Involvement of protein kinase C pathway in UVC-stimulated phospholipase D2 activity in Vero 76 cells

Phospholipase D (PLD) activity is known to be related to oxidant-induced cellular signaling and membrane disturbance. Previously, an induction of PLD activity in various cell lines by X-ray irradiation was observed. In this study, we examined the effect of UVC radiation on the PLD activity in Vero 76 cells. At a dose of 10 kJ/m2 of UVC irradiation, the PLD activity was stimulated approximately 10-fold over the basal activity. This UVC-induced PLD activity was found to be dependent on the presence of extracellular calcium and was inhibited by catalase as well as amifostine-an intracellular thiol antioxidant. Pretreatments with Ro32-0432-a selective inhibitor of protein kinase C (PKC)-and downregulation of PKC by preincubation of phorbol 12-myristate 13-acetate significantly inhibited the UVC-induced PLD activity. UVC-stimulated PLD activity was observed only in murine PLD2 (mPLD2)-transfected Vero 76 cells and not in human PLD1 (hPLD1)-transfected cells. Transient incorporation of PKC with mPLD2 and the phosphorylation of mPLD2 by a and b forms of PKC by UVC irradiation were observed. These results suggest that the UVC-stimulated PLD activity in Vero 76 cells is mediated through transient phosphorylation of PLD2 by the translocation of PKC to PLD2.

Alteration of Oleate-Phospholipase D Activities in Some Cell Lines after Irradiation / 대한암학회지

PURPOSE: Phospholipase D (PLD) catalyzes the hydrolytic cleavage of terminal phosphate diester bond of glycerophopholipids to produce phosphatidic acid (PA). PLD plays an important role in signal transduction and is known to be involved closely in cancer promotion, inflammation, and other cell responses. In order to evaluate radiation effect in tumor cells, various cells were screened for PLD activities and examined their radiation effects on PLD following gamma- ray irradiation. MATERIALS AND METHODS: PLD activities in 19 species of cell were measured by radioactive isotope method with 1,2 - di [1-14C] phosphatidylcholine in the presence of oleate. Among the cell lines examined, VERO 76, L 1210 and P 388 were selected and examined for their effects of metal ions and agonists on PLD activities before and after irradiation by Co-60 teletheraphy unit. RESULTS: The activities of oleate-PLD were observed in 11 species among 19 cell lines examined. VERO 76 and L 1210 cells showed that the PLD activity increased immediately after irradiation and reached to 150~200% of the control levels. The activation of PLD in response to gamma-ray was maximum at 20 Gy. In irradiated VERO 76, the stimulatory effect of Mg2+ was reduced and the activation of PLD by agonists in irradiated cells vary from those of the control cells. CONCLUSION: The activation effect of irradiation on PLD activity observed strongly implies that the PLD activity is closely related to the phenomena of cell necrosis. Therefore the cell lines examined here could provide a good source for the study of radiobiology that cover from cell death to cancer promotion.

Alteration of Phospholipase D Activity in the Rat Tissues by Irradiation / 대한치료방사선과학회지

PURPOSE: Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidyl choline to phosphatidic acid (PA) and choline. Recently, PLD has been drawing much attentions and considered to be associated with cancer process since it is involved in cellular signal transduction. In this experiment, oleate-PLD activities were measured in various tissues of the living rats after whole body irradiation. MATERIAL AND METHODS: The reaction mixture for the PLD assay contained 0.1microCi 1,2-di[1-14C]palmitoyl phosphatidylcholine, 0.5mM phosphatidylcholine, 5mM sodium oleate, 0.2% taurodeoxycholate, 50mM HEPES buffer (pH 6.5), 10mM CaCl2, and 25mM KF. phosphatidic acid, the reaction product, was separated by TLC and its radioactivity was measured with a scintillation counter. The whole body irradiation was given to the female Wistar rats via Cobalt 60 Teletherapy with field size of 10cm x 10cm and an exposure of 2.7Gy per minute to the total doses of 10Gy and 25Gy. RESULTS: Among the tissues examined, PLD activity in lung was the highest one and was followed by kidney, skeletal muscle, brain, spleen, bone marrow, thymus, and liver. Upon irradiation, alteration of PLD activity was observed in thymus, spleen, lung, and bone marrow. Especially PLD activities of the spleen and thymus revealed the highest sensitivity toward gamma-ray with more than two times amplification in their activities. In contrast, the PLD activity of bone marrow appears to be reduced to nearly 30%. Irradiation effect was hardly detected in liver which showed the lowest PLD activity. CONCLUSION: The PLD activities affected most sensitively by the whole-body irradiation seem to be associated with organs involved in immunity and hematopoiesis. This observation strongly indicates that the PLD is closely related to the physiological function of these organs. Furthermore, radiation stress could offer an important means to explore the phenomena covering from cell proliferation to cell death on these organs.

Effect of g-ray Irradiation On the Activities of Monoamine Oxidase in Rat Brain and Liver / 대한치료방사선과학회지

In order to evalute the effects of radiation on mammalian neuronal system, we have examined the effect of gamma-ray radiation on the monoamine oxidase(MAO) activity in monoaminergic neurons. Following the whole body irradiation, MAO activity in the rat brain was measured as well as in the liver for the comparative studies between the neuronal and nonneuronal system. The effects of some radiation protectors and sensitizers were also examined in addition to the O2 effect. The results can be summarized as follows. 1) The MAO activity of rat brain was minimally affected by the radiation dose up to 1,700 cGy. Radiation dose above 2,500 cGy inhibited the brain MAO activity by no less than 10%. MAO-A form was found to be particularly sensitive to radiation. The liver MAO was somewhat inhibited(by about 5%) but hard1y dependent on the dose of radiation. 2) The inhibitory effect on the brain was initiated immediately by the radiation dose of 2,500 cGy. On the contrary, for the liver, the inhibitory effect became apparent only 2 days after irradiation. 3) Two days after a dose of 2,500 cGy, Vmax and Km of the brain mitochondrial MAO decreased. for liver, Vmax decreased while Km increased, which indicates the kinetic patterns for the neuronal and nonneruronal systems are not affected similarly by radiation. 4) The effect of several known radiation protectors and sensitizers on MAO activity was tested but no definite results were obtained. The level of -SH group increased in some degree upon radiation but not by the compounds. 5) MAO activity was not affected by O2 concentration, while an elevated level of lipid peroxidase was found udder the same condition. The results described here indicate that characteristics of MAO, one of the most important central nervous system enzymes, are liable to radiation, which is partially differentiated from the liver MAO. Also indicated are that the -SH groups are hardly related to the effect of radiation but the production of the lipid peroxide seems to be somewhat correlated to the effect of radiation.