differential response of bipolar and normal control lymphoblastoid cell sodium pump to ethacrynic...
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Journal of Affective Disorders 80 (2004) 11–17
Research report
Differential response of bipolar and normal control lymphoblastoid
cell sodium pump to ethacrynic acid
Rena Li1, Rif S. El-Mallakh*
Mood Disorders Research Program, Department of Psychiatry and Behavioral Sciences, University of Louisville School of Medicine,
Louisville, KY 40292, USA
Received 2 May 2002; accepted 30 December 2002
Abstract
Background: While the pathogenesis of manic-depressive, or bipolar, illness is unknown, an excess of intracellular sodium and
calcium concentrations is thought to contribute to the development of the illness. Previous work has demonstrated a reduced
adaptive response of the sodium pump to ethacrynic acid in lymphocytes obtained from bipolar subjects compared to
psychiatrically normal controls.Methods: To further examine this phenomenon, we investigated several aspects of sodium pump
response (transcription, translation, activity, and intracellular ion concentration) in lymphoblastoid cell lines derived from bipolar
subjects and matched normal controls. Cells were treated with ethacrynic acid 100 AM for 3 days. Results: Normal control-derived
cells exhibited an upregulation of sodium pump mRNA synthesis, protein expression, pump-specific binding and activity, and
were able to maintain a normal intracellular sodium concentration. Cells derived from bipolar individuals did not alter sodium
pump parameters in any way, and consequently, had a higher intracellular sodium concentration. Limitations: While bipolar
lymphoblasts were from an inbred Old Order Amish population, the normal controls were from an outbred population.
Conclusions: The results suggest that bipolar illness is associated with an abnormality in cellular sodium homeostatic regulation.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Bipolar disorder; Ethacrynic acid; Na,K-ATPase; Sodium; Sodium pump
Manic-depressive, or bipolar, illness is a relatively
common condition afflicting 1% of the world’s pop-
ulation (Goodwin and Jamison, 1990). Several mod-
els have been presented. These span neurotransmitter,
G protein (Avissar and Schreiber, 1989), second
messenger (Warsh and Li, 1996; Manji et al., 1999;
Emamghoreishi et al., 2000), and ionic regulation
0165-0327/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0165-0327(03)00044-2
* Corresponding author. Tel.: +1-502-852-1124; fax: +1-502-
852-1115.
E-mail address: [email protected]
(R.S. El-Mallakh).1 Present address: Sun Health Institute, Sun City, AZ, USA.
abnormalities (Dubovsky and Franks, 1983; El-Mal-
lakh and Wyatt, 1995). The latter is particularly
promising since it may underlie a common mechanism
of mood stabilization (El-Mallakh and Huff, 2001) and
has led to an interesting animal model of mania
(Ruktanonchai et al., 1998; Decker et al., 2000).
Both mania and bipolar depression have been asso-
ciated with increased intracellular sodium (Na) con-
centrations (Coppen et al., 1966; Shaw, 1966), and a
decrease in erythrocyte sodium and potassium-activat-
ed adenosine triphosphatase (Na,K-ATPase or Na
pump) activity (Looney and El-Mallakh, 1997). Tem-
poral gray matter of post mortem tissue obtained from
Table 1
Lymphoblastoid cell lines used in the studies were matched for age
and gender
Patients Controls
Cell line Age Sex DOI Cell line Age Sex
GM05933A 25 F 1 GM06859A 25 F
GM05977A 26 M ? GM06160 25 M
GM05987B 30 F ? GM05392A 27 F
GM05989B 25 M ? GM07752 17 M
GM05999A 22 F 6 GM07544 22 F
GM07783 25 F 1 GM05377 24 F
GM08825 46 F ? GM05380 32 F
GM08852 34 F ? GM06862 34 F
GM09138 48 M ? GM05398A 44 M
GM09193A 54 M ? GM11500 45 M
GM09732 25 M 6 GM06875 26 M
GM10336A 21 F 6 GM07524 22 F
GM11051B 35 F 17 GM05423 32 F
GM11194 24 F 2 GM06051 26 F
GM11204 53 F 24 GM11120 58 F
DOI, duration of illness in years; F, female; M, male.
R. Li, R.S. El-Mallakh / Journal of Affective Disorders 80 (2004) 11–1712
bipolar subjects expresses less of the a2 isoform of the
Na pump than psychiatrically normal controls (Rose et
al., 1998). It has been purported that a primary increase
of intracellular Na can bring about activation of second
messengers without the presence of a neurotransmitter
signal (El-Mallakh and Li, 1993). Consequently, a
primary Na,K-ATPase abnormality may unify several
pathophysiological hypotheses.
Abnormalities in Na pump regulation in bipolar
subjects have previously been reported by two groups.
In 1981, Naylor and Smith reported that, unlike cells
from normal controls, lymphocytes obtained from
bipolar subjects failed to upregulate Na pump expres-
sion in response to treatment with 100 AM ethacrynic
acid for 3 days. In 1991, Wood et al. replicated Naylor
and Smith’s finding in unmedicated bipolar subjects.
The current study was performed to determine if
lymphoblastoid cell lines might respond in a similar
manner to lymphocytes, and to attempt to replicate and
expand previous reports.
1. Materials and methods
1.1. Lymphoblastoid cell cultures
Lymphoblastoid cell lines were obtained from the
National Institute of General Medical Science’s Hu-
man Genetic Mutant Cell Repository at the Corriell
Institute for Medical Research, Camden, NJ. Bipolar
subjects had a type I illness (as defined by Research
Diagnostic Criteria (Feighner et al., 1972)) and were
members of the Old Order Amish community (Ege-
land et al., 1987; Kelsoe et al., 1989). Control subjects
were screened for medical and psychiatric disease,
matched for age and gender, but were from a hetero-
geneous, outbred community (Table 1). Cells were
grown in RPMI-1640 medium with 15% fetal bovine
serum (FBS), 1% glutamine, at 37 jC and 5% CO2.
All cells were seeded at 0.2 million cells/ml in 75-ml
flasks and split every 2–4 days.
1.2. Cell treatment
Ethacrynic acid (Sigma, St. Louis, MO) was added
at final concentrations of 10�7, 10�6, or 10�5M for 72 h.
Cells were harvested after drug treatment and spun at
250�g for 3 min. The pellets were used for all assays.
1.3. Ouabain binding assay
Lymphoblast cell pellets were resuspended at 2
million cells/ml in K-free Krebs incubation solution
(pH 7.4) which contained 140 mMNaCl, 5.4 mMKCl,
1.2 mM MgSO4, 0.3 mM KH2PO4, 2.8 mM CaCl2,
12.0 mM HCl, 13.7 mM Tris base, 11.1 mM glucose,
and 1% FBS. Cells were incubated with a final con-
centration of 5, 25, 50, 75, or 100 nM [3H]ouabain
(DuPond NEN, Boston, MA) for 6 h at 37 jC. Theincubation was stopped by cooling in ice bath and rapid
vacuum filtration through Whatman GF/B filters
(Brandel, Gaithersburg, MD) followed by three, 5-ml
washes with ice-cold Kreb’s wash solution (same as the
incubation solution minus the glucose and FBS, pH
�7.4). The radioactivity on the filters was counted by a
Multipurpose scintillation counter (Beckman model
LS6500, Arlington Heights, IL). To obtain specific
binding, non-specific binding, determined in the pres-
ence of 10�4 M non-radioisotopic ouabain (Sigma),
were subtracted from the total binding.
1.4. 86Rb uptake assay (Na pump activity assay)
Lymphoblast cell pellets were resuspended in sa-
line solution at 2 million cells/ml. One hundred ml of
5 mM Ringer solution (123 mM NaCl, 1.3 mM CaCl2,
R. Li, R.S. El-Mallakh / Journal of Affective Disorders 80 (2004) 11–17 13
1.2 mM MgSO4, 18.5 mM Na2HPO4, 100 mM KCl,
0.8 mg/ml sucrose, pH 7.4), and 100 ml of 1 ml
ouabain (Sigma) were added and incubated at 37 jCfor 30 min before adding 86Rb (DuPont NEN) at 2
ACi/ml in a total volume of 1 ml. The cell suspension
was then incubated at 37 jC for 1 h and pellets were
collected after spinning at 2700�g for 1 min. The
pellet was washed three times with ice-cold K-free
Ringer solution. Cell pellets were then mixed with
500 ml of 5% trichloroacetic acid solution (Fisher
Scientific, Fair Lawn, NJ), and spun at 5000�g for 15
min. Two hundred ml of the supernatant were added
to 5 ml Econo-Safe scintillation fluid (Research Prod-
ucts International, Mount Prospect, IL), and radioac-
tivity was measured by the Multi-purpose scintillation
counter.
1.5. Intracellular Na, K, and calcium measurements
Lymphoblast cell pellets were washed with isoton-
ic (0.1 M) MgCl2 solution, pH 7.4 (Sigma), three
times and lysed overnight with 30% nitric acid (Fish-
er). A flame atomic absorption spectrophotometer 372
(Perkin-Elmer) was used to measure Na, K, and
calcium (Ca) concentrations. Lamp current, wave-
length, and slit settings are shown in Table 2. Distilled
deionized water was used as background on all
analyzed samples. Data were calculated based on
daily standard curves and equalized per mg protein.
The concentrations of the standards were 10�5 M,
5�10�5 M, and 10�6 M.
1.6. Western blot
Lymphoblast cell pellets were homogenized with a
Microson Ultrasonic Cell Disrupter XL (Heat Sys-
tems, Farmingdale, NY). The protein was measured
by Lowry Assay using a Protein Assay Kit (Bio-Rad
Table 2
Settings for ion measurement on flame atomic absorption
spectrophotometer
Ion Lamp Wavelength Slit
current (k) setting
(V) (nm)
K+ 12 766.5 2.0
Na+ 12 589.0 0.7
Ca2+ 15 422.7 0.7
Laboratories, Hercules, CA). A sample of 15 or 30 mg
of protein was mixed with sample buffer and loaded
into 8% SDS gel (Bio-Rad). The sample buffer
contained 50 mM Tris–HCl (pH 6.8), 5% glycerol,
0.1% SDS, 0.3% mercaptoethanol, and 0.02% bro-
mophenol. The Na pump protein was separated elec-
trophoretically at 60 V for 3 h in running buffer which
contained 25 mM Tris, 192 mM glycine, and 0.1%
SDS. Protein was then transferred overnight from gel
to PVDF nitrocellulose membrane (Millipore, Bed-
ford, MA) at 40 V in a transfer buffer which contained
25 mM Tris, 192 mM glycine, and 0.5% SDS. The
membrane was blotted at 4 jC overnight with 5%
milk (Nestle Food Company, Glendale, CA), and
probed with mouse anti-Na,K-ATPase a1 monoanti-
body (UBI, Lake Placid, NY) for 1 h in 5% milk in
TBS with 0.1% Tween-20 (Sigma). The membrane
was washed with 0.1% Tween-20 in TBS three times
before blotted with horseradish peroxidase-conjugated
goat anti-mouse IgG secondary antibody (Rockland,
Gilbertsville, PA) for 1 h. The membrane was again
washed three times with 0.1% Tween-20 in TBS and
soaked in ECL developing solution mixture (Amer-
sham, Arlington Heights, IL) for 1 min before expo-
sure to Kodak XAR-5 imaging film (Eastman Kodak,
Rochester, NY). The product of the chemilumines-
cence reaction (a horseradish peroxidase system,
ECL, Amersham) was quantified with the Personal
Densitometer SI (Molecular Dynamics, Sunnyvale,
CA). The actual amount of protein loaded was quan-
tified by staining the membrane with Coomassie Blue
R-250/50% methanol–10% acetic acid for 1 min,
which was then destained by immersion in 80%
methanol–10% acetic acid for 3 min, followed by
10 min in 50% methanol–10% acetic acid (Sigma).
The resulting stain was photocopied and the image
quantified on the Personal Densitometer SI. The
number was used for the denominator in all reported
data.
1.7. RNase protection assay
Total RNA (10 ng) was dissolved in the hybrid-
ization buffer (40 mM Pipes, pH 6.4, 80% deionized
formamide, 0.4 M NaCl, 2 mM EDTA) and then
added to the radiolabelled antisense RNA probe
(1�10�6 cpm). (The a1 isoform oligoprobe was
designed using GenBank data with the following
Fig. 1. Effect of 3 days of treatment with ethacrynic acid 10�5 M on several measures of Na,K-ATPase expression and function in
lymphoblastoid cells from bipolar and psychiatrically normal individuals. (A) [3H]Ouabain binding is lower in cells derived from bipolar
subjects (93.8F8.22% (S.E.M.) of baseline) than psychiatrically normal individuals (130.9F13.76% of baseline, P=0.03, F=5.2). (B)
Immunoblot density measures of the a1 subunit increases in cells of normal controls (149.8F17.01% of baseline) but does not change in
bipolar-derived cells (92.8F8.67% of baseline, P=0.009, F=8.36). (C) The mRNA signal of the a1 subunit as measured by RNase protection
increases in response to ethacrynic acid in normal cells (157.2F32.02% of baseline), but drops in bipolar-derived cells (72.7F18.78% of
baseline, P=0.046, F=5.18). (D) Na,K-ATPase activity as measured by 86Rb-uptake increases in normal control-derived cells (123.2F12.41% of
baseline) but decreased in bipolar-derived cells (80.5F4.14% of baseline, P=0.0034, F=11.61). (E) Ethacrynic acid treatment at 10�5 M
increases intracellular Na concentration in bipolar-derived lymphoblasts (128.3F18.12% of baseline) but not in cells derived from
psychiatrically normal controls (92.7F4.82% of baseline, P=0.04, F=4.93).
R. Li, R.S. El-Mallakh / Journal of Affective Disorders 80 (2004) 11–1714
R. Li, R.S. El-Mallakh / Journal of Affective Disorders 80 (2004) 11–17 15
sequence: 5V-CTCAGCTGCACGAGCAGTGT-
TAATCCCCGGCTCAAGTCTGTTCCATA-3V). The
antisense RNA probe was transcribed with T7 RNA
polymerase in the presence of [32P]UTP. The hybrid-
ization mixture was incubated at 45 jC overnight
(>14 h), and then treated with RNase A (40 mg/ml)
and RNase T1 (70 U/ml) at 37 jC for 30 min. The
samples were treated with proteinase K (100 mg/ml)
at 37 jC for 15 min. and then electrophoresed on a 5%
polyacrylamide denaturing gel (8 M urea) at 150 V for
3 h. Gels were dried on Whatmann filter paper (What-
mann, Maidstone, UK) at 80 jC for 3 h and placed on
intensifying screen and exposed to Kodak XAR-5
film (Eastman Kodak) for 24 h at �70 jC.
2. Results
On all measures of Na pump expression or activity
there was a significant difference between bipolar-
and normal control-derived lymphoblasts. Specifical-
ly, cells from control populations responded to 3-day
treatment with ethacrynic acid by increasing Na,K-
ATPase synthesis and activity, while cells from bipo-
lar individuals did not respond (Fig. 1).
[3H]Ouabain binding is a measure of the number of
active Na pump units since ouabain binds only to
enzymes that are actively transporting ions. Ethacrynic
acid, at 100 AM (10�5 M) for 72 h, induced an increase
of 130% above baseline in normal cells. Cell derived
from bipolar subjects failed to show this response, and
remained unchanged at 94.5% (Fig. 1A) (P<0.05).
The affinity of ouabain binding was non-significantly
decreased in bipolar subjects (Kd=4.13F0.67 (S.E.M.)
at baseline to 3.94F0.67 nM after ethacrynic acid
treatment), but not in normal controls (4.94F1.05 to
5.0F1.57 nM). However, the number of ouabain
binding sites was significantly increased in cells from
normal individuals (Bmax increased from 33.5F2.1 to
46.8F1.99, P=0.001, after ethacrynic acid 10�5 M for
72 h), while cells from bipolar subjects showed no
change (36.4F4.99 to 33.6F6.15).
Similarly, the Western blot technique quantifies the
total amount of Na,K-ATPase present in the cells,
including inactive pumps. Ethacrynic acid induced an
increase of a1 isoform protein expression to 151.% in
normal cells (Fig. 1B). Cells from bipolar individuals
did not respond (92.8%) (Fig. 1B). The a2 and a3
isoforms are not expressed in lymphoblasts.
The difference in response to ethacrynic acid
appears to have its origins in the regulation of tran-
scription. In pilot experiments with RNase protection
technique that quantifies a1 isoform mRNA synthe-
sis, treatment with ethacrynic acid increased the
quantity of mRNA in cells from normal individuals
(153.3%) but decreased the quantity in bipolar-de-
rived cell lines (74.8%) (Fig. 1C).
Changes in Na pump activity as measured by 86Rb-
uptake parallel the changes noted in pump expression.
In normal cells ethacrynic acid induced a significant
increase in 86Rb-uptake (156.8%) while a small de-
crease was found in cells from bipolar individuals
(72%) (Fig. 1D).
Changes in intracellular Na concentration ([Na]in)
are consistent with changes seen in pump expression
and activity. Cells from normal individuals showed
essentially no change in [Na]in (92% of baseline) (Fig.
1E). Cells from bipolar individuals showed an in-
crease of [Na]in to 128% of baseline (Fig. 1E). Neither
intracellular K (bipolar 107%, normal control 106% of
baseline), nor total intracellular Ca (bipolar 90.6%,
normal control 90.1% of baseline) were altered.
3. Discussion
Lymphoblastoid cells derived from bipolar subjects
lack the ability to respond to ionic stress induced by
ethacrynic acid. This abnormality is evident at various
levels. First, there is a significant increase in ouabain
radioligand binding in cells from normal individuals
without any observed changes in cells derived from
bipolar individuals (Fig. 1A). Kinetic data indicate
that there is a significant increase in the number of
ouabain binding sites in cells from normal individuals
after ethacrynic acid treatment, while cells from
bipolar subjects show a slight reduction in Bmax.
Second, Na pump activity increased in normal cells
as reflected by 86Rb uptake but not in cells derived
from bipolar subjects (Rb replaces K in K-free sol-
utions) (Fig. 1D). There was no significant difference
in Na pump activity between these two groups prior to
ethacrynic acid treatment. Third, there was a signifi-
cant increase of [Na]in in cells from bipolar patients
after ethacrynic acid stimulation, but there was no
R. Li, R.S. El-Mallakh / Journal of Affective Disorders 80 (2004) 11–1716
change in cells derived from normal individuals (Fig.
1E). Fourth, quantification of protein expression of Na
pump a1 isoform with Western technique indicated
that ethacrynic acid increases protein expression of a1
in cells derived from normal individuals, but not in
cells from bipolar individuals (Fig. 1B). Finally,
mRNA quantification by RNase protection indicates
transcription in normal cells but not in cells derived
from bipolar subjects (Fig. 1C).
The changes seen in the lymphoblastoid cell lines
studied herein reflect those reported previously in
fresh lymphocytes obtained from bipolar patients
and matched normal controls (Naylor and Smith,
1981; Wood et al., 1991). These earlier studies exam-
ined ouabain-binding and Na pump activity. The
current study has extended these observations to the
mRNA level and associated physiological changes in
Na concentration.
These data suggest that individuals with bipolar
illness may be unable to appropriately regulate Na
pump expression and activity in response to etha-
crynic acid challenge. Ethacrynic acid has several
actions on the Na pump. First, ethacrynic acid inhibits
the Na pump by blockade of phosphorylation and
prevention of ADP–ATP exchange. Second, etha-
crynic acid can decrease, slightly, the apparent affinity
for K by stabilizing the phosphorylated intermediate
in the pump exchange cycle. Both Rb-uptake and
ouabain binding are decreased by ethacrynic acid
treatment in the 10�4 to 10�6 M range. Sodium pump
inhibition results in an increase of intracellular Na
concentration. Normal cells respond to these changes
by increasing Na pump expression and activity. Cells
from Old Order Amish bipolar subjects did not exhibit
these responses and showed an increase of intracellu-
lar Na concentrations.
Mood state-associated increases of intracellular Na
concentrations have been reported in both mania and
bipolar depression (Coppen et al., 1966; Shaw, 1966).
Elevations of intracellular Na could bring about ele-
vations of free ionic Ca (Alexander et al., 1986;
Reddy et al., 1992), and could activate second mes-
senger systems in the absence of a first messenger
signal (El-Mallakh and Li, 1993). Higher intracellular
Na concentrations may also reduce the potential
difference across the membrane, resulting in more
easily stimulated neurons, a reduced number of neu-
rotransmitter quantal release with each action poten-
tial, and a slower clearance of free ionic Ca at the
presynaptic terminal (El-Mallakh and Wyatt, 1995;
El-Mallakh and Jaziri, 1990). If the increase in intra-
cellular Na concentration is related to a decrease of
Na,K-ATPase activity, recovery of the neuronal mem-
brane potential may be impaired and indices such as
the H-reflex recovery would be prolonged (El-Mal-
lakh et al., 1996). These events have been previously
purported to be central to the pathogenesis of patho-
logical mood states in bipolar illness (El-Mallakh and
Wyatt, 1995). The observations of the current study
support the idea that an abnormality in regulation of
the Na,K-ATPase may be a characteristic of bipolar-
derived tissues.
However, there are limitations to the current study
that prevent encompassing conclusions. The age of
the subjects is relatively young (31.3 years for
controls, 32.9 years for bipolar individuals). While
normal controls were carefully screened to rule out
psychiatric disease, they are not beyond the age of
risk for developing a mood disturbance. Additionally,
control subjects were not from the Old Order Amish
community (with the exception of two Amish con-
trols). Since this community had been established by
a small number of individuals, the difference in Na
pump response may be a reflection of a founder
effect, rather than a disease effect. Finally, the
utilization of a unique, inbred bipolar population
dictates that caution be exercised in generalization
of the data. These weaknesses are less striking when
one considers that similar observations have been
previously noted in lymphocytes of general commu-
nity-based bipolar samples (Naylor and Smith, 1981;
Wood et al., 1991).
In summary, the current study demonstrates a
divergent and anomalous Na pump response in live
leukocytes possessing the genetic heritage of bipolar
individuals. This abnormality may be central to the
pathogenesis of the disease states in this condition. At
the very least, these studies offer clues to the cellular
abnormalities that underlie bipolar illness.
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