Molecular consequences of cystic fibrosis transmembrane regulator (CFTR) gene mutations in the exocrine pancreas.

BACKGROUND AND AIMS: We tested the hypothesis that the actual or predicted consequences of mutations in the cystic fibrosis transmembrane regulator gene correlate with the pancreatic phenotype and with measures of quantitative exocrine pancreatic function. METHODS: We assessed 742 patients with cystic fibrosis for whom genotype and clinical data were available. At diagnosis, 610 were pancreatic insufficient, 110 were pancreatic sufficient, and 22 pancreatic sufficient patients progressed to pancreatic insufficiency after diagnosis. RESULTS: We identified mutations on both alleles in 633 patients (85.3%), on one allele in 95 (12.8%), and on neither allele in 14 (1.9%). Seventy six different mutations were identified. The most common mutation was DeltaF508 (71.3%) followed by G551D (2.9%), G542X (2.3%), 621+1G-->T (1.2%), and W1282X (1.2%). Patients were categorized into five classes according to the predicted functional consequences of each mutation. Over 95% of patients with severe class I, II, and III mutations were pancreatic insufficient or progressed to pancreatic insufficiency. In contrast, patients with mild class IV and V mutations were consistently pancreatic sufficient. In all but four cases each genotype correlated exclusively with the pancreatic phenotype. Quantitative data of acinar and ductular secretion were available in 93 patients. Patients with mutations belonging to classes I, II, and III had greatly reduced acinar and ductular function compared with those with class IV or V mutations. CONCLUSION: The predicted or known functional consequences of specific mutant alleles correlate with the severity of pancreatic disease in cystic fibrosis.

Molecular determinant for run-down of L-type Ca2+ channels localized in the carboxyl terminus of the 1C subunit.

1. The role of the sequence 1572-1651 in the C-terminal tail of the alpha1C subunit in run-down of Ca2+ channels was studied by comparing functional properties of the conventional alpha1C,77 channel with those of three isoforms carrying alterations in this motif. 2. The pore-forming alpha1C subunits were co-expressed with alpha2delta and beta2a subunits in HEK-tsA201 cells, a subclone of the human embryonic kidney cell line, and studied by whole-cell and single-channel patch-clamp techniques. 3. Replacement of amino acids 1572-1651 in alpha1C,77 with 81 different amino acids leading to alpha1C,86 significantly altered run-down behaviour. Run-down of Ba2+ currents was rapid with alpha1C,77 channels, but was slow with alpha1C,86. 4. Transfer of the alpha1C,86 segments L (amino acids 1572-1598) or K (amino acids 1595-1652) into the alpha1C,77 channel yielded alpha1C,77L and alpha1C,77K channels, respectively, the run-down of which resembled more that of alpha1C,77. These results demonstrate that a large stretch of sequence between residues 1572 and 1652 of alpha1C,86 renders Ca2+ channels markedly resistant to run-down. 5. The protease inhibitor calpastatin added together with ATP was able to reverse the run-down of alpha1C,77 channels. Calpastatin expression was demonstrated in the HEK-tsA cells by Western blot analysis. 6. These results indicate a significant role of the C-terminal sequence 1572-1651 of the alpha1C subunit in run-down of L-type Ca2+ channels and suggest this sequence as a target site for a modulatory effect by endogenous calpastatin.

A sequence in the carboxy-terminus of the alpha(1C) subunit important for targeting, conductance and open probability of L-type Ca(2+) channels.

The role of the 80-amino acid motif 1572-1651 in the C-terminal tail of alpha(1C) Ca(2+) channel subunits was studied by comparing properties of the conventional alpha(1C,77) channel expressed in HEK-tsA201 cells to three isoforms carrying alterations in this motif. Replacement of amino acids 1572-1651 in alpha(1C,77) with 81 non-identical residues leading to alpha(1C,86) impaired membrane targeting and cluster formation of the channel. Similar to alpha(1C, 86), substitution of its 1572-1598 (alpha(1C,77L)) or 1595-1652 (alpha(1C,77K)) segments into the alpha(1C,77) channel yielded single-channel Ba(2+) currents with increased inactivation, reduced open probability and unitary conductance, when compared to the alpha(1C,77) channel. Thus, the C-terminal sequence 1572-1651 of the alpha(1C) subunit is important for membrane targeting, permeation and open probability of L-type Ca(2+) channels.

Gastric metaplasia: a frequently overlooked feature of duodenal biopsy specimens in untreated celiac disease.

BACKGROUND: Duodenal gastric metaplasia is rarely reported in untreated celiac disease, although it is seen in 60% to 100% of duodenal biopsy specimens in nonceliac patients with histologic duodenitis. The low incidence could represent underreporting, a decreased incidence in pediatric patients generally, or the more distal sampling site that is customary for most biopsy specimens that are obtained to diagnose celiac disease. It could also be a unique feature of the inflammatory reaction that characterizes this disease. The purpose of this study was to examine the incidence of gastric metaplasia in duodenal specimens from children with untreated celiac disease with special reference to patient age and biopsy site. METHOD: Formalin-fixed paraffin-embedded specimens of duodenal mucosa were selected from the pathology department's archival material. Sections were either stained histochemically or by immunochemical methods, according to an antigen-retrieval protocol. Forty-four duodenal specimens from untreated patients with celiac disease (n = 22) and control subjects of similar age with normal histology (n = 22) were examined. Ten of each were obtained during upper endoscopy from the proximal duodenum (proximal site) and 12 of each by Crosby capsule near the ligament of Treitz (distal site). RESULTS: All specimens from patients with celiac disease exhibited marked villous atrophy. None had been noted to have gastric metaplasia during routine examination of sections stained by hematoxylin and eosin. Fifteen (68%) of 22 of the celiac specimens and 2 of 22 (9%) control specimens contained gastric metaplasia, identified as patches of gastric-type cells containing MUC5AC (gastric mucin), pS2 (gastric trefoil factor) and neutral (periodic acid-Schiff-positive) mucin. Five of the seven celiac specimens that had no metaplasia showed increased numbers of goblet cells expressing gastric markers. The incidence of gastric metaplasia was not different for endoscopic (70%) or capsule (67%) specimens. Sixty-eight percent (7/11) of patients aged less than 3 years had gastric metaplasia. CONCLUSION: The presence of gastric metaplasia has been previously underreported in celiac disease specimens. Detection would be improved by the routine use of period acid-Schiff/ alcian blue staining. The incidence of gastric metaplasia in celiac disease is not significantly influenced by biopsy site or age at time of the biopsy.

The pathogenesis of duodenal gastric metaplasia: the role of local goblet cell transformation.

BACKGROUND AND AIMS: Gastric metaplasia is frequently seen in biopsies of the duodenal cap, particularly when inflamed or ulcerated. In its initial manifestation small patches of gastric foveolar cells appear near the tip of a villus. These cells contain periodic acid-Schiff (PAS) positive neutral mucins in contrast with the alcian blue (AB) positive acidic mucins within duodenal goblet cells. Previous investigations have suggested that these PAS positive cells originate either in Brunner's gland ducts or at the base of duodenal crypts and migrate in distinct streams to the upper villus. To investigate the origin of gastric metaplasia in superficial patches, we used the PAS/AB stain to distinguish between neutral and acidic mucins and in addition specific antibodies to immunolocalise foveolar cell mucin MUC5AC, the foveolar cell secretory product, gastric trefoil factor (TFF1), the mature goblet cell mucin MUC2, and MUC2 core antigen. RESULTS: Cells in focal patches of gastric metaplasia contained secretory granules of both gastric and goblet cell phenotypes. MUC5AC and TFF1 were present as expected in gastric foveolar cells but in addition, MUC2 core antigen, normally present only in the Golgi of intestinal goblet cells, was expressed in secretory granules. Goblet cells in the vicinity of metaplastic patches also expressed both gastric and intestinal antigens. MUC5AC/MUC2 containing goblet cells were most common near the villus tip but were also seen at the base of crypts. Where crypts and Brunner's gland ducts merged they were always seen on the crypt side of the junction. Goblet cells were the only cells to express gastric antigens in these areas. In advanced metaplastic lesions, dual phenotype goblet cells were less evident and fewer cells expressed intestinal mucin antigens. CONCLUSIONS: We suggest that goblet cells that express both intestinal and gastric antigens may represent local precursors of gastric metaplasia undergoing a transition to foveolar-like cells of mixed phenotype at the site of early metaplastic patches. As metaplasia becomes more widespread, a more pure gastric phenotype emerges. This progression is likely to be controlled by local inflammatory signals.

Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-epsilon.

Treatment of HT-29 cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces MUC2 expression. To investigate the role of PKC in regulating mucin genes in intestinal cells, we examined the regulation of MUC1, MUC2, MUC5AC, MUC5B, and MUC6 expression in two human mucin-producing colonic cell lines, T84 and HT29/A1. T84 and HT29/A1 cells (at 80-90% confluency) were exposed to 100 nM PMA for 0, 3, and 6 h. Twofold or greater increases in mRNA levels for MUC2 and MUC5AC were observed in both cell lines during this time period, whereas the levels of MUC1, MUC5B, and MUC6 mRNAs were only marginally affected. These results indicated that PKC differentially regulates mucin gene expression and that it may be responsible for altered mucin expression. Our previous results suggested that the Ca(2+)-independent PKC-epsilon isoform appeared to mediate PMA-regulated mucin exocytosis in these cell lines. To determine if PKC-epsilon was also involved in MUC2/MUC5AC gene induction, HT29/A1 cells were stably transfected with either a wild-type PKC-epsilon or a dominant-negative ATP-binding mutant of PKC-epsilon (PKC-epsilon K437R). Overexpression of the dominant-negative PKC-epsilon K437R blocked induction of both mucin genes, whereas PMA-induced mucin gene expression was not prevented by overexpression of wild-type PKC-epsilon. PMA-dependent MUC2 mucin secretion was also blocked in cells overexpressing the dominant-negative PKC-epsilon K437R. On the basis of these observations, PKC-epsilon appears to mediate the expression of two major gastrointestinal mucins in response to PMA as well as PMA-regulated mucin exocytosis.

Roles of calreticulin and calnexin during mucin synthesis in LS180 and HT29/A1 human colonic adenocarcinoma cells.

Molecular chaperones are presumed to associate with large secretory mucin glycoproteins during their synthesis in the endoplasmic reticulum (ER), but have not been identified to date. We decided to look for possible involvement of the chaperones calreticulin (CRT) and calnexin (CLN) during synthesis of two similar gastrointestinal mucins, MUC2 and MUC5AC. Pulse-chase labelling of MUC2 and MUC5AC with [(35)S]methionine/cysteine ([(35)S]Promix) was performed using LS180 and HT29/A1 colonic carcinoma cell lines and was followed by immunoprecipitation with anti-mucin and anti-chaperone antibodies. The precipitated labelled mucin precursors were analysed by SDS/PAGE and autoradiography. Using antibodies specific for each mucin, newly synthesized monomeric precursors of both MUC2 and MUC5AC were detected after a 15 min pulse and then disappeared as oligomers were formed during a 2 h chase period. Only homo-oligomers of MUC2 and MUC5AC were present in the cells. Using anti-CRT, the MUC2 monomeric precursor and oligomer were co-precipitated from both cell lines after a 15 min pulse and the oligomer less strongly after a 0.5 h chase, but there was little co-precipitation after a 2 h chase. At this time, MUC2 immunoprecipitated by anti-MUC2 was completely oligomerized and was endo-beta-N-acetylglucosaminidase-resistant, indicating that the mucin had reached the Golgi region. MUC2 co-precipitated with CRT at zero time and 0.5 h was endo-beta-N-acetylglucosaminidase-sensitive; therefore CRT must have associated with MUC2 in the ER. Treatment with tunicamycin (TUN) diminished the binding of MUC2 to CRT, suggesting a requirement for initial N-glycan addition during this process. Using anti-CLN, only a weak co-precipitation of MUC2, compared with that seen with anti-CRT, was detected in LS180 cells. In contrast with the findings for MUC2, there was no co-precipitation of MUC5AC with CRT or CLN from either cell line at the various time points. In conclusion, CRT and CLN appear to be involved in MUC2 synthesis at the stage of folding and oligomerization in the ER. Since no interaction of the chaperones with MUC5AC was detected at a similar stage of synthesis, these two structurally similar secretory mucins seem to have different chaperone requirements in the ER.

Susceptibility of the cysteine-rich N-terminal and C-terminal ends of rat intestinal mucin muc 2 to proteolytic cleavage.

The present study reveals that partial proteolytic degradation of rat Muc 2 mucin can occur rapidly even in the presence of a battery of proteinase inhibitors. During the initial steps of purification from homogenates of intestinal scrapings, degradation was rapid, causing release of the entire 118 kDa C-terminal glycopeptide and, as shown by N-terminal sequencing, a large (200 kDa) N-terminal glycopeptide fragment. Degradation could be prevented by adding 6 M guanidinium chloride provided that its presence was maintained throughout every step of purification. Even after purification, however, the mucin was still vulnerable to partial proteolysis unless it was stored in guanidinium chloride at -20 degrees C. These findings imply that a potent proteinase contaminant remains tightly bound to the mucin through every step of purification, or else that the mucin has autocatalytic properties. Because the C- and N-terminal regions of secretory mucins are required for their assembly into linear mucin polymers that form functional gels, our findings emphasize that extreme care is required to purify structurally intact mucin molecules. They also imply that the specific degradation steps described here are likely to occur rapidly after mucins are secreted into the intestinal lumen and come into contact with the products of sloughed cells.

The carboxyl-terminal sequence of rat intestinal mucin RMuc3 contains a putative transmembrane region and two EGF-like motifs.

A 3' RACE technique was used to establish the nucleotide sequence encoding the C-terminal 379 amino acids of rat intestinal Muc3. Unlike the C-terminus of Muc2 and many secretory mucins, Muc3 contains two EGF motifs and a putative transmembrane domain. The mRNA for rat Muc3 is 7.5-8.0 kb.

Protein kinase C-epsilon is the likely mediator of mucin exocytosis in human colonic cell lines.

The phorbol ester, phorbol 12-myristate 13-acetate (PMA), induces mucin secretion in the colonic tumor cell line T84 in a Ca(2+)-independent manner. To determine whether a specific protein kinase C (PKC) isoform is involved in colonic cells, we compared PMA-dependent mucin secretion by three human colonic tumor cell lines (T84, HT-29/A1, and LS 180) with the expression of PKC isoforms alpha, beta, delta, epsilon, and zeta, previously identified in human colon (L. A. Davidson, Y. H. Jiang, J. D. Derr, H. Aukema, J. R. Lupton, and R. S. Chapkin. Arch. Biochem. Biophys. 312:547-553, 1994). In each cell line PMA (10(-7) M) caused mucin secretion within 30 min. PMA-dependent mucin secretion was three to four times greater from HT-29/A1 and T84 cells than from LS 180 cells. All three-cell lines contained mRNA for PKC-alpha, PKC-epsilon, and PKC-zeta but not PKC-beta or -delta. Each cell line also expressed PKC-alpha, -epsilon, and -zeta protein. PKC-epsilon expression (mRNA and protein) was three to four times greater in HT-29/A1 and T84 cells than in LS 180 cells, correlating with PMA-responsive mucin secretion, whereas all cell lines contained similar levels of PKC-alpha mRNA and protein. When cells were stimulated by PMA, only PKC-epsilon was translocated from cytosol to membrane fractions early enough to stimulate mucin secretion. Because PKC-epsilon is also a Ca(2+)-independent isoform, it is likely to mediate mucin exocytosis in colonic cells.

Shwachman syndrome: exocrine pancreatic dysfunction and variable phenotypic expression.

BACKGROUND & AIMS: Shwachman syndrome is an inherited condition with multisystemic abnormalities, including exocrine pancreatic dysfunction. The aim of this study was to evaluate the occurrence and progression of features in a large cohort of patients. METHODS: Clinical records of 25 patients with Shwachman syndrome were reviewed. RESULTS: Mean birth weight (2.92 +/- 0.51 kg) was at the 25th percentile. However, by 6 months of age, mean heights and weights were less than the 5th percentile. After 6 months of age, growth velocity was normal. Severe fat maldigestion due to pancreatic insufficiency was present in early life (fecal fat, 26% +/- 17% of fat intake; age, < 2 years). Serial assessment of exocrine pancreatic function showed persistent deficits of enzyme secretion, but 45% of patients showed moderate age-related improvements leading to pancreatic sufficiency. Neutropenia was the most common hematologic abnormality (88%), but leukopenia, thrombocytopenia, and anemia were also frequently encountered. Patients with hypoplasia of all three bone marrow cellular lines (n = 11) had the worst prognosis; 5 patients died, 2 of sepsis and 3 of acute myelogenous leukemia. Other findings included hepatomegaly and/or abnormal liver function test results and skeletal abnormalities. CONCLUSIONS: A wide and varied spectrum of phenotypic abnormalities among patients with Shwachman syndrome is described. Pancreatic acinar dysfunction is an invariable abnormality. Patients with severe bone marrow involvement may have a guarded prognosis.

Phenotypic abnormalities in long-term surviving cystic fibrosis mice.

Mouse models for cystic fibrosis (CF) with no CFTR function (Cftr-/-) have the disadvantage that most animals die of intestinal obstruction shortly after weaning. The objective of this research was to extend the lifespan of CF mice and characterize their phenotype. Weanlings were placed on a nutrient liquid diet, and histologic and functional aspects of organs implicated in the disease were subsequently examined. Approximately 90% of Cftr-/- mice survived to 60 d, the majority beyond 100 d. Cftr-/- mice were underweight and had markedly abnormal intestinal histology. The intestinal epithelia did not respond to challenges with agents that raised intracellular cAMP, consistent with the absence of functional CFTR. No lesions or functional abnormalities were evident in the lungs. Liquid-fed Cftr-/- mice were infertile, although some males weaned to a solid diet were fertile before they died. Thus, we have succeeded in using dietary means to prolong the lives of Cftr-/- mice.

Interaction of heparin with synthetic peptides corresponding to the C-terminal domain of intestinal mucins.

Unlike most other mucins described to date, two intestinal mucins, rat MLP (rat Muc 2) and human MUC2 have a C-terminal tail that is enriched in cationic amino acids. The distribution of charge in each case resembles that of several well known heparin binding proteins. Peptides designated E20-14 and F13-15, corresponding to the C-terminal 14 amino acids of the two mucins, were synthesized and shown to bind 3 H-labelled heparin by a process that was saturable and mediated by strong electrostatic interactions, giving Kd values of 10 (-7) to 10 (-8) M. Using turbidometric analyses and native gel electrophoresis, we observed that peptide-heparin mixtures formed polydisperse aggregates that dissociated with a progressive increase in the concentration of heparin. Under certain conditions heparin protected the peptide from proteolysis by trypsin. Both heparin and dextran sulfate, the latter a highly sulfated synthetic polysaccharide, were potent inhibitors of 3 H-heparin binding to peptide E20-14, while less sulfated glycosaminoglycans were poorly- or non-inhibitory. Mucin in tissue dispersions and homogenates, or purified from rat intestine, did not bind to heparin, and failed to interact with an antibody specific for the peptide E20-14. Both mucin samples however, reacted with antibodies that recognize regions upstream of the C-terminal 14 amino acids. Immunofluorescent localization of E20-14 was confined to the basal perinuclear regions of goblet cells, whereas localization of an antibody to a flanking sequence on the N-terminal side of the C-tail, localized to mature mucin storage granules. These findings suggest that the heparin -binding C-tail of the mucin may be removed at an early stage of biosynthesis. Heparin-mucin complexes, if they form in vivo, are thus likely to be confined to the ER and/or Golgi compartments.

Regulated and unregulated pathways for MUC2 mucin secretion in human colonic LS180 adenocarcinoma cells are distinct.

We have shown previously [McCool, Forstner and Forstner (1994) Biochem. J. 302, 111-118] using pulse-chase labelling of mucin with [3H]threonine that LS180 colonic tumour cells synthesize and secrete MUC2 without the addition of secretagogues. Treatment of the LS180 cells with monensin to disrupt Golgi function was also found to inhibit baseline secretion almost completely. In this paper we show that addition of nocodazole to inhibit microtubule assembly reduced baseline secretion by 53% over a 6 h chase period. In contrast, cytochalasin D did not affect the rate of unstimulated mucin synthesis or secretion, suggesting that baseline secretion is not influenced by disruption of actin microfilaments. In addition, regulated mucin secretion by LS180 cells was studied in response to carbachol, phorbol 12-myristate 13-acetate and A23187. Mucin released in response to secretagogues behaved identically on SDS/PAGE to that secreted under baseline conditions. T84 cells and the B6 subclone of the HT29 cell line responded in a similar manner to LS180 cells and secreted high-molecular-mass mucin which included MUC2 and behaved like LS180 mucin on SDS/PAGE. Neither monensin nor nocodazole significantly affected secretagogue-stimulated mucin secretion. Since these compounds inhibited secretion of labelled mucin under baseline conditions, mucin released by secretagogues must have come from a separate, unlabelled mucin pool in stored granules. Cytochalasin D, on the other hand, caused the release of small amounts of stored mucin, suggesting that actin microfilaments participate in regulated exocytosis. Thus two kinds of mucin secretion occur in LS180 cells. Unregulated secretion depends upon continuous transport of mucin granules from Golgi vesicles to the cell surface and does not utilize stored mucin, whereas regulated secretion involves the release of mucin from storage granules and is not affected by microtubule or Golgi disruption.

Longitudinal evaluation of serum trypsinogen measurement in pancreatic-insufficient and pancreatic-sufficient patients with cystic fibrosis.

We studied serial measurements of serum cationic trypsinogen in patients with cystic fibrosis to assess the predictability of changes in individuals and the value of longitudinal measurement in defining pancreatic status. Three hundred twenty-nine patients with cystic fibrosis, aged 3 days to 40 years, had serum levels of trypsinogen measured on 2 to 12 occasions for periods ranging from 1 week to 7 years. Patients were classified into three groups on the basis of 72-hour fecal fat studies performed at the time of diagnosis. Two hundred thirty-three patients had pancreatic insufficiency (PI), 78 had pancreatic sufficiency (PS), and 18 had PS at diagnosis but acquired PI during follow-up (PS-->PI). Infants with PI had greatly elevated serum trypsinogen levels that fell sharply in the first years of life, so that by age 7 years more than 95% had subnormal values; individual patient values followed a predictable course similar to previously reported cross-sectional age-related values. In patients with PS, serum trypsinogen levels generally remained within or above the normal range and, after age 10 years, were well above the upper limit for PI patients. Within-patient variance was significantly greater (p < 0.0001) in patients with PS than in those with PI who were older than 7 years of age. Changes in patients within PS-->PI generally followed the pattern seen in patients with PI, but values in older patients tended to be in the higher range. We concluded that serial measurement of serum trypsinogen is a valuable tool for monitoring the pancreatic status of patients with cystic fibrosis and PS.

Synthesis and secretion of mucin by the human colonic tumour cell line LS180.

Pulse-chase labelling experiments were performed using the mucin-producing colonic carcinoma cell line LS180. Cells were pulsed with [3H]threonine or [3H]glucosamine and chased in complete media without radiolabel for various lengths of time. From cell and media extracts obtained at each time point, mucin proteins were immunoprecipitated with specific anti-mucin antibodies and analysed by SDS/PAGE and fluorography. At short labelling times with [3H]threonine, without chase, a monomeric thiol-reduction-resistant mucin precursor of apparent molecular mass > 670 kDa was identified. The precursor, in contrast to oligomeric species, was not labelled by [3H]glucosamine but exhibited binding to Vicia villosa isolectin B4, suggesting the presence of some core GalNAc residues. Treatment with tunicamycin to inhibit N-glycosylation had no effect on the apparent mass of the precursor. Identity of the mucin antigen with MUC2 mucin was established by immunoprecipitation with antibodies specific for a MUC2 tandem repeat and C-terminal regions. With increasing chase time the precursor was replaced by thiol-reduction-sensitive mucin oligomers that reached peak intracellular radiolabelling with [3H]threonine by 2 h of chase, and then declined. Only oligomeric mucin was secreted into the medium. Secretion of [3H]threonine-labelled mucin was detectable after 2 h of chase and increased as the cytoplasmic mucin label declined. Monensin inhibited [3H]glucosamine incorporation, sialylation and baseline (non-regulated) mucin secretion without affecting initial [3H]threonine incorporation or oligomerization. Oligomerization and Golgi transport are therefore essential early steps in MUC2 mucin secretion. Oligomerization may follow some core O-glycosylation with GalNAc, but precedes elongation of oligosaccharide chains.

Regulation of mucin secretion in T84 adenocarcinoma cells by forskolin: relationship to Ca2+ and PKC.

The relationship between the adenosine 3',5'-cyclic monophosphate-mediated protein kinase A (PKA)-dependent stimulatory pathway for mucin secretion and Ca(2+)-mediated and protein kinase C (PKC)-mediated secretion was studied in T84 cells, using the postreceptor secretagogues forskolin, A-23187, and phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), high- and low-Ca2+ media, and the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Staurosporine (10(-5) M) inhibited both PMA and forskolin at their maximally effective concentrations, whereas H-7 (5 x 10(-5) M) inhibited only PMA. Stimulation of mucin secretion by forskolin (5 x 10(-5) M) was not significantly affected by the reduction of medium Ca2+ to 47 and 129 nM, equivalent to published values for intracellular Ca2+ concentration ([Ca2+]i). Stimulation by forskolin was reduced by preloading cells with BAPTA, but to a much smaller extent than Ca(2+)-dependent stimulation by A-23187. A-23187-mediated mucin secretion from BAPTA-loaded cells was augmented by high doses of forskolin. Similar concentrations of forskolin had no effect on A-23187-stimulated secretion in calcium-replete cells. Our results indicate that forskolin does not stimulate mucin secretion by increasing Ca2+ entry or releasing Ca2+ from intracellular stores. Forskolin can stimulate mucin secretion in a Ca(2+)-independent manner but is apparently inhibited by high levels of intracellular Ca2+ induced by Ca2+ ionophores in 1.0 mM Ca2+ media.(ABSTRACT TRUNCATED AT 250 WORDS)

Suggestive evidence for two different mucin genes in rat intestine.

In the present report we describe the isolation and sequence of a partial cDNA (M2-798) for a rat intestinal mucin designated M2. A rat intestinal lambda ZAP II cDNA library was screened using a polyclonal antiserum which was prepared against deglycosylated high-molecular-mass glycopeptides of the purified mucin. Mucin cDNA clones were found to contain tandem repeats of 18 nt which encoded a threonine- and proline-rich peptide having a consensus sequence of TTTPDV. This is the same sequence reported recently by Gum, Hicks, Lagace, Byrd, Toribara, Siddiki, Fearney, Lamport and Kim [(1991) J. Biol. Chem. 266, 22733-22738] for a rat intestinal cDNA called RMUC 176. A novel feature present in the cDNA M2-798 is a 246 nt unique region at the 3' end which encodes a hydrophobic sequence of 82 amino acids. RNA blots probed with M2-798 cDNA produced a single hybridization band between 7.5 and 9.0 kb in rat small intestine and colon. An identical hybridization pattern was obtained with a PCR-generated cDNA probe corresponding solely to the unique hydrophobic region of M2-798, demonstrating that this region is encoded by the authentic M2 mRNA. Our data suggest that the unique region of M2 has the potential to be either a transmembrane region, or a domain which mediates hydrophobic interactions of the mucin with other molecules. Since we have previously reported another rat intestinal cDNA which encodes the C-terminus of a mucin-like peptide (MLP) [Xu, Wang, Huan, Cutz, Forstner and Forstner (1992) Biochem. J. 286, 335-338], we wished to discover whether M2 was encoded by the same gene. RNA blotting experiments with probes specific for M2 and MLP showed different mRNAs for each. The message for M2 (7.5-8.5 kb) was smaller than that for MLP (> 9.5 kb) and, unlike MLP, gave no signal in human colonic LS174T cells. The results of DNA blots probed with M2-798 and an MLP-probe suggest that M2 and MLP are likely to be single-copy genes. It would appear therefore that normal rat intestine, like human intestine, may express two different mucin genes.

Mucin secretion by T84 cells: stimulation by PKC, Ca2+, and a protein kinase activated by Ca2+ ionophore.

T84 adenocarcinoma cells were stimulated to secrete mucin by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and Ca2+ ionophores A23187 and ionomycin. In Ca(2+)-containing media, maximal stimulation by PMA was significantly inhibited by staurosporine, but maximal A23187-stimulated secretion was not affected. Downregulation of protein kinase C (PKC) reduced maximal PMA-stimulated secretion without affecting the response to A23187. Thus PKC activation is not required for maximal Ca(2+)-mediated mucin secretion. PMA stimulated secretion in low-Ca2+ media, with and without intracellular chelation of Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Surprisingly, Ca2+ ionophores also stimulated secretion under the same circumstances. Persistent A23187-stimulated secretion was strongly inhibited by the protein kinase inhibitors staurosporine and H-7. Secretion in Ca(2+)-containing media was also inhibited at submaximal levels of Ca(2+)-ionophore stimulation. These results indicate that PKC and Ca2+ stimulate mucin exocytosis independently. Ca2+ ionophores also stimulate secretion via a protein-kinase dependent pathway. Enhancement of protein kinase inhibition at lower Ca2+ concentrations suggests that the response could be mediated by a Ca2+ ionophore-induced depletion of an intracellular Ca2+ pool.