1 Department of Medicine, University of California School of Medicine, San Diego, California 92103-8382; 2 Departments of Physiology and Medicine, University of Maryland, Baltimore 21201; and 3 National Institute on Aging, Gerontology Research Center, Baltimore, Maryland 21224
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ABSTRACT |
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Pulmonary vasoconstriction and vascular medial hypertrophy greatly contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension. A rise in cytosolic free Ca2+ ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (Em) regulates [Ca2+]cyt by governing Ca2+ influx through voltage-dependent Ca2+ channels. Thus intracellular Ca2+ may serve as a shared signal transduction element that leads to pulmonary vasoconstriction and vascular remodeling. In PASMC, activity of voltage-gated K+ (Kv) channels regulates resting Em. In this study, we investigated whether changes of Kv currents [IK(V)], Em, and [Ca2+]cyt affect cell growth by comparing these parameters in proliferating and growth-arrested PASMC. Serum deprivation induced growth arrest of PASMC, whereas chelation of extracellular Ca2+ abolished PASMC growth. Resting [Ca2+]cyt was significantly higher, and resting Em was more depolarized, in proliferating PASMC than in growth-arrested cells. Consistently, whole cell IK(V) was significantly attenuated in PASMC during proliferation. Furthermore, Em depolarization significantly increased resting [Ca2+]cyt and augmented agonist-mediated rises in [Ca2+]cyt in the absence of extracellular Ca2+. These results demonstrate that reduced IK(V), depolarized Em, and elevated [Ca2+]cyt may play a critical role in stimulating PASMC proliferation. Pulmonary vascular medial hypertrophy in patients with pulmonary hypertension may be partly caused by a membrane depolarization-mediated increase in [Ca2+]cyt in PASMC.
intracellular calcium; voltage-gated potassium channels; membrane potential
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INTRODUCTION |
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MEMBRANE POTENTIAL (Em) controls cytosolic free Ca2+ concentration ([Ca2+]cyt) mainly by modulating activity of sarcolemmal voltage-dependent Ca2+ channels (39, 56). Membrane depolarization opens voltage-gated Ca2+ channels, increases Ca2+ influx, and raises [Ca2+]cyt in smooth muscle cells (18, 39, 63). Membrane depolarization may also promote Ca2+ entry via the reverse mode of Na+/Ca2+ exchange (Ca2+-entry/Na+-exit mode), which triggers Ca2+-induced Ca2+ release from ryanodine-sensitive Ca2+ stores and further increases [Ca2+]cyt (8, 32, 49). Cytoplasmic ionized Ca2+ serves as a critical signal transduction element in a variety of cell functions, such as contraction (55), migration (43), proliferation (5, 37), and gene expression (15, 29, 34). A rise in [Ca2+]cyt triggers vasoconstriction (55), stimulates smooth muscle cell proliferation (5, 37) and migration (43), and, thus, may serve as a shared signal transduction element for pulmonary vasoconstriction and medial hypertrophy observed in patients with pulmonary hypertension. Persistent membrane depolarization causes sustained increase in [Ca2+]cyt (18) and, thus, should have a constant stimulatory effect on pulmonary vasoconstriction and pulmonary artery smooth muscle cell (PASMC) proliferation. Furthermore, maintenance of sufficient Ca2+ within sarcoplasmic (or endoplasmic) reticulum (SR), a major intracellular Ca2+ store, is necessary for cell growth. Indeed, depletion of the SR Ca2+ stores induces growth arrest in vascular smooth muscle cells (53).
Pulmonary vasoconstriction and vascular medial thickening (due to smooth muscle cell proliferation and migration) greatly contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension (42, 58). PASMC from patients with primary pulmonary hypertension (PPH) have more depolarized Em and higher resting [Ca2+]cyt than cells from normal subjects and patients with normotensive cardiopulmonary diseases and secondary pulmonary hypertension (e.g., congenital heart disease, pulmonary thromboembolic disease). The membrane depolarization and elevated resting [Ca2+]cyt in PASMC from PPH patients might be, at least partly, due to inhibited expression and function of voltage-gated K+ (Kv) channels (62, 66). Furthermore, increases in [Ca2+]cyt and membrane depolarization due to decreased Kv channel activity in PASMC have also been implicated in hypoxic pulmonary vasoconstriction (19, 24, 36, 45, 46, 59, 64).
An imbalanced ratio of endothelium-derived relaxing factors (EDRF) and constricting factors (EDCF) may contribute to the development of pulmonary hypertension (11, 20, 21). EDRF (e.g., NO and prostacyclin) and EDCF (e.g., endothelin-1 and thromboxane A2) both affect Em by modulating K+ channel activity in PASMC (2, 38, 51, 65). Therefore, increases in [Ca2+]cyt induced by membrane depolarization, in addition to triggering muscle contraction, may also play an important role in stimulating cell proliferation (48, 50) and migration (43).
The pulmonary medial (and myointimal) hypertrophy, mainly induced by PASMC proliferation and migration, is a critical contributor to the elevated pulmonary vascular resistance in patients with severe pulmonary hypertension. The rationale of this study was to test the hypotheses that 1) an increase in [Ca2+]cyt due to Ca2+ influx is essential for PASMC growth, and 2) the elevated [Ca2+]cyt results partly from membrane depolarization induced by Kv channel inhibition.
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MATERIALS AND METHODS |
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Cell preparation and culture. Primary cultures of PASMC were prepared from Sprague-Dawley rats (63). The intrapulmonary artery branches as well as the right branch of the main pulmonary artery were incubated for 20 min in Hanks' balanced salt solution with 1.5 mg/ml collagenase (Worthington). After the incubation, a thin layer of adventitia was carefully stripped off, and endothelium was removed by gently scratching the intimal surface. The remaining smooth muscle was digested with 1.75 mg/ml collagenase, 0.5 mg/ml elastase, and 1 mg/ml albumin (Sigma) for 45 min at 37°C. The cells were plated onto 25-mm coverslips in petri dishes and cultured in 10% fetal bovine serum (FBS)-DMEM in a 37°C, 5% CO2, humidified incubator.
Human PASMC (Clonetics) were seeded in flasks at a density of 2,500-3,500 cells/cm2 and incubated in smooth muscle growth medium (SMGM, Clonetics), and the medium was changed after 24 h, followed by every 48 h thereafter. SMGM is composed of smooth muscle basal medium (SMBM) supplemented with 5% FBS, 0.5 ng/ml human epidermal growth factor (hEGF), 2 ng/ml human fibroblast growth factor (hFGF), and 5 µg/ml insulin. Cells were subcultured or plated onto 25-mm coverslips by using trypsin-EDTA buffer (Clonetics) when 70-90% confluence was achieved. The cells at passages 5-8 were used for experimentation. A hemocytometer was used to determine cell counts. The cell number was normalized to the area of the petri dishes (expressed as cells/cm2).Immunofluorescence labeling.
The primary cultured rat PASMC were fixed in 95% ethanol and stained
with the membrane-permeable nucleic acid stain
4',6'-diamidino-2-phenylindole (DAPI; 5 µM; Molecular Probes). The
fluorescence emitted at 461 nm was used to visualize the cell nuclei
and estimate total cell numbers in the cultures. The specific
monoclonal antibody raised against smooth muscle -actin (Boehringer
Mannheim) was used to evaluate the cellular purity of cultures
(54), and a secondary antibody conjugated with
indocarbocyanine (Cy3) (Jackson ImmunoResearch) was used to display the
fluorescence image (emitted at 570 nm). The cells were mounted in 10%
1 M Tris · HCl-90% glycerol (pH 8.5) containing 1 mg/ml
p-phenylenediamine. The cell images were processed by a
MetaMorph Imaging System (Universal Imaging); the Cy3 fluorescence was
colored red and the DAPI fluorescence was colored green to display
images with red-green overlay. The DAPI-stained cells that also
cross-reacted with the smooth muscle cell
-actin antibody were
defined as smooth muscle cells.
Electrophysiological measurements.
Whole cell currents were recorded with an Axopatch-1D amplifier and a
DigiData 1200 interface (Axon Instruments) by using patch-clamp
techniques (23). Patch pipettes (2-4 M) were
fabricated on a Sutter electrode puller with the use of borosilicate
glass tubes and were fire polished on a Narishige microforge.
Step-pulse protocols and data acquisition were performed with pCLAMP
software. Currents were filtered at 1-2 kHz (
3 dB) and digitized
at 2-4 kHz by using the amplifier. All experiments were performed
at room temperature (22-24°C). Em in
single PASMC was measured in current-clamp mode (I = 0)
using the patch-clamp technique. In some experiments,
Em was recorded using an intracellular electrode (30-100 M
) filled with 3 M KCl. The data were acquired by an electrometer (Electro 705; World Precision) coupled to an
IBM-compatible computer and were analyzed using the DATAQ data
acquisition software (Dataq Instruments).
Measurement of [Ca2+]cyt. Cells were loaded with the acetoxymethyl ester form of fura 2 (fura 2-AM; 3 µM) for 30 min at room temperature (24°C) under an atmosphere of 5% CO2 in air. The fura 2-loaded cells were then superfused with standard bath solution for 20 min at 34°C to wash away extracellular dye and permit intracellular esterases to cleave cytosolic fura 2-AM into active fura 2. Fura 2 fluorescence (510-nm emission, 380- and 360-nm excitation) images from the cells and background were obtained with the use of a Gen III charge-coupled device camera (Stanford Photonics) coupled to a Carl Zeiss microscope. Image acquisition and analysis were performed with a MetaMorph Imaging System (Universal Imaging). Video frames containing images of fura 2 fluorescence from cells, as well as the corresponding background images (fluorescence from fields devoid of cells) were digitized at a resolution of 512 horizontal × 480 vertical pixels and an 8-bit gray scale. To improve the signal-to-noise ratio, four to eight consecutive video frames were usually averaged at a video frame rate of 30 frames/s. Images were acquired at a rate of one averaged image every 3 s when [Ca2+]cyt was changing and one every 60 s when [Ca2+]cyt was stable. [Ca2+]cyt was calculated from fura 2 fluorescence emission excited at 380 and 360 nm by using the ratio method (22). In most experiments, multiple cells (usually 6-10) were imaged in single field, and one arbitrarily chosen peripheral cytosolic area (10-12 × 10-12 pixels) from each cell was spatially averaged.
Solution and reagents. A coverslip containing the cells was positioned in the recording chamber (~0.75 ml) and superfused (2-3 ml/min) with the standard extracellular (bath) physiological salt solution (PSS) for recording K+ currents or measuring [Ca2+]cyt. The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, buffered to pH 7.4 with 5 M NaOH or 2 M Tris. In Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2, and 1 mM EGTA was added to chelate residual Ca2+. The internal (pipette) solution for recording whole cell K+ currents contained (in mM) 125 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP, buffered to pH 7.2 with 2 M Tris.
Valinomycin (Sigma) was prepared as a 100 mM stock solution in DMSO. Aliquots of the stock solution were diluted 1:1,000 into 0% FBS-DMEM to make a final concentration of 100 µM valinomycin. Similar dilution of DMSO alone was used as a vehicle control in culture media. 4-Aminopyridine (4-AP; Sigma) was directly dissolved into PSS on the day of use. The pH values of all solutions were checked after the addition of drugs and were readjusted to 7.4.Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed using the unpaired Student's t-test, or analysis of variance, as indicated. Differences were considered to be significant when P < 0.05.
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RESULTS |
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Purity of PASMC in primary cultures.
Pulmonary arteries, isolated from rat lungs, have a trilamellar
structure that is composed of fibroblasts (adventitia), smooth muscle
cells (media), and endothelial cells (intima). Thus smooth muscle cell
cultures are often contaminated with other cells, especially
fibroblasts because of their rapid growth rate. Morphologically, the
primary cultures of fibroblasts and smooth muscle cells are hardly
distinguishable when using phase-contrast microscopy (Fig. 1A, top). In this
study, adventitia was enzymatically removed before the smooth muscle
cell suspension was prepared. In the primary cultures prepared from
pulmonary arteries in which adventitia was intentionally removed,
virtually all of the cells labeled with DAPI cross-reacted with the
smooth muscle -actin antibody (Fig. 1A, left).
This indicates that >99.5% of the cells in cultures were smooth
muscle cells (Fig. 1B). However, in the primary cultures prepared directly from isolated pulmonary arteries, only 43% of the
cells labeled with DAPI cross-reacted with the smooth muscle
-actin
antibody (Fig. 1A, right), indicating substantial
contamination of fibroblasts (Fig. 1B). Thus primary
cultures prepared from the adventitia-free rat pulmonary arteries were
used in this study.
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Serum deprivation inhibits PASMC growth.
Primary cultured rat PASMC were divided into three groups and incubated
in DMEM containing 10%, 0.3%, and 0.1% FBS for 72 h,
respectively. As shown in Fig.
2A, reducing serum
concentration from 10% to 0.1% in culture media significantly
inhibited cell growth, suggesting that the cells cultured in 0.1% FBS
or serum-free (data not shown) DMEM are growth arrested after
24-48 h.
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Resting [Ca2+]cyt in
growth-arrested and proliferating PASMC.
There were no obvious morphological changes between growth-arrested and
proliferating PASMC from rats (Fig.
3A) and humans. However,
resting [Ca2+]cyt, measured in peripheral
cytosolic areas, was significantly higher in proliferating cells than
in growth-arrested rat (Fig. 3B) and human (Fig.
3D) cells. In rat PASMC, the histogram of resting
[Ca2+]cyt indicated a right shift (by ~85
nM) in proliferating cells compared with growth-arrested cells (Fig.
3C). The ratio of [Ca2+]cyt to
intracellularly stored [Ca2+] in the SR
([Ca2+]SR) is ~1:10,000 (6,
7). Therefore, the sustained elevation of
[Ca2+]cyt in proliferating PASMC may cause a
large increase in [Ca2+]SR (6,
7), both of which are essential for mitogen-mediated cell growth
(5, 37, 53).
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Resting Em in growth-arrested and proliferating PASMC.
Em is an important determinant of resting
[Ca2+]cyt in smooth muscle cells because of
the voltage dependence of Ca2+ influx through L-type
voltage-gated Ca2+ channels (18, 39).
Consistent with the results of [Ca2+]cyt,
resting Em in proliferating PASMC was much more
depolarized than that in growth-arrested PASMC from rats and humans
(Fig. 4). In smooth muscle cells, the
voltage window of sarcolemmal L-type voltage-gated Ca2+
channels for sustained elevation of [Ca2+]cyt
ranges from 40 to
20 mV and peaks at
30 mV (18).
Thus the sustained membrane depolarization in proliferating PASMC may produce a constant Ca2+ influx through L-type voltage-gated
Ca2+ channels and may contribute to maintain the elevated
[Ca2+]cyt that is crucial for cell
proliferation (29, 48, 50).
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Whole cell Kv currents in growth-arrested and proliferating human
PASMC.
Resting Em is regulated by activities of
multiple K+ channels, including Kv channels in PASMC
(17, 44, 45, 63). Whether the membrane depolarization in
proliferating cells resulted from inhibition of Kv channels was
examined in human PASMC. Whole cell Kv currents
[IK(V)] were elicited by depolarizing the
cells from a holding potential of 70 mV to a series of test
potentials ranging from
80 to +80 mV in increments of 20 mV (Fig.
5A). The currents appeared to
be activated at potentials close to the resting
Em (approximately
40 mV). In these
experiments, Ca2+-activated (KCa) and
ATP-sensitive (KATP) K+ currents were minimized
by the removal of extracellular (bath) and intracellular (pipette)
Ca2+ (plus 1-10 mM EGTA) and the inclusion of 5 mM ATP
in the pipette solution (12, 13, 44, 57). In proliferating
PASMC, the amplitude of whole cell IK(V) was
significantly reduced and the inactivation of
IK(V) (elicited by a +80 mV test potential) was accelerated compared with growth-arrested cells (Fig. 5). These results
suggest that inhibition of Kv channel function may account for the
membrane depolarization during PASMC proliferation.
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Effects of membrane depolarization on resting
[Ca2+]cyt and ATP-induced
rises in
[Ca2+]cyt.
Cell membrane can be depolarized by either inhibition of K+
permeability through membrane K+ channels or alteration of
the transmembrane K+ concentration gradient. Indeed,
extracellular application of 4-AP, a blocker of Kv channels, reduced
whole cell IK(V) by 58 ± 7% at +60 mV and
57 ± 8% at +80 mV (n = 9, P < 0.001) in rat PASMC (Fig. 6A).
The resultant membrane depolarization induced Ca2+-dependent action potentials (Fig. 6B) and
increased [Ca2+]cyt by 155 ± 11 nM
(n = 17, P < 0.001) (Fig.
6C). These results suggest that activity of Kv channels
plays an important role in the regulation of Em
and [Ca2+]cyt in PASMC.
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Effects of extracellular Ca2+
chelation and K+ ionophore on PASMC
growth in media containing serum.
In addition to activating contractile proteins in the cytosol, a rise
in [Ca2+]cyt due to Ca2+ influx
through the plasmalemmal Ca2+ channels can activate
mitogen-activated protein kinase (MAPK) (10) (which is
part of the phosphorylation cascade that leads to activation of DNA
synthesis-promoting factor) and can rapidly increase nuclear
[Ca2+] (1). These effects would promote cell
proliferation by moving quiescent cells into the cell cycle and
propelling the proliferating cells through mitosis (5,
37). The addition of 2 mM EGTA, a Ca2+
chelator, to culture media decreased the free [Ca2+] from
1.6 mM to ~525 nM and almost abolished rat PASMC growth in the
presence of serum and growth factors (Fig.
8). These data are consistent with our
previous observations (4) in human PASMC that chelation of
extracellular Ca2+ with 2 mM EGTA significantly inhibited
cell growth in media containing 5% FBS and growth factors.
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DISCUSSION |
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Excitable and nonexcitable cells both possess a negative resting Em that is close to the EK. Em has been demonstrated to control electrical excitability (e.g., generation and propagation of action potentials) (39), muscle contraction (55), secretion, apoptosis (61), and gene expression (25, 29, 48, 50). The results from this study demonstrate that whole cell IK(V) was reduced, the cell membrane was depolarized, and resting [Ca2+]cyt was elevated in proliferating PASMC compared with growth-arrested cells. The membrane depolarization apparently resulted from the reduced Kv channel activity and led to the elevated [Ca2+]cyt by activating L-type voltage-gated Ca2+ channels (18, 29, 63). Transition of Na+/Ca2+ exchangers from the Ca2+ exit (forward) mode to Ca2+ entry (reverse) mode may also partially contribute to the increase in [Ca2+]cyt during membrane depolarization (8, 32, 49). An increase in [Ca2+]cyt is believed to play an important role in stimulating cell growth by activating signal transduction proteins in the cytosol and transcription factors in the nucleus that are essential for the progression of the cell cycle (10, 25, 27, 29, 48, 50). The observations from the present study suggest that activity of Kv channels in PASMC may play an important role in modulating cell growth by regulating Em and [Ca2+]cyt.
Role of intracellular Ca2+ in cell
growth.
Reduction of extracellular Ca2+ from 1.6 mM to ~525 nM
with the Ca2+ chelator EGTA significantly inhibits human
and rat (Fig. 8) PASMC growth in media containing 5% FBS and growth
factors (4, 53). This suggests that a constant
Ca2+ influx through sarcolemmal Ca2+ channels
(e.g., voltage-dependent, receptor-operated, and store-operated Ca2+ channels) is involved in PASMC proliferation.
Ca2+ diffuses rapidly between the cytosol and the nucleus
(1). Therefore, a rise in
[Ca2+]cyt can increase nuclear
[Ca2+] within 200 ms (1). Ca2+
in the nucleoplasm promotes cell proliferation by moving quiescent cells into the cell cycle and propelling the proliferating cells through mitosis (5, 25, 37). Ca2+ in the
cytoplasm activates the Ca2+-sensitive signal transduction
proteins that are in the cascade to stimulate cell division. For
example, membrane depolarization-induced Ca2+ influx
activates MAPK, which phosphorylates the downstream protein kinases and
stimulates the cell cycle progression (10, 25, 48, 50).
Expression of many transcription factors (e.g.,
c-fos/c-jun, Ras, nuclear factor-B,
c-myb) that promote the cell cycle and stimulate the cell
division is also Ca2+ dependent (25-27,
48).
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Regulation of [Ca2+]cyt
and Em in PASMC.
In vascular smooth muscle cells, [Ca2+]cyt
can be increased by either Ca2+ influx through
Ca2+ channels in the plasma membrane or Ca2+
release (mobilization) from intracellular stores (mainly SR). Because
of the voltage dependence of the sarcolemmal voltage-gated Ca2+ channels (18, 39),
Em serves as a major regulator of
[Ca2+]cyt in PASMC. The voltage window for
sustained elevation of [Ca2+]cyt of smooth
muscle voltage-gated Ca2+ channels ranges from 40 to
25
mV (18), suggesting that the sustained membrane
depolarization (Fig. 5) may be the cause for the increased resting
[Ca2+]cyt in PASMC during proliferation (Fig.
3).
Divergent effects of membrane depolarization on cell growth in excitable and nonexcitable cells. It has been demonstrated that membrane depolarization induced by attenuating Kv (9, 14, 16) and KCa (28, 30) channel activity inhibits cell proliferation in nonexcitable cells, such as human T lymphocytes (35), melanoma cells (33, 41), and intestinal epithelial cells (60). However, membrane depolarization induced by attenuating K+ channel activity stimulates cell proliferation in excitable cells, such as neurons (29, 50) and smooth muscle cells (e.g., this study). The reason for the opposite effects of membrane depolarization on cell proliferation in nonexcitable (e.g., lymphocytes) and excitable (e.g., neurons and PASMC) cells is that the nonexcitable cells do not express voltage-gated Ca2+ channels.
The transmembrane flux of Ca2+ is mainly determined by the Ca2+ driving force (i.e., the transmembrane electrical potential, ![]() |
ACKNOWLEDGEMENTS |
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We thank Y. Yu, S. S. McDaniel, M. A. Sweeney, and N. Kim for assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-54043 and HL-64945 (to J. X.-J. Yuan) and grants from the American Heart Association Mid-Atlantic Affiliate (to V. A. Golovina). J. X.-J. Yuan is an Established Investigator of the American Heart Association (9740091N).
Address for reprint requests and other correspondence: J. Yuan, Dept. of Medicine, UCSD Medical Center, MC 8382, 200 W. Arbor Dr., San Diego, CA 92103-8382 (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.
Received 4 February 2000; accepted in final form 9 May 2000.
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