Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The calcitonin gene family of peptides consists of calcitonin (CT), two calcitonin gene related peptides (α-CGRP, β-CGRP), adrenomedullin (ADM), amylin (AMY), three calcitonin receptor activating peptides (CRSP1-3) and intermedin/adrenomedullin2 (IMD). These peptides bind to one of two G protein -coupled receptors, the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CRLR). The receptor specificity to different ligands is dependent on the formation of a complex with one of three receptor activity-modifying proteins (RAMP1-3). The aim of this study was to analyse effects of this family of peptides on the formation of osteoclasts and bone resorption, and the expression of the receptor components in bone cells. CT inhibited the formation of multinucleated osteoclasts in spleen cell cultures and in bone marrow macrophage cultures (BMM) without affecting a number of genes important for osteoclast differentiation, activity or fusion of osteoclast progenitor cells. All members of the CT family, except ADM, inhibited osteoclastogenesis in BMM. The inhibitory effect seemed to involve activation of both protein kinase A and the exchange protein directly activated by cyclic AMP (Epac) signalling. BMM expressed the CRLR, RAMP1-3 and the receptor component protein (RCP). AMY, ADM, CGRP and IMD, but not CRSP and CT, increased cyclic AMP (cAMP) levels in these cells, indicating the presence of functional receptors. Stimulation of BMM with RANKL gradually increased the levels of CTR mRNA as well as the capacity of the cells to respond to the stimulation by CRSP and CT. The response to stimulation of ADM was, on the contrary, decreased by RANKL. Stimulation of RANKL caused a transiently enhanced CRLR mRNA expression and transiently decreased RAMP1, but did not affect RAMP2, RAMP3, or RCP mRNA. However, RANKL did not affect protein levels of CRLR or RAMP1-3. CT, CGRP, AMY, ADM, IMD and CRSP all down regulated the CTR mRNA, but none of the peptides caused any effects on the expression of CRLR or any of the RAMPs. All members of the CT family, except ADM, rapidly and transiently, inhibited bone resorption in mouse calvarial bones. CT, CGRP, AMY and CRSP also significantly stimulated cAMP formation in the calvaria. cAMP analogues specifically stimulating the PKA or the Epac pathways did not cause inhibition of bone resorption in the calvaria. An unspecific cAMP analogue, stimulating both pathways did, however, cause inhibition. Analyses of an osteoblastic cell line, MC3T3-E1, showed that these cells express the mRNA for CRLR and all three RAMP proteins. In conclusion, the results of this thesis show that all peptides in CT family of peptides, except ADM, inhibit of bone resorption and osteoclast formation and that these effects involve the adenylate cyclase-cAMP pathway. Furthermore, expressions of CRLR and RAMP1-3 mRNA have been demonstrated on osteoclasts, as well as in an osteoblastic cell line.

Die bakterielle Meningitis (BM) ist trotz antibiotischer Therapie eine Erkrankung mit einer hohen Mortalität und Morbidität. Kopfschmerzen und Meningismus sind Hauptsymtome und ein klinischer Hinweis für die Aktivierung trigeminaler Fasern. Ziel dieser Arbeit war es zu prüfen ob die freigesetzten Neuropeptide einen proinflammatorischen Effekt auf zerebrale Endothelzellen, einen wesentlichem Bestandteil der Blut-Hirn-Schranke haben. Wir verwendeten primär kultivierte zerebrale Kapillarendothelzellen (BMEC) der Ratte und als Stimulus Neuropeptide und/oder Pneumokokkenzellwände (PCW). Beide Neuropeptide, CGRP mehr als SP, verstärken den Effekt von PCW auf die mRNA Expression und Freisetzung von TNF-alpha, IL-1beta, IL-6, IL-10 und MIP-2 aus den BMEC. CGRP und SP haben nur eine geringe Wirkung. PCW regulieren die Dichte der CRLR (CGRP1-R) bzw. NK-1 Rezeptoren und erklären damit die kostimulatorische Wirkung. Zudem untersuchten wir den Effekt von PCW und/oder CGRP auf die Adrenomedullin (AM)- Synthese. AM ist ein vasodilatorisch wirkendes Peptid, dass vorwiegend in Endothelzellen konstitutiv gebildet wird und am CRLR Rezeptor wirkt. PCW und CGRP verstärken die Synthese von AM. Mit dieser Arbeit konnte gezeigt werden, dass PCW zur Hochregulation von Neuropeptidrezeptoren führt und CGRP und SP über diese Rezeptoren einen modulatorischen Effekt auf die Zytokinproduktion in BMEC haben. Ein genaues Verständnis dieser Interaktionen könnte die Entwicklung immunmodulatorischer Interventionen und damit eine Verbesserung der Prognose der bakteriellen Meningitis bewirken. / Despite antibiotic treatment bacterial meningitis is still associated with a high mortality and morbidity. Headache and meningismus as key symptoms, provide clear evidence for the activation of trigeminal nerve fibers. Aim of the study was to test whether the released neuropeptides have a proinflammatory effect in cerebral endothelial cells the major compartment of the blood brain barrier. We used primary brain microvascular endothelial cells of the rat (BMEC) which were stimulated with CGRP, SP and/or pneumococcal cell walls (PCW). Both neuropeptides CGRP more than SP enhanced PCW-induced mRNA expression and the release of TNF-alpha, IL-1-beta, IL-6, IL-10 and MIP-2. Neuropeptides alone were not able to induce these cytokines. PCW upregulate the density of CRLR receptor and regulate the NK-1 receptor and therefore may explain the costimulatory effect. Furthermore the effect of PCW and/or CGRP on adrenomedullin synthesis in BMEC was investigated. Adrenomedullin is a vasodilatatory peptide, which is constitutivly produced by endothelial cells and act on the CRLR receptor. PCW as well as CGRP enhance the synthesis of AM. Our data suggest that PCW upregulate neuropeptide receptors and modulate via these specific receptors the cytokine production. A detailed understanding of these interactions may open new immunmodulatory interventions and therefore may contribute to a better prognosis of bacterial meningitis.