Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Biochimica et Biophysics Acta 875 (1986) 641-653

Elsevier

641

BBA 52164

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid

on prostacyclin formation in bovine aortic endothelial cells

Bernd Mayer a.*, Robert Moser b, Helmut Gleispach b and Walter R. Kukovetz a

“Instltut fiir Pharmakodynamik und Toxikologie, Karl-Franzens-Universitiil Graz, Umuersitiitsplatz 2, A-8010 Graz, and

h Unioersitiirs-Kinderklinik, Auenbruggerplatz 20, A-8036 Grar (Austria)

(Received June 4th. 1985)

(Revised manuscript received November 8th, 1985)

Key words: Arachidonic acid; Prostacyclin; Hydroperoxyeicosatetraenoic acid; Nordihydroguaiaretic acid:

(Bovine aortic endothelial cell)

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A, produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin IZ) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE’s). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F,, as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandms FZa and E, were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETF, 5-HETE, ll-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 19HPETF could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.

Introduction

It has been shown that endothelial cells are able to metabolize arachidonic acid, released from the cellular phospholipids, via the cyclooxygenase

* To whom correspondence should be addressed.

Abbreviations: HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; HHT, 12-hydroxy-

heptadecatrienoic acid; NDGA, nordihydroguaiaretic acid;

TLC, thin-layer chromatography; GC, gas-liquid chromatogra-

phy; MS. mass spectrometry; TMS, trimethylsilyl ether.

pathway to a cyclic endoperoxide, prostaglandin H,, which then is further converted by prostacyclin synthetase to prostacyclin [l-4]. Pros- tacyclin is a physiological antagonist of thrombo-

xane A, both as an inhibitor of platelet aggregation [5] and as a potent vasodilator and vascular smooth muscle relaxant [6].

In various tissues arachidonic acid is the substrate for another group of enzymes, the lipoxygenases, that convert free arachidonic acid into different isomeric hydroperoxyeicosatetraenoic acids that are reduced further by a glutathione-de-

0005.2760/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

642

pendent peroxidase to the corresponding hydroxy compounds [7]. Reticulocytes [8], human eosinophils [9] and human skin fibroblasts [lo], for example, convert arachidonic acid into 1%HPETE via the 15-lipoxygenase pathway. 15HPETE has been described as a potent inhibitor of prostacyclin formation [ll]. The reduced analogue of this compound, 15-HETE, has been reported to inhibit the 5-lipoxygenase-leukotriene pathway in rabbit peritoneal macrophages [12] and platelet

lipoxygenase [13]. Thus, products of 15-lipoxygenase seem to be involved in regulating the activity of other lipoxygenases in various cell types.

Besides prostacyclin which has been shown to be the major metabolite of arachidonic acid in endothelial cells [l-4], this tissue appears to produce another vasodilator substance upon stimu-

lation with various autacoids (acetylcholine, bradykinin, thrombin, ADP), which was named endothelium-derived relaxing factor 114-161 but has not as yet been identified. The possibility was raised by various authors [17-191 that this compound might be a lipoxygenase product of arachidonic acid. There have been many con- tradictory reports concerning the characterization of these metabolites in endothelial cells. Hopkins et al. [20] identified 15-HETE as the only lipoxygenase metabolite formed from human umbili-

cal vein endothelial cells. Kuhn et al. [21] reported that 15-HETE and 12-HETE are synthesized from arachidonic acid by cultured calf aortic endothelial cells. Other workers (22,233 ascribed I5-HETE formation by vascular tissue to cyclooxygenase activity. Henricsson et al. [24] reported 15-HETE synthesis only for artherosclerotic vessel walls in the rat, whereas these authors were unable to detect any lipoxygenase products to be formed by blood vessels from control animals. A frequently cited work published by Greenwald et al. [25] reports 12-HETE as being the only lipoxygenase metabolite formed by vascular tissue. Lagarde et al. [26] very recently failed to observe formation of lipoxygenase metabolites by cultured human um- bilical vein endothelial cells from endogenously released precursor arachidonic acid. Neither of these papers, however, gives a detailed qualitative or, especially, a quantitative mass spectrometric analysis of HETEs formed in endothelial cells. Since we felt that this was necessary in view of the

above-mentioned discrepancies that exist in the literature, we attempted to identify the respective products in freshly isolated bovine aortic endothelial cells by gas chromatography (GC) and both electron impact as well as chemical ionization mass spectrometry (MS). Additionally we performed a quantitative estimation of monohydroxy fatty acids by CC-MS, which is first presented in this paper. It was found that 15HETE was the major lipoxygenase metabolite produced by bovine aortic endothelial cell homogenates as well as by the respective undisrupted cells stimulated with 10 PM Ca ionophore A23187.

Inhibition experiments with NDGA, a we’ll known antioxidant and lipoxygenase inhibitor [27], showed not only a decrease in the biosynthesis of

15HETE but also an enhanced formation of prostacyclin, as measured by its stable non-enzymatic metabolite 6-ketoprostaglandin 5,. This led us to the assumption that 15HPETE, which is an inter- mediary product of the lipoxygenase metabolism of arachidonic acid, might normally exert an inhibitory action on prostacyclin formation. Ad- ditional experiments were performed to test this hypothesis and the results obtained are in support of such an inhibitory function of 15-HPETE.

Materials and Methods

Arachidonic acid, prostaglandin standards, Coomassie brilliant blue G-250, NDGA, soybean

lipoxygenase type IV and Ca ionophore A23187 were obtained from Sigma, Munich, F.R.G. De- uterium-labelled prostaglandins were purchased

from Merck Sharp & Dohme, Munich, F.R.G. This company also provided us with a generous gift of indomethacin (sodium salt). Sephadex LH- 20 was obtained from Pharmacia, Uppsala, Sweden, and silicar CC4 from Mallinkrodt, St. Louis, MO, U.S.A. [1-‘4C]Arachidonic acid (spec. act. 55 mCi/mmol) was from New England Nuclear, Dreieich, F.R.G. It was diluted with unlabelled arachidonic acid to a specific activity of l-10 mCi/mmol and purified by silicic acid chromatography. 15-HPETE was prepared by incubat- ing arachidonic acid with soybean lipoxygenase [28] and quantified by ultraviolet analysis (237 nm, E = 27000). 15-HETE was prepared by NaBH, reduction of 15-HPETE 1281. PtO, was purchased

643

from Ventron, Karlsruhe, F.R.G., methoxamine

hydrochloride in pyridine (2%) and bis(trimethylsilyl)trifluoroacetamide from Pierce Chemical Co., Rockford, IL, U.S.A. Other reagents, TLC plates

(Kieselgel 60, 0.5 mm) and solvents of analytical grade were obtained from Merck, Darmstadt, F.R.G.

Preparation of endothelial cell homogenates

Endothelial cells were obtained from bovine aortae (usually young mature animals) from the local slaughterhouse. Aortae (arches of 25-30 cm length and 2-3 cm width) were removed and immediately freed of fat and connective tissue, then washed in chilled Tyrode’s solution to remove blood components. Upon arrival in the laboratory (always within 30 min) aortae were further rinsed

and cut longitudinally. Endothelial cells were scraped off and placed in 30 ml of chilled Dulbecco’s phosphate-buffered saline. The suspension was centrifuged at 500 x g for 15 min, the cell pellet washed once with 30 ml Dulbecco’s phosphate-buffered saline and finally resuspended in 3 ml of the same buffer. This suspension was ex- amined for purity by light microscopy (greater than 95% endothelial cells) and used for experi-

ments that were carried out in the presence of Ca ionophore A23187 (see below). For all other experiments, the cells were homogenized by means

of a Micro-Dismembrator (Braun-Melsungen, F.R.G.) and protein (0.5-1.5 mg per aortae) was determined by the method of Bradford [29].

Incubation conditions

The cell homogenates were diluted with Dulbecco’s phosphate-buffered saline (pH 7.4) to a concentration of 0.2 mg protein per ml and incubated in 1 ml volumes with 14C-labelled arachidonic acid at 37°C for 10 min. Arachidonic acid was dissolved in Dulbecco’s phosphate- buffered saline containing 0.1 mg bovine serum albumin per ml. The final albumin concentration never exceeded 10 pg/ml. Indomethacin was dissolved in water, NDGA in poly(ethylene glycol) and 15-HPETE in phosphate-buffered saline. The drugs were added to the cells 10 min before the addition of arachidonic acid.

For the GC-MS analysis of the monohydroxy fatty acids, large scale incubations were performed

with 5 ml of an endothelial cell homogenate containing 1 mg of cellular protein per ml. For these experiments [l-‘4C]arachidonic acid was diluted with unlabelled arachidonic acid to a specific activity of 1 mCi/mmol and added to the cell homo-

genate to give a final concentration of 25 PM. For characterization of monohydroxy fatty acids

derived from endogenously released arachidonic acid, 5 ml of an endothelial cell suspension containing 1 mg of protein per ml were incubated at 37°C for 10 min in the presence of 10 PM Ca ionophore A23187. The ionophore was dissolved in ethanol.

All reactions were terminated by addition of

250 ~1 ice-cold ethanol per ml of incubation medium.

Extraction and purification of arachidonic ucid

metabolites

The incubation medium was diluted with 1

volume of water and then 50 ng of tetradeuterated 6-ketoprostaglandin F,, (6-keto[‘H,]prostaglandin F,,), prostaglandin F,, ([ *H,]prostaglandin F,,) and prostaglandin E, ([ *H,]prostaglandin E2) were added as internal standards for the quantitative estimation of the prostaglandins. The incubation medium was then acidified to pH 3.5 with 0.1 M HCl and extracted twice with 2 volumes of diethyl ether. The solvent was removed under nitrogen, the dry residue redissolved in 100 ~1 of diethyl ether/petroleum ether, 75 : 25 (v/v) and applied onto a silicic acid column prepared with

0.3 g silicar CC4 in the same solvent. Unreacted arachidonic acid together with the monohydroxy fatty acids was eluted with 4 ml of diethyl ether/

petroleum ether, 75 : 25 (v/v), and prostaglandins with 3 ml ethyl acetate/methanol, 90: 10 (v/v). The solvent was removed under nitrogen and the residue was redissolved in 1 ml diethyl ether/ methanol, 1 : 1 (v/v).

In most cases 100 ~1 of the material were analyzed by TLC with the organic phase of ethyl acetate/isooctane/ acetic acid/ water, 11 : 5 : 2 : 10 (v/v) as solvent system. Reference compounds (6-ketoprostaglandin F,,, prostaglandin F,a, prostaglandin E,, 15-HETE and arachidonic acid) were spotted separately on the plate and visualized by a phosphomolybdic spray, followed by brief heating. In order to estimate the distribution of the radio-

644

activity on the TLC plates, the silica gel was scraped off in 0.5 cm segments and radioactivity was measured by liquid scintillation counting.

In the experiments where GC-MS analysis of monohydroxy fatty acids was performed, the silicic acid column was prepared with 1.0 g silicar CC4 in diethyl ether/petroleum ether, 25 : 75 (v/v) and unreacted arachidonic acid was eluted with 10 ml of this solvent. Monohydroxy fatty acids were subsequently eluted with 15 ml diethyl ether/ petroleum ether, 75 : 25 (v/v), dried under nitrogen and derivatized for GC-MS.

In order to estimate the distribution of all ra-

dioactive metabolites produced from externally added labeled arachidonic acid, in a few experi-

ments the diethyl ether extract was analyzed by radio TLC without a previous separation by column chromatography. For this purpose, diethyl ether was removed unde rnitrogen, the dry residue redissolved in 100 ~1 diethyl ether/methanol, 1 : 1 (v/v) and analyzed by TLC using the same solvent system as described above.

Derivatization procedure The monohydroxy fatty acids were converted

into the methyl ester derivatives with 1 ml of an ethereal diazomethane solution, the ether was removed 20 min later and the sample was redissolved in 1 ml methanol. One third of the esteri-

fied sample was subjected to catalytic hydrogenation with hydrogen gas and 1 mg PtO, as a catalyst. The suspension was centrifuged and the

supernatant passed through a small Sephadex LH- 20 column. After removal of the methanol, both the unsaturated and saturated methyl esters were dissolved in 50 ~1 of a mixture of bis(trimethylsilyl)trifluoroacetamide and pyridine, 2 : 1 (v/v) and kept at 60°C for 1 h in order to convert the free hydroxy groups into the trimethylsilyl ether (TMS) derivatives. Immediately before GC-MS analysis, the solvent was removed and the residue redissolved in 30-50 ~1 n-hexane.

The fraction containing the prostaglandins was dried under nitrogen and keto groups were converted into the methyloxime derivatives with 50 ~1 methoxamine hydrochloride in pyridine (2%) at 60°C for 2 h. Then pyridine was removed and the samples were methylated and trimethylsilylated as described for the monohydroxy fatty acids.

GC-MS analysis

The instrument used was a Finnigan gas chro-

matograph 9610 coupled to a Finnigan 4000 mass spectrometer with a positive-electron impact and a chemical ionization device combined with an Incas data system. Gas chromatographic separation was performed with a fused silica capillary column coated with SE54 (0.25 mm inner diameter and 30 m length) at a helium flow rate of 2 ml/mm. For separation of the prostaglandins this column was temperature-programmed as follows: after 1 min at 170°C at 40 Cdeg/min and then held at 320°C for 2 min. For separation of the different HETEs, the samples were injected at an initial temperature

of 100°C and the column was temperature programmed as described for the prostaglandins. The ion source was maintained at 250°C, and a scan time of 1 s was chosen for the mass range of 100-700 m/e. For selected ion monitoring, the centroid sampling interval was 0.5 s. Electron impact was performed with an ion voltage of 70 eV and an ion current of 0.2 A. For chemical ioniza-

tion ammonia was used as a reagent gas (0.3 Torr). In this case the ion voltage and the ion current were 120 eV and 0.1 A, respectively.

Quantitative analysis of the prostaglandins was carried out in the chemical ionization mode by measurement of the peak areas for the specific ion current at m/e values given by the M++ 1 and M++ 1, - 90 ions. Tetradeuterated analogues of the prostaglandins were used as internal standards and calibration curves were established with known amounts of the unlabelled compound. Since 13.1% of the added substrate arachidonic acid was i4C- labelled and the prostaglandins were mainly derived from this source, it was necessary to measure the peak areas in the trace of the ions originating from the unlabelled compounds as well as in the trace of the ions given by the 14C-labelled mole-

cules. The final values were calculated with regard to the natural isotopic abundance which was estimated before each series of analysis and gave a rather constant value (3.1 k 0.07 (X + S.D.) for 6-ketoprostaglandin Fi,, I (m/e 540)/I (m/e 542)). On this basis it was also possible to calcu- late the amount of prostaglandins originating from endogenous substate.

Since we lacked deuterated standards, we had to do with a semiquantitative analysis of the

645

monohydroxy fatty acids. Total amounts of these compounds were estimated in terms of radioactiv-

ity recovered after silicic acid chromatography. Additionally, the ratios of the peak areas given by the molecular ions in chemical ionization-NH, mass spectrometry were calculated. These two parameters (total amount and relative ratios) do allow an accurate quantitative estimation. It must be emphasized here, however, that the relative intensities of the molecular ions in the mass spectra of the various hydroxy fatty acids may differ from each other and even slightly vary upon changes in different instrumental parameters. Nev- ertheless, this method is a sufficiently accurate

approach to quantify these metabolites.

Results

Metabolism of arachidonic acid in endothelial cell

homogenates The cell homogenates (0.2 mg protein per ml)

were incubated at 37°C for 10 min in the presence of externally added [l-‘4C]arachidonic acid. Ra- dioactivity was almost completely extracted with

Fig. 1. Thin-layer radiochromatogram of products obtained

after incubation of freshly isolated bovine aortic endothelial

cells with r4C-labelled arachidonic acid: A bovine aortic endo-

thelial cell homogenate (0.2 mg protein per ml) was incubated

with [l-‘4C]arachidonic acid (5 PM; 40 nCi) for 10 min at

37’C. The acidified incubation medium was extracted with

diethyl ether, and this extract was analyzed by radio TLC with the organic phase of ethyl acetate/isooctan/acetic acid/water,

11 : 5 : 2 : 10 (v/v). The silica gel was scraped off in 0.5 cm

segments, and the radioactivity was determined by liquid scin-

tillation counting. The radioactive peaks were tentatively iden-

tified using reference compounds. PG, prostaglandin; AA, arachidonic acid.

diethyl ether (recovery > 90%) and separation of

the various compounds was achieved by TLC. The profile distribution of radioactivity (Fig. 1) re- vealed the presence of great amounts of unreacted arachidonic acid (R, = 0.90) and four more polar components that were tentatively identified using reference compounds. In agreement with other reports [l-4], 6-ketoprostaglandin F,,, the stable

hydrolysis product of prostacyclin, was by far the main metabolite, while prostaglandins F,, and E, were only produced in smaller amounts. In addition, a broad radioactive peak was detected that migrated right behind arachidonic acid (R, =

0.71-0.83) and exhibited the same chromato-

graphic mobility as authentic 15-HETE (R, =

0.81) biosynthesized from arachidonic acid using soybean lipoxygenase as enzyme source.

The prostaglandins and monohydroxy fatty acids were positively identified by GC-MS after previous column chromatography. Prostaglandins were additionally quantified by means of isotope dilution mass spectrometry with stable isotope- labelled compounds as internal standards (see below).

No dihydroxylated metabolites were found in the course of radio TLC, indicating that

arachidonic acid was not metabolized into compounds with similar polarities as leukotriene B4. The possibility of the presence of small amounts of peptide leukotrienes (leukotrienes C,, D,, E, and

F4), however, can not be excluded, since these compounds have been reported to be poorly recovered by conventional extraction procedures [30].

Characterization of the monohydroxy fatty acids Boiled endothelial cell preparations failed to

metabolize [i4C]arachidonic acid which proved that all metabolites obtained were formed en- zymatically. Indomethacin (10 PM) inhibited the formation of prostaglandins, whereas monohydroxy fatty acids were still formed, which suggests the involvement of lipoxygenase activity in the biosynthesis of these compounds.

Large scale incubations were carried out in order to obtain these arachidonic acid metabolites in sufficient yield for the chemical characterization by GC-MS. For this purpose one third of each sample was subjected to catalytic hydrogenation before the derivatization procedure, since the

646

methyl ester-TMS derivatives of the various iso-

merit polyunsaturated hydroxy fatty acids gave very similar fragmentation patterns. The chain fragmentation of the saturated derivatives, on the contrary, is defined by the position of the -0TMS function leading to two intense ions specific for each positional isomer.

Fig. 2 shows the selected ion monitoring of the saturated methyl ester-TMS derivatives of the

monohydroxyeicosatetraenoic acids formed by the endothelial cell homogenates. It can be seen that the compounds were well separated on the capillary column with retention times between 6 and 7 min. The given ions are specific for the II-HETE, 5- HETE and 15-HETE derivatives. Additionally, a peak with m/e of 173 and 301 was observed with

0

I 5.6

i nv. 313

6.0 6.5

Elution Time (mid

Fig. 2. Selected ion monitoring of I5-HETE, II-HETE and

5-HETE produced by freshly isolated bovine aortic endotheliat

cell homogenates. The cells (1 mg proti~n/mI) were incubated

with [1-‘4C]arachidonic acid (25 PM; 0.125 pCi) for 10 min at

37”C, the acidified incubation medium extracted with diethyl ether. and a fraction of the monohydroxy fatty acids was

obtained by column chromatography. The products were

methylated, reduced with H,/PtO,, and converted into the

TMS ether derivatives. The derivatives were analyzed by GC- MS on a 30 m SE54 fused silica capillary column, The oven

temperature was lOO*C for 1 mitt and then increased at 40

Cdeg/min to a final temperature of 320°C. In the course of

mass spectrometric detection, the following ions were moni- tored under electron impact: m/e 173 and m/e 343 for

IS-HETE. m/e 203 and m/e 313 for 5-HETE, and #i/e 229

and m/e 287 for the II-HETE derivative.

a retention time of 5.4 min and identified as methyl ester-TMS derivative of HHT (not shown). No traces of other hydroxy isomers could be detected. The detection limit was about l/50 of the amount of 15-HETE present in the samples.

Mass spectra recorded from the polyunsaturated as well as the fully hydrogenated methyl ester-TMS derivatives in the chemical ionization mode with ammonia as a reagent gas further confirmed the presence of these compounds.

As shown in Table I, 15-HETE, ll-HETE and

HHT were present in approximately equal amounts, whereas the 5-HETE derivative repre- sented only 5% of the total amount of the mono-

hydroxy fatty acids found. Experiments that were carried out in the presence of 10 PM indomethacin

clearly showed more than 90% inhibition of HHT and ll-HETE formation, but no significant effect on the levels of 5- and 15-HETE, indicating that HHT and II-HETE, in contrast to the 5- and 15-HETE, are cyclooxygenase products of arachidonic acid. To confirm the possible involvement of lipoxygenase enzymes in the biosyntheses

of the two latter compounds, incubations were performed in the presence of 10 PM NDGA.

Thereby. the formation of 5- and 15-HETE was

TABLE I

AMOUNTS OF MONOHYDROXY

FORMED FROM ARACHIDONIC

DOTHELIAL CELL HOMOGENATES

FATTY ACIDS

ACID BY EN-

For incubation, preparation and GC conditions see legend of

Fig. 2. The mass spectrometric analysis was done in the chemical

ionization mode with ammonia as reagent gas and the peak

areas given by the M+ + 1 ions of the various compounds were

determined. These ratios together with the total amount of the

radioactive monohydroxy fatty acids were used for the semi-

quantitative analysis of these compounds. Mean values f S.D.

of three (control) and two (inhibitors) experiments. The results

are expressed in terms of ng/0.2 mg protein to allow a better

comparison with the determined prostaglandin values; n.d. not

detectable.

Compound ng/0.2 mg protein

15-HETE

5-HETE

11-HETE

HHT

control

23 +2.5

3.24 1.1

13.0+ 2.3

24.813.4

indome~acin

(10 PM)

22.0 *2.6

3.70*1.5

0.80+0.36

1.7 +0.5

NDGA

(lo PM)

3.2kO.6

n.d.

21.8* 3.6

44.3 + 8.3

inhibited by more than 80%, but the levels of HHT and ll-HETE were increased up to 100% above the values obtained in the control experiments (Table I).

Additionally, we incubated undisrupted endothelial cells in the presence of 10 PM Ca iono-

phore A23187. Qualitative analysis of monohydroxy fatty acids produced under these conditions showed the same pattern of arachidonic acid metabolites as observed in the presence of externally added substrate.

Conversion of arachidonic acid into prostaglandins

by endothelial cell homogenates

In order to study the arachidonic acid metabolite pattern in freshly isolated endothelial cells in more detail and to investigate the influences of enzyme inhibitors, prostaglandin levels were determined by means of isotope dilution mass spec-

trometry. Table II shows the formation of 6-ketopros-

taglandin sa, prostaglandin E, and prostaglandin Fla under various conditions. 6-ketoprostaglandin F,, was the main metabolite of arachidonic acid in this tissue, whereas prostaglandin E, and prostaglandin Fz, were only formed in smaller amounts. On the basis of the isotopic ratios (‘*C/i4C) as

641

obtained from mass spectrometric analysis, the amounts of endogenously formed prostaglandins were also determined (see Table II). The values

obtained are in good agreement with data found after omission of exogenous substrate, thus yield- ing only basal levels of prostaglandins (data not shown). As expected, 10 PM indomethacin significantly inhibited the formation of the prostaglandins. NDGA (10 PM) raised the levels of (i-ketoprostaglandin F,, by 150% and the levels of prostaglandin F,, by 60% above controls. The levels of prostaglandin E, were not significantly changed in the presence of NDGA. 15HPETE (10 PM) in-

hibited the formation of 6-ketoprostaglandin Fi, by approximately 70% and the formation of prostaglandin F2a by about 50%. The prostaglandin E, levels, however, were raised by 70% above control in the presence of 15-HPETE. Since it has been shown that the inactivation of prostacyclin synthetase is mediated by hydroperoxides [31], experiments were performed in the presence of 15-HETE, the reduced hydroxy analogue of 15-HPETE. This

compound failed to show any effect on prostaglandin formation, confirming the requirement of the hydroperoxy moiety for the inhibitory effect

of 15-HPETE on both 6-ketoprostaglandin F,, and prostaglandin F2a formation.

TABLE II

PROSTAGLANDIN FORMATION BY ENDOTHELIAL CELL HOMOGENATES AND ITS MODULATION BY ENZYME

INHIBITORS

Incubations, preparation and GC-MS analysis were carried out as described in the legend to Fig. 3. The amount of the products

derived from the endogenously released arachidonic acid were calculated on the basis of the ‘2C/‘4C ratios in the samples with

regard to the natural isotopic abundance. The inhibitors were added to the cell homogenates 10 min before the addition of arachidonic

acid. Mean values + S.D. (n = 3-4); n.d. not detectable.

Compound ng/ml

control indomethacin

(IO PM)

NDGA

(lo PM)

15HPETE

(lo PM)

6-Ketoprostaglandin F,,

total

endogenous

Prostaglandin E,

total

endogenous

Prostaglandin F2*

total

endogenous

151.1* 12.9 42.3k4.6 42.7* 5.6 12.1 k3.1

26.6k 1.4 1.7kO.5 3.1 f 0.9 n.d.

41.5+ 3.1 2.6 * 0.4 5.3+ 0.7 nd.

384.Ok61.1 43.6 + 6.5

38.7511.1 17.6 f 3.4

21.5k 2.5 45.9 * 3.9 2.8* 0.2 9.3k1.8

68.1 f 13.9 19.1 f 2.2

4.7 (n =I) 1.9kO.7

648

TABLE III

6-KETOPROSTAGLANDIN FiJPROSTAGLANDIN E, AND 6-KETOPROSTAGLANDIN F,,/PROSTAGLANDIN FZa

RATIOS CALCULATED FOR THE PRODUCTS DERIVED FROM THE EXOGENOUSLY ADDED ARACHIDONIC ACID

AND THE RESPECTIVE RATIOS CALCULATED FOR THE ENDOGENOUS PROSTAGLANDINS

The data shown in Table II were used for the calculations. (exogenous = total - endogenous). PG, prostaglandin.

Control

NDGA (10 /.tM)

15-HPETE (10 /.tM)

6-KetoPGFi,/PGE, 6-KetoPGF,,/PGF,,

exogenous endogenous exogenous endogenous

4.1 13.8 3.0 8.0

18.5 13.8 5.4 8.2

0.7 1.9 1.5 9.2

Table II further shows that NDGA, in contrast to 15-HPETE, did not alter the conversion of endogenously released substrate. When comparing the prostacyclin/prostaglandin E, as well as the prostacyclin/prostaglandin F,, ratios of the com-

+ ,5-“PETE IqlYI + HDGA ~WqlY~

~+,5.*p.T. (10 p, +

0

0 1 * 5 ’ 5 IL ’ 8 e ‘O ARKHIclONIC ACID

Fig. 3. Formation of 6-ketoprostaglandin F,, [6-Keto PGF,,]

by endothelial cell homogenates as a function of arachidonic

acid concentration in the absence and presence of inhibitors.

Endothelial cell homogenates were incubated with i4C-labelled

arachidonic acid (8.2 mCi/mmol) at 37°C for 10 min. The

reaction was stopped with ethanol, tetradeuterated prostaglan-

dins were added as internal standards, the acidified incubation medium was extracted with diethyl ether and a fraction con-

taining the prostaglandins was obtained by column chromatog-

raphy. The products were converted into the methyl ester-

methyloxim-TMS derivatives and analyzed by GC-MS. The

oven temperature was 170°C for 1 min and was then increased

at 40 Cdeg/min to a final temperature of 320°C. The mass

spectrometric detection was performed in the chemical ioniza-

tion mode with ammonia as a reagent gas. The inhibitors were

added to the cell homogenates 10 min before the addition of

arachidonic acid. Mean values + S.D. (n = 3-4).

pounds formed from exogenously added 14C- labelled arachidonic acid with respective ratios of the prostaglandins formed from endogenously released arachidonic acid (Table III), it becomes evident that the endogenous arachidonic acid is more preferably metabolized to prostacyclin than the externally added arachidonic acid.

Fig. 3 shows that the conversion rate of arachidonic acid into 6-ketoprostaglandin Fi, in-

creased with increasing substrate concentrations, reaching saturation at about 2 PM arachidonic acid. NDGA (10 PM) again considerably aug- mented 6-ketoprostaglandin F,, formation, whereby the extent of this effect increased with increasing arachidonic acid concentrations.

In the presence of 15-HPETE 6-ketoprostaglandin F,, levels were considerably lowered, almost independent of the exogenous substrate concentration. As also shown in Fig. 3, this inhibitory effect of 15-HPETE was still observed when NDGA (10 PM) was simultaneously present in the incubation medium, i.e., NDGA did not influence 6-ketoprostaglandin Fi, formation in the presence of exogenously added 15-HPETE.

The influence of NDGA on 6-ketoprostaglandin Fi, formation was further studied at different concentrations of the drug. These experiments were carried out at three different substrate concentrations in order to investigate the influence of the arachidonic acid/protein ratios on the effect of NDGA in more detail. The results are shown in Fig. 4. Up to 10 PM NDGA increased 6-ketoprostaglandin F,, formation dose-dependently. This effect, however, was reversed at NDGA concentrations of 50 PM, and 6-keto-prostaglandin F,, formation was considerably inhibited by doses ex-

c

.s 250-

z b

7.5 pw AA

\( 200-

y

t ‘\

a t t

0

6 -:

3.5 AA 5 150 p

Y (b

/ /

p 1 b

El 100 &

‘Z 1, t 1.5 p AA \

m 5 E

:

+I

E

m 50 - 8 // \

+ t ,,!!

0 2.5 5 10[,DGAI EM 20 25 ” 50

Fig. 4. Dose-response curves for the stimulation of 6-ketopros-

taglandin F,, [6-Keto PGF,,] formation by NDGA. Incuba-

tions (in the presence of 1.5 PM, 3.5 PM and 7.5 PM arachidonic

acid (AA)), preparation and GC-MS analysis were carried out

as described in the legend to Fig. 3. Mean values *SD.

(n = 3-4).

ceeding 0.1 mM (data not shown). Comparing the curves obtained with different substrate concentrations it becomes evident that both the maximum

649

of stimulation and the EC,, values for NDGA increased with increasing arachidonic acid con-

centrations. So far we have shown that NDGA did not

influence 6-ketoprostaglandin Fi, formation in the presence of 10 PM exogenously added 15HPETE (Fig. 3). Additionally, we have identified 15HETE

as a lipoxygenase metabolite of arachidonic acid formed by an endothelial cell homogenate. 15- HPETE, the intermediate in the biosynthesis of 15-HETE is a well-known inhibitor of prostacyclin

synthetase. Taken together, these results led us to the idea that 15-HPETE might act as an endogenous inhibitor of prostacyclin synthetase and

NDGA could have raised 6-ketoprostaglandin F,, levels in our experiments by preventing the synthesis of 15-HPETE. Therefore, we studied the formation of 6-ketoprostaglandin F,, and prostaglandin Fza as a function of 15-HPETE concentration in the absence as well as in the presence of 10 PM NDGA. Fig. 5 shows that 15-HPETE inhibited 6-ketoprostaglandin F,, production dose depen-

dently up to 75% with an IC,, value of approx. 3.5 PM (Fig. 5A). Prostaglandin F,, formation was inhibited up to 50% of control, whereby the IC,, value was about 3.0 PM (Fig. 5B). In the presence of NDGA, 15-HPETE blocked the synthesis of 6-keto prostaglandin F,, by 90% and the IC,,

+ NDCA 1OgM z

-80 -

8 N

CONTROL

;j - E

z + NDGA 1OpM -20 -

Fig. 5. Influence of NDGA on the inhibition of 6-ketoprostaglandin F,, (A) and prostaglandin Fza (PGF,,) formation (B) observed in the presence of 15-HPETE. Endothelial cell homogenates were incubated with 14C-labelled arachidonic acid (10 PM; 8.2

mCi/mmol) at 37’C for 10 min. Preparation and GC-MS analysis were carried out as described in the legend to Fig. 3. 15-HPETE

was added to the cell homogenates immediately after the addition of NDGA. Results are expressed as a percentage of the control

experiment where no 15-HPETE was added. NDGA (10 PM) itself increased 6-ketoprostaglandin Ft, levels 2.5-fold as compared to

the experiment performed in the absence of NDGA. Mean values f S.D. (n = 3-4).

650'

value was decreased from 3.5 to 1.5 PM under these conditions. Yet, it should be considered that NDGA alone elevated 6-ketoprostaglandin F,, levels about 3-fold. Prostaglandin FZa formation, in contrast, was not influenced by 15HPETE when NDGA was simultaneously present in the incuba-

tion medium. These latter data suggest that NDGA probably

raised 6-ketoprostaglandin 5, and prostaglandin FZa levels in endothelial cell homogenates by two

different mechanisms.

Discussion

The results presented here confirm previously published data [l-4] which show that, besides smaller amounts of prostaglandin FZa and prostaglandin E,, prostacyclin is the major cyclooxygenase metabolite of arachidonic acid in endothelial cells. Since prostaglandin H, has been reported to break down to prostaglandin E, sponta-

neously [32], our results do not prove the presence of an active prostaglandin E, isomerase. Incuba- tion with radioactive arachidonic acid enabled us

to determine the basal release of prostaglandins under the same conditions, i.e., in the presence of 10 PM externally added arachidonic acid. Similar values were obtained after omission of the exogenous substrate, indicating that endogenous arachidonic acid is metabolized independently of exogenous substrate. Furthermore, we showed that endogenous arachidonate is more preferably metabolized to prostacyclin than externally added arachidonic acid. The same has been reported for cultured mesothelial cells [33] and for ram seminal vesicle microsomes [34]. In the course of the latter

study it was demonstrated that increasing concentrations of arachidonic acid give rise to increased formation of prostaglandin E,, whereas prostacyclin is the major product of arachidonic acid only at very low substrate concentrations. In our study, however, this can not be the only reason for the discrepancy between prostaglandin biosynthesis from exogenous arachidonic acid and endogenously released substrate, since the products derived from the different precursors were determined simultaneously under the same conditions (10 PM arachidonic acid). It is more likely that the enzymes involved in prostaglandin bio-

synthesis are not equally accessible to endogenous and exogenous arachidonic acid, so that prostaglandin H, formed from endogenously released substrate is immediately metabolized by a tightly coupled prostacyclin synthetase. The conversion rate of exogenous prostaglandin H, into pros-

tacyclin might be lower, which might give rise to an accumulation of the endoperoxide, leading to an increased formation of prostaglandin E,. Another explanation of this discrepancy could be

the presence of two pools of different cycloo- xygenases as has been suggested for cultured human vascular endothelial cells [35].

The endothelium is well known to produce

prostacyclin, which inhibits platelet aggregation [5] and acts as a potent vasodilator [6]. Moreover, it seems to mediate the relaxing effect of various autacoids by producing a factor, probably a lipoxygenase metabolite of arachidonic acid [14-191 that relaxes vascular smooth muscle by increasing the formation of cGMP [36]. We performed the chemical characterization of these compounds by means of mass spectrometry. Both cell homogenates as well as undisrupted cells stimulated with Ca ionophore A23187 produced considerable amounts of HHT, ll-HETE and 15-HETE. Be- sides these compounds, small amounts of 5-HETE were also detected. The formation of HHT and ll-HETE as cyclooxygenase products of arachidonic acid has also been described for cultured human skin fibroblasts [lo] and for cultured rat aortic smooth muscle cells [22]. ll-HETE formation seems to represent an incomplete action of cyclooxygenase as has been reported for purified fatty acid cyclooxygenase [37]. NDGA blocked the synthesis of 15- and 5-HETE, indicating that these two compounds are lipoxygenase metabolites of arachidonic acid. The 15-HETE thereby was the major lipoxygenase product of arachidonic acid in this tissue. The presence of 5-lipoxygenase activity, as shown by the detection of small amounts of 5-HETE, would suggest that the endothelial cell preparation also converted arachidonic acid into leukotrienes. Biosynthesis of these compounds, however, must be low or nonexistent in endothelial cell homogenates as studied here, since not even trace amounts of leukotriene B4 or isomeric dihydroxylated metabolites of arachidonic acid could be detected in the course of radio TLC analysis.

651

The absence of these compounds indicates that 5-HPETE was not considerably converted into leukotriene A 4, the common precursor of all

leukotrienes. Our finding that 15-HETE was the major lip-

oxygenase metabolite of arachidonic acid in bovine aortic endothelial cells seems to be consistent with results obtained by Hopkins et al. [20]. Different sources of the investigated tissues and partly the lack of well-suited analytical methods might be the reason for different results of other groups [21-261.

15HETE has been shown to activate a cryptic 5lipoxygenase pathway in the PT-18 mast basophilic cell line [38], as well as to inhibit leukotriene synthesis in rabbit peritoneal macrophages [12] and to inhibit 12-HETE formation in

platelets [13]. Hence, it seems that 15-HETE has manifold functions in regulating the arachidonic acid metabolism in various tissues. Things get even more complex when it is considered that 15-lipoxygenase converts arachidonic acid only into the 15-hydroperoxy derivative which is in most cases rapidly reduced by a glutathione-dependent peroxidase to the corresponding hydroxy compound. The hydroperoxy intermediate itself, but not the reduced form has been demonstrated to be a potent inhibitor of prostacyclin synthetase [II]. The mechanism of action of 15-HPETE still remains unclear, but most likely its inhibitory effect is a consequence of its reduction to 15-HETE. In the course of this reaction hydroxyl radicals are generated, which might be responsible for the inactivation of prostacyclin synthetase [31]. Furthermore, it must be considered that conversion of 15-HPETE

to 15-HETE is associated with glutathione oxida- tion. Most of the glutathione disulfide formed is then reduced by glutathione reductase and GSH is regenerated at the expense of NADPH. A deple- tion of cellular glutathione, however, as a consequence of an accumulation and a subsequent ef- flux of GSSG has been observed. It seems to be due to a decreased NADPH/NADP+ redox level, rather than to insufficient glutathione reductase activity [39]. Since prostacyclin synthetase and other enzymes involved in prostaglandin synthesis have been shown to be GSH dependent [32], decreased glutathione levels may give rise to decreased enzyme activities.

Something must be said in terms of the above-

mentioned endothelium-derived vascular smooth

muscle relaxing factor. This as-yet unknown compound has been described to be very labile and to possibly exert its effect via a radical-involving mechanism [19]. Different hydroxylated eico- satetraenoic acids did not produce a relaxation of endothelium-denuded rabbit aortic strips [40]. However, if the endothelium-derived relaxing factor were in fact a lipoxygenase metabolite of arachidonic acid, it should be expected to be a hydroperoxy intermediate rather than a stable hy-

droxy fatty acid. Finally, the influence of NDGA on pros-

taglandin formation needs to be discussed. In our study it stimulated the formation of cyclooxygenase products with the most pronounced ef-

fect on prostacyclin formation. Since only about 1.5% of arachidonic acid was metabolized into lipoxygenase products, this effect can hardly be explained by a shift from the lipoxygenase to the cyclooxygenase pathway. Since endothelial cell homogenates were capable of converting arachidonic acid into the potent prostacyclin synthetase inhibitor 15-HPETE, a function of this compound as an endogenous inhibitor of prostacyclin formation in these cells seems possible. Certainly, 15-HPETE concentrations needed for a respective inhibition

of prostacyclin synthetase were about lo-fold as compared to the amount of 15-HPETE produced by our cell preparation. It must be remembered, however, that ‘in situ’ generated substances are likely to act in concentrations far below those needed when applied exogenously. The results presented here which show that NDGA did not exert any stimulatory effect on prostacyclin formation when externally added 15-HPETE was simulta-

neously present in the incubation medium (Fig. 3, lowest curve and Fig. 5A) tend to support the idea that 15-HPETE is an endogenous inhibitor of prostacyclin synthetase. Furthermore, inhibition of prostaglandin FZ, formation by 15-HPETE was abolished in the presence of 10 PM NDGA (Fig.

5B). Recently, Beetens and Herman [41] have obtained similar results with vitamin C, which increased prostacyclin formation by aortic rings from various species and prevented the inhibition of prostacyclin formation by 15-HPETE. The authors attributed this observation to an antioxidative effect of vitamin C, which protects cyclooxygenase

652

against inactivation by oxygen radicals [42]. Thus,

NDGA might act by two distinct mechanisms in endothehal cells: (a) as an antioxidant that protects cyclooxygenase against inactivation and (b) as a lipoxygenase inhibitor that blocks the synthesis of 15-HPETE, thus leading to increased prostacyclin synthetase activity. The degree of pros-

tacyclin stimulation was increased with increasing substrate concentrations. Accepting the above-discussed hypothesis of endogenous inhibition of prostacyclin formation by 15-HPETE this may indicate a higher affinity of arachidonic acid to cyclooxygenase as compared to lipoxygenase. The latter enzyme might therefore produce considerable amounts of 15-HPETE only at higher substrate concentrations, leading to a more pronounced inhibition of prostacyclin synthetase in the control experiments.

The fact that NDGA did not influence the metabolism of endogenously released arachidonic acid might indicate that the described endogenous

‘inhibition of prostacyclin formation does not be- come effective under normal physiological conditions, although it is po&ble that under pathophys- iological conditions such as tissue damage which always involves a substantial release of arachidonic acid and therefore high local arachidonic acid concentrations, 15-lipoxygenase activity might be responsible for an endogenous inhibition of pros-

tacyclin synthetase. Regardless, however, of a possible physiological

significance of the observed effects, our results clearly demonstrate that endothelial prostacyclin synthetase activity is considerably underestimated when experiments are carried out in the presence of exogenous arachidonic acid.

Acknowledgements

We wish to thank Dr. F. Brunner for helpful discussions,* Mr. V. Haibel for excellent technical assistance, Mrs. B. Oberer for preparing the illus- trations and Dr. A. Buchmann for typing the manuscript. This work was partly supported by a grant of the Fonds zur Forderung der wissen- schaftlichen Forschung, Vienna, No. 5617.

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Documents

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells