Endothelin-induced biphasic formation of 1,2-diacylglycerol in cultured rabbit vascular smooth muscle cells--mass analysis with a radioenzymatic assay

Mass analysis of 1,2-diacylglycerol (DG) with a radioenzymatic assay revealed that endothelin induced a biphasic formation of DG with an early transient phase peaking at 30 sec and a late sustained phase peaking at 5 min in cultured rabbit vascular smooth muscle cells (VSMCs). The amounts of DG after the 30-sec and 5-min incubation with endothelin were 0.74 +/- 0.08 (mean +/- SE) nmol and 0.87 +/- 0.10 nmol/100 nmol of lipid phosphorus, representing 2.6- and 3.1-fold increases of the resting level, respectively. The EC50 values of endothelin for the early and late phases of DG formation were about 1 nM and 40 nM, respectively. In the 3H] inositol-labeled VSMCs, endothelin induced a rapid transient formation of inositol tris- and bisphosphates which peaked at 30 sec and a sustained formation of inositol monophosphate which peaked at 5 min. The EC50 values for the formation of these inositol phosphates were the same and about 1 nM. These results suggest that the early transient phase of DG is derived from the hydrolysis of polyphosphoinositides, while a large part of the late sustained phase of DG is from the reaction(s) other than the hydrolysis of phosphoinositides but its sources remain to be clarified.