| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1991: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Phosphatidic acid(PA)is known to be increased in response of certain mammalian cells to stimuli. In this study, to investigate the possible role of this phospholipid in stimulus-response coupling, the effect of exogenously added didecanoyl PA(C_<10>PA), 1-stearoyl 2-arachidonoyl PA(SAPA), or phospholipase(PL)D from Streptomyces chromofuscus which generates PA endogenously from membrane phospholipids by its hydrolytic action, on the cell activation, mainly on PLA_2 and PLC activation, was studied using rabbit platelets and rat peritoneal mast cells.
1. Addition of C_<10>PA(-15muM)to the platelets in the presence of Ca^<2+> induced dose-dependently the cell aggregation, arachidonic acid(AA)release, generation of inositol 1, 4, 5-trisphosphate(IP_3)and of diacylglycerol(DG), and increase in the cytosolic Ca^<2+> concentration. PLD(-4U/ml)and SAPA(-60muM)also induced the similar effects. However, in low concentrations(-20muM)of SAPA, the induced aggregation and DG generation were inhibited
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by an inhibitor of AA metabolism, whereas the induced AA release by a PLA_2 inhibitor.
2. In saponin-permeabilized platelets or the membrane fraction from intact platelets, too, these exogenous and endogenous treatments to increase PA caused AA release and generation of IP_3 and DG. Under these conditions, pretreatment with GTPgammaS did not influence the effect. The SAPA-induced AA release and DG generation were not affected by pretreatment of intact platelets with NaF+AlCl_3.
3. In rat peritoneal mast cells, C_<10>PA(- 60muM)caused dose-dependent histamine release, AA liberation and DG generation in the presence of Ca^<2+>. Pertussis toxin did not exert any effect on these responses. In saponin-permeabilized mast cells, C_<10>PA also induced AA liberation and DG generation both of which were not affected by pretreatment of the cells with GDPbetaS, while the AA release was inhibited by a PLA_2 inhibitor.
These results indicate that increased PA content in plasma membranes, caused by incorporation of exogenously-added PA or by hydrolysis of the membrane phospholipids by an added bacterial PLD, may result in activation of phospholipases(A_2, C)in the signal transduction system of the platelets and mast cells. Thus, they suggest that physiologically generated PA in the stimulated cells acts as a positive-feed back regulator to enhance activity of the phospholipases and hence contributes to amplification of the cellular responses. Furthermore, it is suggested that the action of PA is not a receptor-mediated process. Less
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