Les neuropeptides tels que la vasopressine et l’ocytocine, hormis leurs actions périphériques bien connues, régulent de nombreuses fonctions cognitives et comportementales via une action centrale. Récemment, il a été démontré que la vasopressine régule, par le biais des récepteurs centraux V1A, V1B et OT de l’ocytocine, l’humeur, l’apprentissage et les désordres affectifs évoluant dans le système limbique. Néanmoins, les isoformes V1A et V1B restent les moins étudiées, souffrant d’un manque d’outils pharmacologiques sélectifs. Chez le rat, un modèle animal des plus utilisé en laboratoire, aucun agoniste de haute affinité sélectif du récepteur V1A n’a été caractérisé. De la même façon pour le récepteur V1B, nous manquons d’outils sélectifs permettant sa localisation dans le système nerveux central. D’où la nécessité de développer de nouvelles molécules sélectives pour répondre à ces besoins. Ceci nous a conduit, lors de ce travail de thèse, à privilégier deux axes de recherches ciblant : Le récepteur V1A.Cette partie concerne la caractérisation des propriétés pharmacologiques d’un nouveau composé, le FE 201874. Ce composé, dérivé du F-180 a été présenté comme un agoniste sélectif du récepteur V1A humain à courte durée de vie. Les résultats de cette étude ont confirmé que le FE 201874 est un très bon ligand sélectif V1B/V1A et V2/V1A vis à vis des expériences de liaison et peu sélectif OT/V1A, ce composé ayant une affinité non négligeable pour le récepteur OT. Son caractère, agoniste V1A et antagoniste OT lui confère une sélectivité complète V1A pour toute approche fonctionnelle. La validation des résultats s’est faite également par des approches ex vivo et in vivo qui ont confirmé le caractère agoniste sélectif du FE 201874. Notre étude a permis donc à la caractérisation du premier agoniste sélectif du récepteur V1A chez le rat. Ce peptide sélectif serait particulièrement utile pour développer de nouvelles stratégies thérapeutiques ou pour les études pharmacologiques ayant pour but de mieux comprendre les rôles centraux de ces récepteurs dans les pathologies comportementales et les pathologies liées au stress. Le récepteur V1B Cette partie concerne la caractérisation des propriétés pharmacologiques et de marquage de ligands fluorescents dérivés de la d[Leu4,Lys8]VP, le premier agoniste sélectif du récepteur V1B, en vue d’étudier la distribution centrale des récepteurs V1B chez le rat. Les résultats obtenus ont montré que les 3 analogues testés sont des agonistes sélectifs V1A/V1B et V2/V1B et peu si ce n’est pas sélectif OT/V1B. Leur capacité à marquer les récepteurs V1B en système hétérologue de culture cellulaire et en culture cellulaire primaire a également été évaluée, démontrant que ces ligands fluorescents représentent d’excellents outils pour le marquage et l’étude de la localisation des récepteurs V1B. Ces ligands ont ensuite été utilisés sur coupes fraiches de cerveau de rat pour l’étude de la distribution des récepteurs V1B centraux dans les régions impliquées dans les différents processus comportementaux. Plusieurs sites ont été détectés parmi lesquels l’hippocampe, du cortex, de l’amygdale, de l’hypothalamus le bulbe olfactif Enfin, en tenant compte de la nature agoniste de ces ligands fluorescents, l’activation de la voie MAP kinase au niveau des tranches cérébrale a été également mise en évidence. / Neuropeptides such as vasopressin and oxytocin, apart from their well-known peripheral actions, regulate many cognitive and behavioral functions via central action. Recently, it has been demonstrated that vasopressin regulates, through the central receptors V1A, V1B and OT of oxytocin, mood, learning and affective disorders evolving in the limbic system. Nevertheless, the V1A and V1B isoforms remain the least studied, suffering from a lack of selective pharmacological tools. In the rat, an animal model most used in the laboratory, no agonist of high selective affinity of the receptor V1A was characterized. In the same way for the V1B receptor, we lack selective tools allowing its localization in the central nervous system. Hence the need to develop new selective molecules to meet these needs. This led us, in this thesis, to focus on two axes of research aimed at

<p>Background:Pancreas cancer is a most aggressive malignancy. More than 80% of patients diagnosed with pancreas cancer, exhibit such advanced disease, that curative surgery is impossible. Systemic chemotherapy prolongs survival to 5-9 months. High concentrations of chemotherapeutic agents in the abdominal cavity and in the lymphatics draining the area is achieved by intraperitoneal administration. Vasopressin decreases splanchnic blood flow, reducing the intraperitoneal uptake of drugs, thus raising the local and lymphatic dose intensity.</p><p>Aim: The aim of the study was to investigate the feasibility and tumour response of intraperitoneal 5-Fluorouracil (5-FU) treatment in non-resectable pancreas cancer, using vasopressin to improve the pharmacokinetic profile. Further, to study the effect of vasopressin on peritoneal blood flow, altered by intraperitoneal 5-FU or the presence of peritoneal carcinomatosis.</p><p>Methods: In the animal experiments, the 133Xe-clearance technique and as a comparison Laser doppler flow, were used to identify changes of peritoneal blood flow caused by vasopressin in unmanipulated animals and in animals with peritoneal carcinomatosis or animals given intraperitoneal 5-FU. In the clinical studies, 68 (39 women/29 men) patients, with a non-resectable ductal pancreas cancer and a Karnovsky Index ≥70 were included. Patients were treated with 750-1500 mg/m2 5-FU intraperitoneally through a Port-a-cath and Leucovorin 100 mg/m2 intravenously on two consecutive days every 21 days until progression. Seventeen patients, receiving 750 mg/m2 5-FU, were given concomitant vasopressin 0.1 IU/min during 180 minutes, alternatively day 1 or 2.</p><p>Results: In the animal experiments, vasopressin 0.07 IU/kg/min significantly reduced the 133Xe-clearance. Intraperitoneal 5-FU decreased the basal peritoneal blood flow and abrogated the vasopressin effect for 1-2 days. The presence of peritoneal carcinomatosis did not influence the basal peritoneal blood flow, nor the reduction of peritoneal blood flow caused by vasopressin. In the clinical studies, the treatment with intraperitoneal 5-FU was well tolerated, with no WHO Grade 3 or 4 toxicity with doses up to 1250 mg/m2. Thirty patients achieved at least stable disease at three months. The median survival time was 8.0 (range 0.8-54.1) months. There was a significant reduction of 5-FU Cmax on day 2, but no significant reduction of AUC, when vasopressin was given.</p><p>Conclusion: Peritoneal blood flow changes caused by vasopressin can be estimated with the 133Xe-clearance technique. Intraperitoneal 5-FU but not peritoneal carcinomatosis decreases the vasopressin induced 133Xe-clearance reduction, 1-2 days after administration. In patients with non-resectable pancreas cancer, intraperitoneal 5-FU up to 1250 mg/m2 for two days every third week can be given without WHO grade 3 and 4 toxicity. The treatment is well tolerated with few and minor side effects. Tumour responses were observed. Addition of vasopressin does not significantly enhance the pharmacokinetics of intraperitoneal 5-Flurorouracil, but adds toxicity.</p>

Background:Pancreas cancer is a most aggressive malignancy. More than 80% of patients diagnosed with pancreas cancer, exhibit such advanced disease, that curative surgery is impossible. Systemic chemotherapy prolongs survival to 5-9 months. High concentrations of chemotherapeutic agents in the abdominal cavity and in the lymphatics draining the area is achieved by intraperitoneal administration. Vasopressin decreases splanchnic blood flow, reducing the intraperitoneal uptake of drugs, thus raising the local and lymphatic dose intensity. Aim: The aim of the study was to investigate the feasibility and tumour response of intraperitoneal 5-Fluorouracil (5-FU) treatment in non-resectable pancreas cancer, using vasopressin to improve the pharmacokinetic profile. Further, to study the effect of vasopressin on peritoneal blood flow, altered by intraperitoneal 5-FU or the presence of peritoneal carcinomatosis. Methods: In the animal experiments, the 133Xe-clearance technique and as a comparison Laser doppler flow, were used to identify changes of peritoneal blood flow caused by vasopressin in unmanipulated animals and in animals with peritoneal carcinomatosis or animals given intraperitoneal 5-FU. In the clinical studies, 68 (39 women/29 men) patients, with a non-resectable ductal pancreas cancer and a Karnovsky Index ≥70 were included. Patients were treated with 750-1500 mg/m2 5-FU intraperitoneally through a Port-a-cath and Leucovorin 100 mg/m2 intravenously on two consecutive days every 21 days until progression. Seventeen patients, receiving 750 mg/m2 5-FU, were given concomitant vasopressin 0.1 IU/min during 180 minutes, alternatively day 1 or 2. Results: In the animal experiments, vasopressin 0.07 IU/kg/min significantly reduced the 133Xe-clearance. Intraperitoneal 5-FU decreased the basal peritoneal blood flow and abrogated the vasopressin effect for 1-2 days. The presence of peritoneal carcinomatosis did not influence the basal peritoneal blood flow, nor the reduction of peritoneal blood flow caused by vasopressin. In the clinical studies, the treatment with intraperitoneal 5-FU was well tolerated, with no WHO Grade 3 or 4 toxicity with doses up to 1250 mg/m2. Thirty patients achieved at least stable disease at three months. The median survival time was 8.0 (range 0.8-54.1) months. There was a significant reduction of 5-FU Cmax on day 2, but no significant reduction of AUC, when vasopressin was given. Conclusion: Peritoneal blood flow changes caused by vasopressin can be estimated with the 133Xe-clearance technique. Intraperitoneal 5-FU but not peritoneal carcinomatosis decreases the vasopressin induced 133Xe-clearance reduction, 1-2 days after administration. In patients with non-resectable pancreas cancer, intraperitoneal 5-FU up to 1250 mg/m2 for two days every third week can be given without WHO grade 3 and 4 toxicity. The treatment is well tolerated with few and minor side effects. Tumour responses were observed. Addition of vasopressin does not significantly enhance the pharmacokinetics of intraperitoneal 5-Flurorouracil, but adds toxicity.

The aim of this thesis was to determine any association between plasma oxytocin and vasopressin concentrations and renal water and sodium excretion during normal pregnancy. In addition to investigate changes in concentrations of estradiol, progesterone, oxytocin, cortisol, and glucose in the blood before and in the nearest hours after delivery and if treatment with oxytocin affected these concentrations and the fluid balance during the different stages of labour. Oxytocin, vasopressin, estradiol, progesterone, and cortisol were analysed in blood plasma or serum by radioimmunoassay or ELISA: serum glucose, and osmolality, and sodium in plasma and urine were  analysed by standard laboratory techniques. Fifty-seven women were studied during pregnancy and fifty-one during parturition and post partum. The low plasma vasopressin and increasing plasma oxytocin concentrations with unchanged water and sodium excretion indicate that oxytocin assists vasopressin in concentrating urine during pregnancy. Plasma vasopressin concentration continued to be low during parturition and post partum. Urine flow and concentration was unrelated to changes in plasma sodium concentration, indicating regulation of fluid balance during parturition was different to the non-gravid state. Women with weak myometrial contractions during parturition (slow progress of labour) reacted differently than women with normal parturition and a group of women with fast progress of labour. The group with slow labour had lower serum estradiol concentration in the latency phase and became hyponatremic. Pulsatile and continuous oxytocin infusions were both effective in the treatment of slow progress of labour. A lower amount of oxytocin was needed to affect delivery when given as pulsatile infusion. Serum cortisol and glucose concentrations were high during labour and cortisol level remained elevated after delivery and glucose concentration reached the highest levels (12 mmol/L) at the same time. Insulin resistance together with the long time of elevated cortisol concentration partly explained the high glucose concentration. In conclusion, fluid balance is not regulated according to the usual sensitive osmotic and volumetric influence on vasopressin release from the neurohypophysis during pregnancy and parturition. Parturition involves a change from one demanding condition, pregnancy, to another, lactation. Parturition and the hours directly after delivery are a turbulent period involving considerable stress.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are able to change their firing rate and pattern in response to small changes in external osmolality due to the involvement of osmosensitive ion channels. The firing rate and pattern determine the release of vasopressin (VP), a primary hormone regulating osmolality by controlling water excretion from the kidney. Both VP- and oxytocin (OT)-MNCs display irregular and infrequent fire when plasma osmolality is near normal, and they progressively increase the frequency of firing to fast continuous firing with increases in osmolality. VP-MNCs also respond to osmotic stimulation by adopting a phasic pattern of firing, which maximizes neuropeptide secretion. Sustained dehydration also causes structural and functional adaptations in MNCs.<p> Voltage-dependent Ca2+ channels play many important roles not only in the regulation of cell excitability but also in intracellular signal transduction, and L-type Ca2+ channel-mediated Ca2+ signals initiate intracellular signal transduction events that activate long-lasting changes in brain function and behavior. Our electrophysiological and immunocytochemical studies demonstrate that 16-24 h of water deprivation causes a significant increase in the amplitude of L-type Ca2+ current (from 55.5 ± 6.2 to 99.1 ± 10.0 pA) but not in other types of Ca2+ current. This increase occurred in both VP- and OT-MNCs. Such an increase in L-type Ca2+ current may contribute to modulation of firing rate and pattern, regulation of vasopressin release, structural adaptation in MNCs during sustained dehydration.<p> The mechanisms underlying the transition of the electrical behaviour are not completely understood. Ion channels, especially osmosensitive ion channels, play key roles in the modulation of MNC firing. A voltage-gated, 4-AP- and TEA-insensitive slowly activating outward current displayed a significant increase in about 66% of MNCs when the osmolality of the external solution was acutely increased from 295 to 325 mosmol kg-1. The responding cells showed an increase in net outward current from 12.3 ± 1.3 pA/pF to 21.4 ± 1.8 pA/pF. The reversal potential of this current was near the equilibrium for K+ and shifted with changes of K+ concentrations in external solution, suggesting that this current is a K+-selective current. The KCNQ/M current selective blockers linopirdine (150 µM) and XE991 (5 µM) suppressed this current. The IC50 of XE991 blockade was 3.9 ìM. The KCNQ/M channel openers retigabine (10 µM) and flupirtine (10 µM) significantly increased the current and shifted its activation curve toward more negative potentials. E4031, a specific blocker of ERG K+ channels, did not significantly block this current. The results from immunocytochemistry suggest that MNCs express KCNQ2, KCNQ3, KCNQ4, and KCNQ5, but not KCNQ1. These data suggest that this osmosensitive current could be a KCNQ/M current. Studies using single unit extracellular recording in hypothalamic explants showed that 10 µM XE991 increased MNC firing rate and that 20 µM retigabine decreased firing rate or caused a cessation of firing. These data suggest that a KCNQ/M current contributes to the regulation of MNC firing. KCNQ/M channels play key roles in regulating neuronal excitability in many types of central neurons. Slow activation of this current during firing might suppress activity by hyperpolarizing the cells and thus contribute to a transition between fast continuous and burst firing.<p> Our studies will be beneficial to understand the mechanisms that control VP and OT in response to acute changes in osmolality and also the mechanisms underlying MNC adaptation during sustained dehydration.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are able to change their firing rate and pattern in response to small changes in external osmolality due to the involvement of osmosensitive ion channels. The firing rate and pattern determine the release of vasopressin (VP), a primary hormone regulating osmolality by controlling water excretion from the kidney. Both VP- and oxytocin (OT)-MNCs display irregular and infrequent fire when plasma osmolality is near normal, and they progressively increase the frequency of firing to fast continuous firing with increases in osmolality. VP-MNCs also respond to osmotic stimulation by adopting a phasic pattern of firing, which maximizes neuropeptide secretion. Sustained dehydration also causes structural and functional adaptations in MNCs.<p> Voltage-dependent Ca2+ channels play many important roles not only in the regulation of cell excitability but also in intracellular signal transduction, and L-type Ca2+ channel-mediated Ca2+ signals initiate intracellular signal transduction events that activate long-lasting changes in brain function and behavior. Our electrophysiological and immunocytochemical studies demonstrate that 16-24 h of water deprivation causes a significant increase in the amplitude of L-type Ca2+ current (from 55.5 ± 6.2 to 99.1 ± 10.0 pA) but not in other types of Ca2+ current. This increase occurred in both VP- and OT-MNCs. Such an increase in L-type Ca2+ current may contribute to modulation of firing rate and pattern, regulation of vasopressin release, structural adaptation in MNCs during sustained dehydration.<p> The mechanisms underlying the transition of the electrical behaviour are not completely understood. Ion channels, especially osmosensitive ion channels, play key roles in the modulation of MNC firing. A voltage-gated, 4-AP- and TEA-insensitive slowly activating outward current displayed a significant increase in about 66% of MNCs when the osmolality of the external solution was acutely increased from 295 to 325 mosmol kg-1. The responding cells showed an increase in net outward current from 12.3 ± 1.3 pA/pF to 21.4 ± 1.8 pA/pF. The reversal potential of this current was near the equilibrium for K+ and shifted with changes of K+ concentrations in external solution, suggesting that this current is a K+-selective current. The KCNQ/M current selective blockers linopirdine (150 µM) and XE991 (5 µM) suppressed this current. The IC50 of XE991 blockade was 3.9 ìM. The KCNQ/M channel openers retigabine (10 µM) and flupirtine (10 µM) significantly increased the current and shifted its activation curve toward more negative potentials. E4031, a specific blocker of ERG K+ channels, did not significantly block this current. The results from immunocytochemistry suggest that MNCs express KCNQ2, KCNQ3, KCNQ4, and KCNQ5, but not KCNQ1. These data suggest that this osmosensitive current could be a KCNQ/M current. Studies using single unit extracellular recording in hypothalamic explants showed that 10 µM XE991 increased MNC firing rate and that 20 µM retigabine decreased firing rate or caused a cessation of firing. These data suggest that a KCNQ/M current contributes to the regulation of MNC firing. KCNQ/M channels play key roles in regulating neuronal excitability in many types of central neurons. Slow activation of this current during firing might suppress activity by hyperpolarizing the cells and thus contribute to a transition between fast continuous and burst firing.<p> Our studies will be beneficial to understand the mechanisms that control VP and OT in response to acute changes in osmolality and also the mechanisms underlying MNC adaptation during sustained dehydration.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成5年5月14日 雄山博文氏の博士論文として提出された

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2009. / Härtill 4 uppsatser.

Arginine vasopressin (AVP) is a hypothalamic nonapeptide which regulates the hypothalamic-pituitary-adrenal axis response to stress by stimulating the secretion of adrenocorticotropin (ACTH) from corticotroph cells of the anterior pituitary. This effect is mediated by binding of AVP to the pituitary vasopressin receptor (V1bR). The V1bR belongs to the G protein-coupled receptor (GPCR) super family. Repeated stimulation of anterior pituitary cells with AVP has been shown to produce a loss of responsiveness to subsequent AVP stimulation. This phenomenon appears to be mediated by desensitisation of the V1bR, and may be due to phosphorylation of the receptor by G protein-coupled receptor kinase 5 (GRK5). The aim of this research was to establish and validate methods that would allow the role of GRK5 in the desensitisation of V1bR to AVP stimulation to be investigated. As no isoform specific inhibitors for GRK5 were available, HEK293 cells transiently transfected with the rat V1bR were used as a model system for this research. This allowed RNA interference (RNAi) to be used to knockdown GRK5 expression. The protocol for RNAi-mediated knockdown of GRK5 was established as part of this research. Protocols for Western blotting and qRT-PCR were also established to allow the RNAi-mediated knockdown of GRK5 protein and mRNA to be measured. Transfection of HEK293 cells with 10nM GRK5-targeting small interfering RNAs (siRNAs) reduced the expression of GRK5 protein to 53.4% ± 3.4% (mean ± SEM) of that seen in untreated control cells at 84 hours after transfection, while GRK5 mRNA levels were reduced to 28.7% ± 1.9% (mean ± SEM) of that of control cells 48 hours after transfection. An experimental protocol was designed in this research that would coordinate the RNAi-mediated knockdown of GRK5 with transient transfection of the HEK293 cells with the rV1bR. Since, activated V1bRs couple to Gq/11 and stimulate the production of inositol phosphates (IPs), the responsiveness of the V1bR can be determined by measuring the accumulation of [H³]-IPs in cells labelled with [H³]-myo-inositol. In the protocol designed, the effect of GRK5 knockdown on V1bR desensitisation is determined by stimulating HEK293 cells expressing the rV1bR (and previously transfected with GRK5-targeting siRNA) with 0nM or 100nM AVP for 0, 5, 15, 30 or 60 minutes, and comparing the accumulation if IPs over time with that of cells that are not transfected with GRK5-targeting siRNA. This protocol can be used in future to investigate the role of GRK5 in V1bR desensitisation, and may be adapted to determine if other GRK isoforms are involved in V1bR desensitisation.

Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (p. 143-152). Also available via the World Wide Web.