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 3

Key role of DRA in CFTR-independent linaclotide-stimulated duodenal bicarbonate secretion upon loss of CFTR.

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Key role of DRA in CFTR-independent linaclotide-stimulated duodenal bica...
(A and B)To determine the source of linaclotide-stimulated bicarbonate transport in the absence of CFTR function, in vivo experiments were repeated, similar to Figure 2A, except in addition to CFTRinh-172 (2 × 10–5 M, n = 12), one of the following was also added to the luminal perfusate: DRAinh-A250 (10–5 M, n = 10), DIDS (2 × 10–4 M, n = 7), or S3226 (10–5 M, n = 9). (A) Linaclotide 10–7 M, (B) linaclotide 10–5 M. Each point represents a different mouse. *P < 0.05 vs. linaclotide + CFTRinh-172 (2 × 10–5 M) by 1-way ANOVA. (C) Time course with linaclotide dose response (10–9 M, 10–7 M, 10–5 M) in the presence of CFTRinh-172 (2 × 10–5 M) and DRAinh-A250 (10–5 M), as indicated by circles and whiskers (n = 12). Dotted line indicates mean response in the presence of CFTRinh-172 (2 × 10–5 M) only (from Figure 2A). *P < 0.05; **P < 0.01 vs. baseline by 1-way ANOVA. (D) Net peak linaclotide-stimulated (10–7 M, apical) mouse duodenal mucosal bicarbonate secretion (D) and Isc (E) from in vitro experiments, with or without DRA inhibition by DRAinh-A270 (10–5 M, bilateral, n = 5) or DRAinh-4a (10–5 M, bilateral, n = 14). Each point represents a separate piece of duodenum from 5–10 mice. All data are means ± SEM.