PHARMACOLOGICAL COMPARISON OF UTP-INDUCED AND THAPSIGARGIN-INDUCED ARACHIDONIC-ACID RELEASE IN MOUSE RAW 264.7 MACROPHAGES

1 Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+](i)), phosphoinositi de (PI) turnover, and arachidonic acid (AA) release, the causal relati onship between these signalling pathways is still unclear. In the pres ent study, we investigated the involvement of phosphoinositide-depende nt phospholipase C (PI-PLC) activation, Ca2+ increase and protein kina se activation in UTP-induced AA release. The effects of stimulating RA W 264.7 cells with thapsigargin, which cannot activate the inositol ph osphate (IP) cascade, but results in the release of sequestered Ca2+ a nd an influx of extracellular Ca2+, was compared with the effects of U TP stimulation to elucidate the multiple regulatory pathways for cPLA( 2) activation. 2 In RAW 264.7 cells UTP (100 mu M) and thapsigargin (1 mu M) caused 2 and 1.2 fold increases, respectively, in [H-3]-AA rele ase. The release of [H-3]-AA following treatment with UTP and thapsiga rgin were non-additive, totally abolished in the Ca2+-free buffer, BAP TA (30 mu M)-containing buffer or in the presence of the cPLA(2) inhib itor MAFP (50 mu M), and inhibited by pretreatment of cells with pertu ssis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 mu M). By c ontrast, aristolochic acid (an inhibitor of sPLA(2)) had no effect on UTP and thapsigargin responses. 3 U73122 (10 mu M) and neomycin (3 mM) , inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83 % inhibition, respectively) and AA release (76% and 58%, respectively) , accompanied by a decrease in the [Ca2+](i) rise. 4 Wortmannin attenu ated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 mu M), and reduced the UTP-induced AA release in par allel. RHC 80267 (30 mu M), a specific diacylglycerol lipase inhibitor , had no effect on UTP-induced AA release. 5 Short-term treatment with PMA (1 mu M) inhibited the UTP-stimulated accumulation of IP and incr ease in [Ca2+](i), but had no effect on the release of AA. In contrast , the AA release caused by thapsigargin was increased by PMA. 6 The ro le of PKC in UTP- and thapsigargin-mediated AA release was shown by th e blockade of these effects by staurosporine (1 mu M), Ro 31-8220 (10 mu M), Go 6976 (1 mu M) and the down-regulation of PKC. 7 Following tr eatment of cells with SK&F 96365 (30 mu M), thapsigargin-, but not UTP -, induced Ca2+ influx, and the accompanying AA release, were down-reg ulated. 8 Neither PD 98059 (100 mu M), MEK a inhibitor, nor genistein (100 mu M), a tyrosine kinase inhibitor, had any effect on the AA resp onses induced by UTP and thapsigargin. 9 We conclude that UTP-induced cPLA(2) activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activatio n of cPLA(2) by UTP and thapsigargin. The [Ca2+](i)-dependent AA relea se that follows treatment with both stimuli was potentiated by the act ivity of protein kinase C (PKC). A pertussis toxin-sensitive pathway d ownstream of the increase in [Ca2+](i) was also shown to be involved i n AA release.