effects of dynorphins including induction of ACTH release in sheep and in AtT 20 cells, a pituitary-derived mouse cell line. AtT 20 cells do not express opioid receptors and therefore are particularly suitable for search of non-opioid dynorphin receptors. We report here that 3H-dynorphinA-(1-13)-NH2 associates specifically with AtT 20 cells, apparently through an uptake process and a binding site. Within the cell, it binds preferentially to fractions containing secretory vesicles, with a Kd of about 100 nM. DynorphinA-(1-17), and several non-opioid fragments of dynorphin, including A-(2-17), A-(2-16) and A-(2-13), compete with 3H-dynorphinA-(1-13)-NH2 for that site with IC50s ranging from 200 nM to 2 microM. ACTH(1-39) also competes with 3H-dynorphinA-(1-13)-NH2 for the site with an IC50 of about 300 nM. DynorphinA-(2-17) at microM concentrations stimulates release of ACTH from the isolated vesicles. The results indicate the presence of a non-opioid dynorphin binding site on the secretory vesicle fractions of AtT20 cells that might be involved in ACTH release. The ability of ACTH itself to compete for the binding sites associated with the vesicles suggest that those sites may be involved in an autocrine loop., (Copyright 1998 Elsevier Science B.V.)

Morphine administered simultaneously to intracerebroventricular (i.c.v.) and intrathecal (i.t.) sites exhibits synergism, with the antinociceptive potency much greater than would be predicted from a simple addition of the potencies of the same dose administered to either site alone. This synergism was quantified in mice using both a fixed dose method, in which the morphine dose at one site was fixed while the AD50 (antinociceptive dose at 50% effectiveness) of morphine at the other site was determined; and a variable dose method, in which different doses of morphine were administered simultaneously to both sites at a fixed ratio, and the AD50 determined and compared to the AD50 at a single site alone. When animals were made tolerant to morphine by implantation of a 75-mg morphine pellet for 3 days, this synergism was eliminated, so that morphine administered simultaneously to i.c.v. and i.t. sites had an additive effect. However, administration of the peptide DynorphinA-(2-17) i.v. simultaneously to the test doses of morphine in morphine-tolerant animals resulted in a partial restoration of synergism. These results suggest that morphine-induced antinociception is highly dependent on an intact integrated central nervous system system and that the initial tolerance development is the result of a disruption of synergism between the central nervous system sites. Morphine tolerance results not from a reduced sensitivity to morphine at discrete central nervous system sites, but rather from a reduced synergistic interaction of morphine at spinal and supraspinal sites. In support of this conclusion, there was no tolerance observed in morphine-pelleted animals to morphine administered to i.c.v. or i.t. sites alone. DynorphinA-(2-17), a nonopioid peptide has previously been shown to enhance the antinociceptive potency of morphine in morphine-tolerant animals, appears to act by restoring this synergism.

Dynorphin A(1-13) blocks opiate withdrawal in rats without producing dependence, and enhances analgesia in morphine-tolerant animals. Its potential use in humans is therefore of interest. Dynorphin A(1-13) has little toxicity when administered at modest doses IV but has been reported to cause hindlimb paralysis and necrosis of the spinal cord in rats, at the catheter tip, when administered intrathecally. To further evaluate its potential neurotoxicity, we administered dynorphin A(1-13) to rats at very high doses IV. Rats (n = 6-10 per group) received dynorphin A(1-13) as bolus IV doses of 5 mg/kg, or as continuous IV infusions of 40 mg/kg/day for 1 day, with saline controls. The appearance and behavior of all animals was normal. Tail flick latencies remained unchanged (p > 0.5). There were no histologic abnormalities of the spinal cord or brain when examined by light microscopy. Two additional groups received bolus injections of dynorphin A(1-13) 50 or 100 mg/kg IV. Animals receiving 50 mg/kg showed cutaneous flushing, labored respirations, and decreased spontaneous movement, which resolved within 10 min. Histology at 1 week was normal. All six animals receiving 100 mg/kg convulsed and died within minutes. Three animals that received dynorphin A(1-13) 40 mg/kg/day for 7 days had normal behavior and histology. We conclude that the previously observed neurotoxicity of intrathecally administered dynorphin A(1-13) is a local effect that does not occur when dynorphin A(1-13) is administered IV, even at very high doses.

The dynorphin family of peptides stands out among the opioids in that its members are not antinociceptive after central administration in the common antinociceptive assays. In addition, reports of spinal antinociception have been conflicting. We have tested the antinociceptive activity of i.v. dynorphin A-(1-13) in the writhing assay and have found it to be very potent, with an ED50 of 1.0 (0.99-1.02) mumol/kg. Remarkably, [des-tyr1]dyn A-(2-17) was equally active with an ED50 of 1.1 (0.99-1.20). This activity was also retained by several smaller, non-opioid dynorphin A fragments and was not affected by the presence of either 50 mumol/kg naloxone or 20 mumol/kg Nor-BNI. Further, ED50 values were not different in morphine-dependent mice. The peak effect of dyn A-(1-13) and A-(2-17) was observed 5 min after administration and the effect of dyn A-(1-13) or dyn A-(2-17) was still measurable 1 hr after i.v. administration with a 5- to 6-fold increase in ED50 at this time. The ED50 values after i.c.v. and i.t. administration of dyn A-(1-13) were similar to those reported previously. Dyn A(2-17) was also effective by these routes with ED50 values not significantly different from those of dyn A-(1-13). Both dyn A-(1-13) and A-(2-17) were also active when injected i.p., whereas ED50 values increased substantially after s.c. administration.(ABSTRACT TRUNCATED AT 250 WORDS)

A single dose of dynorphin A-(1-13) [dyn A(1-13)] is effective in suppressing the expression of opioid withdrawal and tolerance in morphine-dependent mice. In addition, this modulatory activity is retained by the corresponding non-opioid [des-Tyr1]-dynorphin A peptide [dynA(2-17)]. We have further investigated the non-opioid nature of this activity by comparing the efficacies of dyn A(1-13) and (2-17) under different experimental protocols with a variety of dosing regimens. The effect of dyn A(1-13) on withdrawal and tolerance expression was dose-dependent and could be enhanced by repeated dosing. Thus, the ED50 of naloxone to precipitate withdrawal jumping was increased 1.8-fold when morphine-dependent mice were treated with 4.2 mumol/kg dyn A(1-13) on the fourth day after pellet implantation and 2.4-fold on the sixth day with continued daily dyn A(1-13) treatment. The maximal effect was observed on day 6 when the ED50 of mice treated with 8.4 mumol/kg of dyn A(1-13) was increased nearly 6-fold over that of saline controls. Dyn A(2-17) proved to be nearly as effective as dyn A(1-13).

Previously, we demonstrated that the expression of opiate withdrawal and antinociceptive tolerance can be suppressed by dynorphin (dyn) A-(1-13) in morphine-dependent mice. In this study, it was shown that the normal, endogenous dyn, dyn A-(1-17) also possessed this suppressive property. While using the nonopioid dyn analog, [des-Tyr1]dyn A [dyn A-(2-17)] as a negative control, we discovered unexpectedly that this peptide fragment also suppressed naloxone-induced withdrawal and the expression of morphine tolerance in morphine-dependent mice. Thus, an extensive structure activity relationship was studied using 11 peptide fragments. It was determined that the amino acid sequence of dyn A was required for the suppressive activity because dyn B and alpha-neoendorphin both failed to suppress naloxone-precipitated withdrawal jumping. Of the [des-Tyr1]dyn fragments, the minimal amino acid sequence required to suppress naloxone-induced withdrawal was determined to be dyn A-(2-8), containing the sequence G-G-F-L-R-R-I.

Dynorphin A-(1-13) has been shown to suppress the expression of opiate withdrawal and tolerance dose dependently in morphine-dependent mice when administered i.v. The ED50 of naloxone to precipitate withdrawal jumping was increased by 1.5- and 7-fold when morphine-dependent mice were pretreated with 2.5 and 5.0 mumol/kg of dynorphin A-(1-13), respectively. When dynorphin A-(1-13) (5.0 mumol/kg, i.v.) was administered after the precipitation of withdrawal with naloxone, the ED50 of naloxone was still increased by over 2-fold. Also, the expression of tolerance which was estimated by noting the antinociceptive ED50 of morphine, was inhibited by over 70% with a dynorphin A-(1-13) dose of 2.5 mumol/kg and completely suppressed by pretreatment with 5.0 mumol/kg of dynorphin A-(1-13) i.v. The mechanism by which dynorphin A-(1-13) produces these effects when given i.v. remains to be elucidated.

We previously reported that morphine inhibits macrophage-colony stimulating factor (M-CSF)-induced proliferation of mouse bone marrow cells, both in vivo and in vitro, in a dose-dependent manner. We now report that either dynorphin A-(1-13) or dynorphin A-(1-10) amide, though having no effect on proliferation by themselves at concentrations less than 0.1 mM, can block the inhibitory effect of morphine both in vivo and in vitro, in a dose-dependent manner. The opioid antagonist naloxone can also block morphine's inhibitory effect on bone marrow cell proliferation in vivo, but has no effect in vitro. Dynorphin A-(1-13) was also able to block the dramatic reduction of spleen weight observed in animals chronically treated with morphine. Thus dynorphin, which has previously been shown to antagonize morphine analgesia, is also able to antagonize some of the immunosuppressive effects of morphine.

We previously reported that the opioid peptide dynorphin1-13 improves survival chances in stroked cats. Some evidence also suggests that changes in dopamine and gamma-aminobutyric acid (GABA) uptake may be associated with stroke. In the present study, therefore, we determined binding of the opiate [3H]ethylketocyclazocine (EKC), as well as dopamine and GABA uptake in various brain regions of control, stroked and dynorphin1-13-treated stroked cats. Cats were stroked by middle cerebral artery occlusion. In the EKC binding study, the Kd of the high-affinity site of the occluded cortex was significantly increased, relative to that of both the unoccluded side and control cortex. Dynorphin1-13 treatment reversed this effect, lowering the Kd to control level. In the dopamine uptake study, the Km was decreased and Vmax was increased significantly in unoccluded cortex, compared with that in the occluded cortex or in control cortex. Again, dynorphin1-13 reversed these effects, raising the Km and lowering the Vmax. However, the Km of occluded cortex was also increased so that it became significantly higher than that of control cortex. The Km of unoccluded subcortex in stroked cats treated with dynorphin1-13 was significantly reduced compared with control. In the GABA uptake study, there was no significant change in any parameter. The change in opioid binding observed here and its reversal by dynorphin1-13 are consistent with the notion that the peptide's beneficial effect on stroke is mediated through opiate receptors. Since opioid systems in the brain are known to have association with dopaminergic ones, the change in dopamine uptake could also be the result of an opioid effect.

Like other opioids, the dynorphins play a role in wide variety of physiological parameters, including pain regulation, motor activity, cardiovascular regulation, respiration, temperature regulation, feeding behavior, hormone balance, and the response to shock or stress. The dynorphins are unusual if not unique, however, in that they frequently modulate the activity of other opioids, rather than having direct effects themselves. Thus, they are not analgesic in brain, yet they antagonize opioid analgesia in naive animals and potentiate it in tolerant animals. They have little or no effect by themselves on temperature regulation or respiration, but they enhance the acute effects of morphine on these parameters. Their beneficial effects on stroke are like those of opioid antagonists rather than like agonists. Consistent with such a wide variety of physiological effects, the dynorphins bind to all three of the major opioid receptor types in brain, mu, delta, and kappa, though they exhibit some preference toward kappa sites. They also seem to interact with other physiologically relevant sites; though on the basis of their sensitivity to des-Tyr fragments of dynorphine and/or their insensitivity to naloxone, these sites have been termed "non-opioid". No second messenger systems have been directly associated with dynorphine binding, but several likely candidates exist.

Rats were made tolerant to morphine by subcutaneous implantation of morphine alkaloid pellets. Three days after pellet implantation, withdrawal was induced by pellet removal and was assessed 6 h later. Immediately prior to withdrawal assessment, rats were injected with dynorphin A-(1-13) either i.th. (via a catheter), i.c.v. (via a cannula) or i.v. (via the tail vein). When administered i.th. in the dose range 1.25-5 nmol/rat, dynorphin A-(1-13) attenuated withdrawal over the 40 min observation period. Similarly, dynorphin A-(1-13) administered i.v. (37.5-150 nmol/kg) attenuated withdrawal, though only over the first 20 min following administration. Dynorphin A-(1-13) up to 10 nmol/rat had no effect on withdrawal scores. These data indicate that dynorphin acts at spinal sites to suppress withdrawal in morphine-dependent rats and may play a role in tolerance and dependence mechanisms.

Since the discovery of opiate receptors in the central nervous system (CNS), it has become apparent that endogenous opiate ligands are involved in CNS function. Most attention has focused on their role in modulating pain, but they have also been implicated in various physiological functions and in disease states. We are concerned with evidence that endogenous opioid peptides may also contribute to the neurological deficits arising from cerebral ischaemia. Dynorphin, which is widely distributed in the brain and pituitary, has been reported to produce unusual motor and behavioural effects and may act as a regulatory neuropeptide, not as a classical opiate agonist or antagonist. We have therefore administered to cats in which the right middle cerebral artery had been occluded both dynorphin (1-13) and analogue and control materials. We find that dynorphin (1-13) prolongs survival.

The binding of three tritiated opioid agonists--dihydromorphine, D-ala2-D-leu5-enkephalin and ethylketocyclazocine--was subjected to competition by unlabeled beta-endorphin, dynorphin-(1-13), and various fragments of these peptides, and the results analyzed by a computer program that we developed in an earlier study [11]. Peptides in both groups bound with highest affinity to sites 1 and 3 in our 4-site model, corresponding to the mu and delta sites of conventional classifications, with the dynorphin peptides also interacting with site 2, the kappa site. These results are discussed in relationship to the possible biological roles of these peptides as analgesics or as modulators of analgesia.

Pretreatment of mice with a single injection of morphine, beta-endorphin, Leu-enkephalin, FK33824 or [D-Ala2-D-Leu5]enkephalin, for 3 h increased the ability of naloxone to antagonize the analgesic effects of morphine. However, dynorphin-(1-13) can only antagonize morphine, beta-endorphin or Leu-enkephalin induced increased naloxone efficacy but not FK33824 or [D-Ala2-D-Leu2]enkephalin. Dynorphin itself can also increase naloxone efficacy when treated alone. However, this effect can be prevented when animals are pretreated with dynorphin and naloxone together.

Previous studies from our laboratory have shown that the opioid peptide dynorphin-(1-13), although not analgesic when given by itself, can inhibit morphine-induced analgesia in naive mice and potentiate it in morphine tolerant mice. In the present study, we examined the effect of dynorphin-(1-13) with two other dynorphin-like peptides, alpha-neoendorphin and dynorphin-(1-10) amide, on respiration. Our results show that none of the peptides studied had any significant activity on the respiratory rate in mice when given alone. However, in the presence of morphine, dynorphin-(1-13) antagonized the morphine-induced respiratory rate depression in morphine-tolerant animals; alpha-neoendorphin enhanced the morphine-induced respiratory rate depression in naive but had no effect in morphine-tolerant animals and dynorphin-(1-10) amide had no modulatory effect on the morphine-induced respiratory rate depression in either group of animals.

Circular dichroism was used as a probe for competitive binding of two opioid peptides, dynorphin-(1-13) and beta-endorphin, with cerebroside sulfate, a membrane lipid thought to be part of the morphine receptor complex. The rationale was that bound beta-endorphin is partially helical but bound dynorphin-(1-13) remains unordered, thus making it possible to detect the degree of binding of beta-endorphin. The addition of dynorphin-(1-13) to a cerebroside sulfate solution of beta-endorphin invariably displaced beta-endorphin from the peptide-lipid complex, but the addition of beta-endorphin had little effect on dynorphin-(1-13) bound to the lipid. Similar results were obtained for competitive binding of the two peptides with two other amphiphiles, sodium dodecyl and decyl sulfate. The maximum number of binding sites on dynorphin-(1-13) and beta-endorphin was between five and six, which coincides with the five positively charged side chains plus an alpha NH+3 group at the NH2 terminus on both peptide molecules. The results support our working hypothesis that dynorphin-(1-13) may displace beta-endorphin bound to the receptor, which in turn can account for the inhibition of beta-endorphin-induced analgesia by dynorphin-(1-13).

The role of dynorphin-(1-13) and dynorphin-(1-10)-amide in the neuroendocrine control of primate anterior pituitary hormones was studied in nonrestrained, ovariectomized rhesus monkeys. The effects of these opioids on plasma concentrations of prolactin (PRL), luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyrotropin (TSH), and interactions with naloxone are reported here. Intravenous administration of dynorphin-(1-13), 30 to 120 micrograms/kg, significantly increased plasma PRL levels 3- to 4-fold. These PRL increases occurred within 5 min and levels remained elevated for at least 60 min. Administration of naloxone (1.0 mg/kg i.v.) antagonized the rise in PRL levels. Dynorphin-(1-13) had no significant effect on plasma LH, FSH or TSH levels. Dynorphin-(1-10)-amide (30-120 micrograms/kg) increased plasma PRL levels 2- to 4-fold at 5 to 40 min after administration. Plasma LH levels were significantly depressed 100 to 120 min postdrug. Dynorphin-(1-10)-amide produced no change in plasma FSH or TSH levels. These results indicate that dynorphin is involved in the modulation of PRL and perhaps LH secretion, although not affecting TSH or FSH release.

Porcine dynorphin B-29 was synthesized by solid phase procedures and purified using gel filtration, ion exchange, reverse phase liquid chromatography and preparative high pressure liquid chromatography (HPLC). Binding experiments using brain tissue indicated this peptide displaced mu and k ligands equally well and had significant, though less, affinity for delta. In isolated tissue systems, its potency ranked as follows: guinea pig ileum, mouse vas, rabbit vas, and rat vas. Interestingly, all IC50s were reduced in the presence of peptidase inhibitors, particularly in the rabbit vas. These results indicate that dynorphin B-29 like dynorphin, interacts with multiple receptors in the brain, as well as in isolated tissue systems.

Mice were injected intracerebroventricularly with different doses of dynorphin-(1-13) in vivo and decapitated after different intervals of time; crude P2 fractions were then prepared and used for in vitro binding with [3H]dihydromorphine (DHM) and [3H][D-Ala2,D-Leu5]enkephalin. Dynorphin pretreatment was found to decrease DHM binding in vitro in a both time-dependent and dose-dependent manner. Its maximal effect lasted from 30 min to 3 h and recovery took place in 6-12 h. Analysis of the Scatchard plots showed that dynorphin decreased the number of high affinity-binding site for DHM, while KD of this site was essentially unaltered. Neither Bmax nor KD of low affinity site were much affected. A similar decrease in high affinity Bmax for DADLE was also obtained; however, the recovery was even more rapid. Extra washes of tissue did not significantly increase the binding, but preincubation of tissue in the presence of morphine reversed the dynorphin effect and increased DHM binding significantly to almost control levels. The probable mechanism of dynorphin in decreasing opiate receptor binding is discussed.

This study examines the role of dynorphin-A(1-13) and dynorphin-A(1-10)-amide in the neuroendocrine regulation of anterior pituitary hormones in nonrestrained, adult male rhesus monkeys. The effects of these opioids on plasma concentrations of prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyrotropin (TSH) and growth hormone (GH) were assessed. Intravenous administration of dynorphin-A(1-13), 1-120 micrograms/kg, significantly increased plasma PRL levels. Average maximal increases of 90-230% occurred within 5 min and levels remained significantly elevated for up to 120 min. PRL response reached a plateau following the 30 micrograms/kg dose. Dynorphin-A(1-13) had no observable effects on plasma concentrations of LH, FSH, TSH or GH at any dose level studied. Administration of dynorphin-A(1-10)-amide produced significant dose-dependent increases in plasma PRL concentrations. Dose levels of 1-120 micrograms/kg produced mean peak increases from 100 to 230%, 5-10 min postadministration. Dynorphin-A(1-10)-amide had no significant effect on plasma concentrations of LH, FSH, TSH or GH. The increases in plasma PRL concentrations induced by dynorphin-A were naloxone-reversible. These results indicate a selective effect of dynorphin-A on the regulatory mechanisms of PRL secretion over that of other anterior pituitary hormones.

In order to disassociate the k action of dynorphin from its other actions, seven analogs were synthesized and evaluated for pharmacologic activity in comparison with dynorphin (1-13) and dynorphin amide (1-10). Dynorphin (1-10) was modified by protecting the terminal carboxy group, incorporating thioproline at position 10 and substituting methionine for leucine at position 5. All analogs exhibited the ability to inhibit electrically-induced twitches of the guinea pig ileum and mouse vas deferens in a manner that was dose dependent and naloxone reversible. The decapeptide terminating with a pyrrolidine group showed the highest potency in the ilea and mouse vas deferens. None of the analogs showed analgetic activity by the mouse tail flick test. Binding studies using mouse brain synaptosomes showed that all seven analogs can displace the binding of tritiated dihydromorphine (DHM), ethylketocyclazocine (EKC) and D-Ala-D-Leucine enkephalin (DADL). The alterations in chemical structure affected affinity of the analogs to the opiate receptor and their pharmacologic properties differently, suggesting that different opiate subtypes may be involved.

Dynorphin1-13 is a potent inhibitor of electrically-induced contractions in the guinea pig ileum, where it has the properties of kappa-(ethylketocyclazocine) type opioids. In the brain, however, it has no analgesic potency, yet inhibits that induced by morphine. To gain further insight into its mechanism of action in the latter system, we tested its ability to compete for the binding of several opiates to brain membranes in vitro. Dynorphin1-13 inhibited the binding of all ligands examined, including dihydromorphine, D-Ala2-D-Leu5-enkephalin, ethylketacyclazocine (EKC) and naloxone. In all cases, it reduced the number of high affinity sites and, in the case of EKC, it also increased the Kd. We conclude that the mechanism of dynorphin inhibition is not simple rapidly reversible competition and is certainly not identical with respect to all opiate ligands.