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Am J Physiol Lung Cell Mol Physiol 291: L1169-L1176, 2006. First published July 21, 2006; doi:10.1152/ajplung.00117.2006
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Voltage-gated K+ channels at an early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets

Candice D. Fike, Mark R. Kaplowitz, Yongmei Zhang, and Jane A. Madden

Department of Neurology, Medical College of Wisconsin, Milwaukee, Wisconsin; Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee; and Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Submitted 29 March 2006 ; accepted in final form 27 June 2006


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Our purpose was to determine whether smooth muscle cell membrane properties are altered in small pulmonary arteries (SPA) of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A microelectrode technique was used to measure smooth muscle cell membrane potential (Em) in cannulated, pressurized SPA (100- to 300-µm diameter). SPA responses to the voltage-gated K+ (KV) channel antagonist 4-aminopyridine (4-AP) and the KV1 family channel antagonist correolide were measured. Other SPA were used to assess amounts of KV1.2, KV1.5, and KV2.1 (immunoblot technique). Em was more positive in SPA of chronically hypoxic piglets than in SPA of comparable-age control piglets. The magnitude of constriction elicited by either 4-AP or correolide was diminished in SPA from hypoxic piglets. Abundances of KV1.2 were reduced, whereas abundances of both KV1.5 and KV2.1 were unaltered, in SPA from hypoxic piglets. At least partly because of reduced amounts of KV1.2, smooth muscle cell membrane properties are altered such that Em is depolarized and KV channel family function is impaired in SPA of piglets at an early stage of chronic hypoxia-induced pulmonary hypertension.

membrane potential; membrane depolarization; KV1.2; KV1.5; KV2.1; 4-aminopyridine; correolide

POTASSIUM (K+) is the predominant intracellular ion in vascular smooth muscle. Its movement into and out of the cell through specific membrane K+ channels profoundly influences the membrane potential (Em) (36). Changes in Em lead to changes in intracellular calcium and ultimately to changes in the contractile state of the cell. Most vascular smooth muscle cells have multiple K+ channel subtypes. Within pulmonary artery smooth muscle cells (PASMC), at least three functionally distinguishable K+ channels, including voltage-gated K+ (KV) channels, ATP-sensitive K+ (KATP) channels, and large-conductance Ca2+-activated K+ (BKCa) channels, have been identified (19, 21, 43, 44). Although all of the different K+ channels may influence Em, current evidence suggests that KV channels play the major role in regulating the resting Em in PASMC of adult animals (19, 43, 44).

There is growing evidence that changes in KV channel function and/or expression along with the concomitant influence on Em could contribute to pulmonary hypertension in adults (18, 20). Indeed, KV channel function and expression are altered in PASMC of adult humans with either primary pulmonary hypertension (45) or anorexigen-induced pulmonary hypertension (41). In addition, a number of studies with adult rats have shown that chronic hypoxia impairs PASMC K+ channel current (15, 26, 30, 33), decreases PASMC KV channel expression (15, 26, 30, 39), and depolarizes PASMC (15, 27, 33, 34).

In contrast to the studies in adult animals, the effect of chronic hypoxia on membrane properties of PASMC from newborn animals has received almost no attention (11). Extrapolation of data collected from adults may not be applicable to newborns as the regulation of pulmonary vascular pressure has long been known to be influenced by postnatal age (5, 32, 46). Because of their critical role in regulation of pulmonary vascular tone, the elucidation of PASMC membrane properties in small, resistance-level, pulmonary arteries is of particular importance. We previously showed (12) that pulmonary hypertension develops when newborn pigs are exposed to chronic hypoxia for 3 days and that pulmonary hypertension worsens when hypoxic exposure is extended from 3 to 10 days. The major purpose of this study was to test the hypothesis that in vivo exposure to chronic hypoxia concomitantly depolarizes PASMC Em and alters KV channel function and expression in small, resistance-level, pulmonary arteries of newborn piglets. We evaluated changes that occur with 3-day exposure to hypoxia because of the potential significance for developing therapies to intervene at an early stage and prevent progression of pulmonary hypertension.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Animals. Newborn pigs (2–3 days old) were placed in a hypoxic normobaric chamber for 3 days. Normobaric hypoxia was produced by delivering compressed air and N2 to a plastic 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. Two times per day the chambers were opened, at which time the piglets were cleaned and weighed. The animals were fed ad libitum with an artificial sow milk replacer from a feeding device attached to the chamber. With respect to control pigs, we previously found (12) no differences in vascular responses between piglets raised in a room air environment for 3–4 days and piglets raised on a farm. Therefore, for this study, all but three of the control piglets were studied on the day of arrival from the farm at 5–7 days of age.

On the day of study the piglets were preanesthetized with acepromazine (2 mg/kg im) and ketamine (10 mg/kg im) and then anesthetized with pentobarbital (30 mg/kg 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): 141 Na+, 4.7 K+, 125 Cl–, 2.5 Ca2+, 0.72 Mg2+, 1.7 H2PO4–, 25 HCO3–, and 11 glucose. All procedures involving animals were approved by the Institutional Animal care and Use Committees at Wake Forest University School of Medicine and Vanderbilt University School of Medicine.

Cannulated artery preparation. Immediately before use, segments of 100- to 300-µm-diameter pulmonary arteries were dissected from a lung lobe. The system used to study cannulated arteries was described in detail previously (14). 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 were monitored in all solutions 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).

Cannulated artery and smooth muscle cell Em determination protocols. All arteries were equilibrated for 30–60 min at transmural pressures that approximated in vivo pressures (12, 13): control arteries were equilibrated at a transmural pressure of 15 cmH2O, and hypoxic arteries were equilibrated at a transmural pressure of 25 cmH2O. After equilibration and establishment of basal tone, all arteries were tested for viability by contraction to U-46619 (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). We previously found (14) that although hypoxic arteries constricted to ACh they dilated to the calcium ionophore A-23187, which also requires the endothelium for its function as a dilator. Therefore, changes in vessel diameter to A-23187 were used to check for a functional endothelium in hypoxic arteries.

To measure smooth muscle cell (SMC) Em, techniques described previously were used (1, 17). In brief, arteries from some control and hypoxic piglets were cannulated, pressurized, and equilibrated as described above. SMC were impaled with microelectrodes filled with 3 M KCl and having impedances between 40 and 90 M{Omega}. The microelectrode was connected to a high-impedance biological amplifier (Dagan) that was attached to a digital data acquisition system (PowerLab from ADI Instruments). With a micromanipulator, the electrode was advanced into a 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 was maintained for a minimum of 10 s and returned to the original baseline value when the microelectrode was removed from the SMC. Multiple successful impalements of at least three vascular SMC were averaged for a single artery.

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 ACh and/or A-23817 for the control arteries and by loss of relaxation to A-23187 for the hypoxic arteries. 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 (25 cmH2O) and measured SMC Em in some hypoxic arteries that were pressurized to the transmural pressure used for control arteries (15 cmH2O).

To evaluate KV channel function, we measured changes in vessel diameter in response to pharmacological blockade with the KV channel antagonist 4-aminopyridine (4-AP) in control and hypoxic arteries. For these studies, we continuously monitored the vessel diameter while adding cumulative doses of 4-AP (10–5–10–3 M) to the reservoir. In some control and hypoxic arteries, we measured SMC Em 20–30 min after adding the last dose of 4-AP to the reservoir. The influence of the endothelium on 4-AP responses was assessed by performing studies in other arteries after disrupting the endothelium by air infusion. Effective functional disruption of the endothelium was verified by loss of relaxation to ACh and/or A-23817 for the control arteries and by loss of relaxation to A-23187 for the hypoxic arteries.

The contribution of some other K+ channel families to vascular diameter was also assessed by pharmacological blockage. BaCl2 (10–4 M) was used to block inwardly rectifying K+ (Kir) channels, and iberiotoxin (10–7 M) was used to block BKCa channels. The KV1 family of channels was blocked with the specific antagonist correolide (10–8–10–6 M). For some vessels, 20–30 min after pharmacological block of the KV1 channels with correolide, the effect of pharmacological block of all KV channels with 4-AP (10–3 M) was measured. Vessel viability was tested at the completion of each study by addition of U-46619. In addition, 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- to 600-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 to determine the dynamic range of the immunoblot analysis. An amount of protein that was within the dynamic range of the immunoblot analysis for that protein (for KV1.2 we used 30-µg protein samples; for KV1.5 analysis we used 20-µg protein samples; for KV2.1 we used 30-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- to 600-µm diameter) from control (n = 5) and hypoxic (n = 5) piglets were crushed into a fine powder under liquid N2 in a prechilled mortar and pestle, transferred to a tube containing homogenate buffer, and then sonicated with three 15-s pulses, taking care not to foam the sample. The supernatant was centrifuged, and the protein concentration was determined by protein assay (Bradford). Each vessel homogenate was diluted with phosphate-buffered saline (PBS) to obtain a protein concentration of 1 mg/ml. Aliquots of the protein concentrations were solubilized in equal volumes of denaturing, reducing sample buffer (Novex; 20 mM Tris·HCl, 2.5 mM EDTA, 0.5% Triton X-100, 0.05% SDS, 100 mM NaCl, 1 mM PMSF, 10 ng/ml leupeptin, and 10 µg/ml pepstatin), heated to 80°C for 15 min, and centrifuged for 3 min at 5,600 g in a microfuge. Equal volumes of these supernatants were then applied to Tris-glycine precast 8% polyacrylamide gels (Novex) so that equal amounts of protein were loaded. Electrophoresis was carried out in 25 mM Tris, 192 mM glycine, and 0.1% SDS (pH 8.3) at 125 V for 1.7 h. The proteins were transferred from the gel to a nitrocellulose membrane (Novex) with a Bio-Rad transfer box at 100 V for 1 h in 25 mM Tris, 192 mM glycine, and 20% methanol (pH = 8.3). The membrane was incubated overnight at 4°C in PBS containing 10% nonfat dried milk and 0.1% Tween 20 to block nonspecific protein binding. To detect the protein of interest (KV1.2, KV1.5, or KV2.1), the nitrocellulose membrane was incubated overnight at 4°C with the primary antibody diluted in PBS containing 0.1% Tween 20 and 1% nonfat dried milk (carrier buffer), followed by incubation for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody (Zymed or Jackson Laboratories) diluted in the carrier buffer. The nitrocellulose membrane was washed three times between the first two incubations with the carrier buffer and three times with the carrier buffer plus one time with PBS containing 0.1% Tween 20 after the final incubation. To visualize the antibody, we developed the membranes with 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 smooth muscle {alpha}-actin (Sigma). The bands for each protein were quantified by densitometry.

Statistics. Data are presented as means ± SE. 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 cannulated artery protocols are expressed as final molar concentrations in the vessel bath. ACh, iberiotoxin, and 4-AP were obtained from Sigma. ACh was solubilized in saline, and iberiotoxin was solubilized in H2O. 4-AP was solubilized in water, and pH was titrated to 7.4 with 12 N HCl. A-23187 and U-46619 were from Biomol and were solubilized with DMSO and EtOH, respectively. Correolide was a generous gift from Merck and Co., Inc. and was solubilized in DMSO. The studies in which correolide was used were performed at Wake Forest University School of Medicine. The KV1.2 antibody was obtained from Upstate, and the KV1.5 and KV2.1 antibodies were from Alomone.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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The mean diameter of vessels used for all cannulated artery studies was 210 ± 4 µm for control arteries and 215 ± 5 µm for hypoxic arteries. None of the vehicles used to solubilize the various agents had any effect on arterial diameter.

Transmural pressure and the endothelium do not influence Em in either artery type. Figure 1 summarizes PASMC Em determinations for control and hypoxic arteries measured at different transmural pressures. Regardless of transmural pressure, PASMC Em measurements in hypoxic arteries were less negative (depolarized) compared with control arteries. In addition, for both control and hypoxic arteries, measurements of PASMC Em were similar for air-infused and intact vessels [for control arteries PASMC Em was –51 ± 0.4 mV vs. –50 ± 0.8 mV for intact arteries (n = 26 from 9 piglets) and air-infused arteries (n = 9 from 9 piglets), respectively; for hypoxic arteries PASMC Em was –46 ± 0.5 mV vs. –47 ± 1.4 mV for intact arteries (n = 16 from 9 piglets) and air-infused arteries (n = 6 from 6 piglets)]. Thus neither the presence of the endothelium nor different transmural pressures underlie differences in PASMC Em between control and hypoxic arteries.


Figure 1
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  		Fig. 1. Membrane potential (Em) determinations in 100- to 300-µm-diameter pulmonary arteries of control and hypoxic piglets at transmural pressures of 15 and 25 cmH2O (for control arteries: n = 26 arteries from 9 piglets studied at 15 cmH2O; n = 16 arteries from 9 piglets studied at 25 cmH2O; for hypoxic arteries: n = 9 arteries from 7 piglets studied at 15 cmH2O; n = 16 arteries from 7 piglets studied at 25 cmH2O). All values are means ± SE. *Different from control, 15 cmH2O; #different from control, 25 cmH2O (P < 0.05).

 
Pulmonary arteries from hypoxic piglets show preferential loss of KV channel function. Diameters of both control and hypoxic arteries decreased similarly in response to blockade of BKCa and Kir channels with iberiotoxin and BaCl2 (Fig. 2), respectively. These data suggest that hypoxia did not affect the functional activity of these channels. In contrast, however, pharmacological blockade of KV channels with 4-AP affected the control arteries more than the hypoxic arteries (Fig. 3). Specifically, as shown in Fig. 3A, Em of the control arteries depolarized (became more positive) by 14.1 ± 2.2 mV (from –50.4 ± 0.7 to –36.4 ± 1.9 mV; n = 10) with 4-AP (10–3 M), whereas the hypoxic arteries depolarized only 5.6 ± 1.4 mV (from –45.2 ± 0.8 to –38.8 ± 1.5 mV; n = 5) with 4-AP (10–3 M). Decreases in diameter to 4-AP were also greater for control arteries than for hypoxic arteries (Fig. 3B). Endothelium disruption did not affect the characteristic 4-AP responses for either control or hypoxic arteries (Fig. 3B). Arteries exposed to the specific KV1 channel blocker correolide exhibited responses similar to those seen with 4-AP, i.e., greater constriction of the control than in the hypoxic arteries (Fig. 4). Similar to 4-AP responses, the magnitude of constriction elicited by correolide was greater in control than hypoxic arteries (Fig. 4A). Furthermore, after treatment with correolide control vessels constricted to 4-AP by an additional 14 ± 5%, whereas hypoxic vessels exhibited almost no constriction (constricted by only 1 ± 3%) (Fig. 4B). These results imply that in the control arteries KV channels besides those of the KV1 family contribute to arterial tone.


Figure 2
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  		Fig. 2. Change in diameter to BaCl2 (10–4 M) or iberiotoxin (10–7 M) for control and hypoxic arteries. For BaCl2, n = 11 arteries from 6 control piglets and n = 7 arteries from 5 hypoxic piglets. For iberiotoxin, n = 7 arteries from 5 control piglets and n = 4 arteries from 4 hypoxic piglets. All values are means ± SE.

 

Figure 3
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  		Fig. 3. A: 4-aminopyridine (4-AP, 10–3 M)-induced changes in Em for arteries from control (n = 10) and hypoxic (n = 5) piglets. All values are means ± SE. *Different from control (P < 0.05). B: 4-AP-induced changes in diameter for endothelium-intact and air-infused arteries from control and hypoxic piglets (n = 19 intact arteries from 15 control piglets; n = 9 air-infused arteries from 8 control piglets; n = 25 intact arteries from 20 hypoxic piglets; n = 7 air-infused arteries from 6 hypoxic piglets). All values are means ± SE. *Different from control intact, #different from control air infused (P < 0.05).

 

Figure 4
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  		Fig. 4. A: correolide-induced changes in diameter for control (n = 13 arteries from 11 piglets) and hypoxic (n = 8 arteries from 8 piglets) arteries. All values are means ± SE. *Different from control (P < 0.05). B: 4-AP (10–3 M)-induced changes in diameter after treatment with correolide (10–6 M) for control (n = 5 arteries from 4 piglets) and hypoxic (n = 5 arteries from 4 piglets) arteries. All values are means ± SE. *Different from control (P < 0.05).

 
KV channel proteins are downregulated in hypoxic pulmonary arteries. Immunoblot analyses for KV1.2, KV1.5, and KV2.1 {alpha}-subunits in small pulmonary artery homogenates from control and hypoxic piglets are shown in Figs. 5–7. As determined by densitometry, the mean data for the absorbance of KV1.2 bands were decreased for homogenates of small pulmonary arteries from hypoxic compared with control piglets (Fig. 5). By comparison, there was no difference in the mean data for the absorbance of either KV1.5 (Fig. 6) or KV2.1 (Fig. 7) bands for homogenates of small pulmonary arteries of both groups.


Figure 5
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  		Fig. 5. Voltage-gated K+ channel (KV)1.2 {alpha}-subunit protein in homogenates of 20- to 600-µm-diameter pulmonary arteries from control and hypoxic piglets. Note the lower intensity of bands for the KV1.2 {alpha}-subunit in homogenates of small pulmonary arteries from hypoxic compared with control piglets. Also note that the intensity of bands for smooth muscle {alpha}-actin is similar for homogenates from control and hypoxic arteries. In addition, note the lower densitometry units for the KV1.2 bands for hypoxic compared with control arteries. *Different from control (P < 0.05).

 

Figure 7
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  		Fig. 7. KV2.1 {alpha}-subunit protein in homogenates of 20- to 600-µm-diameter small pulmonary arteries from control and hypoxic piglets. Note the similar intensity of bands for KV2.1 {alpha}-subunit in homogenates of small pulmonary arteries from control and hypoxic piglets. +, Positive control for KV2.1 {alpha}-subunits. Also note that there is no difference in the densitometry units between the KV2.1 bands for hypoxic compared with control arteries.

 

Figure 6
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  		Fig. 6. KV1.5 {alpha}-subunit protein in homogenates of 20- to 600-µm-diameter small pulmonary arteries from control and hypoxic piglets. Note the similar intensity of bands for the KV1.5 {alpha}-subunit in homogenates of small pulmonary arteries from control and hypoxic piglets. Also note that there is no difference in densitometry units between the KV1.5 bands for hypoxic compared with control arteries.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of this study are that PASMC from 100- to 300-µm-diameter pulmonary arteries from piglets exposed to 3 days of chronic in vivo hypoxia are significantly depolarized, KV channel activity is diminished, and KV1.2 channel protein expression is reduced. Neither the endothelium nor the transmural pressure was responsible for the differences in Em between small pulmonary arteries of chronically hypoxic and comparable-age control piglets.

Chronic hypoxia and PASMC Em. There are limited data regarding the effect of chronic hypoxia on PASMC Em in newborns. The only other data we are aware of were from freshly isolated PASMC from second-order branches off the intrapulmonary artery of piglets raised in hypoxia for 3 days (11). Consistent with our findings in the intact pressurized artery preparation, Em in the PASMC were also depolarized (11). Thus, although scarce, the currently available data consistently show that 3 days of chronic in vivo hypoxia alters PASMC membrane properties in small pulmonary arteries of newborns such that Em is less negative in PASMC of hypoxic piglets than in control piglets.

In comparison to the paucity of data in newborns, the effect of chronic hypoxia on PASMC membrane properties in adult pulmonary arteries has received a lot of attention. It was reported over 20 years ago that PASMC isolated from main pulmonary arteries of adult rats exposed to variable lengths of in vivo hypoxia were depolarized within the first 7 days of hypoxic exposure and remained depolarized for up to 60 days of hypoxic exposure (37). More recent studies have found that after 1–4 wk of in vivo chronic hypoxia PASMC in 200- to 1,200-um-diameter pulmonary arteries and cells isolated from these arteries of adult rats were depolarized (10, 27, 30, 34). Even more recently, it was shown that PASMC isolated from resistance pulmonary arteries exposed to moderate levels of hypoxia for only 24 h were depolarized (15). In addition, PASMC obtained from adult normoxic rats and cultured under hypoxic conditions for 48–72 h were depolarized (25). Thus, with exception of one early report in which PASMC isolated from small pulmonary arteries (200-µm diameter) were hyperpolarized for 15 days and gradually returned to control levels by 40 days of chronic hypoxia (37), the majority of the data clearly show that exposure to chronic hypoxia depolarizes PASMC Em in both adults and newborns. Moreover, our findings and those of two others (11, 25) indicate that sustained depolarization occurs in PASMC of resistance-level pulmonary arteries after only a few days of in vivo exposure to chronic hypoxia.

KV channels and newborn PASMC. It is well acknowledged that K+ channels are a major determinant of PASMC Em. Studies with PASMC from adult animals show that of the different K+ channels, the KV channel family plays a significant role in regulation of Em (43, 44). To evaluate function of the KV channel family in newborn pulmonary arteries, we used the KV channel antagonist 4-AP as a pharmacological tool. In normoxic control piglet pulmonary arteries 4-AP elicited constriction and PASMC depolarization. Thus, similar to findings in adults (43, 44), KV channels in 5- to 7-day-old piglets contribute to regulation of resistance-level pulmonary arterial vasomotor tone and PASMC Em. Supportive of the notion that KV channels play a role in regulating tone in the newborn pulmonary circulation, other investigators found that 4-AP elicited vasoconstriction in 700- to 1,200-µm-diameter pulmonary arteries from normoxic newborn piglets either a few hours old or 2 wk old (6). Our results with correolide, a specific blocker of the KV1 channels, implicate this family in resistance arteries of normoxic newborn piglets. Notably, addition of 4-AP after correolide elicited further constriction, thereby suggesting that although KV1 channels play a role, other KV channel families also contribute to the regulation of pulmonary arterial tone in the normoxic newborn piglet. The finding that both iberiotoxin and BaCl2 elicited constriction suggests that both BKCa and Kir channels are also present and functional in neonatal piglet resistance pulmonary arteries.

Chronic hypoxia and KV channels in newborn PASMC. KV channels are regarded as top candidates for the oxygen-sensitive K+ channels in PASMC of adult animals (3, 7, 42). It follows that alterations in KV channels might occur concomitantly with chronic hypoxia-induced changes in PASMC Em. Indeed, our data from the pulmonary resistance arteries of the hypoxic piglets suggest that KV channel function is almost completely abolished after 3 days of hypoxia. Findings with correolide indicate impairments in the KV1 family of channels. In addition, the almost complete lack of arterial constriction to 4-AP after correolide treatment suggests that the function of other KV channels may also be impaired. Of note, pharmacological blockade of the Kir and BKCa channels in the hypoxic piglets produced constrictions similar to those in arteries from normoxic animals. Thus the function of at least these two other K+ channel families remains intact after exposure to 3-day in vivo chronic hypoxia.

Taken together with our immunoblot findings, our data suggest that downregulation of the KV1.2 channel could underlie, at least in part, the PASMC depolarization and impaired KV function that occur during exposure to chronic in vivo hypoxia. Alternatively, it must also be considered that sustained depolarization might have downregulated KV1.2 protein expression as it did expression of KV1.5 channels in GH3 pituitary cells (16). It should also be considered that our findings indicate that, despite preserved amounts of KV1.5 and KV2.1, neither these nor any other KV channels appear to function after 3-day exposure to chronic hypoxia.

Potential mechanisms involved in chronic hypoxia-mediated inhibition of KV channel function include elevated cytosolic calcium levels, oxygen radicals, mitochondrial electron chain transport, NADPH oxidase, redox status, metabolic inhibition, cytochrome P-450 oxidoreductase, cytochrome c, cellular redox status changes, endothelin-1 production, conformational changes, and phosphorylation of {alpha}- and beta-subunits (9, 19). Which, if any, of these mechanisms underlie altered KV function in resistance pulmonary arteries of chronically hypoxic newborn piglets remains to be determined. Furthermore, the limited availability of antibodies and more specific pharmacological agents precludes us from evaluating the function and protein expression of all the KV family channels that might be present and affected by chronic hypoxia in neonatal piglet resistance pulmonary arteries.

Comparing effects of chronic hypoxia on KV channels in PASMC from adults and newborns. Although we are not aware of any other studies using newborn animals, others have examined the effect of chronic hypoxia on K+ channels in the pulmonary circulation of adult animals. Exposure of adult rats to 1–4 wk of in vivo chronic hypoxia augmented the sensitivity to and the magnitude of 4-AP-induced constriction in both first-branch intrapulmonary arteries (10, 22) and 300-µm-diameter pulmonary arteries (27). Isolated, perfused lungs of control rats and rats exposed to hypoxia for 2 days or 3 wk (30) showed similar constrictor responses to 4-AP. Changes in Em induced by 4-AP were augmented in PASMC isolated from first-branch intrapulmonary arteries of chronically hypoxic rats in one study (22) and did not differ between PASMC from normoxic and chronically hypoxic rats in another study (33). Thus, unlike our findings in pulmonary arteries from newborn piglets, KV channels in rat pulmonary arteries appear to remain functional with respect to regulation of Em and vasomotor tone in the pulmonary circulation of hypoxia-exposed adults. It merits comment that although KV channels appear to remain functional in hypoxic adult rats a number of investigators have shown that KV channel currents are actually reduced. Neither the length of hypoxia exposure nor whether exposure was in vivo or in vitro appeared to matter. For example, KV channel currents were decreased in PASMC of adult mice (28) and adult rats exposed to in vivo chronic hypoxia for either 24 h (15) or 2–3 wk (20, 28, 30, 33, 34) and in PASMC of adult rats exposed to 2- to 3-day in vitro chronic hypoxia (25). In contrast, KV channel activity was unchanged in PASMC from adult humans who had been exposed to hypoxia for 4 wk, but KCa activity was attenuated (23). Differences between species, size of arteries used as the source of SMC, and length of hypoxia might account for the disparity in findings between adult humans and rats. Nonetheless, the consistent evidence from studies with rats and mice indicates that chronic hypoxia causes a sustained diminution in KV current but does not abolish the functional contribution of KV channels to regulation of Em or vasomotor tone in the pulmonary circulation of adult animals.

As with newborn piglets, findings with adult models have implicated downregulation of some of the KV1 channel family members during chronic hypoxia-induced pulmonary hypertension. In vitro studies showed that protein expression of KV1.2, KV1.5, and KV2.1 were decreased in PASMC of adult normoxic rats cultured under hypoxic conditions for 24–72 h (25, 38, 39). In intact animals, protein expression of KV2.1 and KV1.5 were decreased in pulmonary arteries isolated from rats exposed to 2–3 wk of chronic in vivo hypoxia (26, 30). A recent, more extensive evaluation of the different KV channel family subtypes revealed that, in addition to KV2.1 and KV1.5, the protein expression of KV1.1, KV1.6, and KV4.3 subunits was also decreased in resistance pulmonary arteries of adult rats exposed to 3 wk of in vivo chronic hypoxia (39). Of note, 3-wk in vivo chronic hypoxia did not alter the protein expression of KV1.2 subunits in resistance pulmonary arteries of adult rats (39). Thus, whereas we found that 3-day exposure to in vivo chronic hypoxia reduced KV1.2 but not KV1.5 or KV2.1 subunit expression in newborn piglet resistance pulmonary arteries, findings with adult rats suggest that more prolonged, 2- to 3-wk exposure to in vivo hypoxia does not reduce KV1.2 protein but instead decreases the protein expression of a number of other KV1 family subunits.

Species differences as well as length of exposure to chronic hypoxia could explain some of the disparity in findings between newborn piglets and adult rats. Segmental and regional diversity in PASMC K+ channel distribution should also be considered. Studies with adult pulmonary arteries have shown that proximal segments contain a larger proportion of KCa-enriched cells, whereas distal segments contain more KV-enriched cells (2). In addition, there is evidence for a maturational change in ion channel expression in the pulmonary circulation (31). In sheep the major K+ channel regulating Em in the pulmonary circulation shifts from KCa channels in the fetus to KV channels in the adult (31), and KV channel expression increases between the newborn period and adulthood (8). Thus it is tempting to speculate that the impaired KV channel expression and function that we found after 3-day exposure to chronic hypoxia represents an attempt to resume the fetal condition. Further work is needed to more extensively evaluate this possibility. Regardless, it should not be surprising that the influence of chronic hypoxia on specific K+ channels varies between newborns and adults.

Limitations of study. Although other types of K+ channels, including KATP channels (4), may play a role in regulation of pulmonary vascular tone and/or PASMC Em in newborns, in this study we focused on KV channels for a number of reasons. First, other investigators have provided evidence that KATP channel function is preserved in the pulmonary circulation of newborn piglets exposed to 3 days of chronic hypoxia (4). Second, KV channels have been implicated in the pathogenesis of pulmonary hypertension in adults (15, 18, 20, 26, 30, 39, 45). Third, evidence from studies with PASMC from adult animals suggests that KV channels are the O2-sensitive K+ channel (3, 7, 42). We evaluated protein expression of KV 1.5 and KV 2.1 because these KV channel family subunits have been shown to be involved with O2 sensing in the postnatal pulmonary circulation (3, 40). We also evaluated protein expression of KV 1.2 because this KV channel family subunit has been implicated in the pathogenesis of chronic hypoxia-induced pulmonary hypertension in adult animals (25, 38, 39). It is possible that K+ channels other than those evaluated in this study are O2-sensitive K+ channels. Moreover, the expression of KV channel family members other than those we evaluated could be altered in PASMC of newborns exposed to chronic hypoxia. These possibilities merit pursuit as more antibodies and specific pharmacological antagonists become available.

It is important to note that the presence of Kir channels in PASMC remains uncertain (7, 29, 35). Because it is known that Kir channels are sensitive to inhibition by extracellular Ba2+, our findings that resistance-level pulmonary arteries from control piglets and piglets exposed to 3-day hypoxia constricted to BaCl2 suggest that these channels are present and functional in PASMC of newborn piglets. However, it is possible that BaCl2 elicited constriction by mechanisms other than PASMC depolarization via inhibition of Kir channels. Thus future investigations are needed, including studies utilizing specific antibodies and more specific Kir pharmacological antagonists and agonists when they become available, to ascertain the presence of and role for Kir channels in regulating PASMC Em and pulmonary vascular tone in the normoxic as well as hypoxic pulmonary circulation of both newborns and adults.

Potential impact of altered KV channel function. Some comments about the potential implications of our findings are warranted. Sustained depolarization such as we found to occur with 3-day exposure to in vivo chronic hypoxia will increase PASMC cytosolic Ca2+ (43), which in turn can stimulate PASMC proliferation (24). Furthermore, reduced KV channel function helps maintain a high concentration of K+ in the cytoplasm, which inhibits apoptosis (19). Thus it is possible that sustained PASMC depolarization and concomitant impaired KV function contribute to the progressive remodeling of the vascular wall that we have shown to occur when hypoxia is extended beyond 3 days (13). It should also be considered that whereas inhibition of KV channels causes depolarization and constriction, activation of KV channels leads to hyperpolarization and dilation. Thus the severely impaired function of KV channels that we found could not only lead to increased basal vascular tone and thereby contribute to the progression of pulmonary hypertension but might have an impact on the effectiveness of dilator therapies used to treat existing pulmonary hypertension.

In summary, our findings show that KV channels are present and contribute to regulation of Em and pulmonary vascular tone in newborn piglets that are less than 1 wk old. Exposure to in vivo chronic hypoxia for only a few days depolarizes PASMC, nearly abolishes KV channel function, and downregulates protein expression of at least one KV1 channel {alpha}-subunit, KV1.2, in resistance pulmonary arteries of newborn piglets. We speculate that development of therapies to counteract these early changes in PASMC properties may help prevent the progressive changes in remodeling and reactivity that occur when hypoxic exposure is extended beyond a few days. Furthermore, that fact that the K+ channels responsible for regulation of vasomotor tone might be different in the hypertensive pulmonary circulation should be taken into account when developing therapies to lower pulmonary vascular resistance in infants with pulmonary hypertension associated with chronic hypoxia.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-68572 (C. D. Fike).


   ACKNOWLEDGMENTS
 
We thank Merck and Co., Inc. for the generous gift of correolide.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. D. Fike, 2215 B Garland Ave., 1125 MRB IV, Vanderbilt Univ. School of Medicine, Nashville, TN 37232-0656 (e-mail: candice.fike{at}vanderbilt.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
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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