Interleukin-1 stimulation of arachidonic acid release from human synovial fibroblasts; blockade by inhibitors of protein kinases and protein synthesis

Abstract

Addition of IL-1 (interleukin-1) to human synovial fibroblasts radiolabelled with [ 3H]arachidonic acid caused a linear dose-dependent increase in arachidonic acid release and a transient rise in labelled diacylglycerol. Protein kinase C activators PMA 4-phorbol 12-myristate 13-acetate and D1C8(1,2-dioctanoyl- sn-glycerol) also increased arachidonic acid release, but the time course observed with PMA was different from that of IL-1. When cultures were treated with PMA for 16–24 h to down regulate protein kinase C, the ability of IL-1 to increase arachidonic acid release persisted to the same extent as in nontreated cultures. In contrast, PMA pretreatment prevented the eight-fold stimulation of arachidonic acid release in response to PMA observed in cultures not previously exposed to PMA. To examine the role of other kinases in IL-1 stimulated arachidonic acid release, cultures were treated with H-7 (1-(5-isoquinolinesulphonyl)2-methylpiperazine dichloride), H-8 ( N-[2-(methylamine) ethyl]-5-isoquinolinesulphonamide dichloride), HA 1004 ( N-(2-guanidoinoethyl)-5-isoquinolinesulphonamide hydrochloride), and staurosporine. IL-1 stimulation of arachidonic acid release was blocked by H-7, H-8 and staurosporine. H-7 was a more potent inhibitor than H-8, suggesting that cAMP dependent kinase did ot mediate IL-1 action. Addition of H-7 at various time following IL-1 decreased IL-1 stimulated arachidonic acid release, suggesting that continued protein kinase activity was necessary for IL-1 action. Cycloheximide and actinomycin D inhibited the stimulation of arachidonnic acid release by IL-1, PMA or DiC8. The addition of cycloheximide or actinomycin D 15–45 min after IL-1 also inhibited IL-1 stimulated arachidonic acid release, indicating that continued protein synthesis was required for IL-1 action. These results suggest that IL-1 stimulation of acylhydrolyase activity in human synovial cells occurs by a mechanism requiring continued protein synthesis and protein kinase activity and that neither protein kinase C nor cAMP dependent protein kinase is involved.