Cellular mechanisms of inhibition of superoxide anion generation in rat neutrophils by the synthetic isoquinoline DMDI

Abstract

This study was undertaken to assess the cellular localization of the inhibitory effect of a chemically synthetic isoquinoline compound 1-(3′,4′-dimethoxybenzyl)-6,7-dichloroisoquinoline (DMDI) on the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat neutrophils. The DMDI concentration dependently inhibited the superoxide anion (O 2 ⋅−) generation and O 2 consumption (IC 50 12.2±4.9 and 15.2±8.4 μM, respectively) of neutrophils. DMDI did not scavenge the O 2 ⋅− generated during the autoxidation of dihydroxyfumaric acid in a cell-free system. DMDI did not elevate cellular cyclic AMP levels. Inhibition of O 2 ⋅− generation by DMDI in neutrophils was not reversed by a cyclic AMP-dependent protein kinase inhibitor, (8 R,9 S,11 S)-(−)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H,8 H,11 H-2,7b,11a-triazadibenzo[ a,g]cycloocta[ cde]trinden-1-one (KT5720). The DMDI concentration dependently inhibited the late plateau phase but not the initial spike of fMLP-induced [Ca 2+] i changes in the presence of extracellular Ca 2+. However, DMDI had no effect on the fMLP-induced [Ca 2+] i changes in the absence of extracellular Ca 2+. In addition, DMDI did not affect the fMLP-stimulated phosphatidylinositol 3-kinase (PI3-kinase) activation. DMDI produced a concentration-dependent reduction in the formation of phosphatidic acid and phosphatidylethanol in the presence of ethanol from fMLP-stimulated neutrophils (IC 50 13.3±4.0 and 9.4±4.3 μM, respectively). On the basis of the immunoblot analysis of the phosphorylation of the mitogen-activated protein (MAP) kinase, DMDI attenuated the fMLP-stimulated MAP kinase phosphorylation in a similar concentration range. Collectively, these results indicate that the inhibition of the respiratory burst by DMDI in rat neutrophils is mediated through the blockade of phospholipase D and MAP kinase signaling pathways.