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R W Gross

Publications and source records attributed to R W Gross.

At least 37 records · Page 2Linked to original sources
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

Long-term potentiation requires activation of calcium-independent phospholipase A2.

The predominant phospholipase activity present in rat hippocampus is a calcium-independent phospholipase A2 (302.9 +/- 19.8 pmol/mg.min for calcium-independent phospholipase A2 activity vs. 14.6 +/- 1.0 pmol/mg.min for calcium-dependent phospholipase A2 activity). This calcium-independent phospholipase A2 is exquisitely sensitive to inhibition by the mechanism-based inhibitor, (E)-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran -2-one (BEL). Moreover, treatment of hippocampal slices with BEL prior to tetanic stimulation prevents the induction of LTP (40.8 +/- 5.6% increase in excitatory post-synaptic potential (EPSP) slope for control slices (n = 6) vs. 5.8 +/- 8.5% increase in EPSP slope for BEL-treated slices (n = 8)). Importantly, LTP can be induced following mechanism-based inhibition of phospholipase A2 by providing the end product of the phospholipase A2 reaction, arachidonic acid, during the application of tetanic stimulation. Furthermore, the induction of LTP after treatment with BEL is dependent on the stereoelectronic configuration of the fatty acid provided since eicosa-5,8,11-trienoic acid, but not eicosa-8,11,14-trienoic acid, rescues LTP after BEL treatment (37.6 +/- 16.1% increase in EPSP slope for eicosa-5,8,11-trienoic acid vs. -3.7 +/- 5.2% increase in EPSP slope for eicosa-8,11,14-trienoic acid). Collectively, these results provide the first demonstration of the essential role of calcium-independent phospholipase A2 in synaptic plasticity.

Animals↗
PubMed1995

Rapid plasmenylethanolamine-selective fusion of membrane bilayers catalyzed by an isoform of glyceraldehyde-3-phosphate dehydrogenase: discrimination between glycolytic and fusogenic roles of individual isoforms.

Recently we demonstrated that the unique stereoelectronic relationships inherent in the structure of plasmenylethanolamine facilitate membrane fusion, and we postulated the existence of a membrane fusion protein which could exploit the propensity of plasmenylethanolamine molecular species to adapt an inverted hexagonal phase [Glaser & Gross (1994) Biochemistry 33, 5805-5812]. We now report a cryptic membrane fusion activity in rabbit brain cytosol, which requires separation from an endogenous inhibitor to express its activity, and demonstrate that vesicle fusion catalyzed by this protein is highly selective for membrane vesicles containing plasmenylethanolamine. The cytosolic protein catalyzing membrane fusion activity was purified to apparent homogeneity by sequential column chromatographies, revealing a single 38-kDa protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Automated Edman degradation demonstrated that the purified protein is an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was confirmed by Western blot analysis utilizing polyclonal antibodies and by solution-state inactivation of membrane fusion activity by a monoclonal antibody directed against GAPDH. Both GTP-affinity and Mono Q chromatographies resolved GAPDH isoforms that catalyzed dehydrogenase activity from the GAPDH isoform that catalyzed membrane fusion activity. The purified fusion protein was calcium-independent, resistant to treatment with N-ethylmaleimide, and possessed an obligatory requirement for plasmenylethanolamine and cholesterol. High-resolution stopped-flow kinetic analysis of plasmenylethanolamine-facilitated membrane fusion demonstrated that one tetramer of the GAPDH isoform catalyzed one fusion event between two vesicles containing plasmenylethanolamine every millisecond (on average). Collectively, these results constitute the first description of a protein which can catalyze the fusion of vesicles at a rate which satisfies the mathematical constraints imposed by the observed rates of fusion of synaptic vesicles with the presynaptic membrane in vivo.

Amino Acid Sequence↗
PubMed1995

Nitric oxide activates the glucose-dependent mobilization of arachidonic acid in a macrophage-like cell line (RAW 264.7) that is largely mediated by calcium-independent phospholipase A2.

Herein, we demonstrate that nitric oxide is a potent (> 20% release) and highly selective inducer of [3H]arachidonic acid mobilization in the macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one resulted in the inhibition of the large majority (86%) of nitric oxide-induced [3H]arachidonic acid release into the medium (IC50 < 0.5 microM) and the concomitant inhibition of in vitro measurable calcium-independent phospholipase A2 activity (92% inhibition) without demonstrable effects on calcium-dependent phospholipase A2 activity. Since nitric oxide is a potent stimulator of glycolysis (and therefore glycolytically derived ATP) and since cytosolic calcium-independent phospholipase A2 exists as a catalytic complex comprised of ATP-modulated phosphofructokinase-like regulatory polypeptides and a catalytic subunit, we examined the role of glucose in facilitating nitric oxide-mediated arachidonic acid release. Nitric oxide-induced release of [3H]arachidonic acid possessed an obligatory requirement for glucose, was highly correlated with the concentration of glucose in the medium, and was dependent on the metabolism of glucose. Thus, [3H]arachidonic acid release is coupled to cellular glucose metabolism through alterations in the activity of calcium-independent phospholipase A2. Collectively, these results identify a unifying metabolic paradigm in which the generation of lipid second messengers is coordinately linked to the signalstimulated acceleration of glycolytic flux, thereby facilitating integrated metabolic responses to cellular stimuli.

Animals↗
PubMed1995

Potassium flux through gramicidin ion channels is augmented in vesicles comprised of plasmenylcholine: correlations between gramicidin conformation and function in chemically distinct host bilayer matrices.

The functional role of distinct phospholipid subclasses and molecular species in modulating gramicidin-mediated K+ flux was characterized through quantification of changes in the fluorescence intensity of ion specific fluorescent probes encapsulated inside vesicles comprised of individual molecular species of plasmenylcholine and phosphatidylcholine. The rate constant of gramicidin-mediated K+ ion flux across bilayers comprised of 1-O-(Z)-hexadec-1'-enyl-2-octadec-9'-enoyl-sn-glycero-3-p hos phocholine (plasmenylcholine) was 18.9 +/- 1.7 s-1, while that present across bilayers comprised of 1-hexadecanoyl-2-octadec-9'-enoyl-sn-glycero-3-phosphocholine (phosphatidylcholine) was 12.3 +/- 1.5 s-1. The observed changes were not due to alterations in the nature of the sn-2 aliphatic chain or the net surface charge present at the membrane interface and were unaltered by the addition of several amphiphilic agents (including charged amphiphiles), suggesting that the observed alterations specifically reflect changes in channel function which result from the covalent alteration of host phospholipid in the proximal portion of the sn-1 aliphatic chain (i.e., phospholipid subclass-specific alterations). Addition of cholesterol to bilayer matrices comprised of plasmenylcholine resulted in dose-dependent attenuation of the rate of gramicidin-mediated K+ flux, but did not alter the rate of gramicidin-mediated K+ flux in membranes comprised of phosphatidylcholine. Gramicidin ion channels experience distinct environments in membranes comprised of phosphatidylcholine and plasmenylcholine host lipids demonstrated by both the different fluorescence anisotropies of endogenous tryptophan residues and the different C=O stretching frequencies of intramonomer carbonyls in gramicidin incorporated into these two choline glycerophospholipid subclasses.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol↗
PubMed1995

Concomitant acceleration of the activation and inactivation kinetics of the human delayed rectifier K+ channel (Kv1.1) by Ca(2+)-independent phospholipase A2.

The electrophysiologic sequelae of arachidonic acid release mediated by the major phospholipase A2 (PLA2) in electrically active tissues (i.e. the 40-kDa Ca(2+)-independent PLA2) were assessed in Sf9 cells expressing the human recombinant delayed rectifier K+ channel Kv1.1. Intracellular administration of Ca(2+)-independent PLA2 increased the rate of activation of the macroscopic current (from tau act = 6.25 +/- 0.76 ms to tau act, PLA2 = 2.78 +/- 0.78 ms at 40 mV) and resulted in channel inactivation (from no observed inactivation to tau inact = 103 +/- 6 ms at 40 mV), which were: 1) dependent on the enrichment of Sf9 cell phospholipids in esterified arachidonic acid; 2) ablated by pretreatment of the PLA2 by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one; and 3) manifest prior to development of alterations in cellular permeability. The bidirectional effects of Ca(2+)-independent PLA2 were indistinguishable from the effects of exogenously applied arachidonic acid (AA), which specifically and reversibly increased the rates of channel activation (from tau act = 5.73 +/- 0.88 ms to tau act,AA = 1.91 +/- 0.39 ms at 40 mV) and inactivation (from no observed inactivation to tau inact = 76.6 +/- 1.4 ms at 40 mV). These electrophysiologic alterations resulted from the effects of arachidonic acid per se since Sf9 cells did not produce oxygenated eicosanoid metabolites, and neither exogenous administration nor in situ generation of other fatty acids resulted in these effects. Collectively, these results unambiguously demonstrate the role of arachidonic acid per se on Kv1.1 electrophysiologic function and suggest the importance of Ca(2+)-independent PLAs as an enzymic modulator of ion channel function in electrically active tissues.

Animals↗
PubMed1994

Phospholipid subclass-specific alterations in the kinetics of ion transport across biologic membranes.

Although the predominance of plasmalogens in electrically-active membranes (e.g., sarcolemma) is well-known, identification of the molecular mechanisms through which the vinyl ether linkage facilitates electrophysiologic function has remained elusive. Herein we demonstrate that the kinetics of both carrier-mediated (i.e., valinomycin) and passive ion transport are substantially different in membranes comprised of plasmalogen molecular species in comparison to their diacyl and alkyl ether choline glycerophospholipid subclass counterparts. The rank order of valinomycin-mediated K+/Na+ exchange in membranes comprised of each choline glycerophospholipid subclass was plasmenylcholine (kappa = (6.1 +/- 0.7) x 10(-2) s-1) > plasmanylcholine (kappa = (1.9 +/- 0.2) x 10(-2) s-1) approximately equal to phosphatidylcholine (kappa = (2.3 +/- 0.5) x 10(-2) s-1). A similar hierarchy of rate constants for valinomycin-facilitated Na+ transport in each subclass was manifest. In contrast, the phospholipid subclass rank order for passive Cl- flux was phosphatidylcholine (kappa = (2.6 +/- 0.4) x 10(-4) s-1) > plasmanylcholine (kappa = (0.8 +/- 0.1) x 10(-4) s-1) approximately equal to plasmenylcholine (kappa = (0.6 +/- 0.2) x 10(-4) s-1). Based upon known differences in the conformation, dynamics, membrane dipole potential, and electron-donating properties of these choline glycerophospholipid subclasses, a model is presented which explains the subclass-induced differences in carrier-mediated and passive ion transport providing a rationale for the predominance of plasmalogens in electrically-active membranes.

Chlorides↗
PubMed1994

Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.

Electrospray ionization mass spectrometry (ESI-MS) was utilized for the structural determination and quantitative analysis of individual phospholipid molecular species from subpicomole amounts of human erythrocyte plasma membrane phospholipids. The sensitivity of ESI-MS was 2-3 orders of magnitude greater than that achievable with fast-atom bombardment mass spectrometry (FAB-MS). Phospholipid structure determination and quantitative analysis with ESI-MS can be performed directly from chloroform extracts of biologic samples, obviating the need for prior chromatographic separation of phospholipid classes which has been necessary in FAB-MS phospholipid analyses. Furthermore, ESI-MS is uncomplicated by differential fragmentation of molecular ions and idiosyncratic surface desorption, allowing the quantitation of phospholipids with coefficients of determination (r2) > 0.99 and accuracies > 95%. More than 50 human erythrocyte plasma membrane phospholipid constituents were identified by direct ESI-MS analysis of chloroform extracts of plasma membranes derived from the equivalent of < 1 microliter of whole blood. The major ethanolamine glycerophospholipid subclass in erythrocyte plasma membranes was plasmenylethanolamine that was highly enriched in polyunsaturated fatty acids at the sn-2 position. Collectively, these results demonstrate that ESI-MS of phospholipids is an enabling strategy for the direct structural determination and quantitative analysis of subpicomole amounts of phospholipids from biologic samples.

Erythrocyte Membrane↗
PubMed1994

Activation of Raf-1 by 14-3-3 proteins.

The protein Raf-1, a key mediator of mitogenesis and differentiation, associates with p21ras (refs 1-3). However, the regulation of the serine/threonine kinase activity of Raf-1 is still not understood. Using the yeast two-hybrid system, we identified two structurally related proteins that interact with the aminoterminal region of Raf-1. These proteins, 14-3-3 zeta (PLA2) and 14-3-3 beta (HS1), are members of the 14-3-3 family of proteins. Expression of 14-3-3 proteins in Xenopus oocytes enhanced Raf-1 activity and promoted Raf-1-dependent oocyte maturation. A dominant negative mutant of Raf-1 blocked the effects of 14-3-3 protein.

14-3-3 Proteins↗
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

The ATP- and CoA-independent synthesis of arachidonoylethanolamide. A novel mechanism underlying the synthesis of the endogenous ligand of the cannabinoid receptor.

Recently, arachidonoylethanolamide was identified as the endogenous ligand of the delta 9 tetrahydrocannabinol receptor. Herein, we demonstrate that the synthesis of arachidonoylethanolamide is catalyzed by a novel CoA- and ATP-independent pathway, which is highly selective for arachidonic acid as the aliphatic constituent, is specific for ethanolamine as the polar moiety and occurs through a mechanism that utilizes a critical sulfhydryl residue. The production of arachidonoylethanolamide was calcium-independent and heat-labile and was selectively catalyzed by brain microsomal and cytosolic proteins among the tissues examined. Four independent lines of evidence demonstrate that the synthesis of arachidonoylethanolamide occurs through an ATP- and CoA-independent process. 1) The depletion of endogenous rabbit brain cytosolic or microsomal CoA and ATP by dialysis, or depletion of ATP by apyrase treatment, did not diminish the production of arachidonoylethanolamide; 2) the addition of exogenous ATP or CoA to cofactor depleted cytosol or microsomes did not stimulate arachidonoylethanolamide production; 3) the synthesis of arachidonoylethanolamide occurred in the absence of detectable arachidonoyl-CoA and did not correlate with either the amount of arachidonoyl-CoA in the incubation medium or the amount of arachidonoyl-CoA incorporated into polar and nonpolar lipids; 4) the addition of a 20-fold molar excess of unlabeled arachidonoyl-CoA to incubations containing [3H]arachidonic acid and ethanolamine did not attenuate the production of [3H]arachidonoylethanolamide by rabbit brain cytosolic or microsomal proteins. Collectively, these results demonstrate a novel chemical paradigm for the ATP- and CoA-independent conjugation of the carboxylate moiety of arachidonic acid to yield arachidonoylethanolamide, the first member of a new class of biologically active eicosanoid second messengers.

Adenosine Triphosphate↗
PubMed1994

Plasmenylethanolamine facilitates rapid membrane fusion: a stopped-flow kinetic investigation correlating the propensity of a major plasma membrane constituent to adopt an HII phase with its ability to promote membrane fusion.

A critical step in membrane fusion involves the formation of a lipid intermediate which shares a conformational similarity with an inverted hexagonal phase (HII). Since plasmenylethanolamines possess a marked propensity for hexagonal phase formation and represent a major lipid constituent of several membrane systems which undergo rapid membrane fusion (e.g., plasma membranes and synaptic vesicle membranes), we compared the relative fusogenicity of lipid vesicles containing plasmenylethanolamine to that of vesicles containing their diacyl phospholipid counterpart (i.e., phosphatidylethanolamine). Vesicles comprised of equimolar mixtures of phosphatidylcholine and phosphatidylethanolamine fused slowly with phosphatidylserine vesicles in the presence of 10 mM CaCl2, as assessed either by lipid mixing (dequenching of octadecyl rhodamine fluorescence, 7.4 Fmax% s-1) or internal contents mixing (fluorescence enhancement from the resultant Tb/dipicolinic acid charge transfer complex, 8.7Fmax% s-1). In stark contrast, vesicles comprised of equimolar mixtures of phosphatidylcholine and plasmenylethanolamine fused three times more rapidly, as assessed by both lipid mixing (22.1 Fmax% s-1) and internal contents mixing (21.4Fmax% s-1) assays. The importance of an HII-like intermediate in membrane fusion was further substantiated by demonstration that plasmenylethanolamines containing arachidonic acid at the sn-2 position (which demonstrate a greater propensity for HII phase formation) exhibited the most rapid rate of membrane fusion (five times greater than phosphatidylethanolamine containing oleic acid at the sn-2 position). Furthermore, vesicles containing plasmenylethanolamines in physiologic ratios with other phospholipids (i.e., PC/PE/PS, 45:45:10, mol/mol) underwent fusion six times more rapidly (4.4Fmax% min-1) than corresponding vesicles in which plasmenylethanolamine was replaced with phosphatidylethanolamine (0.7Fmax% min-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The rapid and reversible association of phosphofructokinase with myocardial membranes during myocardial ischemia.

Myocardial calcium-independent phospholipase A2 (PLA2) activity is mediated by a 400 kDa catalytic complex comprised of a tetramer of phosphofructokinase (PFK) and a 40 kDa catalytic subunit [1,2]. During myocardial ischemia, calcium-independent PLA2 activity rapidly and reversibly translocates from the cytosol to a membrane-associated compartment where it has been implicated as a mediator of ischemic damage [3,4]. Herein we demonstrate that the majority of both PFK mass and activity is translocated from the cytosol to a membrane-associated compartment prior to the onset of irreversible myocytic injury and that translocated PFK is catalytically inactive while membrane-associated. Furthermore, reperfusion of ischemic myocardium, or treatment of membranes derived from ischemic myocardium with ATP results in the conversion of both PFK mass and activity from its membrane-associated state to a soluble, catalytically-competent form. Collectively, these studies demonstrate that the concomitant changes in glycolysis and phospholipid hydrolysis during early myocardial ischemia result, at least in part, from the translocation of a common regulatory polypeptide critical in both processes.

Adenosine Triphosphate↗
PubMed1994

The discordant rates of sn-1 aliphatic chain and polar head group incorporation into plasmalogen molecular species demonstrate the fundamental importance of polar head group remodeling in plasmalogen metabolism in rabbit myocardium.

Although recent studies have demonstrated the existence of the requisite enzymic machinery necessary for the shuttling of vinyl ether linkages through polar head group remodeling, the relative rates of plasmalogen de novo synthesis and polar head group remodeling are unknown. Pulse-chase radiolabeling of perfused rabbit hearts with [1-3H]hexadecanol demonstrated the rapid and progressive incorporation of radiolabel into plasmenylethanolamine (e.g., after 0.5 h of radiolabeling, 10% of [1-3H]hexadecanol incorporated into ethanolamine glycerophospholipid was in plasmenylethanolamine, and after 1.5 h, 21% was in plasmenylethanolamine) with no detectable radiolabeling of plasmenylcholine until 3 h after the pulse. Furthermore, perfusion of hearts with [1',2'-alkyl-3H2]1-O-alkyl-GPC resulted in the rapid incorporation of radiolabel into plasmanylcholine, but not plasmenylcholine, even after extended perfusion intervals. In contrast, both radiolabeled choline and ethanolamine were rapidly incorporated into plasmalogens through polar head group remodeling at rates that were over 300-fold greater than that of plasmalogen de novo synthesis (e.g., an incorporation rate of 31 nmol/gdry.h for ethanolamine but only 93 pmol/gdry.h for hexadecanol into plasmenylethanolamine was manifest). Similarly, sn-2 remodeling of plasmalogen molecular species with arachidonic or oleic acid also occurred at rates that were over 100-fold greater than that of de novo plasmalogen biosynthesis. Collectively, these results underscore the fundamental importance of rapid polar head group remodeling of plasmalogen molecular species in the synthesis and maintenance of plasmenylcholine and plasmenylethanolamine pools in intact contracting myocardium.

Animals↗
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

Dynamics of binary mixtures of plasmenylcholine/arachidonic acid and phosphatidylcholine/arachidonic acid--a study using fluorescence and NMR spectroscopy.

Arachidonic acid is selectively released during signal transduction in many cell types. To examine the effects of physiologically relevant amounts of arachidonic acid on membrane bilayers, alterations in membrane dynamics induced by arachidonic acid were investigated utilizing fluorescence and nuclear magnetic resonance spectroscopy. We demonstrate that decreases in the fluorescence steady-state anisotropy of diphenylhexatriene are induced by incorporation of physiologically relevant amounts (i.e., 5 mol%) of arachidonic acid into either phosphatidylcholine or plasmenylcholine membrane bilayers. Furthermore, examination of the motional dynamics of the bis-allylic protons in arachidonic acid by analyses of their spin-spin relaxation times demonstrated that these protons are more restrained when arachidonic acid is present as a substitutional impurity in plasmenylcholine vesicles than in phosphatidylcholine vesicles. Collectively, these results demonstrate that arachidonic acid, when present in physiologically relevant mole fractions, can modify the molecular dynamics of biological membranes and that the motional dynamics of arachidonic acid in membrane bilayers is influenced by the type of covalent linkage present in the proximal portion of the sn-1 aliphatic chain in host bilayer matrices.

Anisotropy↗