Mucin Release Research Articles

Irinotecan‐induced mucositis manifesting as diarrhoea corresponds with an amended intestinal flora and mucin profile

Chemotherapy-induced diarrhoea is a major oncological problem, caused by the cytotoxic effects of cancer chemotherapy. Irinotecan is linked with severe mucositis and diarrhoea, the mechanisms of which remain poorly understood. Bacterial beta-glucuronidase is thought to be involved in the metabolism of irinotecan, implicating the intestinal flora. Intestinal mucins may also be implicated in the development of chemotherapy-induced diarrhoea. Rats were treated with 200 mg/kg of irinotecan and killed at 96, 120 and 144 h. The rats were monitored for diarrhoea. Pathology and immunohistochemical staining was performed. The samples were cultured and faecal DNA was analysed using real-time polymerase chain reaction. Severe diarrhoea was observed from 72 to 96 h. A decrease in body mass was also observed after treatment. Significant changes in goblet cell numbers (both complete and cavitated cells) were observed in the small and large intestines. Changes in MUC gene expression were observed in the small intestine only. Modifications were observed to the intestinal flora profile, especially Escherichia coli, and an increase in the expression of beta-glucuronidase was detected. In conclusion, irinotecan-induced diarrhoea may be caused by an increase in some beta-glucuronidase-producing bacteria, especially E. coli, exacerbating the toxicity of active metabolites. Accelerated mucous secretion and mucin release may also contribute to the delayed onset of diarrhoea.

Pass the bicarb: the importance of HCO3– for mucin release

Accumulation of thick, sticky mucus is a hallmark of the genetic disease cystic fibrosis (CF) and has a central role in CF pathophysiology. Mutations in the CF transmembrane regulator (CFTR) ion channel are known to result in abnormally thick and sticky mucus; however, why mucus accumulates in CF is still not completely understood. In this issue of the JCI, Garcia and colleagues show that mucin--the heavily glycosylated protein contained within mucus--requires CFTR and bicarbonate in order to be released from mouse intestine (see the related article beginning on page 2613). The authors propose a model whereby CFTR-mediated bicarbonate secretion must be concurrent with mucin exocytosis for proper mucin release.

Advances in Mucous Cell Metaplasia

Mucous cell metaplasia is induced in response to harmful insults and provides front-line protection to clear the airway of toxic substances and cellular debris. In chronic airway diseases mucous metaplasia persists and results in airway obstruction and contributes significantly to morbidity and mortality. Mucus hypersecretion involves increased expression of mucin genes, and increased mucin production and release. The past decade has seen significant advances in our understanding of the molecular mechanisms by which these events occur. Inflammation stimulates epidermal growth factor receptor activation and IL-13 to induce both Clara and ciliated cells to transition into goblet cells through the coordinated actions of FoxA2, TTF-1, SPDEF, and GABA(A)R. Ultimately, these steps lead to up-regulation of MUC5AC expression, and increased mucin in goblet cell granules that fuse to the plasma membrane through actions of MARCKS, SNAREs, and Munc proteins. Blockade of mucus in exacerbations of asthma and chronic obstructive pulmonary disease may affect morbidity. Development of new therapies to target mucus production and secretion are now possible given the advances in our understanding of molecular mechanisms of mucous metaplasia. We now have a greater incentive to focus on inhibition of mucus as a therapy for chronic airway diseases.

Relation of Gallbladder Motility to Viscosity and Composition of Gallbladder Bile in Patients with Cholesterol Gallstones

Background/Aims: Increased viscosity and supersaturation of cholesterol in gallbladder bile, as well as an impaired motility of the gallbladder, are considered to be important factors in the pathogenesis of cholesterol gallstones. However, the relation of these parameters has not yet been determined. Material and Methods: Bile viscosity (mPa·s) was measured by rotation viscosimetry and the composition of gallbladder bile was determined using standard methodology. Gallbladder motility was calculated as ejection fraction in percent of total volume 45 min after a test meal using ultrasonography in patients with gallstones prior to elective cholecystectomy. Results: The study included 35 patients with cholesterol gallstones. Viscosity of gallbladder bile ranged between 0.9 and 12.5 mPa·s (median 2.2 mPa·s) and an ejection fraction of the gallbladder of 55.4 ± 18.3% (mean ± SD) was determined. No significant correlation (r = 0.19, p < 0.2) between the 2 parameters could be calculated. Analysis of the composition of gallbladder bile revealed a positive correlation of all components to biliary viscosity but not to the motility of the gallbladder, with the exceptions of a negative correlation (r = 0.39, p < 0.02) between mucin concentration and the ejection fraction at 45 min after the test meal. Conclusions: The motility of the gallbladder appears to be unrelated to the viscosity of gallbladder bile or gallbladder bile composition. The negative correlation between the ejection fraction of the gallbladder and mucin concentration of gallbladder bile suggests that chronic inflammation of the gallbladder wall is associated with both an impaired motility of the gallbladder and increased mucin release into gallbladder bile.

Increased leukotriene concentration× in submandibular glands from rats with experimental periodontitis

In the present study, we investigated the relation between the inflammatory mediators such as nitric oxide, prostaglandins, and cysteinyl-leukotrienes with mucin release and the sympathetic system in submandibular glands from rats with experimental periodontitis. Submandibular glands from rats with experimental periodontitis. For the first experiment, rats were treated with hydrocortisone sc, 1 mg/kg for 3 days. All other experiments were carried out in isolated submandibular glands from untreated rats. Submandibular glands were treated with cysteinyl-leukotrienes, isoproterenol, NDGA, FPL 55712, L-NMMA, Nio, Nz, AMG, indomethacin, DuP 697 and atenolol. Nitric oxide synthase activity, prostaglandin and cysteinyl-leukotriene productions and mucin secretion were determined. The Newman-Keuls statistical test was applied after analysis of variance. In rats with periodontitis hydrocortisone-induced a 36.6% (P < 0.05) decrease in mucin release. Only cysteinyl-leukotriene production was increased in rats with ligature (79.2%, P < 0.001). Either the inhibition of cysteinyl-leukotriene production or the block of leukotriene receptor abolished the increase in mucin secretion by 25.6% (P < 0.05) and 37% (P < 0.01), respectively, in glands from rats with ligature. On the other hand, the presence of cysteinyl-leukotrienes in the incubation medium induced mucin release from submandibular glands. Atenolol diminished by 24% (P < 0.05), the increase in cysteinyl-leukotrienes observed in rats with periodontitis. Besides, isoproterenol induced cysteinyl-leukotriene production in both groups. In submandibular glands from rats with periodontitis, the increment in mucin release and cysteinyl-leukotrienes production are related events and both are associated with the sympathetic system.

Role of leukotrienes in exercise-induced bronchoconstriction

Exercise-induced bronchoconstriction (EIB) refers to acute airflow obstruction that is triggered by a period of physical exertion. EIB occurs mainly in individuals with other features of asthma but is especially prominent in a subset of asthmatics with pronounced indirect airway hyperresponsiveness. Leukotrienes (LTs) play a critical role in the pathophysiology of EIB. Asthmatics who are susceptible to EIB have increased levels of cysteinyl LTs (cysLTs [ie, LTs C4, D4, and E4]) in induced sputum and exhaled breath condensate. Exercise challenge in individuals susceptible to this disorder initiates the sustained increase in cysLTs in the airways and an increase in the ratio of cysLTs to prostaglandin E(2). The effects of cysLTs leading to secreted mucin release and smooth muscle constriction may be mediated in part through activation of sensory nerves. Therapies that block cysLT production or the cysLT(1) receptor effectively reduce the severity of EIB.

Engineered toxins: New therapeutics

Understanding the structure-function relationship of the botulinum neurotoxins (BoNTs) has enabled creation of novel recombinant proteins in which the binding domain of the neurotoxin is replaced by a ligand to a cell surface receptor. The resulting protein can deliver neurotoxin endopeptidase to a cell based upon its receptor specificity, inhibiting secretion from the selected cell. The approach is exemplified by a protein targeting airway epithelial cells responsible for mucin release. A synthetic gene encoding the endopeptidase and translocation domains of BoNT type C1 together with epidermal growth factor (EGF) was expressed in Escherichia coli . The encoded protein (EGF-C) included a protease cleavage site to allow activation to a di-chain protein. The recombinant protein was purified via an affinity tag. In vitro, EGF-C inhibited both stimulated mucin secretion and mucin gene expression in airway epithelial cell lines at sub-nanomolar concentrations. In vivo, EGF-C inhibited stimulated mucus hypersecretion in a rat model of COPD and this effect was maintained for several weeks following a single administration. These results show that novel proteins incorporating the endopeptidase activity of BoNTs can be produced to inhibit secretion from specified cell types with a prolonged duration of action. This opens up an opportunity to produce novel recombinant proteins for the treatment of chronic diseases not amenable to therapy with the native neurotoxins.

Release of Membrane-Associated Mucins from Ocular Surface Epithelia

Three membrane-associated mucins (MAMs)--MUC1, MUC4, and MUC16--are expressed at the ocular surface epithelium. Soluble forms of MAMs are detected in human tears, but the mechanisms of their release from the apical cells are unknown. The purpose of this study was to identify physiologic agents that induce ocular surface MAM release. An immortalized human corneal-limbal epithelial cell line (HCLE) expressing the same MAMs as native tissue was used. An antibody specific to the MUC16 cytoplasmic tail was developed to confirm that only the extracellular domain is released into the tear fluid or culture media. Effects of agents that have been shown to be present in tears or are implicated in the release or shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor [TNF]), TNF-alpha-converting enzyme, and matrix metalloproteinase-7 and -9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure the efficiency of induced MAM release and surface restoration. Effects of induced release on surface barrier function were measured by rose bengal dye penetrance. MUC16 in tears and in HCLE-conditioned medium lacked the cytoplasmic tail. TNF induced the release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced the release of MUC16 but not of MUC1 or MUC4. Neutrophil elastase removed 68% of MUC16, 78% of which was restored to the HCLE cell surface 24 hours after release. Neutrophil elastase-treated HCLE cells showed significantly reduced rose bengal dye exclusion. Results suggest that the extracellular domains of MUC1, MUC4, and MUC16 can be released from the ocular surface by agents in tears. Neutrophil elastase and TNF, present in higher amounts in the tears of patients with dry eye, may cause MAM release, allowing rose bengal staining.

Regulated Airway Goblet Cell Mucin Secretion

Major advances in understanding regulated mucin secretion from airway goblet cells have been made in the past decade in the areas of pharmacology and basic cell biology. For instance, it is now appreciated that nucleotide agonists acting locally through P2Y purinoceptors on apical membranes of surface goblet cells provide the major regulatory system for mucin secretion. Similarly, Clara cells, the primary secretory cell in the mouse airways (and human small airways), are now recognized as major mucin-secreting cells. In Clara cells, the relative lack of staining for mucosubstances reflects essentially equal baseline rates of mucin synthesis and secretion, with little to no accumulation of mucin granules in storage pools. During mucous metaplasia induced under inflammatory conditions, mucin synthesis is massively upregulated in Clara cells, and stored mucin granules come to dominate the secretory cell phenotype. More importantly, we have seen a transition in the past few years from a pharmacological focus on regulated mucin secretion to a more molecular mechanistic focus that has great promise going forward. In part, these advances are occurring through the use of well-differentiated primary human bronchial epithelial cell cultures, but recent work in mouse models perhaps has had the most important impact. Emerging data from Munc13-2- and synaptotagmin 2-deficient mouse models represent the first direct, molecular-level manipulations of proteins involved in regulated secretory cell mucin secretion. These new data indicate that Munc13-2 is responsible for regulating a baseline mucin secretory pathway in the airways and is not essential for purinergic agonist-induced mucin secretion. In contrast, synaptotagmin 2, a fast Ca2+ sensor for the SNARE complex, is essential for regulated secretion. Interestingly, these early results suggest that there are two pathways for excocytic mucin release from goblet cells.

β-Adrenoceptor alterations coupled with secretory response and experimental periodontitis in rat submandibular glands

In this paper we have studied the influence of a well-established rat model of periodontitis on resting and adrenergic-stimulated mucin secretion from rat submandibular glands. The selective β 1-receptor subtype agonist, dobutamine, induced mucin secretion while the selective β 2-, α 1- and α 2-agonists, soterenol, phenylephrine and clonidine, respectively, did not. In rats subjected to ligature-induced periodontitis mucin release, under unstimulated conditions (basal values), was significantly increased. This increment was abolished in the presence of propranolol and atenolol. Isoproterenol, concentration-dependent, increased mucin release in control and in ligature-induced periodontitis rats. Maximal effect of isoproterenol was decreased in rats with ligature while EC 50 was increased. Neither, the inhibition of NOS by l-NMMA nor the inhibition of COX by indomethacin could revert the effect of ligature on mucin release under unstimulated and isoproterenol-stimulated conditions. The inhibition of adenylyl cyclase by SQ 22536 resulted in a right shift of isoproterenol concentration–response curves in both groups, control and with ligature and returned basal values of rats with ligature to control ones. β-Receptor population was decreased in submandibular gland membranes from rats with ligature without changes in affinity. Potencies of the β-receptor antagonists in the competition studies were similar in both groups under study, control and with ligature. We conclude that in rats subjected to ligature-induced periodontitis unstimulated mucin secretion is increased. The increment seems to be due to an activation of the sympathetic system since it is inhibited by the β-adrenoceptors antagonists and by the inhibition of the adenylyl cyclase. We can speculate that inflammatory mediators from the experimental periodontitis could be involved in the mechanism underlying the activation of the sympathetic system.

Macrolides attenuate mucus hypersecretion in rat airways through inactivation of NF‐κB

To examine the effect of a 14-membered ring macrolide on airway mucus hypersecretion in rats treated with LPS. Mucus hypersecretion in rat airways was induced by intratracheal instillation of LPS. Rats treated with or without LPS were administered roxithromycin (1-10 mg/kg), josamycin (10 mg/kg) or amoxicillin (40 mg/kg), orally for 4 days. Expression of Muc5ac, nuclear factor (NF)-kappaB, and the mitogen-activated protein (MAP) kinases p38 and ERK1/2 in bronchial epithelium were detected by RT-PCR, immunohistochemistry or western blotting. Mucins, IL-1beta, IL-8 and tumour necrosis factor (TNF)-alpha in BAL fluid were assayed by enzyme-linked lectin assay and ELISA. LPS significantly induced the expression of Muc5ac mRNA and protein in bronchial epithelium, increased the release of mucins, IL-1beta, IL-8 and TNF-alpha, and increased neutrophil numbers in BAL. Moreover, LPS increased staining for NF-kappaB in the cytoplasm as well as nuclear translocation of NF-kappaB in airway epithelial cells. Upregulated expression of Muc5ac mRNA correlated positively with NF-kappaB activation and the levels of cytokines (P < 0.05). Roxithromycin (5 and 10 mg/kg) significantly attenuated bronchial Muc5ac expression and NF-kappaB nuclear translocation stimulated by LPS, and reduced neutrophil numbers, mucins and inflammatory cytokines in BAL (P < 0.05). However, LPS-stimulated expression of p38 and ERK1/2 in airway epithelium was not affected by roxithromycin. Josamycin and amoxicillin had no effects on Muc5ac expression, NF-kappaB activation or cytokine release. Roxithromycin inhibits the pulmonary inflammatory response and airway mucus hypersecretion induced by LPS. The inhibitory effect of roxithromycin on airway mucus hypersecretion may be mediated through reduction of NF-kappaB activation, neutrophil infiltration and release of inflammatory cytokines in the lung.

Coordinated release of nucleotides and mucin from human airway epithelial Calu‐3 cells

The efficiency of the mucociliary clearance (MCC) process that removes noxious materials from airway surfaces depends on the balance between mucin secretion, airway surface liquid (ASL) volume, and ciliary beating. Effective mucin dispersion into ASL requires salt and water secretion onto the mucosal surface, but how mucin secretion rate is coordinated with ion and, ultimately, water transport rates is poorly understood. Several components of MCC, including electrolyte and water transport, are regulated by nucleotides in the ASL interacting with purinergic receptors. Using polarized monolayers of airway epithelial Calu-3 cells, we investigated whether mucin secretion was accompanied by nucleotide release. Electron microscopic analyses of Calu-3 cells identified subapical granules that resembled goblet cell mucin granules. Real-time confocal microscopic analyses revealed that subapical granules, labelled with FM 1-43 or quinacrine, were competent for Ca(2+)-regulated exocytosis. Granules containing MUC5AC were apically secreted via Ca(2+)-regulated exocytosis as demonstrated by combined immunolocalization and slot blot analyses. In addition, Calu-3 cells exhibited Ca(2+)-regulated apical release of ATP and UDP-glucose, a substrate of glycosylation reactions within the secretory pathway. Neither mucin secretion nor ATP release from Calu-3 cells were affected by activation or inhibition of the cystic fibrosis transmembrane conductance regulator. In SPOC1 cells, an airway goblet cell model, purinergic P2Y(2) receptor-stimulated increase of cytosolic Ca(2+) concentration resulted in secretion of both mucins and nucleotides. Our data suggest that nucleotide release is a mechanism by which mucin-secreting goblet cells produce paracrine signals for mucin hydration within the ASL.

NPKCε, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y(2) purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP(3)), diacylglycerol, Ca(2+) and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKCalpha, nPKCdelta, nPKCepsilon, and nPKCeta; of these, only nPKCdelta translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149-L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKCalpha, nPKCdelta, and nPKCeta had the same levels of ATPgammaS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKCepsilon (14.6 and 23.5%, for ATPgammaS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKCepsilon exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPgammaS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y(2)-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKCdelta knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKCepsilon KO mice relative to its WT littermates. We conclude that nPKCepsilon is the effector isoform downstream of P2Y(2)-R activation in the goblet cell secretory response. The translocation of nPKCdelta observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology.

Effects of baicalin and wogonin on mucin release from cultured airway epithelial cells

Scutellaria baicalensis Georgi has been used for the treatment of diverse chronic inflammatory diseases including respiratory disease in oriental medicine and its major components - baicalin, baicalein and wogonin - were reported to have various biological effects. This study investigated whether baicalin, baicalein and wogonin affect basal and ATP-induced mucin release from cultured airway epithelial cells. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled using (3)H-glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of each agent to assess the effects on (3)H-mucin release. The results were as follows: (1) Baicalein did not affect both basal and ATP-induced mucin release significantly. (2) Baicalin and wogonin increased basal mucin release at the highest concentrations (10(-3) m). (3) However, baicalin and wogonin significantly inhibited ATP-induced mucin release. It is concluded that baicalin and wogonin can slightly increase basal mucin release whereas they can inhibit ATP-induced mucin release, by directly acting on airway mucin-secreting cells. It is suggested that baicalin and wogonin be further investigated for the possible use as mucoregulators during the treatment of chronic airway diseases.

Notification vs Approval

©2007 Ethis Communications, Inc. The Ocular Surface ISSN: 1542-0124. Novack G. Notification vs approval. 2007;5(3):255-258. hen a pharmaceutical firm wants to begin evaluating their compound in humans in the US, they are required to file an Investigational New Drug (IND) Application (21 CFR 312.1) with the US Food and Drug Administration (FDA). The IND is technically a request for an exemption from US law prohibiting interstate shipment of investigational drugs and assurance that the FDA will not stop (does not reject as unsafe) the initial clinical trial described in the IND. The sponsoring firm provides information on the preclinical biology of the compound (pharmacodynamics, pharmacokinetics, and toxicology), pharmaceutics and manufacturing, and the planned clinical study. Also included is the name and qualifications of the proposed clinical investigator(s). The application must have sufficient safety information at exaggerated doses to allow estimation of the potential benefit-risk ratio. The application is submitted using FDA Form 1571 (http://www.fda.gov/opacom/morechoices/fdaforms). The content of an IND is detailed in 21 CFR 312.23. An interesting, and I believe unique, aspect of the US IND process is that this is a notification (ie, unless we hear from you in 30 days, we will start our clinical study) rather than an approval (ie, we will start the studies after you authorize it; 21 CFR 312.40). This is the difference between the teenager saying “I’m taking the car, bye” and “May I take the car?” With respect to medical devices, the situation has some parallels and some differences from pharmaceuticals. Sponsoring firms wishing to evaluate an investigational medical device in humans in the U.S. must submit an investigational device exemption (IDE), which includes manufacturing and preclinical information on the device. There is also a 30-day review period from the FDA. However, in this case, the sponsor must wait for written approval from the FDA (21 CFR 812.42). Thus, the device review system is an approval, not a notification. There are other categories where only a notification, rather than a prior approval, is required by the FDA. These apply to changes to the formulation or manufacturing process for an approved drug. For some “moderate changes,” the manufacturer may identify these issues as either “Changes Being Effected in 30 Days” (21 CFR 314.70(c)(3)) or “Changes Being Effected” (21 CFR 314.70(c)(7)). In the first case, the FDA has 30 days to contact the sponsor to disallow the change. In the second case, if the FDA disapproves the supplemental application, it may order the manufacturer to cease distribution of the drug product made with the manufacturing change. For some “minor changes,” the manufacturer may make the change and notify the FDA in its next annual report (21 CRF 314.70). Going back to the IND process, as a resident of California, a geographically large state with pharmaceutical manufacturing facilities and experienced clinical investigators, what if I don’t want to technically ship the investigational drug across state lines? Do I still have to file an IND with the FDA? The answer is yes — the California equivalent of the FDA, the Food and Drug Branch (www.dhs.ca.gov/fdb/) will defer an applicant to the federal process. Also of note is that the firm does not have to show that the compound is effective in animal models (eg, this compound cures keratoconjunctivitis in animals), but rather to provide a rationale for the planned human investigation (eg, this compound releases mucin from the conjunctiva, and release of mucin may improve keratoconjunctivitis sicca). Thus, at least for the US, the FDA does not “approve” INDs — they just don’t reject them. In general, the situation is similar in developed nations around the world with respect to the need for application to the regulatory authorities in order to conduct human studies of investigational pharmaceuticals.

Mucin secretion in inflammatory bowel disease: Comparison of a macrophage-derived mucin secretagogue (MMS-68) to conventional secretagogues

We have described a novel macrophage-derived mucin secretagogue (MMS-68) that mediates mucin secretion in colon cancer cell lines and explants of normal and inflammatory bowel disease (IBD) mucosa, we compared MMS-68 induced mucin release with other known intestinal mucin secretagogues in normal colon explants and in the HT-29 colon cancer cell line, and to study the effects of MMS-68 on mucin release from inflamed and uninflamed ulcerative colitis (UC) and Crohn's disease (CD) mucosa. In normal colonic explants and HT-29 cells, each of the secretagogues including MMS-68-induced mucin release two- to fivefold more than culture medium alone. In HT-29 cells, MMS-68 plus leukotriene C4 (LTC4) induced a 50% increase in mucin release over either secretagogue alone, and MMS-68 plus platelet-activating factor (PAF) markedly enhanced mucin release by eightfold over either secretagogue. In colonic explants from patients with UC and CD, the mucin release in response to MMS-68 was similar to that of normal colonic explants. Likewise, in isolated epithelial cells from CD and UC (whether involved or uninvolved), MMS-68-induced release was similar to that of epithelial cells isolated from normal colonic mucosa. The number of MMS-68-producing macrophages was lower in uninflamed UC mucosa compared with inflamed UC mucosa and CD mucosa. The mucin secretagogue activity of MMS-68 is comparable to that of other known secretagogues, and PAF can have a synergistic effect on this activity. Whole tissue explants and isolated colonic epithelial cells from patients with 1BD respond at least as well as their normal counterparts to MMS-68. MMS-68 may play a role in mucin secretion in normal and inflamed colonic tissue.

Potential therapy for mucus hypersecretion in chronic obstructive pulmonary disease

Mucus hypersecretion is a major pathophysiologic feature of chronic bronchitis. Although mucus functions as a barrier and a facilitator of mucociliary clearance, persistent mucus hypersecretion results in airway obstruction and compromised clearance of inhaled bacteria and particles from the airways of patients with chronic obstructive pulmonary disease. Treatment of mucus hypersecretion is a major therapeutic target; however, mechanisms of mucus hypersecretion remain unknown. Herein, we present evidence that human neutrophil elastase (HNE), a pathophysiologically relevant stimulant of mucus hypersecretion in the airways of patients with chronic bronchitis, provokes release of mucin (the glycoprotein component of mucus) by human airway epithelial cells in vitro. Signaling molecules involved in HNE-induced mucin hypersecretion include protein kinase C, specifically the delta isoform, and the myristoylated alanine-rich C kinase substrate protein. These molecules represent potential therapeutic targets for r...

Signaling pathways involved in pilocarpine-induced mucin secretion in rat submandibular glands

We have studied the signaling pathways involved in pilocarpine-induced mucin release in rat submandibular slices. Pilocarpine produced a significant increment of PGE 2 levels and a positive ( r = 0.8870) and significant ( p = 0.0077) correlation between PGE 2 production and mucin released was determined. The participation of PGE 2 was confirmed by the use of indomethacin (indo) and of acetyl salicylic acid (ASA), cyclooxygenase inhibitors, which inhibited pilocarpine-induced mucin release. The muscarinic receptors involved in the regulation of mucin release were identified as M 1 and M 4 by the use of the selective acetylcholine receptors (mAChR) antagonists, pirenzepine, AF-DX 116, 4-DAMP and tropicamide. The secretory process was dependent on both, intracellular and extracellular calcium pools since it was inhibited by thapsigargin and verapamil. Cyclic AMP, nitric oxide synthase and PKC also participated in pilocarpine-induced mucin release. It is concluded that pilocarpine, by activation the M 1 and M 4 mAChR subtypes induces an increase of intracellular Ca 2+ concentration ([Ca 2+] I) and elevates cAMP levels, which in turn stimulates COX, PKC and NOS and promotes mucin exocytosis. PGE 2 released induces cAMP accumulation which, together with PKC are involved in the PGE 2 increased Ca 2+/cAMP-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE 2 production.

A Peptide Against the N-Terminus of Myristoylated Alanine-Rich C Kinase Substrate Inhibits Degranulation of Human LeukocytesIn Vitro

Leukocytes synthesize a variety of inflammatory mediators that are packaged and stored in the cytoplasm within membrane-bound granules. Upon stimulation, the cells secrete the granule contents via an exocytotic process whereby the granules translocate to the cell periphery, the granule membranes fuse with the plasma membrane, and the granule contents are released extracellularly. We have reported previously that another exocytotic process, release of mucin by secretory cells of the airway epithelium, is regulated by the myristoylated alanine-rich C kinase substrate (MARCKS) (Li Y, Martin LD, Spizz G, Adler KB. MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro. J Biol Chem 2001;276:40982-40990; Singer M, Martin LD, Vargaftig BB, Park J, Gruber AD, Li Y, Adler KB. A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma. Nat Med 2004;10:193-196). In those studies, mucin secretion in vitro and in vivo was attenuated by a synthetic peptide identical to the N-terminus of MARCKS, named the MANS peptide (Li and colleagues, 2001). In this study, we used the MANS peptide to investigate possible involvement of MARCKS in secretion of leukocyte granule proteins. In neutrophils isolated from human blood, phorbol 12-myristate 13-acetate-induced myeloperoxidase release was attenuated in a concentration-dependent manner by MANS but not by equal concentrations of a missense control peptide. In additional studies using human leukocyte cell lines, secretion of eosinophil peroxidase from the eosinophil-like cell line HL-60 clone 15, lysozyme from the monocytic leukemia cell line U937, and granzyme from the lymphocyte natural killer cell line NK-92 were attenuated by preincubation of the cells with MANS but not with the missense control peptide. The results indicate that MARCKS protein may play an important role in the secretion of membrane-bound granules from different leukocytes. MARCKS may be an important component of secretory pathways associated with release of granules by different cell types.

Human Neutrophil Elastase Induces Hypersecretion of Mucin from Well-Differentiated Human Bronchial Epithelial Cells in Vitro via a Protein Kinase Cδ-Mediated Mechanism

The presence of mucus obstruction and neutrophil-predominant inflammation in several lung disorders, such as cystic fibrosis, suggests a relationship between neutrophils and excess mucus production. Mechanisms of human neutrophil elastase (HNE)-induced mucin secretion by well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture were investigated. HNE increased mucin secretion in a concentration-dependent manner, with maximal stimulation (more than twofold) occurring within a short (15 minutes) time period. Mucins MUC 5 AC and MUC 5 B, but not MUC 2, were released in response to HNE. Stimulation of mucin secretion required partial elastase enzymatic activity and did not appear to involve a soluble product released by the cells. HNE-stimulated secretion involved activation of protein kinase C (PKC), as HNE exposure rapidly provoked PKC enzymatic activity that was attenuated by the general PKC inhibitors calphostin C and bisindoylmaleimide I. Of the different isoforms, PKCalpha, delta, zeta, lambda, iota, and epsilon were constitutively expressed in NHBE cells while PKCbeta, eta, and mu were PMA-inducible. PKCdelta was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCdelta attenuated HNE-mediated mucin secretion. The results suggest HNE stimulation of mucin release by human airway epithelial cells involves intracellular activation of PKC, specifically the delta isoform.