DOI
https://doi.org/10.25772/RSZW-W547
Defense Date
2009
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Physiology
First Advisor
John Grider
Abstract
The Endocannabinoid system has been found in the gastrointestinal tract, where it plays an important role in gut under both physiological and pathological conditions. Although the major effects of cannabinoids in the gut are mediated through effects on enteric neurons, the role of cannabinoids in the enteric nervous system is poorly understood. In the present study, we have used the primary cultures of myenteric ganglia and a newly developed fetal enteric neuronal cell line to identify whether the endocannabinoid, anandamide, affects ganglionic and neuronal survival and the pathways involved. Anandamide had a biphasic effect on ganglionic survival, increasing survival at low concentrations (1nM-0.1uM) and decreasing survival at high concentrations (1-10uM). Maximal survival (68% increase in number of ganglia surviving) occurred at 0.1uM and the ED50 was 3nM. This effect on promoting survival was inhibited by the CB1 antagonist AM251 (1uM) and by AraC (10uM), but not the CB2 antagonist AM630 (1uM). AM630 (1uM) significantly blocked the decreased survival induced by high concentration anandamide (10uM). The enteric glia was involved in anandamide-induced ganglion survival. Anandamide had no effect on the number of neurons/ganglion in the presence of enteric glia, but decreased the number of neurons/ganglion by 15-20% in absence of enteric glia. This effect was partially reversed by CB1 antagonist, AM251 (1uM) (20%-145% at 1nM-10uM) and by CB2 antagonist AM630 (1uM) (40%-185% at 1nM-10uM). In the fetal enteric neural cell line (IM-FEN), anandamide decreased enteric neuronal survival in a concentration-dependent manner at both 39 and 33 degree (11-45% and 10-22%decrease in survival at 1nM-10uM, respectively). Coculture of astrocytes with the enteric neuronal cells was not able to reverse anandamide-mediated neuronal death. Immunocytochemistry and western blot confirmed that the presence of both CB1 and CB2 receptors in enteric neurons (primary cultures and IM-FEN) and glia (primary cultures). In addition, the PLC-beta inhibitor U73122 (1uM) inhibited anandamide induced ganglia survival significantly. Anandamide also induced increased expression of phospho-P44/42MAPK (13-48% at 1nM-10uM) and phospho-AKT (1-28% at 1 nM-10uM) in IM-FEN. We conclude that anandamide has a differential effect on survival of enteric ganglia and neurons. It promotes ganglionic and neuronal survival by CB1 receptors in the presence of glia and this involves the PLC-beta pathway. Conversely, anandamide promotes neuron death in absence of glia as a result of effects on both the MAPK and PI-3K/AKT pathways. Since the endocannabinoid system is upregulated in inflammatory bowel diseases, these effects may play a role in the pathogenesis of the response to inflammation as well as the recovery and reinnervation of the gut following the acute phase of inflammation. The further significance of this work could contribute to developing new therapeutic methods for treatment of inflammatory bowel disease and related symptoms in clinic practice.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
December 2009