Bioehim$ca el Biophysica Acla, 1081 (lg'911301 307

]01

1991 EIsevi~ Science Publishers BV. (BiomedicalDivision10005-2760/91/$03.50 ADONIS 000527609100091N

Synthesis, structural identification and biological activity of ll,12-dihydroxyeicosatetraenoic acids formed in human platelets Piir Westlund i..

Jan Palmblad

2 J.R. Falck 3 and Sun Lumin ~

, / Department of Ptly~iologicalChemist~, Karolbtsku lnstitelt~,t. St~khvlm ISweden). D~panment of Medicine 3, Karolimka lt~titutet at SSdersjukh~el. Stm'hhohn {Sweden) and j Departalent of Molecular Genetics. Unit,ersit, of Te.t~. South~,e~tern Medical Cenler. Dalh~ TX (U.S A ) "

(Received 30 May 1990) {Revised manu~npt received 30 August 19901 Key words: Structure determination; t 1,12-Dihydroxyeicosatetraenoicacid: Leukotrien¢; Araehldonic acid: (Human platelet) An enantiaspecific route for the synthesis of il,12-dihydroxyeicosatetraenole acids was developed and used to synthesize U , 1 2 - ~ h y d r o x y . 5 ( Z ) , 7 ( E ) , 9 ( E ) , l d ( Z ) - e i c o s a t e t r a s n o i c acids. The li,12-DHETE.~ were synthesized with the stereoehemistry of the hydroxyl group being 11(R),12(S) and I 1 ( S ) , 1 2 ( S ) . The synthetic comgounds were used to elucidate the s m m t m e of II,12-DHETEs formed in human pLltelets by comparison ~ the chromatographic retention time in HPLC and GC as well as their ion fragmentation pattern in GC-MS. T h e major II,12.DHETE formed in human platelets was found to he identical with l l ( R ) , i 2 ( S ) - d i h y d r o x y - 5 ( Z ) , 7 ( E ) , 9 ( E ) , I 4 ( Z )-eicosatetraenoic acid. Two more compounds were tentatively identified as I I ( S ) , 1 2 ( S ) - d i h y d r o x y - 5 ~ Z ) , 7 ( E ) , 9 ~ E ) , 1 4 ( Z ) - e i c o s a f e t r a e n o l c acid and U , 1 2 - d i h y d r o x y - 5 ( E ) , 7 ( E ) , 9 ( E ) , 1 4 ( Z ) . e i c o s a t e t r a e n o l ¢ acid. Fur~hermuce, the ll(S),12(S~.dlhydroxy5 ( Z ) , 7 ( E ) , 9 ( E ) , 1 4 ( Z ) . e i e o s a t a t r a e n o i c acid was | o u , d to possess biological activity on neutrophil functional responses. However, the major compound, 1 1 ( R ) , 1 2 ( S ) - d i h y d r o x y - 5 ( Z ) , 7 ( E ) , 9 ( E ) , 1 4 ( Z ) - e i c o s a t e t r n e n o i c acid, f o r m e d in platdets lacks biological activity in the test systems used. The present data further support that ll,12-dihy0~roxyeicosatetraenolc acids are formed in human #atelets via a leukotrlene like mechanism presumably by the 12-npoxygenase. Furthermore, the blol~ical effects of one of the compounds showed a unique activity profile c o m ~ r n d to other lipoxygenase products.

Introduction The focus of polyunsaturated fatty acid metabolism has during the latest years turned to lipoxygenase pathways and the formation of icukotrienes via 5- and 15-1ipoxygenase catalyzed reactions [1]. A less well understood metabolic pathway in that respect is the 12lipoxygenase in human platelets. Th~-s en?y me converts arachidonic acid to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and the corresponding 12-hydroxy acid (12-HETE) [2]. The biological importance of this path-

Abbreviations: DHETE, dihydroxyelcosatetra~oic acid: RP-HPLC, reversed-phase high-perform~ce liqaid chromatography: SP-HPLC, straight-phase high-performance liquid chromatography: MoM¢~Si, methyl ester-triraethylsilylether; GC-MS, gas chromat~graphy-m~s speClromet~/, Correspondence (= Present address): P. Westlund, Department of Reproductive Endocrinology. Karolinska hospital, Box 6O5OO, S10401 Stockholm,Sweden.

way and its products is still questionable and needs further investigation. However. recently the identification of a group of novel dihydroxy acids formed from arachidonic acid in human plateicts was described [3,4]. The products were found to be formed via initial oxygenation of carbon atom 12, i.e., a 12-1ipoxygenase catalyzed reaction. Among the identified dihydroxy acids were two 11,12dihydroxyeicosatetraenoic acids III,12-DHETEs). These findings might lead to a better understanding of the 12-1ipoxygenase pathway in human platelets and other cell types containing this enzyme activity, It i; thus of importance to further characterize the metabolic pathway, its products and their possible biological function. The present paper describes the synthesis of two 11,12-dihydroxyeicosatetraenoic acid stereoisomers. The synthetic isomers were used to structurally identify the products formed in human platdets by comparison of their chemical and physical properties and to partly characterize their biological activity.

302 H ~

COOMo

i

H-

o

H-,O

2,R:M.

NaOH~4=R=Na

i(Me,Si),Ntl 6

'

3;R=Me

NaOH~5:R =N B

/Id',SlllflLI

7

Fig. 1. Synthetic Scheme for Ihe synthesis of ll(R),]2($I- and lllS).12(s)-dihydroxy-S(Z),7(E),gIE),14(Zl-ei¢osatetraenoic acids.

Material

and Methods

Chemicals Arachidonic acid was from Nu-Chek-Prep (Elysian, MN, U.S.A,). Hanks balanced salt solution (HBSS) was obtained from SBL (Stockholm, Sweden). Lumlnol, human serum albumin (HSA, essential fatty acid free) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were obtained from Sigma Chemical (St. Louis, MO). Leukotriene B4 (LTB4) was a kind gift from J. Rokach (Merck Frosst, Dorval Canada). lonomycin and Fura-2AM were from Calbiochem (La3olla, CA).

Preparation of 11(R),12(S)- and 11(Sg12(S)-dihydroxy5(Z),7(E),9(E).14(Z)-eicvsaletraenoic acids Homochiral 1l(R),12(S)- and II(S)A2(S)-DHETE, 6 and 7, respectively, were prepared by total chemical synthesis as outlined in the Scheme (Fig. l). Sharpless epoxidation [5] of synthetic [6] methyl 12(S)-hydroxy5(Z),8(Z),lO(E),14(Z)-eicosatetraenoate I using vanadyl acetylacetonate (0.03 equiv.) and tert-butyl hydroperoxide (1.1 equiv.) in anhydrous benzene (30 mM) at room temperature for 20 mix generated a chromatographically separable mixture of two epoxyalcobols identified as erythro 2 and threo 3 (7670, 1.4: l ratio) based on spectral analysis and by analogy with prior results iTl. TLC of 2 and 3: SiOa, Et20/hexane ( 3 : 2 / eo.tainlng 1% Et ~N, R F approx. 0.35 and 0.29, respectively. ~H-NMR (300 MHz. CDCI3) of 2 : 6 0.85 (I, J approx. 6.8 Hz, 3H), 1.14-1.42 (m, 6H), 1.68 (tt, J approx. 7.3, 7.3 Hz, 2H), 1.96 2.18 (m, 4H), 2.24-2.42 (m, 4H), 2.8.5 3.02 (m, 3H), 3.63 (s, 3H), 3.70 (d, J

approx. 10.2 Hz, IH), 3.78-3.86 (m, 1H), 5.06 (apparent t, J approx. 10.2 Hz, 1 H), 5.30-5.74 (complex m, 5H). IH-NMR (300 M14z, CDCI3) of_3: ~ 0.86 (L J approx. 6.8 Hz, 3H), 1.20 1.40 (m, 6H), 1.68 (tt, J approx. 7.3, 7.3 Hz, 2H), 1.96-2.16 (m, 4H), 2.30 (t, J approx. 7.2 Hz, 214), 2.38 (t, J approx. 7.2 Hz, 2H), 2.85-3.00 (m, 3H), 3.54-3.74 (m coincident with methoxy singlet at 3.64. 5H), 5.06 (apparent t, J approx. 10.5 14z, 1H), 5.30-5.74 (m, 514). Saponification of 2 in 0.5 M aqueous N a O H / tetrahydrofuran ( 1 : 3 ) at room temperature for 10 h gave the corresponding sodium salt 4_ (90% product yield) which, after thorough evaporation of the organic solvent, was isolated by adsorption onto Bio-Rad SM-2 beads (200 : 1, w/w). Washing the resin with methanol and evaporation of the filtrate gave a viscous oil that was dried azeotropicaUy with anhydrous benzene. To a 25 mM solution of 4 in dry letrahydrofuran at 0 ° C was added lithium bis(trimetbylsilyl)arnide (1 M solution in tetrnhydrofuran, 5 equiv.) [8]. After 12 h, the dark yellow mixture was quenched with saturated aqueous ammonium chloride and the pH adjusted to 4.5 with 0.1 M HCI. Extractive isolation with E t 2 0 and chromatographic purification afforded 6 (42%1. T L C : SiOz, M e O H / C H 2 C I 2 (1:9), R F approx. 0.39. IHN M R (200 MHz, CDCI3): 3 0.88 (t, J approx. 7 Hz, 3141, 1.12-1.47 (m, 6H), 1.66-1.72 (m, 2H), 2.00-2.18 (m, 2H), 2.20-2.45 (m, 6H), 3.49-3.66 (m, 1H), 4.12 (dd, J approx. 5, 7 Hz, 1H), 5.28-5.62 (m, 3H), 5.82 (dd, J approx. 7, 15 Hz, 1H), 6.06-6.37 (m, 2H), 6.44 (dd, J approx. 11, 15 Hz, 1H), 6.50 (dd, J approx. 11, 15 Hz, 1H). Analogous treatment of salt 5 gave 7. IH-NMR (200 MHz, CDCIa): 8 0.88 (t, J approx. 7 Hz, 3H), 1.10-1.48 (m, 6H), 1.64-1.72 (m, 2H), 1.96-2.10 (m, 2H), 2.182.44 (m, 614), 3.43-3.57 (m, 1H), 4.05 (apparent t, J approx. 7 Hz, 11t1, 5.32-5.67 (m, 3H), 5.78 (dd, J approx. 7, i5 Hz, 1H I, 6.08-6.38 (m, 2H), 6.42 (dd, J approx. 11, 15 Hz, 114/, 6.53 (dd, J approx. 11, 15 Hz,

1H). Cell preparation and experiments Neutrophils. Neutrophils were isolated from peripheral venous blood, obtained from healthy volunteers, by a one step separation on discontinuous Percoll gradients [9]. The purity and viability were both more than 95%. Shape changes were followed by interference contrast microscopy. Cells were exposed to the stimulus or HBSS alone for 5 rain. 14ereafter, cells were classified as polarized (i.e., activated) or rounded (i.e., re~tlng) [10]. Chemiluminescence (CL), augmented by himinol (0.17 raM), was assessed essentially as described previously [11,12] by a Chronolog Lumi Aggregometer fitted with a thermostated euvene-holder with stirring and at 37°C. None of the stimuli used here conferred light emission in a cell-free chemiluminescence system. In

303 experiments on the priming effect, neutrophils were first treated with the l l , 1 2 - D H E T E s for 3 rain. Subsequently. LTB 4 was added and CL was followed for an additional 3 min. Those results are reported as the relative light emission when compared with samples treated with buffer alone followed by LTB4. Intracellular Ca 2÷ concentrations were calculated from the change of Fura-2 fluorescence [121- N::utrophils (5 - 106 cells ml i ) in HBSS supplemented with 20 m M Hepes (pH 7.4), were incubated at 3 7 ° C with 0.5 ~tM Fura-2-AM for 30 rain. Loaded cells were washed twice, reconstituted in HBSS (with Ca 2 ~ a: 1.27 raM) and stored on ice until use. Ceils were then warmed at 3 7 ° C with continuous stirring of the cell suspension. Excitation wavelength was set at 340 nm and emission at 510 nm. After a stable baseline had been established, stimulus was added and emitted light recorded until return to baseline, The system was controlled by addition of EGTA, Tris buffer, Triton X-100 and CaCl2 as described [10]; calculations of the calcium concentration were performed according to Metcalf et al. [10]. Standard stimuh were rMLP, LTB4 and ionomycin (0.1, 0.1 and 1 FM, respectively). Chemotaxis was assessed with a modal'led Boyden chamber technique, using a 48 multiwell chamber (Neuroprobe, Cabin John, M D ) [13]. Neutrophils (2-106 m l - l ) , in HBSS supplemented with 0.4% HSA, were allowed to migrate into cellulose nitrate filters (Sertorius. O~ttingen, F.R.G.; 3 .aM pore diameter) for 45 rain at 3 7 ° C . F M L P and LTB4 were used as standard chemotactic stimuli and HBSS as control for spontaneous migration. Migration was assessed as the mean depth, in trma, of penetration into the filter of the leading five cells in each of three microscopic fields in four replicate wells, and the results are given as net migration (i.e., stimulated migration minus spontaneous migration). Statistical analysis was performed with Students" two tailed t-test. Human washed platelets. These were prepared from bully coat as described earlier [14]. The platelets were suspended in Dulbecco's phosphate-buffered saline (PBS) (pH 7.4), 1 . 1 0 ~ platelets m1-1 and then incubated with araehidonie ~,,id (final concentration i50 .aM), at 3 7 ° C for 20 rain. The platelets had been preincubated with aspirin (final concentration 3 mM) for 30 rain in the second washing buffer to inhibit the cyelooxygenase pathway. The incubation was terminated by addition of 3 vol. o[ ethanol,

High~erformance liquid chromatography Two analytical HPLC-columns were used to purify the compounds: Nueleosil 50-5-C~s (250 × 4 ram) and Nucleosil 50-5 (300 × 4 mm) (Machery-Nagel, Diiren, F.R.G.). The solvent systems used were: (A) methanol/ wa~,er/acetic acid ( 6 5 : 3 5 : 0 . 1 . v/v); (B) hexane/ isopropanol/acetic acid (96: 4: 0.0 2 , v/v): and (C) hexane/isopropanol/acetic acid (98 : 2 : 0.02, v/v). The solvent was delivered at 1 ml • min i by an LDC (Laboratory Data Control) HPLC pump (Clearwater, FLI equipped with a Rhe0dyne injector. Ultraviolet absorption was continuously mo~ itored at 270 nm using an LDC Ill variable wavelength detector.

Ultraviolet spectroscopy A Hew!ett-Packard 8450A was used for the recording of ultraviolet spectra. All samples were dissolved in HPLC-grade methanol.

Gas chromatography-mass spectrometO' All compounds were analysed as their methyl estertrimethylsilyl ether derivatives (Me-Me3Si). The analysis was performed using a LKB 9000 equipped with a glass column (1.5 m × 4 ram) packed with 1% SE-30 on Chromosorb W (HP), 80/100 mesh or a VG-7070E equipped with a fused silica column (50 m × 0.32 ram) SE-30 0.15 micron film thickness. The helium flow was 10 m l / m i n , oven temperature was set at 2 0 0 ° C or 220 ° C (VG-7070E) and the energy of the electron beam was set at 22.5 eV, Results The ethyl acetate extracts from the incubations were treated with diazomethane and subjected to an initial purification on SP-HPLC, solvent system B. The region earlier known to contain 11,12- and 14,15-DHETEs [4] was collected and rechromatographed on RP-HPLC. solvent system A (Fig. 2). Four peaks. I to IV, contained material with ultraviolet-absorbing characteristics for compounds containing a conjugated triene structure. Peak II and IV were all known to contain 11,12- and 14,15-DHETEs [4]. The individual peaks and standards wilh known structure were chromatographer on RP-HPLC (~lvent system A) and SP-HPLC (solvent system C), separately or mixed in different combinations. Chromatographic data are compiled in Table 1.

Analysis o[ peak 11 Extraction and purification The s a m p J ~ v,c:,: 51tered and the ethanol was removed by rotary evaporation. The remaining water pltas~, v~as acidified to pH 3.0 and extracted tbxee times with 3 vol. of ethyl acetate. The ethyl acetate was evaporated to dryness and the residue further purified by HPLC.

Material from peak 11 coehromatographed with 11( R ). 12( S )-dihydroxy-5-( Z ),7( E ).9( E ), 14( Z )-eieosatetraenoic acid in RP-HPLC. When material from peak 11 was analysed on SP-HPLC it eluted as two peaks, designated lla and l l b (Fig. 3, upper). Comparison of the retention times for peak lla and l i b and the methyl ester of 11( R/ . 12(S)-DHETE showed that material from

304 11

ua

IV

i ii ii~

H

[ ,

,

0

,

.

lo

i

.20

,

,

,

,

[

to 40 TIME (rain)

,

.

,

.

to

0

Fig. 2. Reversed-phase HPLC purification (system A) of material oblained from Ihe region containing ll.12-DHETEs and 14,15DHE'YES from the initial straight-phase HPLC (system B) purification ztep. All labeled peaks (l-IV) contained material with ultravioIet-eh~acteristics for compounds containing a conjugated tfiene structure.

=

peak l i b c o c h r o m a t o g r a p h e d w i t h this c o m p o u n d (Fig, 3, lower). F u r t h e r cc, m p a r i s o n o f material f r o m peak I l b a n d 11 ( R ) , 1 2 ( S ) - D H E T E o n G C - M S as t h e i r M e - M % S i derivatives s h o w e d t h a t these c o m p o u n d s w e r e identical as j u d g e d b y G C r e t e n t i o n t i m e a n d ion f r a g m e n t r , i o n p a t t e r n . F u r t h e r m o r e , t h e ultraviolet-spectra o t h e c o m p o u n d s were f o u n d to be identical (Table I) t a s e d o n these d a t a c o m p o u n d l i b w a s f o u n d to be identical with ll(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)eicosatetraenoic acid. C o m p o u n d l l a h a s earlier b e e n identified as erythro-14(R),15(S)-dihydroxy-5(Z), 8( Z ) , I 0 ( E ) , I 2 ( E ) - e i c o t a t e t r a e n o i c acid [4].

I~ /

j

z

'

lb

'

~b ' 3~ TIME (rain)

'

~0

'

~0

'

Fig. 3. Straight.phase HPLC purification (system C) of peak II from RP-HPLC (Fig. 2). (upper pan¢l~ chromatography of material from peak llh and the methyl ester of synthetic II(R),12(S)-dihydtoxy5( Z )~7(E ).9( E ),14( Z I-eicosa te tmeuaoie acid (system C) (lower p a n e l ) .

compounds cochromatographed with material from peak I I l i n a n y o f t h e used c h r o m a t o g r a p h i c systems. T h e C - v a l u e of the Me-Me3Si-derivative o f this 11,12D H E T E also differed f r o m t h o s e for t h e s y n t h e t i c c o m p o u n d s ( T a b l e !). H o w e v e r , w h e n t h e s y n t h e t i c c o m p o u n d s were a n a l y z e d o n G C - M S as t h e i r M e - M e 3 S i

Analysis of peak 111 Peak I l l was earlier k n o w n to c o n t a i n material o f a n ~ L 1 2 - D H E T E i s o m e r [4], b u t n o n e o f t h e s y n t h e t i c

Chromatographic, ultraoiolet and GC-MS data of isolated compounds and synOletic I 1,12-dihydroxyeicosatelraenoi¢ acids Compound

RP-HPLC retention times, Methyl esters. system A (mini

SP-HPLC retention times. Methyl esters, system C (min)

Ultraviolet maximum. Methyl esters in HPLC grade methanol. (nm)

GC-M$ data Me-Me~Si derivative l~ SE-30. C-value major ions (m/z)

lla llb Ill IVa IVb II(S)A2(SIDHPSfE 11(R),12(S)DHETE

41 41 43 54 54

39 43 45 41 44

263 262 260 263 262*

273 272 269 272 271

284 283 280 283 282

23.9 23.7 24.7 23.9 na

,194, 479, 463, 394, 321,173 494, 479. 404, 383. 354, 28L 213 494. 479. 404, 383. 354, ?~t, 213 494. 479, 463, 394, 321.173

54

44

262

272 283

23.7

494, 479. 404, 353. 354, 281. 213

41

43

262

272 283

23.7

494. 479, 404, 383, 354, 281,213

= Partly contaminated with compound IVa, na, not analysed.

305 TABLE n

Chemdummescenre responses to II.12-DHETEs and other chemontfractonl~ The figures are mean and S.E. values for the number of separate experiments performed in quadruplicate~g ~ t h cells from different donors (nl. Values under the beadi:lg of "direct effect" refer to the maximal chemiluminescence respoese (in mY/ evoked when Ihe stimulus w ~ added. The "priming elf~t' relat~ Io expenmenLs de. tailed in the Materials and Methods and R~ults ~tion~. where ne.trophils tirgt were treated with tl(S),t2fS)-DHETE or 1I( R ).12(S )-DHEFE (at 1 ~tM) for 5 min and subgcquently activated by 0.t pM LTB4. That latter maximal response is e x p r ~ d as percent of respo~se~ observed fnr cells Ergt treated by HBS$ alone * P < O.05. od - not determined.

i

Stimulus

Concentration (tiM)

II(S).I2(SF DHF.TE

1 e

2o

ao

I

0

lit._+ 6¢g *

4

87 _*17% ad nd

4 10 10

II{R),I2{SI-

_/\ 1o

Treatment type direct priming eff~l cff~t

DHETE LTB~ fMLP

4o

llsm(nan) Fig, 4. Straight-phase HPLC purification {system C) of peak IV from RP-HPLC IFig, 2). (upper panelL Chromatography of peak IV with addiri0n of the methyl e~ter of II(S),12{S)-dihydroxy5{Z ).7( E ),9( E ),14( Z)-eicosaletraenoi¢ acid {system C) {lower panel). derivath, ds in h i g h e r quantities (2 ,ttg b e f o r e derivatizalion), a m i n o r p e a k w i t h a h i g h e r C-value (24.6) s h o w i n g a I I , 1 2 - D H E T E f r a g m e n t a t i o n p a t t e r n was det e e t e r . T h e G C r e t e n t i o n time for this m i n o r p e a k w a s identical w i t h t h e r e t e n t i o n t i m e for t h e I I . 1 2 - D H E T E material f r o m p e a k I l L T h e h i g h e r C-value for this l l , 1 2 - D H E T E indicates t h a t t h e c o n j u g a t e d triene syst e m h a s u n d e r g o n e i s o m e r i z a t i o n to a n all t r a n s c o n t t g u r a t i o n , a well k n o w n p h e n o m e n o n .

Analysis of peak I V M a t e r i a l f r o m p e a k l V h a s earlier been identified as

threo.14( S ),15( S ).di hydro x y. 5( Z ),8( Z ),l O( E ),12( E )eicosatetraenoic acid [4,14].

1 0A 0A

0 820_+130 2300±180

W h e n t h e r e t e n t i o n times f o r material f r o m this peak a n d the m e t h y l ester of synthetic II(S),12(S)-DHETE w e r e c o m p a r e d in R P - H P L C , they were identical. W h e n a n a l y s e d o n S P - H P L C m a t e r i a l f r o m peak I V separated i n t o t w o p o o r l y s e p a r a t e d peaks, designated l V a a n d [ V b (Fig. 4, upper). C o c h r o m a t o g r a p h y of the m e t h y l ester o f ll(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)eicosatetraenoic acid w i t h m a t e r i a l f r o m peak I V s h o w e d t h a t 1 I ( S ),12(S ) - D H E T E c o c h r o m a t o g r a p h e d w i t h 1Vb (Fig, 4, lower). N o m a s s s p e c t r o m e t r i c identification c o u l d b e p e r f o r m e d for material f r o m peak I V b . d u e to lack of material a n d c o n t a m i n a t i o n w i t h material f r o m p e a k [Va, w h i c h h a s a C-value close to t h a t of synthetic 11 ( S ).12( S ) - d i h y d r o x y - 5 ( Z ).7( E ),9( E ), 14( Z )-eicosatetraenoic acid ( T a b l e l). Peak [ V a was identical w i t h the earlier i d e n t i f i e d threo-14(S).15(S)-dihydroxy5(Z).8(Z),lO(E),12(E)-eicosatetraenoie acid [4,14].

lonomycia l ItM Increase of [Ca2+] i n M

, / / ~ I M I . , P 0,1 gM 1

~ I I ( R ) A 2 ( S ) - D H E T E 0

I

2

3

4

0

1

1 ~M

2 3 4 Time, rain Time, rain Fig, 5. Fura-2-thiorescent responds to 11(S),12(S)- and ll(RI.I2(S)-DHETE. {MLP, LTB~ and ionomycin. The figure depic[s an actual tracing. The y-axis gives the increase of [Ca 2+ ]i from the basal level, which was 74_+ l0 nM. Fad rise of (Ca2÷ L conferred by th~ lipoxygenase products was consistem and reproducible in the three s~Lsof experiments, run in quadruplicate, with ceils from different dono~.

306 ;o

J

:/",....

0

,I -10

.

,

.

.9

,

.

-8

=,

. -7

.

,

-6

-5

log M, concentration Fig. 6. Chemotaxisof human neutrophils in response to ll(S)A2(S)([]), and II(R),I2(S~DHETE (O), LTBd (A) and fMLP (z0. The figure gives mean and S.E. values for four separate experiments for the distance to the leading front cells afler subtraction of spontaneous migration (whichusually w~.sapprox. 45/zm). The migratory response la ll(S)A2(S)-DHETE was significantly different from HBSS stimulated cells (P < O.Ol).

that the l l ( S ) - i s o m e r but not the l l ( R ) - i s o m e r enhanced the LTB 4 response (Table II). We next analyzed whether the dihydroxy acids would confer an increase of Fura-2 fluorescence, being a probe for [Ca2+]j. Ionomycin, fMLP and LTB 4 were all more potent in this respect compared to the l l ( S ) - i s o m e r (Table lIl). The kinetics of the [Ca2+L-response were highly characteristic. As shown in Fig. 5, increases were rapid and sustained for ionomyein, transient for LTB4 and biphasic [or fMLP, Fluorescence response to the 11( S )-isomer resembled that of ionomycin. The l l ( S ) , 1 2 ( S ) - D H E T E , but not the l l ( R ) , I 2 ( S ) DHETE, induced stimulated migration in the Boyden chamber. Optimal migration occurred at the same concentration as for LTB4 and fMLP, 100 nM. The distance migrated by ncutrophils exposed to I I ( S ) , 1 2 ( S ) D H E T E was, however, considerably shorter than for the other ehernoattractants (Fig, 6). At supraoptimal concentrations cells reacted with a deactivation pattern to the three stimuli, i.e., cells were highly elongated, did not migrate as far as with optimal concentrations of the stimuli and accumulated at high concentrations approximately 0 - 5 0 / ~ m into the filter. Discussion

Biological properties of synthetic ll,12-dihydroxyeicosatetraenoic acids When nentrophils were exposed to the l l ( S ) , 1 2 ( S ) isomer at 1/tM, they assumed irregular shapes, that also characterized cells activated by LTB4 and fMLP. Neutrnphils responded with a burst of chemiluminescence when exposed to fMLP and LTB4 (Table I1). However, when exposed to the 1!(5')- or the l l ( R ) - i s o mer no chemiluminescence was detected. The question whether the II,12-DHETEs would prime a neutrophil for a subsequent oxidative response to LTB4 was assessed next. The tested substances were incubated with PMNs at 37~C and after 5 min LTBa was added. The ensuing light emission was compared to LTBa-activated controls incubated 5 rain with HBSS and the appropriate concentration of the solvent ethanol. We found

TABLE I11 Elfeels of hpids, ionomycin and fMLP on Fura.2fluorescence The figures are given as the ma.6nlum rise above the lCa2+l~ of resting ceils, which was 74 ~:i0 n M, n deno~s the number of separate experiments, run in quadruplicates, wltb ceils from different donors. Stimulus

Concentration 0tM)

Rise of ICa2+ L (riM)

n

fMLP LTB4 lonomycin II(Shl2(S~-DHETE 11[R).12(S)-DHETE

0l 0.1 1 1 I

+253±55 +248±60 + 381 ±42 +47± 4 O

6 3 3 3 3

One of the major metabolic pathways for arachidonic acid in human platelets is the 12-fipoxygenase pathway. Recently, Wesflund et at. [3,4] showed that dihydroxyeicosatetraenoic acids could be formed via this pathway. The identified acids were 5.12-DHETEs (four isomers) and I I , 1 2 - D H E T E s (two isomers). In the present study we describe the synthesis of two II,12-DHETEs. By the use of these isomers we were able to tentatively identify one more I I , 1 2 - D H E T E isomer formed in huma n platelets and to further characterize the earlier identified II,12-DHETEs. The major isomer found in platelets was identified as 11( R),I 2( S )-dihydroxy-5( Z ),7( E ),9(E )34(Z)-eicosatetraenoic acid based on chromatographic and mass spectrometric data. The formation of this compound probably proceuds via oxygenation by the 12qipoxygenas¢, epoxide formation and enzymatic hydrolysis of the epoxide. Mild el al. [15], recently described the identification of an enzymatically formed 11,12D H E T E , derived from II,12-LTA 4 in several guinea pig tissues. They did not determine the geometry of the 11 and 12 hydroxyl groups, but posttdated it to be l l ( R ) , 1 2 ( S ) . Their and our data further support the hypothesis that I I , 1 2 - D H E T E s can be enzymatically formed via a leukotriene mechanism. However, the enzyme that catalyzes the synthesis of l l , 1 2 - L T A 4 has yet to be established, although it is very likely to be the 12-11poxygenase. The other major l l , 1 2 - D H E T E (compound Ill) was tentatively identified as a ll,12-dihydroxy-5(E),7(E),

307 9(E),14(Z)-eicosatetraenoic acid. The geometry of the hydroxyl groups of this isomer is not known. However, when the D H E T E identified as 1l( R ),12( S )-dihydroxy5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid and compound Ill were analyzed as their Me-Me3Si derivatives after catalytic hydrogenation, saturation of the double bonds, they showed identical C-values [4]. It indica!~,s that compound llI also has the 1I(R )A2(S )-configuration of the hydroxyl groups. Trans-isomers were also formed from the synthetic l l , 1 2 - D H E T E s in minor amounts during the derivatization procedure or GC-MS analysis. However, treatment of the synthetic compounds with the conditions used during the work-up procedure used for the incubations did not lead to formation of the trans-isomcr. The identification of only one trans-isomer also indicates that it might he formed by enzymatic isomerization. The data by Kitamura et at. [16] also support an ~nzymatic formation of this ll,12-DHETE, since their study with acid catalyzed hydrolysis of 11,12-LTAa did not lead to the formation of a major II,12-DHETE trans-isomar. In addition to the earlier described II,12-DHETEs, we were able to identify one more l l , 1 2 - D H E T E , tentatively identified as ll(S),12(S)-dihydroxy-5(Z),7(E), 9(E),14(Z)-eicosatetraenoic acid. This compound was only formed in minor amoums compared to the other two II,12-DHETEs. It appears from the studies on neutrophil functional responses that ll(S),12(S).dthydroxy-5(Z),7(E),9(E), 14(Z)-eicosatetraenoic acid, but not ll(R)A2(S)-dihydroxy-5( Z),7( E ),9( E134( Z)-eiensatetraenoic acid, possesses a cell activation potential. The activity profile is unique. The capacity to induce stimulated migration and shape changes resembles that of LTBa and lipoxin A 4 [11-13[. The ability to confer a rise of the intracellular eoocentration of calcium, but lack of effect on initiation of the oxidative metabolism, as evidenced by the chemiluminescence assay, differs clearly from lipoxin A 4 (which is a weak inducer of chemiluminescence but not of calcium responses [13]), resembles the LTB4 activity profile more. In fact, a number of ether lipids, related to platelet activating factor, have the capacity to induce rises of intracellular calcium, coupled with an inability to activate the oxidase of the neutrophil [17]. The sharp peak of chemoattraetant activity at 100 nM, together with the deactivation pattern at higher concentrations and the polarized cell shape, suggests that the ll(S)-isomer induced chemotaxis by mechanisms similar to a classic chemt~.ttractant, that is by means of surface receptors. These putative receptors appear to be distinct from those of LTBa since the chemiluminescence response to LTB4 was not blunted by a previous treatment of the cells by the ll(S)-isomet. In contrast, the response was primed by this procedure, suggesting that II(S),12(S)-DHETE might have activated the neutrophils in a way that has been noted

for other unrelated stimuli (e.g., platelet activating [actor, fMLP. etc.) [18[. The l l A 2 - D H E T E s have also been tested for proand anti-aggregatory effects on human platelets and were found to he inactive (Westhind, unpublished data). The present data provide further information about the synthesis and biological activity of dihydroxyeicosatetreenoic acids formed in human platelet and might lead to a better understanding of the t2-1ipoxygenase pathway. Acknowled~alents This study was supported by grants from the Swedish Medical Research Council (19P-8884, 19X-5591, 03X05915), the Swedish Association Against Rheumatism, King Gustaf V's 80-Year Fund, USPHS the National Institutes of Health (GM 31278), the Funds of P.A. Hediund, N. Svartz, L Hierta, B. Gustafsson, Karolinska instilutet and StSdersjukhuset. The skillful technical assistance #yen by Mrs. S. Myrin, P. Sp[mgberg and Eva Ohlson is gratefully acknowledged. References221 ! Samuel~on, n., Dahl~n, S.-E.. Lindgren. J.A.. Rouzer. C.A. and Scrhan. C.N~0987) Science237, 1171-1176. 2 Hamburg, M. and Samue!sson. B. (19741 Proc. Natl. Acad. Sci. USA. 71. 3~4]0-3404. 3 Wesaund, P. (19871 Adv. Proslaglandin. Thromboxane Leukomene Rcs. 17, 99104. 4 Westlund, P., Edenius, C. and Lindgrcn. J.A. (19881 Bio~him. Biophys. A¢la962. 1O~-llS. 5 gharpless, K.B.and Vethoeven, T.R, 119791AldnehimieaActa 12. 63-74. 6 Yadagifi, I~. Lumln, S.. Mosset,P.. Capdevila, J. and Falck, J.R. (19861Tctrahedron Lett. 27, 6039-6(M0. 7 Falck,J.R.. Manna, S.. Siddhanta. A.K..Capdcwla.J. and Buynak, J.D. (19831Tetrahedron Lett, 24, 5715-3718. 8 Falck, J.g.. Manna, S., Capdemla. J. and Buyaak. J.D. (19831 Tetrahedron Lett. 24. 571g-5720. 9 Rzngertz.B., Palmblad, J.. R~dmark. O. and Malmsten,C. (!9821 FEBS Lett. 147.180-182. l0 Metcalt,3.A., Oallin, J.[., Nause~f.W.M. and Root, RK. (lgSfi) Laboratory Manual of Neuuophil Function, Raven press, New York. ll Palmblad.J., Gyllenhammar, H., Lind[run. J.A. and Malmslen.C. (19841J. lmmunol. 132~3041-3B45. 12 Palmblad, &. Gyllenlmmmar, H., Ringertz, B., Nilsson, E. and CottulL B. (1988)Biochim.Biophys. Acta 970, 92-102. 13 Palmblad,J., Gyllenhammar, H. and Ringertz,B. (1988lLipoxim /Wong, P.Y.-K. and Serhan. C.N., eds.), pp. 137-145, Plenum Press, New York. 14 Won[. P,Y.-K., Wc~llund,p., HambeTg,M., Granstrbm, E., Chad. P.H.-W. and Samuel~n, B. (198513. Biol.Chem. 26O,9162 9165. 15 Miki, 1., Shimizu. "r., Seyama, Y.. Kilamura, S,, yamaguehi, K., Sand, H., Ueao, H., Hiratsuka,A. and ~iatabe, T. (19881J. Biol. Chem. 264,5799-5805. 16 Kilamura, S.. Shimizu,T.~ Miki. L [zumLT., Kasama. T., Said, A,, Sand, H. and Scyama,Y. 0988) Eur. J. Biochem.176,725-731. 17 Palmblad, J., Sarauel.s,son. J.. Brohulh J. (19901Scand. J. Clin. Lab, Invest. 50, 363-370 18 Gay. CJ.. Beckman. J.K.. brash. A.R., Bates, J.A. and Lukens. J.N. rigs4) Blood 64, 780-785.