~~oe~i~ic~ et ~~ophy~jeu Acta 83.5 (1985) 1-17 Elsevier

BBA 51922

Arachidonic acid metabolism in isolated pancreatic islets. IV. Negative ion-mass spectrometric quantitation of monooxygenase product

synthesis by liver and islets

John Turk a,bqc-*, Bryan A. Wolf ‘, Patricia G. Comens ‘, Jerry Colca ‘***, Barbara Jakschik b and Michael L. McDaniel ’

a Division of Laboratory Medicine and Departments of Medicine, b Pharmacology and’ Pathology, Washington University

School of Medicine, St. Louis, MO 63110 (U.S.A.)

(Received November 27th, 1984)

Key words: Arachidonate metabolite; Monooxygenase; Mass spectrometry; Prostaglandin synthesis: Insulin release: Eicosanoid; (Rat liver and pancreas)

Deute~~-Ia~ll*~ standards of four regionally isomeric e~xyeicosa~enoi~ acids (EETs) and their hydroly- sis products, the dihy~xyeic~~enoic acids (DHETs), have been prepared and analyzed by capillary column gas chromatography (GC)-negative ion (NI)-metbane chemical ionization (MCI)-mass spectrome- try (MS) as the pentafhrorobenzyl esters. As little as 40 pg of these compounds were readily visualized by these methods, and the deuterium-labelled standards were used in a stable isotope dilution mass spectromet- ric assay which was linear from near the detection limit over several orders of magnitude. NADPH-depen- dent synthesis of both EETs and DHETs from ~c~~nate by hepatic rnicro~~ cytochrome P&O-mono- oxygenase activity was demonstrable with these methods and was significantly suppressed by the compound BW755C (500 FM), but not by eicosa-5,8,11,1Ctetraynoic acid (ETYA, 20 PM) or by nordihydroguaiaretic acid (NDGA, 50 @I). All three compounds suppress glucose-induced insulin secretion and lZhydroxy- eicosatetraenoic acid (12HETE) synthesis by isolated pancreatic islets with similar concentration depen- dence. Microsomes derived from isolated pancreatic islets synthesized less than 3% of the EET and DHET hounds as a eom~ble ant of hepatic mierosomes. Intact islets synthesized less than 3% by mass of the EET and DHET compounds compared to the mass of 12HETE produced by the islets. Islets also failed to convert 3H-labelled arachidonate to 3H-labelled EETs or DHETs under conditions where conversion to [3HJ12-HETE and to [3H]prostaglandin E, (but not to [3H]leukotriene C,, D4, or E,) was clearly demonstrable. Neither exogenous EETs nor leukotriene C, stimulated insulin secretion from the isolated islets or reversed the suppression of gluc~~induc~ secretion by the lipoxygenase inhibitor BW755C. The

* To whom correspondence and reprint requests should be addressed at Box 8118, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.

** Present address: The Upjohn Company, Kalamazoo, MI, U.S.A.

Abbreviations: GC, gas chromatography; MS, mass spectrome- try; NI, negative ion; PI, positive ion; EI, electron impact; CL

chemical ionization; HPLC, high performance-liquid chro- mato~aphy; HETE, hy~oxyeicosatetraenoic acid; EET, e~xyei~~t~enoic acid, DHET, ~ydroxyei~sat~enoic acid; ETYA, eicosa-5,8,11,14-tetraynoic acid; NDGA, nordihydro- guaiaretic acid; Hepes, 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid; Mes, 4-morpholineethanesulfonic acid; ME, methyl ester, PFBE, pentafluorobenzyl ester; TMS, trimethyl- silyl ether; MO, methoxime; RIA, radioimmunoassay.

~5-2740/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

2

cytochrome P-4Wmonooxygenase inhibitor, metyrapone (50 PM), did not influence insulin secretion from the isolated islets under conditions where the lipoxygenase inhibitor, NDGA, suppressed glucose-induced secretion. These observations argue against the recently suggested hypothesis that EETs derived from arachidonate by monooxygenase action participate in glucose-induced insulin secretion by isolated pancreatic islets.

Introduction

Oliw et al. [1,2] and other groups [3-51 have

demonstrated that arachidonic acid may be

oxygenated by a microsomal cytochrome P-450-

monooxygenase in the presence of NADPH. The

principal products of this pathway are four region- ally isomeric epoxyeicosatrienoic acids [2] which

are hydrolyzed to the corresponding vicinal diols

[l] by the actions of an epoxide hydrolase enzyme [6]. These metabolites are produced by intact

hepatocytes and renal cells as well as by micro-

somal preparations [7], may be endogenous con-

stituents of liver cell membranes [8], and are bio-

synthesized in an enantioselective manner [9].

Interest in the possibility that these compounds might play a role in the function of endocrine cells

has been generated by the observations that exoge- nous epoxyeicosatrienoic acids provoke secretion

of luteinizing hormone from anterior pituitary cells

[lo], of somatostatin from hypothalamic median

eminence [ll], and of glucagon and insulin from

isolated pancreatic islets [12]. The possible participation of oxygenated

metabolites of arachidonic acid in glucose-induced insulin secretion from pancreatic islets has been

suggested by results from several groups. Isolated islets [13,14] from adult rats and cultured pan- creatic cells from rat neonates [15] synthesize

several arachidonate cyclooxygenase products and

the arachidonate lipoxygenase product, 12-HETE. Concentrations of glucose which stimulate insulin secretion from isolated islets also stimulate islet production of cyclooxygenase products and of the lipoxygenase product, 12-hydroxy-5,8,10,14- eicosatetraenoic acid (12-HETE) [13,14]. The lipo- xygenase and cyclooxygenase inhibitors, eicosa- 5,8,11,14-tetraynoic acid (ETYA), nordihydro- guaiaretic acid (NDGA) and BW755C, all inhibit glucose-induced insulin secretion from isolated pancreatic islets [12,14,16], from cultured neonatal pancreatic cells [15,17-191, and from perfused

pancreas preparations [20]. Selective cyclooxy- genase inhibitors such as indomethacin do not

inhibit glucose-induced insulin secretion in these systems [12,14-201. Taken together, these observa-

tions suggest that 12-HETE or its precursor, 12-

hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-

HPETE), might participate in glucose-induced in- sulin secretion. This hypothesis is supported by the

similar dependence of suppression of islet insulin secretion and of 12-HETE synthesis upon the con-

centration of lipoxygenase inhibitors [21]. At vari- ance with the hypothesis are the facts that 12-lip-

oxygenase products do not completely reverse the effects of lipoxygenase inhibitors on insulin secre-

tion from isolated islets [21] and have relatively

weak insulin secretagogue effects on cultured neonatal pancreatic cells [15,22].

One explanation for these observations is that

lipoxygenase inhibitors might suppress cyto-

chrome P-450-mediam oxygenation of

arachidonate by islets and thereby eliminate the possible insulin secretagogue effects of the epoxyeicosatraenoic acids (EETs) [12]. Islet

synthesis of such compounds has not yet been demonstrated, although initial attempts at detect-

ing EETs derived from islet microsomes have been

unsuccessful [12]. Direct demonstration of endoge- nous production of arachidonate metabolites by islets is complicated by the difficulty in preparing

purified populations of islets in large numbers and by the small mass of the metabolites generated per

islet. To further investigate the possible production of EETs by isolated islets and the role of these compounds in insulin secretion, we have per- formed studies using large numbers of isolated islets and have developed a sensitive and specific method of quantitating EETs and their corre- sponding vicinal diols (DHETs) with negative ion-chemical ionization-mass spectrometry and deuterated internal standards. These methods have been used to compare synthesis of these com- pounds by islet and liver microsomes and to com-

3

pare synthesis by intact islets of lipoxygenase and of cytochrome P-450 products. The influence of lipoxygenase inhibitors on hepatic microsomal cy- tochrome P-450-mediated oxygenation of arachidonate has also been examined.

Materials and Methods

Materials Most materials were obtained from previously

specified sources [13,14]. meta-Chloroperoxyben- zoic acid was obtained from Aldrich (Milwaukee, WI). Metyrapone was obtained from Sigma (St. Louis, MO). Leukotrienes C,, D4 and E, were a gift from Dr. Joshua Rokach (Merck-Frost Canada, Pointe Claire-Dorval, Quebec, Canada).

Methods Liquid chromatography. High-performance

liquid chromatography (HPLC) was performed on a Varian (Walnut Creek, CA) Model 8500 instru- ment in the reverse phase or normal phase. The following columns were obtained from Waters As- sociates (Milford, MA): I (PBondapak C,,, 3.9 mm X 30 cm), II (PPorasil, 3.9 mm X 30 cm), III (PBondapak C,,, 7.8 mm x 30 cm), and IV (PPorasil, 7.8 mm x 30 cm). Column V consisted of two Bio-Sil ODS-5S (250 mm X 4 mm) columns in series obtained from Bio-Rad Laboratories (Richmond, CA). Column VI (250 x 4.6 mm, Nucleosil C,,, 5 micron) was obtained from Al- ltech (Deerfield, IL) and was treated with EDTA before and intermittently during use [38,39]. Thin- layer chromatography (TLC) was performed on Analtech (Newark, DE) plates (silica gel GF, 2.5 X 10 cm, 250 micron). The following solvent mix- tures (v/v) were employed: A (methanol, 73/ water, 27/acetic acid, 0.2), B (hexane, lOO/ isopropanol, 0.3/acetic acid, O.l), C (hexane, lOO/isopropanol, 1.5/acetic acid, 0.2), D (hexane, lOO/isopropanol, 3/acetic acid, 0.2), E (acetonitrile, 70/water, 30), F (ethyl acetate, 25/hexane, 75/acetic acid, O.l), G (methanol, 65/water, 35/acetic acid, 0.08 (pH 6.2) with 10% NH,OH), H (acetonitrile, 30/water, 70/acetic acid, O.l), I (acetonitrile, 53/water, 47/acetic acid, O.l), J (a linear gradient over 120 min from CHCl,, 500/acetic acid, 2, to CHCl,, 5OO/methanol, 50/acetic acid, 2), and K (hexane, lOO/isopro- panol, 0.275).

Extractions. Aqueous media containing arachidonate metabolites derived from incubation with biological materials generally contained methanol used to terminate the incubation. For recovery of EETs and DHETs the medium was adjusted to pH 3.5 and extracted with CH,Cl, as described [7]. Prostaglandin E, and 12-HETE were extracted with disposable octadecyl-silica (ODS) columns [37] as described [13]. For recovery of leukotrienes C,, D4 and E,, the medium was diluted with water to achieve a methanol con- centration of 5% after addition of the internal standards, adjusted to pH 6.2, and applied to a disposable ODS column. The column was washed with water, and then leukotrienes C,, D4, and E, were eluted with methanol/l mM Na,PO, (pH 6.2) (9 : 1).

Derivatization. Carboxyl groups were converted either to (a) methyl esters (ME) with ethereal diazomethane or to (b) pentafluorobenzyl esters (PFBE) with tetramethyl ammonium hydroxide and pentafluorobenzyl bromide in dimethyl- acetamide-methanol as described [21,23]. Hy- droxyl groups were converted to the trimethylsilyl (TMS) ethers with N,O-bis(trimethylsilyl)trifluoro- acetamide in pyridine [21].

Gas chromatography-mass spectrometry. Gas chromatography was performed on a Hewlett Packard 5840A gas chromatograph interfaced with a Hewlett Packard 5985B mass spectrometer. Op- erating conditions were as previously described [13,14,21]. Structural analyses were performed with the mass spectrometer in the positive ion (PI)-electron impact (EI) mode to obtain an infor- mative fragmentation pattern. Quantitative analyses were performed with the mass spectrome- ter in the negative ion (NI)-chemical ionization (CI) mode with methane as reagent gas to obtain the enhanced sensitivity conferred by this method of analysis [24-271.

Preparation of standards. The compound [5,6,8,9,11,12,14,15-*H,larachidonic acid ([ *H,]arachidonic acid) was prepared from ETYA and deuterium gas [28]. The compound [5,6,8,9,11,12,14,15-2H,112-HETE ([*Hs]12- HETE) was prepared from (*Hs]arachidonic acid with CuCl, and H202 [29]. The compound [l- 14C]12-HETE was prepared from [1-14C] arachidonic acid with washed human platelets [13].

4

Regionally isomeric epoxyeicosatrienoic acids

(EETs) were prepared from arachidonic acid of

varied isotopic composition (unlabelled arachidonic acid, [1-i4C]arachidonic acid,

[5,6,8,9,11,12,14,15-3Hs]arachidonic acid,

[5,6,8,9,11,12,14,15-*Hs]arachidonic acid, and various mixtures of these compounds) with meta-

chloroperoxybenzoic acid as described by Oliw et

al. [1,7]. The isomeric epoxides were purified by

reverse phase-HPLC (column III, solvent A) as

described by Oliw et al. [7] and then by normal

phase-HPLC (column II, solvent B) as described

by Chacos et al. [30]. Normal phase-HPLC reten- tion volumes under these conditions at a flow rate

of 1 ml/min were 23 ml for the 14,15-EET; 25 ml for the 11,12-EET; 34 ml for the 8,9-EET; and 54

ml for the 5,6-EET. Yields of the 5,6-EET were generally poor with this procedure. The 5,6-EET

was therefore also prepared by converting

arachidonic acid of varied isotopic composition (TLC R, 0.32, solvent F) to the 6-iodo-5-hydroxy-

8,11,14-eicosatrienoic acid, delta lactone (TLC R,

0.52, solvent F) by treatment with KI and I, as

described by Corey et al. [31]. The iodolactone was

then converted to the 5,6-EET (TLC R, 0.19,

solvent F) by treatment with 0.2 N LiOH in

tetrahydrofuran-water [31], and the 5.6-EET was

purified by normal phase-HPLC as described

above. The purified EET isomers were converted to the

methyl esters (ME) and analyzed by GC-MS (PI-EI) to confirm their structures. The mass

spectra of the EET (ME) compounds derived from

unlabelled arachidonic acid were as reported by Oliw et al. [2].

To prepare the corresponding vicinal diols

(DHET), the purified EET compounds were con- verted to the methyl esters and subjected to hy- drolysis in 10% perchloric acid and tetrahydro-

furan as described by Manna et al. [32]. (The tetrahydrofuran was distilled from LiAlH, just before use.) The methyl esters were then converted to the free acids by hydrolysis in 3 M LiOH/di- methoxyethane (1 : 5) under N, with stirring at 60°C for 90 min. The compounds were then ex- tracted with CH,Cl, after acidification to pH 3.5 with 0.5 M HCl and purified by normal phase- HPLC (column II; 60 cc solvent C, then a linear gradient to solvent D over 5 min; then 100%

solvent D). Under these conditions the retention

volumes were 28 ml for 14,15-DHET; 34 ml for

11,12-DHET; 64 ml for 8,9-DHET; and 104 ml

for 5,6-DHET. Yields of the 5,6-DHET were poor

with this procedure. The 5,6-DHET was therefore

also prepared converting the 5,6-EET to the 5,6-di-

hydroxyeicosatrienoic acid, delta lactone by treat- ment in CH,Cl,/O.S M HCl (4: 1) for 12 h at

room temperature after the method of Oliw (E. Oliw, personal communication). The delta lactone

was then hydrolyzed to the free acid form of

5,6-DHET by alkaline hydrolysis in triethyla-

mine/pyridine/water (1 : 10: 10) for 30 min at

room temperature after the method of Falardeau and Brash [33). The resultant 5,6-DHET was then

purified by normal phase-HPLC as described above.

The purified DHET isomeres were converted to

the methyl ester (ME), trimethylsilyl (TMS) ether

derivatives and analyzed by GC-MS (PI-EI) to confirm their structures. The mass spectra of the

purified DHET (ME, TMS) compound derived from unlabelled arachidonate were as reported by

Oliw et al. [1,2]. The blank values of the deu-

terium-labelled internal standards were about two parts per thousand. Addition of 75 ng of internal

standard therefore resulted in a blank signal of

150 pg. Signals 2-fold or greater than the blank signal were considered the minimum detection

limit.

Isolation, culture and incubation of islets. Pan- creatic islets were obtained under aseptic condi-

tions from rats fed ad libitum as described elsewhere [13,14,21,34-361. For studies examining

islet metabolism of arachidonic acid, isolated islets (approx. 1.5 * 104) were preincubated (30 min, 37’C) in medium (5 mM Hepes, 135 mM NaCl, 24

mM NaHCO,, 5 mM KCl, 1 mM MgCl, and 2.5 mM CaCl,, pH 7.4) supplemented with glucose (3 mM). The islets were then collected by centrifuga- tion, resuspended in fresh medium of the same composition, and divided into groups in individual siliconized tubes. Incubations were initiated by addition of glucose (final concentration 28 mM) and on some occasions ionophore A23187 (final concentration 10 PM). For studies examining ra- diolabelled metabolites, [ 3 H,]arachidonic acid (5 pCi/lOOO islets) was also added at this time. For studies examining the metabolism of endogenous

arachidonate, no exogenous substrate was added. Incubations were continued for 30 min at 37°C and terminated by the addition of 1 vol. of methanol. In most cases, internal standards were contained in the methanol, but in expe~ments involving radioimmunoassay of leukotriene C,, in- ternal standards were omitted. For experiments examining islets metabolism of [ 3H,]arachidonate, internal standards employed were 14C-labelled IZHETE, prostagland~ E,, DHETs and/or EETs (about 50 nCi each) in various combinations and/or unlabelled leukotrienes C,, D4, E, (3 pg each). For experiments examining islet metabolism of endogenous arachidonate, internal standards employed were *H- and 3H-labelled 12-HETE, DHETs, and/or EETs in various combinations and in amounts of about 75 ng (specific activity about 0.2 nCi/ng) per compound per tube. After addition of methanol or the methanolic solution of internal standards, particulate matter was removed by centrifugation (3000 X g, 5 min). Internal standards and islet-derived metabolites were ex- tracted from the supematant as described above. The protein pellet was extracted with 75% ethanol/lS% HCl and assayed for insulin content to ensure that the various samples contained an equal islet mass 1141.

For studies examining the effect of enzyme inhibitors on insulin secretion, 25 islets were ran- domly selected under a stereomicroscope for each sample. The islets were then pre-incubated (30 mix-r, 37°C) in albumin-free medium (of the com- position described above) cont~~ng 3 mM glu- cose and the desired concentration of inhibitor (0 for controls). Inhibitors were added as dimethyl sulfoxide solutions diluted in 150 mM NaCl as described [21]. The dimethyl sulfoxide-saline di- luent without inhibitors was added to controls. At the end of the pre-~cubation period, the medium was removed, replaced with fresh medium of iden- tical composition, removed again (washing step), and replaced with fresh medium containing glu- cose (3 or 28 mM) and the same concentration of inhibitor used in the preincubation period. The experimental incubation then carried out for 30 min at 37°C and was terminated by addition of medium (200 ~1) containing bovine serum albumin (0.2%), mixing, and withdrawal of an aliquot for the radioimmunoassay of secreted insulin as de- scribed [14&O].

For studies examining the effects of exogenous standards of arachidonate oxygenation products on insulin secretion, a protocol similar to that described above was employed. The test com- pound was added after the pre-incubation period and washing, and the addition constituted the beginning of the experimental incubation period. Test compounds were added as ethanol solutions diluted in incubation medium as described 1151. Ethanol-buffer diluent not containing the test compound was added to controls. EET and DHET standards were stored in hexane/pyridine (20 : 1) at -3O’C after the method of Oliw (E. Oliw, personal communication). Shortly before use, each standard was analyzed by normal phase-HPLC, as described above, to insure that decomposition had not occurred. The concentration of mass of the purified standard compound was determined from liquid scintillation counting and the known specific activity (3H dpm/[‘H,,]arachidonate weight) of the starting material. Immediately before use, the desired mass of the purified standard was placed in a silanized 1 ml Reacti-Vial (Pierce, Rockford, IL), concentrated to dryness under nitrogen, re- constituted in 20 ~1 of ethanol and diluted with 180 ,ul of incubation medium. 10 ~1 of this solu- tion was then immediately added to each experi- mental tube receiving the test compounds. Com- plete dissolution and transfer of the test com- pounds under these conditions was documented by liquid scintillation counting of the recipient fluid, which contained 96% of the expected 3H dpm.

Preparation and incubation of microsomes. Islet microsomes were prepared as described elsewhere [34,41]. In brief, isolated islets (approx. (1.2-2.3). 106) were pooled and transferred to a Potter- Elvehjem tissue grinder, size 0019, Kontes Bio- medical Products, Vineland, NJ), washed with a solution of 50 mM Mes/l mM EDTA/250 mM sucrose (pH 7.2) and homogenized with 14 strokes of the motor-driven pestle at 1170 rpm (Polysci- ence RZRlO at setting 3). The homogenate was centrifuged (600 x g, 5 min), and the supemat~t was collected and centrifuged again (20 000 x g, 20 mm). The supernatant was collected and centri- fuged (150000 X g, 90 min) to yield an endo- plasmic reticulum-enriched pellet. The supernatant was decanted, and the pellet was washed with and

6

resuspended in incubation buffer (150 mM NaCl/SO mM Tris/l mM EDTA (pH 7.4). An

aliquot of the microsomal suspension was

withdrawn for the determination of protein con-

tent by the method of Lowry et al. [42]. The

remaining microsomal suspension was distributed

into 4-10 polypropylene test-tubes to which a magnetic stir bar was added. Where appropriate,

enzyme inhibitors were added to some of the tubes

at this point as described above. The microsomes

were then incubated for 30 min at 37’C. At the

end of this period, NADPH and arachidonate were added to achieve a final concentration of 1

mM and 2-100 FM respectively. The concentra-

tion of NADPH was confirmed by spectrophoto-

metric measurement, and dissolution and transfer

of the arachidonate under these conditions were documented by liquid scintillation counting and

the known specific activity (3H dpm/[‘H,,]arach- idonate weight) of the stock arachidonic acid. The

incubation was then continued for 15 min at 37°C with stirring and terminated by the addition of 1

vol. of methanol containing the appropriate mix-

ture of labelled internal standards as described

above. Products were then extracted as described above.

Hepatic microsomes were prepared by a proto-

col similar to that described above except that the

excised liver tissue was washed with 150 mM NaCl

before mincing, and with 150 mM NaCl/SO mM

Tris/l mM EDTA (pH 7.4) after mincing as de-

scribed by Oliw et al. [l]. Thereafter, a protocol identical to that described above for islets was

followed for preparation and incubation of hepatic microsomes and recovery of their products.

Radioimmunoassay of feukotriene C,. Leu- kotriene C, antiserum was kindly provided by Dr.

R. Bell (Riker Laboratories, St. Paul, MN). The

characterization of the antiserum and the assay

procedure have been described elsewhere [49].

Results

Deuterium-labelled analogs of the four region- ally isomeric epoxyeicosatrienoic acids (EETs) were synthesized from deuterium-labelled arachidonic acid as described by Oliw and Moldeus [7], and a portion of each of these compounds was hydro- lyzed to the corresponding vicinal diol (DHET) as

described by Oliw and Moldeus [7] and Falck and

co-workers [32]. The EET compounds were con-

verted to the pentafluorobenzyl esters (PFBE) and the DHET compounds were converted to the PFBE

and then to the trimethylsilyl ether (TMS) derivia-

tives. Both sets of compounds were found to have

excellent vapor phase properties on capillary col-

umn gas chromatography (GC) after derivatiza- tion, and both sets of compounds exhibited very

simple spectra upon negative ion (NI) methane

chemical ionization (MCI) mass spectrometric

(MS) analysis which consisted essentially of a single

ion at an m/z corresponding to the molecular ion (M) minus 181 (loss of the pentafluorobenzyl

group). This ion occurred at m/z 327 for the

deuterium-labelled EET-PFBE compounds and at

m/z 489 for the deuterium-labelled DHET-PFBE,

TMS compounds (Fig. 1).

Eq

0 I I

11 12 13 14

ELUTION TIME (MIN)

Fig. 1. Analysis of the pentafluorobenzyl ester, bis(trimethyl-

silyl) ether derivative of [ *Hs]14,15-dihydroxy-5,8,11-

eicosatrienoic acid by capillary column gas chromato-

graphy-negative ion-methane chemical ionization-mass spec-

trometry. Standard 2H/3H-labelled 14,15-dihydroxy-5,8,11-

eicosatrienoic acid was prepared from *H/‘H-labelled arachidonate of known specific activity, purified by normal

phase-HPLC, and converted to the pentafluorobenzyl ester,

bis(trimethylsily1) derivative as described in the Methods sec-

tion. The derivative was taken up in heptane at a concentration

of 20 ng per J.II (as determined by liquid scintillation counting)

and serial one to ten dilutions in heptane were prepared. 2 1.11

of each solution was introduced into the splitless injector to

initiate the GC-MS analysis. The indicated quantities corre-

sponding to each tracing represent the amount introduced into

the injector.

Dilution experiments such as that illustrated in Fig. 1 indicated that as little as 40 pg of the PFBE derivatives of these compounds could be readily visualized on capillary column GC-NI-MCI-MS analysis. The pro~nent ion at m/z M - 181 and the low detection limits indicate that the PFBE derivatives of the EET and DHET compounds exhibit behavior on CC-NI-MCI-MS analysis similar to that previously reported for other arachidonate metabolites such as prosta~anding f24,25] and hydroxyeicostetraenoi~ acid (HETEs) [13,26,27].

The deuterium-labelled EET and DHET com- pounds could be employed to quantitate un- labelled EET and DHET compounds in stable isotope dilution mass spectrometric analyses as illustrated in Fig. 2. The ion corresponding to M - 181 occurred at m/z 481 for the unlabelled DHET-PFBE, TMS compound, and at m/z 489

Arso=ll0504

for the deuterium-labelled compound. With a con- stant amount of deuterium-labelled compound and increasing amounts of unlabelled compound, the ratio of the ion current at m/z 481 and 489 was a linear function of the mount of unlabelled com- pound over at least three orders of magnitude (Fig. 2).

These methods were used to quantitate the synthesis of both EET and DHET compounds from arachidonate by hepatic microsomes. Hepatic microsomes were incubated with NADPH and unlabelled arachidonate; and the incubation was terminated by addition of a mixture of 3H- and *H-labelled EET or DHET standards in methanol. The products were extracted, isolated by normal phase-HPLC, derivatized and analyzed by GC-N&-MCI-MS as shown in Fig. 3. As il- lustrated in Table I, omission of hepatic mi- crosomes from this system resulted in no product

AMOUNT OF 5,6-WET (no)

ELUTION TIME (min) ELUTION TIME (min)

Fig. 2. Stable isotope dilution mass spectrometric measurement of 5,6-dihydroxy-8,11,14-eicosatrienoic acid. Standard 2H/3H-labeiled 5,6-dihydroxy-8,11,14-ei~at~~oic acid (5,6-DHET) and standard ‘H/3H-labelted 5,6-DHET were prepared as described in the Methods section. A constant amount of the 2H/3H-iabelkd 5,GDHET were introduced into each of eleven 1 ml Reacti-Vials containing varying amounts of ‘H/3H-5,6-DHET. The samples were concentrated to dryness under N,, converted to the PFBE-TMS derivatives, reconstituted in 20 pl of heptane, and analyzed as described in Fig. 1. Results similar to those shown above were obtained in a similar experiment with the 5,6-epoxy-8,11,14-eicosatrienoic acid. Circles indicate raw unlabelled/*H, ratios. Squares indicate values corrected by subtraction of the blank ratio (0.028) of the deuterium-labelled internal standard. The left CC-MS tracing represents point A on the graph and the right tracing point B.

ENDOGENOUS 14,15- DHET (PFBE, TMS)

F ~~~N$GENOuS 5,6 - EET

(CH,&O OSWI,),

EXOGENOUS INTERNAL *’ *“+I *’ STANDARD (2H)8-14,15-DHET (PFBE,TMS)

(CH&SiO OSIICH&

F EXOGENOUS INTERNAL STANDARD (*H)a-S,bEET(PFBE)

1746 - A.DHET FROM LIVER MICROSOMES PLUS

ARACHIDONATE AND NADPH 6 *-a

z : $2

s Z% 8::

c E I I

11 12 13 14

B.DHET FROM BUFFER PLUS ARACHIDONATE AND 67- NADPH

6 +d z SA 2;

z 5a9’- I I

62 Arem=

E 1 ,

I I

11 12 13 14 ELUTION TIME (min)

S49rA.EET FROM LIVER MICROSOMES

E Area =762

I 1 , I

5, 6. EET FROM BUFFER WITHOUT MICROSOMES

1 ;267ppr

E: 11 12 13 14

ELUTION TIME (min)

Fig. 3. Synthesis of dihydroxyeicosatrienoic and epoxyeicosatrienoic acids from arachidonate by liver microsomes in the presence of

NADPH demonstrated by stable isotope dilution mass spectrometric measurements. Rat liver microsomes were prepared and

incubated for 15 min at 37OC in the presence of arachidonate (100 PM) and NADPH (1 mM) as described in the Methods section.

Incubations were terminated by addition of 1 vol. of methanol containing 75 ng of a 2 H/3H-labelled internal standard of 5.6-DHET

(figure left) or 5,6-EET (figure right). The products were extracted, purified by normal phase-HPLC, derivatized. and analyzed by

GC-NI-MCI-MS as described in the Methods section. Tracings similar to those shown above were obtained in other experiments

examining the synthesis of the other DHET (11,12-DHET, 8,9-DHET and 5,6-DHET) and EET (14,15-EET, 11,12-EET and 8,9-EET)

isomers. The peak eluting at 13.2 mm in the figure left upper tracing of m/r 481 vs. time represents endogenous 11,12-DHET (PFBE,

TMS) which was incompletely resolved from the 14,15-DHET isomer on normal phase-HPLC but which is clearly resolved on GC.

formation, which indicates that the products did not arise from auto-oxidation of arachidonate.

Similarly, omission of NADPH prevented product formation, consistent with the known NADPH requirement for cytochrome P-450-catalyzed oxygenation of arachidonate [1,3]. Product forma- tion did occur without exogenous arachidonate, indicating that endogenous arachidonate derived from microsomal lipids could undergo cytochrome P-450-mediated oxygenation. Hepatic microsomes have previously been reported to provide apprecia- ble amounts of arachidonate as substrate for en-

zyme-catalyzed reactions [43]. Product formation

was considerably enhanced by supplementary arachidonate however, consistent with the pre- cursor role of arachidonate in generation of the EET and DHET compounds.

As indicated in Table II, the cytochrome P-450 activity associated with hepatic microsomes could oxygenate each of the four double bonds of arachidonate, and most of the product of oxygena- tion at a given double bond was isolated as the DHET rather than the EET as previously reported [2]. The exception was the 5,6-EET, which was

9

TABLE I

SYNTHESIS OF 14,15-DIHYDROXY-5,8,11-EICOSATRIENOIC ACID FROM ARACHIDONIC ACID BY LIVER MICRO- SOMES IN THE PRESENCE OF NADPH

Microsomes were prepared from rat liver and incubated in the presence or absence of NADPH (1 mM) and exogenous arachidonic acid (100 PM) for 15 min at 37°C as described in Methods. Microsomal protein concentration was 0.8 mg/ml. Incubations were terminated by addition of 1 vol. of methanol containing 75 ng of 2H/3H-labelled 14,15-DHET as an internal standard. Products were extracted, analyzed by normal-phase-HPLC (column II, solvent C), derivatized, and analyzed by GC-NI-MCI-MS as described in Methods.

Entry Liver microsomes

Arachidonic acid

NADPH Amount of 14,15-DHET

(/Jg)

Percent of control

1 + 2 + 3 + 4 + 5 _ 6 - 7 _ 8 _

+ + 6.30 + - 0.06 - _ 0.04 - + 2.27 + + < 0.01 + - < 0.01 - + < 0.01 - _ < 0.01

loo

<l <l 36 0 0 0 0

TABLE II

SYNTHESIS OF REGIONALLY ISOMERIC DIHY- DROXYEICOSATRIENOIC ACIDS AND EPOXYEICO- SATRIENOIC ACIDS FROM ARACHIDONIC ACID BY LIVER MICROSOMES IN THE PRESENCE OF NADPH

Conditions were as described in Table I except that incubations for expts. 1 and 2 were terminated by addition of methanol containing a mixture of the four separate 2H/3H-labelled regionally isomeric DHET internal standards, and expt. 3 was terminated by addition of methanol containing a mixture of the four separate 2H/3H-labeIled regionally isomeric EET stand- ards. After extraction, normal phase-HPLC analysis on column II was performed for DHET compounds (solvent C, then solvent D) and EET compounds (solvent B) as described in the Methods section. Experiments similar to those summarized in the table were performed with varied concentrations of supple- mentary arachidonate (O-100 PM), and results similar to those shown were obtained for DHET (n = 5) and EET (n = 4) anlyses except that the absolute amount of all compounds was reduced at lower arachidonate concentrations.

Experiment

1 and 2

3

Compound Amount ().tg)

14,15-DHET 5.37 + 3.09 11,12-DHET 13.06 f 0.70 8,9-DHET 5.93 f 3.95 5,6-DHET 0.06 f 0.02

14,15-EET 3.17 11,12-EET 0.77 8,9-EET 0.58 5,6-EET 1.83

more abundant than the 5,6-DHET (Table II). This is consistent with the reported observation that the 5,6-EET is a poorer substrate than the other EETs for the epoxide hydrolase [6].

It has been suggested that the suppression of insulin secretion from isolated pancreatic islets by the arachidonate lipoyxgenase inhibitors NDGA and ETYA may reflect reflect inhibition of cy- tochrome P-450-mediated arachidonate oxygena- tion by these compounds [12]. The lipoxygenase inhibitor, BW755C, also suppresses insulin secre- tion from isolated pancreatic islets [21], and a similar mechanism may be postulated. The in- fluence of NDGA, ETYA and BW755C on oxygenation of arachidonate by the cytochrome P-450 associated with hepatic microsomes was therefore determined as illustrated in Table III. BW755C was observed to inhibit significantly the formation of cytochrome P-450 products from arachidonate at a concentration (500 PM) which strongly suppresses glucose-induced insulin secre- tion from isolated pancreatic islets [21]. Neither ETYA (20 PM) nor NDGA (50 PM) was observed to inhibit significantly the formation of cy- tochrome P-450 products from arachidonate, al- though both ETYA and DNGA strongly suppress glucose-induced insulin secretion and 1ZHETE production from isolated islets at the tested con-

10

TABLE III

INFLUENCE OF ARACHIDONATE LIPOXYGENASE IN-

HIBITORS AND METYRAPONE ON SYNTHESIS OF

14,15-DIHYDROXY-5,8,11-EICOSATRIENOIC ACID BY

LIVER MICROSOMES

Conditions were as described in Table I except that the micro-

somes were pre-incubated for 30 min in the presence of the

compounds indicated in the table. The chosen concentrations

of BW755C, ETYA and NDGA result in 90% suppression of

12-HETE synthesis and 74-89s suppression of glucose-in-

duced insulin secretion with isolated islets [14,21]. The chosen

concentration of metyrapone has been reported to inhibit cyto-

chrome P-450-mediated oxygenation of arachidonate by 62%

[4] and to inhibit glucose-induced insulin secretion from iso-

lated pancreatic islets by 50% [12]. Standard errors of the mean

are indicated (n = 3). The P values were calculated with Stu-

dent’s t-test. Similar results were obtained in other experiments

(n = 5) examining the effects of these inhibitors on each of the

other three DHET isomers and on each of the four EET

isomers. n.a., not applicable.

Inhibitor Amount of DHET Percent of Significance

(!Jg) control of difference

amount of from control

DHET

None 2.38 f 0.83

(Control)

BW755C 0.44kO.25

(500 p M) ETYA 1.73 + 0.43

(20 PM) NDGA 2.84k1.32

(50 PM) Metyrapone 1.45 f 0.60

(50 PM)

loo* 0 n.a.

195 6 0.01 < P < 0.02

84+16 0.50 < P i 1.00

114*22 0.50 i P i 1.00

56&13 0.02 i P i 0.05

centrations [14,21]. Others have reported a relative resistance to ETYA of cytochrome P-450-media-

ted oxygenation of arachidonate [5]. Metyrapone (50 PM) was found to inhibit significantly the

formation of cytochrome P-450 products from arachidonate (Table III), and the magnitude of the inhibition is reasonably close to that previously reported for the compound at the tested con-

centration [4]. It was considered possible that oxygenation of

arachidonate by an islet cytochrome P-450 might be more susceptible to inhibition by ETYA or NDGA than that by the hepatic enzyme. Attempts (n = 9) were therefore made to demonstrate synthesis of cytochrome P-450 products from arachidonate by islet tissue as illustrated in Fig. 4.

ELUTION TIME (Mm 1

Fig. 4. Examination of synthesis of 5,6-epoxy-8,11,14-

eicosatrienoic acid by pancreatic islets with stable isotope dilu-

tion mass spectrometric measurements. About 1.5. lo4 pan-

creatic islets were isolated and incubated for 30 min at 37°C in

the presence of 28 mM glucose as described in the Methods

section. A volume of methanol containing 75 ng of *H/‘H-

labelled 5,6-EET and 300 ng of *H/‘H-labelled 12-HETE was

then added, and the products were extracted, analyzed by

sequential reverse phase-HPLC, then normal phase-HPLC, de-

rivatized and analyzed by GC-MS as described in the Methods

section. As illustrated above, no endogenous 5.6-EET was

detectable from the islets, although 43 ng of 12-HETE was

produced under these conditions. In similar experiments none

of the other three EET isomers or any of the four DHET

isomers could be demonstrated from intact islets or from islet

microsomes incubated with arachidonate (100 PM) and

NADPH (1 mM). The 5,6-EET elutes slightly earlier in the

figure above than in Fig. 3, due to removal of a portion of a

loop of the capillary GC column to improve peak shape after 2

months of use.

In each attempt, islets (1.5-2.2. 104) derived from

30-45 rats were employed. On no occasion was enzymatic synthesis of any of the four EET iso-

mers or of any of the four DHET isomers demon- strable either with islet microsomes or with intact

islets even in the presence of 100 PM supplemental arachidonate. Constraints on the amount of DHET that could have been formed and failed to be

detected in the analyses indicated that islet micro- somes synthesized at most 3% of these compounds per pg microsomal protein compared to hepatic microsomes. Similarly intact islets synthesized, at most, 90 pg of DHET per lo3 islets, which is only 3% by mass of the measured 12-HETE production by intact islets [21]. (The possible formation of lactones of the DHET compounds by the islets was not directly examined, but samples were routinely treated with triethylamine/ pyri- dine/water (1: 10 : 10) before derivatization. This treatment hydrolyzes the delta la&one of the 5,6- DHET to the hydroxy acid). No attempts were

made to induce the synthesis of cytochrome P-450 with phenobarbitol.

Previously reported attempts at demonstrating islet synthesis of cytochrome P-450 products from arachidonate have also been unsuccessful [12]. Those studies employed only 200 islets, however, and examined conversion of “C-labelled arachidonate. Approx. loo-times that number of islets was employed in the present investigation. It was considered possible that cytochrome P-450 product formation might be observed with 3H-labelled arachidonate, since that compound has approx. 1000-times the specific activity of 14C- labelled arachidonate. 14C-labelled internal stan- dards of cytochrome P-450 products were in- cluded in these analyses to facilitate chromato- graphic identification of any 3H-labelled products

ELUTION VOLUME (ml)

Fig. 5Synthesis of 3H-labelled dihydroxyeicosatritmoic acids from ‘H-labelled arachidonate by liver ~~rosomes in the presence of NADPH demonstrated by normal phase-HPLC analysis with “C-labelled internal standards. Rat liver rnicro- somes were prepared and incubated (15 min, 37°C) with 3H- labelled arachidonate in the presence of NADPH (1 mM) as described in the Methods section. The incubation was terminated by addition of a volume of methanol containing a mixture of “C-labekd internal standards of the four DHET isomers, and the products were extracted and analyzed by normal phase-HPLC (column II, solvent C for 60 ml, then solvent D). Similar experiments with isolated pancreatic islets revealed no conversion of ‘H-labelled arachidonate to products that co-eluted with i4C-Iabelled internal standards of the DHET isomers.

11

[l]. Formation of ‘H-labelled cytochrome P-450 products from 3H-labelled arachidonate by hepatic microsomes was readily demonstrable with these methods as illustrated in Fig. 5. No formation of any of the four EET or of any of the four DHET compounds by either islet microsomes or by intact islets could be observed under these conditions, however. Intact islets clearly converted [3H] arachidonate to the cyclooxygenase product ~3H]prostagl~din E, and to the 12-lipoxygenase product [3H]12-HETE but not to the 5-lipo- xygenase products [3HJleukotriene C,, D4, or E,

TABLE IV

INFLUENCE OF EXOGENOUS 5,6-EPOXY-8,11,14- EICOSATRIENOIC ACID (5,6-EET) ON INSULIN SECRE- TION FROM ISOLATED PANCREATIC ISLETS

Pancreatic islets were isolated from ten rats on each of three separate days, and the islets were suspended in fresh incubation medium. 25 islets were randomly selected for each sample under a stereo~cro~pe, and three inde~ndent samples were prepared for each experimental condition. The islets were pre- incubated for 30 min at 37’C with shaking in incubation medium (200 ~1) containing 3 mM glucose. The preincubation medium was then removed and replaced by medium (200 ~1) containing glucose (3 or 28 mM). Incubations were initiated by addition of an aliquot (10 ~1) of diluent (see Methods) contain- ing no other additives (controls) or containing the desired concentration of the 5,6-EET. Incubations were then continued for 30 min at 37°C. At the end of this interval, an aliquot (200 ~1) of bovine serum albumin (0.2%) in incubation medium was added, and aliquots of the experimental medium were withdrawn for the radioimmunoassay of insulin. Each sample was measured in triplicate, and three separate incubations were performed at each experimental condition in each experiment. Tabulated secretion rates represent means, and standard errors of the means are indicated (n = 3).

Glucose Concentration Insulin concentration of 5,6-EET secretion @nM) (FM) ( fkJ/min per islet)

3 0.00 0.8310.19 28 6.02 rt 0.75

3 0.01 0.94*0.11 28 5.40 +_ 1.01

3 0.05 0.75 f0.12 28 5.91 f0.77 3 0.25 0.83 i 0.08

28 5.96 i 1.43 3 1.25 0.99 f 0.40

28 6.02 f 0.68 3 6.25 1.15 kO.21

28 5.12* 1.27

12

as assessed by these methods (Fig. 6). (In separate The apparent inability of islets to synthesize experiments, isolated islets also failed to synthesize cytochrome P-450 products from arachidonate in sufficient leukotriene C, (< 50 pg) to be detecta- substantial amounts cast doubt on the possible ble by RIA under conditions where 41 ng of role of these compounds in insulin secretion. The 12-HETE were measured with GC-MS). possibility that endogenously produced EET com-

‘8 i t 16 $

:: 14 -’

:12-, t-7 b

‘I - lo- $ X bl

TC, 1 A 1

0 10 20 30 40 ELUTION VOLUME (ml)

12 Q X

8 !

4 2 2

5 15 25 35 ELUTION

VOLUME (ml)

ELUTION VOLUME (ml)

Fig. 6. Conversion of 3 H-labelled aracbidonate to [ ‘H]12-HETE and to [ 3 Hlprostaglandin E, but not to 3 H-labelled leukotrienes by

pancreatic islets demonstrated by HPLC with internal standards. About 1.5.104 pancreatic islets were isolated and cultured overnight

as described in the Methods section. The islets were then incubated with 3H-labelled arachidonate (30 PCi) in the presence of 28 mM

glucose and 10 gM A23187 for 30 min at 37°C and the incubations were terminated by addition of a mixture of internal standards (3

pg each of unlabelled leukotriene (LT) C,, D, and E, and about 50 nCi each of “C-labelled 12-HETE and prostaglandin (PG) Ez)

in 1 vol. of 80% methanol in water. After separation of particulate matter by centrifugation, the samples were diluted with water to

achieve a final methanol concentration of 5%, adjusted to pH 6.2, and applied to a disposable ODS extraction column. The initial

eluant was collected, acidified to pH 3.5 and extracted with ethyl acetate to recover any prostaglandin E, and 12-HETE which had

not remained on the column. The column was then washed with Hz0 and the leukotrienes were eluted with methanol/l mM Na,PO,

(pH 6.2) (9: 1). The eluant was extracted with ethyl acetate to remove prostaglandin E, and 12-HETE which had eluted from the

column with the leukotrienes. The leukotrienes in the aqueous phase of the eluant were then concentrated to dryness, reconstituted in

500 X of solvent G and analyzed by reverse phase-HPLC (column VI, solvent G) as shown in panel A. The labelled peaks with UV

absorbance at 280 nM correspond to the internal standards of leukotrienes C,, D,, and E,. There is no associated ‘H-labelled

material. The ethyl acetate extracts containing prostaglandin E2 and 12-HETE from above were combined, concentrated to dryness.

reconstituted in solvent H and analyzed by reverse phase-HPLC (column I, solvent H, then solvent I) to separate prostaglandin Ez

and 12-HETE. The peaks corresponding to prostaglandin E, and 12-HETE were collected separately and extracted with ethyl acetate

(prostaglandin Ez) or CHzCl, (12-HETE). The prostaglandin Ez was then analyzed by normal phase-HPLC (column II, solvent J) as

shown in panel C. The 3H-labelled peak co-eluting with the ‘“C-labelled prostaglandin Ez standard represents [ 3H]prostaglandin Ez

formed from [ ‘Hlarachidonate by the islets. The 12-HETE was converted to the methyl ester and analyzed by normal phase-HPLC

(column II, solvent K) as shown in panel B. The 3H-labelled peak co-eluting with “C-labelled 12-HETE standard represents

13H]12-HETE formed from [3H]arachidonate by the islets. In separate experiments (n = 2) about 1.5.104 islets were incubated

without [3H]arachidonate in the presence of 28 mM glucose and 10 PM A23187, and products were extracted without addition of

internal standards. Less than 50 pg of leukotriene C, (by RIA) was produced under these conditions, which result in production of

about 40 ng of 12-HETE [13,14,21].

13

pounds might undergo quantitative conversion to compounds other than DHETs and thereby escape detection could not be excluded, however. The insulin secretagogue properties of exogenous standard EET compounds were therefore ex- amined. As illustrated in Table IV the $6EET (50 nM-6.25 PM) failed to potentiate insulin secretion at substimulatory (3 mM) or maximally stimula- tory (28 mM) glucose concentrations. This is in contrast to the reported observation that exog- enous 5,6-EET stimulates insulin secretion from isolated islets at concentrations within the tested range [12]. Other EET isomers (8,9-EET, 11,12- EET and 14,15-EET) also failed to stimulate in- sulin secretion (Table V), consistent with reported observations [12]. The 5,6-EET (10 PM) also failed to reverse the suppression of glucose-induced in- sulin secretion by BW755C, as did leukotriene C4 (Table VI).

The influence of metyrapone on glucose-in- duced insulin secretion from isolated pancreatic islets was also examined (Table VII). Metyrapone (50 PM) has been demonstrated to inhibit oxygenation of arachidonate by hepatic micro- somal cytochrome P-450 [4] and has been reported to suppress glucose-induced insulin secretion from isolated islets at this concentration [12]. In con- trast to these reported results, we observed no suppression by metyrapone (50 PM) of basal or glucose-induced insulin secretion from isolated

TABLE V TABLE VII

INFLUENCE OF REGIONALLY ISOMERIC EPOXYEICOSATRIENOIC ACIDS ON INSULIN SECRE- TION FROM ISOLATED PANCREATIC ISLETS

EFFECT OF METYRAPONE AND NORDI- HYDRGGUAIARETIC ACID ON INSULIN SECRETION FROM ISOLATED PANCREATIC ISLETS

Conditions were as described in Table IV except that the experimental incubation was initiated by addition of either 8,9-EET or 11,12-EET or 14,15-EET rather than 5,6-EET.

Conditions were as described in Table IV except that the pre-incubation and incubation media contained NDGA (0 or 50 PM) or metyrapone (0 or 50 PM) and the incubation medium was not supplemented with an exogenous archidonate derivative. Glucose

concentration

(mM)

3 28

3 28

3 28

3 28

Potential secretagogue (6.25 PM)

None

14,15-EET

11,12-EET

8,9-EET

Insulin secretion ( pU/min per islet)

1.52f0.13 7.18kO.52 1.00*0.25 7.45 f 0.23 1.17f0.15 7.12 f 0.69 1.42 f 0.05 7.32f0.51

TABLE VI

INFLUENCE OF 5,6-EPOXY-8,11,14-EICOSATRIENOIC ACID (5,6-EET) AND OF LEUKOTRIENE C, (LTC,) ON INSULIN SECRETION FROM ISOLATED PANCREATIC ISLETS IN THE PRESENCE OR ABSENCE OF BW755C

Conditions were as described in Table IV except that (a) the pre-incubation and incubation media contained either 0 pM or 500 /.tM BW755C and (b) the experimental incubation was initiated by addition of either 5,6-EET (10 PM), leukotriene C, (5. lo-’ M), or diluent (controls).

Glucose Inhibitor Potential Insulin concentration secretagogue secretion (mM) ($J/min per islet)

3 28 3

28 3

28 3

28 3

28 3

28

None None

None 5,6-EET

None LTC,

BW755C None

BW755C 5,6-EET

BW755C LTC,

0.69 f 0.09 4.78 f 0.80 0.64 rtO.07 3.63 +z 1.05 0.61+ 0.07 4.01 f 1.25 0.66 f 0.06 0.94 f 0.07 0.63 f0.05 1.26*0.15 0.59*0.10 1.41 zto.13

pancreatic islets, although the lipoxygenase inhibi- tor NDGA (50 PM) clearly suppressed glucose- induced insulin secretion (Table VII). Metyrapone at a concentration of 100 PM also did not in- fluence secretion from isolated islets (not shown).

Glucose concentration

(mM)

3 28 3

28 3

28

Potential inhibitor

(50 PM)

None

Metyrapone

NDGA

Insulin secretion (@J/mm per islet)

1.06*0.17 4.82 0.87 f 1.06+0.20 4.48 1.06 f 0.89 0.27 f 1.36 0.22 f

14

Discussion

These studies demonstrate that capillary col- umn gas chromatographic-negative ion-methane chemical ionization-mass spectrometric methods can be employed to identifiy and quantitate sub- nanogram amounts of arachidonate mono- oxygenase products of cytochrome P-450 action. Other workers have previously demonstrated that such methods represent a sensitive and specific tool for the measurement of other oxygenated arachidonate metabohtes such as prostaglandins and hydroxyeicosatetraenoic acids [24-271. These methods were used to demonstrate that, as re- ported by others [2], hepatic microsomal cyto- chrome P-450 catalyzes the formation of four re- gionally isomeric epoxyeicosatrienoic acids (EETs) from arachidonate and that the corresponding hy- drolysis products (DHETs) are formed from these compounds as well.

A new observation reported here is that BW755C in~bits the cytochrome P-450-catalyzed oxygenation of arachidonate at a concentration (500 PM) which also inhibits cyclooxygenase and 12-lipoxygenase action [21,44,45]. This observation is consistent with the recently suggested hypothesis that suppression of glucose-induced insulin secre- tion from isolated pancreatic islets by hpo- xygenase inhibitors might be due to inhibition of arachidonate monooxygenase product synthesis [12]. At variance with that hypothesis, however, is the observation that the lipoxygenase inhibitors, ETYA (20 PM) and NDGA (50 PM), do not significantly inhibit arachidonate monooxygenase product synthesis but do inhibit insulin secretion from isolated pancreatic islets at the tested con- centrations [21].

Other observations arguing against participa- tion of arachidonate monooxygenase products in glucose-induced secretion from isolated pancreatic islets are that: (a) the cytochrome P-450 inhibitor metyrapone (50 PM) does not suppress glucose-in- duced insulin secretion under conditions where the lipoxygenase in~bitors do so, (b) islet microsomes do not appear to synthesize EET or DHET com- pounds from arachidonate in the presence of NADPH under conditions where synthesis of sub- stantial amounts of these compounds by hepatic microsomes is readily demonstrable, (c) intact iso-

lated islets do not appear to synthesize EET or DHET compounds under conditions where islet synthesis of the cyclooxygenase product pros- taglandin E, and the lipoxygenase product 12- HETE does occur, and (d) exogenous standards of EET compounds do not stimulate insulin secretion from isolated islets under conditions where brisk stimulation of insulin secretion by glucose is ob- served.

Others have reported that metyrapone (50 FM) does suppress glucose-induced insulin secretion from isolated islets and that exogenous 5,6-EET stimulates insulin secretion at concentrations as low as 10 nM [12]. The explanation for this dis- crepancy is not apparent. The 5.6-EET used for the studies reported here was purified by HPLC shortly before each use, and its identity was peri- odically verified by GC-MS to prevent the use of material which had undergone decomposition. The standard compound contained tracer amounts of ‘H label. and complete dissolution of the com- pound in the incubation buffer was documented by liquid scintillation counting. Diluent was also included in control incubations. The isolated islet preparations used in the present studies were clearly capable of responding to secretory stimuli, as indicated by the 5-7-fold increase in insulin secretion observed with 28 mM vs. 3 mM glucose and by the mean stimulated secretion rate of 5.7 $J of insulin per islet per min. It is of interest that in the report suggesting stimulation of insulin secretion by 5,6-EET. insulin secretion rates of only 0.20-0.33 $J of insulin per islet per min were achieved in the presence of the compound [12]. This represents less than 5% of the stimulated insulin-secretion rate achievable with glucose by functioning, viable isolated islets which suggest that the 5,6-EET could exert, at best. a minor in~uence in glucose-induced insulin secretion. The possibilities that exogenous EETs do not enter beta cells, fail to be esterified into membrane phospholipid, or are rapidly metabolized to the inactive DHETs cannot be excluded.

Several groups have now reported that dual inhibitors of arachidonate cyclooxygenase and lipoxygenase such as ETYA, NDGA and BW755C suppress glucose-induced insulin secretion from isolated islets [12-14,161, cultured pancreatic cells from rat neonates [15,17-191 and perfused pan-

15

cress preparations [20]. Selective cyclooxygenase inhibitors do not suppress glucose-induced insulin secretion in these systems [12-201. The effect of lipoxygenase inhibitors to suppress insulin secre- tion has been attributed to the participation of various oxygenated metabolites of arachidonic acid in stimulus-secretion coupling, including products of the 12-lipoxygenase [15,22], of the 5-lipo- xygenase [l&46], and of monooxygenases [12]. In- volvement of specific compounds within those groups of metabolites has been inferred from the secretagogue effects of such compounds obtained from exogenous sources and incubated with pan- creatic cell cultures [15,22], isolated islets [12,16] or perfused pancreas preparations [45]. It is not cer- tain that islets can synthesize all of these com- pounds, however, and local synthesis would be required for an arachidonate metabolite to par- ticipate in glucose-induced insulin secretion from isolated islets.

Of the three candidate pathways mentioned, products of the monooxygenase pathway appear unlikely to participate in glucose-induced insulin secretion from isolated islets in view of the data presented above. Products of the 5-lipoxygenase pathway also seem unlikely to participate in this process on the basis of published observations that: (a) islet synthesis of 5-HETE from endoge- nous arachidonate is not observed under condi- tions where synthesis of 12-HETE is clearly de- monstrable [Zl], (b) exogenous 5-HPETE does not stimulate insulin secretion from isolated islets 1211 or cultured neonatal pancreatic cells [22] under conditions where glucose stimulates and lipo- xygenase inhibitors suppress insulin secretion [21], and (c) isolated islets do not convert 3H-labelled arachidonate to 3H-labelled leukotriene B4 or to its 6-trmts isomers under conditions where conversion to 13H]12-HETE and ~3H~prostagl~din E, is ob- served [l]. New observations presented here that argue against the participation of 5-lipoxygenase products in glucose-induced insulin secretion from isolated pancreatic islets are that: (a) isolated islets do not convert ‘H-labelled arachidonate to 3H- fabelled leukotriene C,, D4 or E, under conditions where conversion to [3H]12-HETE and [3H]pros- taglandin E, is observed, (b) isolated islets convert endogenous arachidonate to less than 50 pg of leukotriene C, (by RIA) under conditions where

more than 40 ng of 12-HETE (by GC-MS) is produced, and (c) exogenous leukotriene C, neither stimulates insulin secretion from isolated islets nor reverses the suppression of secretion by the lipo- xygenase in~bitor, BW755C. Leukotrienes C, and B4 also do not stimulate insulin secretion from cultured neonatal pancreatic cells [22].

Several lines of evidence suggest that an arachidonate 1Zlipoxygenase product may par- ticipate in glucose-induced insulin secretion. Iso- lated pancreatic islets [13,14,21] and cultured neonatal pancreatic cells [15] synthesize 12-HETE both from endogenous arachidonate and exoge- nous, radiolabelled arachidonate. Quantitatively, 1ZHETE is the single, most abundant arachidon- ate metabolite so far measured from isolated islets [13,41,21], and islet production of 1ZHETE is increased by concentrations of D-glucose which stimulate insulin secretion [14]. Production of 12- HETE by isolated islets has also been shown to be stimulated by D-glucose (which stimulates insulin secretion) but not by L-glucose (which does not stimulate insulin secretion) [47]. The compounds ETYA, BW755C and NDGA suppress glucose-in- duced insulin secretion from isolated islets with a concentration dependence similar to that for in- hibition of 1ZHETE synthesis [14,21]. Exogenous 1ZHPETE stimulates insulin secretion from cul- tured neonatal pancreatic ceils [15,22], and exoge- nous 12-HETE partially reverses the suppression of glucose-induced insulin secretion by the lipo- xygenase inhibitor, ETYA, with isolated islets [21].

Observations at variance with the hypothesis that an arachidonate 1Zlipoxygenase product par- ticipates in glucose-induced insulin secretion from isolated islets are that indomethacin (10 PM) par- tially suppresses islet 12-HETE production from endogenous arachidonate under conditions where insulin secretion is unaffected [14] and that exoge- nous 12-HPETE does not reverse the suppression of insulin secretion by lipoxygenase inhibitors with isolated islets [21]. It therefore remains possible that interaction of lipoxygenase inhibitors with some intracellular target distinct from the 12-lipo- xygenase accounts for the suppression of glucose- induced insulin secretion observed with these com- pounds. Neither the arachidonate 5-lipoxygenase nor arachidonate monooxygenase activities seem likely candidates for such a target, however.

16

The arachidonate monooxygenase product 5,6-

EET has been postulated to participate in stimu-

lus-secretion coupling in other endocrine tissues,

including anterior pituitary [lo] and hypothalamic

median eminence [ll]. The quantitative detection

methods described here for the 5,6-EET and its hydrolysis product, 5,6-DHET, should facilitate

evaluation of the possible role of these compounds in these processes. In addition, the demonstration

here that the compound BW775C suppresses

arachidonate monooxygenase product formation suggests that such products be considered as among

the potential mediators in other stimulus-response

events in which BW755C has been demonstrated

to interfere [48].

Acknowledgements

This work was supported in part by a grant to

MLM from the National Institutes of Health (AM 06181) by grants to JT from the Pharmaceutical Manufacturer’s Association Foundation, the

Juvenile Diabetes Foundation, and the National

Institutes of Health (AM 34388), and by grants to BJ from the National Institutes of Health (HL

31922 and HL 21874). The excellent technical

assistance of Richard Thoma, William Thomas Stump, Beverly DeLoach, Deirdre Buscetto and C.

Joan Fink has been greatly appreciated as has the interest and advice of Dr. Jay McDonald, Dr. Paul

Lacy and Dr. Philip Needleman. Special thanks

are due to Jane Huth for the preparation of the manuscript. We are grateful to Dr. Aubrey Morri- son and to Dr. William Sherman for providing

access to the Hewlett-Packard 5985B mass spec- trometer within the Washington University Mass

Spectrometry Resource Center, which is supported by NIH grant RR 00954. We are also, grateful to

Dr. Ernst Oliw for advice regarding chemical synthesis and storage of the epoxy- and dihy- droxyeicosatrienoic acids.

References

Oliw, E., Lawson, J., Brash, A. and Oates, J. (1981) J. Biol.

Chem. 256, 9924-9931

Oliw, W., Guengerich, F. and Oates, J. (1982) J. Biol.

Chem. 257, 3771-3781

Capdevila, J., Parkhill, L., Chacos, N., Okita, R., Masters, B. and Estabrook, R. (1981) B&hem. Biophys. Res. Com-

mun. 101, 1357-1363

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

Capdevila. J., Chacos, N., Wehringloer, J.. Prough, R. and

Estabrook, R. (1981) Proc. Natl. Acad. Sci. USA 9,

5362-5366

Morrison, A. and Pascoe, N. (1981) Proc. Natl. Acad. Sci.

USA 78, 7375-7378

Chacos, N., Capdevila, J., Falck, J.. Manna, S., Martin-

Wixtrom, C., Gill, S.. Hammock, B. and Estabrook, R.

(1983) Arch. B&hem. Biophys. 223, 639-648

Oliw, E. and Moldeus, P. (1982) B&him. Biophys. Acta

721, 135-143

Capdevila, J., Pramanik, B., Napoli, J., Manna, S. and

Falck, J. (1984) Arch. Biochem. Biophys. 231, 511-517

Falck. J., Manna, S., Jacobsen, H., Estabrook, R., Chacos,

N. and Capdevila, J. (1984) J. Am. Chem. Sot. 106.

3334-3336

Snyder, G., Capdevila, J., Chacos, N., Manna, S. and Falck,

J. (1983) Proc. Natl. Acad. Sci. USA 80, 3504-3507

Capdevila. J., Chacos, N., Falck, J.R.. Manna. S.. Negro-

Vilar, A. and Ojeda, S. (1983) Endocrinology 113,421-423

Falck. J., Manna, S., Moltz, J.. Chacos, N. and Capdevila, J.

(1983) Biochem. Biophys. Res. Commun. 114, 743-749

Turk, J.. Colca, J. Kotagal, N. and McDaniel, M. (1984)

Biochim. Biophys. Acta 794, 110-124

Turk. J., Colca, J. Kotagal, N. and McDaniel, M. (1984)

Biochim. Biophys. Acta 794, 125-136

Metz, S., VanRollins, M., Strife, R., Fujimoto, W. and

Robertson, R. (1983) J. Clin. Invest. 71, 1191-1205

Yamamoto, S., Ishii, M., Nakadate, T., Nakaki, T. and

Kato, R. (1983) J. Biol. Chem. 258, 1214412152

Metz, S., Fujimoto, W. and Robertson, R. (1982) Endo-

crinology 111, 2141-2143

Metz, S., Fujimoto, W. and Robertson, R. (1983) Life Sci.

32, 903-910

Metz, S., Little, S., FuJimoto, W. and Robertson, R. (1983)

Adv. Prost. Thromb. Leuk. Res. 12, 271-277

Walsh. M. and Pek, S. (1984) Life Sci. 34. 1699-1706

Turk, J., Colca, J. and McDaniel. M. (1985) Biochim.

Biophys. Acta 834, 23-36

Metz, S.. Murphy, R. and Fujimoto, W. (1984) Diabetes 33.

119-124

Greely, R. (1974) J. Chromatogr. 88, 229-233

Blair, I., Barrow, S., Waddell, K., Lewis, P. and Dollery, C.

(1982) Prostaglandins 23, 579-589

Waddel, K., Robinson, C., Orchard, S., Barrow, S., Dollery,

C. and Blair, I. (1983) Int. J. Mass. Spec. Ion Phys. 48,

233-236

Strife, R. and Murphy, R. (1984) Prost. Leuk. Med. 13, l-8

Strife, R. and Murphy, R. (1984) J. Chrom. 305, 3-12

Taber, D.F., Philips, M.A. and Hubbard, W.C. (1981) Pros-

taglandins 22, 349-352

Boeynaems, J.M., Brash, A.R., Oates, J.A. and Hubbard,

WC. (1980) Anal. Biochem. 104, 259-267 Chacos, N., Falck, J.R., Wixtrom, C. and Capdevila, J.

(1982) B&hem. Biophys. Res. Commun. 104, 916-922

Corey, E.J., Niwa, H. and Falck, J.R. (1979) J. Am. Chem.

Sot. 101, 1586-1587

Manna, S., Falck, J.R., Chacos, N. and Capdevila, J. (1983)

Tetrahedron Lett. 24, 33-36

17

33 Falardeau, P. and Brash, A.R. (1982) Methods Enzymol. 86, 585-592

34 McDaniel, M., Colca, J., Kotagal, N. and Lacy, P. (1983) Methods Enzymol. 98,182-196

35 Lacy, P.E. and Kostianovsky (1967) Diabetes 16, 35-39 36 Shibata, A., Ludvigson, C.W., Naber, S.P., McDaniel, M.L.

and Lacy, P.E. (1976) Diabetes 25, 667-672 37 Powell, W.S. (1982) Methods Enzymol. 86, 467-477 38 Anderson, F., Westcott, J., Zirolli, i and Murphy, R.C.

(1983) Anal. Chem. 55, 1837-1839 39 Metz, S.A., Hall, M.E., Harper, T.W. and Murphy, R.C.

(1982) J. Chromatogr. 233, 193-201 40 Wright, P.H., Mukuli, P.R., Vichick, P. and Susman, K.E.

(1971) Diabetes 20, 33-45 41 McDaniel, M., Colca, J. and Kotagal, N. (1984) Methods in

Diabetes Research l(B) (Lamer, J. and Pohl, S., eds.), pp. 153-166, John Wiley and Sons, New York

42 Lowry, G.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275

43 Majerus, P.W. and Prescott, S.M. (1982) Methods Enzymol. 86.11-17

44 Van Wauwe, J. and Goosens, J. (1983) Prostaglandins 26, 725-730

45 Salari, H., Braquet, P. and Brogeat, P. (1984) Prost. Leuk. Med. 13, 53-60

46 Pek, S.B. and Walsh, M.F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2199-2202

47 Metz, S.A. (1984) Diabetes 33 (Supplement l), 165 48 Sasakawa, N., Yamamoto, S., Kumakura, K. and Kato, R.

(1983) Jpn. J. Pharmacol. 33, 1077-1080 49 Evers, A.E., Murphree, S., Saffitz, J.E., Jakschik, B.A. and

Needleman, P. (1984) J. Clin. Invest. 15, 992-999