Vascular smooth-muscle cells contain AT1 angiotensin receptors coupled to phospholipase D activation

We previously showed that angiotensin II (Ang II) and angiotensin-(2-8)-peptide [Ang-(2-8)] activate a phosphoinositide-specific phospholipase C (PLC) and cause calcium mobilization in rat aortic vascular smooth-muscle cells (VSMC), while Ang II and Ang-(1-7) produce prostaglandins. To define further the signal-transduction mechanisms activated by angiotensin peptides in smooth-muscle cells, we measured diacylglycerol (DAG) accumulation in response to different angiotensin peptides and its inhibition by subtype-selective receptor antagonists. Both an initial (10 s) and secondary (10 min) phase of DAG production in response to 100 nM Ang II were inhibited by 1 microM losartan (DuP 753), an AT1 antagonist, while 1 microM PD 123177, an AT2 antagonist, was ineffective. In contrast, the heptapeptide Ang-(1-7) did not produce DAG in VSMC. Ang II also caused the hydrolysis of phosphatidylinositol and phosphatidylcholine, the formation of phosphatidic acid and the formation of phosphatidylethanol (PEt) in the presence of ethanol, through activation of a PLD and a PLD-induced transphosphatidylation reaction. A similar concentration of Ang-(2-8) also activated PLD; in contrast, Ang-(1-7) was ineffective. PEt production by 100 nM Ang II was significantly attenuated by the AT1 antagonists losartan, its metabolite EXP 3174 or L-158,809 (all at 1 microM), whereas a similar concentration of the AT2 antagonists CGP 42112A or PD 123177 was ineffective. The production of PEt by Ang II was also partially attenuated by the removal of extracellular calcium and potentiated by increasing calcium concentrations, indicating that PLD activity is partially dependent on extracellular calcium. Thus VSMC PLD is coupled to an AT1 receptor and occurs in response to Ang II or Ang-(2-8), but not Ang-(1-7). Since AT1 receptors in VSMC are also coupled to activation of PLC, both PLC and PLD may be coupled to the same or a different AT1 receptor. Alternatively, PLD may be sequentially activated in response to Ang II activation of PLC and a subsequent increase in calcium concentration.