<p>A new group of anabolic androgenic steroid (AAS) users has developed during the last two decades. This group consists primarily of young men interested in improving their physical appearance. Within this group, AAS are sometimes used together with other illicit drugs, alcohol and nicotine. Brutal and violent crimes have been committed under the influence of AAS, possibly because of AAS psychiatric side effects, ranging from increased aggression and psychosis to depression. Unfortunately, the biochemical mechanisms behind these effects are poorly understood.</p><p>In this thesis we used an animal model to study biochemical and behavioural effects of chronic AAS treatment (15 mg/kg/day of nandrolone decanoate for 14 days). The effect on the endogenous opioid peptides and the expression of immediate-early gene protein Fos in various brain regions were studied using radioimmunoassay and immunohistochemistry, respectively. In addition, we studied AAS effect on voluntary alcohol consumption and defensive behaviours, including aggression. The results show that AAS enhance endogenous opioid activity and Fos expression in brain regions regulating reward, aggression and disinhibitory behaviours. An imbalance between two opioid systems with generally opposing effects, the enkephalins with euphoric and the dynorphins with dysphoric effects, was also found. This implies that AAS alter the ability to maintain a stable state of mind and the response to other drugs of abuse. The AAS pre-treated animals enhanced their alcohol intake, were more aggressive and showed lower fleeing and freezing reaction than the controls. In addition, AAS enhanced amphetamine-induced aggression when the amphetamine was given three weeks after the last AAS injection.</p><p>The behavioural and biochemical results found in this thesis, support the hypothesis that use of AAS might lead to the development of dependence and may induce changes in the brain leading to disinhibitory behaviours.</p>

<p>Dyskinesias or abnormal involuntary movements are a debilitating complication of long-term levodopa treatment of Parkinson’s disease (PD) that is widely experienced and may compromise the efficacy of the drug therapy. Tardive dyskinesia is another important adverse effect seen with antipsychotic drug treatment. The neural mechanisms underlying levodopa- and neuroleptic-induced dyskinesia are not clear and involvement of the endogenous opioid neuropeptide system has been implicated. In this thesis, the role of the opioid system is investigated in models of dyskinesia and PD using behavioral, neurochemical and advanced analytical chemistry techniques. In addition, the motor effects of a new partial dopamine agonist with normalizing properties on both reduced and elevated dopamine transmission are studied and a new model for tardive dyskinesia is presented.</p><p>Using microdialysis in combination with micro-electrospray mass spectrometry, the <i>in vivo</i> processing of the opioid neuropeptide dynorphin A(1-17) was studied and 32 metabolites were detected in the striatum. Altered <i>in vivo</i> metabolism of the peptide was found in a model of PD with more metabolites formed in the dopamine-depleted striatum. Moreover, dynorphin A(1-17) was differently processed in levodopa-, bromocriptine and saline-treated animals. </p><p>Levodopa treatment caused an increase in the mRNA expression of the precursor of dynorphin, preproenkephalin-B as well as the precursor of enkephalin, preproenkephalin-A, in all sub-regions of the dopamine-depleted striatum. A non-selective opioid receptor antagonist, naloxone, was found to reduce levodopa-induced dyskinesia with maintained antiparkinsonian response and a normalization of hyperkinesia. Moreover, the new drug GMC1111 showed dopamine stabilizing properties in models of levodopa-induced dyskinesia and PD. This might prove useful in the treatment of PD.</p><p>Altogether, these results suggest that the endogenous opioid system is involved in the pathophysiology of levodopa-induced dyskinesia.</p>

A new group of anabolic androgenic steroid (AAS) users has developed during the last two decades. This group consists primarily of young men interested in improving their physical appearance. Within this group, AAS are sometimes used together with other illicit drugs, alcohol and nicotine. Brutal and violent crimes have been committed under the influence of AAS, possibly because of AAS psychiatric side effects, ranging from increased aggression and psychosis to depression. Unfortunately, the biochemical mechanisms behind these effects are poorly understood. In this thesis we used an animal model to study biochemical and behavioural effects of chronic AAS treatment (15 mg/kg/day of nandrolone decanoate for 14 days). The effect on the endogenous opioid peptides and the expression of immediate-early gene protein Fos in various brain regions were studied using radioimmunoassay and immunohistochemistry, respectively. In addition, we studied AAS effect on voluntary alcohol consumption and defensive behaviours, including aggression. The results show that AAS enhance endogenous opioid activity and Fos expression in brain regions regulating reward, aggression and disinhibitory behaviours. An imbalance between two opioid systems with generally opposing effects, the enkephalins with euphoric and the dynorphins with dysphoric effects, was also found. This implies that AAS alter the ability to maintain a stable state of mind and the response to other drugs of abuse. The AAS pre-treated animals enhanced their alcohol intake, were more aggressive and showed lower fleeing and freezing reaction than the controls. In addition, AAS enhanced amphetamine-induced aggression when the amphetamine was given three weeks after the last AAS injection. The behavioural and biochemical results found in this thesis, support the hypothesis that use of AAS might lead to the development of dependence and may induce changes in the brain leading to disinhibitory behaviours.

Dyskinesias or abnormal involuntary movements are a debilitating complication of long-term levodopa treatment of Parkinson’s disease (PD) that is widely experienced and may compromise the efficacy of the drug therapy. Tardive dyskinesia is another important adverse effect seen with antipsychotic drug treatment. The neural mechanisms underlying levodopa- and neuroleptic-induced dyskinesia are not clear and involvement of the endogenous opioid neuropeptide system has been implicated. In this thesis, the role of the opioid system is investigated in models of dyskinesia and PD using behavioral, neurochemical and advanced analytical chemistry techniques. In addition, the motor effects of a new partial dopamine agonist with normalizing properties on both reduced and elevated dopamine transmission are studied and a new model for tardive dyskinesia is presented. Using microdialysis in combination with micro-electrospray mass spectrometry, the in vivo processing of the opioid neuropeptide dynorphin A(1-17) was studied and 32 metabolites were detected in the striatum. Altered in vivo metabolism of the peptide was found in a model of PD with more metabolites formed in the dopamine-depleted striatum. Moreover, dynorphin A(1-17) was differently processed in levodopa-, bromocriptine and saline-treated animals. Levodopa treatment caused an increase in the mRNA expression of the precursor of dynorphin, preproenkephalin-B as well as the precursor of enkephalin, preproenkephalin-A, in all sub-regions of the dopamine-depleted striatum. A non-selective opioid receptor antagonist, naloxone, was found to reduce levodopa-induced dyskinesia with maintained antiparkinsonian response and a normalization of hyperkinesia. Moreover, the new drug GMC1111 showed dopamine stabilizing properties in models of levodopa-induced dyskinesia and PD. This might prove useful in the treatment of PD. Altogether, these results suggest that the endogenous opioid system is involved in the pathophysiology of levodopa-induced dyskinesia.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser.

The binding of [³H]opioid ligands to homogenates prepared from the spinal cords of rat and other species has been studied. Similar numbers of sites were seen in all areas of the cord when measured in a rostrocaudal direction. There was found to be approximately 2 x higher density of sites in the dorsal half of the cord compared with the ventral half. Binding studies suggested a similar relative distribution of mu, delta and kappa sites in all areas of the cord. The results are discussed in relation to the reported distribution of opioid peptides. In the above study the kappa binding site was defined as the binding of [³H] unselective opioids in the presence of cold ligands to suppress binding to mu- and delta-sites. Competitive binding assays, however, suggested this site did not have the properties of a single homogeneous group. Approximately 50% of the apparent kappa binding was consistent with a classical kappa site. Saturated binding assays afforded Bmax values which suggested lower 'true' kappa site numbers than previously supposed, values which were confirmed using the kappa peptide' [³H]Dynorphin A-(1-9), and the kappa selective [³H]U-69593. Heterogeneity was also seen in other central nervous system tissues. The heterogeneous nature of the kappa site may be due to different sites, due to interactions at a non-opioid site or may represent different conformations of the same site. The second possibility was discounted since observed binding followed the cellular distribution of the plasma marker Na+/K+-ATPase was stereoselective for levorphanol over dextrorphan, and fully displaceable by naloxone. The third possibility was investigated by studying the role of Na+ and MG2+ ions, which are reported to affect receptor conformation in binding assays employing brain tissues. None of the results obtained suggested that conformational changes were responsible for the observed effects, although the experiments were not exhaustive.

No description available.

Les dynorphines sont des neuropeptides importants avec un rôle central dans la nociception et l’atténuation de la douleur. De nombreux mécanismes régulent les concentrations de dynorphine endogènes, y compris la protéolyse. Les Proprotéines convertases (PC) sont largement exprimées dans le système nerveux central et clivent spécifiquement le C-terminale de couple acides aminés basiques, ou un résidu basique unique. Le contrôle protéolytique des concentrations endogènes de Big Dynorphine (BDyn) et dynorphine A (Dyn A) a un effet important sur la perception de la douleur et le rôle de PC reste à être déterminée. L'objectif de cette étude était de décrypter le rôle de PC1 et PC2 dans le contrôle protéolytique de BDyn et Dyn A avec l'aide de fractions cellulaires de la moelle épinière de type sauvage (WT), PC1 -/+ et PC2 -/+ de souris et par la spectrométrie de masse. Nos résultats démontrent clairement que PC1 et PC2 sont impliquées dans la protéolyse de BDyn et Dyn A avec un rôle plus significatif pour PC1. Le traitement en C-terminal de BDyn génère des fragments peptidiques spécifiques incluant dynorphine 1-19, dynorphine 1-13, dynorphine 1-11 et dynorphine 1-7 et Dyn A génère les fragments dynorphine 1-13, dynorphine 1-11 et dynorphine 1-7. Ils sont tous des fragments de peptides associés à PC1 ou PC2. En plus, la protéolyse de BDyn conduit à la formation de Dyn A et Leu-Enk, deux peptides opioïdes importants. La vitesse de formation des deux est réduite de manière significative dans les fractions cellulaires de la moelle épinière de souris mutantes. En conséquence, l'inhibition même partielle de PC1 ou PC2 peut altérer le système opioïde endogène. / Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous Big Dynorphin (BDyn) and Dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild type (WT), PC1-/+ and PC2-/+ animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments Dynorphin 1-19, Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7 and C-terminal processing of Dyn A generates Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even partial inhibition of PC1 or PC2 may impair the endogenous opioid system.

科学研究費補助金 研究種目:一般研究(C) 課題番号:01570633 研究代表者:大磯 ユタカ 研究期間:1989-1990年度

Les peptides appartenant à la famille des tachykinines telle que la substance P (SP) sont des acteurs essentiels contribuant à l’hyperalgésie primaire et secondaire. La SP libérée par les neurones afférents primaires, ne provoque pas à elle seule des décharges nociceptives, mais elle potentialise l’effet de divers neurotransmetteurs tel que le glutamate. Pour ces différentes raisons, de nombreuses recherches ont été effectuées avec des antagonistes des récepteurs neurokinines et en particulier des récepteurs NK1. Cependant, malgré des études pré-cliniques prometteuses, les antagonistes du récepteur NK1 n’ont pas montré d’effet significatif chez l’Homme. La biosynthèse des neuropeptides actifs passe par la maturation protéolytique des pro-neuropeptides. La compréhension des mécanismes de la maturation enzymatique des précurseurs des tachykinines, ainsi que l’étude de la stabilité métabolique de la substance P (SP) permettraient d’élucider des stratégies de traitement innovateur en favorisant l’inhibition du processus de maturation ou la production de fragments peptidiques moins actifs ou inactifs. Le premier objectif de cette étude était d’élucider le rôle de la Proproteine convertase 1 (PC1) et de la Proproteine convertase 2 (PC2) dans la maturation de la protachykinine en utilisant des fractions S9 de la moelle épinière des souris du type sauvage (WT), PC1-/+ et PC2-/+. La caractérisation et la quantification des neuropeptides ont été réalisées à l’aide de la chromatographie liquide à haute performance et de la spectrométrie de masse. Les résultats montrent que PC1 et PC2 interviennent dans la maturation de la protachykinine et ces deux enzymes sont essentielles pour la biosynthèse de la Tachykinine58-71, le précurseur de la SP. Une réduction de plus de 50% de la vitesse de formation dans les fractions S9 de la moelle épinière de souris mutantes PC1 et PC2 a été observée. Les résultats obtenus révèlent que PC1 et PC2 sont impliquées dans la protéolyse de la protachykinine et suggèrent un rôle important de ces enzymes dans la maturation de la protachykinine-1. La protéolyse régule probablement les concentrations extracellulaires de la SP, mais peu d'études ont été menées sur le métabolisme des tachykinines. Dans ce présent travail, nous démontrons que la protéolyse contrôle le niveau de la SP dans la moelle épinière menant à la formation de fragments C-terminaux actifs. La stabilité métabolique de la β-tachykinine58-71 et de la SP était très courte, avec une demi-vie de 5.7 et 3.5 min, respectivement. Plusieurs fragments C-terminaux ont été identifiés, y compris la SP3-11, la SP5-11 et la SP8-11, qui conservent leurs affinités vis-à-vis des récepteurs neurokinines. La stabilité métabolique des fragments C-terminaux était significativement supérieure à celle de la β-Tachykinine58-71 et de la SP. Deux inhibiteurs de Prolyl endopeptidase spécifiques ont été utilisés et ont montré une réduction significative de la vitesse de formation de SP3-11 et de SP5-11. Ainsi, nous avons démontré que le Prolyl endopeptidase est impliqué dans le traitement N-terminal de la SP dans la moelle épinière et dans la formation de la SP3-11 et la SP5-11. Étant donné que la régulation des niveaux endogènes de peptides opioïdes (DynA, Leu-Enk, Met-Enk) et des tachykinines (Tach58-71, SP) dépend fondamentalement de l'activité de PC1 et de celle de PC2, l'analyse des tachykinines et des neuropeptides opioïdes ont été réalisées. Les résultats obtenus révèlent une diminution significative des neuropeptides pro nociceptifs la Tach58-71 (p <0,05), de la SP (p <0,01) et du NKA (P <0,001)), et des neuropeptides opioïdes DynA (p <0,01), de Leu-Enk (p <0,001), de Met-Enk (p <0,001), dans la moelle épinière de souris PC1 - / + et PC2 - / +. Par conséquent, la modulation de l'activité des PCs a un impact important sur les peptides pro-nociceptifs, mais également sur le système opioïde endogène et par conséquent elle affectera significativement les voies modulatrices de la douleur. Ces résultats suggèrent également que la réduction significative des concentrations de peptides pro-nociceptifs peut altérer la réponse du système opioïde endogène. Les analyses des concentrations des peptides opioïdes chez les souris Tac1-/- ont montré spécifiquement que les concentrations en Endomorphine-2 (EM2), en Leu-Enk et en Dyn A sont significativement inférieures que celles obtenues dans la moelle épinière chez les souris WT. Par conséquent, l’absence de la SP a un impact sur les mécanismes endogènes de modulation de la douleur. Mots clés : Tachykinines, substance P, proprotéines convertases, protéolyse, peptides opioïdes, moelle épinière, douleur, chromatographie liquide à haute performance, spectrométrie de masse. / SP is a major proteolytic product of the protachykinin-1 primarily synthesized in neurons and plays a central role in nociceptive transmission. The SP does not acte alone to cause nociceptive discharges, but it potentiates the effect of various neurotransmitters such as glutamate. For these various reasons, much research has been carried out with antagonists of neurokinin receptors and in particular NK1 receptors. However, despite promising pre-clinical studies, NK1 receptor antagonists have not shown significant effect in Humans. The proteolysis control of endogenous protachykinins has a profound impact on pain perception. Proprotein convertases (PCs) are extensively expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue but the role of PCs remains unclear. The first objective of this study was to decipher the role of PC1 and PC2 in the proteolysis of protachykinins using cellular fractions of spinal cords from wild type (WT), PC1 -/+ and PC2 -/+ mices and mass spectrometry. The results clearly demonstrate that both PC1 and PC2 mediate the formation of SP and β-Tachykinin58-71, an important SP precursor, with over 50 % reduction of the rate of formation in mutant PC1 and PC2 mouse S9 spinal cord fractions. The results obtained revealed that PC1 and PC2 are involved in the C-terminal processing of protachykinin peptides and suggest a major role in the maturation of the protachykinin-1 protein. The proteolysis is suspected to regulate extracellular SP concentrations but few studies were conducted on the metabolism of proneuropeptides and neuropeptides. In the present study, we provide evidence that proteolysis controls SP levels in the spinal cord leading to the formation of active C-terminal fragments. The metabolic stability of β-Tachykinin58-71 and SP were very short resulting in half-life of 5.7 and 3.5 min, respectively. Several C-terminal fragments were identified, including SP3-11, SP5-11 and SP8-11, which conserve affinity for the neurokinin receptors. Interestingly, the metabolic stability of C-terminal fragments were significantly superior. Two specific Prolyl endopeptidase inhibitors were used and showed a significant reduction in the rate of formation of SP3-11 and SP5-11 providing strong evidence that Prolyl endopeptidase is involved into N-terminal processing of SP in the spinal cord. The role of proprotein convertases (PCs) in the proteolysis of proneuropeptides was previously established but few studies have shown the direct impact of PCs on the regulation of specific tachykinin and opioid peptides in the central nervous system. This study has determined the relative concentration of targeted neuropeptides in the spinal cord of WT, PC1- / + and PC2- /+ mice to establish the impact of a restricted PCs activity on the regulation of specific neuropeptides. The results revealed a significant decrease of Dyn A (p < 0.01), Leu-Enk (p < 0.001), Met-Enk (p < 0.001), Tach58-71 (p < 0.05), SP (p < 0.01) and NKA (p < 0.001) spinal cord concentrations in both, PC1 -/+ and PC2 -/+ mice. Therefore, the modulation of PCs activity has an important impact on specific pronociceptive peptides (SP and NKA), but the results also showed that endogenous opioid system is hindered and consequently it will affect significantly the pain modulatory pathways. Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1-/- spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. Our results reveal that Endomorphin-2 (EM2), Leu-Enk and Dyn A were down regulated in Tac1-/- spinal cord tissues that strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These results may have insightful impact on future analgesic drug developments and therapeutic strategies. Key words: Tachykinins, substance P, proprotein convertases, proteolysis, opioid peptides, spinal cord, pain, high performance liquid chromatography, mass spectrometry.