Pivotal role of tyrosine phosphatase SHP-1 in AT2 receptor-mediated apoptosis in rat fetal vascular smooth muscle cell

Objective: To examine the possible crosstalk and the roles of angiotensin ( Ang) II type 1 (AT1) and type 2 (AT2) receptors in the control of apoptosis in fetal vascular smooth muscle cells (VSMCs). Methods: Fetal VSMCs were p repared from rat fetal aorta at embryonic day 20. Expression of Ang II rece ptors was measured by a radioligand binding assay. Apoptotic changes were a ssessed by caspase 3 activity and chromatin dye staining. Regulation of ext racellular signal-regulated kinase (ERK) activity via Ang II receptors was analysed by determinating phosphorylated ERK with Western blot. Ang II rece ptor-mediated activation of tyrosine phosphatase SHP-1 was assessed by prot ein tyrosine phosphatase assay. Results: The expression of AT1 and AT2 rece ptors was approximately 70%: 30% per cell. Serum depletion induced apoptosi s in fetal VSMCs and selective AT1 receptor stimulation attenuated the apop totic changes, whereas selective AT2 receptor activation enhanced apoptosis . Ang II increased ERK phosphorylation, which was inhibited by addition of the AT1 receptor-specific antagonist CV11974, but enhanced by addition of t he AT2 receptor-specific antagonist PD123319, suggesting that activation of AT2 receptor attenuated the AT1 receptor-mediated ERK phosphorylation. Mor eover, we demonstrated that AT2 receptor stimulation activated SHP-1 in fet al VSMCs, whereas AT1 receptor stimulation did not. Transient transfection of a dominant-negative SHP-1 mutant into rat fetal VSMCs resulted in a sign ificant decrease of the AT2 receptor-mediated inhibition of ERK phosphoryla tion and attenuated the proapoptotic effect of AT2 receptor. Conclusion: Th ese results indicate that a crosstalk between AT1 and AT2 receptors regulat es the survival of fetal VSMCs and substantiate SHP-1 as a key molecule in AT2 receptor signaling. (C) 2001 Elsevier Science B.V. All rights reserved.