Expression of voltage-dependent K+ channel genes in mesenteric artery smooth muscle cells

Chuanli Xu, Yanjie Lu, Guanghua Tang, and Rui Wang

Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -subunits (Kvbeta 1, Kvbeta 2, and Kvbeta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

To obtain single SMCs for the patch-clamp study, immunocytochemistry study, and RNA isolation, the freshly isolated tissues were cut into 5-mm-long pieces (26). The tissues were then incubated at 37°C in low-Ca2+ PSS (0.1 mM CaCl2) containing 1 mg/ml albumin, 0.5 mg/ml papain, and 1.0 mg/ml dithioerythritol for 30 min and, consecutively, for another 20 min in the nominally Ca2+-free PSS in which Ca2+ was omitted and 1 mg/ml albumin, 0.8 mg/ml collagenase, and 0.8 mg/ml hyaluronidase were added. Single cells were released by gentle triturating through a Pasteur pipette and exhibited long and spindlelike shape under a microscope. For patch-clamp study, cells were stored in the nominally Ca2+-free PSS at 4°C and used within the same day of isolation. For isolation of total RNA, dispersed SMCs were filtered through a fine mesh (pore size of <200 µm) to remove undigested tissue and then centrifuged at 2,000 g for 5 min. The resultant cell pellet was placed in liquid nitrogen and stored at -80°C.

Animal experimental protocols were approved by the Committee on Animal Care and Supply of the University of Saskatchewan.

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 MOmega 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 beta -actin were prepared based on a previous report (14).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Oligonucleotide sequence of primers used for RT-PCR

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 alpha -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Inhibition of the delayed rectifier outward K+ current (IK) in rat mesenteric artery smooth muscle cells (SMCs) by 4-aminopyridine (4-AP). A: representative records of IK in the absence and presence of 4-AP (5 mM). B: current-voltage (I-V) relationships of IK in the absence and presence of 4-AP.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibition of IK in rat mesenteric artery SMCs by tetraethylammonium (TEA). A: representative records of IK in the absence and presence of TEA (10 mM). B: I-V relationships of IK in the absence and presence of TEA.

mRNA detection of Kv alpha - and beta -subunits from rat mesenteric artery. RT-PCR was performed to determine the expression of different members of Kv families (Kv1-Kv4) and regulatory beta -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 beta -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 beta -actin. The amplimers for beta -actin were purposely selected on the basis of reports regarding their ability to specifically amplify rat beta -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.


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 3.   A: RT-PCR amplification of the selected Kv alpha - and beta -subunit mRNA isolated from rat mesenteric artery. Amplified products were displayed in acrylamide gel stained with ethidium bromide. All amplified Kv alpha - and beta -subunits are listed with fragment sizes in parentheses: Kv1.2 (673 bp), Kv1.3 (335 bp), Kv1.4 (271 bp), Kv1.5 (340 bp), Kv2.1 (616 bp), Kv2.2 (398 bp), Kv3.2 (320 bp), Kv3.3 (202 bp), Kv3.4 (202 bp), Kv4.1 (467 bp), Kv4.2 (522 bp), Kv4.3 (270 bp), Kvbeta 1 (150 bp), Kvbeta 2 (141 bp), and Kvbeta 3 (178 bp). No transcripts of Kv1.1, Kv1.6, or Kv3.1 were detected. M, molecular-weight marker (in bp). Data represent 1 of 3 independent experiments. B: RT-PCR amplification of Kv1.1, Kv1.6, and Kv3.1 with RNA isolated from rat brain. C: RT-PCR amplification of transcripts of beta -actin with RNA from mesenteric artery in the absence (-) or presence (+) of murine leukemia virus reverse transcriptase. D: RT-PCR amplification of Kv transcripts from freshly isolated SMC of rat mesenteric arteries. Sizes of amplified fragments of the selected Kv mRNA (Kv1.2, Kv1.3. Kv1.4, Kv1.5, Kv2.1, Kv2.2, Kv3.2, Kv3.3, Kv3.4, Kv4.1, Kv4.2, Kv4.3, Kvbeta 1, Kvbeta 2, and Kvbeta 3) are identical to those depicted in A.

To confirm that the detected Kv transcripts were derived from SMCs rather than from contaminating endothelial cells, RT-PCR analysis of Kv expression was also carried out using RNA prepared from freshly enzymatically isolated SMCs. These cells were stored in the nominally Ca2+-free PSS at 4°C and used within 1 h of isolation. The identity of SMCs was confirmed by their elongated morphology and their intact contractility to the mechanical stimulation or norepinephrine, as described previously (26) (not shown). As shown in Fig. 3D, all the Kv transcripts detected in intact mesenteric artery tissues were also present in the purified single SMCs.

Because the RT-PCR used in this study was not quantitative, these results did not provide information regarding the quantity of the particular Kv channel mRNA.

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 beta -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.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Detection of erg3 mRNA in rat brain and mesenteric artery. Examination of erg3 expression was carried out by RT-PCR analysis using RNA isolated from brain and mesenteric artery with 25 and 35 PCR cycles, respectively. Amplified products for erg3 (294 bp) and beta -actin (626 bp) were fractionated in acrylamide gel. M, molecular-weight marker (in bp).

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.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5.   Western blotting analysis of the expression of Kv1.2 (A), Kv1.3 (B), Kv1.5 (C), Kv2.1 (D), and Kv3.2 (E) channel proteins in rat brain and mesenteric artery. Immunoblots of rat brain and mesenteric membrane proteins (7.5 µg/lane) were incubated with affinity-purified antibodies against Kv1.2, Kv1.3, Kv1.5, Kv2.1, and Kv3.2. Protein markers are shown on left (in kDa). Data shown are representative of 3 independent experiments.

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 alpha -actin (19). Figure 6E shows that all cells were stained with anti-alpha -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.


View larger version (104K):
[in this window]
[in a new window]
 
Fig. 6.   Immunocytochemistry study on cultured rat mesenteric artery SMCs. Single SMCs were exposed to anti-Kv1.2 (A), anti-Kv1.3 (B), anti-Kv2.1 (C), anti-Kv1.5 (D), and anti-actin (E) as described in MATERIALS AND METHODS. Cells in G were treated identically to those in A, C, and E, and cells in F were treated identically to those in B and D, except that the primary antibodies were omitted during experimental procedures. Data represent 1 of 3 independent experiments. Scale bar = 50 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 alpha -subunits as well as IA-facilitating Kv beta -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 alpha -subunits associated with or without cytoplasmic beta -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 beta -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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Archer, S. L., E. Souil, A. T. Dinh-Xuan, B. Schremmer, J. C. Mercier, A. El Yaagoubi, L. Nguyen-Huu, H. L. Reeve, and V. Hampl. Molecular identification of the role of voltage-gated K+ channels, Kv1.5 and Kv2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes. J. Clin. Invest. 101: 2319-2330, 1998[Abstract/Free Full Text].

2.   Barry, D. M., J. S. Trimmer, J. P. Merlie, and J. M. Nerbonne. Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels? Circ. Res. 77: 361-369, 1995[Abstract/Free Full Text].

3.   Christie, M. J., R. A. North, P. B. Osborne, J. Douglass, and J. P. Adelman. Heteropolymeric potassium channels expressed in Xenopus oocytes from cloned subunits. Neuron 4: 405-411, 1990[Medline].

4.   Fan, J., and K. B. Walsh. Mechanical stimulation regulates voltage-gated potassium currents in cardiac microvascular endothelial cells. Circ. Res. 84: 451-457, 1999[Abstract/Free Full Text].

5.   Feng, J., B. Wible, G. R. Li, Z. Wang, and S. Nattel. Antisense oligodeoxynucleotides directed against Kv1.5 mRNA specifically inhibit ultrarapid delayed rectifier K+ current in cultured adult human atrial myocytes. Circ. Res. 80: 572-579, 1997[Abstract/Free Full Text].

6.   Gordienko, D. V., C. Clausen, and M. S. Goligorsky. Ionic currents and endothelin signaling in smooth muscle cells from rat renal resistance arteries. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F325-F341, 1994[Abstract/Free Full Text].

7.   Grissmer, S., A. N. Nguyen, J. Aiyar, D. C. Hanson, R. J. Mather, G. A. Gutman, M. J. Karmilowicz, D. D. Auperin, and K. G. Chandy. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol. Pharmacol. 45: 1227-1234, 1994[Abstract].

8.   Halliday, F. C., P. I. Aaronson, A. M Evans, and A. M. Gurney. The pharmacological properties of K+ currents from rabbit isolated aortic smooth muscle cells. Br. J. Pharmacol. 116: 3139-3148, 1995[Abstract].

9.   Hwang, P. M., C. E. Glatt, D. S. Bredt, G. Yellen, and S. H. Snyder. A novel K+ channel with unique localizations in mammalian brain: molecular cloning and characterization. Neuron 8: 473-481, 1992[Medline].

10.   Michelakis, E. D., H. L. Reeve, J. M. Huang, S. Tolarova, D. P. Nelson, E. K. Weir, and S. L. Archer. Potassium channel diversity in vascular smooth muscle cells. Can. J. Physiol. Pharmacol. 75: 889-897, 1997[Medline].

11.   Moreno, H., C. Kentros, E. Bueno, M. Weiser, A. Hernandez, D. M. Vega-Saenz, A. Ponce, W. Thornhill, and B. Rudy. Thalamocortical projections have a K+ channel that is phosphorylated and modulated by cAMP-dependent protein kinase. J. Neurosci. 15: 5486-5501, 1995[Abstract].

12.   Murakoshi, H., G. Shi, R. H. Scannevin, and J. S. Trimmer. Phosphorylation of the Kv2.1 K+ channel alters voltage-dependent activation. Mol. Pharmacol. 52: 821-828, 1997[Abstract/Free Full Text].

13.   Post, M. A., G. E. Kirsch, and A. M. Brown. Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current. FEBS Lett. 399: 177-182, 1996[Medline].

14.   Raff, T., V. D. Giet, D. Endemann, T. Wiederholt, and M. Paul. Design and testing of beta -actin primers for RT-PCR that do not co-amplify processed pseudogenes. Biotechniques 23: 456-460, 1997[Medline].

15.   Roberds, S. L., and M. M. Tamkun. Cloning and tissue-specific expression of five voltage-gated potassium channel cDNAs expressed in rat heart. Proc. Natl. Acad. Sci. USA 88: 1798-1802, 1991[Abstract].

16.   Schmalz, F., J. Kinsella, S. D. Koh, F. Vogalis, A. Schneider, E. R. Flynn, J. L. Kenyon, and B. Horowitz. Molecular identification of a component of delayed rectifier current in gastrointestinal smooth muscles. Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G901-G911, 1998[Abstract/Free Full Text].

17.   Shi, G., A. K. Kleinklaus, N. V. Marrion, and J. S. Trimmer. Properties of Kv2.1 K+ channels expressed in transfected mammalian cells. J. Biol. Chem. 269: 23204-23211, 1994[Abstract/Free Full Text].

18.   Shi, W., R. S. Wymore, H. S. Wang, Z. Pan, I. S. Cohen, D. McKinnon, and J. E. Dixon. Identification of two nervous system-specific members of the erg potassium channel gene family. J. Neurosci. 17: 9423-9432, 1997[Abstract/Free Full Text].

19.   Skalli, O., P. Ropraz, A. Trzeciak, G. Benzonana, D. Gillessen, and G. Gabbiani. A monoclonal antibody against alpha -smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103: 2787-2796, 1986[Abstract].

20.   Smirnov, S. V., and P. I. Aaronson. Inhibition of vascular smooth muscle cell K+ currents by tyrosine kinase inhibitors genistein and ST 638. Circ. Res. 76: 310-316, 1995[Abstract/Free Full Text].

21.   Suba, E. A., T. M. McKenna, and T. J. Williams. Differential contractile responses of mesenteric and pulmonary artery segments to norepinephrine and phorbol ester in the septic pig. Circ. Shock 37: 164-168, 1992[Medline].

22.   Tang, G., S. T. Hanna, and R. Wang. Effects of nicotine on K+ channel currents in vascular smooth muscle cells from rat tail arteries. Eur. J. Pharmacol. 364: 247-254, 1999[Medline].

23.   Veh, R. W., R. Lichtinghagen, S. Sewing, F. Wunder, I. M. Grumbach, and O. Pongs. Immunohistochemical localization of five members of the Kv1 channel subunits: contrasting subcellular locations and neuron-specific co-localizations in rat brain. Eur. J. Neurosci. 7: 2189-2205, 1995[Medline].

24.   Volk, K. A., J. J. Matsuda, and E. F. Shibata. A voltage-dependent potassium current in rabbit coronary artery smooth muscle cells. J. Physiol. (Lond.) 439: 751-768, 1991[Abstract].

25.   Wang, J., M. Juhaszova, L. J. Rubin, and X. J. Yuan. Hypoxia inhibits gene expression of voltage-gated K+ channel alpha subunits in pulmonary artery smooth muscle cells. J. Clin. Invest. 100: 2347-2353, 1997[Abstract/Free Full Text].

26.   Wang, R., E. Karpinski, and P. K. T. Pang. Two types of calcium channels in isolated smooth muscle cells from rat tail artery. Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H1361-H1368, 1989[Abstract/Free Full Text].

27.   Wu, L., M. A. Mateescu, X. T. Wang, B. Mondovi, and R. Wang. Modulation of K+ channel currents by serum amineoxidase in neurons. Biochem. Biophys. Res. Commun. 220: 47-52, 1996[Medline].

28.   Wu, L., M. A. Mateescu, X. T. Wang, B. Mondovi, and R. Wang. Serum amineoxidase modifies the effect of ceruloplasmin on neuronal K+ channel currents. Ital. J. Biochem. 6, Suppl. 1: 52-56, 1997.

29.   Yuan, J. X., A. M. Aldinger, M. Juhaszova, J. Wang, J. V. J. Conte, S. P. Gaine, J. B. Orens, and L. J. Rubin. Dysfunctional voltage-gated K+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation 98: 1400-1406, 1998[Abstract/Free Full Text].

30.   Yuan, X. J., W. F. Goldman, M. L. Tod, L. J. Rubin, and M. P. Blaustein. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am. J. Physiol. 264 (Lung Cell. Mol. Physiol. 8): L116-L123, 1993[Abstract/Free Full Text].

31.   Yuan, X. J., M. L. Tod, L. J. Rubin, and M. P. Blaustein. Inhibition of cytochrome P-450 reduces voltage-gated K+ currents in pulmonary arterial myocytes. Am. J. Physiol. 268 (Cell Physiol. 37): C259-C270, 1995[Abstract/Free Full Text].

32.   Yuan, X. J., J. Wang, M. Juhaszova, V. A. Golovina, and L. J. Rubin. Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L621-L635, 1998[Abstract/Free Full Text].


Am J Physiol Gastroint Liver Physiol 277(5):G1055-G1063
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society