Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway

Lysosomes can trigger the mitochondrial apoptotic pathway by releasing proteases. Here we report that a 25-kDa protein purified from rat liver lysosomes possesses a long standing potent Bid cleavage activity at neutral pH, and the truncated Bid can in turn induce rapid mitochondrial release of cytochrome c. This protease was revealed as chymotrypsin B by biochemical and mass spectrometric analysis. Although it was long recognized as a digestive protease exclusively secreted by the exocrine pancreas, our data support that it also expresses and intracellularly resides in rat liver lysosomes. Translocation of lysosomal chymotrypsin B into cytosol was triggered by apoptotic stimuli such as tumor necrosis factor-alpha, and intracellular delivery of chymotrypsin B protein induced apoptotic cell death with a potency comparable with cathepsin B, suggestive of a lysosomal-mitochondrial pathway to apoptosis regulated by chymotrypsin B following its release. Noteworthily, either knockdown of chymotrypsin B expression by RNA interference or pretreatment with chymotrypsin B inhibitor N-p-tosyl-L-phenylalanine chloromethyl ketone significantly reduced tumor necrosis factor-alpha-induce apoptosis. These results demonstrate for the first time that chymotrypsin B is not only restricted to the pancreas but can function intracellularly as a pro-apoptotic protease.     

Differences in cathepsin B mRNA levels in rat tissues suggest specialized functions

The tissue distribution of mRNAs encoding two lysosomal proteases, cathepsin B and cathepsin D, was examined using cloned cDNAs to probe Northern and dot blots of RNAs extracted from various rat tissues. Cathepsin B mRNA showed a wide range of variation in expression in the tissues analyzed with the highest concentrations found in spleen and kidney, while the cathepsin D mRNA levels were relatively uniform in these same tissues. Significant quantities of cathepsin B mRNA were detected in total RNA from isolated islets of Langerhans but was not detectable in equivalent amounts of RNA from whole pancreas. The wide variations in tissue levels of cathepsin B mRNA suggest that tissue specific controls may regulate its expression and are compatible with the participation of this protease in specialized cellular functions other than intralysosomal protein degradation.     

Somatostatin depletion of the gut and pancreas induced by cysteamine is not prevented by vagotomy or by dopamine agonists

The role of endogenous somatostatin in the pathogenesis of duodenal was investigated in the present study by using the cysteamine animal model of the disease. Our previous studies showed a rapid and multiorgan depletion of somatostatin immunoreactivity (SIR) in rats given a single dose of duodenal ulcerogen cysteamine. We now determined whether acetylcholinergic and dopaminergic modulation (both known to influence the development of duodenal ulcer) are accompanied by modification of cysteamine-induced SIR depletion in rat organs. Vagotomy performed either 1 or 18 h before cysteamine administration did not interfere with the chemically induced SIR decrease in pancreas, gastric and duodenal mucosa. Vagal denervation alone had no marked influence on SIR levels but if combined with cysteamine, the SIR depletion in the stomach was significantly more pronounced than after the duodenal ulcerogen alone. Pretreatment with the dopamine agonists bromocriptine or lergotrile (known to prevent the chemically induced duodenal ulcers) did not influence the SIR depletion by cysteamine. Thus cysteamine depletes endogenous somatostatin in peripheral organs (e.g., stomach, duodenum, pancreas) by mechanisms independent of both vagus nerve and dopamine agonists. A role of central somatostatin depletion leading to disinhibition of vagus is also considered in the pathogenesis of experimental duodenal ulcer.     

中国大鲵消化系统13种器官的蛋白水解酶种类和活性分析

蛋白水解对生命活动是必不可少的(Vassali et al., 1994),蛋白质的酶解修饰(Xu et al.,1999)、细胞的迁移、组织再生与修复、消化系统对食物中蛋白质的消化等均与蛋白水解酶有关(Baimbridge et al.,1992),许多病理过程也与蛋白水解酶功能失调有关(Teichert et al., 1989; Monard, 1988).因此开展大鲵消化系统各器官的蛋白水解酶种类和性质的研究,对了解大鲵消化系统各器官的功能、演化及大鲵的营养需求、食性、消化生理等是必要的.本文对大鲵消化系统各器官的蛋白水解酶特征进行了初步分析,现将结果报道如下.     

Inflammation of ectopic pancreatic tissue as unusual cause of duodenal perforation--a case report

Ectopic pancreatic tissue, also known as a pancreatic rest, is an uncommon congenital anomaly defined as extrapancreatic tissue located far from the pancreas and without any connection via vascular or anatomical means to it. Such tissue may occur throughout the GI tract but has a propensity to affect the stomach and the proximal small intestine. The majority of patients with pancreatic ectopia are asymptomatic, but when symptoms occur, they can be presented in a variety of ways. We report a patient with acute surgical abdomen due to a duodenal perforation caused by inflammation of ectopic pancreatic tissue in duodenum and stomach. Histology of the resected duodenum and stomach demonstrated heterotopic pancreatic tissue acute inflammation without atypia, suggesting "pancreatitis of the duodenum and stomach". To date, there have been a few reports describing perforation of the stomach due to heterotopic pancreas. Therefore, the present case was considered to be a very rare case of this disorder. To conclude, heterotopic pancreas should always be considered in the differential diagnosis of acute abdomen.     

Radioimmunoassay of pancreatic polypeptide in mammalian and submammalian vertebrates using a carboxyl-terminal hexapeptide antiserum

Pancreatic polypeptide (PP) immunoreactivity in acid-ethanol extracts of the pancreas of representative species of mammals, birds, reptiles, amphibians, and fish was studied by a radioimmunoassay (RIA) that utilizes an antiserum which cross-reacts exclusively with the COOH-terminal hexapeptide of PP (CTPP). PP immunoreactivity in acid-ethanol extracts of rat nonpancreas tissues (stomach, duodenum, skeletal muscle, brain) was also examined. Significant concentrations of PP immunoreactivity were detected in the pancreatic extracts of all species, except fish. Appreciable quantities of PP immunoreactivity were also found in the stomach and duodenum of rats. In all cases, tissue extracts showed parallelism with reference PP (bovine) in the RIA. Gel chromatography (Sephadex G-50sf) of tissue extracts (rat, turtle) demonstrated a major peak of PP immunoreactivity, which eluted in the region of the reference PP. Salamander PP immunoreactivity eluted after bovine PP. In addition, the CTPP RIA can be applied to measure plasma levels of PP in rats, dogs, and humans. By using this PP RIA, we observed that plasma PP levels increase significantly in dogs (P less than 0.05) after intravenous administration of neurotensin. In rats, administration of intravenous bombesin resulted in a significant elevation of plasma PP.     

Morphological analysis of ghrelin and its receptor distribution in the rat pancreas

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.     

Ontogenic pattern of nucleobindin-2/nesfatin-1 expression in the gastroenteropancreatic tissues and serum of Sprague Dawley rats

Nesfatin-1 is a novel metabolic hormone that has glucose-responsive insulinotropic actions. Islet β-cells and gastrointestinal tissues have been reported as abundant sources of nesfatin-1 and its precursor hormone nucleobindin-2 (NUCB2). While nesfatin-1 is emerging as a multifunctional hormone, there are no reports on the developmental expression of NUCB2/nesfatin-1. The main objective of this study was to examine the ontogenic expression of NUCB2 mRNA, and NUCB2/nesfatin-1 immunoreactivity in the pancreas, stomach and duodenum, and the circulating levels NUCB2/nesfatin-1 in Sprague Dawley rats. In addition, we also determined the co-localization of NUCB2/nesfatin-1 and insulin immunoreactivity during development. NUCB2/nesfatin-1 immunoreactivity was found in the rat stomach from postnatal days 13-27. Furthermore, NUCB2/nesfatin-1 immunoreactivity was also detected in the enteroendocrine cells of the duodenum at postnatal days 13 and 27. Duodenal NUCB2 mRNA expression at postnatal day 27 was highest. Serum NUCB2/nesfatin-1 levels on embryonic day 21 and postnatal day 1 were lower than serum NUCB2/nesfatin-1 levels of adults and neonates at postnatal days 13, 20 and 27, gradually increasing with growth, suggesting an increase in its production and secretion from tissues including the gastrointestinal tract and pancreas. Our findings indicate that NUCB2/nesfatin-1 colocalizes with insulin in the islet β-cells at all developmental stages, but the percentage of colocalization varies in an age-dependent manner. These findings suggest that NUCB2/nesfatin-1 has potential age- and tissue-specific role in the developmental physiology of rats during growth.     

Expression of mammalian RF-amide peptides neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and the PrRP receptor in the peripheral tissues of the rat

The mRNA expression of neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and the UHR-1/GPR10 receptor were examined using in situ hybridization in rat peripheral tissues. In the hypophysis, modest expression of PrRP and receptor mRNA were seen in the anterior lobe. The trigeminal ganglion was devoid of expression signals. PrRP and UHR-1/GPR10 receptor mRNA:s were found in the adrenal medulla and PrRP mRNA was found in the pancreas. NPFF mRNA was detected in the spleen. In the testis and epididymis, PrRP and UHR-1/GPR10 receptor mRNA:s were detected. The results suggest a limited expression of mammalian RF-amide peptides in the peripheral organs.     

Identification and regional distribution of the dopamine D(1A) receptor in the gastrointestinal tract

Dopamine (DA) is regarded as an important modulator of enteric function. Recent experiments have suggested that newly cloned DA receptor subtypes are widely expressed in peripheral organs, including the gastrointestinal tract. In the present studies, the D(1A) receptor subtype was identified in rat gut regions through localization of receptor protein by means of light microscopic immunohistochemistry and Western blot analysis and receptor mRNA by RT-PCR and in situ amplification and hybridization (3SR in situ). D(1A) receptor immunoreactivity was shown to have a diverse distribution in the gastrointestinal tract, being present in the gastroesophageal junction, stomach, pylorus, small intestine, and colon. The receptor has a transmural distribution present in both epithelial and muscle layers as well as in blood vessels and lamina propria cells of different gastrointestinal regions. Western blot analysis demonstrated a single 50-kDa band for esophagus, stomach, duodenum, jejunum, and colon. The in situ hybridization signal was localized to the same sites revealed by D(1A) receptor immunoreactivity. RT-PCR revealed an appropriate sized signal in similar regions. This study is the first to identify expression of the central D(1A) receptor throughout the normal mammalian gastrointestinal tract.     

Isolation and sequence of a rat chymotrypsin B gene

A cDNA clone encoding part of chymotrypsin B was isolated from a cDNA library prepared from rat pancreatic mRNA and used as a probe to isolate the chymotrypsin B gene. The nucleotide sequence of this gene is presented. The 4709-base pair transcribed portion of the isolated gene was inferred from the cDNA and gene sequence, and the 5' border was determined by primer extension on pancreatic polyadenylated RNA. The coding portion of the gene is interrupted by six introns. The active site residues His 57, Asp 102, and Ser 195 are encoded by separate exons. Moreover, two regions of the enzyme which form the substrate-binding pocket are also encoded by separate exons. Thus, the substrate specificity and catalytic activity of the enzyme are produced by joining several exons encoding protein segments that are intrinsically catalytically inactive. The number and location of the intron/exon junctions of the chymotrypsin gene as compared to those of other serine protease genes, as well as the location of the genes on separate chromosomes, suggest that the duplication that resulted in the formation of the chymotrypsin gene was an ancient evolutionary event.     

Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract

A remarkable amount, of water is transported in the gastrointestinal (GI) organs to fulfil the secretory and absorptive functions of the GI tract. However, the molecular basis of water movement in the GI epithelial barriers is still poorly known. Important clues about the mechanisms by which water is transported in the GI tract were provided by the recent identification of multiple aquaporin water channels expressed in GI tissues. Here we define the mRNA and protein expression and the cellular and subcellular distribution of aquaporin-8 (AQP8) in the rat GI tract. By semi-quantitative RT-PCR the AQP8 mRNA was detected in duodenum, proximal jejunum, proximal colon, rectum, pancreas and liver and, to a lesser extent, in stomach and distal colon. Immunohistochemistry using affinity-purified antibodies revealed AQP8 staining in the absorptive epithelial cells of duodenum, proximal jejunum, proximal colon and rectum where labeling was largely intracellular and confined to the subapical cytoplasm. Confirming previous results, AQP8 staining was seen at the apical pole of pancreatic acinar cells. Interestingly, both light and immunoelectron microscopy analyses showed AQP8 reactivity in liver where labeling was associated to hepatocyte intracellular vesicles and over the plasma membrane delimiting the bile canaliculi. A complex pattern was observed by immunoblotting with total membranes of the above GI organs incubated with affinity-purified anti-AQP8 antibodies which revealed multiple bands with molecular masses ranging between 28 and 45 kDa. This immunoblotting pattern was not modified after deglycosylation with N-glycosidase F except the 34-kDa band of liver that, as already reported, was partially down-shifted to 28 kDa. No bands were detected after preadsorption of the anti-AQP8 antibodies with the immunizing peptide. The cellular and subcellular distribution of AQP8 suggest physiological roles for this aquaporin in the absorption of water in the intestine and the secretion of bile and pancreatic juice in liver and pancreas, respectively. The large intracellular expression of AQP8 may indicate its recycling between the cytoplasmic compartment and the plasma membrane. The cytoplasmic localization observed may also relate to the involvement of AQP8 in processes of intracellular osmoregulation.     

Localization of GAD-like immunoreactivity in the pancreas and stomach of the rat and mouse.

The aim of this study was to localize cells immunoreactive for glutamate decarboxylase (GAD), the enzyme of GABA synthesis, in pyloric and oxyntic regions of the rat stomach as well as in the rat and mouse pancreas. GAD immunocytochemistry was carried out on polyethylene glycol or cryostat sections of alkaline paraformaldehyde fixed tissue, with simultaneous immunolabelling of various gastro-pancreatic hormones for topographical comparison. In the rat stomach, nerve fibers displaying intense GAD-like immunoreactivity were seen in the myenteric plexus, the circular muscular layer, the submucosa and the lamina propria of the mucosa. But, they were absent from the submucous plexus. Colchicine treatment of the rats allowed to detect some labelled perikarya in the myenteric plexus suggesting that the GABAergic innervation is at least partly intrinsic to the stomach. In the oxyntic and pyloric mucosa, endocrine cells appeared immunostained for GAD. However, the nature of their hormones remained unknown since double immunodetections revealed that they were immunoreactive neither for gastrin nor for somatostatin. In the rat and mouse pancreas, GAD-like immunoreactivity was found in islet cells which corresponded only to insulin-secreting cells. Somatostatin-, glucagon- and pancreatic polypeptide-immunopositive cells were devoid of GAD immunolabelling. No GAD-like immunoreactivity was detected in the exocrine tissue and innervation. These results strenghten the hypothesis that GABA is not only a neurotransmitter in the stomach but that it could also be an endocrine or paracrine factor in the stomach and pancreas.     

Localization of GAD-like immunoreactivity in the pancreas and stomach of the rat and mouse

Summary The aim of this study was to localize cells immunoreactive for glutamate decarboxylase (GAD), the enzyme of GABA synthesis, in pyloric and oxyntic regions of the rat stomach as well as in the rat and mouse pancreas. GAD immunocytochemistry was carried out on polyethylene glycol or cryostat sections of alkaline paraformaldehyde fixed tissue, with simultaneous immunolabelling of various gastro-pancreatic hormones for topographical comparison. In the rat stomach, nerve fibers displaying intense GAD-like immunoreactivity were seen in the myenteric plexus, the circular muscular layer, the submucosa and the lamina propria of the mucosa. But, they were absent from the submucous plexus. Colchicine treatment of the rats allowed to detect some labelled perikarya in the myenteric plexus suggesting that the GABAergic innervation is at least partly intrinsic to the stomach. In the oxyntic and pyloric mucosa, endocrine cells appeared immunostained for GAD. However, the nature of their hormones remained unknown since double immunodetections revealed that they were immunoreactive neither for gastrin nor for somatostatin. In the rat and mouse pancreas, GAD-like immunoreactivity was found in islet cells which corresponded only to insulin-secreting cells. Somatostatin-, glucagon- and pancreatic polypeptide-immunopositive cells were devoid of GAD immunolabelling. No GAD-like immunoreactivity was detected in the exocrine tissue and innervation. These results strenghten the hypothesis that GABA is not only a neurotransmitter in the stomach but that it could also be an endocrine or paracrine factor in the stomach and pancreas.     

Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut

Endocrine cells containing peptide YY (PYY) were numerous in the rectum, colon and ileum and few in the duodenum and jejunum of rat, pig and man. No immunoreactive cells could be detected in the pancreas and stomach. Coexistence of PYY and glicentin was revealed by sequential staining of the same section and by staining consecutive semi-thin sections. Since the PYY sequence is not contained in the glucagon/glicentin precursor molecule the results suggest that the PYY cell in the gut expresses two different genes coding for regulatory peptides of two different families.     

Tissue distribution of pneumadin immunoreactivity in the rat

Pneumadin (PNM) is a decapeptide, originally isolated from mammalian lungs, which exerts a potent stimulating effect on arginine-vasopressin (AVP) release, thereby evoking an antidiuretic effect. We have established a specific radioimmunoassay (RIA) method for rat PNM determination, the sensitivity of which is sufficient for measuring tissue content of the peptide. Moreover, raised antibodies have been used for the immunocytochemical detection of PNM in several rat organs. As expected, high concentrations of PNM were detected by RIA in newborn and adult rat lungs and immunocytochemistry (ICC) localized PNM immunoreactivity (IR) in the bronchial and bronchiolar epithelium. Very high concentrations of PNM were measured by RIA in the prostate, and ICC showed that PNM-IR is contained in the epithelial cells. RIA and ICC demonstrated the presence of low amounts of PNM in the thymus. The highest content of radioimmunoassayable PNM was found in the kidneys and intestinal tract, but dilution test suggested the presence of some interfering substances in these tissues. Accordingly, ICC-detectable PNM-IR was absent in the kidneys and present only in the duodenal criptae and Brunner's glands of the intestinal tract. RIA did not measure sizeable PNM concentrations in the thyroid gland, but ICC showed PNM-IR in C-cells. RIA and ICC did not detected PNM in testes, seminal vesicles, ovaries, uterus, pancreas, liver, spleen, adrenal glands, and heart. Taken together, our findings suggest that PNM, in addition to its role as hypothalamo-pituitary AVP secretagogue, may be involved in the autocrine-paracrine functional regulation of other peripheral organs, like lungs and prostate and perhaps duodenum, thymus and thyroid gland.     

Distribution of glucagon-like peptide I in canine and feline pancreas and gastrointestinal tract

Recently, a putative hormone, glucagon-like peptide I (GLP I), has been identified in the predicted sequences of the precursors to pancreatic glucagon in human, rat, hamster, and ox. The distribution of GLP I immunoreactivity in canine and feline pancreas and gastrointestinal tract was examined immunohistochemically and was compared with that of two other antigenic determinants of pancreatic pro-glucagon, i.e., glucagon and the NH2 terminus of glicentin. All three determinants occurred in the same population of islet cells in normal pancreas and in pancreas consisting predominantly of islet tissue from dogs with canine pancreatic acinar atrophy. Northern blot analysis of mRNA from the latter tissue, using a rat pre-pro-glucagon complementary DNA probe, revealed a single mRNA species similar in size to the pre-pro-glucagon mRNA detected in fetal rat pancreas. The three antigenic determinants of pancreatic pro-glucagon were co-localized also in intestinal L-cells and in canine gastric A-cells. Canine and feline pancreatic pro-glucagons therefore resemble those identified in other mammals and may also occur in gastrointestinal endocrine cells. Although there is evidence that the GLP I sequence is not liberated from pancreatic pro-glucagon, our results raise the possibility that this putative hormone may be a cleavage product of pro-glucagon in the gastrointestinal tract.