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Am J Physiol Lung Cell Mol Physiol 281: L1350-L1360, 2001;
1040-0605/01 $5.00
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Vol. 281, Issue 6, L1350-L1360, December 2001

EDITORIAL FOCUS
Differential expression of KV channel alpha - and beta -subunits in the bovine pulmonary arterial circulation

Elizabeth A. Coppock and Michael M. Tamkun

Department of Physiology and Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha - and beta -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 alpha -subunit and Kvbeta .1, Kvbeta 1.2, and Kvbeta 1.3 accessory subunits dramatically increased along the pulmonary arterial tree. The differential expression of all the beta -subunits but of only one of the putative O2-sensitive alpha -subunits suggests that the alpha -subunits alone are not the O2 sensors but further implicates the auxiliary beta -subunits in pulmonary arterial O2 sensing.

pulmonary artery; oxygen sensor; smooth muscle; voltage-gated potassium channel localization; voltage-gated potassium beta -subunit


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 beta -subunits in cellular O2 sensing has also been suggested because the Kvbeta 1.2 subunit appears to confer O2 sensitivity on the Kv4.2 alpha -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 alpha - and beta -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, Kvbeta 1.1, Kvbeta 1.2, Kvbeta 1.3, and Kvbeta 2.1 subunits in bovine conduit and resistance PASMCs. Surprisingly, we found little difference in the expression of the putative O2-sensitive alpha -subunits Kv1.2, Kv1.5, and Kv2.1. In contrast, expression of the Kv3.1b alpha -subunit and of all the KV beta -subunits (except Kvbeta 2.1, which was not present) was dramatically greater in resistance than in conduit PASMCs.


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

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-Kvbeta 1.1N rabbit polyclonal, anti-Kvbeta 1.2N rabbit polyclonal, and anti-Kvbeta 2.1 mouse monoclonal. Anti-Kv1.2 rabbit polyclonal (15), anti-human Kv1.5 rabbit polyclonal (21), anti-bovine Kv1.5 rabbit polyclonal, anti-Kvbeta 1.2 rabbit polyclonal, and anti-Kvbeta 1.3 rabbit polyclonal antibodies were produced and characterized in the Tamkun laboratory. Anti-alpha -smooth muscle actin mouse monoclonal and Indocarbocyanine (Cy3)-conjugated anti-alpha -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-Kvbeta 1.2, and anti-Kvbeta 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 beta -subunit NH2-terminal domains were selected for isoform-specific production of Kvbeta 1.2 (amino acids 1-72) and Kvbeta 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-Kvbeta 1.2) 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-Kvbeta 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 Kvbeta 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-Kvbeta 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), Kvbeta 1.1N polyclonal (1 µg/ml), Kvbeta 1.2N polyclonal (1 µg/ml), Kvbeta 1.3 polyclonal (1:500), Kvbeta 2.1 monoclonal (1 µg/ml), or alpha -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.

Immunogen block of the Western blot channel signals was performed to demonstrate antibody specificity for the anti-Kv1.5 antibody because it had not been previously characterized. Identical blots were incubated with antiserum that had been preincubated overnight at 4°C with 1 µmol/l of each of the peptides used to generate the antibody or antiserum that was preincubated in the same solution (S2B) minus the peptides (see Fig. 1A).


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  		Fig. 1.   Western blot analysis of voltage-gated K+ (KV) channel alpha - and beta -subunit proteins in bovine resistance pulmonary arterial (PA) smooth muscle cell (SMC) membranes. A: immunoblot with anti-bovine Kv1.5 antibody alone (- peptide) and after preincubation with 1 µmol/l of each Kv1.5 peptide from which the antibody was generated (+ peptide), anti-Kv2.1 antibody, and anti-Kv3.1b antibody alone and after preincubation with 1 µmol/l of Kv3.1b peptide. B: immunoblot binding of anti-Kvbeta 1.1N antibody, anti-Kvbeta 1.2 antibody, anti-Kvbeta 1.3 antibody, and anti-Kvbeta 2.1 antibody. R, bovine resistance PASMC membranes; B, bovine brain membranes; NT, nontransfected L-cell membranes; T, Kvbeta 1.3-transfected L-cell membranes. Nos. at right, molecular mass in kDa.

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 alpha -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunoblot analysis of KV alpha -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 alpha -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 alpha -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 beta -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 alpha -subunits in pulmonary arteries.

Immunoblot analysis of KV beta -subunit expression. Although multiple KV beta -subunit mRNAs have been detected in cultured PASMCs (37, 43), PA beta -subunit protein expression has not been examined. Kvbeta 1.1, Kvbeta 1.2, and Kvbeta 1.3 but not Kvbeta 2.1 were detected in bovine resistance PASMC membranes via immunoblot analysis (Fig. 1B). The anti-Kvbeta 1.1 antibody recognized a single prominent band of ~49 kDa in bovine resistance PASMC membranes (Fig. 1B). The electrophoretic mobility of the Kvbeta 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-Kvbeta 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-Kvbeta 1.3 antibody recognized a single prominent band of ~68 kDa in bovine resistance PASMC membranes (Fig. 1B). The electrophoretic mobility of the PASMC Kvbeta 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 Kvbeta 1.3 (Fig. 1B), indicating that the antibody recognizes Kvbeta 1.3 and that this beta -subunit migrates unusually slowly on SDS gels relative to its predicted molecular mass. Even after extended exposures, there was no evidence of Kvbeta 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 Kvbeta 2.1 is not expressed at significant quantities in bovine pulmonary arteries.

Differential expression of KV alpha -subunits in pulmonary arteries. Kv1.2, Kv1.5, Kv2.1, and Kv3.1b alpha -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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  		Fig. 2.   KV alpha -subunit expression in pulmonary arteries. Left: Western blot analysis of Kv1.5 (n = 5 immunoblots from 2 animals; A), Kv2.1 (n = 4 immunoblots from 3 animals; B), and Kv3.1b (n = 5 immunoblots from 3 animals; C) channel expression in bovine conduit (C), 2nd intralobar, and 3rd and 4th resistance intralobar PASMC membranes. Immunoblots were incubated with antibodies against Kv1.5, Kv2.1, or Kv3.1b and then stripped and reprobed with anti-alpha -smooth muscle actin antibody to control for differences in PASMC loading between samples. Nos. at right, molecular mass. Right: corresponding protein expression in conduit (solid bars) and resistance (pooled 3rd and 4th division; open bars) pulmonary arteries measured in arbitrary units corrected for alpha -smooth muscle actin as determined by Western blot analysis and densitometry. * Significant difference from conduit pulmonary artery, P <=  0.05.

Expression of KV alpha -subunits in pulmonary arteries was also examined qualitatively via immunohistochemistry because a previous report (3) suggested heterogeneous channel expression within the vascular wall. Figures 3 and 4 illustrate Kv1.2 and Kv1.5 immunostaining, respectively, in conduit, second intralobar, and third and fourth resistance intralobar pulmonary arteries. The intensity of Kv1.2 immunostaining at the level of a single myocyte appeared to be fairly equal between conduit and resistance PASMCs and within the vascular wall of both conduit and resistance vessels (Fig. 3). In agreement with the immunoblot analysis in Fig. 2, the intensity of Kv1.5 immunostaining appeared to be modestly greater in resistance than in conduit pulmonary arteries. In contrast to Archer et al. (3), we, like McCulloch et al. (22), found no evidence of K+ channel heterogeneity within the vascular wall of either conduit or resistance vessels (Figs. 2 and 3). This does not rule out heterogeneous expression of subunits that were not examined; however, our results support the more recent data of McCulloch et al. (22).


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  		Fig. 3.   Immunolocalization of Kv1.2 alpha -subunit in bovine pulmonary arteries. Shown are differential interference contrast (DIC) image and binding of anti-Kv1.2 antibody [indocarbocyanine (Cy3) image], respectively, in bovine conduit (A and B), 2nd intralobar (C and D), 3rd intralobar (E and F), and 4th intralobar (G and H) pulmonary arteries. Exposure conditions and magnification for B, D, F, and H were identical.



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  		Fig. 4.   Immunolocalization of Kv1.5 alpha -subunit in bovine pulmonary arteries. Shown are DIC image and binding of anti-human Kv1.5 antibody (Cy3 image), respectively, in bovine conduit (A and B), 2nd intralobar (C and D), 3rd intralobar (E and F), and 4th intralobar (G and H) pulmonary arteries. Exposure conditions and magnification for B, D, F, and H were identical.

Although others have detected Kv2.1 immunostaining in rat lung sections (4) and Kv3.1b immunostaining in isolated rabbit PASMCs (27), we were unable to obtain specific Kv2.1 immunostaining above background or Kv3.1b immunostaining that was blocked by the immunogen in intact bovine PA vessels.

KV beta -subunits have been implicated in PASMC O2 sensing. If this hypothesis is correct, KV beta -subunit expression should be greater in resistance than in conduit PASMCs. Therefore, KV beta -subunit expression was compared between conduit and resistance PASMCs via Western blot analysis and densitometry (Fig. 5). Representative immunoblots demonstrating Kvbeta 1.1, Kvbeta 1.2, and Kvbeta 1.3 expression in PASMCs from the conduit pulmonary artery, second intralobar pulmonary artery, and third and fourth intralobar resistance pulmonary arteries are shown in Fig. 5 along with a quantitative assessment comparing expression levels between conduit and pooled resistance PASMCs. Kvbeta 1.1 and Kvbeta 1.2 expression levels were dramatically greater in both third and fourth intralobar resistance PASMCs than in conduit PASMCs (Fig. 5A, left, and B, left). In contrast, although Kvbeta 1.3 expression appeared dramatically greater in fourth intralobar PASMCs than in conduit PASMCs, there was little difference in Kvbeta 1.3 expression between third intralobar and conduit PASMCs (Fig. 5C, left). Consistent with the representative Western blot data, subunit expression of Kvbeta 1.1, Kvbeta 1.2, and Kvbeta 1.3 were found to be significantly greater (~6.1-, 3.6-, and 2.9-fold, respectively) in resistance (pooled third- and fourth-division vessels) than in conduit PASMCs (Fig. 5, A-C, right).


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  		Fig. 5.   KV beta -subunit expression in pulmonary arteries. Left: Western blot analysis of Kvbeta 1.1 (n = 3 immunoblots from 3 animals; A), Kvbeta 1.2 (n = 6 immunoblots from 4 animals; B), and Kvbeta 1.3 (n = 3 immunoblots from 3 animals; C) channel expression in bovine conduit, 2nd intralobar, and 3rd and 4th resistance intralobar PASMC membranes. Immunoblots were incubated with antibodies against Kvbeta 1.1, Kvbeta 1.2, or Kvbeta 1.3 and then stripped and reprobed with anti-alpha -smooth muscle actin antibody to control for differences in PASMC loading between samples. Nos. at right, molecular mass. Right: corresponding protein expression in conduit (solid bars) and resistance (pooled 3rd and 4th division; open bars) pulmonary arteries measured in arbitrary units corrected for smooth muscle alpha -actin as determined by Western blot analysis and densitometry. * Significant difference from conduit pulmonary artery, P <=  0.05.

Although the anti-Kvbeta 1.1N and anti-Kvbeta 1.3 antibodies failed to stain bovine PA sections, trustworthy Kvbeta 1.2 immunostaining was detected in bovine resistance PASMCs (Fig. 6, A-C). Therefore, Kvbeta 1.2 immunolocalization was compared between conduit, second intralobar, and third and fourth resistance intralobar pulmonary arteries (Fig. 6, D-K). Consistent with the Western blot data of Fig. 5, although present in conduit PASMCs, Kvbeta 1.2 expression was observed to be greater in individual resistance PASMCs (compare Fig. 6, I and K with E and G). Thus increased expression along the PA tree appears to be due to increased expression at the cellular level and not to the lack of Kvbeta 1.2 expression in individual conduit myocytes.


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  		Fig. 6.   Immunolocalization of Kvbeta 1.2 channel subunit in bovine pulmonary arteries. A-C: binding of anti-Kvbeta 1.2 antibody (Cy3 image) in pulmonary resistance vessels (3rd intrapulmonary) incubated with anti-Kvbeta 1.2 antibody alone (A), after preincubation with 1 µmol/l of glutathione S-transferase (GST; B), and after preincubation with 1 µmol/l of GST-Kvbeta 1.2 fusion protein (C). Shown are DIC image and binding of anti-Kvbeta 1.2 antibody (Cy3 image), respectively, in bovine conduit (D and E), 2nd intralobar (F and G), 3rd intralobar (H and I), and 4th intralobar (J and K) pulmonary arteries. Exposure conditions and magnification for A-C and for E, G, I, and K were identical.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of KV channel alpha -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 alpha -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.

Results from the present study demonstrating similar Kv2.1 and Kv1.2 expression levels between conduit and resistance PASMCs are somewhat contradictory to the results of Patel et al. (28), who, using RT-PCR to compare KV channel subunit expression between isolated conduit PASMCs and primary cultured resistance PASMCs, reported increased expression of Kv2.1 and Kv1.2 in conduit PASMCs. It is possible that culturing of the resistance PASMCs in the study by Patel et al. caused a decrease in channel expression. On the other hand, our differences may reflect a species difference between bovine and rat PASMCs or may simply be a matter of what was measured (protein vs. mRNA). Regardless, the results of both studies agree that expression of Kv1.2 and Kv2.1 channel subunits is not greater in the resistance vessels where HPV is thought to occur. Therefore, Kv1.2, Kv1.5, and Kv2.1 alpha -subunit expression levels are not likely to account for the differential response of conduit and resistance pulmonary arteries to hypoxia.

In contrast, Kv3.1b expression was dramatically greater in resistance than in conduit PASMCs. Differential expression of KV channels in the bovine pulmonary vasculature is consistent with the findings of Archer et al. (3) and Albarwani et al. (1) in the rat and of McCulloch et al. (22) in the rabbit. In the rabbit pulmonary vasculature, the tetraethylammonium (TEA)- and glibenclamide-insensitive delayed rectifier K+ current doubled in amplitude on moving down the arterial tree from the main conduit pulmonary artery (8 mm) to the large intrapulmonary artery (>400 µm) and then remained constant through the medium (200- to 400-µm) and small (<200-µm) intrapulmonary arteries. The noninactivating K+ current showed a similar trend but was reduced in the <200-µm intrapulmonary arteries where it was comparable to that in the main conduit pulmonary artery. In the present study, when K+ channel expression was compared between smooth muscle cells of the bovine main conduit PA (25-30 mm) to that of the third (3-5 mm)- and fourth (1-2 mm)-generation pulmonary arteries, there was a dramatic increase in the expression of Kv3.1b (~12-fold greater in resistance than in conduit PASMCs; Fig. 2). Although it is hard to compare intrapulmonary arteries between studies, when compared in terms of the size decrease between conduit and intrapulmonary vessels, the comparison between the conduit pulmonary artery and the large intrapulmonary artery in the rabbit study is similar to the comparison between the conduit pulmonary artery and the third and fourth resistance intrapulmonary arteries in the present study, thus supporting our finding of an increase in Kv3.1b expression. However, in the rabbit study (22), the K+ current that increased from conduit to resistance vessels was TEA insensitive, whereas Kv3.1b was inhibited by TEA. Because heteromeric channel assembly affects K+ channel current pharmacology (15, 32), it is possible, however, that the native PASMC O2-sensitive current includes Kv3.1b subunits in TEA-insensitive heteromeric complexes.

Our results and those of Osipenko et al. (27) showing that Kv3.1b current is significantly inhibited by hypoxia when expressed in transfected L929 cells support a role for Kv3.1b as a molecular component of the resistance PASMC O2-sensitive K+ current. However, because all of the other putative O2-sensitive alpha -subunits were distributed fairly evenly throughout the PA vasculature, it is unlikely that expression of alpha -subunits alone accounts for the differential response of conduit and resistance pulmonary arteries to hypoxia but rather supports a role for the variable expression of an unidentified O2 sensor or perhaps accessory beta -subunits.

Expression of KV channel beta -subunits in pulmonary arteries. Adding to the growing list of possible KV beta -subunit functions is that of a cellular O2 sensor (12, 29). Interestingly, Kvbeta 1.2 has been shown to confer O2 sensitivity on Kvalpha 4.2 in a heterologous expression system (29). Additionally, when the conserved core of the Kvbeta 2.1 subunit was crystallized, bound NADPH was detected in its crystal structure (12), further supporting a role for the beta -subunit in cellular redox sensing. However, because KV beta -subunits usually produce inactivation and the outward K+ current from smooth muscle cells in pulmonary arteries is only slowly inactivating or noninactivating, KV beta -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 beta -induced inactivation (18). Therefore, the presence of KV beta -subunits in the pulmonary arteries does not necessarily demand that the endogenous K+ channels show fast inactivation.

The present study demonstrates that expression of Kvbeta 1.1, Kvbeta 1.2, and Kvbeta 1.3 proteins is significantly greater (~6-, 3.5-, and 3-fold) in resistance than in conduit PASMCs (Figs. 5 and 6), consistent with a role for KV beta -subunits in PA O2 sensing. Furthermore, the Kvbeta 1.2 result was confirmed qualitatively via immunohistochemistry, which shows that Kvbeta 1.2 expression dramatically increases from the conduit to the fourth intralobar pulmonary artery (Fig. 6). Although Kvbeta 1.1 is generally considered to be a neuronal channel, it is likely that the majority of protein detected in the PA smooth muscle layer came from the SMCs. Not only was a very strong Western blot signal obtained, but Kvbeta 1.1 mRNA has been detected by other investigators in acute primary cultures of rat PASMCs (43) and in SMCs of the mesenteric artery (40). Kvbeta 2.1 was not detected in conduit, second intralobar, or third and fourth intralobar resistance PASMCs, although a strong band of ~40 kDa [which corresponds closely to the reported molecular mass of Kvbeta 2.1 in rat brain (24)] was detected in bovine brain (Fig. 1B). This is in contrast to the results of Yuan et al. (43), who reported Kvbeta 2.1 mRNA in primary cultured rat PASMCs with RT-PCR. However, although RT-PCR provides information about gene expression, it does not confirm the presence of an encoded protein. Another possibility is that PASMC culture conditions altered the channel gene expression.

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 alpha -subunits are expressed in both circulations.

The increased expression of KV beta -subunits from the conduit pulmonary artery to the resistance pulmonary artery where HPV occurs supports the idea that beta -subunits act as O2 sensors as does the O2 sensitivity of Kv4.2 in the presence of Kvbeta 1.2. Additional preliminary data in mouse L-cells also show conferred O2 sensitivity on other alpha -subunits by coexpression with a beta -subunit. Kv2.1 channels, which are inhibited by hypoxia in L-cells containing the endogenously expressed Kvbeta 2.1 subunit, are insensitive to hypoxia in HEK293 cells (Sakamoto N and Tamkun MM, unpublished data), which lack this beta -subunit (34). However, when Kvbeta 2.1 is cotransfected into HEK293 cells along with the Kv2.1 alpha -subunit, the Kv2.1 current regains its O2 sensitivity (Sakamoto N and Tamkun MM, unpublished data), suggesting that KV beta -subunits are somehow involved in this response, although the literature argues against the assembly of Kvalpha 2.1 with Kvbeta 2.1 (24).

In summary, this study provides a map for the expression of putative O2-sensitive KV channel subunits in the pulmonary arteries and demonstrates that some of these subunits are differentially expressed between conduit and resistance PASMCs. Specifically, this study confirmed the presence of Kv1.2, Kv1.5, Kv2.1, and Kv3.1b subunit proteins and demonstrated the presence of Kvbeta 1.1, Kvbeta 1.2, and Kvbeta 1.3 but not Kvbeta 2.1 subunit proteins in PASMCs. Importantly, this study demonstrated that the expression levels of the putative O2-sensitive Kv1.2, Kv1.5, and Kv2.1 alpha -subunits are fairly equal between conduit and resistance vessels, thus requiring the differential expression of an O2 sensor to regulate these alpha -subunits. This O2 sensor hypothesis is consistent with the finding that the O2 sensitivity of these alpha -subunits varies with the heterologous expression system used (4, 15, 27, 28). In contrast, the dramatic increase in expression levels of Kv3.1b in O2-sensitive resistance pulmonary arteries suggests that this alpha -subunit may be important in PA O2 sensing. Additionally, the increase in expression of all three KV beta -subunits supports the beta -subunit O2 sensor hypothesis and suggests that multiple mechanisms of O2 sensing are likely to exist in vivo.


    ACKNOWLEDGEMENTS

We thank Dr. Naoya Sakamoto for technical assistance.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 281(6):L1350-L1360
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