Bioscience Reports 3, 631-642 (1983)
Printed in Great Britain
631
A novel method for measuring intracellular pH and
potassium concentration
C. L. BASHFORD, G. ALDER, K. 3. MICKLEM%
and C. A. P A S T E R N A K
Department of Biochemistry, St. George's Hospital
Medical School, Cranmer Terrace, London SWI7 ORE, U.K.
(Received 16 June 1983)
The c o n c e n t r a t i o n of Na + and K + and the pH in the
c y t o p l a s m of L e t t r ~ cells was m e a s u r e d by m o n i t o r i n g
the net flux of H +, Na +, or K + across the p l a s m a
m e m b r a n e which had been r e n d e r e d p e r m e a b l e to t h e s e
ions by t h e a c t i o n of S e n d a l v i r u s . Ion flux was
m e a s u r e d d i r e c t l y by analysis of cell c o m p o s i t i o n , or
indirectly
by o b s e r v i n g t h e c h a n g e in m e m b r a n e
potential
of cells t r e a t e d with a s p e c i f i c ionophore.
C y t o p l a s m i c c o n c e n t r a t i o n s of c a t i o n s w e r e o b t a i n e d by
establishing
t h e c o n c e n t r a t i o n of the c a t i o n in the
m e d i u m at which addition of Sendal virus c a u s e s no
c h a n g e in c y t o p l a s m i c cation c o n t e n t .
The value of
L e t t r ~ - c e l l pH was c o n f i r m e d by d i r e c t m e a s u r e m e n t
e m p l o y i n g 3 t p n u c l e a r m a g n e t i c r e s o n a n c e , and the
values of Na + and K + c o n c e n t r a t i o n w e r e c o n f i r m e d by
analysis of cell c a t i o n and w a t e r c o n t e n t .
L e t t r ~ cells
suspended at 32~
in H e p e s - b u f f e r e d saline at pH 7.3
m a i n t a i n a c y t o s o l i c pH of 7.0 and c o n t a i n 30 mM Na +
and 80 mM K +.
Introduction
Many recent reports have stressed the importance of monovalent
cation content and ion fluxes across the plasma membrane in the
regulation of cell growth and proliferation (Kaplan, 1978). Mitogenic
t r a n s f o r m a t i o n of lymphocytes, for example, is associated with
modified cation ' m e t a b o l i s m ' and is largely abolished by ouabain
(Quastel & Kaplan, 1968), a specific i n h i b i t o r of the plasmamembrane (Na+/K+)ATPase ($kou, 1960, 1965). Indeed it is becoming
clear that t r a n s f o r m a t i o n and stimulation of many cell types is
associated with changes in ion flux and in related parameters such as
membrane potential and intracellular pH (Kiefer et al., 1980; Tsien et
al., 1982; Gerson et al., 1982).
A number of different methods are
available for monitoring cytoplasmic ion contents (Morville et at.,
/973; Pietrzyk et al., 1978; Schummer & $chiefer, 1980) and intracellular pH (Gillies & Deamer, 1979; Hes, 1981).
The procedures
usually involve sophisticated methods for estimating the relative sizes
of different, particularly extracellular, water spaces and do not usually
address the problem of intracellular compartmentation directly.
* Present address:
Department of Biochemistry,
Oxford, South Parks Road, Oxford OXl 3QU, U.K.
University
9 1983 The Biochemical
of
Society
632
BASHFORD ET AL.
In the p r e s e n t c o m m u n i c a t i o n we present a novel and simple
method for measuring cytoplasmic cation activity (concentration) that
is based on the modification of the permeability characteristics of the
plasma membrane of cells exposed to Sendai virus. This virus rapidly
r e n d e r s all cells so far e x a m i n e d completely permeable to lowm o l e c u l a r - w e i g h t compounds and ions; cell lysis does not generally
occur and, under appropriate conditions of virus dose, temperature, and
e x t e r n a l Ca2+ c o n c e n t r a t i o n , the permeabilizing 'channels' can be
r e s e a l e d w i t h i n m i n u t e s ( P a s t e r n a k & M i c k l e m , 1973; Poste &
Pasternak, 197g; Impraim et al., 19g0; Pasternak & Micklem, 19gl;
Forda et al., 1992; gashford et al., 1993). By following the direction
of ion movement as a function of ion concentration in the external
medium it is possible to establish an external ion activity at which
addition of Sendai virus causes no change in cytoplasmic ion content;
in such circumstances the intracellular ion activity (concentration)
equals t h e external ion activity (concentration).
The ionic leaks
induced by Sendal virus may be followed either by chemical analysis of
ceil p e l l e t s or by m o n i t o r i n g p l a s m a - m e m b r a n e potential in the
presence of specific ionophores.
A preliminary account os this work
has been presented elsewhere (Alder et a l , 19g3a).
Theory
In the presence of a specific ionophore the electrical potential
across a cell m e m b r a n e will a p p r o a c h the equilibrium diffusion
potential for that ion in accordance with the Nernst relationship:
V
=
RT
nF
In
lion] medium
[ ion ]cytoplasm
(1)
w h e r e V is the m e m b r a n e potential, R the gas constant, T the
absolute temperature, F the Faraday constant, and n the ionic charge;
the only a s s u m p t i o n , which is g e n e r a l l y i u s t i f i e d , is t h a t the
ionophore-induced permeability greatly exceeds the passive permeability
of all other ionic species.
In such cells, then, V can be positive,
negative, or zero depending on the transmembrane gradient of the ion
in question.
Since Sendal virus causes the membrane potential to move towards
zero (Okada et al., 1975; Fuchs et al., 197g; Impraim et al., 19g0;
Pasternak & Micklem, 19gl; Forda et al., 1992; Bashford et ai., 1993),
i t can be used to establish whether the membrane potential of
ionophore-treated cells under any particular ionic regime is positive,
negative, or zero. Under the latter condition (i.e. zero potential) the
a c t i v i t y (concentration) of the ion in the cytoplasm will equal the
a c t i v i t y (concentration) of the ion in the medium.
M a t e r i a l s and M e t h o d s
Lettr~ cells, which are a line of cells derived from a mouse ascites
tumour, were grown by serial passage in the peritoneal cavity of Swiss
mice, strain TO. Cells were harvested after 7 to 10 days' growth in
mice into a medium containing 150 mM NaCI, 5 mM KCI, 5raM Hepes,
I mM MgCI2, pH adjusted to 7.4 with NaOH (HBS). The suspension
METHOD FOR MEASURING I N T R A C E L L U L A R pH AND K
633
was centrifuged at 700 g for 3 min and the pellet taken up in # vols.
of FIBS to give a 20% v/v ceil suspension, which was kept at room
temperature until required.
Membrane potential was monitored by measuring the absorbance of
oxonol-V (Smith et al., 1976; Bashford et al., 1979a,b) at 630-590 nrn
in a 3ohnson Research F o u n d a t i o n Spectrophotometer/FJuorimeter
(Bashford et al., 19gl), a block diagram of which is shown in Fig. I.
Cell sodium and potassium were determined after diluting aliquots
of the cell suspension five-fold with a medium containing 150 mM
choline chloride, 5 mM Hepes, pH adjusted to 7.4 with Tris, and then
pelleting the cells through oil (2 parts di-n-butylphthalate and i part
dinonylphthalate) in a Beckman microfuge B.
A f t e r the removal of
the wash medium and the oil, the centrifuge tubes were blotted dry
and the pellets were dispersed by sonication in 0.1 M lithium nitrate /
0.2 N H:zSO~ (BDFI), and the ions were assayed by atomic absorption
spectrophotometry.
C e l l w a t e r c o n t e n t was determined as the
d i f f e r e n c e between the wet weight and the dry weight of pellets
prepared as above for cation analysis.
No attempt was made to
estimate the extracellular water space of the pellet as this is reported
to be less than 5% of pellet water when cells are centrifuged through
oil (Kiefer et a]., 19g0; and see below). In ig experiments we found
that the dry wt. of L e t t r d cells was 25 + #% (SD) of the wet wt. of
Thermostaffed
Cell Block
Filter
guide~l
LIght
Chart &A65o-590
RecoVer
Beam
Splitter
I II guidestirrer
I~1 I LigIht
Signal
Processor
Fig. i. Apparatus for monitoring absorbance of cell
suspensions.
Light from a 45-W tungsten lamp was
filtered by a Corning Glass 9782 blue-green filter;
the beam splitter diverts 10% of the transmitted
light to photomultiplier
2 (PMT 2) through an
interference filter with maximum transmission at 590
nm (half band width 5 nm) and passes 90% to
photomultiplier i (PMT i) through an interference
filter with maximum transmission at 630 nm (half
band width 5 nm).
The signal processor outputs the
logarithm of the ratio of the outputs from PMT 2 and
PMT i (i.e. AA630_590) to a strip chart recorder.
63/4-
BASHFORD ET AL.
the cell pellet, which compares with the value of 24% reported for
the closely r e l a t e d Ehrlich ascites tumour cells (Hoffman et al.,
1979).
Valinomyci n and carbonyl cyanide p-trifluoromethoxyphenylhydrazone
(FCCP) were from Sigma Chemical Co.; oxonol-V was a gift from Dr.
B. Chance, 3ohnson Research Foundation, University of Pennysylvania;
other chemicals were of the highest purity commercially available;
S en d al virus, of t h e late-harvest, 't hree-day' type, was grown as
described by Impraim et al. (19g0).
R e s u l t s and D i s c u s s i o n
The response of Lettr~ cells t r e a t e d with oxonol-V to the addition
of valinomycin, a potassium-specific ionophore, or FCCP, a protonspecific ionophore (Harris & Pressman, 1967; Henderson et al., 1969),
in HBS, a conventional isotonic saline, is shown in Fig. 2. Valinomycin
hyperpolarizes the cells, registered by a decrease in absorbance at
630-590 nm, whereas FCCP depolarizes the cells. Subsequent addition
'
2__~M
FCCP
8.0
Q55/~mol
NaOH
.- . . . .
"
pH
(___) 7.5
7.0
.~
O
~
AA63o-~9o
5O
(D
9
0.01
9
Q
4o
9/~M
9
Velinomycin
0
E
c-
6
17 33
KCI
65 rnM
I
b
15
Time ( min )
Fig.
2.
Measurement
of L e t t r ~
cell m e m b r a n e
potential
with oxonol-V.
2 x 106 cells/ml were
suspended at 32 ~ in a medium containing 150 mM NaCI,
5 m M KCI, 1 mM MgCI2, 2 HM oxonol-V and a) 0.5 mM
Hepes, pH adjusted to 7.0 with NaOH or 6) 5 mM Hepes
pH 7.3.
FCCP, valinomycin, and KCI were added to
give the final concentrations indicated.
In a the
pH of the suspension was recorded with a Corning
semi-micro combination electrode and Na0H added as
indicated.
K + content was determined as described
in 'Materials and Methods' for 200-~I aliquots of
the suspension removed at the times indicated.
METHOD FOR MEASURING INTRACELLULAR pH AND K
635
of K+ (for the valinomycin-treated ceils) or OH- (for the FCCPt r e a t e d cells) to the external medium restores the absorbance of the
suspension to the value observed before the addition of the ionophore.
The membrane potential can now be calculated, provided that the
intracellular K+ concent r a t i on and pH are known, by solving for V in
Eq. l using the external ion concentration required to restore the
absorbance to the level observed in the absence of ionophore (Rink et
al., 1980).
The observation that the absorbance of oxonol-treated ceils
responds to diffusion potentials imposed on the plasma membrane by
ionophore-mediated movement of either K+ or H+ in the presence of
the appropriate ionophore strongly suggests that changes in membrane
potential are the principal cause of the changes in absorbance. The
mechanism by which oxonols respond to changes in membrane potentia]
has been discussed elsewhere (Bashford et al. 1979a,b; Bashford, 1981;
Waggoner, 1979) and appears to involve distribution of the oxonol
anion across the membrane in accordance with the Nernst relationship
(Eq. I), and subsequent binding of the dye to hydrophobic sites within
the cell.
Thus the absorbance of the dye reflects the cellular
accumulation of the dye, which in turn registers the transmembrane
potential.
The negative charge of oxonol dyes causes them to be
excluded from mitochondria (Smith et al., 1976), in contrast with the
cationic cyanine dyes which accumulate predominantly in mitochondria
(3ohnson et al., 1981; Cohen et al., 19gl). The sensitivity of oxonol
dyes to membrane potential depends both on the sign of the potential
(inside positive preferred) and on the lipophilicity of the dye
(Bashford et al., 1979b). In Lettr~ cells (potential inside negative)
the lipophilic, phenyl-substituted oxonol-V responds to membrane
potential with greater sensitivity than does oxonol-VI, a less lipophilic,
propyl-substituted analogue (Bashford et al., 1979b; C . L . Bashford,
unpublished observations).
However, in bacterial chromatophores and
submitochondrial particles (potential inside positive) oxonol-VI has
greater sensitivity to membrane potential than oxonol-V (Bashford and
Thayer, 1977; Bashford et al., 1979a).
The effect of Sendai virus on oxonol-treated Lettr~ cells suspended
in HBS in the presence of valinomycin is illustrated in Fig. 3 (lefthand panel, upper trace). After a short lag, Sendai virus causes K+
to leak out of ceils down its concentration gradient (see Fig. 5 below)
and the membrane potential declines to zero as the plasma-membrane
permeability increases. When a similar experiment is performed in a
medium in which extraceilular Na+ is largely replaced by K+ (Fig. 3,
left-hand panel, lower trace), valinomycin depolarizes Lettr6 cells and
subsequent addition of Sendal virus leads to membrane hyperpolarization; this presumably occurs because the K+ gradient has been
reversed. Thus in this situation, in the presence of valinomycin and
!55 mM K+, the membrane has a potential that is inside-positive, and
Sendal virus causes an inward leak of K+ with the potential moving
towards zero, i.e. a change in the direction of hyperpolarization.
The results of a series of such experiments performed in media of
differing cation compositions are presented in Fig. 3 (right-hand panel)
as the maximum rate of change of absorbance caused by Sendal virus,
plotted as a function of extracellular K+ concentration.
The intracellular K+ activity (concentration) is then given by the concentration
636
BASHFORD
v'olinomycinSendai
Ip.g/ml Virus KCI
mM
ET AL.
0.006
0.004
0.002
~>
>
0
IO.OI
~
0~
oJ
o
o~
~o
o
4 rain
3
| ....
-2.0
I
-1.5
log [K+]medium
i
-I.0
Fig. 3.
E f f e c t of Sendal v i r u s on v a l i n o m y c i n t r e a t e d L e t t r ~ cells. 2 x 106 cells/ml were
suspended at 32 ~ in HBS, pH 7.3 (left-hand panel,
upper trace) or a similar medium in which NaCI was
replaced by KCI (left-hand panel~ lower trace) and
containing 2 pM oxonol-V.
Valinomycin and Sendal
virus, final concentration 2 HAU/ml, were added as
indicated.
The rate of change of AA630_590 2 min
after the addition of Sendai virus is presented in
the right-hand panel as a function the K + content of
the medium (HBS in which NaCI was successively
replaced by KCI). The solid line crosses the dashed
(zero) line at -1.16 (69 mM K+).
of extracellular K + at which Sendai virus has no effect on absorbance.
Fig. # shows the results of a similar set of experiments in which
e x t r a c e l l u l a r pH was varied, and the effects of Sendal virus are
monitored in FCCP-treated cells. In this case, at pH values above 7.0
Sendai virus augments the depolarization induced by FCCP; whereas at
pH values below 7.0 Sendal virus counteracts the effects of FCCP.
Using t h e s e t w o m e t h o d s for analysing i n t r a c e l l u l a r c a t i o n s of L e t t r ~
c e i l s we o b t a i n e d values for K+ of g0.3 + 2.9 mM (SEM; n = 6) and
pH of 6.9g + 0.05 (SEM; n = 6).
The o p t i c a l m e t h o d for m e a s u r i n g cellular c a t i o n s d e s c r i b e d a b o v e
can be v a l i d a t e d by a n u m b e r of i n d e p e n d e n t t e c h n i q u e s .
For K+ (and
Na+) it is possible to m o n i t o r the c a t i o n c o n t e n t of cells suspended in
isotonic salines of d i f f e r i n g c o m p o s i t i o n s b e f o r e and a f t e r the addition
of Sendal virus.
Typical results of such an e x p e r i m e n t a r e p r e s e n t e d
in Fig. 5. This shows t h a t in high-Na+ / low-K + m e d i u m Sendal virus
c a u s e s an o u t w a r d leak of K+ and an inward leak of Na+; in a m e d i u m
c o n t a i n i n g 80 mM K+ and 50 mM Na + little m o v e m e n t of e i t h e r K + or
METHOD FOR MEASURING I N T R A C E L L U L A R pH AND K
FCCP Ser~i
2~M
Virus
pH
_2
"
637
0006
0.004
0002
68
0
9- 4
o.ol
O4
0!
8
3
4 rain
t
|
I
!
6.5
7.0
7.5
8.0
pH
Effect of Sendai virus on FCCP-treated
Fig. 4.
Lettr~ cells.
2 x 106 cells/ml were suspended at
32 ~ in HBS, pH 7.8 (left-hand panel, upper trace) or
pH 6.8 (left-hand panel, lower trace) and containing
2 ~M oxonol-V.
FCCP and Sendai virus, 2 HAU/ml
final concentration, were added as indicated.
The
rate of change of AA630_590 2 min after the addition
of Sendal virus is presented in the right-hand panel
as a function of the pH of the medium.
The solid
line crosses the dashed (zero) line at pH 7.01.
Na+ is observed; in high-K+ / low-Na + medium the virus causes Na+ to
leak out from and K + to leak into cells. We conclude that L e t t r ~ - c e l l
cytoplasm contains Na + at 50 mM and K + at g0 mM. It is os interest
to note that in these experiments Sendal virus substantially depolarizes
Lettr~ celis, regardless of the ionic composition of the medium.
This
observation suggests that, in these cells, the membrane potential is
insensitive to changes in cation gradients and arises for reasons other
than diffusion os cations down their concentration gradients (Alder et
al., 1993b).
We have confirmed the above values os Na+ and K + concentration
found in Lettr6 cells by direct analysis of cellular cations and water
content.
The K + content divided by the water content of the ceil
pellet gives a concentration of 79.7 + 3.# mM (SEM; n = 6); a similar
calculation for Na+ gives a concentration of #6.5 + #.1 mM (SEM; n =
6).
These values are the same as those obtained by the other two
methods; they indicate that cell K+ and cell Na+ exist as homogeneous
' p o o l s ' , and t h a t the extracellular water 'space' of cells pelleted
through oil is small, i.e. < 5%.
Kiefer et al. (19g0) have reported
that extraeellular water in pellets of lymphocytes centrifuged through
oil is a small fraction of total pellet water (< 5%).
638
BASHFORD
ET
AL.
(J
%
0,02
E
e-
50
X
+
v
+
O
Z
0.01
O
!
!
o
5
,G
Time After Sendai Virus Addition (rain)
I]3
o
0.02 :>
1>
Oh
c
0
i
ol
50
0.01 o
+
z
0
Time After Sendal Virus Addition (min)
The pH of Lettr~-cell cytoplasm can be calculated directly from
the chemical shift of the cellular inorganic phosphate determined in a
phosphorus-3! nuclear-magnetic-resonance experiment (lles, 19gl; lles
et al., 1982). A typical 3tp n.m.r, spectrum of Lettr~ cells is shown
in Fig. 6. The principal metabolites observed are phosphate monoesters, mostly sugar phosphates (peak A), inorganic phosphate (peak
B), phosphate diesters (peak C), and the y (peak E), (~ (peak F), and
B (peak G) phosphates of ATP.
The chemical shift (p.p.m.) is
presented relative to phosphocreatine, and the pH of the inorganic
phosphate was 7.08 according to the calibration curve of Iles et al.
(1992).
The values for Lettr6-cell cation concentrations determined
using the methods described above are summarized in Table I.
METHOD
FOR
MEASURING I N T R A C E L L U L A R pH AND K
639
C
0.02
O_
[:>
>
O3
0
a
E
= 5(
0.01 o
+
~8
~
0
0
z
Time After Sendai Virus Addition (rnin)
Fig. 5.
The effect of Sendai virus on Lett~e-cell
m e m b r a n e potential and cation content.
2 x 106
c e l l s / m l were s u s p e n d e d at 32~
in media which
contained 40 mM sucrose, 5 mM Hepes, pH adjusted to
7.3 with Na0H, I mM MgCI2, 2 pM oxonol-V and (A) 130
mM NaCI, (B) 80 mM KCI, 50 mM NaCI, (C) 130 mM KCI.
Sendai virus,
2 HAU/ml final concentration 9 was
added as indicated.
Cation contents of 200-~I
aliquots
of the s u s p e n s i o n were m e a s u r e d as
described in 'Materials and Methods'.
Table i.
Cation content of Lettr~ cells
The pH, Na + concentration, and K + concentration of Lettr~-cell
cytoplasm was determined at 32 ~ as described in the text. Results
are presented in the form mean • SEM (numbers of observations); a
dash indicates that a system was not examined.
Method
Na +
(raM)
pH
31p n.m.r.
7.01
(2)
-
Sendai-virusinduced leakage
6.98 • 0.05 (6)
39
Cell content/
Cell water
-
K+
(raM)
(2)
80.3 +- 2.9 (6)
46.5 +_ 4.1 (6)
79.7 • 3.4 (6)
6#0
BASHFORD ET AL.
AB
20
10
C
E
F
G
I
I
0
-10
ppm
I
-20
Fig. 6.
31p nuclear magnetic resonance spectrum of
Lettr~ cells.
1.3 x 10D c^ll s/ml
"
were suspended at
37 ~ in HBS.
The spectrum is the mean of 2048 scans
acquired over 35 min.
The components observed are:
(A) phosphate monoesters (sugar phosphates); (B)
inorganic phosphate, (C) phosphate diesters; (E) y
phosphate,
(F) ~ phosphate, and (G) B phosphate
residues
of ATP.
The chemical
shift of the
inorganic phosphate resonance corresponds to that
found at pH 7.08 according to the calibration curve
of lles et al. (1982).
Conclusion
H a e m o l y t i c Sendai virus discharges ionophore-mediated diffusion
potentials across the cell membrane. By varying the ionic composition
of the medium it is possible to establish a particular ion concentration
at which the addition of Sendai virus causes no change in the potential
of i o n o p h o r e - t r e a t e d cells; under these conditions the cellular ion
a c t i v i t y (concentration) equals the a c t i v i t y (concentration) of the ion
in the medium.
Hence this provides a novel~ easy method for
determining the cation contents of any cell which has a membrane
susceptible to attack by Sendai virus and whose ion permeability can
be modified by specific ionophores; so far all cells studied fall into
this category, and the method as described is applicable.
Acknowledgements
We thank the Cell Surface Research Fund, the SERC, and the
University of London Central Research Fund for financial support.
METHOD FOR MEASURING INTRACELLULAR pH AND K
641
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