Potentiated bradykinin-induced increase of 1,2-diacylglycerol generation and phospholipase D activity in human senescent fibroblasts.

1. The comparative study of the effect of bradykinin (BK) in young and old IMR-90 human fibroblasts shows that old cells are characterized by a reduced increase in 1,2-diacylglycerol (1,2-DAG) generation upon stimulation after short-term treatment and a significant higher increase after long-term agonist treatment. BK-induced activation of phospholipase D (PLD), the major enzyme involved in sustained 1,2-DAG generation, was 2.5-fold higher in old cells, strongly suggesting that it is involved in the potentiated increase of 1,2-DAG formation. The increased activation of PLD by BK in old cells was specific, since in parallel experiments the effect of thrombin was not significantly different in young and old cells. PLD activity in old cells was only reduced by down-regulation of protein kinase C (PKC) activity, in contrast to what was observed in young cells where it was completely abolished. This indicates that the enzyme activity in old cells was partially PKC-independent. BK was also able to increase the release of [14C]ethanolamine, a water-soluble product of hydrolysis of phosphatidylethanolamine (PtdEtn), through PLD activation in young and old cells. The BK effect was significantly higher in old cells and, very likely, PKC-independent, since phorbol 12-myristate 13-acetate failed to induce PtdEtn hydrolysis. 2. The present results indicate that the PLD/1,2-DAG pathway is specifically potentiated by BK in old fibroblasts, demonstrating that the formation of positive effectors of PKC activation is not necessarily decreased in cellular senescence. It remains to be established whether the increased generation of DAG upon BK stimulation plays any role in the altered PKC signalling pathway which characterizes old fibroblasts.