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Am J Physiol Lung Cell Mol Physiol 290: L761-L768, 2006. First published November 11, 2005; doi:10.1152/ajplung.00283.2005
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Oxygen tension modulates the expression of pulmonary vascular BKCa channel {alpha}- and beta-subunits

Ernesto Resnik,1 Jean Herron,1 Rao Fu,1 D. Dunbar Ivy,2 and David N. Cornfield3

1Pediatrics, University of Minnesota, Minneapolis, Minnesota; 2Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado; and 3Pediatrics, Stanford University Medical School, Palo Alto, California

Submitted 30 June 2005 ; accepted in final form 18 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
At birth, the lung environment changes from low to relatively high O2 tension. Pulmonary blood flow increases and pulmonary artery pressure decreases. Recent data suggest that pulmonary vascular calcium-sensitive K+ channel (BKCa) activation mediates perinatal pulmonary vasodilation. Although BKCa channel expression is developmentally regulated, the molecular mechanisms responsible for BKCa expression remain unknown. We tested the hypothesis that the low-O2 tension environment of the normal fetus modulates BKCa channel expression. We analyzed BKCa expression under conditions of hypoxia and normoxia both in vitro and in vivo. BKCa {alpha}-subunit mRNA expression increased twofold in ovine pulmonary artery smooth muscle cell (PASMC) primary cultures maintained in hypoxia. In vivo, BKCa expression was similarly affected by hypoxia. When adult Sprague-Dawley rats were placed in hypobaric hypoxic chambers for 3 wk, hypoxic animals showed an increase of threefold in the expression of BKCa {alpha}- and more than twofold in the expression of BKCa beta1-subunit mRNA. Immunochemical staining was consistent with the genetic data. To assess transcriptional activation of the beta-subunit of the BKCa, both BKCa beta1- and beta2-subunit luciferase (KCa beta:luc+) reporter genes were constructed. Hypoxia increased PASMC KCa beta1:luc+ reporter expression by threefold and KCa beta2:luc+ expression by 35%. Fetal PASMC treated with the hypoxia-inducible factor-1 mimetic deferoxamine showed a 63 and 41% increase in BKCa channel {alpha}- and beta1-subunit expression, respectively. Together, these results suggest that oxygen tension modulates BKCa channel subunit mRNA expression, and the regulation is, at least in part, at the transcriptional level.

hypoxia; oxygen sensing; hypoxia-inducible factor-1

AT BIRTH, THE LUNG ENVIRONMENT changes from being fluid-filled with low oxygen (O2) tension to air-filled with relatively high O2 tension. The increase in O2 tension, ventilation, shear stress, and vasoactive mediators normally causes perinatal pulmonary vasodilation. Pulmonary blood flow increases 8- to 10-fold, and pulmonary arterial pressure declines steadily, over the first several hours of life (34). Despite the clear biological importance of postnatal adaptation of the pulmonary circulation, the mechanisms responsible for perinatal pulmonary vasodilation are incompletely understood.

Recent observations suggest that activation of the calcium-sensitive K+ (BKCa) channel plays a critical role in the sustained and progressive pulmonary vasodilation that normally occurs after birth. Several physiological stimuli that cause perinatal pulmonary vasodilation act, at least in part, through activation of pulmonary vascular BKCa channels. Specifically, an increase in oxygen tension (7), initiation of ventilation (44), and an increase in shear stress (16, 42) cause perinatal pulmonary vasodilation through BKCa channel activation. Even nitric oxide, a molecule that plays a crucial role in perinatal pulmonary vasodilation, acts, at least in part, through BKCa channel activation (36). Indeed, data from our laboratory suggest that BKCa channel expression in the fetal and newborn pulmonary circulation renders the perinatal pulmonary circulation uniquely well adapted, at a biologically critical time point, to respond to vasodilator stimuli. However, the mechanism whereby pulmonary vascular BKCa channel expression is increased in the perinatal, compared with the adult, pulmonary circulation remains unknown.

Large-conductance BKCa play a key role in determining smooth muscle cell tone and, thereby, vascular tone. In pulmonary artery smooth muscle cells (PASMC), activation of the BKCa channel results in K+ efflux, membrane hyperpolarization, closure of voltage-dependent calcium channels, a decrease in cytosolic calcium, and a decrease in tone (5, 26). Each BKCa channel contains four membrane-spanning subunits, the {alpha}-subunit, forming a central pore that is selective for K+ (2, 23, 49). Gating of the channel is modified by binding of the beta-subunits to the intracellular COOH terminus of the {alpha}-subunit (24). The gating characteristics of the BKCa channel is dependent on the specific beta-subunit (46). To date, four different subtypes of the beta-subunit have been identified. There is considerable variation in subunit expression according to anatomical site (22). The beta1-subunit is expressed most abundantly in smooth muscle cells and it confers enhanced calcium sensitivity to the {alpha}-subunit (25, 28). When the {alpha}-subunit is coexpressed with the beta1-subunit, currents possess faster activation kinetics and are larger at negative membrane potentials, indicating a higher "open probability" at those potentials. The membrane potential needed to half-activate the channel shifts from 0 to near –100 mV with coexpression of the {alpha}- and beta1-subunits (22). An acute increase in oxygen tension causes perinatal pulmonary vasodilation, at least in part, through activation of the BKCa channel and pulmonary artery smooth muscle hyperpolarization (7, 29). Thus the observation that open probability of the BKCa channel is increased at more negative membrane potentials may have important implications for the perinatal pulmonary circulation.

The presence of a beta2-subunit has been recently reported in a variety of tissues, including the heart, brain, lung, and kidney. This subunit causes rapid inactivation of the BKCa channel by a "ball and chain" mechanism in which the NH2-terminal region of beta2-subunit can effectively block the channel (47, 48). Because closely circumscribed pulmonary blood flow is essential for pulmonary vascular development (1), the ontogeny of beta2-subunit expression may have significant implications for pulmonary vasculogenesis. Whether the beta2-subunit is expressed in the pulmonary vasculature is currently unknown. Given the distinct and somewhat divergent effects of the different subunits on the physiology of the BKCa channel, insight into factors that modulate BKCa channel subunit expression may have important implications for the role of this channel in both the normal and abnormal perinatal pulmonary circulation. It has recently been shown that differences in expression of the beta1-subunit of BKCa might play a role in the development of vascular dysfunction during hypertension (3, 4). Because the beta1-subunit increases the open probability, and the beta2-subunit more rapidly inactivates the BKCa channel, then, if the low-oxygen tension environment of the normal fetus differentially affects expression of these subunits, the physiology of the BKCa channel may be significantly altered, independently of the effect on the pore-forming {alpha}-subunit.

In recent years, a variety of experiments has demonstrated that a universal response to reduced O2 tension involves the expression and activity of hypoxia-inducible factor 1 (HIF-1), a transcriptional activator of genes involved in O2 delivery or metabolic adaptation to hypoxia (20, 39). Interestingly, an inspection of the regulatory regions in some of the BKCa genes revealed the presence of putative HIF-1 response elements (37). Based on this observation, we hypothesized that the low-oxygen tension environment of the normal fetus increases pulmonary vascular BKCa channel expression through transcriptional regulation that may be mediated by HIF-1. Moreover, we hypothesized that in the pulmonary vasculature, expression of the {alpha}-, beta1-, and beta2-subunits of the BKCa channel is differentially affected by hypoxia.

To test these hypotheses, we measured BKCa channel subunit expression under hypoxic and normoxic conditions in both in vivo and in vitro systems. To assess transcriptional activation of the BKCa genes by hypoxia, BKCa beta-subunit luciferase (KCa beta:luc+) reporter genes were constructed and their expression was determined under both hypoxia and normoxia. To gain insight into the mechanism by which hypoxia increases BKCa channel expression, we performed a series of experiments using deferoxamine (DFX), a chelating agent that binds to the hydroxy-prolyl of HIF-1{alpha}, precluding its degradation and thereby increasing transactivation by HIF-1 (15, 18). Finally, to demonstrate functional significance of the molecular findings, microfluorimetry experiments were performed on hypoxic or normoxic PASMC.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animals. Fetal and adult sheep and adult Sprague-Dawley rats were used according to procedures previously reviewed and approved by the Animal Care and Use Committee at the University of Minnesota Medical School (Minneapolis, MN) and the University of Colorado School of Medicine (Denver, CO). Fetal sheep ages ranged from 135 to 142 days of gestation (term = 147 days).

Cell cultures. The techniques used for the isolation of ovine PASMC have been previously described (29). Distal pulmonary arteries were quickly excised from pentobarbital-anesthetized ovine fetuses and placed in physiological saline solution (120 mM NaCl, 5.9 mM KCl, 11.5 mM dextrose, 25 mM NaHCO3, 1.2 mM NaH2PO4, 1.2 mM MgCl2 and 1.5 mM CaCl2). PASMC were isolated from third-generation resistance pulmonary arteries. Loose connective tissue and adventitia were removed, and the vessels were liberally rinsed with minimal essential medium (MEM; 0.2mM 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 elastase (Sigma, St. Louis, MO), 1 mg/ml collagenase (Worthington Biochemical, Freehold, NJ), 2.0 mg/ml bovine serum albumin, 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 aliquoted onto 25-mm2 glass coverslips coated with poly-L-lysine (Sigma, St. Louis, MO) and into 25-cm2 tissue culture flasks (Falcon Plastics, Oxnard, CA) at a density of 5–10 x 103 cells/cm2. The cells were incubated at 37°C in a humidified 95% air and 5% CO2 atmosphere. Hypoxic cell culture conditions were achieved by incubation at 37°C in a humidified 5% oxygen, 5% CO2, and balance N2 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 day 5 and 14 of culture. Cell density stabilized as subconfluent monolayers after 3–5 days in culture. To verify the cell population, PASMC were routinely stained with {alpha}-actin-specific antibody following 5, 10, and 14 days in culture.

Immunostaining. Fetal PASMC were incubated under either normoxic or hypoxic conditions as described above. Cells grown on glass coverslips were rinsed twice with PBS and then fixed for 10 min in 4% paraformaldehyde in PBS. They were permeabilized for 5 min with 0.1% Triton X-100 in PBS. The coverslips were blocked for 30 min with Superblock (Pierce Chemical, Rockford, IL).

A blocking solution of PBS-Triton with 2% goat serum and 4% bovine serum albumin was applied for 60 min. BKCa subunit primary antibodies (Alomone, Jerusalem, Israel) or negative control IgG were diluted to 3–7 µg/ml in the blocking agent and incubated with the cells overnight at 4°C. After being washed with PBS-Triton, a Cy3-labeled secondary anti-rabbit antibody (Sigma) was applied for 60 min. Coverslips were washed six times and then mounted on glass slides with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Digital photos were taken with a Spot camera (Diagnostic Instruments, Sterling Heights, MI) mounted on a Zeiss Atto Arc (Carl Zeiss MicroImaging, Thornwood, NY) fluorescence microscope.

DFX mesylate treatment. Fetal PASMC were incubated under normoxic and hypoxic conditions as described. PASMC maintained in normoxia were supplemented with 200 µM DFX mesylate (Novartis) for 6 h. After 6 h of exposure to DFX, PASMC were washed and maintained in culture for an additional 18 h. PASMC were collected, and total RNA was extracted as outlined above.

RT-PCR. Lung tissue was extracted and immediately placed on ice. The main intralobar pulmonary arteries 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 powder with a prechilled mortar and pestle. Total RNA was extracted using the guanidium thiocyanate-phenol-chloroform method (TRI reagent, Sigma). After homogenization, the samples were processed according to reagent instructions, and the RNA was dissolved in 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. One to two microliters of this reverse transcription reaction were used in each PCR reaction. Oligonucleotide primers used to amplify BKCa {alpha}- and beta-subunits were based on the homolog human sequences (11, 45, 47). Amplified products were consistent with that expected for human BKCa subunits. In most cases, identity of the product was confirmed with sequence analysis.

18S ribosomal RNA was analyzed in RT-PCR as an internal control. 18S cDNA was amplified with a QuantumRNA primer/competimer set (Ambion) to allow relative quantitation of the ethidium bromide bands. This control band appears at either 489 or 324 bp depending on the primer set used. PCR cocktail consisted of 1x PCR buffer (Perkin Elmer) with 1.5 mM Mg2+, 10 pM each BKCa primer, 10 nM dNTP mixture, 20 pM 18S primer mixture (ratio of 1:9) of 18S primers/competimers, 1 unit 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. Samples without reverse transcriptase were evaluated as controls in PCR runs. Whenever required, the identity of the band was confirmed by sequencing the product. Densitometry analysis was used in relative quantitation assessment of the RT-PCR product (NIH Image; Scion, Frederick, MD). The relative densities of the 18S ribosomal and potassium channel PCR products were compared in each individual gel. RT-PCR was performed at least three times for each mRNA sample.

BKCa reporter genes. Plasmid pKCa beta1:luc+ was constructed by fusing 870 bp of 5' sequences proximal to the KCa beta1-subunit gene (KCNMB1) to the luciferase gene. The promoter sequences were obtained by PCR amplification of a human genomic library (kindly provided by Dr. David Zarkower, Univ. of Minnesota) with KCNMB1-specific primers bKCA1A and bKCA2S. The resulting fragment was purified, analyzed by DNA sequencing, and ligated in front of the luciferase gene carried by the pGL3-Basic vector (Promega). Similarly, plasmid pKCa beta2:luc+ was constructed by fusing 920 bp of 5' sequences proximal to the KCa beta2-subunit gene (KCNMB2) to the luciferase gene.

Transfections-luciferase assays. Fetal PASMC were transfected using cationic lipid reagent Tfx-50 (Promega). Plasmid pRL-TK (Promega), containing the Renilla luciferase gene driven by the simplex virus thymidine kinase promoter, was cotransfected as a control for transfection efficiency. Briefly, PASMC were grown in 24-well plates to 60–80% confluence in cell cultures as described before. A transfecting mix of lipid reagent/DNA (4:1 charge ratio of Tfx reagent to DNA) was then added to the cultures (200 µl of serum-free MEM media + 1 µg plasmid + 0.6 µg pRL-TK plasmid + 7 µl Tfx reagent). After an initial incubation at 37°C for 90 min under normal conditions, cultures were overlayed with 1 ml of serum-supplemented MEM media and placed in normoxic (PaO2 = 120 Torr) or hypoxic (PaO2 = 30 Torr) 37°C incubation chambers for 36–48 h.

Ca2+ imaging. To assess dynamic changes in intracellular Ca2+ concentration ([Ca2+]i) in individual PASMC, the Ca2+-sensitive fluorophore fura-2 AM (Molecular Probes) was used. Subconfluent fetal PASMC on 25-mm2 glass coverslips were placed on the stage of an inverted microscope (Nikon Diaphot). Cells were loaded with 100 nM fura-2 AM and 2.5 mg/ml Pluronic acid (Molecular Probes) for 20 min, followed by a 20-min wash in Ca2+-containing solution to allow for deesterification before the experiment. Ratiometric imaging was performed with excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Imaging was performed with an intensified charge-coupled device camera (Photonic Science, Robertsbridge, UK) using Axon Instruments (Foster City, CA) or Metafluor (Fryer, Bloomington, MN) image capture and analysis software. Ca2+ calibration was achieved by measuring a maximum (with 1 mM ionomycin) and a minimum (with 10 mM EGTA). PO2 was controlled by aerating the recording solution reservoir and the stage microincubator with either 21% O2 with balance N2 (normoxia) or 100% N2 (hypoxia). pH was 7.40 ± 0.05 and did not change during the experiments. [Ca2+]i was calculated assuming a dissociation constant of 220 (14). For each experiment, 10–20 cells were visualized, and ratiometric data were acquired from individual cells.

Statistical analysis. A two-way ANOVA with repeated measures and a Student-Newman-Keuls post hoc test was 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 ± SD. P values <0.05 were considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Hypoxia increases BKCa channel {alpha}-subunit expression. Using primers based on previously described sequences of the BKCa channel subunits (11, 45, 47), RT-PCR demonstrated a robust {alpha}- and beta1- KCa channel subunit expression in PASMC from fetal lambs. We were unable to detect expression of the beta2-subunit in the fetal pulmonary vasculature. We performed quantitative, internally controlled RT-PCR experiments to investigate the effect of hypoxia on mRNA levels of the BKCa {alpha}- and beta1-subunit. When PASMC were incubated under conditions of normal (normoxia, PO2 =120 Torr) or low-oxygen tension (hypoxia, PO2 = 30 Torr), we found that hypoxia results in a significant increase in BKCa {alpha}- and beta1-subunit (Fig. 1).


Figure 1
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  		Fig. 1. Effect of hypoxia on calcium-sensitive K+ channel (BKCa) mRNA levels in fetal pulmonary artery smooth muscle cells (PASMC). RT-PCR was performed on total RNA extracted from fetal PASMC. Specific BKCa {alpha}- and beta1-subunit primers were used. A, top: aggregate data resulting from 6 experiments. BKCa {alpha}-subunit mRNA levels were significantly higher in hypoxia compared with normoxia (P < 0.01). A, bottom: representative gel showing {alpha} BKCa expression and 18S control under hypoxic and normoxic conditions. B, top: aggregate data resulting from 3 experiments. BKCa beta1-subunit mRNA levels were significantly higher in hypoxia compared with normoxia (P < 0.05). B, bottom: representative gel showing BKCa beta1-subunit expression and 18S control under hypoxic and normoxic conditions.

 
Hypoxia increases pulmonary BKCa channel mRNA expression in vivo. To test the effect of hypoxia on BKCa channels in a living mammalian model, adult Sprague-Dawley rats were maintained under conditions of either hypobaric hypoxia (barometric pressure = 410 mmHg; PO2 = 76 mmHg) or normoxia (barometric pressure = 630 mmHg; PO2 =122 mmHg) for 3 wk. Total RNA was extracted from whole lungs of hypoxic and normal animals, and RT-PCR was performed as previously described. In vivo, hypoxia caused a significant increase in the levels of BKCa {alpha}- (Fig. 2A) and beta- (Fig. 2B) subunits. The observations were consistent with the results from in vitro cell system.


Figure 2
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  		Fig. 2. Effect of hypoxia on BKCa {alpha}- and beta1-subunit mRNA levels in vivo. A and B, top: representative gels are shown of RT-PCR performed on total RNA from lungs of adult Sprague-Dawley rats maintained for 3 wk in either hypoxia (animals 1 and 2) or normoxia (animals 3 and 4). A and B, bottom: aggregate data from 3 independent RT-PCR experiments showing an average 3-fold increase in BKCa {alpha}-subunit and more than 2-fold in BKCa beta1-subunit mRNA under hypoxic conditions.

 
Immunocytochemistry of BKCa {alpha}- and beta1-subunit expression in hypoxic and normoxic PASMC. Fetal PASMC maintained in either hypoxia or normoxia were immunostained with specific BKCa {alpha} and beta1 antibodies (Alomone). Staining for BKCa {alpha}- and beta1-subunits appears to be greater in hypoxic compared with normoxic PASMC (Fig. 3).


Figure 3
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  		Fig. 3. Effect of hypoxia on BKCa {alpha}- and beta1-subunit protein expression in fetal PASMC maintained in primary culture. Fetal PASMC were maintained in either normoxia or hypoxia. In A and B, cells were stained with either BKCa {alpha}- or beta1-subunit-specific antibodies or an IgG negative control.

 
DFX, a hypoxia mimetic, increases BKCa {alpha}- and beta-subunit expression. To determine the mechanism whereby hypoxia increases BKCa channel expression, we used a hypoxia mimic that acts by blocking HIF-1{alpha} degradation. PASMC were treated with 200 µM DFX. When cells were exposed to DFX for 6 h and allowed to remain in culture for an additional 18 h before harvesting, BKCa {alpha}- and beta1-subunit mRNA expression was increased by 63 and 41%, respectively (Fig. 4, A and B).


Figure 4
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  		Fig. 4. Effect of deferoxamine (DFX) on BKCa {alpha}- and beta1-subunit expression. Under normoxic conditions, fetal PASMC were incubated in the presence of 200 µM DFX for 6 h. Cells were then washed and replaced in culture for 18 h in normal media. After a total of 24 h, cells were harvested and total RNA was extracted. A, top: relative expression of the BKCa {alpha}-subunit in DFX-treated and -untreated PASMC. *P < 0.03. A, bottom: representative gels of BKCa {alpha}-subunit expression in fetal PASMC treated with either 0, 100, or 200 µM DFX. B, top: relative expression of the BKCa beta1-subunit in DFX-treated and -untreated PASMC. *P < 0.05. B, bottom: representative gels of BKCa beta1-subunit expression in fetal PASMC treated with either 0, 100, or 200 µM DFX.

 
Hypoxia increases BKCa beta-subunit expression at the level of transcription. The noncoding 5' (promoter) sequences flanking the beta1- and beta2-subunit genes of the BKCa channel were cloned by a PCR approach using information from published sequences (22). These sequences were subsequently fused to a luciferase reporter gene to make KCa:luc+ reporter plasmids.

Ovine PASMC in primary cell cultures were transfected with KCa:luc+ reporter plasmids to determine whether the promoter sequences of the BKCa beta-subunit genes contain oxygen-sensitive elements. The expression of these reporter genes was then compared under normal and low oxygen tension. Hypoxia increased BKCa beta1-subunit reporter gene expression by threefold (P < 0.01, vs. normoxia), suggesting a strong sensitivity to O2. While exhibiting a more modest effect, KCa beta2 reporter levels were also increased by hypoxia (35 ± 8% increase, P < 0.01, vs. normoxia; Fig. 5).


Figure 5
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  		Fig. 5. Effect of oxygen on the transcription regulation of beta1- and beta2-BKCa subunit expression. Fetal PASMC transfected with 0.5–1 µg of beta1KCa:luc+ (filled bar) and beta2KCa:luc+ (open bar) plasmids showed increased luciferase expression under hypoxia relative to normoxia. beta1KCa:luc+ and beta2KCa:luc+, BKCa beta1- and beta2-subunit luciferase, respectively.

 
Effect of iberiotoxin on basal [Ca2+]i is greater in hypoxic compared with normoxic fetal PASMC. After obtaining stable baseline values in either hypoxic (PO2 ~30 mmHg) or normoxic HBSS (PO2 ~120 mmHg), we administered iberiotoxin, a selective pharmacological antagonist of the BKCa channel (12), to hypoxic (n = 56 cells; 3 animals) or normoxic (n = 51 cells; 3 animals) fetal PASMC in concentrations ranging from 10–10 to 10–8 M. In hypoxic but not normoxic cells, 5 x 10–10 M increased basal [Ca2+]i (P < 0.01, vs. baseline). Iberiotoxin at 10–9 M caused an increase in [Ca2+]i of 232 ± 19% in hypoxic PASMC, compared with an increase in [Ca2+]i of 104 ± 11% in normoxic PASMC (P < 0.01, hypoxia vs. normoxia; P < 0.01 vs. baseline for hypoxia and normoxia; Fig. 6 ).


Figure 6
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  		Fig. 6. Effect of iberiotoxin on basal cytosolic calcium in hypoxic or normoxic fetal PASMC. Hypoxic fetal PASMC were more sensitive to iberiotoxin than normoxic fetal PASMC. Iberiotoxin caused a greater increase in fetal PASMC intracellular Ca2+ concentration ([Ca2+]i) at all concentrations in hypoxic, compared with normoxic, PASMC. *P < 0.01 vs. baseline; **P < 0.01 vs. normoxia.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
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The present report demonstrates that fetal PASMC express BKCa {alpha}, beta1-, but not beta2-subunits. Moreover, the present data provide evidence that hypoxia increases BKCa channel subunit expression and that hypoxia has a greater effect on BKCa channel {alpha}- and beta1-subunit expression than on beta2. The implication of the present data is that the low-oxygen tension environment of the normal fetus plays a role in preparing the pulmonary vasculature to respond to perinatal vasodilator stimuli by enhancing calcium-sensitive K+ channel expression. To ensure that the use of a single experimental model did not bias the experimental findings, studies were performed in multiple experimental systems. Hypoxia was noted to increase BKCa channel expression in both in vivo and in vitro systems.

The present report provides evidence that pulmonary vascular BKCa channel subunit expression is modulated by oxygen tension. Moreover, the effect of oxygen on BKCa channel subunit expression is not specific to a given developmental stage, as hypobaric hypoxia caused an increase BKCa channel subunit expression in the lungs of adult Sprague-Dawley rats as well as fetal PASMC in primary culture. The increase in beta1-subunit expression in response to hypoxia is, potentially, highly significant. The increase in beta1-subunit expression would be expected to render the BKCa channel more sensitive to an increase in calcium concentration (6, 43), thereby increasing the open probability of the channel in response to an elevation of calcium in the region of the channel.

From a teleological perspective, such an adaptation would be advantageous given the subcellular mechanisms responsible for both oxygen- and nitric oxide-induced perinatal pulmonary vasodilation. Oxygen decreases PASMC [Ca2+]i by causing quantal release ("calcium sparks") of calcium from intracellular ryanodine-sensitive stores (27). The localized increase in calcium in the region of the BKCa channel causes BKCa channel activation, membrane hyperpolarization, closure of voltage-gated Ca2+ channels, a decrease in PASMC [Ca2+]i, and a subsequent vasodilation (19). Thus a mechanism that increases BKCa channel calcium sensitivity would be beneficial in both potentiating and guaranteeing the vasodilatory response of the pulmonary circulation to oxygen. Because nitric oxide has been shown in the perinatal pulmonary circulation to act through a similar subcellular mechanism (36), enhanced BKCa channel sensitivity would likely enhance the perinatal pulmonary response to nitric oxide, a critical vasodilator in the perinatal pulmonary circulation.

Even when the studies we present show that BKCa beta2-subunit expression appears to be negligible in fetal pulmonary smooth muscle cells, they also suggest that the promoter sequences for this gene might be sensitive to oxygen. This observation might be relevant in biological systems in which expression of this subunit is better established (47, 48).

Despite the differential response of the various BKCa subunits to hypoxia, the basis of this differential response remains unclear. Potentially, the promoter of the beta1-, compared with the {alpha}-subunit, of the BKCa channel may contain more, or more efficient, HIF-1 response elements. Alternatively, our results may be partially dependent on the reporter constructs used, as they contain <1 kb of proximal 5' sequences. One or both of our promoter constructs may lack additional regulatory elements that would normally be located relatively far away from the coding sequences.

Whereas short-term changes in O2 tension are thought to result in posttranslational modification of existing proteins (i.e., phosphorylation, dephosphorylation) (32), chronic, more long-lasting changes in O2 tension appear to involve changes in gene expression (38, 41). Hypoxia is increasingly recognized as an important regulator of physiologically relevant genes. Molecular characterization of O2-sensing systems has identified HIF-1 as the main component and master regulator of the molecular response to hypoxia (40). HIF-1 is a heterodimer consisting of subunits {alpha} and beta (also known as the aryl hydrocarbon nuclear translocator). Hypoxia results in the stabilization of HIF-1{alpha} protein, enabling it, upon dimerization with the constitutively expressed HIF-1beta, to bind DNA and activate transcription of target genes. HIF-1 binds to the hypoxia-response element (HRE) commonly located in the promoter region of hypoxia-sensitive genes. At the core of the HRE sits a consensus sequence, 5'-RCGTG-3' (37). The presence of an HIF-1 binding site is necessary but not sufficient for HIF-1-mediated transcriptional activation.

The observation that DFX increases BKCa channel subunit expression suggests that hypoxia increases BKCa mRNA levels through HIF-1{alpha} (13, 21). Interestingly, the increase in BKCa channel expression was not apparent in PASMC that were exposed to DFX for 6 h and harvested immediately thereafter. In contrast, DFX-treated cells that remained in normoxia for 18 h after DFX exposure demonstrated a significant increase in BKCa channel expression. Thus the effects of hypoxia on gene expression appear to be delayed, suggesting hypoxia has effects on both BKCa channel subunit transcription and likely posttranslational modification.

Microfluorimetry data included in the present study provide evidence that the effect of hypoxia on BKCa channel expression possesses functional significance. Hypoxic fetal PASMC were more sensitive to iberiotoxin than normoxic fetal PASMC. The relatively diminished sensitivity to iberiotoxin suggests that with prolonged exposure to normoxia, the BKCa channel no longer determines basal [Ca2+]i and, perhaps, resting membrane potential. This observation is consistent with previous data from our laboratory demonstrating that with maturation the K+ channel that determines resting membrane potential changes from BKCa channel to the voltage-sensitive K+ channel (29). These data offer support for the notion that the low-O2 tension environment of the normal fetus maintains BKCa channel expression at a higher level than it would otherwise, thereby preparing the fetal pulmonary circulation to respond to perinatal vasodilator stimuli (27, 30, 36). Moreover, the data are consistent with the proposition that the increased oxygen tension environment of air-breathing life may be the physiological signal that accounts for the relative increase in voltage-gated K+ channel expression in PASMC with maturation (8), thereby enhancing the capacity for hypoxic pulmonary vasoconstriction.

In recent years, several hypoxia-induced genes that are transactivated by HIF-1 have been identified. These genes, whose products mostly involve O2 delivery or metabolic adaptation to hypoxia, include erythropoietin, glucose transporters, glycolytic enzymes, and vascular endothelial growth factor (31, 33, 37, 38, 41). HIF-1 is also required for embryogenesis, as homozygous HIF-1{alpha}–/– null mice do not develop beyond embryonic day 9 and display severe vascular defects (17, 35). Hence, HIF-1 appears to play an essential role as a master regulator of O2 homeostasis. The hypothesis presented in this manuscript complements very recent data suggesting, for the first time, the involvement of HIF-1 in the genetic regulation of ion channels (9, 10).

In conclusion, we report that the pulmonary vasculature expresses {alpha}- and beta1-KCa subunits. Moreover, hypoxia causes an increase in gene expression of each of these BKCa subunits. The observation that BKCa channel subunit expression is differentially affected by hypoxia suggests that further insight into postnatal adaptation of the pulmonary circulation can be gained by a more detailed understanding of the ontogeny of BKCa channel subunit expression. Moreover, the hypoxia-induced increase in pulmonary BKCa channel {alpha}-subunit expression is observed in both fetal and adult lungs, suggesting that the response, at least in the case of this subunit, is not developmentally regulated. Whether adverse intrauterine stimuli differentially affect BKCa channel subunit expression remains unknown. Further study will be necessary to determine the mechanism whereby low oxygen tension affects BKCa channel subunit expression.


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 ABSTRACT
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This work was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-60784 (D. N. Cornfield) and RO1-HL-70628, an American Heart Association Established Investigator Award (D. N. Cornfield), and Viking Children's Fund (E. Resnik).


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. R. Resnik, Dept. of Pediatrics, Univ. of Minnesota Medical School, MMC 742, 420 Delaware St. S.E., Minneapolis, MN 55455 (e-mail: resni004{at}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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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