Cell Biology International (2005) 29, 731-736 - Peter Bogner and others - Osmotic and diffusive properties of intracellular water in camel erythrocytes: Effect of hemoglobin crowdedness

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Cell Biology International (2005) 29, 731–736 (Printed in Great Britain)

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Osmotic and diffusive properties of intracellular water in camel erythrocytes: Effect of hemoglobin crowdedness

Peter Bogner^a^*, Attila Miseta^b, Zoltan Berente^c, Attila Schwarcz^d, Gyula Kotek^a and Imre Repa^a

^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

Abstract

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.

1 Introduction

Mammalian erythrocytes show remarkable variation in their volume and water content despite similar (&007E;300mOsm) plasma osmolarity. Furthermore, the osmotic resistance of mammalian erythrocytes varies widely, and erythrocytes of camel and camelids are extremely resistant to osmotic challenge (Perk, 1963; Yagil et al., 1974; Bogner et al., 1998). Previously, we showed that camel and llama hemoglobins contain more hydrophilic amino acids when compared to the erythrocytes of other mammalian species (Bogner et al., 1998). Although, both the negatively and positively charged amino acids are present in higher numbers, the net result is higher isoelectric point (pI) for the camel hemoglobin. The high pI of camel hemoglobin results in a lower sodium and potassium content of these cells (according to the Donnan equilibrium) that causes decreased water content as well. The extreme resistance of camel and camelid erythrocytes to osmotic change seems to be the consequence of the presence of a large osmotically non-responsive water fraction, rather than the changes associated with increased plasma membrane's water permeability (Bogner et al., 1998, 2002).

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 100IU Li-heparin/ml blood, samples were shifted to wet-ice and used for experimental analysis within 24h.

Heparinized blood samples were pelleted with a Sorvall RC-5 centrifuge at 3000×g for 10min at 4°C, after which the plasma and buffy coats were carefully removed. Erythrocytes were washed 3 times with 10mM phosphate buffered saline (PBS; pH 7.4). Finally, the cells were transferred to Corex glass tubes and centrifuged for 30min at 15,000×g at 4°C for magnetic resonance measurements. It has previously been shown that after such a centrifugation mentioned before the proportion of extracellular space does not contribute significantly to the diffusion measurements (Latour et al., 1994).

2.2 Osmotic studies

Human erythrocytes were incubated in buffered (10mM Tris) isoosmotic mannitol solution for 24h at 4°C. Since this solution contains no monovalent ions, erythrocytes gradually lose Na⁺ and K⁺ ions, which result in the loss of cellular water, and closer hemoglobin packaging. This process, contrary to the application of hypertonic solutions, prevents excess ions leaking into the cell, and furthermore the cell membrane is highly impermeable to mannitol. After 24h incubation in mannitol solution, most human erythrocytes became crenated spheres. Interestingly the sum of Na⁺ and K⁺ ions in the camel erythrocytes is significantly lower than that in human erythrocytes (&007E;128 and 165mM, respectively). At the end of incubation, dehydrated cells were also transferred to Corex tubes and treated similarly to that of intact cells.

Osmotic resistance was determined after incubating intact or dehydrated erythrocytes in mannitol and NaCl solutions (dehydrated and control cells, respectively) of different osmolality for 30min at room temperature. The osmotic resistance of intact camel cells was also measured in NaCl solution of different osmolality. Water content was determined gravimetrically. Equilibrium water content of cells was plotted against the inverse of osmotic pressure (Π). Extrapolation of the linear regression of data gives the amount of osmotically non-responsive water fraction (Cameron et al., 1997).

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 89mm vertical bore magnet of 9.4T (Oxford Instruments Ltd., UK) using a 35mm inner diameter hollow multinuclear microimaging probe with Litz volume coil and built-in actively shielded gradient system up to 350mT/m (Doty Scientific, Inc., Columbia, SC, USA).

After tuning, whole-volume shimming and calibration of the 4.0ms sinc RF pulses as well as receiver gain, diffusion weighted spin-echo imaging experiments were run at 21°C, with b value arrayed (31 elements, from 0 to 17,684s/mm²). The parameters were as follows: repetition time=8000ms, echo time=28.2ms, field of view=20×20mm, slice thickness=3.0mm, acquisition matrix=16×16 and number of excitations=1. It should be noted that, because of the limitations in gradient strength (Gmax=350mT/m), the diffusion weighting gradients were switched on simultaneously along the 3 axes to reach larger b values. In addition, slice thickness was set to 3mm and TR was elevated to improve the signal to noise ratio (SNR) and to minimize saturation. Diffusion gradient pulses (δ=8.5ms, Δ=15.5ms, gradient strength arrayed from 0 to 300mT/m) were applied along x, y and z axes simultaneously. Images were acquired from horizontal slices.

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°C in water, isopropanol and oil phantoms in order to validate the gradient system. Oil phantom, giving signal even at b=17,000s/mm², showed monoexponential signal decay over the entire b value range (data not shown). Diffusion constants obtained for water (D=2.07±0.02×10⁻³mm²s⁻¹) and isopropanol (D=0.46±0.003×10⁻³mm²s⁻¹) samples are in good agreement with the literature data (Clark and Le Bihan, 2000).

2.5 Data analysis

After completion of the MRI acquisition, all images were reconstructed as 32×32 matrices. Postprocessing (zero filling, 2D Fourier transform and extracting the mean intensities from the whole cross sections) was performed by VNMR 6.1B and Image Browser softwares (Varian, Inc., Palo Alto, CA, USA) on a Sun Ultra 30 workstation (Sun Microsystems, Mountain View, CA, USA). Mean intensities were plotted against b values and the Levenberg–Marquardt non-linear least-squares fitting routine was employed to fit the biexponential function (Marquardt, 1963).

The adequacy of the biexponential fit was examined by Chi-square test. Results of biexponential analysis are expressed as mean±SD, n denotes the sample number. Significant differences among the data were verified with two-tailed Student t test, with p values less than 0.05 considered to indicate a significant difference. All the statistic and fitting procedures were performed by Sigmaplot 5.0 (SPSS Inc Richmond, CA) software package.

3 Results

Human and camel erythrocytes were resuspended in various osmolarity sodium chloride solutions for 30min, and their water content and dry mass were determined as described in “Section 2”. Fig. 1 shows the osmotic response of intact human and camel erythrocytes. The slope is much steeper in case of the human erythrocytes when compared to those of the camel. The y axis intercept values indicate that the camel erythrocytes contain twice as much osmotically non-responsive water when compared to those of the human. Considering the water contents of erythrocytes, the osmotically non-responsive water fraction is &007E;16 and 66% of the total cellular water in human and camel erythrocytes, respectively.

Fig. 1

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 0.33gwater/gdry mass in human hemoglobin solution and in erythrocytes, respectively; 7.56 and 0.68gwater/gdry mass in camel hemoglobin solution and in erythrocytes, respectively).

Fig. 2

Relationship of protein concentration and colloid osmotic pressure. The intercept value indicates the amount of osmotically non-responsive water.

Intact camel erythrocytes contain about 1.02gwater/gdry mass, which is almost exactly half as much as it is in human cells (Table 1). However, incubation in isotonic mannitol solution dehydrates human erythrocytes significantly.

Table 1.

Water content of erythrocytes, diffusion coefficients and volume fractions deduced from biexponential fitting

	g water/g dry mass	ADC_fast, 10⁻⁴ mm² s⁻¹	ADC_slow, 10⁻⁴ mm² s⁻¹	f_slow, %
Intact human erythrocytes	2.08 ± 0.07	3.52 ± 0.20	0.72 ± 0.06	36 ± 2
Dehydrated human erythrocytes	1.11 ± 0.14	3.04 ± 0.25	0.58 ± 0.16	28 ± 8
Intact camel erythrocytes	1.02 ± 0.10	2.90 ± 0.09	0.62 ± 0.06	34 ± 6

Thus, the water content of human erythrocytes decreased to 1.11gwater/gdry mass following 24h mannitol incubation. The osmotic response of dehydrated human erythrocytes is less pronounced when compared to control cells (Fig. 3). The amount of osmotically non-responsive water fraction in the dehydrated human cells increased significantly (0.75gwater/gdry mass).

Fig. 3

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 0–17,684s/mm² in human and camel erythrocytes can be characterized by a biexponential model, the slower component representing about one-third of the cellular water. In intact camel erythrocytes, both the slow and fast diffusing components display low ADCs (p<0.05 and p<0.001, respectively) when compared with normal human erythrocytes. After the dehydration of human erythrocytes their ADC values resemble to that of camel cells, but the ratio of slow diffusing water (fslow) fraction is significantly more (p<0.05) reduced. Despite the 2-fold difference in erythrocyte water content between intact human and camel erythrocytes, the ADC values notably differed by only &007E;15–18%.

The ADC of solvent water was also measured in a 20% solution of camel and human hemoglobins, which gave values of 1.23×10⁻³mm²s⁻¹ and 1.25×10⁻³mm²s⁻¹, respectively.

4 Discussion

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;0.3–0.5gwater/gprotein for a medium sized protein such as human hemoglobin (see also Arosio et al., 2002; Sartor et al., 1995). Unfortunately, no data on camel hemoglobin hydration have been published, but it is known that water-binding is going to be enhanced by its increased hydrophilicity (Zhou, 2001; Jaenicke and Zavodszky, 1990). Theoretically, the osmotic effect of a solute/protein also relates to the water-binding. We demonstrated that the amount of osmotically unresponsive water is higher for camel than it is for human hemoglobin. Considering the quantity of hydration water of hemoglobin (0.3–0.5gwater/gprotein) and osmotically unresponsive water in human erythrocytes (0.33gwater/gprotein) one may conclude that most if not all of hemoglobin hydration water is osmotically unresponsive. Since camel hemoglobin contains significantly higher proportion of charged amino acids, it is conceivable that camel hemoglobin is more hydrated, and in camel erythrocyte too the osmotically unresponsive water roughly equals hydration water. The difference in osmotically unresponsive water between the hemoglobin solution and erythrocyte is explained by the aggregation of protein molecules (Cameron et al., 1988).

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.

Acknowledgements

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 2005

doi:10.1016/j.cellbi.2005.04.008

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