Stimulate Mucin Release Research Articles

Tetraspanin-8 sequesters syntaxin-2 to control biphasic release propensity of mucin granules

Agonist-mediated stimulated pathway of mucin and insulin release are biphasic in which rapid fusion of pre-docked granules is followed by slow docking and fusion of granules from the reserve pool. Here, based on a cell-culture system, we show that plasma membrane-located tetraspanin-8 sequesters syntaxin-2 to control mucin release. Tetraspanin-8 affects fusion of granules during the second phase of stimulated mucin release. The tetraspanin-8/syntaxin-2 complex does not contain VAMP-8, which functions with syntaxin-2 to mediate granule fusion. We suggest that by sequestering syntaxin-2, tetraspanin-8 prevents docking of granules from the reserve pool. In the absence of tetraspanin-8, more syntaxin-2 is available for docking and fusion of granules and thus doubles the quantities of mucins secreted. This principle also applies to insulin release and we suggest a cell type specific Tetraspanin/Syntaxin combination is a general mechanism regulating the fusion of dense core granules.

Gastric Cytoprotection as Basis of Gastrointestinal Mucosa Protection and Repair in Erosive Ulcerative Lesions of Various Aetiologies

Aim.Assessment of efficacy and the mechanism of action of gastrointestinal mucosa (GM) protection in current treatment settings with methylmethionine-sulfonium chloride (vitamin U) to illustrate its applicability in erosive ulcerative lesions of various aetiologies.Key points.Aside to damage prevention in exposure to aggressive agents, gastroprotection implies healing promotion under the preserved level of hydrochloric acid secretion. Prostaglandins (PG) and SH-antioxidants are key mediators of gastroprotection in acute and chronic damage. SH-containing endogenous substances (L-cysteine, D,L-methionine, GSH) and exogenous molecules (methylmethionine-sulfonium chloride (MMSC), N-acetylcysteine) prevent damage due to the ability to absorb/neutralise free radicals released in xenobiotic-triggered cell damage, inhibit TNF-α expression, reduce the aspirin-induced leukocyte-endothelium adhesion and stimulate mucin release. In experiment, MMSC prevented the ethanol-induced GM damage, stimulated mucin release and its redistribution on the GM surface; in clinical trials, MMSC effectively facilitated remission in duodenal ulcer.Conclusion.Preparations exerting a protective effect on gastroduodenal mucosa, such as methylmethionine-sulfonium chloride (vitamin U), may improve basic treatment settings and facilitate remission in erosive ulcerative lesions of upper gastrointestinal tract.

Comparative Evaluation of the Antimicrobial and Mucus Induction Properties of Selected Bacillus Strains against Enterotoxigenic Escherichia coli.

Probiotics have been shown to bind to host receptors, which are important for pathogen adhesion and induce the host’s production of defence factors. They can activate the goblet-cell-derived production of mucins, a major component of the mucus layer and a physical barrier participating in limiting the proximity of microorganisms to the epithelial layer. In the last decade, Bacillus spp. strains have gained interest in human and animal health due to their tolerance and stability under gastrointestinal tract conditions. Moreover, Bacillus spp. strains can also produce various antimicrobial peptides that can support their use as commercial probiotic supplements and functional foods. The present study aimed to evaluate and determine the ability of selected Bacillus spp. strains to inhibit the growth of enterotoxigenic Escherichia coli (ETEC) F4 and to reduce binding of ETEC F4 to HT29-16E (mucus-secreting and goblet-like) human intestinal cells. Moreover, mucus production in the HT29 cells in the presence of the Bacillus spp. strains was quantified by ELISA. Bacillus spp. strains (CHCC 15076, CHCC 15516, CHCC 15541, and CHCC 16872) significantly inhibited the growth of ETEC F4. Moreover, the ability of the probiotic Bacillus spp. strains to stimulate mucin release was highly strain dependent. The treatment with Bacillus subtilis CHCC 15541 resulted in a significant increase of both MUC2 and MUC3 in HT29-16E cells. Therefore, this strain could be an up-and-coming candidate for developing commercial probiotic supplements to prevent infections caused by ETEC F4 and, potentially, other pathogens.

Lubiprostone stimulates small intestinal mucin release

BackgroundLubiprostone 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.MethodsMucin 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.ResultsWhen 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.ConclusionsThese 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.

Effects of Ursolic Acid, Betulin and Sulfur-Containing Compounds on Mucin Release from Airway Goblet Cells

In this study, we investigated whether ursolic acid, betulin and 2 kinds of sulfur-containing compounds--NAC and MESNA--affect mucin release from airway goblet cells and compared the possible activities of these agents with the inhibitory action on mucin release by PLL and the stimulatory action by ATP. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled using 3H-glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of each agent to assess the effects on 3H-mucin release. The results were as follows: ursolic acid, betulin, MESNA and NAC increased mucin release (40-50 % above control) at the highest concentrations (10(-5) M-10(-3) M). We conclude that ursolic acid and betulin can stimulate mucin release by directly acting on airway mucin-secreting cells and suggest that these agents be further investigated for the possible use as mucoregulators in the treatment of chronic airway diseases.

Effects of activation of polymorphonuclear leukocytes on airway goblet cell mucin release in a co-culture system.

Effects of activated PMN on airway goblet cell mucin release were investigated using a co-culture system in which both tracheal surface epithelial (TSE) cells and PMN from hamsters were present. TSE cells were metabolically labeled using (3)H-glucosamine and chased in the presence of PMN under various experimental designs. PMN were obtained from the bronchoalveolar lavage fluid of hamsters following intratracheal instillation of E. coli endotoxin. The amount of (3)H-mucin was measured by Sepharose CL-4B gel-filtration column chromatography. (i). activation of 10(6) PMN by fMLP (0.1 microM) and cytochalasin B (0.1 microM) resulted in production of both the estrolytic ("elastolytic") activity and superoxide, (ii). activation of PMN in the co-culture stimulated mucin release from TSE cells followed by a significant degradation of the released mucins, both of which were blocked in a dose-dependent fashion by pretreatment with alpha1-protease inhibitor, and (iii). generation of varying concentrations of superoxide in the TSE cell culture did not affect mucin release from TSE cells. In the co-culture system, activation of PMN results in release and degradation of mucins, both of which are almost entirely accounted for by serine proteases but not other cellular products such as superoxide.

Effects of polycationic peptides on mucin release from airway goblet cells: relationship between polymer size and activity.

Various sizes of poly-L-lysine (PLL) and poly-L-arginine (PLA) were tested for their possible effects on airway goblet cell mucin release using primary hamster tracheal surface epithelial (HTSE) cells in an attempt to identify the smallest size of the polycationic peptide to suppress mucin release without cytotoxicity. HTSE cells were metabolically labeled using 3H-glucosamine and chased in the presence of varying concentrations of various sizes of the polycationic peptides. The amount of 3H-mucin in the spent media was measured by Sepharose CL-4B gel-filtration column chromatography. Possible cytotoxicity of the peptides was assessed by measuring the release of lactic dehydrogenase (LDH) during the treatment period. (1) PLL (MW 78,000) inhibited whereas PLA (MW 92,000) stimulated mucin release. However, these peptides were cytotoxic at the effective concentrations; (2) Both PLL (MW 9,600) and PLA (MW 8,900) could inhibit mucin release in a dose dependent manner without cytotoxicity; (3) Both PLL and PLA were effective in suppressing mucin release in 20-mer but not in either 10-mer or 5-mer; (4) 14-mers of both PLL and PLA also inhibited mucin release without cytotoxicity; (5) PLL and poly-D-lysine (PDL) of 14-mer were equipotent in its ability to suppress mucin release. Both PLL and PLA are cytotoxic at 'high' molecular weights, but have an ability to suppress mucin release without cytotoxicity at 'low' molecular weights. 14-mer seems to be the small, effective size, if not the smallest, for both PLL and PLA to suppress mucin release without cytotoxicity. The inhibitory effect of these polycationic peptides seems to be determined by the presence and the absolute number of positive charges and also to be independent of optical isomerism.

Selective Involvement of Calcium and Calcium Channels in Stimulated Mucin Secretion from Rat Colon

Background: The cellular mechanisms involved in the mucin secretion of rat colon are unknown. The objective of the present study was thus to determine the role of extracellular calcium and of L-type calcium channels in rat intestinal mucin discharge. Methods: The experiments were conducted using the isolated vascularly perfused rat colon. Mucin secretion was evaluated using an enzyme-linked immunosorbent assay. Results: Intra-arterial bethanechol (200 μM) or luminal deoxycholate (5 mM) produced a significant mucin discharge (609% and 386% of controls, respectively). The colonic mucin output induced by these two secretagogues was significantly inhibited by arterial administration of EGTA (2 mM), verapamil (100 μM) or nifedipine (50 μM). In contrast, luminal EGTA (2 mM) had no inhibitory effect. Intra-arterial infusion of the calmodulin antagonist trifluoperazine (10 μM) also reduced mucin discharge induced by bethanechol or deoxycholate (304% and 223% of controls, respectively). Colonic mucin secretion was significantly stimulated after intra-arterial infusion of 3-isobutyl-methylxanthine (IBMX, 100 μM) or forskolin (2-20 μM). Stimulation by forskolin was unaffected by arterial EGTA, verapamil, nifedipine or trifluoperazine. Conclusion: In the isolated vascularly perfused rat colon, mucin discharge induced by bethanechol or deoxycholate requires extracellular calcium and the activation of voltage-dependent calcium channels of L-type. In contrast, forskolin does not appear to stimulate mucin release by increasing calcium entry.

ATP-induced mucin release from cultured airway goblet cell involves, in part, activation of phospholipase A2.

Adenosine triphosphate (ATP) has been shown to stimulate mucin release by activation of protein kinase C (PKC) following activation of phospholipase C (PLC) coupled to the P2 receptor via G-proteins. The aim of the present study was to investigate pathways downstream to the PKC activation in ATP-induced mucin release from primary hamster tracheal surface epithelial (HTSE) cells. The release of mucin was determined by chromatographic procedure after metabolic labeling of mucin with [3H]-glucosamine. The results were: i) ATP induced the release of arachidonic acid, which caused the release of mucin. Pretreatment with mepacrine (0.3 mM), a phospholipase A2 (PLA2) inhibitor, inhibited the ATP-induced arachidonic acid and mucin release. Oleoyloxyethylphosphocholine, another PLA2 inhibitor, gave similar results. ii) An activator of PKC, 4 beta-phorbol-12 alpha-myristate-13-acetate (PMA, 1 microM) induced mucin release, which was inhibited by mepacrine pretreatment. iii) Downregulation of PKC by prolonged (16 h) PMA treatment caused inhibition of ATP-induced mucin release. Treatment of PKC downregulated HTSE cells with mepacrine did not further decrease the ATP-induced mucin release. These results suggest that PLA2 is involved in ATP-induced mucin release and its activation is sequential to the PLC-PKC pathway.

Mechanism of Calcium on ATP–and Bradykinin-induced Mucin Secretion from Human Pulmonary Mucoepidermoid Carcinoma Cell Lines

We have shown previously that extracellular ATP and bradykinin can stimulate the release of mucin from cultured human pulmonary mucoepidermoid carcinoma cells (NCI-H292) (Kusano et al 1997). Using this same cell culture system, we studied the mechanism by which this ATP- and bradykinin-induced mucin release occurs. We found that the mucin secretory response to ATP was not significantly affected by extracellular Ca2+ depletion, but the response to bradykinin was significantly inhibited by Ca2+-free medium. ATP and bradykinin increased the concentration of free intracellular Ca2+ ([Ca2+]i); the ATP-stimulated response was not affected by extracellular Ca2+ depletion, the bradykinin-stimulated response was prevented in the absence of extracellular Ca2+. ATP and bradykinin enhanced the accumulation of inositol 1,4,5-triphosphate in NCI-H292 cells and pretreatment with pertussis toxin blocked the increase in mucin secretion induced by extracellular ATP or bradykinin. These results suggest that extracellular ATP and bradykinin stimulate mucin release from NCI-H292 cells by a signal transduction that seems to involve ATP- or bradykinin-activated receptors associated with phospholipase C via pertussis toxin-sensitive GTP-binding proteins. These findings may suggest a new direction of research into the regulation of airway mucin secretion.

INHALED ATP CAUSES MUCIN RELEASE FROM GOBLET CELLS OF INTACT RATS

Secretion of mucins from airway epithelial cells has been studied almost exclusively using in vitro cell culture systems. Our understanding of in vivo secretion is greatly limited due to the unavailability of both suitable model systems and adequate assays. It has been reported that ATP induces mucin release from the cultured primary tracheal surface epithelial cell, but there is no clear demonstration of the effect of ATP on mucin release in vivo, which is important to understand the mechanism of mucin release in vivo and also to devise means for regulation of mucin release. The objective of this experiment was to see if inhaled ATP could stimulate airway mucin release in intact rats using both enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. The results were: (1) a new monoclonal antibody (mAbRT03) developed against purified rat mucins specifically recognized high-molecular-mass mucins; (2) ELISA results with conventional gel-filtration assay results are virtually superimposable; (3) inhalation of ATP in intact rats resulted in a dose-independent increase in the amount of mucins in the tracheal lavage fluid with a concomitant decrease in the number of mucin-positive cells in the trachea. We conclude that extracellular ATP can stimulate mucin release from the airway in vivo, and the present rat inhalation system combined with ELISA of the airway secretions should serve a useful model for studying the pharmacology of airway mucin secretion in vivo.

ATP-induced mucin release from cultured airway goblet cells involves, in part, activation of protein kinase C.

Extracellular nucleotides stimulate mucin release by binding to the P2u receptor coupled to phospholipase C via G proteins (Br. J. Pharmacol. 103:1053-1056, 1991; Am. J. Respir. Cell Mol. Biol. 8:121-125, 1993). In the present study, we intended to investigate pathways downstream to the phospholipase C activation which is responsible for adenosine triphosphate (ATP)-induced mucin release in hamster tracheal epithelial cells in primary culture. We have found that: (1) Ca2+ ionophores (A23187 and ionomycin) did not affect mucin release even at 1 microM; (2) thapsigargin (10 microM), either alone or in combination with ATP (20 microM), did not enhance mucin release over its respective control group; (3) pretreatment of hamster tracheal surface epithelial (HTSE) cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) (50 microM) did not inhibit ATP-induced mucin release; (4) 4beta-phorbol 12alpha-myristate 13-acetate (PMA, 1 microM) stimulated mucin release and its effect was completely blocked by protein kinase C inhibitors such as sphingosine (10 microM) and calphostin C (0.1 microM), whereas ATP-induced mucin release was blocked, only in part, by these inhibitors; (5) desensitization of protein kinase C by pretreatment with PMA inhibited the PMA-induced mucin release completely, however, ATP-induced mucin release was inhibited only partially. We conclude that mucin release by ATP does not require an increase in the intracellular Ca2+ level but involves the activation of protein kinase C. The results also suggest the presence of another mechanism separate from the phospholipase C-protein kinase C pathway for the ATP-induced mucin release.

Nucleotide-induced mucin release from primary hamster tracheal surface epithelial cells involves the P2u purinoceptor

Mucin release by airway surface epithelial cells is regulated by extracellular adenosine triphosphate (ATP) via a P2 purinoceptor-mediated mechanism. The objective of the present experiment was to examine the possible involvement of uridine triphosphate (UTP) in this purinergic signal transduction pathway. Using primary hamster tracheal surface epithelial cells, ATP and UTP were compared in their abilities: 1) to displace ATP gamma S35-binding to intact cells; 2) to accumulate inositol phosphates; and 3) to stimulate mucin release. Finally, the presence of a P2u receptor message was examined. Our results showed that: 1) UTP was much less effective than ATP in displacing ATP gamma S35-binding (median inhibitory concentrations (IC50S) 240 vs 2.9 microM); 2) UTP was more potent than ATP in accumulating inositol phosphates (100 vs 43% increase at 2mM); 3) UTP was equipotent with ATP in stimulating mucin release; 4) Northern blot analysis of messenger ribonucleic acids (mRNAs) with a mouse P2u receptor complementary deoxyribonucleic acid (cDNA) probe revealed a single specific band (2.8 kb), partial sequencing of which showed a great homology with those of human or mouse P2u receptors. We conclude that, although both ATP and UTP are equipotent in stimulating mucin release, their binding kinetics to the cell surface are quite different, suggesting the presence of a common binding domain which may be responsible for the mucin release by these nucleotides. We suggest that the P2u purinoceptor is likely to be responsible for mucin release by these nucleotides, probably via activation of phospholipase C.

Binding kinetics of ATP gamma S35 on cultured primary tracheal surface epithelial cells.

Extracellular ATP can stimulate mucin release from primary hamster tracheal surface epithelial (HTSE) cells via a P2 purinoceptor-mediated mechanism, based on agonist potency studies of mucin release (Br. J. Pharmacol. 1991; 103:1053-1056). In the present study, we examined the kinetics of ATP binding to the surface of intact HTSE cells at 4 degrees C using ATP gamma S35 as a radioligand. We found that ATP gamma S35 bound to HTSE cells in a saturable, reversible manner, reaching an equilibrium at about 30 min. Scatchard analysis of equilibrium binding suggested the presence of two binding sites with Kd values of 0.47 and 9.4 microM. Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block ATP gamma S35 revealed a rank order of ATP > ADP > alpha,beta-methylene ATP > 2-methylthio ATP > or = beta, gamma-methylene ATP. Neither AMP nor adenosine could inhibit the ATP gamma S35 binding. A comparison between the ability of nucleotides to compete with ATP gamma S35 binding and their ability to induce mucin release revealed a rather poor correlation (r2 = 0.67) with all of the above nucleotides but a good correlation (r2 = 0.96) without 2-methylthio ATP, indicating the presence of heterogenous ATP binding sites on the HTSE cell surface. UTP, a pyrimidine nucleotide, which is almost equipotent with ATP in its ability to stimulate mucin release, was much less potent than ATP in its ability to displace the ATP gamma S35 binding in these HTSE cells.

Effect of floating a gel matrix on mucin release in cultured airway epithelial cells.

Confluent cultures of primary hamster tracheal surface epithelial (HTSE) cells grown on a thick collagen gel are highly enriched with secretory cells and constitutively release mucins. In the present experiment, we examined the possible effect of mechanical strain of cultured HTSE cells on the release of mucin. The mechanical strain of cells was accomplished by several methods: 1) by floating the gel from the culture dish by rimming; 2) by treatment with EGTA which interrupts intercellular tight junctions; 3) by treatment with collagenase which disrupts the cell-matrix adhesion; and 4) by mechanically flexing the collagen gel matrix. All these conditions caused increases of mucin release without damage on the plasma membrane. We conclude that a number of mechanical strains which might alter cell shape can stimulate mucin release from cultured HTSE cells. Such a mechanism might be operative in the physiological regulation of airway goblet cell mucin secretion where mechanical strains may be induced on epithelial cells by underlying smooth muscles.

Effect of sucralfate on components of mucosal barrier produced by cultured canine epithelial cellsin vitro

The mucous gel maintains a neutral microclimate at the epithelial cell surface, which may play a role in both the prevention of gastroduodenal injury and the provision of an environment essential for epithelial restitution and regeneration after injury. Enhancement of the components of the mucous barrier by sucralfate may explain its therapeutic efficacy for upper gastrointestinal tract protection, repair, and healing. We studied the effect of sucralfate and its major soluble component, sucrose octasulfate (SOS), on the synthesis and release of gastric mucin and surface active phospholipid, utilizing an isolated canine gastric mucous cells in culture. We correlated these results with the effect of the agents on mucin synthesis and secretion utilizing explants of canine fundus in vitro. Sucralfate and SOS significantly stimulated phospholipid secretion by isolated canine mucous cells in culture (123% and 112% of control, respectively). Indomethacin pretreatment significantly inhibited the effect of sucralfate, but not SOS, on the stimulation of phospholipid release. Administration of either sucralfate or SOS to the isolated canine mucous cells had no effect upon mucin synthesis or secretion using a sensitive immunoassay. Sucralfate and SOS did not stimulate mucin release in the canine explants; sucralfate significantly stimulated the synthesis of mucin, but only to 108% of that observed in untreated explants. No increase in PGE2 release was observed after sucralfate or SOS exposure to the isolated canine mucous cells. Our results suggest sucralfate affects the mucous barrier largely in a qualitative manner. No increase in mucin secretion or major effect on synthesis was noted, although a significant increase in surface active phospholipid release was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

P2 purinoceptor regulation of mucin release by airway goblet cells in primary culture

1. The effects of adenine analogues on mucin release by airway goblet cells have been examined in a hamster primary tracheal epithelial cell culture. 2. Adenosine, a P1 receptor agonist, had no effect on mucin release even at 2 mM, while ATP, a P2 receptor agonist, stimulated mucin release in a dose-dependent fashion with an apparent EC50 of 20 microM. The relative potency order among adenine nucleotides was ATP greater than ADP greater than AMP = adenosine. 3. ATP gamma S, a non-hydrolyzable analogue of ATP, was equipotent with ATP in stimulating mucin release. The potency order among some ATP analogues was ATP greater than 2-methylthio ATP greater than alpha, beta-methylene ATP greater than beta, gamma-methylene ATP. Reactive blue 2, a putative P2 gamma-purinoceptor antagonist, did not block the ATP-induced mucin release. 4. The present results indicate that mucin release by airway goblet cells is stimulated by extracellular ATP via P2 receptor-mediated mechanism. We suggest that this mechanism may be important in the physiological regulation of airway goblet cell mucin release in vivo.

The Relationship between Proteases Activity and Glycoprotein Levels in Middle Ear Effusions from Experimental Otitis Media in Cats

The relationship between lysosomal proteases activity (elastase and cathepsin B) and levels of mucous glycoproteins in middle ear effusions (MEEs) was studied using a cat model of otitis media with effusion (OME) induced by Eustachian tube obstruction (ETO). The ratio of cathepsin B activity to total protein concentration (TPC) in MEE was 25.6 +/- 19.4 RFU/g x dl-1 at 1 week after ETO, and increased with the duration of OME. The ratio of elastase activity to TPC had a significant correlation to total leukocyte count. The ratio of fucose levels to TPC, which is one of the parameters reflecting levels of mucous glycoprotein, at 1 week after ETO was significantly higher than that at both 2 and 4 weeks after ETO. The percentage of glycoprotein levels absorbed to wheat germ lectin was highest at 1 week after ETO, and decreased with the duration of OME. In conclusion, mucous glycoproteins in cat occupy a larger portion of glycoproteins in MEE at the early stage of OME, and elastase and other lysosomal proteases may play a role in both stimulation of mucin release from goblet cells and mucin degradation. The balance of these processes seems to be a key factor determining mucin levels in MEEs.

Calcium dependency of adrenergic and muscarinic cholinergic stimulation of mucin release from dog submandibular gland cells

Stimulation of muscarinic cholinergic, α-adrenergic and β-adrenergic receptors elicited mucin release from dispersed dog submandibular cells. The secretory response to acetylcholine was much more pronounced than to adrenergic agonists, and largely dependent on the presence of extracellular Ca 2+, but the dependency on extracellular Na + was slight. Ionomycin also stimulated mucin release. In rat submandibular cells, neither muscarinic cholinergic agonists nor ionomycin were as effective mucosecretagogues as β-adrenergic agonists. α-Adrenoceptor-mediated release was decreased by chelating extracellular Ca 2+ with EGTA. The β-adrenoceptor-mediated response was diminished by extensive exposure of cells to EGTA, due at least in part to the requirement of Ca 2+ for β-adrenoceptor stimulation of cAMP formation. 8-br-cAMP stimulated 45Ca 2+ release from cells preloaded with 45Ca 2+. The 8-br-cAMPinduced mucin release was eliminated in ionomycin-pretreated cells, but not inhibited by chelating extracellular Ca 2+ and by the treatment of the cells with TMB-8 or in the cells loaded with BAPTA. These results suggest that not only the adrenergic system but also the muscarinic cholinergic system may participate in the regulation of mucin release in dog submandibular gland, and also provide the possibility that, in addition to a cAMP-mediated mechanism, Ca 2+-dependent mechanisms may be involved in mucosecretion in dog submandibular acini.

The T84 human colonic adenocarcinoma cell line produces mucin in culture and releases it in response to various secretagogues

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.