[Calcium homeostasis in the thymocyte apoptosis II. Accumulation of calcium in mitochondria and endoplasmic reticulum].

The activity of energy-dependent Ca2+-accumulation systems in rat thymocytes mitochondria and endoplasmic reticulum (ER) in control and at the early stage of X-irradiation or H2O2-induced apoptosis were determined in experiments using the model of digitonin-permeabilized cells with addition of thapsigargin and ruthenium red. The mitochondrial Ca2+-transporting system proved to be more sensitive to both apoptotic stimuli. The stationary level of Ca2+, accumulated in mitochondria and initial rate of Ca2+ accumulation in ER were reduced 15 min after H2O2 treatment. The parameters of mitochondrial Ca2+-accumulation system in irradiated cells were decreased 30 min after irradiation. Cyclosporin A almost completely inhibited DNA fragmentation in irradiated and partly--in peroxide-treated cells. The mitochondrial calcium homeostasis imbalance is suggested to be an early event in thymocytes apoptosis initiation.

[Calcium homeostasis during thymocyte apoptosis. I. Increase in cytosolic Ca2+ concentration at the early stage of apoptosis induced by hydrogen peroxide]. / Kal'tsievyi gomeostaz v timotsitakh pri apoptoze. I. Povyshenie kontsentratsii tsitozol'nogo Ca2+ na rannei stadii apoptoza, indutsirovannogo peroksidom vodoroda.

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.

[Inhibition of lipoxygenase activity reduces radiation-induced DNA fragmentation in lymphocytes]. / Ingibirovanie lipoksigenaznoi aktivnosti snizhaet indutsirovannuiu radiatsiei fragmentatsiiu DNK limfotsitov.

Lypoxygenase activity of lymphocytes towards exogenous substrate linolic acid and the level of endogenous arachidonate metabolite 15-HETE are increased within 6 h and decreased below the control level at 24 h after 1 Gy X-ray irradiation of Wistar rats. DNA fragmentation is stimulated both in vivo within 12 h and during in vitro incubation of lymphocytes, obtained 3 h following irradiation. DNA analysis by agarose gel shows that DNA fragments are multiples of the 200 base pair unit. When 20 microM nordihydroguayaretic acid, a selective inhibitor of arachidonate lypooxidation, was added to the incubation medium, DNA fragmentation was observed to be significantly less, especially at the early steps of incubation.

[Role of inositol triphosphate in the formation of the calcium signal in T-lymphocytes]. / Rol' inozitoltrifosfata v formirovanii kal'tsievogo signala v T-limfotsitakh.

The mechanisms of TCR/CD3 complex and phospholipase C interaction, inositol-1,4,5-triphosphate role in both initial transient and sustained elevation of intracellular Ca2+ and in calcium oscillations driving following T cells stimulation are discussed. Data on IP3-receptors structure and localisation in lymphocytes are summarized.

[Lipoxygenase activity and 15-hete content of rat spleen lymphocytes after exposure to ionizing radiation]. / Lipoksigenaznaia aktivnost' i soderzhanie 15-hete v limfotsitakh selezenki krys pri deistvii ioniziruiushchei radiatsii.

Pretreatment of rat spleen lymphocytes with 0.5 mM A23187 had no influence on the prostaglandins E2 and F2 alpha content, but was followed by the increasing of both 15-HETE and leukotriene B4 levels. Lypoxygenase activity of lymphocytes towards exogenous substrate linoleic acid was increased within 3-12 h after 1 Gy irradiation and decreased below the control level at 24 h. The changes of lypoxygenase activity correlate with that of 15-HETE content. Additional incubation of the cells, obtained at 3 h after irradiation, was followed by the intensification of the chromatin internucleosomal fragmentation and low-molecular DNA fragments accumulation. When 20 mM nordihydroguaiaretic acid, a selective inhibitor of arachidonate lipooxidation, was added to the incubation medium, DNA fragmentation was observed to be significantly less, especially at the early steps of incubation. These results suggest, that metabolites like H(P)ETE are early endogenous mediators of radiation-induced spleen lymphocytes apoptosis.

[Biochemical aspects of radiation-induced apoptosis]. / Biokhimicheskie aspekty vyzvannogo radiatsiei apoptoza.

Radiation-induced activation of the nuclear and cytosolic regulatory pathways, involvement of transcriptional factors and calcium in apoptotic signal transducing, DNA fragmentation at the different stages of apoptosis are discussed. The data on bcl-2 action in preventing calcium signal and apoptosis in lymphoid cells are presented.

[Release of arachidonate from rat spleen lymphocytes after irradiation]. / Vysvobozhdenie arakhidonata iz limfotsitov selezenki krys posle oblucheniia.

After spleen lymphocytes incubation with 14C-arachidonic acid 14C label was found associated predominantly with phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Addition of 0.5 mkM a 23187 to the incubation media with 2 mM CaCl2 caused a 4-fold increase in release of labeled free arachidonate in lymphocytes of the control animals. A dose-dependent increase in release of free arachidonate and membrane Ca(2+)-dependent phospholipase activity towards exogenous 2-14C-arachidonoyl-PE and -PC take place in a period 6-12 h after animal's irradiation in a doses 0.5 and 1.0 Gy. The maximum arachidonate release in 6h following 1 Gy irradiation correlates with activation of more active phospholipase A2, hydrolyzing PE. Free arachidonate is supposed to be mobilized from different phospholipid pools, depending on the content of Ca2+ in the cells after irradiation.

[Phospholipase A2 in the biochemical mechanisms of spleen lymphocyte reaction to x-ray irradiation of animals]. / Fosfolipaza A2 v biokhimicheskikh mekhanizmakh reaktsii limfotsitov selezenki na rentgenovskoe obluchenie zhivotnykh.

Preincubation of rat splenic lymphocytes with nonspecific mitogens and calcium ionophore A23187 led to the activity of phospholipase A2 which hydrolyzed arachidonoyl phospholipids and to increased cellular levels of free arachidonic acid. The effect of some substances was decreased as follows: A23187 > > lipopolysaccharide > phytohemagglutinin > concanavalin A. Dose-dependent activation of phospholipid hydrolysis and arachidonic acid release were observed within 6-12 hrs after X-ray irradiation of animals in doses of 0.5 and 1.0 Gy. The involvement of phospholipase A2 in deterioration of receptor interactions occurring during transmission of an activation signal in splenic lymphocytes in the early postirradiation period is discussed.

[The content of cyclic nucleotides, free cytoplasmic Ca2+ and malondialdehyde in the splenic lymphocytes and thymocytes of rats under the action of moderate doses of radiation]. / Soderzhanie tsiklicheskikh nukleotidov, svobodnogo tsitoplazmaticheskogo Ca2+ i malonovogo dial'degida v limfotsitakh selezenki i timotsitakh krys pri deistvii nevysokikh doz radiatsii.

It has been shown that the malonic dialdehyde (MDA) content in spleen lymphocytes of rats increases after whole-body X irradiation with a dose of 0.5 Gy to reach the maximum level in 24 h. Simultaneously, the concentration of cGMP and free cytosol Ca2+ increases. With a dose of 1 Gy MDA content of cells increases 6 h following irradiation. The maximum drop of the release of viable lymphocytes from the spleen and thymus, observed 24 h and 3 days after irradiation respectively, coincides with the appearance of the second peak of the MDA content. The level of cGMP remains decreased throughout the period of about 6 days. The onset of lymphocyte repopulation in the spleen on day 6 coincides with the decrease in the MDA level and increase in the cytosol Ca2+ concentration.

[Increase in free Ca2+ level determined by quin-2 in spleen lymphocytes from irradiated rats]. / Uvelichenie kontsentratsii svobodnogo Ca2+, opredelennoi s pomoshch'iu kvina-2 v limfotsitakh selezenki obluchennykh krys.

The output of lymphocytes, obtained on a ficoll-verografin gradient and intracellular free Ca2+ concentration were studied in the rat spleen 1, 3 and 6 days after X-ray irradiation in a dose of 0.5 Gy. The amount of lymphocytes decreased by 30% a day after irradiation and turned to the control level 3 days after exposure of animals. Free Ca2+ concentration remains abnormally high during all the examined period, increasing to 411 nM a day after irradiation and approaching to the control value 6 days after irradiation.

[ATP-dependent Ca2+-transporting system in the rat brain during an early period of acute radiation injury]. / ATF-zavisimaia Ca2+transportiruiushchaia sistema mozga krys na rannem étape ostrogo luchevogo vozdeistviia.

The rate of Mg2+, Ca2+-ATPase reaction and ATP-dependent Ca2+ accumulation in a preparation of plasma membranes from brain synaptosomes increases 60 min following whole-body X-irradiation of rats with a dose of 0.21 C/kg, a calcium sensitivity of both processes being increased. A unidirectional change in their kinetics indicates the early radiosensitivity of Ca2+ transfer systems in the brain synaptosome membranes. There is an increase in the availability of SH-groups of membrane preparation proteins for SH-reagents and in the sensitivity of Mg2+, Ca2+-ATPase reaction and ATP-dependent Ca2+ accumulation to trifluoperazine, a calmodulin inhibitor. Both processes lose their ability to be activated by exogenous calmodulin. It is suggested that at an early stage of radiation affection, a change occurs in the molecular organization of the ATPase-calmodulin membrane complex in plasma membranes of rat brain synaptosomes.

[Activity of Mg2+, Ca2+-ATPase and lipid concentration in the plasma membranes rat brain synaptosomes in the early stages of acute radiation injury]. / Aktivnost' Mg2+, Ca2+-ATP-azy i soderzhanie lipidov v plazmaticheskikh membranakh sinaptosom mozga krys na rannikh étapakh ostrogo luchevogo porazheniia.

It is established that an hour after total X-ray irradiation of animals in a dose of 0.21 C/kg the Ca2+ content increases in a homogenate and subcellular fractions of the nervous tissue. The activity of Mg2+, Ca2+-ATP-ase in synaptosome plasma membranes becomes 1.6 times as high as under normal conditions. Changes are also observed in the lipid composition of synaptosome plasma membranes: an increase of the phospholipids and cholesterol contents in the composition of total lipids. The cholesterol phospholipids ratio remains about 1.0. The activation energy of ATP hydrolysis by membrane preparations isolated from the brain tissue of irradiated animals increases as compared to the normal level. It is supposed that variations in characteristics of the lipid bilayer in synaptosome plasma membranes are one of the reasons of changes in the activity of Mg2+, Ca2+-ATP-ase caused by irradiation.

[Nature of the melipramine inhibition of the K+-n-nitrophenyl phosphatase activity of a synaptosomal fraction]. / Kharakter ingibirovaniia melipraminom K+-n-nitrofenilfosfataznoi aktivnosti sinaptosomnoi fraktsii.

The influence of antidepressant melipramine (imipramine) on the K+-paranitrophenyl-phosphatase (K+-n-NFP-ase) activity of the synaptosomal fraction in the bull's brain cortex, treated with sodium iodide, was studied in in vitro experiments. Melipramine (5.10(-5)--10(-3)M) is shown to inhibit the K+-n-NFP-ase activity, competing with potassium ions. With pH changing from acid and neutral to alkaline values the inhibitory action of melipramine gains in intensity, whereas the protective effect of potassium ions declines. It is suggested that one of the mechanisms responsible for the inhibition of the K+-p-NFP-ase activity by melipramine is interaction of the inhibitor in the anionic sections of the enzyme complex.