Interaction between dexamethasone and butyrate in apoptosis induction: non-additive in thymocytes and synergistic in a T cell-derived leukemia cell line.

In thymocytes butyrate and trichostatin A are unable to augment dexamethasone-induced apoptosis. In cultured rat thymocytes the extent of apoptosis induced by dexamethasone alone did not increase by addition of 0.1 - 10 mM butyrate. Even more pronounced was the non-additive interrelationship between dexamethasone and trichostatin A, as trichostatin A-induced apoptosis was not only blocked by the presence of dexamethasone but dexamethasone-induced apoptosis was also partially inhibited in the presence of 0.1 - 0.5 microM trichostatin A. The fact that the non-additive relationship with dexamethasone for apoptosis induction was observed with both histone deacetylase inhibitors suggests that in thymocytes this phenomenon is related to histone acetylation. In contrast to this, in the human T cell-derived leukemia cell line CEM-C7H2, dexamethasone did not block butyrate- or trichostatin A-induced apoptosis; moreover, butyrate, in the concentration range of 0.1 - 1 mM, had a marked synergistic effect on dexamethasone-induced apoptosis. This synergism, however, was not mimicked by trichostatin A, indicating that the effect is not related to histone acetylation but rather due to a pleiotropic effect of butyrate. Furthermore, in CEM-C7H2 cells, at higher concentrations of butyrate (5 - 10 mM) or trichostatin A (0.4 - 0.8 microM), there was a minor but reproducible antagonistic effect of dexamethasone on apoptosis induced by each of the two histone deacetylase inhibitors, suggesting that this antagonistic effect too, is related to histone hyperacetylation.     

Intracellular pH of stimulated thymocytes measured with a new fluorescent indicator

A new fluorescent intracellular pH indicator is described ("quene 1") which is related to the tetracarboxylate Ca2+ indicator based on the quinoline fluorophor ("quin 2"). Quene 1 has excitation and emission maxima at 390 and 530 nm, respectively, and shows a 30-fold increase in fluorescence between pH 5 and 9 with a pK alpha of 7.3. The fluorescence is insensitive to Ca2+ and Mg2+ at free concentrations up to 10(-4) M and to the proportions of Na+ and K+ at total concentrations of Na+ and K+ from 100 to 200 mM. The indicator is loaded into thymocytes using the tetraacetoxymethyl ester derivative which is hydrolyzed in the cells to give the tetracarboxylate anion. Intracellular pH can be measured at intracellular quene 1 concentrations of approximately 0.1 mM and quene 1 does not perturb glycolysis or the ATP level in resting cells at concentrations up to 0.8 mM. The intracellular pH of mouse thymocytes indicated by quene 1 is 7.15 +/- 0.04 and it is insensitive to the concentration of Ca2+ or Mg2+ in the extracellular medium. The intracellular pH decreased when the pH of the medium was lowered by addition of HCl, but was insensitive to NaOH at extracellular pH values up to 8.0. Rapid transient changes in intracellular pH are induced by NH4Cl, NaCO2CH3, or HCO3-/CO2. The thymocytes showed no early changes (within 30 min) in intracellular pH in response to mitogenic concentrations of lectins or 4 beta-phorbol-12-myristate-13-acetate.     

Different roles of spermine in glucocorticoid- and Fas-induced apoptosis

Two experimental systems representative of the mitochondrial and death receptor apoptotic pathways are the dexamethasone-induced programmed cell death in mouse thymocytes and the antibody-mediated cross-ligation of the Fas receptor in the human leukemic T-cell line Jurkat, respectively. In both cell systems, caspase-9, -8, and -3 were activated upon induction of apoptosis and a sub-G(1) peak appeared as a sign of ongoing DNA fragmentation. Addition of 1 mM spermine together with dexamethasone inhibited caspase activation and the appearance of the sub-G(1) peak in mouse thymocytes. In contrast, Fas-induced cell death was totally unaffected by spermine addition. Spermine addition significantly elevated the spermine concentration in both thymocytes and Jurkat cells. Thus, spermine per se did not inhibit the caspases but rather their activation. The fact that spermine inhibited caspase activation only in the thymocytes implies that spermine inhibited dexamethasone-induced apoptosis upstream of caspase-9 activation.     

The Na+-ionophore monensin enhances glucose uptake in mouse thymocytes

Monensin enhanced 2-deoxyglucose uptake and 3-O-methyl glucose transport in mouse thymocytes, but had no effect on L-glucose transport. Cytochalasin B inhibited monensin induced as well as basal glucose uptake. The enhanced 2-deoxyglucose uptake was time and dose-dependent. The increase in the rate of 2-deoxyglucose uptake induced by monensin was more rapid than that of Na+ uptake. Ouabain did not inhibit monensin-enhanced 2-deoxyglucose uptake. Monensin failed to stimulate 2-deoxyglucose uptake at low concentrations of Na+ (13 mM) or K+ (17 mM), higher concentrations of either cation were required for stimulation. Monensin enhanced glucose uptake also in Ca2+-free medium. The data indicate that the stimulation of 2-deoxyglucose uptake by monensin results from activation of carrier-mediated transport.     

Regulation of T lymphocyte apoptosis. Signals for the antagonism between activation- and glucocorticoid-induced death.

Apoptotic death can be induced in T cell hybridomas by glucocorticoids or the stimulation via the TCR/CD3 complex. The two apoptotic processes are mutually antagonistic. We have previously proposed that positive selection of thymocytes for the formation of the T cell repertoire might be based on a similar mechanism. We analyzed the TCR/CD3-mediated signals essential for the regulation of apoptosis in T cell hybridomas. We suggest that both an increase in the intracellular Ca2+ level and an activation of protein kinase C are essential for the TCR/CD3-mediated apoptosis, because we obtained the following results: 1) either reduction of extracellular Ca2+ concentration or addition of a protein kinase inhibitor, 1-(5-isoquinolkinelsulfonyl)-2-methylpiperazine or N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide, inhibited anti-CD3-induced but not dexamethasone-induced DNA fragmentation. 2) The combination of ionomycin and PMA, but neither one alone nor the combination of ionomycin and cyclic nucleotide analogs, induced DNA fragmentation. On the contrary, we suggest that only an increase in the intracellular Ca2+ level is essential for the inhibition of glucocorticoid-induced apoptosis, because ionomycin alone as well as the combination of ionomycin and PMA inhibited dexamethasone- but not anti-CD3-induced DNA fragmentation.     

Azaspiracids modulate intracellular pH levels in human lymphocytes

The azaspiracids (AZAs) are a group of marine toxins implicated in several intoxications whose mechanism of action is unknown. These phycotoxins include the five compounds shown in : AZA-1 (1), AZA-2 (2), AZA-3 (3), AZA-4 (4), and AZA-5 (5). The aim of this work was to study the effects of the five naturally occurring azaspiracids (AZA-1 to -5, Fig. 1) and four synthetic analogues (6-9, Fig. 2) on intracellular pH, and the influence of Ca2+ upon this effect. The AZAs (1-5) were found to modulate cytosolic Ca2+ levels in human lymphocytes, while some of them, but not all, had effects on the intracellular pH. AZA-1 (1) and AZA-2 (2) did not modify intracellular pH in a Ca2+-containing or a Ca2+-free medium. AZA-3 (3) increased intracellular pH by 0.16 units in the presence of extracellular Ca2+, an effect that was blocked when a 1 mM solution of Ni2+ was added. In a Ca2+-free medium, the increase in pH induced by AZA-3 (3) was reduced to 0.08 pH units. AZA-4 (4) inhibited the basal pH increase even in the presence of a 1 mM solution of Ni2+. In a Ca2+-free medium, the inhibition caused by AZA-4 (4) was small, but when Ca2+ was added back to the medium, the pH basal increase was again significantly inhibited. The alkalinization was also inhibited when AZA-4 (4) was added simultaneously, 10 min before or 10 min after thapsigargin (Tg), and also when the Ca2+-influx induced by Tg was inhibited by Ni2+. AZA-5 (5), on the other hand, did not modulate the intracellular pH profile in either a Ca2+-containing or a Ca2+-free medium. Finally, we investigated four synthetic analogues (6-9, Fig. 2) whose structures were based on the four originally proposed structures of azaspiracid-1, with an opened E-ring. Compound 6 induced a small cytosolic Ca2+ increase, but did not modify intracellular pH in saline solution. In a Ca2+-free medium, compound 6 blocked the pH fall when Ca2+ was added back to the medium. Compound 7 also did not modify intracellular pH in saline solutions, however it significantly blocked basal pH increases in a Ca2+-free medium. Compound 8 did not alter intracellular pH, however compound 9 induced a small acidification when Ca2+ was present in the extracellular medium. These results point to a structure-activity relationship in AZAs pH effect that affects the modulation and the coupling of intracellular pH and Ca2+.     

A 31P-NMR study on the recovery of intracellular pH in LLC-PK1/Cl4 cells from intracellular alkalinization

The regulation of intracellular pH (pHi) in a renal epithelial cell line, LLC-PK1/Cl4, during re-acidification from an alkaline load was studied by 31P-NMR. Intracellular alkalinization was induced by 10 mM ammonium glucuronate or by preloading with and subsequent removal of 20% CO2; the rate of re-acidification was found to be 0.047 pH units/min and 0.053 pH units/min, respectively. This rate of re-acidification was inhibited by 83% if Cl- was removed from the extracellular medium. A similar inhibition was found in the presence of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (76% inhibition) and 1 mM bumetanide (81% inhibition). No change in recovery was found after removing sodium from the extracellular medium, indicating that LLC-PK1/Cl4 cells recover from an intracellular alkaline load by a Cl-/HCO3- exchanger, which is SITS- and bumetanide-sensitive and has no requirement for sodium. In addition, the steady-state pHi in Cl4 cells was monitored by 31P-NMR. Removal of Cl- from the extracellular medium introduced an increase in pHi by 0.33 pH units, whereas 1 mM SITS and 1 mM bumetanide caused an increase in pHi by 0.14 or 0.13 pH units. In the presence of 1 mM amiloride, an inhibitor of the Na+/H+ exchanger, the steady-state pHi did not change significantly. These results indicate that at pHo 7.4 the steady-state intracellular pH of LLC-PK1/Cl4 cells strongly depends on the activity of the Cl-/HCO3- exchanger. Under the same conditions the activity of the Na+/H+ exchanger seems to be negligible.     

Dexamethasone-induced gene 2 (dig2) is a novel pro-survival stress gene induced rapidly by diverse apoptotic signals

Glucocorticoid hormones induce apoptosis in lymphoid cells. This process requires de novo RNA/protein synthesis. Here we report the identification and cloning of a novel dexamethasone-induced gene designated dig2. Using Affymetrix oligonucleotide microarray analysis of approximately 10,000 genes and expressed sequence tags, we found that the expression of dig2 mRNA is significantly induced not only in the murine T cell lymphoma lines S49.A2 and WEHI7.2 but also in normal mouse thymocytes following dexamethasone treatment. This result was confirmed by Northern blot analysis. The induction of dig2 mRNA by dexamethasone appears to be mediated through the glucocorticoid receptor as it is blocked in the presence of RU486, a glucocorticoid receptor antagonist. Furthermore, we demonstrated that dig2 is a novel stress response gene, as its mRNA is induced in response to a variety of cellular stressors including thapsigargin, tunicamycin, and heat shock. In addition, the levels of dig2 mRNA were up-regulated after treatment with the apoptosis-inducing chemotherapeutic drug etoposide. Though the function of dig2 is unknown, dig2 appears to have a pro-survival function, as overexpression of dig2 reduces the sensitivity of WEHI7.2 cells to dexamethasone-induced apoptosis.     

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.     

Calcium homeostasis during thymocyte apoptosis. I. Increase in cytosolic Ca2+ concentration at the early stage of apoptosis induced by hydrogen peroxide

The concentration of free cytosolic Ca2+ ([Ca2+]i), 45Ca2+ entry and the level of reduced glutathione (GSH) after x-irradiation in a dose of 4.5 Gy or 0.1 mM H2O2-treatment were investigated in isolated rat thymocytes during the period preceding electrophoretically detected DNA intranucleosomal fragmentation. Using fura-2 it was shown that the level of [Ca2+]i in X-irradiated thymocytes was not changed as compared with the control, while the GSH content was increased. The gradual increase in [Ca2+]i along with GSH level falling was detected in the H2O2-treated cells. 45Ca2+ entry in the cells exposed to apoptogenic stimuli was not enhanced. After addition of H2O2 to the cells previously treated with thapsigargin further [Ca2+]i increase in both normal and nominally calcium-free medium was detected. Cyclosporine A inhibited Ca2+-mobilizing effect of H2O2, but did not prevent it completely. The role of intracellular calcium depots in calcium homeostasis disturbance during oxidative stress and apoptosis is discussed.     

Protein kinase C modulation in apoptotic rat thymocytes: an ultrastructural analysis

Numerous events in the cell, such as gene expression, cell growth and metabolism are regulated by signal transduction pathways involving protein kinase C (PKC). Recent data indicate that a PKC-dependent mechanism also underlies the apoptotic death of cells induced by glucocorticoid hormones. In this report we have analysed the changes of PKC during dexamethasone-induced apoptosis in thymocytes by means of immunocytochemical and immunochemical analysis. The data obtained show an increase and intracellular movement of protein kinase C, which is translocated to the nucleus and linked to the nuclear matrix during the apoptotic process.     

Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (Delta(pH)), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Prostaglandin release but not superoxide production by rat Kupffer cells stimulated in vitro depends on Na+/H+ exchange

The release of the prostaglandins E2 and D2, induced by zymosan and phorbol ester in cultured rat Kupffer cells, was found to depend on the extracellular concentration of Na+. Eicosanoid formation following the administration of the Ca2+ ionophore A23187 or of arachidonic acid, however, did not require the presence of sodium ions in the medium. A half-maximal rate of prostaglandin release by zymosan-treated Kupffer cells was obtained between 4 mM and 5 mM Na+; and a Na+ concentration of greater than or equal to 30 mM was required to maximally stimulate prostaglandin E2 and D2 formation in the cultured liver macrophages. In contrast, the superoxide production following the administration of zymosan or of phorbol ester was quite independent of extracellular Na+. The zymosan and phorbol-ester-stimulated release of prostaglandins E2 and D2 was inhibited by amiloride. Artificial intracellular alkalization enhanced the prostanoid production of unstimulated and of zymosan-stimulated cells whereas artificial intracellular acidification inhibited the zymosan-elicited prostaglandin synthesis. In contrast, the superoxide formation was independent of the pH changes. The data presented here suggest that the prostaglandin production elicited by zymosan or phorbol ester in cultured rat Kupffer cells requires an activated Na+/H+ exchange.     

Changes in pH in the cytoplasm of rat thymocytes after treatment with the mitogen concanavalin A and protein kinase C activator phorbol ester

The intracellular pH (pHi) of rat thymocytes has been measured with the fluorescent probe 2', 7'-bis(carboxyethyl)-5,6-carboxyfluorescein, both in the resting cells and under mitogenic stimulation. Concanavalin A (Con A) has been found to increase pHi from 7.16 +/- 0.02 to 7.30 +/- 0.02 during the first minutes after addition; the phorbol ester TPA raised pHi to 7.25 +/- 0.02. The Con A- and TPA-induced rise of pHi is due to activation of Na+/H+ exchange since it was abolished by amiloride, an inhibitor of Na+/H+ antiport, or in a low-Na+ medium. The elevation of intracellular cAMP level, decrease of cellular ATP, or the lowering of the temperature from 37 degrees down to 25 degrees C inhibited the pHi rise induced by Con A or TPA.     

Effect of different zinc sources and levels on inhibition of the apoptosis induced by glucocorticoid of thymocytes in vitro

This experiment was conducted to investigate the effects of zinc sulfate and zinc methionine (Zn-Met) and their levels on apoptosis induced by glucocorticoid of thymocytes and the possible mechanism. Dexamethasone was used to make the apoptosis model of thymocytes; zinc sulfate and zinc methionine were supplemented to the medium at levels of 0,50, 100, 500, and 1000 μM. The activity of cells,Cu,Zn superoxide dismutase (Cu,Zn-SOD), DNA ladder pattern, intracellular calcium concentration, and the percentage of apoptosis nuclei were determined. Both ZnSO₄ and Zn-Met could modulate apoptosis; they inhibited apoptosis and decreased DNA fragmentation. The regulation was concentration dependent. At levels of 50 and 100 μM, the effect of Zn-Met on inhibiting apoptosis was less efficient than that of ZnSO₄ (p<0.05), but the activity of the cells cultured with Zn-Met was higher than those cultured with ZnSO₄; they showed no difference in modulating apoptosis when added at levels of 500 and 1000 μM to the medium (p>0.05). Intracellular calcium concentrations of cells cultured with Zn-Met were higher than those cultured with ZnSO₄ at the same levels. Zinc supplementation decreased the concentration of intracellular calcium significantly (p<0.05) and increased the activity of Cu,Zn-SOD in the extract of the cells (p<0.05). Both zinc sulfate and Zn-Met could modulate apoptosis of thymocytes induced by glucocorticoid; the mechanism might involve the exchange of intracellular calcium, the redox of cells, and the two forms of zinc might go different ways in the regulations.     

Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

In vitro enhancing effects of thymic dendritic cells on apoptotic cell death of thymocytes

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Nitric oxide protects thymocytes from gamma-irradiation-induced apoptosis in correlation with inhibition of p53 upregulation and mitochondrial damage

Apoptosis plays a crucial role in clonal deletion in the thymus, and NO has been shown to prevent apoptosis in some cell types. Therefore, we examined the effect of NO on gamma-irradiation-induced thymocyte apoptosis. Treatment of 5 Gy gamma-irradiated thymocytes with 1 mM SNAP reduced cell death from 78 to 49% after 8 h incubation (spontaneous cell death in medium control cells was 26%). Coincubation with ZVAD blocked both the spontaneous cell death and the cell death induced by SNAP or gamma-irradiation. The gamma-irradiation-induced increase in caspase 3 and 6 activities was inhibited in the presence of SNAP. The increase in cytosolic cytochrome c as well as the decrease in mitochondrial membrane potential after gamma-irradiation was inhibited in the presence of SNAP. SNAP treatment also decreased the p53 upregulation in gamma-irradiated cells. In summary, we found that NO exerts a protective effect on mouse thymocyte apoptosis induced by gamma-irradiation. The mechanism of this protective effect may involve inhibition of p53 upregulation and reduction in mitochondrial damage, with subsequent inhibition of downstream caspase activation.     

On the interaction of NH+4 and Na+ fluxes in the isolated trout gill.

1. Sodium influx was measured in isolated, previously perfused gill arches of rainbow trout, Salmo gairdneri, by measuring incorporation of 22Na into gill tissue following timed exposure to a 1 mM 22NaCl medium. Transport rates approximated those estimated for intact fish and were linear for at least one min. 2. NH4Cl-containing perfusates at pH 7 and 8 stimulated Na+ influx equally, indicating that only ionized ammonia is important in the transport process. A Na+/NH4+ exchange at basal and/or lateral membranes of the transporting cells is suggested. 3. Low-sodium Ringer perfusate augmented Na+ influx; in one group of gills the transport rate was more than double that of NaCl Ringer controls. The increase in transport induced by internal NH4+ was not additive with the low sodium augmentation. A reduction in intracellular (Na+) is postulated as the mechanism operating in both cases. 4. Ouabain had no appreciable effect on Na+ influx, either with or without NH4+ in the perfusate. Diamox partially blocked the augmented Na+ influx induced by NH4+. Amiloride completely inhibited Na+ influx, both with and without NH4+ in the perfusate.