The study used STC-1 cells, a mouse intestinal secretion cell line described by the researchers as a stable model of native enteroendocrine cells and a commonly used model for CCK secretion. These cells possess characteristics of enteroendocrine cells, and their secretion is linked to increased intracellular calcium. The team measured transmembrane currents with whole-cell patch clamping and used Fura-2 fluorescence at excitation wavelengths of 340 and 380 nanometres to monitor intracellular calcium. CCK released into the culture medium was quantified by ELISA, while immunofluorescence, Western blotting and RT-qPCR assessed protein expression and CCK messenger RNA. Animal work used male C57BL/6 mice aged 6 to 8 weeks. Mice were randomly assigned to control, wrap restraint stress, TRPV4-blocker, NCX1-blocker or CCK-receptor-blocker groups, and data collection and evaluation were performed blind.

Carbachol, a stable analogue of acetylcholine used to stimulate muscarinic receptors, increased transmembrane current and intracellular calcium in STC-1 cells. The current-voltage relationship had a reversal potential near 0 millivolts, consistent with activation of non-selective cation channels, and the measured half-maximal effective concentration of carbachol for the calcium response was about 15 micromolar. The inositol trisphosphate receptor blocker 2-aminoethyl diphenylborinate at 100 micromolar reduced the carbachol-induced current, while xestospongin C at 100 nanomolar also suppressed the current increase. Pretreatment with 2-aminoethyl diphenylborinate also reduced the rise in intracellular calcium. The Orai1 blocker GSK7975A at 10 micromolar inhibited both the enhanced current and calcium response. Together, these observations support involvement of intracellular-store calcium release through IP3 receptors and Orai1-associated signaling in the response to muscarinic stimulation.

TRPV4 protein was detected in STC-1 cells by immunofluorescence and Western blotting. Two TRPV4 agonists, RN-1747 at 20 micromolar and GSK1016790A at 100 nanomolar, increased non-selective cation current and intracellular calcium. Both effects were suppressed by the TRPV4 blocker HC067047 at 10 micromolar. HC067047 also strongly reduced the membrane current and calcium increase induced by 20 micromolar carbachol. These results establish that TRPV4 is expressed and functionally active in this cell model and that it participates in the cholinergic calcium response. They do not establish TRPV4 as the sole or principal membrane channel responsible for that response.

The investigators likewise found evidence for the involvement of NCX1. Raising extracellular calcium with 5 millimolar CaCl2 produced an increase in membrane current that was inhibited by the reverse-mode NCX1 inhibitors KB-R7943 at 30 micromolar and SN6 at 10 micromolar. NCX1 protein was detected in STC-1 cells by immunofluorescence and Western blotting. In cholinergic-stimulation experiments, both KB-R7943 and SN6 reduced the carbachol-induced non-selective cation current, and pretreatment with either compound reduced the associated calcium signal. The findings indicate that reverse-mode NCX1 participates in the membrane-current and calcium-signaling response to carbachol in STC-1 cells.

Experiments designed to link the two proteins provided further evidence of functional coupling. Activating TRPV4 with either RN-1747 or GSK1016790A increased membrane current and intracellular calcium, while pretreating cells with SN6 suppressed both responses. Co-immunoprecipitation detected an interaction between TRPV4 and NCX1 proteins in STC-1 cells. The electrophysiological and imaging results therefore showed functional interdependence, while the co-immunoprecipitation result provided evidence of physical association. On the basis of these observations, the researchers proposed that TRPV4 and NCX1 form a coupled module that supports the elevated calcium signal and calcium-dependent CCK secretion. The experiments do not define the precise molecular steps of that interaction or demonstrate that the two proteins constitute the only route to hormone release.

The hormone link was tested directly by measuring CCK in STC-1 culture supernatants. Carbachol at 20 micromolar significantly increased CCK secretion relative to the control condition. The carbachol-associated increase was markedly suppressed by the TRPV4 inhibitor HC067047 and by the NCX1 inhibitors KB-R7943 and SN6. Thus, TRPV4 and NCX1 were not associated only with generic changes in membrane current or calcium; their inhibition also reduced a measured secretory response. The cellular results place both proteins upstream of part of the carbachol-dependent CCK response under the conditions tested. They do not directly establish that a stress-induced increase in vagal acetylcholine release drives the same sequence in the intact animals.

In the animal experiment, the investigators applied wrap restraint stress for seven consecutive days to male mice as an IBS-D model. After stress, CCK transcription was significantly higher in the duodenum and colon than in controls. Intraperitoneal HC067047 at 10 milligrams per kilogram, KB-R7943 at 20 milligrams per kilogram and the CCK receptor antagonist devazepide at 1 milligram per kilogram each suppressed the stress-associated increase in these transcripts. The study also examined fecal outcomes. Before restraint, there were no significant differences among groups in fecal number, weight or water content. After restraint, mice had significantly more fecal particles, greater fecal weight and increased fecal water content. Administration of each blocker significantly restored these three measures toward control levels, according to the reported results for groups of six mice.

The study provides experimental support for a TRPV4–NCX1–calcium–CCK pathway in intestinal enteroendocrine cells and associates that pathway with diarrhea-like outcomes in a stress-based mouse model. The authors described TRPV4 and NCX1 as potential targets for preventing or treating IBS-D, but that proposal remains preclinical. The evidence comes from one immortalized intestinal cell line and one established mouse model rather than patients with IBS. Female mice were excluded because hormonal cycles could interfere with the stability of the model, leaving the relevance of the results to female mice unresolved. TRPV4 and NCX1 are also present in other gastrointestinal cell types, and the study did not determine whether enteroendocrine-cell-specific deletion would reproduce or prevent the observed fecal effects. Other enteroendocrine hormones, including GLP-1 and PYY, were not assessed as contributors. Human tissue samples, patient cohorts and clinical outcomes were not studied, so the results offer a testable mechanism for IBS-D biology rather than evidence of a diagnostic, preventive or therapeutic benefit in people.

Subject of Research: TRPV4–NCX1 coupling, calcium-dependent CCK secretion and IBS-D-like diarrhea in male mice

Article Title: TRPV4/NCX1 coupling regulates Ca2+‐dependent cholecystokinin secretion from enteroendocrine cells to involve the pathogenesis of irritable bowel syndrome‐diarrhea in male mice

Article References: Wang, J., Chen, X., Tong, S., Qu, C., Lu, W., & Dong, H. (2026). TRPV4 / NCX1 coupling regulates Ca 2+ ‐dependent cholecystokinin secretion from enteroendocrine cells to involve the pathogenesis of irritable bowel syndrome‐diarrhea in male mice. Physiological Reports, 14(20), Article e71149. https://doi.org/10.14814/phy2.71149

Image Credits: AI Generated

DOI: 10.14814/phy2.71149

Keywords: TRPV4, NCX1, cholecystokinin, enteroendocrine cells, calcium signaling, irritable bowel syndrome, IBS-D, male mice, wrap restraint stress, coupling, regulates, dependent