Luminal leptin activates mucin-secreting goblet cells in the large bowel

Leptin has been suggested to be involved in tissue injury and/or mucosal defence mechanisms. Here, we studied the effects of leptin on colonic mucus secretion and rat mucin 2 (rMuc2) expression. Wistar rats and ob/ob mice were used. Secretion of mucus was followed in vivo in the rat perfused colon model. Mucus secretion was quantified by ELISA, and rMuc2 mRNA levels were quantified by real-time RT PCR. The effects of leptin alone or in association with protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) inhibitors on mucin secreted by human mucus-secreting HT29-MTX cells were determined. Leptin was detected in the rat colonic lumen at substantial levels. Luminal perfusion of leptin stimulates mucus-secreting goblet cells in a dose-dependent manner in vivo in the rat. Leptin (10 nmol/l) increased mucus secretion by a factor of 3.5 and doubled rMuc2 mRNA levels in the colonic mucosa. There was no damage to mucosa 24 h after leptin, but the number of stained mucus cells significantly increased. Leptin-deficient ob/ob mice have abnormally dense mucus-filled goblet cells. In human colonic goblet-like HT29-MTX cells expressing leptin receptors, leptin increased mucin secretion by activating PKC- and PI3K-dependent pathways. This is the first demonstration that leptin, acting from the luminal side, controls the function of mucus-secreting goblet cells. Because the gel layer formed by mucus at the surface of the intestinal epithelium has a barrier function, our data may be relevant physiologically in defence mechanisms of the gastrointestinal tract.     

Foot mucus is a good source for non-destructive genetic sampling in Polyplacophora

A non-destructive method for collecting samples for DNA analysis from the mucus of molluscs was successfully adapted for use with the genus Ischnochiton. DNA was extracted using a Chelex-based method and the COI subunit of the mtDNA was amplified and sequenced. Sequences from the mucus were crosschecked against sequences from the foot tissue of the same animal and were found to be identical. This method provides a non-destructive way of carrying out larger studies of the genetics of rare organisms and may be of general use for genetic-based field studies of molluscs.     

Action of serotonin on the gastrointestinal tract

Serotonin is localized in the enterochromaffin cells of the gastrointestinal mucosa and within neurons in the enteric nervous system. It can be released into the blood or into the lumen of the gut. Serotonin inhibits gastric acid secretion and may be an endogenous enterogastrone. It appears to stimulate the production and release of gastric and colonic mucus. When placed on the serosal surface of the rabbit ileum in vitro, serotonin increases short-circuit current and inhibits the mucosal-to-serosal flux of NaCl. Serotonin potentially is involved in the pathogenesis of diarrhea due to amoebae or cholera. As an enteric neurotransmitter, serotonin affects neural modulation of gut smooth muscle function and may act either directly on mesenteric vascular smooth muscle or through enteric nerves to influence gastrointestinal blood flow. Thus, since serotonin may be involved in multiple physiological processes of digestion, this report reviews and summarizes the role of this ubiquitous substance in the major functions of the gastrointestinal system.     

Serotonin and MucXS release by small secretory cells depend on Xpod,a SSC specific marker gene

Mucus secretion and ciliary motility are hallmarks for muco‐ciliary epithelia (MCE). Both, mammalian airways as well as the less complex epidermis of Xenopus embryos show cilia‐driven mucus flow to protect the organism against harmful effects by exogenous pathogens or pollutants. Four cell types set up the epidermal MCE in Xenopus. Multi‐ciliated cells (MCCs) generate an anterior to posterior flow of mucus. Ion secreting cells (ISCs) are characterized by the expression of ion transporters, presumably to maintain a favorable homeostasis. The largest cell type is represented by goblet cells, which cover most of the epidermis and exhibit secretory properties. Additionally, small secretory cells (SSCs) release mucus, antibiotic compounds, and the monoamine serotonin (5‐hydroxytryptamine; 5‐HT). We have recently shown that serotonin regulates flow velocity by acting on ciliary beat frequency. Here, we describe the identification and functional characterization of Xenopus polka‐dots (Xpod). No homologous genes or proteins were found in other vertebrates, including Xenopus tropicalis. We demonstrate that Xpod serves as an SSC‐specific marker, starting to be expressed shortly after SSC specification at neurula stages. Overexpression of a tagged Xpod protein resulted in the localization of secretory granules. Notch signaling induced SSC cell fate, in contrast to its repressing effect on MCC and ISC specification. Xpod loss‐of‐function revealed that mucus and 5‐HT release by SSCs was severely diminished, which impaired the ciliary beating of MCCs. In summary, Xpod specifically marked SSCs and was required for muco‐ciliary secretion in Xenopus laevis.     

Monoamines and their metabolites in the freshwater snail Biomphalaria glabrata

Metabolism of the major monoamines and their functions were studied in the freshwater snail Biomphalaria glabrata. In both juvenile and adult snails, the plasma (cell-free hemolymph) appears to act as a reservoir for most of these monoamines and their metabolites including among others, L-dopa and dopamine as major constituents. Significant quantities of L-tryptophan, precursor of indoleamines, also was found in the plasma. L-dopa, serotonin, homovanillic acid and dopamine were prominently represented in the central nervous system of the snail, while serotonin and its metabolites, 5-hydroxyindole acetic acid and 5-hydroxytryptophol were found in the ovotestis. Catecholamines such as L-dopa, dopamine and homovanillic acid were identified in the albumen gland. Functional aspects of both dopamine and serotonin were studied using in vitro cultures of albumen glands, the site of perivitelline fluid and galactogen synthesis in B. glabrata. Dopamine was found to stimulate the release of secretory proteins when exogenously added to gland cultures and this process was inhibited by chlorpromazine, a dopamine receptor antagonist. Similarly, exogenous serotonin stimulated in vitro protein secretion by albumen glands. Thus, these results suggest that monoamines may play important roles in regulating reproductive activity of this snail and provides an excellent model for studying neurotransmitter function and metabolism in molluscs.     

Proliferation of fibroblasts and endothelial cells is enhanced by treatment with Phyllocaulis boraceiensis mucus

Previously, mucus of some molluscs has been studied as a potential source of new natural compounds capable of inducing cell proliferation and of remodelling tissue. Here, the focus of the study is possible use of mucus released by Phyllocaulis boraceiensis – a compound inducing cell proliferation and enhancing collagen synthesis in dermal fibroblasts and inducing proliferation human endothelial cell cultures. Fibroblasts treated with P. boraceiensis mucus at concentrations below 0.012 μg/μl developed high rates of proliferation, as evaluated using MTT assay; the proliferative effect was dose‐dependent. Production and secretion of extracellular matrix components and collagen type I fibres were enhanced after 24 h of treatment, revealing a hormesis effect, biphasic dose response – low dose for proliferation yet toxic at high dose. No significant change in proliferation was observed in treated endothelial cells and production of lipid polyunsaturated free radicals was low in both cell types. Treatment with P. boraceiensis mucus produced pronounced changes in fibroblast cell number and morphology, and in quantities of well‐ordered collagen deposition. These results support the premise that Phyllocaulis boraceiensis mucus demonstrates proliferative properties in cells involved in the healing process.     

Study of the damaging effects of acetazolamide on gastric mucosa in rats

The present study demonstrated that acetazolamide (100 and 200 mg/kg, s.c.) induced severe gastric hemorrhagic ulceration in rats. The ulceration was aggravated by oral administration of HCl, but was inhibited by NaHCO3. Furthermore, the severity of ulceration was also decreased by pretreatment with methysergide, chlorpheniramine, or cimetidine. These protective effects were accompanied by an increase in serotonin and histamine released from the stomach. Acetazolamide injection also increased the protein level but reduced the sialic acid content in the gastric secretion, indicating that the gastric mucosal barrier may have been damaged. Prostaglandin E2 content of the gastric mucosa was not affected by the drug; however, carbonic anhydrase activity was markedly reduced in a dose-dependent manner. Thus, it is suggested that the ulceration induced by acetazolamide is mainly due to the inhibition of carbonic anhydrase activity and mucus secretion. The increase in serotonin and histamine release also may have been the contributing factors for gastric ulcer formation.     

Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin

Secretion of bicarbonate into the adherent layer of mucus gel creates a pH gradient with a near-neutral pH at the epithelial surfaces in stomach and duodenum, providing the first line of mucosal protection against luminal acid. The continuous adherent mucus layer is also a barrier to luminal pepsin, thereby protecting the underlying mucosa from proteolytic digestion. In this article we review the present state of the gastroduodenal mucus bicarbonate barrier two decades after the first supporting experimental evidence appeared. The primary function of the adherent mucus gel layer is a structural one to create a stable, unstirred layer to support surface neutralization of acid and act as a protective physical barrier against luminal pepsin. Therefore, the emphasis on mucus in this review is on the form and role of the adherent mucus gel layer. The primary function of the mucosal bicarbonate secretion is to neutralize acid diffusing into the mucus gel layer and to be quantitatively sufficient to maintain a near-neutral pH at the mucus-mucosal surface interface. The emphasis on mucosal bicarbonate in this review is on the mechanisms and control of its secretion and the establishment of a surface pH gradient. Evidence suggests that under normal physiological conditions, the mucus bicarbonate barrier is sufficient for protection of the gastric mucosa against acid and pepsin and is even more so for the duodenum. acid-base transporters; cystic fibrosis transmembrane conductance regulator channel; surface pH gradient; mucus gels; trefoil peptides     

Separation of rabbit ileum mucus secretion from electrolyte and water secretion by cholera enterotoxin, verapamil and A23187

Net water, Na+, Cl- and HCO3- fluxes were measured in in vivo rabbit ileal loops, while mucus secretion was assessed by measuring the glycoprotein or total sialic acid secreted into the lumen, or by measuring the luminal fluid viscosity. Inoculating loops with cholera enterotoxin (CT) produced a sustained secretion of electrolytes and water, but a more transient secretion of mucus. A dose of verapamil was found which, when included in the luminal fluid, inhibited or delayed the CT-induced mucus secretion while not affecting the ongoing electrolyte and water secretion. Exposure of the ileal mucosa to the ionophore, A23187, in the presence of 2mM Ca++ resulted in a brief secretion of mucus, with no change in basal water absorption. Verapamil inhibited this A23187-induced mucus secretion. The ionophore was not effective in the absence of luminal Ca++. Thus rabbit ileum mucus secretion can be separated from electrolyte and water secretion by agents that affect Ca++ movement.     

Airway responses to esophageal acidification

The effects of esophageal acidification on airway function are unclear. Some have found that the esophageal acidification causes a small increase in airway resistance, but this change is too small to cause significant symptoms. The aims of this study were to investigate the effects of esophageal acidification on multiple measures of airway function in chloralose-anesthetized cats. The esophagus was cannulated and perfused with either 0.1 M PBS or 0.1 N HCl at 1 ml/min as the following parameters were quantified in separate experiments: diameter of bronchi (n = 5), tracheal mucociliary transport rate (n = 4), tracheobronchial mucus secretion (n = 7), and lung function (n = 6). We found that esophageal acidification for 10-30 min decreased bronchial diameters primarily of the smaller low-resistance airways (10-22%, P < 0.05), decreased tracheal mucociliary transport (53%, 8.7 +/- 2.4 vs. 4.1 +/- 1.3 mm/min, P < 0.05), increased tracheobronchial mucus secretion (147%, 3.4 +/- 0.7 vs. 8.4 +/- 2.6 mg/10 min, P < 0.05), and caused no change in total lung resistance or dynamic compliance (P > 0.05). Considering that tracheal mucociliary transport rate is governed in part by mucus secretion, we concluded that the primary airway response to esophageal acidification observed is increased mucus secretion. Airway constriction may act to assist in rapid secretion of mucus and to increase the effectiveness of coughing while not affecting lung resistance or compliance. Given the buffering capabilities of mucus, esophageal acidification activates appropriate physiological responses that may act to neutralize gastroesophageal reflux that reaches the larynx, pharynx, or lower airways.     

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.     

Changed control of cervical secretion from infertile ewes previously exposed to oestrogenic clover pasture

The amount of cervical mucus recovered from control ovariectomized ewes increased with increasing doses of oestradiol benzoate (OB), while the maximum Spinnbarkeit of mucus occurred at an intermediate dose of OB. Neither the amount nor the Spinnbarkeit of mucus varied with the dose of OB in ewes with permanent infertility caused by grazing oestrogenic pasture (clover-affected ewes). Furthermore, the increase in Spinnbarkeit of cervical mucus seen in normal ewes treated over a 3-day period with OB or with implants containing oestradiol did not occur in affected ewes. In control ewes treated repeatedly with OB, production of mucus declined within 5 days, but no change in secretion was detectable in clover-affected ewes. Therefore, neither the amount nor the duration of oestrogenic stimulation affected the cervical mucus in ewes with clover disease. Affected ewes produced more mucus than did controls in the absence of oestrogenic stimulation. It is concluded that the relatively normal volume of mucus in affected ewes treated with OB results largely from autonomous production. The Spinnbarkeit does not increase in these ewes because the ability of the cervix to respond to oestrogen is impaired.     

Pirenzepine block of ACH-induced mucus secretion in tracheal submucosal gland cells

Muscarinic stimulation of mucus secretion, as measured by the release of [3H]glycoprotein, was studied in explants from the tracheal epithelium of weanling swine. The mucus glycoprotein secretion was transient, ceasing within the first 10 min of a continuous exposure to 100 microM ACh. Increasing the solution's osmotic pressure did not alter basal mucus glycoprotein secretion. Mucus glycoprotein secretion was inhibited by 2-10 microM PZP, indicating that the M3 muscarinic receptors mediate cholinergic stimulation of mucus production.     

Effect of somatostatin on the secretin-induced gastric secretions of pepsin and mucus in cats

The effect of somatostatin 14 on gastric stimulation produced by secretin was determined in 6 conscious cats equipped with a gastric fistula and a denervated fundic pouch. Somatostatin strongly inhibited the basal and secretin-induced pepsin secretion. It did not, however, inhibit the secretin-induced mucus secretion, even though it decreased the basal mucus secretion. During somatostatin administration, the secretagogue effect of secretin on mucus secretion might be dissociated from its stimulatory action on pepsin secretion.     

Autofluorescence as a tool to study mucus secretion in Eisenia foetida

Autofluorescence in living cells is due to the presence of endogenous substances that emit fluorescence upon excitation by incidental light. A type of fluorescence, bioluminescence, has been suggested to be linked to mucus secretion in earthworms; however, the origin and the physiological function of this fluorescence are not clear. The aims of this work were to describe autofluorescence in the earthworm Eisenia foetida by SEM, CLSM, and fluorescence microscopy and to examine the possible mechanism of mucus secretion by video microscopy. Earthworms were stimulated either chemically or electrically to induce the secretion of yellow mucus, which was subsequently studied by video microscopy. Mucus was released from the body wall and near the mouth. This phenomenon was associated with autofluorescence and involved at least four distinct stages: release of vesicles, formation of granules, muscular contraction, and organization of strands. The fluorescent molecules were stored in vesicles bound to the membranes. These vesicles were intact when shed from the body. The vesicles were stable but also changed to a granular material or formed strands. Video analyses demonstrated that secretion was dependent on the type of stimulus.     

Detection of Coxiella burnetii specific DNA in blood samples from Japanese patients with chronic nonspecific symptoms by nested polymerase chain reaction

Sprague Dawley rats, previously infected with Phase-I Bordetella pertussis , developed more severe abnormal respiratory sounds than normal animals, but not coughing, when exposed to aerosolized capsaicin, one of several cough-inducing agents tested. Stethoscope examination suggested that greater production of pulmonary mucus might be occurring after capsaicin challenge of the infected animals, compared to the uninfected controls. Rats of three other strains gave characteristically different responses from the Sprague Dawleys. The administration of capsaicin to B. pertussis -infected rats may provide useful insights into the pathophysiology of excess mucus secretion in human pertussis.     

Stimulation of gastric mucus and protein secretion by cytochalasin E in rats

Effects of cytochalasin E on the secretion of mucus and protein were investigated in gastric fistula rats. Direct exposure of the gastric mucosa to cytochalasin E (5-20 micrograms/ml) significantly stimulated the secretory rate of soluble mucus and protein in pentagastrin-stimulated rats. The amount of surface mucus gel was also increased. The stimulatory effect was increased with increased concentrations of cytochalasin. Histological study suggests that the cytochalasin stimulated the release of mucus from the cell. The increase in secretory rate of protein was not due to an increase of pepsin secretion but rather was the consequence of the increase in mucus secretion.     

VIP augments cholinergic-induced glycoconjugate secretion in tracheal submucosal glands

Using isolated submucosal glands from feline trachea, we examined the effect of vasoactive intestinal peptide (VIP) on mucus glycoprotein secretion and glandular contraction by measuring released radiolabeled glycoconjugates and induced tension, respectively. VIP (10(-10) to 10(-6) M) produced a dose-dependent increase in [3H]glycoconjugate release of up to 300% of controls, which was inhibited by VIP antiserum and not inhibited by atropine, propranolol, or phentolamine. VIP at a low concentration (10(-9) M), which did not produce any significant increases over controls, produced a 2.4- to 5-fold augmentation of the glycoconjugate release induced by 10(-9) to 10(-7) M methacholine (MCh). Atropine or VIP antiserum abolished the augmentation. VIP did not produce any alteration in isoproterenol- or phenylephrine-evoked glycoconjugate secretion. VIP (up to 10(-5) M) did not produce any alteration in the tension, even when the gland had contracted with MCh, or any augmentation of contraction induced by MCh (10(-9) to 10(-7) M). These results indicate that VIP induces mucus glycoprotein release from secretory cells and also that it potentiates the secretion induced by cholinergic stimulation.     

Hyposecretion, not hyperabsorption, is the basic defect of cystic fibrosis airway glands

Human airways and glands express the anion channel cystic fibrosis transmembrane conductance regulator, CFTR, and the epithelial Na(+) channel, ENaC. Cystic fibrosis (CF) airway glands fail to secrete mucus in response to vasoactive intestinal peptide or forskolin; the failure was attributed to loss of CFTR-mediated anion and fluid secretion. Alternatively, CF glands might secrete acinar fluid via CFTR-independent pathways, but the exit of mucus from the glands could be blocked by hyperabsorption of fluid in the gland ducts. This could occur because CFTR loss can disinhibit ENaC, and ENaC activity can drive absorption. To test these two hypotheses, we measured single gland mucus secretion optically and applied ENaC inhibitors to determine whether they augmented secretion. Human CF glands were pretreated with benzamil and then stimulated with forskolin in the continued presence of benzamil. Benzamil did not rescue the lack of secretion to forskolin (50 glands, 6 CF subjects) nor did it increase the rate of cholinergically mediated mucus secretion from CF glands. Finally, neither benzamil nor amiloride increased forskolin-stimulated mucus secretion from porcine submucosal glands (75 glands, 7 pigs). One possible explanation for these results is that ENaC within the gland ducts was not active in our experiments. Consistent with that possibility, we discovered that human airway glands express Kunitz-type and non-Kunitz serine protease inhibitors, which might prevent proteolytic activation of ENaC. Our results suggest that CF glands do not display excessive, ENaC-mediated fluid absorption, leaving defective, anion-mediated fluid secretion as the most likely mechanism for defective mucus secretion from CF glands.     

Mucus secretion and cytoskeletal modifications in cultured nasal epithelial cells exposed to wall shear stresses

The nasal epithelium is continuously subjected to wall shear stresses (WSS) induced by respiratory airflows. An in vitro experimental model was developed to expose nasal epithelial cells cultured under air-liquid interface conditions to steady airflow-induced WSS. Mucus secretion from epithelial goblet cells was quantified using an enzyme-linked lectinosorbent assay, and modifications of the cytoskeletal structure were qualitatively evaluated from fluorescent stains of actin and β-tubulin fibers. The results show increased mucus secretion from cells subjected to WSS of 0.1 and 1.0 dyne/cm² for more than 15 min in comparison with unstressed cells. The integrity levels of β-tubulin fibers were significantly lower in cells subjected to WSS than in unstressed cells. The increased mucus secretion in response to WSS was approximately the same in Taxol-free and Taxol-treated cultures, which indicates that there is no direct connection between β-tubulin fragmentation and mucus secretion. The stressed cells regained their normal cytoskeletal appearance 24 h after the exposure to WSS. The results of this study suggest that WSS have an important role in the mechanical regulation of the nasal surface epithelium function.