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(Circulation Research. 2006;98:1072.)
© 2006 American Heart Association, Inc.

Cellular Biology

Impact of TASK-1 in Human Pulmonary Artery Smooth Muscle Cells

Andrea Olschewski, Yingji Li, Bi Tang, Jörg Hanze, Bastian Eul, Rainer M. Bohle, Jochen Wilhelm, Rory E. Morty, Michael E. Brau, E. Kenneth Weir, Grazyna Kwapiszewska, Walter Klepetko, Werner Seeger, Horst Olschewski

From the Department of Anaesthesiology (A.O., M.E.B.), Intensive Care Medicine, Medical University Graz, Austria; University of Giessen Lung Center (A.O., Y.L., B.T., J.H., B.E., R.E.M., W.S.), Medical Clinic II/V; and Department of Pathology (R.M.B., J.W., G.K.), Justus-Liebig University Giessen, Germany; Minneapolis VA Medical Center and University of Minnesota (E.K.W.); Department of Cardio-Thoracic Surgery (W.K.), Vienna University Hospital, Austria; and Division of Pulmonology (H.O.), Medical University Graz, Austria.

Correspondence to Andrea Olschewski, Department of Anaesthesiology, Intensive Care Medicine, Medical University Graz, Auenbruggerplatz 29, A-8029 Graz, Austria. E-mail andrea.olschewski{at}physiologie.med.uni-giessen.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K⁺) conductances. Although studies suggest that background K⁺ currents carried by 2-pore domain K⁺ channels are important regulators of resting membrane potential in PASMC, their role in human PASMC is unknown. Our study tested the hypothesis that TASK-1 leak K⁺ channels contribute to the K⁺ current and resting membrane potential in human PASMC. We used the whole-cell patch-clamp technique and TASK-1 small interfering RNA (siRNA). Noninactivating K⁺ current performed by TASK-1 K⁺ channels were identified by current characteristics and inhibition by anandamide and acidosis (pH 6.3), each resulting in significant membrane depolarization. Moreover, we showed that TASK-1 is blocked by moderate hypoxia and activated by treprostinil at clinically relevant concentrations. This is mediated via protein kinase A (PKA)-dependent phosphorylation of TASK-1. To further confirm the role of TASK-1 channels in regulation of resting membrane potential, we knocked down TASK-1 expression using TASK-1 siRNA. The knockdown of TASK-1 was reflected by a significant depolarization of resting membrane potential. Treatment of human PASMC with TASK-1 siRNA resulted in loss of sensitivity to anandamide, acidosis, alkalosis, hypoxia, and treprostinil. These results suggest that (1) TASK-1 is expressed in human PASMC; (2) TASK-1 is hypoxia-sensitive and controls the resting membrane potential, thus implicating an important role for TASK-1 K⁺ channels in the regulation of pulmonary vascular tone; and (3) treprostinil activates TASK-1 at clinically relevant concentrations via PKA, which might represent an important mechanism underlying the vasorelaxing properties of prostanoids and their beneficial effect in vivo.

Key Words: pulmonary circulation • potassium channels • TASK-1 • treprostinil • hypoxic pulmonary vasoconstriction

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The membrane potential of pulmonary artery smooth muscle cells (PASMC) is an important regulator of arterial tone. These cells have a resting membrane potential of approximately –65 to –50 mV in vitro, close to the predicted equilibrium potential for potassium (K⁺) ions. The opening of K⁺ channels in the PASMC membrane increases K⁺ efflux, which causes membrane hyperpolarization. This closes voltage-dependent Ca²⁺ channels, decreasing Ca²⁺ entry and leading to vasodilatation. Conversely, inhibition of K⁺ channels causes membrane depolarization, Ca²⁺ entry, cell contraction, and vasoconstriction.

Background or leak K⁺-selective channels, as defined by a lack of time and voltage dependency, play an essential role in setting the resting membrane potential and input resistance in excitable cells. Two-pore domain K⁺ (2-PK) channels have been shown to conduct several leak K⁺ currents. The activity of 2-PK channels is strongly regulated by protons, protein kinases, and hypoxia. Alteration of K⁺ conductance can influence cellular activity via membrane potential changes.

Both RT-PCR and Northern blot analyses performed using mammalian lung tissue identified mRNA transcripts for several 2-PK channels^1,2 Recently, TASK-1, a member of the 2-PK channel family was described in rabbit PASMC³ and in rat pulmonary arteries.⁴ This channel produces K⁺ currents that possess all of the characteristics of background conductances. The activity of TASK-1 is strongly dependent on the external pH and oxygen tension, suggesting that this particular channel might be a sensor of external pH variations and of hypoxia in pulmonary arteries.

To our knowledge, the present report is the first demonstration of the expression of TASK-1 in human PASMC (hPASMC). Our study tested the hypothesis that TASK-1 contributes to the K⁺ current and resting membrane potential by using TASK-1 small interfering RNA (siRNA). We show, moreover, that TASK-1 is sensitive to acute hypoxia and activated by treprostinil, a stable analog of prostacyclin via a protein kinase (PKA)-dependent pathway. Thus it may provide an important mechanism for the prostanoid-induced relaxation of pulmonary arteries.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of Human Primary Pulmonary Artery Smooth Muscle Cells and Cell Culture
Primary SMC were isolated from human pulmonary arteries from 3 unused donor lungs harvested for lung transplantation (see the online data supplement available at http://circres.ahajournals.org). The study protocol for tissue donation was approved by the "Ethik-Kommission am Fachbereich Humanmedizin der Justus-Liebig-Universitaet Giessen of the University Hospital Giessen" in accordance with German law and with Good Clinical Practice/International Conference on Harmonisation guidelines. Written informed consent was obtained from each individual patient or the next of kin of the patient. Cultured hPASMC from 10 donors were purchased from Cambrex (Walkersville, Md) or from Cascade Biologics (Mansfield, UK).

Electrophysiology
The whole-cell patch-clamp technique on hPASMC was used as previously described to measure the resting membrane potential under current clamp and macroscopic K⁺ currents under voltage clamp (see the online data supplement).^3,5,6

Solutions and Chemicals
All compounds were purchased from Sigma Chemical Co (St Louis, Mo). Treprostinil was a gift from Lung RX (Satellite Beach, Fla) (see the online data supplement).

Relative mRNA Quantification
Real-time PCR was used for relative quantification of the TASK-1, TASK-2, TASK-3, PRKCA, and PRKCE mRNA (see the online data supplement).

Design and Transfection of siRNA for Human TASK-1
The target sequence of siRNA is localized 261 bases downstream from the start codon of human TASK-1 (GenBank accession no. AF006823). The forward and reverse strands (UCA CCG UCA UCA CCA CCA U dTdT) and (AGU GGC AGU AGU GGU GGU A dTdT) with two 5' deoxy-thymidine overhangs were commercially synthesized (Eurogentec, Seraing, Belgium) and annealed at a final concentration each of 20 µmol/L by heating at 95°C for 1 minute and incubating at 37°C for 1 hour in annealing buffer (20 mmol/L Na-acetate, 6 mmol/L HEPES–KOH [pH 7.4], and 0.4 mmol/L Mg–acetate). Transfection of siRNA was performed at a final concentration of 40 nmol/L using Oligofectamin (Invitrogen). As a control, a random siRNA sequence (siRNA-ran: UAC ACC GUU AGC AGA CAC C dTdt) prepared as described above was used. RNA and electrophysiological measurements were performed 48 to 72 hours after transfection of siRNAs. For assessment of transfection efficiency, we used an fluorescein isothiocyanate (FITC)-conjugated siRNA (QIAGEN), the intracellular location of which was assessed by direct visualization of the FITC by fluorescence microscopy after transfection.

Immunoprecipitation
Cultured smooth muscle cells were solubilized as described previously⁷ in an extraction buffer supplemented with 1 mmol/L sodium orthovanadate, 10 mmol/L sodium pyrophosphate, 5 mmol/L ß-glycerophosphate, and 50 mmol/L sodium fluoride. The TASK-1 was immunoprecipitated from cell lysates (4 hours; 4°C) using rabbit anti–TASK-1 (Alomone Labs, Jerusalem, Israel).⁸ Antibodies were chemically coupled to protein A–Sepharose beads using a Seize Immunoprecipitation Kit (Pierce, Rockford, Ill). Immunoprecipitates were washed to a final stringency of 470 mmol/L NaCl in extraction buffer. Immunoprecipitates were resolved on a 10% Tris–Tricine SDS-PAGE gel, and blots were probed with rabbit anti–TASK-1 (1:500; Alomone Labs), mouse anti-phosphotyrosine (1:1750; Cell Signaling Technologies, Beverly, Mass), or mouse anti-phospho(Ser/Thr) (1:1000; Cell Signaling Technologies) antibodies.

Immunofluorescence Staining
Immunofluorescence was performed as previously described, using 2 different antibodies directed against unique domains in TASK-1: the first at the amino terminus (Santa Cruz Biotechnology) and the other at residues 252 to 269 in the carboxy terminal end of the protein (Alomone Labs)³ (online data supplement).

Statistical Analysis
Numerical values are given as means±SE of n cells. Intergroup differences were assessed by a factorial analysis of variance with post hoc analysis with Fisher’s least-significant difference test or Student’s unpaired and paired t tests as appropriate. Probability values of <0.05 were considered significant. The mean data at different anandamide concentrations were fitted.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of TASK-1 Channels in hPASMC
The presence of TASK-1 protein was established with 2 anti–TASK-1 antibodies directed against the amino or carboxy terminal regions of the protein (n=4; Figure 1A and 1B). In contrast, staining was absent from control cells treated in the same way but without exposure to TASK-1 antibody (Figure 1C and 1D). PCR studies demonstrated the presence of TASK-1, TASK-2, and TASK-3 in human brain tissue, but only TASK-1 mRNA was detected in primary and in cultured hPASMC as well (Figure 2A).

View larger version (12K): [in this window] [in a new window]   Figure 1. TASK-1 expression in hPASMC. Fluorescence images of hPASMC stained with the Alomone C-terminal TASK-1 and anti-rabbit Alexa 555 antibodies (A) or Santa Cruz N-terminal TASK-1 with anti-goat IgG-FITC antibodies (B). Staining is absent in control cells treated identically but without exposure to primary antibody, as shown by matched fluorescence (C and D, respectively).

View larger version (30K): [in this window] [in a new window]   Figure 2. IKN in primary PASMC. A, Representative gel shows mRNA expression of TASK-1 (91 bp), TASK-1 (94 bp), and TASK-3 (104 bp) in human brain (B), primary hPASMC (F), and cultured hPASMC (K). Only TASK-1 is expressed in primary hPASMC (F) and cultured hPASMC (K). Arrow indicates 100 bp; M, the molecular weight marker used to indicate the size of the PCR fragments; N, no template control. Identical results were obtained with at least 3 additional preparations of RNA. B, top, Effect of 10 µmol/L anandamide on IKN recorded during ramp, voltage protocol inset (left) and difference current trace, obtained by subtracting current amplitudes in the presence of TASK-1 blocker from those obtained under control conditions (right). Difference current reversed close to –84 mV, as expected for a K+-selective conductance under these conditions. Bottom, IKN evoked under symmetrical (155 K+) conditions by ramp in the same cell (voltage protocol above), before (high-K+) and during application of 10 µmol/L anandamide (left) and trace of difference currents trace (right). Difference current was voltage independent and reversed close to 0 mV, as expected for a K+-selective conductance under these conditions. C, Histogram summarizing the effect of 10 µmol/L anandamide on IKN calculated at 0 mV in cultured and primary hPASMC (n=5 each group). I/I0 is the current in the presence of anandamide expressed as a fraction of the current before anandamide application. EK-Nernst indicates Nernst equilibrium potential.

TASK-1 Was Functionally Expressed in hPASMC
Anandamide, a member of endogenous cannabinoids, was recently shown to be a direct and selective blocker of TASK-1 channels.^3,9 As depicted in Figure 2, application of 10 µmol/L anandamide markedly but reversibly inhibited the noninactivating K⁺ current (I_KN) in primary hPASMC from 16±2 to 6±2 pA (P<0.05), recorded at 0 mV after the cells were clamped at 0 mV for least 5 minutes to inactivate voltage-dependent K⁺ channels. Figure 2B shows a representative recording from the holding potential of 0 mV; the voltage was stepped to 60 mV and then ramped to –100 mV over a period of 1.6 seconds. The current during the ramp reflects I_KN parallel with a nonspecific "leak" current and reverses direction at the resting potential of the hPASMC.^3,5 As a consequence of inhibition of the outward current, anandamide also influenced the reversal potential of the current (Figure 2B, top). The "difference" current, obtained by subtracting the current remaining in the presence of anandamide from that obtained under control conditions (Figure 2B, bottom, inset), was reversed close to –84 mV, the calculated Nernst equilibrium potential for K⁺ under these conditions. Maintenance of a resting membrane potential in rabbit PASMC has been proposed to involve TASK-1.³ Indeed, anandamide significantly depolarized hPASMC (9±1 mV; n=5; P<0.05). Under symmetrical K⁺ conditions in the same cell, anandamide caused marked inhibition of K⁺ current (Figure 2B, bottom). The difference current (inset) was linear and reversed at or near 0 mV, the calculated Nernst equilibrium potential for K⁺ under these conditions. This pharmacological and biophysical profile demonstrated the functional expression of the K⁺-selective background channel TASK-1 in primary hPASMC.

In cultured hPASMC, a functional expression of kinetically identical K⁺ channels was recorded (Figure 3). The anandamide concentration-response curve for TASK-1 indicated an apparent IC₅₀ of 0.81±0.08 µmol/L (n=5; Figure 3B). The effect of 10 µmol/L anandamide was reversible (Figure 3C). It is noteworthy that the current block by anandamide recorded at 0 mV was similar in both primary and cultured hPASMC (Figure 2C). Whole-cell K⁺ current (I_K) in cultured hPASMC was also investigated at a holding potential of –80 mV. Current was activated with 250-ms depolarizing pulses from –80 to +70 mV in 10-mV steps. The mean current was measured during the last 150 ms of the voltage-clamp pulse and plotted against the test potential to achieve a steady-state current-voltage relationship. Conductance was obtained from linear regression of the data points between –60 and 0 mV, with correlation coefficients greater than 0.95 in all cases. We focused on the anandamide sensitivity of the whole-cell current. The effect of the drug on I_K is illustrated in Figure 3D. Consistent with the blocking effect of anandamide on I_KN, a significant reduction in conductance resulted when anandamide was applied. Data are given in the Table.

View larger version (21K): [in this window] [in a new window]   Figure 3. IKN in cultured PASMC. A, top, Effect of 10 µmol/L anandamide on IKN recorded during ramp, voltage protocol inset (left), and difference currents trace, obtained by subtracting current amplitudes in the presence of TASK-1 blocker from those obtained under control conditions (right). Difference current reversed close to –84 mV, as expected for a K+-selective conductance under these conditions. Bottom, IKN evoked under symmetrical (155 K+) conditions by ramp in the same cell (voltage protocol above), before (high-K+) and during application of 10 µmol/L anandamide (left) and difference currents trace (right). Difference current was voltage independent and reversed close to 0 mV, as expected for a K+-selective conductance under these conditions. B, Concentration-response curve for anandamide. I/I0 is the current in the presence of anandamide expressed as a fraction of the current before anandamide application. The line is the best fit to the Hill equation using an IC50 of 1.2±0.3 µmol/L and Hill coefficient of 0.9±0.1. C, Effect of 10 µmol/L anandamide on IKN recorded at 0 mV. The current was markedly and reversibly inhibited during application of anandamide. D, Current–voltage relationship for whole-cell K+ current before and after application of 10 µmol/L anandamide, activated from a holding potential of –80 mV during 250-ms depolarizing pulses from –80 to +70 mV. Lines indicate voltage range used for conductance calculation by means of linear regression. The inset shows the kinetics of the anandamide-sensitive current. EK-Nernst indicates Nernst equilibrium potential.

View this table: [in this window] [in a new window]   Table 1. Conductances in Native and siRNA-Transfected hPASMC

The essential property of the TASK-1 channels is their extreme sensitivity to variations in extracellular pH (pH_o) in a narrow physiological range. Figure 4A shows the pH_o dependence of I_KN of primary and cultured hPASMC across the full voltage range over which I_KN is apparent. Modification of I_KN by pH_o was reflected in resting membrane potential. A pH of 8.3 significantly hyperpolarized the cells, whereas acidification caused membrane depolarization in primary hPASMC (–10±1 mV versus 13±2 mV; n=5). Changes in whole-cell conductance caused by variation in pH_o in cultured hPASMC were significant (Table). Overall, we found a functional expression of kinetically and pharmacologically identical background K⁺ current carried by TASK-1 in both primary and cultured hPASMC. To provide more certain evidence for involvement of TASK-1 in I_KN, we tested the effect of tetraethylammonium (TEA) (10 mmol/L), 4-aminopyridine (4-AP) (3 mmol/L), ruthenium red (5 µmol/L), and different concentrations of ZnCl₂ (online data supplement). As expected, TEA and 4-AP failed to inhibit I_KN. In contrast, ruthenium red caused a reduction of I_KN. However, the ruthenium red–sensitive current was linear and the reversal potential varied between 0 and –20 mV, where the calculated Nernst equilibrium potential was –84 mV for K⁺. It is well known that this compound is active at various sites (eg, ryanodine receptors¹⁰ and TRPV channels¹¹); therefore, this effect in primary hPASMC may not be surprising. The divalent zinc cation was also reported to inhibit TASK-1 at higher concentrations. However, in our study, the Zn-sensitive ion current had a reversal potential close to 0 mV, indicating the presence of a nonspecific leak current.

View larger version (26K): [in this window] [in a new window]   Figure 4. Modulation of IKN by pH in primary and cultured hPASMC. A, Representative time-course plot of IKN at different pHo in cultured (top) and in primary (bottom) hPASMC recorded by ramp, activated from a holding potential of 0 mV during 1.6 second from 60 to –100 mV. B, Relationship between pHo and IKN measured at 0 mV. Data were fit with a Boltzmann relationship with pH0.5 7.4 for cultured and pH0.5 7.3 for primary hPASMC.

Taken together, both the potency of anandamide and pH 6.3 for blocking I_KN, and pH 8.3 for activating it, together with their effects on measured resting membrane potential (E_m), strongly suggest that I_KN maintains the resting membrane potential in both primary and cultured hPASMC.

Modulation of I_KN by Treprostinil
Prostaglandins possess a potent vasodilatory activity mediated by the stimulation of adenylate cyclase, with a subsequent increase in intracellular cAMP levels that is associated with the opening of Ca²⁺-activated K⁺ channels (K_Ca). The increased K⁺ conductance results in cell hyperpolarization and block of L-type Ca²⁺ channels, resulting in vasodilation. We used treprostinil, a stable prostacyclin analog, at a clinically relevant concentration, to test whether I_KN in hPASMC is also a treprostinil-sensitive conductance. As illustrated by a single trace in Figure 5A, 10 nmol/L treprostinil enhanced I_KN, recorded at 0 mV (from 10±1 to 18±2 pA; n=6; P<0.01). The treprostinil concentration-response curve for TASK-1 in primary hPASMC gave an IC₅₀ of 1.20±0.11 µmol/L (n=4; Figure 5C). Coapplication of 10 nmol/L treprostinil with 100 nmol/L iberiotoxin, a potent blocker of K_Ca, did not lead to a significant decrease in treprostinil-induced activation of I_KN (from 11±1 to 18±2 pA; n=5; P<0.05; online data supplement). Consistent with the involvement of TASK-1 channels in this activation, it was blocked when anandamide was previously applied earlier (online data supplement). Activation of TASK-1 with 10 nmol/L treprostinil was associated with 9-mV hyperpolarization of the resting membrane potential (from –42±3 to –51±4 mV; P<0.01).

View larger version (28K): [in this window] [in a new window]   Figure 5. PKA-dependent activation of TASK-1 by treprostinil. A and B, Representative time-course plot of treprostinil application (10 nmol/L) on IKN recorded at 0 mV in cultured hPASMC (A) and by ramp in primary hPASMC (B), activated from a holding potential of 0 mV during 1.6 second from 60 to –100 mV. C, Concentration-response curve for treprostinil in primary hPASMC. I/I0 is the current in the presence of treprostinil expressed as a fraction of the current before treprostinil application. The line is the best fit to the Hill equation using an IC50 of 1.2±0.1 µmol/L and Hill coefficient of 0.9±0.1. D, Typical recording of the effects of the cell membrane permeable cAMP analog 8-br-cAMP (100 µmol/L) on IKN recorded at 0 mV in cultured hPASMC. F, Lack of effect of treprostinil on IKN after preincubation with 300 nmol/L KT 5720 in cultured hPASMC. E, Tyrosine (top) and serine/threonine (middle) phosphorylation of TASK-1 in treprostinil-treated and untreated (control) groups. Protein-loading equivalence is shown by probing immunoprecipitates with an anti–TASK-1 antibody in the bottom panel. Cultured hPASMC were used in D through F.

In rat tail artery smooth muscle cells, an activation of K_Ca by iloprost, another prostacyclin analog, was hypothesized to be mediated by cAMP-dependent PKA-induced phosphorylation of the channel.¹² To address the question of whether this pathway is important for TASK-1 activation by treprostinil, we chose a pharmacological approach to investigate the effects of PKA on I_KN. In whole-cell recordings, the direct application of the cell membrane–permeable cAMP analog 8-br-cAMP (an endogenous PKA activator) resulted in a significant increase of I_KN (from 9±2 to 17±2 pA; n=5; P<0.05; Figure 5D). The pretreatment of the cells with KT5720 (300 nmol/L), a specific inhibitor of cAMP-dependent protein kinase, abolished the effect of treprostinil on I_KN and on whole-cell K⁺ current, as shown in Figure 5E and the Table. We sought to investigate the effects of treprostinil exposure on the phosphorylation of TASK-1 in hPASMC. The PASMC were incubated for 1 hour with 10 nmol/L treprostinil. No differences in the tyrosine phosphorylation of TASK-1 were observed when treprostinil-treated and untreated groups were compared (Figure 5F, top). In contrast, a 1-hour incubation with treprostinil (10 nmol/L) stimulated the serine/threonine phosphorylation of TASK-1, as was evident in TASK-1 immunoprecipitates probed with an anti-phospho(Ser/Thr) antibody (Figure 5F, middle). Protein loading equivalence was demonstrated by probing immunoprecipitates with an anti–TASK-1 antibody (Figure 5F, bottom). These results suggest that, in hPASMC, TASK-1 channels are activated by treprostinil primarily through cAMP-dependent pathways.

TASK-1 Knockdown Results in Depolarization of hPASMC
To further confirm the role of TASK-1 channels in regulation of E_m in hPASMC, we knocked down TASK-1 expression in the cells using TASK-1 siRNA. Electrophysiological measurements were performed 24 to 48 hours after transfection of siRNAs. We found that the TASK-1 siRNA efficiently suppressed TASK-1 mRNA levels without affecting other widespread enzyme systems such as protein kinase C (PKC) (Figure 6A). The knockdown of TASK-1 caused a depolarization of resting membrane potential compared with the control cells (–26±1 mV versus –40±2; P<0.05; Figure 6B). Pretreatment of hPASMC with TASK-1 siRNA resulted in lack of significant further suppression of the I_KN by anandamide or acidosis as shown in Figure 6C (from 3±1 to 2±1 pA, from 3±1 to 3±1 pA, respectively). There was no significant activation by alkalosis or treprostinil (from 4±1 to 3±1 pA, from 2±1 to 2±1 pA, respectively). The whole-cell conductance in siRNA-pretreated cells was significantly lower, as illustrated in the Table. Changes in whole-cell conductance under variation of pH_o were not significant (Table). Modulation of I_KN by pH, anandamide, and treprostinil in hPASMC transfected with scrambled sequence TASK-1 siRNA are shown in the online data supplement.

View larger version (21K): [in this window] [in a new window]   Figure 6. Lack of effect of anandamide, pHo, and treprostinil on IKN in siRNA-transfected hPASMC. A, Regulation of human TASK-1 (hTASK-1), human PKC (hPKCA), and human PKC (hPKCE) as measured by real-time RT-PCR (*P<0.05, 1-sample t test with H0: Ct=0). The Ct values are calculated as described in Materials and Methods. Positive values indicate upregulation of the target gene compared with control; negative values indicate downregulation. The Ct values are approximately proportional to the binary logarithm of the fold change. B, Resting membrane potential of native control and transfected cells shown left (*P<0.01). The Em was significantly depolarized in transfected cells. Lack of changes in Em under treprostinil, anandamide, or pHo in transfected cells (n=5 each group). C, Effect of 10 nmol/L treprostinil, 10 µmol/L anandamide, and pHo on remaining IKN recorded at 0 mV.

TASK-1 Is Inhibited by Acute Hypoxia in hPASMC
Because previous work implicated the TASK-1 channels as an important molecular component of the O₂-sensitive K⁺ current in PASMC, we investigated the O₂ sensitivity of this channel in primary hPASMC. Representative traces recorded by ramp, before and after five minutes of exposure to hypoxic solutions, are shown in Figure 7A. The hypoxia-sensitive current reversed close to –84 mV. Consistent with the involvement of TASK-1 channels in this inhibition, it was not further blocked when anandamide was additionally applied (Figure 7A). Under current-clamp conditions, hypoxia caused marked cell depolarization (by 10±1 mV; n=9; P<0.05) Pretreatment of hPASMC with TASK-1 siRNA resulted in lack of significant suppression of the I_KN by hypoxia (Figure 7B). Figure 7C summarizes the mean data confirming that hypoxia significantly inhibited TASK-1 in both primary and cultured hPASMC.

View larger version (24K): [in this window] [in a new window]   Figure 7. Hypoxia blocks TASK-1 in primary and cultured hPASMC. A, Effect of hypoxia on IKN recorded by ramp in primary hPASMC (left) and difference currents trace, obtained by subtracting current amplitudes in the presence of hypoxia from those obtained under control conditions (middle). Difference current reversed close to –84 mV, as expected for a K+-selective conductance under these conditions. Application of 10 µmol/L anandamide under hypoxic conditions did not cause a further change in the current (right), suggesting that the hypoxia-sensitive current is carried by TASK-1. B, Lack of hypoxia effect on IKN in siRNA-transfected hPASMC. (C) Histogram summarizing the effect of hypoxia and anandamide (anan) under hypoxic conditions on IKN calculated at 0 mV in primary, cultured and siRNA-transfected hPASMC (n=5 each group; **P<0.01). EK-Nernst indicates Nernst equilibrium potential. EK-Nernst indicates Nernst equilibrium potential.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Smooth muscle cells have negative resting potentials that are important in regulating the excitability and contractile properties of tissues using these cells for force development. The resting membrane is regulated by K⁺ conductances that maintain the resting membrane potential close to the K⁺ equilibrium potential. At least 3 classes of potassium channels have been identified in PASMC: voltage-dependent potassium channels (K_v),^13–15 calcium-activated potassium channels (K_Ca),^16,17 and ATP-sensitive potassium channels (K_ATP).¹⁸ Although a new group of K⁺ channels, so-called background K⁺ channels, escaped detection at the molecular level for many years, it has now became clear that the newly identified KCNK family of K⁺ channel subunits also contributes to K⁺ current in smooth muscle cells.^3,4 When members of this superfamily of 2-PK channels are functionally expressed, they give rise to K⁺-selective currents that are open at all voltages, in contrast to K_v, K_Ca, or K_ATP channels whose activity is controlled by voltage or metabolic regulation. The decrease in this leak K⁺ conductance leads to cell depolarization that enhances the open probability of L-type Ca²⁺ channels in smooth muscle cells, causing periodic Ca²⁺ entry and vasoconstriction.

In this study, we demonstrated expression of TASK-1 mRNAs and proteins in human pulmonary artery smooth muscle cells. We found an anandamide-sensitive conductance in both primary and cultured hPASMC that had properties similar to TASK-1 channels. The native conductance showed an outward rectification in low external K⁺ solution, was instantaneous and noninactivating, was activated by alkalotic pH, and was blocked by anandamide. Transfection of TASK-1 siRNA into hPASMC significantly depolarized the resting membrane potential and abolished the effect of pH or anandamide. Furthermore, we were able to show the hypoxia sensitivity of the TASK-1 current. We conclude that TASK-1 channels are responsible for the hypoxia- and pH-sensitive, voltage-independent background conductance that sets the resting membrane potential in hPASMC.

The results of our experiments exclude the possibility that classical voltage-sensitive, Ca²⁺-sensitive, and ATP-sensitive K⁺ channels play a significant role in mediating the observed pH-induced changes in the membrane potential. The sensitivity of I_KN to small changes in pH has been reported in many different cell types.^2,19,20 Local changes in pH caused by physiological or pathophysiological conditions such as hypo- or hyperventilation or ischemia in the pulmonary vascular bed may result in acidosis or alkalosis and lead to changes in pulmonary artery pressure. Our data suggest that effects on pH-sensitive ion channels may also explain the acidosis-induced vasoconstriction or the alkalosis-induced vasodilatory responses. Furthermore, anandamide, a selective blocker of TASK-1 significantly reduced the I_KN and caused a depolarization similar to that seen at pH 6.4. This is consistent with observations reported in earlier studies.^3,9,21 With a symmetrical K⁺ distribution, anandamide inhibited a voltage-independent current with a reversal potential close of 0 mV, demonstrating the K⁺ selectivity of the anandamide-sensitive current. To investigate the contribution of TASK-1 to whole-cell K⁺ current, experiments were performed recording the whole-cell current from the holding potential of –80 mV, which allowed the acquisition of voltage-activated and background K⁺ currents. Although the inhibitory effects of acidosis or anandamide were less pronounced, the endocannabinoid anandamide still caused a significant reduction in the cell conductance. Together, these data provide unequivocal biophysical and pharmacological evidence for the functional expression of TASK-1 in both primary and cultured hPASMC.

The application of small interfering RNA provided further evidence to link resting membrane potential and TASK-1. The resting membrane potential and the cell conductances were significantly decreased in transfected cells. Moreover, the loss of the sensitivity of the membrane potential to significant depolarization by anandamide and pH 6.4, or to significant hyperpolarization by pH 8.4 in transfected cells, strongly implicates TASK-1 as a major contributor to the resting membrane potential.

Treprostinil, a stable analog of prostacyclin acts as a potent pulmonary vasodilator. In our experiments, treprostinil enhanced I_KN carried by TASK-1 in hPASMC. The current increase induced by treprostinil exhibited pharmacological saturation and reached a plateau as a consequence. Addition of 8-br-cAMP, a membrane-permeable analog of cAMP, exhibited similar effects. The activation of I_KN was still detected during coapplication of treprostinil with ITX, a selective blocker of Ca²⁺-activated K⁺ channels, but treprostinil did not show any effect after pretreatment with anandamide. The activation of I_KN by treprostinil was abolished after preincubation with KT5720, an inhibitor of cAMP-dependent protein kinase. Moreover, our data indicate that treprostinil can stimulate the serine or threonine phosphorylation of TASK-1 and are consistent with the detection of phosphoserine in TASK-1²² and the presence of consensus serine and threonine PKA and PKC phosphorylation sites in the TASK-1 peptide.² Whereas a tyrosine kinase consensus site is also present in the TASK-1 peptide,² treprostinil did not effect tyrosine phosphorylation of TASK-1. There is accumulating evidence that intracellular protein kinases undertake important modulation of 2-PK channels. Members of the 2-PK superfamily such as TOK1 and TWIK-1 currents are potentiated by activators of PKC, whereas TREK-1 or TREK-2 currents are inhibited.^23–25 When human TASK-1 was expressed in Xenopus oocytes, the current was insensitive to activation of adenyl cyclase by forskolin or IBMX.² In another study, the activation of PKA inhibited TASK-1 cloned from rat cerebellum,²¹ whereas we observed PKA-mediated activation of TASK-1 by treprostinil and by 8-br-cAMP in hPASMC. This disparity in effects between these results could be related to clone specificity (human versus rat) or to preparations (oocytes versus native PASMC) with possible differences between cAMP levels and PKA activity.

Acute hypoxic inhibition of K⁺ channels is a critical step in regulatory processes designed to link lowering of O₂ levels to cellular responses. It is often questioned whether the K⁺ channels are active at sufficiently negative potentials to set the resting membrane potential of PASMC and whether K⁺ channels could mediate hypoxic pulmonary vasoconstriction. TASK-1 with the biophysical profile of a background K⁺ channel could be the perfect candidate for the initiation of hypoxia–induced depolarization in PASMC.

In conclusion, we demonstrated that TASK-1, a member of the 2-PK channel superfamily is expressed in human both primary and cultured hPASMC. TASK-1 controls the resting membrane potential, thus implicating TASK-1 channels in the regulation of pulmonary vascular tone. This channel is activated by c-AMP and may be one of the main targets for the beneficial action of prostanoids in therapy of pulmonary arterial hypertension.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (SFB 547 "Cardiopulmonary Vasculature"). Excellent technical assistance from Brigitte Agari, Barbara Fröhlich, Sabine Gräf-Höchst, Christiane Hild, Esther Kuhlmann, Maria M. Stein, and Caroline Zörb is greatly appreciated.

	Footnotes

 
W.S. and H.O. are consultants for Lung RX (Satellite Beach, Fla).

Original received June 27, 2005; resubmission received December 14, 2005; revised resubmission received March 7, 2006; accepted March 20, 2006.

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