Investigation of the cellular mechanism of inhibition of formyl-methionyl-leucyl-phenylalanine-induced superoxide anion generation in rat neutrophils by 2-benzyloxybenzaldehyde.
The inhibition of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O2(.-)) generation by 2-benzyloxybenzaldehyde (CCY1a) was investigated in rat neutrophils, and the underlying mechanism of this inhibition was assessed. CCY1a concentration-dependently inhibited O2(.-) generation (IC(50)=18.5+/-4.3 microM). In cell-free systems, CCY1a failed to alter O2(.-) generation during dihydroxyfumaric acid autoxidation, in phorbol 12-myristate 13-acetate (PMA)-activated neutrophil particulate NADPH oxidase preparations, or during arachidonic acid-induced NADPH oxidase activation. CCY1a increased cellular cyclic AMP (cAMP) levels in a time- and concentration-dependent manner, and this cAMP-elevating effect was inhibited by the adenylyl cyclase inhibitor 9-(tetrahydro-2'-furyl)adenine (SQ22536), adenosine deaminase (ADA), and the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline. In neutrophils, inhibition of O2(.-) generation by CCY1a was partially reversed by the protein kinase A inhibitor (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT5720). CCY1a did not affect fMLP-induced p38 mitogen-activated protein kinase phosphorylation, but concentration-dependently attenuated the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt (IC(50) about 31.3 and 19.4 microM, respectively). The plateau phase, but not the initial spike, of fMLP-induced [Ca2+](i) changes was inhibited by CCY1a in a concentration-dependent manner. CCY1a inhibition of Ca2+ entry, ERK, and Akt phosphorylation was not prevented by SQ22536 or ADA. fMLP-induced phospholipase D (PLD) activation was inhibited by CCY1a (IC(50)=13.9+/-2.0 microM). ADA and KT5720 did not prevent the inhibition of PLD activation by CCY1a. Collectively, these results indicate that the inhibition by CCY1a of fMLP-induced O2(.-) generation in rat neutrophils can probably be attributed to the increase in cAMP levels, and to the blockade of Ca2+ entry, suppression of Akt, and PLD activation via cAMP-independent mechanisms.
Wang
Jih-Pyang
JP
Department of Education and Research, Taichung Veterans General Hospital, Taichung 407, Taiwan, Republic of China. w1994@vghtc.vghtc.gov.tw
Chang
Ling-Chu
LC
Lin
Yi-Lee
YL
Hsu
Mei-Feng
MF
Chang
Chiung-Yun
CY
Huang
Li-Jiau
LJ
Kuo
Sheng-Chu
SC
D000818:Animals; D001547:Benzaldehydes / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D004347:Drug Interactions; D004789:Enzyme Activation; D066298:In Vitro Techniques; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D013481:Superoxides / Q000378:metabolism*
C468728:2-benzyloxybenzaldehyde; D001547:Benzaldehydes; D011518:Proto-Oncogene Proteins; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; C042932:formylmethionyl-leucyl-phenylalanine methyl ester; D000242:Cyclic AMP; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D010739:Phospholipase D; D002118:Calcium