1 Division of Pediatric Pulmonology and Critical Care Medicine, Department of Pediatrics, and 2 Department of Physiology, University of Minnesota, Minneapolis 55455; and 3 Department of Medicine, Department of Veterans Affairs Medical Center and University of Minnesota, Minneapolis, Minnesota 55417
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ABSTRACT |
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To examine mechanisms underlying developmental changes in pulmonary vascular tone, we tested the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the pulmonary artery (PA) smooth muscle cells (SMCs); 2) voltage-gated K+ (Kv)-channel activity increases with maturation; and 3) O2-sensitive Kv2.1 channel expression and message increase with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of acute hypoxia on cytosolic Ca2+ concentration ([Ca2+]i) in SMCs isolated from adult and fetal PAs. Although PASMCs from both fetal and adult circulations were able to sense an acute decrease in O2 tension, acute hypoxia induced a more rapid and greater change in [Ca2+]i in magnitude in PASMCs from adult compared with fetal PAs. To determine developmental changes in Kv-channel activity, the effects of the K+-channel antagonist 4-aminopyridine (4-AP) were studied on fetal and adult PASMC [Ca2+]i. 4-AP (1 mM) caused PASMC [Ca2+]i to increase by 94 ± 22% in the fetus and 303 ± 46% in the adult. Kv-channel expression and mRNA levels in distal pulmonary arteries from fetal, neonatal, and adult sheep were determined through the use of immunoblotting and semiquantitative RT-PCR. Both Kv2.1-channel protein and mRNA expression in distal pulmonary vasculature increased with maturation. We conclude that there are maturation-dependent changes in PASMC O2 sensing that may render the adult PASMCs more responsive to acute hypoxia.
potassium channels; oxygen sensing; fetus; pulmonary artery; hypoxic pulmonary vasoconstriction; cytosolic calcium
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INTRODUCTION |
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THE MECHANISMS RESPONSIBLE for the maintenance of the high-tone, low-flow fetal pulmonary vasculature and the rapid increase in pulmonary blood flow that occurs at birth remain incompletely understood (29). Evidence suggests that the low O2 tension environment of the normal fetus may contribute to the high fetal pulmonary vascular resistance by direct effects on fetal pulmonary artery (PA) smooth muscle cells (SMCs) K+ channels (13, 27). At birth, pulmonary blood flow increases 8- to 10-fold while PA pressure declines steadily over the first several hours of life (11, 15). Although physical factors and vasoactive products elaborated by the pulmonary vascular endothelium have been clearly shown to be involved in the regulation of perinatal vascular tone (1, 12, 16, 20, 33, 38), sustained and progressive perinatal pulmonary vasodilation requires PASMC K+-channel activation (13, 36).
Recent studies have demonstrated that activation of the Ca2+-sensitive K+ channels (K Ca) play a critical role in perinatal pulmonary vasodilation (13). O2 causes perinatal pulmonary vasodilation through kinase-dependent activation of the K Ca channel. K+-channel inhibition with tetraethylammonium, but not with glibenclamide, blocker of the ATP-sensitive K+ channels, blocks the pulmonary vasodilation caused by ventilation (36), suggesting that ventilation causes sustained and progressive pulmonary vasodilation through activation of tetraethylammonium-sensitive K+ channels. Nitric oxide (NO), a vasoactive mediator that is essential for the normal transition of the pulmonary vasculature (1), causes perinatal pulmonary vasodilation, at least in part, through K Ca-channel activation (5, 10). Recent work demonstrates that NO causes perinatal pulmonary vasodilation through K Ca-channel activation and requires release of intracellular Ca2+ from a ryanodine-sensitive store (31). Even the shear stress response, induced by compression of the ductus arteriosus during fetal life, results from K Ca- and voltage-dependent K+ (Kv)-channel activation (35).
The recent observation that the K+ channel setting of the resting membrane potential (RMP) in the pulmonary circulation changes after birth from a K Ca to a Kv channel suggests developmental regulation of K+ channels in the pulmonary circulation (27). In adult animals, acute hypoxia causes inhibition of Kv-channel activity and membrane depolarization of PASMCs (26, 45), leading to opening of voltage-operated Ca2+ channels, an increase in cytosolic Ca2+ concentration ([Ca2+]i), and vasoconstriction. Previous investigators have identified the Kv2.1 channel as an O2-sensitive K+ channel in the pulmonary circulation alone (8) or as a heterotetramer (24). In this manner, the PASMCs can respond to hypoxia with vasoconstriction, thereby directing pulmonary blood flow away from poorly ventilated portions of the lung and preventing intrapulmonary shunting and systemic hypoxemia (42).
Maturation-related differences in the capacity of the pulmonary vasculature to respond to changes in both O2 tension and endothelium-dependent vasodilator agents have been previously described (2, 22, 46). For example, a 4-6 Torr increase in O2 tension in the late-gestation ovine fetus causes a three- to fourfold increase in pulmonary blood flow (4), whereas in the early-gestation animals, there is no increase in pulmonary blood flow (23). Similarly, in several species, hypoxic pulmonary vasoconstriction has been shown to increase with maturation (17, 28, 39). Pulmonary vasodilation caused by endothelium-dependent nitric oxide also increases with maturation (2). If there is a developmentally regulated change in the K+ channel that sets the RMP and enables the adult pulmonary circulation to respond to an acute decrease in O2 tension with vasoconstriction, then PASMC Kv-channel activity, protein, and expression might be expected to parallel the previously described physiological changes and increase with maturation.
We therefore proposed the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the PASMCs; 2) Kv-channel activity increases with maturation; and 3) Kv2.1-channel protein and mRNA levels increase with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of acute hypoxia on [Ca2+]i of SMCs isolated from adult and fetal PAs. To determine any changes in K+-channel activity with maturation, the effect of a Kv-channel antagonist, 4-aminopyridine, on PASMC [Ca2+]i was studied on SMCs isolated from late-gestation fetal and adult ovine PAs. Kv2.1-channel protein expression and mRNA levels were determined through the use of immunoblotting and semiquantitative RT-PCR.
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METHODS |
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Ca2+ Imaging
Cell culture. The techniques used for isolation and culture of ovine PASMCs have been previously described (14). Distal PAs were quickly excised from pentobarbital sodium-anesthetized ovine fetuses ranging in gestational age from 125 to 142 days (term = 147 days) and placed in physiological saline solution (in mM: 120 NaCl, 5.9 KCl, 11.5 dextrose, 25 NaHCO3, 1.2 NaH2PO4, 1.2 MgCl2, and 1.5 CaCl2). PASMCs were isolated from third-generation resistance PAs. Loose connective tissue and adventitia were removed, and the vessels were liberally rinsed with minimal essential medium (MEM; 0.2 mM Ca2+). Vessel segments were carefully cut into small pieces and placed in 50-ml conical flasks containing 5.0 ml of the enzymatic dissociation mixture, which consisted of 0.125 mg/ml elastase (Sigma, St. Louis, MO), 1 mg/ml collagenase (Worthington Biochemical, Freehold, NJ), 2.0 mg/ml BSA, 0.375 mg/ml soybean trypsin inhibitor (Sigma), and 4 ml of MEM. After incubation at 37°C for 60 min in a shaking bath, the tissue suspension was triturated 10 times every 15 min in a plastic pipette for a total incubation period of 90-120 min. The tissue suspension was then passed through a 100-µm nylon mesh (Nitex, Tetka, Elmsford, NJ) to separate dispersed cells from undigested vessel wall fragments and debris. The filtered suspension was centrifuged (200 g for 10 min), and the cell pellet was resuspended in 10 ml of MEM supplemented with 10% heat-inactivated calf serum. The dispersed cell suspension was divided into aliquots on 25-mm2 glass coverslips coated with poly-L-lysine (Sigma) and in 25-cm2 tissue culture flasks (Falcon Plastics, Oxnard, CA) at a density of 5-10 × 103 cells/cm2. The cells were incubated at 37°C in a humidified 95% air-5% CO2 atmosphere. After 18-24 h, the cultures were washed once with Hanks' balanced salt solution (HBSS) to remove nonadherent cells and debris and refed with fresh medium. Medium was routinely exchanged at 72-h intervals. Cells were studied between days 5 and 14 of culture. Cell density stabilized as subconfluent monolayers after 3-5 days in culture. To verify the cell population, PASMCs were routinely stained with actin-specific antibody after 5, 10, and 14 days in culture.
Solutions. HBSS was used during experimentation. The concentrated HBSS solution contained (in g/500 ml) 2 KCl, 0.15 KH2PO4, 40 NaCl, 1.75 NaHCO3, 0.12 Na2HPO4, and 5 glucose. On the day of experimentation, a physiological saline solution was made by diluting stock HBSS and adding CaCl2, MgCl2, HEPES, and glucose to final concentrations of (in mM) 2 CaCl2, 2 MgCl2, 10 HEPES, and 11.1 glucose. All solutions were made with nanopure distilled water. Osmolality was adjusted to ~300 mosmol/kgH2O, and pH was adjusted to 7.4.
Experimental technique. To assess dynamic changes in [Ca2+]i in individual PASMCs, we used the Ca2+-sensitive fluorophore fura 2-AM (the acetoxymethyl derivative of fura 2; Molecular Probes, Eugene, OR). Subconfluent fetal PASMCs on 25-mm2 glass coverslips were placed on the stage of an inverted microscope (Nikon Diaphot). Cells were loaded with 10 nM fura 2-AM plus 2.5 mg/ml pluronic acid (Molecular Probes) for 20 min in Ca2+-free solution followed by a 20-min wash in Ca2+-containing solution before the start of the experiment. Ratiometric imaging was performed with excitation wavelengths of 340 and 380 nm and an emission wavelength of 560 nm. Imaging was performed with an intensified charge-coupled device camera with Axon Instruments (Foster City, CA) image capture and analysis software. Ca2+ calibration was achieved by measuring a maximum ratio (Rmax; with 1 mM ionomycin) and a minimum ratio (Rmin; with 10 mM EGTA) for each cell. O2 tension was controlled by aerating the heated HBSS reservoir with either 21% O2-balance N2 or 100% N2. A Clark electrode was placed in the HBSS reservoir to continuously monitor PO2. Further control of PO2 was obtained by aerating the stage microincubator with 21% O2-balance N2 or 100% N2. For experiments performed under hypoxic conditions, the temperature of the cells was maintained by using a stage microincubator equipped with a thermocoupling device. pH was 7.40 ± 0.05 and did not change during the experiments.
Intracellular free Ca2+ was calculated with the formula [Ca2+]i (in nM) = Kd × (Fo /Fs) × (RWestern Blotting
Lung tissue was extracted and immediately placed on ice. The main intralobar PAs were exposed by gentle dissection and fourth- and fifth-generation branches were carefully isolated and removed. After adventitial tissue was removed, intralobar pulmonary arteries were removed, frozen in liquid nitrogen, and ground to a powder with a prechilled mortar and pestle. Tissues were homogenized on ice in 0.01 M phosphate-buffered saline, pH 7.4, with protease inhibitors (0.01 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA; all from Sigma) and 0.01 mg/ml leupeptin (Boehringer Mannheim, Indianapolis, IN). The homogenate was spun at 300 g for 10 min to remove large debris. The supernatant was combined with Triton in phosphate-buffered saline to yield a final Triton concentration of 1%, incubated on ice for 1 h, and centrifuged at 16,000 g for 20 min. The protein content of the supernatant was assayed with the bicinchoninic acid method (BCA Protein Assay Kit, Pierce, Rockford, IL). Western blotting was performed with 80 µg crude protein/lane. Protein samples were subjected to SDS-PAGE according to the method of Laemmli (19) in a minigel system (Bio-Rad). Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes for 60 min at 100-V constant voltage using a mini-trans-blot cell. After protein transfer, the PVDF membranes were rinsed in Tris-buffered saline (TBS), and nonantigenic sites were blocked by incubation for 60 min in TBS with 5% dry milk. The blots were incubated overnight at 4°C with a 1:200 dilution of polyclonal rabbit anti-Kv2.1 antibody (Upstate Laboratories, Lake Placid, NY). After two washes with TBS, bound antibody was detected with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin antibodies with a luminol enhanced chemiluminescent detection kit (Pierce). Blots were exposed to X-ray film for time periods ranging up to 4 h and then washed. Densitometry was used to quantify the immunoblot product (NIH Image, Scion, Frederick, MD).RT-PCR
Lung tissue was extracted and immediately placed on ice. The main intralobar PAs were exposed by gentle dissection, and fourth- and fifth-generation branches were carefully isolated and removed. After adventitial tissue was removed, intralobar pulmonary arteries were removed, frozen, suspended in liquid nitrogen, and ground to a powder with a prechilled mortar and pestle. In a separate group of experiments, PASMCs were isolated and placed in primary culture as described above. Cells were harvested after being in culture for 7-12 days before extraction of RNA. Total RNA was extracted with the guanidinium thiocyanate-phenol-chloroform method (Tri Reagent, Sigma). After homogenization, the samples were processed according to the reagent instructions, and the RNA was dissolved in diethyl pyrocarbonate-treated water and stored at18S rRNA was analyzed in RT-PCR as an internal control. 18S cDNA was amplified with a QuantumRNA primer-Competimer set (Ambion) to allow relative semiquantitation of the ethidium bromide bands. This control band appears as 488 bp. Because 18S rRNA is far more abundant than the mRNA under study, the 18S amplification reaction was modulated by the addition of Competimers. These primers are modified to block extension by DNA polymerase. When combined with the functional primers for 18S cDNA, the amplification efficiency is reduced. Pilot experiments determined the correct ratio of primers to Competimers, cycle number, and RT input to yield multiplex PCR products that are all in the linear range of amplification. PCR cocktail consisted of 1× PCR buffer (Perkin-Elmer) with 1.5 mM Mg2+, 10 pM each Kv2.1 primer, 10 nM deoxynucleotide triphosphate mixture, 20 pM of 18S primer mixture (ratio of 1:9) of 18S primers-Competimers, 1 U of AmpliTaq polymerase, and water to make 50 µl. PCR was performed in an MJ Research thermocycler with a heated lid and 0.2-ml thin-walled tubes. The PCR was 2 min at 90°C, followed by 28-32 cycles of 1 min at 94°C, 1 min at 54°C, 2 min at 72°C, and then an extension of 2 min at 65°C. Samples without RT were evaluated in PCR; the products were absent. Identity of the band was confirmed by sequencing the product (>91% homology with known sequences). Densitometry was used in relative semiquantitative assessment of the RT-PCR product with NIH Image (Scion).
Drugs Used
4-AP and ANG II were obtained from Sigma. Fura 2-AM and ionomycin were obtained from Molecular Probes and diluted in DMSO. All drug solutions were adjusted to pH 7.4 before use. All drugs were solubilized in normal saline.Experimental Protocols
Effect of hypoxia on cytosolic Ca2+ in adult and fetal PASMCs. After stable baseline values in normoxic HBSS (21% O2; PO2 ~120 mmHg) were obtained, hypoxic HBSS (100% N2; PO2 ~25 mmHg) was superfused over the cells (n = 45 cells from 8 adult and 41 cells from 6 fetal animals) at 2 ml/min. Measurements of fluorescence were continued for 25 min. ANG II (1 µM) was superfused over the cells at 2 ml/min at the conclusion of the experiment to ensure cellular viability.
Effect of K+-channel
inhibition with 4-AP on cytosolic
Ca2+ in adult and fetal PASMCs.
After obtaining stable baseline values in normoxic HBSS (21%
O2; PO2 ~ 120 mmHg), PASMCs were
treated with 4-AP (a Kv channel blocker). 4-AP was
administered in incrementally increasing doses from 1 µM to 1 mM. In
a separate series of experiments, fetal and adult PASMCs (n = 78 cells from 5 adult and 97 cells from 6 fetal animals) were treated
with ANG II (106 M).
Statistical Analysis
Two-way ANOVA with repeated measures and a Student-Newman-Kuels post hoc test were used to assess differences between and among groups in each experimental protocol. Values are expressed as means ± SE. P < 0.05 was considered significant. ![]() |
RESULTS |
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Effect of Hypoxia on [Ca2+]i in Adult and Fetal PASMCs
Under basal conditions, there was no difference in [Ca2+]i in SMCs from adult and fetal PAs. Hypoxia caused an increase in [Ca2+]i in SMC from both adult and fetal PAs. As shown in Fig. 1, the increase was more rapid in onset and greater in both rate and magnitude in adult PASMCs compared with fetal PASMCs (P < 0.05; see Table 1).
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Effect of 4-AP on Basal [Ca2+]i in Adult and Fetal PASMCs
4-AP had no effect on basal [Ca2+]i in fetal or adult PASMCs at concentrations < 1 mM. 4-AP at 1 mM caused an increase in both fetal and adult PASMC [Ca2+]i. However, fetal PASMC [Ca2+]i increased by 98 ± 24% compared with 308 ± 67% in adult PASMCs (P < 0.05 for fetus vs. adult; P < 0.05 vs. baseline for both fetus and adult; Fig. 2). There was no difference in the response of fetal and adult PASMCs to ANG II. ANG II (10
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Kv2.1 Protein Expression in PAs From Fetuses, Newborns, and Adults
A band was observed at 125 kDa in all groups. The 125-kDa band is consistent with previously published reports. Kv2.1-channel protein expression increased with maturation. Densitometry readings performed on the blots indicated that Kv2.1 channel protein expression increased with maturation. Figure 3 is a representative blot of Kv2.1 protein expression from the fetal and adult pulmonary vasculature. Density measurements of Kv2.1 protein expression per microgram of protein were 0.635 ± 0.29 in the fetus (n = 6), 0.244 ± 0.144 in the newborn (n = 3), and 2.20 ± 0.724 in the adult (n = 4; P < 0.05 vs. fetus and newborn).
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Kv2.1 mRNA Expression in PAs From Fetuses, Newborns, and Adults
A band of the predicted size was observed, ~385 bp. A second band, 488 bp, was observed in each blot and was identified as the 18S ribosomal band. Sequence analysis was performed and indicated 91% homology between the PCR product obtained and human K+ channel Kv2.1, confirming the identity of the observed band. Densitometry was performed for both the 18S band and the PCR product. Figure 4 is a representative blot of the Kv2.1 and 18S ribosomal band PCR products. The Kv2.1 mRNA levels are expressed as a ratio of Kv2.1 to 18S band densitometry measurements. Kv2.1 mRNA levels in fetal (n = 3) and newborn (n = 3) PAs were significantly lower than in adult (n = 3) PAs (P < 0.01). There was no difference in Kv2.1 mRNA levels between fetal and newborn pulmonary arteries. The data are depicted graphically in Fig. 5.
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Kv2.1 mRNA Expression in PASMCs Maintained in Primary Culture From Fetuses and Adults
A 385-bp band was obtained. A second band, 488 bp, was observed in each blot and was identified as the 18S ribosomal band. Sequence analysis was performed and indicated 91% homology between the PCR product obtained and human K+-channel Kv2.1, confirming the identity of the observed band. Densitometry was performed for both the 18S band and the PCR product. Kv2.1 mRNA levels in fetal (n = 4) PASMCs were 64 ± 8% of adult (n = 4) PASMC Kv2.1 mRNA levels (P < 0.05). ![]() |
DISCUSSION |
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The K+ channel that determines RMP in the PASMCs changes from the K Ca channel to the Kv channel with maturation (27). Because previous investigators (17, 28, 39) have found that the ability of the pulmonary circulation to constrict in response to hypoxia, thereby matching ventilation and perfusion, increases with maturation, we tested the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the PASMCs; 2) Kv-channel activity increases with maturation; and 3) Kv2.1-channel protein and mRNA levels increase with maturation. We conclude that there are maturation-dependent increases in the capacity of PASMCs to respond to acute hypoxia, which is likely to be due to developmentally regulated changes in Kv2.1-channel activity and expression.
To address the initial hypothesis, we studied the response of PASMCs to acute hypoxia using microfluorimetry. In response to acute hypoxia, there was a large increase in [Ca2+]i in adult PASMCs and a smaller increase in fetal PAs, indicating that both are capable of directly sensing changes in O2 tension. Although PASMCs from both fetal (13) and adult circulations (30) have previously been shown to respond to acute hypoxia with an increase in [Ca2+]i, this is the first demonstration of a response intrinsic to PASMCs that increases with maturation. The observation that [Ca2+]i changes more rapidly in PASMCs in adult versus fetus implies that the sensitivity of PASMCs to changes in O2 tension increases with maturation. Moreover, the greater rate and magnitude of change in [Ca2+]i from adult vs. fetal PASMCs suggests that PASMCs from the adult circulation possess a greater capacity to respond to hypoxia than those from the fetal pulmonary circulation. Because [Ca2+]i correlates directly with the contractile state of SMCs (40), the relatively greater capacity for the pulmonary vasculature to respond to hypoxia with vasoconstriction in the adult compared with fetus may be the result of properties intrinsic to the PASMCs.
The observation that a Kv-channel antagonist had a greater effect on basal [Ca2+]i in PASMCs isolated from the adult pulmonary circulation compared with the fetal pulmonary circulation is consistent with the proposition that adult PASMCs are more sensitive to hypoxia than fetal PASMCs. To address the possibility that the relatively smaller changes in [Ca2+]i seen in fetal compared with adult PASMCs resulted from a generalized attenuation in Ca2+ transients in fetal PASMCs, cells were treated with ANG II. There was no difference in changes in PASMC [Ca2+]i in response to ANG II between adult and fetal PASMCs. To ensure that these observations were not simply the result of studying PASMCs that had been maintained in primary culture and subject to dedifferentiation, we determined Kv2.1 mRNA levels in PASMCs in primary culture. The results were entirely consistent with those obtained from freshly isolated vascular tissue. Kv2.1 mRNA levels increased with maturation. It is interesting that hypoxia caused an increase in fetal PASMC [Ca2+]i that was 54% of that in adult PASMCs, whereas Kv2.1 mRNA levels in fetal PASMCs in primary culture were 64% of Kv2.1 mRNA levels in the adult PASMCs in primary culture. The correlation between these results lends credibility to the microfluorimetry studies because the fidelity between the results from fresh tissue and PASMCs in primary culture minimizes the likelihood that the O2-sensing properties of the PASMCs had changed while in culture.
Our data indicate that there is relatively more Kv2.1-channel protein and message in the adult than in the fetal and neonatal pulmonary circulation. The relatively greater abundance of the Kv2.1 channel in the adult may represent an adaptation that allows the pulmonary circulation to respond to a specific physiological signal that is relevant to a particular developmental stage. The observation that the O2 sensor in the adult PASMCs is a Kv channel is consistent with this construct because the Kv channel is inactivated by acute hypoxia, causing PASMC depolarization, opening of voltage-operated Ca2+ channels, an increase in [Ca2+]I, and vasoconstriction (44, 45). Thus the maturation-related increase in hypoxic pulmonary vasoconstriction that has been previously reported may derive from the parallel increases in Kv2.1-channel activity, protein, and message increases with maturation.
These observations show that increases in Kv2.1 channel activity parallel the increase in hypoxic pulmonary vasoconstriction that occurs with maturation. Because hypoxia has been reported to inhibit a Kv2.1 channel to initiate pulmonary vasoconstriction (6, 7, 43, 45), the maturation-related increase in the capacity of the pulmonary vasculature to constrict in response to hypoxia may be due to increasing Kv2.1-channel activity with age. There is evidence that chronic hypoxia can downregulate Kv channels in rat PASMCs (32) and decrease KCa-channel activity in cultured human main PASMCs (25). Because the O2 tension in the fetus is normally low, it is possible that prolonged exposure to low levels of O2 tension may modulate the expression of K+ channels to result in a dominant role for K Ca-channel activity in the fetal PASMCs. Furthermore, prolonged depolarization has also been shown to downregulate Kv-channel expression in cultured cell lines (21).
This report taken together with recent data from our laboratory (13) supports the notion that the role of both K Ca and Kv channels in the maintenance of pulmonary vascular tone changes with maturation. Prior work from our laboratory involving K Ca-channel blockade in single fetal PASMCs demonstrated an increase in [Ca2+]i (14) and an inhibition of outward K+ current (13), implying that the K Ca channel plays a central role in the regulation of RMP in fetal PASMCs. The finding that 4-AP has a greater effect on basal [Ca2+]i in adult than in fetal PASMCs is consistent with these reports.
Developmental differences in the contribution of these channels to pulmonary vascular tone might be viewed from a teleological standpoint. Although constriction of the pulmonary vasculature in utero is necessary for normal pulmonary vascular development (6, 9), profound pulmonary vasodilation must occur within the first moments of air-breathing life (15, 29). Perinatal K+-channel activation is essential for normal, sustained pulmonary vasodilation (36). Thus the increase in O2 tension that occurs at birth may result in PASMC K+ efflux via K Ca channels to cause membrane hyperpolarization, closure of voltage-operated Ca2+ channels, decrease in [Ca2+]i, and pulmonary vasodilation (13). In contrast, during air-breathing adult life, Kv channels are necessary to ensure that ventilation and perfusion are appropriately matched (6, 45). It is perhaps noteworthy that this must be a regional regulation of K+-channel expression because Kv channels appear to be O2 sensitive and control RMP in the adjacent ductus arteriosus (37).
The differential response of fetal and adult PASMCs suggests that developmental changes in the PASMCs are, to a significant degree, responsible for the increasing sensitivity to hypoxia with maturation. It is unlikely that PASMC Kv-channel activity is decreased as a result of either posttranslational modification or protein trafficking that prevents the Kv2.1 channel from moving from the cytosol to the cell membrane because protein and message increase in a similar pattern with maturation. The mechanism whereby this alteration in Kv2.1-channel expression occurs is unclear, but the unique environment of the perinatal pulmonary circulation, low blood flow, high resistance, low oxygen tension, and high steroid hormone concentrations (41) may serve to modulate PASMC ion channel expression and activity.
Although hypoxic pulmonary vasoconstriction is critically important during air-breathing life, it may have detrimental effects during the perinatal period when the pulmonary circulation rapidly dilates to accommodate the increase in blood flow that occurs at birth (29). Because the lung of a newborn often has residual fluid in the airways and edema, the likelihood of regional alveolar hypoxemia is quite high. During the neonatal period, hypoxic pulmonary vasoconstriction may be undesirable because it may result in extrapulmonary shunting of blood at the level of the patent foramen ovale and the ductus arteriosus, causing severe central hypoxemia. In other words, the pulmonary circulation of the adult may be adapted to allow for vasoconstriction in response to an acute decrease in O2 tension (42), whereas perinatal pulmonary circulation is uniquely well adapted to vasodilate in response to an acute increase in O2 tension (13, 27).
In summary, these studies suggest that the sensitivity and capacity of PASMCs to respond to hypoxia increases with maturation. These maturation-related increases parallel those described for hypoxic pulmonary vasoconstriction (17, 28, 39). The mechanism whereby these developmental changes occur may be the result of a shift in pulmonary vascular SMC K+-channel activity, with K Ca-channel predominance in the fetus and Kv channel in the adult (27). Because Kv channels set the RMP of PASMCs in the adult pulmonary vasculature and their blockade causes a profound effect on basal tone (7, 45), it is likely that Kv channels play a central role in modulating pulmonary vascular tone in the adult. The increasing capacity of the pulmonary vasculature to respond to hypoxia with maturation may result from a maturation-dependent increase in expression of O2-sensitive K+ channels in SMCs from resistance PAs. The mechanism whereby a maturation-related decrease in K Ca- and increase in Kv-channel activity occurs remains unknown.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-60784 (D. N. Cornfield) and First Award R29-HL-59182-01 (H. L. Reeve); Viking Children's Fund (E. Resnik); and American Heart Association Northland Affiliate Grants-in-Aid (D. N. Cornfield, I. Y. Haddad, and V. A. Porter).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. N. Cornfield, Box 742, Div. of Pediatric Pulmonology and Critical Care, University of Minnesota School of Medicine, 420 Delaware St. S.E., Minneapolis, MN 55455 (E-mail: cornf001{at}tc.umn.edu).
Received 1 April 1999; accepted in final form 4 January 2000.
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