Pregnancy upregulates angiotensin type 2 receptor expression and increases blood flow in uterine arteries of rats

Abstract

Normal pregnancy is associated with decreased uterine vascular contraction and increased blood flow even though angiotensin II (AngII) levels are increased. AngII not only activates the angiotensin type 1 receptor (AT1R) to mediate vasoconstriction but also angiotensin type 2 receptor (AT2R) to cause vasodilation. We hypothesized that upregulation of AT2R expression and function accounts for increased uterine artery blood flow during pregnancy. Virgin, pregnant (at different days of gestation) and post-partum Sprague-Dawley rats were used to determine uterine artery hemodynamics using micro ultrasound and plasma angiotensin II levels by ELISA. Isolated uterine arteries were examined for AT1R and AT2R expression and isometric contraction/relaxation. Plasma AngII levels were steady up to mid-pregnancy, increased as pregnancy advanced, reaching a peak in late pregnancy, and then restored to pre-pregnant levels after delivery. The pattern of increase in AngII levels mirrored a parallel increase in uterine blood flow. AT1R expression did not change, but AT2R expression increased during pregnancy correlating with uterine blood flow increase. Treatment with the AT2R antagonist PD123319 reduced uterine arterial blood flow. Vasoconstriction to angiotensin II was blunted in pregnant rats. Treatment with PD123319 caused greater enhancement of AngII contraction in pregnant than virgin rats. Ex vivo exposure of estradiol to uterine arterial rings dose dependently upregulated AT2R expression, that was inhibited by estrogen receptor antagonist. These results demonstrate that elevated AngII levels during gestation induce an increase in uterine blood flow via heightened AT2R-mediated signaling. Estrogens appear to directly upregulate uterine vascular AT2R independent of any endogenous factors.

Figure 1.

Temporal changes in plasma Ang II levels across pregnancy. Plasma Ang II levels were higher during late pregnancy. Plasma from nonpregnant (NP), pregnant, and post-partum (PP) rats was determined by enzyme immunoassay. n = 6 in each *P < 0.05 vs NP.

Figure 2.

Uterine artery hemodynamics across pregnancy. Doppler ultrasonography was used to measure progressive changes in (A) uterine arterial blood flow, (B) resistance index, and (C) pulsatility index in nonpregnant (NP), pregnant, and post-partum (PP) rats. Peak systolic and end-diastolic velocities were used to calculate resistance and pulsatility indices (see materials and methods section). n = 6 each, *P < 0.05 vs NP.

Figure 3.

Ang II receptor expression in uterine arteries across pregnancy. AT₁R mRNA was not altered but AT₂R expression increased during pregnancy. Quantitative RT-PCR analysis of temporal mRNA levels of (A) AT₁R (both AT_1aR and AT_1bR) and (B) AT₂R normalized to GAPDH in nonpregnant (NP), pregnant (P), and post-partum (PP) rats. (C) Representative western blots for AT₁R and AT₂R and β-actin are shown at top; blot density obtained from densitometric scanning of Ang II receptors normalized to β-actin is shown at bottom. n = 5–6 each, *P < 0.05 vs NP.

Figure 4.

Effect of AT₂R blockade on uterine artery hemodynamics. Doppler ultrasonography was used to measure (A) uterine arterial blood flow, (B) resistance index, and (C) pulsatility index in gestation day 20 pregnant (P) rats after 30 min of vehicle (V) and PD123319 (PD) treatment. Peak systolic and end-diastolic velocities were used to calculate resistance and pulsatility indices (see materials and methods section). n = 6 each, *P < 0.05 vs V.

Figure 5.

Contractile responses in endothelium-intact uterine artery isolated from (A) nonpregnant and (B) pregnant (gestation day 20) rats. (C) Contractile responses in endothelium-denuded uterine arteries. Contractile responses were taken to cumulative additions of Ang II in absence or presence of losartan and PD123319. Values are given as means ± SEM of six rats in each group.

Figure 6.

Estradiol (E₂) upregulates AT₂R expression in ex vivo cultured uterine arteries of pregnant rats. The uterine arterial rings were treated with vehicle (V), E₂, and or ICI 182,780 for 24 h and the rings were processed for AT₂R mRNA and protein quantification. (A) Concentration-dependent E₂-induced increase in AT₂R mRNA was quantified by real-time qPCR and normalized with GAPDH. (B) E₂ (100 nM) induced increase in protein levels of AT₂R. Representative western blots are shown at top; blot density obtained from densitometric scanning normalized to β-actin is shown at bottom. n  =  5–6 each, *P < 0.05 vs V. ^#P < 0.05 vs E₂ 100 nM.