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PubMed2004

A novel soluble protein factor with non-opioid dynorphin A-binding activity.

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1-17), dynorphin A(2-17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1-17), dynorphin A(2-17), and big dynorphin is in the 5-10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6-13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.

Animals↗
PubMed1999

Dynorphin A (1-13) neurotoxicity in vitro: opioid and non-opioid mechanisms in mouse spinal cord neurons.

Dynorphin A is an endogenous opioid peptide that preferentially activates kappa-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1-13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both kappa-opioid and N-methyl-D-aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through kappa-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing kappa-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both kappa-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1-13) on intracellular calcium concentration ([Ca2+]i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1-13) elevated [Ca2+]i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 microM), 2-amino-5-phosphopentanoic acid (100 microM), or 7-chlorokynurenic acid (100 microM)--suggesting that dynorphin A (1-13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (-)-naloxone (3 microM), or the more selective kappa-opioid receptor antagonist nor-binaltorphimine (3 microM), exacerbated dynorphin A (1-13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 microM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 microM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates kappa-opioid receptors and suggests that kappa receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1-13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.

Analgesics, Opioid↗
PubMed2003

Dynorphin A toxicity in striatal neurons via an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor mechanism.

Dynorphin A (1-17) is an endogenous opioid peptide that is antinociceptive at physiological concentrations, but in excess can elicit a number of pathological effects. Both kappa-opioid and N-methyl-D-aspartate receptor antagonists modulate dynorphin toxicity, suggesting that dynorphin is acting directly or indirectly through these receptor types. We found in spinal cord neurons that the neurotoxic effects of dynorphin A and several dynorphin-derived peptide fragments are largely mediated by N-methyl-D-aspartate receptors. Despite these findings, aspects of dynorphin A toxicity could not be accounted for by opioid or N-methyl-D-aspartate receptor mechanisms. To address this issue, neurons enriched in kappa-opioid, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors were isolated from embryonic day-15 mouse striata and the effects of extracellularly administered dynorphin A (1-17) and (13-17) on neuronal survival were examined in vitro. Unlike spinal cord neurons, N-methyl-D-aspartate receptors mature later than alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors in striatal neurons, thus providing a strategy to elucidate non-N-methyl-D-aspartate receptor-mediated mechanisms of toxicity. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A (1-17 or 13-17; 10 microM) caused significant neuronal losses after 48 to 72 hours versus untreated controls. Dynorphin A or A (13-17) toxicity was unaffected by the opioid receptor antagonist naloxone (10 microM) or by dizocilpine (10 microM). In contrast, the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline- 2,3-dione (10 microM) significantly attenuated only dynorphin A (1-17)-induced neuronal losses and not that induced by dynorphin A (13-17). Dynorphin A (1-17) toxicity was accompanied by a proportional loss of R2 and R3 subunits of the AMPA receptor complex, but not non-N-methyl-D-aspartateR1, expressing neurons and was mimicked by the ampakine 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine. Although it is unclear whether dynorphin A activates alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors directly or indirectly via glutamate release, our culture conditions do not support glutamate retention or accumulation. Our findings suggest that dynorphin A (1-17) can exert toxic effects on striatal neurons via an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor mechanism.

Animals↗
PubMedDate not supplied

D-Ala2,F5Phe4-dynorphin amide, an opiate with analgesic and toxic properties.

A novel analog of dynorphin (1-13), D-Ala2,F5Phe4-dynorphin amide, was prepared and its pharmacological spectrum of activity was investigated. In a hot plate test on Swiss Webster and C57Bl mice, a 20 micrograms intracerebroventricular (icv) dose of the analog produced analgesia, which was greater in potency and duration than the parent dynorphin. This action of D-Ala2,F5Phe4-dynorphin amide was antagonized by the opiate receptor antagonist naloxone (2 mg/kg ip), administered either before or after the peptide. In addition to its analgesic action in mice, D-Ala2,F5Phe4-dynorphin amide produced a Straub tail and a catatonic-like state, both of which were also attenuated by naloxone. On the electrically-stimulated mouse vas deferens preparation, in vitro, D-Ala2,F5Phe4-dynorphin amide inhibited contractile activity and had an IC50 of 108.2 +/- 34.7 nM (SEM), about 4-fold weaker than that of dynorphin. This action was also attenuated by naloxone. An icv dose of 150 micrograms of D-Ala2,F5Phe4-dynorphin amide in mice, and a cumulative series of icv doses up to 2600 micrograms in anesthetized rats, failed to produce a lethal effect. No pathological changes were observed in mouse liver and kidney at 24 h after a 50 mg/kg dose of the peptide analog. In rats anesthetized with diallylbarbital (70 mg/kg ip) and urethane (280 mg/kg ip), D-Ala2,F5Phe4-dynorphin amide did not modify blood pressure, heart rate and respiratory rate. However, when mice were injected peripherally with single doses of D-Ala2,F5Phe4-dynorphin amide, convulsive episodes were produced, and lethal effects were observed with an LD50 of 60.0 mg/kg (95% confidence limits: 49.7-70.2 mg/kg) at 48 h. This action of D-Ala2,F5Phe4-dynorphin amide was not attenuated by naloxone (2.0 mg/kg, ip). Although analgesic and behavioral effects of D-Ala2,F5Phe4-dynorphin amide (e.g. Straub tail and catatonic-like state) are opiate-like, the lethal effect may be the consequence of actions of the peptide on non-opiate systems, Thus, the novel fluorinated dynorphin analog, D-Ala2,F5Phe4-dynorphin amide, may be a useful chemical tool for the study of opiate systems and their occasionally unanticipated biological or toxic actions.

Analgesics↗
PubMed1994

Dynorphin-immunoreactive terminals in the rat nucleus accumbens: cellular sites for modulation of target neurons and interactions with catecholamine afferents.

Dynorphin facilitates conditioned place aversion and reduces locomotor activity through mechanisms potentially involving direct activation of target neurons or release of catecholamines from afferents in the nucleus accumbens. We examined the ultrastructural substrates underlying these actions by combining immunoperoxidase labeling for dynorphin 1-8 and immunogold silver labeling for the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH). The two markers were simultaneously visualized in single coronal sections through the rat nucleus accumbens. By light microscopy, dynorphin immunoreactivity was seen as patches of immunoreactive varicosities throughout all rostrocaudal levels of the nucleus accumbens. The dynorphin-immunoreactive terminals identified by electron microscopy ranged from 0.2 to 1.5 microns in cross-sectional diameter, contained numerous small (30-40 nm) clear vesicles, as well as one or more large (80-100 nm) dense core vesicles. From the dynorphin-immunoreactive terminals quantitatively examined in single sections, 74% (173/370) showed symmetric synaptic junctions mainly with large unlabeled dendrites. Of the dynorphin-immunoreactive terminals forming identifiable synapses, approximately 30% contacted more than one dendritic target. In addition, single dendrites frequently received convergent input from more than one dynorphin-labeled terminal. Irrespective of their dendritic associations, dynorphin-immunoreactive terminals also frequently showed close appositions with other axons and terminals; these included unlabeled (41%), TH-labeled (10%) or dynorphin-labeled axons (14%). In contrast to dynorphin-immunoreactive terminals, TH-labeled terminals formed primarily symmetric synapses with small dendrites and spines or lacked recognizable specializations in the plane of section analyzed. In some cases, single dendrites were postsynaptic to both dynorphin and TH-immunoreactive terminals. We conclude that dynorphin-immunoreactive terminals potently modulate, and most likely inhibit, target neurons in both subregions of the rat nucleus accumbens. This modulatory action could attenuate or potentiate incoming catecholamine signals on more distal dendrites of the accumbens neurons. The findings also suggest potential sites for presynaptic modulatory interactions involving dynorphin and catecholamine or other transmitters in apposed terminals.

Animals↗
PubMed1995

The opioid peptide dynorphin modulates AMPA and kainate responses in acutely isolated neurons from the dorsal horn.

In freshly isolated spinal dorsal horn (DH) neurons (laminae I-IV) of the young rat, the effects of dynorphin A1-17, U-50,488H and U-69,593 on inward currents induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) were studied under whole-cell voltage-clamp conditions. When the cells were clamped to a holding potential of -60 mV, co-application of dynorphin A1-17 (10(-6) M) and AMPA (2 x 10(-5) M) reversibly decreased the peak amplitude of the initial transient component of the AMPA-induced current in 72% of the examined cells. In addition, dynorphin (10 microM) in perforated patch-recordings consistently produced a decrease in the steady-state component of the AMPA response. The depressant effect was concentration-dependent (IC50 = 86 nM) and reversible. The dynorphin A1-17-induced depression of the AMPA response was associated with slowing of the response kinetics, including both a 10-90% rise-time and time constant of decay. The AMPA-induced currents were modulated by dynorphin not only during the co-administration but also after the removal of the peptide. Dynorphin increased the initial peak AMPA current in 42% of the examined cells. Similar as with dynorphin A1-17, the peak amplitude of the AMPA-induced current was reversibly suppressed in the presence of 1 microM U-50,488H and U-69,593 in 75% and 86% of the examined cells, respectively. Naloxone and the kappa 1-selective antagonist norbinaltorphimine (nor-BNI) blocked the initial depressant but not late excitatory effects of dynorphin A1-17 and U-50,488H. This antagonistic effect of naloxone and norbinaltorphimine suggests that the depressant effect of dynorphin A1-17 on the AMPA-activated conductance is a true opioid, probably kappa 1-opioid receptor-mediated event. In contrast, the dynorphin-induced late potentiation of AMPA/KA responses appears to be a non-opioid effect since it was not inhibited by nor-BNI, CTAP and naltrindole, the selective kappa-, mu- and delta-opioid receptor blocking agents, respectively. Pretreatment of DH neurons with pertussis toxin blocked the depressant action of dynorphin A1-17, indicating that a Gi- or Go-type G protein was required for this effect on AMPA-activated currents. Intracellular dialysis with a highly specific peptide inhibitor (peptide 6-22) of the cAMP-activated protein kinase (PKA), and with Rp-cAMPS, prevented the depressant effect of dynorphin A1-17. In addition, staurosporine, a nonselective kinase inhibitor, blocked the dynorphin depression of the AMPA response.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Analysis of opioid and non-opioid end products of pro-dynorphin in the substantia nigra of the rat.

The substantia nigra is among the richest pro-dynorphin terminal field regions in the rat brain. We therefore contrasted processing in this area to the known processing in the posterior pituitary. Fractionation of acid extracts of the posterior pituitary by gel filtration followed by analysis by radioimmunoassay indicated that the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) averaged 1:2. The levels of dynorphin A-related end products to alpha-neo-endorphin and bridge peptide (a 2K nonopioid end product of pro-dynorphin) were approximately equimolar; however, the levels of dynorphin B-sized material were 50% lower than dynorphin A levels. Similar analyses of acid extracts of the substantia nigra also indicated that the levels of dynorphin A, alpha-neo-endorphin, and bridge peptide were approximately equimolar. In this terminal field the levels of dynorphin B-sized material were approximately 60% lower than dynorphin A. A striking feature of the nigral system was that the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) averaged 1:16. Thus, in the nigra, dynorphin A(1-17) is primarily a biosynthetic intermediate rather than as an end product.

Animals↗
PubMed1994

The striatonigral dynorphin pathway of the rat studied with in vivo microdialysis--I. Effects of K(+)-depolarization, lesions and peptidase inhibition.

Extracellular levels of dynorphin B were analysed with in vivo microdialysis in the neostriatum and substantia nigra of halothane-anaesthetized rats. Dopamine and its metabolites, 3,4-dihydroxyphenyl-acetic acid and homovanillic acid, as well as GABA were simultaneously monitored. Chromatographic analysis revealed that the dynorphin B-like immunoreactivity measured in perfusates collected under basal and K(+)-depolarizing conditions co-eluted with synthetic dynorphin B. Dynorphin B, GABA and dopamine levels were Ca(2+)-dependently increased by K(+)-depolarization, while 3,4-dihydroxyphenylacetic acid and homovanillic acid levels were decreased. Dopamine and its metabolites, but not dynorphin B or GABA levels, were significantly decreased after a unilateral 6-hydroxydopamine injection into the left medial forebrain bundle. In contrast, following a unilateral injection of ibotenic acid into the striatum, dynorphin B and GABA levels were decreased by > 50% in striatum and substantia nigra on the lesioned side, whereas no significant changes were observed in basal dopamine levels. The inclusion of the peptidase inhibitor captopril (50-500 microM) into the nigral perfusion medium produced a concentration-dependent increase in nigral extracellular levels of dynorphin B. In the striatum, a delayed increase in dynorphin B and GABA levels could be observed following the nigral captopril administration, but this effect was not concentration-dependent. Thus, we demonstrate that extracellular levels of dynorphin B, dopamine and GABA can simultaneously be monitored with in vivo microdialysis. Extracellular dynorphin B appears to originate from neurons, since the levels were (i) increased in a Ca(2+)-dependent manner by K(+)-depolarization, and (ii) decreased by a selective lesion of the striatum, known to contain cell bodies of dynorphin neurons in the striatonigral pathway. Furthermore, (iii) the increase in nigral dynorphin B levels by peptidase inhibition suggests the presence of clearance mechanisms for the released dynorphin peptides.

Animals↗
PubMed2000

Dynorphin B and spinal analgesia: induction of antinociception by the cannabinoids CP55,940, Delta(9)-THC and anandamide.

The endogenous opioid dynorphin B was evaluated for its role in cannabinoid-induced antinociception. Previous work in our laboratory has shown that the synthetic, bicyclic cannabinoid, CP55,940, induces the release of dynorphin B whilst the naturally occurring cannabinoid, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), releases dynorphin A. The dynorphins contribute in part to the antinociceptive effects of both cannabinoids at the level of the spinal cord. The present study compares dynorphin B released from perfused rat spinal cord in response to acute administration of anandamide (AEA), Delta(9)-THC and CP55,940 at two time points, 10 min and 30 min post administration, and attempts to correlate such release with antinociceptive effects of the drugs. Dynorphin B was collected from spinal perfusates of rats pretreated with Delta(9)-THC, CP55,940 or AEA. The supernatant was lyophilized and the concentrations of dynorphin B were measured via radioimmunoassay. At a peak time of antinociception (10 min), CP55,940 and Delta(9)-THC induced significant two-fold increases in the release of dynorphin B. AEA did not significantly release dynorphin B. Upon a 30-min pretreatment with the drugs, no significant dynorphin B release was observed, although antinociceptive effects persisted for CP55,940 and Delta(9)-THC. Previous work indicates that Delta(9)-THC releases dynorphin A while AEA releases no dynorphin A. This study confirms that although all three test drugs produced significant antinociception at 10 min, the endocannabinoid, AEA, does not induce antinociception via dynorphin release. Thus, our data indicate a distinct mechanism which underlies AEA-induced antinociception.

Analgesia↗
PubMed2000

Local inhibitory effects of dynorphin A-(1-17) on capsaicin-induced thermal allodynia in rhesus monkeys.

Although dynorphin A-(1-17) has been characterized in vitro as a high efficacy kappa-opioid receptor agonist, functional studies of dynorphin A-(1-17) following central or systemic administration indicate the involvement of both opioid and non-opioid components. The aim of this study was to investigate whether local administration of dynorphin-related analogs can attenuate capsaicin (8-methyl-N-vanillyl-6-nonenamide)-induced nociception and what type of opioid receptor mediates the local action of dynorphin A-(1-17) in monkeys. Capsaicin (100 microg) was used to evoke a nociceptive response, thermal allodynia, which was manifested as a reduced tail-withdrawal latency in normally innocuous 46 degrees C warm water. Co-administration of dynorphin A-(1-17) (0.3-10 microg) with capsaicin in the tail dose-dependently inhibited thermal allodynia; however, both non-opioid fragments dynorphin A-(2-17) (10-300 microg) and dynorphin A-(2-13) (10-300 microg) were ineffective. Local antiallodynia of dynorphin A-(1-17) was antagonized by a small dose (100 microg) of an opioid receptor antagonist, quadazocine, applied s.c. in the tail. Pretreatment with a selective kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI), s.c. 320 microg in the tail also reversed local antiallodynia of dynorphin A-(1-17). Both locally effective doses of antagonists, when applied s.c. in the back, did not antagonize local dynorphin A-(1-17), indicating that peripheral kappa-opioid receptors selectively mediated the local action of dynorphin A-(1-17) in the tail. In addition, a much larger dose of dynorphin A-(1-17) (1000 microg), when administered s. c. in the back or i.m. in the thigh, did not cause sedative or diuretic effects. These results suggest that in vivo opioid actions of dynorphin-related peptides can be differentiated locally in this procedure. They also indicate that local application of peptidic ligands may be a useful medication for localized pain.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1997

Potentiation of NMDA receptor-mediated responses by dynorphin at low extracellular glycine concentrations.

The effect of dynorphin A(1-13) on N-methyl-D-aspartate (NMDA)-activated currents was investigated in the presence of low extracellular glycine concentrations in Xenopus oocytes expressing recombinant heteromeric NMDA receptors and in cultured hippocampal neurons with the use of voltage-clamp techniques. At an extracellular added glycine concentration of 100 nM, dynorphin A(1-13) (10 microM) greatly increased the amplitude of NMDA-activated currents for all heteromeric subunit combinations tested; on average, the potentiation was: epsilon1/zeta1, 3,377 +/- 1,416% (mean +/- SE); epsilon2/zeta1, 1,897 +/- 893%; epsilon3/zeta1, 4,356 +/- 846%; and epsilon4/zeta1, 1,783 +/- 503%. Potentiation of NMDA-activated current by dynorphin A(1-13) was concentration dependent between 0.1 and 10 microM dynorphin A(1-13), with a half-maximal concentration value of 2.77 microM and an apparent Hill coefficient of 2.53, for epsilon2/zeta1 subunits at 100 nM added extracellular glycine. Percentage potentiation by dynorphin A(1-13) was maximal at the lowest glycine concentrations tested (0.01 and 0.1 microM), and decreased with increasing glycine concentration. No significant potentiation was observed at glycine concentrations > 0.1 microM for epsilon1/zeta1, epsilon2/zeta1, and epsilon4/zeta1 subunits, or at > 1 microM for epsilon3/zeta1 subunits. Potentiation of NMDA-activated currents by dynorphin A(1-13) was not inhibited by 1 microM of the kappa-opioid receptor antagonist nor-binaltorphimine, and potentiation was not observed with 10 microM of the kappa-opioid receptor agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzene-acetamide. Potentiation of NMDA-activated current by dynorphin A(1-13) was inhibited by the glycine antagonist kynurenic acid (50 microM). NMDA-activated current was also potentiated at low glycine concentrations by 10 microM dynorphin A(2-13) or (3-13), both of which have a glycine as the first amino acid, but not by 10 microM dynorphin A(4-13), which does not have glycine as an amino acid. In hippocampal neurons, 10 microM dynorphin A(1-13) or (2-13) potentiated steady-state NMDA-activated current in the absence of added extracellular glycine. The extracellular free glycine concentration, determined by high-performance liquid chromatography, was between 26 and 36 nM for the bathing solution in presence or absence of 10 microM dynorphin A(1-13), (2-13), (3-13), or (4-13), and did not differ significantly among these solutions. The observations are consistent with the potentiation of NMDA-activated current at low extracellular glycine concentrations resulting from an interaction of the glycine amino acids in dynorphin A(1-13) with the glycine coagonist site on the NMDA receptor. Because dynorphin A is an endogenous peptide that can be coreleased with glutamate at glutamatergic synapses, the potentiation of NMDA receptor-mediated responses could be an important physiological regulator of NMDA receptor function at these synapses.

Analgesics, Opioid↗
PubMed1984

Ontogenetic development of the pro-enkephalin B (= pro-dynorphin) opioid peptide system in the rat pituitary.

The postnatal development of several pro-enkephalin-B-derived opioid peptides - dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha-neo-endorphin and beta-neo-endorphin - was examined in rat pituitary lobes. The concentrations of pro-enkephalin-B-derived peptides from the anterior pituitary were between 4- and 12-fold and those from the neurointermediate pituitary between 17- and 122-fold lower in newborn as compared to adult rats. Similarly, the concentrations of vasopressin in the neurointermediate pituitary increased 50-fold between birth and adulthood; those of oxytocin, however, increased more than 540-fold over this period. The molecular weight pattern of dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha- and beta-neo-endorphin-immunoreactive peptides in the anterior and neurointermediate pituitary did not differ between 3-day-old pups and adult rats. In the neurointermediate pituitary, the major immunoreactive components had the same chromatographic properties as synthetic dynorphin 1-17, dynorphin 1-8, dynorphin B, alpha- and beta-neo-endorphin, respectively, on gel filtration and high-performance liquid chromatography (HPLC). This indicates that neonatal rats were already capable of processing the precursor pro-enkephalin B into these various opioid peptides. In newborn rats, however, the amount of dynorphin 1-8 in the neurointermediate pituitary was three times lower than that of its putative intermediate precursor peptide dynorphin 1-17. Similarly, the amount of beta-neo-endorphin was almost four times lower than that of its putative precursor alpha-neo-endorphin. In contrast, in the neurointermediate pituitary of adult rats, dynorphin 1-17 and dynorphin 1-8, in addition to a alpha- and beta-neo-endorphin, occurred in equimolar amounts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Dynorphin and neoendorphin peptides decrease dorsal root ganglion neuron calcium-dependent action potential duration.

Opioid peptides decrease somatic calcium-dependent action potential duration of a subpopulation of mouse dorsal root ganglion (DRG) neurons grown in dissociated cell culture. Based on rank order of potency and naloxone sensitivity, both mu and delta opioid receptors were demonstrated on the somata of DRG neurons and were shown to have a heterogeneous distribution. The purpose of the present investigation was to determine the actions of dynorphin gene products, dynorphin A, dynorphin B, dynorphin A(1-8), dynorphin A(1-9), alpha-neoendorphin and beta-neoendorphin on DRG neuron somatic calcium-dependent action potentials and to compare the actions of dynorphin and neoendorphin peptides to the action of morphiceptin, a mu receptor-selective ligand, and Leu-enkephalin, a delta receptor-preferring ligand. We report that the dynorphin and neoendorphin peptides decreased DRG neuron somatic calcium-dependent action potential duration in a portion of DRG neurons, an action that was dose-dependent and was antagonized by naloxone. DRG neuron responses to the dynorphins and neoendorphins differed from responses to morphiceptin and Leu-enkephalin. First, many DRG neurons responded to dynorphin A but not to morphiceptin or Leu-enkephalin. Second, dynorphin A responses, unlike responses to morphiceptin or Leu-enkephalin, were present after intracellular injection of cesium, a potassium channel blocker. Dynorphin A effectiveness was decreased after deletions at the carboxy-terminus and Leu-enkephalin [dynorphin A(1-5)] was inactive at 10 microM. Thus, on DRG neurons in cell culture, dynorphins and neoendorphins act at opioid receptors distinct from mu and delta receptors, possibly kappa receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1995

The mechanism of action for the block of NMDA receptor channels by the opioid peptide dynorphin.

Dynorphin is one of the endogenous opioids that modulates the excitability of nociceptive (pain-sensing) neurons. We have shown recently that dynorphin blocks NMDA-activated currents directly without the participation of kappa-opioid receptors. In order to understand the mechanism underlying this novel action of dynorphin, we examined, in detail, the interactions between dynorphin and NMDA receptors in isolated trigeminal neurons. Dynorphin reversibly blocks NMDA-activated current (INMDA). The onset and recovery of the block were determined with concentration jump experiments. The association rate (k+) of dynorphin(1-17) is 4.9 x 10(6) sec-1 M-1 and the dissociation rate (k-) is 7.5 sec-1. The apparent dissociation constant (KD) of dynorphin, calculated from these rate constants, is 1.6 microM. Dynorphin does not change the EC50 of NMDA, nor the potentiating action of glycine. The binding site for dynorphin is distinct from that of Zn2+ or H+. Upon treatment with the disulfide reducing agent dithiothreitol (DTT), NMDA receptors become less susceptible to dynorphin block. The affinity of dynorphin for the modified NMDA receptors is reduced by 2.7-fold. In analyses of single NMDA channels in cell-free patches, we found that dynorphin shortens the mean open time, decreases the probability of opening of NMDA channels, but has no effect on the single channel conductance. These results suggest that dynorphin interacts with a site conformationally linked with the redox site(s) on the NMDA receptor, thus altering the gating properties of the channel.

Animals↗
PubMed1993

Dynorphin modulates prolactin secretion in the turkey.

Big dynorphin (prodynorphin 209-240), dynorphin A (prodynorphin 209-225), dynorphin B (prodynorphin 228-240), beta-endorphin (beta-lipotrophin 61-90), or Met-enkephalin, each infused into the third ventricle, were tested for their effect on PRL release in the anesthetized turkey hen. Laying hens that received big dynorphin at the rate of 0.35 nmol/min showed a 5.1-fold increase in serum PRL at the end of a 30-min infusion period. In a second experiment, the big dynorphin-induced PRL increase was 2.6-fold. Nest-deprived, previously incubating hens that received big dynorphin displayed an 8.2-fold increase in serum PRL. Laying and nest-deprived incubating control birds infused with saline displayed no PRL increases. Laying hens that received dynorphin A (0.35 nmol/min) showed a 1.5-fold increase in serum PRL after 30 min of infusion; after 40 min of infusion, this increase rose to 2.7-fold. Infusions of beta-endorphin (0.35 nmol/min), or Met-enkephalin (0.35 nmol/min) failed to evoke PRL increases in either laying or nest-deprived incubating turkeys. Infusion of big dynorphin or dynorphin A for 120 min maintained an elevated PRL level across the period, a level equal to that evoked by electrical stimulation of the medial preoptic nucleus (ES/POM). Infusion of dynorphin B (0.48 nmol/min) or a reduced dose of dynorphin A (0.09 nmol/min) augmented the PRL response evoked by ES/POM. No augmentation was noted for beta-endorphin or Met-enkephalin, nor for saline-infused controls. The dynorphin-induced PRL response appeared to be dose-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Involvement of spinal kappa opioid receptors in the antagonistic effect of dynorphins on morphine antinociception.

The modulatory effects of intrathecally (i.t.) administered dynorphin A(1-17) and dynorphin A(1-13) on morphine antinociception have been studied previously in rats by other investigators. However, both potentiating and attenuating effects have been reported. In this study, the modulatory effects of i.t. administered dynorphin A(1-17) as well as the smaller fragment, dynorphin A(1-8), were studied in mice. In addition, nor-binaltorphimine (nor-BNI), a highly selective kappa opioid receptor antagonist, and naltrindole (NTI), a highly selective delta opioid receptor antagonist, were used to characterize the possible involvement of spinal kappa and delta opioid receptors in the modulatory effects of the dynorphins. Dynorphin A(1-17) and dynorphin A(1-8) administered i.t. at doses that did not alter tail-flick latencies, were both able to antagonize in a dose-dependent manner, the antinociceptive action of s.c. administered morphine sulfate. The antinociceptive ED50 of morphine sulfate was increased 3.9- and 5.3-fold by 0.4 nmol/mouse of dynorphin A(1-17) and dynorphin A(1-8), respectively. Injections of 0.4 and 0.8 nmol/mouse of nor-BNI i.t., but not its inactive enantiomer (+)-1-nor-BNI, inhibited dose-dependently the antagonistic effects of the dynorphins. These doses of nor-BNI alone did not affect the antinociceptive action of morphine sulfate. Intrathecal administration of 5 nmol/mouse of NTI also did not affect the modulatory effects of dynorphins. These observations that dynorphins exert their antagonistic effects on morphine-induced antinociception stereoselectively through spinal kappa opioid receptors may suggest a coupling between spinal kappa and mu opioid receptors.

Animals↗
PubMed1981

Presence of dynorphin-like immunoreactivity in rat pituitary gland and hypothalamus.

Using a highly specific and sensitive radioimmunoassay for dynorphin(1-13), dynorphin-like immunoreactivity (dynorphin-LI) was detected in rat pituitary and hypothalamus. Gel chromatographic studies on Sephadex G-50 revealed three components of dynorphin-LI with molecular weights of approximately 7500-9500 (big dynorphin), 3500-5500 (intermediate dynorphin) and 1600-1900 (small dynorphin), the latter of which eluted at the same position as authentic dynorphin contamination in porcine ACTH extracts (Sigma). Dynorphin-LI in rat anterior pituitary existed mainly as big dynorphin, whereas dynorphin-LI in rat intermediate-posterior pituitary and hypothalamus eluted mainly at the position of authentic small dynorphin.

Animals↗
PubMedDate not supplied

The degradation of dynorphin A in brain tissue in vivo and in vitro.

The demonstration of analgesia following in vivo administration of dynorphin A (Dyn A) has been difficult. In contrast, a number of electrophysiological and behavioral effects reported with in vivo injection of Dyn A can be produced by des-tyrosine dynorphin A (Dyn A 2-17). This suggested the extremely rapid amino terminal degradation of dynorphin A. To test this hypothesis, we examined the degradation of dynorphin A following in vivo injection into the periaqueductal gray (PAG) as well as in vitro using rat brain membranes under receptor binding conditions. In vivo, we observed the rapid amino terminal cleavage of tyrosine to yield the relatively more stable destyrosine dynorphin A. This same cleavage after tyrosine was observed in vitro. Inhibition of this aminopeptidase activity in vitro was observed by the addition of dynorphin A 2-17 or dynorphin A 7-17 but not after the addition of dynorphin A 1-13, dynorphin A 1-8, dynorphin B or alpha-neo-endorphin suggesting a specific enzyme may be responsible. The detection of the behaviorally active des-tyrosine dynorphin A following in vivo injection of dynorphin A suggests that this peptide may play an important physiological role.

Animals↗