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Sodium
and
Potassium
Transmembrane
Concentration
Fluxes
By Gabriel
Cividalli
HILE
blood
have
been
similar
THE
cells
widely
investigated,’5
studies
to sodium
INTRACELLULAR
(RBC),
and their
in white
ratio
in
cells
are
extremely
to
chemical
ing cell
be
very
sensitive
composition
isolation.8
leukocytes
in hypotonic
media
suspension
in artificial
media,
From
variable,
Submitted
A ugust
Supported
by USPHS
Gabriel
Cividalli,
(1-F05-TW-1
Children’s
Cancer
Reprint
Avenue,
1974
Blood,
Hadassah
Mass.
U.S.
University
and
Public
Hospital,
Oncology,
Research
Foundation;
and
towards
the
potassium
less
than
1 to
temperature,
hemolysis)
and
by
incubation
followed
of
Medical
Medicine,
School,
the
International
Service;
Israel.
Hospital
Professor
G.
Center;
Pediatrics,
73.
Research
leave
David
Medical
02115.
and A 1-08/
Medical
of
Hospital
to
8, 1973.
Postdoctoral
on temporary
re-
in order
Mass.
December
durof
subsequent
Children’s
Boston,
NH-NHLI-72-2992B.
Jerusalem,
Children’s
from
environmental
30. 1973; accepted
Health
for
ranging
RBC
M-15322-07,
an
directed
values
and to external
manipulation
studies
have
involved
washing
Department
of
been
cations
in red
membrane,
from
Nathan,
Fellowship
the
Department
M.D.:
Chief,
Pediatrician-in-Chief.
Harvard
Medical
School,
02115.
requests:
Boston,
b
Recipient
the
of Hematology
Boston,
5-RO1-A
M.D.:
781-02)from
Pediatrics,
Division
induce
of Pediatrics, Harvard
Grants
in
sometimes
1, 1973; revised November
has
RBC
only in leukemic
lymphoblasts
which
1). Cation
concentration
in leukocytes
media,
reported
of the
study
Reported
changes
(to
the Division of Hematology
Center, and the Department
of
to
of suspending
All previously
of monovalent
across
the
little
(WBC).
about
4, with a higher
value
of 7.2 found
had been grown
in tissue
culture
(Table
appears
G. Nathan
content
movements
relatively
blood
WBC
David
in rabbit
leukocytes
and in polymorphonuclear
granulocytes
isolated
from
pentoneal
exudate
in guinea
pigs. To determine
the cation
permeability
of these
white
cells,
unidirectional
fluxes of potassium and sodium
were
measured
with
42K and 22Na All of these fluxes were in a
range
from
1.2 to 2.7
meq/liten/min,
values
more than
50 times
faster
than
those observed
in erythrocytes.
When active fluxes
were
inhibited
by ouabain,
potassium
loss and sodium
gain occurred
at an initial
rate of about
0.7 meq/liten/
mm. Marked
changes
also occurred
upon
incubation
at low temperature.
These very
rapid fluxes demand
rigid control
of environmental
conditions
for accurate
measurement
of
intracellular
cations
in
leukocytes.
of blood
were
maintained
in plasma
at
37’C and separated
by differential
centrifugation
in specially
devised
microtubes,
within
less than 30 mm after
sampling.
Intracellular
water
content
was
determined
with tritiated
water,
and trapped
plasma
with
‘4C-sucrose.
Under
these
conditions
the intracellular
concentration
of potassium
in human
leukocytes
was
120 SD ± 7 meq/liter
cell water
and of
sodium
16 SD ± 5 meq/liter
cell water,
and the resultant
ratio was higher than 7.
Values
in the same range
were obtained
W
in Leukocytes
and
Reported
values
for the
potassium
to
sodium
ratio in white
blood cells range
from less than 1 to about
4. This marked
variability
might be explained
by in vitro
cation
shifts
induced
by extraordinarily
high
cation
permeability.
To test
this
hypothesis,
human
leukocytes
from 5 ml
and
(Jrune
David
Mass.
G.
Nathan,
M.D.,
Children’s
Hospital
Medical
Center,
300
Longwood
02115.
& Stratton,
Vol. 43, No. 3 (June), 1974
Inc.
861
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862
CIVIDALLI
Table 1. Published
Source
Rabbit
Values
of Leukocytes
polymorphonuclear
Human,
chronic
Human,
chronic
Human,
mixed
Human,
chronic
Human,
acute
Intra cellular
for
Content
meq/liter
leukemia
meq/10’2
lymphocytic
leukemia
meq/1012
leukocytes
(Na)
water
granulocytic
normal
of Sod ium and Po tassium
Unit of Measurement
leukocytes
cells
meq/liter
AND
NATHAN
in Leukocytes
(K)
(K)/(Na)
Ref.
98 ±
21
95
±
18
0.97
17
28
2
49
±
2
1.8
18
±
cells
18
±
2
23
±
3
1.3
18
water
60
±
6
100
±
6
1.7
19
7
meq/kg
dry weight
162
±
7
418
±
18
2.6
leukemia
meq/kg
dry weight
256
±
7
581
±
37
2.3
7
Lymphoblasts
in tissue culture
mM
19
±
4
136
±
8
7.2
15
Human,
acute
leukemia
20
Human,
normal
lymphocytes
Human,
normal
polymorphonuclear
Human,
mixed
restore
myeloid
leukemia
meq/kg
normal
leuk.
“normal”
examined
the monovalent
them
from their natural
ulation
as possible,
composition.
water
±
8
84
±
7
1.6
water
34
±
3
120
±
3
3.5
8
mmoles/kg
water
30
±
4
118
±
3
3.9
8
34 ±
9
137
±
11
4.0
14
meq/liter
leukocytes
a presumed
53
mmoles/kg
water
cation
cation
plasma
in order
composition.8
composition
environment
to achieve
Unidirectional
cation
In
closer
fluxes
for Determination
Procedures
Heparinized
blood
puncture
formed
in
centrifuged
mostly
an
ambient
for
5 mm
phosphate
buffer
If erythrocyte
was
It
After
was
performed.
capillary
cut
with
tamination
end
of the
and
which
because
capillary
containing
inverted,
and
lithium
the
liters
for
by
it was
the
(by
(Becton
Tn
centrifugation
carbonate
Dickinson,
the
examined
seen
The
15 ml
in
microtubes,
were
sodium,
as
with
of
the
of
and
similarly
transferred
and
and
New
vials
mixer.
least
for sodium
microliters
into
‘4C
3.98
latter
England
plasma,
containing
radioactivities.
1).
collected
capillary
30
end
mm.
Con-
bottom
of
column
then
at
The
(Aquasol,
the
pellet,
analyzed
Twenty
the
RBC
was
a vortex
radioactivities.
scintillator
3H
and
standards
were
WBC
leukocyte
on
erythro(Fig.
for
at
the
Ci/g).
similar
5’
than
and
containing
The
was then
‘4C
Spectrometer.
potassium,
vials
stirring
under
leukocytes
button
of
column
N.J.).
and
red
England
0.25
microtubes
removed,
third
into
by
a
the
thrombocytes
centrifugation
3H
a
Krebs-Ringer
(New
centrifuge
leukocyte
resuspended
for
was
upper
added to samples
content
of the vials
Scintillation
for
was
off.
Rutherford,
was
and
lysate
Liquid
plasma
was
The
photometry)
Carb
of
and
acid
cells.8
of
2 ml WBC
counting
Packard
by
5 mm,
per-
containing
of
less
devised
arterial
immediately
Nuclear,
contained
specially
ear
were
centrifugation
a microhematocrit
contaminated
two-thirds
vials,
nitric
flame
be cut
transferred
lithium-containing
disruption
present,
lower
its content
in
when
iso-
plasma,
5 .tl
England
5-mm
by
were
4C-sucrose
suspension
to
or
supernatant
(New
a second
supernatant
be easily
sometimes
the
carbonate
The
cation
manipulations
samples
freeze-dried
at 1350 g for
centrifuged
all
containing
3H20
transferred
rotor
the
tube
of
20 iCi
leukocyte
then
bucket
microtube.
could
concentrated
content
and
185 g. and
insufficient,
resulting
further
erythrocytes,
tube,
discarded
to
The
in vivo
radioactive
volunteers
and
blood
another
5 Ci
and
re-
with
ouabain
and
magnitude
of cation
human
siliconized,
at
to
added
found
in a swinging
with
capillary
been
mixed
a file
rotor
mCi/mM)
was
thoroughly
centrifugation
in the
had
0.394
sedimentation
conditions
cytes.
which
Mass.,
their
incubation
the relative
Five-milliliter
transferred
have
removing
manip-
METHODS
or
37’C.
bucket
carefully
of
with
of normal
plastic
of
in a swinging
to
Boston,
were
we
Cations
by venipuncture
containers
temperature
was
AND
oflntracellular
obtained
All
leukocytes,
Nuclear,
was
was
in rabbits.
study
representation
measured
topes
and shifts
in cation
composition
during
ethacrynic
acid were also studied
to determine
transport
in leukocytes
compared
to red cells.
MATERIALS
this
of leukocytes
without
and with as little external
was
membranes.
cut
with
ml
micro-
mm
and
were
file,
transferred
Twenty
30
The
a
0.0 15 M
of
now
the
also
allowed
potassium
determined
Nuclear)
in
collected
after
3.98
ml
lithium
a
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TRANSMEMBRANE
FLUXES
IN
LEUKOCYTES
FIg. 1.
Schematic
representation
of equipment.
Separation of WBC was obtained b* centrifugation
of leukocyte-rich
plasma in microtube
A inside plastic holder B. After packing
of leukocytes, the capillary end of the microtube (l.D. approximately 0.4 mm) was cut off, inverted, and passed
through
the cap of the lithium-containing
vial C. WBC were transferred into this vial by further centrifugation.
4
B
C
The
Computations.
v,
(20
volume
of
x l0_6
liters).
3HWBC
plasma
and
‘4C,
lithium-containing
3H1
symbols
of sodium
activity
of
are used
and
in the
potassium
each
calculations:
standard
isotope
in
an
put
in the
aliquot
(2
lithium-containing
ml)
of
WBC
vials
lysate
in
the
lithium-
the
vials.
and
containing
following
and
863
‘4Cp
activities
in
an
equal
aliquot
(2
ml)
of
plasma
diluted
in
vials.
(H2O)wBc,
water
content
of analyzed
WBC
column
(H2O)wBc
Reading,
flame
photometer
reading
3
v
=
(liters):
3
HWBC/
calibrated
to
H1.
correspond
to
concentration
of
standard
(meq/liter).
C, cation
content
(mEq)
in (H2O)WBC:
C
(C)w9c,
cation
concentration
in analyzed
(C)WBC
q. plasma
trapped
in WBC
=
(C)
PL, cation
concentration
cation
column
column
=
as a fraction
in
(meq/liter):
reading.3Hp/3H8.
of its total
H20
content:
14 CpL.HWBC
3
HpL/
WBC
in plasma
concentration
WBC
C/(H20)wsc
q=
(C),-,
reading-v
=
(meq/liter).
WBC,
corrected
for
the
cation
content
in
trapped
plasma
(meq/liter):
corr.
Measurements
Most
of Cation
measurements
after centnifugation
which
was
cleared
were
of blood
of
=
(‘-)wc
-
(‘-)PLJ
q1
-
q
Fluxes
performed
at
thrombocytes
185 g.
with
suspensions
In some
instances
and
erythrocyte
of human
leukocytes
leukocytes
were
by
membranes
in plasma
resuspended
in
prior
centnifugation
obtained
plasma
at
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864
CIVIDALLI
27,000g.
Guinea
after intraperitoneal
studied.
These
0.C.
l0
acid
(Merck,
injection
Sharp
&
influx
Center,
incubated
while
Chemical
Company,
Dohme
was
Research
Lab.,
and adjusted
measured
Tuxedo,
(PMN),
and
(Sigma
N NaOH
granulocytes
of casein9
were
ouabain
in 0.1
Potassium
polymorphonuclear
suspensions
M
centration
Research
pig
by
N.Y.)
to
being
West
to oH 7.0 with
adding
20
a suspension
isolated
resuspended
sCi
in
shaken
St.
Mo.)
Pa.,
freshly
42K
were added
(Union
of leukocytes.
exudate
plasma,
a water
Louis,
NATHAN
peritoneal
autologous
in
Point,
HCL)
from
AND
bath
or
kept
i0
dissolved
in some
Carbide
at
also
37’C
or
M
ethacrynic
at
x 100 con-
experiments.
Corp.,
One-milliliter
were
Sterling
aliquots
Forest
removed
at
various
time intervals
were filtered
through
glass-fiber
filters (Reeve
Angel,
Clifton,
N.J.).
These
filters were first shown to be practically
free of potassium.
The filters were then washed
five times
with cold isotonic
Med2 . The dried filters were transferred
to counting
vials and analyzed
for
42 K radioactivity
in a Packard
Gamma
Spectrometer
and for total potassium
content
by flame
photometry.
To measure
potassium
efflux, a concentrated
(40,000 cu mm) suspension
of leukocytes
in plasma
was incubated
with 20 MCi 42K for nearly
1 hr. At a given time the suspension
was diluted
1 : 20
with
unlabeled
autologous
plasma
which
had
previously
been
centrifuged
at 27,000g.
Two-milliliter
aliquots
were removed
at various
time intervals,
filtered,
washed,
and analyzed
for 42 K radioactivity and total potassium
content
as in the measurement
of influx. Efflux studies
were performed
by dilution
of the cells and resultant
decrease
in specific
activity
of the labeling
isotope
in the
medium
(rather than by washings
as is customary
in studies of RBC fluxes) because
of our endeavor
to avoid loss of intracellular
isotope
and clumping
of cells during centrifugation.
Attempts
to apply the same techniques
to the measurement
of sodium
fluxes revealed
very rapid
flux coefficients
which could
not be measured
accurately.
In addition,
the filters were variably
contaminated
with sodium.
Therefore,
a double-isotope
technique
was applied.
Here, the specific
activity
of 24Na in the medium
and in the cells remained
unchanged,
while 22Na was used to
measure
sodium
fluxes. To measure
sodium
influx,
a suspension
of leukocytes
was equilibrated
for about
30 mm with 150 pCi 24Na. Thirty
microcuries
22Na were then added,
and aliquots
were removed
at rapid
intervals.
These
were quickly
washed
five times
with cold
isotonic
MgCl2. To measure
sodium effiux, 800 pCi 24Na (Union
Carbide
Corp.) was added to a suspension
of leukocytes
in plasma.
The leukocytes
were spun down and concentrated
by resuspension
in I ml
of supernatant
plasma.
Fifteen
microcuries
22Na was now added,
and the leukocytes
were
further incubated
at 37’C to allow equilibration
of both sodium
isotopes.
After about 30 mm, the
WBC suspension
was diluted
1: 20 with the previously
separated
plasma,
containing
24Na but no
22Na, which had meanwhile
been centrifuged
at 27,000 g. Two-milliliter
aliquots
were
filtered
and washed
as in previously
described
studies.
22Na
activity
was
counted
after
most
of the
short-lived
24Na had decayed.
Computations.
The fluxes of tracer cations
in these
experiments,
where
the
cells
were
in a
steady state with respect
to their intracellular
concentration
of sodium
and potassium,
behaved
as in a two-compartment
system.
In our computations
(awac),
represents
the specific
activity of the intracellular
compartment
at time
t,
and
(awBc)
the
specific
activity
of
the same compartment
at isotopic
equilibration.
The rate coefficient
for inward
flux was therefore
represented
by the slope (k,) of the graph
of loge [1 - (awBC),/(awBC)..,]
versus
time (t). 10
Since the volume
of the intracellular
compartment
constituted
in these experiments
is less than
a thousandth
of the extracellular
compartment,
the fraction
of both cations
inside
the latter was
extremely
small.
It was therefore
assumed
that the specific
activity
in the extracellular
compartment
did not change
and that
was equal
to the specific
activity
in plasma
(apL). Therefore,
k, would
be represented
by the slope of the graph of log[l
(awBc),/(apL)J.
The rate coefficient
for outward
flux is represented
by the slope (k0) of the graph of
(
log
versus
time.
versus
time
This
and
slope
1.05
was
since
d loge [(awBc),
e\(a)
approximated
(apL),
-
dt
0.05
0.05 (aw),
(awBc),-(aPL)l
by
(awsc),o,
_ol
the
(apL),o
product
of
a constant,
(l.05)d
the
initial
and
loge
dt
(awBc),
slope
of
loge
(awc),
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TRANSMEMBRANE
FLUXES
IN
In studies
of potassium
fluxes
counts
to potassium
content
(S
tivity
of
24Na
was
865
LEUKOCYTES
the
maintained
specific
activity
of 42 K was proportional
In studies
of sodium
fluxes
cpm 42K/IKI).
-
constant,
and
the
specific
activity
of
Na
to the ratio
the specific
was
therefore
of
acpro-
portional
t the ratio 22Na/24Na.
While
some tracer
isotopes
were lost when
WBC
were
washed
with MgCI2,
these ratios
were assumed
to remain
constant.
Rate coefficients
for influx
(ks) and effiux (k0) of potassium
were obtained
from the slopes of the following
lines calculated
by linear regression:
_s4)
loge(l
k-t
_
and
log5Sw
Those
for sodium
fluxes
were obtained
1
from
k0-0.95-t.
the slope
Iil22 Naw#{176}8e124
for
24 NaPL
,
22
Naw-
L
of the graphs
NaPL
and
k0-0.95-t.
-
Naw-
NaPL
Cation
fluxes were obtained
multiplying
rate coefficients
liter cell water of the respective
cation.
This computation
concentration
of total leukocytes
and the leukocyte
trapped
by the intracellular
concentrations
assumed
identity
between
the
on glass-fiber
filters.
per
cation
RESULTS
The
as
corrected
concentration
outlined
in
the
section,
trapped
plasma.
between
duplicate
determinations.
obtained,
the SD
was usually
Trapped
determinations
was
plasma
5.4
did
of the
meq/liter
are
approaches
shown
in Table
8. The
leukocytes
were
temperature
for
intracellular
for
lowered,
cation
When
potassium
2. The
independent
of
affect
adversely,
however,
sodium
and
ratio of their
shifts
were also
10
M ouabain
and
took
(K)
No influx
m.q/lit.r H20
Mixed
humonlsukocytes
15.7±5.0(9)
120±
Mixed
rabbit
18.9
115
Guin.o
*Mecln
pig p.riton.ol
±
SD (number
PMN
27.4
±
5.6(4)
of determinations).
plasma,
washed
medium,
place
108
No
small
but
gain
even
tion a nd Unidirecti onol Fluxes
Conc.ntra
5.6(5)
in human
sodium
in WBC
to the
sodium
20%, which
duplicate
was
own
loss
of
difference
obtained
from
pig peritoneal
concentration
observed
was added
fofextracellular
±
the
for potassium.
concentrations
cation
potassium
(Na)
leukocytes
percentage
meq/liter
2 hr at 37#{176}C
in their
a marked
Table 2. Sodium and Potassium
calculated
the
in leukocytes
from
guinea
4.9
and potassium
and in PMN
in intracellular
incubated
was
(Fig. 2). Marked
chilled
buffers.
change
was
With a trapped
plasma
of about
difference
between
20 consecutive
The mean concentrations of sodium
human
and rabbit
peripheral
blood,
exudate,
cations in leukocytes,
of monovalent
previous
ffiux
cells
when
when
took
the
place
with various
exchange
of
at 37#{176}C
at
an
in L.ukocyt.s#{176}
K influx
K iffiux
m.q/I’utercellH20/min
±
±
7(9)
1.8(1)
8(5)
-
1.4-2.7(2)
-
5 (4)
-
4.1
(1)
1.3-1.6(3)
1.2(1)
-
-
2.7(1)
-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
866
CIVIDALLI
AND
NATHAN
FIg. 2. Intracellular
concentration of cations during incubation
of leukocytes
(e), at 0#{176}C
(i),
and
at 37#{176}C
at 37#{176}C
with
1O
ouabain
(o)
or
10
M ethacrynic acid (tx).
(Mean
±
SE of four experiments performed
on the blood
of the sam.
donor. Only two
80
experiments
initial
rate
of
about
0.7
meq/liter
acrynic
acid
were obtained
did
tional
fluxes
of sodium
and
shown
in Table
2 and
isotope
in Table
not seem
in rabbit-mixed
2. A rapid
movement
to
with
#{149}thacrynic
acid.)
MINUTES
cell
water/mm.
affect
leukocyte
leukocytes
and
potassium
studies
derived
of the
of sodium
type
and
By
contrast,
l0
M
eth-
cations
(Fig.
2). Similar
results
in guinea
pig PMN.
Unidirecfrom
the
cation
concentrations
shown
in Figs.
3-6,
potassium
across
the
are
set
out
membranes
5PL
Fig. 3. Rate of equilibration of 42K activity
in
WBC and plasma.
The coefficient for potassium
Influx is represented
by the
rate of initial increase in
the ratio of activities
in
WBC and plasma
(S/
or it can be calculated from the slope of the
semilogarlthmic
plot
of
1 - S/SpL
(insert)
as
explained in the text.
SW8C/SPL
MINUTES
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
TRANSMEMBRANE
FIg.
FLUXES
4.
(S)
IN
Semilogarithmic
during
867
LEUKOCYTES
measurement
plot of 42K activity
of potassium
40
MINUTES
efflux.
0.8
0.6
“NQ-
‘NCpL
#{149}
0.4
02
11I
0
2
4
6
I
I
8
10
Fig. 5.
changing
Semiloganthmic
22Na
measurement
of
MINUTES
plot
activity
sodium
of
during
influx.
0.08
0.06
22
0W8C
0.04
Fig. 6.
decreasing
surement
Semilogarithmic
22Na activity
during
plot
of
mea-
0.02
-
0
2
of sodium efflux.
4
6
MINUTES
8
10
12
0.10
Fig. 7.
Effect of i0
ouabain
on
the rate of equilibration
of 42K in
WBC and plasma.
The magnitude
of
the effect
was
more
marked
when
0.08
006
5PL
ouabain
0.04
0.02
0
-=
2
-G
4
6
MINUTES
8
so
was
added
immediately
before
the study
(a) than when
it
had
been
allowed
to
remain
in
previous
contact
with
the leukocytes
for 30 mm (a). The respective
control experiments
are represented
by
filled symbols.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
868
CIVIDALLI
of the
cells
bration
however,
was
observed.
of cation
varied
An
isotopes*
from one
inhibitory
effect
of ouabain
was evident
whenever
experiment
to another.
on
studied
the
AND
rate
NATHAN
of equili-
(Fig.
7).
Its
and
resultant
degree,
DISCUSSION
Our
results
quacy
indicate
ofstandard
very
in WBC.
With the use
ing ofsmall
quantities
of
leukocytes
and
separation.
The
was determined
in RBC1’
seems
and,
changes
presumably,
trapped
with
correction
in other
Reported
data
to leukemic
dium
nitric
and
potassium
nitude.
The flux
in RBC.
While
and
4),
curate,
probably
rapid
turnover
actually
suggest
t = 0 intercept
portion
of this
The
main
percentage
of trapped
cation
in normal
fluxes
fluxes
(Figs.
to
with
when
and
12,13
WBC
faster
were
are
resultant
even
are
higher
restricted
measurements
values
5 and
measure
the
was
leukocytes
show
of error
percentage
chosen
cations
approximately
fifty times
of potassium
fluxes
sodium
source
purpose
‘4C02
Our
their
leukocytes
equilibration
high
plasma
in
leukocytes
during
of the
of intracellular
in tissue
and packmanipulation
analyzed
instant
was
made-
determination
potential
because
14C-sucrose
grown
of
their
conditions
used for this
not liberate
unidirectional
rates were
measurements
measurements
The
necessary
leukocytes.
fluxes
for
devised
for separation
to minimize
external
in WBC.
for determination
ofWBC
cations.8’4
lymphoblasts
cations,
environmental
it has been widely
WBC,
and
does
acid.8
on
in
correction
in packed
necessary
recent
studies
in leukocytes
RBC
of this
small
quantity
of
water,
which
achieves
nearly
also
large
trapped
plasma
because
does
not penetrate
into
disrupted
fluxes
to study
of microtubes
specially
ofcells,
we were able
avoid
the
cation
used
water
content
with tritiated
to lie with
of plasma
rapid
methods,
of
of a similar
than
fairly
those
linear
6) appear
to
somag-
observed
(Figs.
be
less
3
ac-
because
of the lower
intracellular
concentration
and
more
of this cation.
Measurements
of sodium
influx
(Fig.
5) could
the presence
of two rate
coefficients,
since
the curve
had
a
much
lower
than
one, thus indicating
very rapid
loss of a large
of the exchangeable
sodium
curve
could
also be affected
the initial
stage
when
performed
of filtration
quickly,
in leukocytes.
by continuation
and washing
took
considerable
of
However,
of some
the t
0 intercept
22Na influx
during
=
leukocytes.
These
time
in relation
to
processes,
even
the extremely
rapid
turnover
of sodium
in leukocytes.
The slope
in Fig. 5 and the calculation
of the rate coefficient
for sodium
influx
would
not be affected
if that happened,
providing
each sample
is handled
in the same
manner
with respect
to time.
and
Flux coefficients
were calculated
assumed
a steady
state in the
cannot,
therefore,
bain. A decrease
in the presence
apparently
5This
discussed
experiment.
be
dependent
term
later,
applied
in the rate
of ouabain.
is used
as
on
here
a result
the
because
of
to
the
basis
length
of
coefficients
changes
of changes
concentration
measurement
of equilibration
Its magnitude,
flux
the
on the
intracellular
in
of
fluxes
in specific
activity
of cations)#{176} They
after
adding
of the isotope
was always
however,
varied
greatly
time
could
intracellular
interval
not
between
be
measured
concentration
adding
in this
of
case,
cations
oua-
evident
and
was
ouabain
as
during
will
be
the
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
TRANSMEMBRANE
FLUXES
IN
LEUKOCYTES
869
and measuring
the fluxes,
and the resultant
change
in concentration
of intracellular
cations
(Fig. 7). The magnitude
of the ouabain-inhibitable
flux is probably represented
by the initial
net change
in concentrations
of sodium
and
potassium
upon
incubation
with
ouabain
(Fig.
2). It apparently
accounts
for
about
hibited
half of the total
cation
fluxes
by low temperature,
but not
occur
They
in WBC
demand
washed
with
rigid control
ment
of intracellular
chilled
buffers
of environmental
to note
particles
and
that
as life span
one
mount
in the
aspect
an
affected
by marked
intracellular
and decreased
temperature.
such
are
Active
fluxes
acid. The cation
due to
conditions
these
for
were
also inshifts
which
very rapid
fluxes.
accurate
measure-
cations.
It is of interest
to ingest
in leukocytes.
by ethacrynic
of granulocyte
oxidative
attack
monovalent
Whether
dther
6
circulation,
are
tunction,
the
them,
is entirely
against
cation
aspects
markedly
shifts
induced
of granulocyte
affected
capacity
un-
by ouabain
physiology,
by such
shifts
remains
to be determined.
REFERENCES
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the
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mem-
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JC, Welt CG: Pathologic
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The
across
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1974 43: 861-869
Sodium and Potassium Concentration and Transmembrane Fluxes in
Leukocytes
Gabriel Cividalli and David G. Nathan
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