Tendinosis is a condition of chronic pain that afflicts several human tendons, not least the patellar tendon, in which case it is often clinically referred to as ‘jumper’s knee’. The exact mechanisms behind tendinosis are yet not fully understood. One draw-back in the case of patellar tendinosis has been the lack of knowledge of the innervation patterns of the human patellar tendon. It cannot be excluded that the processes of tendinosis are influenced by nerve mediators, released from nerve endings or from stimulated cells inside the tendon. Thus, the studies of the present thesis aimed to 1) map the general, sensory, cholinergic and sympathetic innervation patterns of the human patellar tendon, in both the tendon tissue proper and the loose paratendinous connective tissue surrounding the tendon, and 2) investigate the possible existence of a production of signal substances, traditionally associated with neurons, in non-neuronal tendon cells, and to see if there are signs of local cholinergic and catecholaminergic signaling pathways. Biopsies of both normal pain-free patellar tendons and patellar tendons from patients with chronic painful tendinosis were collected and investigated. The main method utilized was immunohistochemistry, using antibodies directed against synthesizing enzymes for acetylcholine and catecholamines, against muscarinic and adrenergic receptors, and against markers of general and sensory innervation. In situ hybridization (ISH) to detect mRNA for the cholinergic/catecholaminergic synthesizing enzymes was also used. It was found that the loose paratendinous connective tissue of the patellar tendon was rather richly innervated by nerve structures. These consisted of large nerve fascicles, as well as perivascular innervation in the walls of some of the larger arteries and smaller blood vessels. It was found that part of the nerve structures corresponded to sensory afferents, and that some conformed to cholinergic and, especially, sympathetic nerve fibers. The tendon tissue proper was strikingly less innervated than the paratendinous tissue. The sparse innervation that was found in the tendon tissue proper was seen in narrow zones of loose connective tissue and blood vessels, interspersed between the collagen bundles. The overall impression was that the patterns of distribution of the general, sensory, and autonomic innervations of tendinosis tendon tissue were similar to those of normal tendon tissue proper. The most pioneering findings were the immunohistochemical observations of an expression of enzymes related to production of both acetylcholine and catecholamines within the tendon cells (tenocytes) themselves, as well as of a presence of the receptors for these substances on the same cells; features that were predominantly seen in tendinosis tendons. The observations of the synthesizing enzymes for acetylcholine and catecholamines in tenocytes were confirmed by ISH findings of mRNA for these enzymes in the tenocytes. Immunoreactions for muscarinic and adrenergic receptors were also found in blood vessel walls and in some of the nerve fascicles. In summary, this thesis presents novel information on the innervation patterns of the human patellar tendon, in healthy individuals with pain-free tendons as well as in patients with chronic painful tendinosis. Furthermore, it gives the first evidence of the presence of a local, non-neuronal production in the tendon tissue of signal substances normally seen in neurons, and a basis for these substances to affect the tenocytes as these cells also display muscarinic and adrenergic receptors. Thus, the results indicate an existence of autocrine and/or paracrine cholinergic/catecholaminergic systems in the tendon tissue; systems that seem to be up-regulated in tendinosis. This is of great interest as it is known that stimulation of receptors for both catecholamines and acetylcholine can lead to cell proliferation, interfere with pain sensation, influence collagen production, and take part in vasoregulation, as well as, in the case of adrenergic receptors, promote cell degeneration and apotosis. All these processes represent biological functions/events that are reported to be affected in tendinosis. In conclusion, despite the fact that there is very limited innervation within the patellar tendon tissue proper, it is here shown that effects of signal substances traditionally associated with neurons seem to occur in the tissue, via a local production of these substances in tenocytes.

Neurotransmitter receptor types mediating the generation of intracellular signals are of two types; ligand-gated ion channels and G protein coupled receptors. The effector enzyme phosphoinositide-specific phospholipase C (PLC) is modulated by stimulation of G protein coupled receptors, leading to an increased breakdown of inositol phospholipids ("Ptdlns breakdown").In recent years, the receptors in the brain coupled to PLC and modulation of such receptor-mediated Ptdlns breakdown have been characterised. One such modulation is the "potassium effect", whereby an increase in the assay [K+] from 6 to 18 mM potentiates the Ptdlns breakdown response to the muscarinic agonist carbachol in the rat brain. It has been speculated that this effect is one way of enhancing the signal :noise ratio of muscarinic neurotransmission. The mechanisms responsible for the potassium effect have been studied in this thesis.Initial methodological studies indicated that the temperature of the Krebs buffer used after tissue dissection was an important factor regulating the Ptdlns response to receptor stimulation. Expressing the Ptdlns breakdown response as a fraction of the total labelled phosphoinositides was more useful than other ways of expressing the data. Acid extraction of the Lipid fraction was also superior to neutral extraction.Miniprismspreparedfrompig striatum and hippocampus showed qualitative (but not quantitative) similarities with the rat with respect to stimulation by carbachol, noradrenaline and the potassium effect. Dopamine also stimulated Ptdlns breakdown, though probably via a noradrenergic mechanism.The enhancing actions of potassium appeared to be selective for muscarinic Ml-type receptors. Thus glutamate, quisqualate and NaF-stimulated Ptdlns breakdown are not affected by raised [K+].The potassium effect is brought about by two mechanisms. In calcium-free Krebs buffer, the effect could be mimicked by the calcium channel agonist BAY K-8644 and partially antagonised by verapamil. At an assay [Ca2*] of 2.52 mM, however, modulation of calcium uptake had little effect on carbachol-stimulated Ptdlns breakdown at either normal or raised [K+]. The synergy between potassium and carbachol at252 mM Ca?+ is not dependent upon tissue depolarisation perse, since other ways of depolarising the tissue did not enhance the response to carbachol. It is suggested that potassium might have a direct effect on the muscarinic Ml-type receptor - G protein - PLC complex.In order to investigate this possibility, the effect of fluoride ions (which activate G proteins via formation of AlF4) on basal and carbachol-stimulated Ptdlns breakdown was investigated. Fluoride ions inhibited the enhanced breakdown response to carbachol found at raised [K+]. However, this effect is secondary to effects of fluoride on PLC substrate availibility rather than on G protein function. / digitalisering@umu

This work improved the radiosynthesis of a known M2 muscarinic receptor imaging agent, [18F]FP-TZTP, and subsequent syntheses and in vitro evaluation of several novel TZTP derivatives highlighted a lead compound which exhibited M4 potency and selectivity, the thioether fluoro-polyethyleneglycol, which was then adapted for radiolabelling (23% radiochemical yield (uncorrected), >99% radiochemical purity, reaction time of 37 minutes). The present study also seeked to utilize aziridines as intermediates in [18F]-radiolabelling chemistry for the facile radiosynthesis of [18F]-labelled beta-blockers. Novel [18F]-labelled amines were synthesized via ring-opening and deprotection to yield the [18F]-1-fluoro-2-propanamine moiety (85%) favourably over the regioisomer [18F]-2-fluoro-1-propanamine (15%). Subsequent attempts to use these amine synthons in the synthesis of the beta-blocker [18F]Exaprolol resulted in poor radiochemical yields (1-3%). The chemistry of aziridine ring-opening with 19F-fluoride sources was thoroughly explored in order to understand the fundamentals of this chemistry, and the 1-fluoro-2-propanamine moiety was characterized by X-ray crystallography and NMR spectroscopy.

This work improved the radiosynthesis of a known M2 muscarinic receptor imaging agent, [18F]FP-TZTP, and subsequent syntheses and in vitro evaluation of several novel TZTP derivatives highlighted a lead compound which exhibited M4 potency and selectivity, the thioether fluoro-polyethyleneglycol, which was then adapted for radiolabelling (23% radiochemical yield (uncorrected), >99% radiochemical purity, reaction time of 37 minutes). The present study also seeked to utilize aziridines as intermediates in [18F]-radiolabelling chemistry for the facile radiosynthesis of [18F]-labelled beta-blockers. Novel [18F]-labelled amines were synthesized via ring-opening and deprotection to yield the [18F]-1-fluoro-2-propanamine moiety (85%) favourably over the regioisomer [18F]-2-fluoro-1-propanamine (15%). Subsequent attempts to use these amine synthons in the synthesis of the beta-blocker [18F]Exaprolol resulted in poor radiochemical yields (1-3%). The chemistry of aziridine ring-opening with 19F-fluoride sources was thoroughly explored in order to understand the fundamentals of this chemistry, and the 1-fluoro-2-propanamine moiety was characterized by X-ray crystallography and NMR spectroscopy.

The muscarinic acetylcholine type 2 receptor (M2 mAChR), a member of the GPCR superfamily, is found throughout the parasympathetic nervous system where it controls pulmonary, urinary, and cardiac function, and neurotransmission. The molecular mechanisms that regulate M2 mAChR availability at the cell surface are an important component in controlling these physiological events. Since beta-arrestin proteins are known to regulate the activity of other GPCRs, we sought to identify their role in regulating M2 mAChR activity, a topic that remains contentious in the field. To achieve this goal we utilized mouse embryonic fibroblasts (MEFs) derived from beta-arrestin knockout mice lacking one or both isoforms (MEF KO1, KO2, or KO1/2 cells) in addition to exogenous expression of beta-arrestin mutants. This study demonstrates that agonist-induced internalization of M2 mAChR is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles suggesting it behaves as a class B receptor. Next, we sought to identify beta-arrestin s function in regulating the post-endocytic trafficking (down-regulation) of the M2 mAChR. MEF KO1/2 cells were unable to down-regulate M2 mAChRs whereas MEF KO1 or KO2 cells retained the ability to do so. In MEFwt cells, both M2 mAChR and beta-arrestin exhibited basal ubiquitination that increased following agonist stimulation. Receptor degradation appeared to be regulated by the ubiquitination status of beta-arrestin 2, since expression of a chimeric â-arrestin 2 form fused to ubiquitin increased both constitutive and agonist-promoted down-regulation, whereas expression of a beta-arrestin 2 mutant lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, significantly blocked degradation while internalization and stable association remained intact. Upon further analysis, the beta-arrestin 2K18R, K107R, K108R, K207R, K296R mutant blocked delivery of M2 mAChR to the late endosome/lysosome, presumably where degradation occurs. Inhibition of proteasome-dependent recycling of ubiquitin blocked receptor down-regulation without affecting internalization or the ubiquitination state of the M2 mAChR while ubiquitination of beta-arrestin 2 diminished significantly. These results support a role for ubiquitinated beta-arrestin in mediating M2 mAChR sorting and degradation in the lysosome. Collectively, these studies give us new insight on the function of beta-arrestin in regulating the activity of the M2 mAChR.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 132-147). Also available on the World Wide Web.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Made available in DSpace on 2015-05-14T13:00:14Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2331593 bytes, checksum: 773c3f24392a37cc5990eecb7f6f2761 (MD5) Previous issue date: 2010-08-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Imidazolidinic derivates present a wide range of biological activities, such as anti-arrhythmic and anti-hypertensive. The vasorelaxant effect of six Imidazolinic compounds was evaluated in this study. Among of this, the cardiovascular effects induced by IM-7 were evaluated using in vivo and in vitro assays. HPA-01 and IM-3 produced a reduced relaxation of rat mesenteric artery rings contracted by phenylephrine (PHE) (MR = 43.15 ± 7.69%; MR = 54.08 ± 6.59%, respectively). IM-6 e IM-2 showed vasoconstictor effects in rigns with endothelium and vasorelaxant effect when endothelium was removed. Meanwhile, HPA-02 did not induce significant vasorelaxant effect. In normotensive non-anesthetized rats, the pretreatment with L-NAME or hexamethonium did not affect the hypotensive and bradicardic effects induced by IM-7. In isolated rat atrial preparations from normotensive, IM-7 (1pM-1 mM) produced negative chronotropic (MR = 65.45 ± 11.4%; pD2 = 3.94 ± 0.02) and inotropic (MR = 68.58 ± 8,0% ; pD2 = 6.74 ± 0.05) effects. which were attenuated by atropine. In hypertensive non-anesthetized rats IM-7 (1, 5, 10, 20 e 30 mg/Kg, i.v.) induced hypotensive and bradicardic effects. These effects were abolished after pre-treatment with atropine (2 mg/Kg, i.v.). In isolated hypertensive rat mesenteric artery rings IM-7 (1pM-1 mM), elicited concentration-dependent relaxation of PHE-induced contraction (pD2 = 5.64  0.09). After removal of the vascular endothelium IM-7 effects were significantly attenuated (pD2 = 3.8 ± 0.07 p < 0.001), suggesting the participation of endothelium derived relaxant factors in the IM-7 induced vasorelaxant effect. Similar results were obtained in the presence of L-NAME (100 M), ODQ (10μM), or atropine (100 nM). In endothelium denuded rings incubed with KCl 20 mM the relaxant response was not attenuated. Furthermore, in absence of extracellular calcium, IM-7 concentration-dependently depressed the vasoconstrictions derived from CaCl2. Similar results were obtained in contractions induced by 5-HT, cumulative administration of PHE or Na3VO4. These results demonstrate that IM-7 induced best pharmacological profile comparing with the other imidazolidinic derivatives. In normotensive rats, the inotropic e chronotropic effects induced by IM-7 are probably due to activation of muscarinic receptors. In hypertensive rats, the hypotensive, bradicardic and vasorelaxant effects seem to involve muscarinic receptor activation. Furthermore, IM-7 exerts a vasorelaxant effect by inhibiting calcium influx and contractile apparatus. / Derivados imidazolidínicos apresentam uma ampla variedade de atividades biológicas, dentre elas a antiarrítmica e anti-hipertensiva. O efeito vasorerrelaxante de seis derivados hidantoínicos foi avaliado neste estudo. Dentre estas substâncias, os efeitos cardiovasculares induzidos por IM-7 foram avaliados empregando-se abordagens in vivo e in vitro. Em anéis de artéria mesentérica superior de rato, as substâncias HPA-01 e IM-3 apresentaram discreto efeito vasorrelaxante. Concentrações cumulativas de IM-2 e IM-6 induziram vasoconstrição e vasorrelaxamento na presença e na ausência do endotélio, respectivamente, enquanto HPA-02 não causou efeitos significativos. Em ratos normotensos não anestesiados, o bloqueio com hexametônio ou com L-NAME não foi capaz de alterar os efeitos hipotensor e bradicárdico induzidos por IM-7. Em átrio isolado de rato, este derivado hidantoínico causou efeitos cronotrópico (Emáx = 65,45 ± 11,4% ; pD2 = 3,94 ± 0,02 ) e inotrópico (Emáx = 68,58 ± 8,0% ; pD2 = 6,74 ± 0,05 ) negativos, que foram atenuados na presença da atropina. Em ratos com hipertensão L-NAME não-anestesiados, IM-7 (1, 5, 10, 20 e 30 mg/Kg, i.v.) induziu hipotensão e bradicardia, os quais foram abolidos na presença da atropina (2 mg/Kg, i.v.). Em artéria mesentérica superior isolada de rato hipertenso, IM-7 induziu vasorrelaxamento em anéis pré-contraídos com fenilefrina (FEN) (pD2 = 5,64  0,09) e esse efeito foi significantemente atenuado após a remoção do endotélio (pD2 = 3,8 ± 0,07 p < 0,001), sugerindo a participação de fatores relaxantes endoteliais. Resultados similares foram obtidos após a incubação de L-NAME (100 M), ODQ (10 M) ou atropina (100 nM). Em anéis sem endotélio, incubados com KCl 20 mM, o vasorrelaxamento induzido por IM-7 não foi atenuado. IM-7 reduziu contrações induzidas por 80 mM de KCl em anéis sem endotélio (pD2 = 3,84 ± 0,05) de maneira similar ao observado em contrações induzidas por fenilefrina. Concentrações isoladas de IM-7 (10-5, 10-4 e 10-3) reduziram significativamente as contrações induzidas por CaCl2. Resultados similares foram observados nas contrações induzidas por serotonina (5-HT) e pela administração cumulativa de FEN ou Na3VO4. As contrações transientes induzidas por FEN (10 M) não foram alteradas. Estes resultados demonstram que dentre os derivados hidantoínicos avaliados, IM-7 apresentou melhor atividade farmacológica. Em ratos normotensos, os efeitos inotrópicos e cronotrópicos negativos induzidos por esta substância, possivelmente decorrentes da ativação muscarínica, justificam a bradicardia observada em estudos anteriores. O vasorrelaxamento independente de endotélio provavelmente é decorrente da atuação desta substância sobre a maquinaria contrátil na musculatura lisa vascular. Em ratos hipertensos, IM-7 induziu hipotensão, bradicardia e vasorrelaxamento provavelmente de origem muscarínica. Além disso, este derivado hidantoínico também parece ser capaz de inibir o influxo de cálcio e atuar na maquinaria contrátil para promover vasodilatação

Impairment of the cholinergic neurotransmission system is regularly detected in animal models of Alzheimer's disease as well as in human patients suffering from this serious disease. Moreover, there is increasing amount of evidence suggesting that activation of individual mAChR subtypes specifically influences the cleavage of APP, the precursor for β-amyloid. APP can be processed in an amyloidogenic or non-amyloidogenic pathway and a relative abundance of these patways contributes to establishing the final concentration of neurotoxic β-amyloid in the brain. In this work, I have studied the acute and chronic effects of A β1-42 on binding and functional characteristics of mAChR. I have demonstrated that Aβ1-42 present in cell culture expressing the individual subtypes of mAChR negatively and specifically influences the function of the M1 mAChR subtype. I have also detected a decline in muscarinic receptor-mediated signal transduction in brain tissue of young adult APPswe/PS1dE9 mice, a commonly used animal model of Alzheimer's disease. Demonstration of the impairment of muscarinic transmissin in transgenic mice by soluble β-amyloid that occurs earlier than amyloid pathology and behavioral deficit, and its imitation by soluble Aβ1-42 in vitro lend strong support to the notion of the early involvement...