Effect of silica on phospholipase D activity in rat alveolar macrophages

Abstract

Silica may act as a stimulator of pulmonary inflammation and fibrosis. The effect of silica on phospholipase D (PLD) activity assayed as accumulation of [3H]phosphatidylethanol ([3H]PtdEt) was examined in [3H]palmitic acid-labeled primary cultures of rat alveolar macrophages. Silica induced a rapid accumulation of [3H]PtdEt in a time (0, 15, 30 and 45 min)- and concentration (0.5, 1.0, 2.5 and 5.0 mg/ml)-dependent manner indicating PLD activation. This silica-stimulated PLD activity was attenuated by the pretreament with calcium chelator ethylene glycol-bis(βaminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) or/and 1,2-bis(2- aminophenoxy)ethaneN,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA/AM) (EGTA: 54.3 ± 8.6%, BAPTA/AM: 67.5 ±7.8 % and EGTA + BAPTA/AM: 35.8 ± 2.9, respectively). Also, silica-induced PLD activation was partially inhibited by the pretreatment with nonspecific phospholipase C (PLC) and PLD inhibitor (neomycin; 66.4 ± 4.8%) or specific PLC inhibitor (U73122; 70.8 ± 4.6%). Sphingosine as a protein kinase C (PKC) inhibitor did not change silica- induced PLD activity indicating that PKC might not play a role in PLD activation by silica. Based on these results, we concluded that a silica- stimulated phospholipase D activity is present in the rat alveolar macrophages and is predominantly regulated by PLC-mediated intracellular calcium.