摘要:

An in vivo translation system, theXenopus laevisoocyte, was employed to study the synthesis and secretion of pancreatic proteins. RNA was purified from normal and diabetic rat pancreas and normal rat liver by use of guanidine isothiocyanate lysis and cesium chloride gradient centrifugation. The presence of functional mRNA was documented by translation in a reticulocyte lysate that yielded precursors of all major secretory proteins, i.e., slightly higherM, than proteins synthesized in situ by pancreatic acini. MatureX. laevisoocytes were then microinjected with either total RNA or purified mRNA. When oocytes were subsequently incubated with “S-methionine,” pancreatic secretory proteins or hepatic albumin could be immunoprecipitated from oocyte lysate with specific polyclonal antibodies against amylase, trypsin, ribonuclease, and albumin. Amylase was shown to be enzymatically active. Moreover, oocytes released pancreatic secretory proteins into the medium when injected with pancreatic RNA in a time-dependent manner. Only the mature form of amylase was secreted and secretion was not regulated by secretagogues. When a comparison was made after injection of RNA from diabetic pancreas known to contain altered amounts of individual mRNAs, there was a decrease in amylase and an increase in trypsinogen synthesis in oocytes that was comparable to the results of cell free translation. The oocyte expression system, therefore, should be useful not only for studies of protein synthesis but also for processing and secretion.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID

摘要:

A method of measuring amylase secretion from dispersed pancreatic acini is described that utilizes a 20-km nylon mesh to separate cell-associated from secreted amylase. When compared with the standard centrifugation assay, the mesh technique permits easy measurement at multiple, closely spaced time points while reducing the number of samples generated and allowing rapid manipulation of the extracellular suspending medium.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID

摘要:

Study of the products secreted by pancreatic ductal cells and analysis of the mechanisms involved in the discharge of these products have been limited by a lack of in vitro models available to experimentally approach this problem. To this aim, this investigation has been designed to determine if a human pancreatic carcinoma cell line of ductal origin (PANC-1) has maintained some of the differentiated characteristics of normal mammalian pancreatic ductal epithelium. Morphological and immunocytochemical studies indicated that, similar to isolated rat pancreatic ducts, the PANC-1 cell line contained (a) intermediate filaments of the epithelial class, (b) a basolateral plasma membrane localization of Na+, KC+-ATPase, (c) complete tight junctions based on freeze-fracture analysis, (d) a cuboidal morphology when grown on Type I collagen-coated nitrocellulose filters or isolated amnion basement membrane, and (e) normal ductal epithelial ultrastructural features. Biochemical analysis indicated that, also similar to isolated rat and human pancreatic ducts, the PANC-1 cell line contained (a) γ-glutamyltranspeptidase, (b) carbonic anhydrase, and (c) Na+, K+-ATPase based on [3H]ouabain binding assays. Comparative studies with other transformed lines indicated that PANC-1 cells have similarities to ductal lines such as MDCK cells but are markedly different from mesenchymally derived lines such as L cells. In addition, as with isolated rat and human ducts, PANC-1 cells synthesize and secrete sulfated proteins with a MW range of ∼180K to 1 million daltons, with the predominant species being 660K daltons as indicated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results indicate that the PANC-1 cell line has maintained at least some of the differentiated characteristics of normal pancreatic ductal epithelial cells and may be a useful system for study of ductal secretory products as well as the mechanisms involved in the discharge of these products.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID

摘要:

Crude membranes (27,000 g pellets) from five normal human pancreases were prepared. In the presence of GTP, the peptides of the secretin family stimulated adenylate cyclase activity, their order of potency being: secretin >heloderrnin >peptide histidine isoleucinamide (PHI) 2 vasoactive intestinal peptide (VIP) >growth hormone releasing factor (GRF) (1-29)-NH2. In addition, helodermin and PHI were more efficient than secretin. Secretin (3-27) inhibited fully the secretin stimulation and partially only the helodermin and PHI stimulation of the enzyme. Secretin receptors were investigated by the ability of secretin and related peptides to inhibit tracer binding. [1251]Secretin binding was fully inhibited by secretin (Kd0.8 nM), helodermin (d200 nM), and PHI (d250 nM). VIP and GRF(1-29)-NH2induced partial (20%) inhibition at a high 10μM concentration. The fragments secretin (2-27), (3-27), (4-27), and (7-27) showed the same low potency and efficacy based on their ability to stimulate adenylate cyclase and to occupy secretin receptors. The analogues [Va15]secretin and [Ala2]secretin had a higher potency than secretin. Based on this comparison of adenylate cyclase stimulation and [Ala2]secretin binding inhibition, it is tempting to conclude that the human pancreas: (a) possesses highly specific secretin receptors and (b) such receptors could not fully account for the whole pattern of adenylate cyclase activation by related peptides, so that the presence of an added type of “helodermin- PHI-preferring” receptors is suggested.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID

摘要:

Serum levels of glucose, insulin, and gastric inhibitory polypeptide (GIP) in response to intraduodenal and intravenous glucose loads have been examined in rats with exocrine pancreatic atrophy induced by feeding them a copper-deficient diet supplemented with D-penicillamine for 10–12 weeks. Whereas pancreatic weight and protein and trypsin content were drastically reduced as compared with pair-fed controls, insulin content was not significantly different between experimental (1.10 U) and control (I.40 U) rats. The intravenous glucose infusion was given in a dose (1.2 g/kg/h) simulating the glucose concentrations observed in response to an intraduodenal glucose load of 2.0 g/kg body weight. Both basal and stimulated insulin concentrations were lower in experimental animals as compared with controls. However, if the relative insulin response is considered, insulin secretion was almost identical in experimental and control animals. Both groups released significantly more insulin after intraduodenal glucose load than after intravenous glucose application. It is concluded that the entero-insular axis is intact in rats with exocrine pancreatic atrophy.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID

摘要:

The effects of secretin and a cholecystokinin-like peptide alone and in combination on pancreatic protein secretion and synthesis were examined in anesthetized rats. L-[75Se]selenomethionine was given as an i.v. shot (5 μCi) followed by a continuous infusion (1 μCi/h). Pancreatic juice was collected basally for 1 h and then during a 4-h i.v. infusion of 0.5, 2.5, and 12.5 μg/kg/h of Thr28Nle31CCK25–33(CCK-LP) alone or combined with 5 μg/kg/h of secretin, as well as during an infusion of secretin alone. The protein-bound radioactivity in pancreatic juice was taken to indicate secretion of newly synthesized proteins. The free and protein-bound radioactivity remaining in the gland after 2 h of stimulation was also investigated. Secretin alone slightly increased both protein secretion and secretion of newly synthesized proteins. Considerably greater effects were found with CCK-LP. The two lowest doses behaved similarly, whereas the volume and protein responses to 12.5 pg/kg/h were significantly lower and associated with large amounts of free radioactivity in pancreatic juice, suggesting leakage and cell damage. The addition of secretin transiently potentiated protein discharge and secretion of newly synthesized proteins and permanently increased the amount of protein-bound radioactivity per milligram of protein in response to all doses of CCK-LP. Secretin markedly increased volume and reduced the amount of free radioactivity in pancreatic juice with the largest dose of CCK-LP. The peptides were not found to influence amino acid uptake or incorporation of radioactivity into tissue proteins. This study demonstrates an interaction between the effects of secretin and those of CCK-LP on pancreatic protein secretion and synthesis. The findings also suggest that the damage to the pancreas induced by a supramaximal dose of CCK-LP may be reduced by the addition of secretin.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID

摘要:

In this study a radioimmunoassay was developed to measure secreted amylase from the isolated perfused rat pancreas. Using Sephadex G-75 gel chromatography, rat pancreatic amylase was purified to a single migrating protein band as determined by SDS polyacrylamide gel electrophoresis. Specificity of a rat pancreatic amylase antiserum, raised in rabbits, was determined using immunodiffusion, immunoelectrophoresis, and immunoblotting techniques. Secreted amylase concentrations, obtained using the radioimmunoassay, were not significantly different than those measured with the amylase enzyme assay. The rat pancreatic amylase radioimmunoassay was used to measure the amylase secretion in the isolated perfused rat pancreas. Phe- Met-Arg-Phe-amide (FMRF-NH2) immunoreactivity has been shown to be colocalized with pancreatic polypeptide in the rat pancreatic islet, and evidence suggests that islet peptides modulate amylase secretion from the exocrine pancreas. In the present study, FMKF-NH, significantly (p <0.05) suppressed cholecystokinin (CCK)-stimulated amylase secretion by 55%. The average pancreatic amylase secretion in response to CCK was 10.89 ± 2.0 kg/ml/min (n = 6); with the addition of FMKF-NH2, CCK-stimulated amylase secretion was reduced to 4.79 ± 1.6 μg/ml/min (n = 6). These results are consistent with the insuloacinar hypothesis in that an FMRF-NH,-like substance in the islet may act to modulate the exocrine pancreas.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID

摘要:

The neuropeptide galanin is present in intrapancreatic nerve fibers and is known to affect the secretion of the islet hormones. Its most potent effect is thereby the inhibition of insulin secretion. In the present study, we investigated whether galanin influences amylase secretion from isolated rat pancreatic acini. Acini were isolated by the collagenase digestion technique and incubated for 45 min in a Krebs-Henseleit medium with or without addition of the cholinergic agonist carbachol or the C-terminal octapeptide of cholecystokinin (CCK-8) in the presence or absence of galanin. Carbachol, at its optimal concentration M), stimulated amylase secretion to 11.8 ± 0.5% of total amylase content compared to 4.3 2 0.3% in controls (p <0.001). Galanin, (10-8-10-9 M), reduced the carbachol-induced amylase secretion to 10.4 ±0.3% (p <0.01). Galanin at concentration levels below 10−7M had no significant effect. At 10−8M, CCK-8 stimulated amylase secretion to 9.7 ± 0.6% compared with 5.2 ± 0.3% in controls (p <0.01). Galanin (10−7M) reduced this stimulation to 8.0 ± 0.4% (p <0.05). Galanin did not affect basal amylase secretion. It is concluded that the intrapancreatic neuropeptide galanin weakly inhibits carbachol- and CCK-8-induced amylase secretion from isolated rat pancreatic acini. Thus, galanin has the capability to directly affect not only endocrine but also exocrine pancreatic secretion although its effect of inhibiting amylase secretion seems weak.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID

摘要:

Scavengers of toxic oxygen reduction products have been reported to reduce the inflammatory reaction in some models of pancreatitis. In a blinded study, the effect of parenteral pretreatment with superoxide dismutase plus catalase was compared with placebo on pancreatitis induced in rats by infusion of 0.25% or 2% sodium taurocholate into the hepatopancreatic duct. The degree of inflammation was assessed by macroscopic examination of the pancreas, drylwet weight ratios of pancreatic specimens, amylase activity in plasma and peritoneal exudate, the weight of the exudate, and its content of total protein. All parameters were indicative of a more severe inflammation in rats given the higher concentration of sodium taurocholate. The only signifcant effect of the superoxide dismutase plus catalase treatment was a moderate reduction of the drylwet weight ratio, i.e., pancreatic edema, in rats given 2% sodium taurocholate. Our results indicate that toxic oxygen reduction products, available for interception by parenterally administered superoxide dismutase plus catalase, are of only minor importance in the pathogenesis of sodium taurocholate-induced pancreatitis in the rat.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID

摘要:

A new technique to obstruct the pancreatic ducts was developed by injecting zein solution into the common bile duct of the rat. Four weeks after the injection, the fate of the endocrine pancreas was investigated by studying (a) pancreatic content of four pancreatic hormones, (b) histology and immunohistochemistry of the pancreas, (c) i.v. glucose tolerance and i.v. insulin tolerance tests for monitoring plasma glucose, insulin, and pancreatic polypeptide (PP) levels in vivo, and (d) in vitro perifusion of pancreatic tissue slices to assess insulin and PP secretion. In zein-injected rats, the total pancreatic content of insulin, glucagon, PP, and somatostatin was reduced to 80, 70, 40, and 20%, respectively, of the corresponding controls. In response to insulininduced hypoglycemia, the plasma PP levels rose to about one-half that of the controls. By contrast, perifused zein-injected rat pancreases released several times more PP than the controls in response to carbachol stimulation. In zeininjected rats, total pancreatic protein was reduced to 20% of the controls and pancreatic amylase was almost absent, reflecting practically complete loss of acinar tissue. Thus, this experimental model appears to be suitable for producing chronic pancreatic insufficiency in the rat and provides a useful model for studying both endocrine and exocrine functions in the small rodent.

ISSN:0885-3177    

出版商:OVID    

年代:1988

数据来源: OVID