Increased vascular angiotensin type 2 receptor expression and NOS-mediated mechanisms of vascular relaxation in pregnant rats

Stennett AK, Qiao X, Falone AE, Koledova VV, Khalil RA. Increased vascular angiotensin type 2 receptor expression and NOS-mediated mechanisms of vascular relaxation in pregnant rats. Am J Physiol Heart Circ Physiol 296: H745-H755, 2009; doi:10.1152/ajpheart.00861.2008.-Normal pregnancy is associated with reduced blood pressure (BP) and decreased pressor response to vasoconstrictors, even though the renin-angiotensin system is upregulated. Angiotensin II (ANG II) activates both angiotensin type 1 receptors (AT(1)Rs) and angiotensin type 2 receptors (AT(2)Rs). Although the role of the AT(1)R in vascular contraction is well documented, the role of the AT(2)R 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 AT(2)R amount, distribution, and/or postreceptor mechanisms of vascular relaxation. To test this hypothesis, systolic BP was measured in virgin and pregnant (day 19) Sprague-Dawley rats. Isometric contraction/relaxation was measured in isolated aortic rings, and nitric oxide (NO) production was measured using 4-amino-5-methylamino-2',7'-difluorescein fluorescence. AT(1)R and AT(2)R mRNA expression and protein amount were measured in tissue homogenates 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 the aorta of pregnant compared with virgin rats. Treatment with the AT(2)R antagonist PD-123319 caused greater enhancement of Phe contraction, and the AT(2)R agonist CGP-42112A caused greater relaxation of Phe contraction in the aorta of pregnant than virgin rats. ANG II plus the AT(1)R blocker losartan induced greater NO production in the aorta of pregnant than virgin rats. RT-PCR revealed increased mRNA expression of vascular endothelial NO synthase (eNOS), little change in AT(1)Rs, and increased AT(2)Rs in pregnant compared with virgin rats. Western blots revealed an increased protein amount of activated phospho-eNOS, little change in AT(1)Rs, and increased AT(2)Rs in pregnant compared with virgin rats. Immunohistochemistry and immunofluorescence analysis in aortic sections of virgin rats revealed abundant AT(1)R staining in tunica media that largely colocalized with actin in vascular smooth muscle and less AT(2)Rs mainly in the tunica intima and endothelium. In pregnant rats, AT(1)R staining in the smooth muscle layer and adventitia was reduced, and endothelial AT(2)R staining was enhanced. These data suggest an enhanced AT(2)R-mediated vascular relaxation pathway involving increased expression/activity of endothelial AT(2)Rs and increased postreceptor activated phospho-eNOS, which may contribute to the decreased BP during pregnancy.