Lubiprostone stimulates small intestinal mucin release
© De Lisle; licensee BioMed Central Ltd. 2012
Received: 8 May 2012
Accepted: 26 October 2012
Published: 6 November 2012
Lubiprostone is a synthetic bicyclic fatty acid derivative of prostaglandin E1 (PGE1) used for chronic constipation. The best known action of lubiprostone is simulation of Cl- dependent fluid secretion. In a mouse model of the genetic disease cystic fibrosis, we previously showed that in vivo administration of lubiprostone resulted in greater mucus accumulation in the small intestine. The aim of this study was to directly test whether lubiprostone stimulates intestinal mucin release.
Mucin release was measured by mounting segments (4-5 cm) of mouse proximal-mid small intestine in an organ bath, allowing access to the perfusate (luminal) and the bath (serosal) solutions. Nifedipine (10-6 M) and indomethacin (10-5 M) were included in all solutions to inhibit smooth muscle activity and endogenous prostaglandin production, respectively. The tissue was equilibrated under flow for 30 min, using the perfusate collected during the final 10 min of the equilibration period to measure unstimulated release rate. Stimulus was then added to either the perfusate or the bath and the perfusate was collected for another 30 min to measure the stimulated mucin release rate. Mucin in perfusates was quantified by periodic acid-Schiff's base dot-blot assay, using purified pig gastric mucin as a standard.
When applied luminally at 1 μM lubiprostone was ineffective at stimulating mucin release. When added to the serosal solution, 1 μM lubiprostone stimulated mucin release to ~300% of the unstimulated rate. As a positive control, serosal 1 μM prostaglandin E2 increased mucin release to ~400% of the unstimulated rate.
These results support the idea that lubiprostone has prostaglandin-like actions on the intestine, which includes stimulation of mucin release. Stimulation of mucin release by lubiprostone may be protective in gastrointestinal conditions where loss of mucus is believed to contribute to pathogenesis. Thus, in addition to chronic constipation, there is greater potential for the therapeutic applications of lubiprostone.
KeywordsIntestine Lubiprostone Mucin Prostaglandin
Lubiprostone is a synthetic bicyclic fatty acid derivative of prostaglandin E1 used for chronic constipation. The best known action of lubiprostone is simulation of Cl- dependent fluid secretion. The originally proposed mechanism of lubiprostone is activation of the CLC2 chloride channel but this is controversial . Recent work suggests that lubiprostone may act in a prostaglandin-like manner to stimulate cystic fibrosis transmembrane conductance regulator (CFTR) dependent Cl- and fluid secretion .
In the genetic disease cystic fibrosis (CF) there is impaired Cl- and fluid secretion by affected epithelia contributing to the pathophysiology in this disease. Because lubiprostone can stimulate Cl- secretion, we had previously investigated its use as a therapy for CF by testing its effects in a mouse model of CF, a Cftr knockout . Treatment of CF mice with lubiprostone did ameliorate some of the CF related alterations. Lubiprostone treatment of CF mice accelerated gastric emptying, decreased small intestinal bacterial overgrowth, and reduced inflammation. These effects are independent of lubiprostone’s ability to stimulate Cl- secretion because intestinal tissue from CF mice does not secrete Cl- in response to lubiprostone . Unexpectedly, in vivo administration of lubiprostone to CF mice resulted in greater mucus accumulation in the small intestine , mucus accumulation being one of the major phenotypes of CF. This effect on mucus accumulation is consistent with lubiprostone acting through a prostaglandin receptor, as prostaglandin E2 (PGE2) is a known potent stimulus for intestinal mucin release. Therefore, in this project we investigated the ability of lubiprostone to stimulate mouse intestinal mucin secretion, using an ex vivo organ bath approach.
Measurement of intestinal mucin release using an ex vivo system
Quantification of released mucins
Mucin in perfusates was quantified by periodic acid-Schiff's base dot-blot assay, using a kit (Sigma; catalog 395B). Pig gastric mucin (Sigma, St. Louis, MO; catalog M1778) was used as a standard, prepared by suspension at 10 mg mL-1 in PBS using a probe-type sonicator. Serial 2-fold dilutions were prepared and in each assay 125 μg through 0.122 μg of mucin plus a blank were applied by vacuum filtration to nitrocellulose membrane in a dot-blot apparatus (Bio-Rad, Hercules, CA). A standard series of gastric mucins was included on every dot-blot. Perfusate outflow samples (50 μL) were applied in duplicate to the membrane as well. The membrane was removed from the apparatus and sequentially incubated in 3% acetic acid for 10 min, 6X diluted periodic acid for 15 min, 3% acetic acid washing for 10 min, 6X diluted Schiff’s Reagent for 10 min, 5 mg mL-1 sodium metabisulfite in water for 10 min, rinsing in water for 10 min, followed by air drying. The dried blots were scanned with a flatbed scanner (Hewlett Packard, Palo Alto, CA) and relative intensities measured using OptiQuant software (Kodak, Rochester, NY). The standard curve data were analyzed and sample unknowns determined using SigmaPlot software (Jandel Scientific, San Jose, CA; 'Standard Curves' feature in the 'Pharmacology' menu, with ‘Log X-scale’ and ‘Predict unknowns’). The PeakFit program (Jandel Scientific) was used to determine area under the curve (AUC) for 20–30 time points (unstimulated), and the 30–60 min time points (stimulated) which are presented as μg mucin min-1 g-1 tissue.
Data are presented as means ± standard errors. When there are 2 groups of data, a t-test was used. When there are more than 2 groups an ANOVA with post-hoc Tukey’s test was used. P-values <0.05 are considered statistically significant.
Results and discussion
There is disagreement in the literature as to the membrane surface where lubiprostone acts. In some experiments, it was reported that lubiprostone acted when applied to the apical surface of epithelia [1, 5] and in others when it was applied basolaterally . Therefore, we tested application of lubiprostone to either surface. When applied to the luminal surface by addition to the perfusate solution, lubiprostone (1 μM) had no effect on mucin release (Figure 3A). Similar to luminal lubiprostone, luminal application of PGE2 (1 μM) also had no effect on mucin release (data not shown).
We next tested the effect on mucin release of lubiprostone added to the serosal surface (to the bath). For comparison, we used PGE2. As shown in Figure 3B, both PGE2 (1 μM) and lubiprostone (1 μM) stimulated mucin release. PGE2 increased the rate of mucin release to ~400% that of the unstimulated (equilibration) rate (Figure 3B). Lubiprostone also stimulated mucin release to over 300% that of the unstimulated rate (Figure 3B). The difference in stimulation of mucin release between PGE2 and lubiprostone was not statistically significant.
Because lubiprostone also stimulates bicarbonate-rich fluid secretion in the small intestine , it is likely that this effect of lubiprostone facilitates solubilization of exocytosed goblet cell mucin and its expansion to mucus. It has been shown that co-secretion of bicarbonate through a CFTR-dependent pathway is crucial for correct expansion of exocytosed mucins [4, 8]. Since lubiprostone also activates CFTR-dependent bicarbonate secretion, mucin release and bicarbonate will be simultaneously stimulated by lubiprostone.
After this study was submitted, a report was published investigating the effects of lubiprostone stimulation in intestinal loop preparations of rat intestines and ex vivo human colonic samples . They showed that lubiprostone caused a coordinated redistribution of several apical and basolateral ion transporters between cytoplasmic vesicles and the plasma membrane that collectively would support secretion at the same time as suppressing absorption, in villi of the small intestine and in colonic crypts. They also quantified mucin granule exocytosis by morphometric histological analyses and showed that lubiprostone caused depletion of goblet cells mucin granules in both small and large intestine. Our results that lubiprostone stimulates mucin release ex vivo from the mouse small intestine are in agreement with the results of this study.
The results of this study support the idea that lubiprostone has prostaglandin-like actions on the intestine, which includes stimulation of mucin release. It remains to be determined which prostaglandin receptor(s) is/are activated by lubiprostone in the stimulation of mucin exocytosis by small intestinal goblet cells. It is known that mucin exocytosis and bicarbonate-rich fluid secretion are tightly coupled in the intestine  and that fluid secretion is impaired in EP3 PGE2 receptor knockout mice . However, in human duodenum, fluid secretion can be stimulated by an EP4 selective agonist  and an EP4 antagonist blocks bicarbonate secretion in rat duodenum , making the EP4 receptor a likely candidate for mediating lubiprostone's effects on mucin release. Regardless of which prostaglandin receptor is involved, stimulation of mucin release by lubiprostone may be protective in gastrointestinal conditions where loss of mucus is believed to contribute to pathogenesis . Thus, in addition to chronic constipation, there is greater potential for the therapeutic applications of lubiprostone.
Cystic fibrosis transmembrane conductance regulator
Phosphate buffered saline
- PGE2 :
I thank Drs. Ning Yang and Paul Quinton for advice setting up the perfusion system and for supplying the fire polished glass capillaries.
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