Developmental acceleration of bradykinin-dependent relaxation by prenatal chronic hypoxia impedes normal development after birth

Abstract

Bradykinin-induced activation of the pulmonary endothelium triggers nitric oxide production and other signals that cause vasorelaxation, including stimulation of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in myocytes that hyperpolarize the plasma membrane and decrease intracellular Ca²⁺. Intrauterine chronic hypoxia (CH) may reduce vasorelaxation in the fetal-to-newborn transition and contribute to pulmonary hypertension of the newborn. Thus we examined the effects of maturation and CH on the role of BK_Ca channels during bradykinin-induced vasorelaxation by examining endothelial Ca²⁺ signals, wire myography, and Western immunoblots on pulmonary arteries isolated from near-term fetal (∼140 days gestation) and newborn, 10- to 20-day-old, sheep that lived in normoxia at 700 m or in CH at high altitude (3,801 m) for >100 days. CH enhanced bradykinin-induced relaxation of fetal vessels but decreased relaxation in newborns. Endothelial Ca²⁺ responses decreased with maturation but increased with CH. Bradykinin-dependent relaxation was sensitive to 100 μM nitro-l-arginine methyl ester or 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, supporting roles for endothelial nitric oxide synthase and soluble guanylate cyclase activation. Indomethacin blocked relaxation in CH vessels, suggesting upregulation of PLA₂ pathways. BK_Ca channel inhibition with 1 mM tetraethylammonium reduced bradykinin-induced vasorelaxation in the normoxic newborn and fetal CH vessels. Maturation reduced whole cell BK_Ca channel α₁-subunit expression but increased β₁-subunit expression. These results suggest that CH amplifies the contribution of BK_Ca channels to bradykinin-induced vasorelaxation in fetal sheep but stunts further development of this vasodilatory pathway in newborns. This involves complex changes in multiple components of the bradykinin-signaling axes.

regulation of smooth muscle tone in pulmonary arteries during development is a delicate balance of vasoconstrictive and vasorelaxant pathways. Endothelial cells play a crucial role in determining the overall level of vasorelaxation (39, 67), and endothelium-dependent relaxation is partially mediated through bradykinin stimulation (31). Bradykinin is a potent vasodilator that is important in the fetal pulmonary circulation, as well as during inflammation, and its relationship to pulmonary hypertension has been explored (5, 31, 83).

Endothelial bradykinin receptor activation induces vasorelaxation through modulation of several different intracellular signaling pathways that are largely dependent on a rise of endothelial intracellular Ca²⁺ ([Ca²⁺]_i) (67). The most widely studied pathway is bradykinin-induced activation of endothelial nitric oxide (NO) synthase (eNOS), an enzyme that generates NO (64). NO acts on nearby smooth muscle cells to cause downstream stimulation of soluble guanylate cyclase (sGC) pathways that leads to vasorelaxation (3, 45). Previous studies have shown that regulation of vessel relaxation through various receptor signaling systems is altered during pre- and postnatal development, as well as following prenatal chronic hypoxia, which imposes a significant stress on the fetus (9, 10, 35). Evidence suggests that acetylcholine (ACh)-dependent endothelium-mediated relaxation of the pulmonary vasculature is reduced in the fetus relative to the adult (57). Prenatal chronic hypoxia further suppresses ACh-induced relaxation in the fetus, but this is not due to changes in eNOS expression (91). The suppression of ACh-dependent relaxation maintains high pulmonary vascular resistance, which restricts blood flow, which, in turn, is important because the lung is not yet required for gas exchange. However, during the transition at birth from fetus to newborn, the pulmonary vessels dilate rapidly, increasing blood flow to the alveoli, and allow for proper gas exchange in the newborn lung.

Chronic hypoxia is a known risk factor in the development of pulmonary hypertension (67). It can significantly enhance vasoconstriction as well as reduce vasodilatory capacity. Subsequently, these effects elevate pulmonary pressure, which can result in pulmonary hypertension. The risk of pulmonary hypertension is especially prominent among newborns exposed to chronic hypoxia in utero due to pregnancy at high altitude, placental insufficiency, smoking, maternal anemia, or other causes (67). Persistent pulmonary hypertension of the newborn due to hypoxia or other etiologies is an incapacitating disease that can lead to failure of the ductus arteriosus to close, resulting in severe systemic hypoxia. Unfortunately, there are few treatment options and no cures (67). Numerous studies have indicated that endothelium-derived relaxing factors, especially those associated with NO, are crucial in the modifications associated with loss of vasodilatory capacity and development of pulmonary hypertension (1, 30, 48).

Our knowledge regarding the influence of prenatal chronic hypoxia on endothelium-dependent relaxation is limited. The available evidence indicates that there is enhanced eNOS expression (41, 59), reduced sGC (41), and CO-mediated relaxation (41, 59, 60) but enhanced large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel function (42). In fetal lamb, eNOS expression is unchanged (91), but PKG function is enhanced and cGMP function is reduced (26). The diversity in the dysfunctions and compensations associated with high-altitude gestation have led us to design a series of studies to test how prenatal chronic hypoxia affects early postnatal bradykinin-induced vasorelaxation. We tested the specific hypothesis that prenatal chronic hypoxia impairs the normal development of relaxation through eNOS-dependent pathways. This hypothesis was tested in studies on arteries isolated from full-term fetal and newborn sheep housed at low or high altitude, allowing for direct comparative analyses.

METHODS

Experimental animals.

Experimental procedures were performed on sheep arteries, because the developmental progression of ovine lungs and the extent to which prenatal chronic hypoxia affects them are comparable to humans (67). The studies were performed within the regulations of the Animal Welfare Act, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, “The Guiding Principles in the Care and Use of Animals” approved by the Council of the American Physiological Society, and the Animal Care and Use Committee of Loma Linda University (LLU). The tissue preparation, wire myography, and imaging experimental procedures and protocols are based on previously published methodology (10, 35, 37). Sheep were obtained from Nebeker Ranch (Lancaster, CA; 720 m altitude), and normoxic animals were brought to LLU [353 m altitude, 95 ± 5 Torr arterial Po₂ (Pa_O2)] for experimental study. Animals in the chronic hypoxic experimental group were acclimatized to high altitude (3,801 m, 60 ± 5 Torr Pa_O2) at the Barcroft Laboratory, White Mountain Research Station (Bishop, CA) for ∼110 days (35, 37). The hypoxic ewes were then transported to LLU for experimental study. To maintain hypoxic conditions in pregnant sheep, a tracheal catheter was placed in the ewe shortly after the animal arrived at LLU. The tracheal catheter allowed N₂ to reach the animal at a rate adjusted to maintain Pa_O2 at ∼60 Torr, equivalent to the Pa_O2 at the White Mountain Research Station (47). This Pa_O2 was maintained until the time of the experimental study. Ewes in the normoxic and chronic hypoxic groups were allowed to give birth naturally and transported to LLU Animal Care when the lambs were 8–17 days of age, as described previously (10). After arrival and until study, 1–2 days later, hypoxic animals were kept in a chamber supplied with 14–16% O₂ that simulated the altitude at the Barcroft Laboratory. Both groups of lambs were studied between 10 and 20 days after birth.

For the fetal studies, within 1–5 days of arriving at LLU, pregnant sheep were euthanized with an overdose of Euthasol [pentobarbital sodium (100 mg/kg) and phenytoin sodium (10 mg/kg); Virbac, Ft. Worth, TX], the proprietary euthanasia solution. Lungs were removed from the fetuses and used immediately for contractility and imaging experiments. Care was taken during arterial isolation and wire mounting to ensure that the endothelium was not disrupted.

Tissue preparation.

As described elsewhere, fourth- to fifth-branch-order pulmonary arteries with internal diameters of ∼500–700 μm were isolated from full-term fetal (138–141 days) or newborn (10–21 days old) sheep from the different experimental groups (10, 35, 37). The parenchyma was removed carefully from the pulmonary arteries for contractility studies. The arteries were then cut into 5-mm-long rings in ice-cold phosphate-free balanced salt solution (BSS) of the following composition (in mM): 126 NaCl, 5 KCl, 10 HEPES, 1 MgCl₂, 2 CaCl₂, and 10 glucose, with pH adjusted to 7.4 with NaOH. Imaging studies and the contraction studies in which arteries were treated with the NO donor N-nitrosoproline (ProliNO), which releases NO in a 1:2 ProliNO-to-NO molar ratio with a dissociation half-life of ∼2 s at physiological pH and temperature (76), were performed in BSS. All other contraction studies were performed with a modified Krebs-Henseleit (K-H) solution containing (in mM) 120 NaCl, 4.8 KCl, 1.2 K₂HPO₄, 25 NaHCO₃, 1.2 MgCl₂, 2.5 CaCl₂, and 10 glucose. To depolarize the arteries, NaCl was omitted from the BSS or K-H solution and replaced with equimolar KCl.

Contraction studies.

Wire-mounted pulmonary arterial rings were suspended in organ baths (Radnoti Glass Instruments, Monrovia, CA) containing 5 or 10 ml of modified K-H solution or BSS maintained at 37°C. Arteries in modified K-H solution were aerated with 95% O₂-5% CO₂ (pH 7.4), while unaerated HEPES-buffered BSS was used for the ProliNO studies to prevent sparge-induced loss of free NO into the atmosphere. Each ring was suspended between two tungsten wires passed through the lumen: one wire was anchored to the glass hook at the bottom of the organ chamber, while the other was connected to a tissue hook attached to a low-compliance force transducer (Radnoti Glass Instruments) that measured isometric force (10, 35, 37). The transducers were connected to an analog-to-digital data interface (Powerlab 16/30, A/D Instruments, Colorado Springs, CO; or MP100, Biopac, Goleta, CA) attached to a computer. Changes in tension were recorded using Chart 5.5 or 7.0 (AD Instruments) or AcqKnowledge 3.9 (Biopac), and the data were stored on digital media for later analysis.

At the beginning of each experiment, vessels were equilibrated without tension for ≥30 min. Tension of ∼0.75 g was then applied to each vessel and vessel tension was allowed to stabilize, as previously described (10, 35, 37). To allow for comparative evaluation of smooth muscle contraction and relaxation, isolated pulmonary arterial rings bathed in modified K-H solution were stimulated with 125 mM KCl (high-K) to cause membrane depolarization and activate L-type Ca²⁺ channels (35). In some experiments, the tension was normalized to a control response obtained with high K. To evaluate dose-response relaxation characteristics, bradykinin was used to relax the arteries following precontraction with 10 μM phenylephrine (PE). In these experiments, the tension was normalized to the maximum 10 μM PE-precontracted tension (%T_{10 μM PE}).

Confocal microscopy studies.

[Ca²⁺]_i was measured in pulmonary arterial endothelial cells in situ with the Ca²⁺-sensitive dye fluo 4-AM (Invitrogen, Carlsbad, CA) using a laser scanning confocal imaging workstation (model 710 NLO, Zeiss, Thornwood, NY) with an inverted microscope (Zeiss Axio Observer) according to procedures based on those previously described (35, 37). Fluo 4-AM was dissolved in DMSO and added from a 1 mM stock solution to the arterial suspension at a final fluo 4 concentration of 10 μM, along with 0.1% Pluronic F-127, for 1 h at room temperature in darkness in BSS. Arterial segments were then washed with BSS for 30 min to allow for dye esterification and cut into linear strips. The arterial segments were pinned to Sylgard (Ellsworth Adhesives, Germantown, WI) and placed in an open-bath imaging chamber (Warner Instruments, Hamden, CT) mounted on the confocal imaging stage. Cells were illuminated at 488 nm with a krypton-argon laser, and the emitted light was collected using a photomultiplier tube with a band-limited spectral grating in the range 493–622 nm, with full-frame images obtained every 700 ms. To ensure that the endothelial [Ca²⁺]_i was recorded, the pinhole was adjusted to provide an imaging depth of 5.4 μm, and the sample was focused above the internal elastic lamina layer, which has significant autofluorescence when excited at 488 nm in this preparation. This depth is substantially deeper than an individual endothelial cell, on the basis of morphological examination of fixed and live preparations (data not shown), and accounts for sample ruffling, thus allowing for the examination of many more endothelial cells than otherwise would have been achieved. Images were acquired at a 12-bit sampling depth with use of a water-immersion ×40 Plan-Apochromat, 1.0 numerical aperture objective mounted on an InverterScope objective inverter (LSM Tech, Wellsville, PA), which allowed for imaging in an upright configuration. To prevent arterial movement during recordings, arteries were pretreated for 1 h with a cocktail including 10 μM Y27632 to inhibit Rho kinase, 10 μM cytochalasin D to inhibit actin polymerization, and 10 μM W-7 to inhibit myosin light chain kinase (10, 43, 56, 88). The concentrations of these drugs were chosen on the basis of the outcomes of wire myography studies where the influence of the drugs on vessel reactivity were examined individually. Regions of interest were detected automatically post hoc using the LC Pro plug-in for ImageJ (22). For presentation purposes, the fractional fluorescence intensity was automatically calculated using LC Pro.

Background was subtracted from measurements as follows: F/F₀ = F − baseline/F₀ − baseline, where baseline is the intensity from a region of interest with no cells, F is the fluorescence intensity for the region of interest, and F₀ is the fluorescence intensity during a period when there was no Ca²⁺ activity as determined automatically. False positives were excised from the final data set by their relationship to the timing of the application of bradykinin and by visual analysis of the data.

Western immunoblot studies.

To generate whole cell lysates, pulmonary arteries from fetal and newborn sheep were harvested, cleaned, and rapid-frozen in liquid nitrogen and stored at −80°C. Tissues were then homogenized using glass-on-glass in a RIPA extraction buffer containing 10 mM DTT and a protease inhibitor cocktail (catalog no. M1745, Sigma-Aldrich, St. Louis, MO). Samples were centrifuged at 5,000 g for 20 min, and supernatants were collected and analyzed using SDS-PAGE along with reference control samples from pregnant adult sheep middle cerebral arteries. Separated proteins were transferred to nitrocellulose membranes at 200 mA for 90 min in Towbin buffer (25 mM Tris, 192 mM glycine, and 20% methanol). Membranes were blocked using 5% milk in Tris-buffered saline at pH 7.45 for 1 h at room temperature with continuous shaking. Primary antibodies were incubated for 12 h at 4°C using the following dilutions: 300:1 for BK-α (catalog no. APC-021, Alomone, Jerusalem, Israel) and 300:1 for BK-β₁ (catalog no. APC-036, Alomone) (44). For visualization, membranes were incubated for 90 min with a secondary antibody conjugated to DyLight 800 (catalog no. 46422, Pierce Chemical, Rockford, IL). Subsequently, membranes were stripped and reprobed using antibodies against β-actin [monoclonal anti-β-actin produced in mouse, clone AC-74 (catalog no. A2228, Sigma-Aldrich)] as a loading control. Anti-β-actin was diluted 1:5,000 and incubated for 90 min in 5% milk in Tris-buffered saline with 0.1% Tween 20. Membranes were imaged on an infrared imaging system (Odyssey, LI-COR), and individual protein bands were quantified using Image Studio software (LI-COR). Protein abundance was normalized using β-actin as a loading control and expressed as relative abundance compared with an arbitrary reference standard (pooled whole cell lysate obtained from pregnant adult sheep middle cerebral arteries).

Chemicals and drugs.

Most reagents and chemicals were purchased from Sigma-Aldrich; bradykinin, Y-27632, and W-7 were purchased from Tocris (Minneapolis, MN).

Statistical methods and sampling.

All time-series recordings were graphed with IGOR Pro 6.0 (Wavemetrics, Lake Oswego, OR), and summarized data are presented as means ± SE. Statistical analyses were made using Prism 5.0 (GraphPad, La Jolla, CA). Data were evaluated for normality prior to comparative statistical analysis. Dose-response curves were fitted in Prism 5.0 using the Hill equation with nonlinear curve fit analysis (10, 35). A total of 605 arterial segments from 61 sheep were tested for contractility. The Ca²⁺ imaging studies were performed on tissues from six fetal normoxic, four fetal hypoxic, three newborn normoxic, and four newborn hypoxic sheep. Western blot analysis was performed on tissues from nine fetal normoxic sheep and six animals in each of the other three groups. P < 0.05 was accepted as statistically significant, unless otherwise noted.

RESULTS

Endothelial cells of fetal sheep pulmonary arteries.

The first set of studies imaged the endothelium of intact, isolated, ovine pulmonary arteries to demonstrate that our arterial isolation techniques do not disrupt the endothelial layer (Fig. 1). The images of Fig. 1 are important, as Figs. 2–10 show the findings of studies designed to evaluate functional responses of the endothelium in pulmonary arteries from fetal and newborn sheep housed at low or high altitude. Representative images in Fig. 1 illustrate that the endothelial cells have an ellipsoid shape, which is presumed to be along the direction of blood flow, and that the cells are closely associated with one another. No quantitative measurements of endothelial cell shape or structure were performed in the present studies.

Fig. 1.

Representative micrographs of the endothelium in fetal pulmonary arteries. A–D: average fluo 4 fluorescence signals in endothelial cells of pulmonary arterial segments isolated from fetal sheep exposed to normoxia (A) and prenatal chronic hypoxia (B) and newborn sheep exposed to normoxia (C) and prenatal chronic hypoxia (D). Images represent average intensity from 100 frames for animals exposed to normoxia and 120 frames for animals exposed to hypoxia at 1.28 frames per second. Image brightness was adjusted for display purposes. Images were obtained with a ×40 water-immersion Plan-Apochromat objective. Scale bar = 20 μm.

Fig. 2.

Chronic prenatal hypoxia enhances neonatal bradykinin-induced pulmonary arterial relaxation but suppresses postnatal vascular relaxation. A–D: dose-response curves of pulmonary arterial rings exposed to 10 pM–1 μM bradykinin (BK) in an additive manner normalized to maximum 10 μM phenylephrine (PE)-precontracted tension (%T_{10 μM PE}) for fetal and newborn sheep exposed to normoxia and prenatal chronic hypoxia (A and B) and their respective potency (C) and maximum response values (D). Lines show resultant fits with a Hill equation to the dose-response relationships. Values are means ± SE. Data were analyzed by 2-way analysis of variance with a Bonferroni posttest analysis for each dose: *P < 0.05, **P < 0.01, ***P < 0.001, normoxia vs. hypoxia; ††P < 0.01, †††P < 0.001, fetal vs. newborn.

Fig. 10.

Western blot analysis of pulmonary arteries from fetal and newborn sheep shows that maturation alters BK_Ca α- and β₁-subunit expression. A: representative blot showing expression of BK_Ca channel α- and β₁-subunits and β-actin, to which channel subunit expression was normalized for fetal and newborn sheep exposed to normoxia and prenatal chronic hypoxia. B: α-subunit expression. C: β₁-subunit expression. Protein abundance was normalized to β-actin and expressed as relative abundance compared with the reference middle cerebral artery standard. Values are means ± SE. Data were analyzed by 2-way analysis of variance with a Bonferroni posttest analysis: *P < 0.05, **P < 0.01.

Prenatal chronic hypoxia and bradykinin-mediated relaxation.

The first series of functional studies was designed to determine the extent to which bradykinin relaxes pulmonary arteries and the extent to which this is altered by prenatal chronic hypoxia. These studies were based on the general premise that prenatal chronic hypoxia impedes relaxation (20, 21, 87). This proposition was investigated by examining dose-dependent bradykinin-mediated relaxation of 10 μM PE-precontracted vessels. Dose-response curves of the data from these studies and summary results are shown in Fig. 2. Figure 2, A and B, shows that prenatal chronic hypoxia enhances the bradykinin-induced vasorelaxant response in arteries from the fetus but that relaxation is blunted in vessels from the chronic hypoxic newborn. Figure 2C and Table 1 illustrate that the potency is enhanced by postnatal maturity but unaffected by high-altitude chronic hypoxia. Figure 2D and Table 2 show that continued life at high altitude impairs the normal developmental increase in the maximum response to bradykinin, in that relaxation is equivalent to that of vessels from the hypoxic fetus.

Table 1.

Chronic hypoxia, maturation, and potency of bradykinin-induced relaxation

	Normoxia		Hypoxia
	Fetus	Newborn	Fetus	Newborn
Control	−6.86 ± 0.21 (14)	−7.89 ± 0.16* (6)	−6.95 ± 0.15 (14)	−7.91 ± 0.12* (16)
l-NAME	−6.93 ± 1.09 (5)	−8.43 ± 0.26 (4)	−6.83 ± 0.53 (6)	−7.61 ± 0.43 (7)
Indo	−6.38 ± 0.78 (5)	−7.89 ± 0.15 (4)	−6.87 ± 0.74 (3)	−7.04 ± 0.38 (4)
ODQ	−6.89 ± 0.84 (4)	−7.89 ± 0.36 (3)	−6.51 ± 0.51 (3)	−7.86 ± 0.67 (4)
TEA	−6.44 ± 0.90 (5)	−7.65 ± 0.24 (4)	−7.05 ± 0.32 (3)	−7.68 ± 0.23 (4)

Values (means ± SE) are shown as log concentration for the IC₅₀ for bradykinin-induced relaxation for data presented graphically in Figs. 2, 5–7, and 9; number of animals is shown in parentheses. l-NAME, N^G-nitro-l-arginine methyl ester; Indo, indomethacin; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; TEA, tetraethylammonium. Statistical significance as shown using a 95% confidence interval based on the log (inhibitor) vs. response curve fit to the data:

^*significant leftward shift compared with Fetus on the basis of control traces in Fig. 2.

Table 2.

Chronic hypoxia, maturation, and maximum response of bradykinin-induced relaxation

	Normoxia		Hypoxia
	Fetus	Newborn	Fetus	Newborn
Control	83 ± 2 (14)	11 ± 4* (6)	47 ± 3‡ (14)	47 ± 2‡ (16)
l-NAME	91 ± 5 (5)	42 ± 4† (4)	79 ± 5† (6)	76 ± 3†‡ (7)
Indo	85 ± 10 (5)	19 ± 5 (4)	76 ± 12† (3)	66 ± 7†‡ (4)
ODQ	91 ± 5 (4)	44 ± 6† (3)	79 ± 8† (3)	73 ± 5†‡ (4)
TEA	78 ± 14 (5)	36 ± 5† (4)	73 ± 5† (3)	40 ± 5 (4)

Values (means ± SE) are shown as percentage of tension normalized to control response obtained in high-K solution for phenylephrine-induced contraction for data presented graphically in Figs. 2, 5–7, and 9; number of animals is shown in parentheses. Statistical significance as shown using a 95% confidence interval based on the log (inhibitor) vs. response curve fit to the data:

^*significant increase in maximum response compared with Fetus,

^†significant depression in maximum response compared with Control,

^‡significant difference in maximum response compared with Normoxia counterpart.

Prenatal chronic hypoxia and endothelial Ca²⁺ signals.

Bradykinin-induced relaxation is well regarded as being related to Ca²⁺-dependent signaling in endothelial cells (78). Because of the influence of development and chronic hypoxia on bradykinin-induced relaxation of pulmonary vessels presented in Fig. 2, we examined the potential that prenatal chronic hypoxia enhances bradykinin-induced Ca²⁺ signals in fetal endothelial cells but restricts these signals in the newborn. Figure 3 shows Ca²⁺ responses in fetal endothelial cells recorded before and during administration of 1 μM bradykinin in the presence of extracellular Ca²⁺. Figure 3A shows in situ fluorescence images of endothelial cells in the arterial wall isolated from a prenatal chronic hypoxic fetal sheep for various times, as shown for the fluorescence intensity trace in Fig. 3B. Bradykinin rapidly increased cytosolic Ca²⁺ in the pulmonary arterial endothelial cells, and these signals decayed slowly back to baseline. Qualitatively, the increase in Ca²⁺ was similar for all experimental groups examined; thus other representative images are not provided.

Fig. 3.

Chronic prenatal hypoxia increases bradykinin-mediated cytosolic Ca²⁺ responses. A and B: representative images (A) corresponding in time to the trace (B) of fractional fluo 4 fluorescence (F/F₀, where F is fluorescence intensity for the region of interest and F₀ is the fluorescence intensity during a period when there was no Ca²⁺ activity as determined automatically) for 4 pulmonary arterial myocytes from a fetus exposed to prenatal chronic hypoxia in the presence of extracellular Ca²⁺. Lowercase letters (a–d) denote times at which the images occurred. Colored circles in A correspond to the trace of the same color in B. Brightness of the images was adjusted for display purposes. Images were obtained with a ×40 water-immersion Plan-Apochromat objective at 1.28 frames per second. Scale bar = 20 μm.

The magnitude and kinetics of these fluorescence Ca²⁺ signals were then quantified. Figure 4 provides summaries of the quantitative analysis for various aspects of the Ca²⁺ responses. These data demonstrate that the amplitude (Fig. 4A) increases modestly after birth in arterial endothelial cells from control animals. However, prenatal chronic hypoxia enhances the amplitude of the Ca²⁺ signal in the fetus, but not the newborn. The area under the curve (Fig. 4B), event duration (Fig. 4C), and decay (Fig. 4D) decreased following birth but increased somewhat by chronic hypoxia in the newborn. The duration of Ca²⁺ rise (attack, Fig. 4E) became shorter after birth and following prenatal chronic hypoxia in cells from the fetus. Compared with the vasorelaxant influences of bradykinin, the data suggest that postnatal maturation enhances Ca²⁺ sensitivity, while chronic hypoxia augments Ca²⁺ signals, in neonatal endothelial cells but suppresses postnatal sensitivity.

Fig. 4.

Chronic hypoxia augments Ca²⁺ signals in neonates but suppresses sensitivity postnatally. A–E: amplitude of the fractional fluorescence, area under the curve (AUC), duration, decay, and attack of the Ca²⁺ event for regions of interest that were automatically detected during the fluorescence study shown in Fig. 3. Values are means ± SE. **P < 0.01, ***P < 0.001 for effect of chronic prenatal hypoxia (by Kruskal-Wallis nonparametric 1-way analysis of variance with Dunn's multiple-comparison test); †††P < 0.001, effect of maturation.

Prenatal chronic hypoxia and eNOS-dependent relaxation.

Given the uncoupling between endothelial Ca²⁺ signals and vasorelaxation following prenatal chronic hypoxia and postnatal maturation, the next series of studies tested the hypothesis that altered bradykinin-induced eNOS activation mediates this detachment. To evaluate this process, NO synthesis was inhibited with 100 μM N^G-nitro-l-arginine methyl ester (l-NAME) (71). Figure 5 shows that l-NAME successfully reduced bradykinin-induced vasorelaxation in 10 μM PE-precontracted vessels from the hypoxic fetus as well as from normoxic and hypoxic newborns. In addition to the increased sensitivity of fetal vessels to l-NAME following hypoxia, hypoxic fetal vessels displayed greater reductions in overall vasorelaxation. The summary data in Table 1 show that the potency of bradykinin-induced relaxation was unaffected by l-NAME. Table 2 demonstrates that the maximum response was not affected by eNOS inhibition in normoxic fetus but was significantly reduced in the normoxic and hypoxic newborn and the hypoxic fetus. Before birth, prenatal chronic hypoxia increased the role for eNOS, but the change in maximum response was virtually identical to that of hypoxic newborns. Overall, these findings suggest that prenatal chronic hypoxia accelerates development of eNOS-dependent vasorelaxation in the fetus, possibly involving sGC, PKG, or other downstream components of the pathway, but this acceleration restrains the normal development of pulmonary vascular relaxation to bradykinin.

Fig. 5.

Endothelial nitric oxide synthase (eNOS) inhibition reduces bradykinin-induced pulmonary arterial relaxation. A–D: dose-response curves of pulmonary arterial rings exposed to 10 pM–1 μM bradykinin in an additive manner normalized to %T_{10 μM PE} for fetal sheep exposed to normoxia and prenatal chronic hypoxia and newborn sheep exposed to normoxia and prenatal chronic hypoxia in the presence of DMSO (control) for vehicle control and 100 μM nitro-l-arginine methyl ester (l-NAME) for eNOS inhibition. Lines show resultant fits to dose-response relationships with a Hill equation. Values are means ± SE. Data were analyzed by 2-way analysis of variance with a Bonferroni posttest analysis for each dose: *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

Prenatal chronic hypoxia and prostacyclin-dependent vasorelaxation.

Prostacyclin is released by the endothelium and is another significant signaling arm activated by endothelial bradykinin stimulation (11). To test the role of prostacyclin in bradykinin-induced vasorelaxation, 10 μM indomethacin (Indo) was added to inhibit cyclooxygenase (COX)-dependent prostacyclin production. At this concentration, Indo inhibits COX-1 and -2 activity (61, 66). Figure 6 shows dose-dependent relaxation of 10 μM PE-precontracted vessels to bradykinin in the presence of 10 μM indomethacin. As illustrated in Fig. 6, A and B, COX inhibition attenuated the maximal vasorelaxation response in arteries from fetuses following prenatal chronic hypoxia, with no effect on arteries from normoxic fetuses. Figure 6C shows that the relaxation to bradykinin in normoxic newborn arteries was largely unaffected by COX inhibition, although PE vasoreactivity was enhanced. This compares with the data shown in Fig. 6D for hypoxic newborns, which were far more sensitive to Indo. Table 1 shows that COX inhibition failed to alter potency in all groups, but Table 2 shows that normoxic arteries were resistant to Indo, while prenatal chronic hypoxia unmasked prostacyclin-dependent relaxation in arteries from the fetus and the newborn.

Fig. 6.

Cyclooxygenase (COX) inhibition of prostacyclin synthesis suppresses bradykinin-induced relaxation. A–D: dose-response curves of pulmonary arterial rings exposed to 10 pM–1 μM bradykinin in an additive manner normalized to %T_{10 μM PE} for fetal sheep exposed to normoxia and prenatal chronic hypoxia and newborn sheep exposed to normoxia and prenatal chronic hypoxia in the presence of DMSO (control) and 10 μM indomethacin to inhibit prostacyclin production. Lines show resultant fits to the dose-response relationships with a Hill equation. Values are means ± SE. Data were analyzed by 2-way analysis of variance with a Bonferroni posttest analysis for each dose: **P < 0.01, ***P < 0.001 vs. control.

Prenatal chronic hypoxia and sGC relaxation.

sGC is activated by NO in smooth muscle downstream of eNOS stimulation (4). Such activation leads to cGMP formation with subsequent vasorelaxation. To establish a more complete picture of the impact of prenatal chronic hypoxia on the role of sGC, we inhibited this enzyme with 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) prior to endothelial stimulation with bradykinin (29). Figure 7 shows the dose-dependent vasorelaxation to bradykinin in the presence and absence of ODQ. Figure 7, B–D, shows that sGC inhibition suppresses bradykinin-mediated relaxation in the normoxic and hypoxic newborn and the hypoxic fetus, while the meager relaxation in the normoxic fetus was relatively unaffected (Fig. 7A). As summarized in Table 1, ODQ treatment did not affect potency in arteries from fetal or newborn normoxic or hypoxic sheep. However, Table 2 shows that the maximum response was reduced in all groups, except normoxic fetus.

Fig. 7.

Soluble guanylate cyclase inhibition suppresses bradykinin-mediated relaxation. A–D: dose-response curves of pulmonary arterial rings exposed to 10 pM-1 μM bradykinin in an additive manner normalized to %T_{10 μM PE} for fetal sheep exposed to normoxia and prenatal chronic hypoxia and newborn sheep exposed to normoxia and prenatal chronic hypoxia in the presence of DMSO (control) and 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) to inhibit soluble guanylate cyclase. Lines show resultant fits to the dose-response relationships with a Hill equation. Values are means ± SE. Data were analyzed by 2-way analysis of variance with a Bonferroni posttest analysis for each dose: *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

The results of the l-NAME and ODQ studies illustrate that eNOS and sGC are important to bradykinin-dependent relaxation. It is possible that chronic hypoxia accelerates their development in utero but impedes normal development of these or other pathway components after birth. Nonetheless, previous evidence indicates that eNOS expression is unaffected by prenatal chronic hypoxia in our fetal sheep (91). Because of these findings, we performed a series of experiments to examine the direct impact of NO on vessel reactivity. In particular, we evaluated the potential for NO to directly stimulate sGC activity by addition of the NO donor ProliNO (76). Figure 8 shows results from 10 μM 5-HT-preconstricted vessels relaxed with ProliNO in the presence and absence of the sGC inhibitor ODQ, where 5-HT was used because it induces robust contraction (35). Overall, ProliNO effectively relaxed 5-HT-precontracted vessels from normoxic and prenatal chronic hypoxic fetal and newborn sheep (Fig. 8A). sGC inhibition effectively reduced the potency in all groups and reduced the maximum response of ProliNO-induced relaxation in the hypoxic fetus.

Fig. 8.

Nitric oxide (NO)-dependent vasorelaxation is independent of age and chronic hypoxia. A: N-nitrosoproline (ProliNO)-induced isometric tension normalized to maximum 10 μM serotonin (5-HT)-precontracted tension (%T_{10 μM 5-HT}) for fetal sheep exposed to normoxia and prenatal chronic hypoxia (FN and FH) and newborn sheep exposed to normoxia and prenatal chronic hypoxia (NBN and NBH). B–E: fetal and newborn sheep exposed to normoxia and prenatal chronic hypoxia in the absence of ODQ (NO), in the presence of 10 μM ODQ, and in the presence of NaOH (control) as a vehicle- and time-matched control. Lines show resultant fits with a Hill equation to dose-response relationships. Values are means ± SE. Data were analyzed by 2-way analysis of variance with a Bonferroni posttest analysis for each dose: *P < 0.05, **P < 0.01, ***P < 0.001 between groups (A) or relative to the presence of ProliNO (B–E).

BK_Ca channels and bradykinin relaxation.

K⁺ channels are critical for establishing myocyte resting membrane potential and are frequent targets of endogenous and therapeutic vasodilators (52). One primary target of NO-dependent signaling is the BK_Ca channel (79). The BK_Ca channel is stimulated by ryanodine receptor-dependent Ca²⁺ sparks and independently modulated by PKA and PKG downstream of sGC and cGMP (3, 74, 77, 79). To establish the role of BK_Ca channels in bradykinin-mediated vasorelaxation, arteries were treated with tetraethylammonium (TEA) at 1 mM, a concentration that blocks roughly one-half of the current due to BK_Ca channels (44, 46). Figure 9 shows the dose-dependent relaxation of PE-precontracted vessels to bradykinin in the presence and absence of 1 mM TEA. BK_Ca channel inhibition with TEA had no significant effect on bradykinin-induced vasorelaxation in normoxic fetal and hypoxic newborn vessels, as shown in Fig. 9, A and D, respectively. However, Fig. 9B shows that 1 mM TEA impaired vasorelaxation mediated by 1 μM bradykinin in arteries from prenatal chronic hypoxic fetuses, while inhibition of the BK_Ca channel had no effect on bradykinin-induced relaxation in arteries from hypoxic newborns (Fig. 9D). BK_Ca channel inhibition weakened bradykinin-induced relaxation in the normoxic newborn (Fig. 9C), indicating a substantial developmental increase in the role of the BK_Ca channel in bradykinin relaxation. Table 1 shows that 1 mM TEA failed to affect the potency of bradykinin-induced relaxation in any of the groups. Table 2 shows that BK_Ca channel inhibition reduced the maximum response in the prenatal chronic hypoxic fetus and the normoxic newborn. Although chronic prenatal hypoxia promoted BK_Ca-related relaxation in the fetus, it suppressed the normal development of BK_Ca-mediated relaxation in the newborn.

Fig. 9.

Prenatal chronic hypoxia promotes importance of BK_Ca channels to bradykinin-mediated arterial relaxation in the fetus but stunts normal relaxation in the newborn. A–D: dose-response curves of pulmonary arterial rings exposed to 10 pM–1 μM bradykinin in an additive manner normalized to %T_{10 μM PE} for fetal sheep exposed to normoxia and prenatal chronic hypoxia and newborn sheep exposed to normoxia and prenatal chronic hypoxia in the presence of DMSO (control) and 1 mM tetraethylammonium (TEA) to inhibit BK_Ca channels. Lines show resultant fits to the dose-response relationships with a Hill equation. Values are means ± SE. Data were analyzed by 2-way analysis of variance with a Bonferroni posttest analysis for each dose: *P < 0.05, **P < 0.01 vs. control.

BK_Ca channel protein expression.

It has been well established that the BK_Ca channel is a crucial regulator of myogenic tone and blood pressure and is especially important to O₂-induced pulmonary arterial relaxation at birth (12, 70, 86). The BK_Ca channel is made up of four pore-forming α-subunits to which accessory β-subunits can attach (51, 63). Four classes of β-subunits have been identified, although there may be more (62, 84). These accessory β-subunits can regulate voltage and Ca²⁺ sensitivity to specialize BK_Ca channel-opening behavior, with the β₁-subunit being the most common β-subunit in vascular smooth muscle cells (13, 63). In its absence, mouse arterial tone and blood pressure have been shown to increase due to a reduction in BK_Ca channel Ca²⁺ sensitivity and coupling to Ca²⁺ sparks (69). Furthermore, other studies have shown that the activity of the BK_Ca channel is developmentally regulated and is differentially affected by Po₂ (73). To determine whether the increase in relaxation in fetal arteries following prenatal chronic hypoxia was mediated by a change in BK_Ca channel expression, Western immunoblot analysis was conducted for the α- and β₁-subunits. Figure 10 shows the results of this analysis. As shown in Fig. 10B, maturation of the fetus to the newborn significantly decreased α-subunit expression under normoxic conditions. However, α-subunit expression did not decrease further following prenatal chronic hypoxia. Figure 10, C and D, shows that maturation significantly increased β₁-subunit expression, as well as the ratio of β₁- to α-subunit expression, regardless of oxygenation status.

DISCUSSION

These studies are the first to examine the influence of prenatal chronic hypoxia on bradykinin-mediated pulmonary arterial relaxation of the fetus and the newborn and to elucidate the roles of important underlying signaling pathways. A schematic summary of these findings and the relationship to our previous work is presented in Fig. 11 (37). In the pulmonary arterial endothelium, we examined the importance of Ca²⁺ elevations, eNOS, and COX signaling. In smooth muscle, we evaluated subsequent activation of sGC and downstream influences on BK_Ca channels. Our studies demonstrate that prenatal chronic hypoxia accentuates relaxation in the fetal pulmonary vasculature through enhanced pathway development, but this elevation leads to impediments following birth that ultimately impair pulmonary vascular relaxation in the newborn.

Fig. 11.

Influence of early postnatal maturation and intrauterine chronic hypoxia on bradykinin-induced vasorelaxation in fetal and newborn pulmonary vessels. Schematic provides an overview of the findings of the current studies. Data indicate that postnatal chronic hypoxia has an interactive influence on normal development of bradykinin-induced vasorelaxation, with development that involves modification to the function of the endothelium (above horizontal hashed line) and smooth muscle (below horizontal hashed line). Influence of developmental age is shown at the left of each pathway step; effect of chronic hypoxia on the fetus (F) and newborn (N) are shown at the right. Pathways are depicted as a coupled sequence (straight lines) or through a multistep process (dashed lines), where intermediate pathway steps were not evaluated. sGC, soluble guanylate cyclase; RyR, ryanodine receptor (see Ref. 37).

Prenatal chronic hypoxia enhanced bradykinin-mediated pulmonary arterial relaxation in the fetus. This was a surprise, because chronic hypoxia generally increases pulmonary arterial pressure and depresses pulmonary arterial vasodilatory capacity, as was observed in the newborn (Fig. 2D) and in previous studies (40). The findings suggest that fetal sheep adapt in utero to compensate for eventual birth in a high-altitude, low-O₂ environment. On the basis of this line of reasoning, the resultant increase in blood flow at birth in high-altitude-adapted fetuses with heightened bradykinin-mediated arterial relaxation would enhance O₂ diffusion from the alveoli into the bloodstream. O₂ uptake would be improved when the newborn breathes, allowing the animal to thrive during the fetal-to-newborn transition.

This theoretical cause-and-effect relationship was not seen in high-altitude newborn sheep 2 wk after birth. Rather, the normal course of the development of pulmonary vasorelaxation was restricted (Fig. 2D). This loss could potentially contribute to the elevation in pulmonary pressures and increases in pulmonary vascular resistance that we and others have documented in lambs born at high altitude (10, 40). Furthermore, it is possible that the prenatal acceleration of vasorelaxation responses to bradykinin is redistributing blood flow from developing vital organs to the lung, resulting in blood flow distribution issues. Such an alteration of distribution following chronic hypoxia was previously reported in studies on our fetal sheep (47). These studies showed that blood flow to the fetal brain and heart was maintained, despite a significant decline in cardiac output and reduction of flow throughout the body. This preservation of perfusion is indicative of the autoregulatory nature of blood flow to the brain and heart. If chronic prenatal hypoxia also decreases fetal pulmonary vascular resistance and reallocates blood flow to the lung, these effects could, jointly, place additional stress on other vital organ systems that potentially compromises their function and growth.

The improvement in bradykinin potency following birth could be due to various modifications in the bradykinin signaling pathway. On the basis of classical pharmacological principles, there could be changes in receptor density, ligand affinity, or receptor coupling. Changes in bradykinin receptor expression likely underlie the enhancements and reductions in the maximal relaxation response, but not the effects on potency. The modifications in potency more likely reflect improved receptor affinity or coupling of the receptor to intracellular signaling pathways. Receptor glycosylation can influence receptor occupancy and ligand binding affinity (53). Another possibility is that changes in expression of the angiotensin (AT) type 2 (AT₂) receptor can accentuate the potency of bradykinin (7, 92). Indeed, in the mouse lung, AT₂ receptor mRNA expression increases following antenatal hypoxia, while AT₁ receptor expression remains stable (34). This finding mirrors the protein expression changes with ontogeny, where AT₂ receptor expression increases from the neonatal mouse to the adult, while AT₁ receptor expression is steady (25). Another alternative that influences receptor activation is that there could be alterations in tyrosine or serine/threonine phosphorylation, which are important to bradykinin receptor desensitization (53). Ontogeny also could lead to modifications in cell signaling pathways downstream of bradykinin receptor activation. The reduction in Ca²⁺ signaling supports this premise and illustrates that ontogeny enhances Ca²⁺ sensitization of eNOS. However, there also may be changes in MAPK, JAK/STAT, or other signaling systems, such as changes in receptor acylation, which influences G protein receptor coupling (53). Although it is quite plausible that one or more of these options are involved in the changes in bradykinin potency, it was beyond the scope of the present studies to evaluate this in depth. Nonetheless, such changes are important, as they likely facilitate vessel relaxation and are, therefore, important during the transition to air-breathing.

NO signaling is well regarded as being vital to pulmonary vasorelaxation (17, 38, 64, 67). Thus the finding that eNOS was important to bradykinin-induced vasorelaxation was expected. Although the data are not wholly conclusive, the prospect that eNOS inhibition has a greater effect on the arteries of fetal prenatal chronic hypoxic sheep than on the arteries of normoxic fetal sheep is intriguing. These enhancements in eNOS-dependent vasorelaxation are potentially related to the increased endothelial Ca²⁺ signaling observed in prenatal chronic hypoxic vessels and concomitant enhancement in Ca²⁺-dependent activation of eNOS (36, 64).

The discovery that bradykinin Ca²⁺ signals in the endothelium were suppressed following birth, although relaxation was enhanced, is provocative. These data suggest that, after birth, eNOS is more readily activated by Ca²⁺. The improvement of Ca²⁺-eNOS coupling can be due to a number of processes (17, 36, 82). Potentially, amino acid residues that sensitize eNOS to calmodulin, as well as Ca²⁺, are phosphorylated (36). Binding partners may have greater interaction, or there may be changes in the abundances of cofactors such as l-arginine, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin that facilitate NO production directly or indirectly (23, 64, 82). These changes could also include alterations in spatial-temporal coupling based on the characteristics of the Ca²⁺ signals and eNOS cellular distribution (22, 64). Indeed, there was a faster rate of Ca²⁺ rise following chronic hypoxia that paralleled the changes due to ontogeny. In addition, after birth, the decay of Ca²⁺ was more rapid, an effect that was blunted by hypoxia. These findings, coupled with the multifaceted changes in the Ca²⁺ spike amplitude and area under the curve, suggest that there are complex changes in release, influx, and sequestration, as well as extrusion. This includes modifications in the expression or activation of inositol trisphosphate receptors or Ca²⁺-permeable ion channels at the plasma membrane, extrusion of Ca²⁺ from the cytosol by plasma membrane or endoplasmic reticulum Ca²⁺-ATPases, or even storage of Ca²⁺ in the endoplasmic reticulum (32, 33). Resolution of the developmental mechanisms that govern increased bradykinin-mediated relaxation through eNOS-Ca²⁺ coupling and the influence of chronic hypoxia on the developmental process are important but require additional experimentation.

The finding that sGC and eNOS antagonism caused equivalent inhibition of bradykinin-mediated relaxation provides additional evidence that a substantial portion of the bradykinin relaxation is through coupling of eNOS and sGC. Furthermore, the inhibitory studies suggest that the fidelity of coupling between eNOS and sGC is not modified by maturation or chronic hypoxia. Nonetheless, ProliNO greatly intensified and normalized the extent of relaxation across all groups (Fig. 8). Interestingly, sGC inhibition shifted the potency of ProliNO as opposed to reducing the maximal relaxation to bradykinin shown in Fig. 7. Although such differences in how sGC inhibition influences bradykinin vs. ProliNO relaxation are unlikely, they may reflect nonspecific effects of ODQ that attenuate the bradykinin-induced vasodilation pathway at points upstream of sGC activation (6, 29, 80, 85). The differences in the actions of ODQ also suggest that while a major portion of bradykinin-induced relaxation is through cGMP generation, its mechanism of action may also involve NO degradation through phosphodiesterases or NO-independent pathways (6, 85). The ProliNO data further illustrate that bradykinin does not maximally activate relaxation pathways and that there is substantial NO-sensitive and sGC-insensitive reserve. This sGC-insensitive reserve likely includes protein nitrosylation, which is a key mediator of NO-dependent signaling (55).

Although prostacyclin signaling pathways were not fully evaluated, our indomethacin studies were revealing. The lack of a role for COX-dependent vasorelaxation in the fetal normoxic studies was expected and follows from earlier studies that illustrate that prostacyclins have little effect on pulmonary vascular tone until after birth (54). The failure of indomethacin to substantially reduce bradykinin-dependent relaxation in newborns was interesting and compares with previous studies. In studies performed in newborn sheep as well as goats, direct stimulation with prostacyclin agonists and precursors elicits pulmonary vascular vasorelaxation (15, 28). One explanation is that bradykinin does not fully activate resident prostacyclin synthesis pathways. The finding that chronic hypoxia augmented indomethacin-sensitive bradykinin-induced relaxation was also unexpected, as previous work in piglets showed that prostacyclin production is suppressed (20, 21). Resolving whether greater indomethacin sensitivity is due to improved activation of dormant prostacyclin synthesis pathways or whether there are phenotypic changes that increase the expression of key synthesis enzymes or pathway elements in the arterial myocytes that govern prostacyclin-dependent vasorelaxation is worthy of future investigation (20, 21, 28).

The suppression of the NOS-independent component to bradykinin-induced vasodilation following hypoxia in the newborn is intriguing. Restriction of prostacyclin-dependent signaling is unlikely, because our indomethacin data suggest that PLA₂-dependent mechanisms are upregulated following chronic hypoxia. However, chronic hypoxia may suppress the function of other NO synthase-independent pathways important to vasodilation not described in this report, including activation of stored forms of NO (16) and epoxyeicosatrienoic acids (14), as well as myoendothelial gap junctions (24, 50). Resolution of which, if either, of these or other potential ionic or nonionic pathways are suppressed by chronic hypoxia requires additional studies.

Bradykinin-induced relaxation in porcine pulmonary resistance arteries is dependent on the activity of multiple K⁺ channels (5). In particular, BK_Ca channels in vascular smooth muscle are important to fetal bradykinin-induced pulmonary arterial relaxation. Previous work has also shown that BK_Ca channels are important to O₂-dependent relaxation in fetal ovine pulmonary arteries (65, 70). Our findings are consistent with these reports and suggest that prenatal chronic hypoxia augments the role for BK_Ca channels in fetal sheep (Fig. 9B), which may provide a distinct advantage for the lamb during birth. Nonetheless, whatever temporary advantage this increased role for BK_Ca channels may provide during the fetal-to-newborn transition is followed quickly by impaired development of normal BK_Ca channel-related vasorelaxation 2 wk after birth (Fig. 9D).

Impediments in BK_Ca channel function are often caused by chronic hypoxia-induced channel dysfunction or changes in subunit expression. In the present study we inhibited BK_Ca channel activity in myography studies and conducted Western immunoblots to investigate the possibility that BK_Ca channel expression parallels the changes in BK_Ca channel-mediated relaxation (Fig. 9). Such a correlation would indicate that the augmented role for BK_Ca channels in the fetus and the attenuation of normal BK_Ca relaxation in the newborn are brought on by hypoxia-induced changes in channel expression, as we observed in the uterine arteries of pregnant ewes (44). However, the tension and expression studies did not agree with one another. The lack of change in α- or β₁-subunit expression following chronic hypoxia in the fetus or newborn (Fig. 10) was surprising. We expected to see increases in α- and β₁-subunit expression in the prenatal chronic hypoxic fetus to explain the increase in vasorelaxation (Fig. 9B). Indeed, previous studies have shown such a correlation between BK_Ca channel function and expression, where BK_Ca channels in cerebral arteries and aorta of spontaneously hypertensive rat arteries undergo a decrease in Ca²⁺ sensitivity due to a disproportionate expression of α- and β₁-subunits, either by downregulation of the β₁-subunit or upregulation of the α-subunit (2, 58). Furthermore, a study of fetal and adult cultured ovine pulmonary arterial smooth muscle cells showed that short-term hypoxia increased expression of the α- and β₁-subunits (72). The discrepancies between our results and those of previous studies point to a number of possible explanations. The implication is that sustained, intrauterine low-O₂ conditions have a unique effect on the function and expression of the BK_Ca channel compared with the effects seen following induced, short-term hypoxia. However, we have yet to explore activation of whole cell or transient BK_Ca currents by membrane voltage or the coupling to Ca²⁺ sparks, which are vital to how BK_Ca channel-induced membrane hyperpolarization induces vessel relaxation (12, 37).

The increase in BK_Ca channel activity following prenatal chronic hypoxia in the fetus may also be explained by changes in expression or posttranslational modifications that increase BK_Ca channel function. Studies also illustrate that single-nucleotide polymorphisms within the α- or β₁-subunit can confer a gain or loss of function (8, 19). It follows that “gain-of-function” alternative splice variants of the α- and β₁-subunits could be activated during hypoxic stress, thereby conferring an increase in Ca²⁺ sensitivity and vasorelaxation. The location of the subunits is also vital to channel function. For example, in addition to the plasma membrane, BK_Ca channels are also expressed in the mitochondrial membrane of neurons, and this can influence cell excitability (75, 90). Unfortunately, our Western immunoblot analysis only measured whole cell protein content and did not provide information regarding subunit cellular location. Thus it is possible that chronic prenatal hypoxia could modify channel targeting and the proportion of the channels retained in the sarcoplasmic-endoplasmic reticulum vs. those channels transported to the mitochondrial and plasma membranes. An alternative explanation for our findings is that chronic prenatal hypoxia induces changes in the expression of BK_Ca channel γ-subunits, which interact with β₁-subunits. In a key study performed in rat cerebral arterial myocytes, the γ-subunit was found to be important to vasorelaxation, where it facilitated the coupling of BK_Ca channels to Ca²⁺ sparks by shifting voltage dependence and Ca²⁺ sensitivity, assisting in membrane hyperpolarization and concomitant vasorelaxation (18). Thus one potential explanation for the mismatch between data from tension studies and α- and β₁-subunit expression data could be an upregulation of the γ-subunit, which improves BK_Ca channel function. Ultimately, however, resolution of this and other potential explanations requires further investigation.

Although the data suggest that BK_Ca channel-dependent dilation of pulmonary arteries is mediated through the NO-cGMP-PKG signaling axis, PKG also causes vasodilation through other mechanisms. In addition to activating K⁺ channels, hyperpolarizing the plasma membrane, and reducing voltage-dependent Ca²⁺ influx, PKG also functionally decreases the sensitivity of contraction to increases in the cytosolic Ca²⁺ concentration. In particular, PKG can dilate vessels by promoting dephosphorylation of the myosin light chain kinase. This is achieved by phosphorylation and activation of the myosin light chain phosphatase (26, 27, 81). Furthermore, PKG inhibits RhoA activity, which is a key modulator of the Rho kinase pathway (27, 49). This is directly relevant to the current studies, as we previously showed that Rho kinase accounts for roughly half of the arterial tension due to serotonin or membrane depolarization in pulmonary arteries of sheep (10, 68). Future studies are therefore needed to discriminate the interaction between PKG and various pathways.

Perspective.

The present series of studies illustrate that bradykinin-dependent vasorelaxation is mediated through a combination of eNOS-sensitive and -insensitive pathways. From a methodological standpoint, our results demonstrate the importance of conducting whole animal studies, in addition to in vitro experimentation. Sustained stress, such as prenatal chronic hypoxia, can result in complex changes via the interaction of numerous systems not represented by inducing short-term stress in isolated cells. Terrestrial prenatal chronic hypoxia due to living at moderately high altitude influenced the development of eNOS-dependent pathways, causing adaptations that improve pulmonary blood flow and blood oxygenation in the fetus and newborn infant. Presumably, these adaptations allow newborn lambs to survive birth in the rarified environment. Unfortunately, the increased blood flow that results from these adaptations may lessen blood flow necessary for the proper development of other vital organs. On the basis of our data, prenatal chronic hypoxia blunts normal development of bradykinin-mediated vasodilatory pathways, resulting in lessened responsiveness. In turn, this could exacerbate pulmonary pressures and reduce the ability of newborn hypoxic lambs to thrive. On the whole, our results demonstrate that prenatal chronic hypoxia takes a significant toll on proper pre- and postnatal development of the bradykinin-activated, eNOS-BK_Ca channel vasodilatory pathway in sheep. The dramatic effects of hypoxia observed here provide valuable insights into the mechanisms contributing to pulmonary hypertension in the newborn and demonstrate the potential for targeting the eNOS-BK_Ca channel signaling axis in afflicted newborns.

GRANTS

This material is based on work supported by National Science Foundation Grant MRI 0923559 and National Institutes of Health Grants HD-069746 (S. M. Wilson), HL-085887 (M. S. Taylor), R01 NS-076945 (W. J. Pearce), HL-095973 (A. B. Blood), and P01 HD-031226 and R01 HD-003807 (L. D. Longo). Additional funding for research reported in this publication was provided by National Institute of General Medical Sciences Grant 2R25 GM-060507. C. Blum-Johnston was a summer research fellow in the Apprenticeship Bridge to College and Undergraduate Training Programs through the Initiative for Maximizing Student Development Program in the Center for Health Disparities and Molecular Medicine. Additional support for the Advanced Imaging and Microscopy Core was provided by the Loma Linda University School of Medicine.

DISCLAIMERS

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or National Science Foundation.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.B.-J., R.B.T., C.W., M.R., A.R.B., Q.B., R.W., and S.M.W. analyzed the data; C.B.-J., R.B.T., C.W., M.R., A.R.B., Q.B., R.W., A.B.B., M.F., M.S.T., L.D.L., W.J.P., and S.M.W. interpreted the results of the experiments; C.B.-J., R.B.T., C.W., M.R., A.R.B., Q.B., and S.M.W. prepared the figures; C.B.-J., R.B.T., C.W., L.D.L., W.J.P., and S.M.W. drafted the manuscript; C.B.-J., R.B.T., C.W., M.R., A.R.B., Q.B., R.W., A.B.B., M.F., M.S.T., L.D.L., W.J.P., and S.M.W. edited and revised the manuscript; C.B.-J., R.B.T., C.W., M.R., A.R.B., Q.B., R.W., A.B.B., M.F., M.S.T., L.D.L., W.J.P., and S.M.W. approved the final version of the manuscript; R.B.T., A.B.B., M.F., M.S.T., L.D.L., W.J.P., and S.M.W. developed the concept and designed the research; R.B.T., M.R., A.R.B., Q.B., R.W., and S.M.W. performed the experiments.