We confirmed these findings with in situ hybridization ( Figure 3B) and found abundant GC-C expression in the colonic mucosa ( Figure 3C). Although previous studies have also shown GC-C expression localized to specific midbrain neurons, 33 we found that GC-C expression was not detectable in key sensory structures, such as dorsal root ganglia

and spinal cord neurons ( Figure 3D). In order to confirm that inhibition of colonic nociceptors by linaclotide was GC-C dependent, we performed mechanosensitivity studies using GC-C−/− mice. Baseline colonic nociceptor responses were similar to those observed in normal healthy mice; however, the linaclotide-induced inhibition was completely lost ( Figure 4A). Taken together, these data suggest Wortmannin cost that the anti-nociceptive effect of linaclotide is dependent on local activation of GC-C on intestinal epithelial cells. We also show that linaclotide does

not alter colonic muscle contractility, and the membrane permeably 8-bromo-cGMP Natural Product Library does reduce contractility ( Figure 4B). Linaclotide, like other GC-C agonists, elevates intracellular cGMP, which acts as a second messenger in the downstream mediation of intestinal fluid secretion.6, 34 and 35 Linaclotide acts locally with very low systemic bioavailability,34 so is unlikely to activate intestinal nociceptors directly. However, cGMP is released from intestinal epithelial cells upon GC-C activation,9 and 10 and could serve as a downstream mediator for linaclotide-induced effects on colonic nociceptors. In order to further investigate this role of extracellular cGMP, we used a human intestinal Caco-2 cell line, which is known to express GC-C, and stimulated the cells with linaclotide. This stimulation resulted Sodium butyrate in a significant transporter-dependent

basolateral release of cGMP out of the cells, which was concentration-dependently decreased by the cGMP transporter inhibitor, probenecid (Figure 4C). Correspondingly, in colonic nociceptor recordings, linaclotide-induced inhibition of mechanosensitivity ( Figure 4Di) was prevented by probenecid pretreatment ( Figure 4Dii). This finding suggests extracellular cGMP derived from intestinal epithelial cells mediates linaclotide-induced inhibition of colonic nociceptors. To confirm this hypothesis, colonic nociceptor recordings were performed in preparations where the mucosal epithelium had been removed, to abolish the source of GC-C. In these studies, baseline nociceptor mechanosensitivity was normal, however, linaclotide-induced inhibition was significantly diminished in preparations from both healthy ( Figure 4Ei) and CVH mice ( Figure 4Eii).