SNAP23 is selectively expressed in airway secretory cells and mediates baseline and stimulated mucin secretion

Airway mucin secretion is important pathophysiologically and as a model of polarized epithelial regulated exocytosis. We find the trafficking protein, SNAP23 (23-kDa paralogue of synaptosome-associated protein of 25 kDa), selectively expressed in secretory cells compared with ciliated and basal cells of airway epithelium by immunohistochemistry and FACS, suggesting that SNAP23 functions in regulated but not constitutive epithelial secretion. Heterozygous SNAP23 deletant mutant mice show spontaneous accumulation of intracellular mucin, indicating a defect in baseline secretion. However mucins are released from perfused tracheas of mutant and wild-type (WT) mice at the same rate, suggesting that increased intracellular stores balance reduced release efficiency to yield a fully compensated baseline steady state. In contrast, acute stimulated release of intracellular mucin from mutant mice is impaired whether measured by a static imaging assay 5 min after exposure to the secretagogue ATP or by kinetic analysis of mucins released from perfused tracheas during the first 10 min of ATP exposure. Together, these data indicate that increased intracellular stores cannot fully compensate for the defect in release efficiency during intense stimulation. The lungs of mutant mice develop normally and clear bacteria and instilled polystyrene beads comparable to WT mice, consistent with these functions depending on baseline secretion that is fully compensated.     

nPKCepsilon, 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.     

Evidence of Mucin Secretion in Human Lung Adenocarcinoma Cell Lines NCIH650 and NCIH2077 and Effect of Select Secretagogues on Mucin Secretion

Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor ³H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.     

Munc18 and Munc13 regulate early neurite outgrowth

Background information. During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N‐ethylmaleimide‐sensitive fusion protein‐attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth. Results. We tested this possibility in dissociated cell cultures and organotypic slice cultures of two release‐deficient mice (Munc18‐1 null and Munc13‐1/2 double null). Both types of release‐deficient neurons have a decreased outgrowth speed and therefore have a smaller total neurite length during early development [DIV1–4 (day in vitro 1–4)]. In addition, more filopodia per growth cone were observed in Munc18‐1 null, but not WT (wild‐type) or Munc13‐1/2 double null neurons. The smaller total neurite length during early development was no longer observed after synaptogenesis (DIV14–23). Conclusion. These data suggest that the inability of vesicle fusion in the growth cone affects outgrowth during the initial phases when outgrowth speed is high, but not during/after synaptogenesis. Overall, the outgrowth speed is probably not rate‐limiting during neuronal network formation, at least in vitro. In addition, Munc18, but not Munc13, regulates growth cone filopodia, potentially via its previously observed effect on filamentous actin.     

Mucin complexes: characterization of the "link" component of submandibular mucus glycoprotein.

1. Analysis of the submandibular saliva revealed that the secretion consists of mucin complexed with 150 kDa fibronectin fragment and DNA. 2. The kallikreins, secreted by the submandibular gland, appear to be responsible for the fibronectin fragmentation, since an identical peptide was also generated when fibronectin was subjected to incubation with the submandibular saliva or the purified enzyme. 3. The results provide evidence that the 150 kDa glycopeptide so-called salivary mucin "link" component is neither an integral part of the mucin molecule, nor linked to mucin subunits by disulfide bonds, but is a fibronectin fragment which associates with mucin. 4. Using mucin monoclonal antibody (3G12), it was revealed that the nonglycosylated (naked) 8-12 kDa fragment of the mucin molecule is responsible for the interaction of mucin with other components of saliva. 5. Under physiological conditions, the interaction of mucin with fibronectin on the luminal surfaces may be relevant in building mucous barrier and protection of the delicate oral epithelium from damage.     

Inhibition of mucin secretion with MARCKS-related peptide improves airway obstruction in a mouse model of asthma.

Allergic asthma is associated with airway epithelial cell mucous metaplasia and mucin hypersecretion, but the consequences of mucin hypersecretion on airway function are unclear. Recently, a peptide derived from the myristoylated alanine-rich C kinase substrate protein NH(2)-terminal sequence (MANS) was shown to inhibit methacholine (MCh)-induced mucin secretion from airway mucous cells by >90%. We studied the effect of intranasal pretreatment with this peptide on specific airway conductance (sGaw) during challenge with MCh in mice with allergen-induced mucous cell metaplasia. sGaw was noninvasively measured in spontaneously breathing restrained mice, using a double-chamber plethysmograph. Pretreatment with MANS peptide, but not a control peptide [random NH(2)-terminal sequence (RNS)], resulted in partial inhibition of the fall in sGaw induced by 60 mM MCh (mean +/- SE; baseline 1.15 +/- 0.06; MANS/MCh 0.82 +/- 0.05; RNS/MCh 0.55 +/- 0.05 cmH(2)O/s). The protective effect of MANS was also seen in mice challenged with allergen for 3 consecutive days to increase airway hyperresponsiveness, although the degree of protection was less (baseline 1.1 +/- 0.08; MANS/MCh, 0.65 +/- 0.06; RNS/MCh 0.47 +/- 0.03 cmH(2)O/s). Because routine sGaw measurement in mice includes nasal airways, the effectiveness of MANS was also confirmed in mice breathing through their mouths after nasal occlusion (baseline 0.92 +/- 0.05; MANS/MCh 0.83 +/- 0.06; RNS/MCh 0.61 +/- 0.03 cmH(2)O/s). In all instances, sGaw in the MANS-pretreated group was approximately 35% higher than in RNS-treated controls, and mucous obstruction accounted for approximately 50% of the MCh-induced fall in sGaw. In summary, mucin secretion has a significant role in airway obstruction in a mouse model of allergic asthma, and strategies to inhibit mucin secretion merit further investigation.     

Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA

SPOC1 cells, which are a mucin-secreting model of rat airway goblet cells, possess a luminal P2Y2 purinoceptor through which UTP, ATP, and ATPgammaS stimulate secretion with EC50 values of approximately 3 microM. PMA elicits mucin secretion with high EC50 (75 nM) and saturation (300 nM) values. For the first time in airway mucin-secreting cells, the PKC isoforms expressed and activated by a secretagogue were determined using RT-PCR/restriction-enzyme mapping and Western blotting. Five isoforms were expressed: cPKCalpha, nPKCdelta and -eta, and aPKCzeta and -iota/lambda. PMA caused cPKCalpha and nPKCdelta to translocate to the membrane fraction of SPOC1 cells; only nPKCdelta so responded to ATPgammaS. Membrane-associated nPKCdelta and mucin secretion increased in parallel with ATPgammaS concentration and yielded EC50 values of 2-3 microM and maximum values of 100 microM. Effects of PMA to increase membrane-associated cPKCalpha and nPKCdelta saturated at 30 nM, whereas mucin secretion saturated at 300 nM, which suggests a significant PKC-independent effect of PMA on mucin secretion. A prime alternate phorbol ester-receptor candidate is the C1-domain protein MUNC13. RT-PCR revealed the expression of ubiquitous (ub)MUNC13-2 and its binding partner, DOC2-gamma. Hence, P2Y2 agonists activate nPKCdelta in SPOC1 cells. PMA activates cPKCalpha and nPKCdelta at high affinity and stimulates a lower affinity PKC-independent pathway that leads to mucin secretion.     

Influence of autonomic agonists on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into submandibular action of the mouse

The influence of isoproterenol and pilocarpine on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into the proteins of the submandibular glands of the mouse has been investigated during a 10 h period. The total uptake of both labelled precursors into the glands was hardly affected by isoproterenol and pilocarpine during the first 2 h of incubation, thereafter both agonists decreased the uptake slightly. The incorporation of [3H]leucine into secreted proteins was largely similar for the control, isoproterenol and pilocarpine during an incubation of 10 h. [14C]ManNAc incorporation showed a lag period of about 2 h and could be observed in the secreted proteins after 2 h. Particularly after 6 h a strong increase was observed for the control and isoproterenol, whereas pilocarpine showed a much lower increase. The secreted protein components were separated by electrophoresis to study the incorporation of the labelled precursors in separate secretory proteins such as submandibular mucin. Apparently, both agonists increased the incorporation of [14C]ManNAc relative to [3H]leucine into submandibular mucin of the mouse. During a period of 10 h the [14C]ManNAc incorporation into the mucin was enhanced 2-3-fold by isoproterenol and 3-4-fold by pilocarpine. A non-radioactive experiment in vitro showed that the molar ratio of the sugar residues did not change. However, the total amount of sugars relative to the amino acids increased by 50%, pointing to an increase in the degree of glycosylation. This suggests that both adrenergic and cholinergic agonists regulate the total number of carbohydrate chains attached to one and the same polypeptide core of the submandibular mucin of the mouse.     

Cystic fibrosis and the relationship between mucin and chloride secretion by cultures of human airway gland mucous cells

We investigated how cystic fibrosis (CF) alters the relationship between Cl(-) and mucin secretion in cultures of non-CF and CF human tracheobronchial gland mucous (HTGM and CFTGM, respectively) cells. Biochemical studies showed that HTMG cells secreted typical airway mucins, and immunohistochemical studies showed that these cells expressed MUC1, MUC4, MUC5B, MUC8, MUC13, MUC16, and MUC20. Effects of cumulative doses of methacholine (MCh), phenylephrine (Phe), isoproterenol (Iso), and ATP on mucin and Cl(-) secretion were studied on HTGM and CFTGM cultures. Baseline mucin secretion was not significantly altered in CFTGM cells, and the increases in mucin secretion induced by mediators were unaltered (Iso, Phe) or slightly decreased (MCh, ATP). Across mediators, there was no correlation between the maximal increases in Cl(-) secretion and mucin secretion. In HTGM cells, the Cl(-) channel blocker, diphenylamine-2-carboxylic acid, greatly inhibited Cl(-) secretion but did not alter mucin release. In HTGM cells, mediators (10(-5) M) increased mucin secretion in the rank order ATP > Phe = Iso > MCh. They increased Cl(-) secretion in the sequence ATP > MCh ≈ Iso > Phe. The responses in Cl(-) secretion to MCh, ATP, and Phe were unaltered by CF, but the response to Iso was greatly reduced. We conclude that mucin secretion by cultures of human tracheobronchial gland cells is independent of Cl(-) secretion, at baseline, and is unaltered in CF; that the ratio of Cl(-) secretion to mucus secretion varies markedly depending on mediator; and that secretions induced by stimulation of β-adrenergic receptors will be abnormally concentrated in CF.     

Influence of autonomic agonists on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into submandibular mucin of the mouse

The influence of isoproterenol and pilocarpine on the in vitro incorporation of [³H]leucine and $\text{N-}\text{acetyl}\text{[}{}^{14}\text{C}\text{]}\text{mannosamine}$ into the proteins of the submandibular glands of the mouse has been investigated during a 10 h period. The total uptake of both labelled precursors into the glands was hardly affected by isoproterenol and pilocarpine during the first 2 h of incubation, thereafter both agonists decreased the uptake slightly. The incorporation of [³H]leucine into secreted proteins was largely similar for the control, isoproterenol and pilocarpine during an incubation of 10 h. [¹⁴C]ManNAc incorporation showed a lag period of about 2 h and could be observed in the secreted proteins after 2 h. Particularly after 6 h a strong increase was observed for the control and isoproterenol, whereas pilocarpine showed a much lower increase. The secreted protein components were separated by electrophoresis to study the incorporation of the labelled precursors in separate secretory proteins such as submandibular mucin. Apparently, both agonists increased the incorporation of [¹⁴C]ManNAc relative to [³H]leucine into submandibular mucin of the mouse. During a period of 10 h the [¹⁴C]ManNAc incorporation into the mucin was enhanced 2–3-fold by isoproterenol and 3–4-fold by pilocarpine. A non-radioactive experiment in vitro showed that the molar ratio of the sugar residues did not change. However, the total amount of sugars relative to the amino acids increased by 50%, pointing to an increase in the degree of glycosylation. This suggests that both adrenergic and cholinergic agonists regulate the total number of carbohydrate chains attached to one and the same polypeptide core of the submandibular mucin of the mouse.     

nPKC{varepsilon}, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y₂ purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP₃), diacylglycerol, Ca²⁺ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC, nPKC, nPKC, and nPKC; of these, only nPKC 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 cPKC, nPKC, and nPKC had the same levels of ATPS- 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 nPKC (14.6 and 23.5%, for ATPS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y₂-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC KO mice relative to its WT littermates. We conclude that nPKC is the effector isoform downstream of P2Y₂-R activation in the goblet cell secretory response. The translocation of nPKC observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology. protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung     

Mucin secretion by the human colon cell line LS174T is regulated by bile salts

We recently reported that bile salts play a role in the regulation of mucin secretion by cultured dog gallbladder epithelial cells. In this study we have examined whether bile salts also influence mucin secretion by the human epithelial colon cell line LS174T. Solutions of bile salts were applied to monolayers of LS174T cells. Mucin secretion was quantified by measuring the secretion of [3H]GlcNAc labeled glycoproteins. Both unconjugated bile salts as well as taurine conjugated bile salts stimulated mucin secretion by the colon cells in a dose-dependent fashion. Hydrophobic bile salts were more potent stimulators than hydrophilic bile salts. Free (unconjugated) bile salts were more stimulatory compared with their taurine conjugated counterparts. Stimulation of mucin secretion by LS174T cells was found to occur at much lower bile salt concentrations than in the experiments with the dog gallbladder epithelial cells. The protein kinase C activators PMA and PDB had no stimulatory effect on mucin secretion. We conclude that mucin secretion by the human colon epithelial cell line LS174T is regulated by bile salts. We suggest that regulation of mucin secretion by bile salts might be a common mechanism, by which different epithelia protect themselves against the detergent action of bile salts, to which they are exposed throughout the gastrointestinal tract.      

Long-term phorbol ester treatment dissociates phospholipase D activation from phosphoinositide hydrolysis and prostacyclin synthesis in endothelial cells stimulated with bradykinin

Bovine pulmonary artery endothelial cells (BPAEC) were prelabeled with [3H]choline or [3H]myristic acid to selectively label endogenous phosphatidylcholine. BPAEC were stimulated with ATP and bradykinin (BK), and phospholipase D (PLD) activation was detected as a 4-fold increase in [3H]choline in cells prelabeled with [3H]choline or as a 2- to 3-fold increase in [3H]phosphatidylethanol in cells prelabeled with [3H]myristic acid and stimulated in the presence of ethanol. Pretreatment of BPAEC with 0.1 microM phorbol 12-myristate 13-acetate (PMA) for 22 hr completely inhibited agonist-induced PLD activation, whereas prostacyclin synthesis and [3H]phosphoinositide ([3H]PIns) hydrolysis were enhanced in pretreated cells. Long-term PMA treatment thus dissociates agonist-induced PLD activation from [3H]PIns hydrolysis, and agonist-induced prostacyclin synthesis is not dependent upon PLD activation.     

20-HETE对小鼠葡萄糖刺激胰岛素分泌反应的影响

为了考察20-羟基二十碳四烯酸(20-hydroxyeicosatetraenoic acids, 20-HETE)对葡萄糖刺激胰岛素分泌反应的影响,本研究选择CYP4F2转基因小鼠和小鼠胰岛素瘤INS-1E细胞作为研究材料,通过LCMS/MS检测WT和TG小鼠的胰腺20-HETE水平。通过IPGTT测定小鼠葡萄糖耐量,通过ELISA测定小鼠血浆C肽水平来检测胰岛素分泌。通过Western blotting、Real time PCR、免疫组化和免疫荧光来检测小鼠胰腺或INS-1E细胞中Glut2、GSK-3β(Ser9点)和AKT (Ser473点)的磷酸化水平。TG小鼠的20-HETE水平((7.26±2.03) ng/mg蛋白)显著高于WT小鼠((2.14±0.76) ng/mg蛋白)。在用20-HETE合成的选择性抑制剂HET0016处理后,TG小鼠((0.33±0.07) ng/mg蛋白)和WT小鼠((0.27±0.06) ng/mg蛋白)胰腺组织中的20-HETE水平均急剧降低。给予葡萄糖处理30 min后,TG小鼠的血糖水平均显著高于WT小鼠,而血浆C肽水平显著低于WT小鼠(p<0.05)。与WT小鼠相比,TG小鼠的胰腺组织中Glut2 m RNA和蛋白水平显著降低。与WT小鼠相比,CYP4F2转基因小鼠的GSK-3β和AKT磷酸化均显著降低。20-HETE处理可导致INS-1E细胞中AKT/GSK-3β磷酸化水平和Glut2表达水平显著降低(p<0.05)。此外,用17 mmol/L葡萄糖处理INS-1E细胞1 h,20-HETE处理组的胰岛素分泌显著降低。应用GSK-3β选择性抑制剂TWS119预处理INS-1E细胞3 h后,TWS119 (一种GSK-3β选择性抑制剂)预处理显著逆转了Glut2表达水平的降低以及胰岛素分泌的减少。20-HETE主要通过AKT/GSK-3β信号通路来下调Glut2的表达,进而减弱胰岛素分泌,导致胰岛素分泌功能障碍。     

Mucin release and calcium fluxes in isolated rat submandibular acini.

A method is described for preparing isolated rat submandibular acini by collagenase digestion followed by mechanical dispersion. As assessed by Trypan Blue exclusion, phase contrast microscopy, ATP content and release of mucins and lactate dehydrogenase, the acini are morphologically and functionally intact. Secretory function of isolated acini was similar to that of intact tissue in terms of time-course, dose dependence and degree of stimulation of mucin release by adrenergic secretagogues. Mucin release was increased to the same extent (approx. 3-4-fold) by either isoproterenol or noradrenaline at a maximally effective concentration (10 microM). Stimulation of mucin release by isoproterenol (10 microM), noradrenaline (10 microM) or adrenaline (10 microM) was inhibited by propranolol (30 microM) but not by phentolamine (30 microM). Isoproterenol (10 microM) increased both 45Ca2+ uptake and efflux from the acini, which was shown to represent a net release of calcium. However, there was a delay (approx. 10 min) in onset of stimulation of 45Ca2+ mobilization which was not apparent in isoproterenol stimulation of mucin release. Our results indicate that increases in intracellular calcium mobilization in response to a beta-adrenergic secretagogue do not trigger mucin secretion from rat submandibular acini.     

Calcium-dependent Activator Protein for Secretion 1 (CAPS1) Binds to Syntaxin-1 in a Distinct Mode from Munc13-1

Calcium-dependent activator protein for secretion 1 (CAPS1) is a multidomain protein containing a Munc13 homology domain 1 (MHD1). Although CAPS1 and Munc13-1 play crucial roles in the priming stage of secretion, their functions are non-redundant. Similar to Munc13-1, CAPS1 binds to syntaxin-1, a key t-SNARE protein in neurosecretion. However, whether CAPS1 interacts with syntaxin-1 in a similar mode to Munc13-1 remains unclear. Here, using yeast two-hybrid assays followed by biochemical binding experiments, we show that the region in CAPS1 consisting of the C-terminal half of the MHD1 with the corresponding C-terminal region can bind to syntaxin-1. Importantly, the binding mode of CAPS1 to syntaxin-1 is distinct from that of Munc13-1; CAPS1 binds to the full-length of cytoplasmic syntaxin-1 with preference to its “open” conformation, whereas Munc13-1 binds to the first 80 N-terminal residues of syntaxin-1. Unexpectedly, the majority of the MHD1 of CAPS1 is dispensable, whereas the C-terminal 69 residues are crucial for the binding to syntaxin-1. Functionally, a C-terminal truncation of 69 or 134 residues in CAPS1 abolishes its ability to reconstitute secretion in permeabilized PC12 cells. Our results reveal a novel mode of binding between CAPS1 and syntaxin-1, which play a crucial role in neurosecretion. We suggest that the distinct binding modes between CAPS1 and Munc13-1 can account for their non-redundant functions in neurosecretion. We also propose that the preferential binding of CAPS1 to open syntaxin-1 can contribute to the stabilization of the open state of syntaxin-1 during its transition from “closed” state to the SNARE complex formation.     

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and beta-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate.

Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.     

Regulation of insulin exocytosis by Munc13-1

The slower kinetics of insulin release from pancreatic islet beta cells, as compared with other regulated secretory processes such as chromaffin granule secretion, can in part be explained by the small number of the insulin granules that are docked to the plasma membrane and readily releasable. In type-2 diabetes, the kinetics of insulin secretion become grossly distorted, and, to therapeutically correct this, it is imperative to elucidate the mechanisms that regulate priming and secretion of insulin secretory granules. Munc13-1, a synaptic protein that regulates SNARE complex assembly, is the major protein determining the priming of synaptic vesicles. Here, we demonstrate the presence of Munc13-1 in human, rat, and mouse pancreatic islet beta cells. Expression of Munc13-1, along with its cognate partners, syntaxin 1a and Munc18a, is reduced in the pancreatic islets of type-2 diabetes non-obese Goto-Kakizaki and obese Zucker fa/fa rats. In insulinoma cells, overexpressed Munc13-1-enhanced green fluorescent protein is translocated to the plasma membrane in a temperature-dependent manner. This, in turn, greatly amplifies insulin exocytosis as determined by patch clamp capacitance measurements and radioimmunoassay of the insulin released. The potentiation of exocytosis by Munc13-1 is dependent on endogenously produced diacylglycerol acting on the overexpressed Munc13-1 because it is blocked by a phospholipase C inhibitor (U73122) and abrogated when the diacylglycerol binding-deficient Munc13-1H567K mutant is expressed instead of the wild type protein. Our data demonstrate that Munc13-mediated vesicle priming is not restricted to neurotransmitter release but is also functional in insulin secretion, where it is subject to regulation by the diacylglycerol second messenger pathway. In view of our findings, Munc13-1 is a potential drug target for therapeutic optimization of insulin secretion in diabetes.     

Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness

Summary We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467–478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.     

Secretion of serum amyloid protein and assembly of serum amyloid protein-rich high density lipoprotein in primary mouse hepatocyte culture

The serum amyloid protein (apo-SAA) is a unique high density lipoprotein apoprotein exhibiting dramatic increases in plasma concentration following host injury. The events involved in the secretion of apo-SAA and assembly of apo-SAA-rich lipoprotein particles were studied in primary, serum-free culture of adult BALB/c mouse hepatocytes harvested 3 h following administration of the potent apo-SAA inducer, bacterial endotoxin (50 micrograms of intraperitoneally administered Salmonella typhosa lipopolysaccharide). An approximately 3.5-fold increase in the initial rate of apo-SAA secretion was observed over that of hepatocytes isolated from control mice, whereas the rate of apo-A-I secretion was unchanged by endotoxin administration. Sodium dodecyl sulfate-gel electrophoresis and autoradiography of [35S]methionine-labeled cell products indicated the synthesis of both major mouse apo-SAA isotypes by hepatocytes. Essentially all of the secreted apo-SAA chromatographed in Sephadex G-150 with an elution volume corresponding to a molecular weight of approximately 12,000. Approximately 90% of the secreted apo-SAA was recovered in fractions (d greater than 1.21 g/ml) following ultracentrifugal fractionation. In media supplemented with human lipoproteins (100 micrograms/ml), approximately 50% of the secreted apo-SAA was recovered in the high density lipoprotein fraction. These results suggest that mouse apo-SAA is secreted in monomeric form and becomes associated with lipoproteins in the intravascular compartment.