Department of Physiology and Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523
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ABSTRACT |
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Resistance
pulmonary arteries constrict in response to hypoxia, whereas conduit
pulmonary arteries typically do not respond or dilate slightly. One
proposed mechanism for this differential response is the variable
expression of pulmonary arterial smooth muscle cell voltage-gated
K+ (KV) channel subunits (Kv1.2, Kv2.1, Kv1.5,
and Kv3.1b) shown to be O2 sensitive in heterologous
expression systems. In this study, immunoblotting and
immunohistochemistry were used to examine the expression of
KV channel - and
-subunits in the bovine pulmonary arterial circulation to determine whether differential KV
channel subunit distribution is responsible for the distinct
sensitivities of pulmonary arteries to hypoxia. Surprisingly, there was
little difference in the expression levels of Kv1.2, Kv1.5, and Kv2.1 between conduit and resistance pulmonary arteries. In contrast, expression of the Kv3.1b
-subunit and Kv
.1, Kv
1.2, and
Kv
1.3 accessory subunits dramatically increased along the pulmonary arterial tree. The differential expression of all the
-subunits but
of only one of the putative O2-sensitive
-subunits
suggests that the
-subunits alone are not the O2 sensors
but further implicates the auxiliary
-subunits in pulmonary arterial
O2 sensing.
pulmonary artery; oxygen sensor; smooth muscle; voltage-gated
potassium channel localization; voltage-gated potassium -subunit
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INTRODUCTION |
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THE PULMONARY AND SYSTEMIC arterial circulations exhibit vastly different responses to hypoxia. In the systemic circulation, small arteries dilate in response to acute hypoxia to increase blood flow to O2-deprived tissues. In contrast, hypoxia causes pulmonary vasoconstriction (HPV) of the small resistance pulmonary arteries, thereby diverting blood flow away from poorly ventilated alveoli to optimize perfusion-ventilation matching and maintain systemic PO2. The HPV response is likely to be a multifactorial process, with contributions from endothelium-derived vasoactive factors necessary for the full expression of HPV in vivo (36, 38). However, HPV is thought to be initiated, at least partially, through a mechanism intrinsic to pulmonary arterial (PA) smooth muscle cells (SMCs) (39) because responsiveness to hypoxia has been demonstrated not only in isolated lungs (14, 23, 31, 35) but also in PA rings denuded of endothelium (3, 13, 19, 42) and in single PASMCs (3, 8, 9, 20, 26, 28, 30, 31, 41).
In vascular smooth muscle, K+ channels play an important role in the regulation of the resting membrane potential (16, 25). Acute hypoxia inhibits the PASMC outward K+ current (3, 26, 30, 31, 41), leading to membrane depolarization (3, 31, 41) and constriction of small pulmonary arteries (3). Therefore, much attention has been focused on identifying the K+ channels involved in this response. The hypoxia-sensitive K+ currents expressed in most PASMC preparations are voltage-gated K+ (KV) delayed rectifier current types that are sensitive to 4-aminopyridine and insensitive to charybdotoxin (7).
In addition to the divergent response to hypoxia between the pulmonary and systemic circulations, there is also heterogeneity in the response to hypoxia within the pulmonary circulation. Small resistance pulmonary arteries (third intrapulmonary artery or greater) constrict in response to hypoxia (3, 19, 30, 42), whereas large conduit pulmonary arteries (main pulmonary artery and right and left branches) usually do not respond or dilate slightly (3, 19, 30). Hypoxia leads to membrane depolarization (19), an increase in intracellular Ca2+ (33), and contraction (19, 33) of PASMCs isolated from resistance vessels but has little or no effect on conduit PASMCs (19, 33). The regional heterogeneity in response to hypoxia within the pulmonary circulation could reflect the differential expression of K+ channel subtypes between conduit and resistance vessels. Previous electrophysiological studies in the rat (1, 2) and rabbit (22) pulmonary vasculatures demonstrated K+ channel current heterogeneity between cells isolated from the conduit pulmonary arteries and those cells isolated from resistance-size vessels.
Several groups of investigators (15, 27, 28) have studied
the O2 sensitivity of cloned KV channels
expressed in heterologous expression systems in an attempt to identify
potential molecular components of the native PASMC
O2-sensitive KV current. These studies
(3, 27, 28, 43), combined with previous studies in
native cells, support a potential role for the KV
-subunits Kv1.2, Kv1.5, Kv2.1, Kv3.1b, and Kv9.3 which are expressed
in homomeric and/or heteromeric complexes. In addition, a role for the
KV
-subunits in cellular O2 sensing has also
been suggested because the Kv
1.2 subunit appears to confer
O2 sensitivity on the Kv4.2
-subunit (29).
Although past studies have given us insight as to which KV
channel subunits are most likely to make up the native PASMC
O2-sensitive K+ current, it is unknown if these
proteins are expressed primarily in the resistance vessels, where HPV
is thought to occur, or distributed evenly throughout the pulmonary
vasculature. Intuitively, one would expect that those subunits that are
involved in the physiological response of resistance pulmonary arteries
to hypoxia would be expressed more abundantly in resistance than in
conduit PASMCs and, furthermore, that this differential expression
would, at least partially, account for the differential response of
conduit and resistance pulmonary arteries to hypoxia. Therefore, the
primary objectives of the present study were to determine the
expression and localization of SMC KV channel
- and
-subunits in the pulmonary arteries and to determine whether
expression levels of these subunits varied between conduit and
resistance PASMCs. Immunoblotting and/or immunohistochemistry was used
to examine expression of the Kv1.2, Kv1.5, Kv2.1, Kv3.1b, Kv
1.1,
Kv
1.2, Kv
1.3, and Kv
2.1 subunits in bovine conduit and
resistance PASMCs. Surprisingly, we found little difference in the
expression of the putative O2-sensitive
-subunits Kv1.2,
Kv1.5, and Kv2.1. In contrast, expression of the Kv3.1b
-subunit and
of all the KV
-subunits (except Kv
2.1, which was not
present) was dramatically greater in resistance than in conduit PASMCs.
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MATERIALS AND METHODS |
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Antibodies.
Anti-Kv2.1 rabbit polyclonal antibody was purchased from Upstate
Biotechnology (Lake Placid, NY), and anti-Kv3.1b rabbit polyclonal antibody was purchased from Alomone Laboratories (Jerusalem, Israel). The following antibodies were kindly donated by Dr. James Trimmer (State University of New York at Stony Brook, Stony Brook, NY): anti-Kv1.1N rabbit polyclonal, anti-Kv
1.2N rabbit polyclonal, and
anti-Kv
2.1 mouse monoclonal. Anti-Kv1.2 rabbit polyclonal (15), anti-human Kv1.5 rabbit polyclonal
(21), anti-bovine Kv1.5 rabbit polyclonal, anti-Kv
1.2
rabbit polyclonal, and anti-Kv
1.3 rabbit polyclonal antibodies were
produced and characterized in the Tamkun laboratory. Anti-
-smooth
muscle actin mouse monoclonal and Indocarbocyanine (Cy3)-conjugated
anti-
-smooth muscle actin mouse monoclonal antibodies were purchased
from Sigma (St. Louis, MO). Horseradish peroxidase (HRP)-goat
anti-mouse IgG [heavy plus light (H+L)] and HRP-goat anti-rabbit IgG
(H+L) secondary antibodies were purchased from Zymed Laboratories (San
Francisco, CA) and used for immunoblotting. Biotin-SP-conjugated goat
anti-rabbit IgG (H+L) and biotin-SP-conjugated goat anti-mouse IgG
(H+L) secondary antibodies were purchased from Jackson ImmunoResearch
Laboratories (West Grove, PA) and used for immunostaining.
Production of anti-bovine Kv1.5, anti-Kv1.2, and anti-Kv
1.3
antibodies.
Three regions within the NH2 terminus of the bovine Kv1.5
channel reported in GenBank (accession no. AAB32447) (10) were chosen for antibody production. These epitopes were predicted to
be antigenic in the rabbit because there is significant species variation between the bovine and rabbit sequences. The following peptides were produced and purified by the Colorado State University Macromolecular Resources Facility: GGAMTVRGEEEARTT, PAPRRRSGGERG, and ADPGGRPAPPPRQELPQASPRPPEEEDGED. These peptides were
conjugated to KLH, and a combination of the three were used to immunize
two rabbits. Polypeptides from the variable KV
-subunit
NH2-terminal domains were selected for isoform-specific
production of Kv
1.2 (amino acids 1-72) and Kv
1.3 (amino
acids 1-91) antibodies. Rabbit polyclonal antibodies against these
domains were produced with the use of a glutathione
S-transferase (GST) fusion protein expression vector,
pGEX-2T (Pharmacia, Piscataway, NJ) as previously described (17,
21). The antisera were tested by immunoblotting of bovine tissue
with antisera alone, antisera plus peptide, or GST fusion protein
as described in Immunoblotting.
Preparation of PASMC membranes.
A bovine model was chosen for these studies to obtain enough starting
material for Western blot analysis along the PA tree. Lungs obtained
from freshly slaughtered cattle were immediately placed in ice-cold
PBS, and within 1 h, the pulmonary arteries were carefully
exposed, removed from the lungs, and placed in fresh ice-cold PBS. The
vessels were characterized as follows: conduit (main conduit pulmonary
artery before it branches, external diameter 25-30 mm), second
intralobar (second branch off of the main intralobar pulmonary artery,
external diameter 5-10 mm), third intralobar (third branch off of
the main intralobar pulmonary artery, external diameter 3-5 mm),
and fourth intralobar (fourth branch off of the main intralobar
pulmonary artery, external diameter 1-2 mm). Due to limitations on
the number of resistance vessels, resistance pulmonary arteries were
often defined as pooled third and fourth intralobar pulmonary arteries
(see Figs. 2 and 5). The pulmonary arteries were dissected free
of adventitia, cut open longitudinally, and then gently scraped with a
cotton swab to remove the endothelium. Approximately 2-5 g of each
of the resultant PASMC tissue was cut into small pieces and homogenized with a polytron homogenizer in 30-40 ml of 0.32 M sucrose, 5 mM Na2HPO4 with the following protease inhibitors:
0.31 mg/ml of benzamidine, 0.62 mg/ml of N-ethylmaleimide, 1 mg/ml of bacitracin (Sigma), 1 µg/ml of pepstatin, 1 µg/ml of
leupeptin, and 0.07 µg/ml of Pefablock (Boehringer Mannheim). The
tissue was homogenized on ice at high speed, first with a large
(1.75-cm) and then with a small (1.0-cm) diameter probe for 60 s
each. The homogenate was centrifuged at 4°C for 10 min at 3,000 rpm
(Beckman JA 25.5 rotor) to remove large debris and nuclei. The
supernatant was filtered through one layer of cotton gauze and
centrifuged for 1 h at 4°C and 12,000 rpm (Beckman JA 25.5). The
resultant membrane pellet was resuspended in 200-400 µl of
ice-cold PBS and stored at 80°C.
Immunostaining of PA tissue sections.
Main conduit (~25-mm), second intralobar (~8-mm), third intralobar
(~3-mm), and fourth intralobar (~1.5-mm) PA tissue blocks were
immersed in 30% sucrose in PBS for 1 h at 4°C, placed in cryomolds, embedded in Tissue Tek (Sakura Finetechnical, Tokyo, Japan),
and quickly frozen on a slab of dry ice. Cryosections measuring 10 µm
in thickness were collected on gelatin-coated coverslips and then
incubated with primary antibody (1:100 anti-Kv1.2, 1:500
anti-human-Kv1.5, and 1:500 anti-Kv1.2) followed by
biotin-conjugated goat anti-rabbit IgG and Cy3-conjugated streptavidin
(Jackson ImmunoResearch Laboratories) as previously described
(15, 21). Immunogen block of tissue staining was performed
to demonstrate antibody specificity (data not shown because these
antibodies have been previously characterized). Immunogen block is
shown in Fig. 6 for the anti-Kv
1.2 antibody used here for the first time. Staining was performed as described above except that
cryosections were stained with antiserum that had been preincubated
overnight at 4°C with 40 nmol/l (for Kv1.2 and Kv1.5 immunogen block)
or 1 µmol/l (for Kv
1.2 immunogen block) of either GST or the
appropriate GST fusion protein construct as previously described
(15). Binding of the anti-Kv1.2, anti-Kv1.5, or
anti-Kv
1.2 antibodies to bovine resistance pulmonary arteries was
unaffected by preincubation with GST; however, it was almost completely
eliminated after preincubation with the channel-containing fusion protein.
Immunoblotting.
PASMC membrane proteins were fractionated by SDS-PAGE and transferred
overnight to nitrocellulose membrane (Schleicher & Schuell, Dassel,
Germany) with the standard Laemmli method (5). Briefly, SDS sample buffer was added to the isolated membranes, and the samples
were boiled for 5 min before electrophoretic separation on a 10%
polyacrylamide gel with a Bio-Rad minigel system. After overnight
transfer onto nitrocellulose membranes, the samples were stained with
Ponceau S solution (Sigma) to visualize the quality of the transfer,
rinsed with deionized water, and incubated for 1 h at room
temperature in solution 1A [S1A; 50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk] to block nonantigenic sites. The blots were then incubated in S1A at room temperature for 2 h with one of the following primary antibodies: Kv2.1 polyclonal (1:500), Kv3.1b polyclonal (1 µg/ml), Kv1.1N polyclonal (1 µg/ml), Kv
1.2N polyclonal (1 µg/ml), Kv
1.3
polyclonal (1:500), Kv
2.1 monoclonal (1 µg/ml), or
-smooth
muscle actin (1:50,000). The blots were washed three times for 15 min
each with solution 3A [50 mM Tris (pH 7.5), 500 mM NaCl,
and 0.1% Tween 20] and then incubated for 1 h in S1A containing
a 1:3,000 dilution of HRP-conjugated goat anti-rabbit IgG or goat
anti-mouse IgG. The blots were washed three times for 15 min each with
solution 3A, and detection was achieved with the Renaissance
enhanced chemiluminescence (ECL) reagent (NEN Life Science; Boston, MA)
following the manufacturer's instructions. For blots incubated with
anti-bovine Kv1.5, a modified version of the above protocol was used.
Briefly, the blots were incubated overnight at 4°C in solution
1B [50 mM Tris (pH 7.5), 150 mM NaCl, and 10% goat serum (GIBCO
BRL)]. After the blocking step, all subsequent steps were performed at
room temperature. The blots were incubated in solution 2B
[S2B; 50 mM Tris (pH 7.5), 150 mM NaCl, 5% goat serum, and 0.05%
Tween 20] containing a 1:1,000 dilution of anti-bovine-Kv1.5 antibody
for 2.5 h. The blots were washed twice for 15 min in
solution 3B [50 mM Tris (pH 7.5), 500 mM NaCl, 5% NaCl,
and 0.05% Tween 20] and incubated for 1 h in S2B containing a
1:5,000 dilution of HRP-conjugated goat anti-rabbit IgG. The blots were
washed sequentially for 15 min each in solution 1B, S2B, and
solution 3B, and detection was achieved with the Renaissance
ECL reagent.
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Quantification of Western blot signals.
Nitrocellulose blots were exposed to X-ray film for multiple time
periods (generally ranging between 5 s and 5 min) to detect saturation. Nonsaturated films were scanned into Adobe Photoshop, and
densitometry was used to quantify the immunoblot signal (NIH Image,
Scion, Frederick, MD). To compare channel expression between conduit
and resistance PASMC membranes, the average intensity of the channel
signal was multiplied by the number of pixels in that area and then
corrected for the -smooth muscle actin signal present in the same
lane (calculated the same way). Although saturation of the X-ray film
was avoided, given the limited exposure depth of film, these results
should be viewed as semiquantitative.
Statistical analysis. A paired t-test was used to assess the differences in channel expression between conduit and resistance PASMCs. P < 0.05 was considered significant.
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RESULTS |
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Immunoblot analysis of KV -subunit expression.
Kv1.2 (4, 15, 43), Kv1.5 (4, 15, 43), Kv2.1
(4, 28, 43), and Kv3.1b (27) have been
previously reported to be expressed in rat or rabbit (Kv3.1b) PASMCs.
In addition, all of these KV
-subunits have been
reported to be O2 sensitive in heterologous expression
systems (6, 15, 27, 28). In the present study, Kv1.5,
Kv2.1, and Kv3.1b
-subunits were detected in bovine PASMC membranes
via immunoblotting (Fig. 1A).
The anti-Kv1.5 antibody recognized a prominent band at ~70 kDa in
bovine resistance PASMC membranes that was blocked by preincubation
with the peptide sequences from which the antibody was generated. The
molecular mass of the Kv1.5 protein is consistent with that in
rat PASMCs in previous reports (4, 43). A band of similar
size was also detected in rat heart, rat aorta, and L-cells expressing
rat Kv1.5 (data not shown), indicating that this antibody is not bovine specific, although it failed to recognize the human isoform. The anti-Kv2.1 antibody recognized a single band of ~110 kDa in bovine resistance PASMC membranes (Fig. 1A), consistent with
previous reports in rat PASMCs (4, 43). The Kv3.1b channel
was detected as a band of ~70 kDa in bovine resistance PASMCs that
was almost completely blocked by antibody preincubation with the Kv3.1b
immunogen (Fig. 1A). The present study is the first report
of PASMC Kv3.1b expression via immunoblot analysis. At 70 kDa, the
electrophoretic mobility of the PASMC Kv3.1b channel is much lower than
that of the brain channel (90 kDa); however, it is consistent with its predicted protein core molecular mass of ~66 kDa. A 51-kDa band, which was also blocked after preincubation with the Kv3.1b immunogen, was noted in the resistance PASMC lane. It is likely that the 51-kDa
band represents a proteolysis product because it is blocked by the
peptide and it parallels the increase in the 66-kDa band from conduit
to resistance PASMCs (discussed in Differential expression of
KV
-subunits in pulmonary arteries). Although Kv1.2
expression has been reported in rat PASMCs (4, 15, 43,
43), we were unable to obtain a trustworthy immunoblot signal
due to a low signal-to-noise ratio; however, Kv1.2 expression examined
via immunohistochemistry is presented in Differential expression
of KV
-subunits in pulmonary arteries.
Immunoblot analysis of KV -subunit expression.
Although multiple KV
-subunit mRNAs have been detected
in cultured PASMCs (37, 43), PA
-subunit protein
expression has not been examined. Kv
1.1, Kv
1.2, and Kv
1.3 but
not Kv
2.1 were detected in bovine resistance PASMC membranes via
immunoblot analysis (Fig. 1B). The anti-Kv
1.1 antibody
recognized a single prominent band of ~49 kDa in bovine resistance
PASMC membranes (Fig. 1B). The electrophoretic mobility of
the Kv
1.1 subunit was higher in PASMCs than its predicted molecular
mass of ~40 kDa and instead was closer to the 44-kDa band reported in
transfected COS-1 cells (24). The anti-Kv
1.2 antibody
recognized a single prominent band of ~38 kDa in bovine resistance
PASMC membranes (Fig. 1B), which corresponds closely with
its predicted molecular mass. The anti-Kv
1.3 antibody recognized a
single prominent band of ~68 kDa in bovine resistance PASMC membranes
(Fig. 1B). The electrophoretic mobility of the PASMC
Kv
1.3 subunit was much higher than its predicted size of ~40 kDa;
however, a single band of ~61 kDa was detected in L-cells transfected
with Kv
1.3 (Fig. 1B), indicating that the antibody
recognizes Kv
1.3 and that this
-subunit migrates unusually slowly
on SDS gels relative to its predicted molecular mass. Even after
extended exposures, there was no evidence of Kv
2.1 expression in
bovine conduit or resistance PASMC membranes, although a strong signal
at ~40 kDa was readily detected in the bovine brain (Fig.
1B). Therefore, it is likely that Kv
2.1 is not expressed
at significant quantities in bovine pulmonary arteries.
Differential expression of KV -subunits in pulmonary
arteries.
Kv1.2, Kv1.5, Kv2.1, and Kv3.1b
-subunits have all been hypothesized
to be molecular components of the native resistance PASMC
O2-sensitive K+ current. Therefore, expression
levels of these subunits were compared between conduit and resistance
PASMCs with the idea that those subunits involved in the differential
response of conduit and resistance pulmonary arteries to hypoxia would
be differentially expressed between these vessels. Subunit expression
between conduit and resistance PASMCs was examined quantitatively via
Western blot analysis and densitometry. Representative immunoblots
demonstrating Kv1.5, Kv2.1, and Kv3.1b expression in PASMCs from the
conduit pulmonary artery, second intralobar pulmonary artery, and third and fourth intralobar resistance pulmonary arteries are shown in Fig.
2 along with a quantitative assessment
comparing expression levels between conduit and pooled resistance
PASMCs. Kv1.5 expression was only slightly greater in the third and
fourth intralobar resistance PASMCs than in conduit PASMCs (Fig.
2A, left). When examined quantitatively, Kv1.5
expression levels were found to be significantly greater in resistance
(pooled third and fourth division vessels) than in conduit PASMCs,
although the difference was not that large (~1.5-fold greater in
resistance PASMCs; Fig. 2B, right). Kv2.1 expression appeared to be similar between conduit and third and fourth
resistance PASMCs (Fig. 2B, left). Consistent
with these results, quantitative assessment revealed that Kv2.1
expression levels were not significantly different between conduit and
resistance PASMCs (Fig. 2B, right). On the other
hand, Kv3.1b expression was dramatically greater in third and fourth
intralobar resistance PASMCs compared with that of conduit PASMCs and
even second intralobar PASMCs (Fig. 2C, left).
Only after overexposure of the resistance lanes was a Kv3.1b signal
detected in the conduit lane. Consistent with these results, when
examined quantitatively, expression levels of Kv3.1b were found to be
dramatically greater in resistance than in conduit PASMCs (~12-fold
greater in resistance PASMCs; Fig. 2C, right).
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DISCUSSION |
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Expression of KV channel -subunits in pulmonary
arteries.
Because hypoxia significantly depresses KV channel current
in resistance PASMCs but not in conduit PASMCs (3), it
seems logical that the expression of O2-sensitive
KV channel subunits (Kv1.2, Kv1.5, Kv2.1, and Kv3.1b) would
be greater in resistance than in conduit PASMCs. Therefore, it was
surprising that in the present study, the putative
O2-sensing
-subunits Kv1.2, Kv1.5, and Kv2.1 were
distributed fairly equally between conduit and resistance PASMCs. Kv1.5
expression was found to be modestly greater in resistance than in
conduit PASMCs in both immunoblot (Fig. 2) and immunohistochemical
(Fig. 4) experiments; however, because the difference in Kv1.5 protein
expression between conduit and resistance PASMCs measured
quantitatively was fairly small (1.5-fold greater in resistance than in
conduit PASMCs), it may not be very meaningful from a physiological perspective.
Expression of KV channel -subunits in pulmonary
arteries.
Adding to the growing list of possible KV
-subunit
functions is that of a cellular O2 sensor (12,
29). Interestingly, Kv
1.2 has been shown to confer
O2 sensitivity on Kv
4.2 in a heterologous expression
system (29). Additionally, when the conserved core of the
Kv
2.1 subunit was crystallized, bound NADPH was detected in its
crystal structure (12), further supporting a role for the
-subunit in cellular redox sensing. However, because KV
-subunits usually produce inactivation and the outward
K+ current from smooth muscle cells in pulmonary arteries
is only slowly inactivating or noninactivating, KV
-subunits have been largely overlooked in the search for the
molecular components of the PA O2-sensitive K+
current. Recently, however, it was shown that protein kinase A
phosphorylation removes
-induced inactivation (18).
Therefore, the presence of KV
-subunits in the pulmonary
arteries does not necessarily demand that the endogenous K+
channels show fast inactivation.
Study limitations. This study was limited by the efficiency of the available KV channel antibodies. Although the majority of these antibodies were useful in transfected cells and in rat or bovine brain, many of them were not useful against PASMC tissue. Additionally, antibodies that worked well for immunoblotting usually did not work well for immunohistochemistry and vice versa. For example, despite the report by Archer et al. (4) of Kv2.1 immunostaining in rat lung tissue sections, trustworthy Kv2.1 immunostaining could not be obtained in bovine conduit or resistance pulmonary artery, rat femoral artery, or rat lung with multiple Kv2.1 antibodies, although very strong immunoblot signals were generated for these tissues. It is likely that in the vasculature, the Kv2.1 antibody epitope is masked by either cytoskeleton components or unknown channel subunits. Kv9.3 has been hypothesized to be a component of the native resistance PASMC O2-sensitive K+ current (15, 28). Although antibodies against Kv9.3 were produced and characterized, clear consistent results regarding protein expression could not be obtained in tissue preparations. However, Kv9.3 mRNA was detected via RT-PCR and Northern blot analysis in both conduit and resistance vessels (data not shown). Although there was a two- to threefold increase in Kv9.3 transcription levels in resistance PASMCs relative to conduit PASMCs, generation of useful antibodies will be required to determine whether this difference translates into a difference in channel protein expression.
Heteromeric Kv2.1/Kv9.3 (15, 28) and Kv1.2/Kv15 (15) channels have been shown to be O2 sensitive in heterologous expression systems and thus are good candidates for the native resistance PASMC O2-sensitive K+ current. It is possible that although Kv2.1 and Kv1.2 expression levels do not change between conduit and resistance vessels, heteromeric assembly with Kv9.3 and Kv1.5, respectively, is greater in resistance PASMCs. Unfortunately, although multiple immunoprecipitation techniques and antibodies were tried, affinity purifications were not achieved. Therefore, this hypothesis remains untested. However, because Kv1.2 expression does not appear to change at all and Kv1.5 expression changes very little, it is likely that expression of Kv1.2/Kv1.5 heteromeric channels (if they are indeed expressed in the pulmonary arteries) changes very little throughout the PA circulation. On the other hand, increased mRNA expression of Kv9.3 in resistance PASMCs is suggestive of increased Kv2.1/Kv9.3 heteromeric channel formation in the O2-sensitive resistance vessels.Potential mechanisms for the differential response of conduit and
resistance pulmonary arteries to hypoxia.
Differential K+ channel expression is unlikely to account
for the differential hypoxic sensitivities of conduit and resistance pulmonary arteries (3) because O2-sensitive
subunits are expressed throughout the PA tree (1).
However, differential expression of an O2 sensor would
explain why small pulmonary arteries constrict in response to hypoxia
when small arteries in the systemic circulation dilate, although these
-subunits are expressed in both circulations.
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ACKNOWLEDGEMENTS |
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We thank Dr. Naoya Sakamoto for technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-49330 (to M. M. Tamkun); Animal Health and Disease Funds through the College of Veterinary Medicine and Biomedical Sciences at Colorado State University (Fort Collins, CO); and a grant-in-aid from the American Heart Association (to M. M. Tamkun).
Address for reprint requests and other correspondence: M. M. Tamkun, Dept. of Physiology, Colorado State Univ., Fort Collins, CO 80523 (E-mail: tamkunmm{at}lamar.colostate.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.
Received 29 May 2001; accepted in final form 26 July 2001.
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