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1.
Figure 7

Figure 7. Response of mucus release in ΔF508 mice.. From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

Mice homozygous for the ΔF508 mutation showed blunted response to stimulation by PGE2 (10–6 M) and 5-HT (10–5 M). The amount of mucus release did not seem to differ between segments with HCO3 (x’s) and those without HCO3 (circles) for stimulation with PGE2 (PAS, n = 5, P = 0.210; lectin, n = 5, P = 0.185) or with 5-HT (PAS, n = 5, P = 0.475; lectin, n = 5, P = 0.434). Data shown are mean ± SEM.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
2.
Figure 1

Figure 1. Effect of secretagogues on mucus release.. From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

Differences between amounts of mucus released in luminal perfusates are shown before (basal) and after stimulation with PGE2 10–6 M (n = 6, P < 0.001) (A), carbachol 10–4 M (n = 5, P = 0.024) (B), and 5-HT 10–5 M (n = 6, P = 0.03) (C). These agonists significantly increased mucus release. On the other hand, isoproterenol 10–5 M did not stimulate mucus release (n = 4, P = 0.9) (D). Data shown are mean ± SEM.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
3.
Figure 8

Figure 8. Effect of luminal HCO3– on mucus release. . From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

In contrast with serosal HCO3, no significant difference in the amount of mucus release was noted when HCO3 was applied to the luminal side (x’s) compared with segments (n = 8) perfused without luminal HCO3 (circles). PGE2 (PAS, n = 8, P = 0.634; lectin, n = 7, P = 0.576) and 5-HT (PAS, n = 8, P = 0.743; lectin, n = 7, P = 0.762) were added sequentially at 30 minutes and at 39 minutes, respectively. Data shown are mean ± SEM.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
4.
Figure 6

Figure 6. Effect of CFTR inhibition on mucus release.. From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

Application of CFTR inhibitor GlyH-101 inhibited mucus release stimulated by either PGE2 (AB) or 5-HT (CD) even in the presence of HCO3 (x’s) compared with control segments without inhibitor (circles). (A) n = 5, P < 0.001. (B) n = 4, P = 0.002. (C) n = 4, P = 0.036. (D) n = 4, P = 0.056. All segments were bathed continuously in HCO3 solution. Samples were assayed by PAS and lectin binding as noted in Figure 2. Data shown are mean ± SEM.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
5.
Figure 3

Figure 3. Effect of DIDS on mucus release.. From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

Compared with control (x’s), DIDS (circles) applied to the serosal solutions to inhibit HCO3 secretion consistently depressed mucus release stimulated by PGE2 (A and B) and by 5-HT (C and D) compared with controls without DIDS. (A) n = 9, P = 0.001. (B) n = 6, P = 0.002. (C) n = 10, P = 0.002. (D) n = 8, P = 0.002. DIDS application reduced mucus release by 50%–70%. All segments were bathed continuously in HCO3 Ringer solution. Samples were assayed by PAS and lectin binding as noted in Figure 2. Data shown are mean ± SEM.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
6.
Figure 9

Figure 9. Perfusion system.. From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

Each end of a segment of intestine was ligated to a fire-polished glass capillary and suspended vertically inside a perfusion chamber made from a 10-cc plastic syringe. The perfusion chamber was sealed coaxially with rubber gaskets at each end to form a water jacket made from a 60-cc plastic syringe. The segment was perfused at constant flow from bottom to top, and samples were collected from the end of a silastic tube whose exit was held at a level at the top of the intestine to maintain a slightly positive pressure inside the segment to ensure luminal patency. Bathing solutions and gas were introduced through ports in the base of the bathing chamber as shown.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
7.
Figure 4

Figure 4. Effect of bumetanide on mucus release.. From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

Application of NKCC inhibitor bumetanide (circles) to the basolateral side to inhibit fluid secretion consistently decreased stimulated mucus release compared with control without inhibitor (x’s). Mucus release was significantly lower with the inhibitor with each agonist as determined by each assay: PGE2 (A and B) and 5-HT (C and D). (A) n = 3, P = 0.041. (B) n = 4, P = 0.003. (C) n = 3, P = 0.039. (D) n = 4, P = 0.036. All segments were bathed continuously in HCO3 Ringer solution. Samples were assayed by PAS and lectin binding as noted in Figure 2. Data shown are mean ± SEM.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
8.
Figure 5

Figure 5. Effect of PGE2 and 5-HT ± HCO3– on fluid movement in isolated intestinal loops. . From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

In the absence of luminal Na+ and Cl, the intestine demonstrated net secretion (fluid gain) during 30 minutes of incubation. When the intestinal sacs were exposed to PGE2 (10–6 M) and 5-HT (10–5 M) simultaneously, fluid secretion increased dramatically (n = 4, ***P = 0.0001). Removal of HCO3 did not diminish basal (n = 4, P = 0.972) or stimulated fluid secretion (n = 4, P = 0.942), nor did application of DIDS decrease fluid secretion (n = 4, P = 0.922) when compared with stimulation in media with HCO3. However, bumetanide notably inhibited stimulated fluid secretion (n = 4). Luminal NaCl was replaced by the impermeable salt NMDG-gluconate to prevent confounding effects from concurrent fluid absorption. Data shown are mean ± SEM.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
9.
Figure 2

Figure 2. Effect of HCO3– on PGE2- and 5-HT–induced mucus release. . From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

At 20 minutes, the bathing solution on one segment of intestine was changed to HCO3 Ringer (x’s), while the other segment remained in HCO3-free Ringer (circles). Application of PGE2 or 5-HT at 30 minutes caused a marked increase in mucus secretion in both groups, but the amount of mucus released was significantly reduced in the absence of HCO3 with either PGE2 (A and B) or 5-HT (C and D). (A) n = 5, P < 0.002. (B) n = 8, P = 0.026. (C) n = 5, P < 0.001. (D) n = 8, P = 0.001. Samples from protocols represented in panels A and C were assayed with PAS; results of lectin binding assays of the same samples are shown in panels B and D. Data shown are mean ± SEM.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
10.
Figure 10

Figure 10. Conceptual arrangement of mucus cells and enterocytes with components of Cl– and HCO3– secretion and corresponding inhibitors. . From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

In general, HCO3 is thought to be taken up across the basolateral membrane mainly via NBC, which is inhibited by DIDS. HCO3 exits the cell across the apical membrane, possibly via a CFTR-dependent Cl/HCO3 exchanger (also DIDS sensitive) or directly through the CFTR anion conductive channel that is inhibited by GlyH-101. Fluid secretion is dependent upon the uptake of Cl via the NKCC in the basolateral membrane, which is inhibited by bumetanide and on CFTR (or other Cl channels not shown) in the apical membrane. Thus, DIDS should block HCO3 secretion, and bumetanide should block fluid secretion. GlyH-101 is expected to inhibit both CFTR-dependent fluid and HCO3 secretion either directly by blocking CFTR conductance or indirectly by inhibiting a CFTR-dependent Cl/HCO3 exchanger (65). The diagram intentionally suggests close proximity of HCO3-secreting enterocytes, with mucus-secreting goblet cells and possibly enterocytes as a means of maintaining ample HCO3 in the immediate environment of secreted mucin granules. In CF, defunct CFTR would starve the environment of HCO3.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
11.
Figure 11

Figure 11. Possible mechanism for HCO3–-dependent expansion and solubilization of granules of condensed mucins. . From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

Intracellularly, mucins are condensed in granules by high concentrations of Ca2+ and H+, which shield the repulsive forces of the high density of negative charges on the anionic sites of mucin glycoproteins. As granules are secreted, released mucin must be accompanied by rapid dissociation of Ca2+ and H+ from the mucin to unshield the fixed negative sites. The repulsive intramolecular electrostatic forces from exposed anionic sites then expand the mucin molecule extensively. Rapid expansion of the mucin molecule may be important to forming the network of macromolecules that become “normal mucus.” Beyond this simplistic effect at the “moment of birth,” however, HCO3 may be critically involved in other, more complicated processes for disaggregation and unraveling of mucin polypeptide domains and/or further interactions with proteins and other moieties. Thus, the presence of a competing anion such as HCO3 in the extracellular media that efficiently sequesters these mucin-bound cations may optimize mucin expansion and intermolecular disaggregation, while its absence may bear heavily on the abnormal forms of aggregated mucus found in CF.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.
12.
Figure 12

Figure 12. Assay validation for mucus content.. From: Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion.

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.

Mary Abigail S. Garcia, et al. J Clin Invest. 2009 September 1;119(9):2613-2622.

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