Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator–dependent bicarbonate secretion
Mary Abigail S. Garcia, Ning Yang, Paul M. Quinton
Mary Abigail S. Garcia, Ning Yang, Paul M. Quinton
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Research Article

Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator–dependent bicarbonate secretion

Abstract

The mechanisms underlying mucus-associated pathologies in cystic fibrosis (CF) remain obscure. However, recent studies indicate that CF transmembrane conductance regulator (CFTR) is required for bicarbonate (HCO3–) transport and that HCO3– is critical for normal mucus formation. We therefore investigated the role of HCO3– in mucus secretion using mouse small intestine segments ex vivo. Basal rates of mucus release in the presence or absence of HCO3– were similar. However, in the absence of HCO3–, mucus release stimulated by either PGE2 or 5-hydroxytryptamine (5-HT) was approximately half that stimulated by these molecules in the presence of HCO3–. Inhibition of HCO3– and fluid transport markedly reduced stimulated mucus release. However, neither absence of HCO3– nor inhibition of HCO3– transport affected fluid secretion rates, indicating that the effect of HCO3– removal on mucus release was not due to decreased fluid secretion. In a mouse model of CF (mice homozygous for the most common human CFTR mutation), intestinal mucus release was minimal when stimulated with either PGE2 or 5-HT in the presence or absence of HCO3–. These data suggest that normal mucus release requires concurrent HCO3– secretion and that the characteristically aggregated mucus observed in mucin-secreting organs in individuals with CF may be a consequence of defective HCO3– transport.

Authors

Mary Abigail S. Garcia, Ning Yang, Paul M. Quinton

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Figure 12

Assay validation for mucus content.

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Assay validation for mucus content. A set of 8 sequential perfusates fro...
A set of 8 sequential perfusates from each of a pair of intestinal segments was collected and assayed by 4 different methods for mucins. One segment was bathed in HCO3–- containing (2.5 × 10–2 M) and the other in HCO3–-free Ringer. Upper panel: results of assays on perfusate samples from the intestinal segment bathed in HCO3–-buffered Ringer and stimulated with PGE2 after collecting perfusate sample no. 4. Lower panel: same as upper panel except the segment was incubated in the absence of HCO3–. Assays: PAS OD (diamonds): soluble PAS-positive material in liquid samples; PAS dot blot (circles): filtrands assayed for PAS-positive material retained on the Immobilon membrane; WGA-HRP (triangles): filtrands assayed for lectin-binding carbohydrates with HRP-tagged WGA lectin; Muc2 antibody (squares): filtrands assayed for Muc2 mucin with Muc2-specific antibody labeled with HRP-tagged goat anti-rabbit second antibody. Since no assay gave the same quantitative amount of substance present as any other assay, results were normalized as a percentage of the maximal concentration for that assay, which consistently appeared shortly after addition of PGE2 in sample no. 6 in all assays and all experiments. In the lower panel, without HCO3–, all maximum values were less than 60% of the maximum values in the upper panel with HCO3– for each type of assay. Thus, independent of the type of assay, the relative amounts of products were similar for all assays. All showed that released product was maximal after PGE2 stimulation.