Non-opioid dynorphin binding site on secretory vesicles of a pituitary-derived cell line

Accumulating evidence indicates that the endogenous opioid peptides dynorphinA-(1-17) and dynorphinA-(1-13) interact not only with opioid but also with yet poorly characterized non-opioid receptors. The latter have been implicated in a number of the effects of dynorphins including induction of ACTH release in sheep and in AtT 20 cells, a pituitary-derived mouse cell line. AtT 20 cells do not express opioid receptors and therefore are particularly suitable for search of non-opioid dynorphin receptors. We report here that ³H-dynorphinA-(1-13)-NH₂ associates specifically with AtT 20 cells, apparently through an uptake process and a binding site. Within the cell, it binds preferentially to fractions containing secretory vesicles, with a K_d of about 100 nM. DynorphinA-(1-17), and several non-opioid fragments of dynorphin, including A-(2-17), A-(2-16) and A-(2-13), compete with ³H-dynorphinA-(1-13)-NH₂ for that site with IC₅₀s ranging from 200 nM to 2 μM. ACTH(1-39) also competes with ³H-dynorphinA-(1-13)-NH₂ for the site with an IC₅₀ of about 300 nM. DynorphinA-(2-17) at μM concentrations stimulates release of ACTH from the isolated vesicles. The results indicate the presence of a non-opioid dynorphin binding site on the secretory vesicle fractions of AtT20 cells that might be involved in ACTH release. The ability of ACTH itself to compete for the binding sites associated with the vesicles suggest that those sites may be involved in an autocrine loop.     

急性脊髓损伤患者脑脊液和血浆中强啡肽A含量的变化

为探讨强啡肽Ａ（ＤｙｎＡ）在脊髓损伤患者脑脊液和血液中的动态变化及临床意义，作者采用放射免疫测定法（ＲＩＡ）检测了３４例急性脊髓损伤患者脑脊液（ＣＳＦ）和血浆中强啡肽Ａ含量变化，随访６个月至１．５年，对脊髓损伤患者的神经功能恢复进行评价。结果发现：伤后４小时完全性脊髓损伤患者（ｎ＝９）ＣＳＦ和血浆中的ＤｙｎＡ含量（ｐｇ／ｍｌ）分别为：２４０．０２±２２．１７和２０２．０７±２１．５６，不完全性脊髓损伤患者（ｎ＝１６）分别为１８１．７６±２２．８９和１４０．５±２３．４４，无脊髓损伤患者（ｎ＝８）分别为７６．１３±２２．８１和３６．４８±２２．８２。结果揭示：脊髓损伤越重，ＤｙｎＡ升高越明显，恢复至正常的时间越长，预后也越差。本检测对指导急性脊髓损伤的治疗和判断预后具有一定意义。     

Neurotransmitters,neuropeptides and binding sites in the rat mediobasal hypothalamus: effects of monosodium glutamate (MSG) lesions

Summary Indirect immunofluorescence histochemistry and receptor autoradiography were used to study the localization of transmitter-/peptidecontaining neurons and peptide binding sites in the mediobasal hypothalamus in normal rats and in rats treated neonatally with repeated doses of the neurotoxin monosodium-glutamate (MSG). In the arcuate nucleus, the results showed a virtually complete loss of cell bodies containing immunoreactivity for growth hormone-releasing factor (GRF), galanin (GAL), dynorphin (DYN), enkephalin (ENK), corticotropin-like intermediate peptide (CLIP), neuropeptide Y (NPY), and neuropeptide K (NPK). Tyrosine hydroxylase(TH)-, glutamic acid decarboxylase(GAD)-, neurotensin(NT)- and somatostatin(SOM)-immunoreactive (IR) cells were, however, always detected in the ventrally dislocated, dorsomedial division of the arcuate nucleus. In the median eminence, marked decreases in numbers of GAD-, NT-, GAL-, GRF-, DYN-and ENK-IR fibers were observed. The numbers of TH-, SOM-and NPY-IR fibers were in contrast not or only affected to a very small extent, as revealed with the immunofluorescence technique. Biochemical analysis showed a tendency for MSG to reduce dopamine levels in the median eminence of female rats, whereas no effect was observed in male rats. Autoradiographic studies showed high to moderate NT binding sites, including strong binding over presumably dorsomedial dopamine cells. In MSG-treated rats, there was a marked reduction in GAL binding in the ventromedial nucleus. The findings implicate that most neurons in the ventrolateral and ventromedial arcuate nucleus are sensitive to the toxic effects of MSG, whereas a subpopulation of cells in the dorsomedial division of the arcuate nucleus, including dopamine neurons, are not susceptible to MSG-neurotoxicity. The results indicate, moreover that the very dense TH-IR fiber network in the median eminence predominantly arises from the dorsomedial TH-IR arcuate cells, whereas the GAD-, NT-, GAL-, GRF-and DYN-IR fibers in the median eminence to a large extent arise from the ventrolateral arcuate nucleus. Some ENK-and NPK-positive cells in the arcuate nucleus seem to project to the lateral palisade zone of the median eminence, but most of the ENK-IR fibers in the median eminence, located in the medial palisade zone, seem to primarily originate from an area(s) located outside the arcuate nucleus, presumably the paraventricular nucleus. The NPY-positive fibers in the median eminence contain to a large extent immunoreactive dopamine -hydroxylase (DBH), and do not arise from the ventromedial arcuate nucleus. SOM-IR cells in the dorsal periventricular arcuate nucleus do not send major projections to the median eminence. The present findings thus show that MSG treatment represents a valuable tool to clarify the organization of chemically identified neuron populations in the arcuate nucleus-median eminence complex and provide further information for understanding the neuroendocrine effects of neonatal MSG treatment.     

Neuroprotection induced by the adenosine A<Subscript>2A</Subscript> antagonist CSC in the 6-OHDA rat model of parkinsonism: effect on the activity of striatal output pathways

In Parkinsons disease (PD), the striatal dopamine depletion and the following overactivation of the indirect pathway of the basal ganglia leads to very early disinhibition of the subthalamic nucleus (STN) that may contribute to the progression of PD by glutamatergic overstimulation of the dopaminergic neurons in the substantia nigra. Adenosine A_2A antagonism has been demonstrated to attenuate the overactivity of the striatopallidal pathway. To investigate whether neuroprotection exerted by the A_2A antagonist 8-(3-chlorostyryl)caffeine (CSC) correlates with a diminution of the striatopallidal pathway activity, we have examined the changes in the mRNA encoding for enkephalin, dynorphin, and adenosine A_2A receptors by in situ hybridization induced by subacute systemic pretreatment with CSC in rats with striatal 6-hydroxydopamine(6-OHDA) administration. Animals received CSC for 7 days until 30 min before 6-OHDA intrastriatal administration. Vehicle-treated group received a solution of dimethyl sulfoxide. CSC pretreatment partially attenuated the decrease in nigral tyrosine hydroxylase immunoreactivity induced by 6-OHDA, whereas no modification of the increase in preproenkephalin mRNA expression in the dorsolateral striatum was observed. The neuroprotective effect of the adenosine A_2A antagonist CSC in striatal 6-OHDA-lesioned rats does not result from a normalization of the increase in striatal PPE mRNA expression in the DL striatum, suggesting that other different mechanisms may be involved.     

Antinociceptive effect produced by intracerebroventricularly administered dynorphin A is potentiated by p-hydroxymercuribenzoate or phosphoramidon in the mouse formalin test

The antinociceptive effects of intracerebroventricularly (i.c.v.) administered dynorphin A, an endogenous agonist for κ-opioid receptors, in combination with various protease inhibitors were examined using the mouse formalin test in order to clarify the nature of the proteases involved in the degradation of dynorphin A in the mouse brain. When administered i.c.v. 15 min before the injection of 2% formalin solution into the dorsal surface of a hindpaw, 1–4 nmol dynorphin A produced a dose-dependent reduction of the nociceptive behavioral response consisting of licking and biting of the injected paw during both the first (0–5 min) and second (10–30 min) phases. When co-administered with p-hydroxymercuribenzoate (PHMB), a cysteine protease inhibitor, dynorphin A at the subthreshold dose of 0.5 nmol significantly produced an antinociceptive effect during the second phase. This effect was significantly antagonized by nor-binaltorphimine, a selective κ-opioid receptor antagonist, but not by naltrindole, a selective δ-opioid receptor antagonist. At the same dose of 0.5 nmol, dynorphin A in combination with phosphoramidon, an endopeptidase 24.11 inhibitor, produced a significant antinociceptive effect during both phases. The antinociceptive effect was significantly antagonized by naltrindole, but not by nor-binaltorphimine. Phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, bestatin, a general aminopeptidase inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, were all inactive. The degradation of dynorphin A by mouse brain extracts in vitro was significantly inhibited only by the cysteine protease inhibitors PHMB and N-ethylmaleimide, but not by PMSF, phosphoramidon, bestatin or captopril. The present results indicate that cysteine proteases as well as endopeptidase 24.11 are involved in two steps in the degradation of dynorphin A in the mouse brain, and that phosphoramidon inhibits the degradation of intermediary δ-opioid receptor active fragments enkephalins which are formed from dynorphin A.     

强啡肽对脊髓反射抑制作用的受体机制研究

目的 研究鞘内注射强啡肽（Dyn)A(1-13)对脊髓反射抑制作用的受体机制。方法 通过大鼠蛛网膜下腔置管后鞘内注射30nmol DynA(1-13)导致热辐射刺激甩尾反射消失的模型,检测在应用Dyn(A1-13)之前之后鞘内分别注射5、30、150nmol MK-801(NMDA)受体非竞争性拮抗剂）对甩尾反射消失的影响。结果 MK－801在预先给药时对DynA(1-13）所致大鼠甩尾反射消失有剂量相关的保护作用,其中150nmolMK-801的保护率达90％。在迟后给药时则会产生时间相关的保护作用,30、150nmolMK－801的作用峰值分别在15、30min。结论 Dyn致大鼠甩尾反射消失作用至少部分是由NMDA受体介导的。     

Effect of the endogenous κ opioid agonist dynorphin A(1–17) on cocaine-evoked increases in striatal dopamine levels and cocaine-induced place preference in C57BL/6J mice

Rationale Effects of synthetic kappa opioid receptor agonists on cocaine-induced reward have been studied extensively in rats but relatively few studies have used the endogenous kappa agonist dynorphin A(1–17).Objectives Three studies were conducted to examine the effect of the natural sequence dynorphin on cocaine-induced increases in dopamine, on the formation of conditioned place preference and on increases in locomotor activity in C57BL/6 J mice.Methods After implantation of guide cannulae into the caudate putamen, mice were allowed 4–5 days to recover from surgery. In the first study, dynorphin A (0, 1, 2, 4.4 nmol) was infused into the caudate putamen and dopamine levels were measured by in vivo microdialysis in that brain region. Then, the effect of dynorphin A (4.4 nmol) on increases in dopamine levels induced by 15 mg/kg cocaine i.p. was also measured with in vivo microdialysis. The third experiment examined the effect of dynorphin A (4.4 nmol) on conditioned place preference and locomotion induced by 15 mg/kg cocaine.Results Dynorphin A significantly decreased basal dopamine levels in a dose-dependent manner by more than 60% at the highest dose, and this effect was completely blocked by pre-injection of the kappa-opioid receptor antagonist nor-BNI (10 mg/kg). The highest dose of dynorphin (4.4 nmol) blocked increases in dopamine levels, the formation of conditioned place preference and attenuated locomotion induced by 15 mg/kg cocaine.Conclusion The blockade of the cocaine-induced rise in striatal dopamine may contribute to both dynorphins ability to prevent the development of cocaine-induced conditioned place preference and to attenuate the increase in locomotor activity.     

A 35 kDa Fos-related antigen is co-localized with substance P and dynorphin in striatal neurons

The rat striatum after dopamine denervation followed by repeated apomorphine treatment was examined for the co-expression of c-fos and Fos-related antigens with dynorphin, substance P and [Met5]enkephalin using Western blot and immunohistochemical techniques. Administration of apomorphine, a dopamine agonist, elevated the level of 35 kDa Fos-related antigen which co-localized with dynorphin and substance P, but not enkephalin, in striatal neurons.     

Opioid peptides in Parkinson''s disease: effects of dopamine repletion

Neurotransmitters other than dopamine, including neuropeptides, could have important pathophysiologic and therapeutic roles in Parkinson's disease. Both Met-enkephalin, the main transmitter of the striatopallidal pathway, and dynorphin, one of the co-transmitters of the striatonigral pathway display complex anatomic and biochemical interactions with the basal ganglionic dopamine system. In this study, the cerebrospinal fluid content of a proenkephalin derivative, Met⁵ enkephalin-Arg⁶-Gly⁷-Leu⁸ (MERGL), was found in significantly low concentrations in parkinsonian patients following overnight withdrawal of all medications compared with control subjects, and failed to change after at least 16 h of steady-state, optimal doses of levodopa infusion intravenously. MERGL levels increased with advancing age among normal individuals but not among patients with Parkinson's disease. In contrast, dynorphin A(1–8) levels were not different between the two study groups, did not change with levodopa therapy, and failed to correlate with age or any indices of disease progression. These observations, consistent with post-mortem studies on Parkinson brains and contrary to findings in animal models of Parkinsonism, suggest that abnormality of the enkephalin system in this disease is due to involvement of these striatal neurons in the primary pathologic process.     

Kappa opioid receptor activity modulates memory for peck-avoidance training in the 2-day-old chick

To examine the role of kappa opioid receptors in memory formation, 2-day-old chicks were injected intracerebrally with either the endogenous opioid peptide dynorphin_(1–13), the highly kappa selective agonist U-50,488 or the kappa selective antagonist nor-binaltorphimine (nor-BNI), given one-trial peck-avoidance training, and tested 24 h later. Dynorphin_(1–13) impaired memory in a dose dependent manner at 24 h test. Injection of U-50,488 caused a biphasic dose-dependent effect on memory; low doses caused a trend toward enhanced memory and high doses caused significant impairment. Conversely, injection of low doses of nor-BNI caused a trend toward memory impairment, and higher doses caused significant memory enhancement. The results indicate that memory formation for one-trial peck-avoidance training may be modulated by kappa opioid receptor activity.