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Am J Physiol Lung Cell Mol Physiol 291: L993-L1004, 2006. First published June 30, 2006; doi:10.1152/ajplung.00191.2005
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Bone morphogenetic protein-2 upregulates expression and function of voltage-gated K+ channels in human pulmonary artery smooth muscle cells

Ivana Fantozzi,1 Oleksandr Platoshyn,1 Ada H. Wong,2 Shen Zhang,1 Carmelle V. Remillard,1 Manohar R. Furtado,2 Olga V. Petrauskene,2 and Jason X.-J. Yuan1

1Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California San Diego, La Jolla, and 2Applied Biosystems, Foster City, California

Submitted 27 April 2005 ; accepted in final form 28 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activity of voltage-gated K+ (KV) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in control of apoptosis and proliferation in addition to regulating membrane potential and pulmonary vascular tone. Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated KV channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. This study evaluated the effect of BMP-2 on KV channel function and expression in normal human PASMC. BMP-2 (100 nM for 18–24 h) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA10, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3 but downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2, and KCNV2. The most dramatic change was the >10-fold downregulation of KCNG2 and KCNV2, two electrically silent {gamma}-subunits that form heterotetramers with functional KV channel {alpha}-subunits (e.g., KCNB1–2). Furthermore, the amplitude and current density of whole cell KV currents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that K+ currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (2 subunits that were markedly downregulated by BMP-2) are inhibitors of functional KV channels. These results suggest that BMP-2 divergently regulates mRNA expression of various KV channel {alpha}-, beta-, and {gamma}-subunits and significantly increases whole cell KV currents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2-mediated increase in KV channel activity and regulation of KV channel expression. The increased KV channel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP-2 on PASMC.

pulmonary arterial hypertension; patch clamp; membrane potential

ACTIVITY OF VOLTAGE-GATED K+ (KV) channels in vascular smooth muscle cells regulates the resting membrane potential and excitation-contraction coupling (57). The current generated by K+ efflux through KV channels, IK(V), is heavily influenced by numerous vasoactive agonists that control vascular tone (57). In pulmonary arterial smooth muscle cells (PASMC) from animals and humans, downregulated KV channel expression and reduced KV channel function have been linked to pulmonary vasoconstriction triggered by acute hypoxia (33, 70, 79, 104) and to the sustained pulmonary vasoconstriction and severe pulmonary vascular remodeling induced by chronic hypoxia (69, 81, 88). Persistent hypoxic pulmonary vasoconstriction and hypoxia-mediated pulmonary vascular medial hypertrophy increase pulmonary vascular resistance, which contributes to the development of pulmonary hypertension and subsequent right heart failure in patients with chronic obstructive pulmonary disease and congenital cardiopulmonary diseases, as well as in residents living in high-altitude areas.

In addition to contribution to hypoxia-mediated pulmonary hypertension, intimal and medial hypertrophy of small and medium-sized pulmonary arteries is a hallmark of the pulmonary vascular remodeling processes that underlie the development and maintenance of high pulmonary arterial pressure in patients with familial and idiopathic pulmonary arterial hypertension (83). Overgrowth of PASMC in the pulmonary vascular media is particularly important in the development of pulmonary arterial hypertension because 1) sustained PASMC contraction leads to vasoconstriction while excessive PASMC growth enhances the contractile force and 2) PASMC hypertrophy and proliferation cause narrowing of the pulmonary vascular lumen, thereby increasing pulmonary vascular resistance and pulmonary arterial pressure.

Idiopathic pulmonary arterial hypertension is pathologically or histologically characterized by severe pulmonary vascular remodeling (due to smooth muscle and endothelial cell proliferation), obliteration of small arteries, intimal fibrosis, small vessel thrombi, and, sometimes, formation of the plexiform lesion (76). It is generally believed that multiple genetic and environmental factors are involved in the pathogenesis of familial and idiopathic pulmonary arterial hypertension (99), such as mutations of bone morphogenetic protein (BMP) receptor type II (BMPRII) (19, 45, 49, 58), aberrant regulation of matrix metalloproteins (48, 73), downregulation of K+ channels (98, 101, 105), upregulation of Ca2+-permeable channels (95, 97), intake of anorexigens (1, 87), increased production of serotonin and upregulated expression of serotonin receptors and transporter (4, 8, 24, 46), increased angiopoietin-1 production (21, 23, 84), elevated endothelin-1 expression and activity (31), and imbalanced cAMP (5, 60) and cGMP (20, 53) metabolism.

A number of factors can modulate PASMC proliferation and apoptosis, including transcription factors (14, 40, 66), growth factors (47, 55, 97), and mitogenic agonists (35, 63, 89, 107). Increased elastase activity and deposition of the matrix metalloprotein tenascin-C also have been linked with the enhanced proliferation in pulmonary arterial hypertension (39, 73). In some cells, transforming growth factor (TGF)-beta in particular has been shown to modulate the expression of transcription factors, such as c-Myc (29, 93), thereby regulating cell proliferation and/or survival. BMPs, members of the TGF-beta superfamily, also regulate PASMC proliferation, apoptosis, and differentiation (36, 51, 52, 55, 56, 92) via autocrine and paracrine mechanisms. Studies have shown that BMP-2, BMP-4, and BMP-7 can increase the rate of apoptosis and decrease the rate of proliferation of vascular smooth muscle cells (56, 92), including human PASMC (55, 106). In PASMC from patients diagnosed with idiopathic pulmonary arterial hypertension, BMP (BMP-2 and BMP-7)-induced apoptosis was significantly impaired (55, 106). BMP-2-mediated apoptosis was associated with transient phosphorylation of Smad1, a protein signaling element activated on binding of BMPs to their BMPRI and BMPRII (36, 52), and with marked downregulation of Bcl-2, an antiapoptotic protein (106). In an earlier study, Bcl-2 was shown to enhance cell survival or inhibit cell apoptosis by, at least in part, decreasing KV channel activity and downregulating KV channel {alpha}-subunit expression in rat PASMC (25). This supports the mounting evidence that enhanced K+ activity is essential to the onset and progression of PASMC apoptosis (74). The goal of the present study was to test the hypothesis that BMP-2 increases whole cell IK(V) by divergently regulating mRNA expression of various KV channel {alpha}-, beta-, and {gamma}-subunits in human PASMC. In addition, we also examined the effect of BMP-2 on the protein expression of c-Myc, a transcription factor that is upregulated in proliferating cells and downregulated by TGF-beta, and explored the possibility that BMP-2 regulates KV channel expression and promotes cell apoptosis by modulating c-Myc expression.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and preparation. Human PASMC (passages 4–7) from normal subjects (Cambrex, Walkersville, MD) were derived from pulmonary arteries of three individuals. PASMC were cryopreserved at passage 3, replated onto flasks to amplify cell number for two or three passages, and then used for experiments for two or three passages (i.e., at passages 5–7). Cells were seeded and cultured in smooth muscle growth medium (SMGM, Cambrex) in a humidified atmosphere of 5% CO2-95% air at 37°C. SMGM was composed of smooth muscle basal medium (SMBM) supplemented with 5% fetal bovine serum (FBS), 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5 µg/ml insulin. Proliferating (cultured in SMGM) and growth-arrested (cultured in SMBM) cells were treated with BMP-2 for 18 or 24 h before experimentation. Identification of the cells in culture as smooth muscle cells was verified with the smooth muscle {alpha}-actin monoclonal antibody and the nuclear acid stain 4',6-diamidino-2-phenylindole (DAPI, 5 µM). The DAPI-stained cells also cross-reacted with the smooth muscle cell {alpha}-actin antibody, indicating that the cultures were all smooth muscle cells.

Measurement of macroscopic IK(V). Whole cell IK(V) were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments) with conventional voltage-clamp techniques. PASMC plated onto glass coverslips were superfused in a perfusion chamber placed on the microscope stage with Ca2+-free bath solution containing (in mM) 141 NaCl, 4.7 KCl, 3 MgCl2, 1 EGTA, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with 2 M NaOH). Whole cell K+ currents were recorded with a pipette solution containing (in mM) 5 Na2ATP, 135 KCl, 4 MgCl2, 10 EGTA, and 10 HEPES (pH adjusted to 7.2 with 2 M KOH). When cells were superfused with the Ca2+-free bath solution and dialyzed with the 5 mM ATP- and 10 mM EGTA-containing (Ca2+ free) pipette solution, the contribution of outward K+ currents through Ca2+-activated K+ channels and ATP-sensitive K+ channels to the recorded whole cell K+ currents was significantly minimized. It has been demonstrated that 1–3 mM ATP markedly blocked ATP-sensitive K+ channels in PASMC (16), whereas removal or chelation of extracellular and intracellular Ca2+ with a high concentration of EGTA significantly reduced Ca2+-activated K+ channel activity (9, 100, 102, 103).

Series resistance compensation was performed in all whole cell recording experiments. Step-pulse protocols and data acquisition were performed with pCLAMP software (Axon Instruments). Leak and capacitative currents were subtracted with the P/4 protocol in pCLAMP software. Currents were filtered at 1–2 kHz (–3 dB) and digitized at 2–4 kHz. Whole cell IK(V) were recorded during 300-ms voltage steps ranging between –60 and +80 mV (in 20-mV increments) from a holding potential of –70 mV. All experiments were performed at room temperature (22–24°C). Current-voltage (I-V) relationship curves report both current amplitude (in pA) and current density (normalized to cell capacitance; pA/pF).

Real-time reverse transcription-PCR. Total RNA was extracted from human PASMC with an RNeasy Mini Kit (Qiagen) according to previously published methods (106). Sense and antisense primers for the real-time reverse transcription (RT)-PCR experiments were specifically designed by scientists at Applied Biosystems (Foster City, CA) from the coding regions of each KV channel gene; the fidelity and specificity of the sense and antisense primers were examined with a BLAST program. Because of proprietary issues and the policy of Applied Biosystems, the exact primer sequences used for the real-time RT-PCR experiments are not provided but can be requested from the company based on the information (e.g., assay ID, the reference sequence, and the exon boundaries used to design the primers) shown in Table 1.


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  		Table 1. Oligonucleotide primers used for real-time RT-PCR

 
RT was performed with the High Capacity cDNA Archive Kit (Applied Biosystems), which uses random primers. PCR amplification with real-time detection was performed with TaqMan Universal PCR Master Mix with AmpErase (uracil N-glycosylase; UNG) and total RNA of 100 ng/µl. Initial PCR reaction mixtures were prepared in 96-well plates, and then 10-µl aliquots of the reactions were distributed to each of four quadrants on 384-well plates by an automated method on a Biomek FX robotic workstation. Real-time PCR was performed with an ABI PRISM 7900HT Sequence Detection System. Thermal cycling conditions comprised an initial UNG incubation at 50°C for 2 min, AmpliTaq Gold DNA polymerase activation at 95°C for 10 min, 40 cycles of denaturing at 95°C for 15 s, and annealing and extension at 60°C for 1 min. Real-time PCR assays were designed with the Applied Biosystems bioinformatics design pipeline. These assays were all part of the Applied Biosystems TaqMan Assay or Custom TaqMan Assay product lines.

Fold change (FC) calculation was performed with the comparative threshold cycle (Ct) or the {Delta}{Delta}Ct method, based on the formula FC = 2Formula, to calculate normalized FCs in gene expression in test samples relative to a calibrator sample (i.e., treated samples relative to untreated samples) (30). The first step in the FC analysis is normalization of target gene expression signal to endogenous control expression ({Delta}Ct). In our experiments, both 18S and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as housekeeping genes. However, only 18S behaved consistently between samples. Therefore, it was used as the endogenous control signal. The second step is to calculate the difference between normalized target gene expressions in BMP-2-treated and untreated samples ({Delta}{Delta}Ct). FC calculation, which represents the difference of gene expression levels between BMP-2-treated samples and control (or untreated) samples, was carried out for each gene individually, using the formula mentioned above. Each measurement was repeated in three samples from different cell cultures. A limitation of the study is that only one apparently reliable housekeeping gene (18S) was used for data analysis, but despite this the data do show that there are clear changes in the relative abundance of KV channel mRNAs.

Bioinformatics design of real-time PCR assays. Applied Biosystems developed a bioinformatics assay design pipeline that consists of two parts: an assay design engine and an assay evaluation tool. The assay design engine selects primers and probes based on thermodynamic parameters (melting temperature, nucleotide composition, self-complementarity, etc.) to ensure 100% efficiency of the assays. The assay evaluation component of the pipeline scores the potential hybridization of the assay to closely related genes to produce high-specificity assays. To assess cross-hybridization, the concept of forbidden targets (antitargets) and a target sequence table were implemented. The antitargets represent all sequences that should not be detected by an assay. The target sequence table groups redundant sequences for the same target. Validation of more than a thousand assays has demonstrated that the design pipeline generates primers/probe sets that perform with 1) near 100% PCR amplification efficiency and 2) a high degree of specificity for the selective amplification of a targeted gene in the presence of closely related targets (30).

Regular RT-PCR. Total RNA was isolated from human PASMC cultured in SMBM (for 24 h) with the RNeasy Mini Kit (Qiagen). cDNA was synthesized with SuperScriptJ reverse transcriptase (Invitrogen, Carlsbad, CA). RT-PCR was performed with a GeneAmp PCR System (Perkin Elmer, Boston, MA) using a Platinum PCR Supermix (GIBCO). The sequences of sense and antisense primers (Table 2) for the regular RT-PCR experiments were specifically designed from the coding regions of K+ channel {alpha}- and beta-subunit genes. GAPDH was used as an internal control to semiquantify the PCR products and to normalize the PCR products of KV channel subunits. Amplified PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. Results presented are representative of experiments performed in three different PASMC cultures treated or not with BMP-2.


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  		Table 2. Oligonucleotide sequences of primers used for regular RT-PCR

 
Western blot analysis. Human PASMC were first cultured in medium containing PDGF for 6 h to stimulate proliferation. Protein expression of c-Myc was evaluated under control conditions and after treatment with BMP-2 for 1–6 h. Human PASMC were gently washed twice in cold PBS, scraped into lysis buffer [1% Nonidet P-40 (Amaresco), 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, and 30 µl/l aprotinin] and incubated on ice for 30 min. Cell lysates were then sonicated and centrifuged at 12,000 rpm for 10 min, and the insoluble fraction was discarded. In some experiments, cell lysates were treated with the peptide N-glycosidase F (20 U; New England Biolabs) overnight at 4°C. The protein concentration in the supernatant was determined by the bicinchoninic acid protein assay using bovine serum albumin (BSA) as a standard. Ten- to twenty-five-microliter aliquots of protein were mixed and boiled in SDS-PAGE sample buffer for 5 min. The protein samples separated on 10% SDS-PAGE were then transferred to nitrocellulose membranes by electroblotting in a Mini Trans-Blot cell transfer apparatus according to the manufacturer's instructions (Bio-Rad Laboratories). After incubation overnight at 4°C in a blocking buffer [0.1% Tween 20 in phosphate-buffered saline (PBS)] containing 5% nonfat dry milk powder, the membranes were incubated with polyclonal antibodies against c-Myc (Santa Cruz Biotechnology) and {alpha}-actin (Sigma). Finally, the membranes were washed and incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG for 90 min at room temperature. The bound antibody was detected with an enhanced chemiluminescence detection system (Amersham). Band intensity in arbitrary units for both c-Myc and {alpha}-actin as well as the intensity ratio (c-Myc/{alpha}-actin) are reported. To compare protein expression levels of c-Myc and {alpha}-actin, the intensity of light transmission through the blot along a line drawn vertically across the c-Myc and {alpha}-actin bands was determined with Image-Pro Plus analysis software. The intensity of the c-Myc band was then divided by the intensity of the {alpha}-actin band to produce the normalized intensity ratio for comparing relative changes of c-Myc protein expression under control conditions and during treatment with BMP-2.

Chemicals. Chemicals for electrophysiological measurements were purchased from Sigma unless otherwise indicated. 4-Aminopyridine (4-AP) and charybdotoxin (ChTX) were directly dissolved in the bath solution on the day of use; the pH value of the solution was measured after addition of the drugs and readjusted to 7.4 when necessary. Recombinant human BMP-2 (R&D Systems) was prepared in a stock solution in sterile PBS (with 0.1% BSA); aliquots of the stock solution were then diluted into the appropriate culture medium (SMGM or SMBM) to the final concentration on the day of use.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed with a paired Student's t-test or analysis of variance. Differences were considered to be significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
BMP-2 increases whole cell IK(V) in human PASMC. Whole cell K+ currents were recorded in human PASMC superfused with Ca2+-free bath solution and dialyzed with Ca2+-free and ATP-containing pipette solution. Because the Ca2+-activated and ATP-sensitive K+ currents were markedly minimized under these conditions, the remaining outward K+ currents were mainly due to K+ efflux through KV channels. The KV currents, IK(V), measured in these human PASMC had electrophysiological properties and characteristics similar to those depicted in our previous studies (68, 69). The currents were voltage dependent; the threshold for activating the KV channels was between –45 and –55 mV. On depolarizing test potentials, the currents were rapidly activated with a time constant ({tau}act) of <8 ms and slowly inactivated (Fig. 1A).


Figure 1
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  		Fig. 1. Bone morphogenetic protein (BMP)-2 treatment enhances whole cell voltage-gated K+ (KV) channel current (IK(V)) in normal human pulmonary artery smooth muscle cells (PASMC). A: representative currents, elicited by 300-ms step depolarizations at potentials ranging between –60 and +80 mV from a holding potential of –70 mV, in a control PASMC and a PASMC treated with BMP-2 (200 nM for 24 h). B: BMP-2-sensitive IK(V) were obtained by digital subtraction (a) of the currents recorded in BMP-2-treated PASMC from the currents recorded in control PASMC. The current (I)-voltage (V) relationship curve of the BMP-2-sensitive IK(V) is shown in b (means ± SE). C: summarized I-V relationship curves for currents measured in control cells (n = 9) and cells treated with BMP-2 (200 nM for 24 h; n = 9). D: membrane capacitance (Cm) measured in control and BMP-2-treated cells (means ± SE). E: amplitude (a) and current density (b) of IK(V) measured at a test potential of –40 mV in control and BMP-2-treated PASMC. **P < 0.01 vs. control.

 
Treatment of PASMC with BMP-2 (200 nM for 24 h) significantly increased IK(V) (Fig. 1, A and B). Digital subtraction of the currents recorded in control cells from the currents recorded in BMP-2-treated cells describes the BMP-2-sensitive IK(V) (Fig. 1Ba), which was activated by test potentials more negative than –40 mV (Fig. 1Bb). BMP-2 enhanced IK(V) at all test membrane potentials; the percent increase of IK(V) at –60, –40, –20, 0, +20, +40, +60, and +80 mV was 20 ± 10%, 51 ± 13%, 97 ± 26%, 86 ± 19%, 83 ± 19%, 64 ± 10%, 100 ± 8%, and 120 ± 10%, respectively (Fig. 1C). BMP-2 treatment had no effect on membrane capacitance (Fig. 1D), although our previous data (106) indicated that BMP-2 induced significant cell volume decrease (apoptotic volume decrease) and apoptosis. Current amplitude and density were both significantly increased (P < 0.01) by BMP-2 at a test potential of –40 mV (Fig. 1E), suggesting that BMP-2-sensitive IK(V) is active at potentials close to resting membrane potential in cultured human PASMC.

In addition to amplitude and the current density of IK(V), we also examined whether BMP-2 affected channel gating properties by comparing the kinetics (activation, inactivation, and deactivation) of the currents recorded in control cells and BMP-2-treated cells. As shown in Fig. 2, BMP-2 decelerated current activation (P < 0.001) but accelerated current inactivation. The {tau}act in BMP-2-treated PASMC was 5.25 ± 1.96-fold higher (P < 0.001) than {tau}act in control cells, whereas the time constant for current inactivation ({tau}inact) in BMP-2-treated cells was 40% less (P < 0.05) than {tau}inact in control cells (Fig. 2, A–C). However, BMP-2 had a negligible effect on current deactivation; the time constant for current deactivation ({tau}deact) was comparable (P = 0.14) in control and BMP-2-treated PASMC (Fig. 2D). These results indicate that chronic treatment of human PASMC with BMP-2 has different effects on amplitude, current density, and kinetics of IK(V). The BMP-2-mediated significant increase in current amplitude and density of IK(V) might be due to its upregulating effect on the channel expression (see below), whereas the BMP-2-mediated significant inhibition of current activation and slight (but statistically significant) acceleration of current inactivation might result from its potential effect on KV channel gating and/or indirect effect on channel gating via modulation of intracellular signal transduction proteins and KV channel regulatory subunits (e.g., beta-subunits).


Figure 2
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  		Fig. 2. Effect of BMP-2 on IK(V) kinetics in normal human PASMC. A: averaged current traces recorded at a test potential of +80 mV in control cells (n = 9) and cells treated with BMP-2 (200 nM for 24 h;, n = 9). Boxed areas identify time intervals used to calculate activation ({tau}act), deactivation ({tau}deact), and inactivation ({tau}inact) time constants. B–D: normalized currents at +80 mV (a) and summarized data (b) showing {tau}act (B), {tau}inact (C), and {tau}deact (D). In a, gray curve is from control cells and black curve is from BMP-2-treated cells (n = 9 cells for both conditions). *P < 0.05, ***P < 0.001 vs. control.

 
BMP-2-enhanced K+ currents are mainly due to K+ efflux through KV channels. Similar to smooth muscle cells isolated from systemic arteries, PASMC express multiple types of K+ channels, such as KV channels (2, 27, 65, 70, 100), Ca2+-activated K+ channels (2, 65, 103), ATP-sensitive K+ channels (15, 16), and voltage-independent tandem two-pore K+ (KT) channels (28, 32, 61). The whole cell K+ currents in BMP-2-treated PASMC (Fig. 1, A and B) were measured with pCLAMP software; the linear voltage-independent currents, along with the capacitance and leaking currents, were all subtracted by the P/4 protocol. Therefore, the contribution of KT channels to the currents shown in Fig. 1 is negligible.

In PASMC, ATP-sensitive K+ channels are completely inhibited by ~3 mM ATP in the pipette solution (16). The currents in BMP-2-treated PASMC were recorded with a pipette solution containing 5 mM ATP, which would completely block ATP-sensitive K+ channels and significantly limit the contribution of ATP-sensitive K+ channels to the whole cell currents shown in Fig. 1. Furthermore, the Ca2+-free pipette (intracellular) solution used in our study contained 10 mM EGTA, which would considerably reduce free Ca2+ concentration ([Ca2+]) in the cytosol when the whole cell recording configuration was formed. The Ca2+-free bath solution contained 1 mM EGTA, which would completely chelate all residual free Ca2+ in the extracellular solution. Accordingly, the currents recorded in cells superfused and dialyzed with these extracellular (bath) and intracellular (pipette) solutions should be mainly due to K+ currents through KV channels.

To verify that the BMP-2-sensitive outward K+ currents were indeed carried mainly by K+ efflux through KV channels, we examined the acute effects of 4-aminopyriline (4-AP), a relatively selective blocker of KV channels (57), and charybdotoxin (ChTX), a specific Ca2+-activated K+ channel blocker (57), on the currents recorded in BMP-2-treated PASMC. As shown in Fig. 3, extracellular application of 5 mM 4-AP significantly reduced the whole cell K+ currents elicited by depolarizing the cells to a series of test potentials ranging from –60 to +80 mV from a holding potential of –70 mV (Fig. 3A). However, extracellular application of 50 nM ChTX had a negligible effect on the currents (Fig. 3B). These results indicate that the enhanced outward K+ currents in human PASMC treated with BMP-2 are mainly due to increased KV channel activity. It must be emphasized that, although our study indicates that BMP-2 specifically enhanced IK(V), BMP-2 may also increase the activity of other K+ channels (e.g., Ca2+-activated K+ channels, ATP-sensitive K+ channels, and tandem domain two pore channels) in human PASMC; the potential effect of BMP-2 on these channels might be masked by the special experimental conditions and protocols used in this study.


Figure 3
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  		Fig. 3. Effects of KV and Ca2+-activated K+ (KCa) channel blockade on BMP-2-induced currents in human PASMC. A: representative currents (a), elicited by depolarizing the cells from a holding potential of –70 mV to a series of test potentials ranging from –60 to +80 mV (in 10-mV increments), and summarized I-V relationship curves (b, means ± SE) in PASMC previously treated with BMP-2 (200 nM for 24 h). Currents were recorded before (Control), during (4-AP), and after (Washout) extracellular application of 5 mM 4-aminopyridine (4-AP; n = 6). B: representative currents (a) and summarized I-V curves (b; means ± SE) in BMP-2-treated PASMC before (Control), during (ChTX), and after (Washout) transient exposure to 50 nM charybdotoxin (ChTX; n = 9). The subtraction current (inset), obtained by digital subtraction of the currents recorded during ChTX application from the currents recorded before ChTX application, shows that BMP-2 did not stimulate much ChTX-sensitive KCa current.

 
BMP-2 differentially alters KV channel {alpha}-, beta-, and {gamma}-subunit mRNA expression. Amplitude of whole cell IK(V) is predominantly determined by changes in channel function and expression. Increases in KV channel gene expression would thus increase the number of KV channels and subsequently increase whole cell IK(V). Quantitation of changes in transcript levels with real-time RT-PCR, without the need for generating standard curves for each assay, is a useful tool for high-throughput analysis of a large number of genes in cellular regulatory networks. We performed real-time RT-PCR on nucleic acid samples isolated from control proliferating human PASMC and PASMC treated with 100 nM BMP-2 for 18 or 24 h. FCs in transcript levels were determined with the {Delta}{Delta}Ct method (30) and are summarized (means ± SD) in Fig. 4. Statistical significance of the down- and upregulation by BMP-2 was verified with unpaired t-tests. Similar results were obtained when n-fold changes were calculated for samples from proliferating PASMC treated with BMP-2 for 18 h (data not shown), or when data from 18- and 24-h treated cells were averaged (data not shown).


Figure 4
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  		Fig. 4. Quantitative change in mRNA expression of various KV channel subunits measured by real-time RT-PCR. A: representative plot showing the change in KCNG2 (KV6.2) mRNA expression with cycle number in control PASMC and PASMC treated with 100 nM BMP-2 for 24 h. Downregulation is shown by the rightward shift in the mRNA expression curve as a function of cycle number. B: normalized fold change (FC) (BMP-2/Control) mRNA expression is shown for KV channel {alpha}-, beta-, and {gamma}-subunits in cells treated with BMP-2 (100 nM for 24 h). Upward deflections (gray bars) indicate upregulation; downward deflections (black bars) indicate downregulation. The normalized FCs for KCNG2 (KV6.2) and KCNV2 (KV11.1) are –11.17 and –22.66, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, BMP-2-treated samples vs. Control. The data were reproduced in 3 independent experiments. {Delta}Rn, mRNA expression.

 
BMP-2 treatment caused a fourfold or greater increase in the mRNA expression of KCNA7 (KV1.7; P < 0.01) and KCNS3 (KV9.3; P < 0.01) subunits. Greater than twofold increases were observed for KCNA5 (KV1.5; P < 0.01), KCNA10 (KV1.10; P < 0.01), KCNC3 (KV3.3; P < 0.01), KCNC4 (KV3.4; P < 0.001), KCNF1 (KV5.1; P < 0.01), KCNS1 (KV9.1; P < 0.001), and KCNG3 (KV6.1; P < 0.01) subunits. Smaller (≤1.5-fold) enhancement was noted for KCNAB2 (KVbeta2.1; P < 0.001), KCND1 (KV4.1; P < 0.01), KCND2 (KV4.2; P < 0.01), KCND3 (KV4.2; P < 0.01), and KCNG1 (KV6.1; P < 0.001) subunits. BMP-2 treatment also increased KCNA6 (KV1.6) mRNA expression by ~1.25-fold, but the effect was not statistically significant.

BMP-2 treatment caused a decrease in the expression of several other KV channel {alpha}- and beta-subunits. KCNAB1 (KVbeta1.1; P < 0.001), KCNA2 (KV1.2; P < 0.01), KCNG2 (KV6.2; P < 0.001), and KCNV2 (KV11.1; P < 0.001) expression levels were decreased more than twofold by BMP-2. Less than twofold decrease was observed for KCNAB3 (KVbeta3; P < 0.05) and KCNB2 (KV2.2; P < 0.05). BMP-2 treatment also decreased mRNA expression of KCNA1 [KV1.1; not significant (NS)], KCNA4 (KV1.4; NS), and KCNC1 (KV3.1; NS) by one- to twofold, but the effect was not statistically significant.

The most dramatic changes observed were the downregulation of KCNG2 (KV6.2) (Fig. 4A) and KCNV2 (KV11.1) transcripts by BMP-2; the amounts of RT-PCR products for KCNG2 (P < 0.0001) and KCNV2 (P = 0.0003) in BMP-2-treated PASMC were 11.17- and 22.66-fold lower than in control PASMC, respectively (Fig. 4B). Both of these subunits encode for electrically silent KV channel {gamma}-subunits, which may, on coexpression with functional pore-forming KV channel {alpha}-subunits, alter the electrophysiological or biophysical properties of the functional tetrameric channel (62). The possible outcome of this change is discussed further below.

In addition to the quantitative real-time RT-PCR experiments using cDNA isolated from proliferating PASMC (cultured in SMGM), we also determined the effects of BMP-2 on KV channel {alpha}-, beta-, and {gamma}-subunit expression in growth-arrested PASMC (cultured in SMBM), using regular RT-PCR and the primers listed in Table 2. Our results showed that BMP-2 (100 nM for 24 h) slightly downregulated KCNAB1 (KVbeta1) and KCNAB3 (KVbeta3), significantly upregulated KCNA5 (KV1.5), KCNA6 (KV1.6), KCNA7 (KV1.7), KCNA10 (KV1.10), and KCNF1 (KV5.1), and negligibly affected GAPDH.

BMP-2 downregulates c-Myc protein expression. It has been demonstrated that, in various cell types, TGF-beta downregulates the expression of c-Myc, a transcription factor that promotes cell proliferation and/or survival, via a Smad-dependent pathway (29, 93). To assess whether c-Myc might also be involved in the BMP-2-mediated effect on KV channel expression, we examined and compared the protein level of c-Myc in control and BMP-2-treated cells. Human PASMC were first cultured in medium containing PDGF for 6 h to stimulate proliferation. Protein expression of c-Myc was evaluated under control conditions and after treatment with BMP-2 for 1–6 h. As shown in Fig. 5, BMP-2 significantly downregulated the protein expression of c-Myc in a time-dependent manner but had a negligible effect on the protein level of {alpha}-actin. In normal PASMC, our preliminary data also demonstrated that expression of c-Myc during proliferation depends on cytoplasmic [Ca2+] (data not shown). Previously, we had shown (106) that BMP-2-induced human PASMC apoptosis may be due to downregulation of Bcl-2, a membrane-bound antiapoptotic protein whose heightened expression we have also linked (25) to the downregulation of KV channel function and {alpha}-subunit expression. Therefore, these results suggest that BMP-2-mediated enhancement of IK(V) and potential transcriptional regulation on KV channel {alpha}-, beta-, and {gamma}-subunits may result, at least in part, from its downregulating effect on c-Myc (Fig. 5).


Figure 5
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  		Fig. 5. BMP-2 downregulates c-Myc protein expression in normal proliferating human PASMC. A: Western blot showing protein levels of c-Myc and {alpha}-actin in PASMC before (Cont) and after treatment with 500 ng/ml BMP-2 for 1, 3, or 6 h. Cells were cultured in smooth muscle basal medium containing PDGF (10 ng/ml) for 6 h in the continued presence of 2 mM external Ca2+ before BMP-2 treatment. B: intensity of light transmission through the blot along a line drawn vertically across the c-Myc band (top) and {alpha}-actin band (bottom) before (Cont) and after treatment with BMP-2 for 1, 3, and 6 h. C: normalized intensity ratio (intensity of c-Myc band divided by intensity of {alpha}-actin band) showing the relative c-Myc protein expression level under control conditions and during treatment with BMP-2.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
KV channel expression and function play important roles in the development of sustained pulmonary vasoconstriction and vascular remodeling, two major causes for the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension. First, downregulated expression of KV channel {alpha}-subunits and decreased IK(V) amplitude and density cause membrane depolarization such that voltage-dependent Ca2+ channels (VDCC) are activated (100). The latter allow Ca2+ to enter the cell, where it binds to myosin light chain kinase to initiate PASMC contraction and pulmonary vasoconstriction (82). As an important second messenger, Ca2+ also stimulates cell proliferation and DNA synthesis. Indeed, Ca2+ promotes transitions from the quiescent phase to DNA synthesis and mitotic phases of the cell cycle, thereby enhancing cell proliferation (7). Ca2+ is also required to activate many Ca2+-dependent signal transduction proteins (e.g., MAPK and CaM kinase) and transcription factors [e.g., c-Myc, cAMP response element binding protein (CREB), and c-Fos] involved in cell proliferation (6, 34). In the pulmonary vasculature, increased VDCC activity due to KV channel inhibition and sustained membrane depolarization can enhance PASMC proliferation and lead to pulmonary vascular medial hypertrophy (67).

Second, studies have demonstrated that sufficient cytosolic K+ concentration ([K+]cyt) is required to maintain a normal cell volume (11, 50) and to inhibit cytoplasmic caspases and nucleases (10, 37, 54). Increased activity of KV channels or whole cell IK(V) due to upregulated expression of KV channel {alpha}-subunits and/or to increased function of KV channels would enhance K+ efflux or loss and thus accelerate apoptotic volume decrease and facilitate apoptosis. Furthermore, increased K+ efflux or loss as a result of increased IK(V) would also decrease [K+]cyt and relieve the sustained inhibitory effect of K+ on cytoplasmic caspases, thereby promoting apoptosis. Activation of KV channels (and other types of K+ channels) is actually involved in apoptosis induced by many proapoptotic agents, such as staurosporine, nitric oxide, FCCP, and Fas ligand (22, 42, 43, 67, 74). Conversely, downregulation of KV channel {alpha}-subunit expression and inhibition of whole cell IK(V) would thus decelerate apoptotic volume decrease, decrease cytosolic caspase activity, and inhibit apoptosis. In PASMC isolated from patients with idiopathic pulmonary arterial hypertension, downregulated KV channel expression and decreased KV currents may contribute to the inhibited apoptosis (by attenuating apoptotic volume decrease and decreasing cytoplasmic caspase activity) and enhanced proliferation (by causing membrane depolarization and increase in Ca2+ influx through VDCC) (98), which ultimately cause pulmonary vascular medial hypertrophy.

Mutations of the BMPRII gene (BMPR2) have been linked to the development of familial and idiopathic pulmonary arterial hypertension (19, 45, 49, 58), whereas downregulated BMPRI gene expression has been observed in lung tissues and PASMC from patients with pulmonary arterial hypertension (23). The mutant BMPRII prevents its effective dimerization with BMPRI on binding to BMPs and thus inhibits BMPRI activation that is required to phosphorylate the downstream signaling proteins, mainly Smad1, -5, and -8 (59). Many genes encoding for proapoptotic or antiproliferative proteins are regulated by Smad proteins (52, 86). Under normal circumstances (i.e., with wild-type BMPRII isoforms and normal BMP signaling cascade), heterodimerizaton of BMPRI and BMPRII results in signals that induce apoptosis and inhibit proliferation (3, 23, 75, 106) in human PASMC. Therefore, dysfunctional BMP signaling due potentially to BMPRII mutations (19, 45, 49, 58) and BMPRI downregulation (23, 106) will inhibit PASMC apoptosis and enhance PASMC proliferation, as observed in patients with familial and idiopathic pulmonary arterial hypertension (19, 45, 49, 55).

Although there is no doubt that mutations of BMP receptors (e.g., BMPRII) affect pulmonary vascular function (e.g., contractility and reactivity) and PASMC proliferation, there is mounting evidence showing that changes in expression and function of their ligands, the BMPs themselves, also play an important role in the pulmonary vascular remodeling processes relating to pulmonary arterial hypertension. Although there are over 20 different BMPs, only a handful have been associated with vascular proliferation and mammalian development (18), including BMP-2, BMP-4, and BMP-7. Studies have shown that normal (i.e., non-pulmonary arterial hypertension) human lung tissues, pulmonary fibroblasts, and PASMC from patients without pulmonary hypertension (NPH) express BMP-1 through BMP-7 (38, 106). Furthermore, treatment of serum-stimulated PASMC with BMP-2, BMP-4, and BMP-7 significantly enhanced apoptosis (106) and attenuated cell proliferation and DNA synthesis (55). Although the findings were similar for PASMC from NPH and secondary pulmonary hypertensive patients, PASMC from patients with idiopathic pulmonary arterial hypertension were much more resistant to BMP-induced apoptotic effect and BMP-induced antiproliferative effect in these studies (55, 106), suggesting that BMP-mediated apoptotic pathways may be altered (or inhibited) in PASMC from patients with idiopathic pulmonary arterial hypertension.

On the other hand, a recent report by Takeda et al. (85) has suggested that BMP-4 and BMP-6 inhibit mitosis and proliferation in PASMC from patients with idiopathic pulmonary arterial hypertension, whereas mitosis is stimulated by BMP-2 and BMP-7. Even more recently, BMP signaling has also been linked to the pulmonary vascular remodeling associated with sustained hypoxia. Sheares et al. (80) showed that BMP-4 inhibits hypoxic induction of cyclooxygenase-2, an antiproliferative protein, in human PASMC. BMP-4 has also been shown to inhibit mitosis of human fetal lung fibroblasts and to promote their differentiation into a smooth muscle phenotype, both of which contribute to the vascular remodeling observed in idiopathic pulmonary arterial hypertension (38). Therefore, it is still unclear which BMPs mediate PASMC proliferation and apoptosis under normal conditions and in pulmonary vascular disease and which signaling pathway is involved in the development of pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension.

The purpose of this study was to test the hypothesis that, in normal PASMC, BMP upregulates functional KV channels and potentially maintains a relaxant (and/or apoptotic) phenotype of PASMC, to keep the pulmonary vasculature as a low-pressure/resistance and thin vascular wall system. The discussions above clearly show that both BMPs and KV channels are individually involved in modulating PASMC apoptosis and proliferation. The present study provides evidence that BMP signaling may modulate KV subunit expression and channel activity, thereby regulating PASMC proliferation and pulmonary vascular remodeling.

Our data show that 18- to 24-h treatment of normal PASMC with BMP-2 enhances function of KV channels, i.e., increases whole cell IK(V). Furthermore, BMP-2 treatment also differentially up- or downregulates the mRNA expression of numerous KV channel {alpha} (the functional pore-forming subunits)-, beta (the cytoplasmic regulatory subunits)-, and {gamma} (the electrically silent pore-forming subunits)-subunits. Surprisingly, the effect of BMP-2 on the expression of KV channel {alpha} (or {gamma})-subunits that have previously been associated with sustained pulmonary vasoconstriction and pulmonary vascular remodeling, i.e., KCNA2 (KV1.2), KCNA5 (KV1.5), KCNB1 (KV2.1), and KCNS3 (KV9.3) (64, 69, 72), is also different. For example, BMP-2 downregulated KCNA2 (KV1.2) and KCNB1 (KV2.1) but upregulated KCNA5 (KV1.5) and KCNS3 (KV9.3). However, the divergent effects of BMP-2 on mRNA expression of KV channel {alpha}-, beta-, and {gamma}-subunits somehow lead to an increase in whole cell IK(V) in normal PASMC. Consistent with our findings, Young et al. (94) recently reported that the BMP-2-mediated increase in IK(V) was significantly attenuated by anti-KV1.5 antibody. This suggests that 1) KV1.5 is one of the major functional KV channel {alpha}-subunits in PASMC and 2) upregulated KV1.5 expression (as shown in this study) and/or augmented KV1.5 channel function may greatly contribute to the BMP-2-mediated increase in whole cell IK(V).

In this study, we observed significant downregulation of the electrically silent KCNG2 (KV6.2) and KCNV2 (KV11.1) subunits induced by BMP-2 in normal human PASMC. KCNG1–3 (KV6.1–6.3) subunits are known to alter the kinetics of IK(V) currents when coexpressed with the functional pore-forming {alpha}-subunits, e.g., KCNB1 (KV2.1), in vitro (41, 62, 71, 78, 108), resulting in rapid inactivation of the currents and overall decreased steady-state current amplitude. KCNG2 (KV6.2) interaction with KCNB1 (KV2.1) subunits results in delayed rectifier K+ currents whose kinetics and conductance-voltage relationship differ from those mediated by homomultimeric KCNB1 (KV2.1) channels (108). Finally, the association of KCNV2 (KV11.1) and KCNB1 (KV2.1) subunits also causes decreased current amplitude and altered current kinetics (62). These findings imply that KV6 and KV11.1 subunits may suppress the activity of other functional pore-forming KV channel {alpha}-subunits, such as KV2.1. Downregulation of KV6.2 and KV11.1 expression in BMP-2-treated human PASMC may alleviate this suppression, leading to net increase in whole cell IK(V) and, possibly, allowing for accelerated apoptotic cell shrinkage and enhanced apoptosis.

The cellular and molecular mechanisms involved in the BMP-2-mediated effect on mRNA expression of different KV channel {alpha}-, beta-, and {gamma}-subunits remain unclear. However, we believe that BMP-2 (and other BMPs) may regulate gene transcription and expression, along with mRNA and protein degradation, of different KV channel genes through different mechanisms or pathways. For example, BMP-2 may 1) upregulate transcription of KV channel genes that contain Smad-binding sequences by increasing phosphorylated Smad1, -5, and -8 and 2) downregulate transcription of KV channel genes that contain Myc binding sequences by decreasing c-Myc expression (Fig. 6). In prior studies, we showed that 1) BMP-2 treatment caused marked downregulation of Bcl-2, an antiapoptotic protein (106), 2) Bcl-2 decreased KV subunit expression and channel activity and promoted PASMC survival (25), and 3) decreased IK(V) inhibits apoptosis or promotes survival of rat and human PASMC (13, 42–44). By inference, this would suggest that BMP-2, by decreasing Bcl-2 expression, could enhance KV expression and function to promote apoptosis. Our current findings suggest a link between BMP-2 function and IK(V) enhancement.


Figure 6
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  		Fig. 6. Schematic diagram of the proposed mechanism by which BMP-2 regulates KV channel {alpha}-, beta-, and {gamma}-subunit expression. Binding of BMP-2 with BMP receptors (BMPRI, BMPRII) leads to phosphorylation (P) of Smad1, -5, and -8 and downregulation of c-Myc. Smad and c-Myc then regulate gene transcription by directly binding to the gene promoter or indirectly by interacting with other transcription factors and intracellular proteins (e.g., Bcl-2).

 
The link between IK(V) enhancement by BMP-2 and regulation of KV subunit expression is not obvious. BMP-2 clearly has differential effects on different KV channel {alpha}-, beta-, and {gamma}-subunit expression, based on the scattered pattern on FC expression with either real-time or regular RT-PCR, with some subunits upregulated while others are downregulated, and to different extents. It is possible that these differential effects on subunit expression are due to 1) the presence (or absence) of Smad binding sequences and c-Myc binding sequences in the promoter regions of different KV channel genes and 2) differential interactions of Smad and c-Myc, along with other transcription factors (e.g., STAT, AP-1/c-Jun, MAPK, CREB, etc.), in regulating gene transcription of different KV channel subunits (12, 17, 26, 52, 90, 91, 96). We previously showed (96) that overexpression of c-Jun, an AP-1 transcription factor, decreased IK(V) in rat PASMC. We have identified a number of potential Myc-heterodimer binding sites and AP-1 binding sites in many of the KV channel gene promoters (Remillard CV and Yuan JX-J, unpublished observations). Findings from our previous study (106) showed that BMP-2-mediated downregulation of Bcl-2 may account for the PASMC apoptosis produced by BMP-2. In the present study, we clearly show that BMP-2 also downregulates the expression of c-Myc (Fig. 5) in human PASMC. Therefore, the data showing that 1) BMP-2 modulates the activity and/or expression of both c-Myc and Bcl-2 (106), 2) c-Myc may interact directly with Bcl-2 (77), and 3) Bcl-2 overexpression depresses KV function and expression (25) direct us to speculate that BMP-mediated transcriptional and functional regulation of KV channels in normal human PASMC may involve multiple factors including different transcription factors, cytoplasmic modulatory proteins, membrane binding proteins, and intracellular second messengers.

The present study provides evidence that BMP-2-mediated apoptosis (106) may involve activation of KV channels, as well as regulation of their mRNA expression. To substantiate these claims and to further link BMP-2-mediated transcriptional regulation of KV channel genes and functional enhancement of KV channel activity to PASMC apoptosis, we intend to conduct experiments to 1) determine whether BMP-2 also controls KV channel {alpha} (and {gamma})- and beta-subunit protein expression similarly to its effect on mRNA expression, 2) examine whether BMP-2-mediated PASMC apoptosis is susceptible to KV channel blockade (using pharmacological tools, small interfering RNA, antisense oligonucleotides), 3) determine the putative effect of KV6.2 (KCNG2) and KV11.1 (KCNV2) downregulation on native KV currents in PASMC, 4) determine whether Bcl-2 is involved in BMP-2-mediated KV channel expression and function, and, ultimately, 5) examine whether the effect of BMPs on KV channel mRNA/protein expression is different between PASMC from normal subjects and from patients diagnosed with idiopathic pulmonary arterial hypertension.

In summary, BMPs synthesized from lung parenchyma and pulmonary vascular cells serve as an important family of proteins to, by divergently regulating transcription and expression of various KV channel {alpha}-, beta-, and {gamma}-subunits, stimulate and maintain a high level of KV channel activity in normal human PASMC. The BMP-mediated transcriptional regulation of different KV channel {alpha}-, beta-, and {gamma}-subunits may result, at least in part, from its downregulating effect on Bcl-2 (25), an antiapoptotic protein that inhibits apoptosis and downregulates KV channels, and c-Myc, a transcription factor that is upregulated during cell proliferation. In PASMC, activity of KV channels is involved in regulating the resting membrane potential (100–102) and plays an important role in mediating apoptotic volume decrease and apoptosis (10–13, 42–44). Therefore, BMP-mediated increase in KV channel activity in PASMC would contribute to maintaining a relaxing status of pulmonary vessels and to restricting progression of pulmonary vascular medial hypertrophy.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-064945, HL-054043, and HL-066012.


   ACKNOWLEDGMENTS
 
We thank Dr. Pius Brzoska (Applied Biosystems) for bioinformatics support and Ann Nicholson for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. X.-J. Yuan, Division of Pulmonary and Critical Care Medicine, Dept. of Medicine, Univ. of California San Diego, 9500 Gilman Drive, MC 0725, La Jolla, CA 92093-0725 (e-mail: xiyuan{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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