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Reversible cell damage by T-cell perforins. Calcium influx and propidium iodide uptake into K562 cells in the absence of lysis.

ABSTRACT: The non-lethal effects of the lymphocyte-derived pore-forming toxin perforin on the human erythroleukaemia cell line K562 were investigated. By using the fluorescent Ca2+ indicator fura-2, perforin was shown to cause intracellular Ca2+ concentration to rise transiently into the micromolar range in the absence of cell death. By fluorescence-activated cell sorting it was demonstrated that K562 cells took up the membrane-impermeant nuclear stain propidium iodide (PI) when exposed to non-lethal doses of perforin. The permeability to PI was short-lived, confirming the transience of the perforin pore. Analogies with non-lethal effects and recovery processes occurring in nucleated cells exposed to the membrane-attack complex of complement are drawn.

SUBMITTER: Jones J

PROVIDER: S-EPMC1131287 | biostudies-other | 1990 Apr

REPOSITORIES: biostudies-other

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ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis.

Project description:Lymphocytes mediate cytotoxicity by polarized release of the contents of cytotoxic granules toward their target cells. Here, we have studied the role of the calcium release-activated calcium channel ORAI1 in human lymphocyte cytotoxicity. Natural killer (NK) cells obtained from an ORAI1-deficient patient displayed defective store-operated Ca(2+) entry (SOCE) and severely defective cytotoxic granule exocytosis leading to impaired target cell lysis. Similar findings were obtained using NK cells from a stromal interaction molecule 1-deficient patient. The defect occurred at a late stage of the signaling process, because activation of leukocyte functional antigen (LFA)-1 and cytotoxic granule polarization were not impaired. Moreover, pharmacological inhibition of SOCE interfered with degranulation and target cell lysis by freshly isolated NK cells and CD8(+) effector T cells from healthy donors. In addition to effects on lymphocyte cytotoxicity, synthesis of the chemokine macrophage inflammatory protein-1? and the cytokines TNF-? and IFN-? on target cell recognition was impaired in ORAI1-deficient NK cells, as previously described for T cells. By contrast, NK cell cytokine production induced by combinations of IL-12, IL-15, and IL-18 was not impaired by ORAI1 deficiency. Taken together, these results identify a critical role for ORAI1-mediated Ca(2+) influx in granule exocytosis for lymphocyte cytotoxicity as well as for cytokine production induced by target cell recognition.

| S-EPMC3044412 | biostudies-literature

Propylthiouracil increases sodium/iodide symporter gene expression and iodide uptake in rat thyroid cells in the absence of TSH.

Project description:BackgroundPropylthiouracil (PTU) and methimazole (MMI) are drugs that are widely used to treat Graves' disease. Although both exert an antithyroid effect primarily by blocking thyroid peroxidase activity, their molecular structure and other actions are different. We hypothesized that PTU and MMI may have differential effects on thyroid-specific gene expression and function.MethodsThe effects of PTU and MMI on thyroid-specific gene expression and function were examined in rat thyroid FRTL-5 cells using DNA microarray, reverse transcriptase (RT)-polymerase chain reaction (PCR), real-time PCR, Western blot, immunohistochemistry, and radioiodine uptake studies.ResultsDNA microarray analysis showed a marked increase in sodium/iodide symporter (NIS) gene expression after PTU treatment, whereas MMI had no effect. RT-PCR and real-time PCR analysis revealed that PTU-induced NIS mRNA levels were comparable to those elicited by thyroid-stimulating hormone (TSH). PTU increased 5'-1880-bp and 5'-1052-bp activity of the rat NIS promoter. While PTU treatment also increased NIS protein levels, the size of the induced protein was smaller than that induced by TSH, and the protein localized predominantly in the cytoplasm rather than the plasma membrane. Accumulation of (125)I in FRTL-5 cells was increased by PTU stimulation, but this effect was weaker than that produced by TSH.ConclusionsWe found that PTU induces NIS expression and iodide uptake in rat thyroid FRTL-5 cells in the absence of TSH. Although PTU and MMI share similar antithyroid activity, their effects on other thyroid functions appear to be quite different, which could affect their therapeutic effectiveness.

| S-EPMC3407387 | biostudies-literature

Calcium influx-induced lytic cell death disrupts skin immune homeostasis.

Project description: Not available

| S-EPMC10728102 | biostudies-literature

Cytotoxicity of Oleandrin Is Mediated by Calcium Influx and by Increased Manganese Uptake in Saccharomyces cerevisiae Cells.

Project description:Oleandrin, the main component of Nerium oleander L. extracts, is a cardiotoxic glycoside with multiple pharmacological implications, having potential anti-tumoral and antiviral characteristics. Although it is accepted that the main mechanism of oleandrin action is the inhibition of Na+/K+-ATPases and subsequent increase in cell calcium, many aspects which determine oleandrin cytotoxicity remain elusive. In this study, we used the model Saccharomyces cerevisiae to unravel new elements accounting for oleandrin toxicity. Using cells expressing the Ca2+-sensitive photoprotein aequorin, we found that oleandrin exposure resulted in Ca2+ influx into the cytosol and that failing to pump Ca2+ from the cytosol to the vacuole increased oleandrin toxicity. We also found that oleandrin exposure induced Mn2+ accumulation by yeast cells via the plasma membrane Smf1 and that mutants with defects in Mn2+ homeostasis are oleandrin-hypersensitive. Our data suggest that combining oleandrin with agents which alter Ca2+ or Mn2+ uptake may be a way of controlling oleandrin toxicity.

| S-EPMC7570853 | biostudies-literature

Calcium influx mechanisms underlying calcium oscillations in rat hepatocytes.

Project description:UNLABELLED:The process of capacitative or store-operated Ca(2+) entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca(2+) stored in the endoplasmic reticulum, and Orai proteins constitute pore-forming subunits of the store-operated channels. Store-operated Ca(2+) entry is readily demonstrated with protocols that provide extensive Ca(2+) store depletion; however, the role of store-operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store-operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca(2+) influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well-characterized intracellular Ca(2+) oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)-based techniques to investigate the role of store-operated channels in the maintenance of hormone-induced Ca(2+) oscillations in rat hepatocytes. Pharmacological inhibitors of store-operated channels blocked thapsigargin-induced Ca(2+) entry but only partially reduced the frequency of Ca(2+) oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin-induced calcium entry, and more modestly diminished the frequency of vasopressin-induced oscillations. CONCLUSION:Our findings establish that store-operated Ca(2+) entry plays a role in the maintenance of agonist-induced oscillations in primary rat hepatocytes but indicate that other agonist-induced entry mechanisms must be involved to a significant extent.

| S-EPMC2808042 | biostudies-literature

Senescence progression in the normal human fibroblast, WI-38 cells induced with plasma membrane damage, calcium influx, DNA damage and telomere shortening.

Project description:To investigate the comparison of senescence progression in plasma membrane damage-, calcium influx-, DNA damage- and telomere shortening-induced senescence in the normal human fibroblast cells.

2024-01-24 | GSE222400 | GEO

Activation of ryanodine receptors induces calcium influx in a neuroblastoma cell line lacking calcium influx factor activity.

Project description:We have further characterized the Ca2+ signalling properties of the NG115-401L (or 401L) neuroblastoma cell line, which has served as an important cell line for investigating SOC (store-operated channel) influx pathways. These cells possess an unusual Ca2+ signalling phenotype characterized by the absence of Ca2+ influx when Ca2+ stores are depleted by inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase). Previous studies found that Ca2+-store depletion does not produce a CIF (Ca2+ influx factor) activity in 401L cells. These observations have prompted the question whether 401L cells possess the signalling machinery that permits non-voltage-gated Ca2+ influx to occur. We tested the hypothesis that ryanodine-sensitive Ca2+ pools and activation of RyRs (ryanodine receptors) constitute a signalling pathway capable of inducing Ca2+ influx in 401L cells. We found that 401L cells express mRNA for RyR1 and RyR2 and that RyR activators induced Ca2+ release. Activation of RyRs robustly couples with Ca2+ influx responses in 401L cells, in sharp contrast with absence of Ca2+ influx when cells are treated with SERCA inhibitors. Thus it is clear that 401L cells, despite lacking depletion-induced Ca2+ influx pathways, express the functional components of a Ca2+ influx pathway under the control of RyR function. These findings further support the importance of the 401L cell line as an important cell phenotype for deciphering Ca2+ influx regulation.

| S-EPMC1134793 | biostudies-other

Thioridazine requires calcium influx to induce MLL-AF6-rearranged AML cell death.

Project description:In pediatric acute myeloid leukemia (AML), intensive chemotherapy and allogeneic hematopoietic stem cell transplantation are the cornerstones of treatment in high-risk cases, with severe late effects and a still high risk of disease recurrence as the main drawbacks. The identification of targeted, more effective, safer drugs is thus desirable. We performed a high-throughput drug-screening assay of 1280 compounds and identified thioridazine (TDZ), a drug that was highly selective for the t(6;11)(q27;q23) MLL-AF6 (6;11)AML rearrangement, which mediates a dramatically poor (below 20%) survival rate. TDZ induced cell death and irreversible progress toward the loss of leukemia cell clonogenic capacity in vitro. Thus, we explored its mechanism of action and found a profound cytoskeletal remodeling of blast cells that led to Ca2+ influx, triggering apoptosis through mitochondrial depolarization, confirming that this latter phenomenon occurs selectively in t(6;11)AML, for which AF6 does not work as a cytoskeletal regulator, because it is sequestered into the nucleus by the fusion gene. We confirmed TDZ-mediated t(6;11)AML toxicity in vivo and enhanced the drug's safety by developing novel TDZ analogues that exerted the same effect on leukemia reduction, but with lowered neuroleptic effects in vivo. Overall, these results refine the MLL-AF6 AML leukemogenic mechanism and suggest that the benefits of targeting it be corroborated in further clinical trials.

| S-EPMC7509857 | biostudies-literature

Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds.

Project description:To survive daily damage, the formation of actomyosin ring at the wound edge is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that the rapid recruitment of all 3 Drosophila calcium-responding and phospholipid-binding Annexin proteins (AnxB9, AnxB10, and AnxB11) to distinct regions around the wound is regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, while the wound does not close in the absence of calcium influx, we find that reduced calcium influx can still initiate repair processes, albeit leading to severe repair phenotypes. Thus, our results suggest that, in addition to initiating repair events, the quantity of calcium influx is important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair.

| S-EPMC11304956 | biostudies-literature

Hydrogen sulfide maintains dental pulp stem cell function via TRPV1-mediated calcium influx.

Project description:Hydrogen sulfide (H2S), an endogenous gasotransmitter, mediated a variety of biological processes through multiple signaling pathways, and aberrant H2S metabolism has been associated with mesenchymal stem cell (MSC) dysfunction. Here we employed the small interfering RNA treatment for cystathionine ?-synthase (CBS), cystathionine ?-lyase, the main enzymes to synthesize H2S, and CBS-knockout mice to analyze the effect of H2S on dental pulp homeostasis. We showed that H2S deficiency attenuated dental pulp stem cell (DPSC) osteogenic/dentinogenic differentiation in vitro and in vivo with enhanced cell proliferation. Mechanically, H2S facilitated the transient receptor potential action channel subfamily V member 1-mediated calcium (Ca2+) influx, which subsequently activated the ?-catenin pathway. While H2S deficiency decreased Ca2+, resulting in glycogen synthase kinase-3?-mediated ?-catenin degradation, which controls proliferation and differentiation of DPSCs. Consistently, H2S-deficient mice displayed disturbed pattern of dental pulp and less dentin formation. In this study, we identified a previously unknown mechanism by which H2S regulates DPSC lineage determination and dental pulp homeostasis.

| S-EPMC6060166 | biostudies-literature

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