FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2106598416> ?p ?o ?g. }

• W2106598416 endingPage "4593" @default.
• W2106598416 startingPage "4581" @default.
• W2106598416 abstract "HCO3− is required for gel-forming mucins to form normal mucus, but how? Two apparently separate signalling pathways are activated concurrently to bring mucus formation to completion: a Ca2+-mediated pathway mainly directs goblet cell exocytosis, and an independent cAMP-mediated pathway stimulates HCO3− secretion to help discharge exocytosed mucus. cAMP-dependent HCO3− secretion fails, disrupting the normal formation and discharge of mucins in cystic fibrosis (CF) leading to pathologically viscous and tenacious mucus in affected organs. This work advances our understanding of the role of cAMP (CFTR)-dependent HCO3− secretion in forming normal mucus and underscores a new importance of addressing the defect in HCO3− secretion as a critical new therapeutic target in CF. Abstract Evidence from the pathology in cystic fibrosis (CF) and recent results in vitro indicate that HCO3− is required for gel-forming mucins to form the mucus that protects epithelial surfaces. Mucus formation and release is a complex process that begins with an initial intracellular phase of synthesis, packaging and apical granule exocytosis that is followed by an extracellular phase of mucin swelling, transport and discharge into a lumen. Exactly where HCO3− becomes crucial in these processes is unknown, but we observed that in the presence of HCO3−, stimulating dissected segments of native mouse intestine with 5-hydroxytryptamine (5–HT) and prostaglandin E2 (PGE2) induced goblet cell exocytosis followed by normal mucin discharge in wild-type (WT) intestines. CF intestines that inherently lack cystic fibrosis transmembrane conductance regulator (CFTR)-dependent HCO3− secretion also demonstrated apparently normal goblet cell exocytosis, but in contrast, this was not followed by similar mucin discharge. Moreover, we found that even in the presence of HCO3−, when WT intestines were stimulated only with a Ca2+-mediated agonist (carbachol), exocytosis was followed by poor discharge as with CF intestines. However, when the Ca2+-mediated agonist was combined with a cAMP-mediated agonist (isoproterenol (isoprenaline) or vasoactive intestinal peptide) in the presence of HCO3− both normal exocytosis and normal discharge was observed. These results indicate that normal mucus formation requires concurrent activation of a Ca2+-mediated exocytosis of mucin granules and an independent cAMP-mediated, CFTR-dependent, HCO3− secretion that appears to mainly enhance the extracellular phases of mucus excretion. The hallmark of cystic fibrosis (CF) is aggregated mucosal mucus in pancreas, intestines, liver, airways, reproductive organs and other mucus-producing exocrine glands (Oppenheimer & Esterly, 1975; Nicholson, 2002). In CF-affected organs, no uniquely characteristic changes in mucin composition have been identified (Mantle et al. 1990), and only the gel-forming mucins appear to be directly affected in CF pathology, indicating that a common defect caused by CFTR mutations leads to secondary pathogenic changes in these mucins (Quinton, 2001, 2008; Davis & Dickey, 2008; Garcia et al. 2009). Since the disease is caused by mutations in an ion transport gene (CFTR), the inevitable question remains as to how a defect in electrolyte transport results in abnormal mucus formation? Several prevalent hypotheses have been proposed to link the basic defect to the pathogenic, abnormally thick mucus in CF, but recently, defective CFTR-dependent HCO3− secretion was proposed to disrupt normal mucus gel formation (Quinton, 2001, 2008; Garcia et al. 2009; Chen et al. 2010; Muchekehu & Quinton, 2010; Gustafsson et al. 2012). Evidence of impaired HCO3− secretion has been reported in almost all CF-affected organs such as pancreas (Hadorn, 1968; Gaskin et al. 1982; Kopelman et al. 1988), intestine (Hogan et al. 1997; Seidler et al. 1997; Clarke & Harline, 1998), gallbladder (Cuthbert, 2001), reproductive tract (Chan et al. 2006) and airways (Smith & Welsh, 1992; Coakley et al. 2003). The extent to which HCO3− secretion is defective also appears to correlate with the severity of the CF phenotype (Choi et al. 2001; Reddy & Quinton, 2003). We recently showed that normal mucus formation and release requires the presence of CFTR-dependent HCO3−, probably to sequester Ca2+ from condensed mucins as they expand and disperse (Garcia et al. 2009; Chen et al. 2010; Muchekehu & Quinton, 2010). That is, within goblet cell granules, Ca2+ and H+ cations shield the densely packed fixed anionic sites of mucin molecules (Verdugo et al. 1987a,b). HCO3− probably destabilizes the compact mucins in granules by neutralizing H+ and complexing Ca2+ cations allowing the electrostatic repulsion of unshielded anions to expand the macromolecular mucins into a mature mucus matrix. In CF, the defect in HCO3− transport and consequent lack of HCO3− (and CO32−) appears to limit mucin expansion and impede its swelling, transportability and release into the lumen (Garcia et al. 2009; Chen et al. 2010; Muchekehu & Quinton, 2010; Gustafsson et al. 2012). In the present study, we investigated the effects of cAMP and Ca2+-mediated signalling pathways on mucus production to help determine the steps in mucus formation at which CFTR-dependent HCO3− transport becomes crucial. To help delineate how different signals affect mucus gel formation, we arbitrarily considered the intracellular processes up to the point that the mucin granule content emerges from the goblet cell as ‘exocytosis’, and the subsequent extracellular processes of mucin expansion, matrix maturation, and transport into a lumen as mucin ‘discharge’ (Fig. 1; Quinton, 2010). Exocytosis and discharge of mucus In the present study, we arbitrarily consider the intracellular processes up to the point that the granule content begins to leave the goblet cell at the apical membrane as ‘exocytosis’, and the subsequent extracellular processes of mucin expansion, matrix formation, and transport into a lumen as ‘discharge’. Wild-type (WT) adult C57BL/6 mice (20–26 g) from our breeding colony were maintained on standard laboratory chow and allowed free access to food and water. CFTRtm1Kth mice (CF mice) carrying the most common human CFTR mutation, ΔF508, on a C57BL/6 background were purchased (Case Western Reserve University), bred, and raised in our vivarium. CF mice were maintained on Peptamen Junior (Nestle Nutrition) ad libitum with access to Colyte and pellets. Mice were anaesthetized with ketamine (100 mg kg−1) and xylazine (10 mg kg−1) by subcutaneous injection for deep surgical anaesthesia. When the hind limb flexor withdrawal reflex ceased, 12 cm of ileum proximal to the caecum was quickly excised and the animal was immediately killed by cervical dislocation. The University of California San Diego Institutional Animal Care and Use Committee approved all procedures used in this study. The following solutions were used in all experiments and dissection procedures, excluding the HCO3− secretion measurements. NaCl Ringer (HCO3− free) solutions contained in mm: 150 Na+, 4.6 K+, 1 Ca2+, 1 Mg2+, 150 Cl−, 2.5 PO4x− and 10 mm glucose. Glucose-free NaCl Ringer solution was the same except glucose was replaced by mannitol. For HCO3− Ringer solution, 25 mm Cl− was substituted equimolar with HCO3−. All solutions were adjusted to, and maintained at, pH 7.4 by gassing to equilibrium with either 100% O2 (glucose-free NaCl Ringer and NaCl Ringer solution) or 95% O2/5% CO2 gas (HCO3− Ringer solution) as appropriate during all protocols. The osmolality of all solutions was ∼285 mosmol kg−1. In our previous study, when 25 mm HCO3− was present only in the luminal perfusate, we did not detect an effect on stimulated mucus release (Garciaet al. 2009). Under those conditions, sufficient amounts of perfused luminal HCO3− apparently did not effectively reach the immediate sites of mucus secretion in the lumens of the crypts and possibly in inter-villar spaces of the intestinal wall. However, more recently the properties of the CF mouse ileal mucus appeared to normalize when secreted into a high concentration (∼100 mm) of luminal bicarbonate buffer (Gustafsson et al. 2012). Physiologically, HCO3− must be secreted from the contraluminal compartment by the intestinal epithelium to produce effects on mucins and mucus. Therefore, even though we do not know the concentration of HCO3− secreted during mucus formation, it was always present at 25 mm on the basolateral side of the intestine in the present study. After excision, the ilea were placed in NaCl Ringer solution at room temperature and divided into a proximal and distal half (ca 6 cm each; Cook, 1965), which were assigned alternately as control and experimental segments. Nifedipine (1 μm) and indomethacin (10 μm) were included to prevent peristalsis and reduce endogenous prostaglandin release during tissue handling (Garcia et al. 2009). The lumen of each ileal segment was carefully flushed with cold, oxygenated, glucose-free NaCl Ringer solution to remove residual mucosal contents. The segments were mounted and perfused vertically in a custom-designed perfusion chamber (36 ± 1°C). The perfusates were collected at 5 min intervals for basal measurements while incubating in HCO3− or HCO3−-free (NaCl) Ringer solutions during the initial 20 min of perfusion, and then after beginning the experimental protocols at 3 min intervals for an additional 20–30 min. To quantify the mucus content of the mucosal perfusates, we employed a wheat germ agglutinin–horseradish peroxidase (WGA-HRP) binding assay (lectin dot blot assay) on methanol-activated Immobilon-P film (Millipore, MA, USA) as described previously in detail (Garcia et al. 2009). We estimated the effect of pharmacological agents with or without HCO3− on the loss of mucins from goblet cell stores by histological analysis. As described above, mouse ilea (about 12 cm) were excised and immediately divided into proximal and distal halves. For every experiment, each half was divided into three pieces (ca 2 cm each). One piece was freshly dissected and immediately snap frozen, and the other two pieces were treated according to experimental design, i.e. incubated with an agonist in either NaCl or NaHCO3 Ringer solution. Previously, we found that most of the discharged mucus appeared in the perfusate within the 15 min of stimulation (Garcia et al. 2009). Therefore, in the present study, the tissue was stimulated with agonist in the bath for 15 min. After stimulation, the pieces of ileum were placed in OCT (frozen tissue matrix) and snap frozen between cold aluminum blocks (−80°C). Frozen tissue was sectioned at −20°C to 7 μm thickness and stained with Periodic acid–Schiff reaction (PAS) for carbohydrates. The mucin content of goblet cells, which stained intensely with PAS, was easily distinguished among the far more numerous lightly stained enterocytes. Stimulation diminished the amount of stained mucin content since the number as well as the total area of PAS-stained cells decreased. The ratio of the goblet cells and enterocytes along the villar surface is relatively fixed in similar locations (Kudweis et al. 1989). Therefore, we used the length of the villi to standardize and compare the number and area of the goblet cells. That is, we quantified the extent of goblet cell exocytosis along the villar surface by assessing both the decrease in the number of detectable (PAS+) goblet cells per length of villar surface (GCs mm-1) as well as the decrease in total area of detectable (PAS+) goblet cells per length of villar surface (GA mm-1) in each slide examined (Plaisancie et al. 1998). Both the length of villar surface and total area of goblet cells were measured with MetaMorph (Molecular Devices, Sunnyvale, CA, USA). Nine sections (slides) were chosen randomly for each specimen of intestine. For each condition (freshly dissected, stimulated in NaCl Ringer solution, and stimulated in NaHCO3 Ringer solution), we sampled intestines from three mice. Therefore, 27 sections (slides) were analysed for each experimental condition. We compared changes in GCs mm-1 and GA mm-1 of freshly dissected intestine tissue with tissues stimulated in NaCl Ringer and stimulated in NaHCO3 Ringer in WT and CF mice, respectively. We also compared tissues: (1) freshly dissected (unincubated), (2) incubated in NaCl Ringer without stimulation, and (3) incubated in HCO3− Ringer without stimulation, to confirm that there was no significant difference in GCs mm-1 or GA mm-1 between freshly dissected tissues and unstimulated tissues incubated in NaCl Ringer or in HCO3− Ringer solutions (i.e. for GCs mm-1: freshly dissected vs. NaCl Ringer, P= 0.84; freshly dissected vs. HCO3− Ringer, P= 0.60; for GA mm-1: freshly dissected vs. NaCl Ringer, P= 0.35; freshly dissected vs. HCO3− Ringer, P= 0.12). Observers blinded to the source of the tissues independently performed image analyses for corroboration. Immediately after excision, intact ilea from WT and CF mice were immersed in, and rinsed with, ice-cold oxygenated NaCl Ringer solution; the mesentery was removed, and the ileum was slit open along the insertion of the mesentery and mounted between two lucite half-chambers of a Ussing chamber with an exposed area of 0.1 cm2. Experiments were performed under continuous short-circuited conditions (voltage-current clamp, VCC 600; Physiological Instruments, San Diego, CA, USA). The serosal solution contained the following (in mm): 140 Na+, 5.2 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl−, 25 HCO3−, 2.4 HxPO4x−, and 10 glucose. For the mucosal solution the phosphate buffer was deleted and 25 gluconate and 10 mannitol (in mm) replaced HCO3− and glucose, respectively. The unbuffered mucosal solution was bubbled with 100% O2 and the serosal solution was bubbled with 95% O2/5% CO2 at pH = 7.4. Mucosal pH was maintained at 7.4 by a continuous pH-stat titration of an isosmotic solution containing 0.5 mm HCl (radiometer, Copenhagen). The rate of alkalization (HCO3− secretion) was calculated from the volume/time of the HCl-containing titrant solution required to maintain the mucosal pH at 7.4. Basal parameters were measured for 30 min, after which, selected agonists were added to the serosal compartment of the Ussing chamber. Measurements were recorded at 5 min intervals for the subsequent 20–30 min. The rates of mucosal HCO3− secretion were expressed as micromolar per centimetre squared per hour. Mean basal and peak values for consecutive 15 min periods were averaged, and simulated; HCO3− secretion was determined by subtracting mean basal values from mean peak values after adding an agonist. At the end of each experiment, 25 mm glucose was added to test the activity of the glucose–sodium co-transporter and thereby validate tissue viability. The integrity of specimens was shown by transepithelial hyperpolarization (Seidler et al. 1997). Nifedipine was dissolved in dimethyl sulfoxide (DMSO), while indomethacin and prostaglandin E2 (PGE2) were dissolved in ethyl alcohol and added to serosal solutions as needed. All other agents were dissolved in Ringer solution directly. The final concentrations of DMSO and ethyl alcohol in solutions ranged between 1:1000 and 1:10,000 (vol./vol.). All chemicals were obtained from Sigma-Aldrich. All agonists were applied only to the basolateral side of tissues. Values are expressed as means ± standard error of the mean. Statistical comparisons were made using a two-tailed Student's t test for a single value between two groups: a two-tailed ANOVA test for a single value within three groups and a two-way ANOVA test for the mucus discharge results under different experimental conditions over a given period. A value of P < 0.05 was accepted as significantly different. In our earlier study, we did not detect significant mucus discharge from stimulated CF mouse ilea (Garcia et al. 2009), but the step in the process of mucus formation that fails in CF is not resolved. Therefore, we asked whether mucus formation/maturation in CF was impeded before or during mucin discharge. As a measure of induced exocytosis, we used 5–HT (10 μm) plus PGE2 (1 μm) to obtain maximal mucus secretion as described previously (Garcia et al. 2009), and decreases in the cell number and area of PAS-stained goblet cells. In WT ilea incubated in NaCl Ringer solution and stimulated with PGE2 plus 5–HT for 15 min, both the GCs mm-1 and the GA mm-1 were decreased about 20% compared to freshly dissected and frozen (unincubated) tissue (n= 3 mice, P < 0.05; Fig. 2A and B). Moreover, including HCO3− in the Ringer solution decreased both GCs mm-1 and GA mm-1 even further to about 50% of freshly dissected WT intestines (n= 3 mice, P < 0.001; Fig. 2A and B). These observations indicated that in the presence of HCO3−, stimulated WT goblet cell exocytosis is significantly enhanced. Effects of 5-HT plus PGE2 on goblet cell exocytosis in WT and CF ilea with or without HCO3− A, Periodic acid-Schiff (PAS)-stained frozen sections of unincubated WT ileum (left); PGE2 (1 μm) + 5–HT (10 μm)-stimulated WT ileum in Ringer solution without HCO3− (middle); and PGE2+ 5–HT-stimulated WT ileum in Ringer solution with HCO3− (right; scale bar = 50 μm). B, PGE2+ 5–HT significantly decreased both GCs mm-1 (left) and GA mm-1 (right) compared with the unincubated WT ilea. HCO3− significantly further enhanced 5–HT + PGE2-induced goblet cell exocytosis in WT ilea (n= 3 mice, means ± SEM.) C, Periodic acid-Schiff (PAS)-stained frozen sections of freshly unincubated CF ileum (left), PGE2 (1 μm) + 5–HT (10 μm)-stimulated CF ileum in Ringer solution without HCO3− (middle), and PGE2+ 5–HT-stimulated CF ileum in Ringer solution with HCO3− (right; scale bar = 50 μm). D, PGE2+ 5–HT still significantly decreased both GCs mm-1 (left) and GA mm-1 (right) compared with unincubated CF ilea, whereas HCO3− in the bath solution did not enhance 5–HT- and PGE2-induced CF goblet cell exocytosis (n= 3 mice, means ± SEM; NS, no significant difference; P > 0.05). Arrows indicate the PAS-stained goblet cells. GCs mm-1, goblet cell number per length of villar surface; GA mm-1, total goblet cell area per length of villar surface. Inherently, the GCs mm-1 and GA mm-1 were significantly larger in CF than in WT mouse ilea (Fig. 2C and D). As with WT, stimulation with 5–HT plus PGE2 also significantly reduced the number and total area of goblet cells by about 1/3 in CF tissues incubated in NaCl Ringer solution, GCs mm-1 decreased by 28.6% (n= 3 mice, P < 0.001) and GA mm-1 decreased by 36% compared to freshly dissected CF ilea (n= 3 mice, P < 0.001; Fig. 2C and D). But, in contrast to WT, the presence of HCO3− did not enhance goblet cell exocytosis in CF (n= 3 mice, P > 0.05; Fig. 2C and D). These results indicate that CF goblet cells, like normal goblet cells, exocytosed in response to stimulation; however, exocytosed CF mucus does not competently discharge into the lumen since comparatively little mucus was detected in the mucosal perfusates of CF mouse intestines or cervices stimulated similarly (Garcia et al. 2009; Muchekehu & Quinton, 2010). To confirm that 5–HT and PGE2-induced HCO3− secretion in WT mouse small intestine is CFTR dependent, we compared HCO3− secretion in CF ilea lacking CFTR with that of WT ilea. Similar to previous reports (Seidler et al. 1997; Clarke & Harline, 1998), 5–HT plus PGE2 induced an increase in both short circuit current (Isc) and HCO3− secretion in WT, but not in CF ileum (Fig. 3A and B). The basal HCO3− secretion in WT (3.22 ± 0.29, n= 12) was significantly higher than the basal HCO3− secretion in CF (0.99 ± 0.20, n= 11, P < 0.0001). Effects of 5-HT plus PGE2 on short circuit current and HCO3− secretion in WT and CF ilea A, summary of stimulated short circuit current (Isc) in WT and CF ilea. 5–HT (10 μm) plus PGE2 (1 μm) induced an increase in short circuit current in WT ilea (continuous line; n= 7), but not in CF ilea (dashed line; n= 7). B, summary of stimulated HCO3− secretion in WT and CF ilea. 5–HT plus PGE2 induced significant HCO3− secretion in WT ilea (0.72 ± 0.24, n= 6) but not in CF ilea (−0.22 ± 0.20, n= 4). Stimulation with PGE2 plus 5–HT involves both cAMP and Ca2+-mediated signalling pathways (Bonventre & Swidler, 1988; Cooke, 2000; Ning et al. 2004) with the activation of CFTR mainly associated with a cAMP pathway (Anderson et al. 1991; Quinton & Reddy, 1994). To better determine where CFTR-dependent HCO3− secretion contributes in the process of mucus formation and release, we attempted to dissect the effects of Ca2+-mediated simulation from the effects of cAMP-mediated simulation on mucin exocytosis, mucus discharge, and HCO3− secretion. To examine Ca2+-mediated effects, we stimulated the ilea with carbachol (CCH, 100 μm), a cholinesterase-insensitive analogue of the well-established Ca2+-mediated agonist acetylcholine (Fischer et al. 1992). Stimulation with CCH for 15 min significantly reduced detectable (PAS+) goblet cell number (GCs mm-1) and total area of detectable (PAS+) goblet cells (GA mm-1) of WT ileum in NaCl Ringer solution, similar to the effects of 5–HT plus PGE2, i.e by 38% and 32%, respectively, compared to freshly isolated tissue (n= 3 mice, P < 0.001; Fig. 4A and B). However, CCH-stimulated exocytosis in tissues incubated in HCO3− Ringer solution did not increase compared to tissues incubated in NaCl Ringer solution (n= 3 mice; P > 0.05; Fig. 4A and B). In contrast to 5–HT and PGE2, CCH induced only a modest mucus discharge in NaCl Ringer solution, which was similar to mucus discharged from ilea bathed in HCO3− Ringer solution (n= 5, P > 0.5; Fig. 4C). When we assayed for effects on HCO3− secretion, we found that CCH induced a sharp, transient increase in short circuit current without significantly stimulating HCO3− secretion (Fig. 4D). These results indicate that Ca2+-mediated stimulation mainly induces goblet cell mucin exocytosis without stimulating significant HCO3− secretion. Effects of carbachol (CCH) on goblet cell exocytosis, mucus discharge and HCO3− secretion in WT mouse ilea A, Periodic acid-Schiff (PAS)-stained frozen sections of unincubated WT ileum (left), CCH (100 μm)-stimulated WT ileum in Ringer solution without HCO3− (middle) and CCH-stimulated WT ileum in Ringer solution with HCO3− (right; scale bar = 50 μm); Arrows indicate the PAS-stained goblet cells. B, CCH decreased GCs mm-1 (left), and GA mm-1 (right) in WT ilea. HCO3− in the bath solution did not enhance CCH-induced exocytosis (n= 3 mice; mean ± SEM; NS, no significance, P > 0.05). C, comparison of CCH-stimulated mucus discharge with and without HCO3− in the bath solution (left). Comparison of CCH, 5–HT (10 μm) and PGE2 (1 μm)-induced mucus discharge with and without HCO3− in the bath solution (right). Unlike the results of 5–HT or PGE2, there is no significant increase in CCH-induced mucus discharged with HCO3− compared to no HCO3− in the bath. Samples were assayed by lectin binding dot blot (n= 5; P= 0.32). D, CCH induced a sharp and transient increase in short circuit current (left, n= 7); CCH did not increase HCO3− secretion compared to unstimulated control (right, n= 5; mean ± SEM; NS, not significant; P= 0.99). To determine the role of cAMP signalling on goblet cell exocytosis, mucus discharge and HCO3− secretion, we stimulated WT ilea with cAMP-mediated agonist isoproterenol (ISP), and confirmed with another cAMP-mediated agonist, vasoactive intestinal peptide (VIP). Neither ISP (10 μm) nor VIP (100 nm) alone induced significant goblet cell exocytosis, even in the presence of HCO3− (Fig. 5A and B), which was consistent with the results that ISP and VIP did not induce significant mucus discharge from ilea incubated in HCO3− Ringer solution either (n= 4, Fig. 5C). However, ISP and VIP stimulated marked increases in short circuit current and HCO3− secretion, which was not increased by adding carbachol (Fig. 5D). These results show that the effects of cAMP are mainly on HCO3− secretion and apparently occur independently of stimulating goblet cell mucin exocytosis. Effects of isoproterenol (ISP) and vasoactive intestinal peptide (VIP) on goblet cell exocytosis, mucus discharge, and HCO3− secretion in WT mouse ilea Neither ISP (10 μm; A) nor VIP (100 nm; B) significantly reduced GCs mm-1 (left) or GA mm-1 (right) even in HCO3− Ringer solution (n= 3; mean ± SEM; NS, not significant, P > 0.05). C, neither ISP (10 μm) nor VIP (100 nm) induced mucus discharge even in HCO3− Ringer solution (P= 0.13, n= 4). D, left: both ISP (10 μm) and VIP (100 nm) significantly increased short circuit current (n= 6). Right: both ISP and VIP significantly increased HCO3− secretion (n= 6), but adding CCH (100 μm) did not enhance ISP- (n= 4) or VIP- (n= 6) stimulated HCO3− secretion (mean ± SEM; NS, not significant; ISP vs. ISP + CCH, P= 0.98; VIP vs. VIP + CCH, P= 0.85). cAMP- and Ca2+-mediated stimulation appear to have distinct roles in mucus formation and release, i.e. Ca2+ mainly involves activating goblet cell exocytosis, while cAMP mainly activates CFTR-dependent HCO3− secretion. We surmised that the effect of cAMP-activated CFTR-dependent HCO3− secretion must occur during the discharge process independently of the exocytosis process. We tested this notion by combining either ISP or VIP with CCH to stimulate mucus discharge in the presence and absence of HCO3−. We found that both VIP and ISP significantly augmented the CCH-simulated mucus discharge only when HCO3− was present in the bath solution (n= 4–5, Fig. 6A and B), but as shown above (Fig. 5D), CCH did not enhance ISP/VIP-stimulated HCO3− secretion. These observations indicate that Ca2+-mediated mucin exocytosis is relatively independent of cAMP-mediated HCO3− secretion, but competent mucus discharge is highly dependent on concurrent cAMP-mediated HCO3− secretion. Effects of isoproterenol (ISP) and vasoactive intestinal peptide (VIP) on carbachol (CCH)-induced mucus discharge with or without HCO3− in WT mouse ilea A, both ISP (10 μm) and VIP (100 nm) significantly enhanced CCH (100 μm)-induced mucus discharge when HCO3− was present in bath solutions. B, neither ISP nor VIP significantly increased CCH-induced mucus discharge when HCO3− was absent from bath solutions. Samples were assayed by lectin binding dot blot (means ± SEM; n= 4–5; CCH with HCO3− vs. CCH + ISP with HCO3−, P= 0.03; CCH with HCO3− vs. CCH + VIP with HCO3−, P < 0.0001; CCH No HCO3− vs. CCH + ISP No HCO3−, P= 0.90; CCH No HCO3− vs. CCH + VIP No HCO3−, P= 0.91). Although viscous, adherent mucus is pivotal in the development of cystic fibrosis pathology, how mutations in the CFTR gene lead to pathogenic mucus accumulation is only beginning to be understood. Over many years, several hypotheses have been proposed to explain the aetiology of abnormal mucus in CF, but we have observed that normal gel mucus release requires CFTR-dependent HCO3− secretion (Garcia et al. 2009; Muchekehu & Quinton, 2010). HCO3− may sequester Ca2+ from the mucin granule matrix to induce mucin gel dispersion and formation (Chen et al. 2010; Ambort et al. 2012). Gustafsson et al. (2012) very recently found that CF intestinal mucus appeared to form more normally and was less viscous when secreted into solutions of high HCO3− concentrations. Here, we have further examined whether HCO3− secretion and mucus exocytosis are independent processes under separate regulatory controls. Our results showed that with HCO3− present, 5–HT plus PGE2 stimulation induced goblet cell exocytosis, mucus discharge and HCO3− secretion in WT mouse small intestines, but that in CF intestines only exocytosis appeared to occur, suggesting that the intracellular mucin processes in CF goblet cells preceding exocytosis are probably not significantly impaired. Consequently, the abnormal mucus associated with the CFTR defect seems to occur mainly during mucus ‘discharge’ that depends critically on HCO3− secretion. As reported earlier, we used 5–HT and PGE2 to induce robust mucus secretion (Garcia et al. 2009) and stimulate CFTR-dependent HCO3− secretion in mouse small intestine. 5–HT and PGE2 are known to stimulate via both cAMP- and Ca2+-dependent pathways (Bonventre & Swidler, 1988; Cooke, 2000; Ning et al. 2004) as observed in this study. Thus, we attempted to dissect the cAMP-dependent CFTR effects from the Ca2+-mediated effects. Elevation of intracellular Ca2+ has been regarded as a key signal for exocytosis in most exocrine, endocrine and neuronal cells. Modulatory effects of cAMP pathways on Ca2+-induced mucus formation and release have been reported for antral mucous cells and pancreatic ducts (Jung et al. 2010). Nakahari, et al. (2002) found that cAMP accumulation increased the number of primed granules and the sensitivity of granule fusion to Ca2+, which potentiated Ca2+-mediated exocytosis in antral mucous cells (Nakahari et al. 2002). Jung et al. (2010) also reported that Ca2+-dependent exocytosis in cultured pancreatic duct epithelial cells (PDECs) was synergistically amplified by co-activation of cAMP-mediated signalling. However, increased cAMP did not modify the intensity or pattern of Ca2+ influx. Moreover, direct imaging of secretory granules did not reveal any effect of cAMP on the trafficking of these granules to the plasma membrane either, suggesting that the key influence(s) of cAMP may be on processes that take place at or very close to the plasma membrane (Jung et al. 2010). In the present study, when mucin exocytosis is stimulated, the effect of cAMP on mucus discharge seems more likely to be due to concurrent, but separate CFTR-dependent HCO3− secretion based on the following findings: (1) cAMP-mediated stimulation significantly enhanced CCH-stimulated (Ca2+-mediated) mucus discharge only in the presence of HCO3−, indicating that both cAMP signalling and HCO3− are essential for normal mucus ‘discharge’ (Fig. 6), (2) cAMP-mediated HCO3− secretion is CFTR dependent in the mouse ileum (Seidler et al. 1997; Clarke & Harline, 1998), (3) cAMP-mediated agonists by themselves did not induce mucus exocytosis or discharge (Fig. 5), and (4) in CF intestine, goblet cells exocytosed, but mucus discharge was compromised and incomplete even when stimulated in the presence of HCO3− (Fig. 2). These results strongly suggest that concurrent CFTR-dependent HCO3− secretion into the lumen is critical for exocytosed mucins to be ‘discharged’ normally. Within intracellular mucin granules, high concentrations of Ca2+ and H+ cations shield the high density of fixed anionic sites on the oligosaccharides that surround the protein cores of mucin monomers (Verdugo et al. 1987a,b; Perez-Vilar, 2007). During exocytosis, HCO3− is needed to sequester and remove high intragranular [Ca2+] and [H+] to allow normal mucin expansion and discharge (Garcia et al. 2009; Chen et al. 2010; Muchekehu & Quinton, 2010; Gustafsson et al. 2012). In CF, cAMP-activated CFTR-dependent HCO3− secretion is impaired because of dysfunctional or absent CFTR. However, as observed here, the Ca2+-mediated exocytosis process appears to be intact in CF. Thus, exocytosed mucins apparently fail to expand normally when HCO3− is not present in the extracellular fluid into which they are exocytosed, leaving poorly expanded, aggregated mucus adherent to the mucosal surface (Garcia, 2009; Muchekehu & Quinton, 2010; Gustafsson et al. 2012). CF airway submucosal glands appear to retain mucus (Jayaraman et al. 2001; Joo et al. 2002), and the synergistic effects of cAMP on Ca2+-mediated secretion are also absent in these glands (Choi et al. 2007), likewise indicating that cAMP-dependent HCO3− secretion is generally required to discharge Ca2+-mediated exocytosed mucins. Concurrent stimulation of HCO3− secretion may well be an inherent property of exocrine organs that discharge gel-forming mucins. Earlier we proposed that during mucin release the source of HCO3− was most likely to be the enterocytes surrounding the goblet cells in the epithelium (Garcia et al. 2009). In this study, we show that mucin exocytosis from goblet cells is principally mediated by Ca2+. However, in WT intestine stimulated with 5–HT plus PGE2 (mediating both Ca2+ and cAMP signalling), somewhat more goblet cell exocytosis was observed with HCO3− present in the medium than without HCO3− (Fig. 2B). This enhancement was not seen in CF goblet cells (Fig. 2D), suggesting that goblet cell intracellular processes leading to exocytosis might partially depend on HCO3− and CFTR. Presently, the localization of CFTR in goblet cells remains controversial. Some investigators have reported CFTR immunolocalization within gastrointestinal goblet cells from humans (Kalin et al. 1999), pigs (Hayden & Carey, 1996), and mice (Hayden & Carey, 1996; Kuver et al. 2000), while others reported that CFTR is only expressed in neighbouring fluid-transporting epithelial cells (Jakab et al. 2011). Thus, aberrant mucus formation would be secondary to defective HCO3− secretion in enterocytes (Jakab et al. 2011). Yu et al. (2010) reported that a candidate Ca2+-activated Cl− channel protein in the basal membrane, bestrophin–2, may function in concert with a Cl−/HCO3− exchanger localized in the goblet cell apical membrane for transcellular HCO3− transport, which might be regulated by external Cl− supplied by CFTR in adjacent enterocytes (Yu et al. 2010). We cannot exclude the possibility that HCO3− may enter goblet cells and participate in initiating mucin granule exocytosis. Thus, the existence and role of CFTR in goblet cells requires further clarification. Notably, in CF, mucus obstruction in the intestinal tract always occurs in the distal small intestine, not in the colon or duodenum, which suggests that different mechanisms or transport systems for HCO3− may exist in the distal small intestine vs. the colon or duodenum. Besides CFTR, Cl−/HCO3− exchangers, short-chain fatty acid/HCO3− exchange, and basolateral HCO3− transporters also exist in the intestine (Nyberg et al. 1998; Talbot & Lytle, 2010; Bachmann & Seidler, 2011). In a recent study (Xiao et al. 2012), the expression of even small amounts of F508del-CFTR in the brush border membrane significantly enhanced the forskolin-induced HCO3− secretion response in murine duodenum and mid-colon. The secretion of HCO3− was dependent on luminal Cl− in both intestinal segments, and therefore, probably due to Cl−/HCO3− exchange. The results also indicate that Cl−/HCO3− exchangers involve the HCO3− secretion in the duodenum and colon of CF mice, while the present study shows that in the mouse small intestine, CFTR may be the only, or at least the predominant, pathway for HCO3− transport associated with mucus formation in the ileum and that the level of spontaneous, exogenously unstimulated HCO3− secretion in CF ileal tissue is comparatively quite small. Taken together, these results may help explain the pathophysiology of the unique predisposition of distal small intestinal obstruction symptoms in CF mice and patients. In summary, our results in mouse ileal intestine show that two distinct signalling pathways are required for normal mucus formation: a Ca2+-mediated pathway appears mainly to induce goblet cell mucin exocytosis, while an independent cAMP-mediated pathway must concurrently stimulate HCO3− secretion, most likely in separate surrounding enterocytes. Physiologically, CFTR (cAMP)-dependent HCO3− secretion potentiates exocytosed mucin discharge in WT small intestine. However, in cystic fibrosis, cAMP-dependent HCO3− secretion, but not Ca2+-mediated exocytosis, fails and disrupts the normal formation and discharge of mucus forming pathologically more viscous and tenacious mucus in affected organs. The authors have no conflicts of interest to disclose. The experiments were carried out at the University of California, San Diego. N. Y. designed and performed the experiments, analysed and interpreted the data and drafted and revised the manuscript. M.A.S.G. performed some of the Ussing-chamber and pH-stat experiments. P.M.Q. conceptually designed the experiments, critically interpreted the data and critically revised the manuscript. All authors approved the final version of the manuscript. This work was supported by an Elizabeth Nash Memorial Fellowship, CFRI. to N.Y.; MCCC-Cystic Fibrosis Foundation; NIH-RO1 HL084042; Nancy Olmsted Endowment; Jon I. Isenberg Endowed Fellowship to N.Y. The authors thank Dr Hui Dong for the use of his pH stat equipment and his helpful discussions. The authors also thank Kirk Taylor for technical support and thank Dr Guillermo Flores and Laura Molyneux for performing the blinded image analyses. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
• W2106598416 created "2016-06-24" @default.
• W2106598416 creator A5008073510 @default.
• W2106598416 creator A5073519974 @default.
• W2106598416 creator A5079815759 @default.
• W2106598416 date "2013-07-25" @default.
• W2106598416 modified "2023-10-16" @default.
• W2106598416 title "Normal mucus formation requires cAMP-dependent HCO₃⁻secretion and Ca²⁺-mediated mucin exocytosis" @default.
• W2106598416 cites W1493205354 @default.
• W2106598416 cites W1536163743 @default.
• W2106598416 cites W1587143357 @default.
• W2106598416 cites W1651780926 @default.
• W2106598416 cites W1968784553 @default.
• W2106598416 cites W1977487169 @default.
• W2106598416 cites W1982071077 @default.
• W2106598416 cites W1983114544 @default.
• W2106598416 cites W1997783558 @default.
• W2106598416 cites W1997835226 @default.
• W2106598416 cites W2001200618 @default.
• W2106598416 cites W2005269914 @default.
• W2106598416 cites W2013290065 @default.
• W2106598416 cites W2053648906 @default.
• W2106598416 cites W2053875062 @default.
• W2106598416 cites W2066235646 @default.
• W2106598416 cites W2066362211 @default.
• W2106598416 cites W2068812994 @default.
• W2106598416 cites W2071857701 @default.
• W2106598416 cites W2079004999 @default.
• W2106598416 cites W2085700147 @default.
• W2106598416 cites W2086061439 @default.
• W2106598416 cites W2087130195 @default.
• W2106598416 cites W2087645348 @default.
• W2106598416 cites W2090601537 @default.
• W2106598416 cites W2098660493 @default.
• W2106598416 cites W2101710638 @default.
• W2106598416 cites W2113830910 @default.
• W2106598416 cites W2115476038 @default.
• W2106598416 cites W2127920211 @default.
• W2106598416 cites W2133002738 @default.
• W2106598416 cites W2137012287 @default.
• W2106598416 cites W2142781849 @default.
• W2106598416 cites W2144005516 @default.
• W2106598416 cites W2148222883 @default.
• W2106598416 cites W2153835366 @default.
• W2106598416 cites W2155254872 @default.
• W2106598416 cites W2163987523 @default.
• W2106598416 cites W2397862844 @default.
• W2106598416 cites W2416999119 @default.
• W2106598416 cites W2419516382 @default.
• W2106598416 cites W384010788 @default.
• W2106598416 doi "https://doi.org/10.1113/jphysiol.2013.257436" @default.
• W2106598416 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3784200" @default.
• W2106598416 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23818690" @default.
• W2106598416 hasPublicationYear "2013" @default.
• W2106598416 type Work @default.
• W2106598416 sameAs 2106598416 @default.
• W2106598416 citedByCount "75" @default.
• W2106598416 countsByYear W21065984162013 @default.
• W2106598416 countsByYear W21065984162014 @default.
• W2106598416 countsByYear W21065984162015 @default.
• W2106598416 countsByYear W21065984162016 @default.
• W2106598416 countsByYear W21065984162017 @default.
• W2106598416 countsByYear W21065984162018 @default.
• W2106598416 countsByYear W21065984162019 @default.
• W2106598416 countsByYear W21065984162020 @default.
• W2106598416 countsByYear W21065984162021 @default.
• W2106598416 countsByYear W21065984162022 @default.
• W2106598416 countsByYear W21065984162023 @default.
• W2106598416 crossrefType "journal-article" @default.
• W2106598416 hasAuthorship W2106598416A5008073510 @default.
• W2106598416 hasAuthorship W2106598416A5073519974 @default.
• W2106598416 hasAuthorship W2106598416A5079815759 @default.
• W2106598416 hasBestOaLocation W21065984162 @default.
• W2106598416 hasConcept C12554922 @default.
• W2106598416 hasConcept C175969161 @default.
• W2106598416 hasConcept C179264091 @default.
• W2106598416 hasConcept C185592680 @default.
• W2106598416 hasConcept C18903297 @default.
• W2106598416 hasConcept C2777312293 @default.
• W2106598416 hasConcept C49039625 @default.
• W2106598416 hasConcept C55493867 @default.
• W2106598416 hasConcept C86803240 @default.
• W2106598416 hasConceptScore W2106598416C12554922 @default.
• W2106598416 hasConceptScore W2106598416C175969161 @default.
• W2106598416 hasConceptScore W2106598416C179264091 @default.
• W2106598416 hasConceptScore W2106598416C185592680 @default.
• W2106598416 hasConceptScore W2106598416C18903297 @default.
• W2106598416 hasConceptScore W2106598416C2777312293 @default.
• W2106598416 hasConceptScore W2106598416C49039625 @default.
• W2106598416 hasConceptScore W2106598416C55493867 @default.
• W2106598416 hasConceptScore W2106598416C86803240 @default.
• W2106598416 hasIssue "18" @default.
• W2106598416 hasLocation W21065984161 @default.
• W2106598416 hasLocation W21065984162 @default.
• W2106598416 hasLocation W21065984163 @default.
• W2106598416 hasLocation W21065984164 @default.
• W2106598416 hasOpenAccess W2106598416 @default.
• W2106598416 hasPrimaryLocation W21065984161 @default.

• next