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Cell Biology International (2005) 29, 731736 (Printed in Great Britain)
Osmotic and diffusive properties of intracellular water in camel erythrocytes: Effect of hemoglobin crowdedness
Peter Bognera*, Attila Misetab, Zoltan Berentec, Attila Schwarczd, Gyula Koteka and Imre Repaa
aInstitute of Diagnostic Imaging and Radiation Oncology, University of Kaposvár, 7400 Kaposvár, Guba S.u. 40, Hungary
bDepartment of Clinical Chemistry, University of Pécs, Faculty of Medicine, Hungary
cDepartment of Biochemistry and Medical Chemistry, University of Pécs, Faculty of Medicine, Hungary
dDepartment of Neurosurgery, University of Pécs, Faculty of Medicine, Hungary
Camel erythrocytes have exceptional osmotic resistance and is believed to be due to augmented water-binding associated with the high hydrophilicity of camel hemoglobin. In practical terms this means that the proportion of osmotically non-removable water in camel erythrocytes is nearly 3-fold greater than that in human erythrocytes (&007E;65 vs &007E;20%). The relationship between water diffusion and the osmotic characteristics of intracellular water is the subject of this report.
The amount of osmotically inactive water is 2-fold greater in camel hemoglobin solution in vitro compared to that of human, but water diffusion does not differ in camel and human hemoglobin solutions. However, the evaluation of water diffusion by magnetic resonance measurements in camel erythrocytes revealed &007E;15% lower apparent diffusion coefficient (ADC) compared with human erythrocytes. When human erythrocytes were dehydrated to the level of camel erythrocytes, their osmotic and water diffusion properties were similar. These results show that a lower ADC is associated with a more pronounced increase in osmotically inactive water fraction. It is proposed that increased hemoglobin hydrophilicity allows not only augmented water-binding, but also a closer hemoglobin packaging in vivo, which in turn is associated with slower ADC and increased osmotic resistance.
Keywords: Diffusion, Hydration water, Hemoglobin, Osmotic.
*Corresponding author. Tel.: +36 30 9949 907; fax: +36 82 502 020.
Mammalian erythrocytes show remarkable variation in their volume and water content despite similar (&007E;300
Cameron et al. (1997) proposed that the reduced water motion in cells might correlate with the extent of osmotically non-responsive water associated with proteins. Protein–water interaction is a key factor in the restriction of water diffusion within the cell, but it is difficult to study directly because of the fast exchange between the different pools (Waldeck et al., 1997). It is conceivable that both the amount of hydration water and its interaction with the protein surface would influence the apparent diffusion as well as the osmotic behavior of intracellular water. We demonstrated that &007E;20% of intracellular water is directly related to intracellular proteins in terms of osmosis in human erythrocytes (Bogner et al., 2002). It is interesting to note that osmotically non-responsive brain edema develops in certain cases of trauma and ischemia, and water's ADC is frequently found to be reduced by diffusion imaging (Horn et al., 1999; Takayama et al., 2000; Liu et al., 2001).
Because of their general structural and functional similarities, mammalian erythrocytes offer a model in which the diffusion of intracellular water of different osmotic behavior can be studied independently. It is also possible to test whether ADC reflects the osmotic behavior of intracellular water. Since hemoglobin in the camel erythrocytes is less hydrated in vivo, human erythrocytes were dehydrated to clarify the effect of protein hydration per se on intracellular water diffusion.
2 Materials and methods
2.1 Blood samples
Human blood samples were obtained from the cubital vein of young healthy volunteers. Camel blood was drawn from the external jugular vein of the animals in the Budapest Zoo. After the blood was drawn into test tubes containing 100
Heparinized blood samples were pelleted with a Sorvall RC-5 centrifuge at 3000
2.2 Osmotic studies
Human erythrocytes were incubated in buffered (10
Osmotic resistance was determined after incubating intact or dehydrated erythrocytes in mannitol and NaCl solutions (dehydrated and control cells, respectively) of different osmolality for 30
Hemoglobin was isolated after the hypotonic lysis of erythrocytes (Dodge et al., 1962). Ghost-free hemoglobin solutions were lyophilized overnight and hemoglobin solutions were prepared after dissolving lyophilized protein in isotonic buffered NaCl solution.
The colloid osmotic pressures of hemoglobin solutions were measured with a Knauer colloid osmometer (Knauer GmbH, Berlin, Germany). The inverse of concentration was plotted against the inverse of osmotic pressure and the intercept gives the extent of osmotically non-responsive water (Cameron et al., 1997).
Each measurement was repeated >5 times and one characteristic result is shown.
2.3 MR studies
All MR measurements were performed on a Varian UNITYINOVA 400 spectrometer (Varian, Inc., Palo Alto, CA, USA) with a 89
After tuning, whole-volume shimming and calibration of the 4.0
Diffusion anisotropy in red blood cells might be a concern measuring in one direction only, but diffusion anisotropy was proven only in cell suspensions and we and others (P. W. Kuchel, personal communication) could not confirm the existance of diffusion anisotropy in red blood cells after centrifugation. Centrifugation deforms cell shape and vast majority of cells resemble spheres with a flat, triangular tail (Corry & Meiselman, 1978).
2.4 Phantom study
Diffusion measurements were also carried out at 21
2.5 Data analysis
After completion of the MRI acquisition, all images were reconstructed as 32
The adequacy of the biexponential fit was examined by Chi-square test. Results of biexponential analysis are expressed as mean
Human and camel erythrocytes were resuspended in various osmolarity sodium chloride solutions for 30
Osmotic behavior of camel and human erythrocytes incubated in NaCl solutions of various osmolalities. The calculated y axis intercept indicates the osmotically non-responsive water fractions.
To examine the influence of hemoglobin on solvent water properties directly, the colloid osmotic pressures of camel and human hemoglobin solutions (2–10%) were measured (Fig. 2). The intercept values indicate the amounts of osmotically non-responsive water fractions that are significantly higher in the camel compared to human hemoglobin solutions. The absolute values of osmotically non-responsive water in hemoglobin solutions are about an order of magnitude higher than in intact cells (3.57 and 0.33
Relationship of protein concentration and colloid osmotic pressure. The intercept value indicates the amount of osmotically non-responsive water.
Intact camel erythrocytes contain about 1.02
Water content of erythrocytes, diffusion coefficients and volume fractions deduced from biexponential fitting
Thus, the water content of human erythrocytes decreased to 1.11
Osmotic behavior of control and dehydrated and human erythrocytes. Control erythrocytes were incubated in NaCl, and dehydrated erythrocytes in mannitol solutions of various osmolality. The calculated y axis intercept indicates the osmotically non-responsive water fractions.
The diffusion signal decay in the b value range of 0–17,684
The ADC of solvent water was also measured in a 20% solution of camel and human hemoglobins, which gave values of 1.23
The diffusion coefficient for water in biological systems is reduced by a factor of 2–10 compared to bulk water (Tanner, 1983; Hazlewood et al., 1991). Explanations proposed to account for this reduction are: (1) membrane compartmentalization of cellular water; (2) macromolecular structures within cellular compartments serving as local obstructions to diffusion; and (3) interaction of water and macromolecules that affects a substantial fraction of intracellular water. Certainly all the three factors have an effect in living cells but the proportional contribution of the above mentioned factors in intracellular water ADC is hard to define. Camel erythrocytes – in comparison with human erythrocytes – offer a unique model for studying: (1) the influence of water–protein interaction on water diffusion modified by the differing hydrophilicity of human and camel hemoglobin; and (2) the effect of protein hydration/crowdedness on water diffusion in camel and dehydrated human erythrocytes.
Previously we had also examined the cell membrane properties – such as microviscosity, monovalent ion transport and aquaporin 1 water channel protein – of the erythrocytes that can modify the membrane compartmentalization of intracellular water as well as the osmotic behavior of cells (Bogner et al., 2002). Our results did not uncover interspecies differences in membrane properties that explain the unusual osmotic resistance of camel erythrocytes (Bogner et al., 2002). The relationship of erythrocyte water diffusion permeability with membrane characteristics was also studied by Benga and Borza (1995), who found that membrane phosphatidylcholine and sphingomyelin contents were directly related to water diffusion permeability. These compositional features are also related to microviscosity and to passive ion transport. Having studied these parameters, water diffusion permeability does not seem to be significantly different in human and camel erythrocytes (Bogner et al., 2002).
Another aspect of diffusion reduction by the cell membrane in human and camel erythrocytes could be the shape difference, since camel erythrocytes are ellipsoid, wafer-like and human erythrocytes are well-known for their biconcave disc shape (Jain and Keeton, 1974). A relationship between erythrocyte shape and NMR diffusion–diffraction data was found in either osmotically swollen and shrunken cells or in erythrocytes that were treated with NaF (Torres et al., 1998). Although volume change was not measured in the above mentioned experiments, due to ATP depletion caused by the NaF poisoning, ion-pumps stop that concludes in significant Na influx with the consequent cell swelling due to ATP depletion caused by the NaF poisoning (Alberts et al., 2002). So, in these experiments changes in shape were induced along with the change in cell volume/protein hydration and the alteration of macromolecular structures cannot be excluded as well. Clarification of the role of shape itself will need a more elaborate experimental model.
Since 95% of erythrocyte protein is hemoglobin, most water–protein interactions are governed by hemoglobin hydration. Zhou (2001) estimated the hydration water at &007E;0.3–0.5
It is worth of note, that the amount of osmotically unresponsive water in these experiments was significantly lower (&007E;half) than previously described (Cameron et al., 1988; Bogner et al., 1998, 2002). This peculiarity can be explained by the use of the more extensive centrifugation that was performed in order to minimize the effect of extracellular space which in turn reduces also cell water due to increased hydrostatic pressure during centrifugation. This effect is more evident in cells incubated in higher osmolality medium (data not shown).
Translational water diffusion in the human hemoglobin hydration shell, determined by means of dielectric permittivity measurements and nuclear magnetic resonance spectroscopy, proved to be about one-fifth of the corresponding value of bulk water (Steinhoff et al., 1993). Hydration water in human erythrocytes represents &007E;20% of total water content, yet water ADC in these cells is about 10 times slower than in bulk water (Tanner, 1983; Cameron et al., 1988). With the assumption that osmotically unresponsive water in camel erythrocytes equals hydration water, about 60–70% of the cellular water content would be hydration water and water ADC in these cells is about 15% less compared to human erythrocytes i.e. 11–12 times slower than in bulk water. Based on these data, it seems that the amount of hydration water has a limited impact on cellular water ADC in erythrocytes but significantly influences the osmotic response. This is supported by the recent view on hydration water that the residence time of water molecule exists in a preferred hydration site which has narrow time limits, but these particular sites are almost permanently occupied (Zhou, 2001).
Taking into account the diffusion characteristics of hydration water and cellular water content, it is conceivable that influence of molecular obstruction on water diffusion in the cell can be significant. Obstructive effect is dependent on the molecular species of the obstructor on one hand (Garcia-Perez et al., 1999; Hortelano et al., 2001), and on the other hand it seems that the crowdedness of macromolecules is also important (Garcia-Perez et al., 1999; Bon et al., 2002). Recently, Bon et al. (2002) showed that water molecules further away from the surface of the protein in a second hydration layer can have a reduced self-diffusion coefficient approximately by 50-fold compared with bulk solvent. Water molecules in human erythrocytes dehydrated to the level of camel erythrocytes possess similar diffusion constants to that of measured in camel erythrocytes. It is possible that with the dehydration process – and a simultaneous loss of monovalent ions – some protein charges became available to water molecules that would explain the significant increase of osmotically unresponsive water in dehydrated human erythrocytes. Nevertheless, with the same water–protein ratio a similar hemoglobin packing/protein crowdedness could be established in dehydrated human and normal camel erythrocytes that leads to similar ADC values. It is also known that hemoglobin molecules can form aggregates in vitro and in vivo that create the molecular basis of protein crowding (Steinhoff et al., 1993; Arosio et al., 2002). Protein assemblies in general and hemoglobin aggregation are mediated primarily by polar interactions (Elbaum et al., 1976; Takahashi, 1997). Thus, it is proposed that the increased hydrophilicity of camel hemoglobin substantially influences protein aggregation/crowdedness that reduces water ADC in vivo. A secondary effect of increased hydrophilicity is connected with protein hydration that changes the osmotic behavior of water both in vitro and in vivo.
The sponsorship of Hungarian National Science Foundation (OTKA T034200, T032043) is acknowledged. The continuous support of Professors Miklós Kellermayer and Balázs Sümegi is acknowledged. The authors thank the helpful comments of Denys N. Wheatley, Ivan L. Cameron and James C. Clegg. Z.B. is grateful to Hungarian Academy of Sciences for Bolyai János Scholarship (BO/00166/01).
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Received 17 April 2005/18 April 2005; accepted 18 April 2005doi:10.1016/j.cellbi.2005.04.008