A glutamate antagonist blocks perforant path stimulation-induced reduction of dynorphin peptide and prodynorphin mRNA levels in rat hippocampus

Stimulation of the perforant path elicits a behavioral response, wet dog shakes (WDS), and reduction in hippocampal dynorphin A(1-8) immunoreactivity (DYN-IR) and prodynorphin mRNA (DYN mRNA) in rats. This study examined whether glutamate, the proposed endogenous transmitter released by perforant fibers, mediated the above responses. A glutamate antagonist, gamma-D-glutamylglycine (DGG, 25 micrograms/0.5 microliters), or artificial cerebrospinal fluid (ACSF, 0.5 microliters) was injected into the ventral hippocampus 10-20 min prior to acute or daily stimulation of the left perforant path in rats. In acute stimulation experiments, 4 consecutive stimulation trials elicited a total of 73 +/- 4 WDS at an average threshold intensity of 0.46 +/- 0.03 mA in ACSF-treated rats. The hippocampal DYN-IR in these animals decreased by more than 40% in both dorsal and ventral hippocampus relative to sham-stimulated rats. DGG injections significantly elevated the threshold for WDS (0.78 +/- 0.05 mA, P less than 0.01), reduced the number of WDS (45 +/- 6, P less than 0.01), and partially antagonized stimulation-induced reduction of DYN-IR in the ventral, but not dorsal, hippocampus. In daily stimulation experiments, rats received a single trial of stimulation once per day for 6 days. Daily DGG pretreatment almost completely abolished WDS at control threshold intensities, and significantly inhibited stimulation-induced decrease of DYN-IR in both dorsal and ventral hippocampus. In situ hybridization using a 35S-labeled oligodeoxyribonucleotide probe demonstrated a clear depletion of DYN mRNA signal in the dentate granule cell layer of ACSF-treated animals. This depletion was completely prevented in DGG-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Up-regulation of opioid gene expression in spinal cord evoked by experimental nerve injuries and inflammation

Opioid systems modulate nociceptive input at several levels of the CNS. At the spinal cord level neurons are present that express the genes coding for the precursors of the dynorphin and enkephalin opioid peptide families. We found that two conditions in rats, a chronic constriction injury to the sciatic nerve and peripheral inflammation, have a common consequence centrally: they evoke a large, rapid and sustained up-regulation of preprodynorphin mRNA. Both are also characterized by signs of hyperalgesia and increased primary afferent input. In contrast, there is little or no up-regulation of preprodynorphin mRNA following complete transection of the sciatic nerve or sciatic nerve crush. Furthermore, only minor alterations in the levels of preproenkephalin mRNA occur in any of the conditions, except for inflammation where the elevation is relatively small compared to that of preprodynorphin mRNA. These data imply that specific regulatory processes that include stimulation of opioid gene expression are strongly engaged in the spinal cord in certain types of peripheral nerve injuries and inflammation, but not in others. Marked and sustained up-regulation of the spinal cord dynorphin system distinguishes the chronic constriction injury model from other nerve injury models of pain.     

Morphine and dynorphin(1–13) microinjected into the medial preoptic area and nucleus accumbens: effects on sexual behavior in male rats

The effects on sexual behavior of opiate receptor stimulation within A10 and A14 terminal areas were examined in the following experiments. Morphine (0.01–6 nmol) and dynorphin(1–13) (0.01–3 pmol) were microinjected into the medial preoptic area (MPOA). Morphine (10–100 pmol) and dynorphin (10–100 fmol) injected into the MPOA reduced both the latency to ejaculate and the number of intromissions triggering ejaculation. Morphine (6 nmol) produced a failure to resume copulating following the second ejaculation. Morphine (1–10 nmol) injected into the nucleus accumbens (ACC) shortened the latency to the first intromission and lengthened the second postejaculatory interval. Naloxone (3 mg/kg i.p.) reversed the effects of morphine on intromission latency and attenuated the lowering of ejaculatory threshold.     

不同剂量μ受体拮抗剂CTOP对瑞芬太尼诱发痛觉过敏的影响

目的 观察不同剂量的CTOP对瑞芬太尼引起的切口痛大鼠痛觉过敏的影响.方法 采用完全随机法将30只SD雄性大鼠随机为5组(每组6只):正常组(A组);切口痛组(B组);切口痛+瑞芬太尼组(C组);切口痛+瑞芬太尼+CTOP低剂量组(D组);切口痛+瑞芬太尼+CTOP高剂量组(E组).测定各组大鼠基础状态下(T0)的热缩足反射潜伏期(pawwithdrawal thermal latency,PWTL)后,以5％水合氯醛350 mg/kg大鼠腹腔麻醉,A、B、C组尾静脉注射生理盐水0.4 ml,D、E组分别注射CTOP 0.5 μg/kg、0.5 mg/kg,溶于0.4 ml生理盐水内.给药结束10 min后,除A组外全部于右后爪做切口,同时由尾静脉以0.8 ml/h的速度40 μg/kg的剂量分别给A、B组泵生理盐水,C、D、E组泵瑞芬太尼,各30 min.术后2(T1)、24 h(T2)测定PWTL,处死大鼠取脊髓,用酶联免疫法(ELISA)测定强啡肽表达. 结果 T1、T2时间点C组PWTL结果[(10.2±3.0)、(6.2±2.6)s]与A组[(13.3±2.4)、(13.4±2.2)s]、B组[(13.5±2.7)、(11.5±4.1)s]比较,差异有统计学意义(P＜0.05),PWTL时间缩短;且C组强啡肽结果(172±17) ng/L与A、B组(78±9)、(120±10) ng/L比较,差异有统计学意义(P＜0.05),强啡肽表达增多;D、E两组T2的PWTL值和强啡肽结果与C组比较,差异均有统计学意义,PWTL时间延长,强啡肽表达减少;D、E两组强啡肽表达比较,差异有统计学意义(P＜0.05). 结论 在大鼠切口痛模型中,瑞芬太尼导致了切口周围组织痛觉过敏;应用μ受体拮抗剂可以缓解痛觉过敏,低剂量的效果更加显著.     

Decreased Cerebrospinal Fluid β-Endorphin and Increased Pain Sensitivity in Patients with Functional Abdominal Pain

Jørgensen LS, Bach FW, Christiansen P, Raundahl U, Østgaard S, Ekman R. Decreased cerebrospinal fluid β-endorphin and increased pain sensitivity in patients with functional abdominal pain. Scand J Gastroenterol 1993;28:763-766.

We investigated whether central pain mechanisms including the endogenous antinociceptive system are involved in functional abdominal pain–that is, abdominal pain without abnormal findings at routine examinations. β-Endorphin, met-enkephalin immunoreactivity, and dynorphin immunoreactivity were measured in cerebrospinal fluid (CSF) from nine patients with long-lasting functional abdominal pain and nine pain-free controls undergoing minor surgery while under spinal analgesia. Furthermore, pain sensitivity was evaluated with an ischaemic pain test comparing 21 functional abdominal pain patients with two control groups: 1) 24 patients with organic abdominal pain due to duodenal ulcer, gallstone, or urinary tract calculi, and 2) 13 healthy pain-free controls. The CSF β-endorphin concentration was significantly decreased in the functional abdominal pain group as compared with nine matched controls (P = 0.01). Met-enkephalin and dynorphin immunoreactivities were normal. This part of the investigation was suspended after nine patients had been tested, because of post-lumbar-puncture headache. With regard to pain sensitivity, no significant difference between the three groups was shown, but subdivision of the functional abdominal pain group showed that individuals with pain and no symptoms of irritable bowel syndrome (IBS) were significantly more sensitive to pain than functional abdominal pain patients with IBS and healthy controls (P = 0.04).     

Purification and characterization of endoproteases from human choroid plexus cleaving prodynorphin-derived opioid peptides

An endoprotease converting the dynorphins and alpha-neoendorphin has been purified to apparent homogeneity from soluble extracts of human choroid plexus. The purified enzyme was stained as a single band after sodium dodecyl sulphate polyacrylamide gel electrophoresis with an apparent molecular weight of around 54,000 Da. The enzyme potently cleaves dynorphin A, dynorphin B and alpha-neoendorphin at consecutive pairs of basic amino acid residues generating Leu-Enk-Arg6, but it is less active on other neuropeptides containing dibasic stretches. It is optimally active at neutral pH, sensitive to EDTA and slightly affected by the serine protease inhibitors DFP and PMSF. A similar membrane-bound enzyme present in the same tissue was solubilized with 0.5% Triton X-100 and isolated with the same purification procedure. This latter enzyme showed almost identical properties with the soluble peptidase, except for a slightly higher molecular weight.     

Dynorphin A (1–13) potentiates dynorphin A (1–17) on loss of the tail-flick reflex after intrathecal injection in the rat

Dynorphin A (1–13) or dynorphin A (1–17) administered intrathecally to rats induces a dose-dependent loss of the tail-flick reflex and a reversible hindlimb paralysis. Although their potency is comparable, dynorphin A (1–17) is less efficacious in producing loss of the tail-flick reflex. These data suggest that dynorphin A (1–17) acts as a partial agonist or exerts its neurotoxic effect in the presence of an endogenous non-competitive inhibitor. To determine whether these substances act at similar sites we performed isobolographic analysis. This analysis revealed that dose-additivity exists for paralysis whereas deviation from dose-additivity exists for loss of the tail-flick reflex.     

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.     

Dynorphin A-containing neural elements in the nucleus of the solitary tract of the rat. Light and electron microscopic immunohistochemistry

Distribution of dynorphin A (DyA) immunoreactivity in the nucleus of the solitary tract (NTS) was examined in rats after various surgical transections by light and electron microscopic immunohistochemistry. In colchicine-treated animals DyA immunostained were seen in each subdivision of the NTS. In intact rats, dense network of immunopositive nerve fibers was localized light microscopically, and synaptic contacts were found between DyA immunopositive structures (axo-axonic, axo-dendritic synapses), electron microscopicaly. Surgical transections medial, caudal or rostral to the nucleus did not alter the distribution pattern of DyA in the NTS. Lesion immediately lateral to the nucleus resulted in an ipsilateral appearance of immunostained cell bodies. Vagal and glossopharyngeal aferents (including baroreceptor fibers) terminate in the medial and commissural subnucleus of the NTS. Two days after extracranial vagotomy, synaptic contacts between degenerated presynaptic boutons and DyA immunopositive postsynaptic elements were observed in both medial and commissural part of the NTS. These observations provide morphological evidence suggesting that (1) axons of dynorphin A-containing cell bodies form an intrinsic network inside the nucleus; (2) these DyA cells receive direct peripheral inputs through the vagus nerve, and (3) projecting DyA neurons may exist in the NTS, they may innervate medullary, rather than forebrain, higher brainstem or spinal cord neurons.     

Effect of morphine on dynorphin B and GABA release in the basal ganglia of rats

In vivo microdialysis was used to study the effects of systemic, as well as intracerebral administration of morphine and naloxone on dynorphin B release in neostriatum and substantia nigra of rats. The release of dopamine (DA), γ-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) was also investigated. Systemic injection of morphine (1 mg/kg s.c.) induced long-lasting increases in extracellular dynorphin B and GABA levels in the substantia nigra, whereas DA, Glu and Asp levels, measured in the same region, were not significantly affected. No effect on striatal neurotransmitter levels was observed following systemic morphine administration. Local perfusion of the substantia nigra with morphine (100 μM) through the microdialysis probe also increased nigral dynorphin B and GABA levels. Perfusion of the neostriatum with morphine (100 μM) significantly increased GABA and dynorphin B levels in the ipsilateral substantia nigra, but no effect was observed locally. Naloxone blocked the effect of systemic morphine administration on nigral dynorphin B and GABA release, already at a dose of 0.2 mg/kg s.c. Naloxone alone, given either systemically (0.2–4 mg/kg s.c.) or intracerebrally (1–100 μM), did not affect dynorphin B or amino acid levels, either in neostriatum or in substantia nigra. However, naloxone produced a concentration-dependent increase in DA levels. The present results indicate that systemic morphine administration stimulates the release of dynorphin B in the substantia nigra, probably by activating the μ-subtype of opioid receptor, since the effect of morphine on nigral dynorphin B and GABA was antagonized by a low dose of naloxone. The increase in extracellular DA levels produced by high concentrations of naloxone, both in neostriatum and substantia nigra, indicates a disinhibitory effect of this drug on DA release, probably via a non-μ subtype of opioid receptors located on nigro-striatal DA neurones.