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S Ramanadham

Publications and source records attributed to S Ramanadham.

At least 37 records · Page 2Linked to original sources
PubMed1996

Characterization of expression of phosphofructokinase isoforms in isolated rat pancreatic islets and purified beta cells and cloning and expression of the rat phosphofructokinase-A isoform.

Phosphofructokinase (PFK) plays a key role in regulating glycolytic flux, and the mammalian enzyme is a tetramer. Three monomeric isoforms are encoded by separate genes, are differentially expressed in specific tissues, and are designated by tissues in which they are most abundant (A, muscle; B, liver; and C, brain). Glucose-induced insulin secretion from pancreatic islets requires glucose transport into islet beta-cells and glycolytic metabolism. Little is known about islet PFK isozymes, but the possibility that PFK-A is expressed in beta-cells is of interest because that isoform is thought to govern glycolytic oscillations and to interact with a metabolically activated beta-cell phospholipase A2 enzyme. Using as probe a PCR product generated from rat islet RNA with primers designed from the human PFK-A sequence, we have cloned a full-length PFK-A cDNA from a rat islet cDNA library. The rat PFK-A deduced amino-acid sequence is 96% identical to that of human PFK-A, and all residues thought to participate in substrate or allosteric effector binding are conserved between the two sequences. The rat PFK-A amino-acid sequence is 69% and 68% identical to those for rat PFK-B and rat PFK-C, respectively, and differences in residues involved in binding of allosteric effectors were observed among the three isoforms. Rat PFK-A expressed as a glutathione-S-transferase fusion protein was recognized by antibodies raised against a peptide in the PFK-A sequence. Expression of PFK isoform mRNA species was examined by RT-PCR in rat islets, in purified populations of beta-cells prepared by fluorescence-activated cell sorting (FACS), and in RIN-m5F insulinoma cells, all of which expressed mRNA species for PFK-A, -B, and -C isoforms. PFK-A mRNA was expressed at much lower levels in an islet alpha-cell-enriched population. Interleukin-1 impairs islet glucose metabolism and insulin secretion and was found to induce a specific decline in islet expression of PFK-A mRNA. These findings establish the sequence of rat PFK-A, demonstrate that it is expressed in FACS-purified islet beta-cells, and suggest that its expression is regulated by a cytokine which influences insulin secretion.

Amino Acid Sequence↗
PubMed1996

Evidence for association of an ATP-stimulatable Ca(2+)-independent phospholipase A2 from pancreatic islets and HIT insulinoma cells with a phosphofructokinase-like protein.

Glucose-induced insulin secretion from pancreatic islets requires metabolism of glucose within islet beta-cells, and ATP has attracted interest as a messenger of glucose metabolism within beta-cells. Glucose-induced insulin secretion from islets and HIT insulinoma cells is accompanied by activation of an ATP-stimulatable Ca(2+)-independent phospholipase A2 (ASCI-PLA2) enzyme, the catalytic activity of which resides in a 40 kDa protein. An analogous PLA2 enzyme in myocardium was recently found to consist of a complex of a 40 kDa catalytic protein with a tetramer of an isoform of the glycolytic enzyme phosphofructokinase (PFK). Association of the PFK isoform with the myocardial PLA2 catalytic protein was found to confer ATP sensitivity onto the enzyme complex. Here we demonstrate that the majority of HIT cell and islet ASCI-PLA2 catalytic activity elutes from a gel filtration column in a region corresponding to 400 kDa, suggesting that the 40 kDa beta-cell ASCI-PLA2 catalytic protein exists as part of a larger molecular mass complex. Islet and HIT cell ASCI-PLA2 activities were immunoprecipitated by antibodies directed against PFK, and the immunoprecipitates contained 40 and 85 kDa proteins which correspond to the molecular masses of the PLA2 catalytic protein and of a PFK monomer, respectively. Islet and HIT cell ASCI-PLA2 activities were selectively and reversibly adsorbed to affinity matrices containing immobilized PFK but not to similar matrices containing immobilized transferrin or bovine serum albumin. Addition of free PFK prevented binding of HIT cell ASCI-PLA2 activity to immobilized PFK matrices and promoted desorption of activity previously bound to such matrices. These results suggest that beta-cell ASCI-PLA2, like the myocardial enzyme, exists as a complex comprised of a catalytic protein and a PFK-like protein and raise the possibility that the ASCI-PLA2 complex may represent a component of the beta-cell glucose sensor, which links glycolysis, phospholipid hydrolysis, and membrane electrochemical events involved in glucose-induced insulin secretion.

Animals↗
PubMed1996

Interleukin-1 enhances pancreatic islet arachidonic acid 12-lipoxygenase product generation by increasing substrate availability through a nitric oxide-dependent mechanism.

Interleukin-1 (IL-1) impairs insulin secretion from pancreatic islets and may contribute to the pathogenesis of insulin-dependent diabetes mellitus. IL-1 increases islet expression of nitric oxide (NO) synthase, and the resultant overproduction of NO participates in inhibition of insulin secretion because NO synthase inhibitors, e.g. NG-monomethyl-arginine (NMMA), prevent this inhibition. While exploring effects of IL-1 on islet arachidonic acid metabolism, we found that IL-1 increases islet production of the 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid 12-(HETE). This effect requires NO production and is prevented by NMMA. Exploration of the mechanism of this effect indicates that it involves increased availability of the substrate arachidonic acid rather than enhanced expression of 12-lipoxygenase. Evidence supporting this conclusion includes the facts that IL-1 does not increase islet 12-lipoxygenase protein or mRNA levels and does not enhance islet conversion of exogenous arachidonate to 12-HETE. Mass spectrometric stereochemical analyses nonetheless indicate that 12-HETE produced by IL-1-treated islets consists only of the S-enantiomer and thus arises from enzyme action. IL-1 does enhance release of nonesterified arachidonate from islets, as measured by isotope dilution mass spectrometry, and this effect is suppressed by NMMA and mimicked by the NO-releasing compound 3-morpholinosydnonimine. Although IL-1 increases neither islet phospholipase A2 (PLA2) activities nor mRNA levels for cytosolic or secretory PLA2, a suicide substrate which inhibits an islet Ca(2+)-independent PLA2 prevents enhancement of islet arachidonate release by IL-1. IL-1 also impairs esterification of [3H8]arachidonate into islet phospholipids, and this effect is prevented by NMMA and mimicked by the mitochondrial ATP-synthase inhibitor oligomycin. Experiments with exogenous substrates indicate that NMMA does not inhibit and that the NO-releasing compound does not activate islet 12-lipoxygenase or PLA2 activities. These results indicate that a novel action of NO is to increase levels of nonesterified arachidonic acid in islets.

Animals↗
PubMed1995

Pancreatic islet cells express a family of inwardly rectifying K+ channel subunits which interact to form G-protein-activated channels.

Insulin secretion is associated with changes in pancreatic beta-cell K+ permeability. A degenerate polymerase chain reaction strategy based on the conserved features of known inwardly rectifying K+ (KIR) channel genes was used to identify members of this family expressed in human pancreatic islets and insulinoma. Three related human KIR transcript sequences were found: CIR (also known as cardiac KATP-1), GIRK1, and GIRK2 (KATP-2). The pancreatic islet CIR and GIRK2 full-length cDNAs were cloned, and their genes were localized to human chromosomes 11q23-ter and 21, respectively. Northern blot analysis detected CIR mRNA at similar levels in human islets and exocrine pancreas, while the abundance of GIRK2 mRNA in the two tissues was insufficient for detection by this method. Using competitive reverse-transcription polymerase chain reaction, CIR was found to be present at higher levels than GIRK2 mRNA in native purified beta-cells. Xenopus oocytes injected with M2 muscarinic receptor (M2) plus either GIRK2 or CIR cRNA expressed only very small carbachol-induced currents, while co-injection of CIR plus GIRK2 along with M2 resulted in expression of carbachol-activated strong inwardly rectifying currents. Activators of KATP channels failed to elicit currents in the presence or absence of co-expressed sulfonylurea receptor. These results show that two components of islet cell KIR channels, CIR and GIRK2, may interact to form heteromeric G-protein-activated inwardly rectifying K+ channels that do not possess the typical properties of KATP channels.

Amino Acid Sequence↗
PubMed1994

Characterization of an ATP-stimulatable Ca(2+)-independent phospholipase A2 from clonal insulin-secreting HIT cells and rat pancreatic islets: a possible molecular component of the beta-cell fuel sensor.

Isolated pancreatic islets from rats and humans express a plasmalogen-preferring ATP-stimulatable, Ca(2+)-independent phospholipase A2 (ASCI-PLA2) enzyme which participates in the glucose-stimulated hydrolysis of arachidonate from membrane phospholipids and in insulin secretion. Here we report that clonal insulin-secreting HIT beta-cells contain substantial amounts of endogenous plasmalogens and express a similar ASCI-PLA2 activity with the following properties: (1) Enzymatic activity as well as glucose-induced eicosanoid release and insulin secretion are inhibited by a mechanism-based suicide substrate directed towards ASCI-PLA2. (2) HIT cell ASCI-PLA2 is selectively activated and protected against thermal denaturation by ATP. (3) The magnitude of ASCI-PLA2 activation by the nonhydrolyzable ATP analog AMP-PCP is similar to that by ATP. (4) The ATP concentrations required to activate ASCI-PLA2 fall within physiologic ranges in the presence of Mg2+. (5) ADP induces a concentration-dependent attenuation of the activation of ASCI-PLA2 by ATP. HIT cell ASCI-PLA2 exhibited an apparent isoelectric point of 7.5 on chromatofocusing analysis and was quantitatively adsorbed to an ATP-agarose matrix and selectively desorbed from this column by ATP. Mono-Q anion-exchange analysis of the active ATP-agarose eluant yielded a peak of ASCI-PLA2 activity associated with a single protein band with an apparent molecular mass of 40 kDa. Similar chromatographic behavior of the rat pancreatic islet ASCI-PLA2 activity was observed during sequential ATP-agarose and Mono-Q anion-exchange steps. These results indicate that HIT cells express an ASCI-PLA2 similar to the analogous islet enzyme and suggest that expression of this enzyme and of its preferred plasmalogen substrates may be a general property of insulin-secreting beta-cells.

Adenosine Triphosphate↗
PubMed1994

omega-Conotoxin inhibits glucose- and arachidonic acid-induced rises in intracellular [Ca2+] in rat pancreatic islet beta-cells.

Earlier studies suggest that the accumulation of non-esterified arachidonic acid (AA) in islets following stimulation with glucose participates in the glucose-induced secretion of insulin. A possible role for AA might include the facilitation of Ca2+ influx into islet beta-cells. Recently, we demonstrated that AA induces Ca2+ influx into purified rat pancreatic islet beta-cells, prepared by fluorescence-activated cell sorting (FACS). This effect was abolished in the presence of the Ca(2+)-chelator EGTA, but was only partially reduced by the dihydropyridine (DHP) L-type Ca(2+)-channel blocker, nifedipine. This raised the possibility that DHP-insensitive Ca2+ entry mechanisms may exist in pancreatic beta-cells, in addition to the known DHP-sensitive L-type Ca2+ channels. Here we report that omega-conotoxin (CTX), which blocks omega-type Ca(2+)-channels, inhibits AA-induced Ca2+ influx by a magnitude similar to that of nifedipine and that the combination of omega-CTX and nifedipine results in a nearly additive decrement in AA-induced increases in beta-cell cytosolic [Ca2+]. We further demonstrate that bovine serum albumin, which complexes free AA and prevents AA-induced increases in cytosolic [Ca2+], also inhibits the glucose-induced increase in beta-cell [Ca2+]. These results suggest that rat pancreatic FACS-purified islet beta-cells express omega-type (DHP-insensitive) Ca(2+)-channels, in addition to DHP-sensitive Ca(2+)-channels. They further suggest that the glucose-induced accumulation of non-esterified AA in the membranes of beta-cells serves to amplify glucose-mediated Ca2+ influx into the beta-cells.

Animals↗
PubMed1993

Biochemical evidence for nitric oxide formation from streptozotocin in isolated pancreatic islets.

Streptozotocin (STZ) is selectively toxic to insulin-secreting beta-cells of pancreatic islets and induces impairment of islet glucose oxidation and of glucose-induced insulin secretion. Similar effects are induced by Interleukin-1 (IL-1), and the deleterious effects of IL-1 on islets appear to be mediated by nitric oxide (NO). STZ contains a nitroso moiety and may liberate NO by processes analogous to those for the NO-releasing drug nitroprusside. NO is rapidly transformed to nitrite in aqueous solution, and NO activates heme-containing enzymes such as guanylyl cyclase and inhibits iron-sulfur enzymes such as mitochondrial aconitase. Data presented here indicate that incubation of rat islets with STZ at concentrations that impair insulin secretion results in generation of nitrite, stimulation of islet guanylyl cyclase and accumulation of cGMP, and inhibition of islet mitochondrial aconitase activity to a degree similar to that achieved by IL-1. Effects of STZ on beta-cells may be mediated by local liberation of NO from STZ within islets.

Aconitate Hydratase↗
PubMed1993

Mass spectrometric characterization of arachidonate-containing plasmalogens in human pancreatic islets and in rat islet beta-cells and subcellular membranes.

Pancreatic islets, when stimulated with D-glucose, secrete insulin by processes requiring glycolytic metabolism and generation of ATP. Hydrolysis of membrane phospholipids also occurs in glucose-stimulated islets, resulting in accumulation of nonesterified arachidonate, which facilitates Ca2+ entry and the rise in beta-cell [Ca2+] that triggers insulin secretion. Glucose-induced hydrolysis of arachidonate from islet phospholipids is mediated in part by an ATP-stimulated, Ca(2+)-independent (ASCI) phospholipase A2 (PLA2) which prefers plasmenyl over diacyl phospholipid substrates. Here we characterize the endogenous plasmalogen content of islet cells and subcellular membranes. Fast atom bombardment mass spectrometric analyses demonstrated that three of the most abundant molecular species of ethanolamine phospholipids in rat pancreatic islets were plasmalogens with sn-2 arachidonate residues and palmitic, oleic, or stearic aldehyde residues, respectively, in the sn-1 position. Purified populations of beta-cells prepared by fluorescence-activated cell sorting were also found to contain these plasmenylethanolamine molecular species in abundance similar to that in intact islets and greater than that in islet alpha-cells. Both islet plasma membranes (PM) and endoplasmic reticulum (ER) also contained these plasmenylethanolamine species, which accounted for 42% (PM) to 64% (ER) of the ethanolamine phospholipid arachidonate content of these membranes, as measured by stable isotope dilution mass spectrometry. Plasmenylethanolamine species were also abundant constituents of human pancreatic islets (accounting for 58% of their ethanolamine phospholipid arachidonate content) and were hydrolyzed more rapidly than diacyl ethanolamine phospholipid by human islet cytosolic ASCI-PLA2. Both secretagogue-induced eicosanoid release and insulin secretion from human islets were attenuated by an ASCI-PLA2 suicide substrate which sterically resembles plasmalogens. These observations are consistent with the hypotheses that islet beta-cell ASCI-PLA2-catalyzed hydrolysis of arachidonate from endogenous plasmenylethanolamine substrates may occur in membrane compartments which participate in regulation of the beta-cell cytosolic [Ca2+] and that this may be an intermediary biochemical event in the induction of insulin secretion.

Aldehydes↗
PubMed1993

Arachidonic acid release from aortic smooth muscle cells induced by [Arg8]vasopressin is largely mediated by calcium-independent phospholipase A2.

To identify the phospholipase mediating the majority of [Arg8]vasopressin (AVP)-induced release of arachidonic acid in A-10 smooth muscle cells, we exploited the specificity inherent in the mechanism-based inhibitor, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS), which possesses a 1,000-fold selectivity for inhibition of calcium-independent versus calcium-dependent phospholipases A2. Utilizing [3H]arachidonic acid-labeled A-10 smooth muscle cells, one-half of AVP-inducible [3H]arachidonic acid release was inhibited by pretreatment with only 1 microM HELSS and two-thirds of AVP-stimulated [3H]arachidonic acid release was inhibited by 5 microM HELSS. The inhibition of [3H]arachidonic acid release by HELSS was saturable (i.e. no additional inhibition of [3H]arachidonic acid release was present at 10 microM HELSS), specific (i.e. the activities of six intracellular enzymes, as well as the rate of glucose oxidation, were not altered by HELSS treatment), and nontoxic (i.e. HELSS-treated cells excluded trypan blue dye and did not leak intracellular enzymes into the medium). Collectively, these results demonstrate that HELSS blocks AVP-induced arachidonic acid release by specific and irreversible inhibition of calcium-independent phospholipase A2 and underscore the importance of calcium-independent phospholipase A2 in agonist-induced arachidonic acid release in at least some cell types.

Animals↗
PubMed1993

Mass spectrometric identification and quantitation of arachidonate-containing phospholipids in pancreatic islets: prominence of plasmenylethanolamine molecular species.

D-Glucose induces insulin secretion from beta-cells of pancreatic islets by processes involving glycolytic metabolism and generation of ATP. Glucose also induces hydrolysis of beta-cell membrane phospholipids and accumulation of nonesterified arachidonate, which facilitates Ca2+ entry and the rise in beta-cell Ca2+ concentration that is a critical signal in the induction of insulin secretion. Glucose-induced hydrolysis of arachidonate from beta-cell phospholipids is mediated in part by an ATP-stimulated, Ca(2+)-independent (ASCI)-phospholipase A2 (PLA2), which, in vitro, prefers plasmalogen over diacylphospholipid substrates, but it is not known whether islets contain plasmalogens. We have identified and quantitated the major species of arachidonate-containing phospholipids in pancreatic islets by high-performance liquid chromatographic and mass spectrometric analyses. Arachidonate has been found to constitute 30% of the total islet glycerolipid fatty acyl mass. Ethanolamine phospholipids contain 30% of total islet arachidonate, and 44% of that amount resides in three plasmenylethanolamine molecular species with residues of palmitic, oleic, or stearic aldehydes in the sn-1 position. These endogenous islet plasmenylethanolamine species are hydrolyzed more rapidly than phosphatidylethanolamine species by islet ASCI-PLA2 in vitro and are also hydrolyzed in intact islets stimulated with secretagogues. ASCI-PLA2-catalyzed hydrolysis of islet plasmenylethanolamine species in vitro is inhibited by a selective haloenol lactone suicide substrate (HELSS) which is sterically similar to plasmalogens, and HELSS also inhibits all temporal phases of both eicosanoid release and insulin secretion from secretagogue-stimulated pancreatic islets. Islet beta-cell ASCI-PLA2-catalyzed hydrolysis of arachidonate from endogenous plasmenylethanolamine substrates may be an intermediary biochemical event in the induction of insulin secretion.

Animals↗
PubMed1993

Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells.

The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.

Adenosine Triphosphate↗
PubMed1993

Inhibition of arachidonate release by secretagogue-stimulated pancreatic islets suppresses both insulin secretion and the rise in beta-cell cytosolic calcium ion concentration.

Fuel secretagogues induce hydrolysis of esterified arachidonic acid from pancreatic islet cell phospholipids and accumulation of nonesterified arachidonate at concentrations up to 35 microM. Exogenous arachidonate (5-30 microM) amplifies depolarization-induced insulin secretion from islets. Fuel secretagogue-induced hydrolysis of arachidonate from islet phospholipids occurs in Ca(2+)-free medium, suggesting the possible involvement of a Ca(2+)-independent phospholipase. In the companion paper [Gross et al. (1993) Biochemistry (preceding paper in this issue)], we demonstrated that the major islet phospholipase A2 is Ca(2+)-independent, ATP-stimulated, and inhibited by the haloenol lactone suicide substrate (HELSS) (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. Here we demonstrate that HELSS suppressed both release of the arachidonate metabolite prostaglandin E2 and insulin secretion from islets stimulated with D-glucose and the muscarinic agonist carbachol. Both prostaglandin E2 release and insulin secretion were suppressed with similar concentration profiles and time courses. Islet oxidation of [14C]-glucose to [14C]CO2, activities of islet lactate dehydrogenase and alanine and aspartate aminotransferases, and carbachol-induced inositol phosphate accumulation in islets were all unaffected by HELSS. Depolarization of isolated beta-cells with 40 mM KCl induced a rise in cytosolic [Ca2+] that was also unaffected by HELSS. In contrast, the 17 mM D-glucose-induced rise in beta-cell [Ca2+] was inhibited by HELSS in a concentration-dependent manner, but that induced by exogenous arachidonate (15 microM) was not. These results suggest that fuel secretagogues activate the islet Ca(2+)-independent phospholipase A2, resulting in release of nonesterified arachidonate, which facilitates Ca2+ entry into beta-cells and promotes insulin secretion.

Animals↗
PubMed1993

Amplification of insulin secretion by lipid messengers.

D-glucose induces a rise in pancreatic islet beta-cell cytosolic [Ca2+] by processes requiring both glucose metabolism and Ca2+ entry from the extracellular space, and this Ca2+ signal is thought to be critical to the induction of insulin secretion. Insulin secretagogues also induce phospholipid hydrolysis and accumulation of phospholipid-derived mediators in islets, including the lipid messengers DAG, nonesterified arachidonic acid, and arachidonate 12-LO products. This study offers the following viewpoints on potential roles of these lipid messengers in insulin secretion as working hypotheses: 1) the Ca2+ signal provided to the beta-cell by D-glucose induces insulin secretion only in the context of amplifying background signals provided by the beta-cell content of messengers including DAG; 2) muscarinic receptor agonists amplify glucose-induced insulin secretion in part by altering the beta-cell content of DAG; 3) the Ca2+ signal provided by metabolism of D-glucose is amplified by the level of nonesterified arachidonic acid in beta-cell membranes, which acts to facilitate Ca2+ entry; 4) metabolism of glucose induces accumulation of nonesterified arachidonate in beta-cells via activation of a recently identified ASCI-PLA2 enzyme, which may be a component of the beta-cell fuel sensor apparatus; and 5) arachidonate 12-LO metabolites are potential candidates as adjunctive modulators of beta-cell K(+)-channel activity.

Animals↗
PubMed1992

Arachidonic acid metabolism in isolated pancreatic islets. VI. Carbohydrate insulin secretagogues must be metabolized to induce eicosanoid release.

Pancreatic islets stimulated with D-glucose are known to liberate arachidonic acid from membrane phospholipids and release prostaglandin E2 (PGE2). A component of the eicosanoid release induced by D-glucose has been demonstrated to occur without calcium influx and must be triggered by other coupling mechanisms. In this study, we have attempted to identify mechanisms other than calcium influx which might couple D-glucose stimulation to hydrolysis of arachidonate from membrane phospholipids in islet cells. We have found that occupancy of the beta cell plasma membrane D-glucose transporter is insufficient and that D-glucose metabolism is required to induce islet PGE2 release because 3-O-methylglucose fails to induce and mannoheptulose prevents PGE2 release otherwise induced by 17 mM D-glucose. The carbohydrate insulin secretagogues mannose and D-glyceraldehyde have also been found to induce islet PGE2 release, but the non-secretagogue carbohydrates L-glucose and lactate do not. Carbohydrate secretagogues are known to be metabolized to yield ATP and induce depolarization of the beta cell plasma membrane. We have found that depolarization by 40 mM KCl induces PGE2 release only in the presence and not in the absence of extracellular calcium, but exogenous ATP induces islet PGE2 release with or without extracellular calcium. Carbachol is demonstrated here to interact synergistically with increasing concentrations of glucose to amplify PGE2 release and insulin secretion. Pertussis toxin treatment is shown here not to prevent PGE2 release induced by glucose or carbachol but to increase the basal rate of PGE2 release and the islet cyclic AMP content. Theophylline (10 mM) exerts similar effects. Eicosanoid release in pancreatic islets can thus be activated by multiple pathways including muscarinic receptor occupancy, calcium influx, increasing cAMP content, and a metabolic signal derived from nutrient secretagogues, such as ATP.

3-O-Methylglucose↗
PubMed1992

Arachidonic acid induces an increase in the cytosolic calcium concentration in single pancreatic islet beta cells.

The insulin secretagogue D-glucose induces both accumulation of nonesterified arachidonic acid (35 microM) in pancreatic islets and a rise in beta cell cytosolic [Ca++]i. Arachidonate amplifies both voltage-dependent Ca++ entry in secretory cells and depolarization-induced insulin secretion. Here, arachidonate induced a biphasic rise in [Ca++]i of Fura-2AM loaded beta cells which increased with arachidonate concentration (5-30 microM), was reversed upon washout, and was unaffected by the arachidonate oxygenase inhibitor BW755C. The sustained phase of the rise was abolished by removal of extracellular Ca++ and amplified by depolarization with KCl. The accumulation of nonesterified arachidonate in islets stimulated by D-glucose may therefore promote the D-glucose-induced rise in beta cell [Ca++]i.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1992

Quantification of distinct molecular species of the 2-lyso metabolite of platelet-activating factor by gas chromatography-negative-ion chemical ionization mass spectrometry.

The biological activity of platelet-activating factor (PAF) is comprised by a few molecular species of phosphatidylcholine which contain a fatty alcohol connected by an ether linkage to the sn-1 position of the glycerol backbone and an acetate ester at the sn-2 position. The various molecular species of PAF differ in chain length and degree of unsaturation in the fatty alcohol residue side-chain. PAF is rapidly hydrolyzed to lyso-PAF by an acetylhydrolase enzyme which is quite active in a number of cells that synthesize PAF. We describe a method for quantitation of lyso-PAF which involves conversion to its propionate derivative in the presence of an internal standard (deuterium-labelled PAF), digestion to the diglyceride with Bacillus cereus phospholipase C, conversion to the pentafluorobenzoate derivative and capillary column gas chromatographic-negative-ion methane chemical ionization mass spectrometric analysis. Distinct molecular species of lyso-PAF can be individually quantitated at levels of 1 ng or less. These methods are applied to the demonstration of lyso-PAF accumulation in renal tissue from transplanted allografts undergoing acute rejection, in renal tissue from kidneys subjected to cold storage and autotransplantation, and in intestinal mucosa subjected to warm ischemia and reperfusion.

Animals↗
PubMed1992

Selective expression of an arachidonate 12-lipoxygenase by pancreatic islet beta-cells.

The immunohistochemical distribution of arachidonate lipoxygenases in rat pancreas was characterized with specific polyclonal anti-5-lipoxygenase and anti-12-lipoxygenase antibodies. Immunohistochemical analysis of formaldehyde-fixed paraffin-embedded rat pancreas using anti-12-lipoxygenase antibody and biotin-avidin-peroxidase detection demonstrated specific staining of islets and no staining of pancreatic exocrine tissue. Less intense staining of pancreatic vascular myocytes and endothelial cells was also observed. Immunoblotting of isolated pancreatic islet extracts with the anti-12-lipoxygenase antibody demonstrated immunoperoxidase staining of a single protein band which comigrated with purified 12-lipoxygenase (relative molecular weight = 72,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Dispersed cells prepared from isolated islets and then subjected to fluorescence-activated cell sorting and immunostaining exhibited 12-lipoxygenase antigen in beta-cell populations but not in non-beta-cell (predominantly alpha-cell) populations. Assays of enzymatic activity confirmed that the 12-lipoxygenase-catalyzed conversion of arachidonic acid to 12-hydroxyeicosatetraenoic acid methyl ester occurred only with purified beta-cells and not with islet non-beta-cells. No evidence of 5-lipoxygenase antigen or enzymatic activity was found in purified beta-cells or in islet non-beta-cells. We conclude that rat pancreatic islet beta-cells contain an arachidonate 12-lipoxygenase which shares antigenic epitopes with the homologous enzyme contained in tissues from other species. In addition, the selective localization of the 12-lipoxygenase to pancreatic beta-cells and its absence in pancreatic acinar cells and in islet non-beta-cells support observations suggesting that 12-lipoxygenase products may participate in glucose-induced insulin secretion from beta-cells.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗