Enhancement of phagocytosis by dynorphin A in mouse peritoneal macrophages

The effects of the opioid peptide dynorphin A (DynA) on phagocytosis in peritoneal macrophages was examined by flow cytometry (FCM). DynA enhanced phagocytosis in a dose-dependent manner. Leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), β-neo-endorphin (βNeo-End), DynA(9–17) and DynA(13–17) had no such activity, -Neo-endorphin ( Neo-End), dynorphin B (DynB), DynA(l–13) and DynA(6–17) enhanced phagocytosis less effectively than DynA. Naloxone did not inhibit the enhancement of phagocytosis induced by DynA. Unstimulated control phagocytosis was partially suppressed in Ca²⁺-free EGTA-containing solution and even in this solution DynA enhanced phagocytosis. However, the enhancement by DynA was suppressed in EGTA- and BAPTA-AM-containing Ca²⁺-free solution. The present study showed that enhancement of phagocytosis by DynA was independent of extracellular Ca²⁺ ([Ca²⁺]_o) and dependent on intracellular Ca²⁺ ([Ca²⁺]_i). The present results support DynA being one of the mediators from the nervous system that modulates the immune system.     

中药复方“体复康”对运动性疲劳大鼠血乳酸、β-内啡肽、亮氨酸脑啡肽及强啡肽A_(1-13)影响的实验研究

以长时期（共7周）大强度跑台运动（速度由15m／min递增至35m／min，运动时间为20～25min／d）制造大鼠疲劳模型，探索疲劳大鼠外周阿片肽的变化及中药复方的作用机理。结果表明：运动后即刻血乳酸显著高于对照组，血浆β-内啡肽、强啡肽A1-13含量显著下降，而亮氨酸脑啡肽变化不明显；经中药复方治疗后，上述变化均有不同程度的恢复。表明此运动强度下的疲劳大鼠，内源性阿片肽系统受抑制而使外用血中的β-内啡肽、强啡肽A1-13含量下降；中药复方作用后激活了此系统，主要使卜内啡肽水平升高，并对血乳酸有一定的清除作用，从而有利于消除疲劳。     

Selective involvement of kappa opioid and phencyclidine receptors in the analgesic and motor effects of dynorphin-A-(1–13)-Tyr-Leu-Phe-Asn-Gly-Pro

Dynorphin A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro (Dyn Ia; 1-8 nmol) injected intracerebroventricularly in the mouse produces two independent behavioral effects: (1) a norbinaltorphimine (kappa opioid antagonist)-reversible analgesia in the acetic acid-induced writhing test and (2) motor dysfunction characterized by wild running, pop-corn jumping, hindlimb jerking and barrel rolling and antagonized by the irreversible phencyclidine (PCP) and sigma (sigma) receptor antagonist, metaphit and the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, dextromethorphan and ketamine. The specific involvement of the PCP receptor in the motor effects of Dyn Ia is supported by the direct competitive interaction of the peptide with the binding of [3H]MK-801 (Ki: 0.63 microM) and [3H]TCP (Ki: 4.6 microM) to mouse brain membrane preparations.     

强啡肽对脊髓损伤后兴奋性氨基酸含量变化的影响与意义

为阐明脊髓损伤后病理因于强啡肽（Dyn）与兴奋性氨基酸（EAA）的关系，通过蛛网膜下腹内插管，在伤后20min给予不同剂量、不同种类Dyn，应用高效液相分析技术测定大鼠脊髓损伤后伤段脊髓组织中EAA含量的动态变化。结果显示，DunA使脊髓组织EAA含量显著增加，其增加的量和持续时间与DynA剂量有关，且有剂量依赖性，但不受阿片受体拮抗剂影响。相同剂量的非阿片受体激动剂DynA2－17和阿片受体激动剂DynA1-17对脊髓EAA的改变相似，DnyA1-8也产生显著的EAA改变，但程度较小。本实验结果对Dyn的病理作用包括非阿片受体途径的学说提供了进一步的支持，在非阿片途径中EAA的作用可能是最重要的。     

Targeting KNDy neurons to control GnRH pulses

Gonadotropin-releasing hormone (GnRH) is the final output of the central nervous system that drives fertility. A characteristic of GnRH secretion is its pulsatility, which is driven by a pulse generator. Each GnRH pulse triggers a luteinizing hormone (LH) pulse. However, the puzzle has been to reconcile the synchronicity of GnRH neurons with the scattered hypothalamic distribution of their cell bodies. A leap toward understanding GnRH pulses was the discovery of kisspeptin neurons near the distal processes of GnRH neurons, which secrete kisspeptins, potent excitatory neuropeptides on GnRH neurons, and equipped with dual, but opposite, self-modulatory neuropeptides, neurokinin B and dynorphin. Over the last decade, this cell-to-cell communication has been dissected in animal models. Today the 50-year quest for the basic mechanism of GnRH pulse generation may be over, but questions about its physiological tuning remain. Here is an overview of recent basic research that frames translational research.     

Unique biological function of cathepsin L in secretory vesicles for biosynthesis of neuropeptides

Neuropeptides are essential for cell-cell communication in the nervous and neuroendocrine systems. Production of active neuropeptides requires proteolytic processing of proneuropeptide precursors in secretory vesicles that produce, store, and release neuropeptides that regulate physiological functions. This review describes recent findings indicating the prominent role of cathepsin L in secretory vesicles for production of neuropeptides from their protein precursors. The role of cathepsin L in neuropeptide production was discovered using the strategy of activity-based probes for proenkephalin-cleaving activity for identification of the enzyme protein by mass spectrometry. The novel role of cathepsin L in secretory vesicles for neuropeptide production has been demonstrated in vivo by cathepsin L gene knockout studies, cathepsin L gene expression in neuroendocrine cells, and notably, cathepsin L localization in neuropeptide-containing secretory vesicles. Cathepsin L is involved in producing opioid neuropeptides consisting of enkephalin, β-endorphin, and dynorphin, as well as in generating the POMC-derived peptide hormones ACTH and α-MSH. In addition, NPY, CCK, and catestatin neuropeptides utilize cathepsin L for their biosynthesis. The neuropeptide-synthesizing functions of cathepsin L represent its unique activity in secretory vesicles, which contrasts with its role in lysosomes. Interesting evaluations of protease gene knockout studies in mice that lack cathepsin L compared to those lacking PC1/3 and PC2 (PC, prohormone convertase) indicate the key role of cathepsin L in neuropeptide production. Therefore, dual cathepsin L and prohormone convertase protease pathways participate in neuropeptide production. Significantly, the recent new findings indicate cathepsin L as a novel 'proprotein convertase' for production of neuropeptides that mediate cell-cell communication in health and disease.     

Opioid receptor-mediated hyperalgesia and antinociceptive tolerance induced by sustained opiate delivery

Opiates are commonly used to treat moderate to severe pain and can be used over prolonged periods in states of chronic pain such as those associated with cancer. In addition, to analgesic actions, studies show that opiate administration can paradoxically induce hyperalgesia. At the pre-clinical level, such hyperalgesia is associated with numerous pronociceptive neuroplastic changes within the primary afferent fibers and the spinal cord. In rodents, sustained opiate administration also induces antinociceptive tolerance. The mechanisms by which prolonged opiate exposure induces hyperalgesia and the relationship of this state to antinociceptive tolerance remain unclear. The present study was aimed at determining whether sustained opiate-induced hyperalgesia, associated neuroplasticity and antinociceptive tolerance are the result of specific opiate interaction at opiate receptors. Enantiomers of oxymorphone, a mu opioid receptor agonist, were administered to rats by spinal infusion across 7 days. Sustained spinal administration of (-)-oxymorphone, but not its inactive enantiomer (+)-oxymorphone or vehicle, upregulated spinal dynorphin content, produced thermal and tactile hypersensitivity, and produced antinociceptive tolerance. These results indicate that these pronociceptive actions of sustained opiate administration require specific interaction with opiate receptors and are unlikely to be the result of accumulation of potentially excitatory metabolic products. While the precise mechanisms, which may account for these pronociceptive changes remain to be unraveled, the present data point to plasticity initiated by opiate receptor interaction.     

A microdialysis profile of dynorphin A(1-8) release in the rat nucleus accumbens following alcohol administration

BACKGROUND: Pharmacological studies have implicated the endogenous opioid system in mediating alcohol intake. Other evidence has shown that alcohol administration can influence endorphinergic and enkephalinergic activity, while very few studies have examined its effect on dynorphinergic systems. The aim of the present study was to investigate the effect of alcohol administration or a mechanical stressor on extracellular levels of dynorphin A(1-8) in the rat nucleus accumbens-a brain region that plays a significant role in the processes underlying reinforcement and stress. METHODS: Male Sprague-Dawley rats were implanted with a microdialysis probe aimed at the shell region of the nucleus accumbens. Artificial cerebrospinal fluid was pumped at a rate of 1.5 microL/min in awake and freely moving animals and the dialysate was collected at 30-minute intervals. In one experiment, following a baseline period, rats were injected intraperitoneally with either physiological saline or 1 of 3 doses of alcohol, 0.8, 1.6, or 3.2 g ethanol/kg body weight. In a second experiment, following a baseline period, rats were applied a clothespin to the base of their tail for 20 minutes. The levels of dynorphin A(1-8) in the dialysate were analyzed with solid-phase radioimmunoassay. RESULTS: Relative to saline-treated controls, an alcohol dose of 1.6 and 3.2 g/kg caused a transient increase in the extracellular levels of dynorphin A(1-8) in the first 30 minutes of alcohol administration. However, the effect resulting from the high 3.2 g/kg dose was far more pronounced and more significant than with the moderate dose. There was no effect of tail pinch on dynorphin A(1-8) levels in the nucleus accumbens. CONCLUSIONS: In this experiment, a very high dose of alcohol was especially capable of stimulating dynorphin A(1-8) release in the nucleus accumbens. Dynorphin release in the accumbens has been previously associated with aversive stimuli and may thus reflect a system underlying the aversive properties of high-dose alcohol administration. However, the lack of effect of tail-pinch stress in the present study suggests that dynorphin A(1-8) is not released in response to all forms of stressful/aversive stimuli.     

Effect of opioid receptor ligands injected into the rostral lateral hypothalamus on c-fos and feeding behavior

The lateral hypothalamic area (LHa) is an important brain site for the regulation of food intake. Central injection of opioids increases food intake, and the LHa contains mu and kappa opioid receptors, both of which are involved in feeding behavior. It is unclear whether opioids impact feeding when injected directly into the rostral portion of the LHa (rLHa) in rats. We performed a series of studies in which free-feeding rLHa-cannulated rats were injected with opioid agonists (DAMGO, morphine, dynorphin, U-50488H) followed by the measurement of food intake at 1, 2, and 4 h postinjection. To determine whether opioid receptor ligands administered into the rLHa affect neuronal activation in this brain site, we studied cFos immunoreactivity (cFos IR) in response to rLHa stimulation with naltrexone. We found that the only compound that stimulated feeding behavior was morphine. The other agonists had no effect on food consumption. Naltrexone injection into the rLHa increased neural activation in the LHa, indicating the presence of functional opioid receptors in this region. These data suggest that although neuronal activity is affected by opioid agents acting in the rLHa, administration of selective mu and kappa opioid ligands in this subdivision of the LHa does not have a reliable effect on feeding behavior.     

State-dependent changes of brain endogenous opioids in mammalian hibernation

Endogenous opioids belonging to three opioid families were measured in different states of the hibernation cycle in brain of the Columbian ground squirrels. Using high-performance liquid chromatography-EC detection, the hypothalamic and septal concentrations of met-enkephalin were found to be significantly higher (p > 0.05) in the hibernating state than that in the nonhibernating state. In contrast, met-enkephalin content in the medulla decreased significantly during hibernation. Leu-enkephalin content was only increased in the hypothalamus of hibernating animals. Using radioimmunoassay, dynorphin A-like immunoreactivity was observed to increase in the claustrum and striatum, whereas β-endorphin-like peptides showed a significant increase in the hypothalamus during hibernation. It is evident that the changes in endogenous opioids in brain during hibernation are state dependent, type specific and region specific. These specific alterations of various endogenous opioids may imply their different roles in hibernation.