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

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

U73122 completely blocks NEM or SNP-induced SOCE and inhibits PMA-elicited spreading of neutrophils.

<p>(A) representative tracings of [Ca²⁺]_c elevation induced by NEM (100 µM) in the presence or absence of U73122 (10 µM) in Ca²⁺-containing buffer. (B) Statistic results are means ± S.E.M (n = 30 from three independent experiments).^*<i>P</i><0.01, compared with NEM.^#<i>P</i><0.01, compared with SNP. (C) PMA-activated and adherent neutrophils image was obtained by DIC. (D) The effect of U73122 (10 µM) on morphology of PMA-activated neutrophils.</p

NEM or SNP results in SOCE via sulfhydryl modification in PMA-activated neutrophils.

<p>(A) Representative tracing of [Ca²⁺]_c elevation induced by 100 µM NEM in the presence of external Ca²⁺. (B) Representative tracings of 500 µM NEM inhibitory effect on [Ca²⁺]_c change caused by SNP (500 µM) within Ca²⁺-containing medium. (C) Typical tracings of [Ca²⁺]_c responses for PMA-activated neutrophils to 100 µM NEM or 500 µM SNP stimulation in Ca²⁺-free buffer. (D, E), [Ca²⁺]_c of PMA-pretreated neutrophils was triggered with NEM (100 µM) within Ca²⁺-free HBSS followed by readdition of 1 mM external Ca²⁺. (F) 1 mM external calcium was added to Ca²⁺-free PMA-activated neutrophils before application of stimulation, followed by addition of 100 µM NEM.</p

SOCE still occurs but is blocked by 2-APB in PMA-activated human neutrophils.

<p>(A) Spherical resting neutrophils visualised by DIC. (B) When PMA was added (2 µM for 5 min at 37°C), neutrophils were activated and spread on glass surface thereby flattening their morphology as observed by DIC. (C) After calcium store depletion by a calcium pump blocker TG (2 µM), external Ca²⁺ (1<i> </i>mM) was transiently removed 4 min later; then, further addition of 1 mM external Ca²⁺ resulted in obvious calcium entry, revealing the putative response for SOCE. (D) 2 µM TG was added in the absence of Ca²⁺, followed by readdition of 1 mM external Ca²⁺. (E) After calcium stores depletion by 2 µM TG, PMA-activated neutrophils were treated with 75 µM 2-APB. (F) 2 µM TG was added together with 75 µM 2-APB. (G, H) Calcium stores were triggered with G protein-coupled agonist FMLP (2 µM) in the presence/absence of calcium followed by removal/readdition of 1 mM external Ca²⁺.</p

2-APB completely blocks SOCE in response to sulfhydryl modification in PMA-activated human neutrophils.

<p>All experiments were performed with cells in Ca²⁺-containing medium. (A) Typical tracings of [Ca²⁺]_c changes of PMA-activated neutrophils induced by 100 µM NEM in the presence of 2-APB at 0 µM (-□- curve), 75 µM (-○- curve), 150 µM (-△- curve), respectively. (B) Statistic data from three independent experiments (means±SEM, n = 30 cells). ^*<i>P</i><0.01, compared with 2-APB absence. (C, D) [Ca²⁺]_c increase was induced by NEM (100 µM) or SNP (500 µM) in PMA-activated neutrophils, followed by addition of 75 µM 2-APB at t = 197 or t = 314 s, resulting in return to baseline [Ca²⁺]_c.</p

NEM and SNP have no effect on [Ca²⁺]_c of resting human neutrophils.

<p>(A) Representative tracings of [Ca²⁺]_c responses to 100 µM NEM (black line) and 500 µM SNP (gray line). (B) Typical tracing of [Ca²⁺]_c change induced by 1 µM FMLP. For all experiments, resting human neutrophils were performed in suspension (1×10⁶/mL) within Ca²⁺-containing buffer.</p