Sulfhydryl Modification Induces Calcium Entry through IP3-Sensitive Store-Operated Pathway in Activation-Dependent Human Neutrophils

Leiting Pan,Nason Ma’ani Hessari,Xian Wu,Jingjun Xu,Dan Zhao,Imshik Lee,Xinzheng Zhang,David Holowka

doi:10.1371/journal.pone.0025262

Leiting Pan, Nason Ma’ani Hessari + Show 6 more

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https://doi.org/10.1371/journal.pone.0025262

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Journal: PloS one	Publication Date: Oct 3, 2011
Citations: 33	License type: CC BY 4.0

Affiliation: Nankai University

Abstract

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT1MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca2+]c) without external calcium, followed by a second [Ca2+]c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca2+]c change of Ca2+-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca2+]c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP3-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.

Highlights

As the first line of host defense against invasion of pathogenic microbes, neutrophils play a crucial role in a variety of inflammatory responses [1,2]
The present study investigated the calcium entry mechanism induced by the alkylation agent NEM and the exogenous nitric oxide (NO) donor sodium nitroprusside (SNP) in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils
In our experiments, resting human neutrophils were activated by incubation with 2 mM PMA for 5 min at 37uC, which caused adhesion to the glass surface resulting in observable flattening of the cells with differential interference contrast (DIC) microscopy (Fig. 1A and B)

Summary

Introduction

As the first line of host defense against invasion of pathogenic microbes, neutrophils play a crucial role in a variety of inflammatory responses [1,2]. The activation of the human neutrophils leads to a spectrum of responses, including aggregation, stimulation of the respiratory burst and degradation of microbes. As a second messenger, [Ca2+]c executes profound effects across a wide range of physiological functions and signaling transductions in neutrophil immunity responses, including the initiation of cytoskeletal changes, degranulation, adhesion, apoptosis, and oxidative burst [5]. Store-operated calcium entry (SOCE), a critical mechanism of [Ca2+]c regulation in non-excitable cells [6,7,8], plays a crucial role in modulating the immune responses of neutrophils [9,10,11]

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