Pregnancy downregulates actin polymerization and pressure-dependent myogenic tone in ovine uterine arteries

Abstract

Pregnancy is associated with significantly decreased uterine vascular tone and increased uterine blood flow. The present study tested the hypothesis that the downregulation of actin polymerization plays a key role in reduced vascular tone of uterine arteries in the pregnant state. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Activation of protein kinase C significantly increased the filamentous:globular actin ratio and contractions in the uterine arteries, which were inhibited by an actin polymerization inhibitor cytochalasin B. The basal levels of filamentous:globular actin were significantly higher in nonpregnant uterine arteries than those in near-term pregnant sheep. Prolonged treatment (48 hours) of nonpregnant sheep with 17β-estradiol (0.3 nmol/L) and progesterone (100.0 nmol/L) caused a significant decrease in the filamentous:globular actin. In accordance, the treatment of near-term pregnant sheep for 48 hours with an estrogen antagonist ICI 182 780 (10.0 μmol/L) and progesterone antagonist RU 486 (1.0 μmol/L) significantly increased the levels of filamentous:globular actin. Increased intraluminal pressure from 20 to 100 mm Hg resulted in an initial increase in uterine arterial diameter and vascular wall Ca(2+) concentrations, followed by a decrease in the diameter at a constant steady-state level of Ca(2+). Cytochalasin B blocked pressure-induced myogenic constrictions without effect on vascular wall Ca(2+) levels and eliminated the differences in pressure-dependent myogenic tone between nonpregnant sheep and near-term pregnant sheep. The results indicate a key role of actin polymerization in protein kinase C-induced myogenic contractions and suggest a novel mechanism of sex steroid hormone-mediated downregulation of actin polymerization underlying the decreased myogenic tone of uterine arteries in pregnancy.

Figure 1

Effect of cytochalasin B on PDBu-induced contractions. PDBu (10.0 μmol/L)-induced contractions were determined in both nonpregnant and pregnant uterine arteries in the absence or presence of cytochalasin B (Cyt B; 10.0 μmol/L for 20 minutes). Data are mean±SEM of tissues from 6 animals. *P<0.05, PDBu+Cyt B vs PDBu; †P<0.05, pregnant vs nonpregnant animals.

Figure 2

Effect of PDBu on actin polymerization in pregnant uterine arteries. A, Representative images from 3 experiments of freshly isolated smooth muscle cells in the absence (control) or presence of PDBu (10.0 μmol/L). F actin was stained with rhodamine-phalloidin and examined by fluorescence microscopy. B, Uterine arteries were treated in the absence (control) or presence of cytochalasin B (Cyt B; 10.0 μmol/L for 20 minutes) and/or PDBu (10.0 μmol/L for 5 minutes). F actin and G actin were separated by differential sedimentation and determined by Western blots. Data are mean±SEM of tissues from 6 animals. *P<0.05, PDBu vs control; †P<0.05, PDBu+Cyt B vs PDBu.

Figure 3

Effect of pregnancy on PDBu-induced actin polymerization. Uterine arteries of nonpregnant and pregnant sheep were treated in the absence (control, C) or presence of PDBu (10.0 μmol/L). F actin and G actin were separated by differential sedimentation and determined by Western blots. Data are mean±SEM of tissues from 4 animals. *P<0.05, PDBu vs control; †P<0.05, pregnant vs nonpregnant animals.

Figure 4

Effect of steroid hormones on actin polymerization. Uterine arteries of nonpregnant sheep were treated with E₂β (0.3 nmol/L) and P₄ (100.0 nmol/L) or vehicle control for 48 hours. Uterine arteries of pregnant animals were treated with ICI 182 780 (ICI; 10.0 μmol/L) and RU 486 (RU; 1.0 μmol/L) or vehicle control for 48 hours in the presence of E₂β and P₄. F actin and G actin were separated by differential sedimentation and determined by Western blots. Data are mean±SEM of tissues from 4 animals. *P<0.05, E₂β+P₄ vs control; †P<0.05, ICI+RU vs control.

Figure 5

Effect of cytochalasin B on pressure-induced myogenic contractions and vascular wall [Ca²⁺]_i. Uterine arteries of pregnant animals were loaded with fura-2 and pressurized at 20 mm Hg. Arterial diameter and [Ca²⁺]_i (fura-2 fluorescence ratio, R_f340/380) were measured simultaneously in the same tissues. Representative traces of 3 experiments show the effect of increased intraluminal pressure (100 mm Hg) on arterial diameter and [Ca²⁺]_i in the absence (A and B) or presence (C and D) of cytochalasin B (10.0 μmol/L for 20 minutes).

Figure 6

Effect of cytochalasin B on pressure-dependent myogenic tone and vessel wall [Ca²⁺]_i in uterine arteries of nonpregnant and pregnant sheep. Uterine arteries of nonpregnant (NP) and pregnant (P) sheep were loaded with fura-2. Myogenic tone (top) and [Ca²⁺]_i (fura-2 fluorescence ratio, R_f340/380; bottom) were measured simultaneously in the same tissues at given pressures in the absence (control) or presence of cytochalasin B (Cyt B; 10.0 μmol/L for 20 minutes). Data are mean±SEM of tissues from 5 animals. *P<0.05, pregnant vs nonpregnant animals; †P<0.05, Cyt B vs control; repeated-measures, 2-way ANOVA.