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Bin, Anna (2008) Metabolismo dell'adenosina: correlazione tra processi biochimici e contrattilità nella muscolatura liscia intestinale in condizioni normali e patologiche. [Tesi di dottorato]

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Abstract (inglese)

In the gastrointestinal tract continued or repeated exposure to pathogens and non-pathogenic antigens may induce activation of epithelial immune system, potentially through dysregulation of barrier functions (Rijnierse et al., 2007). It is well recognized that an inflammatory response, which is largely restricted to the intestinal mucosa, may cause profound changes in the function of smooth muscle and enteric nerves. Some of these changes also occur at remote non-inflamed sites, producing extensive disturbances in gut physiology (Collins et al., 2001).
In the intestine, cyclic AMP has been shown to play a key role in the control of smooth muscle tension. This compound can be extruded from cells upon activation of adenylate ciclase leading to adenosine accumulation via the extracellular cyclic AMP-adenosine pathway (Giron et al., 2008).
In my PhD research work, I first evaluated the mechanical effects of cyclic AMP on rat isolated ileum. Exogenous cyclic AMP concentration-dependently increased intestinal smooth muscle tension. Pretreatment of the preparations with specific inhibitors of ecto- and endo-phosphodiesterases and ecto-5'nucleotidase demonstrated that extracellular cyclic AMP may act on smooth muscle both directly and indirectly through its degradation products. In particular, it was shown that extracellular cyclic AMP-induced ileum contraction was partially inhibited by adenosine inactivation using adenosine deaminase (ADA) or receptor blockade using 8-phenyltheophilline. Moreover, forskolin, that causes endogenous cyclic AMP accumulation, induced concentration-dependent ileum relaxation, supporting distinct roles for the extracellular cyclic AMP-adenosine pathway as opposed to intracellular-generated cyclic AMP. Accordingly, experiments with [3H]-cyclic AMP revealed that [3H]-cyclic AMP uptake in ileum strips was negligible. These findings corroborate the view that cyclic AMP is not only a second messenger but also a source of adenosine.
In vivo treatment of the animals with reserpine affects both contractile effects and metabolic fate of cyclic AMP in the intestine, indicating that the sympathetic system is involved in the modulation of the extracellular cyclic AMP-adenosine pathway.
Considering the significant role of adenosine in regulating intestinal motility, my attention was then focused on CD73 and ecto-ADA, enzymes which directly control extracellular adenosine metabolism. By monitoring the fate of AMP and the generation of adenosine and inosine in the incubation medium of ileum strips, the presence of highly active CD73 and ADA enzymes was demonstrated and the sympathetic system seems to play an important role in regulating these enzymes. The constitutive but regionally distinctive distribution of CD73, ADA and P1 receptors in the intestinal tissue was confirmed by immunoflurescence experiments performed on frozen ileum sections.
In order to understand the functional role of the CD73 and ADA in the smooth muscle, a method for setting up primary cultures of longitudinal intestinal smooth muscle cells (ISMCs) from rat ileum was developed by using the explant technique. Enzyme activity on ISMC surface was investigated by incubation of these cells with exogenous AMP and HPLC detection of nucleotide metabolites. By using specific inhibitors (alpha,beta-methyleneadenosine 5'-diphosphate, concanavalin A, levamisole) CD73 was confirmed to be the only enzyme able to degrade AMP on ISMC surface. On the other hand, high CD73 activity was not followed by activation of ecto-ADA, based on the experiments evaluating the fate of [3H]-cyclic AMP in the culture medium of ISMCs. These experiments also demonstrated that the extracellular cyclic AMP-adenosine pathway is also present in ISMCs. Immunocytochemical analyses confirmed high CD73 expression on the ISMC surface and revealed that ADA and adenosine receptors are primarily localized in the cytosol.
Finally, I evaluated whether the expression of CD73, ADA and P1 receptors in the rat ileum could be affected by an in vivo latent infection in the enteric nervous system induced by Herpes Simplex Virus type 1 (HSV-1). It has been shown that HSV-1 alters intestinal motility and transit, and is currently under evaluation as a potential animal model of gut motility disorders. Immunofluorescence analysis indicated that alterations in the expression of CD73, ADA and P1 receptors were induced not only by HSV-1 latency in the intestine, but also by the progress of infection. In vivo treatment with HSV-1 appears to be responsible for adenosinergic system modifications and intestinal dysmotility, even if histological examination showed no tissue damage.
In conclusion, this PhD thesis highlights the paramount role of CD73, ADA and adenosine receptors in the control of intestinal motility under physiopathological conditions. Furthermore, these proteins should be regarded as potential novel prognostic markers and/or pharmacological targets in neuromuscular abnormalities of the gastrointestinal tract.


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Tipo di EPrint:Tesi di dottorato
Relatore:Gaion, Rosa Maria
Correlatore:Giron, Maria Cecilia
Dottorato (corsi e scuole):Ciclo 20 > Corsi per il 20simo ciclo > FARMACOLOGIA E TOSSICOLOGIA, TERAPIA RIANIMATIVA E INTENSIVA
Data di deposito della tesi:Gennaio 2008
Anno di Pubblicazione:Gennaio 2008
Parole chiave (italiano / inglese):adenosina, AMP ciclico, via extracellulare AMP ciclico-adenosina, muscolatura liscia intestinale, CD73/ecto-5'nucleotidasi, adenosina deaminasi, recettori dell'adenosina, ileo di ratto
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/14 Farmacologia
Struttura di riferimento:Dipartimenti > Dipartimento di Farmacologia ed Anestesiologia "E. Meneghetti"
Codice ID:422
Depositato il:10 Nov 2008
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