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

Super-Resolution Microscopy Reveals the Native Ultrastructure of the Erythrocyte Cytoskeleton

Summary: The erythrocyte cytoskeleton is a textbook prototype for the submembrane cytoskeleton of metazoan cells. While early experiments suggest a triangular network of actin-based junctional complexes connected by ∼200-nm-long spectrin tetramers, later studies indicate much smaller junction-to-junction distances in the range of 25-60 nm. Through super-resolution microscopy, we resolve the native ultrastructure of the cytoskeleton of membrane-preserved erythrocytes for the N and C termini of β-spectrin, F-actin, protein 4.1, tropomodulin, and adducin. This allows us to determine an ∼80-nm junction-to-junction distance, a length consistent with relaxed spectrin tetramers and theories based on spectrin abundance. Through two-color data, we further show that the cytoskeleton meshwork often contains nanoscale voids where the cell membrane remains intact and that actin filaments and capping proteins localize to a subset of, but not all, junctional complexes. Together, our results call for a reassessment of the structure and function of the submembrane cytoskeleton. : Pan et al. resolve the cytoskeletal organization in native erythrocytes using STORM super-resolution microscopy, revealing an ∼80-nm junction-to-junction distance that is in agreement with relaxed spectrin tetramers and showing that the junctional complexes are partially occupied by actin and its capping proteins. Keywords: erythrocyte, membrane cytoskeleton, spectrin-actin-based cytoskeleton, junctional complex, super-resolution microscopy, native ultrastructure, red blood cel

Ultraviolet light A irradiation induces immunosuppression associated with the generation of reactive oxygen species in human neutrophils

Ultraviolet blood irradiation has been used as a physical therapy to treat many nonspecific diseases in clinics; however, the underlying mechanisms remain largely unclear. Neutrophils, the first line of host defense, play a crucial role in a variety of inflammatory responses. In the present work, we investigated the effects of ultraviolet light A (UVA) on the immune functions of human neutrophils at the single-cell level by using an inverted fluorescence microscope. N-Formyl-methionyl-leucyl-phenylalanine (FMLP), a classic physiological chemotactic peptide, was used to induce a series of immune responses in neutrophils in vitro. FMLP-induced calcium mobilization, migration, and phagocytosis in human neutrophils was significantly blocked after treatment with 365nm UVA irradiation, demonstrating the immunosuppressive effects of UVA irradiation on neutrophils. Similar responses were also observed when the cells were pretreated with H2O2, a type of reactive oxygen species (ROS). Furthermore, UVA irradiation resulted in an increase in NAD(P)H, a member of host oxidative stress in cells. Taken together, our data indicate that UVA irradiation results in immunosuppression associated with the production of ROS in human neutrophils

Ultraviolet irradiation-dependent fluorescence enhancement of hemoglobin catalyzed by reactive oxygen species.

Ultraviolet (UV) light has a potent effect on biological organisms. Hemoglobin, an oxygen-transport protein, plays an irreplaceable role in sustaining life of all vertebrates. In this study we scrutinize the effects of ultraviolet irradiation (UVI) as well as visible irradiation on the fluorescence characteristics of bovine hemoglobin (BHb) in vitro. Data show that UVI results in fluorescence enhancement of BHb in a dose-dependent manner. Furthermore, UVI-induced fluorescence enhancement is significantly increased when BHb is pretreated with hydrogen peroxide (H(2)O(2)), a type of reactive oxygen species (ROS). Meanwhile, The water-soluble antioxidant vitamin C suppresses this UVI-induced fluorescence enhancement. In contrast, green light irradiation does not lead to fluorescence enhancement of BHb no matter whether H(2)O(2) is acting on the BHb solution or not. Taken together, these results indicate that catalysis of ROS and UVI-dependent irradiation play two key roles in the process of UVI-induced fluorescence enhancement of BHb

Mesenchymal Migration on Adhesive–Nonadhesive Alternate Surfaces in Macrophages

Abstract Mesenchymal migration usually happens on adhesive substrates, while cells adopt amoeboid migration on low/nonadhesive surfaces. Protein‐repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to these perceptions, this work discovers a unique locomotion of macrophages on adhesive–nonadhesive alternate substrates in vitro that they can overcome nonadhesive PEG gaps to reach adhesive regions in the mesenchymal mode. Adhering to extracellular matrix regions is a prerequisite for macrophages to perform further locomotion on the PEG regions. Podosomes are found highly enriched on the PEG region in macrophages and support their migration across the nonadhesive regions. Increasing podosome density through myosin IIA inhibition facilitates cell motility on adhesive–nonadhesive alternate substrates. Moreover, a developed cellular Potts model reproduces this mesenchymal migration. These findings together uncover a new migratory behavior on adhesive–nonadhesive alternate substrates in macrophages

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

Elevation of Extracellular Ca²⁺ Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts

<div><p>Aims</p><p>The local concentration of extracellular Ca²⁺ ([Ca²⁺]_o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca²⁺]_o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca²⁺]_o to osteoblastic proliferation.</p><p>Methods</p><p>Cytosolic Ca²⁺ concentration ([Ca²⁺]_c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail.</p><p>Results</p><p>Our data showed that elevating [Ca²⁺]_o evoked a sustained increase of [Ca²⁺]_c in a dose-dependent manner. This [Ca²⁺]_c increase was blocked by TMB-8 (Ca²⁺ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or U73122 (a PLC inhibitor) strongly reduced the [Ca²⁺]_o-induced [Ca²⁺]_c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca²⁺]_o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca²⁺]_o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists.</p><p>Conclusions</p><p>Elevating [Ca²⁺]_o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca²⁺ concentration.</p></div

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