Published online before print June 24, 2004, doi: 10.1161/01.RES.0000137173.42723.fb
© 2004 American Heart Association, Inc.
Cellular Biology |
Preferential Expression and Function of Voltage-Gated, O2-Sensitive K+ Channels in Resistance Pulmonary Arteries Explains Regional Heterogeneity in Hypoxic Pulmonary Vasoconstriction
Ionic Diversity in Smooth Muscle Cells
From the Department of Medicine (Cardiology) and the Vascular Biology Group, University of Alberta, Canada.
Correspondence to Stephen L. Archer, MD, Heart and Stroke Chair in Cardiovascular Research, Cardiology Division, University of Alberta, WMC 2C2.36, 8440 112th St, Edmonton, Alberta T6G 2B7, Canada. E-mail sarcher{at}cha.ab.ca
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Abstract |
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Hypoxic pulmonary vasoconstriction (HPV) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). Kv inhibition depolarizes membrane potential (EM), thereby activating Ca2+ influx via voltage-gated Ca2+ channels. HPV is weak in extrapulmonary, conduit pulmonary arteries (PA) and strong in precapillary resistance arteries. We hypothesized that regional heterogeneity in HPV reflects a longitudinal gradient in the function/expression of PASMC O2-sensitive Kv channels. In adult male Sprague Dawley rats, constrictions to hypoxia, the Kv blocker 4-aminopyridine (4-AP), and correolide, a Kv1.x channel inhibitor, were endothelium-independent and greater in resistance versus conduit PAs. Moreover, HPV was dependent on Kv-inhibition, being completely inhibited by pretreatment with 4-AP. Kv1.2, 1.5, Kv2.1, Kv3.1b, Kv4.3, and Kv9.3. mRNA increased as arterial caliber decreased; however, only Kv1.5 protein expression was greater in resistance PAs. Resistance PASMCs had greater K+ current (IK) and a more hyperpolarized EM and were uniquely O2– and correolide-sensitive. The O2-sensitive current (active at –65 mV) was resistant to iberiotoxin, with minimal tityustoxin sensitivity. In resistance PASMCs, 4-AP and hypoxia inhibited IK 57% and 49%, respectively, versus 34% for correolide. Intracellular administration of anti-Kv1.5 antibodies inhibited correolide’s effects. The hypoxia-sensitive, correolide-insensitive IK (15%) was conducted by Kv2.1. Anti-Kv1.5 and anti-Kv2.1 caused additive depolarization in resistance PASMCs (Kv1.5>Kv2.1) and inhibited hypoxic depolarization. Heterologously expressed human PASMC Kv1.5 generated an O2– and correolide-sensitive IK like that in resistance PASMCs. In conclusion, Kv1.5 and Kv2.1 account for virtually all the O2-sensitive current. HPV occurs in a Kv-enriched resistance zone because resistance PASMCs preferentially express O2-sensitive Kv-channels.
Key Words: immunoelectropharmacology • laser capture microdissection • voltage-gated channels • pulmonary circulation • adenoviral gene transfer
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Introduction |
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The adult pulmonary circulation is a low-resistance circuit designed for gas exchange that is perfused by a thin-walled, afterload-intolerant right ventricle. The pulmonary vasculature consists of large, elastic, extraparenchymal "conduit" arteries and small, muscular intrapulmonary arteries, which control regional distribution of blood flow and largely determine pulmonary vascular resistance (PVR). Hypoxic pulmonary vasoconstriction (HPV) is a widely conserved mechanism for ventilation-perfusion matching.1 With segmental hypoxia (eg, atelectasis), resistance pulmonary arteries (PAs) serving the hypoxic lobes constrict, diverting blood to better-oxygenated segments, thereby optimizing systemic PO2, without increasing PVR.2,3 Microangiography4 and micropuncture5 reveal that HPV primarily occurs in resistance PAs (<200 µmol/L diameter, division 4, and distal), with lesser contributions from larger intrapulmonary arteries and pulmonary veins. Conversely, conduit PAs (division 1 to 2) dilate or fail to constrict to hypoxia.6,7 This anatomical (proximal-distal) and functional (conduit-resistance) distinction extends to the cellular level, with evidence for K+ channel diversity within and between segments.7–9 Whole-cell potassium currents (IK) in conduit PA smooth muscle cells (PASMC) manifest contributions from both voltage-gated (Kv) and large-conductance, calcium-sensitive (BKCa) channels; however, IK in resistance PASMCs predominantly reflect Kv current.3 This diversity may reflect the dual origins of the adult pulmonary circulation from vascular beds with different embryological origins that fuse late in development. Conduit PAs originate from the sixth aortic arch, whereas resistance PAs originate from the mesenchymal lung bud by capillary plexus expansion.10,11
HPV is initiated by inhibition of Kv channels.12–16 Constrictor responses to hypoxia and 4-aminopyridine (4-AP), a Kv channel inhibitor, are greatest in resistance PAs. Likewise Kv current density is greater in resistance PASMCs.7,17 Candidate O2-sensitive Kv channels include Kv1.5,18,19 Kv2.1/9.3,19,20 Kv3.1b,21 and Kv1.2.22 The importance of Kv1.5 and Kv2.1, 4-AP-sensitive channels, and redox-sensitive channels to HPV is emphasized by 2 models that manifest selective suppression of HPV, the chronic hypoxic pulmonary hypertension (PHT) model, and the Kv1.5 knockout mouse. In chronic hypoxia, impairment of HPV results from loss of Kv1.5 and Kv2.1 expression with concordant suppression of O2-sensitive IK.14,23,24 In this model, enhancing expression of Kv1.5, via Kv1.5 adenoviral gene transfer,18 or Kv2.1, via ingestion of dichloroacetate,25 restores Kv expression, O2-sensitive IK and HPV. Mice with targeted Kv1.5 deletions have impaired HPV and reduced PASMC O2-sensitive IK.26 We hypothesized that greater functional expression of Kv1.5, and possibly other O2-sensitive channels, in distal PASMCs accounts for their more robust HPV. The contribution of Kv1.x channels to regional differences in tone and IK was assessed using a naturally occurring pentacyclic triterpenoid Kv1.x inhibitor, correolide.27,28 Segmental expression of Kv genes was quantified on laser capture microdissection (LCM) specimens using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The contribution of Kv1.5 and Kv2.1 to O2-sensitive IK and control of EM was assessed using immunoelectropharmacology19 and conventional patch clamp techniques in conduit and resistance PASMC and Chinese hamster ovary cells (CHO) heterologously expressing either human PA Kv1.5 or rat Kv2.1.
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Methods |
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Definitions
Conduit PAs were defined as the PA up to the point where the artery enters the lung parenchyma (>200-µm diameter). Intrapulmonary resistance PAs were subcategorized based on relationship to airway structures and diameter: large (associated with bronchi, >100 µm), intermediate (associated with alveolar ducts, 40 to 100 µm), and small (surrounded by alveoli, <40 µm).29
Vascular Reactivity (n=25 rats)
Rings were mounted on stainless steel wires in 4-mL baths containing Earle solution (pH 7.43, 37°C, PO2 162±5 mm Hg) at an experimentally determined optimal resting tension of 700 mg (see the online data supplement available at http://circres.ahajournals.org). Vascular reactivity was studied in 12 endothelium-intact and 7 endothelium-denuded conduit and intermediate resistance PAs (diameter 268±11 µm and 74±3 µm, respectively). The endothelium was removed by gentle rubbing of the lumen with a silk suture. Endothelial integrity was assessed in phenylephrine-constricted (PE) (1 µmol/L) rings by measuring relaxation to acetylcholine (0.1 to 10.0 µmol/L). Constriction to 4-AP (1 to 10 mmol/L), barium chloride (1 to 100 µmol/L), correolide (1 to 100 µmol/L), iberiotoxin (100 nM), and 1 hour of hypoxia (achieved by bubbling the bath with 2.5% O2, 5% CO2, pH 7.35 to 7.45, PCO2 27 mm Hg, PO2 
45 mm Hg) was assessed. The dependence of HPV on Kv channel inhibition was assessed in 6 additional endothelium-intact resistance rings preconstricted with 4-AP (10 mmol/L) and then challenged sequentially with hypoxia and phenylephrine.
Electrophysiology
Whole-cell electrophysiology was performed on enzymatically dispersed PASMCs from conduit or resistance PAs (see online supplement).30,31 PASMCs and CHO cells (control or infected with adenovirus for Kv1.5 or Kv2.1,25 see online supplement) were studied at 25°C and 35°C using voltage and current clamp configurations. Currents were evoked by 200-ms test pulses, filtered at 1 kHz, and sampled at 2 to 4 kHz. Two-stepped voltage protocols were used: from –70 to +70 mV in 20-mV steps and from –70 to –10 mV in 5-mV steps (to better-characterize the O2-sensitivity of the current controlling EM while avoiding Kv inactivation). Current density was calculated by dividing average plateau phase IK by the manually measured cell capacitance (pA/pF). To record EM, cells were held at their resting EM in current-clamp mode.
Heterologous Expression of Kv1.5 and Kv2.1
To assess the sensitivity and specificity of the anti-Kv antibodies for their respective channels, CHO cells, which do not express an endogenous Kv current, were infected with replication-deficient serotype-5 adenovirus encoding genes for green fluorescent protein and either human PASMC-derived Kv1.5 or rat Kv2.1 (see online supplement).18,25,31,32
Immunoelectropharmacology was performed in the whole-cell configuration, as previously described.19 In addition, single-channel experiments were performed to identify the O2-sensitive channels using the inside-out patch configuration7 (see online supplement). Preliminary experiments established that 1 µL of Kv1.5 or Kv2.1 antibody in 150 µL of internal pipette solution depolarized PASMCs. The difference in IK between the recording 1 to 2 minutes after membrane rupture and a recording at 10 minutes was defined as the "antibody effect." IgG1 antibodies created from glutathione-S-transferase-fusion proteins were directed against intracellular, carboxyl terminus amino acids (542 to 602 of rat Kv1.5 and 509 to 853 of rat Kv2.1). No "roll-off" of IK was observed in controls for up to 30 minutes. Significant changes in the biophysical stability of the seal or appearance of leak current disqualified cells from analysis.
LCM and qRT-PCR
qRT-PCR was performed on LCM samples from flash-frozen lung33 (see online supplement). Expression was normalized to the SMC-specific reporter, SM22
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Immunoblotting
PAs were frozen in liquid N2 and homogenized in buffer containing an antiprotease cocktail before being placed on a 7.5% gel.19,34 Signal intensity was normalized to actin expression (Santa Cruz Biotechnology, Santa Cruz, Calif).
Drugs and Statistics
Drugs are given in saline and, unless otherwise stated, are from Sigma-Aldrich Inc (St. Louis, Mo). Correolide was a gift from John Obenchain (Merck & Co, Inc, Rahway, NJ). Kir6.1 antibody was a gift from Dr H. Nakayama, Kumamoto, Japan. Antibodies for immunoelectropharmacology were obtained from US Biological (Swampscott, Mass) and Sigma-Aldrich. Values are given as mean±SEM. Intergroup comparisons are made using an ANOVA with post hoc testing using Fisher probable least significant differences test. P<0.05 was considered statistically significant.
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Results |
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HPV Is Strongest in Resistance PAs and Is Endothelium-Independent
Acetylcholine-induced relaxation of PA rings was similar in conduit and resistance PAs (Figure 1 A and 1B). In endothelium-intact rings, contraction to low-dose correolide (1 to 10 µmol/L) was similar in magnitude to HPV and was strongest in resistance PAs. Both the absolute constrictor response to hypoxia (Figure 1C) and the constriction expressed as a percentage of phenylephrine constriction were greatest in resistance PAs (Figure 1E). 4-AP constriction was also strongest in resistance arteries (Figure 1E). Iberiotoxin, a specific BKCa channel inhibitor, did not significantly constrict either segment whereas BaCl, an inhibitor of inward rectifier K+ channels, caused similar constriction in conduit and resistance PAs (data not shown). Endothelial denudation converted acetylcholine relaxation to constriction (Figure 1B) but did not alter the magnitude or onset kinetics of HPV. Hypoxia caused no constriction when superimposed on constriction to 10 mmol/L 4-AP, although phenylephrine constricted these rings normally (Figure 1F), suggesting that the Kv channels mediating HPV are a subset of the resistance PASMC family of 4-AP-sensitive channels.
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Increased Expression of Kv mRNA and Kv1.5 Protein in Resistance PAs
qRT-PCR on LCM-derived specimens demonstrates a stepwise, proximal-to-distal increase in expression of Kv channel mRNA, relative to the SMC reporter, SM22 (Figure 2 A and 2B). However, only Kv1.5 protein was more abundant in resistance PAs (Figure 2C).
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Resistance PASMCs Are Enriched in O2-Sensitive Kv Current
SMCs from resistance and conduit PAs have voltage-gated, rapidly-activating, noninactivating IK (Figure 3). However, resistance PASMCs have greater normoxic current density, despite smaller cell size, as measured by capacitance (Figure 3B). Subtraction analysis shows that Kv current in resistance PASMCs activates at the resting EM of –60 mV (whether measured at 25°C or 37°C, Figures 3D and 4
B), consistent with the participation of Kv channels in setting the PASMC EM. 4-AP and hypoxia reversibly inhibit 57% and 49% of IK, respectively (at 35°C, +70 mV), in resistance PASMCs (Figure 3C and 3D), whereas correolide only inhibits 34% of IK (Figure 5D). Thus, hypoxia inhibits the same channels as 4-AP, whereas the Kv1.x inhibitor correolide leaves approximately one third of the O2-sensitive uninhibited, a component that is attributable to Kv2.1. The O2-sensitive current is not blocked by iberiotoxin but is partially inhibited by tityustoxin (Figure 4C). Hypoxia’s effects onset within 1 minute and are reversible. Conduit PASMCs have virtually no hypoxia-sensitive, correolide-sensitive, or 4-AP-sensitive IK (Figure 5).
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Human PASMC Kv1.5 Channels Are Inhibited by Correolide and Hypoxia
CHO cells lack endogenous Kv current and manifest only an ohmic current (Figure 6A).18 Cells expressing Kv1.5 were selected based on their GFP fluorescence (Figure 6B). This heterologous expression system was used to study the specificity of immunoelectropharmacological current-dissection technique and to characterize the O2-sensitivity of this candidate O2-sensitive channel. The specificity of the anti-Kv1.5 and anti Kv2.1 antibodies was confirmed by showing that they inhibit their respective clonal Kv channels but do not cross-react. Anti-Kv2.1 does not inhibit Kv1.5 current (Figure 6C) and vice versa (see online data supplement). Moreover, antibodies to an unexpressed K+ channel, anti-Kir2.1, had no effect in Kv1.5-CHO cells (Figure 6C). IKv1.5 in infected CHO cells was similar to IK in resistance PASMC (ie, rapidly activating, noninactivating, and reversibly inhibited by hypoxia, 4-AP, and correolide; Figure 6D and 6E).
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Electrophysiological Effects of Correolide Reflect Inhibition of Kv1.5
Resistance PASMCs are relatively hyperpolarized compared with conduit PASMCs and, unlike conduit PASMCs, depolarize in response to correolide in a dose-dependent manner (Figure 7 A and 7B). In the presence of correolide, hypoxia has reduced ability to depolarize the resistance PASMC, indicating a predominant, but not exclusive, role for Kv1.x family channels in hypoxic depolarization (Figure 7A). Similarly, most (but not all) of the hypoxia-sensitive IK is inhibited by anti-Kv1.5 (Figure 7C). To assess the extent to which correolide-induced depolarization was caused by Kv1.5 inhibition, endogenous Kv1.5 was blocked using immunoelectropharmacology. Pretreatment with anti-Kv1.5, but not anti-Kv2.1, eliminated the depolarizing effects of correolide (Figure 7C and 7D).
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Kv1.5 and Kv2.1 Set EM and Account for O2-sensitive IK in Resistance PASMCs
Antibodies against Kv1.5 cause more depolarization than anti-Kv2.1 (Figure 7E and 8A), and the combination of anti-Kv1.5+2.1 caused greater depolarization than either antibody alone. The combination completely blocked hypoxic depolarization of the PASMC (Figure 8A), achieving a greater inhibition of hypoxic depolarization than correolide alone (Figure 7B), indicating a contributory role for the correolide-insensitive channel Kv2.1 (Figure 7A). Single-channel data confirm that the most resistant PASMC patches show activity of only 2 to 3 discrete K+ channel conductances (not shown). Anti-Kv1.5 decreases the open probability of a 33-pS channel (Figure 8B).
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Discussion |
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O2-sensitive K+ channels mediate acute responses to changes in PO2 in the PASMC,2,3 the carotid body type-1 cell,35 adrenomedullary cells,36 neuroepithelial bodies,37 and the ductus arteriosus SMC.31 Although this role for K+ channels is widely conserved, there is significant diversity in the channel types implicated (various Kv channels versus BKCa). Moreover, the downstream response to channel inhibition (vasoconstriction, neurotransmitter release) varies among species, between tissues, and with maturation.3
This study offers 5 findings that increase our understanding of K+ channel diversity in vascular biology. First, resistance PAs constitute an electrically unique zone. HPV occurs here because resistance PASMCs have a virtual monopoly on the functional expression of O2-sensitive Kv channels (Figures 2 through 4
), specifically Kv1.5 and, to a lesser extent, Kv2.1 (Figure 8A). Second, EM in resistance PASMCs is set by Kv1.5 and Kv2.1 (Figures 7 and 8 ), with a minor contribution from a tityustoxin-sensitive channel (Kv1.2 and/or Kv1.3) (Figure 4C). Third, it is the SMC, not endothelial, K+ channel heterogeneity, that accounts for the divergent response to hypoxia and 4-AP. The current study definitively shows that HPV and 4-AP constriction are endothelium-independent (Figure 1E). Fourth, the specificity of the immunoelectropharmacology technique for dissecting the complex mosaic of ionic currents in vascular SMCs was validated, both in PASMCs and in CHO cells heterologously expressing a single Kv channel type (Figure 6C and online Figure I). Finally, human PA Kv1.5 was found to be intrinsically sensitive to hypoxia, 4-AP, and correolide, similar to the O2-sensitive IK in resistance PASMCs (Figure 6).
Hypoxia constricts resistance PAs by an endothelium-independent mechanism, at least for the first hour of hypoxia (Figure 1E and 1F). Although the endothelium modulates HPV,38 the endothelium-independence of the core mechanism has been reported.8,39 Proponents for an obligatory role of the endothelium in HPV acknowledge that the initial phase of HPV is endothelium-independent.40 Many of the studies in which endothelium is found to be "essential" have studied HPV only after first "priming" (precontracting) with agents such as prostaglandin F2. This results in hypoxic responses comprising a transient initial constriction, a relaxation, and then a slowly developing secondary phase. The confounding effects of priming are unknown, but HPV in vivo does not require priming, nor is priming required for robust HPV in healthy, isolated resistance PA rings (Figure 1). HPV increases in strength over 60 minutes to a plateau, without an initial transient contraction-relaxation phase, despite the documented absence of acetylcholine relaxation (Figure 1F), consistent with HPV in humans. Carlson reported, "There is an immediate vasoconstrictor response to hypoxia in the human lung and there is no further potentiation or diminution, of the response during a 60-minute period of hypoxia."41
The current study provides new details of molecular expression profiles in the pulmonary circulation and confirms the view that PASMCs are arrayed in a rich mosaic, in which resistance arterial segments, which control regional blood flow and vascular resistance, have greater levels of expression of Kv channels than do conduit arteries, whose function it is to deliver deoxygenated blood to the entire lung.7 Hypoxic vasoconstriction is solely found in the pulmonary circulation (hypoxia dilates most systemic arteries) and its localization appears to be unique, largely as a result of the preferential presence of hypoxia-inhibited K+ currents in resistance PASMCs. Systemic arterial SMCs, such as canine renal and rodent mesenteric SMCs, lack a hypoxia-inhibited IK.12,13 In this regard, the conduit PA is essentially a systemic artery.
It has been difficult to isolate the molecular species of the Kv channel(s) responsible for setting EM and conducting the O2-sensitive currents. Single-channel studies have demonstrated that hypoxia rapidly and reversibly inhibits several K+ channel conductances (channel types),7 including a 37-pS KDR (now classified as a Kv channel and very similar to the 33-pS conductance we measure for PASMC Kv1.5; see online Figure III). The pharmacology of the O2-sensitive K+ currents in PASMCs (rapidly activating, slowly-inactivating, 4-AP-sensitive, resistant to charybdotoxin and glyburide7,19,26,42) suggests certain candidate O2-sensitive channels, including Kv1.2,22 Kv1.5,18,19,26 Kv2.1,19,20,43 and possibly Kv3.1b.21 Each of these channels is present in PASMCs and each has its proponents. Likewise, this pharmacology tends to exclude a role for 4-AP-insensitive channels (BKCa), rapidly inactivating channels (Kv1.4 and Kv4.3), and charybdotoxin-sensitive channels (eg, homotetrameric Kv1.2 or Kv1.6 channels).
There is direct evidence that HPV is dependent on Kv in that preconstriction with the Kv blocker 4-AP eliminates subsequent HPV, even though the PA constricts vigorously to phenylephrine (Figure 1F). Moreover, the identities of the relevant O2-sensitive Kv -subunits are confirmed to be Kv1.5 and Kv2.1. The strong parallel between correolide and hypoxic constriction suggests that of the candidate Kv channels, a Kv1.x channel is central to the mechanism of HPV. Correolide’s specificity for Kv1.5 channels (at least in PAs) is supported by the finding that its ability to depolarize PASMCs is blocked by pretreatment with anti-Kv1.5, but not anti-Kv2.1, antibodies (Figure 7E). Theoretically, correolide can inhibit Kv1.2; however, tityustoxin, an inhibitor of Kv1.2/Kv1.3,44 inhibits only a small portion of the hypoxia-sensitive IK (Figure 4C). Moreover, after exposure to anti-Kv1.5 plus anti-Kv2.1, little responsiveness to hypoxia remains (Figure 8A). Finally, although mRNA for many Kv channels is more abundant in resistance versus conduit PAs, only Kv1.5 protein is more abundant distally (Figure 2). Although the lack of concordance between mRNA expression, protein expression, and protein function are well known, it is intriguing that there is a gap between the amount of Kv mRNA (which is higher in resistance PAs for most channels) and the amount of protein expressed (only Kv1.5 and perhaps Kv4.3 are increased in resistance PAs; see online Figure II). While the basis for the discrepancies are unexplained, this does provide a cautionary note regarding the reliance on qRT-PCR alone for expression profiling.
Further support for the physiological relevance of Kv1.5 to HPV comes from studies of heterologously expressed Kv1.5. The Kv1.5 channel used in these studies was derived from human PA mRNA, which is important not only because of tissue heterogeneity in formation of heterotetramers and association with ß-subunits but also because of the presence of tissue-specific alternatively spliced variants.45 Overturf et al cloned Kv1.5 from canine colonic SMCs and noted that whereas there was conservation of the channel’s transmembrane S1 to S6 segments with other Kv1.5 species, homology decreased to 74% to 82% in the NH2 and COOH terminal segments, with unproven, but likely, functional consequences. Thus, Kv1.5 isoforms with discrete pharmacology, O2 sensitivity, and slope conductances (10 pS for colonic SMC versus 32 pS for PASMC) exist, highlighting the need to specify the origins of the Kv1.5 isoform being studied. In CHO cells, human PA-Kv1.5 recapitulates the characteristics of the endogenous O2-sensitive IK in PASMCs (voltage-gated, inhibited by 4-AP, correolide, and hypoxia, Figures 4 and 6).
It is often questioned whether the K+ channels are active at sufficiently negative potentials to set the resting EM of PASMCs. We carefully studied this using a protocol that minimized the risk of inactivating Kv channels by avoiding exposure to potentials above –10mV (Figure 4C) at physiological temperatures (35°C). There is an outward Kv current at –65mV, which is inhibited by hypoxia (Figure 4B). Inhibiting this current depolarizes the PASMC. Thus, the consequence of enriched Kv expression in resistance PAs (Figure 2) is a relative hyperpolarization of resistance versus conduit PASMCs (–60 mV versus –35 mV; Figures 4A and 8A). As would be predicted, inhibition of both Kv1.5 and Kv2.1 depolarizes resistance PASMCs to an EM similar to those in control conduit PASMCs (–35 mV) and eliminates hypoxic depolarization (Figure 8A). The relative lack of effect of either anti-Kv antibody or correolide on conduit PASMC EM suggests that other channels, transporters, and/or pumps set EM in conduit PASMC.
Kv2.1, alone or in combination with Kv9.3, has a pharmacological profile and O2-sensitivity, consistent with a role in HPV.19,20,43 Kv2.1 contributes to setting the resting EM in PASMCs and to HPV19 (Figure 8A). The current study suggests that Kv2.1 accounts for the portion of 4-AP-sensitive current that is correolide-insensitive. Hogg et al also used immunoelectropharmacology to demonstrate that anti-Kv2.1 inhibited normoxic IKv (67% at +50 mV) and reduced the hypoxia-responsive IK.43 They did not assess the effects of anti-Kv1.5 and concluded that Kv2.1 plays a crucial role in HPV and in setting EM. The quantitative difference in the importance of Kv2.1 may relate to the dose and specificity of the Kv2.1 antibody used. Moreover, they did not rigorously establish the specificity of the anti-Kv2.1 antibodies. Our data clearly show that the antibodies directly inhibit their target channels without cross-reactivity (Figures 6C and 8 and online Figure I). Smirnov et al, using conventional patch clamp techniques, found 2 populations of conduit PASMCs: one with 4-AP and tetraethylammonium (TEA) sensitivity and the other with only 4-AP sensitivity. In contrast, resistance PASMCs uniformly demonstrated a 4-AP-sensitive current.17 Although they performed no molecular studies, the pharmacology suggested the TEA-sensitive Kv current was Kv2.1, and the exclusively 4-AP-sensitive current was Kv1.5, an interpretation consistent with previous publications.19
Patch-clamp surveys of PASMC electrical phenotypes have revealed greater functional expression of L-type calcium channels46 and Kv channels7,17,43 in resistance PASMCs, and more BKCa current in conduit PASMC.7 In addition to this longitudinal diversity, phenotypically and electrophysiologically distinct cell populations coexist side-by-side within a single segment, constituting an electrical mosaic.7 In the pulmonary circulation, SMC diversity is not restricted to the expression of ion channels. Frid et al identified 4 populations of PASMCs in calf conduit PAs, based on expression of contractile and cytoskeletal proteins (actin, myosin, calponin, desmin, and metavinculin).47 These "phenotypes" responded differently to chronic hypoxia, with increased proliferation exclusively in "meta-vinculin-negative" PASMCs.47 Regional heterogeneity of K+ channel expression has been reported in the heart48 and brain,49 where differences in distributions and properties of K+ channels contribute to regional differences in the seizure susceptibility.
In conclusion, K+ channel diversity is a common but underappreciated phenomenon that needs to be considered in the design and interpretation of vascular biology studies. It is the enrichment of resistance PASMCs with the O2-sensitive Kv channels 1.5 and Kv2.1 that accounts for the localization of HPV to this zone of the pulmonary circulation. Regional ionic diversity permits resistance PAs to match local perfusion to ventilation without increasing PVR and right ventricular afterload. The molecular basis for this ionic diversity requires further study.
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Acknowledgments |
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Drs. Michelakis and Archer are supported by the Canada Foundation for Innovation, the Alberta Heart and Stroke Foundation (H&SF), the Alberta Heritage Foundation for Medical Research (AHFMR), and the Canadian Institutes for Health Research (CIHR). Dr. Archer is supported by NIH-RO1-HL071115. Dr Thébaud is supported by CIHR, H&SF, and AHFMR.
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Footnotes |
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Original received January 23, 2004; revision received June 10, 2004; accepted June 16, 2004.
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References |
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- von Euler U, Liljestrand G. Observations on the pulmonary arterial blood pressure in the cat. Acta Physiol Scand. 1946; 12: 301–320.
- Weir EK, Archer SL. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB. 1995; 9: 183–189.[Abstract]
- Archer S, Michelakis E. The mechanism(s) of hypoxic pulmonary vasoconstriction: potassium channels, redox O(2) sensors, and controversies. News Physiol Sci. 2002; 17: 131–137.
[Abstract/Free Full Text] - Shirai M, Sada K, Ninomiya I. Effects of regional alveolar hypoxia and hypercaprnia on small pulmonary vessels in cats. J Appl Physiol. 1986; 61: 440–448.
[Abstract/Free Full Text] - Kato M, Staub N. Response of small pulmonary arteries to unilobar alveolar hypoxia and hypercapnia. Circ Res. 1966; 19: 426–440.
[Abstract/Free Full Text] - Bennie RE, Packer CS, Powell DR, Jin N, Rhoades RA. Biphasic contractile response of pulmonary artery to hypoxia. Am J Physiol. 1991; 261: L156–L163.[Medline] [Order article via Infotrieve]
- Archer SL, Huang JMC, Reeve HL, Hampl V, Tolarova S, Michelakis ED, Weir EK. Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ Res. 1996; 78: 431–442.
[Abstract/Free Full Text] - Madden J, Vadula M, Kurup V. Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells. Am J Physiol. 1992; 263: L384–L393.[Medline] [Order article via Infotrieve]
- Vadula MS, Kleinman JG, Madden JA. Effect of hypoxia and norepinephrine on cytoplasmic free Ca2+ in pulmonary and cerebral arterial myocytes. Am J Physiol. 1993; 265: L591–L597.[Medline] [Order article via Infotrieve]
- Hall SM, Hislop AA, Pierce CM, Haworth SG. Prenatal origins of human intrapulmonary arteries: formation and smooth muscle maturation. Am J Respir Cell Mol Biol. 2000; 23: 194–203.
[Abstract/Free Full Text] - Hall SM, Hislop AA, Haworth SG. Origin, differentiation, and maturation of human pulmonary veins. Am J Respir Cell Mol Biol. 2002; 26: 333–340.
[Abstract/Free Full Text] - Post J, Hume J, Archer S, Weir E. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am J Physiol. 1992; 262: C882–C890.[Medline] [Order article via Infotrieve]
- Yuan X-J, Goldman W, Tod M, Rubin L, Blaustein M. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am J Physiol. 1993; 264: L116–L123.[Medline] [Order article via Infotrieve]
- Smirnov S, Robertson T, Ward J, Aaronson P. Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells. Am J Physiol. 1994; 266: H365–H370.[Medline] [Order article via Infotrieve]
- Yuan X-J. Voltage gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary artery myocytes. Circ Research. 1995; 77: 370–378.
[Abstract/Free Full Text] - Archer SL, Huang J, Henry T, Peterson D, Weir EK. A redox based oxygen sensor in rat pulmonary vasculature. Circ Res. 1993; 73: 1100–1112.
[Abstract/Free Full Text] - Smirnov SV, Beck R, Tammaro P, Ishii T, Aaronson PI. Electrophysiologically distinct smooth muscle cell subtypes in rat conduit and resistance pulmonary arteries. J Physiol. 2002; 538: 867–878.
[Abstract/Free Full Text] - Pozeg ZI, Michelakis ED, McMurtry MS, Thebaud B, Wu XC, Dyck JR, Hashimoto K, Wang S, Moudgil R, Harry G, Sultanian R, Koshal A, Archer SL. In vivo gene transfer of the O2-sensitive potassium channel Kv1.5 reduces pulmonary hypertension and restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats. Circulation. 2003; 107: 2037–2044.
[Abstract/Free Full Text] - Archer SL, Souil E, Dinh-Xuan AT, Schremmer B, Mercier JC, El Yaagoubi A, Nguyen-Huu L, Reeve HL, Hampl V. 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. 1998; 101: 2319–2330.[Medline] [Order article via Infotrieve]
- Patel AJ, Lazdunski M, Honore E. Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes. EMBO J. 1997; 16: 6615–6625.[CrossRef][Medline] [Order article via Infotrieve]
- Osipenko ON, Tate RJ, Gurney AM. Potential role for kv3.1b channels as oxygen sensors. Circ Res. 2000; 86: 534–540.
[Abstract/Free Full Text] - Hulme JT, Coppock EA, Felipe A, Martens JR, Tamkun MM. Oxygen sensitivity of cloned voltage-gated K(+) channels expressed in the pulmonary vasculature. Circ Res. 1999; 85: 489–497.
[Abstract/Free Full Text] - Reeve HL, Michelakis E, Nelson DP, Weir EK, Archer SL. Alterations in a redox oxygen sensing mechanism in chronic hypoxia. J Appl Physiol. 2001; 90: 2249–2256.
[Abstract/Free Full Text] - Platoshyn O, Yu Y, Golovina VA, McDaniel SS, Krick S, Li L, Wang JY, Rubin LJ, Yuan JX. Chronic hypoxia decreases K(V) channel expression and function in pulmonary artery myocytes. Am J Physiol Lung Cell Mol Physiol. 2001; 280: L801—L812.
[Abstract/Free Full Text] - Michelakis ED, McMurtry MS, Wu XC, Dyck JR, Moudgil R, Hopkins TA, Lopaschuk GD, Puttagunta L, Waite R, Archer SL. Dichloroacetate, a metabolic modulator, prevents and reverses chronic hypoxic pulmonary hypertension in rats: role of increased expression and activity of voltage-gated potassium channels. Circulation. 2002; 105: 244–250.
[Abstract/Free Full Text] - Archer SL, London B, Hampl V, Wu X, Nsair A, Puttagunta L, Hashimoto K, Waite RE, Michelakis ED. Impairment of hypoxic pulmonary vasoconstriction in mice lacking the voltage-gated potassium channel Kv1.5. FASEB J. 2001; 15: 1801–1803.
[Free Full Text] - Vianna-Jorge R, Oliveira CF, Garcia ML, Kaczorowski GJ, Suarez-Kurtz G. Correolide, a nor-triterpenoid blocker of Shaker-type Kv1 channels elicits twitches in guinea-pig ileum by stimulating the enteric nervous system and enhancing neurotransmitter release. Br J Pharmacol. 2000; 131: 772–778.[CrossRef][Medline] [Order article via Infotrieve]
- Davies AM, Batchelor TJ, Eardley I, Beech DJ. Potassium channel KV alpha1 subunit expression and function in human detrusor muscle. J Urol. 2002; 167: 1881–1886.[CrossRef][Medline] [Order article via Infotrieve]
- Archer SL, Johnson GJ, Gebhard RL, Castleman WL, Levine AS, Westcott JY, Voelkel NF, Nelson DP, Weir EK. Effect of dietary fish oil on lung lipid profile and hypoxic pulmonary hypertension. J Appl Physiol. 1989; 66: 1662–1673.
[Abstract/Free Full Text] - Michelakis ED, Hampl V, Nsair A, Wu X, Harry G, Haromy A, Gurtu R, Archer SL. Diversity in mitochondrial function explains differences in vascular oxygen sensing. Circ Res. 2002; 90: 1307–1315.
[Abstract/Free Full Text] - Michelakis ED, Rebeyka I, Wu X, Nsair A, Thebaud B, Hashimoto K, Dyck JR, Haromy A, Harry G, Barr A, Archer SL. O2 sensing in the human ductus arteriosus: regulation of voltage-gated K+ channels in smooth muscle cells by a mitochondrial redox sensor. Circ Res. 2002; 91: 478–486.
[Abstract/Free Full Text] - He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A. 1998; 95: 2509–2514.
[Abstract/Free Full Text] - Archer SL, Gragasin FS, Wu X, Wang S, McMurtry S, Kim DH, Platonov M, Koshal A, Hashimoto K, Campbell WB, Falck JR, Michelakis ED. Endothelium-derived hyperpolarizing factor in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by activating smooth muscle BK(Ca) channels. Circulation. 2003; 107: 769–776.
[Abstract/Free Full Text] - Michelakis E, Rebeyka I, Bateson J, Olley P, Puttagunta L, Archer S. Voltage-gated potassium channels in human ductus arteriosus. Lancet. 2000; 356: 134–137.[CrossRef][Medline] [Order article via Infotrieve]
- Lopez-Barneo J, Lopez-Lopez J, Urena J, Gonzalez C. Chemotransduction in the carotid body: K+ current modulated by PO2 in type I chemoreceptor cells. Science Washington DC. 1988; 242: 580–582.
[Abstract/Free Full Text] - Conforti L, Millhorn DE. Selective inhibition of a slow-inactivating voltage-dependent K+ channel in rat PC12 cells by hypoxia. J Physiol (Lond). 1997; 502: 293–305.[CrossRef][Medline] [Order article via Infotrieve]
- Youngson C, Nurse C, Yeger H, Cutz E. Oxygen sensing in airway chemoreceptors. Nature. 1993; 365: 153–155.[CrossRef][Medline] [Order article via Infotrieve]
- Archer SL, Tolins JP, Reye L, Weir EK. Hypoxic pulmonary vasoconstriction is enhanced by inhibition of the synthesis of an endothelium derived relaxing factor. Biochem Biophys Res Comm. 1989; 164: 1198–1205.[CrossRef][Medline] [Order article via Infotrieve]
- Marshall C, Marshall BE. Hypoxic pulmonary vasoconstriction is not endothelium dependent. Proc Soc Exp Biol Med. 1992; 201: 267–270.[Abstract]
- Ward JP, Robertson TP. The role of the endothelium in hypoxic pulmonary vasoconstriction. Exp Physiol. 1995; 80: 793–801.[Abstract]
- Carlsson AJ, Bindslev L, Santesson J, Gottlieb I, Hedenstierna G. Hypoxic pulmonary vasoconstriction in the human lung: the effect of prolonged unilateral hypoxic challenge during anaesthesia. Acta Anaesthesiol Scand. 1985; 29: 346–351.[Medline] [Order article via Infotrieve]
- Yuan XJ, Wang J, Juhaszova M, Golovina VA, Rubin LJ. Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells. Am J Physiol. 1998; 274: L621—L635.[Medline] [Order article via Infotrieve]
- Hogg DS, Davies AR, McMurray G, Kozlowski RZ. K(V)2.1 channels mediate hypoxic inhibition of I(KV) in native pulmonary arterial smooth muscle cells of the rat. Cardiovasc Res. 2002; 55: 349–360.
[Abstract/Free Full Text] - Rodrigues AR, Arantes EC, Monje F, Stuhmer W, Varanda WA. Tityustoxin-K(alpha) blockade of the voltage-gated potassium channel Kv1.3. Br J Pharmacol. 2003; 139: 1180–1186.[CrossRef][Medline] [Order article via Infotrieve]
- Overturf KE, Russell SN, Carl A, Vogalis F, Hart PJ, Hume JR, Sanders KM, Horowitz B. Cloning and characterization of a Kv1.5 delayed rectifier K+ channel from vascular and visceral smooth muscles. Am J Physiol. 1994; 267: C1231–C1238.[Medline] [Order article via Infotrieve]
- Franco-Obregon A, Lopez-Barneo J. Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries. J Physiol. 1996; 491. 2: 511–518.
- Whorley JD, Frid MG, Orton EC, Belknap JK, Stenmark KR. Hypoxia selectively induces proliferation in a specific subpopulation of smooth muscle cells in the bovine neonatal pulmonary arterial media. J Clin Investigation. 1995; 96: 273–281.[Medline] [Order article via Infotrieve]
- Brahmajothi MV, Morales MJ, Liu S, Rasmusson RL, Campbell DL, Strauss HC. In situ hybridization reveals extensive diversity of K+ channel mRNA in isolated ferret cardiac myocytes. Circ Res. 1996; 78: 1083–1089.
[Abstract/Free Full Text] - Madeja M, Musshoff U, Speckmann EJ. Diversity of potassium channels contributing to differences in brain area-specific seizure susceptibility: sensitivity of different potassium channels to the epileptogenic agent pentylenetetrazol. Eur J Neurosci. 1997; 9: 390–395.[CrossRef][Medline] [Order article via Infotrieve]
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