Biomedical subjects

J Lainé

Publications and source records attributed to J Lainé.

24 records · Page 2Linked to original sources

PubMed1994

Regulation of pancreatic tyrosine kinase and phosphatase activities by cholecystokinin and somatostatin.

Phosphorylation and dephosphorylation of proteins on tyrosyl residues are important reactions involved in cellular activities, namely, those associated with growth and differentiation. Although it is accepted that cholecystokinin (CCK) and somatostatin (SS) stimulate and inhibit pancreatic growth and secretion, the cellular mechanisms by which these two hormones trigger their stimulatory and inhibitory effects are not well known. It has recently been suggested that, in acinar cells, one of the early signals of SS would involve activation of a membrane tyrosine phosphatase, whereas the signal associated with CCK may involve stimulation of protein tyrosine phosphorylation. This study examines the effects of caerulein (Cae) and SMS-201-995 (SMS) on pancreatic growth, particulate and crude cytosolic tyrosine kinase (TRK), and phosphotyrosine phosphatase (PTase) activities. Rats infused intravenously with 0.05% bovine serum albumin (control), Cae (0.25 micrograms.kg-1.h-1), or SMS (5 micrograms.kg-1.h-1) were killed after 0.5, 1, 2, 3, 4, 8, 12, 24, and 48 h of infusion. The pancreas was excised, weighed, and evaluated for contents of DNA and protein and for TRK and PTase activities. The effects of subtotal pancreatectomy on TRK and PTase activities were also examined after 1, 2, 3, 4, and 7 days. In response to Cae, pancreatic growth was evident after 48 h and was accompanied by sustained increases in particulate TRK and particulate PTase. Increases in membrane PTase activities were localized on membranes of the zymogen granules. SMS treatment was associated with increases in pancreatic weight and protein as a result of inhibition of secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Evidence for an apical, nonregulated protein secretion in pig exocrine pancreas.

Secretory proteins are segregated into two pathways out of the trans-Golgi network of regulated secretory cells. To identify proteins specifically secreted by pathways other than the one leading to zymogen granule exocytosis in the exocrine pancreas, conscious permanently cannulated pigs were perfused with atropine to inhibit the regulated fusion of granules. Atropine almost totally inhibited the protein secretion after 1 h of perfusion. The secretion of GP-2, a glycosyl phosphatidylinositol-anchored protein of the zymogen granule membrane, was partially inhibited but was never totally abolished by atropine perfusion. The pattern of proteins secreted under atropine was almost totally different. Soluble GP-2 was the major secretory product. Its specific activity increased 60 times over its normal level in all other conditions. This secretion clearly originated from nonregulated pathways. Results suggest that during the atropine block the apical plasmalemma could be the source of the released GP-2 and that the sustained nature of this release is compatible with a replenishment of the plasmalemma with GP-2 by the continuous exocytosis of vesicles from the nonregulated pathways.

Animals↗

PubMed1993

Reconstitution in vitro of the pH-dependent aggregation of pancreatic zymogens en route to the secretory granule: implication of GP-2.

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. To elucidate the biogenesis of the secretory granule in the exocrine pancreas, we reconstituted in vitro the conditions of pH and ions believed to exist in the TGN using the end product of this sorting process, the zymogen granule contents. Protein aggregation was dependent on pH (acidic) and on the presence of cations (10 mM Ca2+, 150 mM K+) to reproduce the pattern of proteins found in the granule. The constitutive secretory protein IgG was excluded from these aggregates. Zymogen aggregation correlated with the relative proportion of the major granule membrane protein GP-2 in the assay. These results show that the glycosylphosphatidylinositol-anchored protein GP-2 co-aggregates with zymogens in the acidic environment believed to exist in the pancreatic TGN, and thus suggest that GP-2 would function as a membrane anchor for zymogen aggregates, facilitating their entrapment in budding vesicles directed towards the regulated secretory pathway.

Calcium↗

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

Intermediate cells of Lugaro are present in the immature rat cerebellar cortex at an earlier stage than previously thought.

The maturation of the intermediate cell of Lugaro, impregnated by classical silver methods, was studied on parasagittal sections of cerebellar vermis of newborn rat pups. Using morphological criteria characteristic of the adult cell type, immature Lugaro neurons can be recognized from the second part of the first postnatal week on. This implies that these cells differentiate at a much earlier age than previously inferred from studies using tritiated thymidine.

Aging↗

PubMedDate not supplied

Specific interactions of pancreatic amylase at acidic pH. Amylase and the major protein of the zymogen granule membrane (GP-2) bind to immobilized or polymerized amylase.

Regulated secretory proteins are thought to be sorted in the trans-Golgi network towards the secretory granule via acidic aggregation. In the exocrine pancreas, amylase is one of the major zymogens. It is a basic protein of pI 8.6 and does not precipitate in acidic conditions. To identify the mechanism by which amylase aggregates in the acidic cisternae of the pancreatic trans-Golgi network, we have developed an in vitro model in which amylase was fixed to plastic microtiter plates. The fixed amylase was probed with two ligands: amylase itself and GP-2, the major protein of the zymogen granule membrane. Biotinylated amylase bound to fixed amylase in a strict pH-dependent manner with optimal binding between pH 5.0 and 5.7. The affinity of binding was in the nanogram range (Kd approximately 20.0 ng/mL) at pH 5.5. Acid binding of amylase was not reversible by incubation at neutral pH, nor could it be displaced by native amylase. GP-2 binding to fixed amylase was also pH dependent with optimal binding between pH 5.0 and 5.7. As for amylase, it was not reversible by incubation at neutral pH. GP-2 binding sites on fixed amylase appeared to be different from those of biotinylated amylase. While native and biotinylated amylase did not bind to GP-2, polymerized amylase precipitated GP-2 at acidic pH. Taken together these data suggest that slight modifications are sufficient to reveal on the amylase molecule binding sites for GP-2 and for amylase itself. These new binding capacities acquired at acidic pH could be involved in the cascade of reactions that lead to the in vivo formation of the immature secretory granule.

Amylases↗