Regulation of red blood cell filterability by
Ca2+ influx and cAMP-mediated
signaling pathways
Tadahiro
Oonishi,
Kanako
Sakashita, and
Nobuhiro
Uyesaka
Nippon Telegraph and Telephone Corporation Medical Research
Institute, Kanto Teishin Hospital, Tokyo 141; and Department of
Physiology, Nippon Medical School, Tokyo 113, Japan
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ABSTRACT |
To investigate the mechanism of the
regulation of human red blood cell deformability, we examined the
deformability under mechanical stress. Washed human red blood cells
were rapidly injected through a fine needle, and their filterability
was measured using a nickel mesh filter. The decrease in filterability
showed a V-shaped curve depending on the extracellular
Ca2+ concentration; the maximum
decrease was achieved at ~50 µM. The decreased filterability was
accompanied by no change in cell morphology and cell volume, indicating
that the decrease in filterability can be ascribed to alterations of
the membrane properties. Ca2+
entry blockers (nifedipine and felodipine) inhibited the impairment of
filterability under mechanical stress. Prostaglandins
E1 and E2, epinephrine, and
pentoxifylline, which are thought to modulate the intracellular
adenosine 3',5'-cyclic monophosphate (cAMP) level of red
blood cells, improved or worsened the impaired filterability according
to their expected actions on the cAMP level of the cells. These results
strongly suggest that the membrane properties regulating red blood cell
deformability are affected by the signal transduction system, including
Ca2+-dependent and cAMP-mediated
signaling pathways.
calcium entry blockers, prostaglandins, epinephrine,
pentoxifylline; adenosine 3',5'-cyclic monophosphate; calcium ion
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INTRODUCTION |
THE DEFORMABILITY OF red blood cells plays a crucial
role in the maintenance of normal blood flow in the microcirculation (2, 4, 7, 20). Aspects of red blood cell deformability have become
increasingly important in various clinical situations (6, 10, 21, 25,
28, 32). However, the precise molecular mechanism of the regulation of
red blood cell deformability, and, in particular, the mechanism
responsible for changes in the membrane properties associated with the
Ca2+-dependent signal transduction
system in red blood cells (5, 9) remain to be solved. The membrane
properties and thus the deformability of red blood cells are largely
determined by a submembrane reticulum of proteins referred to as the
membrane skeleton. The membrane skeleton of red blood cells has been
characterized in greater detail than that of any other cells, and
considerable information is available regarding the skeleton
composition, ultrastructures, protein-to-protein interactions, membrane
anchorage, and molecular defects (5, 20, 29). Nevertheless, such
information has not been examined in relation to the overall
physiological functions of red blood cells such as deformability,
except for red blood cells from patients with hereditary hemolytic
diseases (20). In the case of platelets, such information is closely
related to the aggregability of platelets through the signal
transduction system in the cell (3).
It is known that red blood cells show a decrease in deformability in
response to a transient elevation of intracellular
Ca2+ under mechanical stress (12,
28). To investigate the relationships between changes in red blood cell
deformability, the Ca2+-dependent
signaling pathways, and the corresponding adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signaling
pathways in the cells, we have developed a new in vitro system that
imposes mechanical stress on red blood cells to decrease their
deformability. The whole cell deformability (filterability) was
measured with a recently developed nickel mesh filtration method that
has been found to be useful in investigating the red blood cell
deformability, both physiologically and clinically (2, 10, 25, 32, 34). In the present study, using these methods, we examined the effects of
extracellular Ca2+ and
Ca2+ entry blockers on red blood
cell deformability with the aim of determining the manner of transient
Ca2+ entry under mechanical
stress. Furthermore, to study the corresponding cAMP-mediated signaling
pathways, we examined the effects of drugs such as catecholamines,
prostaglandins, and xanthine derivatives on red blood cell
deformability; these effects have long been a topic of some controversy
among physiologists (9, 11, 14, 16, 31).
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METHODS |
Preparation of cells.
Venous blood from the anticubital veins of healthy young male
volunteers was collected into disposable syringes with a 22-gauge
needle, with a 1/10 volume of 3.3% trisodium citrate used as an
anticoagulant. After centrifugation at 1,300 g for 10 min at 25°C, the plasma
and buffy coat were removed and replaced with
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid sodium salt (HEPES-Na)-buffered NaCl solution (HBS; 133.5 mM NaCl, 2 mM glucose, and 15 mM HEPES-Na buffer, 287 mosM/kgH2O, pH 7.40) supplemented
with 1 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid-4Na and 0.1% bovine serum albumin (BSA). After centrifugation at
800 g for 10 min at 25°C, the
packed red blood cells were resuspended with HBS supplemented with
0.01% BSA (HBS-BSA) and subsequently washed two times in HBS-BSA by
repeated resuspension and centrifugation at 600 and 500 g for 10 min at 25 °C,
respectively. The red blood cell suspension was prepared with HBS-BSA,
the cell density of which was adjusted to 8 × 109 cells/ml. The red blood cell
suspension thus prepared contained leukocytes at <5 × 105 cells/ml. The
sample was stored at 22°C in an isothermal water bath. All
experiments were performed within 6 h of the collection of the blood.
Red blood cell filterability. The
filtration through a nickel mesh was carried out by the gravity-based,
vertical tube method (2, 25). The nickel mesh (Dainippon Printing,
Tokyo, Japan) is a porous thin metal film produced by means of a
photofabrication technique. The average pore diameter of the nickel
mesh used in this study was 4.4 µm. As described previously (2, 25),
a height (pressure)-time curve was obtained during filtration by gravity using a pressure transducer (P100; Tsukasa Sokken, Tokyo, Japan). The tangent of the height-time curve, made by drawing points
corresponding to different heights (pressures), gives the rate of the
drop of the meniscus in the tube. By multiplying the cross-sectional
area of the tube, the complete set of flow rates and corresponding
pressures, i.e., the pressure-flow rate relationship, is given. We used
a personal computer to collect the data automatically. The experiments
were carried out at 36°C by circulating isothermal water through
the jacket surrounding the vertical tube, the inner diameter of which
was 4 mm. The filtration of the red blood cell suspension was started
at a pressure of 15.0 cmH2O. The
ratio (%) of the flow rate (ml/min) of the red blood cell suspension to that of the suspending medium (HBS-BSA) at 10 cmH2O was used as the index of red
blood cell filterability. Before the filtration experiments, the
suspending medium was filtered through a Millipore filter with a pore
size of 0.1 µm to avoid the otherwise inevitable microdust
contamination. Because the nickel mesh was subject to ultrasonic
washing and was then reused at least 100 times, each experiment was
done with the same nickel mesh.
Injection method for mechanical
stress. Figure 1
illustrates the scheme of our prototype apparatus for imposing the
mechanical stress on red blood cells. Five hundred microliters of the
red blood cell suspension (cell density of 4 × 109 cells/ml) were packed into a
disposable 1-ml tuberculin syringe (4 mm inner diameter; Terumo); air
bubbles were carefully expelled. The cell suspension was then injected
through a 25-gauge disposable needle (30 mm in length and 0.5 mm inner
diameter; Terumo). The injection was performed by placing a weight on
the upper end of the inner piston of the syringe, which was fixed
vertically. The injected red blood cells were caught by a disposable
50-ml polypropylene centrifuge tube (Iwaki Glass, Funabashi, Japan).
The distance from the tip of the needle to the bottom of the centrifuge
tube was 15 mm.
An aliquot of the red blood cell suspension (8 × 109 cells/ml) was mixed with the
same amount of HBS-BSA with an appropriate concentration of calcium
chloride and then was incubated at 36°C for 5 min. Pretreatment
with drugs was done by adding an appropriate concentration of the drug
to the red blood cell suspension and by incubating the cell suspension
at 36°C for 15 min before mixing with the
Ca2+ solution. After undergoing
the mechanical stress, the red blood cells were incubated at 36°C
for 7 min and then diluted with HBS-BSA with an appropriate
concentration of calcium chloride to 1 × 108 cells/ml; next, the cells were
subjected to filterability measurement. The filtration was
started at 10 min after the mechanical stress.
Although the injection device has a disadvantage in that there is no
completely satisfactory way of characterizing the shear stress, some
estimate of the level of the shear stress can be given by using the
following analysis. The flow rate (Q) in the needle is calculated as Q =
VR2, where
V is the falling velocity of the
weight and R (0.2 cm) is the radius of
the syringe; this requires a pressure (P) in the syringe by
Poiseuille's law, P = 8L
Q/
r4 = 8L
VR2/r4,
where L (3 cm) is the length of the
needle,
(0.008 dyn/cm2) is
the fluid viscosity at 30°C, and r
(0.025 cm) is the radius of the needle. As the red blood cell
suspension at 36°C was placed in the syringe at room temperature
(25°C), it is assumed that the cells were mechanically stressed at
~30°C. The resultant upward force (F) on the syringe plunger is
calculated as F =
PR2 = 8
L
R4/Mr4
where M is mass. The net downward
force on the weight is Mg
F,
where M is the mass placed on the
syringe and g (980 cm/s2) is the acceleration due
to gravity; therefore,
dV/dt = g
KV, where K = 8
L
R4/Mr4.
The resultant velocity is V = (g/K)[1
exp(
Kt)] (where
t is time), and the distance fallen
(d) is
d = (g/K){t
[1
exp(
Kt)]/K}. The
mean velocity in the needle is
VR2/r2,
and the wall shear stress in the needle is
= Pr/2L = 4
VR2/r3
by Stokes relation. For the case in which
M = 1,000 g, K = 2.47 s
1, and the plunger reaches
the bottom of the syringe (d = 4 cm) at about t = 0.094 s (94 ms) when the
velocity is 80 cm/s and the wall shear stress is ~6,600
dyn/cm2. In this case, the mean
velocity in the needle reaches ~5,100 cm/s, and the transit time is
then ~0.6 ms. Thus the cells are exposed to relatively high stress
for a very short time.
Other laboratory methods. An aliquot
of the red blood cell suspension was fixed for microscopy with an
isotonic 1% glutaraldehyde solution containing 50 mM phosphate buffer
(pH 7.4) and 24.5 mM NaCl. Specimens were observed in a differential
interference contrast microscope (TMD, Nikon, Tokyo) at ×400
magnification. Cell counting was performed with a hemocytometer
(Coulter Counter model DN; Coulter Electronics, Luton, Beds., UK). The
hematocrit (Hct) value was determined by centrifuging red blood cell
suspensions in the capillary tubes at 14,400 g for 5 min on a Hct centrifuge (model MC-201; Hitachi Koki, Hitachi, Japan). The leukocyte count of the red
blood cell suspensions and mean corpuscular volume (MCV) of the red
blood cells were measured by an automated hematology analyzer (model
NE-800; TOA Medical Electronics, Kobe, Japan). The osmolality of the
suspending medium was measured with a freezing-point depression-type
osmometer (DigiMatic Osmometer model 3D II; Advanced Instruments,
Needham Heights, MA).
Materials. Felodipine
[(±)ethyl methyl
4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate]
and pentoxifylline were provided by Hoechst (Tokyo, Japan). BSA
(essentially fatty acid free) and prostaglandin (PG)
E2 methyl ester (solution in methyl acetate) were purchased from Sigma (St. Louis, MO). Nifedipine was purchased from RBI (Natick, MA).
PGE1 and epinephrine were obtained
as the commercial preparations Prostandin (Ono Yakuhin Kogyo, Osaka,
Japan) and Bosmin (Daiichi, Seiyaku, Tokyo), respectively. Other
chemicals used were of reagent grade.
Statistical analysis. The data are
expressed as means ± SD. Comparisons between the filterability of
red blood cell suspensions treated with and without drugs were made by
paired t-test.
P values <0.01 were considered
significant.
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RESULTS |
Decrease in filterability of mechanically stressed red blood cells
and its dependency on extracellular
Ca2+.
The filterability of the mechanically stressed red blood cells
decreased depending on the weight used for injecting red blood cells
from the syringe in the presence of extracellular
Ca2+; an increase in weight
resulted in a decrease in filterability, which was apparently saturated
when a weight of >1,000 g was used (Fig.
2). Because the motion in the case of the
weight of >1,000 g may be almost as fast as a free
fall and because increasing weight may not change the expected level of
the shear stress much, this apparent saturation may be attributable to
a property of the test system, not the red blood cells. For the
subsequent experiments, a weight of 1,000 g was used. The filterability
of the mechanically stressed red blood cells decreased depending on the
concentration of Ca2+ added to the
suspending medium. The decrease in filterability showed a V-shaped
dependence curve against the extracellular
Ca2+ concentration; the maximum
decrease was observed at ~50 µM (Fig. 3). At extracellular
Ca2+ concentrations ranging from 0 to 1 mM, no change in cell morphology was observed. The Hct values and
the cell numbers before and after the mechanical stress were the same.
The cell volume of the mechanically impaired red blood cells as
evaluated by their Hct and MCV values did not change until 60 min after
the mechanical stress was imposed. Without mechanical stress, no
decrease in filterability was induced by the extracellular
Ca2+. As we reported previously
(2, 25), the filtration experiments were not influenced by contaminated
leukocytes.

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Fig. 2.
Decrease in filterability under mechanical stress. Filterability (%)
of mechanically stressed red blood cells in 50 µM extracellular
Ca2+ concentration was plotted
against the weight (g) used for pressing down the inner piston of the
red blood cell-packed syringe. After mechanical stress, red blood cell
suspensions at 40% hematocrit were incubated for 10 min at 36°C,
diluted to 1% hematocrit, and then filtered through a nickel mesh, as
described in METHODS. Filterability (%) was determined as
the ratio of the flow rate (ml/min) of the red blood cell suspension to
that of the suspending medium (HEPES-Na-bufered NaCl-bovine serum
albumin) at 10 cmH2O. Data are
means ± SD from 3 experiments.
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Fig. 3.
Dependency of the filterability of red blood cells under mechanical
stress on the extracellular Ca2+
concentration. Red blood cell filterability with ( ) and without
( ) mechanical stress is shown, the latter of which was 95.5 ± 2.5%; filterability did not change when the extracellular
Ca2+ concentration was increased
to 1.5 mM. Data are means ± SD from 3 experiments.
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Effects of
Ca2+ entry
blockers on the mechanically impaired filterability.
Pretreatment with a Ca2+ entry
blocker (nifedipine or felodipine) inhibited the impairment in
filterability caused by 50 µM Ca2+ under mechanical stress in a
dose-dependent manner (Figs.
4 and 5).
Because the action of Ca2+ entry
blockers was reported to be modified by solvents (22, 30), the red
blood cells treated with the same concentration of ethanol (0.02%
vol/vol) were used as a reference. The maximum inhibitory effect was
observed at 0.5 µM and higher for nifedipine (from 24.3 ± 10.3 to
42.2 ± 15.6%, P < 0.01) and at
0.1 µM and higher for felodipine (from 23.3 ± 6.0 to 47.8 ± 14.8%, P < 0.01). The improvement
of filterability by the Ca2+ entry
blockers was not accompanied by any change in cell volume. Without
mechanical stress, neither nifedipine nor felodipine changed the red
blood cell filterability when the red blood cells were incubated at
36°C for 60 min at 50 µM or 1 mM of the extracellular Ca2+ concentration.

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Fig. 4.
Effects of nifedipine on mechanically impaired filterability. After a
0.02% volume of the indicated concentrations of nifedipine dissolved
in ethanol was added to the red blood cell suspension and incubated at
36°C for 15 min, the cell suspension was mixed with the same amount
of 100 µM Ca2+ solution and
further incubated for 5 min at 36°C. After undergoing mechanical
stress, the red blood cell suspension at 40% hematocrit was incubated
for 10 min at 36°C, diluted to 1% hematocrit, and then filtered
through the nickel mesh. Mechanically impaired filterability with 50 µM extracellular Ca2+
concentration without nifedipine (a), with 0.02% ethanol (b), and with
0.5 µM nifedipine in 0.02% ethanol (c).
Inset: representative plot of
filterability according to the effects of different concentrations of
nifedipine on mechanically impaired filterability of red blood cells in
50 µM extracellular Ca2+
concentration. Data are means ± SD from 6 sets of experiments.
* P < 0.01 vs. a and b.
Comparison of data between 2 groups was made by paired
t-test.
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Fig. 5.
Effects of felodipine on mechanically impaired filterability.
Experimental conditions are as described in Fig. 4. *P < 0.01 vs. a and b.
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Effects of PGE1,
PGE2, epinephrine, and pentoxifylline.
The effects of several drugs were examined on the mechanically impaired
filterability at the 50 µM extracellular
Ca2+ concentration.
PGE1 improved the impaired
filterability; the maximum improvement (from 21.2 ± 4.5 to 63.7 ± 11.9%, P < 0.01) was observed
at the concentration of ~1 × 10
10 M, showing a
bell-shaped improvement with concentrations ranging from
10
12 to
10
8 M (Fig.
6). Conversely,
PGE2 worsened the impaired
filterability; the maximum deterioration (from 26.3 ± 5.2 to 5.3 ± 1.3%, P < 0.01) was observed
at the concentration of ~1 × 10
10 M, showing a V-shaped
decrease with the concentrations ranging from
10
12 to
10
8 M (Fig.
7).

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Fig. 6.
Effects of prostaglandin E1. Red
blood cells were pretreated with the indicated concentrations of
prostaglandin E1 at 36°C for
15 min. Incubation with Ca2+
solution and filtration of red blood cell suspensions were done in the
same way as that employed in the experiments with nifedipine (Fig. 4).
A: representative plot of
filterability according to the effects of different concentrations of
prostaglandin E2 on mechanically
impaired filterability of red blood cells in 50 µM extracellular
Ca2+ concentration.
B: mechanically impaired filterability
in 50 µM extracellular Ca2+
concentration without prostaglandin
E1 (a) and with
10 10 M prostaglandin
E1 (b). Data are means ± SD
from 6 sets of experiments. * P < 0.01 vs. reference. Comparison of data between two groups was made
by paired t-test.
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Fig. 7.
Effects of prostaglandin E2.
Pretreatment with prostaglandin
E2, incubation with
Ca2+ solution, and filtration of
red blood cell suspensions were done in the same way as that employed
in the experiments with nifedipine (Fig. 4).
A: representative plot of
filterability according to effects of different concentrations of
prostaglandin E2 on mechanically
impaired filterability of red blood cells in 50 µM extracellular
Ca2+ concentration.
B: mechanically impaired filterability
in 50 µM extracellular Ca2+
concentration without prostaglandin
E2 (a), with 0.02% ethanol (b),
and with 10 10 M
prostaglandin E2 in 0.02% ethanol
(c). Data are means ± SD from 4 sets of experiments.
* P < 0.01 vs. a and b.
Comparison of data between 2 groups was made by paired
t-test.
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Epinephrine showed a biphasic effect on the mechanically impaired
filterability: an improvement at
10
10 M (from 22.7 ± 4.5 to 48.8 ± 15.5%, P < 0.01) and
a deterioration at 10
8 M
(from 22.7 ± 4.5 to 12.7 ± 7.8%,
P < 0.01). Red blood cells from some
individuals showed a marked improvement at
10
10 M and a small
deterioration at 10
8 M,
whereas those from other individuals showed a small improvement and a
marked deterioration at the same concentrations (Fig.
8).

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Fig. 8.
Effects of epinephrine. Pretreatment with epinephrine, incubation with
Ca2+ solution, and filtration of
red blood cell suspensions were done in the same way as that employed
in experiments with prostaglandin
E1 (Fig. 6).
A: representative plots of
filterability according to effects of different concentrations of
epinephrine on mechanically impaired filterability of red blood cells
in 50 µM extracellular Ca2+
concentration. , Typical improvement in impaired red cell
filterability at 10 10 M epinephrine; , typical
deterioration at 10 8 M epinephrine.
B: mechanically impaired filterability
in 50 µM extracellular Ca2+
concentration without epinephrine (a) and with
10 10 M (b) and
10 8 M (c) epinephrine. Data
are means ± SD from 6 sets of experiments.
* P < 0.01 vs. a. Comparison
of data between 2 groups was made by paired
t-test.
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Pentoxifylline, a potent inhibitor of phosphodiesterase, markedly
improved the mechanically impaired filterability; the maximum improvement (from 29.2 ± 5.9 to 73.3 ± 9.3%,
P < 0.01) was at the concentration
of ~1 × 10
9 M,
showing a bell-shaped improvement with the concentrations ranging
from 10
12 to
10
7 M (Fig.
9).

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Fig. 9.
Effects of pentoxifylline. Pretreatment with pentoxifylline, incubation
with Ca2+ solution, and filtration
were done in the same way as that employed in the experiments with
prostaglandin E1 (Fig. 6).
A: representative plot of
filterability according to the effects of different concentrations of
pentoxifylline on mechanically impaired filterability of red blood
cells in 50 µM extracellular
Ca2+ concentration.
B: mechanically impaired filterability
in 50 µM extracellular Ca2+
concentration without (a) and with (b)
10 9 M pentoxifylline. Data
are means ± SD from 6 sets of experiments.
* P < 0.01 vs. a. Comparison
of data between 2 groups was made by paired
t-test.
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Both the impairment and the deterioration in the impaired filterability
produced by the drugs described above were accompanied by no changes in
cell volume or morphology. Without mechanical stress, the drugs used
did not change the red blood cell filterability when the red blood
cells were incubated at 36°C for 60 min at 50 µM or 1 mM of the
extracellular Ca2+ concentration.
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DISCUSSION |
It is known that red blood cells have the capacity to change their
deformability in response to various kinds of mechanical stress (12,
27, 28). Usually, the exposure of red blood cells to mechanical stress
in vitro has been achieved by generating shear forces in a viscometer
(12, 17, 27). O'Rear et al. (27) reported that red blood cells after
exposure to subhemolytic shear stress (<1,500
dyn/cm2 for 2 min) in a concentric
cylinder viscometer showed little change in cell morphology. Mechanical
hemolysis occurred at >3,000 dyn/cm2 for 2 min exerted by a
closed concentric cylinder viscometer (26). In contrast, the shear
stress generated by the injection method is relatively high stress
(~6,600 dyn/cm2) for a very
short time (~0.6 ms) as described in METHODS. The actual
shear stress experienced by the cells in the needle varies from zero on
the center line of the flow up to the estimated level for cells near
the wall of the needle and increases with time as the syringe is
emptied. Therefore, the maximum shear stress generated by this method
is much higher than that found in normal physiological conditions.
However, it is noteworthy that the overall impact of the stress on the
red blood cells may be attributable to the time of exposure to the
shear stress, as well as the stress level. Leverett et al. (18) showed
the threshold for extensive shear stress damage by dividing the shear
stress-time domain into two distinct regimes, using the data from
several different methods. Their shear stress-time diagram indicates
that the shear stress of 6,600 dyn/cm2 for 0.6 ms may be a stress
below the threshold for extensive cell damage. In fact, the decrease in
red blood cell filterability caused by the injection method was
accompanied by no morphological change or hemolysis. Thus a
quantitative decrease in the filterability was obtained by this simple
and reproducible method.
The role of intracellular Ca2+ in
regulating the physiological function of red blood cells has been
studied widely, and an elevation of intracellular
Ca2+ has been shown to exert
profound effects on the membrane properties that regulate red blood
cell deformability (29). The mechanical stress given by shear stress of
a physiological magnitude was shown to induce transient elevations of
intracellular Ca2+ (17) and a
decrease in deformability (12, 28). These findings were confirmed in
the present study. Red blood cell deformability has been ascribed to
three physical properties: the surface area-to-volume ratio, the
internal viscosity or mean corpuscular hemoglobin concentration, and
membrane viscoelastic properties (4, 7, 20, 34). The decrease in
filterability without changes in cell volume and morphology obtained in
the present study therefore indicates that the decreased filterability
produced by our method is the result of neither a change in the surface
area-to-volume ratio nor an increased intracellular viscosity from
dehydration but rather of an alteration of the membrane properties.
Under the mechanical stress, the red blood cell filterability markedly
decreased depending on the extracellular
Ca2+ concentration, indicating
that mechanical stress induces an influx of
Ca2+, which causes a decrease in
filterability (Fig. 3). Considering that the
Ca2+ entry blockers inhibited the
decrease in filterability under the mechanical stress (Figs. 4 and 5)
and that, without mechanical stress, no decrease in filterability was
induced by the extracellular Ca2+,
it is possible that transient elevations of intracellular
Ca2+ through a mechanically
activated Ca2+ channel in the red
blood cell membrane play a central role in decreasing the red blood
cell filterability under mechanical stress. Nifedipine and felodipine
did not return the decreased filterability to the level characteristic
of cells mechanically stressed in the absence of extracellular
Ca2+, suggesting that a
dihydropyridine-insensitive channel may also facilitate the
Ca2+ influx. To our knowledge,
this inhibitory effect of Ca2+
entry blockers is the first documentation of the changes induced by
mechanically generated intracellular
Ca2+ elevations in red blood
cells. The physiological meaning of the present observation that red
blood cell filterability under mechanical stress decreased maximally at
~50 µM of the extracellular
Ca2+, which is considerably lower
than the Ca2+ concentration in
normal plasma, is under further investigation. However, the V-shaped
dependency of the impairment on extracellular Ca2+ suggests that there are
threshold and delayed times of response to the intracellular
Ca2+ elevations and that there are
some physiological mechanisms that exert compensatory effects on the
impairment of the filterability. We therefore investigated the role of
cAMP-mediated signaling pathways in the impaired filterability.
Previous studies of the effect of
PGE1 on red blood cell
filterability have yielded somewhat contradictory results (9, 11, 14,
16, 31). In the present study, a marked improvement (from 21.2 ± 4.5 to 63.7 ± 11.9%) in filterability by
PGE1 was demonstrated at very low
concentrations (1 × 10
10 M; Fig. 6). It is
noteworthy that this increase in filterability was strikingly large
compared with increases in previous reports (14, 16, 31). The previous
studies were performed on the "resting" red blood cells that are
not exposed to mechanical stress. Because the filterability of
"normal" red blood cells is essentially unimpaired in the resting
state, it could be quite difficult to increase their filterability. In
some reports (16, 31), a small increase in the filterability of resting
red blood cells was obtained. It is plausible that those cells were
unintentionally exposed to mechanical stress during experimental
procedures such as washing, which is an integral process for measuring
the red blood cell filterability.
The increase in filterability produced by
PGE1 and the decrease produced by
PGE2 in this study (Figs. 6 and 7)
are consistent with the findings of previous reports (14, 31).
Mechanisms of the changes in filterability produced by these drugs have
been proposed (9). Allen (1) proposed that
PGE2 increases the cell volume by
increasing the water content, resulting in the deterioration of
filterability, and that PGE1 has
an effect directly opposite to that of
PGE2. A recent study by Li et al.
(19) suggested that PGE2 impairs
the filterability by decreasing the cell volume through a stimulation
of a Ca2+-dependent
K+ channel. However, the present
results show that PGE1 and
PGE2 are able to alter the
filterability without a significant change in cell volume, indicating
that the effects of these drugs on the filterability under mechanical
stress are exerted by an alteration of the membrane properties.
Adenylate cyclase is reported to be present in human red blood cells
(13, 24), the activity of which is thought to be affected by
PGE1 and
PGE2, as it is in platelets (3, 8, 23). Therefore, it is quite possible that the intracellular cAMP level
is involved in the regulation of red blood cell filterability by
changing the membrane properties through an activation of
cAMP-dependent kinases (5). We tested this hypothesis by observing the
effects of pentoxifylline, which is a potent inhibitor of
phosphodiesterase and hence is expected to increase the intracellular
cAMP level in human red blood cells. We found that pentoxifylline
markedly improved the mechanically impaired filterability with an
incubation at a very low concentration for only 15 min at 36°C
(Fig. 9).
The effects of epinephrine on the impaired filterability (Fig. 8) are
consistent with the presence of adrenergic receptors (33) and adenylate
cyclase in human red blood cells (13, 24), also supporting the
possibility that cAMP-dependent kinases affect the membrane properties
for regulating red blood cell filterability. Furthermore, the biphasic
effect of epinephrine on the impaired filterability is interesting as
to the pathophysiology of the microcirculation (Fig.
8A). Epinephrine worsened the
impaired filterability at the concentration of 1 × 10
8 M, as in the study by
Rasmussen et al. (31). In the present study, at 1 × 10
10 M, which is within the
normal concentration range in plasma (>2.7 × 10
10 M; see Ref. 15),
epinephrine improved the filterability (Fig. 8B), suggesting that epinephrine
plays a beneficial role in maintaining normal microcirculation in vivo
by improving red blood cell filterability. Another important
observation is that the bi-phasic effect of epinephrine differed
somewhat from person to person (Fig.
8A), indicating a difference in the
responsiveness to epinephrine among individuals, which may be related
to predisposing conditions to certain clinical situations such as
hypertension.
It has long been postulated that red blood cell deformability is
regulated by the two main pathways of the intracellular signal transduction system (5, 9), as in many other cell types (3, 23); these
Ca2+-dependent and cAMP-dependent
pathways may evoke various kinds of interacting kinases and
phosphatases, which lead to the phosphorylation and dephosphorylation
of key proteins in the membrane cytoskeleton and regulate red blood
cell filterability. The present results showed that
posttranslational or regulatory modifications of the membrane
properties of red blood cells can be reflected in their filterability,
as in platelet aggregability. In conclusion, the present study clearly
demonstrated the existence of several signal transduction pathways in
human red blood cells.
 |
ACKNOWLEDGEMENTS |
We express appreciation to Dr. H. Shio (Shiga Medical Center for
Adult Diseases) and Dr. E. A. O'Rear (University of Oklahoma) for
critical comments and helpful discussion and to K. Kanezaki (Kanto
Teishin Hospital) for excellent technical assistance. We thank the
journal reviewers especially for helpful suggestions concerning the
manuscript.
 |
FOOTNOTES |
This work was supported in part by grant 05305006 from the Ministry of
Education, Science, and Culture of Japan to N. Uyesaka.
Address for reprint requests: T. Oonishi, NTT Medical Research
Institute, Kanto Teishin Hospital, 5-9-22, Higashi-Gotanda,
Shinagawa-ku, Tokyo 141, Japan.
Received 9 April 1997; accepted in final form 1 August 1997.
 |
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