Submitted on August 6, 2008
Revised on December 29, 2008
Accepted on January 12, 2009
Increased Vascular Angiotensin Type 2 Receptor Expression and NOS-Mediated Mechanisms of Vascular Relaxation in Pregnant Rats
Amanda K. Stennett, Xiaoying Qiao, Anthony E. Falone, Vera V. Koledove, and Raouf A. Khalil1*
1 Brigham & Women's Hospital
* To whom correspondence should be addressed. E-mail: raouf_khalil{at}hms.harvard.edu.
Angiotensin II (AngII) activates both AT1R and AT2R. Although the role of AT1R in vascular contraction is well-documented, the role of AT2R in vascular relaxation, particularly during pregnancy, is less clear. It was hypothesized that the decreased BP and vasoconstriction during pregnancy was, at least in part, due to changes in AT2R amount, distribution and/or postreceptor mechanisms of vascular relaxation. Systolic BP was measured in virgin and pregnant (day 19) Sprague-Dawley rats. Isometric contraction/relaxation was measured in isolated aortic rings, and NO production was measured using DAF-FM fluorescence. AT1R and AT2R mRNA expression and protein amount were measured in tissue homogenate using real-time RT-PCR and Western blots, and their local distribution was visualized in cryosections using immunohistochemistry and immunofluorescence. BP was lower in pregnant than virgin rats. Phenylephrine (Phe) caused concentration-dependent contraction that was reduced in aorta of pregnant compared with virgin rats. Treatment with AT2R antagonist PD123319 caused greater enhancement of Phe contraction, and AT2R agonist CGP42112A caused greater relaxation of Phe contraction in aorta of pregnant than virgin rats. AngII plus AT1R blocker losartan induced greater NO production in aorta of pregnant than virgin rats. RT-PCR and Western blots revealed increased mRNA expression of vascular eNOS and the amount of phospho-eNOS, little change in AT1R, and increased AT2R in pregnant compared with virgin rats. Immunohistochemistry and immunofluorescence analysis in aortic sections of virgin rats revealed abundant AT1R staining in tunica media that largely colocalized with actin in vascular smooth muscle, and less AT2R mainly in tunica intima and endothelium. In pregnant rats, AT1R staining in smooth muscle layer and adventitia was reduced, and endothelial AT2R staining was enhanced. These data suggest an enhanced AT2R-mediated vascular relaxation pathway involving increased expression/activity of endothelial AT2R and postreceptor eNOS actiivity, which may contribute to the decreased BP during pregnancy.
