Intracellular Ca2+ Mobilization by Arachidonic Acid - The Journal of ...

Vol. 261, No.8,Issue of March 15, pp. 3501-3511,1986 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Intracellular Ca2+ Mobilizationby Arachidonic Acid COMPARISON WITH TTLYO-INOSITOL 1,4,5-TRISPHOSPHATEIN

ISOLATED PANCREATIC ISLETS* (Received for publication, July 15,1985)

Bryan A. Wolf$& John TurkSll, William R. Shermanll, and MichaelL. McDanielS From the $Department of Pathology, 7Diuision of Laboratory Medicine and Departments of Medicine and Pharmacology, and 11 Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri 63110

Previous studies have demonstrated thatmyo-inosi- cently been recognized that myo-inositol 1,4,5-trisphosphate to1 1,4,5-trisphosphate (IP,) mobilizes Ca2+ from the (IP3)lacts on the endoplasmic reticulum of a variety of cells endoplasmic reticulum (ER) of digitonin-permeabilized to release sequestered calcium (see Refs. 7 and 8 for reviews). islets and that an increase in intracellular free Ca" We have demonstrated that IP, induced Ca2+efflux from the stimulates insulinrelease. Furthermore, glucose stim- endoplasmic reticulum of normal islets (9). IP, is also released ulates arachidonic acid metabolism in islets. In digi- from membrane phospholipid shortly after agonist stimulatonin-permeabilizedislets, exogenous arachidonic acid tion of a number of cells, including glucose-stimulated panat concentrations between 1.25 to 10 pM elicited sig- creatic islets (10): It is therefore likely that IP, is an endogenificant Ca2+release from the ER at a free Ca2+ con- nous messenger of calcium-mediated events involved in stimcentration of 0.1 PM. Arachidonic acid-induced CaZ+ ulus-secretion coupling in pancreaticislets. release was not due to the metabolites of arachidonic acid. Arachidonic acid induced a rapid release of Ca2+ IP3is generated by phospholipase C-catalyzed hydrolysis of within 2 min. Comparisonof arachidonic acid-induced phosphatidylinositol 4,5-bisphosphate, which results in siCa" release with IP3-inducedCa2+release revealed a multaneous formation of 1,2-diacylglycerols.Diacylglycerol similar molar potency of arachidonic acid and IP,. The has recently been recognized to modulate the activity of a cornbinationof both arachidonic acid andIP3 resulted phospholipid and calcium-dependent protein kinase (protein in a greater effect on Ca2+ mobilization from theER kinase C) (11, 12). Protein kinase C appears to represent than either compound alone. The mass of endogenous another class of regulatory protein kinases, analogous to the arachidonic acid released by islets incubated with 28 CAMP-dependentand calcium/calmodulin-dependent protein mM glucose was measured by mass spectrometric meth-kinases (13). Pancreatic islets havebeen demonstrated to ods and was found to be sufficient to achieve arachi- contain protein kinase C activity, and diacylglycerol accudonic acid concentrations equal to or exceeding thosemulation occurs after glucose-stimulation of isolated panrequired to induce release of Ca2+ sequestered in the creatic isletsfrom neonatal rats (14-18). ER. These observations indicate that glucose-induced DiacylgIyceroIthat accumulates after thrombin stimulation arachidonic acid release could participate in glucose- of platelets consists largely of the l-stearoyl, 2-arachidonoyl induced Ca2+ mobilization and insulin secretion by pan- species, reflecting the predominant fatty acid composition of creatic islets, possibly in cooperation with IPS. phosphatidylinositol and its polyphosphorylated forms in all tissues so far examined (19). In platelets, the bulk of unesterified arachidonic acid liberated after thrombin stimulation appears to derive from the action of neutral lipases on diacThe major stimulus for insulin secretion from pancreatic ylglycerol (20). Metabolites of arachidonic acid are known to islets is D-glucose (1).The biochemical events that couple accumulate in isolated pancreatic islets after glucose stimuglucose stimulation to insulin secretion by islets are incom- lation, andthis presumably reflects an increased rate of pletely understood, but changes in intracellular calcium con- release of arachidonate from islet membrane phospholipid centration appear to play a major role in these processes (see (21-28). Ref. 2 for review). Recent studies in normal islets have sugArachidonic acid has recently been w o r t e d to increase the gested that the endoplasmic reticulum is directly involved in intracellular calcium concentration inrawit neutrophils, and the regulation of intracellular Ca2' concentrations (3-5). Fur- several long chain unsaturated fatty acids have been demonthermore, increases in cytosolic Ca" concentrations in the strated to release calcium sequestered by the sarcoplasmic submicromolar range induce insulin secretion (6). It has re- reticulum of skeletal muscle (29, 30). T o test the possibility that arachidonic acid might participate in the regulation of * This work was supported in part by an Institutional Training intracellular calcium concentration in pancreatic islets, we Grant AM07296-06 (to B. A. W.) from the National Institutes of have examined the effects of exogenous arachidonate on calHealth, National Institutes of Health GrantAM03373 (to M. L. McD.), by grants from the Pharmaceutical Manufacturer's Associa- cium mobilization from isolated, digitonin-permeabilized istion Foundation, the Juvenile Diabetes Foundation, and National lets. We have observed that exogenous arachidonate induces release of calcium sequestered in the endoplasmic reticulum Institutes of Health Grant AM34388 (to J. T.), and byGrant AM20579 from the National Institutes of Health (to W. R. S.). The costs of publication of this article weredefrayedinpartby the payment o f page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed Washington University School of Medicine, Dept. of Pathology, Box 8118, 660 South Euclid Ave., St. Louis, MO 63110.

The abbreviationsused are: IPS,nyo-inositol1,4,5-trisphosphate;

E-HETE, 12-hydroxyeicosatetraenoic acid; 12-HPETE, 12-hydroperoxyeicosatetraenoic acid HPLC, high-performance liquid chromatography; GC, gas chromatography;NI, negative ion; CI, chemical ionization; MS, mass spectrometry; AA, arachidonic acid; Hepes, 4(2-hydroxyethyI)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.

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of these permeabilized islets at micromolar concentrations comparable to those required for IP3 to exert this effect. We have also measured the mass of endogenous arachidonic acid released as a function of time after glucose stimulation of intact isolated pancreatic islets and have found that sufficient arachidonate is released to achieve concentrations equal to or exceeding those required to influence calcium mobilization. EXPERIMENTAL PROCEDURES~

Materials Male Sprague-Dawleyrats (200-300 g) were purchased from Sasco (O'Fallon, MO). Collagenase (CLS IV) was obtained from Cooper Biochemical (Freehold, NJ), 45CaC12(specific activity 800 mCi/ mmol), [3H]arachidonic acid, 3Hz0, Protosol and Aquasol from New England Nuclear, the calcium ionophore A23187 from Behring Diagnostics (La Jolla, CA), sodium vanadate from Fisher, and tissue culture media (CMRL-1066) from Gibco. Arachidonic acid was purchased from NuCheck Prep (Elysian, MN), Pentex bovine albumin (fatty acid-free, fraction V) from Miles Laboratories (Elkbart, IN), BW755c from Wellcome Research Laboratories (Kent, England) through the courtesy of Dr. P. J. McHale, and other chemicals, except as specified, were obtained from Sigma. Methods Isolation and Culture of Islets-For most experiments, pancreatic islets were isolated from nine rats fed ad libitumby collagenase digestion followed by separation on a discontinuous Ficoll gradient (39,40).Islets were then used immediately. For experiments involving determination of unesterified arachidonate levels, approximately 15,000 islets were isolated from 30 rats under aseptic conditions. Isolated islets were then washed extensively with tissue culture medium (CMRL-1066) and selected under astereomicroscope to exclude any contaminating tissues. Isolated islets were cultured overnight C . in tissue culture under an atmosphere of 95% air, 5% COz at 24 O medium CMRL-1006 containing 8 mMD-glUCOSe, 1%L-glutamine, 10% heat-inactivated fetal bovine serum, 0.5% penicillin, and 0.5% streptomycin. After culture, islets were washed extensively in serumfree medium and incubated in Krebs' buffer supplemented with 0.1% fatty acid-free BSA. Determination of Intracellular Volume of Intact Islets-Freshly isolated islets were washed in Hepes/Krebs' buffer (25 mM Hepes, 115 mM NaC1, 5 mM KC1,24mM NaHC03, 1 mM MgCl,,2.5 mM CaC12,pH 7.4) containing 0.1% fatty acid-free bovine serum albumin. Twenty-five islets were randomly counted ina 200-p1 volume in polypropylene microfuge tubes (1.5 ml) and were washed three times in fresh Hepes/Krebs' buffer in the absence of bovine serum albumin. Islets were then preincubated for 30 min in a shaking water bath at 30 "C in 100pl of fresh Hepes/Krebs' buffer containing 3 mM glucose. The medium wasreplaced by 100pl of Hepes/Krebs' buffer containing 3 mM glucose, 5 mM sucrose, 5 mM urea, 1 pCi of [14C]urea (final specific activity: 2.9 mCi/mmol), and 1.5 pCi of [3H]sucrose as an extracellular volume marker (final specific activity, 2.9 mCi/mmol). Arachidonic acid was added in 5 pl to obtain a final concentration . were then incubated at 37 "C for 30 min, between 0 and 38 p ~Islets and the reaction was stopped by centrifugation (10,000 X g, 1 min) in aBeckman microfuge. The supernatantwas removed, and theislet pellet was dissolved in 50 p1of Protosol (New England Nuclear) for 1 h at room temperature. Glacial acetic acid (10 pl) was added. The total content of the tube was transferred to a scintillation vial containing 10 ml of Aquasol and counted in a liquid scintillation spectrometer under a double-isotope setting (14C, 3H). Calculations were performed as described previously (41). Ca2+Uptake and Ca2+Efjlux by Intact Islets-Ca2+ uptake by intact islets was measured with a double-isotope method as described for Portions of this paper (including part of "Experimental Procedures," part of "Results," and Figs. Sl-S5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-2319, cite the authors, and include a check or money order for $4.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal thatis available from Waverly Press.

the determination of the intracellular volume of the islet except that 45Ca (2.5 mM,42 mCi/mmol final specific activity) and [3H]sucrose (5 mM, 6.5 mCi/mmol) were used. The effect of arachidonic acid (020 p M ) was examined during a 30-min incubation period at 37 "C (42).

Ca" efflux was measured with a similar double-isotope method except that intact islets were loaded for 30 min with T a and [3H] sucrose in Hepes/Krebs' buffer containing 28 mM glucose. Ca" efflux was then initiated by replacing the radioactive medium by fresh nonradioactive Hepes/Krebs' buffer containing 3 mM glucose for 5 min (42). Radiolabelingand Perijusion of Intact Islets-Freshly isolated islets were incubated at 37 "C for 90 min in Hepes/Krebs' buffer containing 30 pCi of [3H]arachidonicacid and 28 mM glucose in the absence of BSA. Radiolabeled islets were then washed three times in Hepes/ Krebs' buffer containing 3 mM glucose and 0.1% fatty acid-free BSA. 350 islets were placed in a perifusion chamber and preincubated for 30 min at 37 "C in Hepesmrebs' buffer with 3 mM glucose (43). Islets were then incubated for 30 min in 28 mM glucose and fractions collected (1 ml/min) for determination of [3H]arachidonic acid and insulin release. Permeabilization of Islets-Islets were permeabilized by digitonin treatment as described previously (6). In brief, freshly isolated islets were incubated in a modified Hepes/Krebs' buffer (25 mM Hepes, 115 mM NaC1,24 mM NaHC03, 5 mM KC1, 1 mM MgClz, 1 mM EGTA, 0.1% bovine plasma albumin, pH 6.8) at 37 "C with 20 pg/ml digitonin (Sigma) for 20 min. Permeabilized islets were washed three times in Tris buffer (50 mM Tris, 100 mM KC1, 5 mM MgC12, 0.1% bovine plasma albumin, pH 6.8) prior to use. Ca2+EjjZux in Permeabilized Islets-Permeabilized islets (30/tube) were incubated in the presence of Ca2+and +ATPfor 30 min at 24 "C with 5 pCi of %a2+ and 5 pCi of 3Hz0 as a cell volume marker in 90 pl of a Tris buffer mimicking intracellular conditions (100 mM Tris, 100 mM KCl, 7 mM MgClz, 5 mM ATP, 2.25 pg/ml ruthenium red, pH 7.0). The free Ca2+concentration of this buffer was set at 0.1 p~ by including EGTA at a concentration of 0.147-0.216 mM depending on the specific activity of 45CaC12 (576-1184mCi/mmol) (see below). Ca2+efflux was initiated by the addition of 5 pl of the appropriate effluxing agent and 5 pl of 200 mM Tris, pH7.0, or 5 pl of a vanadate stock solution. Vanadate, 1.25 mM final concentration, was used in some experiments to inhibit Ca2+reuptake by the endoplasmic reticulum. Tubes were included with the same experimental conditions but without ATP. Ca" efflux was stopped after the appropriate time (2-40 min) by centrifugation (10,000 X g, 1 min). The supernatant was removed and theislet pellet processed with Protosol as described above. Calculations were performed by first determining the space associated with 45Ca2+and subtracting the space occupied by 3H20, thus measuring the volume corresponding to the uptake of T a 2 +in the islet. As the non-ATP-dependent 45Ca2+uptake was subtracted and rutheniumred was present to inhibit mitochondrial "Ca" uptake, the final result (picomoles/islet) is a reflection of the Ca" actively stored in the endoplasmic reticulum (9). In some experiments, the free Ca" concentration was set at1, 10, and 40 p~ by diminishing the EGTA concentrations to 0.08, 0.04, and 0 mM, respectively. Mitochondrial Ca2+content was determined under similar conditions except that thefree Ca2+concentration was set at40 p ~and , 5 mM succinate was present. The mitochondrial Ca" content was determined by subtracting the ATP-dependent Ca2+content of the islet in the presence of ruthenium red, i.e. the Ca" content of the endoplasmic reticulum, from the islet ATP-dependent Ca2+content determined in the absence of ruthenium red. In all experiments, the viability of the islets for Ca" efflux was assessed by including a control condition with 2 p~ of the Ca" ionophore A23187. All results are expressed either as picomoles of Caz+/isletfor the Caz+content of the endoplasmic reticulum or of the mitochondria or as picomoles (or per cent of control) of Ca2+released from the endoplasmic reticulum/islet. Previous studies have indicated that under our conditions, 30 islets correspond to 30 pg of protein. Insulin Secretion-Twenty-five islets were randomly selected, washed three times in Krebs'/Hepes' buffer (5 mM Hepes, 115 mM NaC1,24 mM NaHC03, 5 mM KC1, 2.5 mM CaC12, 1 mM MgC12, pH 7.4), and preincubated 30 min at 37 "C in 200plof Krebs'/Hepes buffer supplemented with 3 mM glucose. The preincubation medium was then removed and replaced by 200 pl of fresh medium containing 3, 8, or 28 mM glucose and 0-40 p M arachidonate. Incubations were continued for 30 min at 37 "C.At the end of this interval, an aliquot


of Ca2+in Islets

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of fatty acid-free bovine serum albumin (0.2%, 200 pl) in incubation centration, 74.6 -I 1.8%inhibition of Ca2+uptake by the islets media was added to the incubation media, mixed, and an aliquot was obtained when measured at a free Ca2+concentration of withdrawn for insulin radioimmunoassay (26-28). Experiments with perifused intact islets were performed as previously described in 40 p M and in thepresence of 5 mM succinate. Concentrations up to 9 Gg/ml elicited the same maximal inhibition of the Krebs'/Hepes buffer containing no BSA (43). Other Methods-Sodium vanadate (Na3VO4-(H20),,was dissolved mitochondrial Ca2+pool. This is in good agreement with our in distilled deionized water and adjusted to pH 10.0 with 6 N HC1. A previous estimation of the mitochondrial Ca2+pool representyellow-orange color appeared, indicative of the presence of polymers ing 75% of all ATP-dependentCa2+pools in islets (2). Sodium of vanadate. The solution was boiled until the color disappeared (30- azide in a range of 10-40 mM was found to be as effective as 60 min) and the colorless solution retitrated to pH 10.0 with 10 N NaOH. The solution was then filtered on a 0.2-p Millipore filter and ruthenium red in inhibiting Caz+uptake by the mitochondria stored at 4 "C in astoppered vial. The final concentration of vanadate (data notshown). was checked spectrophotometrically (extinction coefficient = 2,925 Arachidonic acid in a range of 1.25-10 p~ elicited signifiM" .cm", 265 nm) (44). cant Ca" release from the endoplasmic reticulum of permeaATP stocks were prepared from vanadate-free ATP by dissolution bilized islets (Fig. 1).Maximal release of Ca2+by arachidonic in H20/NaOH (2:0.5, v/v). The final pH was adjusted to pH 7.5 with 1 N NaOH and the concentration determined spectrophotometrically acid was observed at 10 p~ and half-maximal release at 2.5 p ~ The . possibility of artifactual binding of CaZ+to arachias described by Lowry and Passoneau (45). Ruthenium red was dissolved in water. The commercial prepara- donic acid under these conditions was assessed by studies tion from Sigma was 45% pure. Spectrophotometric scanning (190- with a Ca2+-specificelectrode. No binding of Ca2+to arachi900 nm) indicated the presence of impurities. No attempt was made donic acid wasdetected by this procedure. Ca2+content measto further purify the preparation. The final concentration of rutheured inthe absence of ATP, i.e. the Ca2+ bound tothe nium red was checked spectrophotometrically (extinction coefficient membranes by non-ATP-dependent processes, was also not = 68,000 M-'.Cm", 535 nm) (46). Arachidonic acid (0.2 mM) was dissolved in 0.1 M NaZCO3,0.15 M affected by the presence of arachidonic acid (0-10 p ~ ) . NaCl which was found to be the most adequate medium for complete The possibility that arachidonic acid-induced Ca2+release dissolution as assessed isotopically with [3H]arachidonic acid. Syn- from the endoplasmic reticulum was attributable toformation thesis and purification of 12-HPETE and 12-HETEwere performed of a metabolite of arachidonic acid was examined. The most as previously described (26). Aqueous solutions of the 12-HPETE and 1 2 HETE were prepared just before use in 10% ethanol, 90% abundant metabolites produced by isolated islets are the 12lipoxygenase products, 12-HETEand12-HPETE (25-28). incubation medium (v/v). Complete dissolution of the 12-HPETE and 12-HETEcompounds under these conditions was demonstrated Neither 12-HPETEnor 12-HETE ina dose range of 0-10 p~ with ultraviolet spectrophotometry. BW755Cwas dissolved in di- had any significant effect on Ca2+ release from the endomethyl sulfoxide, 0.15 M NaCl (1:17, v/v) at a concentration of 19.6 plasmic reticulum of permeabilized islets (Fig. 1).The influmM. Indomethacin was dissolved in the incubation medium with 1 N NaOH at a concentration of 28 mM. IPS was prepared from human T polycythemic red blood cells not over 2 weeks of age as previously described (9). The purity of IP3 was assessed by electrophoresis and Arachidonic acid gas chromatography (47). All of these compounds were diluted to achieve the desired concentration and added as 5-p1 aliquots to 95 pl 0.31 of the incubation medium. The appropriate vehiclewas added to control incubations. Free CaZ+ concentrationswere determined by taking into account the binding of CaZ+to EGTA and ATP, M e to EGTA and ATP, and K+ to ATP. Absolute stability constants were converted to apparent stability constants taking into account the ionic strength, the H+ activity coefficient, and the temperature (using the enthalpy constant) (48, 49). The following absolute stability constants were used (20 "C, ionic strength 0.1 M): [Caz+.EGTA]/[Ca2+].[EGTA] = [Ca"+.HEGTA]/[Ca2+].[HEGTA] = 105.33;[ M e . E G T A ] / [MgZf].[EGTA] = lo5."; [MgZ+.HEGTA]/[M?+]. [HEGTA] = 103.37. [H+.EGTA]/[H+].[EGTA] = [H+.HEGTA]/[H+]-[HEGTA]' = [H+.HzEGTA]/[H+].[HZEGTA] = [H+.H,EGTA]/ [H']. [H3EGTA] = 102.0; [Caz+.ATP]/[Ca2+].[ATP] = lo3."; [Caz+. I ' C""""+" HATP]/[Caz+]. [HATP] = lo2."; [H+. ATP]/[H+].[ATP] = lo6"'; [H+.HATP]/[H+]. [HATP]= 104.06;[Me.ATP]/[MgZ+]. [ATP]= 104.53., [Mg2+.HATP]/[Mg2+].[HATP] = lo2."; [K+.ATP]/[K+]. L I 0 5 10 [ATP] = 10'. Theoretical values were then checked on a Ca2+-specific EICOSANOID (vM) electrode (Orion 93-20 with a double-junction reference electrode 9002)standardized between and loF4M Ca2+. FIG. 1. Dose c u r v e of arachidonic acid, 12-HETE, a n d 12Statistical Analysis-Data were analyzed by one-way ANOVA fol- HPETE on Ca2+ release by digitonin-treated islets. Isolated lowed by multiple comparison between means with the least signifi- islets were permeabilized with digitonin. In brief, islets were incubated cant difference test. All experiments were performed at least three 20 min at 37 "C in a modified Hepes/Krebs' buffer (115 mM NaCl, 5 times in triplicate unless otherwise indicated. mM KCl, 24 mM NaHC03, 1mM MgCl,, 25 mM Hepes, 1 mM EGTA, 0.1% BSA, pH 6.8) with digitonin (20 pg/ml) and then washed three RESULTS times in Tris buffer (50 mM Tris, 100 mM KCl, 5 mMMgC12, 0.1% BSA, pH 6.8). Permeabilized islets (3O/tuhe) werethen loaded for 30 Studieswith Digitonin-permeabitized Pancreatic Isletsmin with 5 pCi of 45Caand 5 pCi of 3H20 as a cell volume marker, at The effect of arachidonic acid on Ca2+ release from the room temperature, in Tris a buffer mimicking intracellular conditions endoplasmic reticulum was studied in situ with digitonin- (100 mM Tris, 100 mM KC1,7 mM MgClZ,5 mM ATP, 0.2 mM EGTA, permeabilized pancreatic islets. The characteristics of this 0.1 p~ free Ca2+,2.25 pg/ml ruthenium red, pH 7.0). Ca2+efflux was model have been previously described (6, 9). Ruthenium red then measured after 10 min in the presence of 1.25 mM vanadate and . (2.25 pg/ml) was present, andthe free Ca2+concentration was the indicated doses of eicosanoid (0-10 p ~ ) Non-ATP-dependent Caz+uptake was determined individually in each experiment for each set at 0.1 p M in order to effectively inhibit mitochondrial Ca2+ condition and subtracted from the data. Results are shown as the uptake. This concentration of ruthenium red is sufficient to mean S.E. of Ca2+released compared to control (pmol/islet) from achieve inhibition of mitochondrial Ca2+uptake. At this con- four experiments performed in triplicate.

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ence of inhibitors of arachidonate metabolism on arachidonic acid-induced Ca2+release was also examined in this system. The cyclooxygenase inhibitor indomethacin (10 p ~ and ) the cyclooxygenase and lipoxygenase inhibitor BW755C (500p ~ were studied at concentrations which completely suppress islet biosynthesis of prostaglandin EZ and of 12-HETE, respectively (25, 27). As illustrated in Table I, neither BW755C nor indomethacin influenced arachidonic acid-induced Ca2+ release from the endoplasmic reticulum when present during the 30-min loading time and/orduring the 10-min efflux period. These observations suggest that the effect of arachidonic acid on Ca2+release from the endoplasmic reticulum is not due to metabolites of arachidonic acid. The effect of other fatty acids on Ca2+release from endoplasmic reticulum was also examined. The saturated fatty acid stearate (0-10 p ~ did ) not influence CaZ+efflux (four experiments in triplicate not shown). The unsaturated fatty acid oleate did stimulate Ca2+ efflux inaconcentration. release dependent manner with a maximal effect at 5 p ~ Ca2+ induced by 5 p~ oleate was 0.17 f 0.05 pmol/islet and that induced by 5 PM arachidonate was 0.23f 0.06 pmol/islet (four experiments in triplicatenot shown). These findings are similar to those reported for sarcoplasmic reticulum, where arachidonate induces calcium efflux somewhat more effectively than oleate, and stearatehas no effect (30). The time course of arachidonic acid-induced Ca2+release from the endoplasmic reticulum is illustrated in Fig. 2. Ara) a rapid and significant release chidonic acid (10 p ~ induced of Ca2+ within 2 min followed by a slow and progressive reuptake as compared to the vehicle. It seemed possible that the slow and progressive reuptake of Ca" by the endoplasmic reticulum could be due to oxygenation or esterification of the exogenous arachidonic acid. The disappearance of 3H-labeled arachidonic acid was assessed by extraction and HPLC analysis relative to a I4C-labeledarachidonate internal standard after various periods of incubation of [3H]arachidonic acid with the permeabilized islet preparationsand subsequent addition of [14C]arachidonicacid. No decline in [3H]arachidonic acid content was detected during the 0-30-min Ca" efflux period, suggesting that the reuptake of Ca" following arachidonate-induced Ca2+ release is not due to the disappearance of free arachidonic acid by oxygenation or esterification. IP3is recognized as asecond messenger for Ca2+-dependent

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st 0.5

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20 TIME (rnin)

30

40

FIG. 2. Time course of arachidonic acid-induced Ca2+ release by digitonin-treated islets. Permeabilized islets were prepared and loaded with 45Caand 3H20as in Fig. 1. Arachidonic acidinduced (10 PM) Ca2+efflux was measured after 2, 5 , 10, 20, and 30 min without any vanadate present (broken line)and compared to its vehicle (solid line). Non-ATP-dependent Ca2+ uptake determined individually for each condition and experiment was subtracted from the data. Results are shown as the mean f S.E. of Ca2+content (pmol/islet) from three experiments performed in triplicate.

hormones (7,8). We haverecently shown, using the digitoninpermeabilized pancreatic islet model, that Ipsinduced a rapid release of Ca2+from the endoplasmic reticulum of islets followed by a slow reuptake of Ca2+(9). In separate studies, we have also observed that anincrease in intracellular Ca2+from 0.1 to 0.2 p~ directly stimulates insulin release in the digitonin-permeabilized islet model (6). We therefore compared arachidonic acid-induced Ca2+release from the endoplasmic reticulum with Ips-induced Ca2+release. As depicted in Fig. ) 34.95 f 5.63% and 39.80 f 3., IP3 (10 and 20 p ~ released 5.08% of the Ca2+ content of the endoplasmic reticulum, respectively. Arachidonic acid induced Ca2+release from the endoplasmic reticulum at concentrations similar to those required for Ips-induced Ca" release. Maximal Ca2+release (19.11 -+ 7.18%) was observed at 10 p M arachidonate. At 20 p~ arachidonic acid, 28.33 f 5.38% Ca2+release was obtained, although a slight decrease in the non-ATP-dependent Ca2+ stores was also observed. The effect of addition of both TABLEI arachidonic acid and IPE on efflux from the endoplasmic Arachidonic acid-induced Ca2+ release in the presenceof B W755C or indomethacin by digitonin-treated islets reticulum is illustrated in Fig. 3. In thepresence of 20 p~ IP3 Permeabilized islets were prepared and loaded with 45Caand 3Hz0 (2-fold the maximal Ca2+-releasingdose), increasing concenas in Fig. 1. Ca2+efflux was measured after 10 min exposure to 10 p~ trations of arachidonic acid (5, 10, and 20 p ~ were ) added arachidonic acid. Inhibitors of arachidonic acid metabolism were simultaneously during a 10-min efflux period. At the highest present either during the efflux period or during both the loading ) arachidonic acid (20 p ~ ) concentration of IPS (20 p ~ and period and efflux period. Non-ATP-dependent Ca2+ uptake deter58.56 f 2.74% Ca" release was obtained. This amount of mined for each condition and experiment was subtracted from the released Ca2+was significantly greater (p < 0.025) than obrelevant data. Results are expressed as the mean f S.E. ofCa" released compared to control (picomoles/islet) from two to four served in the presence of either compound alone. Thus, the experiments performed in triplicate. combination of IP3 and arachidonic acid resulted in an apCaZ+released, proximately additive effect on Ca2+ mobilization from the CaZ+loading period, 3o min Caz+efflux period, 10 min pmol/islet, n endoplasmic reticulum. Under our experimental conditions, mean k S.E. there stillremained Caz+in the endoplasmic reticulum follow0.24 f 0.06 6 Arachidonate ing the efflux period, as assessed by the addition of 2 NM of 0.30 f 0.11 6 BW755C + arachidonate the Caz+ionophore A23187 which released 89.72 k 2.51% of Vehicle Arachidonate 0.17 & 0.04 11 the Ca2+ stored in the endoplasmic reticulum. 0.18 f 0.03 12 BW755C BW755C + arachidonate Our previous observations indicated that one of the char0.36 -t 0.05 12 Arachidonate Indomethacin + arachidonate 0.44 f 0.04 12 acteristics of IPS-induced Caz+release in islets was its Ca2+ 0.41 f 0.03 12 Vehicle Arachidonate dependency since optimal release was observed at a free Ca2+ Indomethacin Indomethacin + arachidonate 0.42 f 0.04 11 concentration of 0.1 PM (9). In experiments performed in the

,

Arachidonate Mobilizationof Ca2’ in Islets

3505

loor r

nIP, I O ~ M Arochidonote 10pM

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A&hid&teluM]

5 IO 20

0 0

5 IO 20

10

2pM

prepared and loaded with 45Caand 3H,0 asin Fig. 1. 45Caefflux was measured after 10 min, withoutany vanadate present,in thepresence of eitherarachidonate(5, 10, 20pM), IPS (10, 20 gM), orIP3 + arachidonate (20 + 5, 20 10, 20 + 20pM, respectively). IP, was prepared in the same buffer as arachidonate (see “Methods”). NonATP-dependent 45Cauptake determined individually for each condition and each experiment was subtracted from the relevant data. Results are shown as the mean f S.E. of CaZ+released (per cent of control) from 12 to 30 observations in 3-6 experiments. Ca2+released was calculated as 100% [Ca2+content of endoplasmic reticulum with compound/Ca2+content of endoplasmic reticulum with vehicle x ~ O O ] . Control Ca2+content of the endoplasmic reticulum with vehicle was 0.82 f 0.05 pmol/islet.

T

+

-

presence of vanadate (to inhibit reuptake by the endoplasmic reticulum), IP, and arachidonic acid both induced Ca2+release more effectively at free Ca2+concentrations below 1pM than at higher free Ca2+concentrations (Fig. 4). At 1pM free Ca”, the effect of IP8 and arachidonic acid on Ca2+ release was barely significant, while at 10 and 40 p~ of free Ca”, neither IP3nor arachidonic acid had adetectable effect on Ca2+release from the endoplasmic reticulum. At 0.1 p~ Ca2+and in the presence of 1.25 mM vanadate, IP3 and arachidonic acid each induced nearly 50% Ca2+release as compared to 35 and 20%, respectively, in the absence of vanadate (Figs. 3 and 4). This observation suggests that a fraction of the Ca2’ released by IP3 and arachidonic acid is rapidly reaccumulated by the endoplasmic reticulum in the absence of vanadate. The effect of arachidonic acid on mitochondrial Ca2+.content was examined at 40 p~ free Cazf and in thepresence of 5 mM succinate. At 0.1 PM free Ca2+,the mitochondria was not recruited for Ca2+handling (data notshown). Arachidonic acid at concentrations of 5 PM or less had no effect on the mitochondria (Table 11). Studies with intact islets-The effect of arachidonic acid on the permeability of intact islets was tested with a doubleisotope method (see “Methods”). Arachidonic acid (0-38 p ~ did not significantly affect the permeability of intact islets since the intracellular volumes measured under those conditions were not different from the control value of 4.35 f 0.45 nl/islet (Table 111). The effect of arachidonic acid on Ca2+fluxes in intactislets was also examined. Arachidonic acid had no significant effect on 45Ca2+uptake by intact islets during a 30-min incubation period. 45Ca2cefflux from pre-loaded intact islets was not significantly changed by the addition of arachidonic acid, as assessed over a 5-min effluxing period (Table IV). In the presence of a 3 mM nonstimulatory concentration of glucose, arachidonic acid (0-40 p ~ stimulated ) insulinrelease in only two of six experiments. At a glucose concentration of

40

1

0.1

FIG. 3. Comparison of IPS and/or arachidonic acid-induced Ca2+release by digitonin-treated islets. Permeabilized islets were

FIG. 4. Ca2’ dependency of IP3 or arachidonic acid-induced Ca” release by digitonin-treated islets. Permeabilized islets were prepared as in Fig. 1. Islets were loaded with 5 pCi of %a and 5 pCi of 3H20 asa cell volume marker at room temperature and in a Tris buffer mimicking intraceIlular conditions (100 mM Tris, 100 mM KC1, 7 mM MgCI2,5 mM ATP, 2.25 pg/ml ruthenium red, pH 7.0) with the following concentrations of EGTA (0.2, 0.08, 0.04, and 0 mM) to obtain free Ca2+concentrations of 0.1, 1, 10, and 40 p ~ Efflux . was initiated by the addition of IP3or arachidonic acid in the presence of 1.25 mM vanadate for 10 min. Non-ATP-dependent Ca2+uptake for each condition and experiment was subtracted from the relevant data. Results are expressed as themean S.E. of Ca2+released (percentage of control values) from three experiments performed in triplicate calculated as in Fig. 3. Control CaZ+contents of the endoplasmic reticulum with vehicle were 0.39 f 0.04, 1.10 f 0.15, 2.68 zk 0.19, and 5.67 +- 0.57 pmol/islet, respectively, at 0.1, 1, 10, and 40 p M free Ca”.

*

TABLE I1 Effect of arachidonic acid on mitochondrial Ca2+pools Islets were permeabilized as in Fig. 1. Permeabilized islets were then loaded 30 min with 5 pCi of 45Ca and 5 pCi of 3H20 asa cell volume marker, a t room temperature, in a Tris buffer (100 mM Tris, 100 mM KCl, 7 mMMgC12, 5 mM ATP, 5 mM succinate, 40 pM free Ca2+,& 2.25 pg/ml ruthenium red, pH 7.0). Efflux was measured after 10 min in the presence of 0, 1, 5, and 10 p M arachidonic acid. Endoplasmic reticulum Ca2+pools were determined in the presence of ruthenium red, and mitochondrial Ca2+pools were calculated by subtracting the Ca2+content of the endoplasmic reticulum from the Ca2+content determined in the absence of ruthenium red. Non-ATPdependent Ca2+uptake determined for each condition and experiment was subtracted from the relevant data. Results are shown as the mean S.E. of 18-39 observations from three to six exDeriments. Ca” content (prnol/islet)

)

Arachidonic acid

Mitochondria -

@M

0 1 5 10 ” p < 0.005 versus control.

7.15 f 0.82 7.18 f 1.21 7.35 f 0.98 3.14 -+ 0.69.

0 or 28 mM, arachidonic acid had no effect on insulin release from batch-incubated islets (data not shown). Studies were also performed examining the effect of arachidonic acid on the dynamics of insulin release using the perifused islet model. Under those conditions, arachidonic acid (20 PM) induced a transient burst of insulin release in the presence of 3 mM glucose (Fig. 5).

3506


of Ca2+in Islets

TABLE I11 Effect of arachidonic acid o n islet permeability Intracellular volume of intact islets was determined using a doubleisotope method as described under "Methods." Results are shown as the mean f S.E. of six observations from two experiments. Islet intracellular volume

Arachidonate

4.42 3.90

"

nl

0 2.5 5 10 20 38

4.35 f 0.45 4.42 f 1.02 5.53 f 1.41 4.81 f 0.75 f 0.66 f 0.63

TABLE IV Lack of effect of arachidonic acid on Ca2" uptake and Ca2+ efflux by intact islets Intact islets were loaded with 45Caand [3H]sucroseas anextracellular space marker for 30 min at 37 "C in Hepes/Krebs' buffer (25 mM Hepes, 115 mM NaC1,5 mM KCl, 24 mM NaHC03, 1 mM MgC12, 2.5 mM CaC12,pH 7.4) containing 3mM glucose for the uptake studies or 28 mM glucose for the efflux studies. &Ca uptake was measured after 30 min loadig while "Ca efflux was determined after a 5-min period in fresh nonradioactive Hepes/Krebs' buffer. Results are shown as the mean S.E. of 15-33 observations from three to four experiments and are expressed as theper centof control Ca2+content of islets. Control Ca2' content of intact islet was 24.12 f 1.75 pmol/ islet for 30 min Ca2+uptake and 4.35 f 0.53 pmol/islet after 5 min efflux.

*

Ca2+content (% of control) Arachidonic acid

30 rnin uptake

I

Z I

0';;

' '

'

" '

'is '

'

'io

' '

25 '

'

'do

FIG. 5. Arachidonate-induced insulin release by perifused intact islets. Intact islets (250-400/chamber) were placed in two perifusion chambers as described under "Methods" and preincubated 30 min with Krebs' buffer containing 3 mM glucose. Incubation was continued for 20 min in fresh Krebs' buffer and 3 mM glucose containing either 20 WM arachidonic acid (closed symbols, solid line) or the control vehicle (open symbols, broken line). At the end of the incubation period, islets were challenged for 10 min with Krebs' buffer containing 28 mM glucose. Oneml/min fractions were collected and assayed by radioimmunoassay for insulin release. Data are representative of three experiments.

5 min efflux TOTAL UNESTERIFIED ARACHIDONATE u 28 mM Glucose

10

100.0 f 6.1 106.0 f 131.7 6.5 107.6 f93.2 4.2 108.1 f 6.7

'io 'i5 . 'do .

FRACTION NUMBER

PM

0 5 10 20

Y

100.0 k 12.2 f 19.1 k 9.8 100.8 f 17.7

Glucose-induced Unesterified Arachidonic Acid Release in Intact Islets-Although several reports suggest that glucosestimulation of isolated pancreatic islets induces release of arachidonate from membrane phospholipid (50) and the appearance of arachidonate metabolites (21, 22, 24, 26), it had not previouslybeen established that a sufficient mass of arachidonate accumulates to exert the effects described above on calcium mobilization. The mass of endogenous unesterified arachidonate accumulating from isolated pancreatic islets as a function of timeafter exposure to 28 mM glucosewas therefore measured by stable isotope dilution mass spectrometric measurements described in detail in the Miniprint Supplement. As illustrated in Fig. 6, the total (medium plus islet-associated) content of unesterified arachidonate was relatively constant in islets incubated with a glucose concentration ( 3 mM) that does not stimulate insulin secretion. Islets incubated with a concentration of glucose (28 mM) that maximally stimulates insulin secretion exhibited an increase in total unesterified arachidonate at 5 min, which was sustained at 15 min, and a furtherincrease at 30 min after exposure to 28 mM glucose. The increase in the mass of unesterified arachidonate induced by 28 mM glucosewas sufficient to achieve a 200 pg increment in the arachidonate levels at 5 min after exposure to 28 mM glucose. The minimal amount of unesterified arachidonate required to achieve an islet intracellular concentration of20 p M is 27 pg (assuming an islet cell water volums of4.4 nl). Although the fraction of the released arachidonate appearing in cell water is not known, in principle the mass of released arachidonate is sufficient to

e-0 3 m M

Glucore

L . -

2 0

5

15 TIME (Minuter)

30

FIG. 6. Time course of glucose-induced unesterified arachidonic acid release in intact islets. Intact islets (2OO/tube) were preincubated 30 min at 37 "C in Krebs' buffer supplemented with 3 mM glucose and 0.1% fatty acid-free bovine serum albumin. Incubations were then continued for 30 min in the presence o f 3 mM glucose (open symbols) or 28 m~ glucose (closed symbols). Incubations were stopped a t 0, 5, 15, and 30 min and unesterified arachidonic acid extracted and measured by gas chromatography-negative ion-chemical ionization-mass spectrometry as described in the Miniprint. Results are shown as the mean 2 S.E. of the total unesterified arachidonic acid in medium and islets for three experiments.

achieve concentrations equal to or exceeding those required to exert the effects on calcium mobilization described above. Further studies were performed to define the precise time course of glucose-induced arachidonic acidrelease. These studies employed intact islets radiolabeled with [3H]arachidonic acid. Islet incorporation of this precursor averaged 50% in these experiments. Unincorporated [3H]arachidonic acid present in the medium was then removed, and islets were resuspended in fresh, [3H]arachidonicacid-free medium. Dynamic studies with a perifusion model using these [3H]arachidonic acid pre-labeled islets indicated that a 28 mM glucose stimulus induced release of insulin concomitant with release of [3H]arachidonicacid within 2 min (Fig. 7). The 3H-labeled material released by glucose stimulation was shown to consist almost entirely of unmetabolized arachidonic acid byreverse-


of Ca2+in Islets

3507

crounits/islet/min) from islets exposed to 28 mM glucose (327 cpm, 0.49 microunit insulin/islet/min) was greater than that 2 from islets exposed to 16 mM glucose (173 cpm, 0.36 microunit insulin/islet/min), or to 8 mM glucose (88cpm, 0.16 microunit insulin/islet/min), or to 3 mM glucose (control). In preliminary experiments, the arachidonate content (by mass) of the major phospholipid species in islet membranes has been examined after thin layer chromatographic separation of the phospholipids, transmethylation to the fattyacid methyl esters in the presence of an internal standard, and quantitative gas chromatographic analysis. While the phosphatidylcholine content of both arachidonate and oleate fell Id9 I after 30 min of incubation with 28 mM glucose, the molar !r 5 15 10 20 25 30 0 5 15 10 20 25 30 decline in esterified arachidonate was nearly 4-fold that of T I M E [min) oleate. Virtually no decline in phosphatidylcholine stearate FIG. 7. Time course of glucose-induced insulin and [3H]ar- content was observed.Molar changes in arachidonate content achidonic acid release by perifused intact islets. Intact islets of phospholipids other than phosphatidylchoIine were minor were radiolabeled with 30 pCi of t3H]arachidonic acid as described under "Methods." Radiolabeled islets (250-450)were placed in a compared to thechange in phosphatidylcholine arachidonate perifusion chamber and washed for 30 min at 37 "Cwith Krebs' buffer ~ o n t e n t . ~

I*

I

I

I

I

supplemented with 3 mM glucose and 0.1% fatty acid-free bovine serum albumin. Perifusion was then continued for 30 min in the presence of 28 mM glucose. One ml/min fractions of the effluent were collected and assayed for insulin and t3H]arachidonic acid release. Insulin release (panel A ) is shown as microunits of insulin/islet/min in the presence of 28 mM glucose. [3H]Arachidonicacid release fpaneE B ) is shown as counts/min [3H]arachidonic acid released inthe presence of 28 mM glucose after subtracting 3 mM glucose values. Data are representative of five experiments.

DISCUSSION

We have demonstrated a Ca2+ mobilizingeffect of arachidonic acid on the endoplasmic reticulum in digitonin-permeabilized pancreatic islets. The characteristics of the permeabilized islets have been recently described (6, 9). In thepresent study, Ca2+ efflux was measured at 0.1 p~ free Ca2+. In digitonin-permeabilized islets, half-maximal insulin release is observed at a free Ca2+ concentration of 0.1 pM (6). This concentration is similar to the resting cytosolic Ca2+concentration observed in RINdFinsulinoma cells (51, 52). ODS COLUMN 301 M ~ O HI u Z O /HOAc Arachidonate-induced Ca" efflux was not observed with 80 / 20 / 0.1 the predominant arachidonate metabolite synthesized by isolated islets (12 HETE) or with its precursor (12-HPETE). Moreover, the effect of arachidonate on calcium mobilization was not influenced by inhibitors of arachidonate metabolism. The effect of arachidonate on calcium mobilization therefore appears to be a direct one and not to be mediated by arachi,. 10 donate metabolites. Exogenous arachidonate (5 b ~ elevates ) cytoplasmic Caz+ in rabbit neutrophils, as assessed by Quin-2 measurements (29). Such measurements are unfortunately not applicable to IO 20 30 40 50 60 70 multicellular aggregates such as islets for technical reasons. ELUTION VOLUME (ml) No significant effect of arachidonate on islet calcium fluxes efflux from endoplasmic FIG.8. Reverse-phase HPLC analysis of '€I-labeled mate- other than enhancement ofCa" rial released from glucose-stimulated, [3H]arachidonatepre- reticulum has been identified. In this study, we failed to labeled islets. Intact islets were radiolabeled with 30 pCi of L3H] observe an effect of arachidonate on 45Ca2+ uptake by intact arachidonic acid as described under "Methods." Unincorporated [3H] islets over 30 min. Arachidonic acid had no effect on Ca2+ arachidonic acid present in the medium wasremoved and islets resuspended in fresh nonradioactive Krebs' buffer containing 28 mM efflux from intact islets. Our previously reported studies inglucose for 5 min at 37°C. Released 3H-labeled material was analyzed dicate that arachidonate does not influence islet plasma memby reverse-phase HPLC with 14C-labeled arachidonic acid as an brane Ca2+-Mg2+-ATPase(53). Although arachidonate at coninternal standard. centrations greater than 5 p~ does decrease mitochondrial CaZ+content, this effect is observed only at free Ca2+concenphase HPLC with 14C-labeled arachidonate as an internal trations considerably higher than those observed in glucosestandard (Fig. 8). Less than 5% of the released 3H-labeled induced insulin secretion. Our observations areconsistent material representedoxygenation products of [3H]arachidonic with the concept that arachidonate-induced Ca2+efflux from acid. Similar observations on the time course of release of 3H- the endoplasmic reticulum may result in elevation of the islet labeled material from perifused islets prelabeled with t3H] intracellular free ea2+level. arachidonic acid and exposed to 28 mM glucose have been Several investigators have shown an effect of arachidonic reported by others (50), but thefact that most of the released acid on hormonal release in other secretory cells (54-56), radiolabeled material represented unmetabolized arachidon- including dispersed cell preparations of endocrine cells from ate was not examined in that report. neonatal rat pancreas. We were unable to demonstrate an The release of [3H]arachidonic acid from islets prelabeled effect of exogenous arachidonic acid on insulin secretion from with this compound has also been examined as a function of intact batch-incubatedislets, but it is not certain how readily glucose concentration at 30 min after changing the glucose concentration from 3 mM to a new concentration. Release of J. Turk, B. A. Wolf, J. Lefkowith, and M. L. McDaniel, manu[3H]arachidonic acid (in counts/min) and of insulin (in. mi- script in preparation.

"i

3508

Arachidonate Mobilization of Ca2+in Islets

0-Glucose

FIG.9. Schematic representation of events involved in insulin secretion. A glucose stimulus results in release of IP, and arachidonic acid (AA) from membrane phospholipids. IP, and arachidonic acid act in cooperation to release ATP-dependent Ca2+pools from the endoplasmic reticulum. The increase in cytosolic Ca2+activates the Ca2+and calmodulin-dependent protein kinase which in turn phosphorylates tubulin and other cytoskeleton proteins resulting in translocation of insulin secretory granules to the plasma membrane, PI, phosphatidylinositol; PIP1, phosphatidylinositol4-phosphate; PIP,, phosphatidylinositol 4,5-bisphosphate; DG, diacylglycerol.

Ca2+-Calmoddin-dependent Protein kinase

granules exogenous arachidonatepenetrates the intracellular space with intact islets. However, studies with perifused islets showed that exogenous arachidonic acid induced a transient burst of insulin release. The effect of arachidonic acid on Ca2+ efflux from the endoplasmic reticulum was directly compared with IP,. Previous studies, in thesame model and under similar conditions, have indicated that IP, mobilized Ca2+from the endoplasmic reticulum of islets (9). It has been proposed that IP3could be a second messenger for glucose-induced insulin secretion (9, 57-59). The characteristics of arachidonic acid-induced Ca2+ efflux were similar to those of IP, (see Ref. 9 for comparison). Both compounds induced maximal Ca2+efflux at 10 p~ and half-maximal Ca2+ release at approximately 2.5 pM. Time course studies indicated that both arachidonic acid and IP3 induced Ca2+efflux within 2 min followed by Ca2+reuptake by the endoplasmic reticulum. Although IPS (in theabsence of vanadate) released a greater fraction of Ca2+sequestered by the endoplasmic reticulum than arachidonic acid, both IP3 and arachidonate (inthe presence of vanadate to inhibit Ca2+ reuptake) induced the same percentage of Ca2+efflux (50%). The Ca2+dependency of Ca2+efflux by arachidonic acid and IPSwas also similar: Ca2+efflux with these compounds was only seen at submicromolar Ca2+concentrations. The possibility that arachidonic acid induced phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate with release of IP3was not examined. However, one recent report has shown that arachidonic acid has no effect on polyphosphoinositide breakdown in neutrophils, although arachidonic acid does elevate cytosolic free Ca2+concentrations in those cells (29). In response to glucose, a specific insulin secretagogue, islets release unesterified arachidonic acid. Glucose-induced arachidonate release is dose-dependent with maximal release observed at 28 mM glucose. Detailed time course studies with [3H]arachidonicacid indicate that arachidonic acid is released

within 2 min. The initial pattern of glucose-induced arachidonate release is similar to that reported for glucose-induced IPSrelease (10). Our own preliminary observations also indicate that glucose induces IP, release within 2 min from isolated pancreatic islets prelabeled with [3H]inositol.4 The mass of arachidonic acid released following glucose stimulus is in excess of that required to induce Ca2+release from the endoplasmic reticulum. This observation is reminiscent of studies by Williamson and colleagues (Ref. 8, for review,see also Ref. 60) who estimated that the agonistinduced release of IP3 in liver was 17-fold higher than the maximal concentration required for IP3-induced Ca2+mobilization. More recent observations in platelets have also shown that thrombin-induced release of IP, is severalfold in excess of the mass of IP3 required for ea2+ release (61). Although the amount of arachidonate released by glucosestimulated islets is severalfold higher thanthe minimal amount which could influence calcium efflux by endoplasmic reticulum, it is substantially less than thatreleased by thrombin-stimulated platelets (62). It is of interest that themass of unmetabolized, unesterified arachidonic acid released by isolated islets in response to glucose is many-fold higher than the mass of any metabolite of arachidonate released under such conditions (25-28). This is consistent with the possibility that arachidonate itself may play some mediator function that does not require its oxygenation to a metabolite. Other investigators have also suggested this possibility based on the large amounts of unmetabolized arachidonate and the relatively small amounts of arachidonate metabolites released by other stimuli in other systems (see Ref. 63 for review and also Ref. 64). In platelets, the predominant phospholipid precursors of released arachidonate maybe phosphatidylinositol species, Manuscript in preparation.

Arachidonate Mobilii:ation of Ca2+i n Islets although phosphatidylcholine also appears to contribute a fraction of arachidonate released in response to thrombin (20). The identity of the phospholipid precursors from which arachidonate is released by glucose-stimulated islets was not addressed in the presentstudy, although our preliminary results suggest that the arachidonate content of islet membrane phosphatidylcholine does decline as a consequence of exposure to 28 mM glucose. Glucose-induced increases in turnover of arachidonate in phosphatidylcholine and phosphatidylinositol in islet membranes have been reported by other investigators (10, 50, 65-72). A schematic representation summarizing events that may be involved in glucose-induced insulin secretion is shown in Fig. 9. The observations reported herein indicate that arachidonic acid per se could have an important cooperative role with IP3 in glucose-induced calcium mobilization and insulin secretion. Acknowledgments-The excellent technical assistance of Joan Fink, Connie Marshall, Richard Thoma, William Thomas Stump, and Beverly DeLoach has been greatly appreciated. We wishto thank Marian Simandland Jennifer Ferrell for expert secretarialassistance and Jane Huthfor the preparation of the miniprint. REFERENCES 1. Hedeskov, C. J. (1980) Physiol. Rev. 6 0 , 442-509 2. McDaniel, M. L., Colca, J. R., Kotagal, N., and Lacy, P. E. (1985) in T h e Diabetic Pancreas (Volk, B. W., and Arquilla, E. R., eds) pp. 213-231, Plenum Press, New York 3. Colca, J. R., McDonald, J. M., Kotagal, N., Patke, C., Fink, C. J., Greider, M. H., Lacy, P. E., and McDaniel, M. L. (1982) J. Biol. Chem. 2 5 7 , 7223-7228 4. Colca, J. R., Kotagal, N., Lacy, P. E., and McDaniel, M. L. (1983) Biochem. J . 2 1 2 , 113-121 5. Colca, J. R., Kotagal, N., Lacy, P. E., and McDaniel, M. L. (1983) Biochim. Biophys. Acta 729,176-184 6. Colca, J. R., Wolf, B.A., Comens, P. G., and McDaniel, M. L. (1985) Biochem. J. 228,529-536 7. Berridge, M. J., and Irvine, R. F. (1984) Nature 3 1 2 , 315-321 8. Williamson, J. R., Cooper, R. H., Joseph, S. K., and Thomas, A. P. (1985) Am. J . Physiol. 2 4 8 , C203-C216 9. Wolf, B. A., Comens, P. G., Ackermann, K. E., Sherman, W. R., and McDaniel, M. L. (1985) Biochem. J. 227,965-969 10. Montague, W., Morgan, N. G., Rumford, G. M., and Prince, C. A. (1985) Biochem. J. 227, 483-489 11. Nishizuka, Y. (1984) Science 225, 1365-1370 12. Nishizuka, Y. (1984) Nature 3 0 8 , 693-698 13. Cohen, P. (1982) Nature 296,613-620 14. Tanigawa, K., Kuzuya, H., Imura, H., Taniguchi, H., Baba, S., Takai, Y., and Nishizuka, Y. (1982) FEBS Lett. 138, 183-186 15. Thams, P., Capito, K., and Hedeskov, C . J. (1984) Biochem. J. 221,247-253 16. Lord, J. M., and Ashcroft, S.J. H. (1984) Biochem. J. 219,547551 17. Hutton, J. C., Peshavaria, M., and Brocklehurst, K. W. (1984) Biochem. J. 224, 483-490 18. Metz, S.,VanRollins, M., Strife, R., Fujimoto, W., and Robertson, R. P. (1983) J. Clin. Invest. 7 1 , 1191-1205 19. Mauco, G., Dangelmaier, C. A., and Smith,J. B. (1984) Biochem. J. 224,933-940 20. Neufeld, E. J., and Majerus, P. W. (1983) J. Biol. Chem. 2 5 8 , 2461-2467 21. Metz, S. A., Murphy, R.C., and Fujimoto, W. (1984) Diabetes 33, 119-124 22. Metz, S.A. (1985) Proc. Natl. Acad. Sci. U. S. A. 8 2 , 198-202 23. Walsh, M. F., and Pek, S.B. (1984) Diabetes 33, 929-936 24. Yamamoto, S.,Ishii, M., Nakadate, T., Nakaki, T., and Kato, R. (1983) J. Biol. Chem. 258, 12149-12152 25. Turk, J., Colca, J. R., Kotagal, N., and McDaniel, M. L. (1984) Biochim. Biophys. Acta 794, 110-124 26. Turk, J., Colca, J. R., Kotagal, N., and McDaniel, M. L. (1984) Biochim. Biophys. Acta 7 9 4 , 125-136 27. Turk, J., Colca, J. R., and McDaniel, M.L. (1985) Biochim. Biophys. Acta834,23-36

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of Ca2+in Islets 69. Laychock, S. G. (1983) Biochem. J. 216, 101-106 Rana, R. S., Mertz, R. J., Kowluru, A., Dixon, J. F., Hokin, L. E., and MacDonald, M. J. (1985) J. Biol. Chem. 260,7861-7867 71. Laychock, S. G. (1983) Diabetes 32,6-13 72. Dunlop, M. E., and Larkins, R. G. (1984) Biochem. Biophys. Res. Commun. 120,820-827

S u p p l e m e n tt o INTRACELLULAR Ca2+ MOBILIZATION m-p-INOSITOL 1,4,5-TRISPHOSPHATE

BY ARACHIDONIC ACID. COMPARISON WITH I N ISOLATE0 PANCREATIC ISLETS

M e a s u r e m e n to fs u b n a n o g r a mq u a n t i t i e so fa r a c h i d o n i ca c i df r o mi s o l a t e d p a n c r e a t i ci s l e t sb yn e g a t i v ei o n mass s p e c t r o m e t r y b yJ o h nT u r k ,B r y a n

A.

Wolfa . n dM i c h a e l

L.

McDaniel

A l t h o u g he x o g e n o u sr a d i o l a b e l l e da r a c h i d o n a t ei sr e a d i l yi n c o r p o r a t e d i n t o membrane p h o s p h o l i p i di nm o s tc e l l s ,m u l t i p l ep o o l s o f t h ei n c o r p o r a t e d (26.65). T haem o u n t a r a c h i d o n a t ee x i s tw i t hd i f f e r i n qS p e c i f i ca c t i v i t i e s o fr a d i o l a b e lr e l e a s e db yv a r i o u is i i m u l in e e dn o tt h e G e f o r ea c c u r a t e l y r e f l e c t t h e mass o f r e l e a s e da r a c h i d o n a t e . S t a b l ei s o t o p ed i l u t i o ng a sc h r o m a t o g r a p h i c( G C ) - m a s ss p e c t r o m e t r i c (MS) m e a s u r e m e n t se m p l o y i n gd e u t e r i u m - l a b e l l e da r a c h i d o n a t ea sa ni n t e r n a l s t a n d a r d r e p r e s e n t - a n ~ e s t a b l i s h e d a n ds p e c i f i cm e t h o do fd e t e r m i n gt i s s u e c o n c e n t r a t i o n so ft h e mass o fu n e s t e r i f i e da r a c h i d o n i ca c i d( 3 1 ) . It h a s r e c e n t l vb e e nd e m o n s t r a t e dt h a tt h eS e n s i t i v i t vo fn e a a t i v ei o nc h e m i c a l i o n i z a t j o n m a s s s p e c t r o m e t r i cm e a s u r e m e n t so ft h ep e n i a f l u o r o b e n z y le s t e r d e r i v a t i v e so fp r o s t a g l a n d i n sa n dm 0 n O h y d r o x y - e i c ~ s a t e t r a e n o i ca c i d si s a b o u tt w oo r d e r so fm a g n i t u d eg r e a t e rt h a nt h es e n s i t i v i t yo fc o n v e n t i o n a l e l e c t r o ni m p a c t m a 5 5 s o e c t r o m e t r i cm e a s u r e m e n t so ft h em e t h v le s t e r so f t h o s ec o m p o u n d s( 3 2 - 3 4 ) . W e h a v ee x t e n d e dt h e s em e t h o d st o - t h em e a s u r e m e n t o ft h er e l a t i v e l ys m a l la m o u n t so fu n m e t a b o l i r e d ,u n e s t e r i f i e da r a c h i d o n i c a c i dp r e s e n ti ni s o l a t e dp a n c r e a t i ci s l e t s . T h ei n t e r n a ls t a n d a r d [5,6,B,9,11,12,14,15-2H8]-a~~chidanic acid ( d g - A A ) was s y n t h e s i z e d f r o m (5,8,11,14)-eicosatetrynoic a c i d ( a g i f to f Or. d a m e s H a m i l t o.no fH. o f f m a nL a R o c h e )b vr e d u c t i o nw i t hd e u t e r i u m 4 1 5 (MG S c i e n t i f i c Gases, C h i c a g o ) a n dp u r i f i e db ys i l i c i ca c i dc h r o m a t o g r a 6 h y as described (35). was c o n v e r t e d t o t h e m e t h y l An a l i q u o t o f t h i s m a t e r i a l e s t e rw i t he t h e r e a ld i a l o m e t h a n ea n ds u b j e c t e dt o GC-MS a n a l y s i s (in a 2 O Y - 1 0 1 o n S u p e l c o p o r tp a c k e d GC c o l u m ni n t e r f a c e dw i t h d f o o t , 2 mm i.d.,2% H e w l e t t - P a c k a r d5 9 8 5 8m a s sS D e c t r o m e t e rO p e r a t e di nt h ee l e c t r o ni m p a c t m o d e T. h ec a r b o nv a l u eu n d e tr h e s e G C c o n d i t i o n s w a s1 9 . 7a, n dt h e mass s p e c t r u m( f i g u r e1 )c o n t a i n e d a p r o m i n e n tm o l e c u l a ri o na t m l z 326. An a l i q u D to ft h e dg-IIA was a l s oc o n v e r t e dt ot h ep e n t a f l u o r o b e n z y le s t e r ( P F B E )w i t hp e n t a f l u o r o b e n z y bl r o m i d e( P i e r c e R , oCkfoPd, I L ) a n dt e t r a m e t h y l ammonium h y d r o x i d e( M a t h e s o n C , o l e m a n a, n dB e l l N , orwood, OH) i n N,N-dimethylacetemida enm d ethanol as d e s c r i b e d ( 3 6 , 2 7 ) . T h i sd e r i v a t i v e was a n a l y z e d on a H e w l e t tP a c k a r dU l t r a p e r f o r m a n c ec a p i l l a r y G C column ( 2 5 rn l e n g t ch r o s s - l i n k e m d e t h y l s i l i c o n ie. 0d . 3 1 mm f i l mt h i c k n e s s 0.17 urn) i n t e r f a c h dw i t ht h eH e w l e t tP a c k a r d5 5 8 5 8 m a s s s p e c I r a m e t e ro p e r a t e di n t h en e g a t i v ei o n - c h e m i c a li o n i z a t i o n( N I - C I ) mode w i t hm e t h a n e as reagent g a s( s o u r c ep r e s s u r e 1.1 t o r r ,i o n i z a t i o nv o l t a g e2 3 0e " ) .I n j e c t i o n so f a h e p t a n es o l u t i o no ft h ed e r i v a t i v e w e r e p e r f o r m e di nt h es p l i t l e s s mode t h e c a p i l l a r yc o l u m n 85'C t o was p r o g r a m m e df r o m ( G r o b - t y p e i n j e c t o r )a n d 240'C a t a r a t e o f Under 30-C p e r m i n s t a r t i n g 0.5 m i n a f t e ri n j e c t i o n . t h e s ec o n d i t i o n zt h er e t e n t i o nt i m e O f t h ed e r i v a t i v e was a b o u t 8.3 m i n u t e s , a n de s s e n t i a l l yt h eo n l yi o no b s e r v e di nt h en e g a t i v ei o n - m e t h a n ec h e m i c a l i o n i z a t i o n mass s p e c t r u m was ( m l z ) 3 1 1 (M-1811, c o r r e s p o n d i n gt ol o s so ft h e p e n t a f l u O P O b e n z y 1g r o u p T . h ea n a l o g o u si o ni nt h eN I - C I m a s s I p e c t r u mo f ( m l r ) 303. W i t h t h e PFBE d e r i v a t i v eo fn a t u r a l l vO C C U r r i n oa r a c h i d a n a t ei s t h ed e u t e r i u m - l a b e i l e dp r a d u c t , - t h er e l a t i v ea b u n d a n c eo ft h ei b n sa t mlr 303 a n d 3 1 1 i n t h e N I - C l s p e c t r u mo ft h e PFBE d e r i v a t i v e w a s a b o u0t . 0 0 2 3 ( b l a n kv a l u e ) . ~~

~

AMOUNT OF do-ARACHIDONIC ACID I N G I ~i~~~~ 2. s t a b l ei s o t o p ed i l u t i o n a r a c h i d o n i ca c i d .

F o re x p e r i m e n t si n v o l v i n gm e a s u r e m e n to ff r e ea r a c h i d o n a t ef r o mi s o l a t e d i s l e t s ,b a t c h e so f 200 i s o l a t e di s l e t sw e r eh a n d - p i c k e da n dp l a c e di y e a c h o f1 6s i l i c o n i z e dg l a s st u b e s .T h ei s l e t sW e r ep r e i n c u b a t e df o r 30 m l n a t 37'C W i t hs h a k i n gi n 1 m l o f K r e b s - R i n g e rb i c a r b o n a t eb u f f e r (KRB) s u p p l e m e n t e dw i t hg l u c o s e ( 3 m M ) a n d 0.1% ( w t . I v o 1 )b o v i n e serum albumxn (BSA, f a t t y s c i d f r e e , f r a c t i o n V f r o mM i l e sL a b o r a t o r i e s ) .A tt h ee n do f t h i sp e r i o d , a n a l i q u o t (100 V I ) o f t h em e d i u m was r e m o v e d f o r t h e u l )o f fresh i n c u b a t i o n A l i q u o t 5( 1 0 0 radioimmunoassay (RIA) o f - i n s u l i n . m e d i u mw e r et h e na d d e dw h l c hc o n t a i n e d2 5 0 mM g l u c a s e , o r 3 m M g l u c o s e .T h i s 28 m M o r a d d i t i o nr e s u l t e di n a f i n a lg l u c o s ec o n c e n t r a t i o ni nt h et u b e so f 3 mM. ( T h eh i g h e rg l u c o s ec o n c e n t r a t i o nm a r k e d l ys t i m u l a t e si n s u l i n s e c r e t i o nf r o mt h e g ei s o l a t e di s l e t sb u tt h el o w e rg l u c o s ec o n c e n t r a t i o n d o e sn o (t 2 5 , 2 6 1 ) T. h ei n c u b a t i o n s ;ere t h e nc o n t i n u e da t 3 7 ' C w i t hs h a k i n g f o ri n t e r v a l s Of 0 5 15 o r 30 m i n A . t h ee n do tf h ei n c u b a t i o np e r i o d a, n a l i q u o t ( 1 0 0 V I ) 0 : i h e ' r n e d l u m was w i t h d r a w nf o rR I Ao fi n s u l i n ,a n dt h e r e m a i n d e ro ft h em e d i u m was r e m o v e df r o mt h ei s l e t sa n dp l a c e di n 3 ml of m e t h a n o cl o n t a i n i n g an i n t e r n a sl t a n d a r d silanized glass vials. One rnl 3H8-AA was t h e n a d d e d t o m i x t u r eo f 1 0 0 n go f dg-AA a n d 30 n C io f t h e s ev i a l s . To t h ei s l e t sr e m a i n i n gi nt h ei n c u b a t i o nt u b e , 1 ml O f m e t h a n o cl o n t a i n i n gt h ei n t e r n a sl t a n d a r dm i x t u r e was a l s oa d d e d T . h ei s l e t s u s p e n s i o nw a sv o r t e x e df o r 30 s e ca l l o w e dt os t a n da t room t e m p e r a t u r ef o r 30 m i n ,v o r t e x e da g a i nf o r 3 0 s e c , ' a n dc e n t r i f u g e da tl o ws p e e dt os e d i m e n t t h ei s l e td e b r i s .T h es u p e r n a t a n t w a s t h e nr e m o v e da n dp l a c e di n a m e d i af r o mt h ei s l e ti n c u b a t i o n sa n d. t h e s i l a n i r e dg l a s vs i a lT. h e i s l e t s were t h e np r o c e s s e ds e p a r a t e l ya n d m e t h a n o l i ce x t r a c t so ft h e content. a n a l y z e df o ra r a c h i d a n a t e T h em e d i a were a c i d i f i e d t o p H 3.5 ( 0 . 1 N H C I ) ,a n de x t r a c t e dt w i c ew i t h t w ov o l u m e so C f H P C l ? T. h ee x t r a c t sw e r ec o n c e n t r a t e dt od r y n e s su n d e r Np r e c 0 n s t i t u t e d ' i n . C H 2 C l z (500 " 1b) a c ek x t r a c t e w d itH h z0 ( 1 6 0 p l )c o n c e n t r a t e dt od r y n e s s ,c i n v e r t e dt ot h e PFBE a s d e s c r i b e da b o v e , e.x t t - a c t e i . c o n c e n t r a t e d .a n dr e c o n s t i t u t e di nm e t h a n o l ( 2 0 " 1 ) a n d 80 u l o f s o l v e n tA ' [ a c e t o n i t r i l e . 8 0 l R 1 02 0 ) .M e t h a n o l i ce x t r a c t s from t h ei s l e t s w e r ec o n c e n t r a t e dt od r y n e s s - u n d e r N2. t e c a n s t i t u t e d i n C H Z C I Z( 5 0 0 " 1 b) a c ke x t r a c t e dw i t h Hz0 ( l o o u l ) , c o n c e n t r a t i d , c o n v e r t e d t o t h e es.lal m p l e s A a s a b o vA PFBE, a n d r e c o n s t i t u t e di nm e t h a n o la n dS o l v e n t w e r et h e na n a l y z e db y HPLC i n s o l v e n t A ( 2 m l l m i n ) O n a W a t e r yp s o n d a p a kc 1 8 c o l u m n ( 3 . 9 mm x 30cm 1 0 ~ ) . As i l l u s t r a t e di nf i g u r e 3, t h er e t e n t i o n v o l u m eo f p e n t a f l u o r o ~ e o z y l - a r a c h i d o n a t e ( A A - P F B E )u n d e rt h e s ec o n d i t i o n si s T h eA A - P F B E w a s l o c a t e db yl i q u i ds c i n t i l l a t i o n counting o f a n a h n u t 34 m l . ( 1 0 0 " 1 )o fe a c hf r a c t i o n ,e x t r a c t e df r o mt h e HPLC s o l v e n tw i t h C H 2 C l z .c o n c e n t r a t e dt od r y n e s s ,a n dr e c o n s t i t u t e di nh e p t a n e . ~

M;326

mass s p e c t r o m e t r i cm e a s u r e m e n ot f

~~~~

iiyiiot

RP-HPLC ANALYSIS OF PENTAFLUOROEENZYL ARACHIDONATE ODs Column

ACCN 1101H20 20 0

F i q u r e 1.

E l e c t r o ni m p a c t

mass s p e c t r u mo f

h

octadeutero-methyl-arachidonate.

When a c o n s t a n ta m o u n to f dg-AA a n d a v a r i e da m o u n to fu n l a b e l l e d AA ( d o - A A ) -w e r ec o n v e r t e dt o PFBE a n da n a l y z e db y GC-NICl-MS u n d e rt h ea b o v e c o n d i t i o n st h er a t 7 0 o f t h e i o n c u r r e n ta tm l r 303 t o t h a t a t m l z 3 1 1 was a l i n e a rf u n i t i o no ft h ea m o u n to fu n l a b e l l e da r a c h i d o n a t eo v e rs e v e r a lo r d e r s om f agnitude as i l l u s t r a t e d i n f i g u r e 2. As l i t t l e as 2 0p go uf n l a b e l l e d a r a c h i d o n a t ec o u l db ee a s i l yv i s u a l i z e dw i t ht h e s em e t h o d s .

ELUTION VOLUME F i g u r e 3.

(dl

HPLC a n a l y s i s o f p e n t a i l u o r o b e n z y l - a r a c h i d o n a t e .

Arachidonate Mobilization then performed a n a l y s e s o f t h e sampleswere C a p i l l a r yc o l u m n GC-NICI-MS The r a t i o o f as i l l u s t r a t e d i n f i g u r e 4. u n d e rc o n d i t i o n sd e s c r i b e da b o v e t h ei o nc u r r e n ta t m l r 303 ( e n d o i e n o u sa r a c h i d o n a t e ) to t h a t a t m l z 311 ( d e u t e r i u m - l a b e l l e di n t e r n a ls t a n d a r da r a c h i d o n a t e )r e f l e c t e dt h e a r a c h i d o n a t ec o n t e n to ft h es a m p l e . The l o w e rt w op a n e l so ff i g u r e 4 r e p r e s e n tt r a c i n g so b t a i n e df r o m a s a m p l eo fm e d i u mi n c u b a t e dw i t h o u ti s l e t s f o r 30 m i ni np a r a l l e lw i t ht h ei s l e ti n c u b a t i o n s .T h i s sample was p r o c e s s e di np a r a l l e lw i t h and i n a manner i d e n t i c a lt oi s l e t - d e r i v e d s a m p l e sa n de x h i b i t s an i o n c u r r e n t r a t i o a t ( m l z ) 3031311 e s s e n t i a l l y i d e n t i c a tl ot h es t o c ki n t e r n a sl t a n d a r dF o u rs u c hb l a n ks a m p l e s were p r o c e s s e di ne a c he x p e ~ i m e n ti nw h i c hi s l e ta r a c h i d o n a t ec o n c e n t r a t i o n s were measured.

of

Ca2+in Islets

3511

o ifinnccrreeaassiinnggs e c rseetcorreyrt aotreyf r o am t ef r o m 1 5 m i nbbeeyyoonndd 30 rnin (37). It i s oo ff i n t e r e ss ttt thhaattt o t at ol(ti as l(l e i acti ea dt p u lsu s medium) a r a c h i d o n a tt eer ereleased l e a s e db y b v i st l-eats- sa o s sc o e ldo medium1 g l u c o s effoolllloow wssrroouugghhl lyyt ht hee same p a t t e r n as i l l u si tt rr aa tt ee ddiinnffi igguurree 55"" p a n e l E. I nbbootthp hpaanneel lss o ff f i g u rr ee 5 t h e ' p a i n t srr ee pp rr ee ss ee nn ttthheeddi if ff feerri innccee i ni n p a r a m e t e rv a l u ef o rii ss ll ee tt ss i n c u b iantceudbwai t eh 2 8 i t h2 8 dw mmM M g l u c o s em m ii nnuusstthhaattf foorr i s l e t sii nn cc uu bb aa tt ee ddwwiitthh 3 mM u g l u c o s e as a f u n c t i o n O f f t i m e .IInnhhi ibbi it toor rsosof f a r a c h i d o n a t er e l e a s eh a v eb < e e nr e p o r t e d t o s u p p r e s sgg ll uu ccoossee--iinndduucceeddi innsi u l i n s ee cc rr ee tt ii oo nnbb yyii ss oo ll aa tt ee ddpp aa nn cc rr ee aa tt ii ccii ss ll ee tt ss (38,671. s u g g e s t i n gt h ep o s s i b i l i t y S (38,671, t h a tt h eg l u c o s e - i n d u c e da r a c h i d o n a t er e l e a s ed e m o n s t r a t e dh e r e may b e an obliu g a t o r vye v e n ti nu g l u c o s e - i n d u c e di n s u l i ns e c r e t i o nf r o mi s o l aft ew dm i s o l a t e d n p a n c ri e a t ii cti s l ee tt ss..

~I

TIME (Minuter)

BJGlucose-InducedTotal Arachidonate Releare

F i g u r e 4.

Gas c h r o m a t o q r a p h i cm a s s - s p e c t r o m e t r i cm e a s u r e m e not f I s l e t - d e r l v e d arachidonic a c i d .

4 E x a m i n a t i o n o r t h et i m e CDUrSe o f a r a c h i d o n a t ea c c u m u l a t i o ni nt h e i n c u b a t i o n mediumof i s l e t si n c u b a t e dw i t h 3 mM g l u c o s ee x h i b i t e d n o s t a t i s t i c a l l ys i g n i f i c a n ti n c r e a s ei n medium a r a c h i d o n a t e (118+25 p g l i s l e t ) a ta n yt i m ep o i n t .I s l e t si n c u b a t e dw i t h 28 mM g l u c o s ee x h i b i t e d a s i g n i f i c a n t (0.001 < p < 0.005 b yS t u d e n t ' st w o - t a i l e d t t e s t )i n c r e a s ei n 5 m i n (150+10 p g l i s l e t ) compared t o 0 m i n (50*6 medium a r a c h i d o n a t ea t pglislet) a s m a l l e ri n c r e a s eb e t w e e n 5 and 1 5 min (210*13 p g l i s l e t ) and a 1 5 and 30 rnin (390*65 pglislet). The change in l a r g e ri n S r e a s eb e t w e e n i s l e t - a s s o c i a t e da r a c h i d o n a t ec o n t e n t i n t h e same e x p e r i m e n t s was s i m i l a r t o a n da b o u t w o t h a t o f medium a r a c h i d a n a t ec a n t e n t , thirdsof thetotal a r a c h i d o n a t er e m a i n e di s l e t - a s s o c i a t e d . The t i m ec o u r s e o f gluose-induced i n s u l i ns e c r e t i o ni nt h e s ee x p e r i m e n t si si l l u s t r a t e di nf i g u r e 5 (panel A) and r e f l e c t st h ew e l lr e c o g n i z e db i p h a r i cs e c r e t o r yr e s p o n s et og l u c o s e , w h i c hi sC h a r a c t e r i z e db y a f i r s t p h a s ep e a k i n ga t 5 mi", a f a l l i n s e c r e t o r yr a t et o w a r d sb a s a l e v e l sb e t w e e n 5 and 1 5 min,and a secondphase

Q,

0

5

15

30

TIME (Minutes) F i g u r e 5.

T i m e - c o u r s e o f q l u c o s e - i n d u c e di n s u l i ns e c r e t i o na n da r a c h i d o n a t e r e l e a s eb yi s o T a t e dp a n c r e a t i ci s l e t s .