Testosterone alters maternal vascular adaptations: role of the endothelial NO system

Abstract

Sex steroid hormones estradiol and progesterone play an important role in vascular adaptations during pregnancy. However, little is known about the role of androgens. Plasma testosterone (T) levels are elevated in preeclampsia, mothers with polycystic ovary, and pregnant African American women, who have endothelial dysfunction and develop gestational hypertension. We tested whether increased T alters vascular adaptations during pregnancy and whether these alterations depend on endothelium-derived factors, such as prostacyclin, endothelium-derived hyperpolarizing factor, and NO. Pregnant Sprague Dawley rats were injected with vehicle (n=12) or T propionate [0.5 mg/Kg per day from gestation day 15-19; n=12] to increase plasma T levels 2-fold, similar to that observed in preeclampsia. Telemetric blood pressures and endothelium-dependent vascular reactivity were assessed with wire-myograph system. Phospho-endothelial NO synthase and total endothelial NO synthase were examined in mesenteric arteries. Mean arterial pressures were significantly higher starting from gestation day19 until delivery in T-treated dams. Endothelium-dependent relaxation responses to acetylcholine were significantly lower in mesenteric arteries of T-treated dams (pD(2) [-log EC(50)]=7.05±0.06; E(max)=89.4±1.89) compared with controls (pD(2)=7.38±0.04; E(max)=99.9±0.97). Further assessment of endothelial factors showed NO-mediated relaxations were blunted in T-treated mesenteric arteries (E(max)=42.26±5.95) compared with controls (E(max)=76.49±5.06); however, prostacyclin- and endothelium-derived hyperpolarizing factor-mediated relaxations were unaffected. Relaxation to sodium nitroprusside was unaffected with T-treatment. Phosphorylations of endothelial NO synthase at Ser(1177) were decreased and at Thr(495) increased in T-treated mesenteric arteries without changes in total endothelial NO synthase levels. In conclusion, increased maternal T, at concentrations relevant to abnormal clinical conditions, cause hypertension associated with blunting of NO-mediated vasodilation. T may induce the increased vascular resistance associated with pregnancy-induced hypertension.

Figure 1

Mean arterial pressure (MAP) and heart rate in control and testosterone-treated pregnant rats. MAP (A) and heart rate (B) were continuously monitored via telemetry catheters in femoral artery from gestational day (GD) 14 until delivery in control and testosterone-treated (0.5 mg/kg/day, s/c from GD 15–19) pregnant rats. MAP and heart rate values are presented in 12-h intervals showing circadian variation; nighttime periods are shaded. Data points represent the mean±SEM of measurements in 7 rats in each group. *P≤0.05 vs control.

Figure 2

Endothelium-dependent relaxation in mesenteric arterial rings. A submaximal phenylephrine contraction (EC₈₀) was elicited, acetylcholine (ACh) was added, and the percent relaxation of phenylephrine contraction was measured. Data points represent the mean±SEM of measurements in 18 to 24 vascular rings from 9 rats of each group. *P≤0.05 vs control.

Figure 3

PGI2-, EDHF-, and NO-mediated endothelium-dependent relaxation in mesenteric arterial rings. Submaximal phenylephrine contraction (EC₈₀) was elicited, acetylcholine (ACh) was added to arterial rings in presence of selective inhibitors as described in methods and Table 1, and then the percentage of relaxation to phenylephrine contraction was measured to determine (A) PGI2-, (B) EDHF-, and (C) NO-mediated mesenteric arterial relaxation. Data (mean±SEM) represent measurements from 14 to 24 vascular rings from 7–9 rats per treatment group. *P≤0.05 vs control.

Figure 4

Endothelium-independent relaxation in mesenteric arteries. Submaximal phenylephrine contraction (EC₈₀) was elicited in endothelium-denuded vascular rings, increasing concentrations of sodium nitroprusside (SNP) were added, and then the percentage of relaxation to phenylephrine contraction was measured. Data points represent mean±SEM of measurements in 10 to 12 mesenteric arterial rings from 6 rats of each group.

Figure 5

Endothelial nitric oxide synthase (eNOS) expression. (A) Quantitative RT-PCR analysis of eNOS expression in mesenteric arteries isolated from testosterone-treated and control pregnant rats. (B) Total eNOS protein expression in mesenteric arteries of testosterone-treated and control pregnant rats. Representative Western blots for eNOS and actin are shown at top; blot density obtained from densitometric scanning of eNOS normalized to actin is shown at bottom. Values are given as means±SEM of 5 to 7 rats in each group. *P≤0.05 vs control.

Figure 6

Phosphorylation of eNOS in mesenteric arteries isolated from control and testosterone-treated pregnant rats. Tissue lysates were immunoblotted with antibodies recognizing Ser¹¹⁷⁷-, Ser⁶³⁵,- or Thr⁴⁹⁵-phosphorylated eNOS, and blots were reprobed with anti-eNOS antibody. (A) Representative Western blots of phospho-eNOS and total eNOS. The level of (B) Ser¹¹⁷⁷, (C) Ser⁶³⁵ or (D) Thr⁴⁹⁵ phosphorylation of eNOS was quantified by scanning densitometry and normalized to total eNOS. Values are means±SE; n = 5 for each group. *P≤0.05 vs control.