title: Distinct effects of N-ethylmaleimide on formyl peptide- and cyclopiazonic acid-induced Ca2+ signals through thiol modification in neutrophils.
abstract: In this study, we demonstrate that N-ethylmaleimide (NEM), a cell permeable thiol-alkylating agent, enhanced the [Ca2+]i rise caused by stimulation with cyclopiazonic acid (CPA), a sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibitor, in rat neutrophils. In addition, NEM attenuated the formyl-Met-Leu-Phe (fMLP)-induced [Ca2+]i rise whether NEM was added to cells prior to or after fMLP stimulation. Moreover, application of NEM after fMLP activation in the absence of external Ca2+ inhibited the Ca2+ signal upon addition of Ca2+ to the medium. Similar patterns were also obtained by using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a cell impermeable dithiol-oxidizing agent, which replaced NEM in the CPA- and fMLP-induced [Ca2+]i rise experiments. Treatment with dithiothreitol (DTT), a cell permeable dithiol-reducing agent, N-acetyl-l-cysteine (NAC), a cell permeable monothiol-reducing agent, and tris-(2-carboxyethyl)phosphine (TCEP), a cell impermeable reductant without a thiol group, all rescued the fMLP-induced Ca2+ signal from NEM. Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) C6, inositol trisphosphate receptor (IP3R) 2 and IP3R3. NEM had no effect on the mitochondrial membrane potential. NEM could restore the polarization and F-actin accumulation of fMLP-treated cells to those of the control. In the absence of external Ca2+, NEM rendered the CPA-induced [Ca2+]i elevation persistently but inhibited the fMLP-induced Ca2+ spike, which was reversed by tris-(2-cyanoethyl)phosphine (TCP), a cell permeable reductant without a thiol group. DTNB did not affect the Ca2+ spike caused by fMLP. These results indicate that through protein thiol oxidation, NEM affects the receptor-activated and the store depletion-derived Ca2+ signals in an opposing manner.
authors.affiliation: Department of Biochemistry, School of Medicine, China Medical University, Taichung, Taiwan, ROC.
authors.forename: Mei-Feng
authors.initials: MF
authors.lastname: Hsu
authors.forename: Shu-Ping
authors.initials: SP
authors.lastname: Sun
authors.forename: Yu-San
authors.initials: YS
authors.lastname: Chen
authors.forename: Chi-Ren
authors.initials: CR
authors.lastname: Tsai
authors.forename: Li-Jiau
authors.initials: LJ
authors.lastname: Huang
authors.forename: Lo-Ti
authors.initials: LT
authors.lastname: Tsao
authors.forename: Sheng-Chu
authors.initials: SC
authors.lastname: Kuo
authors.forename: Jih-Pyang
authors.initials: JP
authors.lastname: Wang
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D015220:Calcium Channels / Q000032:analysis; D020013:Calcium Signaling / Q000187:drug effects*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000378:metabolism*; D010720:Phosphines / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000235:genetics
chemical_list: D000199:Actins; D015220:Calcium Channels; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide; D007211:Indoles; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010720:Phosphines; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D013438:Sulfhydryl Compounds; D050052:TRPC Cation Channels; C492145:Trpc6 protein, rat; C080938:tris(2-carboxyethyl)phosphine; C000543:cyclopiazonic acid