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Mass Spectrometric Evidence That Agents That Cause Loss of Ca²⁺ from Intracellular Compartments Induce Hydrolysis of Arachidonic Acid from Pancreatic Islet Membrane Phospholipids by a Mechanism That Does Not Require a Rise in Cytosolic Ca²⁺ Concentration¹

Mass Spectrometry Resource, Divisions of Diabetes, Endocrinology, and Metabolism and of Laboratory Medicine, Departments of Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: John Turk, Washington University School of Medicine, Box 8127, 660 S. Euclid Avenue, St. Louis, Missouri 63110. E-mail: jturk{at}imgate.wustl.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may amplify the glucose-induced Ca²⁺ entry into islet ß-cells that triggers insulin secretion. Ca²⁺ loss from ß-cell intracellular compartments has been proposed to induce both Ca²⁺ entry and events dependent on arachidonate metabolism. We examine here effects of inducing Ca²⁺ loss from intracellular sequestration sites with ionophore A23187 and thapsigargin on arachidonate hydrolysis from islet phospholipids. A23187 induces a decline in islet arachidonate-containing phospholipids and release of nonesterified arachidonate. A23187-induced arachidonate release is of similar magnitude when islets are stimulated in Ca²⁺-replete or in Ca²⁺-free media or when islets loaded with the intracellular Ca²⁺ chelator BAPTA are stimulated in Ca²⁺-free medium, a condition in which A23187 induces no rise in ß-cell cytosolic [Ca²⁺]. Thapsigargin also induces islet arachidonate release under these conditions. A23187- or thapsigargin-induced arachidonate release is prevented by a bromoenol lactone (BEL) inhibitor of a ß-cell phospholipase A₂ (iPLA₂), which does not require Ca²⁺ for catalytic activity and which is negatively modulated by and physically interacts with calmodulin by Ca²⁺-dependent mechanisms. Agents that cause Ca²⁺ loss from islet intracellular compartments thus induce arachidonate hydrolysis from phospholipids by a BEL-sensitive mechanism that does not require a rise in cytosolic [Ca²⁺], and a BEL-sensitive enzyme-like iPLA₂ or a related membranous activity may participate in sensing Ca²⁺ compartment content.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
NORMAL pancreatic islet ß-cells maintain euglycemia by secreting insulin when the blood glucose concentration rises above 5 mM. Because this response is defective in type II diabetes mellitus (1), mechanisms underlying glucose-induced insulin secretion are biomedically important. Early events in this process include glucose transport into ß-cells, its phosphorylation by glucokinase, and subsequent metabolism (2), which yields signals that inactivate plasma membrane ATP-sensitive K⁺ channels (K_ATP)¹ (3, 4). This causes membrane depolarization, opening of voltage-operated Ca²⁺ channels, Ca²⁺ influx, and a rise in cytosolic [Ca²⁺] that triggers insulin exocytosis (5, 6, 7). Glucose also induces hydrolysis of arachidonic acid from islet membrane phospholipids, and the resultant accumulation of nonesterified arachidonate (8, 9) may amplify depolarization-induced Ca²⁺ influx (10) and insulin secretion (9).

How glucose induces hydrolysis of arachidonate from islet phospholipids is incompletely understood, but some findings raise the question of whether a decline in Ca²⁺ content of subcellular compartments could participate in initiating this process. Glucose-stimulated ß-cells first exhibit a transient decline in cytosolic [Ca²⁺] (11, 12, 13). It has been proposed that this is followed by Ca²⁺ release from intracellular sequestration sites (14, 15, 16) that results in activation of an inward cationic current across the plasma membrane (17, 18) that interacts cooperatively with K_ATP channel closure to induce membrane depolarization and voltage-operated Ca²⁺ channel opening (19). It is not known what signal(s) generated by Ca²⁺ loss from intracellular sites might influence plasma membrane ion fluxes. Because arachidonate affects activities of several ion channels (20, 21, 22, 23, 24) and promotes Ca²⁺ influx into ß-cells (10, 25), it is one candidate for a diffusible mediator that might convey signals from internal membranes to the plasma membrane, if it were released as a consequence of Ca²⁺ loss from intracellular sites.

That such release might occur is suggested by a report that agents that cause Ca²⁺ loss from intracellular sites induce apoptosis in ß-cells by a mechanism that requires generation of arachidonate lipoxygenase products and is blocked by lipoxygenase inhibitors and mimicked by the lipoxygenase product 12-HETE (26). Although not directly demonstrated, this implies that agents that induce Ca²⁺ loss from intracellular sites trigger hydrolysis of arachidonate from ß-cell phospholipids because that is the rate-limiting event in producing arachidonate metabolites (27), and nonesterified arachidonate levels govern islet 12-HETE production (28). Induction of apoptosis in this system does not require a rise in cytosolic [Ca²⁺] and occurs in Ca²⁺-free medium and in ß-cells loaded with the Ca²⁺ chelator BAPTA (26). This implies that hydrolysis of arachidonate from ß-cell phospholipids might also be triggered by a mechanism that does not require a rise in cytosolic [Ca²⁺] but is activated by Ca²⁺ loss from intracellular sites. The study reported here tests this hypothesis under conditions of short-term exposure of islets to agents that induce Ca²⁺-loss from intracellular sequestration sites, which may be more germane to acute signal transduction events than the longer term exposure required to induce apoptosis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Dipentadecanoyl-glycerophosphoethanolamine [(15:0/15:0)-GPE] and dimyristoyl-glycerophosphocholine [(14:0/14:0)-GPC] were obtained from Avanti Polar Lipids (Birmingham, AL) and L--1-palmitoyl-2-[¹⁴C]linoleoyl-phosphatidylcholine (50 mCi/mmol) from New England Nuclear (Boston, MA). [²H₈]-arachidonic acid was prepared by catalytic reduction of eicosa-(5, 8, 11, 14)-tetraynoic acid with [²H₂] gas (8). Male Sprague-Dawley rats (180–220 g) were obtained from Sasco (O’Fallon, MO); collagenase from Boehringer Mannheim (Indianapolis, IN); tissue culture medium CMRL-1066, penicillin, streptomycin, and HBSS from Gibco (Grand Island, NY); Pentex BSA (fatty acid free, fraction V) from Miles Laboratories (Elkhart, IN); rodent Chow 5001 from Ralston Purina (St. Louis, MO); ATP-agarose, calmodulin-sepharose, calmodulin, ampicillin, propranolol, and kanamycin from Sigma Chemical Co. (St. Louis, MO); D-glucose from the National Bureau of Standards (Washington, D.C.); and the bromoenol lactone suicide substrate (BEL) [(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one] from Cayman Chemical (Ann Arbor, MI). BAPTA-AM, A23187, and 4-bromo-A23187 were obtained from Calbiochem (La Jolla, CA) and FURA 2-AM from Molecular Probes (Eugene, OR). Media included KRB (Krebs-Ringer bicarbonate buffer: 25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO₃, 5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂); cCMRL (CMRL-1066 supplemented with 10% heat-inactivated FBS, 1% L-glutamine, 1% penicillin, 1% streptomycin); and HBSS supplemented with 0.5% penicillin-streptomycin.

Isolation of pancreatic islets and incubation with A23187 and other additives
Islets were isolated from male Sprague-Dawley rats as described (29) by collagenase digestion of excised, minced pancreas, density gradient isolation, and manual selection under microscopic visualization. To examine responses to A23187 in Ca²⁺-replete medium, islets (300 per condition) were placed in silanized glass tubes containing 0.4 ml KRB (with 6 mM glucose and 0.1% BSA) and ethanol only (0.1%) or A23187 (10 µM) in ethanol. Incubations (30 min, 37 C) were performed under 5% CO₂/95% air and terminated by adding chloroform/methanol (2 ml, 1/1, vol/vol). Islet lipids were extracted and analyzed as described below. Similar experiments were performed with thapsigargin (5 µM) in dimethylsulfoxide vehicle. To examine responses to agonists in Ca²⁺-free medium, preincubations (15 min) and experimental incubations (30 min) were performed in zero-calcium buffer (25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO₃, 5 mM KCl, 1 mM MgCl₂, 6 mM glucose, 0.1% BSA) containing 1 mM EGTA and no added Ca²⁺. To examine effects of BAPTA, BEL, or propranolol, islets were preincubated (15 min) with BAPTA-AM (100 µM), BEL (10 µM), or propranolol (250 µM) in cCMRL medium. Medium was then removed and islets rinsed thrice in zero-calcium buffer before experimental incubations (30 min). When propranolol was used, experimental incubation media also contained the agent (250 µM). Under all loading and incubation conditions, cell viability exceeded 98% by trypan blue exclusion. Islet insulin content was determined as described (30) by RIA after extraction with 75% ethanol/1.5% HCl.

Extraction of islet lipids and addition of internal standards
To examine islet phospholipids, the chloroform/methanol (2 ml, 1/1, vol/vol) solution used to terminate incubations contained the internal standards (15:0/15:0)-GPE (3 nmol) and (14:0/14:0)-GPC (3.7 nmol). After removing incubation medium and adding this solution, islets were sonicated (30 sec, on ice, 30% power, Branson Sonifier, Danbury, CT). Chloroform/methanol (2 mL, 1/1, vol/vol) and water (1.8 mL) were then added and samples vortex-mixed and centrifuged (900 x g, 5 min). The chloroform phase was placed in a silanized glass tube, concentrated to dryness, reconstituted, and analyzed by electrospray ionization (ESI) mass spectrometry (MS). To examine release of nonesterified arachidonate, the chloroform/methanol (2 ml, 1/1, vol/vol) solution used to terminate incubations contained acetic acid (0.3%) and [²H₈]-arachidonic acid (3 nmol) internal standard. After adding this solution, samples were vortex-mixed and centrifuged (900 x g, 5 min). The chloroform phase was placed in a silanized glass tube, concentrated to dryness, reconstituted, and analyzed by RP-HPLC and GC/MS.

Electrospray ionization mass spectrometric analysis of islet phospholipids
ESI/MS was performed on a Finnigan (San Jose, CA) TSQ-7000 triple stage quadrupole mass spectrometer equipped with an ESI source controlled by Finnigan ICIS software operated on a DEC alpha workstation. Phospholipids were dissolved in methanol/chloroform (9/1, vol/vol) containing LiOH (2 nmol/µl) to facilitate formation of glycerophosphoethanolamine (GPE) (M-H)^- ions for negative ion analyses and of Li⁺ adducts of glycerophoshocholine (GPC) for positive ion analyses. Samples were infused (1 µl/min) into the ESI/MS system with a Harvard syringe pump and analyzed under described instrumental conditions (31, 32). For tandem MS, precursor ions selected in the first quadrupole were accelerated (32–36 eV collision energy) into a chamber containing argon (2.3–2.5 mtorr) to induce collisionally activated dissociation (CAD) and product ions analyzed in the final quadrupole. To identify arachidonate-containing GPE species, negative ion tandem MS scanning was performed to identify parent ions that yielded arachidonate anion (m/z 303) as a product upon CAD. To identify arachidonate-containing GPC species, positive ion tandem MS scanning was performed to identify parent ions that yielded a product ion at m/z 473, which represents the arachidonoyl-glycerophosphoethanol moiety derived from GPC-Li⁺ adducts (31).

Reverse phase HPLC isolation and derivatization of nonesterified arachidonic acid
Islet lipid extracts containing [²H₈]-arachidonic acid were analyzed by RP-HPLC (Ultrasphere ODS column, 4.6 x 250 mm, 5 µ particle size, Alltech, Deerfield, IL) in the solvent system (flow 2 ml/min, column temperature 40 C) methanol/water/acetic acid (80/20/0.1) with flow-through UV monitoring (203 nm). The arachidonic acid peak (retention time 20 min) was extracted by adding chloroform (2 ml) and 2% acetic acid in water (2 ml), vortex-mixing, and centrifugation (900 x g, 5 min). The chloroform phase was removed and concentrated to dryness and arachidonate and [²H₈]-arachidonate converted to pentafluorobenzyl ester (PFBE) derivatives with pentafluorobenzyl bromide and tetramethylammonium hydroxide (33).

Gas chromatographic/mass spectrometric quantitation of arachidonic acid
Heptane solutions of arachidonate-PFBE were introduced into a Hewlett-Packard 5890 gas chromatograph (GC) via a Grob-type injector (temperature 225 C) in splitless mode and analyzed on an DB-1 capillary column (15 m length, cross-linked methylsilicone, i.d. 0.20 mm, film thickness 0.33 micron, J&W Scientific, Rancho Cordoba, CA) interfaced with a Hewlett-Packard 5988B mass spectrometer. Helium was carrier gas (total flow 10 ml/min, head pressure 4 lb/in²). Injector and interface temperatures were 225 C. At 0.5 min after injection, GC oven temperature was increased from 180–220 C over 1 min (arachidonate-PFBE retention time 10 min). The mass spectrometer was operated in methane (source pressure 1.5 torr, source temperature 120 C) negative ion chemical ionization mode with a Hewlett-Packard RTE-A data system. Selected monitoring of carboxylate anions from PFBE derivatives of arachidonate (m/z 303) and [²H₈]-arachidonate (m/z 311) was performed to quantitate arachidonate by reference to a standard curve (8, 33).

Isolation of ß-cells and measurement of cytosolic [Ca²⁺]
Single cell suspensions prepared from islets with dispase (25, 34) were analyzed by fluorescence-activated cell sorting (FACS) on a FACS-Vantage instrument (25, 35) to yield a population of predominantly (>90%) ß-cells, as verified by immunocytochemical staining for insulin (36). Beta cell cytosolic [Ca²⁺] was measured as described (10, 25, 37) after attaching cells (10⁵/ml) to 25 mm diameter Cell-Tak-coated glass coverslips and incubation (37 C, overnight, under 5% CO₂ in air) in cCMRL medium, which was then replaced with 6 mM glucose in KRB medium. Beta cells were incubated (30 min, room temperature, in the dark) with Fura-2 AM (10 µM) (10, 25) and rinsed twice with 6 mM glucose in KRB medium. In some cases, ß-cells were then incubated (15 min, room temperature) with BAPTA-AM (100 µM) in 6 mM glucose in KRB medium and rinsed as above. Coverslips with attached ß-cells were placed in the incubation chamber of a PTI DeltaScan instrument with a Nikon microscope and FURA lens attachment. The chamber contained either 6 mM glucose in KRB medium or buffer (25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO₃, 5 mM KCl, 1 mM MgCl₂, 6 mM glucose) with 1 mM EGTA and no added Ca²⁺. Dual wavelength excitation (340 and 380 nm) was performed, and the ratio of emission (505 nm) intensities at these wavelengths was monitored as an index of cytosolic [Ca²⁺] until a stable baseline was achieved. Changes in this ratio were then monitored after adding 4-bromo-A23187 (10, 25).

Preparation of an islet complementary DNA (cDNA) library and isolation of cDNA encoding islet iPLA₂
A cDNA library was constructed from rat islet poly(A)-RNA with a ZAP Express cDNA Gigapack Gold Cloning Kit (Stratagene), and a full-length cDNA encoding rat islet iPLA₂ was isolated from the library in pBK-CMV plasmid and sequenced (38).

Expression of islet iPLA₂ in Sf9 cells
Spodoptera frugiperda (Sf9) insect cells were used as expression host because they express relatively large amounts of recombinant protein encoded by a cDNA insert when infected with baculovirus vectors (39). Sf9 cells also perform eukaryotic posttranslational modifications and are used to generate appropriately posttranslationally modified, catalytically active Group IV PLA₂ (cPLA₂) from mammalian cPLA₂ cDNA in a baculovirus vector (40). The islet iPLA₂ cDNA insert was released from pBK-CMV plasmid by digestion with endonucleases EcoRI and XhoI and subcloned into EcoRI and XhoI sites of pBAC-1 baculovirus transfer plasmid (Novagen). Sf9 cells were cotransfected with this transfer plasmid and linearized baculovirus DNA (BacVector-2000, Novagen) to construct a recombinant baculovirus with iPLA₂ cDNA insert. Ready-Plaque Sf9 cells (3 x 10⁶) in Sf-900II SFM medium were placed in 60-mm tissue culture dishes as a monolayer and incubated (room temperature, 2 h). Recombinant transfer plasmid and linearized BACVector-2000 DNA were incubated (room temperature, 15 min) with Eufectin Transfer Reagent, and Sf-900 II SFM medium was added to this mixture, which was then placed on the Sf9 cell monolayer. After incubation (room temperature, 1 h), agarose/Sf-900II SFM mixture (6 ml) was added to plates containing Sf9 cells. After agarose solidification, Sf-900II SFM medium (3 ml) was placed atop the agarose, and plates were incubated (28 C, 72 h). Plaques were purified by iterations of this process with serial dilutions of recombinant virus. After determination of titer, recombinant baculovirus was used to infect (multiplicity of infection 1.0) Sf9 cells grown in suspensions in 80-ml spinner flasks in Grace’s medium (41). At 48 h after infection, Sf9 cells were collected by centrifugation (500 x g, 10 min), washed with and resuspended in buffer (250 mM sucrose, 25 mM imidazole, pH 8.0), and disrupted by sonication (10 one sec bursts, Vibra Cell sonicator). Cytosol was prepared by sequential centrifugations (10,000 x g for 10 min and 100,000 x g for 60 min) and the second supernatant used for PLA₂ activity assays and chromatographic analyses.

Phospholipase A₂ activity assays
Sf9 cell cytosolic protein was determined by Bio-Rad assay and PLA₂ activity measured in aliquots of cytosol (20 µg protein) added to assay buffer (200 mM Tris-HCl, pH 7.0, total assay volume 200 µl) containing either 5 mM EGTA or 10 mM CaCl₂. Some aliquots of cytosol were pretreated (2 min) with BEL (0.1–10 µM) before PLA₂ assay. Reactions were initiated by injecting substrate (L--1-palmitoyl-2-[¹⁴C]linoleoyl-phosphatidylcholine, specific activity 50 Ci/mol, final concentration 5 µM) in ethanol (5 µl). Assay mixtures were incubated (3 min, 37 C, with shaking) and reactions terminated by adding butanol (0.1 ml) and vortexing. After centrifugation (2000 x g, 4 min), products in the butanol layer were analyzed by silica gel G TLC in petroleum ether/ethyl ether/acetic acid (80/20/1). The TLC plate region containing free linoleate (Rf 0.58) was identified with iodine vapor and scraped into scintillation vials. Released [¹⁴C]-linoleate was measured by liquid scintillation spectrometry and converted to a PLA₂ specific activity (42).

Chromatographic analysis of iPLA₂ activity in cytosol from Sf9 cells infected with recombinant baculovirus containing islet iPLA₂ cDNA
Cytosol from infected Sf9 cells was supplemented with 1 mM EGTA and applied to a DEAE-Sephacel column previously equilibrated with buffer A (250 mM sucrose, 25 mM imidazole, pH 8.0, 1 mM EGTA). The column was washed with buffer B (25 mM imidazole, pH 8.0, 1 mM EGTA), and iPLA₂ activity was eluted with a linear NaCl gradient (0–1 M) in buffer B. Fractions with activity were pooled and applied to an ATP-agarose column previously equilibrated with buffer B. The column was washed with buffer B, 10 mM AMP in buffer B, buffer B, and 1 mM ATP in buffer B. Activity eluted in the ATP solution, and these fractions were pooled, supplemented with 5 mM CaCl₂, and applied to a 0.5 ml calmodulin-sepharose column, which was washed sequentially with buffer C (25 mM imidazole, pH 8.0) supplemented with CaCl₂ (0.5 mM) and with buffer C containing 4 mM EGTA and no added Ca²⁺. The iPLA₂ activity was measured in aliquots of eluant.

Statistical analyses
Student’s t test was used for comparisons between only two groups. For experiments involving multiple groups, comparisons were performed by one-way ANOVA with posthoc Newman-Keuls analyses. The n values specified in the description of data sets refer to measurements performed on separate incubations rather than repeated measurements on different aliquots from the same experiment.

	Results

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Ionophore A23187 is an important tool for examining effects of Ca²⁺ release from intracellular compartments (43, 44). A23187 induces Ca²⁺ movement down its concentration gradient across cell membranes, and this results in Ca²⁺ loss from membrane-bound intracellular sequestration sites (43, 44). A23187 has been reported to have little influence on release of [³H]-arachidonate metabolites from prelabeled islets (30), and if A23187 does not induce islet phospholipid hydrolysis, loss of Ca²⁺ from intracellular compartments would be unlikely to contribute to islet phospholipase activation. To more rigorously examine this issue, mass spectrometric methods were employed to determine whether A23187 might induce islet phospholipid hydrolysis.

Islets were incubated without or with A23187 and their phospholipids extracted, mixed with internal standard phospholipids, and analyzed by ESI/MS. Figure 1A is a negative ion ESI/MS profile of glycerophosphoethanolamine (GPE) species from islets incubated without A23187. The ion at m/z 662 represents the internal standard (15:0/15:0)-GPE (M-H)^- ion. The three most abundant native GPE species are represented by ions at m/z 722, 750, and 766. As discussed further below, each of these species contains arachidonate as sn-2 substituent. Figure 1B illustrates that treating islets with A23187 induced a decline in the abundance of these species relative to the internal standard.

View larger version (30K): [in this window] [in a new window]   Figure 1. Electrospray ionization mass spectrometric analysis of pancreatic islet phospholipids. Isolated islets (300/condition) were incubated in 6 mM glucose KRB medium in the absence (panels A and C) or presence (panels B and D) of ionophore A23187 (10 µM). Phospholipids were then extracted, mixed with internal standards, processed, and analyzed by ESI/MS. GPE species were visualized by negative ion analyses (panels A and B), and the ion at m/z 662 represents (15:0/15:0)-GPE internal standard. GPC species were visualized by positive ion analyses (panels C and D), and the ion at m/z 684 represents (14:0/14:0)-GPC internal standard.

ESI/tandem MS scanning experiments were performed to identify arachidonate-containing parents in islet phospholipid mixtures. Negative ion analyses were performed to identify parent GPE ions that yield arachidonate anion (m/z 303) as a product upon CAD, an ion observed in the tandem spectra of all arachidonate-containing GPE species (32). Such scans indicated that all three major native GPE species in Fig. 1, A and B, contain arachidonate (not shown). For species represented by the ions at m/z 722, 750, and 766, the sn-1 substituents are residues of palmitic aldehyde, stearic aldehyde, and stearic acid, respectively (32). Positive ion analysis were performed to identify parent GPC-Li⁺ ions that yield an ion an m/z 473 as a product upon CAD, which represents the arachidonoyl-glycerophosphoethanol moiety and is common to the tandem spectra of all arachidonate-containing GPC-Li⁺ adducts (31). Such scans indicated that the parent ions at m/z 788 and 816 in the native islet GPC mixtures in Fig. 1C represent arachidonate-containing species (not shown). The former ion represents (1-palmitoyl, 2-arachidonoyl)-GPC-Li⁺ and the latter (1-stearoyl, 2-arachidonoyl)-GPC-Li⁺ (31, 32).

Table 1 summarizes a series of experiments like that in Fig. 1 to examine effects of A23187 on the abundance of major GPE and GPC species in islets. A23187 induced a significant decline in islet arachidonate-containing GPE species but had little effect on the abundance of GPC species. The A23187-induced decline in islet arachidonate-containing GPE species could reflect activation of a phospholipase enzyme that hydrolyzes these molecules. Among the candidate phospholipases are a group IV cytosolic phospholipase A₂ (cPLA₂) (45), a group I secretory PLA₂ (sPLA₂) (46), and a group VI cytosolic PLA₂, (iPLA₂), which has recently been cloned from islets (38). Islet iPLA₂ does not require Ca²⁺ for catalytic activity and is sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate (38), which is not an effective inhibitor of either cPLA₂ or sPLA₂ (47, 48, 49, 50, 51, 52). Table 2 illustrates that the A23187-induced decline in arachidonate-containing GPE species was prevented by pre-treating islets with BEL.

View larger version (20K): [in this window] [in a new window]   Figure 2. Isotope dilution gas chromatographic/mass spectrometric quantitation of nonesterified arachidonate from pancreatic islets. Islets were treated with vehicle only (upper panel) or 10 µM BEL (lower panel) and incubated (300/condition) with 10 µM A23187 in 6 mM glucose KRB medium. Lipids were extracted, mixed with [2H8]-arachidonic acid internal standard, and analyzed by RP-HPLC to isolate arachidonate, which was extracted and converted to a PFBE derivative. This derivative was analyzed by GC/NICI/MS with selected monitoring of carboxylate anions of endogenous arachidonate (m/z 303) and [2H8]-arachidonate internal standard (m/z 311).

View larger version (69K): [in this window] [in a new window]   Figure 3. Ionophore A23187 induces accumulation of nonesterified arachidonic acid in islets even in the presence of Ca2+ chelators. Islets were pre-incubated (15 min) with vehicle (leftmost four bars) or BAPTA-AM (rightmost two bars). After removing preincubation medium and washing, islets were incubated (300/condition) in Ca2+-replete medium (leftmost two bars) or in Ca2+-free, EGTA-containing medium (rightmost four bars) in the absence (first, third, and fifth bars) or presence (second, fourth, and sixth bars) of A23187 (10 µM). Lipids were extracted, mixed with [2H8]-arachidonic acid internal standard, and analyzed by RP-HPLC to isolate arachidonate, which was derivatized, analyzed by GC/MS, and quantitated relative to the internal standard by reference to a standard curve. Ordinate values represent µg of endogenous arachidonate per 28 nmol of acid-extractable insulin, and SEM are indicated (n = 24). Statistical analyses performed by ANOVA with a posthoc Newman-Keuls test yielded the following P values for comparisons among conditions: first bar vs. second, fourth, and sixth bars (all <0.001) and vs. fifth bar (<0.05); second bar vs. first and third bars (both <0.001) and vs. sixth bar (<0.01); third bar vs. second, fourth, and sixth bars (all <0.001) and vs. fifth bar (<0.05); fourth bar vs. first and third bars (both <0.001); fifth bar vs. sixth bar (<0.01) and vs. first and third bars (both <0.05); sixth bar vs. first and third bars (both <0.001) and vs. second and fifth bars (both <0.01). The P values for all other comparisons exceeded 0.05.

View larger version (50K): [in this window] [in a new window]   Figure 4. BEL prevents A23187-induced accumulation of nonesterified arachidonic acid in islets even in the presence of Ca2+ chelators. Islets were preincubated (15 min) with vehicle (first, third, fifth, and seventh bars) or with 10 µM BEL (second, fourth, sixth, and eighth bars). Islets were then placed in fresh medium and preincubated (15 min) with vehicle (leftmost four bars) or 100 µM BAPTA-AM (rightmost four bars). After removing preincubation medium and washing, islets (300/condition) were incubated (30 min) in Ca2+-free, EGTA-containing medium in the absence (first, second, fifth, and sixth bars) or presence (third, fourth, seventh, and eighth bars) of A23187 (10 µM). Lipids were extracted and nonesterified arachidonate quantitated by isotope dilution GC/MS as in Fig. 3. Ordinate values represent µg of endogenous arachidonate per 28 nmol of acid-extractable insulin, and SEM are indicated (n = 14). Statistical analyses performed by ANOVA with a posthoc Newman-Keuls test yielded the following P values for comparisons among conditions: first bar vs. third and seventh bars (both <0.001), vs. second and fifth bars (both <0.01), and vs. fourth, sixth, and eighth bars (all <0.05); second bar vs. third, fifth, and seventh bars (all <0.001) and vs. first bar (<0.01); third bar vs. first, second, fourth, sixth, and eighth bars (all <0.001) and vs. fifth bar (<0.01); fourth bar vs. third, fifth, and seventh bars (all <0.001) and vs. first bar (<0.05); fifth bar vs. second, fourth, sixth, seventh, and eighth bars (all <0.001) and vs. first and third bars (both <0.01); sixth bar vs. third, fifth, and seventh bars (all <0.001) and vs. first bar (<0.05); seventh bar vs. first, third, fourth, fifth, sixth, eighth bars (all <0.001) and vs. third bar (<0.05); eighth bar vs. third, fifth, and seventh bars (all <0.001) and vs. first bar (<0.05). The P values for all other comparisons exceeded 0.05.

View larger version (28K): [in this window] [in a new window]   Figure 5. Effects of Ca2+ ionophore on islet ß-cell cytosolic [Ca2+]. Purified populations of islet ß-cells were isolated, attached to glass coverslips, and loaded with FURA 2-AM. Beta cells were then incubated with vehicle (tracings A and B) or with BAPTA-AM (tracing C). Beta cells were then placed in fresh buffer (25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 6 mM glucose) that contained either 2.5 mM CaCl2 (tracing A) or EGTA and no added Ca2+ (tracings B and C). Beta cells were then placed in a microfluorimetry apparatus and the ratio of emission intensity (505 nm) produced by excitation at 340 nm and 380 nm was determined as an index of cytosolic [Ca2+]. After a stable baseline was achieved, Ca2+ ionophore was added at the time indicated by the arrow. This experiment is representative of three.

View larger version (51K): [in this window] [in a new window]   Figure 6. Thapsigargin induces accumulation of nonesterified arachidonic acid in islets in the absence or presence of Ca2+ chelators, and BEL prevents this response. Islets were preincubated (15 min) with vehicle (first, third, fifth, and seventh bars) or with 10 µM BEL (second, fourth, sixth, and eighth bars). Islets were then placed in fresh medium and preincubated (15 min) with vehicle (leftmost fourth bars) or with 100 µM BAPTA-AM (rightmost four bars). After removing medium and washing, islets (300/condition) were incubated (30 min) in Ca2+-replete medium (leftmost four bars) or in Ca2+-free, EGTA-containing medium (rightmost four bars) in the absence (first, second, fifth, and sixth bars) or presence (third, fourth, seventh, and eighth bars) of thapsigargin (5 µM). Lipids were extracted and nonesterified arachidonate quantitated by isotope dilution GC/MS. Ordinate values represent µg of arachidonate per 28 nmol of acid-extractable insulin, and SEM are indicated (n = 6). Statistical analyses performed by ANOVA with a posthoc Newman-Keuls test. yielded the following P values for comparisons among conditions: first bar vs. third, fifth, seventh (all <0.001); second bar vs. third, fifth, and seventh bars (all <0.001); third bar vs. first, second, fourth, sixth, and eighth bars (all <0.001), vs. seventh bar (<0.01), and vs. fifth bar (<0.05); fourth bar vs. third, fifth, and seventh bars (all <0.001); fifth bar vs. first, fourth, sixth, seventh, and eighth bars (<0.001) and vs. third bar (<0.05); sixth bar vs. third, fifth, and seventh bars (all <0.01); seventh bar vs. second, fourth, fifth, sixth, and eighth bars (all <0.001) and vs. third bar (<0.01); eighth bar vs. third, fifth, and seventh bars (all <0.001). All other comparisons yielded P values > 0.05.

Islet iPLA₂ is BEL-sensitive and does not require Ca²⁺ for catalytic activity (38), but it is not known whether the islet enzyme might be negatively modulated by Ca²⁺. To explore this possibility, the cDNA encoding islet iPLA₂ was placed in a baculovirus vector (41), and the enzyme was then expressed in Sf9 cells (40). As illustrated in Fig. 7A, assays conducted in the absence of Ca²⁺ revealed little PLA₂ activity in the cytosol of native Sf9 cells, but infection of the cells with recombinant baculovirus containing islet iPLA₂ cDNA (pBAC-iPLA₂) resulted in expression of such activity. The iPLA₂ activity expressed in infected Sf9 cells was sensitive to inhibition by BEL (Fig. 7B) and was higher when assayed in Ca²⁺-free, EGTA-containing buffer than in Ca²⁺-supplemented buffer, illustrating the lack of a Ca²⁺ requirement for catalytic activity (Fig. 7C).

View larger version (60K): [in this window] [in a new window]   Figure 7. Expression of iPLA2 activity in Sf9 cells infected with recombinant baculovirus containing the islet iPLA2 cDNA. A, Cytosol was prepared either from native Sf9 cells (left bar) or from Sf9 cells infected with recombinant baculovirus containing the islet iPLA2 cDNA insert (pBAC-iPLA2, right bar). Aliquots of cytosol were assayed for PLA2 activity with radiolabeled phospholipid substrate in an EGTA-containing buffer with no added Ca2+. B, Cytosol from Sf9 cells infected with pBAC-iPLA2 was pretreated (5 min) with vehicle (first bar) or with varied concentrations (0.1, 1, or 10 µM) of BEL (second, third, and fourth bars), and then PLA2 activity was measured in Ca2+-free, EGTA-containing buffer. C, Cytosol from Sf9 cells infected with pBAC-iPLA2 was assayed for PLA2 activity either in Ca2+-free, EGTA-containing buffer (left bar) or in buffer supplemented with CaCl2 and no EGTA (right bar).

View larger version (17K): [in this window] [in a new window]   Figure 8. Calmodulin inhibits recombinant islet iPLA2 activity in a Ca2+-dependent manner. Cytosol was prepared from Sf9 cells infected with recombinant baculovirus containing the islet iPLA2 cDNA as insert and assayed for PLA2 activity in Ca2+-free, EGTA-containing buffer (triangles) or in buffer supplemented with CaCl2 and no EGTA (circles). Varied amounts of calmodulin (0–5 µg) were added to individual assay solutions, as indicated on the abscissa.

View larger version (24K): [in this window] [in a new window]   Figure 9. Recombinant islet iPLA2 activity adsorbs to a calmodulin-sepharose matrix and is desorbed by EGTA-containing buffer. Cytosol was prepared from Sf9 cells infected with recombinant baculovirus containing the islet iPLA2 cDNA insert and analyzed by chromatography on DEAE-Sephacel and then ATP-agarose. Fractions containing Ca2+-independent PLA2 activity that eluted from the ATP-agarose column in ATP-containing buffer were pooled, supplemented with 5 mM CaCl2, and applied to a calmodulin-sepharose column, which adsorbed 97% of applied activity. The column was then washed sequentially with 25 ml of buffer containing 0.5 mM CaCl2 and then with 12 ml of buffer containing 4 mM EGTA and no added Ca2+. The iPLA2 activity was measured in aliquots of column eluant.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Islet iPLA₂ is sensitive to inhibition by BEL and does not require Ca²⁺ for catalytic activity. Recombinant islet iPLA₂ expressed in Sf9 cells is both partially inhibited by and can physically associate with calmodulin by Ca²⁺-dependent mechanisms. A myocardial PLA₂ of unknown primary structure is also inhibited by Ca²⁺/calmodulin (59), and a recombinant PLA₂ cloned from CHO cells with a primary structure similar to islet iPLA₂ also interacts with calmodulin affinity matrices (60), suggesting that negative modulation by Ca²⁺/calmodulin is not confined to islet iPLA₂. Such negative modulation might explain how the decline in ß-cell cytosolic [Ca²⁺] induced by BAPTA activates hydrolysis of arachidonate from islet phospholipids.

It is not clear how the Ca²⁺ content of intracellular sequestration sites could influence interactions of iPLA₂ and calmodulin, but islets and insulinoma cells also contain substantial amounts of membrane-associated, BEL-sensitive PLA₂ activity that does not require Ca²⁺ for catalysis (42, 61, 62) but that might be influenced by the Ca²⁺ content of membrane-bound Ca²⁺ sequestration sites in some manner. Many aspects of the relationships between the cytosolic and membrane-associated BEL-sensitive PLA₂ activities in islets and the details of their regulation remain to be clarified, but the findings here raise the possibility that such activities could be involved in sensing Ca²⁺ loss from cytosol and membrane-bound sequestration sites. Regulation of these activities is likely to be complex, and the cytosolic activity may be affected by concentrations of ATP and ADP (62), by interacting proteins (63), by posttranslational modifications (60), and by other factors that remain to be identified.

BEL also inhibits a cytosolic, Mg²⁺-dependent isozyme of phosphatidic acid phosphohydrolase (PAPH) (52). Islets express such PAPH activity (53), but inhibition of islet cell PAPH activity with propranolol has been reported to stimulate rather than to suppress both arachidonate release from islet cell phospholipids and insulin secretion (54). Propranolol also does not suppress A23187-induced arachidonate release from islets. These observations do not establish PAPH as an obvious candidate for mediating the Ca²⁺-store depletion-induced hydrolysis of arachidonate from islet phospholipids that is prevented by BEL.

Regardless of the identity of the BEL-sensitive target(s) that participate in hydrolysis of arachidonate from islet phospholipids, the fact that such hydrolysis can be triggered by Ca²⁺ loss from intracellular compartments could have implications for islet signaling mechanisms. The earliest change in cytosolic [Ca²⁺] in glucose-stimulated islets is a transient decline to below resting values (11, 12, 13), and this response is dependent on glucose metabolism, as is hydrolysis of arachidonate from islet phospholipids (64, 65). It has been proposed that Ca²⁺ is then released from islet intracellular sequestration sites (14, 15, 16) and that the decline in Ca²⁺ content of these sites activates an inward cationic current across ß-cell plasma membranes (17, 18) that interacts cooperatively with K_ATP channel closure to induce membrane depolarization and opening of voltage-operated Ca²⁺ channels (19). This model is similar to the phenomenon of capacitative Ca²⁺ entry, which involves activation of Ca²⁺ influx mechanisms in response to Ca²⁺ loss from intracellular stores (66, 67, 68, 69).

How the filling state of Ca²⁺ stores is communicated to the plasma membrane is unknown. One possibility is that a diffusible mediator is generated as a consequence of Ca²⁺ loss from intracellular compartments and traverses cytosol to interact with plasma membrane targets. Observations in differentiated U937 cells, which exhibit capacitative Ca²⁺ entry, suggest that arachidonic acid might represent one such mediator (43). Both A23187 and thapsigargin induce Ca²⁺ loss from U937 cell intracellular sequestration sites and hydrolysis of arachidonate from U937 cell phospholipids (43). Inhibition of arachidonate release with a nonselective PLA₂ inhibitor prevents capacitative Ca²⁺ entry in U937 cells, and exogenous arachidonate induces Ca²⁺ influx (43). Exogenous arachidonate also induces Ca²⁺ influx into islet ß-cells (10, 25), and BEL attenuates glucose-induced Ca²⁺ entry into ß-cells (25). In various cells, arachidonic acid amplifies Ca²⁺ entry through both voltage-operated (20) and receptor-operated (21) Ca²⁺ channels and influences activities of other ion channels that may participate in regulating cytosolic [Ca²⁺] (22, 23, 24).

Ca²⁺-store depletion-induced hydrolysis of arachidonic acid from membrane phospholipids could also be involved in ß-cell injury mechanisms. Prolonged (24–48 h) exposure to agents which deplete internal Ca²⁺ stores induces apoptosis in ß-cells (26). This effect occurs even under experimental conditions where no rise in ß-cell cytosolic [Ca²⁺] occurs, but it is dependent on generation of arachidonate metabolites (26). These observations imply that depletion of ß-cell Ca²⁺ stores induces hydrolysis of arachidonate from ß-cell phospholipids by a mechanism that does not require a rise in cytosolic [Ca²⁺]. The findings reported here provide a direct demonstration that this occurs, but additional study is required to establish the full functional consequences of Ca²⁺-store depletion-induced hydrolysis of arachidonate from islet membrane phospholipids and details of the mechanism by which it occurs.

Under the conditions of the studies performed here, treatment of islets incubated in Ca²⁺-replete medium with Ca²⁺ ionophore A23187 or thapsigargin induced an increment in insulin secretion. The ratio to control values was 2.42 ± 0.03 for A23187-treated islets and 3.92 ± 0.21 for thapsigargin-treated islets, and the increment in secretion in induced by these agents was not prevented by pretreating the islets with BEL. In Ca²⁺-free, EGTA-containing medium, the corresponding ratios to control insulin secretion values were 0.79 ± 0.17 for A23187-treated islets and 1.36 ± 0.08 for thapsigargin-treated islets, suggesting that insulin secretion in response to these agents is largely dependent on Ca²⁺ influx from the extracellular space. In contrast, Ca²⁺ influx is not required for induction of ß-cell apoptosis in islets treated with thapsigargin or the Ca²⁺ ionophore ionomycin, although generation of arachidonate 12-lipoxygenase products appears to participate in induction of apoptosis by these agents even under conditions where Ca²⁺ influx cannot occur (26). Studies are in progress to determine whether treating islets with thapsigargin or Ca²⁺ ionophores augments production of arachidonate 12-lipoxygenase products, an effect observed in other circumstances where arachidonate release from islet phospholipids is enhanced (28).

	Acknowledgments

 
We acknowledge the excellent technical assistance of Dr. Mary Mueller and Sheng Zhang and the excellent secretarial assistance of Anita Zinna.

	Footnotes

 
¹ This work was supported by U.S. Public Health Service grants PO1-HL57278, P41-RR-00954, and S10-RR-11260 and by an American Diabetes Association Career Development Award (S.R.).

Received March 2, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Porte D 1991 Beta cells in diabetes mellitus. Diabetes 40:166–180[Abstract]
2. Meglasson MD, Matschinsky FM 1986 Pancreatic islet glucose metabolism and regulation of insulin secretion. Diabetes Metab Rev 2:163–214[Medline]
3. Cook D, Hales CN 1984 Intracellular ATP directly blocks K⁺ channels in pancreatic ß-cells. Nature 311:271–273[CrossRef][Medline]
4. Arkhammar P, Nilsson T, Rorsman P, Berggren PO 1987 Inhibition of ATP-regulated K⁺ channels precedes depolarization-induced increase in cytoplasmic free Ca²⁺ concentration in pancreatic ß-cells. J Biol Chem 262:5448–5454[Abstract/Free Full Text]
5. Gylfe E 1988 Glucose-induced early changes in cytoplasmic calcium of pancreatic ß-cells studied with time-sharing dual-wavelength fluorometry. J Biol Chem 263:5044–5048[Abstract/Free Full Text]
6. Misler S, Barnett D, Gillis K, Pressel D 1992 Electrophysiology of stimulus-secretion coupling in human ß-cells. Diabetes 41:1221–1228[Abstract]
7. Misler S, Barnett D, Gillis K, Scharp D, Falke L 1992 Stimulus-secretion coupling in ß-cells of transplantable human islets of Langerhans. Evidence for a critical role for Ca²⁺ entry. Diabetes 41:662–670[Abstract]
8. Wolf BA, Turk J, Sherman WR, McDaniel ML 1986 Intracellular Ca²⁺ mobilization by arachidonic acid. Comparison with myo-inositol 1,4,5-trisphosphate in isolated pancreatic islets. J Biol Chem 261:3501–3511[Abstract/Free Full Text]
9. Wolf BA, Pasquale SM, Turk J 1991 Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion. Biochemistry 30:6371–6379
10. Ramanadham S, Gross RW, Turk J 1992 Arachidonic acid elevates cytosolic free calcium concentration in individual pancreatic islet ß-cells. Biochem Biophys Res Commun 184:647–653[CrossRef][Medline]
11. Rorsman P, Abrahamson H, Gylfe E, Hellman B 1984 Dual effects of glucose on the cytosolic Ca²⁺ activity of mouse pancreatic beta cells. FEBS Lett 170:196–200[CrossRef][Medline]
12. Roe MW, Lancaster MW, Mertz RJ, Worley JF, Dukes ID 1994 Thapsigargin inhibits the glucose-induced increase of intracellular Ca²⁺ in mouse islets of Langerhans. Am J Physiol 266:E852–E862
13. Chow RH, Lund PE, Loser S, Panten E, Gylfe E 1995 Coincidence of early glucose-induced depolarization with lowering of cytoplasmic Ca²⁺ in mouse pancreatic beta cells. J Physiol 485:607–617[Medline]
14. Kikuchi M, Wollheim CB. Siegel EG, Renold AE, Sharp GWG 1979 Biphasic insulin release in rat islets of Langerhans and the role of intracellular Ca²⁺ stores. Endocrinology 105:1013–1019[Medline]
15. Roe MW, Lancaster ME, Mertz J, Worley JF, Dukes ID 1993 Voltage-dependent intracellular calcium release from mouse islets stimulated by glucose. J Biol Chem 268:9953–9956[Abstract/Free Full Text]
16. Roe MW, Campbell G, Spencer B, Lancaster ME, Worley JF, Dukes ID 1995 Glucose transiently activates cytosolic Ca²⁺ release in mouse islets by a phospholipase C-dependent mechanism. Biophys J 68:A235 (Abstract)
17. Worley JF, McIntyre M, Spencer B, Dukes ID 1994 Depletion of intracellular Ca²⁺ stores activates a maitotoxin-sensitive non-selective cationic current in ß-cells. J Biol Chem 269:32055–32058[Abstract/Free Full Text]
18. Worley JF, McIntyre MS, Spencer B, Mertz RJ, Roe MW, Dukes ID 1994 Endoplasmic reticulum calcium store regulates membrane potential in mouse islet ß-cells. J Biol Chem 269:14359–14362[Abstract/Free Full Text]
19. Dukes ID, Roe MW, Worley JF, Philipson LH 1997 Glucose-induced alterations in ß-cell cytoplasmic Ca²⁺ involving the coupling of intracellular Ca²⁺ stores and plasma membrane ion channels. Curr Opin Endocrinol Diab 4:262- 271
20. Vacher P, McKenzie J, Dufy B 1989 Arachidonic acid affects membrane ionic conductances of GH₃ pituitary cells. Am J Physiol 257:E203–E211
21. Miller B, Sarantis M, Traynelis SF, Atwell D 1993 Potentiation of NMDA receptor currents by arachidonic acid. Nature 355:722–725
22. Petrou S, Ordway R, Singer J, Walsh J 1993 A putative fatty acid-binding domain of the NMDA receptor. Trends Biochem Sci 18:41–42[CrossRef][Medline]
23. Meeves H 1994 Modulation of ion channels by arachidonic acid. Prog Neurobiol 43:175–186[CrossRef][Medline]
24. Eddlestone GT 1995 ATP-sensitive K channel modulation by products of PLA₂ action in the insulin-secreting HIT cell line. Am J Physiol 268:C181–C190
25. Ramanadham S, Gross RW, Han X, Turk J 1993 Inhibition of arachidonate release by secretagogue-stimulated pancreatic islets suppresses both insulin secretion and the rise in ß-cell cytosolic calcium ion concentration. Biochemistry 32:337–346[CrossRef][Medline]
26. Zhou Y-P, Teng D, Dralyuk F, Ostrega D, Rose MW, Philipson L, Polonsky KS 1998 Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca²⁺ stores and arachidonic acid metabolism. J Clin Invest 101:1623–1632[Medline]
27. Needleman P, Turk J, Jakschik BA, Morrison AR, Lefkowith JB 1986 Arachidonic acid metabolism. Annu Rev Biochem 55:35–68[CrossRef][Medline]
28. Ma Z, Ramanadham S, Corbett JA, Bohrer A, Gross RW, McDaniel ML, Turk J 1996 Interleukin-1 enhances pancreatic islet arachidonic acid 12- lipoxygenase product generation by increasing substrate availability through a nitric oxide-dependent mechanism. J Biol Chem 271:1029–1042[Abstract/Free Full Text]
29. McDaniel ML, Colca JR, Kotagal N, Lacy PE 1983 A subcellular fractionation approach for studying insulin release mechanisms and calcium metabolism in islets of Langerhans. Methods Enzymol 98:182–200[Medline]
30. Turk J, Colca JR, Kotagal N, McDaniel ML 1984 Arachidonic acid metabolism in isolated pancreatic islets II. The influence of glucose and of inhibitors of arachidonate metabolism on insulin secretion and metabolite synthesis. Biochim Biophys Acta 794:125–136[Medline]
31. Hsu FF, Bohrer A, Turk J 1998 Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry. J Am Soc Mass Spectrom 9:516–526[CrossRef][Medline]
32. Ramanadham S, Hsu FF, Bohrer A, Nowatzke W, Ma Z, Turk J 1998 Electrospray ionization mass spectrometric analyses of phospholipids from rat and human pancreatic islets and subcellular membranes. Comparison to other tissues and implications for membrane fusion in insulin exocytosis. Biochemistry 37:4553–4567[CrossRef][Medline]
33. Ramanadham S, Bohrer A, Mueller M, Jett P, Gross RW, Turk J 1993 Mass spectrometric identification and quantitation of arachidonate-containing phospholipids in pancreatic islets: prominence of plasmenylethanolamine molecular species. Biochemistry 32:5339–5351[CrossRef][Medline]
34. Ono J, Takaki R, Fukama M 1977 Preparation of single cells from pancreatic islets of adult rats by the use of dispase. Endocrinol Jpn 24:265–270[Medline]
35. Van De Winkel M, Maes E, Pipeleers DB 1982 Islet cell analysis and purification by light scatter and autofluorescence. Biochem Biophys Res Commun 107:525–532[Medline]
36. Wang JL, McDaniel ML 1990 Secretagogue-induced oscillations of cytoplasmic Ca²⁺ in single ß- and - cells obtained from pancreatic islets by fluorescence-activated cell-sorting. Biochem Biophys Res Commun 166:813–818[CrossRef][Medline]
37. Tsien RY, Rink T, Poenie M 1985 Measurement of cytosolic free Ca²⁺ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 6:145–157[CrossRef][Medline]
38. Ma Z, Ramanadham S, Kempe K, Chi XS, Ladenson J, Turk J 1997 Pancreatic islets express a Ca²⁺-independent phospholipase A₂ enzyme that contains a repeated structural motif homologous to the integral membrane protein binding domain of ankyrin. J Biol Chem 272:11118–11127[Abstract/Free Full Text]
39. Becker GW, Miller JR, Kovacevic S, Ellis R, Louis AI, Small JS, Stark DH, Roberts EF, Wyrick TK, Hoskins J, Chiou G, Sharp JD, McClure DB, Rioggin RM, Kramer RM 1994 Characterization by electrospray mass spectrometry of human Ca²⁺-sensitive cytosolic phospholipase A₂ produced in baculovirus-infected insect cells. Biotechnology 12:69–74[CrossRef][Medline]
40. deCarvalho MS, McCormack AL, Olsen E. Ghomashchi F, Gelb MH, Yates JR, Leslie CC 1996 Identification of phosphorylation sites of human 85 kDa cytosolic phospholipase A₂ expressed in insect cells and present in human monocytes. J Biol Chem 271:6987–6997[Abstract/Free Full Text]
41. O’Reilly DR, Miller LK, Luckow VA 1992 Baculovirus Expression Vectors: A Laboratory Manual. WH Freeman & Co, New York
42. Gross RW, Ramanadham S, Kruszka KK, Han X, Turk J 1993 Rat and human pancreatic islet cells contain a calcium-independent phospholipase A₂ activity selective for hydrolysis of arachidonate which is stimulated by ATP and specifically localized to islet beta cells. Biochemistry 32:327–336[CrossRef][Medline]
43. Rzigalinski BA, Blackmore PF, Rosenthal MD 1996 Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells. Biochim Biophys Acta 1299:342–352[Medline]
44. Wolf