Short Communication Mediators of Inflammation, 9, 31–34 (2000)

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.


Introduction
The stimulation of ce lls by diverse agonists leads to the liberation of arachidonic acid from membrane phosp holipids. Arachidonic ac id c an be metabolize d through the c ycloox ygenase or lipox ygenase pathw ays to form the eic osanoids, including prostaglandins and leukotrie nes. These are important mediators of acute inflammatory proce sse s. There is substantial e videnc e that this arachidonic acid re lease is me diate d by the Ca 2 + -sensitive group IV cytosolic phosp holipase A 2 (cPLA 2 ). [1][2][3][4] The ac tivity of the cPLA 2 , w hich is pre se nt in many mammalian c ells, can be regulate d by tw o important mechanisms. The first appears to be a rise in the intrac ellular Ca 2+ conc entration, w hich c ause s translocation of the c PLA 2 from the cytosol to the inte rnal membranes w here it binds through a Ca 2 +depende nt lipid binding domain. A second mechanism of re gulation of cPLA 2 is its phosphorylation by prote in kinases. 1,4 So the phorbol ester 12-O-tetrade canoylphorbol-13-ac etate (TPA), w hich is know n to ex hibit its physiologic al activitie s via stimulation of prote in kinases, 5 induce d an inc rease in phosphorylation and c atalytic ac tivity of cPLA 2 as w ell as arachidonic ac id re le ase in macrophage s, ne utrophile s, keratinocyte s and glomerular mesangial ce lls. 6 -10 We have previously re ported that TPA is able to stimulate the liberation of arachidonic acid also in platelets. 11 In these c ells different protein kinases have bee n found: prote in kinase C, 12 p44 MAP kinase (also name d ERK1 ), p42 MAP kinase (also named ERK2 ) 13 -15 and p38 MAP kinase . 16 The aim of our prese nt w ork w as to inve stigate, w hich of them are involve d in the TPA-induced arachidonic ac id release in bovine p late le ts by using specific inhibitors of the p rote in kinases.

Cells
Imme diate ly after the death of the animal, bovine blood (1 litre ) w as c ollec te d in a polyprop ylene ve sse l containing a solution of 0.077 M EDTA-Na 2 in 0.2% (w /v ) saline (0.1 litre per 1 litre blood ). After dilution of the blood w ith 0.5 litre of 0.9% (w /v ) saline the platelets w e re is olated by centrifugation as previously desc ribe d. 1 8 The plate lets w ere stored at + 4°C. All ex pe riments w e re performed w ithin 8 h after isolation of the platelets.

Measurement of inhibition of cPLA 2 -activity
Inhibition of c PLA 2 w as dete rmined by measuring the TPA-or c alc ium ionop hore A23187-induc ed arachidonic ac id release from bovine plate le ts w ith HPLC/ UV-detection as pre viously desc ribed. 1 1,18 Briefly, to a solution of ETYA in DMSO, w hich inhibits formation of arachidonic ac id me tabolite s in platelets , w as added the DMSO solution of a te st compound or DMSO alone (in c ase of the control te sts and the kinetic ex perime nts ) follow ed by the plate let susp ension and a solution of calcium chloride at 37°C (final platelet conce ntration: about 8 ´10 8 c ells /2 ml). Then the c PLA 2 w as activated by TPA (2 m M) or A23187 (20 m M). The solution of TPA w as freshly pre pare d each time. Whe n using TPA as stimulant the incubation time w as variable (kine tic ex perime nts ) or 60 min (ex pe rime nts w ith e nzyme inhibitors ). In the ex pe riments w ith A23187 the incubation time w as 1 min. Afte r te rminating the enzyme reaction the produce d arachidonic ac id w as cle aned up by solidphase ex traction and quantified w ith HPLC/UV-dete ction at 200 nm.

Solubility of the test compounds
All compounds w ere soluble under the te st conditions.

Cell lytic potency of the test compounds
The ce ll lytic potenc y of the test c omp ounds w as me asured by turbidime try ac cording to a proce dure previously desc ribed. 19 The compounds did not show ce ll lytic prope rties at the conc entrations applied.

Results
Rec ently, w e have reporte d that TPA stimulates the liberation of arachidonic acid in w ashed bovine platelets. 11 The time c ourse of arachidonic ac id release w as sigmoid reaching a plate au afte r about 15 min. To avoid metabolism of arachidonic ac id via the c ycloox ygenase-1 and the 12-lipox ygenase pathw ays, the dual cycloox ygenase-1 /12-lip ox yge naseinhibitor 5,8,11,14-e ic osatetraynoic ac id (ETYA) w as added to the platele ts in these ex periments. We now have found that a sec ond e ve n more pronounce d arachidonic acid liberation occurs if the inc ubation w as ex te nded (Fig. 1 ). How ever, this se cond ste p of arachidonic acid re lease only c ould be me asure d w hen fre sh (1-2 days old) plate lets w ere used, w hile the first ste p still appe are d 3-4 days after the isolation of the platelets from bovine blood.
Contribution of the cPLA 2 to the arachidonic acid release in TPA-treated platelets The arachidonic ac id release in A23184-treate d platelets is catalyse d by group IV cytosolic p hospholipase A 2 (c PLA 2 ). 2 0 -23 To inve stigate, w hether cPLA 2 is also predominantly responsible for the arachidonic ac id liberation induced by TPA, w e monitored the inhibition of the w hole arachidonic ac id production after TPA stimulation (incubation time 60 min ) by se veral PLA 2 inhibitors. Me thyl arachidonyfluorophosphonate (MAFP), 2 4,2 5 w hich is know n as dual inhibitor of cPLA 2 and group VI Ca 2 + -inde pendent phospholipase A 2 (iPLA 2 ), 2 6 blocked the arachidonic rele ase to about 82% at 10 m M (Table 1 ). Ex p eriments w ith higher MAFP conc entrations have not be en performed, sinc e w e had found that MAFP show s c ytotox ic properties at c once ntrations not far above 10 m M. So a significant loss of lactate de hydroge nase from the cells could be dete rmined at 33 m M. 1 9 A re cent study has demonstrated that both cPLA 2 and iPLA 2 are involved in p rote in kinase depe nde nt arachidonic acid libe ration in macrophage-like P388D 1 ce lls. 27 There fore, w e also ex amined the effect of the selec tive iPLA 2 inhibitor bromoe nol lac tone (BEL) on TPA-induce d arachidonic ac id release . At a c oncentration of 5 m M BEL, at w hich a max imal effect on iPLA 2 -activity has be en asce rtaine d, 27 the arachidonic acid libe ration w as decre ased by about 27%. On the other hand, w ith the cPLA 2 inhibitor ML 3116, 28 w hich is able to block the arachidonic acid release in A23187-stimulated platelets nearly completely, the TPA-induce d arachidonic acid liberation could only be inhibited to about 70%. The increase of ML 3116 conce ntration from 10 m M to 33 m M did not cause a further de crease of arachidonic acid re le ase.
Re ce ntly it w as suggested, that in rat liver mac rophage s TPA leads to an arachidonic acid release via an activation of PLC and DAG lipase. 2 9 If at all, in platelets this pathw ay does not play a gre ate r role, sinc e the arachidonic acid formation w as not affec te d by the DAG lipase inhibitor RHC 80267 (c onc entration: 100 m M).
In conclusion, w e propose that tw o diffe re nt PLA 2 s, cPLA 2 and iPLA 2 , are mainly responsible for the liberation of arachidonic acid from plate le t phospholipids after stimulation w ith TPA.

Effect of protein kinase inhibitors on the TPA-induced liberation of arachidonic acid in platelets
To inve stigate the role of the diffe rent prote in kinase s present in platelets on the TPA-induce d phospholipase A 2 activation, w e me asure d the inhibition of the arachidonic acid formation by the p ote nt broad spe ctrum prote in kinase inhibitor staurosporine, the PKC inhibitors GF 109203X and Ro 31-8220, the p44 /p42 MAP kinase inhibitor PD 98059 and the p38 MAP kinase inhibitor SB 202190. The inhibitors w e re applie d at conce ntrations, at w hich a full inhibition of the appropriate protein kinase had bee n achieve d in intact c ells acc ording to data in the literature. 6 ,7,30 -33 Staurosporine strongly inhibited arachidonic ac id formation (Table 1 ). Similar re sults w ere obtained w ith the PKC inhibitors GF 109203X and Ro 31-8220, and w ith the p38 MAP kinase inhibitor SB 202190. How e ve r, the ir inhibition value s w ere a little bit low e r than that of staurosporine. In c ontrast, the p42 /44 MAP kinase inhibitor PD 98059 ex hibited only a w e ak inhibition of the arachidonic ac id re le ase (about 26% at 33 m M).

Effect of the protein kinase inhibitors on the A23187-induced liberation of arachidonic acid in platelets
Staurosporine , GF 109203X, Ro 31-8220, SB 202190 and PD 98059 did not inhibit the arachidonic ac id release in A23187-stimulate d bovine platelets at the conc entrations ap plie d in the ex perime nts w ith TPA, thus p rote in kinase s do not se em to be involve d in the A23187-induce d arachidonic acid liberation. The ine ffec tivene ss of the DAG lipase inhibitor RHC 80267 (conc entration 100 m M) in these ex periments c onfirm the re sults of previous studie s, w hich demonstrate d that the PLC/DAG lip ase pathw ay is not involved in the arachidonic ac id liberation occ urring after the stimulation of plate lets w ith A23187. 2 0,22

Discussion
The ex perime nts w ith the PLA 2 inhibitors propose that c PLA 2 and to a lesser ex tent iPLA 2 are re sponsible for the TPA-induc ed arachidonic ac id release in bovine platelets.
As far as the ap plie d protein kinase inhib itors have the spec ific ity ascribe d in lite rature, the pronounce d inhibition of TPA-induce d arachidonic acid re le ase by the PKC inhibitors GF 109203X and Ro 31-8220 and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA 2 s in platele ts by TPA is me diated via an activation of PKC and the p 38 MAP kinase. Although the e ffec t of PD 98059 on TPAinduc ed arachidonic ac id rele ase w as signific antly le ss pronounc ed than that of the othe r prote in kinase inhibitors investigated, p42 /p44 MAP kinase may also be involved in the ac tivation of the PLA 2 s.
In c onclusion, the results indicate that the TPAinduc ed arachidonic acid liberation is me diate d via othe r me chanisms than the arachidonic ac id re lease observe d afte r stimulation w ith A23187. Further investigations w ill be nec essary to elucidate the se que nce of the e ve nts occ urring after TPA-stimulation and the reasons for the biphasic arachidonic ac id liberation obse rved.