Heterogeneity of [H]neurotensin binding: studies with dynorphin, L-156,903 and levocabastine

The binding of [3H]neurotensin (NT) to membranes from rat forebrain was complex, exhibiting 'high' affinity (Kd approximately 0.5 nM) and 'low' affinity (Kd approximately 5.0 nM) binding components. Dynorphin A(1-13) (DYN A(1-13] and L-156,903 (N-oxo-3-(10H-phenothiazine-10-yl)propyl-1- arginyl-1-prolyl-1-phenylalanine) potently inhibited [3H]NT binding to brain with shallow biphasic competition curves. Saturation binding studies conducted in the presence or absence of DYN A(1-13) or L-156,903 indicated that these compounds, like levocabastine, exhibited substantial selectivity for 'low' affinity NT site. Structure-activity studies indicated rigid structural requirements for the NT binding activity of DYN A(1-13) and L-156,903. In contrast to the results using brain tissue, DYN A(1-13), L-156,903 and levocabastine were very weak or inactive to inhibit [3H]NT binding to rat uterus. These studies further characterize the heterogeneity of [3H]NT binding in vitro and demonstrate clear tissue differences in binding within a given species.     

Regulation of the concentration of dynorphin A1–8 in the striatonigral pathway by the dopaminergic system

The purpose of this study was to explore the dopaminergic control of the striatonigral dynorphin system by measuring the levels of dynorphin A1-8-like immunoreactivity (DN-LI) after repeated injections of a dopaminergic receptor agonist or antagonist. Seven daily injections of different doses of apomorphine (0.5, 1.0, 2.5 and 5.0 mg/kg, s.c.) caused a significant dose-related increase of DN-LI in the striatum (26, 34, 63, 85% over control at each corresponding dose). Similar increases were observed in the substantia nigra (22, 52, 50 and 62% over control). In another experiment, rats received 5 mg/kg of apomorphine for 1, 3, and 7 days. There was a significant time-related increase in DN-LI both in the striatum (37, 50 and 85% over control at each corresponding period) and in the substantial nigra (32, 78 and 62%). Repeated administration of haloperidol (1 mg/kg, i.p.) failed to change the striatal level of DN-LI, but, when given at the same time as apomorphine, significantly attenuated the effect of apomorphine. These results suggest that dopamine exerts a modulatory influence on the metabolism of dynorphin in the striatonigral pathway.     

Effect of long-term haloperidol treatment on striatal neuropeptides: Relation to stereotyped behavior

Behavioral and biochemical responses to D₁ and D₂ dopamine (DA) agonists were used to evaluate the participation of striatal peptidergic mechanisms in the motor function alterations that attend chronic neuroleptic treatment. Rats, given haloperidol (1 mg/kg, s.c.) for 21 consecutive days, were randomly allocated to one of the following treatments: the D₁ agonist SKF 38393, the D₂ agonist quinpirole, their combination or saline. Stereotyped behavior and neuropeptide levels were evaluated after 5 days treatment and 4 days washout. Haloperidol increased most oral behaviors including licking, chewing and biting as well as striatal enkephalin and somatostatin levels. Subsequent treatment with SKF 38393 diminished the haloperidol-induced increase in licking and chewing; quinpirole reduced chewing behavior. The administration of both agonists together decreased chewing and biting. Neither DA agonist alone, nor their combination, reduced the haloperidol-induced increase in enkephalin levels. Both SKF 38393 and quinpirole, when given alone, tended to decrease the haloperidol-induced increase in somatostatin levels; when both the D₁ and D₂ agonists were administered together, somatostatin levels declined significantly. These results suggest that somatostatin- but not enkephalin-containing striatal neurons contribute to the expression of haloperidol-induced stereotypies.     

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.     

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.     

Cholecystokinin-8S increases dynorphin B, aspartate and glutamate release in the fronto-parietal cortex of the rat via different receptor subtypes

The effect of sulphated cholecystokinin-8 (CCK-8S) on extracellular dynorphin B, aspartate, glutamate and GABA levels in the rat fronto-parietal cortex was investigated with in vivo microdialysis. The peptide was infused through the microdialysis probe trying to mimic local CCK-8S release. Basal levels of dynorphin B were around 20pM, aspartate 100nM, glutamate 600nM and GABA 30nM. CCK-8S (10μM) induced a ≈3-fold increase in extracellular dynorphin B, aspartate and glutamate levels, while GABA levels were only slightly increased. The effect of CCK-8S was restricted to the stimulated neocortex. Systemic pretreatment with the CCK_B antagonist, L-365, 260, but not with the CCK_A antagonist, L-364, 718, significantly antagonised the effect of CCK-8S on cortical dynorphin B and aspartate release. However, both CCK_A and CCK_B antagonists inhibited the increase in cortical glutamate levels. Thus, the present results indicate that cortical CCK release exerts a stimulatory modulation on cortical dynorphin B and aspartate release via the CCK_B receptor subtype, and on glutamate release via both CCK_A and CCK_B receptor subtypes. Considering electrophysiological evidence that CCK increases neuronal firing rates in many brain regions, it may be suggested that CCK represents a stimulatory system modulating the function of the neocortex. Received: 25 September 1996 / Accepted: 25 January 1997     

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.