Calcium-mediated mechanisms in chemically induced cell death.
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Animals↗
Biomedical subjects
S Orrenius
Publications and source records attributed to S Orrenius.
Deletion and depletion: the involvement of viruses and environmental factors in T-lymphocyte apoptosis.
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Animals↗
Cellular signaling in thymocyte apoptosis.
Induction of apoptosis (programmed cell death) in response to T cell receptor triggering is now thought to be involved in the process of negative selection in the thymus, and current work is therefore aimed at investigating how apoptosis is regulated within the cells. To this end, recent work has implicated several of the well-known signal transduction pathways already known to regulate T cell activation in the regulation of apoptosis in thymocytes. In particular, elevations of the cytosolic Ca2+ level or increases in cAMP can trigger thymocyte apoptosis, whereas activation of protein kinase C appears to inhibit apoptosis in response to either induction pathway. Moreover, crosslinking of Thy-1, CD4, or CD8 leads to potentiation of T cell receptor-mediated cell death, effects that appear to involve protein tyrosine kinase activation. These observations may be relevant to the question of how T cell receptor occupancy can mediate both differentiation and death during intrathymic T cell development.
Animals↗
Comparison between the effects of the microsomal Ca(2+)-translocase inhibitors thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone on cellular calcium fluxes.
The effects of two inhibitors of the microsomal Ca(2+)-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone, were compared in hepatocytes and in a T-cell line (JURKAT). Both compounds mobilized the same intracellular Ca2+ pool, which contained the Ins(1,4,5)P3-sensitive store, in hepatocytes and in JURKAT cells. The mobilization of the internal Ca2+ store with either compound activated Mn2+ entry in JURKAT cells, but not in hepatocytes. This suggests different properties of the bivalent-cation entry pathway between these cell types.
Animals↗
Mobilization of the hormone-sensitive calcium pool increases hepatocyte tight junctional permeability in the perfused rat liver.
Hepatocyte tight junctional permeability has been shown to be regulated by hormones that exert their effects via phospholipase C activation. However, the precise transduction pathway involved in this effect is not known. The present study has employed the selective inhibitor of microsomal Ca2+ sequestration, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), to examine the effect of the mobilization of the endoplasmic reticular Ca2+ pool on tight junctional permeability in the perfused rat liver. Infusion of tBuBHQ followed by a bolus infusion of horseradish peroxidase (HRP) resulted in a significant increase in the first peak of biliary HRP, a measure of junctional permeability, whereas transcellular (vesicular) transport of HRP was not affected. Therefore, we conclude that the effect of hormones on tight junctional permeability is mediated, at least in part, by the mobilization of intracellular Ca2+.
Animals↗
Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells.
Chronic lymphocytic leukemia of B cell type (B-CLL) is a neoplastic disorder characterized by the accumulation of small resting lymphocytes in the periphery. The phenotype of these cells suggests that they are "frozen" at an early stage of maturation. Glucocorticoid hormones are commonly used to treat patients with B-CLL, resulting in a reduction in the peripheral lymphocyte count by an undefined mechanism. Here we report that glucocorticoids stimulate DNA fragmentation characteristic of a suicide process known as apoptosis or programmed cell death (PCD) in suspensions of cells from patients with B-CLL. The effects can be mimicked by Ca2+ ionophore and involve a sustained increase in the cytosolic Ca2+ concentration. Specific antibodies binding to membrane-associated IgM on the leukemic cells can also induce PCD by a similar mechanism. Phorbol esters block DNA fragmentation and cell killing in response to all of the agents, suggesting that activation of protein kinase C desensitizes the cells to PCD. Targeting the B-CLL cells with antibodies that induce an unbalanced, sustained Ca2+ increase may therefore represent a rational strategy for the destruction of leukemic cells.
Antibodies, Monoclonal↗
Role of calcium in toxic and programmed cell death.
An uncontrolled and sustained increase in cytosolic Ca2+ concentration has been implicated as an early event in the development of anoxic or toxic cell injury. More recently it has become clear that an elevation of cytosolic Ca2+ is also involved in programmed cell death in the immune system. Here, we review some of our recent studies and provide further evidence for the role of Ca2+ in cell killing.
Animals↗
Spermine prevents endonuclease activation and apoptosis in thymocytes.
Glucocorticoid hormones, Ca2+ ionophores, and some toxic chemicals activate a suicide process in thymocytes, known as apoptosis or programmed cell death. A crucial event in apoptosis is the activation of a Ca(2+)- and Mg(2+)-dependent endonuclease that promotes extensive DNA fragmentation. In this study, we investigated the effect of various polyamines on endonuclease activation leading to thymocyte apoptosis. We found that both glucocorticoid- and Ca2+ ionophore-induced DNA fragmentation and apoptosis were prevented by spermine. Other polyamines such as putrescine or spermidine had moderate or no effect. Moreover, spermine, and to a lesser extent spermidine, but not putrescine, prevented endonuclease activation in permeabilized liver nuclei incubated in the presence of Ca2+ and Mg2+, indicating that spermine efficiency in blocking DNA fragmentation was related to the interaction of this polyamine with the endonuclease or its substrate, DNA. Experiments with the fluorescent dye, ethidium bromide, and a purified preparation of liver endonuclease revealed that the protective effect of spermine on DNA fragmentation was related to its ability to modify the chromatin arrangement. Thymocytes incubated with methyl glyoxal bis(guanylhydrazone) to deplete intracellular spermine exhibited spontaneous DNA fragmentation, which suggests that modulation of the intracellular polyamine content and regulation of chromatin structure may play a critical role in the early phases of apoptosis. Finally, these results demonstrate that inhibition of DNA fragmentation also prevents the onset of apoptosis, directly linking endonuclease activation and cell death.
Animals↗
Modifications of cell signalling in the cytotoxicity of metals.
Many metals act on biological systems at low concentrations and recent epidemiological and experimental research indicates that toxic effects of certain metals occur at levels only marginally higher than those found in healthy subjects. Despite a large number of studies describing metal cytotoxicity, the molecular mechanisms involved are still poorly understood. However, it now seems evident that several metals can interact with enzyme functional groups and that proteins involved in signal transduction, including Ca2+ channels and pumps, may be especially sensitive to this interaction. Impairment of the ability of cells to adequately respond to the stimulation by hormones and growth factors may result in the loss of important cell functions or activation of mechanisms that compromise cell survival. In the following sections we will briefly describe the effects of various metals on cell signalling and present our recent findings on the mechanism by which inorganic mercury affects signal transduction.
Animals↗
Tributyltin stimulates apoptosis in rat thymocytes.
Treatment of rat thymocytes with micromolar concentrations of tributyltin caused a rapid increase in the cytosolic free Ca2+ concentration that was inhibited by Ni2+, which blocks Ca2+ influx through membrane channels. The elevation of cytosolic Ca2+ was associated with extensive DNA fragmentation, which was prevented by pretreatment of the cells with either of the intracellular Ca2+ chelators quin-2 or 1,2-bis(2-amino-phenoxy)ethane-N',N',N',N',-tetraacetic acid. Loss of thymocyte viability, which followed DNA fragmentation, was also prevented by the two Ca2+ chelators or by removing extracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. The pattern of DNA fragmentation was characteristic of that produced by agents which activate a Ca2(+)- and Mg2(+)-dependent endogenous endonuclease during apoptosis or programmed cell death. Additional studies showed that other organotin compounds, including trimethyltin, triphenyltin, and dibutyltin had minimal effects on cytosolic Ca2+, DNA fragmentation, and cell viability. These results are consistent with a greater susceptibility of thymocytes to tributyltin and provide a basis for understanding its selective immunotoxicity in vivo.
Animals↗
Receptor-operated calcium influx in rat hepatocytes. Identification and characterization using manganese.
Agonist-stimulated divalent cation entry was studied in fura-2-loaded hepatocytes. In the presence of extracellular Mn2+, the Ca2(+)-mobilizing hormone vasopressin produced a severalfold stimulation of the basal rate of fura-2 fluorescence quenching as a result of Mn2+ influx; this effect was blocked by the presence of Ni2+ in the incubation medium. Half-maximum and maximum stimulation of Mn2+ influx was observed with 0.1 and 0.8 nM vasopressin, respectively. Agonist-stimulated Mn2+ influx was also seen with angiotensin II, ATP, phenylephrine, and the combination of AlCl3 and NaF. The stimulation of Mn2+ influx did not occur immediately after addition of Ca2(+)-mobilizing agents, but was characterized by a latency period of 20-30 s. In contrast to vasopressin, glucagon did not stimulate Mn2+ influx into hepatocytes, but produced both a 3-fold enhancement of the rate of vasopressin-stimulated Mn2+ entry and the abolishment of the latency period. The effects of glucagon were mimicked by forskolin and dibutyryl cAMP. Pretreatment of hepatocytes with pertussis toxin or depolarization of the cells altered neither the basal rate of Mn2+ entry nor the ability of vasopressin to stimulate this rate. Emptying of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store by treatment with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) did not enhance Mn2+ entry into hepatocytes; however, exposure of the cells to tBuBHQ for 2 min markedly enhanced the ability of vasopressin, alone or in combination with glucagon, to increase the rate of Mn2+ influx. Furthermore, pretreatment with tBuBHQ for 2 min abolished the latency of vasopressin-stimulated Mn2+ influx. It is concluded that Ca2(+)-mobilizing hormones stimulate Ca2+ influx in hepatocytes, possibly through receptor-operated Ca2+ channels. The stimulation of divalent cation entry is transduced by a G protein, and the rate of influx appears to be controlled both by the intracellular level of cAMP and the empty state of an intracellular Ca2+ pool that may be inositol 1,4,5-trisphosphate-insensitive.
Angiotensin II↗
Agents that elevate cAMP stimulate DNA fragmentation in thymocytes.
Increases in the cAMP level are often inhibitory in mature T lymphocytes and may be involved in the development of tolerance to self Ag. In this report, agents inducing an increase in the cAMP level by independent mechanisms were found to stimulate DNA fragmentation, characteristic of a suicide program known as apoptosis, in isolated thymocytes. Data obtained with cAMP analogs known to act synergistically to stimulate protein kinase A suggested that the latter directly mediated endonuclease activation. Agents previously shown to stimulate protein kinase C and to inhibit Ca2(+)-dependent, TCR-mediated thymocyte apoptosis, including IL-1, also blocked both DNA fragmentation and cell death in response to cAMP, suggesting interactions ("cross-talk") between the two protein kinase systems. As it has been proposed that apoptosis mediates negative cell selection in the thymus, our results indicate that cAMP may play a role in the development of functional mature T lymphocytes.
Animals↗
Interleukin 1 inhibits T cell receptor-mediated apoptosis in immature thymocytes.
T cell receptor signaling has been implicated in an intrathymic process of cell selection in which potentially harmful autoreactive precursors are deleted before they emigrate to the periphery. Recent work has shown that immature thymocytes undergo apoptosis, or programmed cell death, upon stimulation via the T cell antigen receptor, and it has been suggested that this mechanism may mediate deletion of the self-reactive clones during T cell development. Here we report that the accessory cell-derived T cell growth hormone interleukin-1 prevents T cell receptor-mediated thymocyte apoptosis by a mechanism that appears to involve protein kinase C activation. This effect may be relevant to the positive signaling that spares the appropriate precursors during the generation of functional T lymphocytes.
Antibodies, Monoclonal↗
Calcium-dependent DNA fragmentation in human synovial cells exposed to cold shock.
Exposure of confluent human synovial McCoy's cells to near-freezing temperatures followed by rewarming at 37 degrees C resulted in endonuclease activation and cell death characteristic of a suicide process known as apoptosis. Both DNA fragmentation and cell killing were dependent on a sustained increase in the cytosolic Ca2+ concentration. Sensitivity to cold shock-induced endonuclease activation was critically dependent on the cell cycle (proliferative) status and limited to confluent cells, whereas cells in the logarithmic growth phase were completely resistant. However, DNA fragmentation was promoted in the proliferating McCoy's cells pretreated with H-7 or sphingosine, inhibitors of protein kinase C. In addition, phorbol ester, known to activate PKC, inhibited DNA fragmentation in the confluent cells. Our findings indicate that cold shock-induced DNA fragmentation in McCoy's cells is dependent on a sustained Ca2+ increase, and sensitivity to the process appears to be regulated by the status of protein kinase C.
Aminoquinolines↗
Rapid turnover of endogenous endonuclease activity in thymocytes: effects of inhibitors of macromolecular synthesis.
Previous work has shown that inhibitors of protein or mRNA synthesis block endonuclease activation in thymocytes undergoing programmed cell death. In the present study we used isolated nuclei to investigate the effects of cycloheximide and actinomycin D, inhibitors of protein and mRNA synthesis, respectively, on endogenous endonuclease activity in thymocytes. We observed a rapid loss of Ca2(+)-dependent endonuclease activity in nuclei isolated from thymocytes treated with these inhibitors. In contrast, pretreatment of cells with antipain and leupeptin, inhibitors of proteases, prevented the depletion of endonuclease activity in the nuclei, suggesting that proteolysis was involved. The effects of cycloheximide and actinomycin D were mimicked by incubating thymocytes with treatments known to exert their effects via activation of protein kinase C. Our results suggest that endonuclease activity in thymocyte nuclei undergoes rapid, spontaneous turnover. Agents interfering with macromolecular synthesis may therefore block DNA fragmentation in thymocytes by depleting nuclei of endogenous endonuclease activity.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Modulation of cellular glutathione and protein thiol status during quinone metabolism.
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Animals↗
Cellular signalling in programmed cell death (apoptosis).
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Animals↗
An inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in liver nuclei.
Recent studies in our laboratory have revealed the existence of an ATP- and calmodulin-dependent Ca2+ uptake system in rat liver nuclei that can promote increases in the free Ca2+ concentration in the nuclear matrix. In the present investigation we show that liver nuclei possess a marked ability to sequester and buffer Ca2+, suggesting a potential role for the nucleus in the regulation of the cytosolic free Ca2+ concentration. In addition, we demonstrate that the intracellular messenger, inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3], stimulates the release of a fraction of the nuclear Ca2+ and transiently lowers the intranuclear free Ca2+ concentration. The Ins(1,4,5)P3-stimulated Ca2+ release is followed by Ca2+ reuptake into an inositol phosphate-insensitive nuclear compartment. Together, these results demonstrate that liver nuclei contain, at least, two Ca2+ pools, one of which is releasable by Ins(1,4,5)P3. These findings are consistent with a role for the nucleus in the modulation of the cytosolic free Ca2+ level by agonists and suggest that the control of the nuclear Ca2+ load by second messengers may participate in the regulation of intranuclear Ca2(+)-dependent processes by hormones and other agents.
Animals↗