Figure 3. Elevated cGMP levels activate... | Rockefeller University Press

Figure 3.

$Elevated cGMP levels activate CFTR and inhibit Nhe3 in vivo. (A) Signaling events regulated by GC-C in the IEC. Phosphorylation of CFTR by PKGII increases chloride and bicarbonate secretion. Phosphorylation of NHE3 inhibits Na+ uptake. Image created using BioRender. (B) Transcript levels of genes downstream of GC-C. Each value represents data from an individual mouse, and the mean ± SD is shown. P values obtained from individual unpaired, two-tailed t tests with Welch’s correction are shown. Data are from seven female mice across two experiments. (C) GE and small intestinal transit after oral gavage with vehicle (TPGS) or CFTR(inh)-172. Values shown are from individual mice, and the mean ± SD is indicated. Two-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was performed, and adjusted P values are shown. Data are from experiments performed twice with a total of eight male and eight female mice of each genotype. (D) GE and small intestinal transit after oral gavage with buffer or linaclotide. Values shown are from individual mice, and the mean ± SD is indicated. Two-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was performed, and adjusted P values are shown. Data are from experiments performed twice with a total of eight male and eight female mice of each genotype. (E) Western blot showing Nhe3 expression in the membrane fraction of ileal (left) and colonic (right) epithelial cells. For samples from the ileum, 150 µg of protein was loaded, and for the colon, 80 µg of protein was loaded. The Nhe3 levels were normalized to villin. Graphs shown below depict densitometric analysis and represent the mean ± SD with data analyzed by an unpaired, two-tailed t test. Data are representative of three female mice. (F) Luminal pH in the ileum and the colon. Values shown are mean ± SD with data from individual mice, and P values shown are from an unpaired, two-tailed t test with Welch’s correction. Data are shown from 17 male and 9 female mice of both genotypes across experiments performed thrice. (G) Luminal sodium levels in the ileum and the colon. Values shown are mean ± SD with data from individual mice, and P values shown are from an unpaired, two-tailed t test with Welch’s correction. Data are from eight male and nine female mice, and experiments were performed twice. (H) Sodium content in fecal pellets collected. Values shown are the mean ± SD with data shown from individual mice, and P values shown are from an unpaired, two-tailed t test with Welch’s correction. Data from wild type mice are from 9 male and 12 female mice and from 12 male and 13 female S839I mice across experiments performed thrice. (I) GE and small intestinal transit after oral gavage with vehicle (TPGS) or the Nhe3 inhibitor (tenapanor). Values shown are from individual mice, and the mean ± SD is indicated. Two-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was performed, and adjusted P values are shown. Experiments were performed twice with a total of eight male and six female mice of each genotype.$

Elevated cGMP levels activate CFTR and inhibit Nhe3 in vivo. (A) Signaling events regulated by GC-C in the IEC. Phosphorylation of CFTR by PKGII increases chloride and bicarbonate secretion. Phosphorylation of NHE3 inhibits Na⁺ uptake. Image created using BioRender. (B) Transcript levels of genes downstream of GC-C. Each value represents data from an individual mouse, and the mean ± SD is shown. P values obtained from individual unpaired, two-tailed t tests with Welch’s correction are shown. Data are from seven female mice across two experiments. (C) GE and small intestinal transit after oral gavage with vehicle (TPGS) or CFTR(inh)-172. Values shown are from individual mice, and the mean ± SD is indicated. Two-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was performed, and adjusted P values are shown. Data are from experiments performed twice with a total of eight male and eight female mice of each genotype. (D) GE and small intestinal transit after oral gavage with buffer or linaclotide. Values shown are from individual mice, and the mean ± SD is indicated. Two-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was performed, and adjusted P values are shown. Data are from experiments performed twice with a total of eight male and eight female mice of each genotype. (E) Western blot showing Nhe3 expression in the membrane fraction of ileal (left) and colonic (right) epithelial cells. For samples from the ileum, 150 µg of protein was loaded, and for the colon, 80 µg of protein was loaded. The Nhe3 levels were normalized to villin. Graphs shown below depict densitometric analysis and represent the mean ± SD with data analyzed by an unpaired, two-tailed t test. Data are representative of three female mice. (F) Luminal pH in the ileum and the colon. Values shown are mean ± SD with data from individual mice, and P values shown are from an unpaired, two-tailed t test with Welch’s correction. Data are shown from 17 male and 9 female mice of both genotypes across experiments performed thrice. (G) Luminal sodium levels in the ileum and the colon. Values shown are mean ± SD with data from individual mice, and P values shown are from an unpaired, two-tailed t test with Welch’s correction. Data are from eight male and nine female mice, and experiments were performed twice. (H) Sodium content in fecal pellets collected. Values shown are the mean ± SD with data shown from individual mice, and P values shown are from an unpaired, two-tailed t test with Welch’s correction. Data from wild type mice are from 9 male and 12 female mice and from 12 male and 13 female S839I mice across experiments performed thrice. (I) GE and small intestinal transit after oral gavage with vehicle (TPGS) or the Nhe3 inhibitor (tenapanor). Values shown are from individual mice, and the mean ± SD is indicated. Two-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was performed, and adjusted P values are shown. Experiments were performed twice with a total of eight male and six female mice of each genotype.

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