Involvement of different protein kinases and phospholipases A₂ in phorbol ester (TPA)‐induced arachidonic acid liberation in bovine platelets

Abstract

THE effe ct of various p h o s p h o lip as e A 2 and p r otein kin as e in h ibito rs o n th e arach ido nic acid libe ration in bo vin e p latele ts in duced by IntroductionThe 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 ). 1-4The 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. 1,4So 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. Materials and MethodsReagents MAFP (methyl arachidonylfluorophosp honate ), BEL ((E)-6-(bromomethylide ne )-3-(1-naphthyl)-3,4,5,6-te trahydro-2H-pyran-2-one ), RHC 80267 (1,6-bis (c yclohex ylox iminoc arbonylamino )hex ane ), PD 98059 (29 -amino-39 -me thox yflavone ), 12-O-te trade canoylphorbol-13-ac etate (TPA) (Biomol, Hamburg ); SB 202190 31 Short CommunicationMediators of Inflammation, 9, 31 -34 (2000 ) (DMSO) (Merck, Darmstadt), ML 3116 (1-(7-c arbox yhe ptyl)-3-dodecanoyl-1H-indole-2-carbox ylic ac id ) w as synthe sized by the published proce dure; 1 7 bovine blood (local slaughte rhouse ). CellsImme 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 ...

HPLC assay with UV spectrometric detection for the evaluation of inhibitors of cytosolic phospholipase A2

“…Contrary, the sPLA 2 inhibitor AR-C67047MI (4) [42] did not effect enzyme activity at 10 M, indicating that sPLA 2 isoforms are not present or active, respectively, in the enzyme preparation. As already mentioned, for inactivation of iPLA 2 , which is a putative constituent of platelet cytosol [41,46], the irreversible iPLA 2 inhibitor bromoenol lactone was added to the cPLA 2 preparation.…”

Phospholipase A2-catalyzed hydrolysis of plasmalogen phospholipids in thrombin-stimulated human platelets

“…To date, there is little evidence for the involvement of a Ca 2+ -independent PLA 2 (iPLA 2 ) in human platelets. However, Lehr and Griessbach [4] have recently reported a decrease in arachidonic acid production in 12-O-tetradecanoylphorbol-13-acetate stimulated platelets pretreated with the iPLA 2 inhibitor bromoenol lactone (BEL). In addition, both TxA 2 and arachidonic acid release was observed in collagen stimulated platelets isolated from cPLA 2 -/-/sPLA 2 -IIA -/-mice, suggesting the involvement of another PLA 2 isoform possibly iPLA 2 [5].…”

Phospholipase A2 inhibitors in inflammation

“…Experiments with cells that had been engineered to overexpress cPLA 2 and experiments with macrophages isolated from cPLA 2 knockout mice showed that the A23187-mediated arachidonic acid release is predominantly catalysed by cPLA 2 [18][19][20][21]. Furthermore, protein kinases are not involved in the activation mechanism of cPLA 2 by A23187 when short incubation times are applied, since the A23187 response is not inhibited by protein kinase inhibitors under such conditions [58]. However, when using intact cells it has to kept in mind that certain compounds including lipophilic acids and bases, can readily cause cell lysis and loss of viability in vitro, which may erroneously indicate enzyme inhibition [60][61][62].…”

“…In some cases metabolites of arachidonic acid, such as prostaglandin D 2 or E 2 , are used to determine the inhibition of cPLA 2 activity [121,135]. A weakness of these assays is the evidence that other isoforms of the enzyme may be present in the cells, such as sPLA 2 -V, sPLA 2 -X or iPLA 2 s. These could be involved in substrate cleavage [21,28,31,33,34,57,58]. Furthermore, if agonists that stimulate cPLA 2 activity via the activation of protein kinases are used (e.g., TPA, zymosan, IL-1 [59]), protein kinase inhibitors could also cause a reduction of arachidonic acid release.…”