Compartmentalization of Ca2+ in sickle cells

Cell Calcium 6: 397-411,

1985

COMPARTMENTALIZATION OF Ca2+ IN SICKLE CELLS. M.D. RHODA., F. GIRAUD*,C.T. CRAESCU,Y. BEUZARD Unite INSERM U-91, Hapital Henri Mondor, 94010 CRETEIL,France. "UA CNRS 646, Physiologie de la nutrition, bbt. 447, Universitd Paris XI, 91405 ORSAY Cedex, France, to whom correspondenceshould be addressed.

SUMMARY Control (AA) and sickle cell anemia (SS) erythrocyteswere loaded with Ca-chelator(Quin2 or Benz2) to increasethe cellularexchangeable Ca2+ pool and to measure the Ca2+ exchange fluxes and the cytosolic ionized Ca2+ ([Cali) (Lew et al., 1982, Nature, 298, 478). The chelator incorporationinduced a decrease in the ATP content which was smaller in SS than in AA cells and partiallyreversibleupon reincubationin a chelator-freemedium. The amount of trapped chelatorwas determined by two methods: 1) 45Ca binding to the chelator in Ca-iopophore treated cells in Ca-EGTA buffers and 2) [3H]Quin2incorporation.A slight overestimationof the chelator content was found with the second method but incorporationwas the same in both types of cells. The kinetics of 45Ca equilibrationand 45Ca release were used to measure Ca2+ fluxes and [Ca]i in oxygenatedchelator-loadedcells. SS cells, as compared to AA cells, exhibited a moderate increase in Ca2+ fluxes (30-75 $1 but [ Ca] i remained in the same range (about 20 nM). Thus the excess of Ca2+ found in SS cells is not availablefor the Ca2+ pump or the K+ channel a conclusionin agreementwith that of Bookchin et al. (1984, Cell Calcium, 5, 277). Analysis of the 45Ca kinetics showed that in AA cells, exchangeableCa2+ behaved as one compartment. In SS cells, the existenceof a second slowly-exchangeable Ca2+ compartment was demonstrated.This latter (3-5 urn0111 cells) was independent of the concentration of the chelator and thus could represent exchanwithin the intracellular inside-out vesicles geable Ca2+ enclosed recently observed in SS cells (Williamsonet al., 1984, J. Cell. Biol.,

99, 430a). Alternatively,these two kinetic pools could reflect'heterogeneity of the SS cell population. INTRODUCTION It is now largely accepted that the sickle cells have a high Ca2+ content and an increased Ca2+ uptake on deoxygenation.and sickling (1,2). The cellular dehydrationand the membrane rigidity observed in these cells and particularlyin the irreversiblysickled cells (ISC) 397

have been often attributed to their high Ca2+ content (3.4). Since this hypothesis is not unanimously accepted (51, we have tried to improve the data about the distribution of Ca2+ by a kinetic analysis of the 45Ca exchanges in oxygenated SS cells compared to oxygenated AA cells. For this study, the exchangeable Ca2+ pool was increased by loading non-disruptively the cells with Ca2+ selective chelators (6.7). The use of the intracellular chelator permits both the determination of the cytoplasmic free Ca2+ level ( [Ca2+ Ii) and the measurements of the Ca2+ fluxes (influx-efflux). Under the conditions used in this work (1 mM ‘OCaC12 in the incubation medium in all the experimental periods including the chelator loading), internal Ca2+ was in steady state and the true Ca2+ fluxes could be calculated from the equilibration kinetic of the cell Ca2+ with the tracer 45Ca. Oxygenated SS cells showed a small but significant increase in Ca2+ influx but [Ca2+ ]i (in the nanomolar range) was not higher than in normal cells. In addition it was found that exchangeable Ca2+ in AA cells behaves as one compartment whereas in SS cells a second slowly exchangeable Ca2* compartment was demonstrated. MATERIALSAND METHODS Benz 2-AM was a gift from Dr V.L. Lew. [3H]Quin 2-AM and Quin 2-AM were from Amersham (France). 45Ca was from CEA (France). All the other reagents were of analytical grade. Preparation

of erythrocytes

Blood samples from HbSS patients were collected into heparinized The anemic patients were at vacutainer tubes during routine visits. distance of transfusion and crisis. Normal blood from nonanemic volunteers was collected in a similar manner. The erythrocytes were washed 140 mM-NaCl, 5 mM-KCl, 1 mMthree times in the following solution: 10 mM-HEPES(pH 7.4) (buffer A) and MgCl2, 1 mM-CaC12, 10 mM-glucose, kept overnight at 4OC in the same medium at an haematocrit of about 20 %. Chelator

loading

and Ca2+flux

measurements

The experimental protocol was essentially similar to that described by Lew et al. (7) except that all the steps including chelator loading were carried. out in the presence of 1 uWCaC12. All the experiments were carried out under oxygenated conditions. Before chelator loading, erythrocytes were incubated in ATP regenerating system ( 2 mM-adenine, 10 mM-inosine in buffer A, 45 min, 37OC, haematocrit 15 %) and then washed three times in buffer A. Chelator loading: Ca2+ chelators (Benz2-AM or Quin2-AM f [3H] Quin2AM) in DMSO (0.85 % max. final concentration) were added to the cell in buffer A (37OC, haematocrit suspensions after 10 min preincubation to 30 X, 50 to 75 min). The use of [3H]Quin2-AM gives the possibility determine the intracellular chelator content. The kinetic of [3H]Quin2 At different times aliqUOts of incorporation was studied as follows. the cell suspensions (100-200 ~1) were added to 10 ml of ice-cold This washing procedure was repeated twice buffer A and centrifuged. more. The packed cells were lysed in 1 ml 6 % trichloroacetic acid and 398

the [3H] radioactivity was measured from an aliquot of the trichloroacetic acid supernatant mixed with Triton X-lOO/scintillation fluid (3:7 by vol.). Measurement of the specific activity was obtained from an aliquot of the cell suspension treated directly with trichloro acetic acid. When chelator-unloaded cells were required, they were incubated in parallel in the absence of chelator but in the presence of DMSO. Chelator-loaded and unloaded cells were washed 3 times in buffer A and resuspended in the same buffer. Ca2+ influx: cell suspensions were preincubated for 10 min (37”C, haematocrit 10 %) before 45Ca addition. At different times (up to 210 min) aliquots of the suspension (100-200 ~1) were treated as above of trichloroacetic acid and 45Ca (3 washes in 10 ml buffer A, addition counting by liquid scintillation). Ca2+ efflux: a sample of the cell suspension, preincubated at least for 30 min in the presence of 45Ca was washed three times with ice-cold buffer A, resuspended in the same buffer and reincubated for 120 to 210 min (37’C, haematocrit 10 %). Aliquots of the suspension were taken at different times and treated as for the influx studies (except that only one wash was done) for the determination of cell 45Ca content. ATP and cell

volume measurement

ATP was determined with a UV test-combination (Boehringer-Mannheim GmbHDiagnostica, RFA). Cell volume was estimated from the haematocrit (microhaematocrit centrifuge) or from the haemoglobin content (absorbance reading at 540 nm, after cell lysis in water). Both methods gave the same results. Calculations

and kinetic

analysis

Quin2 content (umol/l cells) was calculated from the cell associated 3H radioactivity (cpm/l cells) divided by the specific activity of 3H Quin2-AM in the external medium (cpm/umol). The kinetics of were analysed by plotting: Log (Qeq-Qt) as a 13Hi Quin2 incorporation function of time. Qeq is the Quin2 content at equilibrium and Qt the Quin2 content at time t. The true intracellular concentration of Quin2 ([chelli) was calculated from the extrapolation at zero time of the linear part of the curve. Justification of this procedure will be discussed below. Under our experimental conditions, the Ca2+ exchange system was in steady-state since all the steps including the chelator loading were carried out in the presence of 1 mM-CaC12 and thus 45Ca movements can be analysed assuming that cell Ca2+ constituted a one-open compartment sys tern. Influx experiments were used to determine the total exchangeable Ca2+ (umol/l cells). It was calculated from the cellular 45Ca radioactivity at isotopic equilibrium (cpm/l cells) divided by the specific activity of 45Ca in the external medium (cpm/umol) . This value will be referred in the text as to 45Ca and similarly, the values obtained from the cellular ‘(5Ca radioactievqity at each time normalized to the specific activity of external 45Ca will be referred as to ‘I5Cat. Kinetic analysis of the movements of 45Ca were done by plotting Log (45Caeq - 45Cat) as a function of the straight of time. The slopes lines obtained gave the rate constants for Ca2+ influx (k, h-l). Influx 399

was calculated from: (k= 45Caeq, umol/l cells/h). In SS cells, under certain conditions, these plots were biphasic. In these cases, extrapolation of the linear part of the curve at zero time permitted to calculate the size and the rate constant of the slowly exchangeable comparmethod was used to calculate these parameters for tment . Curve-peeling of these compartthe rapidly exchangeable Compartment. Identification ments will be discussed below. Efflux experiments were analysed by plotting Log 45Cat as a function equilibrium in these cases, Only of time. As 45Ca was not at isotopic the rate constants determined as above, were used for the calculation of Ca2+ efflux, the size of the exchangeable Ca2+ pool being obtained from influx experiments. The free cytosolic Ca2+ concentration ([Cali, nmol/l cells) was calculated from the relation: [Ca]i = KD*4SCaeq/[chel]i-4SCaeq in which KD is the dissociation constant of the Ca-chelator complex, Q5Caeq and [chel]i (kmol/l cells) having the same meaning as above. This relation is valid because there is no other Ca-buffering System than the chelator in the erythrocytes (7) and only if 4SCaeq>> [ Ca]i. The value of Kb was taken as 115 nM for Quin2 (8) and was also determined in one experiment (described in the text) and found to be about 100 nM.

RESULTSAND DISCUSSION ATP content Tiffert et al. (9) have reported that the intracellular incorporation of Ca chelators (Benz2 or Quin2) into erythrocytes induces substantial and irreversible loss of ATP which, in turn, causes Ca2+ pump inhibition. Because our experimental conditions were not exactly the same as those of these authors, we have measured the ATP content of the cells (AA and SS) at each step of the experiments (Table I). After the first step which consists of an incubation of the cells with adenine the ATP content was 0.55 f 0.05 and and inosine (see methods), 0.70 f 0.05 mmol/l cells respectively in AA and SS cells. After 75 min of chelator loading, the ATP content fell in both types of cells and The decrease was smaller with Benz2 than with with both chelators. because of the intracellular Benz2 concentration was Quin2, probably smaller than that of Quin2 (about 100 and 250 l,mol/l cell respectivethe decrease in ATP level was smaller in SS ly) . For both chelators than in AA cells. After 180 min reincubation of chelator-loaded cells the Benz2-loaded as well as the Quin2-loa(end of Ca2+ flux studies), ded SS cells had nearly recovered their initial ATP levels, whereas the Quin2-loaded AA cells had only partially recovered this level. Thus at least when ATP depletion was only moderate, it did not seem to be irreversible. On the other hand, SS cells were less sensitive to this process. ATP depletion has been ascribed to the action of formaldehyde, itself produced by the hydrolysis of the acetoxymethylester derivative of the chelator (9). As the level of intracellular free chelator was the same in AA and SS erythrocytes (see below), the concentration of formaldehyde might be the same. Thus a modification of the metabolism 400

of formaldehydein SS cells could be responsiblefor the smaller effect of the chelator on the ATP depletion. In all conditions the level of ATP was sufficientto maintain normal Ca2+ pump activity. TABLE I: THE EFFECT OF Ca-CHELATORINCORPORATIONON THE ATP CONTENT OF-SS ERYTHROCYI'ES. CHELATOR

CONDITIONS A

Quin2

AA CELLS

SS CELLS

100

100

B

54.2 -e3.6 (5)

69.6 f 1.4 (5)

C

64.0 + 7.9 (5)

82.2 k 7.1 (4)

B

62.3 + 12.2 (3)

89.7 it7.0 (4)

C

119

106 (2)

Benz2 (2)

The ATP content at each step is expressedas a percentageof its value before chelator loading (A). It was measured on washed chelator-loaded cells (B) and after 180 min of reincubationof the same cells at the end of the Ca flux experiments(C). Internalchelator content was about 200-250 pmOl/l cells for Quin2 about 100 umOl/l Cells for Benz2. Values are means + S.E. of 3 to 5 experiments(number in parenthesis)or means of 2 experiments.

Chelatorcontent The intracellularconcentrationof the chelator was usually determined by measuring [3H]Quin2AM incorporation(see below). To validatethis procedure,the method of Lew et al. (7) was used at the same time in one experiment.Quin2-loadedAA and SS cells were ATP-depletedand incubated in the presence of ionophore A23187 and 45Ca-EGTA buffers giving free Ca2+ concentrations in the nanomolar range ([Cal,).At the end of the incubation,the pH of the medium (log l/[Hle) and of the washed freezed-thawedcells (log l/[H]i) were measured. The free cytosolicionizedCa2+ concentration([Cal.) was calculated from the equation: [Ca]i= [Ca]e*([H]i/[H]e)2. The &a content of the washed cells was alSO measured to determine the concentrationof total intracellularCa2+ (45Caeq). The relation methods) be transformed + l/[chelq:: The plot of 1i45Caeq 401

allows to determine graphically KD and [chel]i (Fig. 1). The values found for KD of Quin2 (109 and 74 nM) well agreed with the reported values of the literature (8 1. The values of [ chel]i were 36 and 34 UrnoWl cells respectively for AA and SS cells. The corresponding values obtained on the same cells with the [3H]Quin2 extrapolation method were 47 and 40 pmOl/l Cells.

0

0.025 l/tCIli (nmol/l

0.050 cells)

0.075 -1

Fig. 1: Determination of the intracellular Quin2 content and of the in situ dissociation constant of the Ca-Quin2 i?oU@eX. The method is exactlv that described by Lew et al. (7). Chelator-loaded (80 pmol/l cells), ATP-depleted AA ( 6 ) and SS ( 0 1 ‘cell suspensions (haematocrit 8 5) were incubated in a medium containing (mM): NaC1, 75; KCl, 75; Tris-Cl (pH 7.6 at 37’c), 10; MgC12, 0.2; 45CaC12, 0.1. The Ca ionophore A23187 (75 pmol/l cells) was added and after 10 min, the suspension was distributed to tubes containing EGTA solutions calibrated to give final free Ca2+ concentrations in the nanomolar range ([Cal,). After 90 and 100 min duplicate aliquots were added to ice-cold buffer A containing Tris-EGTA (0.1 mM) in place of CaC12 and albumine (0.25 %), centrifuged and washed twice without albumine and processed as described in methods for the determination of 45Ca radioactivity. From this latter and the specific activity of 45Ca of the medium the total Ca content at equilibrium (45Caeq) was calculated (see methods). At the end of the experiment the pH of the suspension and that of the freeze-thawed cell pellet were measured at 37°C. [Gale was calculated knowing the total EGTA and Ca concentrations of the medium and the dissociation constant of the Ca-EGTA complex (250 IJM). [Cal, was calculated from [Ca]e and the pH values (see text). The plots shown in the

402

figure were straight lines indicating that the data are well described by a one-to-one binding of Ca to 35 urn0111 Cells of chelator with about 100 nM dissociation constant for Ca2+.

The [3H]Quin2 method consists of an incubation of the cells with Quin2 AM and [3H]Quin2 AM (both from Amersham and delivered on the same day) for 50 to 75 min. The kinetics of incorporation was the same in both AA and SS erythrocytes (Fig. 2A). In the first minute of incuba-

0B

600 500 500 300 400 fu 0 Y

3ocI

100

-

f = 200I CU .g 0

y

50

-

0

100

y” 20

t1 0

la

20 TIME

30 ( minutes

40 1

50

0

u)

20

so

TIME (lninutet I

Fig. 2: Kinetics of Quin2 loading. AA ( 0 1 and SS ( 0 1 erythrocyte suspensions were incubated in the presence of Quin2-AM (500 umol/l cells) and [3H]Quin2-AM (200 uCi/l cells). At different times aliquots were taken for measurement of 3H radioactivity (see methods). A) amount of Quin2 associated with the cells as a function of time (Qt, calculated from the cell associated radioactivity divided by the specific activity of [3H]Quin2-AM of the medium). B) semilogarithmic plot of the difference between Qeq (equilibrium value of Qt) and Qt as a function of time. Only the plot of AA cells is presented, that corresponding to SS cells being identical. Extrapolation at time 0 of the linear part of this plot permits to calculate the true intracellular concentration of Quin2. In this experiment, the Quin2 content was 300 umol/l cells e.g. a chelator incorporation yield of 60 $.

tion there was already an important amount of radioactivity associated with the cells. After about 50 min an equilibrium Value was reached. the data were plotted When according to the equation: LOg(Qeq-Qt) = f(t) (see methods) a biphasic curve was obtained indica-

403

ting the existence of two compartments of radioactivity (Fig. 2B). It was first postulated that the rapid compartment corresponded to [3H]Quin2-AM not washed by the aqueous buffer and remaining associated to the external surface of the cells, Attempts were made to remove this hypothetical external fraction by: 1) centrifugation through dibutylphtalate oil, 2) addition of albumin (1 5) to the wash medium, 3) addition of “intralipide” (25 5) to the wash medium (this mixture is normally used as a vehicle for intravenous injection of hydrophobic compounds in humans; its composition is: 20 g soya oil, 1.2 g egg phosphat idylchol ine, 2.2 g glycerol in 100 ml distilled water). All these treatments were inefficient in removing any radioactivity when compared to the normal wash medium. The complex kinetic of the radioactive uptake of [3H] Quin2-AM could be also explained by an heterogeneity of the Cell population or by the presence of [3H] degradation products of Quin2. The fact that the size of the rapid component increased when the time of conservation of [3H] Quin2-AM increased, was in favor of the second hypothesis. Accordingly, only the slow component corresponded to the incorporation of unaltered Quin2 and the intracellular concentration of Quin2 was obtained by extrapolation of the linear part of the plot of Fig. 28. The yield of incorporation was variable, from 80 % to 40 $ depending on the age of Quin2-AM. Ca2+ influx Chelator-loaded and unloaded AA and SS erythrocytes were incubated under oxygenated conditions in buffer A containing 45Ca. A typical experiment is presented on Fig. 3A. In the absence of chelator, SS cells took up about 5 pmol/l cells of 45Ca whereas under the same conthe uptake in AA cells ticas very low (
0

3060 TIME

90

;1

aJ1M~80

(minukr

;o io 6;

TIME (minutes)

1

%Za uptake by Qu1n.Floaded and SS ( 0 ) Cells.

(-

) and unloaded

f -----)

AA

Tells were incubated without or with QuinZ-AM (500 pmol/l cells) in buffer A (1 mM CaC12), washed and reincubated in the same buffer conaliquots were taken at different times and taining 45Ca. Duplicate processed as described in methods for the determination of b5Ca radioaCtiVity. A) 45Ca content as a function of time (45Cat, calculated from this radioactivity and the specific activity of 45Ca in the medium). B) semi-logarithmic plots of the difference between 45Caeq (equilibrium value Of 45Cat) and 45Cat as a function of time for QuinZ-loaded AA and SS cells. The slope of the straight lines obtained permit to calculate the values of the Ca2+ influx (see methods). In this experiment Ca2+ influx was 32.7 and 46.1 kmol/l cells/h respectively for AA and SS cells.

size of the slowly exchangeable compartment was independent of the chelator concentration and amounted always to about 5 umol/l cells, a value corresponding to that measured in the absence of chelator. 45Ca influx into this pool was 3 vmol/l cells/h. In AA cells, the 45Ca influx was 24.9 and 22.1 kmol/l cells/h respectively for the low and high chelator content.

405

I

0

50

60

TIME

90

120 1!50 180 210

0

so TIME

( minutmsI

60

90

l20

bO l80

( mhutn 1

Fig. 4: 45Ca uptake by AA ( 0 , A ) and SS ( 0 , A ) cells loaded with different amounts of. Benz2. Cells were loaded with Benz.?!-AM either 500 @01/l cells ( 0 , 0 ) or 100 umol/l cells ( A , A ). The measurement of 45Ca uptake, the meaning of the plots A and B were explained in Fig. 3 legend. For calculation of Ca2+ influxes see methods. In AA cells Ca2+ influx was independent of the amount of the chelator (24.9 and 22.1 umol/l cells/h respectively for the low and high chelator-containing cells). In high chelatorcontaining SS cells, Ca2+ influx was 33.5 umol/l cells/h. However in low chelator-containing SS cells, the semi-logarithmic plot in B was biphasic revealing the existence of two compartments of exchangeable Ca2+. Graphical analysis of this plot permit to calculate the characteristics (sizes and rate constants kl and k2) and thus the Ca2+ influx into each of them. In this experiment Ca2+ influx was 3 umol/l cells/h into the slowly-exchangeable compartment (5 umol/l cells) and 29.7 urn0111 cells/h into the rapidly-exchangeable one, a value identical to that measured in the high chelator-containing SS cells,

Ca2+ efflux Low chelator containing AA and SS cells were loaded with 45Ca for 30 A and reincubated in the same buffer. min, washed 3 times in buffer 45Ca content of the cells was measured for up to 210 min (Fig. 5A). The

406

semi-logarithmic was linear for

8 0

plot for the efflux (see methods) in these AA cells but semi-linear in SS cells (Fig.

, , 30 60 TIME

r

I

90

I

I

I

120 150 180 2x3

( minutes

1

* 0

. 30

. 60

. 90

TIME

conditions

58).

This

. . PO l50 180 I

(minutes

1

AA ( 0 > and SS ( 0 > Fig. 5: 45Ca efflux from Win2 and 45Ca-loaded cells. m were loaded with Quin2-AM (100 urn0111 cells) in buffer A (1 mM CaCl2), washed and reincubated for 30 min in the same buffer containing

4SCa. The cell suspensions were washed again and incubated in buffer A (see methods). A) 45Ca remaining into the cells as a function of time (45Cat). B) semi-logarithmic plot of 45Cat as a function of time. This plot was linear in AA cells but biphasic in SS cells revealing the existence of two compartments of exchangeable Ca2+ with rate constant kl and k2. 45Ca uptake was also measured on the same Cells (not shown) in order to determine at isotopic equilibrium the sizes of the slowly and of the rapidly-exchangeable compartments (see Fig. 4 legend). From these values Ca2+ effluxes were calculated and found to be equal to Ca2+ influxes indicating that the system was in steady-state (2.5 and 29 pmol/l cells/h respectively for the slow and rapid compartment). In AA cells, exchangeable Ca2+ measured by the efflux of 4SCa, as well as by the influx (not shown, but see Fig. 3 and 4) behaved as one compartment in steady-state (influx equal to efflux, 16 pmol/l cells/h).

confirmed that SS internal Ca2+ behaved as a two-compartment system as already observed during influx studies. The sizes of each compartment were determined from the influx experiments carried out in parallel from each compartment were found to be equal (not shown). 4SCa efflux 407

to 45Ca influx into these compartments (respectively 2.5 and 29 umol/l cells/h for the slow and the rapid compartment). In AA cells, influx and efflux were equal (16 umol/l cells/h) and these movements were described by a one-open compartment system. The similarity between the two fluxes indicated that exchangeable Ca2+ was in steady state during the whole experimental period of flux measurement (about 4 hours). It has been observed that SS cells loaded with 45Ca by a sickleulse or addition of the Ca2+ ionophore A23187 exhibited a biphasic e 5Ca extrusion: at first 45Ca efflux was very rapid and then ceased leaving a small fraction of 45Ca apparently retained within the cells (2, 10). Similar observations were made on resealed SS cells (2, 11 ). Under these conditions [Ca]i was greatly increased inducing a stimulation of the Ca2+ pump and causing the large efflux observed during the first minutes. Relatively to this first phase, the second slow phase was attributed by these authors to a non-exchangeable Ca2+ pool. In our experiments 45Ca efflux was measured on cells having a normal [Ca]i and for longer periods (210 min) than those used in the other works, conditions permitting the detection of the slowly-exchangeable pool.

From the values of Caeq and of [ chel]i, one can calculate the free cytosolic Ca2+ concentration [Ca]i (see methods). This was done in some experiments in which [3H]Quin2 was used to determine [chel]i (Table II). No significant difference in [Ca]i was observed between AA and SS cells. Because [chel]i seems to be overestimated by [3H]Quin2 method, the [Ca]i values of Table II may be slightly underestimated. TABLE II:

Ca INFLUX AND CYTOSOLICFREE Ca CONCENTRATION ( [Cali)

IN AA

AND SS CHELATOR-LOADEDERYTHROCYTES. CHELATOR

AA CELLS

SS CELLS

Ca influx

(umol/l

cells/h)

Benz2

23.2

+ 1.5

(6)

40.8

+ 3.9

(7)

Quin2

29.8

f 2.6

(9)

39.1

f 2.7

(9)

18.9

+ 1.8

(6)

JCAIi [3H] QuinZ

19.4

+ 2.7

(MIO~/~

(5)

cells)

Benz2 and Quin2 internal concentration were those indicated in Table I legend. Values are means f SE of 5 to 9 experiments (number in parenthesis).

408

coNcLusxoNs As shown in this report there is a significant but moderate increase in the Ca2+ influx in oxygenated SS cells compared to AA cells, but there is no differences between their [Ca]i (Table II). Neither this moderate increase in Ca2+ permeability of oxygenated SS cells nor the larger increase encountered in deoxy SS cells by Eaton (1); Palek (2) and Bookchin et al. (12) can explain the Ca2+ accumulation found in SS cells. Because the excess of Ca2+ in SS cells was ionophore mobilizable within (10) , Bookchin and Lew have proposed that it is sequestrated inside-out vesicles (IOVs) and thus unavailable for Ca2+ exchanges, activation of the Ca2+ pump or for the Ca2+- activated K channel (13). A same conclusion was also reached by Williamson et al, (14) in a preliminary report. The existence of the Ca2+ se uestration into IOV is in agreement with both the maintenance of the 1 Ca 1i in the normal range and with the moderate increase of the Ca2+ exchan e rate in oxygenated SS cells (Table II). With kinetic analysis of the Q5Ca exchanges measured under our various experimental conditions (absence or presence of different concentrations of chelator) we provide informations on Ca2+ compartmentation in SS cells. in the absence of chelator, Firstly, 45Ca uptake after 180 min was hardly measurable in AA cells whereas it amounted to 3-5 pmol/l of SS cells (Fig. 3)) in agreement with previous measurements (10). Secondly, at low chelator concentration exchangeable Ca2+ behaved as one-compartment in AA cells but in SS cells the kinetics of 45Ca uptake and realease were well described by a two-compartment model (Fig. 4 and 5). The size of the rapidly-exchangeable compartment was dependent on the amount of chelator whereas that of the slowly-exchangeable one was not and amounted to about 3-5 umol/l of SS cells, a figure similar to that observed in the absence of chelator. at high chelator content, both in AA and SS cells, the Thirdly, content and exchangeable Ca2+ pool was much larger than at low chelator behaved as one-compartment system (Fig. 3). Its rate of exchange in SS cells was equal to that of the rapidly-exchangeable compartment. These results suggest that the slowly-exchangeable compartment, not accessible to the chelator, can correspond to a fraction of the Ca2+ enclosed within the IOVs and conversely that the rapidly-exchangeable whose size increased with the amount of chelator, can compartment, represent cytosolic Ca2+. If the volume of the vesicles is 2.5 5 of the total cell volume (131, the concentration of vesicular exchangeable Ca2+ (3.5 umol/l cells) will be 100-200 umol/l vesicles and the Ca2+ fluxes through the IOVs (3 umol/l cell/h) will be 120 urn0111 vesicles/h. This explanation implies that the kinetic pools represent Ca2+ compartmentation within each cell. Knowing that SS cells are highly heterogeneous (13). an alternative hypothesis would be that these pools correspond to two subpopulations of SS cells with different rate constants for Ca2+ exchange and different permeabilities for the chelator. The present experiments do not allow to rule out this possibility and more work is needed to ascertain our first hypothesis. 409

ACKNOWLEDGMENTS We thank

for

providing

Dr F. Calacteroa. samples of blood

INSERM U91 HaDital from his patients.

Henri

Mondor

Creteil,

REFERENCES

J.W., 1. Eaton, H.S. (1973). Nature, 246, 2.

Skelton, Elevated 105-106.

J. (1977). Palek, cium movements in Clinical Medicine,

T.D., Swofford, H.S., erythrocyte calcium

Kolpin, C.E. and Jacob, in sickle .cell disease.

Red cell calcium content and tranamembrane sickle cell anemia. Journal of Laboratories 89, 1365-1374.

3. Clader,

B.E. and Nathan, D.C. (1978). Cation permeability tions during sickling: Relation to cation composition and hydration ofirreversibly sickled cells. Blood, 51, 983-989.

4. Clark,

M.R., Morrison, C.E. cation transport in irreversibly Investigation, 62, 329-337.

and

Shohet, S.B. sickled cells.

(1978). Journal

V.L. and Bookchin, R.M. (1980). A Ca2+ refractory Ca-sensitive K+ permeability mechanism in sickle cell cells. Biochimica and Biophyaica Acta, 602, 196-200.

5. Lew,

6. Taien,

fers

R.Y. (1981). and indicators

caland

alteracellular

Monovalent of Clinical

state of the anaemia red

A non-disruptive technique-for loading into cells. Nature, 290, 527-528.

Ca buf-

7. Lew,

V.L., Tsien R.Y., Miner, C. and Bookchin, R.M. (1982). Physiological [ Ca2+ ji 1 eve1 and pump-leak turnover in intact red cells measured using an incorporated Ca-chelator. Nature, 298, 478-481.

8. Taien,

R.Y., Pozzan, T. and Rink, T.T. (1982). T Cell cause early changes in cytoplasmic free Ca2+ and membrane in lymphocytes. Nature, 295, 68-71.

9.

mitogena potential

Tiffert. T.. Garcia-Sancho, T. and Lew. V.L. (1984). Irreversible ATP depletion caused by iow concentration of formaldehyde and of calcium-chelator esters in intact human red cells. Biochimica and Biophyaica Acta, 773, 143-156.

10. Bookchin,

R.M. and Lew, V.L. (1980). Ca pump and Ca:Ca exchange in sickle 563.

Progressive red cells.

inhibition Nature,

of 284,

the 561-

11. Litosch,

I. and Lee, K.S. (1980). Sickle cell calcium metabolism: Studies on Ca2+-Mg2+ ATPaae and Ca-binding properties of sickle red cells membranes. American Journal of Hematology, 8, 377-387.

12. Bookchin,

in sickle

R .M., ‘Ortiz, cell anemia

O.T. and Lew, V.L. (1984). red cells. Cell Calcium, 5, 410

State 277

of

calcium

13. Bookchin,

R.M. and Lew, V.L. (1983). Red cell membrane abnormalities in sickle cell anemia, in Progress in Hematology, vol. XIII (eds, E.B. Brown, M.D.), pp. l-23, Grune h Stratton, Inc.

R. (1984). Internal CaB+14. Williamson, P., Rubin, L. and Schlegel, containing vesicles in sickle cell erythrocytes. Journal of Cellular Biology, 99, 43Qa.

Received 10.6.85 Revised version received

and accepted

411

13.9.85