A role for phospholipase D activation in the lipid signalling cascade generated by bradykinin and thrombin in C2C12 myoblasts

Abstract In the present study evidence is provided for a rapid activation of lipid signalling pathways induced by thrombin and bradykinin (BK) in C2C12 myoblasts. Both agonists were able to increase [ 3 H]inositol phosphates (InsP), 1,2-[ 3 H]diacylglycerol (DAG) and [ 3 H]phosphatidic acid (PtdOH) levels. In particular [ 3 H]PtdOH values were rapidly increased and maintained at significantly high values at prolonged times of incubation. BK and thrombin were able to activate phospholipase D (PLD) in vivo as demonstrated by the accumulation of [ 3 H]phosphatidylethanol (PtdEtOH) through the transphoshatidylation reaction catalyzed by the enzyme in the presence of ethanol. The observation that ethanol could significantly reduce [ 3 H]PtdOH formation in myoblasts stimulated with BK and thrombin indicates that stimulation of PLD has a major role. The two agonists appear to stimulate PLD activity through a common molecular mechanism, involving the activation of protein kinase C (PKC). In addition, BK and thrombin appear able to activate DAG kinase at early times of incubation and also this pathway may contribute to determine the increase in [ 3 H]PtdOH levels. This is the first report which describes activation of lipid signalling pathways by BK and thrombin in myoblast cells and it is possible that these early signals may have an important role in mediating the biological effects of the two agonists.