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Am J Physiol Lung Cell Mol Physiol 293: L1171-L1182, 2007. First published August 24, 2007; doi:10.1152/ajplung.00126.2007
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Perinatal hypoxia triggers alterations in K+ channels of adult pulmonary artery smooth muscle cells

M. Marino,1,2 J. L. Bény,1 A. C. Peyter,2 R. Bychkov,1 G. Diaceri,2 and J. F. Tolsa2

2Neonatal Research Laboratory, Department of Pediatrics, University Hospital Center and University of Lausanne, Lausanne, Switzerland; and 1Laboratory of Vascular Cell Physiology, Department of Zoology, University of Geneva, Geneva, Switzerland

Submitted 30 March 2007 ; accepted in final form 21 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adverse events during the perinatal period, like hypoxia, have been associated with adult diseases. In pulmonary vessels, K+ channels play an important role in the regulation of vascular tone. In the fetus, Ca2+-activated K+ channels (KCa) are predominant, whereas from birth voltage-gated K+ channels (KV) prevail in the adult. We postulated that perinatal hypoxia could alter this maturational shift and influence regulation of pulmonary vascular tone in relation to K+ channels in adulthood. We evaluated the effects of perinatal hypoxia on KV and KCa channels in the adult main pulmonary artery (PA) using a murine model. Electrophysiological measurements showed a greater outward current in PA smooth muscle cells of mice born in hypoxia than in controls. In controls, only KV channels contributed to this current, whereas in mice born in hypoxia both KV and KCa channels were implicated. KV channel activity was even higher in mice born in hypoxia than in controls. Therefore, perinatal hypoxia results in increased KCa and KV channel activity in adult PA. Moreover, PA of adults born in hypoxia displayed higher large-conductance KCa {alpha}-subunit and KV1.5 {alpha}-subunit protein expression than controls. Interestingly, relaxation induced by nitric oxide (NO) donors [S-nitroso-N-acetyl-D,L-penicillamine, 2-(N,N-diethylamino)-diazenolate-2-oxide] in isolated PA of control mice was not mediated by KCa channels and only slightly by KV channels, whereas following perinatal hypoxia both KCa and KV channels contributed to this relaxation. Thus perinatal hypoxia results in altered expression and activity of different K+ channels in the adult main PA, which could contribute to modifications of pulmonary vasoreactivity.

mouse; potassium ion channel activity; potassium ion channel blockers; nitric oxide donors

ADVERSE EVENTS IN THE PERINATAL period may predispose to pathological responses later in life. In utero growth restriction and perinatal hypoxia have been involved in the development of adult diseases such as coronary artery disease, systemic hypertension, stroke, and non-insulin-dependent diabetes mellitus (4, 5, 12).

Several lines of evidence indicate that chronic pulmonary vascular diseases and altered pulmonary vascular reactivity in adulthood may be associated with a perinatal hypoxic insult (13, 15, 20, 31, 42, 44). Human and animal studies showed that individuals having suffered from a transient hypoxic insult in utero and/or soon after birth displayed exaggerated pulmonary vasoconstrictive responses when reexposed to hypoxia later in life (9, 14–18, 42, 45). The mechanisms underlying the exaggerated pulmonary vasoconstrictor responsiveness and the resulting altered lung circulation in adulthood following a perinatal hypoxic event have not been elucidated so far.

The regulation of the pulmonary circulation undergoes dramatic changes from the fetus to the newborn and to the adult. Indeed, the pulmonary circulation of the normal newborn is adapted to respond to an acute increase in O2 tension, whereas the pulmonary circulation of the adult is adapted to respond to an acute decrease in O2 tension. During fetal life, pulmonary circulation is characterized by a low O2 tension and elevated vascular resistance with limited blood flow. At birth, the transition from gas exchange by the placenta to the lungs requires important changes in the pulmonary circulation (19, 46). Postnatal survival depends upon the ability of the pulmonary circulation to undergo rapid vasodilatation during the first minutes after birth. Among the different factors implicated into this transition, the nitric oxide (NO)/cGMP pathway and potassium (K+) channels have predominant roles. NO induces pulmonary artery smooth muscle cell (PASMC) relaxation by activating cGMP production, which, in turn, stimulates protein kinase G (PKG). PKG induces relaxation through a cascade of various mechanisms, including the activation of K+ channels (1). Besides PKG, K+ channels could also be directly activated by different agents, including O2 and NO (6, 49). K+ channel activation leads to smooth muscle cell (SMC) hyperpolarization, inducing closure of voltage-gated Ca2+ channels and a decrease in intracellular Ca2+ concentration ([Ca2+]i), resulting finally in vasorelaxation. K+ channels are expressed in almost all cells and play a major role in the regulation of resting membrane potential (Em; see Ref. 8). Four types of K+ channels have been identified in arterial SMC: voltage-gated K+ channels (KV), Ca2+-activated K+ channels (KCa), ATP-sensitive K+ channels (KATP), and inward-rectifier K+ channels, which contribute to the regulation of arterial SMC Em (29). In PASMC, mainly three different types of K+ channels have been identified using electrophysiological techniques : KV, KCa and KATP channels (1, 29, 56).

Maturational changes have been found in K+ channel activity and expression in the pulmonary circulation between the fetus and the adult (39). The K+ channels controlling Em in the hypoxic environment of the developing fetus appear to be predominantly KCa channels, whereas KV channels prevail in normoxic adult PASMC (39). This maturational shift from KCa to KV channels is mainly triggered by changes in O2 tension at birth (34, 40, 41). Because O2 tension is normally low in the fetus and increases after birth, it is possible that perinatal hypoxia disturbs the maturational shift in K+ channels and therefore KV and KCa channel activity later in life.

Interestingly, numerous aspects of K+ channel function were previously found to be affected by chronic hypoxia in fetal ovine cerebral vessels (27).

Therefore, we postulated that perinatal hypoxia could alter the maturational shift from KCa to KV channels at birth and influence mechanisms of pulmonary vasorelaxation in relation to K+ channels in adulthood.

We developed an experimental model of perinatal hypoxia in mice and evaluated in adulthood the activities of KV and KCa channels at the level of the main pulmonary artery (PA) by techniques of electrophysiology and their contribution to the regulation of pulmonary vascular tone by pharmacological studies.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Animal model. C57BL/6 mice from Harlan (Horst, The Netherlands) were used. All experimental procedures were approved and carried out in accordance with the Swiss Veterinarian Animal Care Office. Pregnant mice were placed in hypoxic conditions (13% O2) 5 days before delivery (day 21) and left under hypoxia with their litter for 5 days after birth. Pups were then raised in normoxia (21% O2) until adulthood. The timing of exposure to hypoxia was chosen to cover the lung vasculogenesis period in mice, during which the functional units of gas exchange develop (53). The degree of hypoxia was chosen according to previous studies in rodents (17, 18). Pups born and grown in normoxic conditions were used as controls. Male mice were studied as adults between the 18th and 25th week.

Adult mice were administrated a lethal dose of pentobarbital sodium (1 g/kg ip), and the main PA was immediately harvested, resulting in a ring of 1.5–2.0 mm length and 0.5–1.0 mm diameter.

Cell preparation and isolation. Isolation of PASMC was performed as previously described (8) with minor modifications. The main PA was dissected and placed in an isolated Ca2+-free cell solution (in mM: 137 NaCl, 5.4 KCl, 0.44 KH2PO4, 0.42 NaH2PO4, 0.2 MgCl2, 6 H2O, 4.17 NaHCO3, 1.8 EGTA, and 10 HEPES) containing 1 mg/ml BSA and 2 mg/ml glucose at room temperature during 10 min. During this time, the vessel was cleaned and prepared for enzymatic digestion, which was performed during 20 min in an incubator at 37°C using a second solution (in mM: 137 NaCl, 5.4 KCl, 0.44 KH2PO4, 0.42 NaH2PO4, 0.2 MgCl2·6 H2O, 4.17 NaHCO3, 0.2 CaCl2·2 H2O, 0.05 EGTA, and 10 HEPES) containing collagenase (1.6 mg/ml), elastase (0.5 mg/ml) and glucose (2 mg/ml). As soon as enzymatic digestion was finished, the tissue was rinsed with the isolated cell solution during 1 min at room temperature. Next, the tissue was flushed with a micropipette (up-and-down pipetting) to release PASMC. Most cells isolated by this technique are PASMC, as demonstrated by immunocytochemistry (see below). Isolated PASMC were placed in culture plates, stored at 4°C, and used 1 h after their isolation. Only elongated, smooth, attached on culture plates and optically refractive cells were used for patch-clamp measurements.

Isolated PASMC immunostaining. Freshly isolated PASMC were plated in an eight-chamber glass slide (Lab-Tek Chamber Slide System; Nalge Nunc International, Naperville, IL) and left during 1 h at 4°C to allow attachment. Cells were then fixed by incubation in 1:1 acetone-PBS during 10 min at room temperature followed by 10 min in acetone at –20°C. Immunoreactivity to smooth muscle-specific forms of actin ({alpha}-actin) and myosin, as well as to endothelial markers like von Willebrand factor and CD-31 (platelet endothelial cell adhesion molecule-1), was tested using specific antibodies (Sigma, St. Louis, MO) diluted 1:50 (actin, myosin and von Willebrand factor) or 1:30 (CD-31) and the Vectastain Universal ABC-AP Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Coloration was obtained using the Vector Red Alkaline Phosphatase Substrate Kit I (Vector Laboratories) according to the manufacturer's instructions. This substrate produces a red reaction product that can be seen using either brightfield or fluorescent microscopy.

Patch-clamp recording. Membrane currents were recorded at room temperature using the nystatin perforated patch configuration with a patch amplifier (EPC7; Heka, Lambrecht/Pfalz, Germany) and Digidata 1322A (Molecular Devices, Sunnyvale, CA). Nystatin was used to permeabilize the cellular membrane and provide an electrical continuity between the pipette and the cytoplasm, allowing diffusion of monovalent ions without dialysis of cytoplasmic high-molecular-weight compounds (22). Nystatin was dissolved in dimethyl sulfoxide (DMSO) and diluted in the pipette solution to obtain a final concentration ranging from 50 to 100 µg/ml. Patch-clamp electrodes of borosilicate capillary glass were pulled using a microelectrode puller (model P-2000; Sutter Instruments, Novato, CA). Electrodes had a resistance of 5–7 M{Omega} and were filled with 130 mM KCl and 10 mM HEPES.

Cell preparation was bathed with a physiological solution (in mM: 130 NaCl, 5.6 KCl, 1 MgCl2·6H2O, 2 CaCl2·2H2O, 10 glucose, and 8 HEPES, pH 7.5) and continuously perfused through a peristaltic pump (Minipuls 3; Gilson, Middleton, WI).

Voltage-clamp protocols were generated using the Pclamp 9 software (Molecular Devices) and data were analyzed with the Clampfit software (Molecular Devices). Outward net currents were elicited by voltage-step pulses with an increment of 10 mV (200-ms duration) from a holding potential of –80 mV.

The contribution of KCa and KV channels to the outward current measured in PASMC from control mice and mice born in hypoxia was investigated by addition of the specific K+ channel inhibitors 4-aminopyridine (4-AP), charybdotoxin (CTX), and iberiotoxin (IbTX). Dose responses to these inhibitors were performed in PASMC of both groups. According to these dose responses and previous studies performed in murine PASMC (3, 23), 2 x 10–3 M 4-AP, 10–7 M CTX, and 10–7 M IbTX were used for further experiments.

The cell capacitance was measured manually by dividing the integration of capacitive currents from the amplitude of 10-mV voltage steps. Current density was expressed as picoAmpere per picoFarad.

Western blotting analysis. Groups of 10 main PA of adult mice were crushed in a cryogenic mortar and homogenized in a lysis buffer {50 mM HEPES, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM dithiothreitol, 5 µg/ml pepstatin, 3 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 1 mM sodium vanadate, 50 mM NaF, and 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate}. The homogenates were centrifuged at 3,000 g during 10 min at 4°C. Protein concentration of the supernatant was quantified using a BCA protein assay kit (Pierce, Rockford, IL). Thirty micrograms of proteins per lane were fractioned by SDS-PAGE (7.5%) and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). Blots were then blocked overnight at 4°C in Tris-buffered saline plus 0.05% Tween 20 (TBS-T) containing 2% nonfat dry milk. The membranes were immunoblotted for 1 h at room temperature using two different antibodies, targeted against the large-conductance KCa (BKCa) {alpha}-subunit or KV1.5 {alpha}-subunit proteins (Alomone Laboratories, Jerusalem, Israel) diluted in TBS-T containing 2% nonfat dry milk 1:300 and 1:400, respectively. Investigation of KV1.5 {alpha}-subunit was performed because KV1.5 channels are known to be sensitive to acute and chronic hypoxia (2, 37, 52). Immunoblotting of beta-actin protein with an antibody (Sigma) diluted 1:250 in TBS-T with 2% nonfat dry milk was also performed to normalize the protein content of each lane. After incubation with the primary antibodies, blots were washed in TBS-T and incubated with ECL peroxidase-labeled anti-rabbit antibody (Amersham Biosciences, Buckinghamshire, UK), diluted 1:10,000 in TBS-T. After being washed in TBS-T, specific proteins were detected by chemiluminescence using the ECL Western blotting analysis system (Pierce) and exposition to X-ray film. To determine the relative K+ channel subunit expression, intensity of each band was quantified with the UN-SCAN-IT gel software (Silk Scientific, Orem, UT), and normalization to beta-actin content was done. Relative expression of K+ channel subunits in PA homogenates of mice born in hypoxia was reported to the content measured in controls.

beta-Actin has been previously used to normalize Western blots in other animal models with hypoxia, e.g., in PA of fetal lambs exposed to chronic high-altitude hypoxia (13). However, we cannot exclude that perinatal hypoxia has some effect on this structural protein in the main PA of adult mice. Therefore, we validated the use of beta-actin as an internal control in our model by comparison with {alpha}-tubulin and {alpha}-actinin protein contents by immunoblotting using specific antibodies (Sigma). In our murine model, beta-actin protein content was demonstrated to be uninfluenced by perinatal exposure to hypoxia (data not shown).

Isolated vessel tension studies. Vessel rings were suspended in organ chambers filled with 10 ml of modified Krebs-Ringer bicarbonate solution (in mM: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and 11.1 glucose) maintained at 37°C and aerated with 95% O2-5% CO2 (pH 7.4; see Ref.48). Each ring was suspended by two stirrups (0.1 mm diameter) passed through the lumen. One stirrup was anchored to the bottom of the organ chamber, and the other one was connected to a strain gauge (PowerLab/8SP; ADInstruments, Hastings, UK) for the measurement of isometric force in grams. Vessels were brought to their optimal resting tension after 2 stretches of 0.5 g. After equilibration, indomethacin (10–5 M), an inhibitor of cyclooxygenase activity (50), and NG-nitro-L-arginine (L-NA; 10–4 M), an inhibitor of NOS activity (28), were added in the organ chambers to exclude possible interference of prostanoids and NO, respectively. The K+ channel inhibitors 4-AP (2 x 10–3 M) and CTX (10–7 M), inhibiting KV and KCa channels, respectively, were also added to study the role of K+ channels in the relaxation of PA induced by NO donors. The vessels were then contracted with phenylephrine (10–5 M) at a level corresponding at least to the maximal response to K+ (10–3 M KCl). Finally, cumulative dose responses of PA to S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 10–9 M to 10–4 M) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA NONOate, DEA/NO, 10–10 M to 10–4 M) were evaluated. Changes in tension induced by SNAP or DEA/NO were expressed as a percent of the initial contraction induced by phenylephrine. The half-maximal effective concentrations (EC50) of SNAP or DEA/NO were calculated with a dose-response curve fit, and results were expressed in molarity.

Drugs. Reagents were all obtained from Sigma Chemicals, except 4-AP (Fluka, Buchs, Switzerland) and SNAP, DEA/NO, and L-NA (Alexis Biochemicals, San Diego, CA). Unless otherwise indicated, all stock solutions were prepared in bidistilled H2O. SNAP was dissolved in DMSO and DEA/NO in NaOH (10 mM). Indomethacin was prepared in equal molar solution with Na2CO3 (47).

Data analyses. Data were expressed as means ± SE. Unpaired Student's t-test was used to compare the control and perinatal hypoxia groups in electrophysiological and Western blot results. Paired Student's t-test was used in control and perinatal hypoxia groups to analyze the effect of K+ channel inhibitors on the outward current. One-way ANOVA with Fisher post hoc test was used to analyze isolation vessel studies. Statistical significance was accepted when the two-tailed P value was <0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
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Immunocytochemistry. Freshly isolated PASMC displayed a positive staining to the smooth muscle-specific forms of actin ({alpha}-actin) and myosin, but a negative staining to the endothelial markers von Willebrand factor and CD-31 (Fig. 1, A–D). These results clearly demonstrate that most isolated cells were SMC, and not endothelial cells or fibroblasts.


Figure 1
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  		Fig. 1. Immunochemical characterization of freshly isolated pulmonary artery smooth muscle cells (PASMC). The cellular specificity of freshly isolated cells from the main pulmonary artery (PA) was tested by immunocytochemistry. These cells display positive staining (red) to smooth muscle-specific forms of actin ({alpha}-actin; A) and myosin (B) but negative staining to endothelial markers like von Willebrand (vW) factor (C) and CD-31 (D). Negative control is also shown (E).

 
Whole cell current measurements in freshly isolated PASMC. PASMC capacitance was 13% higher in control mice than in mice born in hypoxia (39.0 ± 1.9 and 34.0 ± 1.6 pF, respectively, P < 0.05; n = 15–20 cells; data not shown).

Outward currents were significantly higher in PASMC from mice born in hypoxia compared with PASMC from controls (15.5 ± 1.1 and 11.8 ± 0.9 pA/pF at +40 mV, respectively; P < 0.05; n = 17–21 cells; Fig. 2).


Figure 2
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  		Fig. 2. Current (I)-voltage (V) curves of PASMC from mice born in normoxia or in hypoxia. Outward current was measured in freshly isolated PASMC from adult mice born in normoxia (Ctr) or in hypoxia (PH) in the perforated-cell configuration. Left: outward membrane current density elicited by a series of 10-mV depolarizing steps (–80 to +80 mV) from a holding potential of –80 mV. Right: steady-state I/V relationships of voltage-dependent outward currents. Data are expressed as means ± SE (n = 17–21 cells). *Significant difference between the Ctr and PH groups (P < 0.05, unpaired Student's t-test).

 
To better characterize the difference observed between control mice and mice born in hypoxia, we applied in the bath solution the K+ channel blockers 4-AP, CTX, and IbTX. Dose responses to these drugs were performed, and K+ channel activity was measured by depolarizing PASMC in the perforated patch configuration. Dose-dependent inhibition of the outward current was observed in both groups in the presence of 4-AP, a selective blocker of KV (Fig. 3A). This confirms the presence of KV channels in these cells. Inhibition of the outward current was significantly higher in PASMC of mice born in hypoxia compared with controls. However, EC50 did not differ between PASMC of mice born in hypoxia or in normoxia (1.62 ± 0.96 x 10–3 M and 1.37 ± 0.26 x 10–3 M at +40 mV, respectively; n = 4–13 cells), indicating that sensitivity to 4-AP is similar in both groups. A single dose of 2 x 10–3 M 4-AP was used for further experiments. The steady-state current-voltage relationships displayed in both groups a significant decrease of the current density in the presence of 4-AP (Fig. 4). As observed in dose-response experiments, 4-AP-induced inhibition of the outward current was significantly more important in PASMC from mice born in hypoxia compared with PASMC of control mice (31.9 ± 5.4 and 15.6 ± 4.6% at +40 mV, respectively; P < 0.05, n = 9–13 cells).


Figure 3
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  		Fig. 3. Effects of 4-aminopyridine (4-AP), charybdotoxin (CTX), and iberiotoxin (IbTX) application on the outward current of freshly isolated PASMC. Dose responses to 4-AP (10–6 to 10–1 M, n = 4–13 cells; A), CTX (10–10 to 10–6 M, n = 4–8 cells; B), and IbTX (10–10 to 10–6 M, n = 4–8 cells; C) were tested in the perforated-cell configuration on freshly isolated PASMC from adult mice born in normoxia (Ctr) or in hypoxia (PH). I/Imax represents the ratio between the outward current (I) measured in the presence of a K+ channel blocker and the maximum outward current without inhibitor (Imax). The curves correspond to the best sigmoidal fits. Data are expressed as means ± SE. *Significant difference between the Ctr and PH groups (P < 0.05, unpaired Student's t-test).

 

Figure 4
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  		Fig. 4. I-V curves of PASMC from mice born in normoxia or in hypoxia in the presence of the voltage-dependent K+ channel (KV) inhibitor 4-AP. Outward current was measured in freshly isolated PASMC from adult mice born in normoxia (Ctr) or in hypoxia (PH) in the perforated-cell configuration. Left: outward membrane current density elicited by a series of 10-mV depolarizing steps (–80 to +80 mV) from a holding potential of –80 mV. Right: steady-state I/V relationships of voltage-dependent outward currents. Effect of 4-AP on the outward current was measured in PASMC of mice born in normoxia (A) or in hypoxia (B). Data are expressed as means ± SE (n = 8 cells). *Significant difference with and without 4-AP (P < 0.05, paired Student's t-test).

 
CTX, a blocker of large- and intermediate-conductance channels expressed in vascular SMC, was used to evaluate the activity of KCa channels in PASMC. Dose-dependent inhibition of the outward current by CTX was observed in PASMC of mice born in hypoxia but not in controls (Fig. 3B). A single dose of 10–7 M CTX was used for further experiments. In steady-state current-voltage relationships, CTX application also failed to inhibit the outward current measured in PASMC from control mice (Fig. 5A). In contrast, CTX induced a significant decrease of the outward current in PASMC from mice born in hypoxia (Fig. 5B). CTX-induced inhibition of the outward current was then significantly higher in the perinatal hypoxia group than in controls (28.6 ± 5.3 and 6.7 ± 4.5% at +40 mV, respectively; P < 0.05, n = 8 cells).


Figure 5
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  		Fig. 5. I-V curves of PASMC from mice born in normoxia or in hypoxia in the presence of the Ca2+-activated K+ channel (KCa) inhibitor CTX. Outward current was measured in freshly isolated PASMC from adult mice born in normoxia (Ctr) or in hypoxia (PH) in the perforated-cell configuration. Left: outward membrane current density elicited by a series of 10-mV depolarizing steps (–80 to +80 mV) from a holding potential of –80 mV. Right: steady-state I/V relationships of voltage-dependent outward currents. Effect of CTX on the outward current was measured in PASMC of mice born in normoxia (A) or in hypoxia (B). Data are expressed as means ± SE (n = 8 cells). *Significant difference with and without CTX (P < 0.05, paired Student's t-test).

 
Similar results were obtained with IbTX, a specific inhibitor of BKCa channels. This drug induced a dose-dependent inhibition of the outward current in PASMC of mice born in hypoxia but not in controls (Fig. 3C). A single dose of 10–7 M IbTX was used for further experiments. In steady-state current-voltage relationships, IbTX application also did not modify the current density of PASMC from control mice but induced a significant decrease in PASMC from mice born in hypoxia (18.8 ± 3.3 and 1.5 ± 2.1% at +40 mV, respectively; P < 0.05, n = 7–8 cells; Fig. 6).


Figure 6
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  		Fig. 6. I-V curves of PASMC from mice born in normoxia or in hypoxia in the presence of the large-conductance KCa channel (BKCa) inhibitor IbTX. Outward current was measured in freshly isolated PASMC from adult mice born in normoxia (Ctr) or in hypoxia (PH) in the perforated-cell configuration. Left: outward membrane current density elicited by a series of 10-mV depolarizing steps (–80 to +80 mV) from a holding potential of –80 mV. Right: steady-state I/V relationships of voltage-dependent outward currents. Effect of IbTX on the outward current was measured in PASMC of mice born in normoxia (A) or in hypoxia (B). Data are expressed as means ± SE (n = 7–8 cells). *Significant difference with and without IbTX (P < 0.05, paired Student's t-test).

 
Therefore, KCa channels appear to contribute to the outward current only in PASMC of mice born in hypoxia, whereas KV channels are active in PASMC in both groups with a higher activity in mice born in hypoxia.

Western blotting analysis. The BKCa {alpha}-subunit and KV1.5 {alpha}-subunit proteins were detected in the main PA of controls and of mice born in hypoxia. The relative BKCa {alpha}-subunit protein content was higher in mice born in hypoxia than in control mice (1.50 ± 0.07 and 1.12 ± 0.08, respectively; P < 0.05, n = 3–4 groups of 10 PA; Fig. 7A). The relative KV1.5 {alpha}-subunit protein content was also significantly higher in mice born in hypoxia than in mice born in normoxia (1.13 ± 0.043 and 0.81 ± 0.10, respectively; P < 0.05, n = 3 groups of 10 PA; Fig. 7B).


Figure 7
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  		Fig. 7. Western blotting analysis of K+ channel proteins in adult main PA. Western blotting analysis of BKCa {alpha}-subunit (A) and KV1.5 {alpha}-subunit (B) protein content in adult main PA of mice born in normoxia (Ctr) or in hypoxia (PH). Data are expressed as means ± SE (n = 3–4 pools of 10 PA) of the ratio between K+ channel subunit expression (normalized with the beta-actin protein content) found in PA of mice born in hypoxia and in control PA. *Significantly different compared with controls (P < 0.05, unpaired Student's t-test).

 
Isolated vessel tension studies. K+ channel inhibitors were applied during isolated vessel tension studies on the adult main PA at basal tension. CTX (10–7 M) did not change the basal tension in both groups, whereas 4-AP (2 x 10–3 M) induced a transient contraction, which was similar in control mice (15.5 ± 1.9%, n = 23 mice) and in mice born in hypoxia (14.4 ± 1.8%, n = 32 mice; data not shown).

Pulmonary vascular rings were preconstricted with phenylephrine (10–5 M) in the presence of 10–5 M indomethacin and 10–4 M L-NA. Phenylephrine-induced contractions showed no significant difference between all groups, with or without addition of the K+ channel inhibitors (data not shown).

The NO donors SNAP and DEA/NO induced a dose-dependent relaxation in preconstricted PA, with a maximal effect at 10–4 M in all groups (Figs. 8A and 9A).


Figure 8
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  		Fig. 8. Pharmacological responses of isolated main PA to the nitric oxide (NO) donor S-nitro-N-acetyl-D,L-penicillamine (SNAP). Cumulative dose responses to SNAP (10–9 to 10–4 M) were tested on adult main PA preconstricted with phenylephrine (10–5 M) in presence of indomethacin (10–5 M) and NG-nitro-L-arginine [L-NA (NLA); 10–4 M]. A: dose-dependent relaxation was induced by SNAP in mice born in normoxia (Ctr) or in hypoxia (PH). B: influence of CTX (10–7 M) or 4-AP (2 x 10–3 M) on the SNAP-induced vasorelaxation was tested in controls. C: influence of CTX (10–7 M) or 4-AP (2 x 10–3 M) on SNAP-induced relaxation was tested in mice born in hypoxia. Changes in tension are expressed as %initial contraction. Data are expressed as means ± SE (n = 7–14 mice). *Significant difference with and without CTX; {dagger}significant difference with and without 4-AP (P < 0.05, one-way ANOVA).

 

Figure 9
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  		Fig. 9. Pharmacological responses of isolated main PA to the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA)/NO. Cumulative doses of DEA/NO (10–9 to 10–4 M) were tested on the adult main PA preconstricted with phenylephrine (10–5 M) in presence of indomethacin (10–5 M) and L-NA (10–4 M). A: dose-dependent relaxation was induced by DEA/NO in mice born in normoxia (Ctr) or in hypoxia (PH). B: influence of CTX (10–7 M) or 4-AP (2 x 10–3 M) on DEA/NO-induced relaxation was tested in controls. C: influence of CTX (10–7 M) or 4-AP (2 x 10–3 M) on DEA/NO-induced relaxation was tested in mice born in hypoxia. Changes in tension are expressed as %initial contraction. Data are expressed as means ± SE (n = 5–9 mice). *Significant difference with and without CTX; {dagger}significant difference with and without 4-AP (P < 0.05, one-way ANOVA).

 
In the absence of K+ channel blockers, SNAP induced similar relaxation in control mice and in mice born in hypoxia (Fig. 8A). In controls, SNAP-induced relaxation was not influenced by the presence of CTX or 4-AP (Fig. 8B). In contrast, in PA of mice born in hypoxia, SNAP-induced relaxation was inhibited significantly by 4-AP or CTX (Fig. 8C).

Similarly, in the absence of K+ channel blockers, DEA/NO-induced relaxation of PA was similar in control mice and mice born in hypoxia (Fig. 9A). In controls, the presence of CTX did not influence DEA/NO-induced relaxation, whereas 4-AP slightly but significantly decreased this relaxation (Fig. 9B). In mice born in hypoxia, DEA/NO-induced relaxation was significantly decreased in the presence of 4-AP or CTX (Fig. 9C).

EC50 of SNAP- and DEA/NO-induced relaxations are presented in Table 1. For both NO donors, EC50 did not differ between controls and mice born in hypoxia in the absence of K+ channel inhibitors. In controls, 4-AP and CTX did not influence EC50 of SNAP- and DEA/NO-induced relaxation. However, in PA of mice born in hypoxia, 4-AP and CTX led to a significant increase of EC50 of SNAP- and DEA/NO-induced relaxation. EC50 of DEA/NO-induced relaxation was increased significantly in the presence of CTX in mice born in hypoxia compared with controls but not in the presence of 4-AP. However, EC50 of SNAP-induced relaxation was also increased significantly in the presence of 4-AP in mice born in hypoxia compared with controls.


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  		Table 1. Half-maximal efficient dose of SNAP or DEA/NO in the absence or presence of K+ channel inhibitors

 
Therefore, in controls, KCa and KV channels do not appear to be implicated in the mechanism of SNAP-induced relaxation, whereas KV channels contribute slightly to DEA/NO-induced relaxation. In contrast, in mice born in hypoxia, both KCa and KV channels contribute partially to SNAP- and DEA/NO-induced relaxation.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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Adverse events in utero are associated with the occurrence of diseases in adulthood. In the lung, altered pulmonary vascular reactivity in adulthood could be associated with a perinatal hypoxic insult (9, 13, 15–17, 20, 31, 42, 45). Here we show that prolonged exposure to hypoxia in the perinatal period results in modifications of regulatory mechanisms of the pulmonary vascular tone in adulthood, in particular in alterations of different K+ channel expression and activity.

Our electrophysiological results showed that, in adult control PASMC, cell capacitance, reflecting cell membrane surface, was greater than that of mice born in hypoxia. Interestingly, in cerebral arteries, cell capacitance was also found to be higher in adults than in fetal SMC (25). Moreover, in our model, the outward current measured in the perforated-cell configuration was greater in PASMC from mice born in hypoxia than in controls. Similar observation was also done in cerebral arteries between fetal and adult SMC, respectively (25). These data suggest that, in our model, perinatal hypoxia could disturb the transition between fetal and adult life and that adult PASMC from mice born in hypoxia could maintain some characteristics of fetal PASMC.

In mice born in normoxia, the outward current measured in adult PASMC was significantly decreased in the presence of 4-AP but not of CTX or IbTX. This demonstrates the contribution of KV channels but not BKCa channels to this outward current. Similar observations were recently reported in adult mice of a different strain (23). Western blotting analysis of KV1.5 {alpha}-subunit and BKCa {alpha}-subunit expression showed that both proteins were present in the main PA of adult mice born in normoxia, suggesting that, in basal conditions, KCa channels are present but not active. These results are in accordance with others who found a maturational transition between KCa and KV channels during development, with an important decrease of the role and activity of KCa and a predominance of KV in distal PA of adult sheep (39). In our murine model, a similar shift seems to occur in the main PA. Such maturational transition could explain why CTX- and IbTX-sensitive current was absent in PASMC of adult controls. However, the presence of BKCa {alpha}-subunit protein in the main PA of adult controls suggests that the developmental shift would decrease BKCa activity or sensitivity rather than BKCa expression. It is also possible that the presence of BKCa {alpha}-subunit protein in PA homogenates is due to its expression in endothelial cells rather than in SMC.

In contrast, in mice born in hypoxia, 4-AP, CTX, and IbTX were able to decrease the outward current measured in freshly isolated PASMC, demonstrating the contribution of both KV and KCa channels to this current. Interestingly, the inhibition observed in the presence of 4-AP was higher in mice born in hypoxia than in controls, suggesting an increased KV activity following perinatal hypoxia. The detection of a CTX- and IbTX-sensitive current demonstrates the presence of active BKCa channels in PASMC of adults born in hypoxia, whereas no BKCa activity was found in controls. Western blotting analysis of KV1.5 {alpha}-subunit and BKCa {alpha}-subunit expression confirmed the presence of both proteins in the main PA of adults born in hypoxia. The level of these proteins was even higher than in controls. Taken together, our data show that, in the adult main PA, perinatal hypoxia induces an increase in activity and protein content of both KV and KCa channels. Such modifications of K+ channel protein expression and activity could result in an altered regulation of adult PA vasomotor tone.

Isolated vessel tension studies showed that, in the absence of K+ channel inhibitors, the adult main PA of both normoxic and hypoxic groups displayed similar dose-dependent relaxations to the NO-donors SNAP or DEA/NO. In controls, SNAP-induced relaxation of the adult main PA was not influenced by the presence of 4-AP or CTX, whereas DEA/NO-induced relaxation was slightly but significantly inhibited only by 4-AP. In contrast, SNAP- and DEA/NO-induced relaxations of the main PA of mice born in hypoxia were decreased significantly in the presence of both 4-AP or CTX. Therefore, in controls, relaxation of adult main PA by NO donors seems to be only slightly mediated by KV channels but not by KCa channels, whereas both KV and KCa channels are implicated in mice born in hypoxia. These results are in accordance with previous data showing that, in normal C57BL6 adult mice, the relaxation induced by nitroglycerin, another NO donor, was not mediated by a hyperpolarization of the smooth muscle (48). Our present data suggest, however, that, in mice born in hypoxia, smooth muscle hyperpolarization could be implicated in NO-mediated relaxation of the adult main PA.

Our results showed that KCa channels appeared to contribute to the outward current only in PASMC of mice born in hypoxia, whereas KV channels were active in PASMC of both groups with a higher activity in mice born in hypoxia. Moreover, we observed greater BKCa and KV channel expression in adult PASMC of mice born in hypoxia than in mice born in normoxia. These data suggest that perinatal hypoxia could disturb the transition between fetal and adult life and that PASMC from mice born in hypoxia could then likely maintain some characteristics of fetal PASMC.

In the pulmonary vasculature, the relative contribution of KCa and KV channels varies between the fetal hypoxic and the adult normoxic environments. In fetal life, the physiological hypoxic environment causes inhibition of KCa channels, resulting in PASMC depolarization contributing to the constricted state of PA (11). Prolonged depolarization has been shown to downregulate KV channel expression (24), providing an additional pathway by which KV channel activity may be modulated in the constricted fetal pulmonary circulation. At birth, O2 induces PASMC hyperpolarization by activation of KCa channels (10), leading to a decrease in [Ca2+]i (34, 41) and to PA vasodilatation (10, 34). Although acute normoxia activates KCa channels to initiate membrane hyperpolarization, long-term normoxia may allow the upregulation of KV channels and development of mechanisms by which acute hypoxia is sensed in the adult. Moreover, KCa channel protein and mRNA expression were found to decrease in the pulmonary vasculature between fetal and adult life (41). The maturation-related increase in the capacity of the pulmonary vasculature to respond to hypoxia may therefore be due to an increase in KV channel activity with age and a decrease in both KCa channel protein and mRNA expression. Persistence of a low O2 tension after birth, like in perinatal hypoxia, could therefore disturb the normal adaptation to neonatal life and particularly the developmental shift observed in K+ channels, with a persistence of the activity of KCa channels. In our model, the detection of a CTX- and IbTX-sensitive current in PASMC of mice born in hypoxia and not in controls is in accordance with the hypothesis that perinatal hypoxia could result in a persistence of a fetal phenotype. Oxygen is thought to play a key role in the normal developmental shift observed in K+ channels (34, 40, 41). This hypothesis is sustained by the perturbation of this maturational change observed in our animal model when the transition from hypoxia to normoxia occurs at the end of the pulmonary vasculogenesis period instead of at birth.

Our data showed a greater KV channel activity and expression in adult PASMC of mice born in hypoxia than in mice born in normoxia. In pulmonary vessels, KV channels can be inhibited by hypoxia and then lead to depolarization of SMC, resulting in a rise in [Ca2+]i and vasoconstriction (56). Electrophysiological studies suggest that whole cell K+ currents are decreased in both chronic (21, 43) and acute hypoxic conditions (30, 35, 55). Chronic hypoxia downregulates mRNA and protein expression of KV channel {alpha}-subunits in PASMC (33, 52). In particular, KV1.5 channels are known to be sensitive to acute and chronic hypoxia (2, 37, 52). In addition, suppression of KV1.5 channel expression in knocked-out mice attenuated hypoxic pulmonary vasoconstriction (2). Moreover, in vivo gene transfer of KV1.5 channels reduced pulmonary hypertension in rats under chronic hypoxia while restoring the vasoconstrictive response to acute hypoxia (37). This underlines the importance of KV channels in the response of the adult pulmonary vasculature to hypoxia. In our model, both KV channel-dependent outward current and KV1.5 {alpha}-subunit expression were increased by perinatal hypoxia in adult PA. This could contribute to the enhanced vasoconstrictive response observed in other animal models with reexposure to hypoxia in adulthood (16–18).

In pulmonary vessels, effects of hypoxia on KCa channels are controversial and depend on the species studied. Acute hypoxia has been shown to induce depolarization and extracellular Ca2+ concentration entry, activating KCa channels, which results in a relaxing feedback (38). In contrast, it has been shown that acute hypoxia could inhibit canine and fetal ovine PASMC KCa channels (11, 36). In addition, chronic hypoxia decreased CTX-sensitive KCa channels in human PASMC (32). In our murine model, perinatal exposure to hypoxia results in increased BKCa channel activity and protein content in adults.

Several mechanisms could be implicated in the persistence of fetal characteristics in adult PASMC of mice born in hypoxia, implicating an abnormally elevated BKCa channel activity. KCa channel activity was found to be modulated by their phosphorylation state. In cerebral arteries, the greater electrical activity observed in fetal than in adult cells (25) was attributed to a higher extent of BKCa channel phosphorylation in fetal myocytes, resulting in a greater activation of these channels (26). Such modulation of phosphorylation extent could also be implicated in the increased BKCa activity observed in adult PASMC of mice born in hypoxia compared with controls.

It is also possible that perinatal hypoxia influences KCa channel activity by modification of their subunit composition. In PASMC, BKCa channels are composed of pore-forming {alpha}-subunits and accessory beta-subunits, the latter playing a key role by modulating the sensitivity to Ca2+ (7). Several isoforms have been described for BKCa {alpha}- and beta-subunits, which appeared to be influenced by hypoxia. In fetal ovine PASMC, hypoxia induced an increased expression of BKCa {alpha}-, beta1-, and beta2-subunit mRNA (40). The different beta-subunit isoforms differently regulate BKCa channel activity. BKCa beta2-subunit causes rapid inactivation of BKCa channels, whereas beta1-subunit increases the channel opening probability (51, 54). Therefore, in the case of prolonged hypoxia during fetal life and after birth, BKCa beta-subunit expression may be altered and disturb the activity of BKCa channels independently of the effect on pore-forming {alpha}-subunits. These data lead to the hypothesis that, in our model, BKCa channel {alpha}- and beta-subunit expression could be modified in adult PASMC of mice born in hypoxia and influence the physiological role of BKCa channels. Our Western-blotting data showed that perinatal hypoxia increased BKCa {alpha}-subunit protein expression in adult PA. Further investigations of beta-subunit expression could also be of interest.

It should be noted that the results of our study are related to the main PA. Indeed the overall reactivity differs between the large-conduit PA and the more distal resistant pulmonary vessels. However, the modifications observed in the main PA of mice born in hypoxia are already significant. Therefore, other alterations could be expected in more distal vessels.

In conclusion, we showed that, as previously described in other animal models, KV channel activity was predominant compared with KCa channel activity in PASMC of adult mice born in normoxia. Perinatal exposure to hypoxia resulted in increases in protein expression and activity of both BKCa and KV channels in adult PA. This suggests that perinatal hypoxia interferes with the maturational shift of K+ channels normally observed in the pulmonary vasculature between the fetus and the adult. These perturbations could contribute to modifications of pulmonary vasoreactivity and predispose or participate in pulmonary vascular pathologies in adulthood. Further investigations of the physiological significance of such changes in the K+ channel subpopulations could help to better understand mechanisms implicated in long-term effects of perinatal hypoxia on the lung vasculature.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Leenaards Foundation, the Swiss National Foundation for Scientific Research (Grant no. 3200-067046), the Emma Muschamp Foundation, the Fern Moffat Foundation, and the Eagle Foundation.


   ACKNOWLEDGMENTS
 
We thank Prof. Sergio Fanconi and the Department of Pediatrics of the University Hospital of Lausanne for support and G. Zufferey for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. F. Tolsa, Neonatal Research Laboratory, Dept. of Pediatrics, 1011 Lausanne-CHUV, Switzerland (e-mail: Jean-Francois.Tolsa{at}chuv.ch)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Archer SL, Huang JM, Hampl V, Nelson DP, Shultz PJ, Weir EK. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci USA 91: 7583–7587, 1994.[Abstract/Free Full Text]
  2. Archer SL, London B, Hampl V, Wu X, Nsair A, Puttagunta L, Hashimoto K, Waite RE, Michelakis ED. Impairment of hypoxic pulmonary vasoconstriction in mice lacking the voltage-gated potassium channel Kv1.5 FASEB J 15: 1801–1803, 2001.[Free Full Text]
  3. Archer SL, Reeve HL, Michelakis E, Puttagunta L, Waite R, Nelson DP, Dinauer MC, Weir EK. O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase. Proc Natl Acad Sci USA 96: 7944–7949, 1999.[Abstract/Free Full Text]
  4. Barker DJ. In utero programming of chronic disease. Clin Sci (Lond) 95: 115–128, 1998.[Medline]
  5. Barker DJ, Osmond C, Golding J, Kuh D, Wadsworth ME. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. Br Med J 298: 564–567, 1989.[Abstract/Free Full Text]
  6. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850–853, 1994.[CrossRef][Medline]
  7. Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT, Aldrich RW. Vasoregulation by the beta1 subunit of the calcium-activated potassium channel. Nature 407: 870–876, 2000.[CrossRef][Medline]
  8. Bychkov R, Gollasch M, Ried C, Luft FC, Haller H. Regulation of spontaneous transient outward potassium currents in human coronary arteries. Circulation 95: 503–510, 1997.[Abstract/Free Full Text]
  9. Caslin A, Heath D, Smith P. Influence of hypobaric hypoxia in infancy on the subsequent development of vasoconstrictive pulmonary vascular disease in the Wistar albino rat. J Pathol 163: 133–141, 1991.[CrossRef][Web of Science][Medline]
  10. Cornfield DN, Reeve HL, Tolarova S, Weir EK, Archer S. Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel. Proc Natl Acad Sci USA 93: 8089–8094, 1996.[Abstract/Free Full Text]
  11. Cornfield DN, Stevens T, McMurtry IF, Abman SH, Rodman DM. Acute hypoxia causes membrane depolarization and calcium influx in fetal pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 266: L469–L475, 1994.[Abstract/Free Full Text]
  12. Fowden AL, Giussani DA, Forhead AJ. Intrauterine programming of physiological systems: causes and consequences. Physiology (Bethesda) 21: 29–37, 2006.[CrossRef][Medline]
  13. Gao Y, Portugal AD, Negash S, Zhou W, Longo LD, Usha RJ. Role of Rho kinases in PKG-mediated relaxation of pulmonary arteries of fetal lambs exposed to chronic high altitude hypoxia. Am J Physiol Lung Cell Mol Physiol 292: L678–L684, 2007.[Abstract/Free Full Text]
  14. Goldberg SJ, Levy RA, Siassi B, Betten J. The effects of maternal hypoxia and hyperoxia upon the neonatal pulmonary vasculature. Pediatrics 48: 528–533, 1971.[Abstract/Free Full Text]
  15. Grover RF, Vogel JH, Voigt GC, Blount SG Jr. Reversal of high altitude pulmonary hypertension. Am J Cardiol 18: 928–932, 1966.[CrossRef][Web of Science][Medline]
  16. Hakim TS, Mortola JP. Pulmonary vascular resistance in adult rats exposed to hypoxia in the neonatal period. Can J Physiol Pharmacol 68: 419–424, 1990.[Web of Science][Medline]
  17. Hampl V, Herget J. Perinatal hypoxia increases hypoxic pulmonary vasoconstriction in adult rats recovering from chronic exposure to hypoxia. Am Rev Respir Dis 142: 619–624, 1990.[Web of Science][Medline]
  18. Herget J, Kuklik V. Perinatal lung injury extends in adults the site of hypoxic pulmonary vasoconstriction upstream. Physiol Res 44: 25–30, 1995.[Web of Science][Medline]
  19. Heymann MA. Control of the pulmonary circulation in the fetus and during the transitional period to air breathing. Eur J Obstet Gynecol Reprod Biol 84: 127–132, 1999.[CrossRef][Web of Science][Medline]
  20. Himmelmann AW, Speich R, Real F, Russi EW. Unusual course of pulmonary hypertension of vascular origin. Respiration 60: 292–294, 1993.[Web of Science][Medline]
  21. Hong Z, Weir EK, Nelson DP, Olschewski A. Subacute hypoxia decreases voltage-activated potassium channel expression and function in pulmonary artery myocytes. Am J Respir Cell Mol Biol 31: 337–343, 2004.[Abstract/Free Full Text]
  22. Horn R, Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92: 145–159, 1988.[Abstract/Free Full Text]
  23. Ko EA, Burg ED, Platoshyn O, Msefya J, Firth AL, Yuan JX. Functional characterization of voltage-gated K+ channels in mouse pulmonary artery smooth muscle cells. Am J Physiol Cell Physiol 293: C928–C937, 2007.[Abstract/Free Full Text]
  24. Levitan ES, Gealy R, Trimmer JS, Takimoto K. Membrane depolarization inhibits Kv1.5 voltage-gated K+ channel gene transcription and protein expression in pituitary cells J Biol Chem 270: 6036–6041, 1995.
  25. Lin MT, Hessinger DA, Pearce WJ, Longo LD. Developmental differences in Ca2+-activated K+ channel activity in ovine basilar artery. Am J Physiol Heart Circ Physiol 285: H701–H709, 2003.[Abstract/Free Full Text]
  26. Lin MT, Longo LD, Pearce WJ, Hessinger DA. Ca2+-activated K+ channel-associated phosphatase and kinase activities during development. Am J Physiol Heart Circ Physiol 289: H414–H425, 2005.[Abstract/Free Full Text]
  27. Longo LD, Pearce WJ. Fetal cerebrovascular acclimatization responses to high-altitude, long-term hypoxia: a model for prenatal programming of adult disease? Am J Physiol Regul Integr Comp Physiol 288: R16–R24, 2005.[Abstract/Free Full Text]
  28. Mulsch A, Busse R. NG-nitro-L-arginine {N5-[imino(nitroamino)methyl]-L-ornithine} impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine. Naunyn Schmiedebergs Arch Pharmacol 341: 143–147, 1990.[Web of Science][Medline]
  29. Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799–C822, 1995.[Abstract/Free Full Text]
  30. Olschewski A, Hong Z, Nelson DP, Weir EK. Graded response of K+ current, membrane potential, and [Ca2+]i to hypoxia in pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol 283: L1143–L1150, 2002.[Abstract/Free Full Text]
  31. O'Neill D, Morton R, Kennedy JA. Progressive primary pulmonary hypertension in a patient born at high altitude. Br Heart J 45: 725–728, 1981.[Free Full Text]
  32. Peng W, Hoidal JR, Farrukh IS. Role of a novel KCa opener in regulating K+ channels of hypoxic human pulmonary vascular cells. Am J Respir Cell Mol Biol 20: 737–745, 1999.[Abstract/Free Full Text]
  33. Platoshyn O, Yu Y, Golovina VA, McDaniel SS, Krick S, Li L, Wang JY, Rubin LJ, Yuan JX. Chronic hypoxia decreases KV channel expression and function in pulmonary artery myocytes. Am J Physiol Lung Cell Mol Physiol 280: L801–L812, 2001.[Abstract/Free Full Text]
  34. Porter VA, Rhodes MT, Reeve HL, Cornfield DN. Oxygen-induced fetal pulmonary vasodilation is mediated by intracellular calcium activation of KCa channels. Am J Physiol Lung Cell Mol Physiol 281: L1379–L1385, 2001.[Abstract/Free Full Text]
  35. Post JM, Gelband CH, Hume JR. [Ca2+]i inhibition of K+ channels in canine pulmonary artery. Novel mechanism for hypoxia-induced membrane depolarization. Circ Res 77: 131–139, 1995.[Abstract/Free Full Text]
  36. Post JM, Hume JR, Archer SL, Weir EK. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am J Physiol Cell Physiol 262: C882–C890, 1992.[Abstract/Free Full Text]
  37. Pozeg ZI, Michelakis ED, McMurtry MS, Thebaud B, Wu XC, Dyck JR, Hashimoto K, Wang S, Moudgil R, Harry G, Sultanian R, Koshal A, Archer SL. In vivo gene transfer of the O2-sensitive potassium channel Kv1.5 reduces pulmonary hypertension and restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats. Circulation 107: 2037–2044, 2003.[Abstract/Free Full Text]
  38. Reeve HL, Archer SL, Weir EK. Ion channels in the pulmonary vasculature. Pulm Pharmacol Ther 10: 243–252, 1997.[CrossRef][Web of Science][Medline]
  39. Reeve HL, Weir EK, Archer SL, Cornfield DN. A maturational shift in pulmonary K+ channels, from Ca2+ sensitive to voltage dependent. Am J Physiol Lung Cell Mol Physiol 275: L1019–L1025, 1998.[Abstract/Free Full Text]
  40. Resnik E, Herron J, Fu R, Ivy DD, Cornfield DN. Oxygen tension modulates the expression of pulmonary vascular BKCa channel{alpha}- and beta-subunits. Am J Physiol Lung Cell Mol Physiol 290: L761–L768, 2006.[Abstract/Free Full Text]
  41. Rhodes MT, Porter VA, Saqueton CB, Herron JM, Resnik ER, Cornfield DN. Pulmonary vascular response to normoxia and KCa channel activity is developmentally regulated. Am J Physiol Lung Cell Mol Physiol 280: L1250–L1257, 2001.[Abstract/Free Full Text]
  42. Sartori C, Allemann Y, Trueb L, Delabays A, Nicod P, Scherrer U. Augmented vasoreactivity in adult life associated with perinatal vascular insult. Lancet 353: 2205–2207, 1999.[CrossRef][Web of Science][Medline]
  43. Shimoda LA, Sylvester JT, Sham JS. Chronic hypoxia alters effects of endothelin and angiotensin on K+ currents in pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 277: L431–L439, 1999.[Abstract/Free Full Text]
  44. Sime F, Penaloza D, Ruiz L. Bradycardia, increased cardiac output, and reversal of pulmonary hypertension in altitude natives living at sea level. Br Heart J 33: 647–657, 1971.[Free Full Text]
  45. Tang JR, Le Cras TD, Morris KG Jr, Abman SH. Brief perinatal hypoxia increases severity of pulmonary hypertension after reexposure to hypoxia in infant rats. Am J Physiol Lung Cell Mol Physiol 278: L356–L364, 2000.[Abstract/Free Full Text]
  46. Tolsa JF. Physiologic aspects of lung circulation in adjustment to extra-uterine life. Arch Pediatr 7, Suppl 2: 269s–270s, 2000.
  47. Tolsa JF, Gao Y, Sander FC, Souici AC, Moessinger A, Raj JU. Differential responses of newborn pulmonary arteries and veins to atrial and C-type natriuretic peptides. Am J Physiol Heart Circ Physiol 282: H273–H280, 2002.[Abstract/Free Full Text]
  48. Tolsa JF, Marino M, Peyter AC, Beny JL. Role of membrane potential in endothelium-dependent relaxation of isolated mouse main pulmonary artery. J Cardiovasc Pharmacol 47: 501–507, 2006.[CrossRef][Web of Science][Medline]
  49. Tristani-Firouzi M, Reeve HL, Tolarova S, Weir EK, Archer SL. Oxygen-induced constriction of rabbit ductus arteriosus occurs via inhibition of a 4-aminopyridine-, voltage-sensitive potassium channel. J Clin Invest 98: 1959–1965, 1996.[Web of Science][Medline]
  50. Vane JR. Inhibitors of prostaglandin, prostacyclin, and thromboxane synthesis. Adv Prostaglandin Thromboxane Res 4: 27–44, 1978.[Medline]
  51. Wallner M, Meera P, Toro L. Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: a transmembrane beta-subunit homolog. Proc Natl Acad Sci USA 96: 4137–4142, 1999.[Abstract/Free Full Text]
  52. Wang J, Juhaszova M, Rubin LJ, Yuan XJ. Hypoxia inhibits gene expression of voltage-gated K+ channel alpha subunits in pulmonary artery smooth muscle cells. J Clin Invest 100: 2347–2353, 1997.[Web of Science][Medline]
  53. Warburton D, Schwarz M, Tefft D, Flores-Delgado G, Anderson KD, Cardoso WV. The molecular basis of lung morphogenesis. Mech Dev 92: 55–81, 2000.[CrossRef][Web of Science][Medline]
  54. Xia XM, Ding JP, Lingle CJ. Molecular basis for the inactivation of Ca2+- and voltage-dependent BK channels in adrenal chromaffin cells and rat insulinoma tumor cells. J Neurosci 19: 5255–5264, 1999.[Abstract/Free Full Text]
  55. Yuan XJ, Goldman WF, Tod ML, Rubin LJ, Blaustein MP. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am J Physiol Lung Cell Mol Physiol 264: L116–L123, 1993.[Abstract/Free Full Text]
  56. Yuan XJ, Wang J, Juhaszova M, Golovina VA, Rubin LJ. Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 274: L621–L635, 1998.[Abstract/Free Full Text]



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