THE REGULATION OF PHOSPHOLIPASE-D ACTIVITY AND ITS ROLE IN SN-1,2-DIRADYLGLYCEROL FORMATION IN BOMBESIN-STIMULATED AND PHORBOL 12-MYRISTATE 13-ACETATE-STIMULATED SWISS 3T3 CELLS

被引:65
作者
COOK, SJ [1 ]
BRISCOE, CP [1 ]
WAKELAM, MJO [1 ]
机构
[1] UNIV GLASGOW,INST BIOCHEM,MOLEC PHARMACOL GRP,GLASGOW G12 8QQ,SCOTLAND
基金
英国惠康基金;
关键词
D O I
10.1042/bj2800431
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) to quiescent Swiss 3T3 cells resulted in a sustained increase in sn-1,2-diradylglycerol (DG) mass and [H-3]DG in [H-3]palmitate-labelled cells where phosphatidylcholine was the major labelled phospholipid. This occurred in the absence of inositol phosphate accumulation. In [H-3]palmitate-labelled cells both bombesin and PMA stimulated the formation of phosphatidylbutanol ([H-3]PtdBut) in the presence of 0.3% (v/v) butan-1-ol. The kinetics of [H-3]PtdBut formation were consistent with phospholipase D (PLD) activation preceding sustained DG formation. The inclusion of butan-1-ol inhibited 70% of PMA-stimulated DG formation but only 30% of the bombesin response. The ability of bombesin and PMA to stimulate the accumulation of [H-3]PtdBut was completely abolished in Swiss 3T3 cells which had been pre-treated with 400 nM-PMA for 48 h to down-regulate protein kinase C activity. PMA-stimulated [H-3]PtdBut formation was inhibited by 90% by the protein kinase C inhibitor Ro-31-8220 (10-mu-M) but bombesin-stimulated PtdBut accumulation was inhibited by at most 50% by the same concentration of inhibitor. Cyclic AMP-elevating agents, i.e. forskolin, dibutyryl cyclic AMP and isobutylmethylxanthine, did not inhibit bombesin stimulation of PLD activity. Bombesin-stimulated PLD activity was inhibited by 50 % by buffering of the extracellular Ca2+ concentration to 150 nM, but combination of this treatment with Ro-31-8220 addition was less than additive. Ionophore A23187 alone was able to stimulate PLD activity, but this response was inhibited 50 % by Ro-31-8220. Thapsigargin was unable to stimulate PLD activity and had no modulatory effect upon bombesin-stimulated PLD activity at any agonist concentration. The results are discussed in terms of the role of PLD in DG generation and the regulation of PLD activity both by bombesin and by PMA.
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页码:431 / 438
页数:8
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