The effect of U-50,488H tolerance-dependence and abstinence on the levels of dynorphin (1–13) in brain regions, spinal cord, pituitary gland and peripheral tissues of the rat

Male Sprague-Dawley rats were rendered tolerant to and physically dependent on U-50,488H, a κ-opiate agonist, by injecting 25 mg/kg of the drug intraperitoneally twice a day for 4 days. Two sets of rats were used. Rats labeled as tolerant-dependent were injected with U-50,488H (25 mg/kg) 1 h before sacrificing on day 5, whereas the abstinent rats were sacrificed on day 5 without the injection of U-50,488H. Of all the tissues examined, the pituitary gland had the highest level of dynorphin (1–13), whereas the heart had the lowest level. The levels of dynorphin (1–13) increased in the hypothalamus, hippocampus and pons/medulla of U-50,488H tolerant-dependent rats, whereas in abstinent rats the levels of dynorphin (1–13) were elevated only in the midbrain. The levels of dynorphin (1–13) in the pituitary gland of U-50,488H tolerant-dependent or abstinent rats were unchanged. In peripheral tissues, the levels of dynorphin (1–13) in the heart of U-50,488H tolerant-dependent rats were increased. In the abstinent rats they were elevated in the adrenals, spleen, and the heart but were decreased in the kidneys. Compared to morphine tolerant-dependent and abstinent rats, significant differences in the levels of dynorphin (1–13) in tissues of 50,488H tolerant-dependent and abstinent rats were observed and may explain many pharmacological differences in the μ- and κ-opiate induced tolerance-dependence and abstinence processes.     

Effect of chronic treatment with morphine, midazolam and both together on dynorphin(1–13) levels in the rat

We have recently reported that midazolam, a benzodiazepine receptor agonist that is also a short acting anesthetic and analgesic drug, can produce analgesia and decrease morphine tolerance and dependence in the rat by interacting with the opioidergic system. This study was designed to investigate the chronic effect of midazolam and/or morphine on the levels of dynorphin(1–13) in the pituitary gland, different brain regions, spinal cord and peripheral tissues of the rat. Four sets of animals were used: (I) saline-saline; (II) midazolam (0.03, 0.3 or 3.0 mg/kg, body wt., i.p.)-saline; (III) saline-morphine (10.0 mg/kg, body wt., s.c.); and (IV) midazolam-morphine (0.03, 0.3 or 3.0 mg/kg midazolam+10.0 mg/kg morphine) groups. The first saline or midazolam injection was given i.p. and after 30 min, the second injection of saline or morphine was given s.c. daily for 11 days. Animals were sacrificed on the 11th day, 60 min after the last injection and dynorphin(1–13) was measured in indicated tissues by radioimmunoassay method. The midazolam treated animals showed a significant decrease in dynorphin(1–13) levels in the cortex, cerebellum, cervical region of spinal cord, heart and adrenals, and a significant increase in the hypothalamus, striatum and lumbar region of the spinal cord. The morphine treated animals showed a significant decrease in dynorphin(1–13) levels in the pituitary gland, hypothalamus, hippocampus, striatum, cerebellum, pons, medulla, kidneys, adrenals and spleen, and a significant increase only in the lumbar region of the spinal cord. When both drugs were injected together there was no effect on pituitary gland, kidneys and spleen. These drugs antagonize each other's effect on dynorphin(1–13) in the hypothalamus, striatum, cerebellum, pons, medulla and heart. However, the midazolam-morphine combination significantly increases dynorphin(1–13) levels in the hippocampus, cortex, midbrain, cervical and lumbar regions of the spinal cord, and adrenals. These results suggest the involvement of dynorphin(1–13) in the inhibition of morphine-induced tolerance and dependence by midazolam in the rat. These results may also help us in understanding the intrinsic mechanisms involved in narcotic tolerance and dependence.© 1997 Elsevier Science B.V. All rights reserved.     

β-endorphin and methionine-enkephalin levels in discrete brain regions, spinal cord, pituitary gland and plasma of morphine tolerant-dependent and abstinent rats

The effect of morphine tolerance-dependence, protracted and naloxone-precipitated abstinence on the levels of β-endorphin and methionine-enkephalin in discrete brain regions, spinal cord, pituitary gland and plasma was determined in the male Sprague-Dawley rats. Among the brain regions examined, the levels of β-endorphin in descending order were: hypothalamus, amygdala, midbrain, hippocampus, corpus striatum, pons and medulla and cortex. The levels of β-endorphin in midbrain, hypothalamus, and pituitary of morphine tolerant-dependent rats were decreased significantly. During protracted withdrawal β-endorphin levels were decreased in amygdala, spinal cord and pituitary. During naloxone-precipitated abstinence β-endorphin levels were increased in corpus striatum, midbrain and cortex. In addition, in naloxone-precipitated abstinence β-endorphin levels were decreased in pituitary gland and hippocampus but increased in plasma. The levels of methionine-enkephalin in brain regions in decreasing order were: corpus striatum, pons and medulla, amygdala, hypothalamus, midbrain, hippocampus and cortex. The levels of methionine-enkephalin in pons and medulla, amygdala, hippocampus and pituitary gland were decreased in morphine tolerant-dependent rats. During protracted abstinence from morphine, methionine-enkephalin levels in spinal cord, amygdala, pons and medulla, midbrain, cortex, corpus striatum and pituitary gland were decreased. The levels of methionine-enkephalin in hypothalamus and corpus striatum of naloxone-precipitated abstinent rats were increased but were decreased in amygdala and pituitary gland. These results suggest that during morphine tolerance-dependence and during protracted abstinence β-endorphin and methionine-enkephalin levels in discrete brain regions and pituitary gland are decreased. During precipitated abstinence β-endorphin levels are increased in brain regions (except hippocampus) and plasma but decreased in pituitary, whereas methionine-enkephalin levels in amygdala and pituitary gland are decreased except in corpus striatum and hypothalamus where they are increased. The pituitary levels of β-endorphin where reduced in all three conditions. However, the levels after withdrawal were not significantly different from those in tolerant—dependent animals.     

Beta-endorphin and methionine-enkephalin levels in discrete brain regions, spinal cord, pituitary gland and plasma of morphine tolerant-dependent and abstinent rats

The effect of morphine tolerance-dependence, protracted and naloxone-precipitated abstinence on the levels of beta-endorphin and methionine-enkephalin in discrete brain regions, spinal cord, pituitary gland and plasma was determined in the male Sprague-Dawley rats. Among the brain regions examined, the levels of beta-endorphin in descending order were: hypothalamus, amygdala, midbrain, hippocampus corpus striatum, pons and medulla and cortex. The levels of beta-endorphin in midbrain, hypothalamus, and pituitary of morphine tolerant-dependent rats were decreased significantly. During protracted withdrawal beta-endorphin levels were decreased in amygdala, spinal cord and pituitary. During naloxone-precipitated abstinence beta-endorphin levels were increased in corpus striatum, midbrain and cortex. In addition, in naloxone-precipitated abstinence beta-endorphin levels were decreased in pituitary gland and hippocampus but increased in plasma. The levels of methionine-enkephalin in brain regions in decreasing order were: corpus striatum, pons and medulla, amygdala, hypothalamus, midbrain, hippocampus and cortex. The levels of methionine-enkephalin in pons and medulla, amygdala, hippocampus and pituitary gland were decreased in morphine tolerant-dependent rats. During protracted abstinence from morphine, methionine-enkephalin levels in spinal cord, amygdala, pons and medulla, midbrain, cortex, corpus striatum and pituitary gland were decreased. The levels of methionine-enkephalin in hypothalamus and corpus striatum of naloxone-precipitated abstinent rats were increased but were decreased in amygdala and pituitary gland. These results suggest that during morphine tolerance-dependence and during protracted abstinence beta-endorphin and methionine-enkephalin levels in discrete brain regions and pituitary gland are decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of morphine in brain regions, spinal cord and serum following intravenous injection to morphine tolerant rats

In order to determine the possible contribution of altered distribution of morphine in the morphine tolerance process, the distribution of morphine was studied in brain regions and spinal cord, following its intravenous administration. Male Sprague-Dawley rats were made tolerant to morphine by implanting 6 morphine pellets, each containing 75 mg of morphine base, for 7 days. Seventy-two hours after the removal of the pellets, a time when serum morphine levels were negligible or absent and yet tolerance to the pharmacological effects of morphine was present, morphine (10 mg/kg, i.v.) was injected in placebo and morphine pellet implanted rats. At various times (5, 30, 60, 120 and 360 min) after the injection of morphine, brain regions (hypothalamus, cortex, hippocampus, midbrain, pons and medulla, striatum and amygdala), spinal cord and serum were collected. The level of morphine in the tissues was determined by using a highly sensitive and specific radioimmunoassay (RIA) method. Five minutes after morphine injection, the concentration of morphine was the highest in the hypothalamus and the lowest in amygdala. The concentration of morphine in hypothalamus, pons and medulla, hippocampus and midbrain of morphine tolerant rats was smaller than in placebo pellet implanted rats. The tissue to serum ratio of morphine in the hypothalamus, hippocampus, striatum, midbrain and cortex were also smaller in morphine tolerant than in non-tolerant rats. The concentration of morphine in brain regions with time did not exhibit linearity. At other time intervals like 30 and 60 min, the concentration of morphine in several brain regions and spinal cord was significantly higher in morphine tolerant than in non-tolerant rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diazepam binding inhibitor-like immunoreactivity(51–70): distribution in human brain, spinal cord and peripheral tissues

We have used specific radioimmunoassay to describe the distribution of diazepam binding inhibitor-like immunoreactivity (DBI-IR(51–70)) in human post-mortem tissues. In brain, highest concentrations were found in the cerebellum, amygdala, hippocampus, hypothalamus and substantia nigra. In the spinal cord, DBI-IR(51–70) was evenly distributed. In peripheral tissues, highest concentrations were found in the liver and kidney. Chromatographic analysis revealed several molecular forms of DBI-IR(51–70) the major form being of greater molecular weight and hydrophobicity than the synthetic fragment peptide. In peripheral tissues, but not in the CNS, a small peak of immunoreactivity was indistinguishable form the synthetic peptide. DBI-IR(51–70) is therefore widespread, but tissue processing may be different.     

Suppression of morphine withdrawal by electroacupuncture in rats: dynorphin and κ-opioid receptor implicated

Our previous work has demonstrated that 100-Hz electroacupuncture (EA) or 100-Hz transcutaneous electrical nerve stimulation (TENS) was very effective in ameliorating the morphine withdrawal syndrome in rats and humans. The mechanism was obscure. (1) Rats were made dependent on morphine by repeated morphine injections (5–140 mg/kg, s.c., twice a day) for eight days. They were then given 100-Hz EA for 30 min 24 h after the last injection of morphine. A marked increase in tail flick latency (TFL) was observed. This effect of 100-Hz EA could be blocked by naloxone (NX) at 20 mg/kg, but not at 1 mg/kg, suggesting that 100-Hz EA-induced analgesia observed in morphine-dependent rats is mediated by κ-opioid receptors. (2) A significant decrease of the concentration of dynorphin A (1–17) immunoreactivity (-ir) was observed in the spinal perfusate in morphine-dependent rats, that could be brought back to normal level by 100-Hz EA. (3) 100-Hz EA was very effective in suppressing NX-precipitated morphine withdrawal syndrome. This effect of EA could be prevented by intrathecal administration of nor-BNI (2.5 μg/20 μl), a κ-opioid receptor antagonist, or dynorphin A (1–13) antibodies (25 μg/20 μl) administered 10 min prior to EA. In conclusion, while the steady-state spinal dynorphin release is low in morphine-dependent rats, it can be activated by 100-Hz EA stimulation, which may be responsible for eliciting an analgesic effect and ameliorating morphine withdrawal syndrome, most probably via interacting with κ-opioid receptor at spinal level.     

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 κ-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 κ-opioid and N-methyl- -aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through κ-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 κ-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both κ-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 μM), 2-amino-5-phosphopentanoic acid (100 μM), or 7-chlorokynurenic acid (100 μM)—suggesting that dynorphin A (1–13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (−)-naloxone (3 μM), or the more selective κ-opioid receptor antagonist nor-binaltorphimine (3 μM), exacerbated dynorphin A (1–13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 μM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 μM). 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 κ-opioid receptors and suggests that κ 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.     

Evidence for the behavioral supersensitivity of dopamine D2 receptors without receptor up-regulation in morphine-abstinent rats

The effect of morphine tolerance-dependence and abstinence on the characteristics of dopamine D₂ receptors in brain regions and spinal cord was determined in the rat. Male Sprague-Dawley rats were implanted s.c. under light ether anesthesia with 6 morphine pellets for a 7-day period, each containing 75 mg of morphine free base. Rats implanted with placebo served as controls. This procedure resulted in the development of tolerance to morphine as evidenced by decreased analgesic response to a challenge dose of morphine. Similarly, the development of physical dependence was evidenced by a decreased in body weight and colonic temperature after morphine pellet removal (withdrawal). The binding characteristics (B_max andK_d values) of [³H]spiroperidol to dopamine D₂ receptors were determined in the tissues of morphine-tolerant and morphine-abstinent rats. In the tolerant rats, the pellets were left intact at the time of sacrificing, whereas, in the abstinent rats the pellets were removed 18 h prior to sacrificing. The binding of [³H]spiroperidol was determined in membranes prepared from brain regions (hypothalamus, hippocampus, cortex, pons and medulla, midbrain, corpus striatum and amygdala) and spinal cord of rats from various treatment groups. [³H]Spiroperidol bound to brain regions and spinal cord at a single high affinity site. TheB_max or theK_d values in brain regions and spinal cord of morphine-tolerant and -abstinent rats did not differ from their respective placebo controls. The behavioral responses to a selective dopamine D₂ receptor agonist, 2-bromo-α-ergocryptine were also determined in the morphine-abstinent rats. In morphine-abstinent rats, increased behavioral activity, such as total distance travelled, number of movements, and the number of stereotypic movements was seen as compared to placebo controls. The dose of 2-bromo-α-ergocryptine which by itself had no effect on any type of behavioral activity in placebo-treated rats, increased the total distance travelled, horizontal activity, number of movements, and movement time in morphine-abstinent rats. Although in morphine-tolerant or morphine-abstinent rats, the characteristics of [³H]spiroperidol binding to dopamine D₂ receptors in brain regions and spinal cord were unchanged, the supersensitivity was observed to behavioral responses of 2-bromo-α-ergocryptine, a selective dopamine D₂ receptor agonist. These results provide an evidence for behavioral responses of 2-bromo-α-ergocryptine, a selective up-regulation in morphine abstinent rats. Previously, we have show that dopamine D₁ receptors are unaffected in morphine tolerant rats but are modified in morphine-abstinent rats. Thus, in the morphine abstinent process a significant difference was noted in the biochemical characteristics of dopamine D₁ and D₂ receptors.     

Levels of pro-neo-endorphin/dynorphin-derived peptides in the hypothalamo-posterior pituitary system of male and female Brattleboro rats

Immunoreactivities for leucine-enkephalin and the related opioid peptides α-Neo-endorphin, dynorphin(1–17) and dynorphin(1–) were measured in hypothalamus and poterior-intermediate pituitary extracts from male and female Brattleboro rats homozygous (unable to produce vasopressin) and heterozygous(able to produce vasopressin) for daibetes insipidus. In hypothalamus no differences were found in peptide levels among the 4 groups of animals. In contrast, striking, but variable differences were found in posterior intermediate pituitary. In homozygous male animals dynorphin(1–17) immunoreactivity was not significantly different from levels measured in heterozygous male animals. Immunoreactivities for the 3 other peptides, however, showed greatly reduced levels in homozygous male animals compared to heterozygous male animals. Female homozygous animals had greatly reduced levels of all 4 peptides (including dynorphin(1–17) compared to female heterozygous controls. In addition, female heterozygous animals had considerably higher levels of all peptides than male heterozygous animals. In contrast, no sex differences were found in normal Long-Evans rats of the strain from which Brattleboro rats were derived.The following conclusions could be drawn from these findings. (1) Homozygous Brattleboro rats have reduced levels of Leuenkephalin-related opioid peptides in posterior pituitary due to defect or alteration of secretion or turnover but dot due to a defect in biosynthesis. (2) This defect is partially sex dependent and is directly or indirectly linked to the vasopressin deficiency. (3) Since normal rats do not show sex differences in peptides levels, the partial sex dependence of the opioid peptide defect in posterior pituitary seems to be X-chromosomally linked to the vasopressin deficiency.     

Loss of antiallodynic and antinociceptive spinal/supraspinal morphine synergy in nerve-injured rats: restoration by MK-801 or dynorphin antiserum

The co-administration of morphine at spinal (i.th.) and supraspinal (i.c.v.) sites to the same rat produces antinociceptive synergy, a phenomenon which may underlie the clinical analgesic utility of this drug. In animals with peripheral nerve injury, however, the antinociceptive potency and efficacy of i.th. morphine is significantly decreased. Here, the possible loss of spinal/supraspinal morphine antinociceptive synergy and relationship to elevation of spinal dynorphin content was studied. Ligation of lumbar spinal nerves resulted in elevated dynorphin in the ipsilateral lumbar and sacral spinal cord. In sham-operated rats supraspinal/spinal co-administration of morphine produced synergistic antinociception which was unaffected by i.th. MK-801 or dynorphin A((1-17)) antiserum. In nerve-injured rats, i.th. morphine was inactive against tactile allodynia and showed diminished in potency against acute nociception without supraspinal/spinal antinociceptive synergy. Antiserum to dynorphin A((1-17)) or the non-competitive NMDA antagonist MK-801 increased the antinociceptive potency of i.th. morphine, restored supraspinal/spinal morphine antinociceptive synergy and elicited a dose-related i.th. morphine antiallodynic action. These agents did not demonstrate antinociceptive or antiallodynic activity alone and did not alter morphine actions in sham-operated animals. The loss of spinal/supraspinal antinociceptive synergy and lack of antiallodynic activity of spinal morphine appear to be due to the elevation across multiple spinal segments of dynorphin following nerve injury. Pathological actions of elevated dynorphin may directly or indirectly modulate the NMDA receptor, result in a loss of supraspinal/spinal morphine synergy and may thus account for the decreased clinical analgesic efficacy of morphine in peripheral neuropathies.     

Intrathecal dynorphin A1–13 and dynorphin A3–13 reduce rat spinal cord blood flow by non-opioid mechanisms

Radiolabeled microspheres were used to examine the effects of paralytic intrathecal doses of dynorphin A (Dyn A_1–13) and Dyn A_3–13 on rat brain and spinal cord blood flows and cardiac output. Dyn A_1–13 produced significant dose-related reductions in blood flow to lumbosacral and thoracic spinal cord without altering cardiac output and blood flow to brain and cervical spinal cord. Naloxone failed to block these effects. Dyn A_3–13, which lacks opioid activity, also significantly reduced blood flow in lumbosacral spinal cord. Thus, the paralytic effects of Dyn A in the rat may involve reductions in spinal cord resulting from non-opioid actions of Dyn A.     

Inhibition of morphine tolerance and dependence by diazepam and its relation to μ-opioid receptors in the rat brain and spinal cord

We have recently observed that concomitant administration of diazepam to morphine pellet implanted rats results in the inhibition of the development of morphine tolerance and dependence. We have now analyzed μ-opioid receptors in rats treated with morphine and diazepam for 5 days by using -DAMGO for binding studies. Male Sprague–Dawley rats were made tolerant and dependent by subcutaneous (s.c.) implantation of six morphine pellets (two pellets on the first day, and four on the second day). Diazepam (0.25 mg/kg b.wt) was injected once daily intraperitoneally (i.p.) for 5 days. Control rats were implanted with placebo pellets and injected once daily with saline or diazepam (i.p.). Animals were administered s.c. naloxone (10 mg/kg) to induce naloxone-precipitated withdrawal syndrome on the final day of the experiment (day 5). There was an up-regulation of μ-receptor (B_max increased) in the spinal cord of morphine tolerant (+139%) and dependent (+155%) rats compared to saline treated animals. Diazepam treatment abolished the up-regulation of μ-receptors in spinal cord of morphine treated rats. In the cortex, B_max was not affected in morphine tolerant or dependent rats but it decreased by 38% in morphine tolerant and 65% in morphine dependent rats treated with diazepam. The K_d of μ-receptors increased in the cortex, striatum and hypothalamus of morphine dependent rats. Diazepam treatment decreased the K_d of μ-receptors in the cortex of morphine tolerant and hypothalamus of morphine-dependent rats. These results suggest that diazepam treatment antagonizes the up-regulation of CNS μ-receptors observed in morphine tolerant rats. In addition, morphine tolerance and dependence may be associated with conversion of μ-opioid receptors to μ*-constitutive opioid receptors that are less active, and this conversion is prevented in the brain of animals treated with diazepam.     

μ-, δ-, κ- and ε-Opioid receptor modulation of the hypothalamic-pituitary-adrenocortical (HPA) axis: subchronic tolerance studies of endogenous opioid peptides

In opiate-naive rats, the endogenous opioid peptides, β-endorphin, dynorphin(1–13) and Met---Enk---Arg---Phe (MEAP) and the synthetic enkephalin analogue -Ala²- -Leu⁵-Enk (DADLE) potently stimulated plasma corticosterone in a dose-dependent, naloxone-reversible manner. To characterize their in vivo affinities, the effects of these peptides on plasma corticosterone release were tested in rats made tolerant to morphine, U50488H, DADLE/morphine or β-endorphin. These cross-tolerance studies showed that dynorphin and MEAP exerted their action on plasma corticosterone release at κ-opioid receptors. The action of DADLE occurred at δ-opioid receptors, while the action of β-endorphin occurred principally at another receptor site. These results indicate that there is independent modulation of the hypothalamic-pituitary-adrenal axis by endogenous opioid peptides at μ-, δ- and κ-opioid receptors. In addition, there may be modulation by β-endorphin at a separate site that we suggest could be a central ε-receptor site. This cross-tolerance paradigm, using a neuroendocrine model, provides in vivo evidence for the action of centrally active endogenous opioid peptides at multiple and independent opioid receptors.     

Characterization and localization of immunoreactive dynorphin, α-neo-endorphin, met-enkephalin and substance P in human spinal cord

By use of specific antisera, the distributions of immunoreactive dynorphin (ir-DYN), α-neo-endorphin (ir-α-NEO), Met-enkephalin (ir-MET) and substance P (ir-SP) were evaluated in discrete regions of human spinal cord and spinal ganglia. The relative concentrations of immunoreactive peptides in particular regions were as follows: sacral > lumbar > cervical > thoracic. Concentrations of ir-DYN, ir-α-NEO and ir-SP were 2–10-fold, but of ir-MET 1–2-fold, higher in the dorsal as compared to the ventral parts of cervical, lumbar and sacral cord.The concentrations of all peptides (when examined in discrete areas of thoracic cord) were found to be highest in the substantia gelatinosa. All peptides were present in the gray matter but only ir-MET was found in white matter.Gel-permeation chromatography of dorsal sacral spinal cord extracts revealed two major ir-DYN peaks. The smaller molecular weight peak, eluted at the position of synthetic dynorphin_1–17. ir-α-NEO and ir-SP comigrated exactly with their respective synthetic marker peptides. Substantial amounts of ir-SP and also, as confirmed by high pressure liquid chromatography, ir-MET, were found in the dorsal and ventral roots and spinal ganglia, and very low concentrations of ir-DYN or ir-α-NEO were also detected in these tissue.These results suggest that dynorphin and α-neo-endorphin, in addition to enkephalins, may be involved in transmission of somatosensory information in the human spinal cord.     

The influence of chronic stress on multiple opioid peptide systems in the rat: pronounced effects upon dynorphin in spinal cord

Recurrent exposure to intermittent electrical foot-shock (30 min, twice daily) for 7 days caused an increase in immunoreactive (ir) dynorphin and ir-alpha-neo-endorphin in lumbar and cervical (but not thoracic) spinal cord as measured 16 h following the final session. At this time the level of ir-Met-enkephalin-Arg6-Gly7-Leu8 (MEAGL) was also increased at the lumbar level. An acute foot-shock depleted spinal cord dynorphin in chronically stressed but not in naive rats. No alterations in levels of ir-dynorphin or ir-MEAGL were seen in discrete brain tissues. In contrast to the brain, where no effects were seen, the levels of beta-endorphin increased in both lobes of the pituitary. This change, however, was not accompanied by an alteration in levels of beta-endorphin in plasma. These data show that chronic foot-shock stress selectively influences particular pools of opioid peptides, predominantly those derived from proenkephalin B in the spinal cord and from proopiomelanocortin in the anterior pituitary. It is suggested that alterations observed in the spinal cord reflect enhanced activity of the proenkephalin B system in response to chronic nociceptive stimulation.     

Enkephalinergic and dynorphinergic neurons in the spinal cord and dorsal root ganglia of the polyarthritic rat - in vivo release and cDNA hybridization studies

Complex and contradictory data have been reported regarding the changes in spinal opioidergic systems associated with chronic inflammatory pain in the rat. In an attempt to solve these discrepancies, the in vivo release of met-enkephalin and dynorphin and the expression of the corresponding propeptide genes were investigated at the spinal level in arthritic rats and paired controls. A dramatic increase in the concentration of prodynorphin mRNA (+300–550%) and a less pronounced elevation of that of dynorphin-like material (+40–50%) were found in the dorsal part of cervical and lumbar segments of the spinal cord in rats rendered arthritic by an intradermal injection of Freund's adjuvant four weeks prior to these measurements. In addition, the spinal release of dynorphin-like material (assessed through an intrathecal perfusion procedure in halothane-anaesthetized animals) was approximately twice as high in arthritic rats as in controls. In spite of significant elevations in the levels of both met-enkephalin (+30–70%) and proenkephalin A mRNA (+40−50%) in the dorsal part of cervical and lumbar segments, the spinal release of met-enkephalin-like material was decreased (−50%) in arthritic rats as compared to paired controls. Proenkephalin A mRNA (but not prodynorphin mRNA) could be measured in dorsal root ganglia, and its levels were dramatically reduced in ganglia at the lumbar segments in arthritic rats. Such parallel reductions in the spinal release of met-enkephalin-like material and the levels of proenkephalin A mRNA in dorsal root ganglia of arthritic rats support the idea that the activity of primary afferent enkephalinergic fibres decreases markedly during chronic inflammatory pain.     

Effect of Chronic Treatment with Morphine, Midazolam, and Both Together on β-Endorphin Levels in the Rat

We have recently reported that a short-acting anesthetic and analgesic drug midazolam can produce analgesia and decrease morphine tolerance and dependence in the rat by interacting with the opioid system. This study was designed to investigate the effect of midazolam, morphine, and both together on β-endorphin levels in the rat. Male Sprague-Dawley rats were divided into four groups: (1) saline-saline; (2) saline-morphine; (3) midazolam-saline, and (4) midazolam-morphine groups. First, saline or midazolam injection was given IP and after 30 min a second injection of saline or morphine was given subcutaneously once daily for 11 days. Animals were sacrificed on 11th day 60 min after the last injection, to measure β-endorphin by radioimmunoassay. Saline-morphine-treated animals showed a significant increase in β-endorphin levels in the cortex, pons, medulla, lumbar spinal cord, adrenals, and spleen, and a decrease only in its level in pituitary. Midazolam-saline-treated animals showed a significant increase in β-endorphin levels only in the medulla, and a decrease in its levels in hippocampus, striatum, and adrenals. Saline-morphine-treated animals did not show any changes in plasma β-endorphin, but animals treated with midazolam-saline had a significant decrease in plasma β-endorphin. In rats treated with morphine and midazolam together, β-endorphin levels in cortex, lumbar spinal cord, and spleen decreased to the similar levels observed in rats treated with saline-saline; in pons and cervical spinal cord the levels were even lower than that found in saline-saline group. The decrease in pituitary β-endorphin in morphine-midazolam-treated rats was due to morphine's own activity, whereas the decrease in plasma β-endorphin in hippocampus in the morphine-midazolam group was a synergistic effect of morphine and midazolam. The β-endorphin level in adrenal glands in the morphine-midazolam-treated animals was not different from that found in rats treated with morphine alone but was still higher than that in the saline-saline group. In general, it appears that chronic treatment with morphine stimulates the β-endorphinergic system. A concomitant treatment with midazolam abolishes the stimulatory effect of morphine on the β-endorphinergic system. These results may help us in understanding the intrinsic mechanisms involved in narcotic tolerance and dependence. Copyright © 1996 Elsevier Science Inc.     

Release of immunoreactive met-enkephalin from the spinal cord by intraventricular β-endorphin but not morphine in anesthetized rats

Effect of β-endorphin and morphine injected intraventricularly on the release of immunoreactive Met-enkephalin, Leu-enkephalin and dynorphin_1–13 from the spinal cord was studied in anesthetized rats. Intraventricular β-endorphin, 16 μg, caused a marked spinal release of immunoreactive Met-enkephalin and to a much lesser extent, of immunoreactive Leu-enkephalin while intraventricular morphine, 40 μg, did not cause any significant release of immunoreactive enkephalins. The release of immunoreactive Met-enkephalin was not blocked by the pretreatment with 5 mg/kg naloxone, i.p. Immunoreactive dynorphin_1–13 was not released by either β-endorphin or morphine. High performance liquid chromatographic analysis indicated that immunoreactive Met-enkephalin released by β-endorphin had a retention time identical to [³H]Met-enkephalin. These findings in conjunction with previous pharmacological studies suggest different modes of pharmacological action for β-endorphin and morphine.     

Brain dynorphin and enkephalin systems in Fischer and Lewis rats: effects of morphine tolerance and withdrawal

Lewis rats are more likely to self-administer various drugs of abuse than Fischer rats. Here these two strains of rats were compared with regard to basal brain opioid peptide levels and the response to chronic morphine treatment and to naloxone-precipitated withdrawal. Lewis rats had lower basal dynorphin peptides in the substantia nogra, striatum (not Leu-enkephalinArg⁶) and VTA (not dynorphin B) and the pituitary gland. Leu-enkephalinArg⁶ levels were also lower in these structures (with the exception of striatum which had higher levels) and in the nucleus accumbens. There were also strain differences in the response to chronic morphine treatment; in the nucleus accumbens, morphine treatment increased dynorphin A levels in Fischer rats only, in the ventral tegmental area effects were opposite with increased dynorphin levels in Fischer and decreased levels in Lewis rats, in the hippocampus dynorphin levels were markedly reduced in Lewis rats only. In Fischer rats, chronic morphine strongly affected peptide levels in the substantia nigra and striatum, whereas Lewis rats responded less in these areas. Leu-enkephalin, which derives from both prodynorphin and proenkephalin, and Met-enkephalin, which derives from proenkephalin, were effected by chronic morphine mainly in Fischer rats, increasing levels in most of the brain areas examined. The results in this study show (1) strain differences in basal levels of prodynorphin-derived opioid peptides, (2) the prodynorphin system to be differently influenced by morphine in Lewis rats than in Fischer rats and 3) the proenkephalin system to be influenced by chronic morphine in brain areas related to reward processes only in Fischer rats.