Published online before print October 9, 2003, doi: 10.1161/01.RES.0000099883.68414.61
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© 2003 American Heart Association, Inc.
Cellular Biology |
Two-Pore Domain K Channel, TASK-1, in Pulmonary Artery Smooth Muscle Cells
From the Department of Physiology and Pharmacology (A.M.G., R.J.T.), University of Strathclyde, Glasgow, UK; Quintiles Scotland Ltd (O.N.O., K.M.M.), Heriot-Watt University Research Park, Edinburgh, UK; Institute of Biomedical and Life Sciences (D.M.), University of Glasgow, Glasgow, UK; Argyll and Clyde NHS (F.E.J.K.), Royal Alexandra Hospital, Paisley, UK.
Correspondence to Prof Alison M. Gurney, Department of Physiology and Pharmacology, University of Strathclyde, 27 Taylor St, Glasgow, UK G4 0NR. E-mail a.m.gurney{at}strath.ac.uk
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Abstract |
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Pulmonary vascular tone is strongly influenced by the resting membrane potential of smooth muscle cells, depolarization promoting Ca2+ influx, and contraction. The resting potential is determined largely by the activity of K+-selective ion channels, the molecular nature of which has been debated for some time. In this study, we provide strong evidence that the two-pore domain K+ channel, TASK-1, mediates a noninactivating, background K+ current (IKN), which sets the resting membrane potential in rabbit pulmonary artery smooth muscle cells (PASMCs). TASK-1 mRNA was found to be present in PASMCs, and the membranes of PASMCs contained TASK-1 protein. Both IKN and the resting potential were found to be exquisitely sensitive to extracellular pH, acidosis inhibiting the current and causing depolarization. Moreover, IKN and the resting potential were enhanced by halothane (1 mmol/L), inhibited by Zn2+ (100 to 200 µmol/L) and anandamide (10 µmol/L), but insensitive to cytoplasmic Ca2+. These properties are all diagnostic of TASK-1 channels and add to previously identified features of IKN that are shared with TASK-1, such as inhibition by hypoxia, low sensitivity to 4-aminopyridine and quinine and insensitivity to tetraethylammonium ions. It is therefore concluded that TASK-1 channels are major contributors to the resting potential in pulmonary artery smooth muscle. They are likely to play an important role in mediating pulmonary vascular responses to changes in extracellular pH, and they could be responsible for the modulatory effects of pH on hypoxic pulmonary vasoconstriction.
Key Words: two-pore domain K channel • pulmonary artery myocyte • smooth muscle • resting potential
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Introduction |
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The membrane potential of pulmonary artery smooth muscle cells (PASMCs) is an important factor in the control of pulmonary vascular tone. This is reflected in the vasoconstrictor effect of agents causing membrane depolarization1–3 and the vasodilator action of drugs causing hyperpolarization.4,5 There is general agreement that the resting potential of PASMCs is determined largely by a noninactivating K+ conductance (IKN) that is sensitive to inhibition by 4-aminopyridine, albeit at relatively high (>1 mmol/L) concentrations.6 Thus, both the resting potential and IKN are abolished when pipette K+ is replaced with equimolar Cs+, whereas equimolar substitution of Cs+ for extracellular K+ has little effect.7 In addition, both the resting potential and IKN display low sensitivity to the drugs quinine and Ba2+ and are insensitive to tetraethylammonium ions, glibenclamide, and a range of Ca2+-activated K+-channel blockers.7,8 This pharmacological profile is reminiscent of the two-pore domain K+ channel, TASK-1, which was recently shown to act as a background K+ conductance regulating resting potential in neuronal,9–11 cardiac,12 and adrenal glomerulosa13 cells. Because an important characteristic of the resting conductance in PASMCs is that it is inhibited by hypoxia, leading to membrane depolarization and Ca2+ influx,3,7,14–16 we were especially interested in the finding that hypoxia inhibits recombinant TASK-1 channels.17 Moreover, channels with TASK-like properties have been found to underlie oxygen-sensing K+ currents in chemosensitive cells of the carotid body, airways, and central nervous system.18–20 This raises the possibility that TASK-1 channels might be the molecular correlate of IKN and be responsible for setting the resting membrane potential in PASMCs. Indeed, mammalian lung has been shown to express mRNA for TASK-1,21 although it is unclear in which of the many cell types present in lung it is located.
A defining characteristic of TASK-1 channels is their high sensitivity to changes in extracellular pH.22 Although specific and selective inhibitors of TASK-1 channels have yet to be identified, the channels do display a distinctive pharmacological profile. Thus, in addition to the drug sensitivities described above, TASK-1 channels are characteristically activated by volatile anesthetics like halothane23 and inhibited by low concentrations of anandamide24 and Zn2+.25 These properties were exploited in the present study to investigate the expression of TASK-1 channels in rabbit PASMCs and their contribution to the background K+ current, IKN, and the resting membrane potential. The results provide the first demonstration of an important functional role for a two-pore domain K+ channel in vascular smooth muscle.
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Materials and Methods |
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New Zealand White rabbits (2 to 3 kg; Harlan UK Ltd, Bicester, UK) were euthanized by sodium pentobarbitone overdose (80 mg/kg IV), according to the UK Animals (Scientific Procedures) Act, 1986. The main pulmonary artery and its right and left branches were dissected free and placed in physiological salt solution (PSS) of the following composition (in mmol/L): NaCl 124, KCl 5, CaCl2 1.8, MgCl2 1, NaH2PO4 0.5, KH2PO4 0.5, glucose 10, 5-N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) 15; adjusted to pH 7.3 with NaOH. Smooth muscle cells were dissociated by overnight incubation with papain as previously described,26 or by incubating 1-mm strips of artery for 30 minutes at 37°C with 12 mg collagenase (Sigma type 1A), 1 mg protease (Sigma type XXIV), 10 mg trypsin inhibitor (Sigma), and 10 mg bovine serum albumin (BSA) in 5 mL dissociation medium (DM) of the following composition (in mmol/L): NaCl 110, KCl 5, NaHCO3 15, CaCl2 0.16, MgCl2 2, NaH2PO4 0.5, KH2PO4 0.5, glucose 10, HEPES 15, phenol red 0.04, ethylenediaminetetraacetic acid (EDTA) 0.49, taurine 10; adjusted to pH 7.0 with NaOH. Resting potential and IKN amplitude were not significantly different in cells obtained with either method.
Reverse transcription (RT) PCR was preformed on total RNA extracted from suspensions of rabbit PASMCs as previously described27 or single PASMCs harvested under microscope control and aspirated directly into the RT-PCR mix. Four sets of primer pairs (PP) were designed against conserved sequences in mouse (GenBank accession No. AB013345), rat (AB048823), and human (NM_002246) TASK-1 channels. They were as follows: PP1, 5'-TCCATGTGCTCGTGCCTCTG-3', 5'-AGCTCCTGCGCTT-CATGAGG-3'; PP2, 5'-GGCAAGGTGTTCTGCATGTTCTACG-3', 5'-AAGCCGATGGTGGTGAGGGTG-3'; PP3, 5'-CCTTCTACTT-CGCCATCACCGTCATC-3', 5'-CATGTTGGCCATGGACACCTCG-3'; PP4, 5'-TCATCGTGTGCACCTTCACCTACCTG-3', 5'-CATGTT-GGCCATGGACACCTCG-3'. PP2, PP3, and PP4 target overlapping regions of the TASK-1 template (see Figure 1C). Cycle parameters were typically 95°C for 5 minutes, followed by 20 to 35 cycles at 95°C for 1 minute, 52°C to 56°C for 30 seconds, and 68°C for 2 to 4 minutes. Reverse transcriptase was omitted from control cDNA reactions. Products were resolved by agarose gel electrophoresis, purified, and verified by sequencing.
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Immunofluorescence was performed as previously described,27 using two different antibodies directed against unique domains in TASK-1, one 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). Cells adhered to glass coverslips were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 minutes then permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. After blocking with 1% BSA for 1 hour, cells were incubated with anti-TASK-1 antibody (1:100 dilution) for 1 hour then probed for 1 hour with a secondary antibody conjugated to fluorescent Alexa 488, or a biotinylated secondary antibody followed by streptavidin-Alexa 488. Duplicates were processed without primary antibody for controls. Fluorescence was imaged at wavelengths >500 nm with a BioRad Radiance 2000MP confocal microscope, using the 488-nm line of an argon laser and a Nikon x60 (N.A. 1.0) objective.
For electrophysiological experiments, cells were superfused at 
0.5 mL/min at room temperature with PSS supplemented with 10 mmol/L tetraethylammonium chloride (TEA) and 10 µmol/L glibenclamide to block BKCa and KATP channels, respectively. The whole-cell patch-clamp technique was used as previously described to measure the resting membrane potential under current clamp and macroscopic K+ currents under voltage clamp.8 Unless otherwise indicated, pipettes were filled with the following: (in mmol/L) KCl 130, MgCl2 1, ethyleneglycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) 1, HEPES 20, Na2GTP 0.5; pH adjusted to 7.2 with KOH. Where indicated, 10 mmol/L 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was substituted for EGTA. Pipette resistance was 2 to 4 M
. To isolate IKN from other voltage-dependent K+ currents, cells were clamped at 0 mV for at least 5 minutes. To suppress Cl- currents in some experiments, all but 10 mmol/L of the Cl- in both the PSS and pipette solution was replaced with methanesulphonate. Currents were filtered at 0.5 to 5 kHz and digitized at 0.62 to 20 kHz and were not leak subtracted. Current amplitudes were normalized against cell capacitance, measured from the current response to -5 or -10 mV steps applied from a -80 mV holding potential. Drugs were applied from a manually operated, gravity fed, multibarrel perfusion pipette positioned close to the cell, which changed the solution around the cell in a few seconds.
The pH of the PSS was set with 1 mol/L NaOH. The order of the change in pH was randomized for each experiment and had no effect on the results. Halothane was dissolved in dimethylsulphoxide (DMSO) to 1 mmol/L, diluted in PSS, and used immediately. Other drug solutions were prepared from frozen aliquots. Glibenclamide (10 mmol/L) was dissolved in DMSO and anandamide (10 mmol/L) in 50% ethanol in water. Effects of these drugs were determined by comparison with vehicle controls. Other drugs were dissolved in deionized water. Data are expressed as mean±SEM of n cells. Statistical comparisons used one-way analysis of variance (ANOVA), with probability values corrected by Tukey’s pairwise comparison, or Student’s paired or unpaired t test as appropriate. A value of P<0.05 was considered significant.
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Results |
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Expression of TASK-1 Channels in PASMCs
We first established the presence of TASK-1 mRNA in rabbit PASMCs. RT-PCR experiments with four specific primer pairs for TASK-1 indicated that mRNA for the channel is present in suspensions of cells isolated from pulmonary arteries (Figure 1A). These results were reproduced in separate RT-PCR reactions on RNA from at least three rabbits. Although the suspensions contained predominantly smooth muscle cells, they were almost certainly contaminated with endothelial and other cells. To verify the smooth muscle origin of the amplified mRNA, further RT-PCR experiments were performed on single, identified PASMCs. Figure 1B shows reproducible amplification of a PCR product of the predicted size from a single PASMC, using primer pair PP3. Immunocytochemistry provided further evidence for TASK-1 expression in PASMCs. The presence of TASK-1 protein was established with two anti-TASK-1 antibodies directed against either the amino or carboxy terminal regions of the protein. Both antibodies resulted in fluorescent labeling in PASMCs from at least six separate preparations (Figures 2A and 2B). In contrast, staining was absent from control cells treated in the same way, but without exposure to TASK-1 antibody (Figures 2C and 2D). Confocal microscopy of cells stained with both antibodies showed the presence of TASK-1 staining at the surface membrane and in the cytoplasm.
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K+ Current Modulation by pH
A defining hallmark of the TASK family of channels is their sensitivity to extracellular pH (pHo). As shown in Figure 3A, varying the pH of the extracellular solution caused reversible changes in the resting membrane potential of isolated rabbit pulmonary artery myocytes. The relationship between resting potential and pHo is illustrated in Figure 3B, where the resting potentials measured at pH 6.3, 7.3, and 8.3 were significantly different (P<0.001, ANOVA). Increasing pHo from 7.3 to 8.3 caused membrane hyperpolarization of 8±2 mV (n=8), whereas acidification to pH 6.3 depolarized cells by 20±5 mV (n=5). These changes in membrane potential were reflected in the effects of pHo on the amplitude of the noninactivating K+ current, IKN, recorded at 0 mV (Figure 3C). In 10 cells, the mean amplitude of the current measured at pH 6.3 was 48±7% of that measured at pH 7.3, whereas the current at pH 8.3 was 163±14% of that at pH 7.3. The modulation of IKN and resting potential by pHo was usually apparent within seconds of changing the perfusion solution, consistent with a direct effect of pHo on the channels rather than mediation by slow changes in intracellular pH. Figure 3D illustrates the effect of pHo across the full voltage range over which IKN is resolved. From the holding potential of 0 mV, the voltage was stepped to 60 mV and then ramped to -10 mV over a period of 1 second. The current during the ramp reflects IKN in parallel with a nonspecific "leak" current and reverses direction at the resting potential of the cell.8 The effect of pHo was restricted to membrane potentials of -60 mV or above, where IKN is active. It had little effect at more negative potentials where leak current predominates. As a consequence of modulating outward current, pHo also influenced the reversal potential of the current recorded during voltage ramps. Consistent with the depolarizing effect of acidification, a positive shift of 5±2 mV (n=3) resulted when pHo was reduced from 7.3 to 6.8, whereas increasing pHo to 7.8 caused a shift of -9±3 mV (n=3). Figure 3E shows the pHo dependence of IKN measured at 0 mV. Fitting a Boltzmann relationship to the data indicates that the pHo giving 50% inhibition (pH0.5) was pH 7.3.
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Varying the extracellular pH also modulated IKV, the K+ current activated during short (250 ms) depolarizing steps applied from a holding potential of -80 mV. This is illustrated in Figure 4A where the main effect appears to be on current inactivation, which was accelerated by acidification and slowed by alkalinization. Thus, at pH 8.3, the peak amplitude of IKV at 40 mV, seen as an early transient component that has been likened to A-current,26 was unchanged compared with that at pH 7.3 (106±12%, n=3). In contrast, the sustained component of current measured at 200 ms in the same cells, attributed to delayed rectifier channels,26 was significantly enhanced at pH 8.3 compared with pH 7.3 (147±12%, n=3, P<0.05). Reducing the pH from 7.3 to 6.3 suppressed both components of current, although inhibition of the sustained component (56±13%, n=3, P<0.01) was usually more pronounced than the early peak current (42±12%, n=3, P<0.05). As shown in Figure 4B, the effect of pHo on the sustained IKV appeared within a few seconds of changing the solution and was fully reversible. Modulation was apparent across the voltage range over which IKV was activated, with similar percentage changes measured at all potentials (Figure 4C). It is clear from Figure 4C that acidification reduced the current at -40 mV, implying that inhibition of IKV could have contributed to its depolarizing effect.
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Modulation by Halothane, Anandamide, and Zn2+
Another marker of TASK channels is their sensitivity to volatile anesthetics, such as halothane, which cause facilitation of channel activity. As illustrated in Figure 5A, 1 mmol/L halothane caused facilitation of the noninactivating current at 0 mV in 12 out of 14 cells. The mean facilitation observed was 19±4% (n=14), halothane significantly increasing IKN at 0 mV from 15±3 to 18±4 pA (n=14, P<0.01). This effect was fully reversible, although it sometimes took a few minutes for the current to return to the control level after washout. Consistent with the involvement of TASK-1 channels in this facilitation,28 it was blocked at acid pHo (Figure 5B). Thus, when cells were bathed in PSS at pH 6.3, the mean current was 4±3 pA before and 3±3 pA (n=9) after applying halothane (1 mmol/L). The stimulatory effect of halothane was restricted to potentials positive of -60 mV, with no effect on the leak current at more negative potentials (Figure 5C). In five of the cells studied at pH 7.3, the facilitation caused by halothane was transient. Thus in the continuous presence of halothane, the response in these cells appeared biphasic, and in some the current eventually declined to below the control level (not shown). This may reflect an additional inhibitory effect of halothane, because a small inhibition was observed in the two cells that failed to show facilitation in response to halothane. The mean reduction in current amplitude amounted to 16±6% of the maximum (facilitated) current (n=7). The inhibitory effect became more pronounced at higher halothane concentrations. Thus at 2 or 5 mmol/L halothane, facilitation was observed in only three of eight cells and inhibition, amounting to a decrease in the current amplitude of 37±9% (n=8), was predominant. The effects of halothane on the resting membrane potential mirrored its effects on IKN. Thus, 1 mmol/L halothane caused a small but significant hyperpolarization (Figure 5D) of -2.7±0.7 mV (n=7, P<0.01), whereas at higher halothane concentrations (2 to 5 mmol/L), depolarization from -42±1 to -34±5 mV (n=3) was predominant. A biphasic effect was observed in some cells. In contrast to its effects on IKN, up to 5 mmol/L halothane had no consistent effect on the amplitude of the transient or sustained IKV, measured over a range of test potentials (Figure 5E).
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Although the effects of pH and the facilitatory action of halothane on membrane potential and IKN are indicative of a possible role for TASK channels, these agents discriminate poorly among different members of the TASK family and even other K channels. One agent that is able to inhibit TASK-1 at concentrations well below those affecting other TASK channels is Zn2+.25,29 As illustrated in Figure 6A, IKN recorded at 0 mV was reversibly inhibited by Zn2+ at relatively low concentrations in 11 out of 13 cells tested. At 100 µmol/L, Zn2+ reduced the current by 32±11% (n=6; P<0.05) and at 200 µmol/L inhibition increased to 53±10% (n=6; P<0.05). This effect of Zn2+ was restricted to membrane potentials above -60 mV, where IKN is active (Figure 6B). As shown in Figure 6C, the inhibition of IKN was associated with 10 mV depolarization of the resting potential from -37±4 mV to -27±6 mV (n=3). Unfortunately, the effect of Zn2+ was not selective, because as illustrated in Figure 6D (inset), it also inhibited the transient component of IKV, effectively slowing the rate of activation of KV current. Except at steps to -20 mV, Zn2+ (200 µmol/L) did not, however, alter the sustained component of IKV (Figure 6C).
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The endocannabinoid anandamide was recently shown to be a direct and selective blocker of TASK-1 channels, low micromolar concentrations inhibiting these channels but not other TASK or two-pore domain channels.24 Figure 6E shows the effect of 10 µmol/L anandamide on the noninactivating current recorded at 0 mV. At this concentration, anandamide reduced IKN on average by 25±3% (n=8) from 13±4 to 10±3 pA (P<0.05), although in two cells it had no measurable effect.
IKN Lacks Sensitivity to Cytoplasmic Ca2+
Studies on recombinant TASK-1 channels have shown that intracellular Ca2+ has no effect on their activity,22 yet there is evidence that Ca2+ inhibits the resting K+ conductance in dog PASMCs.30 To test whether or not IKN in rabbit is a Ca2+-sensitive conductance, we first studied the effect on the current of increasing Ca2+ influx with the Ca2+ ionophore, A23187. This often induced inward current at negative potentials and increased outward current at positive potentials, probably due to the activation of Cl- channels by the rise in submembrane [Ca2+].31 To avoid this and its potential interference with IKN, the experiments were repeated using low Cl- (10 mmol/L) PSS and pipette solutions and measurements were confined to 0 mV, the Cl- equilibrium potential. At a concentration evoking contraction of rabbit PASMCs, A23187 (1 µmol/L) had no clear effect on the amplitude of IKN measured at 0 mV in these conditions (Figure 7A, n=3). In three other cells studied in normal recording solutions, the removal of extracellular Ca2+ also failed to change the amplitude of IKN at 0 mV (Figure 7B). This was despite the presence of 1 mmol/L EGTA in the bath solution to ensure that extracellular Ca2+ was buffered to a minimal level. An alternative method used to modulate the intracellular Ca2+ concentration was to vary the nature and concentration of the Ca2+ chelator used in the internal, pipette solution. The amplitude of IKN measured at 0 mV was not found to differ significantly when the pipette contained 0.05 to 0.1, 1 or 5 mmol/L EGTA, or 10 mmol/L BAPTA (Figure 7C).
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Discussion |
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We have demonstrated that the noninactivating K+ current, IKN, previously shown to underlie the resting potential of rabbit PASMCs,7 has all the properties expected for a background K+ channel belonging to the TASK family of two-pore domain channels. Pharmacological characterization previously demonstrated that IKN is insensitive to block by TEA, glibenclamide, Cs+, and a range of KCa channel blockers and is inhibited only at high concentrations of 4-aminopyridine and quinine.7,8 The present study shows that IKN is additionally sensitive to pHo, halothane, Zn2+, and anandamide, but not to intracellular Ca2+. Thus, the pharmacological profile of IKN and the resting potential of PASMCs closely matches that of heterologously expressed TASK-1 channels.21,32 The pHo at which 50% of IKN was inhibited, pH 7.3, is the same as that found for TASK-1 channels.22 Furthermore, the high sensitivity of IKN to inhibition by Zn2+ and anandamide strongly implicates TASK-1 as a major contributor to the current.24,25 The finding that pHo and Zn2+ also modulated IKV is consistent with a role for IKV in their effects on membrane potential. It is unlikely to be a major role though, because our previous studies showed that IKV (both the transient and sustained components) contributes little to the normal maintenance of resting potential.7 The lack of effect of halothane on IKV, although producing membrane hyperpolarization and facilitation of IKN, provides further evidence to link resting potential regulation to IKN and TASK-1 channels, rather than IKV. Previous studies showed that mRNA for TASK channels is expressed in the lung.21 We now extend these observations to show that TASK-1 mRNA and protein are expressed in PASMCs. Moreover, antibody staining showed TASK-1 expression at the surface membrane, compatible with a functional role in mediating membrane K+ currents.
The sensitivity of IKN to small changes in pHo means that extracellular acidification depolarizes PASMCs, thereby facilitating voltage-gated Ca2+ entry and contraction. Thus, modulation of IKN and membrane potential may contribute to acidosis-induced pulmonary vasoconstriction33 and the well known pulmonary vasodilator effect of alkalosis, which is inhibited by 10 mmol/L 4-aminopyridine and by manipulations that suppress the transmembrane K+ gradient and cause depolarization.34 Because IKN inhibition and membrane depolarization may contribute to the vasoconstrictor response of pulmonary arteries to hypoxia,6 pHo-dependent modulation of IKN may also explain the stimulatory effect of acidosis and inhibitory effect of alkalosis on hypoxic pulmonary vasoconstriction.35 The exquisite sensitivity of IKN to changes in pHo suggests that pHo-dependent modulation of IKN plays a functional role in physiological conditions, but it may become particularly important in pathological conditions where substantial changes in pHo can occur.36
The finding that acidosis inhibited the facilitation caused by halothane supports the hypothesis that halothane was acting on pH-sensitive TASK-1 channels. It is important to note, however, that the effects of halothane, Zn2+, and anandamide on IKN and membrane potential were not always as expected for modulation of TASK-1 channels. The failure of a small proportion of cells to respond to these agents suggests that the expression of TASK-1 channels may vary among PASMCs. Variable expression of K+ channels has been reported before and is thought to reflect phenotypic diversity among PASMCs.37,38 The inhibitory effect of halothane seen in some PASMCs seemed to obscure the facilitation at high concentrations and probably limited it at lower concentrations. This effect could be due to a separate action on additional channels that contribute to IKN. Halothane was recently found to inhibit an O2-sensitive background K+ current in glossopharyngeal neurons, which has a pharmacological profile compatible with THIK, another family of two-pore domain K+ channels.39 Like IKN, this current was insensitive to TEA and inhibited by millimolar quinidine, but it was only weakly modulated by pHo and not inhibited by 5 mmol/L 4-aminopyridine. It is therefore interesting that although acidosis inhibited IKN, it did not completely abolish it, and previous studies have shown that maximally effective concentrations of 4-aminopyridine only partially inhibit IKN.7 Thus several observations suggest that IKN may reflect the activity of more than a single population of K+ channels with differential sensitivity to pHo and pharmacological agents, and that the relative expression of these channels may vary among PASMCs. This is consistent with other cell types, where multiple K+ channels contribute toward the background K+ conductance that controls the resting potential.40
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Acknowledgments |
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Acknowledgments
Funding for this study was provided by the Biotechnology and Biological Sciences Research Council and the British Heart Foundation.
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Footnotes |
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Original received May 20, 2003; revision received September 10, 2003; accepted September 25, 2003.
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References |
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