Variations in the activity of digestive enzymes along the intestine of the burbot Lota lota expressed by different methods.

The activities of major digestive hydrolases (proteases, amylase, lipase and esterases) along the intestine were studied in the burbot Lota lota (L.) using different methods of activity expression. The enzyme activities were determined both in the whole gut segments and in the isolated mucosa, and then expressed in terms of tissue mass and protein content in the samples. Further, the cumulative activities of these enzymes in the pyloric caeca were compared with those in the rest of the intestine to estimate the overall contribution of these regions to digestion. The data obtained suggest the essential role of the pyloric caeca in the digestion of the burbot. In addition, the variations in the pH values along the intestine and the changes in the enzyme activities with incubation temperature were examined. The study proved the method of enzyme activity expression to be a key factor influencing the outcome of the experiment.

Myosin light chain kinase is involved in the mechanism of gastrointestinal dysfunction in diabetic rats.

BACKGROUND: It is well established that smooth muscle contractility is regulated by an elevation of cytosolic Ca(2+) via myosin light chain phosphorylation, which is activated by myosin light chain kinase (MLCK). Recently, MLCK has been demonstrated to play an important role in smooth muscle contraction and normal gastrointestinal motility. AIMS: The aim of our study is to investigate whether MLCK is involved in the mechanism of gastrointestinal dysfunction and the ameliorating effects of insulin on gastrointestinal dysfunction in diabetic rats. METHODS: A diabetic rat model was established by an intravenous injection with streptozotocin. Rats were randomized into three groups: control group, diabetic group, and insulin-treated group. The gastrointestinal functions were assessed in terms of gastric emptying and intestinal transit. The expression of MLCK in the pylorus and ileum of the three groups was determined by real-time polymerase chain reaction (PCR) and Western blot methods. RESULTS: The diabetic group exhibited a significant delay in gastric emptying and intestinal transit than the control group. Insulin treatment significantly ameliorated the gastric emptying and intestinal transit in diabetic rats. The expression levels of MLCK in the pylorus and ileum of the diabetic group were both significantly decreased compared with the control group, and the changes of MLCK expression in these tissues of diabetic rats were partially reversed after treatment with insulin. CONCLUSIONS: Decreased expression of MLCK in gastrointestinal tissues could be a possible cause for gastrointestinal dysfunction. Insulin may partly ameliorate gastrointestinal dysfunction by restoring the expression of MLCK.

Evaluation of the relationship between lesions in the gastroduodenal region and cyclooxygenase expression in clinically normal dogs.

OBJECTIVE: To determine whether clinically normal dogs have lesions in the pylorus and duodenum and to examine the expression of cyclooxygenase (COX) isoforms in the pylorus and duodenum of these dogs. ANIMALS: 27 clinically normal dogs. PROCEDURES: Physical examination was performed on clinically normal dogs from animal shelters and research projects; the dogs were then euthanized. After the dogs were euthanized, the pylorus and duodenum were photographed and scored for gross appearance of lesions. Samples were obtained for histologic evaluation and determination of COX expression via western blot analyses. Tissues from the pylorus and duodenum were categorized as normal, inflamed, or eroded on the basis of histologic analysis. Each histologic category of tissue was then evaluated to determine the correlation with gross appearance and COX expression. RESULTS: Of the 27 dogs, 5 had unremarkable histologic findings in the pylorus and duodenum. Inflammation was found in the pylorus of 10 dogs and in the duodenum of 5 dogs. Epithelial erosion was detected in the pylorus of 1 dog and in the duodenum of 3 dogs. Gross appearance was not significantly correlated with histologic appearance. Expression of COX-1 was not upregulated by inflammation, whereas COX-2 expression was increased by inflammation or erosion. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs that appear to be clinically normal may have underlying gastroduodenal lesions associated with upregulation of COX-2. Because of the inability to determine this during routine physical examination, practitioners should be aware of this potential situation when prescribing COX inhibitors.

Purification and biochemical characterization of an acid-stable lipase from the pyloric caeca of sardine (Sardinella aurita).

A lipolytic activity was located in the sardine digestive glands (pyloric caeca), from which a sardine digestive lipase (SaDL) was purified. Pure SaDL has a molecular mass of 43 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The enzyme was found to be more active on short-chain triacylglycerols than on long-chain ones. SaDL does not present the interfacial activation phenomenon. Control experiments were performed under the same experimental conditions, with dromedary and turkey pancreatic lipases and showed a positive interfacial activation phenomenon. Sodium deoxycholate (NaDC) has an inhibitory effect on the lipase activity. The pure enzyme lost 40% of its activity in presence of 8 mM NaDC. SaDL was found to be mostly stable at low pH values. Interestingly, no colipase was detected in the sardine pyloric caeca. Analogous results were reported for the scorpion and the crab digestive systems. This is in line with the idea that colipase might has evolved in mammal animals simultaneously with the appearance of an exocrine pancreas. No similarity was found between the NH(2)-terminal amino acid residues of SaDL and those of lipases from the digestive tract of other species. Altogether, these results suggest that SaDL is a member of a new group of lipases belonging to aquatic species.

Trypsin from the pyloric ceca of pectoral rattail (Coryphaenoides pectoralis): purification and characterization.

Trypsin from the pyloric ceca of pectoral rattail (Coryphaenoides pectoralis) was purified and characterized. Purification was carried out by ammonium sulfate precipitation, followed by column chromatographies on Sephacryl S-200, DEAE-cellulose and Sephadex G-50. The enzyme was purified 89-fold with a yield of 2.2%. Purified trypsin had an apparent molecular weight of 24 kDa when analyzed using SDS-PAGE and size exclusion chromatography. Optimal profiles of pH and temperature of the enzyme were 8.5 and 45 degrees C, respectively, using N(alpha)-p-tosyl-l-arginine methyl ester hydrochloride as a substrate. It was stable in a wide pH range of 6-11 but unstable at a temperature greater than 40 degrees C. Trypsin was stabilized by calcium ion. The activity of purified trypsin was effectively inhibited by soybean trypsin inhibitor and TLCK and was partially inhibited by EDTA. Activity continuously decreased with increasing NaCl concentration (0-30%). The kinetic trypsin constants K(m) and K(cat) were 0.15 mM and 210 s(-1), respectively, while the catalytic efficiency (K(cat)/K(m)) was 1400 s(-1) mM(-1). The N-terminal amino acid sequence of trypsin was determined to be 12 residues (IVGGYECQEHSQ), and the sequence showed high homology to other fish trypsins.

Digestive enzymatic profile of Dentex dentex and response to different dietary formulations.

Digestive physiology of on-growing common dentex (Dentex dentex), including protease, amylase and lipase activity in stomach, pyloric caeca, anterior and posterior intestine, was evaluated. The influence of dietary macronutrient balance on these digestive processes was also assessed. Four experimental diets with different protein:lipid:carbohydrate ratios (43/16/28; 43/24/4; 38/19/28 and 38/24/13) were formulated. The highest activity for acid proteases was located in the stomach at pH 1.5. Alkaline proteolytic activities showed the highest values in the pyloric caeca and posterior intestine at pH 8.5-9.0. Dentex showed substantial amylase activity in the pyloric caeca and posterior intestine. Lipase activity was higher in the pyloric caeca, anterior and posterior intestine and was not detected in the stomach. Feed composition influenced alkaline protease activity in the anterior and posterior intestine and was higher for the diet with less protein and more carbohydrates. Enhanced amylase activity was observed in the pyloric caeca and posterior intestine in those groups fed on higher carbohydrate and lower lipid level diets. High dietary carbohydrate levels produced the highest lipase activity but this only occurred in the anterior intestine. We can conclude that the digestive tract of dentex adapts well to protein digestion and possesses a high potential for digesting the other dietary macronutrients, too. Dietary carbohydrate content seems to induce changes in protease, amylase and lipase activity.

Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix).

Trypsin was purified from the pyloric caeca of bluefish (Pomatomus saltatrix) by ammonium sulfate precipitation, acetone precipitation and soybean trypsin inhibitor-Sepharose 4B affinity chromatography. Bluefish trypsin migrated as a single band using both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE and had a molecular mass of 28 kDa. The optima pH and temperature for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide (BAPNA) were 9.5 and 55 degrees C, respectively. The enzyme was stable over a broad pH range (7 to 12), but was unstable at acidic pH, and at temperatures greater than 40 degrees C. The enzyme was inhibited by specific trypsin inhibitors: soybean trypsin inhibitor (SBTI), N-p-tosyl-l-lysine chloromethyl ketone (TLCK) and the serine protease inhibitor phenylmethyl sulfonylfluoride (PMSF). CaCl2 partially protected trypsin against activity loss at 40 degrees C, but NaCl (0 to 30%) decreased the activity in a concentration dependent manner. The N-terminal amino acid sequence of trypsin was determined as IVGGYECKPKSAPVQVSLNL and was highly homologous to other known vertebrate trypsins.

Phenotypic flexibility of digestive system in Atlantic cod (Gadus morhua).

This study examined the restoration of the digestive capacity of Atlantic cod (Gadus morhua Linnaeus) following a long period of food deprivation. Fifty cod (48 cm, 1 kg) were food-deprived for 68 days and then fed in excess with capelin (Mallotus villosus Müller) on alternate days. Ten fish were sampled after 0, 2, 6, 14 and 28 days and the mass of the pyloric caeca, intestine and carcass determined. Two metabolic enzymes (cytochrome c oxidase and citrate synthase) were assayed in white muscle, pyloric caeca and intestine, and trypsin activity was measured in the pyloric caeca. A delay of 14 days was required before body mass started to increase markedly, whereas most of the increase in mass of both the pyloric caeca and intestine relative to fish length occurred earlier in the experiment. By day 14, the activities of trypsin and citrate synthase in the pyloric caeca as well as citrate synthase in the intestine had reached maxima. The growth of the digestive tissues and restoration of their metabolic capacities thus occur early upon refeeding and are likely required for recovery growth to take place. The phenotypic flexibility of the cod digestive system is therefore remarkable: increases in trypsin activity and size of pyloric caeca resulted in a combined 29-fold increase in digestive capacity of the fish during the refeeding period. Our study suggests that Atlantic cod are able to cope with marked fluctuations in food availability in their environment by making a rapid adjustment of their digestive capacity as soon as food availability increases.

Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea.

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.

Carboxypeptidase E in rat antropyloric mucosa: distribution in progenitor and mature endocrine cell types.

Processing of most gut hormones involves cleavage between dibasic amino acids followed by carboxypeptidase-catalyzed removal of the COOH-terminal basic residue, resulting in peptides with a COOH-terminal glycine. Such peptides may subsequently be converted to amidated peptides or can be directly secreted. It is believed that carboxypeptidase E (CPE) is involved in gut hormone processing but its presence in gut endocrine cells has never been studied. We have analyzed the distribution of CPE in the antropyloric mucosa of rat stomach and report that gastrin cells and progenitor gastrin-somatostatin (G/D) cells express CPE while mature somatostatin cells and the majority of serotonin cells fail to express CPE. These data indicate that immature G/D cells are able to process gastrin to glycine-extended forms and that CPE-mediated processing is not a characteristic of mature somatostatin and serotonin cells.

Distribution of heme oxygenase-2 in nerves and interstitial cells of Cajal in the normal pylorus and in infantile hypertrophic pyloric stenosis.

CONTEXT: Interstitial cells of Cajal (ICCs) are pacemaker cells, which are of fundamental importance in regulating gastrointestinal motility. Recent evidence suggests that carbon monoxide is a neurotransmitter involved in neurotransmission between ICC and smooth muscle cells. Heme oxygenase-2 (HO-2) is the major physiological mechanism for the generation of carbon monoxide in the enteric nervous system. OBJECTIVE: To investigate the immunocolocalization of HO-2 and ICCs in the normal pylorus and in infantile hypertrophic pyloric stenosis (IHPS). DESIGN: Specimens from 18 infants with IHPS and 8 control specimens were examined using double-immunostaining with c-Kit and HO-2 antibodies. The immunolocalization was detected with the help of confocal laser scanning microscopy. RESULTS: Abundant HO-2 immunoreactivity was found in ICCs in the smooth muscle layer of normal pylorus. There was a decrease in the number of ICCs and lack of HO-2 immunoreactivity in ICCs in IHPS. CONCLUSIONS: The results of the present study provide the first evidence for the presence of HO-2 in ICCs in the normal human pylorus. The lack of ICCs and HO-2 in IHPS suggests impaired intracellular communication between ICCs and smooth muscle cells, contributing to motility dysfunction in IHPS.

Isolation and characteristics of trypsin from pyloric ceca of the starfish Asterina pectinifera.

Trypsin was purified from pyloric ceca of the starfish Asterina Pectinifera by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 28000. Optimum pH and temperature of A. pectinifera trypsin for hydrolysis of N(alpha)-p-Tosyl-L-arginine methyl ester hydrochloride were approximately pH 8.0 and 55 degrees C, respectively. A. pectinifera trypsin was unstable at above 50 degrees C and below pH 5.0, and was not activated by adding Ca(2+). The N-terminal amino acid sequence of A. pectinifera trypsin, IVGGHEF, was found.

Site-specific gene expression of nNOS variants in distinct functional regions of rat gastrointestinal tract.

5' mRNA variants of neuronal nitric oxide synthase (nNOS) are generated either by alternative promoter usage resulting in different mRNAs that encode for the same protein (nNOSalpha) or alternative splicing encoding NH(2)-terminally truncated proteins (nNOSbeta/gamma) that lack the PDZ/GLGF domain for protein-protein interaction of nNOSalpha. We studied the expression of 5' nNOS mRNA forms and nNOS-interacting proteins (postsynaptic density protein-95; PSD-95) in the rat gastrointestinal tract and analyzed the more distinct localization of nNOS protein variants in the duodenum by immunohistochemistry with COOH- and NH(2)-terminal nNOS antibodies. 5' nNOS mRNA variants showed a site-specific expression along the gastrointestinal tract with presence of all forms (nNOSalpha-a, -b, -c; nNOSbeta) in the muscle layer of esophagus, stomach, duodenum, longitudinal muscle layer of jejunum/ileum, proximal colon, and rectum. In contrast, a lack of nNOSalpha-a and nNOSbeta mRNA was observed in pylorus, circular muscle layer of jejunum/ileum, and cecum. Expression of nNOSalpha and nNOSbeta cDNAs revealed proteins of ~155 kDa and 135/125 kDa, respectively. Immunohistochemistry showed a differential distribution of COOH- and NH(2)-terminal nNOS immunoreactivity in distinct layers of rat duodenum, suggesting a cell-specific expression and distinct compartmentalization of nNOS proteins. Observed distribution of 5' nNOS mRNA variants and proteins argue for a complex control of nNOS expression by usage of separate promoters, cell- and site-specific splicing mechanisms, and translational initiation. These mechanisms could be involved in gastrointestinal motor diseases and may explain the phenotype of nNOSalpha knockout mice with gastric stasis and pyloric stenosis, due to a total loss of nNOS in the pyloric sphincter region.

Time-course changes in the expression of Na+, K+-ATPase in gills and pyloric caeca of brown trout (Salmo trutta) during acclimation to seawater.

Changes in protein and mRNA expression of Na(+),K(+)-ATPase in gills and pyloric caeca of brown trout were investigated on a detailed time course after transfer from freshwater to 25 ppt seawater (SW). A transient deflection in plasma osmolality and muscle water content lasting from 4 h until day 3 was followed by restoration of hydromineral balance from day 5 onward. Gills and pyloric caeca responded to SW transfer by increasing Na(+),K(+)-ATPase activity from days 5 and 3, respectively, onward. In both tissues, this response was preceded by an increase in alpha-subunit Na(+), K(+)-ATPase mRNA as early as 12 h posttransfer. The similarity of the response in these two organs suggests that they both play significant physiological roles in restoring hydromineral balance after abrupt increase in salinity. Further, SW transfer induced a slight, though significant, increase in primary gill filament Na(+), K(+)-ATPase immunoreactive (NKIR) cell abundance. This was paralleled by a marked (50%) decrease in secondary lamellar NKIR cell abundance after less than 1 d in SW. Thus, SW acclimation in brown trout is characterised by a lasting decrease in overall NKIR cell abundance in the gill. We propose that SW transfer stimulates Na(+),K(+)-ATPase enzymatic activity within individual chloride cells long before (<1 d) it becomes apparent in measurements of whole-gill homogenate enzymatic activity. This is supported by the early stabilisation (12 h) of hydromineral balance.

Purification and characterization of pancreatic elastase from North Atlantic salmon (Salmo salar).

An elastase I-like enzyme was purified to homogeneity from the pyloric caeca of North Atlantic salmon (Salmo salar) and compared with porcine elastase I. The molecular weight and isoelectric point were estimated to be 27 kDa and over 9.3, respectively. The pH optimum was between 8.0 and 9.5, and the enzyme was unstable at pH values below 4. Kinetic properties examined using Suc-(Ala)3-p-nitroanilide showed that the catalytic efficiency of salmon elastase was about 2.5 times higher than that of porcine elastase. Furthermore, the salmon enzyme was less stable at lower pH values and temperatures than the porcine enzyme. The preference for amino acids at the primary binding site was found to be different from that of the porcine elastase. The salmon elastase binding pocket seems to prefer more branched aliphatic residues than the porcine elastase.

Nitric oxide synthase-containing nerve elements in the pylorus of the cat.

Combined nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) immunocytochemistry were used to study the distribution of NOS- and VIP-containing nerve elements in the feline pylorus. A large number of stained multipolar neurons was found in the myenteric plexus. However, some NADPH-d and NOS positive neurons were also observed in the submucous plexus and in the internal part of muscular layer. A few stained perikarya were found in the tunica mucosa, in a very close situation to the blood vessels. A large number of thin varicose fibres, with intense reaction for all markers were seen around or in close contact with the unstained perikarya to the blood vessels and some of them around the pyloric glands. The density of NOS and NADPH-d positive nerve elements was much higher than that of VIP-immunoreactive (IR) nerve elements. Our results suggest that nitric oxide (NO) might act as a regulatory neurotransmitter of the pyloric sphincter, blood flow and secretion in this region.

Purification and characterization of tissue kallikrein-like proteinases from the black sea bass (Centropristis striata) and the southern frog (Rana berlandieri).

Serine proteinases were isolated from the pyloric caeca of the black sea bass (Centropristis striata) and the pancreas of the Southern frog (Rana berlandieri) and were purified to apparent homogeneity by aprotinin affinity column chromatography, reverse phase high performance liquid chromatography and gel filtration FPLC liquid chromatography to produce products with molecular masses of approximately 27,000 Da and isoelectric points from 4.2 to 5.0. Both enzymes were kallikrein-like and were bound by diisopropylfluorophosphate; had pH optima from 9 to 10; showed high specificity for the hydrolysis of arginine peptide bonds and low to moderate affinity for lysine bonds at the P1 substrate recognition sites; were inhibited by aprotinin, benzamidine, leupeptin, and soybean trypsin inhibitor; generated kinin from kininogen and were highly stable at room temperature. Differences between the enzymes were observed relative to their hydrophobicities, substrate specificities, stabilities at acidic pHs in the presence and absence of calcium, and the amounts of kinin generated from kininogen. Many of the fish trypsins, previously identified as anionic trypsins, may actually be more kallikrein-like.

[The pepsinogen of the gastric mucosa in the red-cheeked suslik Citellus erythrogenys]. / Pepsinogen slizistoi obolochki zheludka krasnoshchekogo suslika Citellus erythrogenys.

Studies have been made on the isozymic composition of pepsinogen (polyacrylamide gel electrophoresis) and peptidase activity in gastric mucosa of the ground squirrel at various stages of hibernation. Eight pepsinogen isoforms were found in acid-producing zone of the mucosa, pyloric mucosa lacks first three of them, its peptidase activity being two times lower than in other parts of the stomach. During hibernation, no significant changes were found in fractional composition of pepsinogen, peptidase activity being decreased twofold only in the fundulus of the stomach, remaining practically constant in other parts of the latter. This finding indicates that the stomach is ready for the activity irrespectively of food uptake.

[Effects of qualitatively different food on acidity and proteolytic activity in different parts of the stomach of patients with duodenal ulcer]. / Vliianie kachestvenno razlichnoi pishchi na kislotnost' i proteoliticheskuiu aktivnost' v raznykh otdelakh zheludka u bol'nykh s duodenal'noi iazvoi.

Using an original technique, the authors studied acid-proteolytic activity in the stomach at night in 44 patients with duodenal ulcer before and after 12-h fasting; after meat, starch gel, vegetable oil meals. It was found that long-term fasting does not result in changed acid-proteolytic activity in the stomach body, though reduces a mean acid concentration in the stomach antral-pyloric portion 2-fold. Proteins and carbohydrates do not influence the acidity and produce multidirectional action on proteolytic activity of the stomach content: proteins inhibit, but carbohydrates enhance proteolysis. Vegetable oil results in a moderate decrease of the acidity.