Pulmonary vascular response to normoxia and KCa channel activity is developmentally regulated

Michael T. Rhodes1, Valerie A. Porter1,2, Connie B. Saqueton3, Jean M. Herron1, Ernesto R. Resnik1, and David N. Cornfield1

1 Division of Pediatric Pulmonology and Critical Care Medicine, Department of Pediatrics, and 2 Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455; and 3 Department of Pediatrics, University of Nevada, Las Vegas, Nevada 89102


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To address developmental regulation of pulmonary vascular O2 sensing, we tested the hypotheses that 1) fetal but not adult pulmonary artery smooth muscle cells (PASMCs) can directly sense an acute increase in O2, 2) Ca2+-sensitive K+ (KCa) channel activity decreases with maturation, and 3) PASMC KCa channel expression decreases with maturation. We used fluorescence microscopy to confirm that fetal but not adult PASMCs are able to sense an acute increase in O2 tension. Acute normoxia induced a 22 ± 2% decrease in cytosolic Ca2+ concentration ([Ca2+]i) in fetal PASMCs and no change in [Ca2+]i in adult PASMCs (P < 0.01). The effects of K+ channel antagonists were studied on fetal and adult PASMC [Ca2+]i. Iberiotoxin (10-9 M) caused PASMC [Ca2+]i to increase by 694 ± 22% in the fetus and caused no change in adult PASMCs. KCa channel expression and mRNA levels in distal pulmonary arteries from fetal and adult sheep were examined. Both KCa channel protein and mRNA expression in the distal pulmonary vasculature decreased with maturation. We conclude that maturation-dependent changes in PASMC O2 sensing render the fetal PASMCs uniquely sensitive to an acute increase in O2 tension at a biologically critical time point.

ontogeny; smooth muscle cells; ion channel; cytosolic calcium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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. 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 cell (SMC) K+ channels. At birth, pulmonary blood flow increases 8- to 10-fold, whereas pulmonary arterial pressure declines steadily over the first several hours of life (22). 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), sustained and progressive perinatal pulmonary vasodilation requires PASMC K+ channel activation (30).

Recent studies have demonstrated that activation of the Ca2+-sensitive K+ (KCa) channel plays a critical role in perinatal pulmonary vasodilation. O2 causes perinatal pulmonary vasodilation through kinase-dependent activation of the KCa channel (8). K+ channel inhibition with tetraethylammonium (TEA), but not with glibenclamide, a blocker of the ATP-sensitive K+ channels, blocks the pulmonary vasodilation caused by ventilation (30), suggesting that ventilation causes sustained and progressive pulmonary vasodilation through activation of TEA-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 KCa channel activation (2, 4). Recent work (24) demonstrates that NO causes perinatal pulmonary vasodilation through KCa channel activation and requires the release of cytosolic Ca2+ from a ryanodine-sensitive store. Even the shear stress response induced by compression of the ductus arteriosus during fetal life involves KCa and voltage-dependent K+ (KV) channel activation (29).

The observation that the K+ channel, which determines the resting membrane potential (RMP) in the pulmonary circulation, changes after birth from a KCa to a KV channel suggests that K+ channel expression in the pulmonary circulation is developmentally regulated (21). Recent data (10) indicate that there are maturation-dependent increases in the capacity of PASMCs to respond to acute hypoxia, likely due to increases in Kv2.1 channel activity and expression with maturation. In adult animals, acute hypoxia causes inhibition of KV channel activity and depolarization of PASMCs (34), leading to the opening of voltage-operated Ca2+ channels, an increase in cytosolic Ca2+ concentration ([Ca2+]i), and vasoconstriction. 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 (33).

Because hypoxic pulmonary vasoconstriction is critically important during air-breathing life, the adult pulmonary circulation is adapted to respond to an acute decrease in O2 tension. In contrast, perinatal pulmonary vasodilation in response to an acute increase in O2 tension is biologically imperative. The fetal pulmonary circulation is exquisitely sensitive to small increases in O2 tension. An increase in fetal O2 tension of only 6-8 Torr results in a threefold increase in pulmonary blood flow in the late-gestation ovine fetus (3, 18). The human fetal pulmonary vasculature demonstrates a similar sensitivity to small increases in O2 tension (20). Because there is a developmentally regulated change in the K+ channel that sets RMP (21) and enables the perinatal pulmonary circulation to respond to an acute increase in O2 tension (8) with vasodilation, PASMC KCa channel activity, protein, and expression might be expected to decrease with maturation.

We therefore proposed the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to an acute increase in O2 tension are intrinsic to the PASMCs, 2) KCa channel activity decreases with maturation, and 3) KCa channel protein and mRNA levels decrease with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of an acute increase in O2 tension on the [Ca2+]i of SMCs isolated from adult and fetal PAs. To determine any changes in K+ channel activity with maturation, the effects of K+ channel antagonists (5) iberiotoxin (a selective KCa channel blocker) and TEA (a preferential KCa channel blocker) on PASMC [Ca2+]i were studied in SMCs isolated from late-gestation fetal and adult ovine PAs. KCa channel protein expression and mRNA levels were determined with immunoblotting and semiquantitative RT-PCR.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture

The techniques used for isolation and culture of ovine PASMCs have been previously described (11). 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 a physiological saline solution containing (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 fourth- or fifth-generation resistance PAs. Loose connective tissue and adventitia were removed, and the vessels were liberally rinsed with MEM (0.2 mM Ca2+). Vessel segments were carefully cut into small pieces and placed into 50-ml conical flasks containing 5.0 ml of the enzymatic dissociation mixture, which consisted of 0.125 mg/ml of elastase (Sigma, St. Louis, MO), 1 mg/ml of collagenase (Worthington Biochemical, Freehold, NJ), 2.0 mg/ml of bovine serum albumin, 0.375 mg/ml of 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 40-80 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% calf serum. The dispersed cell suspension was divided into aliquots on 25-mm2 glass coverslips (Sigma) and 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 10% O2-5% CO2-balance N2 atmosphere or in humidified 95% air-5% CO2 (hypoxia). 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 48-h intervals. Cells were studied between day 5 and day 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 alpha -actin-specific antibody (Sigma) after 5, 10, and 14 days in culture.

Solutions

Recording solutions consisted of (in mM) 10 HEPES, 10 glucose, 135 NaCl, 5.6 KCl, 1.8 CaCl2, and 1.0 MgCl2. All solutions were made with nanopure distilled water. Osmolality was adjusted to ~300 mosM, and pH was adjusted to 7.4.

Ca2+ Imaging

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). The cells were loaded with 100 nM fura 2-AM plus 2.5 mg/ml of 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 (Photonic Science, Robertsbridge, UK) with Axon Instruments (Foster City, CA) image capture and analysis software. Ca2+ calibration was achieved by measuring a maximum (Rmax; with 1 mM ionomycin) and a minimum (Rmin; with 10 mM EGTA) fluorescence ratio for each cell. O2 tension was controlled by aerating the HBSS reservoir with either 21% O2-balance N2 or 100% N2. O2 tension was monitored throughout with an O2 electrode (Microelectrodes, Bedford, NH). Further control of PO2 was obtained by aerating the stage microincubator with 21% O2-balance N2 or 100% N2. The pH was 7.4 ± 0.05 and did not change during the experiments. Intracellular free Ca2+ was calculated by assuming a dissociation constant (Kd) of 220 (28). For each experiment, 5-12 cells were visualized, and cytosolic Ca2+ measurements were made from individual cells.

Western Blotting

To determine KCa channel protein expression in the pulmonary circulation, vascular structures were isolated from the lungs of fetal and adult sheep. Tissue was obtained from the distal (greater than or equal to fourth-generation) PAs, frozen with liquid N2, and processed in radioimmunoprecipitation assay buffer with protease inhibitors aprotinin, EDTA, leupeptin, 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, calpain 1 and 2 inhibitors, and benzamidine (Sigma). The KCa channel antibody, 3 µg/reaction, was combined with 0.5 ml of Gammabind Plus Sepharose beads (Pharmacia) in PBS and rotated at room temperature for 60 min. The mixture was washed in 0.2 M sodium borate (pH 9) and resuspended in borate and dimethylpimelimidate and rotated for 30 min. The beads were spun, washed with 0.2 M ethanolamine (pH 8), resuspended, and washed for an additional 2 h. One milligram of processed protein tissue was added to 50 µl of antibody-coupled beads in 1 ml of PBS and protease inhibitors at 4°C, then incubated and rotated overnight. The beads were collected by centrifugation at 14,000 g for 1 min. The pellet was washed three times with cold PBS (with protease inhibitors), resuspended in SDS-PAGE sample buffer, boiled for 5 min, microfuged for 5 s, and placed on ice. Western blotting was performed with 20 µg protein/lane in a 7.5% polyacrylamide gel (Ready Gel, Bio-Rad). Protein samples were subjected to electrophoresis according to the method of Laemmli (16) in a Mini-Gel system (Bio-Rad). Proteins were transferred onto polyvinylidene difluoride membranes for 60 min at 100-V constant voltage with a Mini Trans-Blot cell. After protein transfer, the polyvinylidene difluoride membranes were rinsed in Tris-buffered saline (TBS) and incubated for 60 min in TBS with 6% dry skim milk. The KCa channel antibody targeted to the alpha -chain of the KCa channel (Alomone, Jerusalem, Israel) was diluted in TBS-milk and incubated overnight with the blots at 4°C. After two washes with TBS, the secondary antibody, goat anti-rabbit IgG, was added for 2 h. After the blots were washed, chemiluminescence reagents (Pierce Chemical) were applied, and the blots were exposed to X-ray film for time periods ranging from 2 min to overnight.

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 the adventitial tissue was removed, the intralobar PAs were removed, frozen in liquid N2, and ground to a powder with a prechilled mortar and pestle. 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 (DEPC)-treated water and stored at -70°C. Optical density was measured to determine the RNA concentration. One microgram of RNA was added to 11 µl of first-strand cDNA synthesis reagent (Pharmacia), with random hexamers as primers in a final volume of 33 µl. Two microliters of this RT reaction were added to each PCR. The oligonucleotide primers used to amplify KCa channel cDNA were based on the human sequence (31) and were (forward) 5'-CTACTGGGATGTTTCACTGGTGT-3' and (reverse) 5'-TGCTGTCATCAAACTGCATA-3', which yielded a product consistent with that expected for human KCa channels. Identity of the product was confirmed with sequence analysis.

18S rRNA was analyzed with 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 324 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/competimers, cycle number, and RT input to yield multiplex PCR products, which are all in the linear range of amplification. The PCR cocktail consisted of 1× PCR buffer (PerkinElmer) with 1.5 mM Mg2+, 10 pM each KCa channel primer, 10 nM deoxynucleotide triphosphate mixture, 20 pM 18S primer mixture (ratio 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-wall tubes. The PCR was for 2 min at 90°C followed by 28-32 cycles of 1 min at 94°C, 1 min at 55°C, 2 min at 72°C, and then an extension for 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 (NIH Image, Scion, Frederick, MD). The relative densities of the 18S ribosomal and K+ channel PCR products were compared in each individual gel. RT-PCR was performed three times for each mRNA sample.

Drugs

Angiotensin II and TEA were obtained from Sigma. Fura 2-AM and ionomycin were obtained from Molecular Probes and diluted in DMSO. Iberiotoxin was obtained from Alomone Laboratories. All drug solutions were adjusted to pH 7.4 before use. All drugs were solubilized in normal saline.

Experimental Protocols

Basal cytosolic Ca2+ in hypoxic adult and fetal PASMCs. Fetal (n = 113 cells; 6 animals) and adult (n = 99 cells; 4 animals) PASMCs were isolated and maintained in primary culture, loaded with fura 2, and [Ca2+]i was determined, all under hypoxic conditions.

Effect of an acute increase in O2 tension on cytosolic Ca2+ in adult and fetal PASMCs. After stable baseline values in hypoxic recording solution (100% N2; PO2 ~25 mmHg) were obtained in adult (n = 45 cells; 8 animals) and fetal (n = 41 cells; 6 animals) PASMCs, normoxic recording solution (21% O2; PO2 ~120 mmHg) was superfused over the cells at a rate of 2 ml/min. Measurement of fluorescence was continued for 10 min.

Effect of iberiotoxin on basal [Ca2+]i in PASMCs. After stable baseline values in normoxic HBSS (21% O2; PO2 ~120 mmHg) were obtained, iberiotoxin was administered to adult (n = 56 cells; 6 animals) and fetal (n = 51 cells; 5 animals) PASMCs in concentrations ranging from 10-11 to 10-6 M. Concentrations were increased in log increments. TEA was administered to the cells in a similar manner in concentrations ranging from 10-7 to 10-3 M. Angiotensin II (1 µM) was superfused over the cells at 2 ml/min at the conclusion of the experiment to ensure cellular viability

Statistical Analysis

A two-way analysis of variance (ANOVA) with repeated measures and a Student-Newman-Keuls post hoc test were used to assess differences between and among groups in each experimental protocol. To compare densitometry results, a paired t-test was used. Values are expressed as means ± SE. P values < 0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Basal Cytosolic Ca2+ in Hypoxic Adult and Fetal PASMCs

Under hypoxic conditions, [Ca2+]i in fetal PASMCs was 100 ± 7 nM compared with 63 ± 6 nM in adult PASMCs (P < 0.01, fetal vs. adult; Fig. 1).


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Fig. 1.   A: comparison of cytosolic Ca2+ concentration ([Ca2+]i) in fetal (n = 113 cells; 6 animals) and adult (n = 99 cells; 4 animals) pulmonary artery smooth muscle cells (PASMCs) maintained in hypoxia. Under hypoxic conditions, basal [Ca2+]i was significantly higher in fetal compared with adult PASMCs. B: effect of an acute increase in O2 tension on [Ca2+]i.

Effect of an Acute Increase in O2 Tension on Fetal and Adult PASMC [Ca2+]i

An acute increase in O2 tension caused a decrease in [Ca2+]i in fetal but not in adult PASMCs. As shown in Figs. 1 and 2, the decrease was rapid in onset in fetal PASMCs. Adult PASMC [Ca2+]i did not change with an acute increase in O2 tension (P < 0.01 vs. fetal).


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Fig. 2.   Effect of an acute increase in O2 tension on [Ca2+]i of fetal (n = 113 cells; 6 animals) and adult (n = 99 cells; 4 animals) PASMCs maintained in hypoxia. Acute normoxia caused a progressive decrease in [Ca2+]i in PASMCs from fetal but not from adult animals.

Effect of Iberiotoxin and TEA on Basal [Ca2+]i in Adult and Fetal PASMCs Under Normoxic Conditions

Iberiotoxin had no effect on basal [Ca2+]i in adult PASMCs at concentrations <10-6 M. Iberiotoxin at 10-9 M caused an increase of 224 ± 25% in fetal PASMC [Ca2+]i (P < 0.01, fetal vs. adult; P < 0.01 vs. baseline for fetal; Fig. 3). Log order increases in iberiotoxin concentrations caused a dose-dependent increase in fetal but not in adult PASMC [Ca2+]i (Fig. 3). TEA had no effect on basal [Ca2+]i in adult PASMCs at concentrations <10-3 M. TEA at 10-7 M caused an increase of 124 ± 17% in fetal PASMC [Ca2+]i (P < 0.01, fetal vs. adult; P < 0.01 vs. baseline for fetus). TEA at 10-6 M caused an increase of 312 ± 32% in fetal PASMCs. There was no difference in the response of fetal and adult PASMCs to angiotensin II. Angiotensin II (10-6 M) caused a 154 ± 24 nM increase in [Ca2+]i in fetal PASMCs compared with a 172 ± 31 nM increase in [Ca2+]i in adult PASMCs.


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Fig. 3.   Effects of K+ channel inhibition with iberiotoxin, a specific Ca2+-sensitive K+ (KCa) channel antagonist, on smooth muscle cell [Ca2+]i in cells isolated from fetal and adult pulmonary arteries. n, No. of cells from indicated no. of animals. At a concentration of 10-9 M, iberiotoxin had no effect on [Ca2+]i in PASMCs from adults but caused a substantial increase in [Ca2+]i in those from fetuses (P < 0.01 vs. baseline). Iberiotoxin had no effect on adult PASMC [Ca2+]i in concentrations as high as 10-7 M, whereas increasing concentrations of iberiotoxin caused a dose-related increase in fetal PASMC [Ca2+]i (P < 0.01, fetal vs. adult).

KCa Channel Protein Expression

A band was observed at 125 kDa in all groups (n = 5 fetuses, 2 blots; n = 5 adults, 2 blots). The 125-kDa band was consistent with a previously published report (15). Densitometry readings performed on the blots indicated that KCa channel protein expression decreased with maturation. Figure 4 is a representative blot of KCa channel protein expression from the fetal and adult pulmonary vasculature. A 180-kDa band was noted in the fetal pulmonary vasculature.


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Fig. 4.   Western blot of adult and fetal samples from the distal pulmonary artery (dPA) obtained with anti-KCa channel polyclonal antibody. Each lane was loaded with 20 µg of immunoprecipitated protein. The predicted bands of ~125 kDa were observed in both adult and fetal samples. Intensity of the band indicates decreasing KCa channel protein levels with postnatal maturation. The 125-kDa band is consistent with a previously published report (15). In the fetal samples, an additional band of ~180 kDa was consistently observed. The band is absent in samples from the adult pulmonary circulation and the adult mouse brain. Protein extracted from the adult mouse brain served as the positive control. Coincubation with both the antigen and antibody served as the negative control.

KCa Channel mRNA Expression in PAs

A band of the predicted size (~446 bp) was observed in fetal (n = 3 animals, 9 gels) and adult (n = 3 animals, 9 gels) PAs. A second band, 324 bp, was observed in each blot and was identified as the 18S ribosomal band. Sequence analysis was performed and indicated 94% homology between the PCR product obtained and the human KCa channel, confirming the identity of the observed band. Densitometry was performed for both the 18S band and the PCR product. Figure 5 is a representative blot of the KCa channel and 18S ribosomal band PCR products. The KCa channel mRNA levels are expressed as a ratio of KCa channel to 18S band densitometry measurements. KCa channel mRNA levels in fetal PAs were significantly higher than those in adult PAs (P < 0.05). The data are depicted in Fig. 6.


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Fig. 5.   Representative gel of a RT-PCR analysis of KCa channel mRNA expression during development. The KCa band (~446 bp) was compared with the control 18S band (~324 bp) in fetal and adult samples. KCa channel band intensity was determined by densitometry and normalized to that of the 18S band. KCa channel mRNA levels decreased with maturation.



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Fig. 6.   KCa channel mRNA levels in the distal pulmonary vasculature decrease with maturation. Band intensities were determined by densitometry. KCa channel band intensity was normalized to that of the 18S band. mRNA was extracted directly from pulmonary arteries isolated from fetal (n = 3 animals; 9 gels) and adult (n = 3 animals; 9 gels) sheep. RT-PCR was performed 4 times for each mRNA sample.

KCa Channel mRNA Expression in PASMCs Maintained in Primary Culture

In fetal (n = 8 animals) and adult (n = 8 animals) PASMCs maintained in primary culture, a 446-bp band was obtained. A second band, 324 bp, was observed in each blot and was identified as the 18S ribosomal band. Sequence analysis was performed and indicated 94% homology between the PCR product obtained and the human KCa channel, confirming the identity of the observed band. Densitometry was performed for both the 18S band and the PCR product. KCa channel mRNA levels were 2.39 ± 0.47 in fetal PASMCs and 1.54 ± 0.24 (P < 0.05) in adult PASMCs. The data are depicted graphically in Fig. 7.


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Fig. 7.   KCa channel mRNA levels in PASMCs maintained in primary culture decrease with maturation. Band intensities were determined by densitometry. KCa channel band intensity was normalized to that of the 18S band. mRNA was extracted from fetal (n = 8 coverslips, 4 animals) and adult (n = 8 coverslips, 4 animals) PASMCs derived from the distal pulmonary vasculature of fetal and adult sheep. RT-PCR was performed 3 times for each mRNA sample.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we report that under conditions of chronically low tension, [Ca2+]i in fetal PASMCs is greater than that in adult PASMCs. Moreover, fetal but not adult PASMCs can respond directly to an acute increase in O2 tension with a decrease in [Ca2+]i and to iberiotoxin with an increase in basal [Ca2+]i. The observation that pulmonary vascular KCa channel mRNA and protein expression decrease with maturation is consistent with these results.

The present studies provide evidence that fetal PASMCs directly sense and respond to an acute increase in O2 tension with a decrease in [Ca2+]i. Because [Ca2+]i is proportional to the contractile state of SMCs, a decrease in PASMC [Ca2+]i is consistent with PASMC relaxation (32). The observation that under hypoxic conditions fetal PASMC [Ca2+]i is greater than that in adults implies that elevated fetal pulmonary vascular resistance may result from direct effects of the normally low O2 tension environment of the fetus on PASMCs. Although recent data (10) suggest that adult PASMCs are more sensitive to acute hypoxia than fetal PASMCs, the present study addresses [Ca2+]i in PASMCs maintained under low O2 tension conditions for a sustained time period. Thus direct effects on the fetal PASMCs may contribute to both elevated fetal pulmonary vascular resistance and O2-induced perinatal pulmonary vasodilation. Whereas previous studies (11, 17) have demonstrated the ability of PASMCs to sense an acute decrease in O2 tension, the present study provides the first evidence that fetal PASMCs can respond to an acute increase in O2 tension.

We report that in PASMCs maintained under hypoxic conditions while in primary culture, [Ca2+]i is higher in fetal than in adult cells. This result is especially interesting because our laboratory has previously reported (10) that acute hypoxia causes [Ca2+]i to increase more rapidly and to a greater degree in adult compared with fetal PASMCs. This observation implies that adult but not fetal PASMCs adapt to chronic hypoxia with a decrease in [Ca2+]i after exposure to hypoxia for at least 5 days. The absence of a response to an acute increase in O2 tension in adult PASMCs supports the notion that developmental differences intrinsic to the PASMC determine the effects of chronic hypoxia on PASMC O2 sensing and [Ca2+]i regulation. The response of vascular SMCs to chronic hypoxia has previously been shown to be differentially regulated at the level of both the specific cell types within the vessel wall (14) and the vascular bed of origin (19, 26, 27). The present findings suggest that the response of PASMCs to chronic hypoxia may be developmentally regulated.

Adult PASMC [Ca2+]i does not change in response to an acute increase in O2 tension. From a developmental standpoint, the inability of adult PASMCs to respond to an increase in O2 tension is not surprising because further dilation of the normally low-resistance pulmonary vasculature is unnecessary. Perinatal pulmonary vasodilation is of critical importance because blood must reach the alveoli or severe hypoxemia would result. The ability to vasodilate in response to an acute increase in O2 tension is biologically imperative. An indication of the importance of this response is that the perinatal pulmonary vasculature responds to an acute increase in O2 tension with both an increase in NO production from the endothelial cells (25) and a decrease in SMC [Ca2+]i. Whereas both SMCs and endothelial cells respond individually to an acute increase in O2 tension, the coordinated response of each cell may serve to potentiate pulmonary vasodilation. For example, NO produced by the endothelium will cause SMC relaxation, which, in turn, will increase pulmonary blood flow and endothelial shear stress, further augmenting NO production (6).

Previous work (13) has demonstrated that the K+ channel that determines RMP in PASMCs changes with maturation. Data from our laboratory (21) indicate that RMP is determined by the KCa channel in fetal and the KV channel in adult PASMCs. Indeed, the capacity of PASMCs to respond to an acute decrease in tension increases with maturation as Kv2.1 channel protein and mRNA expression increase (10). The present study provides evidence that KCa channel activity and expression decrease with maturation. The observation that iberiotoxin increases fetal but not adult PASMC [Ca2+]i supports the notion that under basal conditions, the KCa channel determines the contractile state of the PASMC. Because iberiotoxin causes inactivation of the KCa channel, an increase in [Ca2+]i indicates that the KCa channel is open under basal conditions. The absence of a change in adult PASMC [Ca2+]i indicates that these channels are not open under basal conditions in the adult. This observation is consistent with previous data from our laboratory (12) in which charybdotoxin, a selective KCa channel blocker, caused a significant increase, whereas ATP-sensitive K+ channel blockade with glibenclamide had no effect on fetal PASMC [Ca2+]i. Interestingly, such a result is consistent with data from our laboratory in whole animal studies (7, 30) in which blockade of the ATP-sensitive K+ channels had no effect on basal tone in the ovine fetal pulmonary circulation.

Both protein and mRNA expression of the pulmonary arterial KCa channel decrease with maturation. The presence of a 125-kDa band is consistent with a previous report (23). Because these experiments were performed on pulmonary arterial tissue, no distinction in KCa channel expression can be made between PASMCs and endothelial cells. Western blots clearly demonstrated that pulmonary arterial KCa channel protein expression decreases with maturation. The presence of a 180-kDa band in the fetal pulmonary arterial preparation is noteworthy because it is absent in samples derived from nonfetal sources such as mouse brain and the adult ovine pulmonary circulation. To ensure that the 180-kDa band was not antibody specific, the experiment was performed with two different antibodies directed against the KCa channel alpha -chain (kindly provided separately by I. Levitan, Brandeis University, Waltham, MA, and H. G. Knaus, University of Innsbruck, Innsbruck, Austria). The 180-kDa band was consistently present in protein derived from the fetal pulmonary circulation and absent in all other tissues studied. The significance of this observation remains unknown.

The observation that pulmonary arterial KCa channel gene expression is greatest in the fetal pulmonary circulation is supportive of physiological data from our laboratory. Specifically, activation of the KCa channel has been shown to mediate the perinatal pulmonary vasodilation caused by an acute increase in O2 tension (8), NO (24), shear stress (29), and ventilation (30). Because these physiological stimuli are critically important in mediating the postnatal adaptation of the pulmonary circulation, a decrease in pulmonary arterial KCa channel expression may compromise the transition of the pulmonary circulation that occurs at birth. A recent report from our laboratory (9) indicating that KCa channel gene expression is decreased in an ovine model of persistent pulmonary hypertension of the newborn is consistent with this construct. Thus a decrease in pulmonary arterial KCa channel expression may compromise perinatal pulmonary vasodilation that occurs in response to birth-related physiological stimuli.

These experiments provide support for the concept that developmental regulation of O2-induced pulmonary vasodilation derives from maturation-related differences in pulmonary vascular KCa channel expression. However, several limitations inherent in these studies warrant consideration. First, physiological experiments were performed on PASMCs maintained in primary culture, whereas molecular studies were performed on freshly isolated ovine tissue. The fact that the results reported here are internally consistent despite the use of both freshly isolated tissue and cells in primary culture seems to lend further credibility to these studies. Moreover, similar changes in KCa channel mRNA levels were noted even if mRNA was isolated from PASMCs maintained in primary culture. Similar experiments for protein expression could not be performed due to the relatively limited amount of KCa channel protein expression in PASMCs in primary culture.

In summary, these experiments demonstrate that fetal but not adult PASMCs can respond to an acute increase in O2 tension with a decrease in [Ca2+]i. The capacity to respond to an acute increase in O2 tension corresponds to a developmentally regulated increase in KCa channel activity and protein and gene expression. The relatively greater pulmonary vascular KCa channel expression in the fetus compared with that in the adult may represent a developmental adaptation that increases the likelihood of perinatal pulmonary vasodilation. The biological mechanism whereby KCa channel expression peaks in the perinatal pulmonary circulation remains unknown, but it may represent a novel therapeutic target for infants at risk for the development of persistent pulmonary hypertension of the newborn.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-60784 (to D. N. Cornfield), the Viking Children's Fund (to E. Resnick), and a Parker B. Francis Family Foundation Fellow Award (to V. A. Porter).


    FOOTNOTES

Address for reprint requests and other correspondence: D. N. Cornfield, Div. of Pediatric Pulmonology and Critical Care, Univ. of Minnesota School of Medicine, 420 Delaware St. SE, MMC 742, Minneapolis, MN 55455 (E-mail: cornf001{at}tc.umn.edu).

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. Section 1734 solely to indicate this fact.

Received 4 August 2000; accepted in final form 14 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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