Anna Bin

Control of enteric neuromuscular functions by purinergic A3 receptors in normal rat distal colon and experimental bowel inflammation

BACKGROUND AND PURPOSE Adenosine A3 receptors mediate beneficial effects in experimental colitis, but their involvement in enteric neuromuscular functions during bowel inflammation is undetermined. This study investigated the regulatory role of A3 receptors on colonic motility in the presence of experimental colitis.

Herpes Simplex Virus Type 1 Infection of the Rat Enteric Nervous System Evokes Small-Bowel Neuromuscular Abnormalities

BACKGROUND & AIMS Infectious agents, such as neurotropic viruses, are proposed to disrupt the enteric neuromuscular system, leading to dysmotility, although the mechanisms are unknown. Our purpose was to assess whether herpes simplex virus type-1 (HSV-1) establishes an enteric-neuronal infection and induces gut dysmotility. METHODS Rats were inoculated with HSV-1 intranasally and after 4 weeks intragastrically. After 1-10 weeks, infection was determined by molecular analysis whereas neuromuscular function was evaluated by pharmacologic/electrical stimulation of longitudinal ileal segments and by gastrointestinal transit and by [(3)H]acetylcholine release measurements. Inflammation in the neuromuscular layer was assessed by myeloperoxidase and cytokine levels and by anti-CD3(+) immunohistochemistry. RESULTS After 1-10 weeks of intragastric inoculation, HSV-1 latency-associated messenger RNA transcripts were detected in the brain and in ileal neurons with no signs of illness or histologic gut abnormalities. By using a recombinant HSV-1 carrying the lacZ gene, HSV-1 virions were localized in myenteric ganglia by in situ X-gal staining. Interleukin-2 and IFN-gamma levels were increased significantly 1 and 6 weeks after inoculation. CD3(+) cells were found around the myenteric ganglia 6 weeks after inoculation. Smooth muscle responses to carbachol, CaCl(2), and gut transit were increased significantly after 1 and 6 weeks, whereas KCl- and electrical field stimulation-mediated contractions were modified significantly only 1-2 weeks after HSV-1 administration. The release of [(3)H]acetylcholine was reduced significantly in ileum segments after 1 and 6 weeks. CONCLUSIONS After intragastric inoculation, HSV-1 establishes a latent infection in the rat myenteric ganglia, which leads to gut dysmotility.

Adenosine-Mediated Enteric Neuromuscular Function Is Affected during Herpes Simplex Virus Type 1 Infection of Rat Enteric Nervous System

Adenosine plays an important role in regulating intestinal motility and inflammatory processes. Previous studies in rodent models have demonstrated that adenosine metabolism and signalling are altered during chronic intestinal inflammatory diseases. However, the involvement of the adenosinergic system in the pathophysiology of gut dysmotility associated to a primary neurodysfunction is still unclear. Recently, we showed that the neurotropic Herpes simplex virus type-1 (HSV-1), orally inoculated to rodents, infects the rat enteric nervous system (ENS) and affects gut motor function without signs of systemic infection. In this study we examined whether changes in purinergic metabolism and signaling occur during permanent HSV-1 infection of rat ENS. Using isolated organ bath assays, we found that contraction mediated by adenosine engagement of A1 or A2A receptors was impaired at 1 and 6 weeks post-viral administration. Immunofluorescence studies revealed that viral infection of ENS led to a marked redistribution of adenosine receptors: A1 and A2B receptors were confined to the muscle layers whereas A2A and A3 receptors were expressed mainly in the myenteric plexus. Viral-induced ENS neurodysfunction influenced adenosine metabolism by increasing adenosine deaminase and CD73 levels in longitudinal muscle-myenteric plexus with no sign of frank inflammation. This study provides the first evidence for involvement of the adenosinergic system during HSV-1 infection of the ENS. As such, this may represent a valid therapeutic target for modulating gut contractility associated to a primary neurodysfunction.

The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

Adenosine is a versatile signaling molecule recognized to physiologically influence gut motor functions. Both the duration and magnitude of adenosine signaling in enteric neuromuscular function depend on its availability, which is regulated by the ecto-enzymes ecto-5′-nucleotidase (CD73), alkaline phosphatase (AP), and ecto-adenosine deaminase (ADA) and by dipyridamole-sensitive equilibrative transporters (ENTs). Our purpose was to assess the involvement of CD73, APs, ecto-ADA in the formation of AMP-derived adenosine in primary cultures of ileal myofibroblasts (IMFs). IMFs were isolated from rat ileum longitudinal muscle segments by means of primary explant technique and identified by immunofluorescence staining for vimentin and α-smooth muscle actin. IMFs confluent monolayers were exposed to exogenous 5′-AMP in the presence or absence of CD73, APs, ecto-ADA, or ENTs inhibitors. The formation of adenosine and its metabolites in the IMFs medium was monitored by high-performance liquid chromatography. The distribution of CD73 and ADA in IMFs was detected by confocal immunocytochemistry and qRT-PCR. Exogenous 5′-AMP was rapidly cleared being almost undetectable after 60-min incubation, while adenosine levels significantly increased. Treatment of IMFs with CD73 inhibitors markedly reduced 5′-AMP clearance whereas ADA blockade or inhibition of both ADA and ENTs prevented adenosine catabolism. By contrast, inhibition of APs did not affect 5′-AMP metabolism. Immunofluorescence staining and qRT-PCR analysis confirmed the expression of CD73 and ADA in IMFs. Overall, our data show that in IMFs an extracellular AMP-adenosine pathway is functionally active and among the different enzymatic pathways regulating extracellular adenosine levels, CD73 and ecto-ADA represent the critical catabolic pathway.