dynorphins and dynorphin-amide-(1-10)

There is experimental evidence that indicates that the endogenous opioid system of the central nucleus of the amygdala (CeA) may mediate some of the reinforcing effects of ethanol. However, the precise interactions of ethanol with the endogenous opioid system at the level of the CeA have not been investigated.. The aim of the current study was to investigate the hypothesis that acute systemic ethanol administration will increase the release of endogenous opioid peptides at the level of the CeA in a time- and dose-dependent manner.. Rats were implanted with a unilateral guide cannula to aim microdialysis probes at the CeA. Intraperitoneal injections of saline and various doses of ethanol (0.8, 1.6, 2.0, 2.4, and 2.8 g ethanol/kg body weight) were administered to the rats. Dialysate samples were collected at 30-min intervals at distinct time points prior to and following treatment. Radioimmunoassays specific for beta-endorphin, met-enkephalin, and dynorphin A1-8 were used to determine the effect of ethanol on the content of the opioid peptides in the dialysate.. We report that the 2.8-g/kg dose of ethanol induced a long-lasting increase in beta-endorphin release from 60 min onwards following administration and, later, an ongoing increase in dynorphin A1-8 release. None of the ethanol doses tested elicited significant changes in dialysate met-enkephalin content compared to the saline treatment.. Acute systemic ethanol administration induced a dose- and time-dependent increase in beta-endorphin and dynorphin A1-8 release at the level of the CeA, which may be involved in ethanol consumption.

Topics: Amygdala; Animals; beta-Endorphin; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Methionine; Ethanol; Injections, Intraperitoneal; Male; Microdialysis; Opioid Peptides; Peptide Fragments; Peptides; Photomicrography; Rats; Rats, Sprague-Dawley

It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibly to the opioid receptors in vitro. Recently a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gly chloromethyl ketone (Dynorphin(1-10)-Gly11 chloromethyl ketone, DynCMK) was synthesized, and its binding characteristics to frog (Rana esculenta) brain membranes were evaluated. In competition experiments, the product shows a relatively high affinity for the kappa-opioid binding sites labelled by [3H]ethylketocyclazocine (Ki is approximately equal to 200 nM), whereas its binding to the 1 ([3H]dihydromorphine) and to the delta sites ([3H]D-Ala2-Leu5]enkephalin) is weaker. Preincubation of the frog brain membranes with DynCMK at micromolar concentrations results in a washing-resistant and dose-dependent inhibition of the [3H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 microM DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine compared to the control values. The kappa-preferring binding properties of the compound suggest that it could serve as an affinity label for the kappa-type of opioid receptors.

Topics: Affinity Labels; Amino Acid Chloromethyl Ketones; Analgesics, Opioid; Animals; Binding, Competitive; Brain Chemistry; Dihydromorphine; Dynorphins; Enkephalin, Leucine-2-Alanine; Ethylketocyclazocine; Membrane Proteins; Peptide Fragments; Radioligand Assay; Rana esculenta; Receptors, Opioid; Receptors, Opioid, kappa; Tritium

The analgesic effects of intracerebroventricularly (i.c.v.) administered dynorphin A(1-13) and its analog dynorphin A(1-10)amide using the hot plate test were studied in mice. Both dynorphins applied i.c.v. by the freehand method had an analgesic effect but no effect was seen when applied i.c.v. through an implanted cannula. Moreover, freehand i.c.v. injection of saline increased the time of immobility in the forced swimming test and glycaemia levels compared with intact mice. In contrast to the freehand injection, saline administration through an implanted cannula did not influence the immobility of animals in forced swimming test when compared with the intact controls. These results suggest that 1) the freehand method is very stressful procedure of administration which could influence the effects of dynorphins in the hot plate test and 2) dynorphins exert an analgesic effect in the hot plate test only when combined with a stressor (freehand i.c.v. injection).

Topics: Analgesics, Opioid; Animals; Dynorphins; Immobilization; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Pain Threshold; Peptide Fragments; Stress, Physiological; Swimming

The biotransformation in human blood in vitro of three dynorphin A (Dyn A) peptides was studied by matrix assisted laser desorption mass spectrometry to determine whether the natural peptide, Dyn A(1-17), is biotransformed differently from Dyn A (1-13), the natural sequence shortened form used in numerous neurobiological and pharmacological studies. In addition to studies of Dyn A(1-17), a natural product from prodynorphin and Dyn A(1-13), a natural sequence truncation of Dyn A(1-17), Dyn A(1-10)amide, a synthetic analogue of Dyn A(1-17) presumed to be protected from rapid biotransformation was also studied Synthetic Dyn A peptides were incubated in freshly drawn blood for various periods of time prior to mass spectrometric analysis. Several peptide products were identified from each precursor; the time profiles of appearance and disappearance of the major products were followed. Substantial differences in products and especially in the rate of biotransformation were observed between the processing of Dyn A(1-17) and the two shorter Dyn A peptides, Dyn A(1-13) and Dyn A(1-10)amide. Significant amounts of the natural Dyn A(1-17) survived 4 h of incubation (half-life 3 h). Dyn A (2-17), a major processed product of Dyn A(1-17) in blood, continued to accumulate during the 4-h incubation period. By contrast, both Dyn A(1-13) and Dyn A(1-10) amide were biotransformed very rapidly with half-lives of < 1 min and 10 min, respectively. Most of the products from these two peptide precursors were also further processed rapidly, with the exception of Dyn A(4-12) and Dyn A(4-10)amide, which were detected for over 2 h. Dyn A(1-6) was found as a minor biotransformation product from all three precursor peptides. These findings suggest that an important function of the four C-terminal amino acid residues of the natural form, Dyn A(1-17) [compared to Dyn A(1-13)], is to stabilize or protect the peptide from biotransformation by enzymes, by preserving a natural hairpin structure possibly near the carboxyl-terminus.

Topics: Adult; Analgesics; Biotransformation; Blood; Chromatography, High Pressure Liquid; Dynorphins; Female; Humans; Kinetics; Male; Middle Aged; Peptide Fragments; Sequence Analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Dynorphin A (1-13) and its analog dynorphin A (1-10) amide were applied intracerebroventricularly in male ICR mice. Both dynorphins did not reveal any analgesic activity in tail-flick test under normal (non-stressed) conditions. However, in combination with stress (forced swimming or whole body vibration) both dynorphins prolonged tail-flick latencies when compared with stressed saline controls. Naloxone inhibited the effect of dynorphins in forced swimming test. Neither dynorphin A (1-13) nor dynorphin A (1-10) amide increased tail-flick latencies when combined with weak immobilization stress. Our results suggest that the analgesic effects of dynorphins are potentiated by strong stressors.

Topics: Analgesia; Analgesics, Opioid; Animals; Dynorphins; Immersion; Immobilization; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Naloxone; Narcotic Antagonists; Pain Measurement; Peptide Fragments; Stress, Psychological; Swimming; Vibration

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.

Topics: Analgesics; Animals; Bone Marrow; Cell Division; Colony-Forming Units Assay; Dynorphins; Macrophage Colony-Stimulating Factor; Mice; Morphine; Naloxone; Organ Size; Peptide Fragments; Spleen

A kappa-opioid receptor subtype was purified from a digitonin extract of frog brain membranes, using affinity chromatography. The affinity resin was prepared by coupling dynorphin (1-10) to AH Sepharose 4B. The purified receptor binds 4,750 pmol [3H]ethylketocyclazocine (EKC) per mg protein (5,600-fold purification over the membrane-bound receptor) with a Kd of 9.1 nM. The addition of cholesterol-phosphatidylethanolamine (2:1) enhanced 3.6-fold the binding activity of the purified material, which gives a purification very close to the theoretical. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows one major band (65,000 Mr) in sodium dodecyl sulfate (SDS) gel electrophoresis.

Topics: Animals; Chromatography, Affinity; Dynorphins; Peptide Fragments; Rana esculenta; Receptors, Opioid; Receptors, Opioid, kappa; Subcellular Fractions

Intravenous injection of dynorphin A-(1-10) amide (Dyn, 81-324 nmol/kg) induced a dose-dependent hypotensive effect in the rat. This effect was antagonized by pretreatment with immunoglobulin G, purified from a specific antiserum raised against alpha-human atrial natriuretic peptide (anti-hANP-IgG), as well as by high doses of naloxone (2 or 10 mg/kg). In addition, a 12-fold increase in plasma level of atrial natriuretic peptide-like immunoreactivity (ANP-IR) was found following Dyn administration, which was accompanied by a significant decrease of atrial ANP-IR. These results suggest that the stimulated release of ANP-IR from the atrium may constitute one of the mechanisms for the depressor effect of dynorphin peptides.

Topics: Animals; Atrial Natriuretic Factor; Dose-Response Relationship, Drug; Dynorphins; Heart Atria; Hypotension; Immunoglobulin G; Male; Myocardium; Peptide Fragments; Rats; Rats, Inbred Strains; Time Factors

Topics: Animals; Antihypertensive Agents; Atrial Natriuretic Factor; Blood Pressure; Dynorphins; Male; Peptide Fragments; Rats; Rats, Inbred Strains

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.

Topics: Animals; Dynorphins; Follicle Stimulating Hormone; Growth Hormone; Luteinizing Hormone; Macaca mulatta; Male; Naloxone; Peptide Fragments; Pituitary Gland, Anterior; Pituitary Hormones, Anterior; Prolactin; Thyrotropin

In vitro lipid-mobilizing activities of morphine (MO) and dynorphin-(1-10) amide (DYA) were compared using adipocytes of young adult rats (body weight 170-200 g, age 50-60 days, diameter of adipocytes 54 +/- 1.12 microns) and of 20-month-old rats (body weight 500-690 g, diameter of adipocytes 99.5 +/- 3.0 microns). The adipokinetic activities of adenosine deaminase (ADA) and dynorphin-(1-13) (DY) were also tested. In the experiments 0.16 units of ADA did not influence basal or stimulated lipolysis, whereas DY exerted a slight but statistically significant adipokinetic effect. In three experimental series the EC50 of MO ranged between 0.89 and 14.60 mumol l-1, the EC50 of DYA in young rats was estimated as 0.8 mumol l-1 and in old animals, 1.3 mumol l-1. Statistically significant differences in the lipid-mobilizing potency between DYA and MO could be observed only in one experimental series in young rats. Expressed in percent of maximum lipolysis induced by isoprenaline, the maximum lipolytic response to DYA in young animals was significantly lower (Emax 26.1 +/- 1.9) when compared to the maximum effect of MO (Emax 76.3 +/- 8.5 and 68.9 +/- 3.5, respectively). Adipocytes of old rats seemed to be more sensitive not only against MO but also against DYA. When studying lipolysis no signs of competitive dualism could be observed in the interaction between MO and DYA. The possibility of two or more independent lipid-mobilizing mechanisms in the effect of the two opioids compared cannot be excluded.

Topics: Adenosine Deaminase; Adipose Tissue; Animals; Dynorphins; In Vitro Techniques; Lipid Mobilization; Lipolysis; Male; Morphine; Peptide Fragments; Rats; Rats, Inbred Strains

Topics: Animals; Atrial Natriuretic Factor; Dynorphins; Heart; Male; Myocardium; Peptide Fragments; Radioimmunoassay; Rats; Rats, Inbred Strains

A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol [3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Brain Chemistry; Chromatography, Gel; Cyclazocine; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Membranes; Molecular Weight; Naloxone; Peptide Fragments; Pyrrolidines; Rana esculenta; Receptors, Opioid; Receptors, Opioid, kappa; Solubility

Diode-array UV detection has been adapted for analysis of opioid peptides and their metabolic fragments differing in aromatic amino acid content. In combination with high-performance liquid chromatography, the technique allowed a direct and rapid discrimination between peptides containing phenylalanine, tryptophan and tyrosine, or a combination of these residues. Enkephalin fragments with either tyrosine or phenylalanine, or both, were identified after digestion of the pentapeptide with proteolytic activity recovered from human cerebrospinal fluid. The N-terminal tyrosine-containing fragment of dynorphin A was identified after hydrolysis of the peptide by a cerebrospinal fluid endopeptidase. The study was extended to the analysis of some non-opioid peptides. The Tyr1 analogue of delta-sleep-inducing peptide was easily distinguished from the authentic compound with a tryptophan at the N-terminus. Results indicated that the technique was useful for discriminating between dipeptides differing in aromatic residues that were unresolved by high-performance liquid chromatography.

Topics: Amino Acids; Chromatography, High Pressure Liquid; Dynorphins; Endopeptidases; Endorphins; Enkephalin, Leucine; Humans; Neprilysin; Peptide Fragments; Peptides; Spectrophotometry, Ultraviolet

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.

Topics: Animals; Dynorphins; Female; Macaca mulatta; Naloxone; Peptide Fragments; Pituitary Hormones, Anterior; Receptors, Opioid; Thyrotropin

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.

Topics: Animals; Brain; Drug Tolerance; Dynorphins; Guinea Pigs; Ileum; In Vitro Techniques; Male; Mice; Nociceptors; Peptide Fragments; Receptors, Opioid; Synaptosomes; Vas Deferens

The effectiveness of beta-endorphin, dynorphin-(1-13), dynorphin-(1-10) amide, alpha-neoendorphin and [D-Ala2,D-Leu5]enkephalin in suppressing withdrawal in heroin addicts was compared in this study. Groups of six patients were stabilized overnight in the hospital and were treated with either saline or peptide when withdrawal symptoms began to appear the following morning. Withdrawal was scored before and after treatment by the patient himself and an independent observer. Peptides were administered in a bolus dose of 60 micrograms/kg body weight. The patient, the observer and the physician who administered the injection were all blind to the nature of the compound given. All treatments, including those with saline, produced an overall reduction of withdrawal score. However, by statistical analysis, only treatments with beta-endorphin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13) were effective in producing a significant decrease of withdrawal symptoms. The length of relief brought about by the different peptides varied from less than an hour to a maximum of 5 h in one case. The average period of relief brought about by beta-endorphin, dynorphin-(1-13) and [D-Ala2,D-Leu5]enkephalin was 44, 46 and 60 min, respectively. Of the five peptides administered [D-Ala2,D-Leu5]enkephalin produced the largest number of side-effects.

Topics: Adult; Animals; beta-Endorphin; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Heroin; Humans; Male; Mice; Peptide Fragments; Protein Precursors; Substance Withdrawal Syndrome

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.

Topics: Animals; Drug Tolerance; Dynorphins; Endorphins; Male; Mice; Mice, Inbred ICR; Morphine; Peptide Fragments; Protein Precursors; Respiration