Comparison of the effects of nitric oxide and peroxynitrite on the 12-lipoxygenase and cyclooxygenase metabolism of arachidonic acid in rabbit platelets

Prostaglandins, Leukotdenesand Essential Fatty Acids (1998) 59(2), 95-10O © HarcourtBrace & Co. Ltd 1998

Comparison of the effects of nitric oxide and peroxynitrite on the 12. lipoxygenase and c y c l o o x y g e n a s e m e t a b o l i s m of arachidonic acid in rabbit platelets Y. Fujimoto, S. Tagano, K. Ogawa, S. Sakuma, T. Fujita Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka, 569-1094, Japan

Summary The effects of a new type of nitric oxide (NO)-releasing compound, 1-hydroxyl-2-oxo-3-(N-methyl-3aminopropyl)-3-methyl-1-triazene (NOC7), and peroxynitrite (ONOO-) on the formation of 12-hydroxy-5,8,10,14eicosatetraenoic acid (12-HETE), thromboxane (TX) B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) from exogenous arachidonic acid in washed rabbit platelets have been compared. At concentrations of 5 #M and below, NOC7 inhibited 12-HETE formation (56.5-98.8% inhibition). Moreover, NOC7 inhibited TXB 2 and HHT formation at concentrations ranging from 5 to 20 #M (TXB2, 62.2-88.1% inhibition; HHT, 11.6-62.2% inhibition). ONOO- had little or no effect on the production of these three metabolites at concentrations of up to 50 #M. Experiments utilizing a new class of NO antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1 -oxyl 3-oxide, revealed that the observed effects of NOC7 are caused by NO. The effects of NO were reversed by addition of the superoxide generating system (xanthine plus xanthine oxidase and catalase), indicating that superoxide is a vital modulator of the action of NO. These results suggest that NO, but not ONOO- (up to 50 #M), can be a potent dual inhibitor of the 12-1ipoxygenase and cyclooxygenase activities in platelets and that superoxide is an important regulator of the action of NO. INTRODUCTION

Considerable attention has been focused on multiple functions of a labile inorganic radical species, nitric oxide (NO), in a variety of biological systems? NO plays an important role in the cardiovascular system, not only by controlling the vascular tone but also by inhibiting platelet aggregation and platelet adhesion. 2,3In the vascular system, NO can be generated by endothelial cells, neutrophils and macrophages. It has been shown that platelets also produce NO from L-arginine by a Ca2+dependent mechanism and that this pathway plays a regulatory role in platelet function. 4,5 On the other hand, unstimulated and stimulated platelets generate superoxide (Q'-)Y NO and 02"- react together at near the diffusion limit (6.7 x 109 M-~ s-l) s to yield peroxynitrite (ONOO-). The production of ONOO- has been recently Received 1 May 1998 Accepted 2 July 1998 Correspondence to: Yohko Fujimoto, Tel: +81 726 90 1055; Fax: +81 726 90 1005

demonstrated or proposed in a variety of pathophysiological conditions, including arthritis, 9 atherosclerosis 1° and ischemia-reperfusion. H ONOO- has also been reported to affect platelet aggregation. 12 In platelets, arachidonic acid (AA) is converted into thromboxane (TX) A2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) by the cyclooxygenase pathway and into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12HETE) by the 12-1ipoxygenase pathway) 3 TXA2 is a potent vasoconstrictor and inducer of platelet aggregation and rapidly breaks down to form the stable end-product, TXB2. 12-HETE has been reported to give rise to platelet aggregation, ~4'~5 and neutrophil ~6 and aortic smooth muscle cell migrationY So, it seems likely that 12-HETE as well as TXA2 is involved in the initiation and propagation of thrombotic and atherosclerotic disorders. Recently, Nakatsuka and Osawa ~s have reported the selective inhibition of the 12-1ipoxygenase pathway of AA metabolism by NO in platelets. However, little information is available concerning the action of ONOO- on the metabolism of AA via cyclooxygenase and 12-lipoxygenase in platelets. The 95

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present study was undertaken to investigate the effect of ONOO- on the two enzymatic pathways of AA in platelets in comparison to that of NO. NO was generated from NO-releasing compound, 1-hydroxyl-2-oxo-3-(N-methyl3-aminopropyl)-3-methyl-l-triazene (NOC7), which releases NO spontaneously at ambient temperatures without the requirement of enzyme activation or biotransformation.19 MATERIALS AND METHODS Materials

TXB2, sodium salt of AA and xanthine were purchased from Sigma (St Louis, MO, USA). HHT was purchased from Cayman (Ann Arbor, MI, USA) and 12-HETE was from Cascade Biochem (Berkshire, UK). NOC7 and carboxy2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO) were supplied from Dojindo Laboratories (Kumamoto, Japan). Xanthine oxidase (from cow milk) and catalase (from bovine liver) were obtained from Boehringer Mannheim (Mannheim, Germany) and 9anthryldiazomethane (ADAM) was from Funakoshi Pharmaceutical (Tokyo, Japan). All other reagents were of analytical grade. ONO0- synthesis

The preparation of ONOO- was performed by the method previously described. 11 Solutions of (a) 0.6 M NaNOz and (b) 0.7M HC1 plus 0.6M H202 were pumped at 6ml/15 s into a tee-junction and mixed in a 3 mm diameter by 2.5 cm glass tube. The acid-catalyzed reaction of nitrous acid with H202 to form ONOOH was quenched by pumping 1.5 M NaOH at the same rate into a second teejunction at the end of the glass tubing. Residual H202 was degraded by the reaction with M n Q for one hour, which was subsequently removed by centrifugation. The resulting ONOO- was stored at -80°C. The concentration of ONOO- was determined by applying a molar extinction coefficient of 1670 M-1 cm-1 at A302~.

buffer, pH 7.4 (buffer A) containing 1 mM EDTA, and then resuspended in buffer A. Incubation of platelets

The washed platelet suspension (3 x l0 s platelets) was preincubated for 5 rain at 3 7°C in 1 ml of buffer A with or without the indicated concentrations of NOC7, ONOO-, carboxy-PTIO, xanthine plus xanthine oxidase or catalase. AA (40 gM) was subsequently added to the platelet suspension, and the mixture was incubated at 37°C for 5 rain. The reaction was terminated by quickly adding an appropriate amount of 0.25 M HC1 to bring the pH of the reaction mixture to 3.0. Measurement of 12-HETE, TXB2, HHT and AA

After incubation, the reaction mixture was extracted with 3 ml of ethyl acetate. 12-HETE, TXB2, HHT and AA in the extracted lipid were simukaneously determined by a high-performance liquid chromatographic (HPLC) method described in our recent studies.2°,21 Briefly, 12HETE and HHT were separated in normal-phase chromatography and simultaneously quantitated by employing a UV spectrophotometric detector. TXBz and AA were measured after esterification using ADAM. TXB2 and AA esterified with ADAM were separated in reverse-phase chromatography and simultaneously quantitated by employing a fluorescence spectrofluorometer. Our previous study utilizing indomethacin, an inhibitor of cyclooxygenasey and quercetin, an inhibitor of lipoxygenase, 2~'24has demonstrated the capacity of the present in vitro system to simultaneously detect changes in the activities of platelet cyclooxygenase and lipoxygenase.25 Statistical analysis

Results are means + SEM. Statistical significance was determined by Student's t-test. RESULTS

Preparation of platelets

Blood was withdrawn into a 3.8% solution of trisodium citrate (9:1, v/v) from the abdominal aorta of male rabbits (2-2.5kg) under sodium pentobarbital anaesthesia. Platelets were then collected by differential centrifugation. Whole blood was centrifuged for 10 rain at 200 × g at room temperature and the platelet-rich plasma was withdrawn from above the pelleted erythrocytes. After addition of EDTA (to a final concentration of 1 mM), the platelet-rich plasma was cooled to 0°C and centrifuged at 2000 × g for 10 min. The platelet pellet was washed twice with 134 mM NaC1, 5 mM glucose, 15 mM Tris-HC1

Figure 1 illustrates the effects of various concentrations of NOC7 and ONOO- on the formation of 12-HETE, TXB2 and HHT from exogenous AA in washed rabbit platelets. By being pre-incubated with the platelets for 5 min before an addition of AA, NOC7 inhibited the formation of 12HETE, TXB2 and HI-IT at concentrations ranging from 1 to 20 ~tM (Fig. 1A). At concentrations of 5 ~tM and below, NOC7 caused a concentration-dependent inhibition of the platelet 12-lipoxygenase pathway, as shown by decreased formation of 12-HETE (56.5-98.8% inhibition). NOC7 at 1 or 2 ~tM did not affect the platelet cyclooxygenase pathway (measured as TXB2 and HHT formation), whereas it significantly inhibited this pathway at concen-

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(2), 95-100

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Effects of NO and ONO0- on 12-1ipoxygenase and cyclooxygenase in rabbit platelets

,ol,

A

3

2

Ii b 0 0

1

2

5

10

20

1

I

I

I

0

10

20

0

50

100

Concentration (gM)

Fig, 1 Effects of NOC7 and ONOO- on the formation of 12-HETE, TXB2 and HHT in washed rabbit platelets. Platelets (3 x 108/ml) were preincubated with or without various concentrations of NOC7 or ONOO- for 5 rain at 37°C prior to the incubation with arachidonic acid (40 gM) for 5 rain at 37°C. (A) NOC7; (B) ONOO-. Values are means ± SEM (n = 4), a p < 0.05; compared with the corresponding value in the absence of NOC7 or ONOO-. bp < 0.01; compared with the corresponding value in the absence of NOC7 or ONOO-.

trations of 10 and 20 gM (TXB2, 78.9 and 88.1% inhibition; HHT, 45.7 and 62.2% inhibition). These results imply that the cyclooxygenase pathway is less sensitive to the inhibitory effect of NOC7 than the 12-lipoxygenase pathway. In contrast to the inhibitory effect of NOC7

97

on the formation of 12-HETE, TXB2 and HHT, ONOOshowed little or no effect on the formation at concentrations of up to 50 gM (Fig. 1B). At a concentration of 100 ~M, the formation of 12-HETE, TXB2 and HHT was inhibited significantly by ONOO- (12-HETE, 38.9% inhibition; TXB2, 40.5% inhibition; HHT, 31.7% inhibition). Thus, ONOO- showed rather weaker inhibition on the 12lipoxygenase and cyclooxygenase pathways as compared with NOC7. In addition, the amount of AA remaining after incubation was quantified by HPLC. In the control experiment, when the platelets were preincubated in the absence of NOC7 or ONOO- followed by the addition of AA (40 gM), 21.6 + 1.4 nmol AA was detected (n= 4). Raising the NOC7 concentration from 0 to 20 gM or the ONOO- concentration from 0 to 100 ~M increased the amount of AA from 21.6 + 1.4 to 33.0 + 1.6 nmol or from 21.6 + 1.4 to 26.2 + 2.0 nmol (n=4). These results lessen the possibility that NOC7- and ONOO--induced reduction of 12-HETE, TXB2 and HHT formation can be ascribed to decreased availability of AA by their peroxidative action. NOC7 gives two molecules of NO upon thermal decomposition. 19 It has been shown that a new class of NO antidote, i.e. carboxy-PTIO scavenge NO via a unique radical radical reaction with NO. 2627 Therefore, we determined the effect of NOC7 in the presence of carboxyPTIO on the production of ! 2-HETE, TXB2 and HHT in rabbit platelets (Table 1). Preincubation of the platelets with carboxy-PTIO (5 and 50 gM) alone showed no effect on the formation of 12-HETE, TXB2 and HHT. The inhibitory effect of 2 gM NOC7 on 12-HETE formation could be antagonized by the addition of carboxy-PTIO at concentrations of 5 and 10 p~V[.Addition of carboxy-PTIO significantly elevated levels of cyclooxygenase metabolites when compared with the platelets pre-incubated with 20 ~VI NOC7 (carboxy-PTIO 20 pM) or restored to almost the control levels (carboxy-PTIO, 50 gM). So, it may safely be assumed that NO was responsible for the decrease in AA metabolism.

Table I Effect of NOC7 in the presence of carboxy-PTIO on the formation of 12-HETE, TXB2 and HHT in washed rabbit platelets

Control Carboxy-PTIO (5 gM) Carboxy-PTIO (50 gM) NOC7 (2 gM) NOC7 (2 gM) + Carboxy-PTIO (5 gM) + Carboxy-PTIO (10 gM) NOC7 (20 gM) NOC7 (20 gM) + Carboxy-PTIO (20 gM) + Carboxy-PTIO (50 gM)

12-HETE

TXB2 (nmol)

HHT

6,46 6.28 6.78 0.75 4.51 4,81 0.07 0.19 1.00

2.39 2.40 2.43 2.37 2.29 2.35 0.34 1.44 1.90

1.92 1.87 1.82 2.19 2.23 2.25 0,74 1.64 1.90

_+0.42 ± 0.33 ± 0.22 ± 0.07 ± 0.27 a ± 0.34 a ± 0.01 ± 0.02 b ± 0.09 °

+ 0.22 _+0.22 _+0.21 _+0.18 _+0.08 + 0.20 ± 0.02 ± 0.10 ° +_0,15 c

± 0.16 ± 0.15 _+0.15 ± 0.19 ± 0.21 ± 0.21 ± 0.07 ± 0.12 b ± 0.15 °

Platelets (3 x 10a/ml) were preincubated with or without NOC7 or carboxy-PTIO for 5 min at 37°C prior to the incubation with arachidonic acid (40 gM) for 5 min at 37°C. Values are means ± SE (n = 3). a p < 0.01; compared with NOC7 (2 gM) alone, b p < 0.02. ° P < 0.01; compared with NOC7 (20 gM) alone.

© Harcourt Brace & Co. Ltd 1998

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NO reacts rapidly with 02"- at diffusion control rate to give ONOO-. 8 Table 2 shows the effects of additions of a reactive oxygen species generating system and catalase during the pre-incubation of rabbit platelets with NOC7. When the platelets were preincubated with xanthine (0.5 mM) plus xanthine oxidase (50 mU/ml), the formation of 12-1ipoxygenase and cyclooxygenase metabolites was markedly inhibited. The reaction of xanthine plus xanthine oxidase is known to generate 02"- and H202.2s Previously, we have shown that H202 inhibits platelet 12-1ipoxygenase and cyclooxygenase activities. 29 In the present experiments, the effect of H202 could be blocked by the addition of catalase (which metabolizes H~O2 to H20 and 02, 1000U/ml). The combination of xanthine plus xanthine oxidase and catalase blocked the inhibitory action of 10~[M NOC7 on 12-HETE, TXB2 and HHT production. DISCUSSION

Important interactions between NO and AA metabolism appear to exist. However, results regarding the influences of NO on cyclooxygenase activity are difficult to interpret because it has been shown to activate, 3°,31 inhibit, 32 or have no effect3~ on this enzyme activity. It has been reported that NO inhibits the 12-lipoxygenase pathway with little effect on the cyclooxygenase pathway in platelets, TM but few reports indicate the effect of ONOOon platelet 12-lipoxygenase and cyclooxygenase activities. The purpose of the present paper is to describe the action of NO and ONOO- on the metabolism of AA via 12-1ipoxygenase and cyclooxygenase in platelets. NOC7 releases NO spontaneously and acts as NO donor. In the present study, NOC7 was a potent inhibitor of platelet 12-1ipoxygenase activity. In addition to 12lipoxygenase, NOC7 elicited inhibitory activity on

platelet cyclooxygenase, although the inhibition was a little weaker. NO was the responsible molecule for the inhibition by NOC7 of platelet 12-1ipoxygenase and cyclooxygenase, as demonstrated by the use of a new NO scavenger carboxy-PTIO. These results suggest that NO can be the naturally occurring inhibitor of both platelet 12-lipoxygenase and cyclooxygenase activities. TXA2 is a potent vasoconstrictor and inducer of platelet aggregation. This AA metabolite is involved in the initiation and propagation of thrombotic and atherosclerotic disorders. The lipoxygenase metabolites as well as the cyclooxygenase metabolites have also been focused as mediators of thrombosis and atherosclerosis. A 12-lipoxygenase product, 12-HETE, has been reported to amplify thrombin-induced aggregation of platelets and counteract the inhibitory effect of a cAMP elevator PGE1 on platelet aggregation. 14,I5 12-HETE has also been reported to induce migration of smooth muscle cells from media to intima, Iz which event is a significant process in the genesis of atherosclerosis. These reports imply that dual inhibition of the 12-lipoxygenase and cyclooxygenase pathways in platelets may be beneficial to vascular diseases such as thrombosis and atherosclerosis. Inhibition of platelet aggregation by NO is thought to occur through stimulation of guanylate cyclase.34 Our data suggest that, in addition to its stimulatory effect on soluble guanylate cyclase, NO inhibits platelet aggregation by inhibiting both platelet 12-lipoxygenase and cyclooxygenase activities. In contrast to the above observations, Nakatsuka and Osawa is reported that L-arginine or sodium nitroprusside, a generator of NO, had little effect on the cyclooxygenase pathway in platelets at concentrations up to 100 ~M. It is possible that the difference between the present work and that of Nakatsuka and Osawa TM may be related to the nature of the NO-generating agent employed.

Table 2 Effects of additions of a reactive oxygen species generating system and catalase during the preincubation of washed rabbit platelets with NOC7

12-HETE

TXB2 (nmol)

HHT

Control

6.33 + 0.40

2.22 ± 0.20

1.85 ± 0.06

X (0.5 mM) plus XO (50 mU/ml)

1.78 ± 0.15

0.69 ± 0.06

0.54 ± 0.04

X (0.5 mM) plus XO (50 mU/ml) + Catalase (1000 U/ml)

6,16 ± 0.35

2.29 ± 0.24

2.01 ± 0.19

NOC7 (10 ~M)

0.08 + 0.01

0.52 ± 0.03

0.79 ± 0.07

NOC7 (10 pM) + X (0.5 mM) plus XO (50 mU/ml)

5.49 ± 0.55 a

1.94 ± 0.18a

1.83 ± 0.14a

+ Catalase (1000 U/ml) Platelets (3 × 108/ml) were preincubated with or without xanthine (X) plus xanthine oxidase (XO), catalase or NOC7 for 5 min at 37°C prior to the incubation with arachidonic acid (40 ~M) for 5 min at 37°C. Values are means ± SE (n = 3). " P < 0.01; compared with NOC7 (10 ~M) alone,

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(2), 95-100

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Effects of NO and ONO0- on 12-1ipoxygenase and cyclooxygenase in rabbit platelets

NO b e c o m e s a g o o d t a r g e t for 0 2 - b e c a u s e of t h e h i g h rate c o n s t a n t for r e a c t i o n a n d also b e c a u s e NO a n d 02"c a n b e p r o d u c e d b y platelets, 4-7 t h u s favoring b i m o l e c u lar collision to form ONOO-. In this s t u d y we f o u n d t h a t ONOO- was a w e a k i n h i b i t o r of t h e 12-1ipoxygenase a n d c y c l o o x y g e n a s e activities in platelets. Thus, t h e biological activity of NO m a y b e a b r o g a t e d via a r e a c t i o n w i t h 02"-. To test this possibility, a n a d d i t i o n a l s u p p l y of 02"- g e n e r a t e d b y x a n t h i n e p l u s x a n t h i n e oxidase was a d d e d to NOC7. T h e s e e x p e r i m e n t s were p e r f o r m e d in t h e presence of catalase (1000 U/ml) to n e u t r a l i z e H202. As s h o w n in this study, t h e p o t e n c y of NO as a n i n h i b i t o r of platelet 12-1ipoxygenase a n d c y c l o o x y g e n a s e was d e c r e a s e d in t h e p r e s e n c e of 0 2 - . Platelets are e x p o s e d to low levels of NO all of t h e time. W e m u s t c o n s i d e r t h a t b a s a l synthesis of NO in vivo is c o n t i n u o u s u p o n s t i m u l a t i o n and, t h e r e fore, m a y afford e x t e n d e d i n h i b i t i o n of t h e platelet 12-1ipoxygenase a n d c y c l o o x y g e n a s e p a t h w a y s u n d e r n o r m a l p h y s i o l o g i c a l conditions. In s o m e p a t h o l o g i c a l situations, excess p r o d u c t i o n of 02"- m a y cause loss of t h e i n h i b i t o r y a c t i o n of NO o n platelet 12-lipoxygenase a n d c y c l o o x y g e n a s e activities a n d at t h e s a m e t i m e y i e l d ONOO- w h i c h h a s a m u c h less i n h i b i t o r y effect o n t h e s e e n z y m e activities. It s e e m s likely t h a t 02"- is a n i m p o r t a n t r e g u l a t o r of t h e a c t i o n of NO o n p l a t e l e t 12-1ipoxygenase a n d c y c l o o x y g e n a s e activities u n d e r p h y s i o l o g i c a l a n d p a t h o l o g i c a l conditions. O u r p r e s e n t r e s u k s s u g g e s t t h a t NO can b e a p o t e n t d u a l i n h i b i t o r of t h e 12-1ipoxygenase a n d c y c l o o x y g e n a s e activities in platelets b u t t h a t ONOO- is a p o o r i n h i b i t o r of t h e s e e n z y m e activities. Therefore t h e b a l a n c e of NO a n d 02"- c o u l d b e critical for t h e n o r m a l f u n c t i o n i n g of platelets. D i s t u r b a n c e of this b a l a n c e c o u l d h a v e disastrous pathophysiological consequences.

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