Fluid and amylase secretion by perfused parotid gland: physio-morphological approach.
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Whole gland perfusion technique was applied to rat parotid glands to assess whether amylase affects fluid secretion. Control perfusion without any secretagogue evoked no spontaneous secretion. Carbachol (CCh 1 microM) induced both amylase and fluid secretion with distinctive kinetics. Fluid secretion occurred constantly around 60 microL/g-min, whereas amylase secretion exhibited an initial peak, followed by a rapid decrease to reach a plateau. Isoproterenol (Isop 1 microM) alone did not induce fluid secretion although it evoked amylase secretion as measured in isolated perfused acini. Addition of Isop during CCh stimulation evoked a rapid and large rise in amylase secretion accompanied by small increase in oxygen consumption. Morphological observations carried out by HR SEM and TEM revealed exocytotic profiles following Isop stimulation. CCh stimulation alone seldom showed exocytotic profiles, suggesting a low incidence of amylase secretion during copious fluid secretion. Combined stimulation of CCh and Isop induced both vacuolation and exocytosis along intercellular canaliculi. These findings suggest that control of salivary fluid secretion is independent of the amylase secretion system induced by CCh and/or Isop. (+info)
Sphincter of Oddi dysfunction produces acute pancreatitis in the possum.
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BACKGROUND: Sphincter of Oddi dysfunction has been implicated as a cause of various forms of acute pancreatitis. However, there is no direct evidence to show that sphincter of Oddi dysfunction can cause obstruction of trans-sphincteric flow resulting in acute pancreatitis. AIMS: To determine if induced sphincter of Oddi spasm can produce trans-sphincteric obstruction and, in combination with stimulated pancreatic secretion, induce acute pancreatitis. METHODS: In anaesthetised possums, the pancreatic duct was ligated and pancreatic exocrine secretion stimulated by cholecystokinin octapeptide/secretin to induce acute pancreatitis. In separate animals, carbachol was applied topically to the sphincter of Oddi to cause transient sphincter obstruction. Sphincter of Oddi motility, trans-sphincteric flow, pancreatic duct pressure, pancreatic exocrine secretion, plasma amylase levels, and pancreatic tissue damage (histology score) were studied and compared with variables in ligation models. RESULTS: Acute pancreatitis developed following stimulation of pancreatic exocrine secretion with peptides after pancreatic duct ligation (p<0.05). Neither pancreatic duct ligation nor stimulation of pancreatic exocrine secretion with cholecystokinin octapeptide/secretin alone resulted in acute pancreatitis. Topical carbachol stimulated sphincter of Oddi motility abolished trans-sphincteric flow, and increased pancreatic exocrine secretion (p<0.05) and pancreatic duct pressure to levels comparable with pancreatic duct ligation (p<0.001). Carbachol application (with or without combined peptide stimulation) elevated plasma amylase levels (p<0.01) and produced pancreatic tissue damage (p<0.05). Decompression of pancreatic duct ameliorated these effects (p<0.05). CONCLUSION: Induced sphincter of Oddi dysfunction when coupled with stimulated pancreatic secretion causes acute pancreatitis. This may be an important pathophysiological mechanism causing various forms of acute pancreatitis. (+info)
Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis.
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Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue-induced pancreatitis, the trypsin activity in the pancreas of ctsb(-/-) animals was more than 80% lower than in ctsb(+/+) animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb(-/-) animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis. (+info)
Effects of gamma-aminobutyric acid on secretagogue-induced exocrine secretion of isolated, perfused rat pancreas.
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Because GABA and its related enzymes have been determined in beta-cells of pancreas islets, effects of GABA on pancreatic exocrine secretion were investigated in the isolated, perfused rat pancreas. GABA, given intra-arterially at concentrations of 3, 10, 30, and 100 microM, did not exert any influence on spontaneous or secretin (12 pM)-induced pancreatic exocrine secretion. However, GABA further elevated CCK (10 pM)-, gastrin-releasing peptide (100 pM)-, or electrical field stimulation-induced pancreatic secretions of fluid and amylase dose dependently. The GABA (30 microM)-enhanced CCK-induced pancreatic secretions were completely blocked by bicuculline (10 microM), a GABA(A) receptor antagonist, but were not affected by saclofen (10 microM), a GABA(B) receptor antagonist. The enhancing effects of GABA (30 microM) on CCK-induced pancreatic secretions were not changed by tetrodotoxin (1 microM) but were partially reduced by cyclo-(7-aminoheptanonyl-Phe-D-Trp-Lys-Thr[BZL]) (10 nM), a somatostatin antagonist. In conclusion, GABA enhances pancreatic exocrine secretion induced by secretagogues, which predominantly induce enzyme secretion, via GABA(A) receptors in the rat pancreas. The enhancing effect of GABA is partially mediated by inhibition of islet somatostatin release. (+info)
Chemostat enrichments of human feces with resistant starch are selective for adherent butyrate-producing clostridia at high dilution rates.
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Resistant starch (RS) enrichments were made using chemostats inoculated with human feces from two individuals at two dilution rates (D = 0.03 h(-1) and D = 0.30 h(-1)) to select for slow- and fast-growing amylolytic communities. The fermentations were studied by analysis of short-chain fatty acids, amylase and alpha-glucosidase activities, and viable counts of the predominant culturable populations and the use of 16S rRNA-targeted oligonucleotide probes. Considerable butyrate was produced at D = 0. 30 h(-1), which corresponded with reduced branched-chain fatty acid formation. At both dilution rates, high levels of extracellular amylase activity were produced, while alpha-glucosidase was predominantly cell associated. Bacteroides and bifidobacteria predominated at the low dilution rate, whereas saccharolytic clostridia became more important at D = 0.30 h(-1). Microscopic examination showed that within 48 h of inoculation, one particular bacterial morphotype predominated in RS enrichments at D = 0.30 h(-1). This organism attached apically to RS granules and formed rosette-like structures which, with glycocalyx formation, agglomerated to form biofilm networks in the planktonic phase. Attempts to isolate this bacterium in pure culture were repeatedly unsuccessful, although a single colony was eventually obtained. On the basis of its 16S rDNA sequence, this RS-degrading, butyrate-producing organism was identified as being a previously unidentified group I Clostridium sp. A 16S rRNA-targeted probe was designed using this sequence and used to assess the abundance of the population in the enrichments. At 240 h, its contributions to total rRNA in the chemostats were 5 and 23% at D = 0.03 and 0.30 h(-1), respectively. This study indicates that bacterial populations with significant metabolic potential can be overlooked using culture-based methodologies. This may provide a paradigm for explaining the discrepancy between the low numbers of butyrate-producing bacteria that are isolated from fecal samples and the actual production of butyrate. (+info)
Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp. strain A28.
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The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M(w)-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N'-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30 degrees C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium. (+info)
Adult human cytokeratin 19-positive cells reexpress insulin promoter factor 1 in vitro: further evidence for pluripotent pancreatic stem cells in humans.
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Human pancreatic cells with a typical ductal phenotype and potential to proliferate can be obtained in vitro, but the differentiation capacity of these putative human pancreatic stem cells remains to be documented. We investigated the protein and mRNA expression of insulin promoter factor 1 (IPF-1) (or pancreas/duodenal homeobox 1), a transcription factor critical for pancreatic development and endocrine cell neogenesis, in human pancreatic ductal cells derived from cultured exocrine tissue. In vitro, exocrine cells rapidly adhered (within 12 h) and were de-/transdifferentiated to ductal cells after 3 days with a dramatic loss of amylase protein (n = 4, 92 +/- 3.3%, P < 0.05 vs. day 1) and a simultaneous increase of ductal cytokeratin 19 protein (n = 4, 3.4-fold on day 3 and 7-fold on day 9, P < 0.05 vs. day 1). IPF-1 protein and mRNA levels were low to undetectable in exocrine preparations before culture. After 2 days of culture, a 3.2-fold increase in IPF-1 protein was observed, corresponding to the characteristic 46-kDa protein in Western blots. Reverse transcriptase-polymerase chain reaction confirmed a 10.5-fold increase in IPF-1 mRNA levels after 3 days of culture (n = 5, P < 0.001 vs. day 1). Double immunocytochemistry showed direct evidence that IPF-1 appeared during culture in these exocrine-derived ductal cells (cytokeratin 7-positive) and was not merely in contaminating endocrine cells (chromogranin A-positive). In conclusion, we describe herein the first converging evidence on both the molecular and protein level that human cells with a typical ductal phenotype derived ex vivo from pancreatic exocrine tissue (obtained from healthy donors) can reexpress IPF-1 in culture, suggesting their pancreatic precursor/stem cell potential. (+info)
The influence of sensory nerves and CGRP on the pancreatic regeneration after repeated episodes of acute pancreatitis in rats.
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Stimulation of capsaicin sensitive nerves or administration of calcitonin gene-related peptide (CGRP) before induction of acute pancreatitis (AP) attenuates pancreatic damage, whereas CGRP administration after development of AP aggravates lesion of pancreatic tissue. The aim of this study was to determine the effect of prolonged activity of sensory nerves or CGRP administration on the pancreatic repair after repeated episodes of AP. Five episodes of acute caerulein-induced pancreatitis (10 microg/kg/h for 5 h s.c.) were performed at weekly intervals in rats receiving either vehicle or capsaicin at the sensory nerve stimulatory dose (0.5 mg/kg, 3 times daily), or CGRP (10 microg/kg, 3 times daily). Two weeks after the last induction of AP morphological signs of pancreatic damage, pancreatic blood flow (PBF), serum and pancreatic amylase activity, fecal chymotrypsin activity, pancreatic weight, pancreatic RNA and DNA content, as well as, serum interleukin-1beta (Il-1beta ) were assessed. Pancreata of animals receiving vehicle alone showed almost full recovery within two weeks after last episode of pancreatitis induction. In capsaicin-treated group of rats, we observed the increase in PBF by 44% and in serum Il-1beta concentration by 91%. The pancreatic amylase activity, fecal activity of chymotrypsin, pancreatic nucleic acids content and DNA synthesis were decreased. In rats treated with CGRP the alterations in PBF, serum Il-1beta concentration, as well as, in pancreatic and fecal activity of enzymes were similar to capsaicin treated group but less pronounced. We conclude that prolonged activity of capsaicin-sensitive sensory nerves and the presence of their main mediator-CGRP during pancreatic regeneration after AP leads to pancreatic functional insufficiency typical for chronic pancreatitis. (+info)