Reactive Oxygen Species Scavengers Improve Voltage-Gated K + Channel Function in Pulmonary Arteries of Newborn Pigs with Progressive Hypoxia-Induced Pulmonary Hypertension

Animals

Newborn pigs were obtained from the vendor on day of life 2 and raised in a hypoxic, normobaric environment until either day of life 5 (3 days of hypoxia) or day of life 12 (10 days of hypoxia), following our previously described methods.^19,30 Normobaric hypoxia was produced by delivering compressed air and N₂ to a chamber so that oxygen content was maintained at 10%–11% O₂ (PO₂: 60–72 Torr). CO₂ was maintained at 3–6 Torr by absorption with soda lime. The chamber was opened twice each day for cleaning and to weigh the animals. The animals were fed ad libitum with an artificial sow milk replacer from a feeding device attached to the chamber. Normoxic, age-matched control animals were either 5 or 12 days old when obtained from the vendor and were studied on the day of arrival, i.e., at the same postnatal ages as the hypoxic piglets. We have previously found no differences in vascular responses between piglets raised in a room-air environment and piglets raised on a farm. Hereafter the piglets are referred to as the 3-day exposure group (piglets raised in hypoxia for 3 days and comparable-age control piglets) and the 10-day exposure group (piglets raised in hypoxia for 10 days and comparable-age control piglets).

On the day of study, the piglets were preanesthetized with acepromazine (2 mg/kg intramuscularly [im]) and ketamine (10 mg/kg im) and then anesthetized with pentobarbital (30 mg/kg intravenously [iv]). All animals were given heparin (1,000 IU/kg iv) and then exsanguinated. The thorax was opened, and the lungs were removed and placed in cold (4°C) physiological saline solution (PSS) until use. The PSS had the following composition (in mM): Na⁺, 141; K⁺, 4.7; Cl⁻, 125; Ca²⁺, 2.5; Mg²⁺, 0.72; H₂PO_4-, 1.7; HCO_3-, 25; and glucose, 11. All procedures involving animals adhered to the National Institutes of Health guidelines for the use of experimental animals and were approved by the Institutional Animal Care and Use Committee at Vanderbilt University School of Medicine.

Cannulated-artery preparation

Immediately prior to use, segments of 100–300-μm diameter pulmonary arteries were dissected from a lung lobe. The system used to study cannulated arteries has been described in detail previously. ³² The exterior of the artery was suffused with PSS from a reservoir at 37°C and aerated with a gas mixture containing 21% O₂, 5% CO₂, and 74% N₂. The arterial lumen was filled from a syringe containing PSS, aerated with the same gas mixture as the reservoir, and connected to the cannula with polyethylene tubing. Gas concentrations and pH in all solutions were monitored with a blood gas analyzer. Inflow pressure was adjusted by changing the height of the reservoir. The artery was observed continuously with a video system containing a color camera (Hitachi VCC151) and a color television monitor. Vessel diameters were measured with a videoscaler (FORA IV).

All arteries were equilibrated for 30–60 minutes at transmural pressures that approximated in vivo pressures previously measured in normoxic and hypoxic piglets: ³¹ control arteries were equilibrated at a transmural pressure of 11 mmHg, and the hypoxic arteries were equilibrated at a transmural pressure of 18 mmHg. Following equilibration and establishment of basal tone, all arteries were tested for viability by contraction to U46619 (10⁻⁸ M). The presence of a functional endothelium was determined in control arteries by dilation of the vessel to acetylcholine (ACh, 10⁻⁶ M; average dilation: 10% ± 3%). We previously found that although hypoxic arteries constricted to ACh, they did dilate to the calcium ionophore A23187 (10⁻⁶ M; average dilation: 6% ± 2%), ³² which also requires the endothelium for its function as a dilator. Therefore, changes in vessel diameter to A23187 were used to check for a functional endothelium in hypoxic arteries.

Smooth muscle cell membrane potential (SMC Em) determination

We previously found that SMC Em is more depolarized (less negative) in PRAs of hypoxic than in those of normoxic control piglets of the 3-day exposure group. ¹⁹ For this study, we wanted to determine the effect on SMC Em of a longer, 10-day exposure to in vivo hypoxia. With our previously published methods, arteries from some control and hypoxic piglets of the 10-day exposure group were cannulated, pressurized, and equilibrated as described above. SMCs were impaled with microelectrodes filled with 3 M KCl and having impedances between 40 and 90 MΩ. Each microelectrode was connected to a high-impedance biological amplifier (Dagan) that was attached to a digital data acquisition system (PowerLab, ADI Instruments). By means of a micromanipulator, the electrode was advanced into an SMC from the adventitial side of the artery. Criteria for a successful impalement included an abrupt drop in potential to a new steady state value that is maintained for a minimum of 20 seconds and returns to the original baseline value when the microelectrode is removed from the SMC. Multiple successful impalements of at least three vascular SMCs were averaged for a single artery.

We also evaluated the influence of transmural pressure on SMC Em. To do this, we measured SMC Em in some control arteries that were pressurized to the transmural pressure used for hypoxic arteries, 18 mmHg, and in some hypoxic arteries that were pressurized to the transmural pressure used for control arteries, 11 mmHg. To assess the influence of the endothelium, SMC Em was measured in some arteries from control and hypoxic piglets after the endothelium was disrupted with air infusion. Effective functional disruption of the endothelium was verified by loss of relaxation to A23187. To evaluate the influence of Kv channels on SMC Em, in some other control and hypoxic arteries, we measured SMC Em before and 20–30 minutes after adding the Kv channel antagonist 4-aminopyridine (4-AP, 10⁻³ M) to the reservoir.

We also performed studies to determine whether ROS-removing agents might restore the SMC Em measured in hypoxic arteries to levels similar to those measured in control arteries. SMC Em was measured before and 20–30 minutes after adding to the reservoir either (1) a combination of a cell-permeable superoxide dismutase mimetic, M40403 (3 μg/mL), which dismutates superoxide to hydrogen peroxide (H₂O₂), and a cell-permeable H₂O₂-decomposing enzyme, polyethylene glycol-catalase (PEG-CAT, 250 U), or (2) apocynin (APO, 10⁻⁶ M) to inhibit NADPH oxidase (NOX), a potential enzymatic source of ROSs.

Cannulated-artery protocols to assess Kv channel function

To evaluate the effect of in vivo chronic hypoxia on Kv channel function, we measured changes in vessel diameter in response to pharmacologic blockade with the Kv channel antagonist 4-AP, 10⁻⁵ to 10⁻³ M, in PRAs from control and hypoxic piglets. The influence of the endothelium on 4-AP responses was assessed in PRAs after disruption of the endothelium with air infusion. Functional disruption of the endothelium was assessed as loss of relaxation to the calcium ionophore A23187. To assess the influence from ROSs on Kv channel function, a combination of M40403 (3 μg/mL) and PEG-CAT (250 U) was added to the reservoir and allowed to circulate for 20 minutes, after which changes in vessel diameter to cumulative doses of 4-AP were measured. To evaluate the influence of NOX, an enzymatic source of ROSs, on 4-AP responses, we measured changes in vessel diameter to 4-AP after adding the NOX inhibitor APO (10⁻⁶ M) to the reservoir. To assess whether exogenous ROSs might convert 4-AP responses in control arteries to levels similar to those in hypoxic arteries, we measured responses to 4-AP after adding the ROS generator menadione (10⁻⁵ M)^33,34 to the reservoir of control arteries.

Cannulated-artery protocols to assess other K⁺ channel function

The influence of chronic hypoxia on another K⁺ channel family, K_ATP channels, was assessed by pharmacologic activation with levcromakalim. For these studies, vessel tone was elevated by 40%–50% with either U46619 or endothelin-1 prior to measurement of changes in vessel diameter in response to levcromakalim, 10⁻⁹ to 10⁻⁵ M.

Vessel viability was tested at the completion of all cannulated-artery studies by addition of U-46619. In some studies, vessel responses to the vehicle used for solubilization of each agent were evaluated.

Immunoblot analyses of Kv1.2, Kv1.5, and Kv2.1

Pulmonary arteries (20–500-μm diameter) were dissected from lungs of control and hypoxic piglets, frozen in liquid nitrogen, and stored at −80°C until use for immunoblot analysis.

For each protein, we performed preliminary studies with different amounts of total protein in order to determine the dynamic range of the immunoblot analysis. An amount of protein within the dynamic range of the immunoblot analysis for that protein (for Kv1.2 and Kv1.5 analyses we used 20-μg protein samples, and for Kv2.1 analysis we used 50-μg protein samples) was then used to compare protein abundance between homogenates of small pulmonary arteries from control and hypoxic piglets as follows.

Frozen samples of small pulmonary arteries (20–500-μm diameter) from control and hypoxic piglets were crushed under liquid N₂ in a prechilled mortar and pestle into a fine powder, transferred to a tube containing homogenate buffer, and then sonicated with three 15-second pulses; care was taken not to foam the sample. The supernatant was centrifuged, and the protein concentration was determined by protein assay (Bradford). With our previously described methods, supernatants were applied to trisglycine precast 8% polyacrylamide gels (Novex) so that equal amounts of protein were loaded. Electrophoresis was carried out, and the proteins were transferred from the gel to a nitrocellulose membrane (Novex). The membrane was incubated for 1 hour at room temperature in phosophate-buffered saline (PBS) containing 10% nonfat dried milk and 0.1% Tween-20 to block nonspecific protein binding. To detect the protein of interest, the nitrocellulose membrane was incubated overnight at 4°C with the primary antibody (Kv1.2, 1: 500 from Upstate; Kv1.5 and Kv2.1, 1 :500 from Alomone) diluted in PBS containing 0.1% Tween-20 and 1% nonfat dried milk (carrier buffer), followed by incubation for 1 hour at room temperature with a horseradish peroxidise-conjugated secondary antibody (Zymed) diluted in the carrier buffer. To visualize the antibody, the membranes were developed using enhanced chemiluminescence reagents (ECL, Amersham), and the chemiluminescent signal was captured on x-ray film (ECL Hyperfilm, Kodak). Similar procedures were followed to reprobe the membranes for β smooth muscle actin (Sigma). The bands for each protein were quantified via densitometry.

Statistics

Data are presented as means ± SEM. An unpaired t test or ANOVA with post hoc multiple-comparison test was used to compare data between groups as appropriate; P < 0.05 was considered significant.

Materials

Concentrations for each drug listed in cannulatedartery protocols are expressed as final molar concentrations in the vessel bath. The 4-AP, PEG-CAT, and menadione were obtained from Sigma Chemical and were solubilized, respectively, in water and pH titrated to 7.4 with 12 N HCl, distilled water, and ethanol (EtOH). The M40403 was a generous gift from Activbiotics (Lexington, MA) and was solubilized in 26-mM NaHCO₃ buffer. The apocynin was from EMD Biosciences and was solubilized in dimethyl sulfoxide (DMSO). The A23187 and U46619 were from Biomol and were solubilized, respectively, in DMSO and EtOH. The levcromakalim was from Tocris and was solubilized in DMSO.

PASMCs are depolarized after 10 days of exposure to in vivo hypoxia

As shown in Table 1, regardless of transmural pressure, PASMC Em measurements in PRAs from piglets exposed to 10 days of hypoxia were less negative (depolarized) than those in PRAs from piglets exposed to 10 days of normoxia. These differences in PASMC Em between PRAs isolated after 10 days of normoxic versus hypoxic exposure (Table 1) mirrored the differences in PASMC Em measured in PRAs from piglets exposed to 3 days of normoxia (–51 ± 0.4 mV) versus 3 days of hypoxia (−46 ± 0.5 mV) in our previous study. ¹⁹ Moreover, findings of PASMC Em in arteries from piglets exposed to 10 days of either normoxia or hypoxia were similar for air-infused and intact vessels (for normoxic, control arteries, PASMC Em was −51 ± 0.8 and −50 ± 1.7 mV for intact [n = 7] and air-infused [n = 7] arteries, respectively; for hypoxic arteries, PASMC Em was −47 ± 1.5 and −47.7 ± 2.1 mV for intact [n = 5] and air-infused [n = 5] arteries, respectively). Thus, neither the presence of the endothelium nor differing transmural pressures explain differences in PASMC Em between control and hypoxic arteries. Furthermore, PASMC Em measurements in hypoxic PRAs were not significantly changed by treatment with ROS-removing agents: PASMC Em was −49 ± 1, −46± 1, and −49 ± 2 mV, respectively, for untreated arteries (n = 10), M40403+PEG-CAT-treated arteries (n = 5), and apocynin-treated arteries (n = 5).

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Table 1.

Membrane potential (Em) determinations in 100–300-μm-diameter pulmonary arteries of normoxic and hypoxic piglets from the 10-day exposure group measured at transmural pressures of 11 and 18 mmHg

Group	Transmural pressure, mmHg	n	Mean Em ± SEM, mV
10-day normoxic	11	23	-55 ± 1
10-day normoxic	18	5	-58 ± 1.5
10-day hypoxic	11	6	-51 ± 0.8a
10-day hypoxic	18	18	-49 ± 1a

a

Significantly different (P < 0.05) from normoxic piglets at both 11 and 18 mmHg.

Kv channels are dysfunctional after 10 days of exposure to in vivo hypoxia

Pharmacological blockade of Kv channels with 4-AP affected the Em of control arteries more than that of the hypoxic arteries. Specifically, as shown in Figure 1A, the Em of the control arteries depolarized (became more positive) by 15.0 ± 1.9 mV (from −54.2 ± 1.2 to −39.3 ± 1.4 mV; n = 13) with 4-AP (10⁻³ M), whereas the Em of the hypoxic arteries depolarized only 5.9 ± 1.3 mV (from −48.0 ± 1.1 to −42.8 ± 1.7 mV, n = 11) with 4-AP (10⁻³ M). These findings support Kv channel dysfunction in the PRAs of piglets exposed to 10 days of hypoxia.

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Figure 1.

A, 4-aminopyridine (4-AP; 10⁻³ M)-induced change in membrane potential (Em) for arteries from normoxic (n = 13) and hypoxic (n = 11) piglets from the 10-day exposure group. An asterisk indicates significant difference from normoxia (P < 0.05). B, 4-AP-induced changes in diameter for endothelium-intact and air-infused arteries from normoxic and hypoxic piglets from the 10-day exposure group (for normoxic arteries, n = 39 intact arteries and n = 24 air-infused arteries; for hypoxic arteries, n =32 intact arteries and n =15 air-infused arteries). An asterisk indicates significant difference from normoxic intact arteries, and a plus sign (B) indicates significant difference from normoxic air-infused arteries (P < 0.05). In both panels, values are mean ± SEM.

Also supportive of Kv channel dysfunction, PRAs from piglets exposed to 10 days of hypoxia constricted less with 4-AP than did PRAs from comparable-age normoxic, control piglets (Fig. 1B). Of note, 4-AP responses were also lower in air-embolized PRAs from hypoxic arteries than in those from normoxic arteries (Fig. 1B), indicative that PASMCs and not endothelial cells are the cellular source of Kv dysfunction.

ROSs contribute to Kv dysfunction after 10 days of exposure to in vivo hypoxia

Treatments to remove ROSs and to inhibit NOX, a potential enzymatic source of ROSs, increased the constrictor response to 4-AP in PRAs from piglets of the 10-day hypoxic exposure group (Fig. 2). In contrast, treatments to remove ROSs and to inhibit NOX had inhibitory effects on the constrictor response to 4-AP in PRAs from comparable-age normoxic, control piglets (Fig. 2). Moreover, addition of the ROS generator menadione reduced constriction to 4-AP in normoxic, control arteries from the 10-day exposure group (Fig. 3). Thus, exogenous ROSs impair Kv channel function in control PRAs, and ROS-removing agents improve Kv channel function in PRAs following exposure to hypoxia for 10 days.

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Figure 2.

A, Effect of treatment with the superoxide dismutase mimetic M40403 and the H₂O₂-decomposing agent polyethylene glycol catalase (PEG-CAT) on responses to 4-aminopyridine (4-AP) in arteries from normoxic and hypoxic piglets of the 10-day exposure group (for normoxic arteries: n = 39 untreated arteries and n = 12 M40403+PEG-CAT-treated arteries; for hypoxic arteries: n = 32 untreated arteries and n = 15 M40403+PEG-CAT-treated arteries). B, Effect of treatment with the NADPH oxidase inhibitor apocynin on responses to 4-AP in arteries from normoxic and hypoxic piglets of the 10-day exposure group (for normoxic arteries: n = 13 untreated arteries and n = 12 apocynin-treated arteries; for hypoxic arteries: n = 12 for untreated arteries and n = 11 apocynin treated arteries). In both panels, values are mean ± SEM, an asterisk indicates a significant difference from untreated normoxic arteries, and a plus sign indicates a significant difference from untreated hypoxic arteries (P < 0.05).

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Figure 3.

Effect of treatment with the ROS generator menadione on responses to 4-aminopyridine (4-AP) in arteries from normoxic piglets of the 10-day exposure group (n = 12 untreated arteries and n = 11 menadione-treated arteries). All values are mean ± SEM. An asterisk indicates a significant difference from untreated arteries (P < 0.05).

ROSs do not contribute to Kv dysfunction after 3 days of exposure to in vivo hypoxia

We previously showed that functional activity of Kv channels was lower in PRAs of piglets exposed to 3 days of hypoxia than in those of comparable-age normoxic piglets. ¹⁹ Based on our findings with PRAs from the 10-day group, we wanted to determine whether ROSs might contribute to Kv dysfunction in PRAs from piglets exposed to a shorter, 3-day duration of in vivo hypoxia. Unlike the longer duration of hypoxia (Fig. 2), treatments to remove ROSs or to inhibit NOX had no impact on constrictor responses to 4-AP in pulmonary arteries of piglets exposed to hypoxia for only 3 days (Fig. 4). Findings for control PRAs (Fig. 4) were similar to those from the older age group of piglets (Fig. 2).

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Figure 4.

A, Effect of treatment with the superoxide dismutase mimetic M40403 and the H₂O₂-decomposing agent polyethylene glycol catalase (PEG-CAT) on responses to 4-aminopyridine (4-AP) in arteries from normoxic and hypoxic piglets of the 3-day exposure group (for normoxic arteries: n = 19 untreated arteries and n = 10 M40403+PEG-CAT-treated arteries; for hypoxic arteries: n = 14 untreated arteries and n = 10 M40403+PEG-CAT-treated arteries). B, Effect of treatment with the NADPH oxidase inhibitor apocynin on responses to 4-AP in arteries from normoxic and hypoxic piglets of the 3-day exposure group (for normoxic arteries: n = 26 untreated arteries and n = 8 apocynin-treated arteries; for hypoxic arteries: n = 24 for untreated arteries and n = 7 apocynin-treated arteries). In both panels, all values are mean ± SEM, and an asterisk indicates a significant difference from untreated normoxic arteries (P < 0.05).

Selective Kv channel proteins are downregulated after 10 days of exposure to in vivo hypoxia

ROS removal did not completely restore to control levels the Kv channel function in PRAs of piglets exposed to 10 days of hypoxia (Fig. 2A). We therefore investigated whether expression of Kv channels was reduced during the 10-day exposure to hypoxia. Immunoblot analyses for Kv1.2, Kv1.5, and Kv2.1 α subunits in small pulmonary artery homogenates from control and hypoxic piglets of the 10-day exposure group are shown in Figure 5. As determined by densitometry, Kv1.2 expression was decreased in pulmonary arteries from hypoxic piglets (Fig. 5A), while expression levels of Kv1.5 and Kv2.1 were similar for hypoxic and control pulmonary arteries (Figs. 5B and 5C, respectively).

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Figure 5.

A, Immunoblot results and corresponding densitometry for Kv1.2 α subunit protein relative to β actin in pulmonary arteries from normoxic and hypoxic piglets of the 10-day exposure group. The plus sign denotes positive control (rat brain). An asterisk indicates a significant different from normoxic piglets (P < 0.05). B, Immunoblot results and corresponding densitometry for Kv1.5 α subunit protein relative to β actin in pulmonary arteries from normoxic and hypoxic piglets of the 10-day exposure group. C, Immunoblot results and corresponding densitometry for Kv2.1 α subunit protein relative to β actin in pulmonary arteries from normoxic and hypoxic piglets of the 10-day exposure group. The plus sign denotes positive control (rat brain). In all panels, all values are mean ± SEM.

K_ATP channel function is preserved following 3 or 10 days of exposure to in vivo hypoxia

We explored the possibility that in addition to alterations in Kv channel expression and function, exposure to in vivo hypoxia might impair the function of other K⁺ channels. As shown in Figure 6, diameters of both control and hypoxic arteries from both 3- and 10-day exposure groups responded similarly to the K_ATP channel agonist levcromakalim.

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Figure 6.

A, Levcromakalim-induced changes in diameter in arteries with elevated tone from normoxic (n = 11) and hypoxic (n = 7) piglets of the 3-day exposure group. B, Levochromakalin-induced changes in diameter in arteries with elevated tone from normoxic (n = 6) and hypoxic (n = 5) piglets of the 10-day exposure group. In both panels, all values are mean±SEM.