Decreased dynorphin A (1-17) in the spinal cord of spastic rats after the compressive injury

Spasticity in rat hindlimbs was induced by compressing cervical spinal cord with a wax ball. Ashworth score and H-reflex were measured 1 week after the surgery. The results showed that: (1) muscle spasm was detected in the hindlimbs a week after the operation and maintained at least 8 weeks, (2) in the spastic animals, dynorphin A (1-17)-ir decreased significantly in thoracic and lumbar segments of the spinal cord and (3) peripheral administration of kappa receptor agonist U50488H and electrical stimulation at 100 Hz effectively relieved the muscle spasm. Our data supported the note that the reduction of endogenous dynorphin A (1-17) might play an important role in the pathogenesis of spinally induced muscle spasticity and the replenishment of its shortage might relieve the spasticity.     

Effects of synchronous or asynchronous electroacupuncture stimulation with low versus high frequency on spinal opioid release and tail flick nociception

Electroacupuncture stimulation (EAS) is known to change brain neurotransmitter release. In the present study, we investigated the effects of synchronous or asynchronous electroacupuncture stimulation with low versus high frequency on spinal opioid release and tail flick nociception. Rats were given "2/100 Hz" EAS, which stands for an asynchronous mode of stimulation, in which 2 Hz was alternated with 100 Hz, each lasting for 3 s, or "(2 + 100) Hz" EAS, a mode of stimulation in which 2 Hz stimulation was applied to the left hind leg simultaneously with 100 Hz stimulation on the right hind leg. The rats were subjected to the same total number of electrical stimulations in these two modes. Results were as follows: (1) 2/100 Hz EAS was 40% more potent than (2 + 100) Hz EAS (P < 0.01) in producing an anti-nociceptive effect. (2) Intrathecal (i.t.) injection of the mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) blocked in a dose-dependent manner the anti-nociceptive effect produced by 2/100 Hz EAS but not by (2 + 100) Hz EAS, whereas i.t. injection of the kappa-opioid receptor antagonist norbinaltorphimide (Nor-BNI) blocked the anti-nociceptive effect induced by both modes of EAS. (3) I.t. injection of endomorphin-2 antiserum blocked in a dose-dependent manner the anti-nociceptive effect of 2/100 Hz EAS but not that of (2 + 100) Hz EAS, whereas i.t. injection of dynorphin antiserum blocked the anti-nociceptive effect induced by both modes of stimulation. (4) 2/100 Hz EAS increased the release of both endomorphin-2 and dynorphin, whereas (2 + 100) Hz EAS increased the release of dynorphin but not of endmorphin-2. We conclude that the more potent anti-nociceptive effect induced by 2/100 Hz EAS, as compared with that of (2 + 100) Hz EAS, was due, at least partly, to the synergistic interaction of endomorphin-2 and dynorphin in rat spinal cord.     

Naloxone potentiates the release of oxytocin induced by systemic administration of cholecystokinin without enhancing the electrical activity of supraoptic oxytocin neurones

Summary Studies performed in conscious female rats confirmed that iv injection of cholecystokinin octapeptide (CCK; 20µ/kg) increased the circulating concentration of oxytocin but not that of vasopressin, and confirmed that the stimulation of oxytocin release was markedly facilitated after iv administration of naloxone (1mg/kg), indicating attenuation of oxytocin release by endogenous opioids. To investigate the site of action of the endogenous opioids, the electrical activity of putative oxytocin neurones in the supraoptic nucleus was recorded in urethaneanaesthetised female rats. Oxytocin neurones responded to CCK injection with an increase in firing rate lasting 5–15 min, but this response was not facilitated by prior injection of naloxone. The results suggest that the opioid influence upon CCK-induced oxytocin release operates at the level of the neurosecretory terminals in the neurohypophysis rather than centrally. Since CCK does not elevate vasopressin release, it appears unlikely that dynorphin, the opioid peptide co-existing with vasopressin, is responsible in these circumstances for the cross-inhibition of oxytocin release. It is suggested that products of proenkephalin A, the met-enkephalin precursor present in the supraoptic nucleus and in the neurohypophysis itself, may be active in the regulation of oxytocin release.     

Aversive properties of naloxone in non-dependent (naive) rats may involve blockade of central β-endorphin

The present study examines the influence of destruction of the medio-basal arcuate hypothalamus (MBH), the primary site of synthesis of central pools of -endorphin (-EP), upon the aversive properties of naloxone in a conditioned place preference paradigm. Bilateral radiofrequency lesions of the MBH resulted in a pronounced fall in levels of immunoreactive -EP in the brain. Lesioned rats, in contrast to non-operated animals, showed a clear reduction in the conditioned place aversion produced by naloxone. However, they showed no loss of the conditioned preference produced by the mu-selective opioid receptor agonist, morphine, or the conditioned aversion produced by the kappaselective agonist, U50-488. In contrast to the effect of the lesions, suppression of circulating -EP by dexamethasone treatment failed to influence conditioning produced by naloxone. Thus, the data indicate that the aversive properties of naloxone are attenuated by disruption of central (but not peripheral) -EP activity. We suggest that these properties of naloxone reflect an antagonism of -EP activity in the brain. In addition, the data indicate that differing mechanisms underlie the aversive actions of naloxone as compared to U50-488.     

RELATIONSHIP BETWEEN ACUPUNCTURE ANALGESIA AND MET- ENKEPHALIN OR DYNORPHIN

subjective: The effect of 4～5 Hz electroacupuncture (EA) on alterations of both met-enkephalin (MEK) and dynorphin (Dyn) in the patient plasma or mouse spinal cord and its relation with analgesic effect were studied. Methods: In acupuncture clinic 10 patients with acute pain were treated with 4 Hz EA at Zusanli(ST 36) and/or Hegu(LI 4) acupoints for 30 min. 20 BALB/C mice were randomly divided into 2 groups: a. EA group(n=10), treated with 4～5 Hz EA at bilateral “Zusanli“(ST 36) for 15 min; b. control group(n=10) treated with no EA, but also restrained for 15 min. Before and after EA or restraining acupoints, the pain threshold of the patients or mice was detected. 10 μI of the patient plasma before and after EA and each mouse spinal cord suspension, of the 2 groups were blotted onto nitrocellulose membrane (NCM) respectively. The protein dot blot signals were detected by immunoreactivity (IR) and using Shimadu TLC Scanner and analyzed statistically. Results: The results showed that an increase in patient plasma MEK-IR or Dyn-IR and a decrease in mouse spinal MEK-IR or Dyn-IR could be detected, and the alteration of plasma or spinal MEK-IR was more significant than that of plasma or spinal Dyn-IR. There was a positive correlation in alteration between plasma or spinal MEK-IR and plasma or spinal Dyn-IR with respective parallel levels in individuals. The increased plasma MEK-IR or the decreased spinal MEK-IR was positively or negatively correlated with the analgesic effect, while the correlation between plasma or spinal Dyn-IR and analgesic effect was insignificant. Conclusion: The results suggest that under lower frequency EA the met-enkephalin may play an important role in analgesia.     

诱导型一氧化氮合酶在强啡肽致脊髓损伤中的作用

目的：探讨诱导型一氧化氮合酶（ｉＮＯＳ）在强啡肽致脊髓损伤中的作用。方法：［３Ｈ］－左旋精氨酸转化法测定腹侧和背侧脊髓ｉＮＯＳ活性,原位杂交法观测脊髓ｉＮＯＳｍＲＮＡ表达及其细胞分布。结果：大鼠蛛网膜下腔注射（ＩｎＩ）强啡肽Ａ１－１７（Ｄｙｎ）２０ｎｍｏｌ引起持久性截瘫和迟发性神经元死亡;在Ｄｙｎ致瘫后２～３ｈｉＮＯＳｍＲＮＡ表达开始增多增强,４ｈ达高峰,２４ｈ和４８ｈ仍见广泛表达,其分布以胶质细胞和大运动神经元为主;腹侧脊髓ｉＮＯＳ活性在Ｄｙｎ致瘫后４ｈ显著升高,并持续至２４ｈ和４８ｈ;提前１０ｍｉｎＩｎＩ选择性ｉＮＯＳ抑制剂氨基胍１μｍｏｌ可显著对抗Ｄｙｎ２０ｎｍｏｌ引起的持久瘫及伤后４ｈ腹侧脊髓ｉＮＯＳ活性升高。结论：ｉＮＯＳ持续性高表达与Ｄｙｎ致脊髓损伤机制有关     

Acute nicotine changes dynorphin and prodynorphin mRNA in the striatum

RATIONALE: Nicotine displays rewarding and aversive effects, and while dopamine has been linked with nicotine's reward, the neurotransmitter(s) involved with aversion remains speculative. The kappa-dynorphinergic system has been associated with negative motivational and affective states, and whether dynorphin (Dyn) contributes to the behavioral pharmacology of nicotine is a pertinent question. OBJECTIVE: We determined whether administration of a single dose of nicotine alters the biosynthesis of Dyn in the striatum of mice. RESULTS: Nicotine free base, 1 mg/kg, sc, induced a biphasic, protracted increase of striatal Dyn, an initial rise by 1 h, which declined to control levels by 2 h, and a subsequent increase, between 6 and 12 h, lasting over 24 h. At 1 h, the nicotine effect was dose dependent, with doses>or=0.5 mg/kg inducing a response. Prodynorphin mRNA increased by 30 min for over 24 h, and in situ hybridization demonstrated elevated signal in caudate/putamen and nucleus accumbens. The nicotinic antagonist mecamylamine prevented the Dyn response, and a similar effect was observed with D1- and D2-like dopamine receptor antagonists, SCH 23390, sulpiride, and haloperidol. The glutamate NMDA receptor antagonist MK-801 reversed the nicotine-induced increase of Dyn, while the AMPA antagonist NBQX had a marginal effect. CONCLUSIONS: We interpret our findings to indicate that acute nicotine enhances the synthesis and release of striatal Dyn. We propose that nicotine influences Dyn primarily through dopamine release and that glutamate plays a modulatory role. A heightened dynorphinergic tone may contribute to the aversive effects of nicotine in naive animals and first-time tobacco smokers.     

携带前强啡肽基因的Ad5 F35重组腺病毒制备及鉴定

目的 用Ad5F35载体制备携带前强啡肽基因的重组腺病毒Ad5F35-PDP并对其进行鉴定.方法 利用NheI和Agel分别对合成的目的 基因prodynorphin聚合酶链反应(PCR)产物和pDC316-LacZ-a载体质粒进行双酶切.将PDP目的 基因片段和线性化的pDC316连接,克隆构建pDC316-PDP.pDC316-PDP与腺病毒骨架pBHG-fiber5/35共转染293细胞制备Ad5F35-PDP重组腺病毒,收获并纯化病毒,检测重组病毒滴度,运用PCR进行鉴定.结果 编码PDP的基因序列经测序鉴定证实与Gene Bank中的一致,pDC316-PDP构建成功.pDC316-PDP与腺病毒骨架共转染293细胞后见明显的毒斑,说明两者在293细胞中同源重组并包装成功.经PCR证实Ad5F35-PDP重组腺病毒构建完成.病毒滴度为1×1012v.P./ml.结论 本实验成功制备了含PDP全序列的高滴度、高纯度的Ad5F35-PDP重组腺病毒,为下一步进行该重组病毒生物安全性能和生物学功能研究及今后应用Ad5F35-PDP重组腺病毒对吗啡成瘾患者进行基因治疗奠定了基础.     

The role of nociceptin and dynorphin in chronic pain: implications of neuro-glial interaction

Nociceptin-opioid peptide (NOP) receptor, also known as opioid receptor like-1 (ORL1), was identified following the cloning of the kappa-opioid peptide (KOP) receptor, and the characterization of these receptors revealed high homology. The endogenous ligand of NOP, nociceptin (NOC), which shares high homology to dynorphin (DYN), was discovered shortly thereafter, and since then, it has been the subject of several investigations. Despite the many advances in our understanding of the involvement of NOC and DYN systems in pain, tolerance and withdrawal, the precise function of these systems has not been fully characterized. Here, we review the recent literature concerning the distribution of the NOC and DYN systems in the central nervous system and the involvement of these systems in nociceptive transmission, especially under chronic pain conditions. We discuss the use of endogenous and exogenous ligands of NOP and KOP receptors in pain perception, as well as the potential utility of NOP ligands in clinical practice for pain management. We also discuss the modulation of opioid effects by NOC and DYN. We emphasize the important role of neuro-glial interactions in the effects of NOC and DYN, focusing on their presence in neuronal and non-neuronal cells and the changes associated with chronic pain conditions. We also present the dynamics of immune and glial regulation of neuronal functions and the importance of this regulation in the roles of NOC and DYN under conditions of neuropathic pain and in the use of drugs that alter these systems for better control of neuropathic pain.