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Monensin-induced-suicidal-erythrocyte-death 2010 Cellular-Physiology-and-Biochemistry, biochemia, ...

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Original Paper
Cellular Physiology
Cellular Physiology
and Biochemistr
Cell Physiol Biochem 2010;25:745-752
Accepted: February 26, 2010
y
and Biochemistr
Monensin Induced Suicidal Erythrocyte Death
Shefalee K. Bhavsar, Matthias Eberhard, Diwakar Bobbala and
Florian Lang
Department of Physiology, University of Tübingen, Germany
Key Words
Phosphatidylserine • Monensin • Scrambling • Cal-
cium • Cell volume • Eryptosis • Glucose depletion •
Apoptosis
annexin V-binding. Glucose depletion was followed
by decreased forward scatter and increased cytosolic
Ca
2+
concentration and annexin V-binding. The effect
on forward scatter was partially reversed, the effect
on cytosolic Ca
2+
concentration and annexin V binding
augmented by additional treatment with monensin. In
conclusion, monensin dissociates the alterations of
cell membrane and cell volume in suicidal erythrocyte
death.
Abstract
Eryptosis, the suicidal erythrocyte death, is
characterized by cell membrane scrambling and cell
shrinkage. Eryptosis may be triggered by excessive
hyperosmotic or isosmotic cell shrinkage leading to
increase of cytosolic Ca
2+
concentration. Eryptosis is
further stimulated by the K
+
ionophore valinomycin,
which leads to exit of KCl and osmotically obliged
water, or by energy (glucose) depletion, which
compromises the function of the Na
+
/K
+
ATPase thus
increasing cytosolic Na
+
concentration. The present
study explored whether the Na
+
ionophore monensin
affects erythrocyte cell volume and eryptosis. The cell
membrane scrambling was estimated from binding of
annexin V to phosphatidylserine at the erythrocyte
surface, cell volume from forward scatter in FACS
analysis, cytosolic Ca
2+
concentration from Fluo3
fluorescence and the cytosolic ATP concentration from
a luciferase-based assay. Within 24 hours, exposure
to monensin (0.1-10 µg/ml) significantly increased
forward scatter, cytosolic Ca
2+
concentration and
Copyright © 2010 S. Karger AG, Basel
Introduction
Similar to apoptosis of nucleated cells the suicidal
erythrocyte death or eryptosis is characterized by cell
membrane scrambling and cell shrinkage [1]. Both events
are triggered by increase of cytosolic Ca
2+
concentration
due to Ca
2+
entry through Ca
2+
-permeable cation chan-
nels [2-9]. The enhanced cytosolic Ca
2+
concentration
activates Ca
2+
-sensitive K
+
channels [10, 11], resulting in
exit of KCl with osmotically obliged water and thus in
cell shrinkage [12]. Increased Ca
2+
concentration fur-
ther stimulates phospholipid scrambling of the erythro-
Prof. Dr. Florian Lang
Physiologisches Institut, Universität Tübingen
Gmelinstr. 5, 72076 Tübingen (Germany)
Tel. +49 7071 29 72194, Fax +49 7071 29 5618
E-Mail florian.lang@uni-tuebingen.de
745
© 2010 S. Karger AG, Basel
1015-8987/10/0256-0745$26.00/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
Accessible online at:
www.karger.com/cpb
cyte membrane with exposure of phosphatidylserine at
its surface [9, 13-16]. Erythrocytes are sensitized to the
scambling effect of Ca
2+
by ceramide [17].
Phosphatidylserine-exposing erythrocytes are phagocy-
tosed and rapidly cleared from circulating blood thus lead-
ing to anemia [18-20].
While excessive cell shinkage is well known to trig-
ger eryptosis [21], little is known about the effect of cell
swelling. In nucleated cells, Na
+
entry and subsequent
cell swelling may be elicited by monensin (rumensin), a
well known Na
+
ionophore, which thus stimulates necro-
sis rather than apoptosis [22, 23]. The effect of monensin
may be due to mitochondrial damage [23] and/or weak-
ening of the antioxidative defence [24]. Monensin intoxi-
cation leads to severe rhabdomyolysis and acute renal
failure with ultimate death of the patients [22, 25]. The
excessive Na
+
entry following monensin intoxication
stimulates the Na
+
,K
+
-ATPase [26], which may in turn
lead to energy depletion, another well known trigger of
eryptosis [27].
The present study explored the effect of monensin
on erythrocyte cell volume and cell membrane asymme-
try. As a result, monensin treatment of erythrocytes leads
to cell membrane scrambling and cell swelling and thus
dissociates the alterations of the cell membrane and cell
volume during erythrocyte death.
wavelength of 488 nm and an emission wavelength of 530 nm
on a FACS calibur (BD, Heidelberg, Germany).
Measurement of intracellular Ca
2+
After incubation 50 µl erythrocyte suspension were
washed in Ringer solution and then loaded with Fluo-3/AM
(Calbiochem, Bad Soden, Germany) in Ringer solution
containing 5 mM CaCl
2
and 2 µM Fluo-3/AM. The cells were
incubated at 37°C for 20 min and washed twice in Ringer solution
containing 5 mM CaCl
2
. The Fluo-3/AM-loaded erythrocytes
were resuspended in 200 µl Ringer. Then, Ca
2+
-dependent
fluorescence intensity was measured in fluorescence channel
FL-1 in FACS analysis.
Measurement of hemolysis
After 24 hours of incubation at 37°C, the samples were
centrifuged (3 min at 400 g, RT), and the supernatants were
harvested. As a measure of hemolysis, the hemoglobin (Hb)
concentration of the supernatants was determined
photometrically at 405 nm. The absorption of the supernatant
of erythrocytes lysed in distilled water was defined as 100%
hemolysis.
Determination of intracellular ATP concentration
For determination of erythrocyte ATP, 90 µl of erythro-
cyte pellets were incubated for 24 h at 37°C in Ringer solution
with or without monensin (final hematocrit 5%). All manipula-
tions were then performed at 4°C to avoid ATP degradation.
Cells were lysed in distilled water, and proteins were precipi-
tated by addition of HClO
4
(5%). After centrifugation, an aliquot
of the supernatant (400 µl) was adjusted to pH 7.7 by addition
of saturated KHCO
3
solution. After dilution of the supernatant,
the ATP concentrations of the aliquots were determined utiliz-
ing the luciferin-luciferase assay kit (Roche Diagnostics) on a
luminometer (Berthold Biolumat LB9500, Bad Wildbad, Ger-
many) according to the manufacturer’s protocol. ATP concen-
trations are expressed in mmol/l cytosol of erythrocytes.
Materials and Methods
Erythrocytes, solutions and chemicals
Leukocyte-depleted erythrocytes were kindly provided
by the blood bank of the University of Tübingen. The study is
approved by the ethics committee of the University of Tübingen
(184/2003V).
Erythrocytes were incubated
in vitro
at a hematocrit of
0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1
MgSO
4
, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
(HEPES), 5 glucose, 1 CaCl
2
; pH 7.4 at 37°C for 24 hours. Where
indicated, monensin (Axxora, Lörrach, Germany) was added at
the indicated concentrations. In Ca
2+
-free Ringer, 1 mM CaCl
2
was substituted for 1 mM ethylene glycol tetraacetic acid
(EGTA).
Statistics
Data are expressed as arithmetic means ± SEM. Statistical
analysis was made using paired ANOVA with Tukey’s test as
post-test, as appropriate. n denotes the number of different
erythrocyte specimens studied. Since different erythrocyte
specimens used in distinct experiments are differently
susceptible to eryptotic effects, paired comparison was
employed.
Results
FACS analysis of
annexin V-binding and forward scat-
ter
After incubation under the respective experimental
condition, 50 µl cell suspension were washed in Ringer solution
containing 5 mM CaCl
2
and then stained with Annexin-V-Fluos
(1:500 dilution; Roche, Mannheim, Germany) in this solution
for 20 min under protection from light. In the following, the
forward scatter of the cells was determined, and annexin V
fluorescence intensity was measured in FL-1 with an excitation
Treatment of erythrocytes with a Na
+
ionophore is
expected to enhance the entry of Na
+
together with Cl
-
and osmotically obliged water leading to cell swelling.
Alterations of erythrocyte volume should be reflected by
the respective changes of forward scatter (FSC) in FACS
analysis. As illustrated in Fig. 1, exposure of erythrocytes
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Cell Physiol Biochem 2010;25:745-752
Bhavsar/Eberhard/Bobbala/Lang
Fig. 2.
Effect of monensin on erythrocyte ATP content.
Arithmetic means ± SEM (n = 4) of the ATP concentration after
a 24 hours incubation in Ringer solution without (white bar) or
with (black bars) monensin at the indicated concentrations or
in glucose free Ringer solution (mGlu, grey bar) as a positive
control. *** indicate significant difference (p<0.001 ) from
control (absence of monensin and presence of glucose).
Fig. 1
.
Effect of monensin on erythrocyte forward scatter. A.
Original histogram of the forward scatter of erythrocytes
following exposure for 24 hours to Ringer solution without (-,
black line) and with (+, red line) 1 µM monensin. B. Arithmetic
means ± SEM (n = 8) of erythrocyte forward scatter following
exposure for 24 hours to Ringer solution without (white bar) or
with (black bars) monensin at the indicated concentrations. *,
*** (p<0.05, p<0.001) indicates significant difference from the
respective value without exposure to monensin. C. Arithmetic
means ± SEM (n = 8) of the percentage of hemolysed
erythrocytes exposed for 24 hours to Ringer solution without
(white bar) or with (black bars) monensin at the indicated
concentrations.
Fig. 3.
Effect of monensin on cytosolic Ca
2+
concentration in
erythrocytes. A. Histogram of Fluo3 fluorescence in a
representative experiment of erythrocytes exposed for 24 hours
to Ringer solution without (-, black line) and with (+, red line) 1
µM monensin. B. Arithmetic means ± SEM (n = 8) of the geo
means of Fluo3 fluorescence in erythrocytes exposed for 24
hours to Ringer without (white bar) or with (black bars)
monensin. **, *** indicates significant difference (p<0.01,
p<0.001) from the respective value in the absence of monensin.
for 24 hours to Ringer solution with monensin (= 0.1 µM)
was indeed followed by an increase of FSC, an effect
reaching statistical significance at higher monensin
concentrations (1 µM; Fig. 1). Additional experiments
were performed to explore, whether the increase of cell
volume was sufficient to trigger hemolysis. As demonstra-
ted in Fig. 1, at the monensin concentrations and exposure
times, monensin did not elicit significant hemolysis.
Monensin-induced Eryptosis
Cell Physiol Biochem 2010;25:745-752
747
Fig. 4.
Effect of monensin on phosphatidylserine exposure of
erythrocytes. A. Histogram of erythrocyte annexin V-binding
in a representative experiment of erythrocytes exposed for 24
hours to Ringer solution without (-, black line) and with (+, red
line) 1 µM monensin, M1 is marker indicating
phosphatidylserine exposing cells. B. Arithmetic means ± SEM
(n = 8) of the percentage of phosphatidylserine-exposing
erythrocytes following exposure for 24 hours to Ringer solution
without (white bar) or with (black bars) monensin. *, *** (p<0.05,
p<0.001) indicates significant difference from the respective
value without exposure to monensin. C. Arithmetic means ±
SEM (n = 6) of the percentage of phosphatidylserine-exposing
erythrocytes following exposure for 24 hours to Ringer solution
in the presence (+Ca
2+
, left bars) or absence (-Ca
2+
, right bars)
of extracellular Ca
2+
without (white bars) or with (black bars) 1
µM monensin. * (p<0.05) indicates significant difference from
the respective value without exposure to monensin. ## (p<0.001)
indicates significant difference from the respective value in the
presence of extracellular Ca
2+
.
Excessive Na
+
entry is expected to stimulate Na
+
/
K
+
ATPase activity, which should enhance the ATP
consumption and thus decrease cytosolic ATP
concentration. Accordingly, additional experiments were
performed to determine, whether exposure to monensin
influences ATP concentrations in erythrocytes. As shown
in Fig. 2, a 24 hours exposure of human erythrocytes to
monensin (
≥
5 µM) led to a significant decrease of ATP
concentration. Energy depletion by incubation in the
glucose free Ringer solution (mGlu) also led to significant
decrease of ATP concentration.
ATP depletion is known to increase cytosolic Ca
2+
concentration in erythrocytes [28]. Thus, Fluo 3
fluorescence has been utilized to elucidate whether
monensin influences erythrocyte Ca
2+
concentration. As
shown in Fig. 3, monensin exposure indeed increased the
Fluo3 fluorescence, pointing to an increase of cytosolic
Ca
2+
concentration.
An increase in cytosolic Ca
2+
concentration is known
to stimulate cell membrane scrambling with
phosphatidylserine exposure at the cell surface, which
could be identified by determination of annexin V-binding.
As shown in Fig. 4, the percentage of annexin V binding
erythrocytes was markedly increased following exposure
of erythrocytes for 24 hours to Ringer solution containing
monensin (0.1 µM to 10 µM).
To test whether monensin-induced cell membrane
scrambling is due to increase of cytosolic Ca
2+
concentration, erythrocytes were exposed to monensin
Fig. 5.
Effect of monensin on erythrocyte forward scatter
following glucose depletion. A. Original histogram of the forward
scatter of erythrocytes following exposure for 24 hours to
glucose free Ringer solution without (-, black line) and with (+,
red line) 1 µM monensin. B. Arithmetic means ± SEM (n = 8) of
erythrocyte forward scatter following exposure for 24 hours to
Ringer solution (white bar) or glucose free Ringer solution (black
bar) *** (p<0.001) indicates significant difference from the
respective value in the presence of glucose. C. Arithmetic means
± SEM (n = 8) of erythrocyte forward scatter following exposure
for 24 hours to glucose free Ringer solution without (white bar)
or with (black bars) monensin at the indicated concentrations
in the absence of glucose. *, ** (p<0.05, p<0.01) indicates
significant difference from the respective value without
exposure to monensin.
748
Cell Physiol Biochem 2010;25:745-752
Bhavsar/Eberhard/Bobbala/Lang
Fig. 6.
Effect of monensin on cytosolic Ca
2+
concentration in erythrocytes following glucose
depletion. A. Histogram of Fluo3 fluorescence in a
representative experiment of erythrocytes exposed for
24 hours to Ringer solution without (-, black line) and
with (+, red line) 1 µM monensin in the absence of
glucose. B. Arithmetic means ± SEM (n = 8) of the geo
means of Fluo3 fluorescence in erythrocytes exposed
for 24 hours to Ringer without (white bar) or with (black
bars) monensin in the absence of glucose. **, ***
(p<0.01, p<0.001) indicates significant difference from
the respective value in the absence of monensin. C.
Arithmetic means ± SEM (n = 8) of the geo means of
Fluo3 fluorescence in erythrocytes exposed for 24
hours to Ringer solution (white bar) or glucose free
Ringer solution (black bar). ** (p<0.01) indicates signifi-
cant difference from the respective value in the presence
of glucose. D. Arithmetic means ± SEM (n = 8) of the
geo means of Fluo3 fluorescence in erythrocytes
exposed for 24 hours to Ringer solution (white bars) or
glucose free Ringer solution (black bars) in the presence
of respective concentrations of monensin. **, ***
(p<0.01, p<0.001) indicates significant difference from
the respective value in the presence of glucose.
Fig. 7.
Effect of monensin on phosphatidylserine exposure of
erythrocytes following glucose depletion. A. Histogram of
erythrocyte annexin V-binding in a representative experiment
of erythrocytes exposed for 24 hours to glucose free Ringer
solution without (-, black line) and with (+, red line) 1 µM
monensin in the absence of glucose, M1 is a marker indicating
phosphatidylserine exposing cells. B. Arithmetic means ± SEM
(n = 8) of the percentage of phosphatidylserine-exposing
erythrocytes following exposure for 24 hours to Ringer solution
(white bar) or glucose free Ringer solution (black bar). **
(p<0.01) indicates significant difference from the respective
value in the presence of glucose. C. Arithmetic means ± SEM
(n = 8) of the percentage of phosphatidylserine-exposing
erythrocytes following exposure for 24 hours to Ringer solution
without (white bar) or with (black bars) monensin in the absence
of glucose. *, ** (p<0.05, p<0.01) indicates significant difference
from the respective value without exposure to monensin.
in the absence of extracellular Ca
2+
. As shown in Fig. 4,
the stimulation of annexin V binding by monensin was
significantly blunted in the absence of extracellular Ca
2+
.
ATP depletion, increase of cytosolic Ca
2+
concentration, and subsequent stimulation of cell
membrane scrambling are known features of energy
depletion by omission of glucose [27]. However, glucose
depletion leads to cell shrinkage rather than cell swelling
[27]. Accordingly, additional experiments were performed
to explore whether monensin interacts with the response
of erythrocytes to glucose depletion. According to forward
scatter, exposure of the erythrocytes to glucose free
solutions for 24 hours led to pronounced cell shrinkage
(Fig. 5). The additional treatment with monensin partially
reversed the effect of glucose depletion on forward scatter
(Fig. 5).
Glucose depletion further increased cytosolic Ca
2+
concentration in erythrocytes. As illustrated in Fig. 6,
glucose depletion increased the Fluo3 fluorescence, an
effect augmented in the presence of monensin.
Accordingly, glucose depletion and monesin synergized
to enhance cytosolic Ca
2+
concentration.
Monensin-induced Eryptosis
Cell Physiol Biochem 2010;25:745-752
749
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