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Hsu MF, Sun SP, Chen YS, Tsai CR, Huang LJ, Tsao LT, Kuo SC, Wang JP.
Distinct effects of N-ethylmaleimide on formyl peptide- and
cyclopiazonic acid-induced Ca2+ signals through thiol
modification in neutrophils.Biochem Pharmacol. 2005 Nov
1;70(9):1320-9.
Department of Biochemistry, School of Medicine, China Medical
University, Taichung, Taiwan, ROC.
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 (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 cyclopiazonic
acid (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 cyclopiazonic acid (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.
PMID: 16143313
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