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The Discocyte-Echinocyte
Transformation:
Comparison
of N ormal
and ATP-enriched
Human
Erythrocytes
By Claude
J. F#{233}o
and
This work studies the relationship
existing
between
intracellular
AlP levels and the
discocyte-echinocyte
shape equilibrium
in
normal
as well
as AlP-enriched
human
red blood cells. Fresh erythrocytes
metabolically
depleted
by incubation
in a glucosefree buffer undergo
echinocytic
transformation
(crenation)
as intracellular
ATP
levels decline below
50% of their original
value. When ATP is regenerated
by further
incubation
of the same cells in the presence of glucose,
inosine,
and
adenine
(GIA),
concentrations
required
for
the
complete
recovery
of the discocytic
shape
are lower
than those which
were
neceswry
to maintain
this shape
in the first
place.
Addition
of inorganic
phosphate
(P1) to the GIA medium
results in elevation of fresh
cell ATP to supranormal
T
HE
RELATIONSHIP
and
the
between
intracellular
Pierre
levels. When
such ATP-enriched
cells are
then depleted
in the presence
of the same
concentration
of
inorganic
phosphate,
echinocytic
transformation
occurs
much
more rapidly
than
in normal
fresh
cells
not previously
incubated
with
P1 despite
a similar
rate of AlP depletion
in beth
cases. It is suggested
that
the intrinsic
mechanism
responsible
for shifting
the
discocyte-echinocyte
equilibrium
in the
human
erythrocyte
is dependent
on one or
more intracellular
or intramembrane
factors occurring
in conlunction
with
AlP
depletion
or repletion
rather
than to the
absolute
concentration
of this nucleotide
within
the cell. This additional
factor(s)
appears
to be temperature
dependent
and
is influenced
by the concentration
of Pi in
the medium.
the
adenosine
F. Leblond
discocyte-echinocyte
triphosphate
transformation
(ATP)
concentration
of
the
human
red blood
cell was first established
by Nakao
and co-workers.’-3
Weed
et al.,45 a few years
later, emphasized
the importance
of intracellular
calcium
as
well as ATP
in determining
the shape
and deformability
of intact
erythrocytes
and
ghosts.
In order
morphologic
to
incubation
in a glucosefree
subsequent
inosine,
study
events
this
which
incubation
and adenine.
medium
containing
relationship
occur
during
medium
and
of the cells
Erythrocytes
inorganic
in greater
detail,
we
the depletion
of red
during
repletion
in a medium
enriched
with
phosphate
(Pi)
and
have
examined
the
cell ATP
following
of this
nucleotide
supplemented
with
ATP
by preincubation
then
depleted
deprivation
were also examined.
It appears
from
these
experiments
that the discocyte-echinocyte
of the human
red cell is more
critically
influenced
by the presence
in intracellular
ATP per se, or that of intracellular
factors
occurring
the Institut
From
Submitted
Supported
National
Address
Blood,
and Lung
for
reprint
94270-Le
© 1974
Vol.
/4.
1974;
by I.N.S.E.R.
Heart
Bic?tre,
de Pathologie
March
by Grune
44,
No.
Cellulaire,
accepted
M.
(France).
institute
grant
requests.-
5 (November),
20.
by the
No.
Claude
Kremlin-Bict5tre.
& Stratton,
Unite
May
INSERM
48.
Hopital
by
glucose,
in a
by
glucose
equilibrium
of a change
in conjunc-
de Bic#{234}tre. Bic#{234}tre, France.
1974.
Medical
Research
Council
of
Canada.
and
by
L1.S.
Hopital
de
HL-06241.
J.
F#{233}o,institut
de
Pathologie
Cellulaire,
France.
Inc.
1974
639
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640
FEC
tion
with
this
nucleotide
ATP
depletion
or repletion,
itself.
The
exact
rather
nature
than
of
by absolute
these
factors
AND
LEBIOND
concentrations
remains
to
of
be
deter-
mined.
MATERIALS
Venous
was
blood
was
immediately
buffy
coat
10 mm.
was
The
removed
saline
heparin
the
was
(15
cells
as completely
examination.
300 g for
under
from
supernatant
microscopic
buffered
collected
separated
AND
by
For
as possible
stored
at
Erythrocytes
incubation,
4’C
were
as determined
with
then
cells
were
a micro-electrode
A TPdepletion.
than
Depletion
the
saline
sodium
incubation
pH
fluoride
in a new
washed
three
times
10
The
mm
at
at
suspending
the
at pH
7.4
by
plasma
4’C.
of
was obtained
by simply
for
cold,
for
Tris-
centrifugation
hematocrit
at
ranging
Copenhagen)
g
cells
between
pH was initially
adjusted
to 7.6 with
to 7.3 at the end of the incubation
(Radiometer,
The
2500
red
in 4 volumes
at a final
meter
medium
the
to
ATP-enriched
as used
inorganic
normal
with
a small
period,
a precision
of
10
in
by
resynthesis
glucose,
10 mM
sodium
were
(Fig.
ATP
inosine,
was
and
at pH
10, 50,
ATP.
induced
2 mM
100,
and
to
inhibit
an
levels
almost
adequate
After
by
further
adenine
sulfate
7.4.
Dipyridamole,
ATP
more
authors.68
180mM
inorganic
GIA-enriched
possible
buffer
concentra-
inhibitor
in stored
completely
of
blood
the
adenosine
910was
effect
found
of
Pi
when
I).
subsequently
or
of
of
in a glucosefree,
other
or Na2 F1P04 added
to the
it was found
that
the highest
erythrocyte
but
the cells
considered
by various
chloride
1, concentrations
intracellular
itself,
utilized
15 hr,
Tris-buffered
in Fig.
incubating
a procedure
depleted
fresh
cells
in
Cell
Morphology
by
a glucosefree
incubation
in
medium
the
same
containing
in
glucosefree
addition
buffer
160
mM
phosphate.
of Red
Evaluation
Red
of
in preserving
medium
cells
for
rise
effect
to the incubation
method
of either
NaH2PO4
15 hr at 37’C,
and
highest
no
inhibition
5 mM
with
to be useful
have
milliosmols),
containing
As shown
found
300
periods
to isotonicity
gave
only
added
for
(Pi) in the form
for a period
of
also
7.4,
enzymatic
AiP-enrichedeells.
deaminase
(pH
medium
completed
phosphate
were tried
of ATP
solution
in this
incubation
(GIA)
cell
according
ogous
for
15 mM)
donors.
for
recentrifuged
used
The
close
g
plasma
later
suspended
adult
500
Media
Tris-buffered
not
the
methane,
the
healthy
at
U.
Incubation
tion
from
and
and
IS’,, and 2S
as determined
by the micromethod.
volume
of 15 mM Tris so as to obtain
a value
0.01
lU/mI)
centrifugation
(Tris-(hydroxymethyl)-amino
S mm.
METHODS
morphology
to
or
a previously
ABO-compatible
was
evaluated
described
plasma
in
wet
preparations
method.”
and
The
immediately
under
cells
were
examined
lightly
between
a
phase-contrast
suspended
glass
slide
microscope
in
fresh
and
cover
Pi
(mM)
‘I,
a)
U
E
a)
)V)
‘a
a,
U
Fig. 1. AlP
increase
in fresh
human
erythrocytes
incubated
for 15 hr at 37’C
in a medium
containing
inorganic
phosphate.
.,
incubation
in
Tns-buffered
NaCI
supplemented
with
glucose
(5
mM),
inosine
(10
mM),
and
adenine
sulfate
(2 mM)
(GIA).
, same
incubation medium
with 0.1 mM dipyridamole
added. Each point
represents
the mean
of two experiments.
C
4
autolslip.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
DISCOCYTE-ECHINOCYTE
care
being
taken
different
echinocyte
types
2 and
of membrane
was
measured
0.1 ml of the
boiling
cell
water
This
by
allowing
3 were
for
some
according
to
30-60
distinct
distinguished
the
degree
of
rouleau
formation.
classification
of Bessis,12
spicules
regularly
arranged
from
echinocytes
method
accord,ng
type
I and
The
in which
over
the
discocytes
by
flicker.13
was
by
for
then
placing
proportions
of
Mo.).
emission
Light
integrated
the
period
established
solution,
5.4x
IO7andl.8x
at
blood
to
of deioniLed
extract
remaining
firefly
as the
each
10 ml
stable
new
for
of firefly
signals
obtained
from
known
salt,
extracted
from
horse
the
ATPase
Chemicals
of
assay
ATP
was
suitable
Co.,
St.
Louis,
tube
a new
in a
activity.
The
photomultiplier
concentrations
Exactly
placed
containing
extract
Sigma
and
months.
(Sigma
lantern
muscle,
abolish
several
from
et al.14
water
a preparation
extracts
current
bottle
and
tube
lantern
output
to Aledort
distilled
protein
photomultiplier
and
measured
For
order
l8’C,
-
cell
in
over
calibration
an
curve
(adenosine-5’-triphos-
Chemicals
Co.)
varying
between
l08M.
to
verify
the
specificity
salt,
disodic
of
Merck
salt.
the
Co.,
Merck
Co.)
luciferin-luciferase-ATP
reaction,
ADP
Darmstadt,
West
Germany),
and
were
tested
at the
same
concentrations
of red
blood
cells
as derived
(adenosine-S’-
AMP
(adenosine-S’used
for
calibra-
ATP.
Intracellular
ATP
in
a shielded
of 45 sec.
monophosphate,
curve
mm
under
was
trisodic
with
bioluminescence
hemolyzed
stored
disodic
diphosphate,
firefly
was
I-2
red
with
phate
In order
by the
suspension
bath
extract
performed
tion
compression
Measurement
ATP
was
avoid
Echinocytes
absence
TP
A
to
erythrocyte
shapes
were identified
type
3 is a spherical
cell having
surface.
their
TRANSFORMATION641
and
ATP
after
as pM/g
change
was
expressed
correction
for
of hemoglobin
in the mean
as MM/mI
hematocrit
since
corpuscular
and
neither
volume
dilution.
It was
anticoagulation
nor
not
from
considered
incubation
the
calibration
necessary
induced
to express
any
significant
of the cells.
RESULTS
Erythroetes
As
With
shown
in Fig.
occurred
during
3 hr, no change
fresh autologous
fallen
Normal
below
2, a progressive
decrease
glucosefree
incubation
over
in morphology
was observed
plasma.
Starting
at the 4th
of
5#{216}0/
echinocytes
A TP Content
was
its
observed
original
with
in
concentration,
a marked
red
cell
ATP
concentration
a period
of 12 hr.
when
the cells were
hr, when
intracellular
progressive
_-___
acceleration
in the
For
the
examined
ATP
first
in
had
appearance
rate
of
of
this
shape
50
/
Fig. 2.
Depletion
cytes in glucosefr;e
relation
to
mation.
Each
mean
of two
cell
of fresh erythrobuffer
and resyn
echinocytic
point
experiments.
02
12
transfor-
represents
the
Hours
__z
---------
Deplet
i on
Resynthesis
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FO
642
AND
LEBIOND
3. Control
to Figs. 2
Fig.
ment
experiand 4.
Erythrocytes
incubated
for
15 hr in GIA,
but without
Pi, do not lose or gain significant
amounts
of AlP
nor
undergo
disc-.chinocyte
transformation
until
placed
in
a glucosefree
medium
(depletion).
Each
Preincubation
change
until
This
shape
and
the
the
12th
population
GIA
cocytic
change
hr where
transformation
shape
when
qualitatively
at any
intermediate
stages
cells,
preincubated
medium
(Fig.
of the
9O%_l0O0/
was,
examined
posed
of various
type 3. Control
the
point
is the
experiments(±
Depletion
one
time
between
in saline
3), maintained
cells
their
cells in the
morphologic
become
a gradual
was
initial
echinocytes.
phenomenon,
heterogeneous,
the discocyte
supplemented
ATP
for a period
of 15 hr before
undergoing
later transferred
into the glucosefree
tion of these
ATP-depleted
a dramatic
reversion
of the
had
speaking,
mean
of four
1 SD).
i.e.,
concentration
and
depletion
and
buffer.
Subsequent
GIA-supplemented
picture
after
com-
and the echinocyte
with glucose
or
echinocytic
incuba-
medium
2 hr (Fig.
only
in
dis-
resulted
2). At
in
this
time, virtually
all cells had returned
to a disc configuration
while
intracellular
ATP
had risen
from
0.2 to 0.45 zM/ml.
It should
be noted
that
during
the
depletion
incubation,
when
the mean
ATP
concentration
reached
this level,
it
was accompanied
by 15#{176}/-2O%echinocytes
in the cell population.
In order
to
explain
tration,
this difference
a duplicate
of either
CaCl2
depletion
In both
phase
cases,
same
and
set
(2
in morphology
of similar
mM)
or
for the
incubations
EGTA
(10
was maintained
this
the rate of echinocytic
corresponded
to similar
With
Supranormal
As seen in Fig. 4, the
medium
that also contained
mean
ATP
incubated
concentration
for
the
same
mM)
to
intracellular
carried
out
the
concentrations
was
of ATP
obtained
utilized,
throughout
preincubation
160 mM
of the
period
of fresh
Pi resulted
population
of time
over
its original
in absence
ATP,
the
cells
would
ATP.
echinocytic
of Pi (Fig.
however
could
be reduced
(1%-3%)
after
blood
collection
was carried
with
the
incu-
of added
cells
for 15 hr in the GIA
in a threefold
increase
in the
tion period;
of the cells
loaded
The
for 3 hr.
was the
A TP Content
in intracellular
underwent
Once
concenaddition
media.
repletion
reversion
for an influence
on this phenomenon.
did not show
such a rise
few cells (5#{176}-10,) always
their number
immediately
ATP
with
incubation
time for 20 hr and the
transformation
and
bations;
thus
no conclusive
evidence
extracellular
calcium,
at the concentration
Ervthrocvtes
same
was
Despite
change
be maintained
level.
Control
cells
3, preincubation)
this high
ATP
level,
during
the preincuba-
in the
if the preparation
out more
quickly.
discocytic
shape
a
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DISCOCYTE-ECHINOCYTE
TRANSFORMATION
643
Fig. 4.
Depletion
of erythrocytes
previously
enriched with AlP. Loading
is obtained by preincubating
for 15 hr in
GIA containing
160 mM Pi. Depletion
occurs
out
in glucosefree
Pi.
Each
experiments
for
up
buffer
point
(
±
is the
mean
of
five
1 SD).
Ov.r$omding
cells
if their
elevated
extrinsic
echinocytic
environment.’5
No
after
serial
were
dilution
in 24-hr
content
transformation
difference
was
of
the
usually
between
occurs
these
to
4) resulted
incubated
morphologic
only 20#{176}
of the population
ml: the rate of echinocytic
after
12 hr when
change
increased
than
18 hr with
0.5
5#{176}O of
the
cells
after
in saline
and
in saline
alone,
unmask
in
in slow
against
a difference
glucosefree
evolution
cells in this
fresh
cells
in
sensitivity
change
from
to
ATP
to
immediate
buffer
with
during
depletion.
echinocytic
intracellular
fresh
plasma
the
to normal
and normal
ATP
had fallen
rapidly
thereafter
a mean
with
intracellular
ATP
present
in freshly
autologous
that
found
Incubation
(Fig.
incubated
them
plasma
medium
of time the
the amount
protect
were
their
alone
incubation
would
between
the two cell populations.
Finally,
ATP-enriched
cells
without
added
Pi to examine
in saline
the
this period
i.e., twice
suspended
ATP
D.piiiion
replacing
or without
Pi added.
Over
equal
to or above
2 zM/ml,
drawn
cells.
ATP-enriched
even
I
with-
to 40 hr at 37#{176}C
by simply
GIA
with
remained
see
alone,
$
or
involving
4 to 1 tM/
reach
more
falling
below
zM/ml.
Incubation
change
than
Pi (Fig.
but
5) resulted
without
in a much
significant
more
difference
rapid
in
echinocytic
the
rate
of
Fig.
5.
Same
incubation
conditions
as in Fig. 4, but the
depletion
180 mM
medium
contains
Pi. Note that despite
a similar
rate of ATP depletion,
transformation
ocmore rapidly.
echinocytic
curs much
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
644
FEO
ATP
depletion.
Thus,
there were 50%
P1 was absent.
for
a concentration
echinocytes
in the
of
presence
1 zM/ml
of
ATP,
of Pi, compared
AND
for
to only
LEBIOND
instance,
10%
when
DISCUSSION
defined
and
transformation
Bessis
and
Lessin
of the
normal
and
pathologic
erythrocyte
to the
artifacts
that
can
arise
examination
possible
under
the
phase-contrast
ble morphologic
or intrinsic
to
surfaces,’7”8
ber
The
‘
pH,’8”9
experiments
generally
possible
agree
echinocytes
5000
in the
explanation
for
this
population
to
date
cells
this
as being
listed
the
and
attention
blood
determine
used
reversi-
either
effect
that
extrinsic
of glass
of a num-
in appropriate
is intracellular
Nakao
and
on
relationship
the
co-workers’
ATP
observations
between
which
is within
the range
cells,
can correspond
population
depending
closer,
this
although
perimental
evidence,
density
delicate,
be one
red
cell
obtained
by
to anywhere
on
the
the
to
the
valid,
condi-
erythrocytes
transformation
use,
for
individual
example,
indeed
be
of 100#{176},,
of
at a mean
ATP
individual
cell be-
threshold
to
cell
be
value.
supported
separation
centrifugation.
of the type
recently
developed
enough
to measure
ATP
inside
may
to cell
concen-
however,
it is conin which
case the
back
to 100#{176},,
discs
cell
have
of
heteroge-
If, as suggested
varies
according
measured
mean
have
occurred
at which
each
would
or angle-head23
natural
donor.
depletion
has occurred,
become
more
homogeneous,
reversed
the morphology
time,
An
This
by
ex-
techniques
even
by Weed
individual
better,
but
and
Bessis24
erythrocytes.
be the role played
by intracellular
calcium
during
as it is possible
that,
in one way or the other,
or distribution
of intracellular
calcium
in erythromeasured
in our experiments,
no strong
argument
for the occurrence,
ratio which
could
possibility,
may
in
ATP
the
of fresh
echinocytic
incubation
actual
threshold
making
Another
possibility
would
the course
of these
incubations,
Pi may influence
the amount
cytes.
Since calcium
was not
can be made
ATP/calcium
a normal
of intracellular
variation
around
probably
gradient22
technique
sensitive
lies
from
in a population
of averaging,
substratefree
than
the
ATP
interpretation,
A third
red
plasma,’5
phenomenon
obtained
echinocytic.
Once
profound
that the population
had
of substrate
could
have
at a mean
more
would
with
of I zM/ml,
drawn
normal
of this nucleotide
large.
Hence,
because
on
of
which
-echinocyte
called
drugs’8’2#{176}when
factor
information
of ATP
freshly
red cell
the cells during
which
was lower
based
and
incubation.
of any
comes
ceivable
addition
incubated
intrinsic
by LaCelle
et al.,2’ the amount
age, it is expected
that the actual
tration
quite
factors
discocyte
by showing
that a given
absolute
concentration
of ATP
correspond
to a constant
morphologic
picture
within
the
equilibrium.
As can be seen by comparing
Figs.
2, 4, and
for
0#{176},,
and
of the
One
of 24-hr
known
additional
5, a concentration
our technique
neity
The
amphiphilic
only
ATP and morphology
does not necessarily
discocyte-echinocyte
tions
from
the
-
contribute
between
in detail
are generally
regarded
extrinsic
factors
are
or noncharged
concentration.
Our
microscope.
of elevated
depletion.
described
transformation
the cell. Among
of anionic
and
‘
as proposed
for instance,
of a change
explain
the observed
data.4’5
recently
by Shohet
and
in the
Haley,25
intracellular
would
be
a
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
DISCOCYTE-ECHINOCYTE
decrease
TRANSFORMATION
in the
brane
ATP-dependent
phospholipids.
here
were
645
acylation
This,
incubated
and
however,
in absence
can
of plasma
or
nied
that
even
washed
red cells
can
membrane
lysolecithin,
the concentration
various
manipulations
and incubations
that ATP-enriched
formation
when
cells
suspended
of the
observation
to an
require
added
load
of
a finite
period
suspension
ATP,
even
by
in the
in high
echinocytic”
were
agent
et al.26 that
lysolecithin,
and our cells
echinocytic
inducers.
The effect of Pi on red
investigators.2729
The
experiments
that
reported
of altered
spherocytic
of
phatemic
mal
and
Our
been
results
ATP
held
become
Pi is present
in the
observed
in the
lowered
after
15-hr
Pi (Fig.
membrane
of the fact
From
this
sponsible
study,
for
the
closely
related
in conjunction
itself.
fluenced
be
when
of
did
it can
echinocytic
only
be
mentioned
by passive
to one or more
intracellular
with ATP depletion
than
Such
by
a factor(s)
the
does
not
concentration
required
to
the
the
nor-
of
intracellular
provided
was
not
temperature
was
examined
was
immedi-
prevented
Pi-containing
either,
from
medium
even
after
permeability
above
diffusion.33
that
below
shape.
was
of the
of the
is unlikely
intrinsic
human
a
cell
in view
mechanism
re-
erythrocyte
is more
or intramembrane
factors
occurring
to the absolute
amount
of this nucleo-
operate
at reduced
of Pi in the
elucidate
hypophos-
is also
incubation
in the
or
in the
phenomenon
to the
echinocytic
concluded
echinocytic
substrate,
ATP
to
reported
incubated
levels
This
in which
transformation
are
without
difference
low temperatures
the cell mostly
only
in erythrocyte
the
dipyridamole
not become
in our
similar
The
ATP
morphology
Cells
by numerous
in profoundly
cell
4).
when
extrinsic
obtained
is the
high
(Fig.
cell
at 37#{176}C.A dramatic
to Pi at the
that Pi enters
red
incubated
5),
phosphate.
by addition
Methods)
studied
cells
in vivo
containing
when
of
Pi is essentially
when
change
that
ATP
morphology
or
cells
that
intracellular
an intrinsic
“anti-
as
been
and
however,
medium
of
of the
incubation
will
red
incubation
absence
becoming
elevated
(see Materials
and
tide
that
echinocytic
addition
cell
for the
morphologically
seem
as
Rubenstein.3#{176}
in vitro
cases,
(4#{176}C
or 22#{176}C),or even
ately
red
occurs
responsible
indicate
rapidly
in GIA
plasma,29
In both
has
transin view
intact
erythrocytes
immediately
after
pathway
and
Pi on
hypophosphatemic
has
to adapt
in intracellular
cells
that
cells
it is recog-
echinocytic
surprising
metabolically
were
examined
metabolism
Warrendorf
extracellular
patients.31’32
in order
same
increase
the
by
transformation
presence
the
energy
by preincubating
recently
effect
cell
the
Nevertheless,
From
this,
it would
cannot
be considered
threefold
mem-
since
from
immediate
plasma
is not
extrinsic
of time,
through
erythrocyte
invoked
retain
small
amounts
of endogenous
of which
might
have
risen
with
the
to which
they were submitted.
The fact
Lichtman
acting
of
be
serum.
not protected
in 24-hr
incubated
aged
plasma.
concentration,
turnover
hardly
temperatures
incubation
exact
nature
medium.
of
this
and
Further
intrinsic
is instudies
echinocytic
inducer.
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1974 44: 639-647
The Discocyte-Echinocyte Transformation: Comparison of Normal and
ATP-enriched Human Erythrocytes
Claude J. Féo and Pierre F. Leblond
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