GEA3162, a nitric oxide-releasing agent, activates non-store-operated Ca2+ entry and inhibits store-operated Ca2+ entry pathways in neutrophils through thiol oxidation

We demonstrated that 5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium (GEA3162), a nitric oxide (NO)-releasing agent, stimulated [Ca2+](i) rise in rat neutrophils. This Ca2+ response was prevented by the thiol reducing agents, 2-mercaptoethanol, N-acetyl-L-cysteine, dithiothreitol, 2,3dimercaptopropane-l-sttlfonic acid (DMPS) and tris-(2-carboxyethyl)phosphine (TCEP), but slightly reduced by the antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). GEA3162 also increased the formation of cellular reactive oxygen intermediates and decreased the cellular content of low molecular thiols. These responses were greatly reduced by Trolox, dithiothreitol and N-acetyl-L-cysteine. GEA3162 stimulated the protein tyrosine phosphorylation in neutrophils. The [Ca2+]; rise caused by formyl-Met-Leu-Phe (fMLP) and cyclopiazonic acid (CPA) was suppressed by GEA3162. TCEP prevented the inhibition of fMLP-induced [Ca2+]; rise by GEA3162. In the absence of external Ca2+, GEA3162 inhibited the CPA-induced [Ca2+](i) rise, whereas it only slightly affected the fMLP-induced mobilization of the Ca2+ store. Application of GEA3162 after the stimulation with fMLP or CPA suppressed the robust Ca2+ entry followed by the readdition of Ca2+ into medium. Moreover, the Ca2+ entry was more susceptible to inhibition by treatment with GEA3162 prior to than after the fMLP stimulation. GEA3162 had no effect on the mitochondrial membrane potential. GEA3162 induced actin reorganization and condensed filament network at the cell periphery. These results indicate that GEA3162 exerted both the stimulation of Ca2+ entry and the inhibition of the store-operated Ca2+ entry in rat neutrophils. The dual effects of GEA3162 on the regulation of the external Ca2+ entry are mainly through the thiol modification of target protein (s) residing on the outside of the plasma membrane. (c) 2006 Elsevier B.V All rights reserved.