1 Division of Pediatric Pulmonology and Critical Care Medicine, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota 55455; and 3 Departments of Pediatrics and 2 Cardiothoracic Surgery, University of California- San Francisco and Cardiovascular Research Institute, San Francisco, California 94143
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ABSTRACT |
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K+ channels play an important role in mediating pulmonary vasodilation caused by increased oxygen tension, nitric oxide, alkalosis, and shear stress. To test the hypothesis that lung K+ channel gene expression may be altered by chronic increases in pulmonary blood flow, we measured gene and protein expression of calcium-sensitive (KCa) and voltage-gated (Kv2.1) K+ channels, and a pH-sensitive K+ channel (TASK), in distal lung from fetal lambs in which an aortopulmonary shunt was placed at 139 days gestation. Under baseline conditions, animals with an aortopulmonary shunt showed elevated pulmonary artery pressure and pulmonary blood flow compared with twin controls. Hypoxia caused a greater increase in pulmonary vascular tone in shunt animals compared with controls. Alkalosis caused pulmonary vasodilation in control but not shunt animals. To determine lung K+ channel mRNA levels, we performed quantitative RT-PCR. In comparison with control animals, lung KCa channel mRNA content was increased in shunt animals, whereas TASK mRNA levels were decreased. There was no difference in Kv2.1 mRNA levels. Channel protein expression was consistent with these findings. We conclude that, in the presence of elevated pulmonary blood flow, KCa channel expression is increased and TASK is decreased.
pH-sensitive potassium channel; pulmonary hypertension; calcium-sensitive potassium channel; vascular reactivity
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INTRODUCTION |
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CONGENITAL HEART DISEASE remains a major source of morbidity and mortality in children. Although congenital heart disease is often associated with immunological, renal, or gastrointestinal abnormalities, pulmonary vascular disease is among the most frequent, and least well understood, pathological condition that complicates the management of children with congenital heart disease (19). As perinatal pulmonary vascular resistance decreases after birth, pulmonary blood flow increases (26). If pulmonary blood flow remains elevated, as is the case in many forms of congenital heart disease, pulmonary vascular smooth muscle extends into the periphery of the pulmonary circulation, extracellular matrix deposition increases (21), and endothelial-dependent pulmonary vasodilation is attenuated (9).
Recent evidence suggests that chronic elevations in pulmonary blood flow lead to an alteration in the production of vasoactive mediators from the pulmonary endothelium, with the production of vasodilator agents such as nitric oxide (NO) and prostacyclin diminished (17) and the production of the vasoconstrictor molecules thromboxane and endothelin increased (2, 3). Despite the lack of mechanistic understanding of abnormal pulmonary vasoreactivity associated with congenital heart disease, the physiology and histology have been well characterized. Initially, the ratio of pulmonary to systemic blood flow (Qp/Qs) is elevated. Over time, as intimal proliferation and smooth muscle cell hypertrophy occur (21), pulmonary vascular resistance increases and Qp/Qs approaches one. Concomitant with the decrease in pulmonary blood flow, the response of the pulmonary circulation to vasodilator stimuli such as oxygen, adenosine, NO, and alkalosis is attenuated (4). The absence of the pulmonary vasodilator response may be so pronounced that surgical repair of the congenital heart disease is no longer feasible. Even when surgical repair of the heart lesion can be undertaken, postoperative management of children with congenital heart disease is frequently complicated by sudden increases in pulmonary vascular resistance (16).
Recent studies have demonstrated that pulmonary vascular K+ channels modulate the response of the pulmonary circulation to several critically important physiological stimuli. For example, in the perinatal pulmonary circulation, oxygen causes pulmonary vasodilation through kinase-dependent activation of Ca2+-sensitive K+ channels (KCa; see Ref. 13). K+ channel inhibition with tetraethylammonium (TEA), but not glibenclamide, an ATP-sensitive K+ channel antagonist, blocks the pulmonary vasodilation caused by ventilation (32), suggesting that ventilation causes sustained and progressive pulmonary vasodilation through activation of TEA-sensitive K+ channels. NO, a vasoactive mediator that is essential for the normal transition of the pulmonary vasculature (1), causes perinatal pulmonary vasodilation, at least in part, through KCa channel activation (6, 29). Even the shear stress response, induced by compression of the ductus arteriosus during fetal life, involves KCa and voltage-dependent K+ (Kv) channel activation (31). In the adult pulmonary circulation, vasoconstriction in response to hypoxia is mediated by inactivation of a voltage-sensitive K+ channel (36).
Given these observations, we hypothesized that chronically elevated pulmonary blood flow 1) attenuates pulmonary vasodilation in response to oxygen and alkalosis; 2) modulates K+ channel expression; and 3) potentiates hypoxic pulmonary vasoconstriction. To test these hypotheses, an ovine model of congenital heart disease was utilized, physiological studies were performed, and pulmonary K+ channel mRNA levels were determined.
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METHODS |
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Surgical Preparations and Care
Ewes. Twelve late twin-gestation mixed-breed Western pregnant ewes were operated under sterile conditions, as previously described (22). Ten animals, five shunt and five twin controls, were studied for hemodynamic measurements. Pulmonary tissue for molecular studies was obtained from 10 different animals, 5 shunted and their twin controls. Pulmonary vascular tissue for protein studies was obtained from two shunt and two twin control animals. Through a left lateral fetal thoracotomy, an 8.0-mm Goretex vascular graft (~2-mm length; W. L. Gore and Associates, Milpitas, CA) was anastomosed between the ascending aorta and main pulmonary artery of the fetus with 7.0 proline (Ethicon, Somerville, NJ) using a continuous suture technique, as previously described (22). After recovery from anesthesia, the ewe was returned to the cage with free access to food and water. Antibiotics (2 million units of penicillin G K+ and 100 mg gentamicin sulfate) were administered to the ewe during surgery and daily thereafter until 2 days after spontaneous delivery of the lamb (shunted lamb).
Lambs. After spontaneous delivery, antibiotics (1 million units of penicillin G K+ and 25 mg im gentamicin sulfate) were administered for 2 days. The lambs were weighed daily, and the respiratory rate and heart rate were obtained. Furosemide (1 mg/kg im) was administered daily to all lambs. Elemental iron (50 mg im) was given weekly. At 8 wk of age, five shunted lambs and their twin controls were killed by an intravenous injection of pentobarbital sodium (Euthanasia CII; Central City Medical, Union City, CA) followed by bilateral thoracotomy. An autopsy was performed to confirm patency of the vascular graft. The lungs were removed, and sections from each lobe were frozen in liquid nitrogen to allow extraction of RNA. All procedures and protocols were approved by the Committee on Animal Research of the University of California, San Francisco.
Tissue Preparation
The heart and lungs were removed en bloc. The lungs were dissected with care to preserve the integrity of the vascular endothelium. Sections (2-3 g) from each lobe of the lung were removed. These tissues were snap-frozen in liquid nitrogen and stored atPhysiological Studies
At 2 mo of age, 10 additional lambs (5 shunted and 5 age-matched controls) had polyvinyl catheters placed in an artery and vein of one hindleg under local anesthesia with 1% lidocaine hydrochloride. These catheters were advanced to the descending aorta and the inferior vena cava, respectively. The lambs were then anesthetized with ketamine hydrochloride (~0.3 mg · kgControl Lambs
After a 45-min recovery period from surgery, baseline measurements of the hemodynamic variables (pulmonary and systemic arterial pressure, heart rate, left pulmonary blood flow, left and right atrial pressures) and systemic arterial blood gases and pH were measured. An infusion of U-46619 (a thromboxane A2 mimetic) was then begun in the inferior vena cava. The dose (~1-2 µg · kgShunted Lambs
After a 45-min recovery period from surgery, baseline measurements of the hemodynamic variables and systemic arterial blood gases and pH were measured. Hyperoxia, alkalosis, and alveolar hypoxia were administered in random order, as described above. All protocols and procedures were approved by the Committee on Animal Research of the University of California, San Francisco. All animals were killed using appropriate methods as described in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.Drug Preparation
U-46619 (9,11-dideoxy-9-epoxymethano-PGF2Physiological Measurements
Pulmonary and systemic arterial and right and left atrial pressures were measured using Statham P23Db pressure transducers (Statham Instruments, Hato Rey, PR). Mean pressures were obtained by electrical integration. Heart rate was measured by a cardiotachometer triggered from the phasic systemic arterial pressure pulse wave. Left pulmonary blood flow was measured on an ultrasonic flowmeter (Transonic Systems). All hemodynamic variables were recorded continuously on a Gould multichannel electrostatic recorder (Gould, Cleveland, OH). Systemic arterial blood gases and pH were measured on a Corning 158 pH/blood gas analyzer (Corning Medical and Scientific, Medfield, MA). Hb concentration and oxygen saturation were measured by a hemoximeter (model OSM 2; Radiometer, Copenhagen, Denmark). Qp/Qs was calculated using the Fick equation. Pulmonary vascular resistance was calculated using standard formulas.RT-PCR
Lung tissue was extracted and immediately frozen in liquid nitrogen and ground to powder with a prechilled mortar and pestle. Total RNA was extracted using the guanidium thiocyanate-phenol-chloroform method (Trireagent; Sigma). After homogenization, the samples were processed according to the reagent instructions, and the RNA was dissolved in diethyl pyrocarbonate-treated water and stored atOligonucleotide primers used to amplify the -subunit of the
maxi-KCa channel were based on the human sequence and were
5'-CTACTGGGATGTTTCACTGGTGT-3' (forward) and 5'-TGCTGTCATCAAACTGCATA-3'
(reverse), which yielded a product consistent with the expected 446-bp
fragment for the human maxi-KCa. Identity of the product
was confirmed with sequence analysis.
Oligonucleotide primers used to amplify Kv2.1 cDNA were based on the human sequence and were 5'-ACAGAGCAAACCAAAGGAAGAAC-3' (forward) and 5'-CACCCTCCATGAAGTTGACTTTA-3' (reverse), which yielded a product consistent with that expected for human Kv2.1 of 385 bp. Identity of the product was confirmed with sequence analysis.
Oligonucleotide primers used to amplify Twik-related acid-sensitive K+ channel (TASK) cDNA were based on the human sequence and were 5'-TCATCGTGTGCACCTTCACCTA-3' (forward) and 5'-GGTACCTCACCAAGGTGTTGAT-3' (reverse), which yielded a product consistent with that expected for human TASK, 386 bp. Identity of the product was confirmed with sequence analysis.
18S rRNA was analyzed in RT-PCR as an internal control. 18S cDNA was amplified with a QuantumRNA primer/competimer set (Ambion) to allow relative quantitation of the ethidium bromide bands. This control band appears as 488 bp. Because 18S rRNA is far more abundant than the mRNA under study, the 18S amplification reaction was modulated by the addition of "competimers." These primers are designed to regulate amplification by DNA polymerase. When combined with the functional primers for 18S cDNA amplification, efficiency is reduced in a linear fashion. Pilot experiments determined the correct ratio of primers to competimers, cycle number, and RT input to yield multiplex PCR products that are all in the linear range of amplification. PCR cocktail consisted of 1× PCR buffer (Perkin-Elmer) with 1.5 mmol/l Mg2+, 10 pmol/l each primer, 200 µmol/l dNTP mixture, 20 pmol/l 18S primer mixture (ratio of 1:9) of 18S primers/competimers, and 1 unit AmpliTaq polymerase and water to make 50 µl. PCR was performed in an MJ Research thermocycler with a heated lid and 0.2 ml thin-walled tubes. The PCR reaction was 2 min at 90°C followed by 30-40 cycles of 1 min at 94°C, 1 min at 53°C, 2 min at 72°C, and then an extension of 2 min at 65°C. Samples without RT were evaluated in PCR; the products were absent. Identity of the bands was confirmed by sequencing the product. Densitometry was used in quantitiative, internally controlled assessment of the RT-PCR product (NIH Image; Scion, Frederick, MD). The relative density of the 18S ribosomal and K+ channel PCR products were compared in each individual gel. RT-PCR was performed three times for each mRNA sample.
Western Blot Analysis
Frozen pulmonary artery was ground to powder in liquid N2 with a mortar and pestle. This was homogenized with a Dounce tissue grinder in RIPA buffer with protease inhibitor cocktail and 0.1% Triton X-100. The homogenate was sonicated briefly and then centrifuged at 1,000 g for 5 min. The supernatant protein concentration was determined with the bicinchoninic protein assay (Pierce). Protein (75 µg) was combined with SDS-PAGE reducing sample buffer and electrophoresed in a 4-20% gradient gel. The proteins were electroblotted on a polyvinylidene difluoride membrane (Bio-Rad). Skim milk (6%) in 20 mM Tris-buffered saline was used for blocking and washing the membranes. Antibodies against maxi-KCa channel and TASK channel (Alomone) were diluted 1:200 in milk-TBS, and membranes rotated in the solutions overnight at 4°C. In blocking experiments, the antibody was incubated 1 h, as recommended with specific antigen provided with the antibodies, and then diluted 1:200. Second antibody was anti-rabbit IgG horseradish peroxidase conjugate (Jackson), 1:3,000 in milk-TBS with 0.01% Tween 20, rocked for 2 h at room temperature. After being washed, the membranes were incubated for 10 min at room temperature with Super Signal West Pico chemiluminescent reagent (Pierce). Kodak X-Omat fs-1 film was exposed for 10 min. Bands for TASK and KCa channels were seen at 55 and 125 kDa, respectively, and antibody binding to these bands was blocked by antigen preincubation. Separate gels were prepared and loaded with 75 µg protein and probed with an antibody againstStatistical Analysis
The means ± SD were calculated for the baseline hemodynamic variables and systemic arterial blood gases and pH. The effects of each vasoactive agent were compared with their previous steady-state condition by the paired t-test using the Bonferroni correction when necessary. The percent change in pulmonary vascular resistance induced by these agents was compared between study groups by the unpaired t-test or ANOVA for repeated measures with multiple comparison testing. P < 0.05 was considered statistically significant.For the molecular data presented, values are expressed as means ± SE. Statistical significance was tested with the Student's t-test (paired or unpaired, as appropriate). P values <0.05 were considered significant.
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RESULTS |
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Under baseline conditions, animals with an aortopulmonary shunt
showed elevated pulmonary artery pressure (PAP) and pulmonary blood
flow compared with twin controls. In the control group, LPA blood flow
was 0.8 l/min, PAP was 25% that of systemic pressure, and the
Qp/Qs was 1:1. In shunted animals, LPA blood
flow was 2.1 l/min, PAP was 45% that of systemic pressure, and
Qp/Qs was 3.1:1 (Table
1). Mean PAP was 18 ± 3 mmHg in control animals compared with 28.3 ± 13.5 mmHg in shunt
animals (Table 1; P < 0.05 vs. control).
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In response to acute hypoxia, pulmonary blood flow was unchanged in
both control and shunt animals. PAP increased in both shunt and control
animals. Acute hypoxia caused pulmonary vascular resistance to increase
to a greater degree in shunt compared with control animals (Fig.
1 and Tables 2 and
3;
P < 0.05 shunt vs. control). Aortic
pressure was unaffected. In control lambs, neither systemic arterial pH
(from 7.41 ± 0.02 to 7.43 ± 0.02) nor
PCO2 (from 35.7 ± 3.3 to 33.6 ± 4.1 torr) changed. Systemic arterial PO2 decreased
from 75.7 ± 10.8 to 28.4 ± 3.5 torr (P < 0.05). Similarly, in shunt lambs, neither arterial pH (from 7.41 ± 0.02 to 7.41 ± 0.03) nor PCO2 (from
35.9 ± 3.3 to 37.6 ± 4.3 torr) changed. Systemic arterial
PO2 decreased from 70.0 ± 5.6 to
28.0 ± 2.7 torr (P < 0.05; Tables 1 and
2).
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In response to an acute increase in oxygen tension, both shunt and
control animals demonstrated a decrease in PAP. Pulmonary blood flow
did not change. Pulmonary vascular resistance decreased by 12 ± 4% in control animals and 22 ± 3% in shunt animals (Tables 1
and 2; Fig. 2; P = not
significant vs. baseline for both shunt and control animals). In
control lambs, neither systemic arterial pH (from 7.41 ± 0.01 to
7.39 ± 0.01) nor PCO2 (from 35.8 ± 5.4 to 37.0 ± 7.1 torr) changed. Systemic arterial
PO2 increased from 58.8 ± 13.8 to
369.2 ± 62.4 torr (P < 0.05). Similarly, in
shunt lambs, neither systemic arterial pH (from 7.40 ± 0.03 to
7.39 ± 0.04) nor PCO2 (from 39.2 ± 4.1 to 41.2 ± 3.7 torr) changed. Systemic arterial
PO2 increased from 64.8 ± 8.7 to
349.2 ± 104.2 torr (P < 0.05).
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Acute alkalosis caused pulmonary vasodilation in control animals but
not in shunt animals. In control animals, acute alkalosis caused a
decrease in both PAP and pulmonary vascular resistance. Pulmonary blood
flow did not change. In shunt animals, acute alkalosis had no effect on
PAP, pulmonary artery blood flow, or pulmonary vascular resistance
(Fig. 3 and Tables 2 and 3). Pulmonary
vascular resistance decreased by 39 ± 3% in control animals
(P < 0.05), whereas there was no change in shunt
animals (Tables 2 and 3). In control lambs, systemic arterial
PO2 (from 61.2 ± 17.6 to 63.8 ± 12.6 torr) was unchanged. Systemic arterial pH increased from 7.36 ± 0.05 to 7.53 ± 0.06 (P < 0.05), and
PCO2 decreased from 39.6 ± 5.3 to
30.0 ± 3.7 torr (P < 0.05). Similarly, in shunt lambs, systemic arterial PO2 (from 74.3 ± 9.4 to 75.0 ± 8.3 torr) was unchanged, systemic arterial pH
increased from 7.41 ± 0.02 to 7.55 ± 0.02 (P < 0.05), and PCO2 decreased
from 38.3 ± 4.7 to 30.1 ± 3.0 torr (P < 0.05).
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Pulmonary parenchymal tissue expressed the KCa and Kv2.1 channels as well as the TASK channel. RT-PCR yielded a 446-bp fragment of the ovine KCa channel transcript. Similarly, a 385-bp fragment was obtained for the ovine Kv2.1 channel. Both KCa and Kv channel mRNA were highly homologous to the human sequences of the same molecules. Neonatal lamb TASK mRNA was >92% homologous to its human counterpart.
KCa channel mRNA expression was increased in shunt compared
with control animals, as determined by relative quantitative, internally controlled RT-PCR. A representative gel of KCa
channel expression and 18S ribosomal mRNA expression in both control
and shunt animals is provided in Fig. 4A. The ratio of
KCa channel to 18S mRNA levels was 0.81 ± 0.06 in the
shunt animals compared with 0.60 ± 0.14 in controls
(P = 0.039; Fig.
4B). Lung KCa mRNA expression was increased by 34% (134 ± 6% that of the control) in shunt compared with control animals (P < 0.05; Fig.
5).
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Lung TASK mRNA expression was decreased in shunt animals. The ratio of
TASK channel to 18S mRNA levels was 0.57 ± 0.06 in the shunt
animals compared with 0.76 ± 0.04 in the control animals (P < 0.03; Fig.
6B). Relative to control
animals, lung TASK mRNA expression was decreased by 28% (72 ± 6% of control; P < 0.05). A representative gel of
TASK channel expression and 18S ribosomal mRNA expression in both
control and shunt animals is provided in Fig. 6A. The
presence of an aortopulmonary shunt had no effect on Kv2.1
channel mRNA levels (P = 0.16, Fig.
7, A and B).
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KCa channel and TASK protein expression were analyzed in
vascular tissue derived from two control and two shunt animals.
KCa bands of ~125 kDa were observed in both control and
shunt samples. The 125-kDa band is consistent with previously published
reports (20, 25, 28). Expression of the KCa
channel protein was increased in shunt animals compared with controls.
TASK protein was also expressed in pulmonary vascular tissue for both
control and shunt animals. The 55-kDa band is consistent with
previously published reports. TASK expression was decreased in shunt
animals compared with controls (Fig. 8).
Bands were not evident when samples were coincubated with both antigen
and antibody, demonstrating antibody specificity. Separate gels
incubated with -smooth muscle cell actin antibody indicated similar
amounts of protein in shunt and control samples. The results were
entirely consistent with the mRNA expression experiments reported
above.
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DISCUSSION |
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Pulmonary vascular disease frequently complicates both surgical and medical management of congenital heart disease (18). Although pulmonary hypertension and abnormal pulmonary vascular reactivity are frequently associated with congenital heart disease (21), our understanding of both the physiology and the molecular mechanisms that underlie the pathophysiology remains incompletely understood. We report that chronic elevation (for 8 wk) in pulmonary blood flow in a neonatal ovine model of congenital heart disease results in attenuated pulmonary vasodilation in response to alkalosis, enhanced hypoxic pulmonary vasoconstriction, and preservation of oxygen-induced pulmonary vasodilation. In association with these changes in pulmonary vasoreactivity, pulmonary K+ channel expression changes as KCa channel mRNA expression increases and TASK mRNA expression decreases. Parallel changes were noted in KCa and TASK channel protein expression. These observations support the notion that alterations in blood flow and oxygen tension, given the relatively high oxygen content in the pulmonary arterial blood of shunt animals, may modulate K+ channel expression in the pulmonary vasculature.
The observation that pulmonary blood flow is increased in the present model is consistent with previous reports that an aortopulmonary shunt placed in fetal lambs leads to an increase in pulmonary blood flow during postnatal life (22). Qp/Qs is increased, and pulmonary artery blood pressure is at one-half the systemic levels. Previous studies using this model have identified histological alterations that are similar to those observed in children with pulmonary hypertension associated with congenital heart disease (22). At 2 mo of age, the oxygen-induced pulmonary vasodilation was similar in both control animals and in animals with an aortopulmonary shunt. In contrast, the response to alkalosis was absent in shunt animals, whereas the response to hypoxia was enhanced. The pattern of responses observed argues that pulmonary vasoreactivity in animals with a shunt was not globally diminished, since the vasodilator response to oxygen was retained. This is consistent with a previous report in this model demonstrating that the response to inhaled NO is preserved (23).
Because K+ channels have been shown to mediate the response of the pulmonary vasculature to several physiological stimuli (6, 13, 31, 37), we determined the mRNA levels of specific K+ channels. The results of the physiological studies were consistent with the mRNA measurements. First, the preservation of the response to an acute increase in oxygen tension might be explained, in part, through the increase in KCa channel mRNA expression. Activation of the KCa channel in pulmonary vascular smooth muscle cells has been shown to cause an increased K+ efflux, membrane hyperpolarization, and a decrease in cytosolic calcium, resulting in relaxation. Because KCa channels have been shown to mediate oxygen-induced perinatal pulmonary vasodilation (13), the increase in KCa channel expression in shunt animals may enable the pulmonary vasculature to dilate in response to oxygen despite pulmonary vascular smooth muscle cell hypertrophy. The fact that the ratio of KCa to 18S mRNA increases argues against the possibility that increases in mRNA expression derive from an overall increase in pulmonary vascular smooth muscle. Because KCa, but not Kv2.1, channel mRNA expression is affected in shunt animals, it seems unlikely that the present results derive from nonspecific changes in K+ channel mRNA expression.
Recently, a K+ channel that is sensitive to changes in extracellular pH has been identified. The TASK channel is a background channel that is constitutively open. The channel is thought to be voltage insensitive. However, with an increase in extracellular pH to 7.7, K+ current through the channel increases to 90% of its maximum. With a decrease in pH to 6.7, the K+ current through the channel decreases to ~10% of its maximum (24). Hence, the changes in acid-base status might affect pulmonary vascular tone through K+ efflux via the TASK channel. Presumably, alkalosis increases pulmonary artery smooth muscle cell K+ efflux through the TASK channel, leading to membrane hyperpolarization, closure of the voltage-operated calcium channels, and a decrease in cytosolic calcium. The decrease in pulmonary artery smooth muscle cell cytosolic calcium concentration results in pulmonary vasodilation (34).
Although the TASK channel has been described in vitro (24), the present report suggests that the TASK channel may have physiological relevance. The absence of a pulmonary vasodilator response to alkalosis, in combination with a decrease in TASK channel mRNA expression, lends credibility to the concept that the channel senses changes in extracellular pH. The absence of pulmonary vasodilation in response to alkalosis cannot be attributed simply to a decreased capacity for pulmonary vasodilation, since the response of shunt animals to an increase in oxygen tension is preserved.
Previous investigators have demonstrated that alkalosis-induced pulmonary vasodilation is mediated by endothelium-dependent NO release, prostacyclin, and KCa channel activation (35). The present study suggests that TASK activation also plays a role in alkalosis-induced pulmonary vasodilation. Because alkalosis induces pulmonary vasodilation through NO, prostacyclin release, and KCa channel activation (35), the role of the TASK channel may lie in sensing the change in extracellular pH. With the TASK channel conferring sensitivity to changes in extracellular pH, the pulmonary vasculature can respond with elaboration of vasoactive mediators from the pulmonary endothelium and effect KCa channel activation and pulmonary vasodilation (13, 29).
Among the limitations of this study is the inability to distinguish
between alterations in endothelial compared with vascular smooth muscle
K+ channel expression. Multiple lines of evidence suggest
that vascular endothelium express both KCa and Kv channels
(11, 30, 31, 33). Because RNA was extracted from whole
lung homogenate, whether the changes in K+ channel mRNA
levels result from alterations in the endothelium, vascular smooth
muscle cells, or a combination is unknown. However, given that this
model leads to pulmonary vascular remodeling and not remodeling of the
airways, it is reasonable to conclude that the changes in mRNA
expression between the shunt and control animals result from changes in
K+ channel expression in the vasculature as opposed to the
airway smooth muscle or airway epithelial cells. The protein studies were performed on pulmonary vascular tissue, as opposed to whole lung
homogenate, providing further support for specific alterations in
pulmonary vascular K+ channel expression in the present
model. To ensure fidelity between alterations in K+ channel
mRNA levels and K+ channel protein expression, Western
analysis was performed. The present data support the notion that
increases in mRNA expression of the -subunit of the
maxi-KCa channel lead to increases in protein expression of
the
-subunit of the KCa channel. Similarly, the decrease
in TASK mRNA expression correlated with a decrease in TASK protein
expression. We speculate that a slight change in ion channel expression
can have a significant effect on vascular endothelial and smooth muscle
cell physiology (20, 28, 33, 34).
Given the accentuation of hypoxic pulmonary vasoconstriction in the shunt compared with the control animals, Kv2.1 channel mRNA levels might be expected to be greater in shunt animals. Several investigators have demonstrated that hypoxia causes pulmonary artery smooth muscle cell depolarization through inactivation of Kv channels (7, 37). The observation that Kv2.1 channel mRNA expression is similar in both shunt and control animals suggests that an alternative mechanism, such as proliferation of pulmonary vascular smooth muscle cell and extracellular matrix, might augment the contractile strength of the pulmonary vasculature. Alternatively, production of vasodilator compounds by the pulmonary endothelium, such as prostacyclin or NO, may be compromised, rendering the pulmonary vasculature more responsive to hypoxia. An additional explanation may be that increases in pulmonary blood flow enhance the sensitivity of the pulmonary artery smooth muscle contractile apparatus to calcium.
There is precedent for the notion that expression of vascular K+ channels is dynamic, changing with both maturation (15) and in response to hypertension (20, 28). Recent reports from our laboratory indicate that pulmonary vascular KCa channel expression decreases with maturation (25), whereas Kv2.1 channel expression increases with maturation (15). Systemic hypertension increases KCa channel expression in the systemic vasculature (20). In a model of chronic intrauterine pulmonary hypertension, KCa channel expression is decreased (14). Although the present findings may seem to diverge from those reported previously, it is essential to consider several important differences between the two models. With ligation of the ductus arteriosus, the pulmonary blood flow in the fetal lamb remains low, <10% of the biventricular output, whereas PAP is higher than systemic blood pressure, and oxygen tension is quite low. In contrast, the pulmonary blood flow in the neonatal lamb with an aortopulmonary shunt is significantly greater than systemic blood flow, PAP is less than one-half systemic pressure, and oxygen tension in the blood entering the pulmonary arteries is relatively high. These important physiological differences, superimposed on alterations in the pulmonary circulation during fetal as opposed to neonatal life, likely influence vascular K+ channel expression. Indeed, consideration of the changes in K+ channel expression in the present model compared with the model of chronic intrauterine hypertension may provide insight into the application of specific treatment modalities based on the etiology of the pulmonary hypertension. It is also important to recognize that the study animals were only 8 wk old at the time of study and subsequent death. With more pronounced pulmonary hypertension, the changes in K+ channel mRNA expression may have been greater.
Despite the apparent changes in vascular K+ channel expression associated with an increase in pulmonary blood flow, the mechanisms responsible for alterations in K+ channel expression remain completely speculative. Perhaps, the relatively oxygen-rich blood that is present in pulmonary arteries of shunt animals provides a signal that modulates K+ channel expression. Alternatively, the increase in pressure in the pulmonary arteries of shunt animals may be the critical stimulus for modulating K+ channel expression. Because endothelial dysfunction occurs in neonatal pulmonary hypertension (5, 10), and in this model (8, 23), alterations in the production of vasoactive mediators such as endothelin or NO may modulate K+ channel expression.
In conclusion, we report that pulmonary vascular reactivity is altered in neonatal lambs with an aortopulmonary shunt. After 2 mo, shunt animals demonstrate no pulmonary vasodilator response to alkalosis, an accentuated response to hypoxia, and an intact vasodilator response to oxygen. In association with the changes in pulmonary vasoreactivity, pulmonary K+ channel expression is altered. In particular, TASK channel mRNA expression is decreased, Kv2.1 channel expression is unchanged, and KCa channel mRNA expression is increased. The specific alterations in K+ channel expression argue that pulmonary K+ channels mediate the response of the pulmonary circulation to alkalosis, hypoxia, and an acute increase in oxygen tension. In children with congenital heart disease, pharmacotherapy targeted to pulmonary vascular K+ channels may represent a novel therapeutic strategy.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants RO1 HL-60784 (D. N. Cornfield) and RO1 HL-61284 (J. R. Fineman), the Viking Children's Fund (E. R. Resnik), and an American Heart Association Established Investigator Award (D. N. Cornfield).
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. N. Cornfield, Div. of Pediatric Pulmonology and Critical Care Medicine, Univ. of Minnesota School of Medicine, 420 Delaware St. S.E., MMC 742, Minneapolis, MN 55455 (E-mail: cornf001{at}umn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 23, 2002;10.1152/ajplung.00428.2001
Received 31 October 2001; accepted in final form 29 May 2002.
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