American Journal of Respiratory Cell and Molecular Biology

Multiple types of ATP-sensitive potassium (KATP) channels have been described in smooth muscle, including those inhibited by ATP and those activated by nucleotide diphosphate (KNDP). The molecular identities of these channels have been proposed to be SUR2B/Kir6.2 and SUR2B/Kir6.1, respectively. However, subunit expression is largely unknown in vascular muscle, and the native channel has not been reported previously in human tissue. We used the patch-clamp technique to examine KATP channel properties in cultured human pulmonary artery smooth muscle cells (HPASMC). Under physiological recording conditions, levcromakalim (10 μ M) hyperpolarized cells ( ∼ 25–30 mV) and activated a glibenclamide-sensitive, background K+ current, which was smaller in proliferating cells. Lowering ATP from 1 to 0.1 mM significantly enhanced responses to levcromakalim in HPASMC but not in HEK-293 cells stably transfected with SUR2B/Kir6.1. In both cell types, levcromakalim activated a 28–29 pS channel, which, upon patch excision, required the presence of nucleotide diphosphate for significant openings. Transcripts for SUR2B and Kir6.1, but not Kir6.2, were found by reverse transcription-polymerase chain reaction in HPASMC and in rat pulmonary arterial tissue. We conclude that KATP channels are expressed in human pulmonary artery, and whereas data are consistent with the presence of nucleotide diphosphate–activated potassium channels, native whole-cell regulation cannot be reconstituted fully in heterologous expression systems.

ATP-sensitive K+ (KATP) channels are distributed widely throughout the cardiovascular system and are characteristically activated by declining cytosolic ATP or elevated nucleotide-diphosphate (NDP) concentrations, thus providing a link between metabolism and membrane excitability (1-3). These channels are inhibited by sulfonylurea agents like glibenclamide and opened by K+ channel openers (KCO) such as pinacidil and levcromakalim, which in smooth muscle results in membrane hyperpolarization and vasorelaxation (3, 4). Since their initial identification in rabbit mesenteric artery, KATP channels and macroscopic currents have now been identified in several different vascular tissues of animal origin (4), although so far currents have only been described in human mesenteric artery (5). In general, whole-cell currents are time- independent, displaying little voltage dependence, and in most tissues, lowering pipette ATP can significantly increase currents (6-10). Similarly, responses to KCO are enhanced at low intracellular ATP concentrations (4, 8, 10), including in pulmonary artery (9). In a number of blood vessels, KATP channels contribute to the maintenance of the resting membrane potential and can be regulated in opposing fashion by endogenous vasoactive agents that activate either cyclic adenosine 5′ monophosphate-dependent protein kinase A or protein kinase C (4). Thus, these channels are likely to play an important role in the regulation of arterial tone.

At the molecular level, the KATP channel is a complex of at least two subunits, the sulfonylurea receptor (SUR) and a pore-forming subunit of the inward rectifier (Kir) family (Kir6.x) (2, 3). Functional channels are formed by the assembly of four SUR and four channel-forming subunits. The pore is thought to confer ATP inhibition and determine conductance, whereas the SUR is considered the primary target for sulfonylureas, KCO, and NDP (2). The identities of the pancreatic (SUR1/Kir6.2) and cardiac (SUR2A/Kir6.2) channels seem established (2, 3, 11). Recently, two smooth muscle–like clones were identified, SUR2B/Kir6.2 and SUR2B/Kir6.1. The reconstituted SUR2B/ KIR6.2 channel has a conductance of 80 pS and is inhibited by ATP, whereas the SUR2B/Kir6.1 channel has a lower conductance of 33 pS and is activated by NDP and ATP (12-14). It is possible that these channels underlie the classic KATP and the NDP-dependent K+ (KNDP) channels, which have both been found in rat and rabbit portal vein myocytes (15-17), although these clones do not describe fully the properties of channels found in other tissues (4). Our own data provide strong biochemical and electrophysiologic evidence that Kir6.2 or Kir6.1 can form mixed heteromultimers (18), possibly explaining the intermediate conductances, and variable sensitivities to ATP and NDP found in native smooth muscle tissues (4). So far molecular characterization is limited to visceral smooth muscle, where mRNA for SUR2B and Kir6.2, but not Kir6.1, has consistently been reported in smooth muscle cells (19-21). In guinea-pig bladder, the presence of SUR1 has also been detected (20). Thus, it will be important to determine the relative expression of KATP channel components in vascular smooth muscle that might give rise to the native current. Differences in expression are likely to give rise to diverse physiological roles and pharmacological properties of these channels.

The aim of the present study was to identify and examine the properties of KATP channels in cultured human pulmonary artery smooth muscle cells (HPASMC) and to determine the expression of KATP channel components. So far there has been little characterization of KATP channels in human tissue, and no single-channel data exist for pulmonary artery from any species. To further elucidate the molecular identity of the native currents, we compared whole-cell regulation of the smooth muscle clone SUR2B/ Kir6.1 expressed in HEK-293 cells under similar experimental conditions. Some of the preliminary observations have been presented to the American Physiological Society in abstract form (22).

Cell Culture

Cultured HPASMC (fourth passage) were obtained from Clonetics (Walkersville, MD) and plated in smooth muscle growth medium (SmGM; Clonetics) supplemented with gentamicin (50 μg/ ml), human epidermal (0.5 μg/ml) and fibroblast growth factors (1.0 μg/ml), insulin (5 μg/ml), and 5% fetal bovine serum. Cells were grown at 37°C in a humidified atmosphere of 5% CO2. After reaching confluence, cells were dislodged from the dish by treatment with accutase (TCS Biologicals, Buckingham, UK) and transferred to T75 flasks for further passage. Smooth muscle cells were characterized by immunohistochemical staining using a mouse anti-human α-actin monoclonal antibody (Boehringer, Ingelheim, Germany). For electrophysiologic experiments, cells (eighth passage) were plated onto coverslips in six-well plates at a density of 30,000 cells/well, and at 24 h the growth medium was replaced by serum-free Dulbecco's modified Eagle's medium (without antibiotics) to inhibit cell proliferation. Only single smooth muscle cells were used for patch-clamp studies. Most experiments were performed on cells starved of serum for at least 24 h, although some were performed on cells within 8 h after growth medium was replaced by serum-free medium. HEK293 cells (human embryonic kidney cell line) were cultured under a humidified atmosphere in 5% CO2 in minimal essential medium supplemented with Earle's Salts, 2 mM l-glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin (from a stock of 10,000 U/ml penicillin and 1 mg/ml streptomycin). Cells were transfected, and the stable cell line expressing SUR2B/Kir6.1 was generated as described previously (23).

Molecular Biology

For the purpose of identifying KATP channel subunits in HPASMC and rat pulmonary artery by molecular methods, reverse transcription-polymerase chain reaction (RT-PCR) was used. For messenger RNA isolation from human pulmonary artery, HPASMC were grown in three T175 flasks to confluence and washed twice with cold phosphate-buffered saline (made in diethylpyrocarbonate H2O). For RNA isolation from rat pulmonary artery, first order branches of left and right lung from 18 Sprague Dawley rats (220–250 g) were dissected out and cleaned of fat and connective tissue. Using an RNA isolation kit (Fast-track2.0; Invitrogen, Groningen, The Netherlands) and following the manufacturer's instructions, polyA RNA was isolated using the oligo dT cellulose column provided. Synthesis of cDNA was performed using 1 μl polyA RNA (100 ng) and oligo dT primers and following the manufacturer's instructions (cDNA cycle kit, Invitrogen). For PCR, 2 μl of cDNA was used in a 50 μl reaction and the following added: 5 μl PCR buffer (×10, containing 1.5 mM MgCl2) (Boehringer Mannheim), 25 mM of each deoxynucleoside-5′-triphosphate, 1 U Taq DNA polymerase (Boehringer Mannheim), and 1 μM of each specific primer. Primers for human were: SUR1 (GenBank accession no. AF087138) forward (5′-CGATGCCATCATCACAGAAG-3′), SUR1 reverse (5′-CTGAGCAG CTTCTCT GGCTT-3′), SUR2A (AF061320) forward (5′-ATATGGTCAAATCTCTACCTG GAGG-3′), SUR2A (AF061323) reverse (5′-GTTGGTCAT CACCAAAGTGGAA AAG-3′), SUR2B (AF061324) forward same as SUR2A forward, SUR2B reverse (5′-CATGTCTGCGCGA ACAAAAGAAGC-3′), Kir6.1 (E12830) forward (5′-TTGGCCAGAAAGAGTATCCCG GAG-3′), Kir6.1 reverse (5′-CATTCCACTTTTCTCCATGTAA GC-3′), Kir6.2 (E12832) forward (5′-TGTCCCGCAAGGGCAT CATCCCCG-3′), and Kir6.2 reverse (5′-TAGTCA CTTGGAC CTCAATGGAG-3′). Primers for rat were as described previously (24): SUR1 (L40624) forward (5′-GTTCCAGCAGAAGCT CCTAG-3′), SUR1 reverse (5′-CTGTCATAGCGTACACTCAG G-3′), SUR2B (AF087838) forward (5′-CGAAAGAGCAGCAT ACTCATTA-3′), SUR2B reverse (5′-CCTCTCTTCATCACAA TGACC-3′), Kir6.2 (D96039) forward (5′-AGGTACCGTAC TCG GGAGAGG-3′), Kir6.2 reverse (5′-GCCGTTTTCATGAAGAT GCAGCC-3′), Kir6.1 (D42145) forward (5′-TAC ATGGAGAAA GGCATCACGG-3′), and Kir6.1 reverse (GCCGTCTTCATGAA GATGCAGCC-3′). The SUR1, SUR2A, and SUR2B primer sets were designed to span a number of exons. The cycling conditions were the following: for HPASMC 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min, for 30 cycles; for rat pulmonary artery 94°C for 3 min followed by 35 cycles of 94°C for 45 s, 60°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 10 min. As positive controls, brain cDNA was used as a template for SUR1, and cardiac cDNA for SUR2A and Kir6.2. As a negative control, 2 μl of the RT-PCR product synthesized in the absence of avian myeloblastosis virus-transcriptase was used as a template. PCR products were electrophoresed on a 1.3% agarose gel. The sequence of all PCR products was confirmed by DNA sequencing using the dRhodamine Terminator cycle sequencing kit (Applied Biosciences, Warrington, UK) and an automatic sequencer (ABI377; Perkin-Elmer, Foster City, CA). Poly A RNA for human heart and whole adult brain were obtained from Clontech (Basingstoke, UK).

Electrophysiological Recording

Current and membrane potential were recorded in the whole-cell and cell-attached configuration of the patch-clamp technique using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Current signals were filtered at 1–2 kHz (eight-pole low-pass Bessel), digitized at 5 kHz using a Digidata 1200 interface, and saved onto computer for later analysis. Patch pipettes from either thin- or thick-walled 1.5-mm O.D. borosilicate glass capillaries (Clark Electromedical; Pangbourne, UK) were pulled and fire-polished using a DMZ-universal puller (Zietz Instruments, Müchen, Germany). Pipettes had resistances of 2–4 MΩ for whole-cell recordings and 6–8 MΩ for single-channel recordings when filled with electrolyte solution. Electrode capacitance was reduced by coating pipettes with a parafilm/mineral oil suspension and compensated electronically. Series resistance during whole-cell recording was compensated to at least 75% using the amplifier circuitry. The cell capacitance and input resistance were calculated under voltage clamp from the current response to a 10-mV hyperpolarizing step applied from a holding potential of −80 mV. The former was calculated from the area under the capacity transient and the latter from the change in the leakage current. Electrophysiological data were analyzed using pClamp (version 6) and Axoscope (Axon Instruments), SATORI v3.2 (Intracel, Coventry, UK) and Origin (version 6; Microcal Software, Northampton, MA) software. The single-channel current amplitude was measured by constructing amplitude histograms. The data were fitted using a Gaussian function, and the two peaks subtracted to obtain the current amplitude.

Statistical Analysis

Values are given as means ± standard error of the mean (SEM), and n indicates the number of cells. Statistical significance was assessed using a paired or unpaired Student's t test or one-way analysis of variance (ANOVA) with correction for multiple comparisons between different groups of cells. P values < 0.05 were considered to be statistically significant.

Solutions and Drugs

For whole-cell recordings in both HPASMC and HEK-293 cells, the standard bath solution was an extracellular physiological salt solution (PSS) containing (millimolar) 137 NaCl, 5 KCl, 0.4 KH2PO4, 0.3 NaH2PO4, 2 NaHCO3, 1 MgCl2, 1.8 CaCl2, 10 N-2-hydroxythylpiperazine-N′-2-ethane-sulfonic acid (HEPES), and 5.5 glucose (pH 7.4 with NaOH). A high potassium bath solution (60 mM K+) was obtained by replacing NaCl in the PSS with an equimolar concentration of KCl. The basic pipette solution contained (millimolar) 130 KCl, 1 MgCl2, 1 ethylene glycol-bis(aminoethylether)-tetraacetic acid (EGTA), and 15 HEPES (pH 7.2 with KOH). Adenosine triphosphate-Na2 was added to the pipette solution at the concentration indicated in the text (0.1–10 millimolar), and the pH of the solutions was readjusted. For single-channel recordings, the pipette and bath solution for cell- attached experiments contained (millimolar) 140 KCl, 1.0 CaCl2, 1.0 MgCl2, 5.5 glucose, and 10 HEPES (pH 7.4 with NaOH). For the inside-out patch-clamp configuration, the solution bathing the cytosolic face of the patch was (millimolar) 140 KCl, 1.0 MgCl2, 1 EGTA, and 10 HEPES (pH 7.2 NaOH).

Glibenclamide, BaCl2, adenosine triphosphate-Na2, and uridine 5′ diphosphate (UDP) were obtained from Sigma Chemical Co. (Poole, Dorset, UK). Levcromakalim was a gift from SmithKline Beecham Ltd (Harlow, Essex, UK). Levcromakalim was stored as a 10-mM stock in dimethyl sulfoxide and glibenclamide as a 10-mM stock in a 50:50 solution of dimethyl sulfoxide and polethylene glycol (Sigma). All other stock solutions were made up in distilled water. On the day of the experiment, stock solutions were diluted in the bath solution to give the desired concentration.

Membrane Properties of Cultured HPASMC

Cultured human smooth muscle cells have routinely been used as a model system for biochemical studies, although the impact of culture on the electrophysiological properties of cells is not fully understood. At least four types of K+ current have been described previously in pulmonary arterial cells: delayed rectifier K+ (KDR) current, fast transient outward K+ current, Ca2+-activated K+ (KCa) current, and KATP current (25-27). We therefore investigated the effect of serum withdrawal, which is known to inhibit cell growth, on the expression of whole-cell K+ currents and passive membrane properties in cultured HPASMC. For these experiments, cells were bathed in standard PSS and dialysed with the basic pipette solution containing 1 mM ATP and 1 mM EGTA, and currents activated by depolarizing pulses ranging from −70 to +80 mV from a holding potential of −80 mV. In cells in which the serum and growth factors in the culture medium had been removed for less than 8 h, depolarizing steps elicited small currents, the size of the steady-state component averaging ∼ 90 pA at +60 mV (Figure 1A). At negative potentials, current was usually time-independent; it became increasingly noisy at positive potentials, presumably resulting from the random release of Ca2+ from stores activating KCa channels (25). In some cells, a small rapidly activating and inactivating outward current could also be observed at potentials above 0 mV. With longer culture times in serum-free media, the magnitude of outward currents became progressively larger, although this was not associated with any significant change in input resistance (2.0 ± 0.4 GΩ, n = 15 at < 8 h, compared with 2.1 ± 0.4 GΩ, n = 62 at > 48 h). After 48 h in serum-free media, time- and voltage- dependent currents were observed in the majority of cells, being ∼ 3- to 5-fold larger in size (Figure 1A). Some cells displayed a prominent transient outward current, which activated fully within 5–10 ms after the start of the depolarizing step and inactivated rapidly to a steady-state level, whereas other cells showed currents with little inactivation during the 300-ms voltage step.

The transient outward current was almost completely suppressed by the voltage-gated K+ (Kv) channel inhibitor, 4-aminopyridine (4-AP; 5 mM), essentially leaving a rapidly activating, noninactivating noisy current at positive potentials in the presence of the inhibitor. For example, at +60 mV, 4-AP significantly (P < 0.005, n = 9; paired t test) inhibited current measured within the first 10 ms by 46% from 273.0 ± 54.6 to 148.6 ± 27.6 pA, whereas the steady-state current was less (P < 0.05) affected by 4-AP, being reduced by 27% from 175.0 ± 42.7 to 126.9 ± 35.2 pA. In contrast, in the presence of the nonselective KCa channel inhibitor, tetraethylammonium (10 mM), the transient current became the prominent current and the noisy component was no longer visible. Current activated at the start of the voltage step at +60 mV was reduced by 20% (205.8 ± 34.2 to 165.0 ± 30.9; n = 9), whereas the steady-state component was reduced by 45% (P < 0.001, paired Student's t test, n = 9) to 65.0 ± 5.0 pA. Thus, the transient current component has similar properties to Itran and Ito described previously in pulmonary artery (25, 26), whereas both KDR and KCa channels probably underlie a significant fraction of the steady-state current.

Mean effects of serum withdrawal on current are shown in Figure 1B, where steady-state outward current activated at +60 mV has been normalized to cell size (capacitance). It can be seen that current density increased as incubation time in serum-free medium lengthened, although increases were significant only at 48 but not at 24 h. Similarly, there were significant (P < 0.01) increases in the magnitude of the current component activated within 5–10 ms, which increased from 5.26 ± 0.69 pA/pF (n = 16) at < 8 h to 14.68 ± 2.34 pA/pF (n = 31) at > 48 h after serum withdrawal. Such changes in current density were associated with a significant decrease in cell capacitance, which averaged 38.4 ± 3.2 pF (n = 15) and 27.2 ± 1.2 pF (n = 32), respectively (Figure 1C).

K+ channels are known to be important determinants of resting membrane potential in smooth muscle (11). To assess whether the higher K+ channel density was associated with membrane hyperpolarization, the resting membrane potential was measured under current clamp as the zero-current potential (I = 0) at different times in serum-free medium. When measurements were made within 8 h, cells had a resting membrane potential of ∼ −16 mV (Figure 1D). This became progressively more hyperpolarized with a longer incubation time in serum-free medium, reaching −41 mV at > 48 h. In addition, the resting membrane potential became significantly (P < 0.01) more hyperpolarized (−55.8 ± 2.4 mV; n = 10) at > 48 h, if cells were dialysed with higher ATP (3 mM) and EGTA (10 mM) concentrations. At present, it is unclear which channel type is responsible for these more hyperpolarized resting potentials.

Properties of ATP-Sensitive K+ Current in HPASMC

Whole-cell KATP current has been identified previously in isolated pulmonary smooth muscle cells from rabbit (9, 28), but has so far not been investigated in human pulmonary artery. Moreover, it is unclear whether KATP currents are affected by cell growth. To assess the presence of KATP channels, the effects of levcromakalim on whole-cell current and membrane potential were investigated in voltage- and current-clamp, respectively. In a cell incubated in serum-free medium for > 48 h, under conditions in which the bathing solution contained physiological Ca2+ (1.8 mM) and K+ (5 mM) concentrations and the pipette solution contained 1 mM ATP, application of 10 μM levcromakalim hyperpolarized the cell from −33 to −52 mV (Figure 2A). Following washout of levcromakalim, the membrane potential slowly recovered to its previous level after about 5 min. Figure 2B summarizes the mean change caused by levcromakalim. It can be seen that levcromakalim induced a significant (P < 0.001) hyperpolarization (∼ 24 mV) in membrane potential. In the presence of the KATP channel inhibitor, glibenclamide (10 μM), the hyperpolarizing action of levcromakalim was essentially abolished (−40.2 ± 9.5 mV under control conditions and −39.0 ± 12.0 mV in the presence of glibenclamide, n = 5; P > 0.05). The effects of levcromakalim on membrane potential were also investigated in cells incubated in serum-free medium for less than 8 h. Under these conditions, application of levcromakalim induced a similar (∼ 29 mV) change in the resting membrane potential (Figure 2C).

The currents underlying the hyperpolarizing effects of levcromakalim were investigated in voltage-clamp experiments in the whole-cell configuration. Cells were held at −60 mV and a series of voltage steps ranging from −120 to +40 mV applied. Application of 10 μM levcromakalim induced a significant increase in a time-independent, background current at all potentials, an effect reversible on washout (not shown), and blocked in the presence of glibenclamide (10 μM). Mean current–voltage (IV) relationships of currents measured during the last 30 ms of the voltage step are shown in Figure 3B. Levcromakalim activated current at all potentials, increasing current at +40 mV 3-fold from 56.6 ± 10.1 to 171.3 ± 20.1 pA (n = 8), an effect fully inhibited by glibenclamide. However, the magnitude of the current activated by levcromakalim was reduced by more than 50% (P < 0.05) in cells starved of serum for less than 8 h (Figure 3C).

To test the selectivity of the levcromakalim-sensitive current for K+ ions, experiments were performed where the extracellular K+ concentration was increased from 5 to 60 mM. In these experiments, voltage ramps from −120 to +40 mV (107 mV/s) were used to obtain the reversal potential of the levcromakalim-sensitive current, being −75 ± 4 mV (n = 6) with 5 mM extracellular K+ and −20 ± 1 mV (n = 6) with 60 mM extracellular K+. The calculated EK under these conditions is −21.0 and −81.8 mV for 60 and 5 mM extracellular K+, respectively. Thus, the levcromakalim-sensitive current shifted when K+ was altered and reversed near the expected EK, indicating that currents were indeed through K+-selective channels.

Single-Channel Recordings

The properties of the channels activated by levcromakalim in HPASMC and HEK-293 cells were investigated with single-channel experiments in the cell-attached and inside-out configuration, where recordings were made in symmetric K+ (140/140 mM). In 5/27 cells from human pulmonary artery, application of levcromakalim (20 μM) in the cell-attached mode caused channel openings to be observed (NPO = 0.0504 ± 0.0163, n = 5), having an amplitude of 2.0 ± 0.1 pA (n = 5) at −60 mV (Figures 4A and 4C). The channels activated by levcromakalim showed burst-like openings. In HEK-293 cells expressed with SUR2B/Kir6.1, occasional channel openings with an amplitude of 2.02 ± 0.08 pA (n = 5) at −60 mV were observed in cell-attached patches (NPO = 0.00098 ± 0.00077), the activity of which could be greatly enhanced by levcromakalim (NPO = 0.2133 ± 0.0569, n = 7) (Figure 4E). The mean IV relationship for these channels is shown in Figure 4B. The slope conductance, calculated from the IV relationship, was 28.3 ± 1.5 pS (n = 5) in HPASMC, essentially identical to the value obtained under the same experimental conditions for SUR2B/Kir6.1 (29.2 ± 1.7 pS, n = 6). Glibenclamide inhibited (P < 0.05) the channels activated by levcromakalim, reducing NPO to 0.0035 ± 0.0021 (n = 5) and 0.0046 ± 0.0008 (n = 7) in HPSMC and HEK-293 cells, respectively. In excised inside-out patches, application of levcromakalim failed to cause channels to open in HPASMC. However, in 4/51 patches, application of UDP (3 mM) to the intracellular surface of the patch in the continued presence of levcromakalim activated a channel of similar magnitude to that opened by levcromakalim in cell-attached patches. The single-channel amplitude in the presence of UDP was 1.90 ± 0.03 pA (n = 4) at −60 mV, and NPO was 0.4068 ± 0.3610. Essentially similar results were obtained with cells expressing SUR2B/ Kir6.1. In excised patches, few openings occurred in the continued presence of levcromakalim (NPO = 0.00085 ± 0.00045, n = 7), although channel activity was greatly enhanced by application of UDP (NPO = 0.5293 ± 0.2107, n = 7), activating a channel of 1.99 ± 0.07 pA (n = 10) at −60 mV (Figure 4F). Thus, the single channel properties of the channel activated by levcromakalim in HPASMC resembles the KNDP channel described previously in rat and rabbit smooth muscle (16, 29) and its cloned counterpart, SUR2B/Kir6.1.

ATP Sensitivity of Levcromakalim Responses in HPASMC and HEK-293 Cells

It has been demonstrated previously that lowering the intracellular ATP concentration in rabbit pulmonary arterial myocytes enhances the magnitude of responses to levcromakalim (9). We therefore investigated whether such regulation occurred in HPASMC and in HEK-293 cells stably expressing SUR2B/Kir6.1, the putative clone for the KNDP channel. In HPASMC, the magnitude of levcromakalim-sensitive current in a physiological K+ gradient increased 2.5-fold at +20 mV when the pipette ATP was lowered from 1 to 0.1 mM (Figure 5A). Similar effects were obtained in cells bathed in 60 mM K+, where the current activated by levcromakalim at −60 mV was −153 ± 21 pA (n = 8) in the lower ATP, compared with −19 ± 8 pA (n = 5; P < 0.001) at the higher ATP. These results indicate that ATP has an inhibitory action on the whole-cell responses to levcromakalim. In contrast, under similar experimental conditions, the magnitude of the levcromakalim-induced current in cells expressing SUR2B/Kir6.1 was insensitive to the intracellular ATP concentration over the range 0–5 mM. However, at higher ATP concentrations (10 mM), responses to levcromakalim were significantly (ANOVA, P < 0.05) suppressed when compared with those obtained with 1 mM ATP (Figure 5B).

RT-PCR Analysis

RT-PCR analysis of KATP channel subunits was performed in cultured HPASMC grown to confluence and in rat pulmonary artery. PCR was performed in the presence of specific primers for SUR1, SUR2A, SUR2B, Kir6.1, and Kir6.2. In HPASMC, reaction products corresponding to the expected fragment sizes for SUR2B and Kir6.1, but not SUR1, SUR2A, and Kir6.2, were detected after RT-PCR from the polyA RNA extracted from cultured HPASMC (Figure 6A). Similar results were obtained on two other occasions. For positive controls, cardiac cDNA was used to confirm PCR fragments for SUR2A and Kir6.2, and brain cDNA to detect SUR1. Each of the positive PCR products was confirmed by DNA sequence analysis and found to be correct. The RT-PCR was repeated on two other occasions with similar results. To determine if lack of expression of Kir6.2 in HPASMC was a culturing artifact, a species difference, or a fundamental difference between pulmonary artery and other smooth muscle cell types, RT-PCR was also performed in rat pulmonary artery. Reaction products corresponding to the expected fragment sizes for SUR2B, SUR1, and Kir6.1, but not Kir6.2, were detected after RT-PCR from the polyA RNA (Figure 6B) in intact pulmonary artery.

KATP channels are expressed in varieties of vascular tissues of animal origin, including rabbit pulmonary smooth muscle (9, 28). Our results show that KATP channels also exist in human pulmonary cells and that the properties of these channels in culture are similar to those described in freshly isolated cells from pulmonary artery (9, 28) and other vascular tissues (4), although KATP current density was decreased in proliferating cells. This conclusion is based on the following observations: (1) In current clamp, levcromakalim caused substantial hyperpolarization of the membrane potential, an effect blocked by glibenclamide, a potent blocker of KATP channels; (2) in voltage clamp, levcromakalim activated a time-independent background current, the magnitude of which was decreased in proliferating cells and increased when intracellular ATP concentration was reduced from 1 to 0.1 mM; (3) the reversal potential of the levcromakalim- or glibenclamide-sensitive current was close to the expected EK and shifted when the extracellular K+ ion concentration was altered; (4) single-channel studies revealed that levcromakalim activated a 28-pS K+ channel that required the presence of UDP for activation in excised patches; and (5) RT-PCR analysis confirmed the presence of the SUR2B and Kir6.1 in HPASMC, both putative subunits for the KNDP channel described in a number of vascular tissues (3, 4).

Effect of Cell Growth on Electrophysiological Properties

It is well known that smooth muscle cells grown in culture undergo substantial changes in their morphological and biochemical properties, which is generally described as a transition from a contractile to a synthetic (proliferating) phenotype (30). There have, however, been few studies on the effect of cell growth on the electrophysiological properties in smooth muscle. A membrane potential of −55 mV was reported in primary cultured and short term (5–7 d) subcultured HPASMC (27), close to the membrane potential (−53 mV) recorded in freshly isolated cells from rabbit main pulmonary artery (25). In other reports where subcultured HPASMC up to the eighth passage (continually cultured in serum-containing medium) were used, a less negative membrane potential of between −18 and −33 mV was described (26, 31). In the present studies, we recorded significantly different membrane potentials in proliferating (−16 mV) and growth-arrested (−41 mV) HPASMC. It seems that in primary culture HPASMC retain the resting membrane potential similar to that recorded in native cells, and following longer term culture, cells gradually become depolarized, possibly because of the transition from contractile to proliferative phenotype. The changes in membrane potential can be reversed, at least partially, by arresting cell growth with culture medium depleted of growth-stimulating factors. This is supported by the recent study showing that cultured adult vascular smooth muscle cells can convert reversibly between contractile and noncontractile phenotypes by changing the medium between the serum-contained and the serum-free ones (30). The physiological relevance of the change in membrane potential is not obvious but may have significant effects on functions of the noncontractile phenotype.

In our studies, serum withdrawal appeared to cause a generalized increase in K+ current density. These effects could not be attributed to changes in leak current, because there was no difference in the input resistance in proliferating and nonproliferating cells. Both the outward transient and the steady-state current components were enhanced, probably reflecting increases in fast transient outward potassium current and KDR and KCa currents, currents previously described in human (26, 27, 31) as well as rabbit (25) pulmonary arterial cells. In addition, we found responses to levcromakalim to be enhanced in serum-starved cells, suggesting increased KATP channel activity. Because KATP channels do contribute to the resting potential in pulmonary artery (28), this may explain in part the more hyperpolarized resting potentials in growth-arrested cells (11, 31). Previous studies have shown that a noninactivating Kv current was depressed in proliferating human pulmonary artery cells, and that this was associated with membrane depolarization and elevated intracellular Ca2+ concentrations (31). As cell growth in these studies could be inhibited by chelating extracellular Ca2+ or by application of a K+ ionophore, this suggests that Ca2+ influx is an important stimulus for smooth muscle cell growth, whereas K+ efflux, through prevention of cell depolarization, may inhibit growth. Moreover, smaller Kv currents and decreased expression of Kv subunits have been found in pulmonary cells obtained from patients with primary pulmonary hypertension, a disease known to be associated with medial hypertrophy and cell proliferation (32, 33).

The mechanism responsible for changes in K+ channel activity remains unclear at this stage and could be related to inhibition of channel activity directly and/or decreases in channel expression. K+ channels are known to be under the tight control of kinases and phosphatases that regulate the phosphorylation state and activity of the channel both in the short term and in the long term. One possibility is that the activity of the Ca2+-dependent phosphatase, calcineurin, is high in proliferating cells, thus promoting channel dephosphorylation. We have shown recently that this enzyme inhibits smooth muscle KATP channel activity and that regulation is steeply dependent on intracellular Ca2+ range in the range 100–300 nM (10). Consistent with this concept is our observation that, under conditions favoring phosphorylation (3 mM ATP and high intracellular Ca2+ buffering), cells were significantly more hyperpolarized.

Regulation of Native and Cloned KATP Channels

A well-known feature of the cardiac KATP channel is competitive inhibition by ATP of responses to KCO (1, 4). In smooth muscle, this has generally been used as one of the criteria to confirm the presence of the classic type of KATP channel, together with basal activation of whole-cell current in low intracellular ATP (4). As in our study, whole-cell responses to levcromakalim can be enhanced ∼ 3- to 5-fold by reducing the ATP intracellular concentration from 1–3 to 0.1 mM in smooth muscle cells isolated from coronary, pulmonary, and aortic tissues (8-10). Likewise, whole-cell KATP currents are significantly activated under similar conditions in the majority of smooth muscle cells in which this has been investigated (4). In contrast, neither were responses to levcromakalim in rabbit portal vein sensitive to the intracellular ATP concentration, nor could whole-cell KATP current be activated by lowering ATP (15). Such results were presumed to be related to the presence of the KNDP subtype in this preparation. Recent studies on the reconstituted KATP channel, SUR2B/Kir6.1, have shown ATP to have a biphasic effect on channel activity in excised patches, with lower concentrations of ATP activating the channel, whereas higher concentrations (> 3 mM) inhibit the channel (14). Pinacidil shifts the bell-shaped concentration–response curve by a log unit so that inhibition starts to occur above 300 μM. In our stable line expressing SUR2B/Kir6.1, intracellular ATP had little effect on whole-cell responses to levcromakalim, except at high concentrations (10 mM), where the magnitude of the current was significantly suppressed.

How, then, do we reconcile data suggesting a more classic channel controlling the macroscopic KATP current in native cells with the molecular data showing widespread distribution of both SUR2B and Kir6.1 and a more limited expression of Kir6.2 (3)? Moreover, recent studies in Kir6.2−/− knockout mice show essentially unchanged whole-cell current and relaxant responses to pinacidil in aorta (34). The simplest interpretation is that SUR2B/Kir6.1 does underlie the KATP current in vascular smooth muscle, but that ATP-sensitivity of the channel in the whole cell is altered by cytosolic factors. Our own data do support this notion, because the single channel conductance and kinetic properties of the channel activated by levcromakalim are remarkably similar to SUR2B/ Kir6.1 (29 pS) recorded under comparable experimental conditions. However, it is known that vascular KATP channels show considerable heterogeneity with respect to conductance and ATP and NDP sensitivity, suggesting that multiple types of channels do indeed exist in smooth muscle (4, 11). Broadly speaking, channels have either a low to intermediate conductance (15–70 pS) or a relatively high conductance (> 150 pS) in symmetrical K+ (140–150 mM) (4, 11, 17). This contrasts with the channel found in pancreatic β cells, cardiac myocytes, and skeletal muscle, whose conductance is ∼ 70 pS under similar ionic conditions (3). Perhaps some of the confusion in the data obtained in smooth muscle might be related to mixed populations of SUR and Kir6.0 subunits forming hybrid channels (18). So far, both Kir6.1 and Kir6.2 have yet to be found in the same smooth muscle preparation, although single-channel experiments clearly report both the classic KATP and KNDP channels in the same smooth muscle preparation (16, 17). Moreover, the conductance of some KNDP channels in high symmetric K+ (140 mM) is ∼ 42–44 pS (17, 21), which approaches that of a dimer linking Kir6.1 to Kir6.2, measuring 47 pS with the same K+ gradient (18).

Molecular Characterization of KATP Channels

Our molecular studies in cultured HPASMC demonstrated the presence of SUR2B and the inward rectifier Kir6.1, but not SUR2A, SUR1, or Kir6.2. These data are consistent with Northern blot analysis in rat showing expression of Kir6.1 (35), but not Kir6.2 (12), in whole-lung tissue and with the widespread expression of SUR2B reported in all tissues examined so far, including smooth muscle (13). Of the other known isoforms of SUR, SUR2A in rat is found predominately in heart, skeletal muscle, and bladder, but not in lung (13), whereas SUR1 is found almost exclusively in the pancreas and brain (12). To date, molecular studies in smooth muscle have detected SUR2B but not SUR2A in murine colonic (19) and guinea-pig bladder (20) myocytes, although in the latter study SUR1 was also detected, raising the possibility that SUR1 and SUR2B might form heteromultimers in some smooth muscles. We also detected SUR1 in rat pulmonary artery, although it is not clear at this stage if SUR1 is actually expressed in smooth muscle cells or in other cell types that are known to exist in the endothelial and adventitial layers of blood vessels. In intestinal and urinary smooth muscle, Kir6.2, but not Kir6.1, has been reported consistently (19– 21). In contrast, we found no evidence for the expression of Kir6.2 either in human or rat pulmonary artery. This strongly suggests that lung and visceral smooth muscle differ in the expression of pore-forming subunits. This is likely to have important physiological and pathophysiological consequences because organ responses to metabolic inhibition, and perhaps hypoxia, will vary depending on the relative expression of Kir6.1 and Kir6.2. Indeed, application of metabolic inhibitors activates glibenclamide-sensitive currents in bladder (20), but not in pulmonary smooth muscle cells (9). Thus, it will be important in the future to clarify the distribution of SUR and Kir subunits in other vascular smooth muscles using preparations containing a homogeneous population of cells.

Although our molecular data suggest that SUR2B/Kir6.1 is likely to underlie the 28 pS channel activated by levcromakalim in HPASMC, we cannot exclude the possibility that the channel may be composed of additional or different regulatory subunits. This may in part explain why the ATP sensitivity of whole-cell currents carried by SUR2B/ Kir6.1 differs from that observed for KATP currents in cultured HPASMC and many other native smooth muscles recorded under similar conditions (4). Alternatively, expression of the channel in HEK-293 cells may alter the ATP sensitivity of the channel, perhaps through changes in the basal phosphorylation state of the channel. On the other hand, Kir6.1 could combine with other Kir subunits, which could potentially lead to a KATP channel with hybrid properties (18). Clearly, further work is required to distinguish between these possibilities. Interestingly, in colonic smooth muscle, a small-conductance (27 pS in symmetric 140 mM K+) KATP channel was reported, though Kir6.1 was not detected in this preparation (19). This may suggest the presence of another pore-forming subunit, and certainly, KATP channels with even smaller conductances (∼ 15 pS) have been reported in some vascular tissues (4), thus seemingly not relating to either Kir6.1 or Kir6.2.

In summary, we conclude that KATP channels are expressed in human pulmonary artery and that the properties of these channels in culture are similar to that observed in other freshly isolated cells from other species. However, the activity of these channels (and indeed other K+ channels) appears suppressed in proliferating cells. In contrast to visceral muscle, our molecular and single-channel data point to the KNDP subtype as the predominant channel in this preparation and possibly in pulmonary artery from other species. However, the properties of the whole-cell current in pulmonary artery appears altered from that generated by the KNDP channel clone expressed in HEK-293 cells, suggesting other factors governing the regulation of this channel in the native environment.

This work was funded by the British Heart Foundation (PG/ 99176), the MRC (G117/180), United Therapeutics (USA), and the Wellcome Trust. L.H.C. is a Medical Research Council Senior Research Fellow in Basic Science and A.T. is a Wellcome Trust Senior Research Fellow in Clinical Science.

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Address correspondence to: Lucie H. Clapp, Centre for Clinical Pharmacology, Department of Medicine, 4th floor, Rayne Institute, University College, 5 University Street, London WC1E 6JJ, UK. E-mail:

Abbreviations: 4-aminopyridine, 4-AP; analysis of variance, ANOVA; ethylene glycol-bis(aminoethylether)-tetraacetic acid, EGTA; N-2-hydroxythylpiperazine-N′-2-ethane-sulfonic acid, HEPES; human pulmonary artery smooth muscle cells, HPASMC; current–voltage relationship, IV relationship; ATP-sensitive potassium, KATP; Ca2+-activated potassium, KCa; potassium channel openers, KCO; delayed rectifier, KDR; inward rectifier, Kir; nucleotide diphosphate regulated potassium, KNDP; voltage-gated potassium, Kv; physiological salt solution, PSS; sulfonylurea receptor, SUR; reverse transcription-polymerase chain reaction, RT-PCR; uridine 5′diphosphate, UDP.


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