DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function
Jessica B. Sarthi, Annie M. Trumbull, Shayda M. Abazari, Vincent van Unen, Joshua E. Chan, Yanfen Jiang, Jesse Gammons, Marc O. Anderson, Onur Cil, Calvin J. Kuo, Zachary M. Sellers
Jessica B. Sarthi, Annie M. Trumbull, Shayda M. Abazari, Vincent van Unen, Joshua E. Chan, Yanfen Jiang, Jesse Gammons, Marc O. Anderson, Onur Cil, Calvin J. Kuo, Zachary M. Sellers
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Research Article Gastroenterology

DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function

Abstract

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Authors

Jessica B. Sarthi, Annie M. Trumbull, Shayda M. Abazari, Vincent van Unen, Joshua E. Chan, Yanfen Jiang, Jesse Gammons, Marc O. Anderson, Onur Cil, Calvin J. Kuo, Zachary M. Sellers

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Figure 1

Linaclotide stimulates duodenal bicarbonate secretion in mice and humans.

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Linaclotide stimulates duodenal bicarbonate secretion in mice and humans...
(A) In vivo measurement of linaclotide-stimulated (10–9 M, 10–7 M, 10–5 M) duodenal bicarbonate secretion in C57BL6/J mice (n = 9). After 30-minute baseline measurements, each linaclotide dose was sequentially perfused for 30 minutes. *P < 0.05; **P < 0.01 vs. baseline by 1-way ANOVA. (B) Net peak (max – baseline) bicarbonate secretion. Linaclotide values were from A. For STa (10–9 M, 10–7 M), separate experiments were performed (n = 11). Each point represents a different mouse. (C) In vivo effect of linaclotide (10–9 M, 10–7 M, 10–5 M) with NHE3 inhibitor S3226 (10–5 M) (n = 8). Dotted line is the mean response to linaclotide (from A). *P < 0.05; **P < 0.01 vs. baseline by 1-way ANOVA. (D) Representative trace for in vitro bicarbonate secretion in mouse duodenum. Y axis is titrated HCl, which reflects base secreted by the tissue. During baseline there are few peaks. Upon stimulation, the number of titration events and/or volume of titrant increases. (E) In vitro baseline and linaclotide-stimulated (10–7, 10–5 M, apical) mouse duodenal mucosal bicarbonate secretion. Each point (n = 8–13) is a separate piece of duodenum from 10 mice. *P < 0.05 vs. baseline by 1-way ANOVA. (F) Representative short-circuit current (Isc) trace for in vitro mouse experiments. (G) In vitro baseline and linaclotide-stimulated (10–7, 10–5 M, apical) mouse duodenal Isc. Each point represents a separate piece of duodenum from the same mice in E. ***P < 0.001 vs. baseline by 1-way ANOVA. (H) In vitro transepithelial resistance measurements from E and G. *P < 0.05 vs. baseline by 1-way ANOVA. (I–K) Human duodenal mucosal bicarbonate secretion (I), Isc (J), and transepithelial resistance (K) from endoscopic biopsies. Experiments were performed and data expressed similar as murine experiments in D–H. Each point (n = 9–10) represents a different biopsy. **P < 0.01 vs. baseline by 1-way ANOVA. All data are means ± SEM, unless stated otherwise.