digitonin and Lymphoma

Respiration characteristics of mitochondria of the parental and giant cells of murine NK/Ly (Nemeth-Kellner lymphoma) were studied. The giant cell-enriched ascites were obtained by serial intraperitoneal injections of vinblastine in tumour-bearing mice. Ascites containing >70% giant cells were used. Their diameter of was over 17 μm (~2800 μm(3)), while the diameter of the parental cells was 12.7 μm (1100 μm(3)). The respiration rate of mitochondria in situ was measured by oxygen consumption in intact and digitonin-permeabilized NK/Ly cells. Endogenous respiration of intact giant NK/Ly cells was three times higher compared to the parental ones, roughly in agreement with the volume change. The giant NK/Ly cells were far more resistant to permeabilization with digitonin than the parental cells, as shown by Trypan Blue and LDH (lactate dehydrogenase) release tests. After digitonin permeabilization, oxygen consumption was reduced to a minimal level (0.06 ng atom O/(s × 106 cells) in both types of cells. Addition of α-ketoglutarate or succinate to the incubation medium increased oxygen consumption in the parental cells by 46 and 164% respectively. In the giant NK/Ly cells, the corresponding increases were 164 and 276%. Addition of ADP to α-ketoglutarate- or succinate-supplemented medium further stimulated oxygen consumption of the permeabilized NK/Ly cells; however, the effect of ADP was more pronounced in the giant cells. In addition, indices of respiratory control were significantly higher in the giant cells. Oligomycin suppressed considerably the respiration of the intact giant cells but had a much weaker effect on parental cells. Thus, giant NK/Ly cells possess much higher respiration rates and show tighter coupling between the respiration and oxidative phosphorylation compared with parental cells.

Topics: Adenosine Diphosphate; Animals; Cell Line, Tumor; Cell Membrane Permeability; Cell Size; Digitonin; Giant Cells; Ketoglutaric Acids; L-Lactate Dehydrogenase; Lymphoma; Mice; Mice, Inbred C57BL; Mitochondria; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Respiration; Vinblastine

L5178Y-R mouse lymphoma (LY-R) cells undergo rapid apoptosis when treated with photodynamic therapy (PDT) sensitized with the silicon phthalocyanine Pc 4. In this study we show that cytochrome c is released into the cytosol within 10 min of an LD99.9 dose of PDT. Cellular respiration is inhibited by 42% at 15 min, and 60% at 30 min after PDT treatment, and caspase 3-like protease activity is elevated by 15 min post-PDT. In digitonin-permeabilized cells addition of cytochrome c to the respiration buffer reverses PDT-induced inhibition of state 3 respiration via Complex I by 40-60%, and via Complex III by 50-90%. In contrast, extramitochondrial cytochrome c does not stimulate respiration in permeabilized control cells, and catalyzes only a low rate of oxygen consumption via electron transfer to cytochrome b5 on the outer mitochondrial membrane. These results demonstrate that PDT-induced inhibition of respiration is primarily due to leakage of cytochrome c into the cytosol rather than to damage to the major enzyme complexes of the electron transport chain. Whether or not inhibition of respiration influences the time course or extent of Pc 4-PDT-induced apoptosis in LY-R cells is not clear at the present time.

Topics: Animals; Apoptosis; Caspases; Cell Membrane Permeability; Cell Respiration; Cytochrome c Group; Digitonin; Electron Transport; Electron Transport Complex III; Enzyme Activation; Indoles; Lymphoma; Mice; NAD(P)H Dehydrogenase (Quinone); Organosilicon Compounds; Oxygen Consumption; Photochemotherapy; Radiation-Sensitizing Agents; Silanes; Tumor Cells, Cultured

L1210 lymphoma cells were permeabilized with digitonin, and the ability of Ins(2,4,5)P3 and Ins(1,3,4,5)P4 to mobilize intracellular Ca2+ was studied. At high doses of Ins(2,4,5)P3 Ca2+ was rapidly released from intracellular stores, and prior or subsequent addition of Ins(1,3,4,5)P4 had no discernible effect. However, the Ca2(+)-mobilizing action of low (threshold or just above) concentrations of Ins(2,4,5)P3 was markedly enhanced by Ins(1,3,4,5)P4, which alone caused no mobilization of Ca2+; this phenomenon was shown not to be due to protection of Ins(2,4,5)P3 by the Ins(1,3,4,5)P4 against hydrolysis. The ability of the pre-addition of Ins(1,3,4,5)P4 to enhance subsequent Ins(2,4,5)P3-induced Ca2+ mobilization was always seen whether or not the free Ca2+ concentration was low (pCa = 7) or high (pCa = 6). However, at low Ca2+, Ins(1,3,4,5)P4 could cause a further mobilization if added after the Ins(2,4,5)P3, whereas at higher Ca2+ values Ins(1,3,4,5)P4 was only able to affect Ca2+ if added before Ins(2,4,5)P3. These effects of Ins(1,3,4,5)P4 were not, at the same concentration, mimicked by a random mixture of InsP4 isomers obtained by partial acid hydrolysis of phytic acid, by Ins(1,3,4)P3 or by Ins(1,3,4,5,6)P5, and they were shown not to be due to enzymic generation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 by (a) the absence of any detectable production of Ins(1,4,5)P3 if radiolabelled Ins(1,3,4,5)P4 was used, or (b) the observation that Ins(1,3,4,5,6)P5 could mimic Ins(1,3,4,5)P4 provided that higher doses were used; this inositol phosphate, when added radiolabelled, yielded only trace quantities of D/L-Ins(1,4,5,6)P4, which itself does not mobilize Ca2+. We interpret these results overall to mean that in these cells there is a small proportion of the Ins(2,4,5)P3-mobilizable Ca2+ pools which can only be mobilized in the presence of Ins(1,3,4,5)P4 [or at the least, Ins(1,3,4,5)P4 can help Ins(2,4,5)P3 to gain access to them]. The significance of this conclusion is discussed in the light of current concepts of the second messenger function of Ins(1,3,4,5)P4.

Topics: Animals; Calcium; Cell Membrane Permeability; Chromatography, High Pressure Liquid; Digitonin; Drug Synergism; Inositol Phosphates; Lymphoma; Mice; Tumor Cells, Cultured

S49 Mouse lymphoma wild-type cells were grown in spinner cultures of 40 liters to a density of approximately 3 million cells/ml. Growth of cells to high density (2-3 million cells/ml) required that the cell suspensions be bubbled with oxygen. Cells from 40 liter cultures were collected by centrifugation and disrupted by nitrogen cavitation. Highly purified membranes (0.35 g membrane protein) that were rich in beta-adrenergic receptor (0.4-0.7 pmol receptor/mg membrane protein) were prepared by differential centrifugation and then solubilized with the plant glycoside, digitonin (1.5% digitonin at 3 mg of membrane protein/ml). Beta-adrenergic receptors were isolated and purified by sequential affinity chromatography, ion-exchange chromatography, and steric exclusion high-pressure liquid chromatography. The extract was subjected to affinity chromatography on a derivatized Sepharose-4B CL column to which the high-affinity, beta-adrenergic antagonist (-)alprenolol had been immobilized. Following extensive washing, the receptor bound to this matrix was eluted using a 0-100 micromolar linear gradient of (-)alprenolol. The receptor eluted as a sharp peak at 30 micromolar ligand and displayed a specific activity of 280 pmol receptor/mg of protein. Ion-exchange chromatography on DEAE-Sephacel increased the specific activity to 950 pmol/mg of protein. The final step in the purification, steric-exclusion high-pressure liquid chromatography on two TSK-3000 and one TSK-2000 columns, tandem linked, resulted in a beta-adrenergic receptor preparation with a specific activity of 6700 pmol/mg of protein (15,900-fold purification). Autoradiography of the radioiodinated pure receptor, the receptor photolabeled with [125I]iodoazidobenzylpindolol or silver-staining of chemical amounts of protein revealed that the Mr of the pure receptor is 66,000 upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate under reducing conditions. The receptor is a beta2-subtype adrenergic receptor.

Topics: Animals; Autoradiography; Cell Line; Cell Membrane; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Digitonin; Electrophoresis, Polyacrylamide Gel; Iodocyanopindolol; Lymphoma; Mice; Mice, Inbred BALB C; Molecular Weight; Pindolol; Receptors, Adrenergic, beta; Solubility

28Mg2+ influx studies in S49 murine lymphoma cells indicate that only 2-3% of total cell Mg2+ content can be exchanged at isotopic equilibrium, implying compartmentation of the newly transported Mg2+. The nature of this compartmentation was examined using selective permeabilization of the plasma membrane with the detergent, digitonin. Control experiments demonstrated that the digitonin permeabilization procedure did not release mitochondrial and lysosomal components, alter mitochondrial respiration, or significantly change cell morphology. Thus, under appropriate conditions, the digitonin permeabilization technique allows determination of the amount of a particular cell constituent within the solute space of the cytoplasm. In nonproliferating cells at an extracellular Mg2+ concentration of 0.1 mM, newly transported Mg2+ equilibrates within 2 h with a small cytoplasmic Mg2+ pool comprising about 3% of the total cytoplasmic Mg2+ (about 2% of total cell Mg2+). The pool of Mg2+ does not equilibrate with bulk cytoplasmic or cellular Mg2+ for at least 16 h. The Mg2+ pool size is dependent on extra-cellular Mg2+ concentration, is saturable with increasing extracellular Mg2+, and reaches a maximal size of 6-7% of total cell Mg2+ at 2 mM extracellular Mg2+. Unlike Mg2+, newly transported Ca2+ is quickly sequestered in noncytoplasmic compartments. In proliferating cells, however, newly transported Mg2+ exchanges extensively with cytoplasmic Mg2+ over the course of 4 h, suggesting that compartmentation of Mg2+ may be dependent on proliferative status.

Topics: Animals; Autoradiography; Biological Transport; Calcium; Cell Compartmentation; Cell Line; Digitonin; L-Lactate Dehydrogenase; Lymphoma; Magnesium; Mice; Microscopy, Electron; Oxygen Consumption; Rubidium; Time Factors