Membrane lipid diffusion and band 3 protein changes in human
erythrocytes due to acute hypobaric hypoxia
Gloria
Celedón1,
Gustavo
González2,
Carlos P.
Sotomayor2, and
Claus
Behn3
1 Departamento de
Fisiología, Facultad de Ciencias, Universidad de
Valparaíso, Valparaíso;
2 Instituto de Química,
Facultad de Ciencias Básicas y Matemáticas, Universidad
Católica de Valparaíso, Valparaíso; and
3 Departamento de
Fisiología y Biofísica, Facultad de Medicina,
Universidad de Chile, Santiago 1, Chile
 |
ABSTRACT |
Because it has been reported that hypoxia in rats may promote
lipid peroxidation and other free radical reactions that could modify
membrane lipids and proteins, the effect of acute hypobaric hypoxia on
human erythrocyte membranes was investigated. 12-(1-Pyrene)dodecanoic acid fluorescent probe was used to assess short-range lateral diffusion
status in the membrane bilayer. Membrane protein modification was
detected by SDS-PAGE. Healthy young men were exposed for 20 min to the
hypobaric hypoxia, simulating an altitude of 4,500 m. Under this
condition, erythrocyte membrane lipids reached a state of higher
lateral diffusivity with respect to normobaric conditions and membrane
band 3 protein was modified, becoming more susceptible to
membrane-bound proteinases. These observations suggest that acute
hypobaric hypoxia may promote an oxidative stress condition in
the erythrocyte membrane.
hypobaric pressure; membrane proteinases; membrane lipid dynamics
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INTRODUCTION |
IT IS WELL KNOWN THAT oxidative stress results in
damage to cells, which may lead to the loss of cell function. It has
been suggested that biochemical changes taking place during acute
hypoxia may make cells particularly susceptible to oxidative injury (5, 10). Thus increased levels of lipid peroxides were reported in brain,
aorta, and serum (12, 18) of rats subjected to acute or short-term
hypoxia. In the absence of oxygen as an electron acceptor, cell
components become more reduced, and, after reoxygenation, they may
donate electrons directly to oxygen or to low-molecular-weight mediators, initiating free radical processes (10). Thus hypoxia may
promote free radical reactions by activating oxygen to reactive species
(5). The objective of the present study is to evaluate the effects of
acute hypobaric hypoxia on human erythrocyte membranes in terms of
membrane bilayer dynamics and susceptibility of the membrane proteins
to degradation.
 |
MATERIALS AND METHODS |
Materials.
12-(1-Pyrene)dodecanoic acid (PDA) was obtained from Molecular Probes.
Phenylmethylsulfonyl fluoride (PMSF) and Coomassie brilliant blue R 250 (CBB) were obtained from Sigma. Other chemicals were from standard
commercial sources.
Blood samples and erythrocyte membrane preparation.
Three healthy male volunteers aged between 19 and 23 yr were placed in
a low-barometric-pressure chamber (model HHB 01-20, INDURA) in Santiago
(580 m altitude). After 15 min, the chamber pressure was 433 mmHg
(4,500 m altitude). Exposure time to this pressure condition was 20 min, and chamber recompression was carried out in 15 min. Blood samples
were obtained before volunteers entered the chamber (normobaric
condition) and 10 min after recompression (posthypobaric condition) by
venipuncture in the presence of heparin. The subjects fasted 12 h
before the study and gave their informed consent to participate.
Erythrocytes were separated by centrifugation at 3,000 g for 10 min and, after three washings
with PBS (pH 7.4), were lysed for membrane isolation according to the
method of Dodge et al. (6). Membrane protein was determined by the
method described by Peterson (15).
Membrane bilayer dynamics.
The pyrene-derivative probe PDA, incorporated in lipid bilayers in the
micromolar analytical concentration range, produces, after appropriate
illumination, intermolecularly excited dimers (excimers) through a
diffusion-controlled reaction between an excited molecule and one in
the ground state. Pyrene excimers emit a structureless
red-shifted band with respect to the monomer fluorescence, the
excimer-to-monomer fluorescence quantum yield ratio and hence the
corresponding excimer-to-monomer fluorescence intensity ratio
(Iexcim/Imon)
being proportional to the excimer formation constant. This constant
gives information on the immediate probe environment in the sense that
it is a measure of the microscopic diffusion of membrane lipid
components (2). PDA excimer formation, measured by
Iexcim/Imon,
is used in this work to obtain information regarding short-range
lateral diffusion in the membrane bilayer.
PDA was incorporated at 5 µmol/l by addition of small aliquots
(<0.5% total volume) of concentrated solutions of the probe in DMSO
to membranes suspended in PBS (pH 7.4; protein concentration 0.25 mg/ml) and incubated at 37°C for 60 min. Fluorescence spectra were
obtained with a Fluorolog photon-counting spectrofluorometer from Spex,
interfaced to a personal computer, employing ISS software for data
acquisition. Membrane suspension spectra were recorded at 37°C
using square quartz cuvettes with a 5-mm path length. Fluorescence
intensities were evaluated at 374 nm for maximum monomer emission
(Imon) and at 480 nm for maximum
excimer emission (Iexcim), the
excitation wavelengths being 344 nm for direct excitation and 289 nm
for excitation through resonance energy transfer from membrane
proteins. Lipid phase state next to proteins can be monitored by
excitation at 289 nm, since only probe molecules in close proximity to
the protein surface will be excited, due to the rapid decrease of
energy transfer efficiency with distance. Control suspensions without
probe were used to correct for background light scattering.
Membrane protein susceptibility to degradation.
Membrane protein profiles were evaluated by SDS-PAGE in the
discontinuous buffer system of Laemmli (11), with a 7.5% separating gel and a 3.5% stacking gel under reducing conditions. Protein bands
were visualized by staining with CBB. Densitometric profiles of stained
SDS-PAGE gels were obtained with a Genius scanner and then analyzed as
described in Ref. 3. Protein bands were quantitated in terms of
percentage of total proteins present in the profile. Membrane
electrophoresis experiments for each subject were performed in
duplicate. Less than 1% difference between them was found for spectrin
and band 3 protein bands. Membrane protein degradation was assessed by
incubation of isolated membranes resuspended in PBS (pH 7.4; 1.67 mg
protein/ml) at 37°C for 6 h before SDS-PAGE.
 |
RESULTS AND DISCUSSION |
Iexcim/Imon
of PDA incorporated into isolated red blood cell membranes of
individuals before and after exposure to 4,500-m simulated altitude
showed significant increases (~30%) of values in the posthypobaric
condition over those in the normobaric condition (Table
1). These results indicate that probe
lateral diffusion in the membrane plane was enhanced when the
individuals were exposed to the simulated altitude, indicating that in
the posthypobaric condition the membrane lipids are in a state of
significantly higher lateral diffusivity both in the bilayer body and
in the region near proteins. Similar changes in lateral diffusivity of lipids were reported in studies by Galla and Luisetti (9) on temperature dependence of the excimer-to-monomer ratio of pyrene decanoic acid incorporated into human erythrocyte membranes. They found
a similar 30% increase in the excimer-to-monomer ratio when temperature was increased from 37 to 43°C.
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Table 1.
Effect of posthypobaric condition on PDA lateral diffusion in
erythrocyte membranes measured through formation of PDA excimers
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Erythrocyte membranes of individuals exposed to 4,500-m simulated
altitude (posthypobaric condition) were subjected to SDS-PAGE. Protein
bands appeared to be unchanged when visualized by CBB staining (data
not shown) compared with membranes of individuals under normobaric
conditions. However, when membranes of individuals subjected to
hypobaric conditions were incubated at 37°C for 6 h and subjected
to SDS-PAGE, CBB staining of the gel showed that band 3 protein was
decreased in comparison with that from membranes collected before
individuals entered the hypobaric chamber (normobaric condition; Fig.
1). Analysis of band 3 protein
in relation to all membrane proteins showed that 6 h of incubation at
37°C decreased its amount, and it is also apparent that band 3 protein is more susceptible to degradation in the posthypobaric
condition (Table 2). Under the previously
described conditions, only some of the subjects showed spectrin
decrements and the appearance of high-molecular-mass proteins (>200
kDa).

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Fig. 1.
Densitometric scan of erythrocyte membrane protein gel electrophoresis.
A: posthypobaric condition membranes
with no incubation. B: posthypobaric
condition membranes with 6 h of incubation at 37°C.
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Table 2.
Effect of posthypobaric condition and proteinase inhibitors on amount
of band 3 protein after 6-h incubation at 37°C
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Proteinase inhibitors partially protected band 3 protein, indicating
that membrane proteinases are involved in band 3 degradation (Table 2).
This is in accordance with recent studies demonstrating that cells have
lipolytic, DNA repair, and proteolytic systems that prevent the
formation or accumulation of oxidatively damaged phospholipids, DNA,
and proteins (4). The presence of proteinases in erythrocyte and
reticulocyte extracts (8, 16) that are specific to the oxidatively
damaged intracellular proteins has also been demonstrated. Involvement
of membrane-bound proteinases in the degradation of oxidatively damaged
band 3 protein has been described (1, 3). It can also be observed that
protection by proteinase inhibitors is not complete (Table 2). This is
probably due to the existence of membrane proteinases not susceptible
to PMSF and EDTA or to the possibility of having nonproteolytic band 3 fragmentation due to cell processes triggered by the hypobaric exposure, leading, for example, to increased lipid peroxides. This
increase has been reported for rat serum and cells that have been
subjected to acute hypobaric hypoxia (12, 18).
To our knowledge, this is the first report of damage to an erythrocyte
membrane protein and modifications of membrane lipid dynamics caused by
exposure to acute hypobaric conditions. Band 3 protein modification was
not evident by direct SDS-PAGE of erythrocyte membranes after the
hypobaric condition but was clearly observed after incubation of the
isolated membranes. However, changes in the lateral diffusivity of
lipids were readily observed after the hypobaric condition. These
changes could be a prelude to more extensive damage such as that
caused by chronic hypoxia, which leads to an increased oxidative stress
as reported in Ref. 13.
Although at present we cannot interpret the increase of lipid lateral
diffusivity in terms of lipoperoxidation, a process known to be
associated with membrane lipid fluidity modification (7, 14), it cannot
be ruled out that alterations can occur simultaneously in membrane
proteins and lipids and that a modified lipid-protein interaction may
promote lipid reorganization. In this regard, it is interesting to note
the observed increase in PDA excimer formation in regions near proteins
(Table 1). In erythrocyte membranes, lipoperoxidation and band 3 protein degradation have been described in conditions associated with
oxidative stress promoted by radicals derived from azo compounds (3,
17). Experiments are needed to directly demonstrate that acute
hypobaric hypoxia gives rise to an oxidative stress condition in
erythrocyte membranes.
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ACKNOWLEDGEMENTS |
We thank Carlina Tapia for skillful technical assistance and Dr.
Mario Sandoval, Hospital del Trabajador, Asociación Chilena de Seguridad, for providing facilities to use the
low-barometric-pressure chamber.
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FOOTNOTES |
This work was supported by Fondo Nacional de Ciencia y
Tecnología Grant 1950454.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: G. González, Instituto de
Química, Universidad Católica de Valparaíso,
Valparaíso, Chile.
Received 20 March 1998; accepted in final form 7 August 1998.
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