Cellular mechanism of sodium oleate-stimulated secretion of cholecystokinin and secretin

Long-chain fatty acids are potent stimulants of secretin and CCK release. The cellular mechanisms of fatty acid-stimulated secretion of these two hormones are not clear. We studied the stimulatory effect and mechanism of sodium oleate (SO) on secretin- and CCK-producing cells. SO stimulated the release of secretin or CCK from isolated rat mucosal cell preparations enriched in either secretin- or CCK-producing cells, respectively. SO also time- and dose-dependently stimulated secretin and CCK release from STC-1 cells. In STC-1 cells, SO-stimulated secretin and CCK release was potentiated by IBMX and inhibited by a protein kinase A-selective inhibitor and a cAMP-specific antagonist. SO-stimulated releases of the two hormones were also inhibited by downregulation or inhibitors of protein kinase C, a calmodulin antagonist and an inhibitor of calmodulin-dependent protein kinase II. Chelating of extracellular Ca(2+) or addition of an L-type calcium channel blocker diminished SO-stimulated hormone releases. SO caused an increase in intracellular Ca(2+) concentration that was partially reversed by diltiazem but had no effect on production of cAMP, cGMP, or inositol-1,4,5-triphosphate. These results indicate that SO acts on secretin- and CCK-producing cells. Its stimulatory effect is potentiated by endogenous protein kinase A and mediated by activation of Ca(2+) influx through the L-type channels and of protein kinase C and Ca(2+)/calmodulin-dependent protein kinase II.     

A secretin releasing peptide exists in dog pancreatic juice

Canine pancreatic juice has been shown to stimulate exocrine pancreatic secretion in the dog. In the present study we investigated whether there is a secretin-releasing peptide in canine pancreatic juice. Pancreatic juice was collected from the dogs with Thomas gastric and duodenal cannulas while pancreatic secretion was stimulated by intravenous administration of secretin at 0.5 microg/kg/h and CCK-8 at 0.2 microg/kg/h, respectively. The pancreatic juice was separated into three different molecular weight (MW) fractions (Fr) by ultrafiltration (Fr 1; MW > 10,000, Fr 2; MW=10,000-4,000 and Fr 3; MW < 4,000), respectively. All the fractions were bioassayed in anesthetized rats. Fraction 3 dose-dependently and significantly stimulated pancreatic juice flow volume from 78.0% to 99.4% (p<0.05) and bicarbonate output from 128.9% to 202.1% (p<0.01), respectively. Plasma secretin concentration also increased from 1.2 +/- 0.5 pM to 5.0 +/- 0.8 pM and 6.0 +/- 1.0 pM (p<0.05). None of these fractions increased pancreatic protein secretion or plasma CCK level. The stimulatory effect of Fraction 3 on pancreatic secretion and the release of secretin was completely abolished by treatment with trypsin (1 mg/ml for 60 min at 37 degrees C) but not by heating (100 degrees C, 10 min). Intravenous injection of a rabbit anti-secretin serum, which rendered plasma secretin almost undetectable in rat plasma, also abolished Fr 3-stimulated pancreatic secretion of fluid and bicarbonate secretion. These observations suggest that a secretin-releasing peptide exists in the canine pancreatic juice. It is trypsin-sensitive and heat-resistant. This peptide may play a significant physiological role on the release of secretin and regulation of exocrine pancreatic secretion.     

MEK inhibits secretin release and pancreatic secretion: roles of secretin-releasing peptide and somatostatin

We investigated the mechanism of action of methionine enkephalin (MEK) on HCl-stimulated secretin release and pancreatic exocrine secretion. Anesthetized rats with pancreatobiliary cannulas and isolated upper small intestinal loops were perfused intraduodenally with 0.01 N HCl while bile and pancreatic juice were diverted. The effect of intravenous MEK on acid-stimulated secretin release and pancreatic exocrine secretion was then studied with or without coinfusion of naloxone, an anti-somatostatin (SS) serum, or normal rabbit serum. Duodenal acid perfusate, which contains secretin-releasing peptide (SRP) activity, was collected from donor rats with or without pretreatment with MEK, MEK + naloxone, or MEK + anti-SS serum, concentrated by ultrafiltration, and neutralized. The concentrated acid perfusate (CAP), which contains SRP bioactivity, was infused intraduodenally into recipient rats. MEK increased plasma SS concentration and inhibited secretin release and pancreatic fluid and bicarbonate secretion dose-dependently. The inhibition was partially reversed by naloxone and anti-SS serum but not by normal rabbit serum. In recipient rats, CAP increased plasma secretin level and pancreatic secretion. CAP SRP bioactivity decreased when it was collected from MEK-treated donor rats; this was partially reversed by coinfusion with naloxone or anti-SS serum. These results suggest that in the rat, MEK inhibition of acid-stimulated pancreatic secretion and secretin release involves suppression of SRP activity release. Thus the MEK inhibitory effect appears to be mediated in part by endogenous SS.     

Casein binds to the cell membrane and induces intracellular calcium signals in the enteroendocrine cell: a brief communication

Dietary protein but not amino acids stimulates cholecystokinin (CCK) secretion in rat mucosal cells. However, the dietary protein sensory mechanisms and the intracellular signal pathway in the enteroendocrine cells have not yet been clarified. The relationship between dietary protein binding to cell membrane and intracellular calcium responses were examined in the CCK-producing enteroendocrine cell line STC-1. The binding of solubilized STC-1 cell membrane to proteins was analyzed using a surface plasmon resonance sensor. Intracellular calcium concentrations of STC-1 cell suspensions loaded with Fura-2 AM were measured using a spectrafluorophotometer system with continuous stirring. Intracellular calcium concentrations in STC-1 cells were increased by exposure to alpha-casein or casein sodium, but not to bovine serum albumin. Solubilized STC-1 membranes bound to alpha-casein and casein sodium but did not bind to bovine serum albumin. alpha-Casein demonstrated higher membrane binding and intracellular calcium stimulating activities than casein sodium. Thus, protein binding to the STC-1 cell membrane and intracellular calcium responses were correlated. Intracellular calcium responses to alpha-casein were suppressed by an L-type calcium channel blocker. These results suggest that casein, a dietary protein, binds to a putative receptor on the CCK-producing enteroendocrine cell membrane and elicits the subsequent intracellular calcium response via an L-type calcium channel.     

Presence of biologically and immunologically active secretin-like substance in the mammalian brain

The present study involves the isolation and characterization of secretin-like immunoreactivity from the brains of pigs, rats and dogs. Secretin-like immunoreactivity was extracted with 0.1 N HCl and subjected to SP-Sephadex ion exchange chromatography and gel filtration on a Sephadex G-50 superfine column. The average amounts of secretin-like immunoreactivity in the extracts of 2 pigs, 7 rats and 6 dog brains were 0.25 ng/g, 2.4 +/- 0.2 ng/g and 0.34 +/- 0.07 ng/g fresh tissue weight, respectively. The secretin-like immunoreactivities in the brain extracts exhibited the same retention coefficient as natural porcine secretin on gel filtration and were eluted in the same salt gradient from the SP-Sephadex column. A partially purified secretin-like immunoreactivity isolated from canine brain exhibited the same bioactivity as natural porcine secretin to stimulate pancreatic volume flow in anesthetized rats (n = 4). These results indicated that secretin-like immunoreactivities from brain extracts possess the same molecular size and charge as natural porcine secretin and the secretin-like immunoreactivity isolated from dog brain is active in stimulating pancreatic secretion in anesthetized rats.     

Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic isozyme. The effects on folding, stability and enzymatic properties.

Secretory phospholipases A2 (PLA2s) are small homologous proteins rich in disulphide bridges. These PLA2s have been classified into several groups based on the disulphide bond patterns found [Dennis, E. A. (1997) Trends Biochem. Sci. 22, 1-2]. To probe the effect of the various disulphide bond patterns on folding, stability and enzymatic properties, analogues of the secretory PLA2s were produced by protein engineering of porcine pancreatic PLA2. Refolding experiments indicate that small structural variations play an important role in the folding of newly made PLA2 analogues. Introduction of a C-terminal extension together with disulphide bridge 50-131 gives rise to an enzyme that displays full enzymatic activity having increased conformational stability. In contrast, introduction of a small insertion between positions 88 and 89 together with disulphide bridge 86-89 decreases the catalytic activity significantly, but does not change the stability. Both disulphide bridges 11-77 and 61-91 are important for the kinetic properties and stability of the enzyme. Disulphide bridge 11-77, but not 61-91, was found to be essential to resist tryptic breakdown of native porcine pancreatic PLA2.     

Solution structure of porcine pancreatic phospholipase A2.

The lipolytic enzyme phospholipase A2 (PLA2) is involved in the degradation of high-molecular weight phospholipid aggregates in vivo. The enzyme has very high catalytic activities on aggregated substrates compared with monomeric substrates, a phenomenon called interfacial activation. Crystal structures of PLA2s in the absence and presence of inhibitors are identical, from which it has been concluded that enzymatic conformational changes do not play a role in the mechanism of interfacial activation. The high-resolution NMR structure of porcine pancreatic PLA2 free in solution was determined with heteronuclear multidimensional NMR methodology using doubly labeled 13C, 15N-labeled protein. The solution structure of PLA2 shows important deviations from the crystal structure. In the NMR structure the Ala1 alpha-amino group is disordered and the hydrogen bonding network involving the N-terminus and the active site is incomplete. The disorder observed for the N-terminal region of PLA2 in the solution structure could be related to the low activity of the enzyme towards monomeric substrates. The NMR structure of PLA2 suggests, in contrast to the crystallographic work, that conformational changes do play a role in the interfacial activation of this enzyme.     

Interaction of Gila monster venom with secretin receptors in rat pancreatic membranes

The stimulatory effect of Gila monster venom on adenylate cyclase activity in rat pancreatic membranes was compared to that of porcine secretin and porcine VIP. The maximal effect exerted by the venom was identical to that of VIP but significantly lower than that of secretin. The effect of Gila monster venom could, however, be attributed to its interaction with secretin receptors rather than with VIP receptors, at variance with its previously described action on guinea pig pancreatic acini. Adenylate cyclase activation by both Gila monster venom and secretin in rat pancreatic membranes was, indeed: (1) dose-dependently inhibited by two secretin fragments secretin-(4-27) and secretin-(7-27), and (2) more severely depressed than VIP stimulation, after pretreating pancreatic membranes with dithiothreitol (DTT).     

Possible role of PEPT1 in gastrointestinal hormone secretion

Oligopeptides originating from ingested meal stimulate the secretion of various gastrointestinal hormones, but the mechanism is unknown. In this study, we show that transfection of oligopeptide transporter 1 (PEPT1) in STC-1 cells, a murine enteroendocrine cell line, evokes di-peptide-stimulated hormone secretion in a pH-dependent manner. Measurement of membrane potentials shows that PEPT1- transfected STC-1 cells are depolarized by di-peptide glycyl-glycine but not by glycine monomer. Glycyl-glycine stimulation induces a rise in the intracellular calcium concentration in PEPT1-transfected STC-1 cells. The secretion induced by glycyl-glycine in PEPT1-transfected STC-1 cells was blocked by nifedipine, a Ca(2+) channel blocker, suggesting that the secretion is triggered by Ca(2+) influx through L-type voltage-dependent Ca(2+) channels. These data suggest that PEPT1 mediates oligopeptide-induced hormone secretion in enteroendocrine cells.     

Bacterial expression and characterization of starfish phospholipase A(2)

Phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA(2) from porcine pancreas. To investigate enzymatic properties of the starfish PLA(2) in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA(2) cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A(2) from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA(2) protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-beta-D(-)-thiogalactopyranoside. The recombinant PLA(2) produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris--HCl buffer (pH 8.0). Renatured PLA(2) was purified by subsequent column chromatographies on DEAE--cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA(2) was replaced by an Ala in the recombinant PLA(2), the recombinant enzyme showed essentially the same properties as did the native PLA(2) with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca(2+) requirement.     

The STC-1 cells express functional orexin-A receptors coupled to CCK release

Orexins are newly discovered neuropeptides regulating feeding and vigilance and have been detected in neuroendocrine cells of the gut. Potential neuroendocrine functions of orexin are unknown. Therefore, the effects of orexin-A on the intestinal neuroendocrine cell line, STC-1, were investigated as a model system. RT-PCR demonstrated the presence of both OX(1) and OX(2) receptors. Stimulation with orexin-A produced a dose-dependent release of cholecystokinin (CCK), which was abolished by removal of extracellular Ca(2+) or the presence of the voltage-gated L-type Ca(2+)-channel blocker diltiazem (10 microM). Orexin-A (Ox-A) elevated intracellular Ca(2+), which was dependent on extracellular Ca(2+). Furthermore, orexin-A caused a membrane depolarization in the STC-1 cells. Ox-A neither elevated cAMP levels nor stimulated phosphoinositide turnover in these cells. These data demonstrate a functional orexin receptor in the STC-1 cell line. Ox-A produces CCK release in these cells, by a mechanism involving membrane depolarization and subsequently activation of L-type voltage-gated Ca(2+)-channels.     

Different actions of secretin and Gly-extended secretin predict secretin receptor subtypes

Only one secretin receptor has been cloned and its properties characterized in native and transfected cells. To test the hypothesis that stimulatory and inhibitory effects of secretin are mediated by different secretin receptor subtypes, pancreatic and gastric secretory responses to secretin and secretin-Gly were determined in rats. Pancreatic fluid secretion was increased equipotently by secretin and secretin-Gly, but secretin was markedly more potent for inhibition of basal and gastrin-induced acid secretion. In Chinese hamster ovary cells stably transfected with the rat secretin receptor, secretin and secretin-Gly equipotently displaced (125)I-labeled secretin (IC(50) values 5.3 +/- 0.5 and 6.4 +/- 0.6 nM, respectively). Secretin, but not secretin-Gly, caused release of somatostatin from rat gastric mucosal D cells. Thus the equipotent actions of secretin and secretin-Gly on pancreatic secretion appear to result from equal binding and activation of the pancreatic secretin receptor. Conversely, secretin more potently inhibited gastric acid secretion in vivo, and only secretin released somatostatin from D cells in vitro. These results support the existence of a secretin receptor subtype mediating inhibition of gastric acid secretion that is distinct from the previously characterized pancreatic secretin receptor.     

The structural and functional contribution of N-terminal region and His-47 on Taiwan cobra phospholipase A2.

Modification of His-47 and removal of the N-terminal octapeptide caused a different effect on the structure of Naja naja atra (Taiwan cobra) phospholipase A2 (PLA2). Unlike native enzyme, Ca2+ induced an alteration in the structural flexibility of His-modified PLA2. Moreover, the spatial positions of Trp residues in His-modified PLA2 were not properly rearranged toward lipid-water interface in the presence of Ca2+. CD spectra and fluorescence measurement showed that the dynamic properties of Trp residues and the gross conformation of N-terminally truncated PLA2 were totally different from native enzyme. Although a precipitous drop in the enzymatic activity was observed with modified PLA2, His-modified PLA2 and N-terminally truncated PLA2 retained cytotoxicity on inducing necrotic death of human neuroblastoma SK-N-SH cells. Our data suggest that structural perturbations elicited by the chemical modification cause a dissociation of enzymatic activity and cytotoxicity of PLA2.     

Localization of phospholipase A2 in normal and ras-transformed cells

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.     

Secretin N-terminal hexapeptide potentiates insulin release in mouse islets

Peptides representing the N-terminal part of secretin were synthesized and their effects tested on column-perifused isolated mouse pancreatic islets. Insulin release induced by D-glucose was potentiated by the two peptides His-Ser-Asp-Gly-Thr-Phe-OMe (S1-6) and Ser-Asp-Gly-Thr-Phe-OMe (S2-6). The consecutive smaller N-terminal peptides Asp-Gly-Thr-Phe-OMe (S3-6) and Gly-Thr-Phe-OMe (S4-6) had no effects while the dipeptide ester Thr-Phe-OMe (S5-6) also potentiated the release of insulin. The results suggest that the N-terminal part of secretin may be involved in the marked in vitro glucose-dependent insulin release induced by secretin.     

棉酚抗生育新机制——对磷脂酶A_2活力的抑制作用(英文)

 采用微孔比色法及荧光分析法 ,研究抗男性生育化合物棉酚与猪胰腺磷脂酶A2 (phospho lipaseA2 ,PLA2 ,EC3 1 1 4 )温育并透析前后对酶活力及荧光的影响 .结果表明 ,棉酚与PLA2 不可逆地结合明显地降低了PLA2 活力及荧光强度 .棉酚对酶活力抑制作用的IC50 为 35μmol L ;当其浓度达到 80 μmol L时 ,能够完全抑制PLA2 ( 4 11μmol L)对合成底物 2 硫代十六酰乙基磷酸胆碱(HEPC ,0 .2 5mmol L)的水解作用 .PLA2 的最大激发波长与发射波长分别为 2 75nm ,34 3nm ,荧光强度与酶浓度呈良好的线性关系 .棉酚对PLA2 的荧光具有较强的淬灭作用 .由于PLA2 与男性生育密切相关 ,棉酚对PLA2 活力的影响可能是其避孕作用及伴随的副作用的一种新的重要机制     

The edema inducing activity of phospholipase A2 enzymes

The edema inducing activity of phospholipase A2 (PLA2) enzymes from snake venoms and porcine pancreas was investigated using mouse paw as experimental model. All ten PLA2 enzymes exhibited potent edema inducing activity. PLA2, however, is generally not the major edema inducing component of snake venom. Chemical modification studies indicated that enzymatic activity of PLA2 was required for its edema inducing activity. All PLA2 enzymes examined displayed a rapid onset edema which was suppressed by pretreatment of the mice with antihistamine. Dexamethasone pretreatment also inhibited edemas elicited by some PLA2 enzymes.     

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2.

The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.