Ca2+ transport and permeability in inside-out red cell membrane vesicles after freezing

CRYOBIOLOGY23, 134-140 (1986)

Ca*+ Transport

A. RUBINACCI,

and Permeability in Inside-Out Vesicles after Freezing B. FULLER,*

E WUYTACK,t

AND

Red Cell Membrane

W. DE’LOECKER

Afdelingen Biochemie en TFysiologie, Departement Humane Biologie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, B-3000 Leuven, Belgium, and *Academic Department of Surgery, Royal Free Hospital School of Medicine, University of London, London NW.? 2QG, England To evaluate the effects of freezing and thawing on Ca*+ transport and permeability, inside-out red cell membrane vesicles (IORCMV) are examined. Exposure to the cryoprotectant Me,SO as well as different cooling regimes on unprotected and cryoprotected vesicles do not affect the membrane Ca?’ transport. However, freezing and thawing increase the membrane permeability to sucrose. o 19x6 AcademicPress,Inc

It has been suggested that cell membrane damage is an early event in the destruction of cells during freezing (6, 12). The pathology of freeze-induced damage in cells is demonstrated by leakage of intracellular solutes and proteins into extracellular spaces, uptake of high molecular weight dyes (e.g., trypan blue), and, in the most severe form, by lysis (15). However, it has been suggested that, as opposed to gross membrane disruption, freezing causes subtle changes in membrane function which then precipitate a cascade of events leading to the more generally observed pathological phenomena (8, 16). To be able to separate primary and secondary events, model systems of cell membrane freezing damage have been sought. In particular, liposomes, derived entirely from synthetic materials or from materials extracted from biological tissues, have been studied during freezing (17, 23). These have provided much useful information although it is difficult to extrapolate these observations to cellular systems. We have chosen to investigate the effects of freezing on calcium transport in inside-out red cell membrane vesicles Received May 20, 1985; accepted August 26, 1985.

(IORCMV) for two complementary reasons: (a) the ability to transport calcium across the membrane requires functional maintenance of the enzyme unit (including specific binding sites for calcium and ATP), its relationship with the bilayer structure, and normal low permeability of the bilayer to passive diffusion of the divalent cation (4); (b) the IORCMV is a good model system because, unlike intact cells, the vesicles do not require preloading with calcium and they are of simple anatomy. The choice of IORCMV in no way supposes any difference in expression of damage to the cytoplasmic (inner) face of the bilayer, when compared with the outer face during freezing. However, IORCMV provide an excellent model for direct spectroscopic examination of membrane transport after these stresses, allowing the extrapolation of the experimental data of the damaging effects of particular freezing regimes to intact erythrocytes (11). In addition, changes in membrane permeability during the freezing process are investigated by monitoring leakage of labeled sucrose out of preloaded IORCMV. The effects of two different freezing regimes and the addition of cryoprotectant dimethylsulfoxide are assessed. 134

001 l-2240186 $3 .OO Copyright0 1986by AcademicPress,Inc. All rightsof reproductionin any form reserved.

Ca2 + TRANSPORT

AFTER

FREEZING

135

MO.; and 0.5 vol of 1 mM arsenazo III Na salt (Sigma Co.). To perform the measurements 200+.1 aliquots of IORCMV resusInside-out red cell membrane vesicles (IORCMV) from human packed red cells pended in vesiculation medium were mixed were prepared by the method according to with 450 ~1 of incubation medium and 350 Steck et al. (25). One hundred ml of packed ~1 of distilled water to make up I ml and were maintained at 37°C. red cells were washed 3 times with 5 vol of the cuvettes Na phosphate buffered saline, pH 8.0 [I50 Readings were obtained in a dual wavemM NaCl; 5 mM NaH,PO, (Merck, Darm- length (DW 2a UV/VIS) spectrophotometer stadt, Germany)], followed by centrifuga- (American Instrument Co., Silver Spring, tion at 2300g for 10 min (Sorvall RC-2, Nor- Md.) at 685-675 nm. After stabilization of walk, Conn.). The washed cells were lysed the baseline reading, 10 p.1of 1 mM calcium by rapid dilution with 40 vol of 5 mM Na chloride (Merck) was added, and the opphosphate buffer, pH 8.0, and the ghost tical density recorded. A second 10 p.1of 1 membranes were pelleted at 22,000g for 10 mM calcium chloride was added to check min. To initiate vesiculation the ghosts were sensitivity of the reading, followed by the diluted into 40 vol of 0.5 mM Na phosphate addition of 10 pJ of 50 mM ATP (disodium buffer, pH 8.0, and stored overnight on ice. salt, Sigma Co.) to initiate active calcium The IORCMV were recovered by centrif- uptake. The rate of uptake was expressed ugation at 28,000g for 30 min and diluted in terms of the total protein content of 1:1 in 0.5 mM phosphate buffer. The whole IORCMV (13). For freezing studies two conditions were procedure was carried out at 4°C. To complete vesiculation the IORCMV were considered (Fig. 1). Under the first condition, “unprotected IORCMV,” 1 ml of vespassed through a 27-gauge hypodermic needle. The separation of IORCMV from icles suspended in 0.5 mM Na phosphate resealed ghosts by dextran gradient centrif- buffer was cooled in polypropylene cryougation was omitted because of the risk of tubes (Nunc 38 x 12.5 mm, Gibco additional damage to cell membrane en- Europe, Gent, Belgium). Under the seczyme activity and because of the relative ond one, “dimethylsulfoxide protected reproducibility of the percentage (30-50%) IORCMV,” vesicles suspended in 0.5 mM of IORCMV, the remaining being right- Na phosphate buffer were exposed to the side-out vesicles which do not contribute to cryoprotectant Me$O (Merck) at a final calcium transport measurements (1, 26). concentration of 1.5 M for 10 min on ice. Active calcium uptake was measured by The tubes were then cooled in alcohol baths means of a metallochrome indicator, ar- to their respective temperatures of ice cryssenazo III. Under our conditions arsenazo tallization ( - 2°C for unprotected and - 7°C III has been selected as the indicator of for Me,SO-protected vesicles), nucleation choice because of its high calcium affinity was induced by clamping each tube with in the range 5-20 ~.LMof ionized calcium in forceps precooled in liquid nitrogen, and 10 solution (22). Calcium transport was regis- min were allowed for dissipation of the latered by changes in the optical density of tent heat of ice crystallization. Samples the medium, which, in turn, is a function of from both unprotected and Me,SO-proionized calcium concentration. The incu- tected vesicles were then cooled to - 196°C bation medium contained 1 vol of 10 mM by two different regimes. In the fast MgCI, (Merck); 1 vol of 50 mM NaN, (BDH freezing regime the tubes were immediately Chemicals Ltd., Poole, GB), 1 vol of 1 M submerged in liquid nitrogen (cooling rate KC1 (Merck); 1 vol of 300 mM imidazoleapproximately -200”Cmin). In the slow HCl buffer (pH 6.8) (Sigma Co., St. Louis, freezing regime, tubes were cooled at 1°C MATERIALS

AND

METHODS

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RUBINACCI

ET AL.

Freshly-prepared

IORCW Condition

Ice

2

4

control C-(unprotected)

J

Fast freezing regime

Additional vesicubtion procedure

1

\

Slow free2 regime

Additional vesiculation procedure

.Addltional vesiculation

! C

n

[i

Addltibnal vesiculatian procedure

procedure

r

1 I;

J II

1. Schematic representation of the experimental protocol to obtain the various control and experimental groups of inside-out red cell membrane vesicles (see Materials and Methods). FIG.

min in a controlled refrigerated alcohol bath (Fryka-Therm, FT800, Kaletechnic, Denmark) to - 35°C before transfer to liquid nitrogen. All tubes were rapidly thawed in a water bath at 37°C. For both conditions, before calcium uptake measurements, the samples underwent a further washing step with the vesiculation medium and the vesicles were passed again through the hypodermic needle. To assess the experimental effect of this additional step, another two groups were added in which thawed unprotected IORCMV were used for calcium uptake measurements without undergoing this additional revesiculation procedure. For controls an unprotected and a Me,SO-protected group were kept on ice without freezing. To measure permeability changes during freezing, samples of IORCMV were prepared in which 0.2 p.Ci of [U-r4C] sucrose (sp radioactivity >350 mCi/mmol; The Radiochemical Centre, Amersham Bucks., UK) was added to 1 ml of the vesiculation medium. To remove untrapped radioactivity the IORCMV, containing the permeability marker, were washed twice in 0.5 mM Na phosphate buffer, centrifuged at 28,000g for 30 min, and the pellet was resuspended in an equal volume of 0.5 mM Na phosphate buffer. The IORCMV containing [ “C]-sucrose were frozen slowly

(unprotected) as described above, and subsequently thawed. The control samples were processed identically and were kept on ice. Aliquots of 1 ml of ice-maintained and frozen-thawed IORCMV were further diluted in 4 ml of 0.5 mM Na phosphate buffer and centrifuged at 28,0001: for 30 min. The supernatants were removed for counting and the pellets of IORCMV were resuspended in an equal volume of 0.5 mM Na phosphate buffer. Samples of 200 p,l from the resuspended IORCMV and from the supernatants were dissolved in 2 ml of Solulyte (J. T. Baker Chemicals, Deventer, Holland) by heating overnight in an oven at SO”C, and after adding 15 ml of Lipofluor scintillation fluid (J. T. Baker Chemicals) were counted in a liquid scintillation spectrometer (Rack Beta, LKB, Wallace, OY, Turku, Finland). Statistical evaluation of differences between experimental groups were performed using Student’s t test for unpaired data and significance assumed at p values of 0.01 or less (24). RESULTS

Calcium uptake for the eight experimental groups is shown in Table 1. It can be seen that for unprotected IORCMV, cooling by both fast (Group C) and slow

137

Ca2+ TRANSPORT AFTER FREEZING TABLE I Ca*’ Uptake and Sucrose Permeability in Inside-Out Red Cell Membrane Vesicles (IORCMV) MeSO-protected vesicles

Unprotected vesicles Groups

Ca?- uptake”

Groups

Ca?+ uptake

Controls A (n = 28) Cooled fast C (n = 13) Cooled slowly E (n = 18) Cooled fast without additional vesiculation procedure D (n = IO) Cooled slowly without additional vesiculation procedure F (n = I I)

6.25 t 0.71 6.39 i 0.78 4.89 +- 0.50

Controls B (n = I I) Cooled fast C (n = IO) Cooled slowly H (n = 13)

4.06 t 0.64 6.56 2 1.07 5.62 -+ 0.50

5.91 -t 0.50 3.89 k 0.57 Sucrose leakage”

Ice stored (n = 3) Cooled slowly (n = 5)

13.2 -+ 0.5% 29.0 2 3.1%’

<’Ca?- uptake (expressed as means ? SEM in nmoliminimg protein) in groups of IORCMV after different freezing regimes. For explanation of the various groups (A-H) see Fig. I. h Sucrose leakage expressed as percentage ? SEM of the trapped sucrose which appeared in the medium after treatment.

resulted in complete recovery of calcium uptake (N.S. for both groups compared to unprotected controls A). Neither did addition and removal of Me,SO (Group B) change calcium uptake (N.S. compared to A), while cooling fast (Group G) and slowly (Group H) in the presence of Me,SO again resulted in total recovery of calcium uptake. Reformation of IORCMV during the washing procedure after thawing did not affect recovery since omission of this step in unprotected IORCMV cooled fast (Group D) or slowly (Group F) did not change calcium uptake. There was some suggestion that activity was reduced in Group F compared to unprotected controls (Group A), but this did not reach statistical significance. When considering the permeability of IORCMV to sucrose, it was seen that during 2 hr in ice, and harvesting by centrifugation, 13.2% + 0.5 (n = 3) of the 14Clabeled marker appeared in the supernatant. Our results are in agreement with those of Nash and Meiselan (19) who showed that in resealed red cell ghosts there was a slow transmembrane diffusion of sucrose at 0°C. This leakage was greatly (Group

E) regimens

increased to 29.0% 2 3.1 (n = 5) during slow cooling of the vesicles to - 196°C. DISCUSSION

From the results described above, it is evident that calcium uptake by IORCMV is not greatly affected by cooling to - 196”C, in either the absence or presence of the protectant compound Me,SO. Small differences in mean calcium uptakes are seen between individual groups and this may be attributable to differences in uptake activity between preparations of IORCMV from different batches of blood. It has been reported previously that there may be changes in activity of IORCMV from various blood collections, although the mechanisms remain obscure (21). None of the experimental groups (B-H) are statistically different from the controls (Group A), although in the Me$O-exposed control Group B and in the slowly-without cryoprotectant-cooled Group E the trend is for a small reduction in calcium uptake. The exposure to the cryoprotectant Me,SO without freezing does not seem to have any significant effect on the Ca2+ transport mechanism.

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RUBINACCI

These results contrast sharply with the observed effects of freezing in intact erythrocytes. Lovelock (11) demonstrated that complete lysis of red cells occurs after exposure to temperatures below - lO”C, a fact confirmed by others (18). Lysis is total whether slow or fast cooling rates are used. Some small percentage of cells do survive at cooling rates in excess of lo3 “C per minute, but this is much faster than any of the rates used in the present study. Not until protective agents such as glycerol and Me,SO are added does survival improve. Almost complete recovery of intact erythrocytes after freezing at - 196°C is achieved using Me,SO and cooling rates of 200-300 “Urnin, equivalent to our “fast” regime (18). Thus in the present study it is demonstrated that the activity of the calcium-ATPase is unaffected by freezing under conditions which cause membrane breakdown in intact erythrocytes. Here the addition of Me,SO proves irrelevant since recovery of calcium uptake is complete even in its absence. Frozen-thawed IORCMV also show typical permeability to calcium, since the calcium sequestered by the IORCMV remains inside the vesicles without demonstrable “leak back” during the periods of observation. It has been shown previously that changes in membrane permeability to calcium, induced by the ionophore A23187, cause abrupt release of the divalent cation (20). There is some evidence that membrane bound enzyme in other systems are resistant to freeze-thaw damage. Ashwood-Smith (2) demonstrated that rat liver microsomes could be frozen to - 196°C and exhibit activity of the P-450 mixed function oxidase system after thawing without additional benefit after the addition of Me,SO. More recently plasma membrane vesicles isolated from rye protoplasts are shown to retain the activity of the Mg’+ -dependent K+ stimulated ATPase after cooling to - 196°C (7). The only detectable change brought about by freezing in these studies is that of

ET AL.

an alteration in IORCMV permeability to [14C]sucrose during the freeze/thaw process. This is demonstrated by the much greater release of sucrose (which would normally cross cell membranes only slowly) from the preloaded IORCMV than from control IORCMV maintained on ice for several hours. A similar pattern of membrane permeability change is reported by Morris (16) who demonstrated that glucose is released during freezing and thawing of liposomes constructed from lipid extracts of whole erythrocytes. In this system he concludes that the liposome permeability to glucose quickly reverts to control values after thawing. This is particularly interesting since it relates to our observation that IORCMV maintain normal permeability characteristics to calcium after thawing. Other workers investigated release of hemoglobin, reintroduced as a solution into reconstituted erythrocyte membrane vesicles, and reported that in samples cooled below - 15°C there was a considerable hemoglobin release into the extracellular medium after thawing (9). It has been previously shown that isolated liver cells retain the ability to react osmotically after two freezing/thawing steps, even though the cells are metabolically inactive after the first step (8). The concensus of the previous studies, and of our current observations, suggests that during freezing/thawing alterations take place in the permeability characteristics of biomembranes which are potentially reversible. The present work clearly demonstrates that whatever molecular changes take place in the IORCMV membrane during freezing, they revert in an orderly fashion in such a way that transmembrane calcium movement is subsequently normal. How can this be related to the known lytic effect of freezing in intact erythrocytes? It is well understood that as solutions freeze, ice separates as pure water, causing increasing concentration of solutes in the unfrozen liquid. Ice normally crystallizes first

Cal + TRANSPORT AFTER FREEZING

139

in the extracellular medium, resulting in os- permeability (5). However, it is not known motic dehydration of cells during freezing whether this can occur during freezing. We have described experiments which (14). At some point this would appear to change membrane permeability character- suggest that during freezing, cell memistics, which in our model system return to branes undergo changes which in many renormal after removal of the stress. For in- spects are reversible. It would seem likely tact erythrocytes (or other cells) frozen in that in erythrocytes cell death by freezing plasma or tissue culture medium, the results from alterations in the intracellular system is essentially different because the compartment as a consequence of the trancompositions of intracellular and extracel- sient changes in membrane permeability. lular solutions are dissimilar. The changes The gross damage seen as cell lysis could in membrane permeability allow redistribe secondary to these events. bution of concentrated extracellular (essenACKNOWLEDGMENTS tially sodium chloride) and intracellular solThe authors are indebted to the Belgian National utes. Upon thawing the erythrocyte membrane regains, at least partly, normal Foundation for Medical Research (F.G.W.O.) for a permeability, thus “sealing in” the atypical grant to the laboratory, to the Nationale Loterij for a research grant. and to Mrs. F. De Wever, Miss C. solutes at high concentrations, which re- Wittevrongel, and Mrs. C. Brees for their valuable sults in osmotic lysis of the erythrocytes. technical assistance. In the IORCMV, since freezing takes place REFERENCES using the same buffer solution as used for the formation of the vesicles, internal and I. Akyempon, C. K., and Roufogalis, B. D. The stoichiometry of the Ca?’ jumps in human external solutes are identical and thus this erythrocyte vesicles: Modulation by Ca?+, “redistribution-lysis” does not occur. A Mg’- and calmodulin. Cell Calcium 3, l-17 piece of additional evidence for the “redis(1982). tribution” theory is that in mammalian cells 2. Ashwood-Smith. M. J. Stability of microsomal enin which transmembrane pump activities zymes associated with the conversion of carcinogens to bacterial mutagens to freezing and are reduced by cooling to 4”C, there is a thawing. Cpobiology 14, 240-244 (1977). slow exchange of solutes across the mem3. Berthon, B., Claret. M., Mazet, J., and Poggioli, brane resulting in cell swelling, even J. Volume and temperature dependent permewithout the concentrating effect of ice forabilities in isolated liver cells. J. Physiol. mation (3). (London) 305, 267-277 (1980). The nature of the membrane changes 4. Borle, A. B. Control. modulation, and regulation of cell calcium. Rev. Physid. Biochern. Phurbrought about by the dehydrative effects of mucol. 90, 54- 153 (1981). freezing remains obscure. It was originally 5. Crowe, L., Mouradain. R., Crowe, J., Jackson, suggested that elution of lipoproteins by the S., and Womersley, C. Effects of carbohydrates high salt concentrations is the damaging on membrane stability at low water activities. Biochim. Biophys. Ada. 769, 141-150 (1984). factor (12) although more recent evidence on this is equivocal. It was also suggested 6. Daw. A., Farrant, J., and Morris, G. J. Membrane leakage of solutes after thermal shock or that “atypical” disulfide bonds could be freezing. Cqobiolo~y 10, 126- 133 (1973). formed leading to modified tertiary struc7. del Campilla, E., and Steponkus, P. L. Cryostatures and thus affecting the membrane libility of the plasma membrane ATPase of rye leaves. Cr.whio/ogy 21, 683 (1984). [Abstract] poproteins and enzyme systems (10). Re8. Fuller, B. J., Grout, B. W., and James, E. Cell cent evidence from membranes studied membrane surface area changes related to metduring extreme dehydration at ambient abolic and osmotic behaviour in isolated rat hetemperatures suggests that removal of patocytes after freezing and hypertonic expowater from bilayers could result in a switch sure. Cpo-Lerr. 5, 239-254 (1984). to hexagonal II state, which might change 9. Gulevsky. A. K., Volkova. L. A.. Ryzhov, V. G.,

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