Cook S J, Briscoe C P, Wakelam M J
Molecular Pharmacology Group, University of Glasgow, Scotland, U.K.
Biochem J. 1991 Dec 1;280 ( Pt 2)(Pt 2):431-8. doi: 10.1042/bj2800431.
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 [3H]DG in [3H]palmitate-labelled cells where phosphatidylcholine was the major labelled phospholipid. This occurred in the absence of inositol phosphate accumulation. In [3H]palmitate-labelled cells both bombesin and PMA stimulated the formation of phosphatidylbutanol ([3H]PtdBut) in the presence of 0.3% (v/v) butan-1-ol. The kinetics of [3H]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 [3H]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 [3H]PtdBut formation was inhibited by 90% by the protein kinase C inhibitor Ro-31-8220 (10 microM), 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.
将佛波酯佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）添加到静止的瑞士3T3细胞中，导致在以磷脂酰胆碱为主要标记磷脂的[³H]棕榈酸酯标记细胞中，sn - 1,2 - 二酰甘油（DG）质量和[³H]DG持续增加。这一过程在肌醇磷酸积累缺失的情况下发生。在[³H]棕榈酸酯标记细胞中，铃蟾肽和PMA在存在0.3%（v/v）丁醇 - 1的情况下均刺激了磷脂酰丁醇（[³H]PtdBut）的形成。[³H]PtdBut形成的动力学与在持续DG形成之前磷脂酶D（PLD）的激活一致。加入丁醇 - 1抑制了70%的PMA刺激的DG形成，但仅抑制了铃蟾肽反应的30%。在已用400 nM - PMA预处理48小时以下调蛋白激酶C活性的瑞士3T3细胞中，铃蟾肽和PMA刺激[³H]PtdBut积累的能力完全被消除。PMA刺激的[³H]PtdBut形成被蛋白激酶C抑制剂Ro - 31 - 8220（10 μM）抑制了90%，但相同浓度的抑制剂对铃蟾肽刺激的PtdBut积累最多抑制50%。环磷酸腺苷升高剂，即福斯可林、二丁酰环磷酸腺苷和异丁基甲基黄嘌呤，并未抑制铃蟾肽对PLD活性的刺激。将细胞外Ca²⁺浓度缓冲至150 nM可使铃蟾肽刺激的PLD活性抑制50%，但将此处理与添加Ro - 31 - 8220相结合的抑制作用小于两者相加的效果。单独的离子载体A23187能够刺激PLD活性，但这种反应被Ro - 31 - 8220抑制了50%。毒胡萝卜素无法刺激PLD活性，并且在任何激动剂浓度下对铃蟾肽刺激的PLD活性均无调节作用。本文根据PLD在DG生成中的作用以及铃蟾肽和PMA对PLD活性进行调节的相关内容对结果进行了讨论。
