Measurement of phospholipase d activation in vascular smooth muscle cells

Phospholipase D (PLD), which hydrolyzes phospholipids (primarily phos-phatidylcholine) to generate phosphatidic acid, is an essential component in cellular signal transduction (1,2). Phosphatidic acid and its dephosphorylated product 1,2 diacylglycerol, are important intracellular second messengers that play critical roles in various cell types including vascular smooth muscle cells (3,4). The human PLD gene 1 has been cloned and expressed. The expressed mammalian PLD has a molecular weight of approx 120 and has both catalytic and transphosphatidylation activities with phosphatidyl cho-line as substrate (5). PLD activation can be initiated by various agonists that fall into two main categories: (1) agents that signal through tyrosine kinase-dependent pathways, e.g., growth factors, and (2) agents that signal through seven transmembrane receptors, acting through trimeric membrane G proteins, e.g., angiotensin II and endothelin-1 (6-9). The molecular mechanisms by which Ang II receptors couple to PLD have recently been identified. The Gβγ subunits as well as their associated Gα(12) subunits, mediate Ang II-induced PLD activation via Src-dependent mechanisms in vascular smooth muscle cells. Small molecular-weight G protein RhoA is also involved in these signaling cascades (10).