Guanylyl cyclase C ameliorates visceral pain: an unsuspected link

Visceral pain associated with irritable bowel syndrome afflicts 15% of the US population. Although treatments are limited, guanylyl cyclase C (GUCY2C) agonists alleviate pain and constipation. Until now, it was assumed that the activation of GUCY2C and production of cGMP in enterocytes stimulated fluid secretion and reduced visceral sensation. The recent discovery that a subtype of enteroendocrine cells (EECs) known as neuropod cells synapse with submucosal neurons unveiled a pathway for communicating gut signals to the nervous system. In this issue of the JCI, Barton et al. report that GUCY2C is enriched in neuropod cells and is involved with sensory nerve firing. Selective deletion of GUCY2C in mouse models suggests that defective GUCY2C neuropod–cell signaling underlies visceral pain. These studies introduce possibilities for dissociating the secretory and analgesic effects of GUCY2C agonism. Although further work remains, unveiling the role of neuropod cells is a major step in understanding visceral pain.

It has been estimated that up to 50% of visits to a gastroenterologist are for evaluation and treatment of unexplained visceral pain, commonly referred to as irritable bowel syndrome (IBS) (1, 2). IBS is a constellation of chronic symptoms including abdominal pain associated with alterations in stool frequency and consistency such as diarrhea and/or constipation (3). The pathophysiological basis of IBS is unknown, but heightened visceral nociception has been demonstrated in clinical investigations (4). Historically, treatments for IBS have focused on symptom management; only recently have specific pharmacological therapeutics been directed at molecular targets to ameliorate symptoms. Among the most notable examples have been drugs that activate the receptor/enzyme guanylyl cyclase C (GUCY2C), a product of the GUCY2C gene on intestinal mucosal cells and stimulate intestinal fluid secretion and motility to treat constipation-predominant IBS. GUCY2C is expressed on the apical surface of enterocytes throughout the intestine, and, upon ligand binding, converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). cGMP increases intestinal fluid secretion by (i) activating cystic fibrosis transmembrane conductance (CFTR) and (ii) inhibiting the apical Na⁺/H⁺ exchanger 3 (NHE3). During the development of GUCY2C agonist drugs — e.g., linaclotide and plecanatide — for the treatment of constipation, it was noted that they also reduced visceral pain in both animal models and humans. Since cGMP is released from enterocytes and cGMP reduces the firing of nociceptive neurons, it was naturally assumed that both the secretory and nociceptive effects of GUCY2C agonists were mediated by enterocytes (Figure 1). However, a problem remained.

Figure 1

An updated model for the regulation of visceral pain involves neuropod GUCY2C. (A) Previously, it was believed that GUCY2C agonists, such as linaclotide and plecanatide, modulated visceral pain by activating GUCY2C on enterocytes to generate cGMP. The prior model proposed that cGMP released from the basal surface of enterocytes diffused to submucosal neurons to diminish neuronal firing. (B) Barton, et al. (12), demonstrated that GUCY2C agonists act on neuropod cells, which are connected to sensory neurons to ameliorate visceral pain by a non-cGMP mechanism.

Although cGMP is indeed released from enterocytes following GUCY2C activation, apical secretion greatly exceeds basolateral release (5). Thus, the concentrations of cGMP in the gut lumen are much higher than in the submucosa where neurons reside. In fact, using microdialysis techniques, one study demonstrated that the concentration of cGMP in the submucosa was four orders of magnitude lower than that required to affect sensory neuron firing (5).

Enteroendocrine cells (EECs) are scattered throughout the gut mucosa with their apical surface exposed to the lumen. In this orientation, EECs detect and respond to ingested nutrients by secreting hormones from their basal surface into the paracellular space, where the hormones are taken up by blood vessels to reach the systemic circulation. Although originally named for their production of hormones, recently, it has been recognized that EECs possess many neuron-like properties (6). When viewed in three dimensions, cholecystokinin-producing (CCK-producing) and peptide YY–producing (PYY-producing) EECs were found to have basal processes that extended to other cells (7). These processes contained axon-like neurofilaments; mitochondria; and both a large, dense core and small, clear secretory vesicles, consistent with hormone and neurotransmitter vesicles, respectively (8). Moreover, these cells expressed both pre and post-synaptic proteins and were found to synapse with submucosal neurons (9). Based on their neuron-like features, these processes were named neuropods. Following this discovery, enterochromaffin cells, a subtype of EECs that secretes serotonin, have also been shown to synapse with neurons (10). Thus, direct EEC-to-neuron synaptic communication seems to be common to many EEC subtypes. The term neuropod cell has been used to refer to EECs that functionally connect with neurons (9, 11).

Capitalizing on these observations, Barton et al., in the current issue of the JCI, reveal how GUCY2C agonists ameliorate visceral pain (12). First, the authors demonstrated that Gucy2c was enriched in a subset of intestinal cells possessing neuropods and that these cells expressed both endocrine and neural markers consistent with neuropod cells, thus providing the initial hint that EECs might be a target for GUCY2C agonists. Using intestinal organoids that contained neuropod cells, the authors demonstrated that sensory neurons isolated from dorsal root ganglia grew toward and connected with GUCY2C enriched cells. In this in vitro model, the GUCY2C agonist linaclotide elevated the DRG-neuron rheobase and reduced action potential firing, whereas cGMP administered to the solution bathing organoids and neurons did not affect excitability. Notably, the effects of linaclotide were not seen in Gucy2c^–/– cells. These findings indicated that neuropod cells mediated the linaclotide effects on sensory neurons through a mechanism that could not be reproduced by exogenous cGMP (12).

Importantly, Gucy2c^–/– mice exhibited spontaneous visceral pain that was refractory to linaclotide. The selectivity of this phenomenon for neuropod cells was demonstrated by deletion of Gucy2c only in CCK cells. These animals also developed spontaneous visceral pain (12).

Thus, GUCY2C agonists ameliorated visceral pain through neuropod cell signaling (12). Not only is this observation important to our understanding of the pharmacology of GUCY2C agonists, but it provides insights into the pathophysiology of visceral pain. Although it has long been suspected that some disturbance in visceral pain signaling and perception was at the root of IBS, a role for EECs was not evident. The discovery of Gucy2c in neuropod cells provided an initial clue, and linking agonist stimulation to a reduction in sensory nerve firing provided mechanistic explanations for both the pain and therapeutic response to GUCY2C-targeted drugs (12). It is interesting to speculate that visceral pain in IBS is due to deficient GUCY2C signaling — either through the lack of sufficient guanylin, and/or uroguanylin (13), or neuropod cell unresponsiveness. These possibilities are important for understanding the fundamental defects underlying unexplained abdominal pain.

Cyclic GMP is the only second messenger signaling pathway known to be activated by GUCY2C. The observation that, in neuropod-connected DRG neurons, action potential firing was suppressed only through GUCY2C activation of neuropod cells and not by exogenous cGMP (12) raises the possibility that cGMP is not the transmitter that reduces DRG firing, and that another, yet unknown, transmitter is involved. Except for L cells that produce both glucagon-like peptides and PYY, when EECs were discovered, the general dictum was that one cell produced one hormone. This concept has since been disproved (14), and it is well accepted that an EEC may express multiple hormones and neurotransmitters (15). In the case of GUCY2C-expressing neuropod cells, identifying this transmitter could unveil another potential target for treating visceral pain.

Although CCK cells are a major EEC type, they represent only a subset of all EECs (16). It is somewhat surprising that deletion of Gucy2c only in CCK cells was sufficient to induce spontaneous visceral pain (12). This finding suggests that small changes in neuropod cell GUCY2C signaling can produce pain. Perhaps this mechanism accounts for the large number of individuals afflicted with IBS.

CCK cells are also concentrated in the proximal small intestine. Even though the colon is a site of IBS pain, GUCY2C agonists exert their pain-reducing effects by targeting cells in the small intestine (17, 18). The current study suggests that the neuropod-DRG connection provides an initial link from the proximal intestine to the spinal cord to modulate visceral pain pathways from the distal bowel (12).

GUCY2C agonists ameliorate constipation and IBS pain; however, IBS has multiple manifestations and is commonly associated with diarrhea. Even the same patient may alternate between constipation and diarrhea. Because of their intestinal secretory effects, linaclotide and plecanatide are not used for diarrhea-predominant IBS. It is not known if the pain associated with diarrhea-predominant IBS has the same pathophysiological basis as constipation-predominant IBS, i.e., neuropod GUCY2C–signaling deficiency. However, based on the effects of linaclotide on visceral pain in mice it is tempting to think GUCY2C agonists could reduce pain in IBS regardless of its association with diarrhea or constipation. The discovery by Barton et al. provides a route to dissociate the effects of GUCY2C on pain from intestinal secretion (12). To avoid activating GUCY2C on enterocytes and worsening diarrhea, an attractive strategy to treat visceral pain would be to selectively target neuropod cells.

The author receives grant support from the NIH (DK120555, DK124474, and DK125308), the Department of Veterans Affairs, and Aligning Science Against Parkinson’s (grant no. 020527) through the Michael J. Fox Foundation for Parkinson’s Research (MJFF).

Address correspondence to: Rodger A. Liddle, 1033A Genome Science Research Building 1, 905 LaSalle Street, Duke University Medical Center, Durham, North Carolina 27710, USA. Phone: 919.681.6380; Email: rodger.liddle@duke.edu.

Conflict of interest: The author is an author for and receives royalty payments from UpToDate.

Copyright: © 2023, Liddle et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License.

Reference information: J Clin Invest. 2023;133(4):e166703. https://doi.org/10.1172/JCI166703.

See the related article at Intestinal neuropod GUCY2C regulates visceral pain.

1. Corsetti M, Whorwell P. The global impact of IBS: time to think about IBS-specific models of care? Therap Adv Gastroenterol. 2017;10(9):727–736.
   View this article via: CrossRef PubMed Google Scholar
2. Black CJ, Ford AC. Global burden of irritable bowel syndrome: trends, predictions and risk factors. Nat Rev Gastroenterol Hepatol. 2020;17(8):473–486.
   View this article via: CrossRef PubMed Google Scholar
3. Drossman DA. Functional gastrointestinal disorders: history, pathophysiology, clinical features and Rome IV. [published online February 19, 2016]. Gastroenterology. https://doi.org/10.1053/j.gastro.2016.02.032.
   View this article via: PubMed Google Scholar
4. Simren M, et al. Cumulative effects of psychologic distress, visceral hypersensitivity, and abnormal transit on patient-reported outcomes in irritable bowel syndrome. Gastroenterology. 2019;157(2):391–402.
   View this article via: CrossRef PubMed Google Scholar
5. Jin XH, et al. Compartmentalization of extracellular cGMP determines absorptive or secretory responses in the rat jejunum. J Clin Invest. 1999;103(2):167–174.
   View this article via: JCI CrossRef PubMed Google Scholar
6. Bohorquez DV, et al. Neuroepithelial circuit formed by innervation of sensory enteroendocrine cells. J Clin Invest. 2015;125(2):782–786.
   View this article via: JCI CrossRef PubMed Google Scholar
7. Bohorquez DV, Liddle RA. Axon-like basal processes in enteroendocrine cells: characteristics and potential targets. Clin Transl Sci. 2011;4(5):387–391.
   View this article via: CrossRef PubMed Google Scholar
8. Bohorquez DV, et al. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy. PLoS One. 2014;9(2):e89881.
   View this article via: CrossRef PubMed Google Scholar
9. Kaelberer MM, et al. A gut-brain neural circuit for nutrient sensory transduction. Science. 2018;361(6408):eaat5236.
   View this article via: CrossRef PubMed Google Scholar
10. Bellono NW, et al. Enterochromaffin cells are gut chemosensors that couple to sensory neural pathways. Cell. 2017;170(1):185–198.
    View this article via: CrossRef PubMed Google Scholar
11. Buchanan KL, et al. The preference for sugar over sweetener depends on a gut sensor cell. Nat Neurosci. 2022;25(2):191–200.
    View this article via: CrossRef PubMed Google Scholar
12. Barton JR, et al. Intestinal neuropod cell GUCY2C regulates visceral pain. J Clin Invest. 2023;132(4):e165578.
13. Waldman SA, et al. Blunted evoked prouroguanylin endocrine secretion in chronic constipation. Clin Transl Gastroenterol. 2019;10(7):e00016.
    View this article via: CrossRef PubMed Google Scholar
14. Egerod KL, et al. A major lineage of enteroendocrine cells coexpress CCK, secretin, GIP, GLP-1, PYY, and neurotensin but not somatostatin. Endocrinology. 2012;153(12):5782–5795.
    View this article via: CrossRef PubMed Google Scholar
15. Haber AL, et al. A single-cell survey of the small intestinal epithelium. Nature. 2017;551(7680):333–339.
    View this article via: CrossRef Google Scholar
16. Gribble FM, Reimann F. Enteroendocrine cells: chemosensors in the intestinal epithelium. Annu Rev Physiol. 2016;78:277–299.
    View this article via: CrossRef PubMed Google Scholar
17. Weinberg DS, et al. Bioactivity of oral linaclotide in human colorectum for cancer chemoprevention. Cancer Prev Res (Phila). 2017;10(6):345–354.
    View this article via: CrossRef PubMed Google Scholar
18. Shailubhai K, et al. Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis. World J Gastrointest Pharmacol Ther. 2015;6(4):213–222.
    View this article via: CrossRef PubMed Google Scholar