The seven transmembrane α-helices of the human 5 -HT_1A , 5-HT_1Dα and 5-HT_1Dβ receptors have been modelled using the 3-dimensional coordinates of the seven transmembrane a-helices of the bacterial protein bacteriorhodopsin as a template. The probable 5-HT binding site was identified between helices 3, 4, 5 and 6. Interactions between the natural ligand 5-HT (A) and the receptor models are described in detail and the agonist binding site is further validated by the docking of four known 5-HT receptor ligands. The models are able to account qualitatively for the receptor binding affinities of the studied ligands. Small molecule similarity studies suggest that a thieno[2,3-b]pyridine analog (B) of 5-HT could possibly act as a bio-isostere for 5-HT. This was further corroborated when (B) was docked into the 5-HT receptor models and was found to be accommodated easily in the 5-HT binding site participating in the same interactions as observed for 5-HT. Thieno[2,3-b]pyridines similar to (B) were thus identified as synthetic target compounds. Furthermore, the models were used to provide suggestions for the design of novel, more selective, 5-HT receptor agonists. The thieno[2,3-b]pyridine ester (C; R=C0₂Et) was reduced to the hydroxymethyl derivative (C; R=CH₂0H) but the methylthio group could not be successfully removed in the presence of the thiophene ring. Using a different approach the thieno[2,3-b]pyridine (D; R=CO₂Me, X=CN) was synthesised as a model compound and converted through to the <i>t</i>-BOC protected amines (D, R=NHCO₂C(CH₃)₃, X=CN) and (D, R=NHCO₂C(CH₃)₃ , X=CONH₂). The same reactions were applied to ethyl-3-(5-cyanothieno[2,3-b] pyridin-3-yl)propanoate (E, R=CO₂Et, X=CN) but this unfortunately could not be converted through to the required <i>t</i>-BOC protected 5-HT analog (E, R=NHCO₂C(CH₃)₃, X=CN).

Platelet activation and aggregation are important processes in hemostasis resulting in reduction of blood loss upon vessel wall injury. However, platelet activation can lead to thrombotic events causing myocardial infarction and stroke. A more detailed understanding of the regulation of platelet activation and the subsequent formation of thrombi is essential to prevent thrombosis and ischemic stroke. Cations, platelet surface receptors, cytoskeletal rearrangements, activation of the coagulation cas-cade and intracellular signaling molecules are important in platelet activation and thrombus formation. One such important molecule is serotonin (5 hydroxytryptamin, 5 HT), an indolamine platelet agonist, biochemically derived from tryptophan. 5 HT is secreted from the enterochromaffin cells into the gastrointestinal tract (GI) and blood. Blood borne 5 HT has been proposed to regulate hemostasis by acting as a vaso-constrictor and by triggering platelet signaling through 5 HT2A receptor. Although platelets do not synthetize 5 HT, they take it up from the blood and store it in their dense granules which are secreted upon platelet activation. To identify the molecu-lar composite of the 5 HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke, 5 HT transporter knock out mice (5Htt / ) were analyzed in different in vitro and in vivo assays and in a model of is-chemic stroke. In 5Htt / platelets, 5 HT uptake from the blood was completely abol-ished and agonist-induced Ca2+ influx through store operated Ca2+ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein (GP) VI and C type lectin-like receptor 2 (CLEC 2) were reduced. These observed in vitro defects in 5Htt / platelets could be normalized by the addition of exogenous 5 HT. Moreover, reduced 5 HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt / mice. Surprisingly, in the transient middle cerebral artery occlusion model (tMCAO) of ischemic stroke 5Htt / mice showed near-ly normal infarct volumes and a neurological outcome comparable to control mice. Although secreted platelet 5 HT does not appear to play a crucial role in the devel-opment of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and thus plays an important role in thrombus stabilization. To further investigate the role of cations, granules and their contents and regulation of integrin activation in the process of thrombus formation, genetically modified mice were analyzed in the different in vivo thrombosis models. Whereas Tph1 / mice (lacking the enzyme responsible for the production of 5 HT in the periphery), Trpm7KI (point mu-tation in the kinase domain of Trpm7 channel, lacking kinase activity) and Unc13d / /Nbeal2 / mice (lacking α granules and the release machinery of dense granules) showed a delayed thrombus formation in vivo, MagT1y/ mice (lacking a specific Mg2+ transporter) displayed a pro thrombotic phenotype in vivo. Trpm7fl/fl Pf4Cre (lacking the non specific Mg2+ channel) and RIAM / mice (lacking a potential linker protein in integrin “inside out” signaling) showed no alterations in thrombus formation upon injury of the vessel wall. / Thrombozytenaktivierung und Aggregation sind wichtige Schritte der Hämostase, die zur Reduktion des Blutverlustes bei Gefäßwandverletzung führen. Jedoch kann die Aktivierung von Thrombozyten zur Thrombose führen, wodurch Herzinfarkt und Schlaganfall entstehen kann. Ein besseres Verständnis der Regulierung der Throm-bozytenaktivierung und die darauf folgende Thrombusbildung sind notwendig, um Thrombose und Hirninfarkte zu vermeiden. Kationen, Thrombozy-ten Oberflächenrezeptoren, Zytoskelett Reorganisation, Aktivierung der Koagulati-onskaskade und intrazellulare Signalmoleküle sind wichtig in der Thrombozytenakti-vierung und Thrombusbildung. Solch ein wichtiges Molekül ist Serotonin (5 hydroxytryptamin, 5 HT), ein Indolamin Thrombozyten-Agonist, welcher aus Tryp-tophan synthetisiert wird. 5 HT wird aus den Enterochromaffinzellen in den Gastroin-testinaltrakt (GI) und das Blut abgegeben. 5 HT aus dem Blut wirkt als Regulator der Hämostase durch die Wirkung als Vasokonstriktor und die Auslösung der Throm-bozyten-Signalwege durch den 5 HT2A Rezeptor. Thrombozyten synthetisieren kein 5 HT, sondern nehmen es aus dem Blut auf und speichern es in den dichten Granu-la, die nach der Thrombozyten-Aktivierung freigesetzt werden. Um die molekulare Zusammensetzung des 5 HT Aufnahmesystems in Thrombozyten zu identifizieren und die Rolle des 5 HT aus Thrombozyten in Thrombose und ischämischem Schlag-anfalls zu klären, wurde eine 5 HT Transporter-defiziente Mauslinie (5Htt / ) in ver-schiedenen in vitro und in vivo Untersuchungen und im Model des ischämischen Schlaganfalls analysiert. In 5Htt / Thrombozyten ist die Aufnahme von 5 HT aus dem Blut vollständig geblockt und Agonisten-induzierter Ca2+ Fluss durch Speicher-abhängigen Ca2+ Einstrom (SOCE), Integrinaktivierung, Degranulierung und Aggre-gation abhängig von Glykoprotein (GP) VI und C type lectin-like receptor 2 (CLEC 2) waren reduziert. Diese in vitro beobachteten Defekte in 5Htt / Thrombozyten konnten durch Zugabe von 5 HT normalisiert werden. Zudem wurden reduzierte 5 HT Werte im Plasma, eine erhöhte Blutungszeit und die Bildung von instabilen Thromben ex vivo unter Fluss und in vivo in der abdominalen Aorta und der Carotis von 5Htt / Mäusen beobachtet. Überraschenderweise zeigten die 5Htt / Mäuse nach transientem Verschluss der A. cerebri media (tMCAO), einem Modell des ischämi-schen Schlaganfalls, ein normales Infarktvolumen und einen unveränderten neurolo-gischen Endzustand im Vergleich zu Kontrollmäusen. Obwohl sekretiertes 5 HT aus Thrombozyten keine wesentliche Rolle in der Entwicklung eines Reperfusionsscha-dens nach einem Schlaganfall spielt, ist es essentiell in der Verstärkung der zweiten Phase der Thrombozytenaktivierung durch SOCE und spielt eine wichtige Rolle in der Thrombusstabilität. Um die Rolle von Kationen, Granula und deren Bestandteile und der Regulierung in der Integrinaktivierung im Prozess der Thrombusbildung zu untersuchen, wurden genetisch veränderte Mäuse in den verschiedenen in vivo Thrombosemodellen ge-testet. Während Tph1 / Mäuse (denen das Enzym zur Produktion von 5 HT in der Peripherie fehlt), Trpm7KI (Punktmutation in der Kinasedomäne des Trpm7 Kanals, Fehlen der Kinase Aktivität) und Unc13d / /Nbeal2 / Mäuse (denen die α Granula und die Freisetzungsmaschinerie der dichten Granula fehlt) und keine oder eine verlang-samte Thrombusbildung zeigten, wiesen MagT1y/ Mäuse (denen der spezifische Mg2+ Transporter fehlt) einen prothrombotischen Phänotyp auf. Trpm7fl/fl Pf4Cre Mäuse (denen der nicht spezifische Mg2+ Kanal fehlt) und RIAM / Mäuse (denen ein potenti-elles Linker Protein im Integrin “inside out” Signal fehlt) zeigten keine Veränderung in der Thrombus Bildung nach Verletzung der Gefäßwand.

Veränderungen des zentralen serotonergen Systems können mit diversen psychiatrischen Krankheiten wie z. B. Depressionen, Aufmerksamkeitsdefizit/ Hyperaktivitäts-Störung (ADHS), Phobien oder Panik- und Angststörungen assoziiert werden. Die fortlaufende Untersuchung des Neurotransmitters Serotonin (5-HT) und seine Bedeutung für physiologische und verhaltens- bezogene Prozesse ist daher unerlässlich. Tiermodelle, die auf Ausschaltung elementarer oder assoziierter Gene des serotonergen Systems beruhen, sind infolgedessen eine ausgezeichnete Möglichkeit anatomische, (patho)physiolo- gische und verhaltensbezogene Auswirkungen eines fehlgeleiteten serotoner- gen Systems zu untersuchen und zu analysieren. Aufgrund ihrer großen Be- deutung für Lern- und Gedächtnisprozesse steht die Hirnregion des dorsalen Hippocampus im Fokus dieser Dissertation. Die Analyse umfasste jeweils die gesamte Hirnstruktur des Hippocampus bzw. seine Unterregionen, Gyrus dentatus (DG), Cornu Ammonis (CA)1 und CA3. Die Zielsetzung dieser Arbeit war die Untersuchung zellulärer bzw. molekularer Veränderungen von konstitutiven Tryptophanhydroxylase 2 (Tph2) knockout (KO) Mäusen. Durch die Inaktivierung von Tph2 und damit dem geschwindig- keitsbestimmenden Enzym (TPH2) der Serotoninsynthese, wurde im zentralen Nervensystem (ZNS) der KO Mäuse ein Mangel von 5-HT festgestellt. Der dorsale Hippocampus wurde auf zellspezifische Veränderungen nach dem Furchtkonditionierungstest analysiert. Die Reaktion der Neurone in den drei Unterregionen der Hirnstruktur wurde durch Immunofluoreszenzfärbung des „immediate-early“ Genprodukts c-fos bzw. des Calcium-bindenden Proteins Parvalbumin untersucht. Es wurde dabei zum einen die absolute Zellzahl in den Strukturen erfasst und zum anderen die Analyse bezüglich des Volumens vorgenommen. Die Zelldichte von c-Fos wies signifikante Unterschiede zwischen den Gruppen im gesamten dorsalen Hippocampus und bei genauerer Betrachtung in der Unterregion des DG auf. Die Tph2-/- Mäuse zeigten nach dem Furchtkonditionierungstest eine prägnante Erhöhung der aktivierten Zellen. Es scheint, dass 5-HT eine zu starke Aktivierung des dorsalen Hippocampus verhindert um schlechte kontextbezogene Gedächtnisinhalte nicht zu verfesti- gen. Dabei inhibiert 5-HT Zellen im DG und der CA1 Region die nicht zu den Parvalbumin-immunoreaktiven GABAergen Interneuronen gehören. / Although multiple lines of evidence implicate brain serotonin (5-HT) system dysfunction in the pathophysiology of anxiety disorders such as the panic syndrome, the neural processes are not well understood. Here, we investigated the impact of constitutively deficient 5-HT synthesis on fear response-related network function in the hippocampus using tryptophan hydroxylase-2 (Tph2) mutant mice. A double immunostaining of c-Fos and Parvalbumin, a calcium-binding protein expressed in a subpopulation of the GABAergic interneurons of the hippocampus, was established. Tph2-/- mice showed increased c-Fos expression after fear conditioning in the dorsal hippocampus while the number of c-Fos/Parvalbumin-positive cells was not changed. This data support the view that 5-HT is necessary to inhibit neuronal activity within fear acquisition in the dorsal hippocampus possibly excluding Parvalbumin - positive cells.

No description available.

The serotonin receptor 2A (5-HT₂[A]) is a member of the G-protein coupled receptor family and is of interest due to its role in physiological functions such as smooth muscle contraction, platelet aggregation, thermoregulation, learning and memory. More importantly, 5-HT₂[A] has also been implicated in CNS disorders including schizophrenia, depression and anxiety. A yeast two-hybrid screen had previously been carried out to identify proteins that interacted with 5-HT₂[A] and therefore may modulate intracellular function. The cytoskeletal actin-binding protein filamin-C was identified as a possible 5-HT₂[A] interacting partner. The aim of the research in this thesis was to further investigate the potential interaction between 5-HT₂[A] and filamin-C and to investigate functional roles for the interaction. A fragment of human filamin-C, aa 2162-2725, was shown to interact with the C-terminus of human 5-HT₂[A] using two in vitro techniques, the yeast-two hybrid system and a GST capture assay. The region of filamin-C needed to bind to 5-HT₂[A] was narrowed to the start of repeat 20, aa 2251, through to aa 2424 at the beginning of repeat 22 and comprises 182 residues. The 5-HT₂[A] region needed to bind to filamin-C was ascertained via yeast two-hybrid to be 31 amino acids between 394-423. Work was performed to determine whether FLNC mRNA was expressed in neural and glial cells and whether FLNC and HTR2A mRNA were co-expressed in any cells. FLNC mRNA was identified in seven out of eight neural and glial cell lines and western blot analysis confirmed this finding at the protein level. Two cell lines, U-118MG and A172, were found to contain both HTR2A and FLNC mRNA. Co-immunoprecipitation experiments showed endogenous filamin-C bound to endogenous 5-HT₂[A] and this complex could be precipitated using anti-filamin-C antibody. Additionally, a GST-5-HT₂[A] fusion complex was found to bind to endogenous filamin-C from U-118MG cells. Immunofluorescent labelling of cells was used to study filamin-C and 5-HT₂[A] proteins in vivo. U-118MG cells showed staining for 5-HT₂[A] around the membrane of the cell, as well as in the cytoplasm, whereas filamin-C staining occurred in the cytoplasm. Co-localisation analysis identified some areas of overlap between 5-HT₂[A] and filamin-C in the cytoplasm of U-118MG cells. The functional role for the 5-HT₂[A]/filamin-C colocalisation was investigated. It was postulated that filamin-C may be involved in the internalisation of 5-HT₂[A]. To test this hypothesis, an in vivo model system was used to investigate whether disruption of the filamin-C/5-HT₂[A] interaction affects internalisation of the receptor. The key preliminary findings of this study, which used expression of a competitor peptide, to disrupt and co-interact, suggested that the filamin-C/5-HT₂[A] interaction is not essential for the internalisation of receptors in response to ligand binding. However, this interaction was important for delivery or maintenance of 5-HT₂[A] to the cell membrane, and expression of the competing peptide caused an accumulation of cytoplasmic 5-HT₂[A].

The serotonergic system regulates numerous behaviours and disruptions in this system have been associated with disorders of mood and mind. Although molecular genetic analysis has dissected many of the genes involved in the specification of the serotonergic system, relatively little is known about the mechanisms that promote axonal outgrowth from serotonin-producing neurons and how these projections are directed to innervate and form synapses with their appropriate targets. The mouse anorexia mutation causes hypersprouting of serotonergic projections in target fields and has provided us the unique opportunity to examine the crucial events that lead to the establishment of these complex serotonergic networks. Through positional cloning, I have identified a candidate gene that is upregulated during a time in which innervation and synaptogenesis of serotonergic neurons are maximal. I have assessed the expression of this candidate gene in the brain and have found striking differences in the pattern of expression between the normal and the mutant mouse. Furthermore, by using transgenic methods, I have partially rescued several hallmark behavioural phenotypes in the mutant mouse. Thus, this candidate almost certainly represents the “Anorexia” gene.

The serotonergic system regulates numerous behaviours and disruptions in this system have been associated with disorders of mood and mind. Although molecular genetic analysis has dissected many of the genes involved in the specification of the serotonergic system, relatively little is known about the mechanisms that promote axonal outgrowth from serotonin-producing neurons and how these projections are directed to innervate and form synapses with their appropriate targets. The mouse anorexia mutation causes hypersprouting of serotonergic projections in target fields and has provided us the unique opportunity to examine the crucial events that lead to the establishment of these complex serotonergic networks. Through positional cloning, I have identified a candidate gene that is upregulated during a time in which innervation and synaptogenesis of serotonergic neurons are maximal. I have assessed the expression of this candidate gene in the brain and have found striking differences in the pattern of expression between the normal and the mutant mouse. Furthermore, by using transgenic methods, I have partially rescued several hallmark behavioural phenotypes in the mutant mouse. Thus, this candidate almost certainly represents the “Anorexia” gene.

The central nervous system shows sexual dimorphism in the structure and functions in vertebrates. Brain sexual differentiation is resulted from the neural development. The neural development of the brain is influenced by the genetic factors and the external environmental factors. Serotonin (5-HT) functions as a differentiation signal in the early developing period. There are seven 5-HT receptors (5-HT1¡B5-HT2¡B5-HT3¡B5-HT4¡B5-HT5¡B5-HT6¡B5-HT7 receptor) . The 5-HT cell is one of the earliest differentiated neurons in the brain. In the present study, the role of central 5-HT system for the temperature-related neural development was investigated. These results showed that the proliferation of neurons was enhanced by 5-HT. The elevated temperature-induced proliferation of 5-HT cell might be mediated by the 5-HT receptor 1A type. On the other hand, estrogen receptor £\ (ER£\) is involved in the estrogen-enhanced proliferation of 5-HT cell in the neural culture. The ER£\ antagonist suppressed the elevated temperature-induced proliferation of 5-HT cell. It is suggested that the ER£\ plays a role in the mechanism of high temperature-induced the proliferation of 5-HT cell.

Thesis--Ph. D., University of Hong Kong, 1985.

Chronic obstructive pulmonary disease (COPD) is a progressive disease and characterized by persistent airflow limitation. Pathophysiologically it involves many components, including oxidative stress and inflammation of the airways and lungs. Although the primary cause of COPD is smoking, acute exacerbation due to infections can accelerate disease progression, which is a significant cause of morbidity, mortality and burden on healthcare costs. One-half of all acute exacerbations of COPD are associated with bacterial infection, with non-typeable Hemophilus influenzae being the most common pathogen. Staphylococcus pneumonia, one of the most common Gram-positive bacterial pathogens, is also involved in airway infections, either primary or subsequent to viral diseases. To COPD patients the common respiratory pathogens include Gram-negative and Gram-positive bacterial species. Lipopolysaccharide (LPS), a major component of the outer membrane of all Gram-negative bacteria, which is the predominant inducer of inflammatory responses, has been widely studied. On the other hand, less is known about Gram-positive bacteria, which do not contain LPS but express lipoteichoic acid (LTA) as an important proinflammatory constituent in their cell wall. Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that plays an important role in regulating pulmonary function and pathogenesis of inflammation. In this study, we hypothesize that the serotoninergic system may be involved in LPS- and LTA-induced inflammation in COPD. Since monocyte recruitment to lung is a key step in COPD, this study aims to investigate the effects of LPS or LTA alone and in combination of 5-HT pretreatment on the release of pro-inflammatory cytokines in undifferentiated (i.e. monocytes) and differentiated THP-1 cells (i.e. macrophages). LPS, LTA or 5-HT alone induced the release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in both monocytes and macrophages. Our findings also showed that 5-HT pretreatment suppressed the LPS-induced IL-8 and MCP-1 release, suggesting that 5-HT might act as an anti-inflammatory mediator. On the other hand, 5-HT pretreatment enhanced the LTA-induced IL-8 and MCP-1 release, indicating that 5-HT might also act as a pro-inflammatory mediator. These results demonstrate that 5-HT may be involved in the differential modulation of inflammatory processes during Gram-negative or Gram-positive infections in COPD. / published_or_final_version / Medicine / Master / Master of Medical Sciences