Intestinal Neuropod GUCY2C regulates visceral pain

Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options. Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP which regulates luminal fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we reveal that intestinal GUCY2C is selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons, in mice and humans. GUCY2CHigh neuropod cells associate with co-cultured dorsal root ganglia neurons and induce hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient, but not GUCY2C-deficient, neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling and VP that was comparable to chemically-induced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes. Overall design: This data presents and compares transcriptomic profiles of GUCY2C High small intestinal "neuropod cells" to GUCY2C medium small intestinal cells. RNAsequencing was performed on small intestines of 5, 10-week-old litter-matched Gucy2c-GFP mice that were FACS sorted into GUCY2CHigh and GUCY2CMed groups at 20,000 cells per group. mRNA was extracted using Qiagen RNeasy Micro Plus kit. Library preparation, sequencing, alignment, and read counting was performed by the Novogene Corporation (Tianjin, China). Libraries were prepared using NEBNext Ultra™ II RNA Library Prep Kit (New England Biosciences (NEB), Ipswich, MA, E7760). 50 sequencer according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on the same machine and paired-end reads were generated. STAR software was then used to align and map clean reads to the mouse reference genome (mm9). Subsequent RNAsequencing analysis was performed in R (R Foundation for Statistical Computing, Vienna, Austria). For analysis, only genes with counts per million (cpm) >0.5 in at least 3 of the samples were included (15,689 genes). Limma (v3.40.6) and edgeR (v3.26.8) packages were used to determine differential gene expression (adj P value <0.01).