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

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 (AT1Rs) and angiotensin type 2 receptors (AT2Rs). Although the role of the AT1R in vascular contraction is well documented, the role of the 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. 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. AT1R and AT2R 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 AT2R antagonist PD-123319 caused greater enhancement of Phe contraction, and the AT2R agonist CGP-42112A caused greater relaxation of Phe contraction in the aorta of pregnant than virgin rats. ANG II plus the AT1R 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 AT1Rs, and increased AT2Rs in pregnant compared with virgin rats. Western blots revealed an increased protein amount of activated phospho-eNOS, little change in AT1Rs, and increased AT2Rs 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 AT2Rs mainly in the tunica intima and endothelium. In pregnant rats, AT1R staining in the 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 AT2Rs and increased postreceptor activated phospho-eNOS, which may contribute to the decreased BP during pregnancy

Gender-Specific Reduction in Contraction is Associated with Increased Estrogen Receptor Expression in Single Vascular Smooth Muscle Cells of Female Rat

Gender differences in the incidence of cardiovascular disease have been related to plasma estrogen levels; however, the role of vascular estrogen receptor (ER) subtypes in these sex differences is less clear. We tested whether the gender differences in vascular smooth muscle (VSM) function reflect differential expression/activity of ERα, ERβ and the newly-identified GPR30. Single aortic VSM cells (VSMCs) were freshly isolated from male and female Sprague-Dawley rats, and their contraction to phenylephrine (PHE, 10(−5) M), Angll (10(−7) M) and membrane-depolarization by KCl (51 mM) was measured in the absence or presence of 10(−6) M 17β-estradiol (E2, stimulant of most ERs), PPT (ERα agonist), DPN (ERβ agonist), and ICI 182,780 (an ERα/ERβ antagonist with GPR30 agonistic properties). The cells were fixed and fluorescently labeled with ERα, ERβ or GPR30 antibody, and the subcellular distribution of ERs was examined using digital imaging microscopy. The mRNA expression and protein amount of aortic ER subtypes was examined using RT-PCR and Western blots. PHE, AngII, and KCl caused less contraction in VSMCs of females than males. Pretreatment of VSMCs with E2 reduced PHE-, AngII- and KCl-induced contraction in both males and females. PPT caused similar inhibition of PHE-, AngII- and KCl-induced contraction as E2, suggesting a role of ERα. DPN mainly inhibited PHE and KCl contraction, suggesting an interaction between ERβ and Ca(2+) channels. ICI 182,780 did not reduce aortic VSMC contraction, suggesting little role for GPR30. RT-PCR and Western blots revealed greater expression of ERα and ERβ in VSMCs of females than males, but similar amounts of GPR30. The total immunofluorescence signal for ERα and ERβ was greater in VSMCs of females than males, and was largely localized in the nucleus. GPR30 fluorescence was similar in VSMCs of males and females, and was mainly in the cytosol. In PPT treated cells, nuclear ERα signal was enhanced. DPN did not affect the distribution of ERβ, and ICI 182,780 did not significantly increase GPR30 in the cell surface. Thus, ER subtypes demonstrate similar responsiveness to specific agonists in VSMCs of male and female rats. The reduced contraction in VSMCs of females could be due to gender-related increase in the expression of ERα and ERβ