Biomedical subjects

S Orrenius

Publications and source records attributed to S Orrenius.

At least 199 records · Page 11Linked to original sources

PubMed1990

NK cell-induced cytotoxicity is dependent on a Ca2+ increase in the target.

In previous work we showed that programmed cell death (PCD) in thymocytes is mediated by a sustained increase in cytosolic Ca2+ concentration, resulting in the activation of an endogenous endonuclease, DNA fragmentation, and cell death. In this study we investigated the roles of Ca2+ and DNA fragmentation in target cell killing by natural killer (NK) cells. The effector cells induced a rapid, sustained increase in cytosolic Ca2+ concentration in Jurkat target cells. Buffering the target cell cytosolic Ca2+ with the Ca2(+)-selective dye, quin-2, prevented target cell killing. Extensive DNA fragmentation was associated with killing in every target tested, and this response was also blocked by quin-2. The endonuclease inhibitor, aurintricarboxylic acid, inhibited both DNA fragmentation and killing without influencing the Ca2+ increase in target cells. Thus, it is concluded that NK cell killing depends on a Ca2+ increase and appears to involve endogenous endonuclease activation in target cells.

Aminoquinolines↗

PubMed1990

Release of Ca2+ from the endoplasmic reticulum is not the mechanism for bile acid-induced cholestasis and hepatotoxicity in the intact rat liver.

The hypothesis that monohydroxy bile acids exert their cholestatic and hepatotoxic effects via a sustained elevation of cytosolic [Ca2+] was tested in the isolated perfused rat liver. Infusion of the specific inhibitor of microsomal Ca2+ sequestration, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) (25 microM for 10 min) produced efflux of Ca2+ from the liver and a sustained (20 min) increase in cytosolic [Ca2+] as indicated by the threefold increase in hepatic glucose output. Release of the endoplasmic reticular Ca2+ pool was demonstrated by the complete abolition of vasopressin- and phenylephrine-induced Ca2+ exchange between the liver and perfusate. Despite the profound perturbation of intracellular Ca2+ homeostasis produced by tBuBHQ, there was no decrease in bile flow and no evidence of hepatocellular injury (for 60 min), as indicated by lactate dehydrogenase release. In contrast, lithocholic acid (25 microM for 10 or 30 min) or taurolithocholic acid (5 microM for 10 or 30 min) produced an 80-90% inhibition of bile flow and a progressive increase in perfusate lactate dehydrogenase activity. During and after bile acid infusion, there was no change in Ca2+ fluxes between liver and perfusate, no stimulation of glucose output from the liver, and hormone-stimulated Ca2+ responses were preserved. It is concluded that the mechanisms for bile acid-induced cholestasis and hepatotoxicity in the intact liver are not attributable to changes in intracellular Ca2+ homeostasis, and especially not to prolonged release or depletion of Ca2+ sequestered in the endoplasmic reticulum.

Bile↗

PubMed1990

2,5-Di(tert-butyl)-1,4-benzohydroquinone--a novel mobilizer of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool.

Isolated hepatocytes and the isolated perfused rat liver have been used to study the alterations of cytosolic free Ca2+ concentration ([Ca2+]i) produced by 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a potent inhibitor of hepatic microsomal Ca2+ sequestration (Moore, G.A., McConkey, D.J., Kass, G.E.N., O'Brien, P.J. and Orrenius, S. FEBS Lett., 224, 331-336), (1987). Addition of tBuBHQ to isolated hepatocytes caused a rapid increase in [Ca2+]i which was similar in magnitude to the [Ca2+]i elevation induced by the Ca2+ mobilizing hormone, vasopressin. In contrast with vasopressin which caused a Ca2+ transient, tBuBHQ elevated [Ca2+]i to a new steady state that was maintained for up to 15-20 min. When vasopressin was administered during the tBuBHQ-induced period of elevated [Ca2+]i, [Ca2+]i rapidly returned to basal levels. Similarly, if vasopressin was administered just prior to tBuBHQ, the resultant tBuBHQ-dependent change in [Ca2+]i was transient, and not sustained. The hydroquinone mobilized the same intracellular Ca2+ pool as inositol 1,4,5-trisphosphate, but tBuBHQ did not produce any detectable inositol polyphosphate accumulation. tBuBHQ stimulated glucose release from perifused hepatocytes, mimicking the effect of vasopressin. In the perfused liver, tBuBHQ infusion produced a single, slow and prolonged release of Ca2+ into the perfusate and inhibition of subsequent vasopressin-induced Ca2+ effluxes. Inhibition of the response to vasopressin was reversed over time, and closely correlated with the extent of inhibition of both Ca2+ sequestration and (Ca2+-Mg2+)-ATPase activity in microsomes isolated from the isolated perfused liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

The cytoskeleton as a target in quinone toxicity.

The exposure of mammalian cells to toxic concentrations of redox cycling and alkylating quinones causes marked changes in cell surface structure known as plasma membrane blebbing. These alterations are associated with the redistribution of plasma membrane proteins and the disruption of the normal organization of the cytoskeletal microfilaments which appears to be due mainly to actin cross-linking and dissociation of alpha-actinin from the actin network. The major biochemical mechanisms responsible for these effects seem to involve the depletion of cytoskeletal protein sulfhydryl groups and the increase in cytosolic Ca2+ concentration following the alkylation/oxidation of free sulfhydryl groups in several Ca2+ transport systems. Depletion of intracellular ATP is also associated with quinone-induced plasma membrane blebbing. However, ATP depletion occurs well after the onset of the morphological changes, and thus it does not seem to be causatively related to their appearance. Thiol reductants, such as dithiothreitol, efficiently prevent the oxidation of cytoskeletal protein thiols, the increase in cytosolic free Ca2+ concentration and cell blebbing induced by redox cycling, but not alkylating, quinones. These results demonstrate that alkylating and redox cycling quinones cause similar structural and biochemical modifications of the cytoskeleton by means of different mechanisms, namely alkylation and oxidation of critical sulfhydryl groups.

Actinin↗

PubMed1990

Effects of xenobiotics on signal transduction and Ca2+ mediated processes in mammalian cells.

Cellular responses to hormones and growth factors that act through the hydrolysis of membrane-bound inositol phospholipid are mediated by at least two second messengers--diacylglycerol and inositol 1, 4, 5-trisphosphate. While diacylglycerol stimulates protein kinase C activity, inositol 1, 4, 5-trisphosphate mobilizes intracellular Ca2+. Studies in our laboratory have demonstrated that xenobiotics can interfere with signal transduction at different levels with a resulting loss of normal Ca2+ responses to hormones and growth factors. Effects of toxicants on ion channels, receptors, G proteins, and other enzymes involved in cell signalling have been investigated. Our recent studies have shown that mild oxidative stress can activate protein kinase C which may in turn stimulate cell proliferation. In addition, toxic agents can directly affect intracellular Ca2+ homeostasis by interfering with Ca2+ pumps and Ca2+ channels. This can compromise the ability of the cell to buffer Ca2+ changes and result in a sustained elevation of the cytosolic Ca2+ concentration with the subsequent activation of various Ca(2+)-dependent degradative processes. Thus, the alteration of cell signalling by toxicants may affect several important cell functions, including cell differentiation and proliferation, and may ultimately lead to cell death.

Animals↗

PubMed1989

Ca2(+)-mobilizing hormones stimulate Ca2+ efflux from hepatocytes.

Treatment of hepatocytes with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a novel mobilizer of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool, produces a sustained elevation of [Ca2+]i (Kass, G. E. N., Duddy, S. K., and Orrenius, S. (1989) J. Biol. Chem. 264, 15192-15198). Exposure of hepatocytes to the Ca2(+)-mobilizing hormones, vasopressin, angiotensin II, or ATP following [Ca2+]i elevation by tBuBHQ produced a rapid return of [Ca2+]i to basal or near basal levels. Release of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool by tBuBHQ following pretreatment with vasopressin or angiotensin II resulted in a [Ca2+]i transient and not the sustained [Ca2+]i elevation observed in the absence of the Ca2(+)-mobilizing hormones. The G-protein activator, NaF plus AlCl3, mimicked both effects of the Ca2(+)-mobilizing hormones on [Ca2+]i. The mechanism for Ca2+ removal from the cytosol by Ca2(+)-mobilizing hormones did not involve cyclic nucleotides nor did it require protein kinase C activation or cyclo- and lipoxygenase-dependent metabolites of arachidonic acid. Furthermore, the hormone-mediated decrease in [Ca2+]i did not involve the pertussis toxin-sensitive Gi-protein. Removal of the tBuBHQ-mobilized Ca2+ from the cytosol of hepatocytes by Ca2(+)-mobilizing hormones was mediated by stimulation of a Ca2+ efflux pathway. Thus, in addition to initiating [Ca2+]i transients by releasing Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ store and stimulating Ca2+ influx, Ca2(+)-mobilizing hormones also regulate the termination of the [Ca2+]i transient by stimulating a Ca2+ efflux pathway.

Animals↗

PubMed1989

Quantitation of DNA fragmentation using fiberglass filters.

Many types of physiological and toxicological cell killing are mediated by extensive DNA fragmentation. To date, most assays used to detect DNA fragmentation have relied on cumbersome techniques to separate intact chromatin from cleaved DNA. Here we describe a filtration assay for quantitation of DNA fragmentation. Fiberglass filtermats were used to separate intact chromatin from DNA fragments. Analysis of the separation showed that intact chromatin consistently remained on filters, while DNA fragments of all (random) sizes were consistently found in the filtrates. The assay was adaptable to different DNA detection procedures and generated results comparable to those obtained using established methods in standard model systems.

Animals↗

PubMed1989

Calcium-dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex.

Development of tolerance to self Ag occurs during a negative cell selection process in the thymus. This selection process is thought to involve interactions between Ag-specific thymocyte receptors and self Ag presented by the MHC proteins on accessory cells, resulting in deletion of potentially harmful self-reactive precursors. However, the mechanisms underlying this clonal deletion have not been identified. In confirmation of previous findings (C. A. Smith, G. T. Williams, R. Kingston, E. J. Jenkins, and J. J. T. Owen, 1989. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337:181), we have found that an anti-CD3 antibody stimulated DNA fragmentation, characteristic of a suicide mechanism known as apoptosis or programmed cell death (PCD), in suspensions of human thymocytes. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin. Although the magnitude and duration of the Ca2+ increase were similar to those observed in response to Con A, the mitogen did not stimulate DNA fragmentation or cell death. Phorbol ester prevented Ca2+-dependent DNA fragmentation and cell killing in response to anti-CD3 or other agents that stimulated PCD, suggesting that activation of protein kinase C abrogated cell suicide. Disappearance of CD4+CD8+ immature thymocytes was generally observed in response to all agents that stimulated PCD, whereas mature PBL were insensitive to stimulation of PCD. Our results suggest that antibody-mediated stimulation of immature thymocytes via the TCR complex results in Ca2+-dependent, endonuclease-mediated cell killing, depending on the activation status of protein kinase C.

Antibodies, Monoclonal↗

PubMed1989

2,5-Di-(tert-butyl)-1,4-benzohydroquinone rapidly elevates cytosolic Ca2+ concentration by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool.

2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca2+ sequestration (Moore, G. A., McConkey, D. J., Kass, G. E. N., O'Brien, P. J., and Orrenius, S. (1987) FEBS Lett. 224, 331-336), produced a concentration-dependent, rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated rat hepatocytes (EC50 = 1-2 microM). The amplitude of the [Ca2+]i increase was essentially identical with that produced by vasopressin, but the tBuBHQ-stimulated [Ca2+]i increase remained sustained for 15-20 min. Vasopressin added 2-3 min after tBuBHQ caused [Ca2+]i to rapidly return to basal levels; however, tBuBHQ added after vasopressin resulted in a Ca2+ transient rather than a sustained [Ca2+]i elevation. Ca2+ influx was not stimulated in tBuBHQ-treated hepatocytes, but was markedly enhanced upon addition of vasopressin. Depletion of the endoplasmic reticular Ca2+ pool by the addition of vasopressin to hepatocytes incubated in low Ca2+ medium virtually abolished the tBuBHQ-mediated [Ca2+]i rise and vice versa. In saponin-permeabilized hepatocytes, tBuBHQ released Ca2+ from the same nonmitochondrial, ATP-dependent Ca2+ pool which was released by inositol 1,4,5-trisphosphate. Furthermore, tBuBHQ-induced Ca2+ release in saponin-permeabilized cells was not inhibited by neomycin, and tBuBHQ did not produce any apparent accumulation of inositol phosphates in intact hepatocytes. The rate of passive efflux of Ca2+ from Ca2+-loaded hepatic microsomes was unaltered by tBuBHQ. Thus, tBuBHQ inhibits ATP-dependent Ca2+ sequestration via a direct effect on the endoplasmic reticulum Ca2+ pump, resulting in net Ca2+ release and elevation of [Ca2+]i. Taken together, our results show that in the absence of hormonal stimuli, excess Ca2+ is only slowly cleared from the hepatocyte cytosol, indicating that the basal rate of Ca2+ removal by the plasma membrane Ca2+ pump and mitochondria is slow. Furthermore, Ca2+-mobilizing hormones appear to stimulate an active process of Ca2+ removal from hepatocyte cytosol which does not depend on re-uptake into the endoplasmic reticulum.

Animals↗

PubMed1989

Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C.

Glucocorticoid hormones and Ca2+ ionophores stimulate a suicide process in immature thymocytes, known as apoptosis or programmed cell death, that involves extensive DNA fragmentation. We have recently shown that a sustained increase in cytosolic Ca2+ concentration stimulates DNA fragmentation and cell killing in glucocorticoid- or ionophore-treated thymocytes. However, a sustained increase in the cytosolic Ca2+ level also mediates lymphocyte proliferation, suggesting that apoptosis is blocked in proliferating thymocytes. In this study we report that phorbol esters, which selectively stimulate protein kinase C (PKC), blocked DNA fragmentation and cell death in thymocytes exposed to Ca2+ ionophore or glucocorticoid hormone. The T cell mitogen, concanavalin A, which stimulates thymocytes by a mechanism that involves PKC activation, caused concentration-dependent increases in the cytosolic Ca2+ level that did not result in DNA fragmentation, but incubation with concanavalin A and the PKC inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) resulted in both DNA fragmentation and cell death. Phorbol ester directly inhibited Ca2+-dependent DNA fragmentation in isolated thymocyte nuclei. Our results strongly suggest that PKC activation blocks thymocyte apoptosis by preventing Ca2+-stimulated endonuclease activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1989

Activation of hepatocyte protein kinase C by redox-cycling quinones.

The effects of quinone-generated active oxygen species on rat hepatocyte protein kinase C were investigated. The specific activity of cytosolic protein kinase C was increased 2-3-fold in hepatocytes incubated with the redox-cycling quinones, menadione, duroquinone or 2,3-dimethoxy-1,4-naphthoquinone, without alterations in particulate protein kinase C specific activity or Ca2+- and lipid-independent kinase activities. Redox-cycling quinones did not stimulate translocation of protein kinase C; however, activated protein kinase C was redistributed from cytosol to the particulate fraction when quinone-treated hepatocytes were exposed to 12-O-tetradecanoylphorbol 13-acetate (TPA). Quinone treatment did not alter cytosolic phorbol 12,13-dibutyrate (PDBu) binding capacity, and the cytosol of both control and quinone-treated hepatocytes exhibited a Kd for PDBu binding of 2 nM. Quinone-mediated activation of cytosolic protein kinase C was reversed by incubation with 10 mM-beta-mercaptoethanol, dithiothreitol or GSH, at 4 degrees C for 24 h. Furthermore, protein kinase C specific activity in control cytosol incubated in air increased by over 100% within 3 h; this increase was reversed by thiol-reducing agents. Similarly, incubation of partially-purified rat brain protein kinase C in air, or with low concentrations of GSSG in the presence of GSH, resulted in a 2-2.5-fold increase in Ca2+- and lipid-dependent kinase activity. In contrast with the effects of the redox-cycling quinones, when hepatocytes were treated with the thiol agents N-ethylmaleimide (NEM), p-benzoquinone (pBQ) or p-chloromercuribenzoic acid (pCMB), the cytosolic Ca2+- and lipid-dependent kinase activity was significantly inhibited, but the particulate-associated protein kinase C activity was unaffected. The Ca2+- and lipid-independent kinase activity of both the cytosolic and particulate fractions was significantly stimulated by NEM, but was unaffected by pBQ and pCMB. These results show that hepatocyte cytosolic protein kinase C is activated to a high-Vmax form by quinone-generated active oxygen species, and this effect is due to a reduction-sensitive modification of the thiol/disulphide status of protein kinase C.

Animals↗

PubMed1989

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) kills glucocorticoid-sensitive thymocytes in vivo.

The effect of in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on glucocorticoid- and calcium ionophore-induced DNA fragmentation in rat thymocytes was investigated. TCDD dose-dependently abolished DNA fragmentation in response to both agents after 7 days of exposure. Analysis of the time dependence of the effect revealed that after 1 or 2 days TCDD potentiated DNA fragmentation in untreated and glucocorticoid-treated thymocyte suspensions relative to controls. The DNA fragmentation in untreated thymocyte suspensions from TCDD-treated rats was completely prevented by inhibitors that block glucocorticoid-induced thymocyte suicide. Our results suggest that TCDD-induced thymic atrophy is due to Ca2+-dependent endonuclease activation.

Animals↗

PubMed1989

Cytoskeletal alterations in human platelets exposed to oxidative stress are mediated by oxidative and Ca2+-dependent mechanisms.

The metabolism of the redox-active quinone, menadione (2-methyl-1,4-naphthoquinone), in human platelets was associated with superoxide anion production, oxidation and depletion of intracellular glutathione, and modification of protein thiols. The cytoskeletal fraction extracted from menadione-treated platelets exhibited a dose-dependent increase in the amount of cytoskeleton-associated protein and a concomitant loss of protein thiols. These alterations were associated with oxidative modifications of actin, including beta-mercaptoethanol-sensitive crosslinking of actin to form dimers, trimers, and high-molecular-weight aggregates which also contained other cytoskeletal proteins, i.e., alpha-actinin and actin-binding protein. In addition, analysis of the cytoskeletal fraction from platelets treated with high concentrations (greater than or equal to 100 microM) of menadione by polyacrylamide gel electrophoresis under reducing conditions revealed a net decrease in the relative abundance of the individual cytoskeletal polypeptides. Under the same incubation conditions the platelets exhibited a sustained increase in cytosolic Ca2+ concentration. The presence of glucose, or the omission of Ca2+ from the incubation medium, prevented both the increase in cytosolic Ca2+ and the decrease in the relative amounts of cytoskeletal proteins. The latter effect was also largely prevented in platelets loaded with Quin-2 tetraacetoxymethyl ester to buffer the menadione-induced elevation of cytosolic Ca2+. Finally, the presence of a protease inhibitor, leupeptin, in the incubation medium prevented the menadione-induced decrease in the amount of actin-binding protein but not the decrease in the other cytoskeletal proteins. Our findings demonstrate that the multiple effects of oxidative stress on the platelet cytoskeleton are mediated by oxidative as well as by Ca2+-dependent mechanisms.

Actins↗

PubMed1989

Calcium-activated DNA fragmentation in rat liver nuclei.

Incubation of isolated rat liver nuclei with ATP, NAD+, and submicromolar Ca2+ concentrations resulted in extensive DNA hydrolysis. Half-maximal activity occurred with 200 nM Ca2+, and saturation of the process was observed with 1 microM Ca2+. ATP stimulated a calmodulin-dependent nuclear Ca2+ uptake system which apparently mediated endonuclease activation. Ca2+-activated DNA fragmentation was inhibited by the inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide, and was associated with poly(ADP-ribosyl)ation of nuclear protein. The characteristics of this endonuclease activity indicate that it may be responsible for the Ca2+-dependent fragmentation of DNA involved in programmed cell death (apoptosis) and in certain forms of chemically induced cell killing.

Adenosine Triphosphate↗

PubMed1989

Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca2+ concentration.

Glucocorticoid hormones kill immature thymocytes through the induction of a suicide process commonly referred to as "apoptosis." A characteristic marker for this process is the stimulation of endogenous endonuclease activity which results in the extensive cleavage of cell chromatin. In an attempt to characterize the biochemical events involved in this process, we studied the role of Ca2+ in glucocorticoid-induced DNA fragmentation and cell killing in thymocytes. Treatment of thymocytes from immature rats with the synthetic glucocorticoid methylprednisolone resulted in extensive DNA fragmentation which was preceded by an early, sustained increase in cytosolic Ca2+ concentration. This increase in Ca2+ level was blocked by cycloheximide and actinomycin D, inhibitors of de novo protein and mRNA synthesis, respectively. Prevention of the Ca2+ increase by buffering cytosolic Ca2+ with quin-2, or through incubation of the thymocytes in a "Ca2+-free" medium, prevented endonuclease activation and cell killing. Inhibitors of calmodulin also prevented DNA fragmentation without inhibiting the glucocorticoid-stimulated elevation of cytosolic Ca2+ concentration. The Ca2+ increase appeared to be due to the action of a heat-labile cytosolic factor, synthesized in response to glucocorticoids, which facilitated the influx of extracellular Ca2+. Our findings suggest that glucocorticoids induce thymocyte suicide through an elevation of cytosolic Ca2+ concentration resulting in endonuclease activation, DNA fragmentation, and cell death.

Animals↗

PubMed1989

Rapid microfilament reorganization induced in isolated rat hepatocytes by microcystin-LR, a cyclic peptide toxin.

The cyclic heptapeptide hepatotoxin microcystin-LR from the cyanobacterium Microcystis aeruginosa induces rapid and characteristic deformation of isolated rat hepatocytes. We investigated the mechanism(s) responsible for cell shape changes (blebbing). Our results show that the onset of blebbing was accompanied neither by alteration in intracellular thiol and Ca2+ homeostasis nor by ATP depletion. The irreversible effects were insensitive to protease and phospholipase inhibitors and also to thiol-reducing agents, excluding the involvement of enhanced proteolysis, phospholipid hydrolysis, and thiol modification in microcystin-induced blebbing. In contrast, the cell shape changes were associated with a remarkable reorganization of microfilaments as visualized both by electron microscopy and by fluorescent staining of actin with rhodamine-conjugated phalloidin. The morphological effects and the microfilament reorganization were specific for microcystin-LR and could not be induced by the microfilament-modifying drugs cytochalasin D or phalloidin. Using inhibition of deoxyribonuclease I as an assay for monomeric actin, we found that the microcystin-induced reorganization of hepatocyte microfilaments was not due to actin polymerization. On the basis of the rapid microfilament reorganization and the specificity of the effects, it is suggested that microcystin-LR constitutes a novel microfilament-perturbing drug with features that are clearly different from those of cytochalasin D and phalloidin.

Actin Cytoskeleton↗

PubMed1989

Role of Ca2+ in toxic cell killing.

Recent work has shown that a sustained increase in cytosolic Ca2+ concentration is often linked to the onset of cytotoxicity. Sten Orrenius and colleagues describe several biochemical mechanisms that are stimulated by such a Ca2+ increase and can directly mediate cell death by causing disruption of the cytoskeleton, DNA fragmentation and extensive damage to other cell components.

Animals↗

PubMed1989

ATP stimulates Ca2+ uptake and increases the free Ca2+ concentration in isolated rat liver nuclei.

Addition of ATP to a highly purified fraction of rat liver nuclei incubated with submicromolar concentrations of Ca2+ and trace amounts of 45Ca2+ resulted in the rapid accumulation of 45Ca2+ in the nuclei. This was associated with an increase in intranuclear free Ca2+ concentration as measured with the fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-a mino-5'- methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (fura-2). Inhibitors of microsomal and mitochondrial Ca2+ translocases had no effect on nuclear Ca2+ sequestration, indicating that it was distinct from previously known intracellular Ca2+-transporting systems. Ca2+ uptake and the associated increase in intranuclear free Ca2+ concentration were prevented by calmidazolium, a potent calmodulin antagonist. Partial characterization of the ATP-stimulated nuclear Ca2+ uptake showed that maximal rates of Ca2+ uptake and increase in intranuclear free Ca2+ level occurred at concentrations of Ca2+ normally present in the cytosol of mammalian cells. Together, these results show that a distinct, ATP- and calmodulin-dependent Ca2+ uptake system exists in liver nuclei. This system may play an important role in the regulation of intranuclear Ca2+-dependent processes.

Adenosine Triphosphate↗