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
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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 ...