Published online before print April 27, 2001, doi: 10.1161/hh0901.090300
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© 2001 American Heart Association, Inc.
Cellular Biology |
Lysophosphatidic Acid Positively Regulates the Fluid Flow–Induced Local Ca2+ Influx in Bovine Aortic Endothelial Cells
From the Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Correspondence to Hisayuki Ohata, PhD, Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. E-mail ohata{at}pharm.showa-u.ac.jp
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Abstract |
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Abstract—Using real-time confocal microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid existing in plasma, positively regulates fluid flow–induced [Ca2+]i response in fluo 4–loaded, cultured, bovine aortic endothelial cells. The initial increase in [Ca2+]i was localized to a circular area with a diameter of <4 µm and spread concentrically, resulting in a mean global increase in [Ca2+]i. The local increase often occurred in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca2+]i were dependent on both the concentration of LPA (0.1 to 10 µmol/L) and the flow rate (25 to 250 mm/s). The response was inhibited by removing extracellular Ca2+ or by the application of Gd3+, an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticular Ca2+-ATPase. It was also inhibited by 8-bromo-cGMP, and the inhibition was completely reversed by KT5823, an inhibitor of protein kinase G (PKG). These results suggest that the [Ca2+]i response arises from Ca2+ influx through Gd3+-sensitive MS channels, which are negatively regulated by the activation of PKG. The spatiotemporal properties of the [Ca2+]i response were completely different from those of a Ca2+ wave induced by ATP, a Ca2+-mobilizing agonist. Therefore, we called the phenomenon Ca2+ spots. We conclude that LPA positively regulates fluid flow–induced local and oscillatory [Ca2+]i increase, ie, the Ca2+ spots, in endothelial cells via the activation of elementary Ca2+ influx through PKG-regulating MS channels. This indicates an important role for LPA as an endogenous factor in fluid flow–induced endothelial function.
Key Words: endothelium • mechanotransduction • Ca2+ imaging • ion channels • phospholipids
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vascular endothelial cells are constantly exposed to various types of mechanical stress induced by the blood flow. It is well known that endothelial cells sense and respond to fluid flow and that they play an important role in the maintenance of hemodynamic homeostasis1 through their release of NO,2 3 4 prostacyclin,5 6 and endothelin-1.7 In particular, this mechanotransduction system is considered to be an essential mechanism for local hemodynamic control. In addition, delayed responses, such as reorganization of the cytoskeleton8 9 and changes in cell morphology,10 11 are also dependent on fluid flow. An increase in [Ca2+]i is considered to be a major mediator in the onset of these functions.12 13 14 15 16 17 An ATP-dependent [Ca2+]i response to fluid flow was observed by several investigators.12 13 16 Recently, P2X4 purinoceptors, a subtype of ATP-operated cation channels, were proposed as candidates of shear transduction.17 However, the [Ca2+]i response to fluid flow was also observed in the absence of ATP.14 15 On the other hand, integrins, which are adhesion molecules located at the cell surface, are likely to be key mechanosensors.18 19 Furthermore, mechanosensitive (MS) ion channels may also transduce mechanical stretch.20 21 22 Thus, the mechanism by which endothelial cells transduce fluid flow to cellular events is still controversial.
We have reported previously that lysophosphatidic acid (LPA), a bioactive phospholipid, sensitizes [Ca2+]i changes to mechanical stress in cultured smooth muscle cells from the ileum,23 cultured lung epithelial cells,24 and cultured lens epithelial cells.25 In addition, using real-time confocal microscopy, we recently demonstrated that LPA enhanced the mechanical stress–induced local increase in [Ca2+]i, termed Ca2+ spots, in cultured lens epithelial cells.26 Considering those facts, we have proposed that the Ca2+ spot is an elementary Ca2+-influx event through MS ion channels that is directly coupled with the first step in mechanoreception, and we have suggested that LPA functions as an endogenous factor affecting mechanotransduction systems.
LPA is a lipid mediator with diverse biological activities in various cell types27 via LPA receptors, termed the endothelial differentiation gene (Edg) receptor subfamily,28 and is present in serum at physiologically relevant concentrations.29 30 LPA is present at concentrations of 0.29 µmol/L and 3.21 µmol/L in normal human plasma and in the plasma of patients with multiple myeloma, respectively.31 In addition, it has been demonstrated that LPA accumulates in human atherosclerotic lesions, suggesting a role of LPA as an atherogenic/thrombogenic molecule.32 Therefore, it is possible that LPA affects the fluid flow–induced cellular response in endothelial cells. In the present study, using real-time confocal microscopy, we demonstrate for the first time that LPA positively regulates fluid flow–induced local Ca2+ influx (Ca2+ spots) by modulating MS ion channels in cultured bovine aortic endothelial cells. Our results suggested that LPA may function as an endogenous factor affecting the sensitivity of fluid flow reception and resultant cellular responses in endothelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Culture
Established cultured bovine aortic endothelial cells were provided by Dr Shimizu (School of Pharmaceutical Sciences, Showa University, Tokyo, Japan) and were cultured in MEM containing 10% FBS under humidified conditions (95% air/5% CO2) at 37°C.33 For determination of [Ca2+]i, the cells were subcultured on glass coverslips 25 mm in diameter inside 35-mm Petri dishes. The confluent cells from the 5th to 30th passages were used for the experiments.
Confocal Imaging of
[Ca2+]i With Fluo
4
Cultured endothelial cells on
coverslips were loaded with 5 µmol/L fluo 4-AM with 0.03% cremophor
EL in physiological saline solution consisting of
(mmol/L) NaCl 137, KCl 2.7, CaCl2 1.8,
MgCl2 1.0, glucose 5.6, and HEPES 8.4 (pH 7.4)
for 1 hour at room temperature. Fluorescence images of fluo 4
were collected by use of a real-time confocal imaging system as
previously described.26 With
use of the imaging system, fluo 4 images with 60x60 pixels with x-y
resolution of 1.3 to 2 µm per pixel were acquired with 55- to 110-ms
exposure at 32°C. The intensity of fluo 4 fluorescence for
each region of interest was divided by the averaged resting
fluorescence intensity of >10 frames before stimulation of the
same region, after which the background fluorescence was
subtracted. The resultant relative fluorescence intensity
(F/F0) was used as an indicator of
[Ca2+]i. Image
processing and analysis were performed by use of NIH Image
(version 1.62) and macro programs that we
developed.
Application of Mechanical Stress
With the use of a Perista pump (Atto Corp), the bath
solution was spritzed from a pipette placed parallel to the coverslip
at a distance of 300 µm from and 25 µm over the cells of interest
at an appropriate constant flow rate (25 to 250 mm/s) for 4 to 6
seconds or several minutes. The inside diameter of the pipette tip was
500 µm. Exact placement of the tip of the pipette was achieved in
each experiment by use of a focus control unit. The
homogeneous distribution of nonturbulent flow over the
observed cells (15 to 60 cells per microscopic field) was confirmed by
measuring calcein-loaded blood cells eluted from the tip of the
pipette. This fluid flow did not affect acquisition of the
fluorescent images and induced a reproducible response. The
maximum value of shear stress (
) to the cells under this condition
was estimated from the following
equation34 :
=4µQ
-1r-3,
where µ represents the fluid viscosity (0.009 poise),
Q is the flow volume (0.005 to 0.05 mL/s), and r is the radius of the
pipette tip. Estimated values of ranged from 3.67 to 36.7
dyne/cm2. These values of the shear stress
occur within the physiological range or are
slightly higher.
Materials
Fluo 4-AM and calcein-AM were obtained from Molecular
Probes, Inc. 8-Bromo-cGMP (8-Br-cGMP) and KT5823 were obtained from
Calbiochem-Novabiochem Co. LPA oleoyl was purchased from Avanti Polar
Lipids, Inc. Thapsigargin was obtained from Wako Pure Chemicals. All
other chemicals were commercial products of the highest grade
available.
Statistical Analysis
Statistical analysis among multiple groups
and between 2 groups was evaluated by 1-way ANOVA and Student
t test,
respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fluid Flow–Induced [Ca2+]i Increase in the Presence of LPA
Using a real-time confocal imaging system, we monitored [Ca2+]i changes in cultured bovine aortic endothelial cells loaded with fluo 4 during the application of fluid flow to the cells in the absence and in the presence of LPA (Figure 1
). Neither the addition of 3 µmol/L LPA nor the
application of fluid flow at 250 mm/s for 10 seconds affected
[Ca2+]i, as shown
in
Figure 1B. In the presence of LPA, the fluid flow caused
local increases in
[Ca2+]i from the
region near the cell edge
(Figure 1A). Each local increase in
[Ca2+]i occurred
independently in >80% of the cells observed in a microscopic field
after a lag time of a few seconds during the application of fluid flow.
Subsequently, a continuous application of fluid flow caused a mean
global increase in
[Ca2+]i. The time
course of the increase in the starting region (2 µmx2 µm) revealed
a stepwise or repetitive increase pattern in >50% of the cells, which
responded as shown in
Figures 1A and 1B in cells 1, 2, and 3. Most repetitive
increases in
[Ca2+]i in
restricted regions were considered to result from summation of
[Ca2+]i increases
originating in distinct neighboring starting regions within a cell, as
illustrated in
Figure 1C. The
[Ca2+]i response
was maintained for at least 20 minutes under flow conditions in the
presence of LPA and was returned to the baseline level within a minute
by stopping the fluid flow or washing out the LPA (data not shown).
Additionally, this phenomenon could be observed repeatedly in the same
cells for a few hours. It was clear that the
[Ca2+]i response
was not an artifact due to localization of fluo 4, because the same
phenomena were also observed by fura 2 ratiometric imaging (data not
shown).
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Dependence of LPA Concentration and Fluid Flow
Rate on Fluid Flow–Induced
[Ca2+]i
Response
To clarify the dependence of LPA concentration and
fluid flow rate on fluid flow–induced
[Ca2+]i response,
endothelial cells were stimulated via a stepwise
increase in the rate of fluid flow (25, 60, and 250 mm/s) at
2-minute intervals in the presence of 5 distinct concentrations of
LPA (from 0.1 to 10 µmol/L). Temporal patterns of the
[Ca2+]i response in
the starting region and statistical evaluation of the dependence of LPA
concentration and fluid flow rate on fluid flow–induced
[Ca2+]i response
with the use of linear regression analysis are
presented in
Figures 2 and 3, respectively. Cells responding to the fluid
flow were defined as those cells in which the
F/F0 of fluo 4 increased by >20% of the
resting level within 1 second. Percentages of cells responding to fluid
flow, the peak level of 
F/F0 in responding
cells, and the averaged increase in the level of
F/F0 during the 2 minutes after the onset or
change in the rate of fluid flow were significantly increased with
rising LPA concentration under all 3 flow conditions
(Figures 3A, 3B, and 3C, respectively). In contrast, the
percentage of cells that responded to the fluid flow
(Figure 3D) and the averaged level of increase
(Figure 3F), but not the peak level of increase
(Figure 3E), were significantly increased with an elevation
of the rate of fluid flow in the presence of LPA. These results clearly
indicate that fluid flow–induced
[Ca2+]i response is
dependent on both the LPA submicromolar concentration and the
physiological rate of fluid
flow.
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Effect of ATP on Fluid Flow–Induced
[Ca2+]i
Response
The dependence of ATP concentration and fluid flow rate
on the fluid flow–mediated increase in
[Ca2+]i was
examined to establish whether LPA plays a pivotal role in the fluid
flow–mediated increase in
[Ca2+]i. As shown
in
Figure 4, a fluid flow–induced increase in
[Ca2+]i was
observed in the majority of the cells even at a low rate of fluid flow
(25 mm/s) in the presence of 0.3 µmol/L ATP. Peak levels were
not increased by elevations in the concentration of ATP in the range of
0.3 to 3 µmol/L. Furthermore, subsequent increases in the fluid flow
rate to 250 mm/s induced only slight increases in
[Ca2+]i. These
results suggest that a fluid flow–induced increase in
[Ca2+]i in the
presence of ATP is not dependent on the rate of fluid flow under these
conditions.
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Mechanisms of Fluid Flow–Induced
[Ca2+]i Increase in
the Presence of LPA
Fluid flow induction of
[Ca2+]i increase
was examined to determine whether it arose via
Ca2+ influx through plasma membrane channels
or via Ca2+ release from intracellular
stores
(Figure 5). The percentage of cells responding to fluid flow
of 250 mm/s in the presence of 3 µmol/L LPA and the rising rate
and peak level of
[Ca2+]i at the
starting region in responsive cells were markedly decreased in cells
exposed to Ca2+-free medium containing 0.1
mmol/L EGTA. In addition, pretreatment with 1 and 3 µmol/L
Gd3+, an inhibitor of MS
channels,35 36
decreased the percentage of cells responding to fluid flow and the
rising rate of
[Ca2+]i at the
starting region in a dose-dependent fashion. On the other hand,
pretreatment with 3 µmol/L thapsigargin, an inhibitor of
the endoplasmic reticular Ca2+-ATPase
pump,37 did not affect the
percentage of cells that responded to the fluid flow. Moreover, the
temporal pattern of increase in
[Ca2+]i at the
starting region was unaffected. We have confirmed that the treatment of
thapsigargin abolished the
[Ca2+]i response to
subsequent applications of 10 µmol/L ATP (data not shown), indicating
that the function of intracellular Ca2+
stores was completely inhibited by the thapsigargin treatment. These
results indicate clearly that the fluid flow–induced increases in
[Ca2+]i arise from
Ca2+ influx through the
Gd3+-sensitive MS channels but not from
Ca2+ release from intracellular
stores.
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Recently, it has been reported that a protein kinase G
(PKG)-sensitive channel mainly mediates flow-induced
Ca2+ influx into cultured
endothelial
cells.22 To elucidate the
channels involved in the Ca2+ influx
enhanced by LPA, the effects of 8-Br-cGMP, a membrane-permeable
activator of PKG, and KT5823, a selective PKG
inhibitor,38 on
fluid flow–induced Ca2+ influx enhanced by
LPA were examined
(Figure 6). Pretreatment with 8-Br-cGMP (2 mmol/L)
significantly decreased the percentage of cells that responded to fluid
flow in the presence of 3 µmol/L LPA. Moreover, the peak level and
the duration of increase in
[Ca2+]i at the
starting region in responsive cells decreased. Inhibition of
Ca2+ influx and the change in the temporal
pattern by 8-Br-cGMP were reversed by pretreatment with KT5823 (1
µmol/L), whereas KT5823 alone did not affect the
Ca2+ influx in the presence of LPA (data not
shown). Taken together, these results indicate that the fluid
flow–induced Ca2+ influx enhanced by LPA
occurs through Gd3+-sensitive MS channel,
which is negatively regulated by the activation of PKG in
endothelial cells.
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Spatiotemporal Properties of Fluid
Flow–Induced
[Ca2+]i Increase in
the Presence of LPA
To determine the exact starting region for fluid
flow–induced local
[Ca2+]i increase in
the presence of LPA, we analyzed the spatiotemporal properties
of the increase in
[Ca2+]i more
closely at higher x-y spatial resolution (1.3 µm per pixel)
(Figure 7). As shown in the sequential fluo 4
F/F0 images in
Figure 7B, this increase in
[Ca2+]i started
locally in a circular area with a diameter of <4 µm and then spread
concentrically. In addition, the rising rate of
[Ca2+]i in the
starting region (1.3 µmx1.3 µm) was highest
(Figure 7C). Also,
[Ca2+]i in the
starting region of the response reached the peak within 0.1 second
after the onset of the response and was always higher than the levels
in the surrounding area during the response to fluid flow
(Figures 7B and 7D). These spatiotemporal properties were also
observed in confocal x-y images of LPA-induced
Ca2+ spots in cells pretreated with 3
µmol/L thapsigargin
(Figure 7E). These results indicate that the fluid
flow–induced local increase in
[Ca2+]i reflects
Ca2+ supplied only to a localized starting
region, which then diffuses passively into the surrounding area. We
called the local increase in
[Ca2+]i the
Ca2+ spot, which is specific
[Ca2+]i response to
fluid flow.
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) plotted against each rising rate in the starting regions.
The spatiotemporal properties of the LPA-induced
Ca2+ spots were completely different from
those of Ca2+ waves induced by the addition
of 10 µmol/L ATP, a Ca2+-mobilizing
agonist, to endothelial cells
(Figure 7F
). Rising rates of
[Ca2+]i in the
starting region and the surroundings were analyzed. As shown in
Figure 7G, in 15 of 18 Ca2+ spots
induced by LPA, the ratio of the rising rate of
[Ca2+]i in the
starting region to that in the region at distance of 10 µm from the
starting region was >5.0, and the averaged ratio was 8.90±1.56
(mean±SE), whereas in all 11 Ca2+ waves
induced by the addition of 10 µmol/L ATP, the ratio was <3.0, and
the averaged ratio was 1.67±0.18 (mean±SE,
P<0.001 versus the
Ca2+ spot described above). In addition, the
rising rates in the starting region of the
Ca2+ spots were significantly higher than
those of Ca2+ waves. These results also
indicate that the Ca2+ spot is a localized
[Ca2+]i
response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, using real-time confocal microscopy, we confirmed that LPA positively regulates the fluid flow–induced increase in [Ca2+]i in cultured bovine aortic endothelial cells loaded with fluo 4. Several lines of evidence indicate that this increase in [Ca2+]i is due to Ca2+ influx through recently identified PKG-regulating MS channels stimulated by fluid flow: (1) The percentage of cells responding to the elevation in fluid flow and the averaged increase in the [Ca2+]i response increased with elevation of both the concentration of LPA (from 0.1 to 10 µmol/L) and the rate of fluid flow in the range of 25 to 250 mm/s (from 3.67 to 36.7 dyne/cm2), which corresponded to in vivo arterial blood flow and shear stress (Figures 2
and 3). In addition, the peak level in the
[Ca2+]i response
increased with rising LPA concentration but not with the rate of fluid
flow. These results suggest that increases in
[Ca2+]i as a
consequence of elevation of the flow rate should be mainly ascribed to
increases in duration and/or frequency of the
[Ca2+]i response;
however, elevation of LPA concentration increased the peak level in
addition to the duration and/or the frequency. (2) The
[Ca2+]i response
was inhibited in Ca2+-free medium containing
0.1 mmol/L EGTA or by an addition of
Gd3+, an inhibitor of MS
channels,35 36
but not by thapsigargin, an inhibitor of the endoplasmic
reticular
Ca2+-ATPase37
(Figure 5). (3) The
[Ca2+]i response
was also inhibited by 8-Br-cGMP, and the inhibition was reversed by
pretreatment with KT5823, a selective PKG
inhibitor38
(Figure 6), suggesting that the
Ca2+ influx occurs through a recently
identified MS channel, which is negatively regulated by the activation
of PKG in endothelial
cells.22 This channel is
considered to be the main Ca2+ entry pathway
mediating fluid flow–induced Ca2+ influx in
endothelial
cells.22 (4) The
spatiotemporal properties of the Ca2+
response indicate that Ca2+ supplied from
the restricted starting region diffuses passively into the surrounding
area
(Figure 7). These results are the first visualized data
indicating that fluid flow–induced elementary
Ca2+ influx through MS channels occurs in
endothelial cells. These pharmacological and
spatiotemporal properties of Ca2+ spots,
elementary MS Ca2+ influx events, were
recently observed in mechanically stimulated bovine lens epithelial
cells in the presence of the same concentration of
LPA.26 The occurrence of
Ca2+ spots must be an elementary event
underlying mechanical stress–induced cellular responses, including
fluid flow–induced endothelial responses. MS channel density is estimated effectively at 0.1/µm2 in endothelial cells.21 Because the density of Ca2+ spots was 1 to 3 Ca2+ spots per 200 to 400 µm2, 3% to 10% of MS channels would apparently respond to fluid flow. Furthermore, the number of Ca2+ spots must be underestimated because of the limitations of the spatiotemporal performance of the real-time confocal system that was used. A large Ca2+ spot may mask neighboring Ca2+ spots. Consequently, undetectable Ca2+ spots may occur. On the other hand, cells are stimulated by homogeneous application of fluid flow; however, the mechanical force may focus on specific regions depending on the relationship between cell shape, distribution of the cytoskeleton, and the direction of fluid flow. Therefore, those MS channels localized exclusively in the region in which the mechanical force is focused may be activated. Such local [Ca2+]i increases are considered to be physiologically suitable properties for detecting the strength and direction of mechanical stress and also to be a stimulant with a vector, such as fluid flow, in contrast to the response to chemical stimulants. Further studies are required to clarify the relationship between distribution of sites of local increases in [Ca2+]i, cell shape, and the direction and strength of fluid flow.
No endogenous substances that affect fluid
flow–stimulated Ca2+ influx are known
except ATP, although there have been a number of examples showing that
fluid flow stimulates Ca2+ influx into
endothelial cells. P2X4 purinoceptor–mediated
Ca2+ signaling would be one candidate, and
the shear stress–dependent pathway would be another; however, the
physiological role remains
unclear,17 because
Ca2+ signaling as a response to fluid flow
was also observed in the absence of
ATP.14 15 In this
respect, LPA would be another important endogenous factor
affecting fluid flow–stimulated Ca2+ influx
in endothelial cells. The percentage of cells
responding to fluid flow, the peak level, and the averaged level of
increase in [Ca2+]i
were increased by elevating the concentration of LPA
(Figures 3A through 3C), and the range of the concentration
corresponded to the human plasma concentration in the range of the
physiological level to the
pathophysiological
level.31 On the other hand,
the [Ca2+]i
responses to fluid flow in the presence of ATP were not dependent on
the rate of fluid flow under conditions identical to those with LPA
(Figure 4). Therefore, there is a possibility that LPA
functions as an essential factor for a fluid flow–mediated
endothelial cell response at the normal submicromolar
level and that LPA can affect the progress of vascular pathogenesis via
endothelial dysfunction at the
pathophysiological level. LPA accumulates in human
atherosclerotic lesions31 or
is released from activated
pletelets29 and may
subsequently contribute to several vascular responses, such as changes
in endothelial cell wound
healing39 and
permeability.40 The positive
regulating effect of LPA on the fluid flow–induced
Ca2+ influx may be involved in these
processes in endothelial cells. Our results support a
physiological and
pathophysiological role of LPA in
endothelial cell–related
cardiovascular function, although the subsequent
endothelial cellular responses and intracellular
signaling cascade after the Ca2+ influx
through PKG-regulating MS channels in the presence of LPA must be
clarified.
Although the mechanisms by which LPA enhances Ca2+ influx through PKG-regulating MS channels remain unclear, this occurrence was not considered to be due to activation of phospholipase C by LPA itself, because application of LPA actually increased [Ca2+]i via its G-protein–coupled receptor27 only at 10 µmol/L, but not at 3 µmol/L. These results also indicated that the mechanism of fluid flow–stimulated Ca2+ influx in the presence of LPA was not explained as a direct action of LPA itself on [Ca2+]i. We have previously confirmed that the enhancing effect was not due to the amphipathic action of LPA at the same range of the concentration in cultured lens epithelial cells.25 Elucidation of the LPA receptor subtypes and of the signal transduction pathway associated with the enhancing effect may contribute to the understanding of regulatory mechanisms modulating fluid flow–stimulated Ca2+ influx through MS channels.
In conclusion, LPA positively regulated Ca2+ influx through the PKG-regulating MS channel in fluid flow–stimulated endothelial cells at a range from the physiological level to the pathophysiological level. It is likely that this effect of LPA in endothelial cells plays an important role in the maintenance of local hemodynamic homeostasis.
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Acknowledgments |
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This study was supported in part by a grant-in-aid for a Drug Innovation Science Project (to Dr Momose) from the Japan Health Science Foundation and a grant-in-aid (to Drs Ohata and Yamamoto) for General Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. We thank Dr S. Shimizu (Pharmaceutical Sciences, Showa University) for kindly providing cultured bovine aortic endothelial cells. We are grateful to Dr T. Kawanishi (National Institute of Health Sciences) for insightful comments.
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Footnotes |
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Original received January 3, 2001; revision received March 2, 2001; accepted March 19, 2001.
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References |
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