Characterization of maleimide-activated Ca2+ entry in neutrophils.
N-Ethylmaleimide (NEM), a thio-alkylating agent, concentration-dependently stimulated the elevation of [Ca(2+)](i) in rat neutrophils in the presence of external Ca(2+). This effect was not observed in Ca(2+)-free medium and was abrogated by dithiothreitol pretreatment. The application of NEM after cyclopiazonic acid (CPA) stimulated the store-emptying activation of Ca(2+) entry. Unlike CPA-induced cation entry, NEM showed poor uptake of Ba(2+) and Sr(2+) and did not induce Mn(2+) influx. NEM diminished CPA-induced Mn(2+) influx, an effect that was blocked by dithiothreitol. Both Ni(2+) and La(3+) attenuated the elevation of [Ca(2+)](i) in response to NEM; however, greater resistance was observed to Ni(2+) inhibition of NEM-induced Ca(2+) influx than inhibition of store-operated Ca(2+) entry. Both cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A) and 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), Ca(2+) channel blockers, and calyculin A, an inhibitor of protein serine/threonine phosphatases 1/2, diminished the NEM-induced Ca(2+) entry. Treatment of cells with genistein, a general tyrosine kinase inhibitor, or with wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), phosphatidylinositol 3-kinase inhibitors, had no appreciable inhibitory effects on the action of NEM. However, 2-aminoethyldiphenyl borate, an inositol trisphosphate receptor antagonist, enhanced rather than inhibited the [Ca(2+)](i) change in response to NEM. These results indicate that NEM stimulates Ca(2+) entry and regulates Ca(2+) signaling through direct thiol oxidation, bypassing the cellular signal transduction pathway. The NEM-regulated Ca(2+) signal demonstrates characteristics that distinguish it from the store-emptying operation in neutrophils, and therefore represents two distinct modes of Ca(2+) regulation.
Wang
Jih-Pyang
JP
Department of Education and Research, Taichung Veterans General Hospital, 160, Chung Kang Road, Sec. 3, Taiwan, Republic of China. w1994@vghtc.vghtc.gov.tw
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D020013:Calcium Signaling / Q000187:drug effects*; D002412:Cations / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D066298:In Vitro Techniques; D008301:Maleimides / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D000477:Alkylating Agents; D015220:Calcium Channels; D002412:Cations; D008301:Maleimides; C043592:maleimide; D005033:Ethylmaleimide; D002118:Calcium