Patients with inflammatory bowel disease (IBD), which afflicts at least one million people in the US, suffer from chronic visceral pain and hypersensitivity that is often poorly managed. The ultimate long-term objective of the proposed project is to elucidate mechanisms of inflammatory visceral hypersensitivity (VH) and its attenuation with the hope of providing new directions for pain treatment in IBD patients. Transient receptor potential vanilloid 4 (TRPV4), which has been implicated in inflammatory hyperalgesia and visceral pain and is upregulated in the colons of IBD patients, is activated by phosphorylation. Agonists of 12-adrenoceptors (AR) have analgesic effects for a variety of pain types, including neuropathic pain and VH. Further, 12-AR activation ultimately leads to a decrease in intracellular kinase activity, thereby reducing protein phosphorylation. The hypothesis of this proposal is that VH induced by colon inflammation is attenuated following 12-AR activation in part through modulation of TRPV4 channels expressed on visceral afferents. This will be the first study to show TRPV4 involvement in a model of IBD and the first study to relate the analgesic effects of 12-ARs to decreased TRPV4 phosphorylation.
Two specific aims are proposed to address the hypothesis: (1) VH associated with colon inflammation correlates with increased TRPV4 expression and phosphorylation in primary visceral afferents and (2) 12-ARs inhibit VH in rats with TNBS-induced colon inflammation partly by modulating TRPV4 phosphorylation.. Colon inflammation will first be induced in adult male rats by intraluminal administration of the hapten 2,4,6-trinitrobenzenesulfonic acid, and VH will be assessed in treated and control rats by measuring visceromotor reflexes (VMR) to colorectal distension (CRD) using electromyographic recording. To address aim 1, TRPV4 protein expression in dorsal root ganglia (DRG) will be determined by Western blot, and phosphorylation will be evaluated by running immunoprecipitation-purified TRPV4 samples on an SDS-PAGE gel and comparing phosphoprotein gel staining to total protein staining. The effect of TRPV4 knockdown on VH will be determined by injecting intervertebrally (L6-S1) either anti-TRPV4 siRNA or mismatch and comparing VMR between siRNA groups. To address aim 2, VMR will be assessed following i.p. administration of clonidine (an 12-AR agonist), and retrograde tracing and immunohistochemistry will be utilized to show co-expression of 12-ARs and TRPV4 in colon-innervating DRG neurons. TRPV4 phosphorylation and protein expression will be evaluated as described above following administration of either clonidine or vehicle to rats with inflammation-induced VH. Finally, calcium imaging will be performed to show the effect of clonidine on TRPV4-mediated calcium influx in DRG from sensitized rats.
More than one million people in the US suffer from inflammatory bowel disease (IBD), which causes chronic pain and hypersensitivity. When adjusted for productivity losses, IBD is estimated to cost more than two billion dollars annually. By revealing mechanisms of pain associated with IBD, in addition to how this pain can be alleviated, this project could contribute to improved treatment options for patients with IBD.
