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Arachidonic acid metabolites by cytochrome P-450 dependent monooxygenase pathway in bovine adrenal fasciculata cells
PROSTAGLANDINS
ARACHIDONIC ACID METABOLITES BY CYTOCHROME P-450 DEPgWDE KONOOXYGENASE PATRWAY IN BOVINE ADRENAL FASCICULATA CELLSNT M. Nishimura, A. Hirai, M. Omura, Y. Tamura, and S. Yoshida The Second Department Of Internal Medicine, School Of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan
[1-14C]Arachidonic acid was incubated with microsomes of bovine adrenal fasciculata cells in the presence of 1 mM NADPH for 30 min The metabolites were separated and purified by reverse at 37°C. phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry. Identified metabolites were four dihydroxyeicosatrienoic acids (DHTs) (5,6-, 8,9-, 11,12-, 14,15DHTs), 20-hydroxyeicosatetraenoic acid and eicosatetradioic acid. The formation of these metabolites was dependent on NADPH and inhibited by SKF-525A. 14,15-DHT was also formed by isolated bovine adrenal fasciculata cells. These results indicate that cytochrome P-450 dependent arachidonate monooxygenase pathway may exist in bovine adrenal fasciculata cells. Addition of the chemically synthesized epoxyeicosatrienoic acids (EETs) to isolated bovine adrenal fasciculata cells stimulated cortisol production. Among four regioisomeric EETs, 14,15-EET was most potent and stimulated steroidogenesis in a dose-related manner over a range of 0.5 to 5.0 uM.
Introduction Arachidonic acid has been shown to be metabolized by cytochrome P-450 dependent monooxygenase pathway in various organs and tissues such as: the kidney (l-3), the liver (4-8), the cornea (9,101, the hypothalamus (11) and the pituitary gland (12). This pathway converts arachidonic acid to several oxygenated metabolites, i.e. monohydroxyeicosatetraenoic acids (HETEs), w-, w-l-oxidation products and four regioisomeric epoxyeicosatrienoic acids (EETs) which are further converted to corresponding dihydroxyeicosatrienoic acids (DHTs) by epoxide hydrolase. These metabolites have been shown to have various biological activities, i.e. stimulation of secretion of peptide hormones (11,13-17), relaxation of blood vessels (18) and inhibition or stimulation of Na-K-ATPase (19-21).
lpreliminary data were presented at Taipei Conference on Prostaglandin and Leukotriene Research, Taipei, Taiwan, R.O.C. during April, 1988.
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It is well known that steroidogenesis in the adrenal cortex involves the sequential action of four distinct forms of cytochrome P-450. Among these enzymes, 17a-hydroxylase and 21-hydroxylase are localized in microsomes. In adrenal fasciculata cells, arachidonic acid has been shown to be metabolized by cyclooxygenase (22) and lipoxygenase pathways (23). However, no studies have been reported concerning the metabolites of arachidonic acid by cytochrome P-450 dependent pathway in adrenal cortex. Therefore, the present study was performed firstly to determine whether the bovine adrenal fasciculata cells may have the capacity to metabolize arachidonic acid via the cytochrome P-450 dependent monooxygenase pathway, and secondly, to examine the effect of the metabolites of this pathway on the production of cortisol by isolated bovine adrenal fasciculata cells. Materials
and Methods
1. Materials [l-14C]Arachidonic acid (56 mCi/mmol) was obtained from New England Nuclear (Boston, MA, U.S.A.) and arachidonic acid (99.9% pure) from Nu-Chek Prep. (Elysian, Mine., U.S.A.). N,O-Bis(trimethylsilyl)trifluoroacetamide, N-trimethylsilyl imidazole and all solvent for high performance liquid chromatography (HPLC) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Eagle's minimum essential medium (MEM) was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). N-methyl-N'-nitro-Nnitrosoguanidine, collagenase type I, deoxyribonuclease type II and bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). NADPH was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). SKF-525A was a generous gift from Smith Kline and French (Philadelphia, Penn., U.S.A.). Cortisol Kit II was purchased from the First Radioisotope Co. (Tokyo, Japan). Aldosteron RIA Kit was purchased from Dinabot Co. (Tokyo, Japan). COAT-A-COUNT 17a-OH progesterone RIA Kit was purchased from Diagnostic Products Corporation (CA, U.S.A.). 2. Preparation
of authentic
standards
of EETs,
DHTs
and HETEs
Standards of EETs and DHTs were prepared by non-selective epoxydation of arachidonic acid as described by Chung and Scott The epoxide mixture was separated by straight phase (SP)-HPLC (24). using a HPLC pump 880-PU [Japan Spectroscopic Co., Ltd. (JASCO), Tokyo, Japan] equipped with a silica column (Develosil 5 pm, 20 mm x 500 mm, Nomura Chemical Co., Ltd., Seto, Aichi, Japan), with a solvent system of n-Hexane/isopropanol/acetic acid (100:0.6:0.05 The UV absorption of column effluent was monitored at by vol.). 206 nm using a UV detector 875-UV (JASCO, Tokyo, Japan). The corresponding DHTs were obtained by acidic aqueous hydrolysis followed by the purification by reverse phase (RP)-HPLC.
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Hydroperoxyeicosatetraenoic acids (HPETEs) were synthesized by photo-oxidation method (25) with minor modifications. Synthesized HPETEs were purified by SP-HPLC and RP-HPLC as previously reported (26). HETEs were prepared by the reduction of HPETEs by NaBH4 (27) followed by SP-HPLC purification. 3. Animal and tissue preparation Fresh bovine adrenal glands were obtained from local abattoir and brought to the laboratory within 1 hour after the animals were slaughtered. Bovine adrenal fasciculata zone were prepared according to the method of Hadjian et al. (28) and Karlmar (29) with minor modifications. The adrenals were bisected at their largest diameter and the medulla extracted thoroughly. Starting from the inner zone of the cortex, thin slices (0.5 mm) were cut using a slicer (Natsume Seisakusho, Tokyo, Japan); the first two slices were discarded and the next two slices were pooled and stored in MEM containing 0.1% BSA. Microscopic examination of the pooled slices showed that tissue contained mainly fasciculata cells and no glomerulosa cells were present. After four washes with this buffer, the slices were minced and incubated in MEM containing 0.1% BSA, 0.25% collagenase type I and 0.05 mg/ml deoxyribonuclease type II at 37°C for 30 min in 5% CO, and 95% 0,. Incubation mixtures were centrifuged at 100 x g for 10 min at room temperature. The cells were resuspended in Krebs-Ringer bicarbonate (KRB) buffer containing 0.2% glucose and 0.1% BSA and filtered through cotton gaze and centrifuged at 100 x g for 10 min at room temperature. The cellular pellet was suspended in the same buffer for washing. This washing procedure was repeated three times. Finally 2 x lo5 cells were suspended in 1 ml of KRB buffer containing 0.2% glucose. The formation of cortisol, 17a-hydroxyprogesterone and aldosterone by isolated bovine adrenal fasciculata cells was determined by the use of specific radioimmunoassays (30,31) according to the method of Hadjian et al. (28) with minor modifications. The isolated cells produced cortisol and 17a-hydroxyprogesterone in response to the stimulation with ACTH or angiotensin II, while no detectable formation of aldosterone was observed. For the preparation of microsomes, all procedure was carried out at 0 to 4°C. Slices of the bovine adrenal fasciculata zone prepared as described above was pooled and stored in ice-cold buffer containing 30 mM potassium phosphate, pH 7.4, 0.25 M sucrose and 0.1 mM EDTA (buffer A). After four washes with buffer A, the slices were weighed and chopped into small pieces and homogenized with 2 volumes of buffer A. The homogenate was centrifuged at 10,000 x g for 60 min at 4°C. The 10,000 x g supernatant was centrifuged at 105,000 x g for 60 min. The resulting microsomal pellet was resuspended in 100 mM potassium phosphate buffer, pH 7.4 (2 mg protein/ml).
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4. Protein
determination
The microsomal protein was determined by the method et al. (32) with bovine serum albumin as standard. 5. Metabolism
of arachidonic
of Lowry
acid
(a) Metabolism of arachidonic fasciculata cells
acid by microsomes
of bovine
adrenal
[l-14C]Arachidonic acid (900 ug, 3 PCi) was incubated with microsomes (60 mg protein) in the presence or absence of 1 mM NADPH for 30 min at 37°C. The reaction was terminated by the addition of 2 volume of ice-cold methanol. The methanolic incubation mixture was acidified to pH 3.5 with 2 N citrate and centrifuged at 1,500~ g for 10 min at 4°C. Clear supernatant was diluted with water to make a 10% methanol-aqueous solution and applied to a prewetted octadecyl silica (ODS) mini column (Bond Elute CI8 6 cc, Analytichem International, Harbor City, CA, U.S.A.) followed by a wash with water. Arachidonic acid metabolites were eluted by 6 ml of ethyl acetate. The ethyl acetate fraction was evaporated under nitrogen gas stream and resolved in 50% methanol. microsomes were preincubated In experiments using inhibitors, with agents for 10 min before the addition of NADPH and [1-14C]arachidonic acid. An incubation of [l-'4C]arachidonic acid with inactivated microsomes (boiled microsomes) was performed to assess possible non-enzymatic autooxidation of arachidonic acid. (b) Metabolism of arachidonic fasciculata cells
acid by isolated
bovine
adrenal
Isolated bovine adrenal fasciculata cells were suspended in KRB buffer containing 1 mM calcium (Caa+) and 0.2% glucose (5 x lo6 cells/l0 ml), and incubated with [l-14C]arachidonic acid (17.5 ug, 3 UCi) at 37°C for 15 min. The termination of the reaction and extraction of the metabolites were performed in the same way as described in the method of arachidonate metabolism by microsomes. Extracted metabolites were analyzed by RP- and SP-HPLC. 6. HPLC
analysis
of arachidonic
acid metabolites
The extracted radioactive metabolites were separated by RP-HPLC using a 880-PU liquid chromatograph (JASCO, Tokyo, Japan) equipped with an ODS column (Develosil 5 pm, 4.6 mm x 150 mm, Nomura Chemical The chromatography was performed at Co., Ltd., Seto, Aichi, Japan). 30°C with isocratic elution with a solvent system of methanol/ acetonitrile/water/acetic acid (45:25:30:0.01 by vol.). The flow rate was 1 ml/min. Radioactivity in eluate from HPLC was monitored by a Beckman 171 radioisotope detector (Beckman Instruments, Inc., Fullerton, CA, U.S.A.). Elution times for 14,15-DHT, 11,12-DHT,
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8,9-DHT and 5,6-DHT were 14.4,16.3,18.2 and 22.4 min, respectively. Production of the metabolites was expressed as "pmol of arachidonic acid (AA) converted/mg of protein/30 min" according to the method of Schwartzman et al. (20). Each of the radioactive products separated by RP-HPLC was collected and converted to the methyl ester derivative by the reaction with ethereal diazomethane and further purified by SP-HPLC using a JASCO 880-PU liquid chromatograph equipped with a silica column (Unisil Q 3 urn,4.6 x 150 mm, Gasukuro Kogyo Inc., Tokyo, Japan). The flow rate was 1 mljmin and the column was maintained at 30°C. Separation was carried out using a gradient of isopropanol in n-Hexane containing 0.05% acetic acid. The initial solvent was 1.5% isopropanol in n-Hexane. After 25 min the concentration of isopropanol was linearly increased to 5% over 10 min and held at this concentration for an additional 15 min. Elution times of the methyl ester derivatives of 14,15-DHT, 11,12-DHT, 8,9-DHT and 5,6-DHT were 17.9, 15.6, 20.2 and 35.0 min, respectively. 7. Derivatization for GC/MS analysis The methyl ester derivatives purified by SP-HPLC were silylated with N,O-bis(trimethylsilyl)trifluoroacetamideand N-trimethylsilylimidazole (1:2 by vol.) for 60 min at room temperature. 8. GC/MS analysis Gas chromatography-mass spectrometry (GC/MS) analysis was performed on a Hitachi M-80 GC/MS system (Hitachi, Tokyo, Japan). The GC column was 1.5% OV 101 (2 m) and operated isothermically at 21OOC. Electron impact was 70 eV. C values were determined from the retention times of fatty acid methyl esters (C1B-C24) according to the method of Miwa et al. (33) with minor modifications. 9. Cell incubation and cortisol assay Collagenase-dispersed bovine adrenal fasciculata cells were resuspended in 1 ml of KRB buffer containing 1 mM Ca2+ and 0.2% glucose. Aliquots (950 ~1) of cell suspension (2 x lo5 cells/ml) were distributed in randomized plastic dishes and preincubated at 37°C in 5% CO, and 95% 0s. After 15 min of preincubation, the reaction was started by the addition of 50 ~1 of an aqueous vehicle (KRB buffer containing 4% ethanol) in which the various concentration of four regioisomeric EETs were dissolved and incubated for 30 min. Control incubations were carried out by the addition of 50 ~1 of vehicle solution alone. Cortisol was measured by a specific radioimmunoassay (30). Results were expressed as net cortisol production (ng/2 x lo5 cells/30 min), after subtraction of the corresponding control value. 10. Statistical analysis Comparisons were made using the non-paired Student's t-test.
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Results 1. HPLC analysis of the arachidonate metabolites by microsomes of bovine adrenal fasciculata cells RP-HPLC resolved several radioactive peaks (Fig. 1). The polar metabolites which eluted between 3 to 7 min co-chromatographed with prostaglandins and they were not further characterized in this study. The elution times of Peak I, Peak II, Peak III were identical to those of the authentic standards of 14,15-DHT, 11,12DHT and 8,9-DHT. The elution time of Peak IV was a little later than that of the authentic standards of 8,9-DHT, and the elution time of Peak V was a little bit earlier than that of the authentic standard of 5,6-DHT. Two minor radioactive peaks were eluted at the retention times of 5- and 15-HETE. However, both were also formed
14, 15-11,12DHT
DHT
8*9-
5.6)Hl r
DHT
11
1 ,x0.2
I
I
lJJ II
Ill
/
5
10
15 retention
time
20
‘
25 (mid
Fig. 1. Separation of [l-'4C]Arachidonate Metabolites Produced by Microsomes of Bovine Adrenal Fasciculata Cells. Products were extracted and analyzed as described in "Materials and Methods". The radioactive peaks were named Peaks I, II, III, IV and V as indicated. Each peak was collected and methylated and further purified by SP-HPLC. The arrows denote the retention times of the authentic standards.
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to similar extents in a 30 min control incubation of [1-14C]arachidonic acid with boiled microsomes. Small radioactive peaks were observed near the retention times of the authentic standards of EETs. However, they were not identified because of sample limitation. The methyl ester derivatives of Peak I, Peak II, Peak III and Peak IV were eluted as single peaks on SP-HPLC. The methyl ester derivative of Peak V was separated into two radioactive peaks by SP-HPLC (Fig. 2). The materials eluted at 16.0 min and 35.0 min were referred to as the methyl ester derivatives of Peaks VA and VB, respectively. The elution time of the methyl ester derivative of Peak VB was identical to that of the authentic standards of the methyl ester derivative of 5,6-DHT.
5,6-DHT-Me
1 I--
I’
-----_------___
10 retention
20 time
30
(min)
Pig. 2. Separation of the Methyl Ester Derivative of Peak V. The methyl ester derivative of Peak V was analyzed by SP-HPLC as described in "Materials and Methods". It was resolved to two radioactive peaks and named PeaksVA and VB as indicated. The arrow denotes the retention time of the authentic standard.
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2. GC/MS
analysis
Metabolites of arachidonic acid by microsomes fasciculata cells were identified by GC/MS.
of bovine
adrenal
Mass spectrum of the methyl ester trimethylsilyl derivative of Peak I is shown in Fig. 3. This compound showed prominent ions at Signals were also noted at 496 (tic), 481 (e-15) 173, 275 and 323. The mass spectrum of this compound was identical and 465 (Mf-31). with that of the authentic standard of 14,15-DHT and the material in Peak I was identified as 14,15-DHT. Mass spectrum of the methyl ester trimethylsilyl derivative of Peak II is shown in Fig. 4. This compound showed prominent ions at The mass spectrum of this compound 315, 295 (M+-111-90) and 213. was identical with that of the authentic standard of 11,12-DHT and the material in Peak II was identified as 11,12-DHT. Mass spectrum of the methyl ester trimethylsilyl derivative of Peak III is shown in Fig. 5. This compound showed prominent ions at 243, 253 and 255 (345-90). Signals were also noted at 345 and 355. The mass spectrum of this compound was identical with that of the authentic standard of 8,9-DHT and the material in Peak III was identified as 8,9-DHT. Mass spectrum of the methyl ester trimethylsilyl derivative of Peak VB is shown in Fig. 6. This compound showed prominent ions at Signals were also noted at 496 203, 215 (305-90), 293 and 305. CM+), 481 (M+-15), 465 (I@-31) and 406 (k?-90). The mass spectrum of this compound was identical with that of the authentic standard of 5,6-DHT and the material in Peak VB was identified as 5,6-DHT. Mass spectrum of the methyl ester trimethylsilyl derivative of Peak VA is shown in Fig. 7. The material in Peak VA was identified as 20-hydroxyeicosatetraenoic acid and distinguished from (a) This 19-hydroxyeicosatetraenoic acid by following criteria. compound showed ions at 406 (tit), 391 (I@-15), 375 c&-31), 316 W-90) and 103, and lacks strong signals in lower mass range including 117 which is characteristic of 19-hydroxyeicosatetraenoic acid. (b) C value of the methyl ester trimethylsilyl derivative of Peak VA was 22.6. This was identical with that of 20-hydroxyeicosatetraenoic acid reported by Oliw et al. (3). Mass spectrum of the methyl ester derivative of Peak IV showed The mass signals at 362 (M+), 247, 234, 221, 208 and 194 (Fig. 8). spectrum of this compound was identical with published spectrum of 5,8,11,14-eicosatetradioic acid (3) and the material in Peak IV was identified as 5,8,11,14-eicosatetradioic acid.
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(x)
OCTOBER 1989 VOL. 38 NO. 4
A$!suaau!
an!aela.d
421
150
zo200
250
I, I q&
215
(305-90)
203
1
m/z
r
m/z
350
233 203 _-_T__-
x10
450
500
III
of Peak VB
Y5
bJl+
of Peak
PA+-31
Derivative
hl+-W-l
400
r
Ester Trimethylsilyl acid).
300
293
305
355
.yl Derivative
253 243 ---T---
“JMSO~OTMS
305 c___
Ester Trimethylsi acid).
q 1 I
6. Mass Spectrum of the Methyl (5,6-dihydroxy-8,11,14-eicosatrienoic
Pig.
!!_
f ._ G
6040-
80-
6
CI .E
loo-
C;
Fig. 5. Mass Spectrum of the Methyl (8,9-dihydroxy-5,11,14-eicosatrienoic
25 4
(345-00) 255
PROSTAGLANDINS
(%) b!sUWU! eA!aelaJ
OCTOBER 1989 VOL. 38 NO. 4
(%) r($!SUWl!
tM!alZleJ
423
PROSTAGLANDINS 3. Studies on the formation of Peaks I through V by microsomes of bovine adrenal fasciculata cells The amounts of [l-14C]arachidonic acid converted to Peaks I through V were 10.422.2, 5.020.8, 1.720.3, 34.0+6.6 and 168.3235-l pmol of AA convertedlmg of protein/30 min, respectively (mean *S.E., n=4). No detectable formation of Peaks I through V was observed in the absence of NADPH. SKF-525A (50-200 pM) dose dependently reduced the formation of Peaks I through V (data not shown). 4. HPLC analysis of the arachidonate metabolites by isolated bovine adrenal fasciculata cells Among Peaks I through V, the formation of Peak I alone was observed in experiment using isolated bovine adrenal fasciculata cells. The amount of [l-'4C]arachidonic acid converted to Peak I was 28.3i5.3 pmol of AA converted/lo6 cells/l5 min (mean+S.E., n=4).
**
T control
5,6EET
8.9EET
11,12EET
14,15EET
Fig. 9. Effect of EETs (5 PM) on Steroidogenesis in Bovine Adrenal Fasciculata Cells. Vertical bars indicate mean + S.E. (n = 6). *: pco.05, **: pcO.01, vs control.
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5. Effect of EETs on steroidogenesis The effect of the addition of chemically synthesized EETs (5 uM) on steroidogenesis of bovine adrenal fasciculata cells are shown in Fig. 9. Among four regioisomeric EETs, 14,15-EET was most potent. And as shown in Fig. 10, 14,15-EET stimulated steroidogenesis dose dependently over the concentration of 0.5-5.0 uM.
0
0.5
2.5
14,15-EET
5.0
10.0
(PM)
Effect of 14,15-EET (0.5-10.0 nM) on Steroidogenesis in Bovine Adrenal Fasciculata Cells. Vertical bars indicate mean 2 S.E. (n = 6). *: ~~0.05, **: p
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Discussion The cytochrome P-450 dependent enzyme system is well known for steroidogenesis in the adrenal cortex and have been intensively investigated (34,35). However, no studies have been reported concerning cytochrome P-450 dependent monooxygenase pathway for arachidonic acid in adrenal fasciculata cells. Therefore, the present study was performed in order to elucidate whether this metabolic pathway may exist in bovine adrenal fasciculata cells. [1-14ClArachidonic acid was incubated with microsomes of bovine adrenal fasciculata cells in the presence of NADPH. The metabolites were separated and identified by GC/MS. 20-Hydroxyeicosatetraenoic acid, eicosatetradioic acid and four regioisomeric DHTs (5,6-, 8,9-, 11,12- and 14,15-DHTs) were identified. The formation of these metabolites was dependent on NADPH and inhibited by the addition of SKF-525A, a specific inhibitor of cytochrome P-450 dependent monooxygenase pathway. Detectable formation of EETs was not observed in this study. EETs have been reported to be unstable and rapidly converted to corresponding DHTs by epoxide hydrolase which exists in microsomes and cytosol (6). Therefore, it seems to be likely that four regioisomeric DHTs might be formed via unstable intermediates, EETs. It has been reported that arachidonic acid is also converted to HETEs (36) and 19-hydroxyeicosatetraenoic acid (3) by cytochrome P-450 dependent monooxygenase. Two minor radioactive peaks which co-chromatographed with the authentic standards of 5- and 15-HETE were found in this study. However, both were also formed to similar extents in a 30 min control incubation of [1-14Clarachidonic acid with boiled microsomes. Therefore, it seems less likely that these peaks might be formed enzymatically. No detectable formation of 19-hydroxyeicosatetraenoic acid was observed in this study, suggesting that further study might be required. In the experiment using isolated bovine adrenal fasciculata cells, only Peak I was formed among Peaks I through V. This means that there was a discrepancy between the profiles of arachidonate metabolites formed by the intact cells and the microsomes. Similar observation was also described by Schwartzman et al. in the experiment using bovine cornea1 epithelium (20), though the exact mechanism of the discrepancy still remains indeterminate yet. From the results of the present study using microsomes and isolated cells, it is quite likely that cytochrome P-450 dependent arachidonate monooxygenase pathway may exist in bovine adrenal fasciculata cells. Arachidonic acid and its metabolites have been shown to play an important role in the signal transduction in various cells including endocrine cells. In adrenal fasciculata cells, it has been reported that arachidonic acid was metabolized by cyclooxygenase (22) and lipoxygenase pathways (23). We previously reported that one of the 5-lipoxygenase metabolites, "5-HPETE", may play an important role in ACTH-stimulated steroidogenesis in rat adrenal fasciculata cells (23). As mentioned above, the present study demonstrated the presence of cytochrome P-450 dependent monooxygenase pathway for arachidonic acid in bovine adrenal fasciculata cells. Therefore we investigated whether the metabolites of arachidonic acid by cytochrome P-450 dependent monooxygenase may have any biological effects on bovine adrenal fasciculata cells. A notable finding was
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that EETs, active metabolites of cytochrome P-450 dependent monooxygenase pathway, stimulated cortisol production by bovine adrenal fasciculata cells. Among four regioisomeric EETs, 14,15-EET was most potent and stimulated steroidogenesis at the concentration of 0.5-5.0 pM. Several metabolites of arachidonic acid by cytochrome P-450 dependent monooxygenase pathway have been shown to have various biological activities, i.e. stimulation of secretion of peptide hormones (11,13-17), relaxation of blood vessels (18) and inhibition or stimulation of Na-K ATPase (19-21). The present finding that EETs can stimulate cortisol production in bovine adrenal cortex is the first observation for steroidogenic effect of EETs. Further study seems to be required to elucidate the mechanism of the action of EETs on adrenal steroidogenesis. Acknowledgment
The authors are most grateful to Dr. Yasuo Shida for his valuable contribution to GC/MS analysis. And the authors would like to thank Mr. Yoshio Yamauchi and Mr. Tsutomu Tomita for helpful advices in developing HPLC procedures. This study was partly supported by a Grant-in-Aid from the Ministry of Health and Welfare and a research grant (63570522) from the Scientific Research Fund of the Ministry of Education, Japan. References 1.
Morrison, A.R., and N. Pascoe. Metabolism of arachidonate through NADPH-dependent oxygenase of renal cortex. Proc. Natl. Acad. Sci. U.S.A. 78~7375. 1981.
2.
Schwartzman, M.L., N.G. Abraham, M.A. Carroll, R.D. Levere, and J.C. McGiff. Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney. Biochem. J. 238:283. 1986.
3.
Oliw, E.H., J.A. Lawson, A.R. Brash, and J.A. Oates. Arachidonic acid metabolism in rabbit renal cortex. J. Biol. Chem. 256:9924. 1981.
4.
Capdevila, J., N. Chacos, J. Werringloer, R.A. Prough, and R.W. Estabrook. Liver microsomal cytochrome P-450 and the oxidative metabolism of arachidonic acid. Proc. Natl. Acad. Sci. U.S.A. 78:5362. 1981.
5.
Oliw, E.H., and P. Moldeus. Metabolism of arachidonic acid by isolated rat hepatocytes, renal cells and by some rabbit tissues. Biochim. Biophys. Acta 721:135. 1982.
6.
Oliw, E.H., F.P. Guengerich, and J.A. Oates. Oxygenation of arachidonic acid by hepatic monooxygenases. J. Biol. Chem. 257:3771. 1982.
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Chacos, N., J.R. Falck, C. Wixtrom, and J. Capdevila. Novel epoxides formed during the liver cytochrome P-450 oxidation of Biochem. Biophys. Res. Commun. 104:916. arachidonic acid. 1982.
8.
Schwartzman, M.L., K.L. Davis, J.C. McGiff, R.D. Levere, and N.G. Abraham. Purification and characterization of cytochrome P-450-dependent arachidonic acid epoxygenase from human liver. J. Biol. Chem. 263:2536. 1988.
9.
Schwartzman, M.L., J. Masferrer, M.W. Dunn, J.C. McGiff, and N.G. Abraham. Cytochrome P450, drug metabolizing enzymes and Current arachidonic acid metabolism in bovine ocular tissues. Eye Res. 6:623. 1987.
10.
Schwartzman, M.L., N.G. Abraham, J. Masferrer, M.W. Dunn, J.C. McGiff. Cytochrome P450 dependent metabolism of arachidonic acid in bovine cornea1 epithelium. Biochem. Biophys. Res. Commun. 132:343. 1985.
and
11.
Capdevila, J., N. Chacos, J.R. Falck, S. Manna, A. Negro-Vilar, and S.R. Ojeda. Novel hypothalamic arachidonate products stimulate somatostatin release from the median eminence. Endocrinology 113:421. 1983.
12.
Capdevila, J., G.D. Snijder, and J.R. Falck. Epoxygenation arachidonic acid by rat anterior pituitary microsomal fractions. FEBS 178:319. 1984.
13.
Negro-Vilar, A., G.D. Snyder, J.R. Falck, S. Manna, N. Chacos, and J. Capdevila. Involvement of eicosanoids in release of oxytocin and vasopressin from the neural lobe of the rat pituitary. Endocrinology 116:2663. 1985.
14.
Snyder, G., F. Lattanzio, P. Yadagiri, J.R. Falck, and J. Capdevila. 5,6-Epoxyeicosatrienoic acid mobilizes Caz+ anterior pituitary cells. Biochem. Biophys. Res. Commun. 139:1188. 1986.
of
in
15.
Luini, A.G., and J. Axelrod. Inhibitors of the cytochrome P-450 enzymes block the secretagogue-induced release of corticotropin in mouse pituitary tumor cells. Proc. Natl. Acad. Sci. U.S.A. 82:1012. 1985.
16.
Cashman, J.R., D. Hanks, and R.I. Weiner. Epoxy derivatives of arachidonic acid are potent stimulators of prolactin secretion. Neuroendocrinology 46~246. 1987.
17.
Falck, J.R., S. Manna, J. Moltz, N. Chacos, and J. Capdevila. Epoxyeicosatrienoic acids stimulate glucagon and insulin release from isolated rat pancreatic islets. Biochem. Biophys.
428
OCTOBER 1989 VOL. 38 NO. 4
PROSTAGLANDINS
Res.
Commun.
114:743.
1983.
18.
Carroll, M.A., M. Schwartzman, .I. Capdevila, J.R. Falck, Vasoactivity of arachidonic acid epoxides. J.C. McGiff. J. Pharmacol. 138:281. 1987.
19.
Schwartzman, M., N.R. Ferreri, M.A. Carroll, E. Songu-Mize, and J.C. McGiff. Renal cytochrome P450-related arachidonate metabolite inhibits (Na++K+) ATPase. Nature 314:620. 1985.
20.
Schwartzman, M.L., M. Balazy, J. Masferrer, N.G. Abraham, J-C. McGiff, and R.C. Murphy. 12(R)-Hydroxyeicosatetraenoic A cytochrome P450-dependent arachidonate metabolite that acid: Proc. Natl. Acad. Sci. inhibits Na+, K+-ATPase in the cornea. U.S.A. 84:8125. 1987.
21.
Escalante, B., J.R. Falck, P. Yadagiri, L. Sun, and 19(S)-Hydroxyeicosatetraenoic acid is a M.L. Schwartzman. potent stimulator of renal Na+-K+-ATPase. Biochem. Biophys. Res. Commun. 152:1269. 1988.
22.
Swartz, adrenal
23.
Hirai, A., K. Tahara, Y. Tamura, and S. Yoshida. Involvement of 5-lipoxygenase metabolites in ACTH-stimulated corticosteroidogenesis in rat adrenal glands. Prostaglandins 30:749. 1985.
24.
Chung, S-K., and A.I. Scott. Synthesis of (+)-eicosa-cis-8,9(11,12- and 14,15-)epoxy-cis-11,14-(8,14and 8,11-)dienoic acids and attempted bioconversion to prostaglandins. Tetrahedron Lett. 35:3023. 1974.
25.
Porter, N.A., J. Logan, and V. Kontoyiannidou. purification of arachidonic acid hydroperoxides importance. J. Org. Chem. 44:3177. 1979.
26.
Takenaga, M., A. Hirai, T. Terano, Y. Tamura, H. Kitagawa, and S. Yoshida. Comparison of the in vitro effect of eicosapentaenoic acid (EPA)-derived lipoxygenase metabolites on human platelet function with those of arachidonic acid. Thromb. Res. 37:373. 1986.
27.
Camp, R.D.R., A.I. Mallet, P.M. Woollard, S.D. Brain, A.K. Black, and M.W. Greaves. The identification of hydroxy fatty acids in psoriatic skin. Prostaglandins 26:431. 1983.
28.
Hadjian, A.J., C. Guidicelli, and E.M. Chambaz. Cholinergic muscarinic stimulation of steroidogenesis in bovine adrenal cortex fasciculata cell suspensions. Biochim. Biophys. Acta
Role S.L., and G.H. Williams. steroidogenesis. Endocrinology
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of prostaglandins 113:992. 1983.
and Eur.
in
Preparation and of biological
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714:157.
1982.
29.
Karlmar, K-E. In vitro conversion of cholesterol into aldosterone and cortisol in different zones of the bovine adrenal cortex. Acta Endocrinol. 101:436. 1982.
30.
Ishizuka, T., K. Miura, S. Nagao, and Y. Nozawa. Differential redistribution of protein kinase C in human aldosteronoma cells and adjacent adrenal cells stimulated with ACTH and angiotensin II. Biochem. Biophys. Res. Commun. 155:643. 1988.
31.
Granoff, A.B., F.I. Chasalow, and S.L. Blethen. 17-Hydroxyprogesterone responses to adrenocorticotropin in children with premature adrenarche. J. Clin. Endocrinol. Metab. 60:409. 1985.
32.
Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. 1951.
33.
Miwa, T-K., K.L. Mikolajczak, F.R. Earle, and I.A. Wolff. Gas chromatographic characterization of fatty acids. Analytical Chem. 32:1739. 1960.
34.
Kominami, S., K. Shinzawa, and S. Takemori. Purification and some properties of cytochrome P-450 specific for steroid 17cr-hydroxylationand CI7-C20 bond cleavage from Guinea pig adrenal microsomes. Biochem. Biophys. Res. Commun. 109:916. 1982.
35.
Kominami, S., H. Ochi, Y. Kobayashi, and S. Takemori. on the steroid hydroxylation system in adrenal cortex microsomes. J. Biol. Chem. 255:3386. 1980.
36.
Capdevila, J., L.J. Marnett, N. Chacos, R.A. Prough, and R.W. Estabrook. Cytochrome P-450-dependent oxygenation of arachidonic acid to hydroxyeicosatetraenoic acids. Proc. Natl. Acad. Sci. U.S.A. 79:767. 1982.
Editor:
430
J. Roberts
II
Received:
10-4-88
Studies
Accepted:
8-7-89
OCTOBER 1989 VOL. 38 NO. 4
ARACHIDONIC ACID METABOLITES BY CYTOCHROME P-450 DEPgWDE KONOOXYGENASE PATRWAY IN BOVINE ADRENAL FASCICULATA CELLSNT M. Nishimura, A. Hirai, M. Omura, Y. Tamura, and S. Yoshida The Second Department Of Internal Medicine, School Of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan
[1-14C]Arachidonic acid was incubated with microsomes of bovine adrenal fasciculata cells in the presence of 1 mM NADPH for 30 min The metabolites were separated and purified by reverse at 37°C. phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry. Identified metabolites were four dihydroxyeicosatrienoic acids (DHTs) (5,6-, 8,9-, 11,12-, 14,15DHTs), 20-hydroxyeicosatetraenoic acid and eicosatetradioic acid. The formation of these metabolites was dependent on NADPH and inhibited by SKF-525A. 14,15-DHT was also formed by isolated bovine adrenal fasciculata cells. These results indicate that cytochrome P-450 dependent arachidonate monooxygenase pathway may exist in bovine adrenal fasciculata cells. Addition of the chemically synthesized epoxyeicosatrienoic acids (EETs) to isolated bovine adrenal fasciculata cells stimulated cortisol production. Among four regioisomeric EETs, 14,15-EET was most potent and stimulated steroidogenesis in a dose-related manner over a range of 0.5 to 5.0 uM.
Introduction Arachidonic acid has been shown to be metabolized by cytochrome P-450 dependent monooxygenase pathway in various organs and tissues such as: the kidney (l-3), the liver (4-8), the cornea (9,101, the hypothalamus (11) and the pituitary gland (12). This pathway converts arachidonic acid to several oxygenated metabolites, i.e. monohydroxyeicosatetraenoic acids (HETEs), w-, w-l-oxidation products and four regioisomeric epoxyeicosatrienoic acids (EETs) which are further converted to corresponding dihydroxyeicosatrienoic acids (DHTs) by epoxide hydrolase. These metabolites have been shown to have various biological activities, i.e. stimulation of secretion of peptide hormones (11,13-17), relaxation of blood vessels (18) and inhibition or stimulation of Na-K-ATPase (19-21).
lpreliminary data were presented at Taipei Conference on Prostaglandin and Leukotriene Research, Taipei, Taiwan, R.O.C. during April, 1988.
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It is well known that steroidogenesis in the adrenal cortex involves the sequential action of four distinct forms of cytochrome P-450. Among these enzymes, 17a-hydroxylase and 21-hydroxylase are localized in microsomes. In adrenal fasciculata cells, arachidonic acid has been shown to be metabolized by cyclooxygenase (22) and lipoxygenase pathways (23). However, no studies have been reported concerning the metabolites of arachidonic acid by cytochrome P-450 dependent pathway in adrenal cortex. Therefore, the present study was performed firstly to determine whether the bovine adrenal fasciculata cells may have the capacity to metabolize arachidonic acid via the cytochrome P-450 dependent monooxygenase pathway, and secondly, to examine the effect of the metabolites of this pathway on the production of cortisol by isolated bovine adrenal fasciculata cells. Materials
and Methods
1. Materials [l-14C]Arachidonic acid (56 mCi/mmol) was obtained from New England Nuclear (Boston, MA, U.S.A.) and arachidonic acid (99.9% pure) from Nu-Chek Prep. (Elysian, Mine., U.S.A.). N,O-Bis(trimethylsilyl)trifluoroacetamide, N-trimethylsilyl imidazole and all solvent for high performance liquid chromatography (HPLC) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Eagle's minimum essential medium (MEM) was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). N-methyl-N'-nitro-Nnitrosoguanidine, collagenase type I, deoxyribonuclease type II and bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). NADPH was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). SKF-525A was a generous gift from Smith Kline and French (Philadelphia, Penn., U.S.A.). Cortisol Kit II was purchased from the First Radioisotope Co. (Tokyo, Japan). Aldosteron RIA Kit was purchased from Dinabot Co. (Tokyo, Japan). COAT-A-COUNT 17a-OH progesterone RIA Kit was purchased from Diagnostic Products Corporation (CA, U.S.A.). 2. Preparation
of authentic
standards
of EETs,
DHTs
and HETEs
Standards of EETs and DHTs were prepared by non-selective epoxydation of arachidonic acid as described by Chung and Scott The epoxide mixture was separated by straight phase (SP)-HPLC (24). using a HPLC pump 880-PU [Japan Spectroscopic Co., Ltd. (JASCO), Tokyo, Japan] equipped with a silica column (Develosil 5 pm, 20 mm x 500 mm, Nomura Chemical Co., Ltd., Seto, Aichi, Japan), with a solvent system of n-Hexane/isopropanol/acetic acid (100:0.6:0.05 The UV absorption of column effluent was monitored at by vol.). 206 nm using a UV detector 875-UV (JASCO, Tokyo, Japan). The corresponding DHTs were obtained by acidic aqueous hydrolysis followed by the purification by reverse phase (RP)-HPLC.
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Hydroperoxyeicosatetraenoic acids (HPETEs) were synthesized by photo-oxidation method (25) with minor modifications. Synthesized HPETEs were purified by SP-HPLC and RP-HPLC as previously reported (26). HETEs were prepared by the reduction of HPETEs by NaBH4 (27) followed by SP-HPLC purification. 3. Animal and tissue preparation Fresh bovine adrenal glands were obtained from local abattoir and brought to the laboratory within 1 hour after the animals were slaughtered. Bovine adrenal fasciculata zone were prepared according to the method of Hadjian et al. (28) and Karlmar (29) with minor modifications. The adrenals were bisected at their largest diameter and the medulla extracted thoroughly. Starting from the inner zone of the cortex, thin slices (0.5 mm) were cut using a slicer (Natsume Seisakusho, Tokyo, Japan); the first two slices were discarded and the next two slices were pooled and stored in MEM containing 0.1% BSA. Microscopic examination of the pooled slices showed that tissue contained mainly fasciculata cells and no glomerulosa cells were present. After four washes with this buffer, the slices were minced and incubated in MEM containing 0.1% BSA, 0.25% collagenase type I and 0.05 mg/ml deoxyribonuclease type II at 37°C for 30 min in 5% CO, and 95% 0,. Incubation mixtures were centrifuged at 100 x g for 10 min at room temperature. The cells were resuspended in Krebs-Ringer bicarbonate (KRB) buffer containing 0.2% glucose and 0.1% BSA and filtered through cotton gaze and centrifuged at 100 x g for 10 min at room temperature. The cellular pellet was suspended in the same buffer for washing. This washing procedure was repeated three times. Finally 2 x lo5 cells were suspended in 1 ml of KRB buffer containing 0.2% glucose. The formation of cortisol, 17a-hydroxyprogesterone and aldosterone by isolated bovine adrenal fasciculata cells was determined by the use of specific radioimmunoassays (30,31) according to the method of Hadjian et al. (28) with minor modifications. The isolated cells produced cortisol and 17a-hydroxyprogesterone in response to the stimulation with ACTH or angiotensin II, while no detectable formation of aldosterone was observed. For the preparation of microsomes, all procedure was carried out at 0 to 4°C. Slices of the bovine adrenal fasciculata zone prepared as described above was pooled and stored in ice-cold buffer containing 30 mM potassium phosphate, pH 7.4, 0.25 M sucrose and 0.1 mM EDTA (buffer A). After four washes with buffer A, the slices were weighed and chopped into small pieces and homogenized with 2 volumes of buffer A. The homogenate was centrifuged at 10,000 x g for 60 min at 4°C. The 10,000 x g supernatant was centrifuged at 105,000 x g for 60 min. The resulting microsomal pellet was resuspended in 100 mM potassium phosphate buffer, pH 7.4 (2 mg protein/ml).
OCTOBER 1989 VOL. 38 NO. 4
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4. Protein
determination
The microsomal protein was determined by the method et al. (32) with bovine serum albumin as standard. 5. Metabolism
of arachidonic
of Lowry
acid
(a) Metabolism of arachidonic fasciculata cells
acid by microsomes
of bovine
adrenal
[l-14C]Arachidonic acid (900 ug, 3 PCi) was incubated with microsomes (60 mg protein) in the presence or absence of 1 mM NADPH for 30 min at 37°C. The reaction was terminated by the addition of 2 volume of ice-cold methanol. The methanolic incubation mixture was acidified to pH 3.5 with 2 N citrate and centrifuged at 1,500~ g for 10 min at 4°C. Clear supernatant was diluted with water to make a 10% methanol-aqueous solution and applied to a prewetted octadecyl silica (ODS) mini column (Bond Elute CI8 6 cc, Analytichem International, Harbor City, CA, U.S.A.) followed by a wash with water. Arachidonic acid metabolites were eluted by 6 ml of ethyl acetate. The ethyl acetate fraction was evaporated under nitrogen gas stream and resolved in 50% methanol. microsomes were preincubated In experiments using inhibitors, with agents for 10 min before the addition of NADPH and [1-14C]arachidonic acid. An incubation of [l-'4C]arachidonic acid with inactivated microsomes (boiled microsomes) was performed to assess possible non-enzymatic autooxidation of arachidonic acid. (b) Metabolism of arachidonic fasciculata cells
acid by isolated
bovine
adrenal
Isolated bovine adrenal fasciculata cells were suspended in KRB buffer containing 1 mM calcium (Caa+) and 0.2% glucose (5 x lo6 cells/l0 ml), and incubated with [l-14C]arachidonic acid (17.5 ug, 3 UCi) at 37°C for 15 min. The termination of the reaction and extraction of the metabolites were performed in the same way as described in the method of arachidonate metabolism by microsomes. Extracted metabolites were analyzed by RP- and SP-HPLC. 6. HPLC
analysis
of arachidonic
acid metabolites
The extracted radioactive metabolites were separated by RP-HPLC using a 880-PU liquid chromatograph (JASCO, Tokyo, Japan) equipped with an ODS column (Develosil 5 pm, 4.6 mm x 150 mm, Nomura Chemical The chromatography was performed at Co., Ltd., Seto, Aichi, Japan). 30°C with isocratic elution with a solvent system of methanol/ acetonitrile/water/acetic acid (45:25:30:0.01 by vol.). The flow rate was 1 ml/min. Radioactivity in eluate from HPLC was monitored by a Beckman 171 radioisotope detector (Beckman Instruments, Inc., Fullerton, CA, U.S.A.). Elution times for 14,15-DHT, 11,12-DHT,
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8,9-DHT and 5,6-DHT were 14.4,16.3,18.2 and 22.4 min, respectively. Production of the metabolites was expressed as "pmol of arachidonic acid (AA) converted/mg of protein/30 min" according to the method of Schwartzman et al. (20). Each of the radioactive products separated by RP-HPLC was collected and converted to the methyl ester derivative by the reaction with ethereal diazomethane and further purified by SP-HPLC using a JASCO 880-PU liquid chromatograph equipped with a silica column (Unisil Q 3 urn,4.6 x 150 mm, Gasukuro Kogyo Inc., Tokyo, Japan). The flow rate was 1 mljmin and the column was maintained at 30°C. Separation was carried out using a gradient of isopropanol in n-Hexane containing 0.05% acetic acid. The initial solvent was 1.5% isopropanol in n-Hexane. After 25 min the concentration of isopropanol was linearly increased to 5% over 10 min and held at this concentration for an additional 15 min. Elution times of the methyl ester derivatives of 14,15-DHT, 11,12-DHT, 8,9-DHT and 5,6-DHT were 17.9, 15.6, 20.2 and 35.0 min, respectively. 7. Derivatization for GC/MS analysis The methyl ester derivatives purified by SP-HPLC were silylated with N,O-bis(trimethylsilyl)trifluoroacetamideand N-trimethylsilylimidazole (1:2 by vol.) for 60 min at room temperature. 8. GC/MS analysis Gas chromatography-mass spectrometry (GC/MS) analysis was performed on a Hitachi M-80 GC/MS system (Hitachi, Tokyo, Japan). The GC column was 1.5% OV 101 (2 m) and operated isothermically at 21OOC. Electron impact was 70 eV. C values were determined from the retention times of fatty acid methyl esters (C1B-C24) according to the method of Miwa et al. (33) with minor modifications. 9. Cell incubation and cortisol assay Collagenase-dispersed bovine adrenal fasciculata cells were resuspended in 1 ml of KRB buffer containing 1 mM Ca2+ and 0.2% glucose. Aliquots (950 ~1) of cell suspension (2 x lo5 cells/ml) were distributed in randomized plastic dishes and preincubated at 37°C in 5% CO, and 95% 0s. After 15 min of preincubation, the reaction was started by the addition of 50 ~1 of an aqueous vehicle (KRB buffer containing 4% ethanol) in which the various concentration of four regioisomeric EETs were dissolved and incubated for 30 min. Control incubations were carried out by the addition of 50 ~1 of vehicle solution alone. Cortisol was measured by a specific radioimmunoassay (30). Results were expressed as net cortisol production (ng/2 x lo5 cells/30 min), after subtraction of the corresponding control value. 10. Statistical analysis Comparisons were made using the non-paired Student's t-test.
OCTOBER 1989 VOL. 38 NO. 4
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PROSTAGLANDINS
Results 1. HPLC analysis of the arachidonate metabolites by microsomes of bovine adrenal fasciculata cells RP-HPLC resolved several radioactive peaks (Fig. 1). The polar metabolites which eluted between 3 to 7 min co-chromatographed with prostaglandins and they were not further characterized in this study. The elution times of Peak I, Peak II, Peak III were identical to those of the authentic standards of 14,15-DHT, 11,12DHT and 8,9-DHT. The elution time of Peak IV was a little later than that of the authentic standards of 8,9-DHT, and the elution time of Peak V was a little bit earlier than that of the authentic standard of 5,6-DHT. Two minor radioactive peaks were eluted at the retention times of 5- and 15-HETE. However, both were also formed
14, 15-11,12DHT
DHT
8*9-
5.6)Hl r
DHT
11
1 ,x0.2
I
I
lJJ II
Ill
/
5
10
15 retention
time
20
‘
25 (mid
Fig. 1. Separation of [l-'4C]Arachidonate Metabolites Produced by Microsomes of Bovine Adrenal Fasciculata Cells. Products were extracted and analyzed as described in "Materials and Methods". The radioactive peaks were named Peaks I, II, III, IV and V as indicated. Each peak was collected and methylated and further purified by SP-HPLC. The arrows denote the retention times of the authentic standards.
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OCTOBER 1989 VOL. 38 NO. 4
PROSTAGLANDINS
to similar extents in a 30 min control incubation of [1-14C]arachidonic acid with boiled microsomes. Small radioactive peaks were observed near the retention times of the authentic standards of EETs. However, they were not identified because of sample limitation. The methyl ester derivatives of Peak I, Peak II, Peak III and Peak IV were eluted as single peaks on SP-HPLC. The methyl ester derivative of Peak V was separated into two radioactive peaks by SP-HPLC (Fig. 2). The materials eluted at 16.0 min and 35.0 min were referred to as the methyl ester derivatives of Peaks VA and VB, respectively. The elution time of the methyl ester derivative of Peak VB was identical to that of the authentic standards of the methyl ester derivative of 5,6-DHT.
5,6-DHT-Me
1 I--
I’
-----_------___
10 retention
20 time
30
(min)
Pig. 2. Separation of the Methyl Ester Derivative of Peak V. The methyl ester derivative of Peak V was analyzed by SP-HPLC as described in "Materials and Methods". It was resolved to two radioactive peaks and named PeaksVA and VB as indicated. The arrow denotes the retention time of the authentic standard.
OCTOBER 1989 VOL. 38 NO. 4
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PROSTAGLANDINS
2. GC/MS
analysis
Metabolites of arachidonic acid by microsomes fasciculata cells were identified by GC/MS.
of bovine
adrenal
Mass spectrum of the methyl ester trimethylsilyl derivative of Peak I is shown in Fig. 3. This compound showed prominent ions at Signals were also noted at 496 (tic), 481 (e-15) 173, 275 and 323. The mass spectrum of this compound was identical and 465 (Mf-31). with that of the authentic standard of 14,15-DHT and the material in Peak I was identified as 14,15-DHT. Mass spectrum of the methyl ester trimethylsilyl derivative of Peak II is shown in Fig. 4. This compound showed prominent ions at The mass spectrum of this compound 315, 295 (M+-111-90) and 213. was identical with that of the authentic standard of 11,12-DHT and the material in Peak II was identified as 11,12-DHT. Mass spectrum of the methyl ester trimethylsilyl derivative of Peak III is shown in Fig. 5. This compound showed prominent ions at 243, 253 and 255 (345-90). Signals were also noted at 345 and 355. The mass spectrum of this compound was identical with that of the authentic standard of 8,9-DHT and the material in Peak III was identified as 8,9-DHT. Mass spectrum of the methyl ester trimethylsilyl derivative of Peak VB is shown in Fig. 6. This compound showed prominent ions at Signals were also noted at 496 203, 215 (305-90), 293 and 305. CM+), 481 (M+-15), 465 (I@-31) and 406 (k?-90). The mass spectrum of this compound was identical with that of the authentic standard of 5,6-DHT and the material in Peak VB was identified as 5,6-DHT. Mass spectrum of the methyl ester trimethylsilyl derivative of Peak VA is shown in Fig. 7. The material in Peak VA was identified as 20-hydroxyeicosatetraenoic acid and distinguished from (a) This 19-hydroxyeicosatetraenoic acid by following criteria. compound showed ions at 406 (tit), 391 (I@-15), 375 c&-31), 316 W-90) and 103, and lacks strong signals in lower mass range including 117 which is characteristic of 19-hydroxyeicosatetraenoic acid. (b) C value of the methyl ester trimethylsilyl derivative of Peak VA was 22.6. This was identical with that of 20-hydroxyeicosatetraenoic acid reported by Oliw et al. (3). Mass spectrum of the methyl ester derivative of Peak IV showed The mass signals at 362 (M+), 247, 234, 221, 208 and 194 (Fig. 8). spectrum of this compound was identical with published spectrum of 5,8,11,14-eicosatetradioic acid (3) and the material in Peak IV was identified as 5,8,11,14-eicosatetradioic acid.
420
OCTOBER 1999 VOL. 38 NO. 4
PROSTAGLANDINS
(x)
OCTOBER 1989 VOL. 38 NO. 4
A$!suaau!
an!aela.d
421
150
zo200
250
I, I q&
215
(305-90)
203
1
m/z
r
m/z
350
233 203 _-_T__-
x10
450
500
III
of Peak VB
Y5
bJl+
of Peak
PA+-31
Derivative
hl+-W-l
400
r
Ester Trimethylsilyl acid).
300
293
305
355
.yl Derivative
253 243 ---T---
“JMSO~OTMS
305 c___
Ester Trimethylsi acid).
q 1 I
6. Mass Spectrum of the Methyl (5,6-dihydroxy-8,11,14-eicosatrienoic
Pig.
!!_
f ._ G
6040-
80-
6
CI .E
loo-
C;
Fig. 5. Mass Spectrum of the Methyl (8,9-dihydroxy-5,11,14-eicosatrienoic
25 4
(345-00) 255
PROSTAGLANDINS
(%) b!sUWU! eA!aelaJ
OCTOBER 1989 VOL. 38 NO. 4
(%) r($!SUWl!
tM!alZleJ
423
PROSTAGLANDINS 3. Studies on the formation of Peaks I through V by microsomes of bovine adrenal fasciculata cells The amounts of [l-14C]arachidonic acid converted to Peaks I through V were 10.422.2, 5.020.8, 1.720.3, 34.0+6.6 and 168.3235-l pmol of AA convertedlmg of protein/30 min, respectively (mean *S.E., n=4). No detectable formation of Peaks I through V was observed in the absence of NADPH. SKF-525A (50-200 pM) dose dependently reduced the formation of Peaks I through V (data not shown). 4. HPLC analysis of the arachidonate metabolites by isolated bovine adrenal fasciculata cells Among Peaks I through V, the formation of Peak I alone was observed in experiment using isolated bovine adrenal fasciculata cells. The amount of [l-'4C]arachidonic acid converted to Peak I was 28.3i5.3 pmol of AA converted/lo6 cells/l5 min (mean+S.E., n=4).
**
T control
5,6EET
8.9EET
11,12EET
14,15EET
Fig. 9. Effect of EETs (5 PM) on Steroidogenesis in Bovine Adrenal Fasciculata Cells. Vertical bars indicate mean + S.E. (n = 6). *: pco.05, **: pcO.01, vs control.
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OCTOBER 1969 VOL. 38 NO. 4
PROSTAGLANDINS
5. Effect of EETs on steroidogenesis The effect of the addition of chemically synthesized EETs (5 uM) on steroidogenesis of bovine adrenal fasciculata cells are shown in Fig. 9. Among four regioisomeric EETs, 14,15-EET was most potent. And as shown in Fig. 10, 14,15-EET stimulated steroidogenesis dose dependently over the concentration of 0.5-5.0 uM.
0
0.5
2.5
14,15-EET
5.0
10.0
(PM)
Effect of 14,15-EET (0.5-10.0 nM) on Steroidogenesis in Bovine Adrenal Fasciculata Cells. Vertical bars indicate mean 2 S.E. (n = 6). *: ~~0.05, **: p
OCTOBER 1989 VOL. 38 NO. 4
425
PROSTAGLANDINS
Discussion The cytochrome P-450 dependent enzyme system is well known for steroidogenesis in the adrenal cortex and have been intensively investigated (34,35). However, no studies have been reported concerning cytochrome P-450 dependent monooxygenase pathway for arachidonic acid in adrenal fasciculata cells. Therefore, the present study was performed in order to elucidate whether this metabolic pathway may exist in bovine adrenal fasciculata cells. [1-14ClArachidonic acid was incubated with microsomes of bovine adrenal fasciculata cells in the presence of NADPH. The metabolites were separated and identified by GC/MS. 20-Hydroxyeicosatetraenoic acid, eicosatetradioic acid and four regioisomeric DHTs (5,6-, 8,9-, 11,12- and 14,15-DHTs) were identified. The formation of these metabolites was dependent on NADPH and inhibited by the addition of SKF-525A, a specific inhibitor of cytochrome P-450 dependent monooxygenase pathway. Detectable formation of EETs was not observed in this study. EETs have been reported to be unstable and rapidly converted to corresponding DHTs by epoxide hydrolase which exists in microsomes and cytosol (6). Therefore, it seems to be likely that four regioisomeric DHTs might be formed via unstable intermediates, EETs. It has been reported that arachidonic acid is also converted to HETEs (36) and 19-hydroxyeicosatetraenoic acid (3) by cytochrome P-450 dependent monooxygenase. Two minor radioactive peaks which co-chromatographed with the authentic standards of 5- and 15-HETE were found in this study. However, both were also formed to similar extents in a 30 min control incubation of [1-14Clarachidonic acid with boiled microsomes. Therefore, it seems less likely that these peaks might be formed enzymatically. No detectable formation of 19-hydroxyeicosatetraenoic acid was observed in this study, suggesting that further study might be required. In the experiment using isolated bovine adrenal fasciculata cells, only Peak I was formed among Peaks I through V. This means that there was a discrepancy between the profiles of arachidonate metabolites formed by the intact cells and the microsomes. Similar observation was also described by Schwartzman et al. in the experiment using bovine cornea1 epithelium (20), though the exact mechanism of the discrepancy still remains indeterminate yet. From the results of the present study using microsomes and isolated cells, it is quite likely that cytochrome P-450 dependent arachidonate monooxygenase pathway may exist in bovine adrenal fasciculata cells. Arachidonic acid and its metabolites have been shown to play an important role in the signal transduction in various cells including endocrine cells. In adrenal fasciculata cells, it has been reported that arachidonic acid was metabolized by cyclooxygenase (22) and lipoxygenase pathways (23). We previously reported that one of the 5-lipoxygenase metabolites, "5-HPETE", may play an important role in ACTH-stimulated steroidogenesis in rat adrenal fasciculata cells (23). As mentioned above, the present study demonstrated the presence of cytochrome P-450 dependent monooxygenase pathway for arachidonic acid in bovine adrenal fasciculata cells. Therefore we investigated whether the metabolites of arachidonic acid by cytochrome P-450 dependent monooxygenase may have any biological effects on bovine adrenal fasciculata cells. A notable finding was
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that EETs, active metabolites of cytochrome P-450 dependent monooxygenase pathway, stimulated cortisol production by bovine adrenal fasciculata cells. Among four regioisomeric EETs, 14,15-EET was most potent and stimulated steroidogenesis at the concentration of 0.5-5.0 pM. Several metabolites of arachidonic acid by cytochrome P-450 dependent monooxygenase pathway have been shown to have various biological activities, i.e. stimulation of secretion of peptide hormones (11,13-17), relaxation of blood vessels (18) and inhibition or stimulation of Na-K ATPase (19-21). The present finding that EETs can stimulate cortisol production in bovine adrenal cortex is the first observation for steroidogenic effect of EETs. Further study seems to be required to elucidate the mechanism of the action of EETs on adrenal steroidogenesis. Acknowledgment
The authors are most grateful to Dr. Yasuo Shida for his valuable contribution to GC/MS analysis. And the authors would like to thank Mr. Yoshio Yamauchi and Mr. Tsutomu Tomita for helpful advices in developing HPLC procedures. This study was partly supported by a Grant-in-Aid from the Ministry of Health and Welfare and a research grant (63570522) from the Scientific Research Fund of the Ministry of Education, Japan. References 1.
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Editor:
430
J. Roberts
II
Received:
10-4-88
Studies
Accepted:
8-7-89
OCTOBER 1989 VOL. 38 NO. 4