Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5
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ABSTRACT |
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Molecular basis
of native voltage-dependent K+
(Kv) channels in smooth muscle cells (SMCs) from rat mesenteric
arteries was investigated. The whole cell patch-clamp study revealed
that a 4-aminopyridine-sensitive delayed rectifier
K+ current
(IK) was the
predominant K+ conductance in
these cells. A systematic screening of the expression of 18 Kv channel genes using RT-PCR technique showed that six IK-encoding genes
(Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv2.2, and Kv3.2) were expressed in
mesenteric artery. Although no transient outward Kv currents
(IA) were
recorded in the studied SMCs, transcripts of multiple
IA-encoding
genes, including Kv1.4, Kv3.3, Kv3.4, Kv4.1, Kv4.2, and Kv4.3 as well
as
IA-facilitating
Kv -subunits (Kv
1, Kv
2, and Kv
3), were detected in
mesenteric arteries. Western blot analysis demonstrated that four
IK-related Kv
channel proteins (Kv1.2, Kv1.3, Kv1.5, and Kv2.1) were detected in
mesenteric artery tissues. The presence of Kv1.2, Kv1.3, Kv1.5, and
Kv2.1 channel proteins in isolated SMCs was further confirmed by
immunocytochemistry study. Our results suggest that the native
IK in rat
mesenteric artery SMCs might be generated by heteromultimerization of
Kv genes.
Kv channels; peripheral artery; reverse transcription-polymerase chain reaction; Western blot; immunocytochemistry
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INTRODUCTION |
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THE RESTING MEMBRANE potential of many types of smooth muscle cells (SMCs) is regulated by voltage-dependent K+ (Kv) channels (1, 32). The activation of Kv channels increases K+ efflux, resulting in membrane hyperpolarization. This leads to the closing of voltage-dependent Ca2+ channels and a decreased Ca2+ influx, and vasodilation ensues. It is therefore well conceived that the normal expression and function of Kv channels are prerequisites for the maintenance of the physiological contractile state of vascular SMCs. By the same token, altered Kv channel expression and function have been linked to many pathophysiological vascular conditions. For example, hypoxia-induced pulmonary vasoconstriction was coupled to the altered function and expression of specific oxygen-sensitive Kv channels (1, 25). In addition, dysfunction of Kv channels has been described in pulmonary arterial SMCs from patients with primary pulmonary hypertension (29).
Two major types of Kv channel currents have been recorded in vascular SMCs, i.e., the delayed rectifier outward K+ current (IK) and the transient outward K+ current (IA). IK has a delayed activation and inactivates slowly and is present in almost all types of vascular SMCs. Within the physiological range of membrane potential and with normal intracellular Ca2+ concentration, IK represents the dominant repolarizing conductance, thus setting the resting membrane potential. IK is sensitive to tetraethylammonium (TEA) and/or 4-aminopyridine (4-AP), depending on the cell type studied. IA activates and inactivates very rapidly. This current has been undetectable in most vascular SMCs. However, IA does coexist with IK in a few types of vascular SMCs, including those from renal microvascular bed (6), rat pulmonary (31), and rabbit pulmonary arteries (20). IA can be reversibly blocked by 4-AP but is not sensitive to TEA.
As a representative of peripheral vasculatures, mesenteric artery has been extensively used in physiological and pharmacological studies on the regulation of peripheral vascular resistance. In contrast to pulmonary artery in which the expression of multiple Kv genes at either the mRNA or protein level was elucidated (1, 32), little is known about the molecular basis of the native Kv channels in mesenteric artery SMCs. This gap of knowledge also extends to the majority of peripheral vascular SMCs. Knowing the molecular basis of Kv channels in peripheral vascular SMCs would greatly improve our understanding of the structure-function relationship of Kv channels as well as their regulating mechanisms. Unfortunately, the molecular identity of Kv channels in pulmonary artery SMCs cannot be simply extrapolated to their counterparts in peripheral vascular SMCs because pulmonary circulation often responds to vasoactive substances or stimuli differently from peripheral circulation (21). For example, hypoxia was reported to reduce Kv currents in cultured rat pulmonary, but not in mesenteric, arterial myocytes (30).
The present study was carried out to systematically investigate the expression of all known Kv genes to provide a comprehensive framework of the molecular basis of the native Kv channels in peripheral vascular SMCs. After identification of the predominant IK in rat mesenteric artery SMCs using the whole-cell configuration of the patch-clamp technique, multiple cellular and molecular biology techniques were employed, including RT-PCR, Western blot, and immunocytochemistry, to screen the expression of various IK-encoding Kv genes in these cells at the mRNA and protein levels.
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MATERIALS AND METHODS |
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Isolation of rat mesenteric artery and preparation of single SMCs.
Male Sprague-Dawley rats (120-150 g) were anesthetized by
intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt).
Small mesenteric arteries below the second branch off the main
mesenteric artery were dissected out. Only small arteries and
arterioles were kept in the ice-cold physiological salt solution (PSS),
which contained (in mM) 137 NaCl, 5.6 KCl, 0.44 NaH2PO4, 0.42 Na2HPO4,
4.17 NaHCO3, 1 MgCl2, 2.6 CaCl2, 10 HEPES, and 5 glucose,
with pH adjusted to 7.4 with NaOH. Connective tissues were gently
removed under a dissecting microscope with surgical tweezers. For total
RNA and protein isolation, the isolated small mesenteric arteries were
immediately placed in liquid nitrogen and stored at 80°C.
Electrophysiological recording.
The whole cell Kv currents were recorded as described previously (22).
Briefly, two to three drops of the cell suspension were added to the
perfusion chamber inside a petri dish that was mounted on the stage of
an inverted phase-contrast microscope (Olympus IX70). Cells were left
to stick to the glass coverslip in the experimental chamber for
15-20 min before an experiment was started. Pipettes were pulled
from soft microhematocrit capillary tubes (Fisher, Nepean, ON) with tip
resistances of 2-4 M when filled with the pipette solution.
Currents were recorded with an Axopatch 200-B amplifier (Axon
Instruments), controlled by a Digidata 1200 interface and pCLAMP
software (version 6.02, Axon Instruments). Membrane currents were
filtered at 1 kHz with a four-pole Bessel filter, digitized, and
stored. At the beginning of each experiment, junction potential between
pipette solution and bath solution was electronically adjusted to zero
(26, 27, 28). Test pulses were made with a 10-mV increment from
50 to +50 mV. The holding potential was set at
80 mV at
which Kv channels are not inactivated. Current-voltage curves were
constructed using the sustained current amplitude at the end of 600-ms
test pulses. The bath solution contained (in mM) 140 NaCl, 5.4 KCl, 1.2 MgCl2, 10 HEPES, 1 EGTA, and 10 glucose (pH adjusted to 7.3 with NaCl). The pipette solution was
composed of (in mM) 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, 5 glucose, and 2 Na2ATP (pH adjusted
to 7.3 with KOH). Unless otherwise indicated, cells were continuously superfused with the bath solution containing tested chemicals at
desired final concentrations. The data were collected and processed using pCLAMP software (version 6.01) from Axon Instruments. All experiments were conducted at room temperature (20-22°C).
Isolation of total RNA. Total RNA was isolated using TRIREAGENT (Molecular Research Center) containing guanidine thiocyanate and phenol. Briefly, 0.4 g of mesenteric arteries or SMC pellet from six mesenteric arteries was homogenized in 2 ml of TRIREAGENT using a Polytron homogenizer, and the lysates were centrifuged at 12,000 g for 5 min to remove tissue debris. After addition of 0.4 ml of chloroform, tubes were centrifuged at 12,000 g for 15 min at 4°C. RNA in the supernatant was precipitated by adding equal volume of ice-cold isopropanol and pelleted by centrifugation at 12,000 g for 8 min at 4°C and washed by 70% ethanol. To eliminate residual contaminating genomic DNA, the RNA preparation was further treated with RQ1 RNase-free DNase (1 U/5 µg RNA; Promega) for 45 min at 37°C and then repurified by phenol-chloroform extraction and ethanol precipitation.
RT-PCR analysis.
Amplification of first-strand cDNA was performed in a 20-µl reaction
mixture containing 1 µg of total RNA, 1 µl of 50 µM random hexamers, 1 mM dNTP, 50 units of murine leukemia virus reverse transcriptase, and 1× reaction buffer
(Perkin-Elmer). The reaction was carried out at room temperature for 15 min and at 42°C for 1 h. Five microliters of the first-strand cDNA
reaction mixture were used in a 50-µl PCR reaction mixture consisting
of 0.4 µM each 5' and 3' primers, 10 mM
Tris · HCl (pH 8.3), 50 mM KCl, 2 mM
MgCl2, 0.2 mM each dNTP and 2 units of Vent DNA polymerase (New England Biolabs). After
PCR, amplified products in a 10-µl aliquot were subjected to
electrophoresis in a 6% acrylamide gel in Tris-borate-EDTA buffer and
visualized with ethidium bromide. All primers were designed based on
the common region for different splicing isoforms of the selected Kv
genes (Table 1). Primers to amplify a
626-bp fragment of rat -actin were prepared based on a previous
report (14).
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Membrane protein preparation and Western blot. Membrane proteins were prepared as previously described with modifications (32). Briefly, mesenteric artery was homogenized with a Polytron homogenizer in 3 ml of Tris-buffered saline [20 mM Tris · HCl (pH 7.4), 0.25 M sucrose, and 1 mM EDTA] containing protease inhibitor mixture (2 µl of 1 M phenanthroline, 300 mM iodoacetamide, 10 mM phenylmethlsulfonyl fluoride, 1 mg/ml antipain, 1 mg/ml leupeptin, 1 mM pepstatin A, and 1 M benzamidine). The homogenate was centrifuged at 6,000 g for 15 min at 4°C to remove nuclei and undisrupted cells. The supernatant was further centrifuged at 40,000 g for 1 h at 4°C. The resulting pellets were then washed and resuspended with the same Tris-buffered saline without sucrose. Protein concentration was determined using Bio-Rad protein assay solution with BSA as standard. For Western blot, membrane proteins were loaded and run on standard 7.5% SDS-polyacrylamide gel in Tris-glycine electrophoresis buffer [25 mM Tris, 200 mM glycine (pH 8.3), and 0.1% SDS]. Proteins were transferred onto nitrocellulose membrane in 192 mM glycine, 25 mM Tris (pH 8.3), and 20% methanol at 100 V for 1.5 h in a water-cooled transfer apparatus. The membrane was blocked in a blocking buffer, PBS containing 3% nonfat milk, at room temperature for 1 h. The membrane was then probed overnight at 4°C with either monoclonal antibodies against Kv1.2 and Kv2.1 (Upstate Biotechnology) or the affinity-purified polyclonal antibody against Kv1.5 (Upstate Biotechnology) or Kv1.3 and Kv3.2 (Alomone Labs) in the blocking buffer. After the membrane was washed five times in PBS, the membrane was subsequently incubated with either goat anti-mouse IgG for Kv1.2 and Kv2.1 or goat anti-rabbit IgG for Kv1.3, Kv1.5, or Kv3.2 conjugated with horseradish peroxidase diluted to 1:4,000 in the blocking buffer for 2 h at room temperature. Bound antibodies were detected using chemiluminescent substrate kit (NEN Life Sciences).
Immunocytochemistry.
SMCs isolated from mesenteric arteries were cultured in 48-well plates
with DMEM containing 10% fetal bovine serum at 37°C in a
humidified atmosphere of 5% CO2.
After incubation for 72 h, cells were fixed and permeabilized for 30 min at room temperature with 3% paraformaldehyde-0.1% Triton X-100.
Nonspecific staining was blocked for 1 h at room temperature with PBS
containing 4% normal horse or monkey serum in which secondary antibody
against mouse IgG or rabbit IgG was made, respectively. After they were washed five times with PBS, cells were incubated overnight at 4°C
with the monoclonal primary antibody against Kv1.2 or Kv2.1 or
-smooth muscle actin (Sigma) or polyclonal antibody against Kv1.3 or
Kv1.5 in PBS containing 1% BSA and 0.03% Triton X-100. After they
were washed five times in PBS, cells were incubated for 30 min with
biotinylated horse anti-mouse IgG antibody or biotinylated monkey
anti-rabbit IgG antibody (1:200; Vector Laboratories). After they were
washed five times in PBS, cells were exposed to Vector ABC reagent
(1:100; avidin coupled to biotinylated horseradish peroxidase) for 30 min. Cells were washed again in PBS and visualized by incubating with
horseradish peroxidase substrate containing 0.02% diaminobenzidine,
0.3% nickel ammonium sulfate, and 0.002% hydrogen peroxide (Vector
Laboratories). The appearance of reaction product was monitored and
photographed under bright-field illumination.
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RESULTS |
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Voltage-dependent
K+ channel
currents in rat mesenteric artery SMCs.
A delayed rectifier
IK was found to
be the predominant Kv current in freshly isolated rat mesenteric artery
SMCs (n = 46). IK was activated
at membrane potential positive to 20 mV with a slow onset and
noninactivating kinetics over the stimulation period (600 ms). The
transient and fast-inactivating
IA was not detectable in these cells.
IK was inhibited
by 4-AP. As shown in Fig. 1, 4-AP at 5 mM
significantly inhibited the amplitude of
IK over the
entire voltage range. The concentration dependence of the 4-AP-induced
inhibition of IK
was demonstrated with an IC50 of
5.06 ± 0.88 mM (n = 6).
IK in rat
mesenteric artery SMCs was also inhibited by TEA (Fig.
2). However, the sensitivity of IK in these cells
to TEA was very low, with an IC50
of 9.9 ± 1.2 mM (n = 7). The
inhibitory effects of both 4-AP and TEA were fully reversible (not
shown).
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mRNA detection of Kv - and
-subunits
from rat mesenteric artery.
RT-PCR was performed to determine the expression of different members
of Kv families (Kv1-Kv4) and regulatory
-subunits utilizing the
primer pairs specifically designed based on the known cDNA sequences
(Table 1). The result in Fig. 3 shows that
Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv2.2, Kv3.2, Kv3.3, Kv3.4, Kv4.1,
Kv4.2, Kv4.3, and three Kv
-subunits were reproducibly amplified
from mesenteric artery RNA with 30 PCR cycles. However, transcripts of
Kv1.1, Kv1.6, and Kv3.1 were not detected, even though they were
detected in brain tissue (Fig. 3B).
The successful elimination of genomic DNA by the DNase treatment of
mesenteric artery RNA was exemplified by the RT-PCR analysis of the
transcriptional expression of
-actin. The amplimers for
-actin
were purposely selected on the basis of reports regarding their ability
to specifically amplify rat
-actin reverse-transcribed mRNA but not
pseudogene genomic DNA sequence (14). As shown in Fig.
3C, a single 626-bp product was
amplified from reverse-transcribed mesenteric artery RNA only in the
presence of RT, indicating that mesenteric artery RNA was free of
genomic DNA.
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Expression of neuronal specific RNA
erg3.
Sympathetic neurons are embedded in peripheral vasculature. To exclude
the neuronal contamination of our results obtained from isolated
mesenteric artery tissues, the expression of
erg3 gene was tested by RT-PCR. As a
reliable neuron-specific marker, erg3
gene is exclusively expressed in brain and many peripheral neurons such
as superior cervical ganglia, celiac ganglia, and superior mesenteric
ganglia (18). Figure 4 shows that
transcripts of erg3 were readily
detected in brain tissue with 25 PCR cycles but was not detected in rat
mesenteric artery tissues with 35 PCR cycles in RT-PCR analysis. As a
positive control, transcripts of -actin were expressed in both brain
and mesenteric artery tissues. These results suggest that our detected
Kv transcripts from mesenteric artery tissue were virtually free of
contamination from sympathetic neurons.
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Western blot analysis of delayed rectifier
IK channel proteins.
To examine whether the transcriptional expression of Kv channels was
reconciled with the corresponding protein expression, Western blot
studies were carried out on all
IK-encoding genes detected at the mRNA level by RT-PCR, except on Kv2.2 because of the
lack of supply of anti-Kv2.2 antibody. The results in Fig. 5A show
that anti-Kv1.2 antibody recognized a wide band in brain membrane
fraction, extending over the molecular mass range of 75-105 kDa.
This result is similar to the report that the size of Kv1.2 in brain
may vary from 75 to 85 kDa (2). Two faint bands at 75 and 90 kDa were
detected in mesenteric artery. The size of Kv1.2 is similar to that
from heart and pulmonary artery (2, 32). The anti-Kv1.3 antibody
recognized a single band at 88 kDa from brain and two bands at 88 and
70 kDa from mesenteric artery (Fig.
5B). The size of these two bands is
consistent with previous results (23, 32) and those provided by
researchers at Alomone Labs. The 70-kDa band may be due to the protein
degradation or the presence of a new isoform. The anti-Kv1.5 antibody
recognized a single band at 75 kDa in both brain and mesenteric artery
(Fig. 5C). The anti-Kv2.1 antibody
detected a broad band ranging from 90 to 110 kDa in brain (Fig.
5D). The size of brain Kv2.1 was similar to the size described previously (12). However, a band at 95 kDa was detected in mesenteric artery. A previous study on Kv2.1
channels in neurons from rat brain has indicated that a different
status of phosphorylation was the main mechanism for the dramatic
difference in molecular mass of Kv2.1 (12). Correspondingly, the
difference in the sizes of Kv2.1 between mesenteric artery and brain
suggests that a dephosphorylated Kv2.1 channel might be the major form
in the mesenteric artery. The anti-Kv3.2 antibody recognized a single
band at 116 kDa in brain but not in mesenteric artery (Fig.
5E). The size of this band is in
good agreement with the results from Alomone Labs but is larger than
the size of Kv3.2 channels identified by Moreno et al. (11). This
discrepancy in the size of Kv3.2 may be due to different experimental
procedures in the extraction of membrane protein or in the protocols of
Western blot.
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Immunocytochemical identification of Kv channel proteins.
To determine if Kv subunits detected by Western blot in mesenteric
artery tissues were actually present in purified SMCs, cultured rat
mesenteric artery SMCs were fixed and probed with the selected
antibodies. Because Kv 3.2 protein was not detected in the intact
mesenteric artery tissue by Western blot (Fig.
5E), examination of the
corresponding proteins on SMCs by immunocytochemistry was not performed
in this study. The results of a typical immunocytochemical experiment
on cultured SMCs are shown in Fig. 6.
Specific antibodies against Kv1.2, Kv1.3, Kv1.5, and Kv2.1 were found
to bind to cultured rat mesenteric artery SMCs to different extents. To
confirm that the stained cells were indeed SMCs, cultured cells were
also exposed to the antibody against smooth muscle-specific -actin
(19). Figure 6E shows that all cells
were stained with anti-
-actin antibody. In contrast, the cells that
were only exposed to the secondary antibodies show no staining.
Together, the localization of these specific Kv channel proteins in
mesenteric artery SMCs was demonstrated.
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DISCUSSION |
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Vascular tone of peripheral arteries is an important determinant of
circulation resistance and blood pressure. By setting up the resting
membrane potentials of vascular SMCs, Kv channels participate in the
regulation of vascular tone. Now that this pivotal role of Kv channels
has been recognized, the molecular nature of the native Kv channels in
peripheral vascular SMCs becomes the focus of investigation. This
knowledge is essential for the understanding of the structure-function
relationship of Kv channels and will guide efforts to manipulate the
native Kv channels under different situations. Unfortunately, the
molecular characterization of the native Kv channels in peripheral
vascular SMCs remains largely unknown except for a few scattered
reports (15). Our study represents the first endeavor for screening the
expression of various Kv genes in peripheral artery SMCs. The major
discoveries of this study are the following.
1) Among cloned Kv genes that encode
the delayed rectifier
IK, mRNAs of
Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv2.2, and Kv3.2, but not those of Kv1.1,
Kv1.6, and Kv3.1, were detected in rat mesenteric artery tissues.
2) Channel proteins for Kv1.2,
Kv1.3, Kv1.5, and Kv2.1, but not Kv3.2, were identified in mesenteric
artery tissue and in purified single mesenteric artery SMCs.
3) Three Kv -subunits were
expressed at the mRNA level in rat mesenteric arteries.
4) Although an
IA was not
readily detectable in rat mesenteric artery SMCs, multiple
IA-encoding genes, Kv1.4, Kv3.3, Kv3.4, Kv4.1, Kv4.2, and Kv4.3, were expressed at
the mRNA level.
Our results clearly strengthened the difficulty to simply extrapolate
the molecular traits of Kv channels in neurons or other types of
vascular SMCs, such as those from pulmonary artery or other conduit
systemic arteries, to Kv channels in peripheral artery SMCs. For
example, transcripts of Kv1.1 and Kv1.6 were detected in aorta (15) and
pulmonary artery SMCs (1, 29) but not in mesenteric artery SMCs as
shown in our present study. The recognized diversity of SMCs in
different tissues, such as in cell phenotype and expression of
cytoskeletal proteins (10), thus should logically be extended to the
molecular basis of Kv channels. This realization should greatly improve
our understanding of the cell type-specific properties of Kv channels
in peripheral vascular SMCs. Equally important is that our results
present an expression pattern of Kv genes in SMCs from a peripheral
vasculature. Against this expression pattern, many variations of Kv
subunit expression with physiological stimuli or under
pathophysiological conditions can be compared, identified, and
selectively manipulated. In this context, the absence of
IA but the
expression of
IA-encoding Kv
-subunits as well as
IA-facilitating
Kv
-subunits in rat mesenteric artery SMCs may be revealing. In many
types of SMCs including mesenteric artery SMCs,
IA cannot be
detected. Although detected in vitro in some SMCs,
IA presents a
relatively smaller fraction of total outward Kv currents and is largely
inactivated at physiological membrane potentials. However, the detected
IA-encoding Kv
subunits may serve as a regulatory mechanism. When subjected to
different environmental changes, the peripheral vascular SMCs may
upregulate these
IA-encoding Kv
subunits at mRNA or protein levels to such an extent that a functional
IA may emerge.
Subsequently, the cellular excitability would be altered. Because of
the limited availability of the specific antibodies against these
IA-encoding Kv
subunits, we were not able to test the expression of these Kv subunits
in mesenteric artery SMCs at the protein level. In any case, this
hypothesis merits further exploration.
The observed expression of Kv genes at mRNA and protein levels from mesenteric artery in our study may be questioned for contamination from non-SMC cell types. Several precautions have been taken in dealing with this concern. First, the RNA contamination from peripheral neurons in our RT-PCR studies was addressed by the examination of a neuron-specific marker, erg3, in mesenteric artery tissues. The absence of erg3 transcripts in mesenteric artery tissues (Fig. 4) suggested that RNA isolated from mesenteric artery is free of neuronal RNA. Second, the endothelial contamination in our RT-PCR analysis of Kv expression needs to be carefully evaluated. Although no study has detected the cloned Kv genes in endothelial cells from peripheral vascular tissues, the expression of Kv genes has been reported in pulmonary artery endothelial layer (1) and in cardiac microvascular endothelial cells (4). However, the endothelial contamination of our RT-PCR analysis of the expression of Kv transcripts is remote. When the freshly isolated pure SMCs were used in our study as the source for RNA in RT-PCR analysis, the expression pattern of Kv transcripts was identical to that of the intact vascular tissues (Fig. 3D). Finally, to ensure that the products of Kv genes detected in mesenteric artery tissues were localized in single SMCs, immunocytochemistry study was carried out on isolated rat mesenteric artery SMCs. This study confirmed that Kv1.2, Kv1.3, Kv1.5, and Kv2.1 proteins were present in rat mesenteric artery SMCs (Fig. 6). Because the same Kv genes that were expressed at the protein level in cultured SMCs were expressed at the mRNA level in freshly isolated SMCs, the alteration in Kv gene expression, at least qualitatively, during a short-term cell culture does not appear significant. Nevertheless, the quantitative change in the expression levels of Kv genes in vascular SMCs during culture should not be excluded yet.
The pharmacological properties of the native Kv currents (IK) in rat mesenteric artery SMCs are in line with IK in other types of vascular SMCs with respect to their relative high sensitivity to 4-AP (8) and low sensitivity to TEA (24). On the other hand, the pharmacological properties of IK in rat mesenteric artery SMCs were somehow different from those of Kv currents expressed by different IK-encoding genes. The Kv1.5 channel, which contributes to most of the delayed rectifier IK in human cardiomyocytes or in rat pulmonary artery SMCs (1, 4), has an IC50 of TEA of 330 mM (7). This value is ~30-fold greater than the IC50 of TEA (9.9 mM) in rat mesenteric artery SMCs. In addition, the Kv1.5 channel was very sensitive to 4-AP (IC50 of 200 µM) (4), whereas the native IK in rat mesenteric artery SMCs was moderately sensitive to 4-AP (IC50 of 5.06 mM). The Kv1.2 channel, on the other hand, was reported to be extremely sensitive to TEA (IC50 of 560 nM) and to 4-AP (IC50 of 590 µM) (7). Therefore, it also does not fit in the pharmacological profile of the native IK in rat mesenteric artery SMCs. Kv1.3 and Kv2.1 have IC50 of TEA values (5-10 mM) that are the closest to the native IK in rat mesenteric artery SMCs, even though their IC50 of 4-AP values (195-500 µM) do not match perfectly with those of the native IK (7). In a recent study, Kv2.2 mRNA was identified in SMCs found in all regions of the canine gastrointestinal tract and in several vascular tissues (16). The functional expression of this Kv2.2 channel in Xenopus laevis oocytes resulted in a slowly activating IK. This current was inhibited by TEA (IC50 of 2.6 mM), 4-AP (IC50 of 1.5 mM at +20 mV), and quinine (IC50 of 14 µM) and was insensitive to charybdotoxin. It appears that Kv2.2 may contribute to the native Kv current in gastrointestinal and some vascular SMCs. In contrast to the above study (16), the cloned Kv2.2 was found to have a low sensitivity to TEA (IC50 of 7.9 mM) (9), which was similar to the native IK in rat mesenteric artery SMCs in our study.
Pharmacological tools and biophysical traits may help but are not
sufficient to determine the molecular components of native IK. Several
factors need to be considered when comparing the pharmacological characteristics of the expressed Kv channels from different expression systems. 1) Kv current may exhibit
distinct pharmacological and electrophysiological properties, depending
on whether its encoding Kv genes are expressed in
Xenopus oocytes or mammalian cell
lines. This is because the modulation (phosphorylation and
glycosylation) of a Kv channel in the expression systems is often
different from that in native cellular milieu (12, 17).
2) The native
IK may result
from the heteromultimerization of different Kv channel -subunits
associated with or without cytoplasmic
-subunits. The heteromeric
expressed Kv channels often possess pharmacological sensitivities
different from those of homomeric expressed Kv channels (3, 13). In rat
mesenteric artery SMCs, several members of the Kv1 family and three
-subunits were detected. This may provide the molecular basis for
the heteromultimerization of the native IK channels.
Therefore, our results suggest that the native
IK channels in
rat mesenteric artery SMCs may be the tetramers of multiple
IK-encoding Kv
genes. Kv1.2, Kv1.3, Kv1.5, Kv2.2, and Kv2.1 may all participate in the
heteromultimerization, with Kv2.1, Kv2.2, and Kv1.3 having potentially
greater contributions.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. Wang, Dept. of Physiology, College of Medicine, Univ. of Saskatchewan, Saskatoon, SK, Canada S7N 5E5 (E-mail: wangrui{at}duke.usask.ca).
Received 8 March 1999; accepted in final form 2 August 1999.
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