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Mechanisms of Amp hi pat h-Induced Stomatocytosi s in Human Erythrocytes
By S.L. Schrier, A. Zachowski, and P.F. Devaux
We studied stomatocytosis induced in human red blood cells
(RBC) by vinblastine and chlorpromazine, monitoring the
movements of spin-labeled phosphatidylcholine (PC*) and
sphingomyelin (SM*) by electron spin resonance (ESR) spectroscopy. This technique allows determinationof the fraction
of labeled lipids, respectively, on the external leaflet, on the
cytosol face, or trapped in endocytic vacuoles. Both vinblastine and chlorpromazine produce a time- and concentrationdependent stomatocytic shape change, which is paralleled
by a shift of approximately 10% to 33% of outer leaflet SM*
and PC* inward. Of this amount, 8% to 12% was trapped in
endocytic vacuoles and 8% to 19% had flipped to the inner
leaflet. Vanadate, while inhibiting the stomatocytosis, did
not block the flip of either SM* or PC* to the inner leaflet. To
explain the inhibiting effect of vanadate, as well as the
adenosine triphosphate (ATP) requirementfor drug-induced
stomatocytosis, we propose the following model: (1) addition of amphipath partially scrambles the bilayer; and (2) the
flop of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer leaflet provides substrate for the
aminophospholipid translocase (APLT), which flips back PS
and PE inward faster than PC or SM can diffuse outwardthereby producing inner layer expansion or stomatocytosis.
This role of APLT accounts for the vanadate inhibition of
amphipath stomatocytosis. However, the vanadate effect
can be overcome by increasing the amphipath concentration,
which at such levels probably passively expands the inner
leaflet.
0 1992by The American Society of Hematology.
T
amphipath into the outer leaflet sufficient to cause an
expansion of as little as 0.6% to 1.0% will produce outward
buckling or echinocytosis.5.’ Conversely, if a cationic amphipath, for example, were to be incorporated into the
inner leaflet, the result would be inward buckling leading to
stomato~ytosis.’~~
Thus, the bilayer couple hypothesis implies that passive
insertion of a charged amphipath into an appropriate
leaflet is both necessary and sufficient to produce the shape
change. However, we have previously shown that while
adenosine triphosphate (ATP) is required for full expression of amphipath-induced stomatocytosis and endocytosis,”,” the requirement is not absolute and depends considerably on the specific cationic amphipath sed.'^-'^ We next
showed that vanadate, a potential inhibitor of ATPases,
blocked amphipath-induced stomatocytosis, but not
echinocytosis.” One could overwhelm the inhibitory action
of vanadate by adding more amphipath. These observations
suggest that an ATPase is involved in amphipath-mediated
stomatocytosis and endocytosis, but not echinocytosis. Since
APLT can mediate the conversion to stomatocytosis in
ghosts’ and is sensitive to vanadate inhibition, it is reasonable to hypothesize that APLT is the ATPase involved.
In a previous study, we showed that, in parallel with
amphipath-induced stomatocytosis, PE (but not PS) becomes more accessible to phospholipase degradation, consistent with a flop of PE from inner to outer leaflet.I6
Simultaneously, PC and SM become less accessible, but it
could not be determined by the phospholipase method
whether PC and SM had flopped to the inner leaflet or had
become trapped in the endocytic vacuoles of the spherostomatocyte.16 If the experiment was conducted at low temperature, where all the transmembrane motions of phospholipids are slowed down, it was possible to detect an appearance
of PS on the outer leaflet.”
These experiments were designed to evaluate the possible role of APLT in amphipath-induced stomatocytosis,
while at the same time tracking the transbilayer movements
of the phospholipids by using spin-labeled probes and
electron spin resonance (ESR) spectrometry.
Spin-labeled probes of PC and SM containing a short p
fatty acid chain were used. They are therefore sparingly
HE MECHANISMS by which mature adult red blood
cells (RBC) change their shape under physiologic and
pathologic conditions has been the object of considerable
study.’ There are many pathologic variations in RBC shape
and for at least two, the echinocyte and the stomatocyte,
there are good in vitro surrogates.’ Study of the echinocytediscocyte-stomatocyte interconversion in vitro has led to
the bilayer couple hyp~thesis,~
which is based on the fact
that the lipids of the mature human RBC are confined to
the membrane where the phospholipids form a coupled
bilayer. Each of two layers is approximately equal in size;
however, the bilayer is asymmetric with the zwitterionic
phospholipids sphingomyelin (SM) and phosphatidylcholine (PC) being present in the outer leaflet, whereas the
aminophospholipids phosphatidylethanolamine (PE) and
the negatively charged phosphatidylserine (PS) are the
main constituents of the inner leaflet? The asymmetry of
the bilayer is controlled by the aminophospholipid translocase (APLT), a Mgz’-adenosine triphosphatase (ATPase)
that actively transports PS and PE from the outer to the
inner leaflet (flip).’
Since these asymmetric layers are coupled, an imbalance
or an intercalation of a molecule into one of the leaflets of
the bilayer will cause buckling. Thus, the intercalation of an
From the Division of Hematology, Stanford University School of
Medicine, Stanford, CA; and the Institut de Biologie PhysicoChimique, Pans, France.
Submitted January 4, 1991; accepted September 24, 1991.
Supported by US Public Health Service Grant No. R 0 1 DK13682 to
S.L.S., and by grants from the Centre National de la Recherche
Scientifique (URA 526) and the UniversitePans VII.
This work was done while S.L.S. was on sabbatical leave at the
Institute de Biologie Physico-Chimique supported by a grant from the
Centre International des Etudiants et Stagiaires, France.
Address reprint requests to S.L. Schner, MD, Division of Hematology, S-161, Stanford University Medical School, 300 Pasteur Dr,
Stanford, CA 943055112,
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971I92 l7903-0121$3.00/0
782
Blood, Vol79, No 3 (February 1). 1992: pp 782-786
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783
AMPHIPATH-INDUCED STOMATOCYTOSIS
PHOSPHATIDYLCHOLINE(PC).
U
it
CHz-OPO-(CHz)z-N+(CH3)3
NO
\/
CH3 -&(CH
0
2)2
I
I
0
-COO HC
CHs-(C H2 ) 14-COOH2 C
SPHINGOMYELIN(SM).
U
it
0
CHz-OPO-(CHz)z-N+(CH3)3
NO
\/
I
1
0
CHs-C-(CH 2)z-COHNHC
CH3 -(CH
2)
lz-CH=CH-CHOH
Fig 1. Structural formulas of the single short-chain phospholipid
probes used. The nitroxide group that provides the ESR signal is
subject to reduction, which destroys the signal.
water soIuble'~'*(Fig 1) and easily incorporated into RBC;
when in the outer leaflet, they are quantitatively extracted
by delipidated bovine serum albumin (BSA) solutions.'*
Thus, during induction of stomatocytosis, the amount of
SM probe (SM*) or PC probe (PC*) remaining in the outer
leaflet is conveniently and quantitatively determined by
BSA extraction. SM* or PC* crossing the bilayer would be
reduced by cytosolic compounds, destroying the nitroxide
group and causing loss of the ESR signal.I8 SM* or PC*
incorporated within RBC, which is neither extractable into
BSA (outer leaflet) nor reducible by cytosolic processes
(inner leaflet), is probably trapped in the intravesicular
leaflet of endocytic vacuoles.'"."
MATERIALS AND METHODS
Materiuls. The single short-chain spin-labeled probes PC* and
SM* labeled in the p position were synthesized as previously
described5,'* (Fig 1). Chloroform or chloroform-methanol (21)
solutions of the probes were evaporated to dryness in a stream of
argon. Delipidated BSA, di-isopropyl-fluorophosphate (DFP),
chlorpromazine, and vinblastine were obtained from Sigma, St
Louis, MO. Fresh stock solutions of amphipath were made just
before use and stored in foil-coated vessels to prevent lightinduced degradation. All other reagents were of the best grade
available.
Methods. Blood was obtained by venipuncture into EDTA
from normal laboratory volunteers after they gave informed
consent according to the code of ethics of the World Medical
Association. The RBC used in these experiments were either
freshly drawn or 1 day old and refrigerated at 4°C.
In a typical experiment, RBC were centrifuged, the plasma was
removed, and the RBC were washed five times in triple volumes of
buffered saline potassium glucose (BSKG) consisting of 145
mmol/L NaCI, 5 mmol/L KC1, 6 mmol/L glucose, and 10 mmol/L
phosphate buffer, pH 7.4. Then, 5 mmol/L DFP was added to
prevent the hydrolysis of the p acyl chain when the PC* probe was
used.I8The argon-dried spin-labeled probe to be used in an amount
equal to 1% to 3% of total phospholipid was vortexed for 20
seconds in 50 to 100 pL of BSKG and then added to 1- to 2-mL
suspensions of washed, packed RBC. After a brief period of
incorporation, the hematocrit was adjusted to 50 with BSKG.
Reactions with drugs were performed at 37°C in 200-pLvolumes
at a hematocrit of 45 containing either no drug or 0.5 to 1.5mmol/L
vinblastine or 0.5 to 1mmol/L chlorpromazine. At varying times of
incubation, 150-pL aliquots were removed and added to 40 p,L of
10% BSA, mixed, allowed to stand for 1 minute, centrifuged at
7,600g for 30 seconds, and the supernatant quantitatively removed
and added to 10 pL of 0.1 mol/L potassium ferricyanide to keep
the nitroxide group oxidized.I8The supernatant was then analyzed
for ESR intensity in a Varian E 109 spectrometer (Palo Alto, CA)
to indicate quantity of the probe extractable from the outer leaflet.
To determine if any of the SM* or PC* had flipped to the inner
leaflet, aliquots of RBC were centrifuged and the supernatant was
discarded. The packed RBC were then quantitatively transferred
to the ESR spectrometer cuvette to provide the direct readings for
total amounts of spin-labeled probe incorporated into the RBC.
Readings were repeated every 2 to 4 minutes, holding the cuvette
at 37°C. The rate of reduction of the ESR signal was recorded
generally for 20 to 40 minutes, after which the curves showed a
sharp break and plateau. This point indicated the amount of
spin-labeled probe that had flipped to the inner leaflet. When the
reduction was completed, the RBC were removed from the cuvette,
extracted twice more with 2% BSA to remove any spin label
remaining in the outer leaflet, and then quantitatively returned to
the cuvette for determination of spin-labeled probe that was
neither reduced (located in the cytoplasmic leaflet) nor extractable
into BSA (remaining in the outer leaflet).
RBC were fixed in 1% glutaraldehyde in phosphate-buffered
saline (PBS) and then morphology was evaluated by Nomarski
interference-phase microscopy at magnification of 6 5 0 ~and
1 , 0 0 0 ~ .When required, a modified numerical scoring system
quantifying the echinocyte-discocyte-stomatocyteinterconversion
was used?
RESULTS
Vinblastine. Washed, intact RBC were labeled with
either SM* or PC* in amounts varying from 1% to 3% of
total phospholipid. This incorporation invariably produced
echinocytosis as previously reported,' and the amount of
vinblastine used was adjusted to overcome the echinocytosis and produce the desired amounts of spherostomatocytosis. The concentration dependency of vinblastine was studied with both PC* and SM* and is recorded in Table 1 as
the percent of phospholipid extracted into BSA from the
outer leaflet. The results obtained with identically treated
RBC incubated in parallel without addition of amphipathic
drug were set at 100%. Reactions were performed for 5
minutes at 37°C. With increasing amounts of drug, there
was a progressive increase in spherostomatocytosis paralTable 1. Percent of IncorporatedSpin-Labeled PC or SM Extracted
Into BSA
Vinblastine
Concentration
(mmol/L)
PCf
SM'
0.75
1.oo
1.25
100
79
-
1.50
71
-
89
78
69
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784
SCHRIER. ZACHOWSKI, AND DEVAUX
w
m
2n
3
60
S
B
s40
W
0
K
W
n
20
0
OUTER
LAYER
INNER
LAYER
ENDOCYllC
VESICLES
OUTER
LAYER
INNER
LAYER
ENDOCYTIC
VESICLES
PROBE LOCATION
PROBE LOCATION
Fig 2. Location in the membrane of the phospholipidprobes after
incubationwith 1.5 mmol/L vinblastine.
Fig 3. Location in the membrane of the phospholipid probes after
incubationwith 1.0 mmol/L chlorpromazine.
leled by a decrease in outer leaflet PC* and SM*. The
detailed redistribution of the probe was monitored following the addition of 1.5 mmol/Lvinblastine in three separate
experiments. In the experiment shown in Fig 2, there was a
disappearance of 29% of PC* and 31% of SM* from the
outer leaflet, and a transbilayer movement of approximately 19% of both PC* and SM* to the inner leaflet, with
10% of PC* and 12% of SM* not accessible from either side
and therefore very likely entrapped in endocytic vacuoles
(Fig 2). In a repeat experiment, the values for PC* were
32% loss from the outer leaflet with a transbilayer movement of 18%, a recovery of 9% trapped in endocytic
vacuoles, and 5% unaccounted for. The SM* results were
23% loss from outer leaflet, a transbilayer movement of
15%, and a recovery of 8% in endocytic vacuoles.
Chlorpromazine. To confirm that the results were not
limited to a single cationic amphipath"*'*." experiments
were repeated using chlorpromazine, usually at a concentration of 1 mmol/L. The reactions were performed on RBC
labeled with PC* and SM* and then incubated at 37°C. The
amount of probe extracted into BSA was related to the
control sample incubated without drug, the value of which
was established at 100%. There was a rapid onset of
chlorpromazine action, which was essentially completed
after approximately 1 minute (Table 2). The distribution of
the probe during chlorpromazine-induced shape change is
shown in Fig 3 for one of two similar experiments. Seventeen percent of PC* disappeared from the outer leaflet,
with 9% moving to the inner leaflet, while 8% was trapped
in endocytic vacuoles. The values for SM* were 27% loss
from the outer leaflet, with 14% moving across the bilayer,
and 13% being trapped in vacuoles. For comparison purposes, the results for a second experiment were as follows:
for PC*, 19% disappeared from the outer leaflet, with 11%
moving transbilayer, while 8% was trapped in endocytic
vacuoles; with SM*, 31% disappeared from the outer
leaflet, with 17% crossing the bilayer, and 14% being
trapped in endocytic vacuoles.
Effect of vanadate. RBC were preincubated for 90
minutes at 37°C with and without 150 pnol/L vanadate,
after which SM* was incorporated into the outer leaflet of
the RBC. That concentration of vanadate was chosen
because when studied it completely inhibits the translocation of aminopho~pholipids.'~
These RBC were then incubated with vinblastine (0.75 to 1.5 mmol/L) and the
movements of the spin-labeled probe were traced in parallel with morphologic assessment of RBC shape (Table 3).
The inhibition by vanadate of the stomatocytic shape
change was readily confirmed" morphologically and by the
amount of SM* trapped in the endocyticvacuoles (Table 3),
which are a concomitant of the spherostomatocytic shape.
Whereas 1.0 mmoULvinblastine caused 10% of SM* to be
trapped in endocytic vacuoles, the addition of vanadate
reduced that to 1%. However, increasing the concentration
of vinblastine to 1.5 mmol/L almost overwhelmed the
vanadate inhibition, producing extensive spherostomatocytosis as indicated by the increasing morphologic score and
by the increase in SM* trapped in endocytic vacuoles to 8%
in contrast to the vanadate free control value of 12% (Table
3). From this experiment, one could suppose that there was
some vanadate inhibition of SM* flip. However, in the
absence of vanadate, much more of the probe is trapped in
endocytic vacuoles, reflecting the greater extent of
Table 2. Percent SM* Extracted at the Indicated Time of Incubation
Wkh Chlorpromazine(1 mmol/L)
Time of Incubation ( 8 )
% Extraction
30
60
120
93
82
84
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AMPHIPATH-INDUCED STOMATOCYTOSIS
785
Table 3. Effect of 150 pmol/L Vanadate on the Movement of SM*
Control
Vanadate
Location of Probe (%)
Location of Probe (%)
Endocytic
Vacuole
Inner
Leaflet
Morphology
(mmolIL1
Outer
Leaflet
0.75
1 .o
1.5
84
81
77
-
-
10
12
9
11
Vinblastine
Morphology score (200RBC counted): spherostomatocyte
Score
Outer
Leaflet
Endocytic
Vacuole
Inner
Leaflet
Morphology
Score
300
390
400
100
91
84
-
-
1
8
8
8
90
220
280
= 4;stomatocyte 111 =
spherostomatocytosis with concomitant endocytic vacuole
formation. The actual amount of SM* that flipped to the
inner leaflet is derived by subtracting the amount trapped in
endocytic vacuoles from the amount of probe that disappeared from the outer leaflet. When that correction is
made, it becomes apparent that vanadate had virtually no
effect on the amount of SM* that flipped to the inner leaflet
as a consequence of incubation with vinblastine (Table 3).
Bilayer status after addition of amphipath. We reasoned
that if a cationic amphipath resulted in partial or complete
scrambling of the bilayer, then addition of SM* at varying
times after addition of the stomatocytogenic amphipath
should produce a pattern of distribution different than the
greater than 95% of SM* ordinarily remaining in the outer
leaflet. Accordingly, identical amounts of SM* were added
to aliquots of suspensions of RBC at varying intervals after
addition of chlorpromazine, and the amount of SM* in the
outer leaflet was referred to the value in the sample
incubated without drug. Results in Table 4 indicate that the
bilayer asymmetry appears to be altered for approximately
120+ seconds after chlorpromazine addition.
DISCUSSION
Experiments were designed to trace the detailed movements of the phospholipids of the human RBC as it
undergoes amphipath-induced stomatocytosis. It was proposed that these experiments might explain an apparent
aberration in the bilayer couple hypothesis, namely, the
inhibitory role of vanadate15(Table 3).
Amphipath-induced stomatocytosis by either chlorpromazine or vinblastine is a time- and concentration-dependent
process, and the addition of the several concentrations of
the two drugs used produced a shift of approximately 10%
to 33% outer leaflet SM* or PC*, depending considerably
on the extent of shape change achieved (Tables 1 and 2 ) . Of
this amount, approximately 8% to 12% was trapped in the
intravesicular leaflet of endocytic vacuoles and approximately 8% to 19%flipped to the inner leaflet (Figs 2 and 3).
Table 4. Percent SM* in Outer Leaflet
Time After Addition of
0.5 mmoliL Chlorpromazine (s)
0
EXP. 1
Exp. 2
100
100
10
94
30
60
120
63
79
82
85
78
480
-
-
loo+
Incubation performed at 37°C. Chlorpromazine was added at time
zero and SM* was added at the indicated times.
3; stomatocyte II = 2;stomatocyte I = 1; discocyte = 0.
Note that the nonaccessibility to BSA of spin-labeled lipids
located in endocytic vacuoles indicates that lipids on the
intravesicular leaflet of these vacuoles are not free to
diffuse laterally out of the vacuoles. A barrier to diffusion
must exist at the point where the vacuoles are sealed.
Thus, the cationic amphipaths studied can partially
scramble the bilayer. Previous observations showed that
chlorpromazine induced a partial scrambling of endogenous PE16 and of analogues of all the phospholipid^.'^^'^
Furthermore, when we did the experiment in reverse,
adding SM* at varying times after chlorpromazine addition
(Table 4), it appeared that the bilayer was partially scrambled and remained so, from 10 seconds to 120 seconds after
chlorpromazine addition.
The role of vanadate in inhibiting amphipath stomatocytosis was studied and showed that preincubation of RBC
with 150 kmol/L vanadate did not block amphipathinduced scrambling of phospholipids as defined by the
amount of probe transferred into the cytoplasmic membrane leaflet (Table 3). Therefore, amphipath-induced
scrambling is not dependent on ATPases and by inference,
the APLT.
The question then is, what is the role of vanadate,15
and ATPases~~'~l'
in amphipath-induced stomatocytosis, but not echinocytosis? We propose that some
cationic amphipaths produce a rapid scrambling of the
bilayer with PC and SM moving inward while PE moves
outward along with PS (as previously
PS arriving
at the outer leaflet is immediately flipped back to the inner
leaflet by the APLT, thus creating an imbalance between
the two halves of the membrane. As little as 0.6% enrichment' of one of the leaflets of the bilayer is sufficient to
produce an appropriate bulging and shape change. Thus,
vanadate inhibition of APLT accounts for the vanadate
inhibition of the amphipath-induced stomatocytosis. The
requirement for ATP is explained by the fact that ATP is an
obligatory substrate for APLT.'.'' Thus, stomatocytosis
produced by low concentrations of amphipath does not
reflect solely a preferential incorporation of the drug into
the cytoplasmic leaflet of the bilayer, but is also dependent
on a rearrangement of the membrane phospholipids. The
idea that amphipaths may produce nonbilayer structures in
the membrane has also been suggested as an explanation of
the fact that the charge on an amphipath did not invariably
determine the shape change it produced.20
Therefore, stomatocytogenic amphipaths produce at least
two different membrane alterations: partial scrambling of
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SCHRIER, ZACHOWSKI, AND DEVAUX
786
the bilayer, and intercalation into the inner cytoplasmic
leaflet. An agent like primaquine, that has an absolute
requirement for ATP,10,1’,13,14
would act primarily by scrambling the bilayer and less by intercalation into the inner
leaflet. Conversely, an agent like chlorpromazine that
requires little ATP for shape
would produce
somewhat less phospholipid scrambling and act more directly by intercalating itself into the inner leaflet. In fact,
chlorpromazine does bind more avidly to the inner leaflet
than to the outer leaflet.” This hypothesis also provides an
explanation of why the vanadate inhibition of stomatocytosis can be overcome by increasing the concentration of
amphipath used’’ (Table 3). Vanadate at a concentration of
150 ~ m ~ l /completely
L’~
blocks aminophospholipid translocation and also completely inhibits the partly purified
APLT.” Thus, at very high concentration, the cationic
amphipath acts passively according to the bilayer couple
hypothesis, intercalating itself (slightly) more into the inner
layer.
The central role of APLT in amphipath-induced stomatocytosis is supported by the observation that neonatal RBC,
which undergo substantially increased amphipath-induced
~tomatocytosis,~~
have increased APLT levels sufficient to
account for the increased stomatocytosis and endocytosis
seen (personal communication, Dr Sophie Cribier, Institute
de Biologie Physico-Chimique, Paris, France).
ACKNOWLEDGMENT
The authors wish to thank Paulette Heme for superb technical
skills in providing the probes that formed the basis of our analytic
work.
REFERENCES
1. Bessis M: Red cell shapes. An illustrated classification and its
rationale. Nelle Rev Fr Hematol72:721,1972
2. Deuticke B: Transformation and restoration of biconcave
shape of human erythrocytes induced by amphiphilic agents and
changes of ionic environment. Biochim Biophys Acta 163:494,1968
3. Sheetz M, Singer S: Biological membranes as bilayer couples.
A molecular mechanism of drug-erythrocyte interaction. Proc Natl
Acad Sci USA 71:4457,1974
4. Verkleij AJ, Zwaal RFA, Roelofsen B, Comfurius P, Kasto D,
Van Deenen LLM: The asymmetric distribution of phospholipids
in the human red cell membrane. A combined study using
phospholipases and freeze-etching electron microscopy. Biochim
Biophys Acta 323:178,1973
5. Seigneuret M, Devaux PF: ATP dependent asymmetric distribution of spin-labelled phospholipids in the erythrocyte membrane: Relation to shape changes. Proc Natl Acad Sci USA
81:3751, 1984
6. Ferrell J, Lee K-J, Huestis W Membrane bilayer balance and
erythrocyte shape: A quantitative assessment. Biochemistry 24:
2849,1985
7. Allan D, Hagelberg C, Kallen K-J, Haest CWM: Echinocytosis and microvesiculation of human erythrocytes induced by insertion of merocyanine 540 into the outer membrane leaflet. Biochim
Biophys Acta 986:115,1989
8. Ferrell J, Huestis WH: Phosphoinositide metabolism and the
morphology of human erythrocytes. J Cell Biol98:1992,1984
9. Daleke DL, Huestis WH: Incorporation and translocation of
aminophospholipids in human erythrocytes. Biochemistry 24:5406,
1985
10. Ben-Bassat I, Bensch KG, Schrier S L Drug-induced erythrocyte membrane internalization. J Clin Invest 51:1833,1972
11. Schrier SL, Junga I, Seeger M: The mechanism of druginduced erythrocyte vacuole formation. J Lab Clin Med 83:215,
1974
12. Feo C, Mohandas N: Clarification of role of ATP in red-cell
morphology and function. Nature 265:166,1977
13. Zarkowsky H, Rinehart J: Endocytosis in adenosine triphosphate-depleted erythrocytes. Biochim Biophys Acta 84:242,1979
14. Schrier SL, Junga I, Krueger J, Johnson M: Requirements of
drug-induced endocytosis by intact human erythrocytes. Blood
Cells 4353, 1978
15. Schrier SL, Junga I, Ma L Studies on the effect of vanadate
on endocytosis and shape changes in human red blood cells and
ghosts. Blood 68:1008,1986
16. Schrier SL, Chiu DT-Y, Yee M, Sizer K: Alteration of
membrane phospholipid bilayer organization in human erythrocytes during drug-induced endocytosis. J Clin Invest 72:1698,1983
17. Rosso J, Zachowski A, Devaux PF: Influence of chlorpromazine on the transverse mobility of phospholipids in the human
erythrocyte membrane: Relation to shape changes. Biochim Biophys Acta 942:271,1988
18. Morrot G, Herve P, Zachowski A, Fellmann P, Devaux P F
Aminophospholipid translocase of human erythrocytes: Phospholipid substrate specificity and effect of cholesterol. Biochemistry
283456,1989
19. Bitbol M, Fellmann P, Zachowski A, and Devaux, P: Ion
regulation of phosphatidylserine and phosphatidylethanolamine
outside-inside translocation in human erythrocytes. Biochim Biophys Acta 904:268,1987
20. Isomaa B, Hagerstrand, Paatero G: Shape transformations
induced by amphiphiles in erythrocytes. Biochim Biophys Acta
899:93, 1987
21. Elferink JGR: The asymmetric distribution of chlorpromazine and its quaternary analogue over the erythrocyte membrane.
Biochem Pharmacol26:2411,1977
22. Morrot G, Zachowski A, Devaux P F Partial purification and
characterization of the human erythrocyte Mg2’-ATPase. A candidate aminophospholipid translocase. FEBS Lett 266:29, 1990
23. Matovcik LM, Junga IG, Schrier SL: Drug-induced endocytosis of neonatal erythrocytes. Blood 65:1056,1985
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1992 79: 782-786
Mechanisms of amphipath-induced stomatocytosis in human
erythrocytes
SL Schrier, A Zachowski and PF Devaux
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