Chronic hypoxia increases pressure-dependent myogenic tone of the uterine artery in pregnant sheep: role of ERK/PKC pathway

Abstract

Chronic hypoxia during pregnancy has profound effects on uterine artery (UA) contractility and attenuates uterine blood flow. The present study tested the hypothesis that chronic hypoxia inhibits the pregnancy-induced reduction in pressure-dependent myogenic tone of resistance-sized UAs. UAs were isolated from nonpregnant ewes (NPUAs) and near-term pregnant ewes (PUAs) that had been maintained at sea level (approximately 300 m) or at high altitude (3,801 m) for 110 days. In normoxic animals, the pressure-dependent myogenic response was significantly attenuated in PUAs compared with NPUAs. Hypoxia significantly increased myogenic tone in PUAs and abolished its difference between PUAs and NPUAs. Consistently, there was a significant increase in PKC-mediated baseline Ca(2+) sensitivity of PUAs in hypoxic animals. Hypoxia significantly increased phorbol 12,13-dibutyrate (PDBu)-induced contractions in PUAs but not in NPUAs. Whereas the inhibition of ERK1/2 by PD-98059 potentiated PDBu-mediated contractions of PUAs in normoxic animals, it failed to do so in hypoxic animals. Hypoxia decreased ERK1/2 expression in PUAs. PDBu induced membrane translocation of PKC-alpha and PKC-epsilon. Whereas there were no significant differences in PKC-alpha translocation among all groups, the translocation of PKC-epsilon was significantly enhanced in NPUAs compared with PUAs in normoxic animals, and hypoxia significantly increased PKC-epsilon translocation in PUAs. In the presence of PD-98059, there were no significant differences in PDBu-induced PKC-epsilon translocation among all groups. Treatment of PUAs isolated from normoxic animals with 10.5% O(2) for 48 h ex vivo significantly increased PDBu-induced contractions and eliminated its difference between PUAs and NPUAs. The results suggest that hypoxia upregulates pressure-dependent myogenic tone through its direct effect in suppressing ERK1/2 activity and increasing the PKC signal pathway, leading to an increase in the Ca(2+) sensitivity of the myogenic mechanism in the UA during pregnancy.

Fig. 1.

Effect of high-altitude hypoxia on pressure-dependent myogenic tone in uterine arteries (UAs). Pressure-dependent myogenic tone was determined in UAs obtained from nonpregnant and pregnant ewes with normoxia (control) and long-term high-altitude hypoxia treatment. Myogenic tone was calculated as the percent difference between passive and active diameter at given pressures, as described in methods. Data are expressed as means ± SE of tissues from 4–8 animals/group. *P < 0.05, significant difference between control and hypoxic groups, as determined by repeated-measures two-way ANOVA.

Fig. 2.

Effect of high-altitude hypoxia on phorbol 12,13-dibutyrate (PDBu)-induced contractions in UAs. Cumulative concentration-response curves to PDBu were determined in UAs obtained from nonpregnant and pregnant ewes with normoxia and long-term high-altitude hypoxia treatment in the absence or presence of PD-98059 (PD; 30 μM, pretreatment for 20 min). Data are expressed as percentages of contraction generated by 120 mM KCl, and each point represents the mean ± SE of tissues from 4–10 animals.

Fig. 3.

Effect of high-altitude hypoxia on the maximal response (E_max) and potency (pD₂) of PDBu-induced contractions in UAs. E_max and pD₂ values were determined from the PDBu concentration-response curves shown in Fig. 2 by computer-assisted nonlinear regression to fit the data using GraphPad Prism. Data are expressed as means ± SE of tissues from 4–10 animals. ^aP < 0.05, pregnant vs. nonpregnant animals; ^bP < 0.05, hypoxia vs. normoxia.

Fig. 4.

Effect of high-altitude hypoxia on ERK1/2 and phosphorylated (p-)ERK1/2 protein abundance in UAs. ERK1/2 and p-ERK1/2 protein abundance were determined in UAs obtained from normoxic nonpregnant ewes (NNUAs), normoxic pregnant ewes (NPUAs), high-altitude hypoxic nonpregnant ewes (HNUAs), and high-altitude hypoxic pregnant ewes (HPUAs). N, normoxia; H, hypoxia. Data are means ± SE of tissues from 5–6 animals. ^aP < 0.05, nonpregnant vs. pregnant animals; ^bP < 0.05, hypoxic vs. normoxic.

Fig. 5.

Effect of PKC activation on the Ca²⁺-force relation in β-escin-permeabilized UAs in high-altitude hypoxic animals. Concentration-response curves of Ca²⁺-induced contractions were determined in β-escin-permeabilized UAs obtained from nonpregnant and pregnant long-term high-altitude hypoxic animals in the absence (control) or presence of PDBu (3 μM, pretreatment for 20 min). Data are expressed as percentages of maximal KCl-induced contractions of the same tissue before permeabilization to normalize the tissue size and are means ± SE of tissues from 5–14 animals. pD₂ and E_max values are presented in results.

Fig. 6.

Effect of high-altitude hypoxia on PDBu-induced membrane translocation of PKC isozymes in UAs. PDBu-induced membrane translocations of PKC-α and PKC-ɛ were determined in NNUAs, NPUAs, HNUAs, and HPUAs in the absence or presence of PD-98059 (30 μM, pretreatment for 20 min). PBDu-induced increases in the ratio of the particulate to cytosolic distribution of PKC-α and PKC-ɛ are expressed as fold changes of basal levels of each isozyme blotted in the same membrane. Lane 1, basal; lane 2, PD-98059; lane 3, PDBu; lane 4, PDBu + PD-98059. Data are means ± SE of tissues from 5–6 animals. ^aP < 0.05, PDBu vs. basal; ^bP < 0.05, PDBu + PD98059 vs. PDBu alone; ^cP < 0.05, all groups vs. NPUAs.

Fig. 7.

PDBu-induced membrane translocation of p-PKC-α and p-PKC-ɛ in UAs. PDBu-induced membrane translocations of p-PKC-α and p-PKC-ɛ were determined in UAs obtained from four pregnant ewes.

Fig. 8.

Effect of ex vivo hypoxia on PDBu-induced contractions in UAs. UAs isolated from normoxic nonpregnant and pregnant ewes were incubated at 37°C in a humidified incubator with either 21% or 10.5% O₂ for 48 h before they were subjected to contractions induced by increased concentrations of PDBu. Data are expressed as percentages of contraction generated by 120 mM KCl, and each point represents the mean ± SE of tissues from 7–12 animals.

Fig. 9.

Effect of ex vivo hypoxia on the E_max and pD₂ of PDBu-induced contractions in UAs. UAs isolated from normoxic nonpregnant and pregnant ewes were incubated at 37°C in a humidified incubator with either 21% or 10.5% O₂ for 48 h before they were subjected to contractions induced by increased concentrations of PDBu in the absence or presence of PD-98059 (30 μM, pretreatment for 20 min). E_max and pD₂ values were determined from PDBu concentration-response curves by computer-assisted nonlinear regression to fit the data using GraphPad Prism. Data are expressed as means ± SE of tissues from 7–12 animals. ^aP < 0.05, 10.5% vs. 21% O₂; ^bP < 0.05, with (+) PD-98059 vs. without (−) PD-98059; ^cP < 0.05, nonpregnant vs. pregnant animals.