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D LeBel

Publications and source records attributed to D LeBel.

At least 19 recordsLinked to original sources
PubMed1999

Specific localization of membrane dipeptidase and dipeptidyl peptidase IV in secretion granules of two different pancreatic islet cells.

Endocrine cells require several protein convertases to process the precursors of hormonal peptides that they secrete. In addition to the convertases, which have a crucial role in the maturation of prohormones, many other proteases are present in endocrine cells, the roles of which are less well established. Two of these proteases, dipeptidyl peptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19), have been immunocytochemically localized in the endocrine pancreas of the pig. Membrane dipeptidase was present exclusively in cells of the islet of Langerhans that were positive for the pancreatic polypeptide, whereas dipeptidyl peptidase IV was restricted to cells positive for glucagon. Both enzymes were observed in the content of secretory granules and therefore would be released into the interstitial space as the granules undergo exocytosis. At this location they could act on secretions of other islet cells. The relative concentration of dipeptidyl peptidase IV was lower in dense glucagon granules, where the immunoreactivity to glucagon was higher, and vice versa for light granules. This suggests that, in A-cells, dipeptidyl peptidase IV could be sent for degradation in the endosomal/lysosomal compartment during the process of granule maturation or could be removed from granules for continuous release into the interstitial space. The intense proteolytic activity that takes place in the endocrine pancreas could produce many potential dipeptide substrates for membrane dipeptidase. (J Histochem Cytochem 47:489-497, 1999)

Animals↗
PubMed1998

Membrane dipeptidase in the pig exocrine pancreas. Ultrastructural localization and secretion.

The GPI-anchored membrane dipeptidase is the major peptidase activity of the secretory granule membrane in the exocrine pancreas. The enzyme is also found in the granule content and in pancreatic secretions. Immunocytochemical localization confirmed its location in the granule membrane and in the acinar cell apical plasma membrane. In the endoplasmic reticulum and Golgi, membrane dipeptidase was strictly membrane-bound. There was no membrane dipeptidase in duct cells. The release of membrane dipeptidase from the membrane starts in the immature granule. To identify the mechanism responsible for its release, secretions were collected from cannulated conscious pig under basal conditions and atropine perfusion. The latter treatment caused complete inhibition of protein secretion but had a negligible effect on membrane dipeptidase activity in the secretions. In secretions, membrane dipeptidase partitioned into the detergent-rich phase on phase separation in Triton X-114, whereas treatment with bacterial phosphatidylinositol-specific phospholipase C caused the peptidase to partition into the aqueous phase, indicating that the secreted enzyme could come from shedding of membrane fragments at the apical surface or via the action of a previously characterized phospholipase A activity.

Animals↗
PubMed1998

Ontogeny of insulin-like growth factors (IGF), IGF binding proteins, IGF receptors, and growth hormone receptor mRNA levels in porcine pancreas.

We examined the ontogeny of mRNA levels of IGF-I and -II, IGF type 1 (IGFI-R) and type II receptors (IGFII-R), IGF binding protein-1 and -3 (IGFBP-1 and -3), GH receptor (GHR), and tissue concentrations of IGF and IGFBP in the pancreas of pigs. Tissues were collected from fetuses at 90 and 110 d of gestation and from pigs at 1, 21, 90 and 180 d of age. Northern blots were performed using total RNA hybridized with 32P-labeled cDNA probes (human IGF-I and human IGFI-R) and cRNA probes (rat IGF-II, human IGFII-R, human IGFBP-1, pig IGFBP-3, and pig GHR). There were two accelerated growth stages of the pancreas: the first one at 90 d of fetal life, which is characterized by cell hyperplasia (high ratio of DNA to body weight), and the second one at postnatal 90 d, which is attributed to cell hypertrophy (high ratios of pancreatic weight, RNA, and protein to DNA). The level of IGF-II mRNA and its tissue concentration were predominant during fetal life and low thereafter. The IGF-I mRNA level was high during fetal and early postnatal life and decreased thereafter. Messenger RNA levels of IGFI-R, IGFBP-3, and GHR and concentrations of IGFBP-1 and -2 were abundant during fetal and early postnatal life. In conclusion, IGF may be involved in various physiological periods of pancreatic development in pigs.

Aging↗
PubMed1997

Ontogeny of epidermal growth factor (EGF), EGF receptor (EGFR) and basic fibroblast growth factor (bFGF) mRNA levels in pancreas, liver, kidney, and skeletal muscle of pig.

Epidermal growth factor (EGF), EGF receptor (EGFR), and basic fibroblast growth factor (bFGF) messenger RNA (mRNA) levels were examined by Northern blot analysis in four tissues (pancreas, liver, kidney, and skeletal muscle) of pig from fetal 90 d to postnatal 180 d of age. The present study shows for the first time that EGF mRNA increased with advancing age in the kidney and skeletal muscle of pig. A high level of EGF mRNA was observed in the kidney compared with the liver and skeletal muscle. In the pancreas, high levels of EGF mRNA were found in fetuses and newborns and were low in older pigs. Pancreatic EGFR mRNA level parallelled its EGF mRNA, whereas in the kidney and skeletal muscle, patterns of EGFR mRNA were reversed to their EGF mRNA levels. In the liver, EGFR mRNA was abundant but EGF mRNA was undetected. In the pancreas and skeletal muscle, the highest levels of bFGF mRNA were found in fetuses of 90 d of age and then decreased with advancing age. In the liver and kidney, there were no major changes in bFGF mRNA levels during the examined developmental periods. These results show that EGF, EGFR, and bFGF mRNA levels are developmentally and tissue specifically regulated in pig. In the pancreas, mRNA levels of EGF, EGFR and bFGF were high in fetal and neonatal life and low thereafter. In the kidney and skeletal muscle, EGF mRNA increased with advancing age. EGF may play a role in muscle growth and maintenance in growing pigs during the later stage of development.

Aging↗
PubMed1996

Development of GP-2 and five zymogens in the fetal and young pig: biochemical and immunocytochemical evidence of an atypical zymogen granule composition in the fetus.

To uncover the mechanisms involved in the biogenesis of secretory granules, we studied development of the exocrine pancreas in the pig from the fetus up to the mature animal by following the enzyme activities and expression (Northern blot) of five zymogens and GP-2, the major protein of the granule membrane. Fetal pancreas mainly contained chymotrypsinogen and barely detectable amounts of amylase, trypsin, lipase, and elastase. GP-2 was not notably expressed before the Day 21 of life. Ultrastructural examination of the fetal tissue embedded in Epon with osmium postfixation or in Lowicryl at -20 degrees C without postfixation showed dense granules with an irregular shape but also showed that most granules had uncondensed contents, with the aspect of immature granules, or had a dense core surrounded by light material. With immunogold cytochemistry, the concentration of chymotrypsinogen was directly associated with the acquisition of electron density by the granule matrix. These observations suggest that fetal granules have a slower rhythm of zymogen condensation and an irregular shape that could be due to the particular composition of the matrix and the absence of GP-2. We conclude that, in the exocrine pancreas, secretory granules can be formed under various conditions, even with a matrix containing a ratio of components very different from that of the normal mature animal.

Animals↗
PubMed1993

Simultaneous kinetic determinations of lipase, chymotrypsin, trypsin, elastase, and amylase on the same microtiter plate.

Micromethods are described to determine in 10 min the activity of the five most common pancreatic zymogens: amylase, lipase, trypsin, chymotrypsin, and elastase. Progress of the reactions is monitored at 405 nm, allowing the kinetic determination of the five enzymes on a single 96-well microtiter plate. Amylase activity is measured by the release of p-nitrophenol from a chemically defined substrate. Linearity of the assay is from 10 to 360 U/L of amylase, and activities as low as 0.4 U/L can be easily measured by extending the period of incubation up to 24 h. Chymotrypsin, trypsin, and elastase activities are monitored by the release of p-nitroanilide from specific substrates, and activities are from 25 to 6,500, 15 to 260, and 20 to 600 U/L, respectively. Finally, lipase is determined by the clearing of a commercially available stabilized emulsion of triolein. The lipase determination can be performed from 90 to 3,600 U/L. When microplate methods were compared with conventional procedures, a perfect correspondence was found between the two types of procedure. Factors necessary to convert microplate results to those of conventional assays are provided. These microassays make possible the rapid and simultaneous determination of the three main types of pancreatic hydrolases (a glycohydrolase, three proteases, and a lipase) with < 5 microliters of pancreatic juice by kinetic analysis. They could be easily adopted as routine assays in most research laboratories.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of the apical plasma membrane of the rat pancreatic acinar cell.

A method is described for the rapid purification of the apical plasma membrane from the rat pancreatic acinar cell. It makes use of wheat germ agglutinin affinity chromatography to selectively bind vesicles with N-acetyl glucosamine present at their surface. Particular conditions (150 mM NaCl) had then to be used to keep membrane vesicles in the coveted orientation, i.e. as right-side-out vesicles. Due to its specific apical location in many epithelial cells, gamma-glutamyltranspeptidase was chosen to monitor the purification procedure. The final fraction was enriched in gamma-glutamyltranspeptidase by a factor of 75 relative to the homogenate. Na,K-ATPase, a strict basolateral membrane marker, was not detectable in the fraction. No membranes originating from other compartments, more particularly expected from zymogen granules, or from other cell types, did contaminate the preparation. As expected for an epithelial cell apical plasmalemma, lipid composition showed a very high ratio of glycolipids (37.5%). The absence of membrane-bound GP-2, and the exceptionally high specific activity of gamma-glutamyltranspeptidase suggest that the apical membrane would not be made up by the exocytosis of secretory granule, but instead by the fusion of specialized secretory vesicles very likely originating from the constitutive secretory pathway. In conclusion, this report describes a method of obtaining a fraction highly enriched in the secretory apex of the pancreatic exocrine cell that would be directly involved in exocytosis with zymogen granules and also in local anion transport.

Animals↗
PubMed1991

In resting conditions, the pancreatic granule membrane protein GP-2 is secreted by cleavage of its glycosylphosphatidylinositol anchor.

GP-2 is the major membrane protein of the exocrine pancreatic secretory granule. It is an integral protein which is anchored by a phosphatidylinositolglycan. In addition to being present in the soluble contents of the granule, GP-2 is also actively secreted by the pancreas. Although 93% of the GP-2 in the resting secretions of anaesthetized rats could be pelleted, Triton X-114 phase extraction showed that 70% of this GP-2 had lost its hydrophobic properties. Proteases have been postulated to release GP-2 from the membrane, but phospholipases also have the capacity to release the protein from the membrane by hydrolysis of its peculiar glycosylphosphatidylinositol membrane anchor. These studies show the presence of inositol 1,2-(cyclic)monophosphate on the secreted hydrophilic GP-2, confirming the involvement of an endogenous phospholipase C in the solubilization of GP-2 by the exocrine pancreas. It is therefore concluded that most of the GP-2 secreted by the pancreas of anaesthetized rats under resting conditions is released from the membrane by a phospholipase C which hydrolyses the phosphodiester bond linking GP-2 to its diradylglycerol anchor.

Animals↗
PubMed1989

A competition enzyme-linked immunosorbent assay (ELISA) for the measurement of pancreatic GP-2 glycoprotein.

A competition enzyme-linked immunosorbent assay has been developed for the quantitative detection of soluble and membrane-bound GP-2, a glycoprotein which is confined to the exocrine pancreas. Zymogen granule membranes fixed to microtiter plates with poly-L-lysine were used as the source of antigen. Detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS] was added to the assay in order to reveal all the antigens, more particularly in membranous samples. Presence of detergent at concentrations as high as 0.5% did not interfere with any particular steps of the ELISA. This competition ELISA can detect 10 ng of GP-2 and will be useful for measuring soluble as well as membrane GP-2 in order to elucidate its role in the secretory process of the pancreas as well as in certain pathologies such as cystic fibrosis.

Animals↗
PubMed1989

Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis.

GP-2 is the major membrane glycoprotein characteristic of the pancreatic zymogen granule membrane. When granules are lysed in the presence of DTT, GP-2 becomes completely and specifically degraded. This proteolysis was reproducible with the same characteristics in the purified granule membrane. The protease was purified from this source using hydrophobic interaction chromatography. The proteolytic activity was identified as a 29-kDa protein because, in a reconstituted system containing both the purified GP-2 and the 29-kDa protein, the proteolytic degradation of GP-2 was sensitive to the same spectrum and concentrations of inhibitors or reducing agents as in the membrane. The activity was characteristic of a serine protease. It was also shown that GP-2 only becomes sensitive to proteolytic digestion when its disulfide bonds are reduced, and that DTT does not activate the protease. Seven intramolecular disulfide bonds were identified on GP-2. All of them are located in a 65-kDa tryptic fragment that is very resistant to exogenous proteases under nonreducing conditions. Because of the quite specific degradation of GP-2 under reducing conditions, we believe that the 29-kDa protease must be closely associated with GP-2 on the membrane. This protease could be responsible, in part, for the solubilization of the GP-2 from the membrane into the zymogen granule content and its resulting secretion by the pancreas.

Alkylation↗
PubMed1989

Alterations of pancreatic growth and of GP-2 content in the reserpinized rat model of cystic fibrosis.

The chronically reserpinized rat is an experimental model for cystic fibrosis. In this study, we report the effects of two doses of reserpine (0.5 and 1.0 mg.kg-1.d-1) on the growth of the pancreas and on its content of the glycoprotein GP-2, a characteristic protein of the zymogen granule. An assessment of the effects of secondary malnutrition induced by the drug was also performed by adding a group of pair-fed animals. During the 7 d of treatment, body wt and food intake were monitored. These two parameters were significantly affected from the 4th d on. Pancreatic wt, DNA, protein, and activity of amylase and chymotrypsinogen were measured after 4 and 7 d of treatment; lipase activity and GP-2 content, after 7 d. Although the DNA content never did change, total protein diminished by 27% at the higher dose of reserpine. Pancreatic wt, amylase activity and GP-2 content were reduced by the treatment, while chymotrypsinogen and lipase activities were increased. Effects on pancreatic wt, amylase, chymotrypsinogen, and GP-2 were dose-dependent. Malnutrition had effects similar to reserpine on body wt, protein, amylase, and chymotrypsinogen. Pancreatic wt, lipase, and GP-2, however, were specifically altered by the chronic reserpine treatment. It is concluded from these results that reserpine induces, in the pancreas, specific alterations that are distinguishable from the accompanying malnutrition. These findings support the use of pancreatic wt, lipase, and GP-2 as specific markers of the effects of the drug on the pancreatic tissue in the chronically reserpinized rat model for cystic fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Light and electron microscopy of the exocrine pancreas in the chronically reserpinized rat.

The effects of reserpine injections were studied on the morphology of the pancreas in an experimental model for cystic fibrosis, the chronically reserpinized rat. A detailed examination of the tissue was carried out at the light and electron microscopic levels. The nonspecific effects of secondary malnutrition induced by the drug were assessed with a group of animals pair fed with the treated animals. In a companion paper, we show that pancreatic wt, lipase, and GP-2 contents also are affected by reserpine treatment. In this study, we report that no morphologic differences were observed between the exocrine pancreatic tissue of control and pair-fed animals. By contrast, reserpine induced an accumulation of zymogen granules in 60% of the treated animals and a concomitant decrease of the area occupied by the rough endoplasmic reticulum in the same cells. Finally, in all treated animals, at the light and electron microscopic levels, it was observed that some particular regions of the pancreatic tissue were strongly affected. In these regions, numerous autophagic bodies and lysosomes were observed. Cisternae of the Golgi complex were also more distended. Some acinar cells were in the process of lysis. Several vacuolar inclusions were present in some intralobular duct cells. Cellular material was seen in acinar and intralobular duct lumina. In these same regions, distended intralobular ducts and acinar lumina were observed. These last two features have never been reported in the reserpinized rat but are important manifestations of the pathology in cystic fibrosis patients where obstructions of ducts are believed to trigger focal destruction of the pancreatic tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

The major protein of pancreatic zymogen granule membranes (GP-2) is anchored via covalent bonds to phosphatidylinositol.

GP-2, the major integral protein characteristic of the pancreatic zymogen granule membrane can be released from the membrane by the action of a phosphatidylinositol specific phospholipase C (PI-PLC). In a hydrophobic/hydrophilic phase separation system using the non-ionic detergent Triton X-114, the membrane-bound form of the protein went from the detergent phase into the hydrophilic phase upon action of the phospholipase. PI-PLC solubilization of GP-2 unmasked an antigenic determinant similar to the cross-reacting determinant of the trypanosome variant surface glycoproteins. This determinant being a distinctive feature of the glycan moiety of phosphatidyl-inositol anchored membrane proteins, it established the glycosyl-phosphatidyl-inositol nature of the GP-2 membrane anchor. Since soluble GP-2 is also found in the contents of the granule and is secreted intact into the pancreatic juice, it is likely that one of the mechanisms responsible for its release could be a specific phospholipase. GP-2 is the first glycosyl-phosphatidyl-inositol-anchored protein that is integral to the membrane of an organelle and not located at the surface of the cell.

Animals↗
PubMed1986

Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[alpha-32P]ATP.

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[alpha-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 microM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 microM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A-Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-beta-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.

Adenosine Triphosphate↗
PubMed1986

Reducing conditions induce a total degradation of the major zymogen granule membrane protein in both its membranous and its soluble form. Immunochemical quantitation of the two forms.

The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 X 10(3) daltons (Da) which amounts to 25% of the total proteins of this membrane. When zymogen granule membranes were prepared in presence of 5 mM dithiothreitol (DTT), this glycoprotein specifically vanished from the membrane preparation. During membrane purification two other fractions were produced out of the purified granules: a soluble fraction of zymogens referred to as granule content and a dense pellet. The possibility that DTT could release the 92-kDa protein from the membrane to these other fractions has been rejected. Altogether, addition of DTT during the lysis of the granules induced a total degradation of the 92-kDa protein. This hydrolysis could be inhibited by phenylmethylsulfonyl fluoride but not by N-alpha-p-tosyl-L-lysine chloromethyl ketone or L-1-tosylamide-2-phenylethylchloromethyl ketone. In the course of these experiments, using gel filtration of the granule content, it was found that the 92-kDa protein was also present in the granule content in the form of an aggregate of 300 kDa. A protease was present in this aggregate and could hydrolyse the 92-kDa protein upon addition of DTT. From immunoblotting studies and rocket immunoelectrophoresis, it was found that the soluble 92-kDa protein was antigenically similar to the membrane protein and that 44% of the immunoreactive glycoprotein of the granule was soluble in the content. A cross-reacting fragment of 65 kDa has been observed in all the fractions, yet at different levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Different patterns of proteins are secreted by the pig pancreas when stimulated by secretin alone or in combination with caerulein.

The protein compositions of pig pancreatic secretions collected under stimulation by secretin alone or in combination with caerulein were compared by SDS polyacrylamide gel electrophoresis. Different sets of proteins were observed in these two different conditions. One of the major proteins secreted under secretin alone was immunologically similar to the 92 kDa glycoprotein characteristic of the pig zymogen granule membrane. Since its proportion in the two secretions was drastically different and since this protein is exclusively found in the acinar cell, these observations support the view that the proteins released by the pig pancreas under secretin stimulation alone, and under the combination of secretin + caerulein do not originate from the same intracellular pool of the acinar cell and that the secretin-induced secretion does not derive from zymogen granules.

Animals↗
PubMed1984

The integral and peripheral proteins of the zymogen granule membrane.

This study reports the lipid and protein composition of purified pig zymogen granule membranes. Lipids made up more than 70% of the membrane dry weight and phosphatidylserine constituted 17% of the total phospholipids. The membrane was shown to contain nine major proteins. A protein of Mr 92 000 was the major constituent accounting for more than 25% of the Coomassie blue staining on gels. Six glycoproteins, including the latter, were revealed by periodic acid-Schiff staining and concanavalin A binding. A phase separation technique using partition of intrinsic and extrinsic membrane proteins in Triton X-114 solutions has shown that most of the proteins were integral membrane proteins. The ATP diphosphohydrolase, which is distinctive of the zymogen granule membrane, segregated with integral proteins. These data constitute a detailed description of a purified membrane fraction from pig pancreatic zymogen granules.

Animals↗