Myung Sun Choi

Elevation of oleate-activated phospholipase D activity during thymic atrophy

Various phospholipases are thought to be associated with the in vitro apoptosis of thymocytes. In the present study, the in vivo phospholipase D (PLD) activity of rat thymus was studied after whole‐body X‐irradiation or injection of dexamethasone (DEX). Using exogenous [14C]dipalmitoyl phosphatidylcholine (PC) as the substrate, an elevation of oleate‐activated PLD activity was observed during thymic atrophy. The activity increases were sevenfold at 48 hr after 5‐Gy irradiation and fourfold at 72 hr after injection of 5 mg/kg DEX. The elevation of PLD activity appeared to parallel extensive thymus shrinkage. An increased level of thymic phosphatidic acid (PA), the presumed physiological product of PLD action on PC, was also detected. By comparing the acyl chains of PA with those of other phospholipids, PA appeared to originate from PC. To assess the role of PLD during thymic atrophy, thymocytes and stromal cells were isolated. Although thymocytes themselves exhibited significant PLD activation, the major elevation in PLD activity (greater than fourfold) was found in isolated stromal cells. PLD was also activated during in vitro phagocytosis of apoptotic thymocytes by the macrophage‐like cell line P388D1. This in vitro phagocytosis was significantly inhibited by PLD action blockers, such as 2,3‐diphosphoglycerate and 1‐butanol. These observations strongly suggest that the alteration of oleate‐activated PLD activity is part of an in vivo event in the progression of thymic atrophy, including phagocytic clearance of apoptotic thymocytes.

Alteration of lipid composition of rat thymus during thymic atrophy by whole-body X-irradiation

Purpose:Thymic atrophy induced by irradiation is well known, but in vivo lipid metabolism during the atrophy has not been studied in detail. We determined the lipid composition of rat thymus during the progress of thymic atrophy induced by whole-body X-irradiation. Materials and methods:The lipid analysis of total lipid of rat thymus after 5 Gy whole-body X-irradiation was performed by high performance liquid chromatography and gas chromatography equipped with mass spectrometry. Results:Major changes observed were a 16.2-fold elevation of cholesterol ester (CE) during a 48-h post-irradiation period and a 6.1-fold increase of alkyldiacylglycerol (ADG) at 24 h. Other significant changes detected were an increase in lysophosphatidylcholine and a transient increase in ceramide and phosphatidic acid. Acyl chain analysis revealed a substantial elevation of arachidonate composition of CE and an unusually high content of polyunsaturated fatty acids (71.5%, mol/mol) in ADG. Conclusion:Lipid analysis shows that the thymic atrophy by X-irradiation was accompanied by a significant change in thymic lipids. This in vivo result opens up new vistas of the role of lipids in apoptosis and phagocytosis during thymic atrophy.

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.