Long-Term High-Altitude Hypoxia and Alpha Adrenoceptor-Dependent Pulmonary Arterial Contractions in Fetal and Adult Sheep

Abstract

Autonomic innervation of the pulmonary vasculature triggers vasomotor contractility predominately through activation of alpha-adrenergic receptors (α-ARs) in the fetal circulation. Long-term hypoxia (LTH) modulates pulmonary vasoconstriction potentially through upregulation of α₁-AR in the vasculature. Our study aimed to elucidate the role of α-AR in phenylephrine (PE)-induced pulmonary vascular contractility, comparing the effects of LTH in the fetal and adult periods on α-AR subtypes and PE-mediated Ca²⁺ responses and contractions. To address this, we performed wire myography, Ca²⁺ imaging, and mRNA analysis of pulmonary arteries from ewes and fetuses exposed to LTH or normoxia. Postnatal maturation depressed PE-mediated contractile responses. α₂-AR activation contracted fetal vessels; however, this was suppressed by LTH. α_1A- and α_1B-AR subtypes contributed to arterial contractions in all groups. The α_1D-AR was also important to contractility in fetal normoxic vessels and LTH mitigated its function. Postnatal maturity increased the number of myocytes with PE-triggered Ca²⁺ responses while LTH decreased the percentage of fetal myocytes reacting to PE. The difference between myocyte Ca²⁺ responsiveness and vessel contractility suggests that fetal arteries are sensitized to changes in Ca²⁺. The results illustrate that α-adrenergic signaling and vascular function change during development and that LTH modifies adrenergic signaling. These changes may represent components in the etiology of pulmonary vascular disease and foretell the therapeutic potential of adrenergic receptor antagonists in the treatment of pulmonary hypertension.

Keywords: adrenergic receptor; calcium; contraction; fetus; hypoxia; pulmonary artery; sheep.

Figure 1

Phenylephrine-mediated contraction of pulmonary arteries from sheep is depressed in the adult period and enhanced by LTH. (A) Dose-response curves of pulmonary arterial rings exposed to 1 nM to 100 μM of phenylephrine in an additive manner from normoxic (solid lines) and LTH (dashed lines) fetal (gray lines and boxes) and adult (black lines and circles) sheep. Curves are plotted in relation to the maximal contraction induced by initial stimulation of 125 mM K⁺ Krebs-Henseleit solution (%T_Kmax). (B) Maximum contraction relative to %T_Kmax. (C) Log EC₅₀ for phenylephrine induced contraction. (D) Area under the dose-response curve relative to high K⁺ contraction. (E) Absolute force developed for 100 μM phenylephrine-induced contraction. Points and bars are mean values and error bars indicate ± S.E.M. Comparisons between groups were made using a two-way ANOVA with a Bonferroni post hoc analysis (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). Numbers of animals for each group are provided in the “Methods” section.

Figure 2

Phenylephrine-induced contraction is largely dependent on α₁-adrenergic contraction. (a) Dose-response curves of pulmonary arterial rings exposed to 1 nM to 100 μM phenylephrine in an additive manner from normoxic and LTH, fetal (A,B) and adult (C,D) sheep. Solid lines with circles indicate vehicle-control (DMSO) while long dashes and squares indicate 10 nM prazosin and triangles and short dashes indicate 100 nM yohimbine. Log agonist vs. response curves are plotted in relation to the maximal contraction induced by initial stimulation of 125 mM K⁺ Krebs-Henseleit solution (%T_Kmax). (b) Log EC₅₀ for phenylephrine-induced contraction. (c) Maximum contraction relative to %T_Kmax. (d) Area under the dose-response curve relative to high K⁺ contraction. Points and bars are mean values while error bars indicate ± S.E.M. Comparisons of drug treated arteries to control were made using a one-way ANOVA with a Newman-Keuls multiple comparison test (^**p < 0.01, ^***p < 0.001). Numbers of animals for each fetal and adult group are provided in the “Methods” section and in (b).

Figure 3

Dexmedetomidine causes contraction and relaxation of normoxic fetal pulmonary arteries. Dose-response curves of pulmonary arterial rings exposed to 100 pM to 10 μM of dexmedetomidine in an additive manner from normoxic fetal sheep in the absence (A) and presence (B) of 10 μM serotonin. Gray lines and triangles are for dexmedetomidine contraction (A) or relaxation (B) in the presence of yohimbine, and black lines and boxes are for dexmedetomidine contraction (A) or relaxation (B) in the presence of vehicle-control (DMSO). Solid lines indicate log agonist vs. response curves that are plotted in relation to the maximal contraction induced by initial stimulation of (A) 125 mM K⁺ Krebs-Henseleit solution (%T_Kmax) or (B) 10 μM serotonin. Points and error bars indicate mean ± S.E.M. Numbers of animals for each group are provided in the “Methods” section.

Figure 4

α_1A-AR and α_1B-AR antagonists preferentially block phenylephrine-induced pulmonary vascular contractility. (a) Dose response curves of pulmonary arterial rings exposed to 1 nM to 100 μM phenylephrine in an additive manner from normoxic and LTH, fetal (A,B) and adult (C,D) sheep. Curves of log agonist vs. response were repeated in the absence (circles and solid lines) or presence of 10 μM CEC (α_1B-AR blocker, squares and long dashed lines), 100 nM WB (α_1A-AR blocker, triangles and short dashed lines) or 100 nM BMY (α_1D-AR blocker, upside down triangles and dashed and dotted lines) for normoxic and LTH, fetal (A,B) and adult (C,D) sheep pulmonary arteries. (b) Log EC₅₀ for phenylephrine induced contraction. (c) Maximum contraction relative to high K⁺. (d) Area under the dose-response curve relative to high K⁺ contraction. Points and bars are mean values while error bars indicate ± S.E.M. Comparisons of drug treated arteries to control were made using a one-way ANOVA with a Newman-Keuls Multiple Comparison Test (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). Numbers of animals for each fetal and adult group are provided in the “Methods” section and (b).

Figure 5

LTH in the fetal and adult periods have unique impact on mRNA expression of alpha adrenergic receptors. Presented are the raw Cycle threshold (Ct) values from real time quantitative PCR measurements for (A) Alpha 1a AR, (B) Alpha 1b AR, (C) Alpha 1d AR, and (D) GAPDH. Normalization was to input RNA, all PCR reactions used input cDNA derived from 5 ng of total RNA. Bars and error bars represent the mean ± S.E.M. Significant differences between groups were established using a two-way ANOVA with a Bonferroni post hoc analysis (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). Numbers of animals for each group are provided in the methods section.

Figure 6

Impact of LTH in the fetal and adult periods on unstimulated and PE-mediated whole-cell Ca²⁺ responses in pulmonary arterial myocytes. Shown are representative fluorescent images at time points shown by the arrow and the traces of fractional Fluo-4 fluorescence for unstimulated (A) and 10 μM phenylephrine stimulated (B) arteries. (C) Percentage of myocytes in a 1,000 μm² area that had Ca²⁺ responses to 10 μM PE under unstimulated (open circles) and stimulated (filled circles) conditions based on the number of runs examined. Colored circles on each image correspond to the placement of regions of interest for the traces of the same color in the tracing. About 10 μM phenylephrine was present during the time period denoted by the horizontal bar. Images were made with a 20× non-immersion objective (NA 0.8). Scale bar is 25 μm. Numbers of animals for each group are provided in the “Methods” section.