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
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Abstract
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Materials & Methods
Results & Discussion
References

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

    INTRODUCTION
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Materials & Methods
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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
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Materials & Methods
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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
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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

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

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.

    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.

    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.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

1.   Beppu, M., M. Inoue, T. Ishikawa, and K. Kikugawa. Presence of membrane-bound proteinases that preferentially degrade oxidatively damaged erythrocyte membrane proteins as secondary antioxidant defense. Biochim. Biophys. Acta 1196: 81-87, 1994[Medline].

2.   Celedón, G., C. Behn, Y. Montalar, M. Bagnara, and C. P. Sotomayor. Transbilayer asymmetry of pyrene mobility in human spherocytic red cell membranes. Biochim. Biophys. Acta 1104: 243-249, 1992[Medline].

3.   Celedón, G., V. Lips, C. Alvarado, M. Cortés, E. A. Lissi, and G. González. Protein degradation in red cells exposed to 2,2'-azo-bis(amidinopropane) derived radicals. Biochem. Mol. Biol. Int. 43: 1121-1127, 1997[Medline].

4.   Davies, K. J. A. Intracellular proteolytic systems may function as secondary antioxidant defenses: an hypothesis. Free Radic. Biol. Med. 2: 155-173, 1986.

5.   Degroot, H., and A. Littauer. Hypoxia, reactive oxygen and cell injury. Free Radic. Biol. Med. 6: 541-551, 1989[Medline].

6.   Dodge, J. T., C. Mitchell, and D. J. Hanahan. The preparation and chemical characterization of hemoglobin-free ghosts of human erythrocytes. Arch. Biochem. Biophys. 100: 119-130, 1963.

7.   Eichenberger, K., P. Böhni, K. Winterhalter, S. Kawatoy, and C. Richter. Microsomal lipid peroxidation causes an increase in the order of the membrane lipid domain. FEBS Lett. 142: 59-62, 1982[Medline].

8.   Fagan, J. M., and L. Waxman. Purification of a protease in red blood cells that degrades oxidatively damaged hemoglobin. Biochem. J. 277: 779-786, 1991[Medline].

9.   Galla, H. J., and J. Luisetti. Lateral and transversal diffusion and phase transitions in erythrocyte membranes. An excimer fluorescence study. Biochim. Biophys. Acta 596: 108-117, 1980[Medline].

10.   Jones, D. P. The role of oxygen concentration in oxidative stress: hypoxic and hyperoxic models. In: Oxidative Stress. London: Academic, 1985, p. 151-195.

11.   Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

12.   Minyailenko, T. D., V. P. Pozharov, and M. M. Seredenko. Severe hypoxia activates lipid peroxidation in the rat brain. Chem. Phys. Lipids 55: 25-28, 1990[Medline].

13.   Nakanishi, K., F. Tajima, A. Nakamura, S. Yagura, T. Ookawara, H. Yamashita, K. Susuki, N. Taniguchi, and H. J. Ohno. Effects of hypobaric hypoxia on antioxidant enzymes in rats. J. Physiol. (Lond.) 489: 869-876, 1995[Abstract].

14.   Ohyashiki, T., T. Ohtsukay, and T. Mohri. A change in the lipid fluidity of the porcine intestinal brush-border membranes by lipid peroxidation. Studies using pyrene and fluorescent stearic acid derivatives. Biochim. Biophys. Acta 861: 311-318, 1986[Medline].

15.   Peterson, G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83: 346-356, 1977[Medline].

16.   Sacchetta, P., P. Battista, S. Santarone, and D. D. Cola. Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family. Biochim. Biophys. Acta 1037: 337-343, 1990[Medline].

17.   Sato, Y., S. Kano, T. Takahashi, and Y. Susuki. Mechanism of free radical-induced hemolysis of human erythrocytes: hemolysis by water-soluble radical initiator. Biochemistry 34: 8940-8949, 1995[Medline].

18.   Yoshikawa, T., Y. Furukawa, S. Wakamatsu, H. Takemura, H. Tanaka, and M. Kondo. Experimental hypoxia and lipid peroxide in rats. Biochem. Med. 27: 207-213, 1982[Medline].


Am J Physiol Cell Physiol 275(6):C1429-C1431
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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