Purification of carboxyl ester lipase (bile salt-stimulated lipase) from human milk and pancreas.
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Carboxylesterase↗
Biomedical subjects
R D Duan
Publications and source records attributed to R D Duan.
Bacterial adherence and mucosal cytokine responses. Receptors and transmembrane signaling.
By attaching to cells or secreted mucosal components, microbes are thought to avoid elimination by the flow of secretions that constantly wash mucosal surfaces. The attached state enhances their ability to trap nutrients and allows the bacteria to multiply more efficiently than do unattached bacterial cells. Attachment is therefore regarded as an end result in itself, and emphasis has been placed on the role of adherence for colonization of mucosal surfaces. Specific adherence was shown to be essential for the tissue tropism that is to guide microbes to their respective sites of colonization/infection. Attachment is not only a mechanism of tissue targeting but also a first step in the pathogenesis of many infections. The attaching bacteria engage in a "cross-talk" with the host cells through the mutual exchange of signals and responses. Enteropathogenic E. coli induce attaching and effacing lesions (Finley et al., this issue). Shigella and Listeria sp. invade the cells and cause actin polymerization (Sansonetti et al., this issue). This review describes the ability of bacteria to trigger mucosal inflammation through activation of cells in the mucosal lining. The results suggest that receptors for bacterial adhesins bind their ligands with a high degree of specificity and that ligand-receptor interactions trigger transmembrane signaling events that cause cell activation. Receptors for microbial ligands thus appear to fulfill also the same criteria as those used to define receptors for other classes of ligands such as hormones, growth factors, and cytokines.
Bacterial Adhesion↗
Protein phosphatase inhibitors potentiate Ca2+/calmodulin-dependent protein kinase II activity in rat pancreatic acinar cells.
Cholecystokinin (CCK) is known to rapidly and transiently increase both [Ca2+]i and autonomous CaM kinase II activity in rat pancreatic acini. Because induction of autonomous activity may involve intramolecular autophosphorylation, the effects of protein phosphatase inhibitors were examined. None of the inhibitors tested (okadaic acid, calyculin A, and cyclosporin A) affected basal activity. Okadaic acid, a potent inhibitor of PP2A and weaker inhibitor of PP1, increased the peak autonomous activity by 30% over the level normally induced by CCK alone, while calyculin A, a potent inhibitor of both PP1 and PP2A, showed an even greater increase of 97%. Both inhibitors also delayed the decline of autonomous activity and calyculin A had a more potent effect than okadaic acid. Cyclosporin A, an inhibitor of PP2B, had no effect. The data indicate that PP1 may be involved in the dephosphorylation of CaMK II and decline of autonomous activity.
Amino Acid Sequence↗
Identification of an alkaline sphingomyelinase activity in human bile.
The hydrolysis of sphingomyelin has been found to generate important signals regulating cell proliferation, differentiation and apoptosis. However, the enzymes responsible for digestion of dietary sphingomyelin have not been well documented. This study demonstrates the occurrence of a sphingomyelinase (SMase) in both human hepatic bile and gallbladder bile. The enzyme was equally found in both bacteria negative and positive bile samples and in samples obtained from patients with or without gallbladder diseases. A bacteria-free gallbladder bile was used for characterization. It was found that bile SMase hydrolyzed sphingomyelin to phosphorylcholine and ceramide with negligible activity against either phosphatidylcholine or p-nitrophenyl phosphate. The enzyme preferred an alkaline condition and the optimal pH was 9. The activity of this alkaline SMase was bile salt dependent and was fully activated by 4-6 mM bile salts. Triton X-100, the non-ionic detergent did not activate bile SMase. Ca2+ and Mg2+ ions had no significant effect at optimal bile salt concentration. The molecular mass of this enzyme was about 85 kDa as measured by Sephadex G200 gel chromatography. In conclusion, we demonstrated a SMase in bile which differs markedly from the known acid and neutral SMase. Its potential important roles in sphingomyelin digestion and gallbladder diseases require further investigation.
Alkalies↗
Role of the ceramide-signaling pathway in cytokine responses to P-fimbriated Escherichia coli.
Escherichia coli express fimbriae-associated adhesins through which they attach to mucosal cells and activate a cytokine response. The receptors for E. coli P fimbriae are the globoseries of glycosphingolipids; Gal alpha 1-->4Gal beta-containing oligosaccharides bound to ceramide in the outer leaflet of the lipid bilayer. The receptors for type 1 fimbriae are mannosylated glycoproteins rather than glycolipids. This study tested the hypothesis that P-fimbriated E. coli elicit a cytokine response through the release of ceramide in the receptor-bearing cell. We used the A498 human kidney cell line, which expressed functional receptors for P and type 1 fimbriae and secreted higher levels of interleukin (IL)-6 when exposed to the fimbriated strains than to isogenic nonfimbriated controls. P-fimbriated E. coli caused the release of ceramide and increased the phosphorylation of ceramide to ceramide 1-phosphate. The IL-6 response to P-fimbriated E. coli was reduced by inhibitors of serine/threonine kinases but not by other protein kinase inhibitors. In contrast, ceramide levels were not influenced by type 1-fimbriated E. coli, and the IL-6 response was insensitive to the serine/threonine kinase inhibitors. These results demonstrate that the ceramide-signaling pathway is activated by P-fimbriated E. coli, and that the receptor specificity of the P fimbriae influences this process. We propose that this activation pathway contributes to the cytokine induction by P-fimbriated E. coli in epithelial cells.
Animals↗
Distribution of alkaline sphingomyelinase activity in human beings and animals. Tissue and species differences.
The alkaline sphingomyelinase (SMase) was first found in rat intestinal brush border. The important roles of this enzyme in digestion of sphingomyelin and in mucosal cell proliferation have been suggested. In the present work, the distribution of the alkaline SMase in the tissues of human beings and animals have been studied. By assaying the enzyme activity in human biopsy samples, we found that the alkaline SMase activity was absent in the stomach, increased in the duodenum, present at high levels in the small intestine, and slightly declined in the colon and rectum. High activities were found similarly in the intestinal contents of the healthy adults and infants. The activities were also found in the intestinal mucosa of rats, normal and germ-free mice, and hamsters with the same distribution pattern as in humans, but not in the intestinal mucosa of guinea pigs. Apart from the intestinal tract, a SMase activity preferring alkaline pH was identified in human and guinea pig bile, but not in the bile of rat, pig, sheep, and cow. No activity was found in either pancreatic tissue or pancreatic juice in all species tested, and none was detected in human urine and milk. In conclusion, alkaline SMase exists predominantly in the digestive system with considerable tissue and species differences.
Adult↗
Alkaline sphingomyelinase activity in rat gastrointestinal tract: distribution and characteristics.
Previous studies indicated that there was an alkaline sphingomyelinase (SMase) activity in small intestine, but its properties have not been studied in detail. In the present work, we studied the distribution of this enzyme activity in rat gastrointestinal tract and characterized it in intestinal mucosal homogenates. Little alkaline SMase activity was detected in the stomach and the duodenum. The activity in both mucosa and intestinal content increased in the small intestine and reached the maximum at the distal jejunum, then declined in the ileum and slightly increased again in the colon. The activity distribution pattern differed markedly from those of acid SMase and alkaline phosphatase. Little alkaline SMase activity could be found in bile, liver and pancreas before or after treatment with trypsin. The optimum pH of the alkaline SMase was 9. It specifically hydrolyzed sphingomyelin (SM), not phosphatidylcholine, to ceramide and phosphocholine. The alkaline SMase was bile salt dependent and was optionally activated by 3 mM bile salts. Triton X-100 could not mimic the effect of bile salt, rather dose-dependently inhibited the enzyme activity. Ca2+, Mg2+ did not change the alkaline SMase activity in the presence of bile salts, and reduced the activity in the absence of bile salt. Trypsin inactivated acid SMase in pancreas, liver and duodenum but had no influence on intestinal alkaline SMase activity. In conclusion, the intestinal alkaline SMase has a specific distribution pattern and the characters of it differ in several respects from the known acid and neutral SMases.
Alkaline Phosphatase↗
Activation of MAP kinase kinase (MEK) and Ras by cholecystokinin in rat pancreatic acini.
Cholecystokinin (CCK) has recently been shown to activate mitogen-activated protein (MAP) kinase in rat pancreatic acini [Duan and Williams, Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G401-G408, 1994]. To evaluate the mechanism of MAP kinase activation, we studied the effects of CCK on MAP kinase kinase (MEK) in rat pancreatic acini. Two forms of MEK were identified by immunoblotting, using antibodies specific to MEK1 and MEK2. MEK activity in acinar extracts and after immunoprecipitation with anti-MEK was detected using a recombinant fusion protein, glutathione S-transferase-MAP kinase, as a substrate. MEK activity rapidly increased after stimulation of acini by CCK, with significant stimulation at 1 min and a maximal effect at 5 min, followed by a slow decline to slightly above control levels after 30 min. The threshold concentration of CCK was approximately 10 pM, and the maximal effect was induced by 1 nM CCK, which increased MEK activity by 120%. In addition to CCK, bombesin and carbachol, but not secretin or vasoactive intestinal peptide, enhanced MEK activity. Phorbol ester mimicked the effect of CCK, whereas ionomycin and thapsigargin failed to activate MEK. We further studied the activation of Ras, an important component leading to activation of MEK by growth factors. Ras in acini was immunoprecipitated and identified by Western blotting. CCK and 12-O-tetradecanoylphorbol-13-acetate stimulated the incorporation of GTP into Ras, a requirement for its activation, reaching a maximum at 10 min of approximately 120% over control. In conclusion, the activation of MAP kinase by CCK can be explained by activation of MEK and may involve the activation of Ras by a protein kinase C-dependent mechanism.
Animals↗
Conversion to Ca(2+)-independent form of Ca2+/calmodulin protein kinase II in rat pancreatic acini.
CCK rapidly converted Ca2+/calmodulin kinase II (CaMK II) to a Ca(2+)-independent form with peak action at 30 sec followed by decline to the basal level at 10 min. The threshold concentration of CCK for this action was 30 pM and maximum effect occurred at 1 nM, which induced a 6-10-fold increase. Bombesin and carbachol similarly induced CaMK II autonomous activity, whereas secretin and JMV 180 did not. Ionomycin induced a more stable elevation of CaMK II autonomous activity and the intracellular Ca2+ chelator, BAPTA/AM, blocked the effect of CCK. In conclusion, pancreatic CaMK II is rapidly activated by a large increase in [Ca2+]i generated by either stimulation of phosphatidylinositol pathway or by an influx of extracellular Ca2+.
Amino Acid Sequence↗
Multiple inhibitory effects of genistein on stimulus-secretion coupling in rat pancreatic acini.
Genistein, a tyrosine kinase inhibitor, inhibited cholecystokinin (CCK)-induced maximal amylase release from rat pancreatic acini by 18, 31, and 46% at concentrations of 100, 300, and 750 microM, respectively, after 30 min preincubation. Genistein similarly decreased amylase release stimulated by bombesin but not that stimulated by secretin or vasoactive intestinal peptide. The steps of stimulus-secretion coupling affected by genistein were further evaluated. We found genistein dose dependently suppressed CCK-as well as NaF-induced polyphosphoinositide hydrolysis with a 50% inhibitory concentration of 380 and 400 microM, respectively, but only slightly suppressed the increase of intracellular Ca2+ concentration in response to either low or high concentrations of CCK. Genistein at 300 microM also decreased incorporation of [3H]inositol into phosphatidylinositol 4,5-bisphosphate. Most strikingly, 300 microM genistein inhibited Ca(2+)-stimulated amylase release by 85% in streptolysin O-permeabilized acini and thapsigargin-stimulated amylase release by 69% in intact acini. Daidzein, the inactive analogue of genistein, had no effect on any of the above parameters. Genistein, up to 750 microM, did not affect amylase release in response to phorbol ester. To relate these inhibitory effects of genistein to its inhibition of tyrosine phosphorylation, Western blotting was performed with an anti-phosphotyrosine monoclonal antibody. Genistein at 100 microM partly and at 300 microM completely inhibited CCK-induced tyrosine phosphorylation. In conclusion, genistein inhibits amylase release at multiple stages of stimulus-secretion coupling. These effects most likely involve both tyrosine kinase-dependent and -independent mechanisms.
Amylases↗
Cholecystokinin rapidly activates mitogen-activated protein kinase in rat pancreatic acini.
The existence and activation of mitogen-activated protein (MAP) kinase in isolated pancreatic acini have been demonstrated. Immunoblotting and immunoprecipitation revealed two forms of MAP kinase in pancreatic acini, with relative molecular masses of approximately 42 and 44 kDa. Both forms of MAP kinase were activated by cholecystokinin (CCK). The threshold concentration of CCK was approximately 3 pM, and the maximal effect occurred at 1 nM, which enhanced MAP kinase activity by 2.5-fold, as determined in polyacrylamide gel copolymerized with substrate myelin basic protein. Activation of MAP kinase by CCK was rapid, reaching a maximum within 5-10 min that subsequently declined. Bombesin and carbachol but not secretin or vasoactive intestinal peptide also activated MAP kinase. CCK-induced activation of MAP kinase may be mediated by protein kinase C, since 12-O-tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of CCK and staurosporine concentration dependently inhibited the action of CCK. Treatment of acini with thapsigargin, ionomycin, or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not influence MAP kinase, indicating that mobilization of intracellular calcium by CCK is not important in activation of acinar MAP kinase. CCK and TPA increased tyrosine phosphorylation of both 42- and 44-kDa forms. Genistein and tyrphostin 23, the inhibitors of tyrosine kinase, suppressed the activation of MAP kinase by CCK. In conclusion, MAP kinase in pancreatic acini is activated by agonists related to hydrolysis of phosphoinositide, via a mechanism involving protein kinase C and tyrosine kinase.
Animals↗
Is there a specific lysophospholipase in human pancreatic juice?
The existence of a specific lysophospholipase in human pancreatic juice was evaluated. The proteins were separated by a series of chromatographic steps including Sephacryl S-200, cholate-Sepharose 4B, Sephadex G-100 and CM-Sephadex G-50. The enzyme activities against 1-palmitoyl lysolecithin (LL) as well as tributyrin (TB) and p-nitrophenyl butyrate (PNPB) were determined in all the fractions of these purification procedures. Enzyme activity against LL was always eluted in parallel with activities against TB and PNPB, and no unique activity against LL could be found. The specific activity against LL was 40-times lower than that against PNPB and 200-times lower than that against TB. It is concluded that there is no unique lysophospholipase in human pancreatic juice and that the hydrolysis of lysolecithin is most likely performed by carboxyl ester lipase.
Butyrates↗
Decrease in contents of pancreatic carboxyl ester lipase, phospholipase A2, and lingual lipase in rats with streptozotocin-induced diabetes.
The changes in contents of pancreatic carboxyl ester lipase, phospholipase A2, and lingual lipase in rats with streptozotocin (STZ)-induced diabetes have been studied. The contents of pancreatic carboxyl ester lipase and phospholipase A2 decreased by 40% and 45%, respectively, 5 days after injection of STZ, whereas pancreatic lipase steadily increased to 100% over control. The content of lingual lipase decreased sharply by more than 90% 2 days after STZ injection, followed by a tendency to recover slightly. Insulin treatment at a dose abolishing the urine glucose in diabetic rats for 3 days restored the contents of pancreatic lipase, carboxyl ester lipase, and lingual lipase but not pancreatic phospholipase A2. The results indicate that lack of insulin action induces an anticoordinate change in gastrointestinal lipolytic enzymes, with decreases in pancreatic carboxyl ester lipase, phospholipase A2, and lingual lipase contents and an increase in pancreatic lipase content.
Animals↗
Effect of emeriamine on exocrine and endocrine pancreatic function in normal and diabetic rats.
The effects of emeriamine, a new anti-diabetic drug, on exocrine and endocrine pancreatic function in normal and diabetic rats have been studied both in vivo and in vitro. It was found that emeriamine dose-dependently normalized the symptoms of hyperingestion and hyperposia in streptozotocin (STZ)-induced diabetic rats, with fasting glucose levels significantly decreased and insulin levels not changed. In STZ-induced diabetic rats, there was a significant increase in pancreatic lipase and trypsin contents and a sharp decrease in amylase content. These changes in lipase and trypsin, but not in amylase were normalized by administration of emeriamine. In the normal rat, emeriamine had no effect on either serum glucose or insulin levels, but significantly decreased the pancreatic amylase, lipase as well as trypsin contents by 68%, 58% and 51%, respectively. In vitro, emeriamine (10(-8) - 10(-4) mol l-1) had no effect on enzyme release from pancreatic acini either under basal or carbachol-stimulated conditions. Emeriamine inhibited glucose-induced insulin release from isolated pancreatic islets. In conclusion, emeriamine has an inhibitory effect on synthesis of pancreatic enzymes and on glucose-stimulated insulin release.
Amylases↗
The effect of pretranslational regulation on synthesis of pancreatic colipase in streptozotocin-induced diabetes in rats.
A significant increase in synthesis of pancreatic colipase in streptozotocin (STZ)-induced diabetes in rats has been demonstrated previously. The aim of the present study was to identify whether this change in colipase synthesis was related to a pretranslational or translational regulation. The levels of colipase, lipase, and amylase mRNA were determined by Northern blot hybridization. The enzymatic activities and synthesis rates for these proteins were determined. One week after injection of STZ, the mRNA levels for both colipase and lipase were increased by about 100% over control, with accompanying increases in enzyme synthesis rates and enzymatic activities. The amylase mRNA, amylase synthesis rates, and amylase activity decreased by 95%. Insulin injection at a dose of 2 U/100 g/day for 5 days restored enzyme mRNA levels as well as enzyme activities. Kinetic studies revealed that lipase mRNA rapidly increased after induction of diabetes, closely followed by increases in lipase synthesis rates and lipase content. Colipase mRNA also rapidly increased, with values 60, 85, and 82% over control 1, 2, and 3 days after STZ injection, respectively. But the colipase synthesis rate increased slowly, being only 10, 20, and 40% over control 1, 2, and 3 days after STZ treatment, respectively. Colipase content did not increase until 4 days after STZ injection (3 days after the increase in colipase mRNA). The decrease in amylase mRNA was paralleled by decreases in amylase synthesis rates and amylase content. In conclusion, the increase in colipase content in STZ-induced diabetes in rats is a consequence of enhanced transcriptional or pretranslational regulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Amylases↗
Gastric inhibitory polypeptide stimulates pancreatic lipase and colipase synthesis in rats.
Effects of short-term infusion and long-term injection of gastric inhibitory polypeptide (GIP) on changes in pancreatic lipase and colipase contents in rats were studied, and mRNAs encoding for lipase and colipase were determined by Northern blot hybridization with specific cDNA probes. GIP infused at a dose of 3 micrograms/h for 24 h significantly increased the pancreatic lipase content by 34% (P less than 0.05) but had no significant effect on colipase and amylase contents. No change in mRNAs encoding for these proteins was found after infusion of GIP for 24 h. Injection of GIP (5-60 micrograms/kg) three times a day for 5 days dose dependently increased the contents of lipase and colipase, with the increase in colipase being more prominent. Injection of GIP for 5 days at a dose of 30 micrograms.kg-1.day-1 increased colipase and lipase contents by 52 and 25%, and their corresponding mRNAs by 60 and 160%, respectively. The amylase mRNA was not changed by injection of GIP. It is concluded that GIP has a specific stimulatory effect on the synthesis of pancreatic lipase and colipase at both pretranslational and translational levels.
Animals↗
Evidence of a stimulatory effect of cyclic AMP on pancreatic lipase and colipase synthesis in rats.
The effects of endogenous and exogenous cyclic AMP on the synthesis of pancreatic lipase, colipase, and amylase were studied. Pancreatic lobules were prepared and incubated with forskolin, dibutyryl cyclic AMP (dbcAMP), and dibutyryl cyclic GMP (dbcGMP), respectively, in the presence of 35S-cystine. The individual pancreatic enzymes were isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactive cystine into lipase, colipase, and amylase was determined. Incubation with forskolin (25 microM) rapidly increased lipase synthesis rate within 30 min, followed by an increase in colipase synthesis rate after 60 min of incubation. Amylase synthesis rate did not change during the 1st h of incubation but decreased slightly when incubated for 2 h. Incubation of pancreatic lobules with dbcAMP (1 mM) for 1 h also stimulated the incorporation of cysteine into lipase and colipase by 21% and 25%, respectively, whereas incubation with dbcGMP had no effect on the synthesis rates of lipase and colipase. Neither dbcAMP nor dbcGMP had any effect on synthesis rate of amylase. It is concluded that cyclic AMP might be an important intracellular signal for the synthesis of pancreatic lipase and colipase in the rat.
Amylases↗