Selective Loss of Calcium Permeability on

Maturation of Reticulocytes

JAMES S. WILEY and CALVIN C. SHALLER

From the Hematology-Oncology Section, Department of Medicine, Hospital of the University ofPennsylvania, Philadelphia, Pennsylvania 19104

A B S T RA C T Calcium and sodium permeability ofhuman reticulocytes have been studied and comparedto mature erythrocytes. Mature erythrocytes had ex-tremely low Ca2+ permeability which was less than0.1% of values published for squid axon or HeLacells. Calcium entry was markedly increased in retic-ulocyte-rich suspensions and the uiptake was linearlyrelated to the percentage of reticulocytes present.The data suggest that reticulocytes are 43-fold morepermeable to Ca2+ than mature cells although theirCa2+ concentration is not increased. Sodium influxinto reticulocyte-rich suspensions was also increasedin direct proportion to the percent of reticulocytespresent. Reticulocytes are sixfold more permeable toNa+ than mature cells so the ratio of Ca2+:Na+permeability falls by sevenfold as the reticulocytechanges to an erythrocyte. [3H]Ouabain binding wasincreased in reticulocyte-rich cell suspensions and thecorrelation suggested a value of about 4,000 sites perreticulocyte compared with 362+69 per mature cell.Maturation of the human reticulocyte produces dis-proportionate changes in cation permeability and inparticular a selective loss of Ca2+ permeability.

INTRODUCTION

Reticulocytes lose their ribosomes within 36 hafter entering the peripheral blood, although this ma-turation time may be almost doubled in conditionsof erythropoietic stress (1, 2). During the maturationprocess, reticulocyte volume decreases by 20-30%and several studies have shown that cytoplasmic hemo-globin and membrane lipids are decreased in the same

This work has appeared in preliminary form in 1974. Fed.Proc. 33: 266.

Dr. Wiley's present address is the Department of Medicine,Austin Hospital, Melbourne 3084, Australia.

Received for publication 22 November 1976 and in revisedform 22 February 1977.

proportion (3, 4). The two major lipids of the membrane,phospholipid and cholesterol, are both lost in paral-lel while changes in osmotic fragility confirm thatmembrane area is reduced (3). Major alterations inmembrane transport occur during this maturationprocess. Reticulocytes possess a Na+-dependent trans-port system for alanine and a-aminoisobutyric acidwhich disappears on cell maturation (5). Active trans-port of Na+ is also lower in mature erythrocytescompared with reticulocyte-rich populations (6),although cation fluxes have not been correlated withreticulocyte numbers. In this study the fluxes ofCa2+ and Na+ as well as the binding of [3H]ouabainhave been measured in reticulocyte-rich erythrocytes.Results show a major reduction in Ca2+ permeabilityon maturation of reticulocytes which is almost anorder of magnitude greater than the Na+ permeabilitychanges.

METHODSMaterials. NaCl, KC1, sodium EGTA,' SrCl2 were Analar

grade from Fisher Scientific Co., Pittsburgh, Pa.; LiClwas from K and K Laboratories, Inc., Plainview, N. Y.;inosine, hexokinase, and glucose-6-phosphate dehydrogenasewere from Boehringer, Mannheim, W. Germany; iodoaceticacid and sodium iodoacetamide were from Eastman Kodak Co.,Rochester, N. Y.; ATP, NADP+, and Tris-HCl were fromSigma Chemical Co., St. Louis, Mo.; and 45CaCl2 and 22NaClwere from New England Nuclear, Boston, Mass. [3H ]-Ouabain, 5 Ci/mM ethanol-benzene solution, New EnglandNuclear lot no. 184-196, was evaporated to dryness undera stream of nitrogen and redissolved in 20-fold excess ofunlabeled ouabain to give a stock solution of 10 juMouabain in water of activity 12.5 ,ICi/ml. The exact con-centration of ouabain was confirmed by spectrophotometricanalysis with a molar extinction coefficient of 1.54 x 104at the absorption peak of 220 nm. (7). This stock solu-tion was stable at 4°C for several months. Silica cruci-bles (high-form, 5 ml capacity) were from Thermal American

1Abbreviations used in this paper: EGTA, ethylene glycol-bis(13-aminoethyl ether)N,N,N',N'-tetraacetate.

TheJournal of Clinical Investigation Volume 59 June 1977 1113-1119 1113

Ftused Quartz Co., Montville, N. J.; standard solutions of'Ca2+ (5-50 ,uM) were prepared by dilution of commercial1,000 ppm Ca (NO3)2 (i.e., 25 mM) from Fisher ScientificCo. in a solution of 0.1 N HCI plus 10 mMSrCl2.

High-reticuilocyte blood donors. Patients were selectedwith peripheral blood reticulocytosis of 2.0-25% resultingf'rom chronic autoimmune hemolytic anemia, pyruivate kinasedeficiency hemolytic anemia, chronic gastrointestinal bloodloss, or during hematinic therapy of' a deficiency anemia.Patients with renal disease were excluded because of theinhibitory effects of uremic toxins on cation transport inthe erythrocyte (8). Likewise, patients withl liver disease,uintreated megaloblastic anemia, or alcoholism were ex-chided because of their increased erythrocyte size and stur-f'ace area (9). Sodium and Ca21 influix were studied simul-taneously in 8 of the 20 patients with reticulocytosis while12 patients were studied with only one flux measurement.

Preparation of erythrocytes. Venous blood was collectedinto heparin and erythrocytes were separated from leukocytesby thrice washing at 40C in a mediuim of composition 145mM NaCl, 5 mMKCl, plus 20 mM imidazole Cl, pH7.5. Care was taken to remove all buiffy coat since bothleukocytes and platelets contain large amounts of calcium.This basic medium was used f'or all incubations since theimidazole buffer does not associate with added Ca2+ ions.

Reticulocyte enumeration. During washing of erythro-cytes and removal of buffy coat uip to one-fourth of thereticulocytes may be lost. Therefore, the percentage ofreticulocytes was always measured after washing. Onedrop of washed erythrocytes was mixed with three dropsof autologous plasma and stained for 15 min with an equalvolume of freshly-filtered 1.0% new methylene blue (10).The number of reticulocytes in 1,000 erythrocytes wascounted.

Calcium influx. Washed erythrocytes were depleted ofATP by preincubation for 90 min in medium plus 1 mMiodoacetate and 10 mMinosine (11). The erythrocytes werethen washed twice and added to prewarmed media of com-position 145 mMNaCl, 5 mMKCI, 20 mMimidazole Cl,pH 7.5, plus 1.5 mM45CaC12 (1 ,uCi/ml). Samples were takenafter 15 min and after 1, 2, 3, and 4 h, and washed fourtimes in cold 150 mM NaCl plus 1 mM Na EGTA.Each cell pellet was hemolyzed in 0.01 N NH40H, andpart of the hemolysate was deproteinized with 6% (wt/vol)perchloric acid plus 1 mMNa EGTAand 45Ca21 in the super-nate was measured by liquid scintillation counting. To con-vert the uptake of radioactive calcium from nanomoles permicromole Hb into nanomoles per milliliter cells, the meancorpuscular hemoglobin concentration was measured in eachexperiment both for the abnormal and nonnal cells. Na+influx was measured in cells depleted of' ATP by the aboveprocedure to find if the iodoacetate treatment had induceda generalized permeability increase. No increase in Na+ in-flux was found.

Total cell calcium cotncentration. Calciuim was measuredby atomic absorption spectroscopy of a dry ashed sample of'erythrocytes. Erythrocytes, whichl had been washed free ofbuffy coat were further washed f'otir times at 40C in 150mMNaCl or 150 mMLiCl either with or without 1 mMNa EGTA. The final pellet of washed cells was hemolyzedwith 6 ml of 0.01 N NH40H (verified Ca2+-free), and 5 mlof' the hemolysate was added to a silica crtucible, slowlyevaporated to dryness under an infrared lamp, and com-busted at 5000C overnight in a muffle ftirnace. It was im-portant that the sample was completely dry bef'ore combus-tion, since any trace of water led to uincontrollable bub-bling when the sample was heated toward 500°C. Theresidue in each crucible was extracted with dilute acid by

addling 3 ml of 0.1 N HCI pluis 10 mMSrCl2 to the cruci-ble aiid shaking for 2 h at room temperatuire. The conitenitsof the crucible were decanted into polypropylenie tubes, cndthe iron oxide was allowed to settle. Calcitum in the super-nate was analyzed on a Varian Techtron 1200 Atomic Absorp-tion Spectrophotometer (Varian Associates, Walnuit Creek,Calit. at 422.6 nm withi acetylene futel plus comi)ressledair. Ca"2 coitaminant in HCl-SrCl2 solution (solvent blank,was 2 p\.. A 1-ml aliqulot of the hemolysate wvas takeifor measturemenit of hemiioglobin concenitrationi, so tlhat tleCa21 p)resent in each cruicible couild be expressed as n1an1o-moles per milliliter of cells.

Precauitions to excluide calciuim contaniinationi. IsotoniicNaCl and LiCl contain 1-4 ,uM Ca2 contaminant whicichwas removed by filtering these soltutions throuigh al 8 x 1.5-cm column-lll of Chelex-100 ion-exchange resin (Bio-RadLaboratories, Richmnond, Calif.). These soltutions and othersused in Ca24 measuirements were stored in polypropylenebottles since both glass and some polystyrene andl poly-ethylene containers contribute small and variable amiiouintsof Ca2+ to a soltution. Whlen Pyrex glassware wNas uised.it was acid washed and kept immersed in deioniized wateruintil immediately before uise, when it was againl rinsedin deionized water. All crucibles had close-fittinig lids, andafte r combustion the lids were duisted free of ref'ractorvbrick (lust bef')re opening. Recovery stuidies of Ca2+ addedto crucibles anid dry aslhed at 5000C established that nosignificanit amotunits of Ca2+ were retained b-y the silica crtuci-bles. 45CaC12 (1.25, 2.5, 3.75, 5, and 74 iomlol) was addedto crucibles, 5 ml of an erythrocyte hemolysate was addedand the contenits were dry ashed and extracted in diluiteacid as above. The recovery of 45Ca was estimated by liqtuidscintillation counting and was 81, 85, 86, 98, and 101%,respectively, for each of the above amouints of added Ca2 .

Sodium influx. This was measuired from the uiptake ofradioactivity by cells incubated 10 and 20 min in mediacontaining 22NaCl as described by Wiley and Cooper (12).Media always contained glutcose (10 mnM) plus ouiabain(50 ,uM).

ATP estimation. The cell stuspensioni was deproteinizedwith 2 vol of 0.6 N-perchloric acid, centrifutged at O'C,and the supernate adjuisted to pH 6-7 with K2CO3. ATPin the netutralized extract was estimated spectrophotometri-cally by measuiring the redtuction of NADP+by a coupledhexokinase-glucose-6-phlosphate dehydrogenase reaction.

Creatinie. This was meastured in a neuitr-alize(d perclhloricacid extract of washed cells (13).

[3H]OHtabain biniding to erytlhrocytes. Biniding of ouia-bain was measured essentially as described by Hoffllmani (14)and Wiley et al. (15). Washed erythrocvtes were incubatedwith 20 nM [3H ]ouabain for 30 or 60 min atter whicilhcells were washed aind 0.5-ml portions of p)acked cellswere added to 15 ml Aquafluior (New Englandl Ntuclear)anid cotunted after storage for 72 h in the (lark. Another- por-tion was taken for erythirocyte enulmerationi with the Couiltermodel ZB electronic particle couinter (Coumlter ElectrollicsInc., IHialeah, Fla.). Potassiu-m influx was also imeasuire(d toassess the fractional inhibition of the catioin pumInp. 42KC1was added to the basic inctubation mediulm to give a fina'K+ concentration of 5.6-7.2 mMand influx measuiredl over .sl-h incubation. The total nutmber of otuabaiui-binding sitesper cell was equated to the number of [3 H]ouiabain mnole-cules bouind per cell when there was 100%4 inhibitionof active K+ influix.

Statistics. Mean values+ 1 SDare shown ulnless otherwisenoted. Regression lines and correlation coefficients were cal-culated by the method of least squares anid differencesbetween sample means analyzed by a t test.

1114 J. S. Wiley anid C. C. Shalller

TABLE ICalcium Concentration oJ Normal and Reticulocyte-Rich Erythrocytes

Diagnosis Reticulocytes Washing medium Mean calcium SE Range

% nmollml cells

Normals < 1.0 NaCl 5.7 0.8 3.6-7.4Normals <1.0 NaCl + EGTA 6.5 1.4 3.1-11.2Normals < 1.0 LiCl 5.2 1.1 2.1-8.9Normals <1.0 LiCl + EGTA 6.3 0.6 5.0-8.3G. I. blood loss 11.4 NaCl + EGTA 3.7 0.6 2.7-5.7Folate deficiency on therapy 5.6 NaCl + EGTA 2.7 0.2 2.3-3.2Autoimmune hemolytic anemia 24 NaCl + EGTA 9.2 0.7 8.4-10.1Autoimmune hemolytic anemia 25 NaCl + EGTA 9.3 0.2 9.0-9.6

Normal group was three males and three females and the results with different washing media were always obtained withpaired observations on the same cells on the same day. Measurements on the four patients with elevated reticulocytes wereobtained from quadruplicate samples.

RESULTS

Erythrocyte calcium concentrations. The meancalcium content of erythrocytes from six normal donorswas 5.9+1.8 nmol/ml cells after four washes in averified Ca2+-free medium. Identical values were ob-tained whether the medium was isotonic NaCl orLiCl or whether the chelating agent, EGTA, waspresent or absent (Table I). The range of values ob-tained for replicate determinations on one individualis shown in Table I and usually fell within a two-or threefold range. The calcium content of erythro-cytes from patients with a reticulocytosis (5.6-25%)did not differ significantly from normal (Table I)

Calcium exchangeability. Erythrocyte Ca2+ ex-changed with isotopic Ca2+ added to a physiologicalmedium in which the cells were incubated at 37°C.This exchange was complete by 5 min since theuptake of isotopic Ca2+ was the same in samplestaken at 5, 10, 15 and 60 min. Concurrent measure-ments of 45Ca2+ uptake by liquid scintillation count-ing plus total cell Ca2+ concentration by atomic ab-sorption showed that isotopic Ca2+ exchanged withonly a fraction of total cell Ca2+. Two normals ex-changed 15 and 40% of their total Ca2t, while thepatient with 25% reticulocytes showed an 88% ex-change. This greater exchangeability of reticulocyteCa2+ was studied by measurements of unidirectionalCa2+ influx.

Calciulm influx into normal erythrocytes. Unidirec-tional 45Ca influix was studied in erythrocytes depletedof ATP to inhibit the outward pumping of Ca2+ions (11). To deplete the ATP the washed erythrocyteswere preincubated with 10 mMinosine plus either1 mM iodoacetate or 5 mM iodoacetamide. Theseinhibitors were equally effective in combination withinosine since ATP fell from 1.2 ,mol/ml cells initiallyto less than 0.1 ,umol/ml cells after 90 min (Fig. IA).

Cells were again washed and incubated without inhibi-tors in media containing 1.5 mM45Ca2t ion. A rapidinitial uptake of isotopic Ca2+ occurred within 15 minfollowed by a slower uptake which was linear between15 min and 4 h (Fig. IB). In cells depleted by ino-sine plus iodoacetate the initial uptake was 2.3 nmolCa2+/ml cells and the linear phase averaged 0.8nmol Ca2+/ml cells every hour to reach 5.4±+1.5 nmolCa2+/ml cells after a 4-h incubation. Calcium uptakeby cells depleted of ATP by incubation with inosineplus iodoacetamide was not significantly differentto the values above (Fig. 1B). Analysis of total Ca2+in one experiment confirmed a net increase in thiscation.

Calcium influx into reticulocytes. Calcium uptakeis increased into erythrocytes from patients with a retic-ulocytosis. Cell suspensions with reticulocytes of 5.0or 11.4% allowed approximately 6- or 10-fold greaterCa2+ uptake, respectively (Fig. 2). Similar uptakes wereobtained for the same cells depleted of ATP by threedifferent combinations of inhibitors: inosine plus iodo-acetate, inosine plus iodoacetate plus cyanide, or ino-sine plus iodoacetamide. The combination of inosineplus iodoacetate was chosen to deplete cell ATPfor all subsequent studies. The greater Ca2+ uptakeby reticulocytes was confirmed with blood from anormal donor. The erythrocytes were washed freeof buffy coat and fractionated by centrifugation intoreticulocyte-rich and reticulocyte-poor fractions (16).Table II shows that the top fraction with 2.4%reticulocytes showed a twofold greater Ca2+ uptakethan the reticulocyte-poor fractions.

Comparison of calcium and sodium influx. Thelarge influx of Ca2+ into ATP-depleted reticulocytessuggests that the reticulocyte membrane is verypermeable to this cation. Sodium influx was also meas-ured in reticulocyte-rich cells for comparison with theCa2+ fluixes. Fig. 3 shows that Na+ infltux was increased

Calcitum Permeability of Reticulocytes 1115

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FIGURE 1 (A) ATPconcentration of erythrocytes incubated at37°C with 10 mMinosine plus 1 mMiodoacetate (0) orinosine plus 5 mMiodoacetamide (0). (B) Calcium uptakeby erythrocytes depleted of ATP by either inosine plusiodoacetate (0) or iodoacetamide (0). Mean values are for22 or 6 normal donors, respectively, and show±+l SE by thevertical bars.

in direct proportion to the percent reticulocytes present(r = 0.88, P < 0.001) and the influx was twofold normalfor cells with 20% reticulocytosis. In contrast the Ca2+uptake was elevated 11-fold normal in cells with 20%reticulocytosis (Fig. 3). A linear relation existed be-tween Ca2+ uptake and percent reticulocytes (r = 0.71,P < 0.01) although the regression line did not passthrough the mean for normal erythrocytes. Calcium

uptake showed no correlation with erythrocyte crea-tine which was measured in 12 reticulocyte-rich cellsuspensions as an index of the mean erythrocyteage. Sodium influx was measured into erythrocytesboth before and after depletion of ATP with iodo-acetate plus inosine. No increase in Na+ influx wasfound in depleted cells in three separate experiments.

[3H ]Ouabain binding to reticulocytes. Reticulo-cytes bound more ouabain than normal cells and therewas a direct relation between the number of ouabainmolecules bound per cell and the percentage ofreticulocytes present (Fig. 4). Eight patients werestudied with reticulocytosis between 4 and 20%,and the number of binding sites per cell increasedabout twofold over this range. Seven normal subjectsgave a value 362±69 for the mean number of ouabain-binding sites per erythrocyte. One subject (C. S.)gave repeated values of 320, 326, 278, 332, and 322sites/cell at intervals over 2 yr and other subjectsalso gave reproducible results.

Potassium influx. The ouabain-sensitive (active)component of K+ influx was increased for the eightpatients with reticulocytosis (2.28±0.83 ,ueq/ml cellsper h) when compared with normals (1.46±0.20 ,ueq/ml cells per h; P < 0.01). However there was no dif-ference in ouabain-insensitive K+ influx between thetwo groups (0.47±0.17 ,ueq/ml cells per h for patientsand 0.46±0.10 ,ueq/ml cells per h for normals).

DISCUSSION

The influx of Ca2+ into human erythrocytes can onlybe adequately studied under conditions which inac-tivate the outward Ca2+ pumping. Selective inhibitionof the pump is difficult to achieve since rutheniumred, La3+ ions, or chlorpromazine2 block not only theoutward pumping of Ca2+ but also the passive inwardmovement of this cation. Rapid depletion of ATPhowever inhibits the Ca2+ pump and allows a measur-able uptake of Ca2+ ions (11). Circumstantial evidencesuggests that this uptake measures the passive Ca2+permeability of energy-replete cells. First, cells pre-treated with two different metabolic inhibitors (iodo-acetate and iodoacetamide) gave the same values forCa2+ uptake (Figs. 1B and 2) which suggests that Ca2+entry resulted from ATP depletion and not from amembrane effect of the inhibitor to increase Ca2+ per-meability. Second, Na+ influx into ATP-depleted cellswas about the same as for ATP-rich cells which agreeswith a previous study (17) and shows that iodoacetateor iodoacetamide pretreatment does not increase pas-sive permeability to cation. A rapid initial uptake of1-2 nmol 45Ca/ml cells occurs within the first 5 min ofincubation of erythrocytes with isotopic Ca2+. This

2Wiley, J. S., and C. C. Shaller. Unpublished observa-tions.

1116 J. S. Wiley and C. C. Shaller

8 12RETICULOCYTES 7

rmls FIGURE 3 Comparison of Ca2+ uptake with Na+ influx incells with reticulocytosis from 2 to 20%. Regression lineshave been fitted by the method of least squares with r

A = 0.71 for Ca2+ uptake and r = 0.88 for Na+ influx. Mean3 4 for normals-+ SDare shown for Na+ influx (upper rectangle)

and Ca2+ uptake (lower rectangle).

FIGURE 2 Calcium uptake by reticulocyte-rich erythrocytes,with either 11.4% reticulocytes (Retics.) (uppercurve) or 5.0% reticulocytes (lower curve). Cells were de-pleted of ATP by preincubation with inosine plus iodoace-tate (0), inosine plus iodoacetamide (0), or inosine plusiodoacetate plus cyanide (U). Uptake for normal erythrocytes+2SD is shown by the cross-hatching.

initial uptake is the same for either ATP-rich orATP-depleted cells and its rate is one to two ordersof magnitude faster than the subsequent uptake ofCa2+ by ATP-depleted cells. Such rapid kinetics sug-gest that the initial uptake is a distinct process fromtransmembrane Ca2+ movement and may represent anexchange of membrane 40Ca with 45Ca in the medium.The slow uptake of Ca2+ between 15 min and 4 hcan be shown with either 45Ca or total Ca2+ analysisand measures transmembrane movement. Values forthis flux confirm the very low Ca2+ permeability oferythrocytes, shown by Schatzmann and Vincenzi

TABLE IICalcium Uptake by Reticulocyte-Enriched

Normal Erythrocytes

Fraction Reticulocytes Ca2+ uptake

% nmol/ml celLsM4 h

Top 2.4 22.1Middle 0.8 9.2Bottom 0.3 8.5

Washed erythrocytes were fractionated by centrifugation at28,000 g for 60 min at 30°C. Cell ATP was then depleted byincubation for 90 min in media containing inosine plusiodoacetic acid. Finally Ca2+ uptake was measured over a4-h incubation in saline media containing 1.5 mM45Ca C12.

(18) for fresh cells stored at 40C in plasma and byothers for ATP-depleted cells at 370C (11, 19).

The main finding is that reticulocytes have a

Ca2+ permeability which is many fold greater than thatof mature erythrocytes. Both Na+ and Ca+ influx wereincreased in the presence of reticulocytosis but whileNa+ flux increased 2-fold, the Ca2+ flux increased11-fold for cells with 20%reticulocytosis. Extrapolationof these values suggest that reticulocytes have a 6-fold greater Na+ influx and 43-fold greater Ca2+uptake than mature cells. Two factors contribute tothis difference between reticulocytes and mature cells;first, the permeability to Ca2+ per square micrometerof membrane, and second, the membrane surface area.

Although the membrane area cannot be directly meas-ured, a relative estimate has been provided by the

-, 1200-

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RETICULOCYTES %

FIGURE 4 Correlation between ouabain binding sites percell and percent reticulocytes in the suspension. Regres-sion line has been fitted by the method of least squareswith r = 0.70. Mean for normals+1 SD is shown in theshaded rectangle.

Calcium Permeability of Reticulocytes 1117

40

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lipid antialyses of' Shattil anid Cooper (3). (C'oI memIl-branes of reticuilocytes lose 35%o of their c ihlesterolandl phospholipid on matutrationi which pr iilahx 'ic-compan-ies a decrease in memil)rane siirt`aI- irea !A'similar managnittude. Reticuilocyte volume also dlecreasesabouit 25-30% on matuirationi so that the i)temob)ranearea of reticulocytes may be ap)proxi mately It' O)Ir freaterthan that of mature cells when compared o)ni a oinitvolumnle basis. Although this estimcate is noVi Approxi-mate it does show that the many-fold reductiont Iln cationlfluixes on matuiration of the reticuilocyte canll b)e at-tribuite(d largely to a decrease in memlhranie p)erme-alility. The ratio of C(2":Na' flutxes .s mite in-dependent of' relative membranie area. TIhle a': Na'flulx ratio f;alls by a factor of about 7 as the :eticulo-cyte changes to an erythrocvte so that inaturation of'the reticiulocyte leads to a selective loss of Ca2;permeability.

Several lines of' evidence suiggest that reticuilocyteslose their high Ca2+ permeability on matuiration ratherthan diuring erythrocyte aging. First, the Ca2- influtxcorrelates well with percentage of' reticuilocvtes (Fig.3) btut this flutx does not correlate with ervthrocytecreatine which reflects the ImleaIn cell age (13, 20).Second, when erythrocytes are separated by cenitrifuiga-tion into fractions of varying ages, the fraction withold cells showed the same Ca2+ uiptake as the 1)1ulk ofthe red cell population. The mechanismn by which Ca2+permeability is selectively lost is not known althouglhit may relate to the disappearanice of membranie pro-teins specific to the reticulocyte (21). Absoliute valuesfor the Ca2+ influx may be caleulated fromii the Ca2+uptake of ATP-depleted erythrocytes (5.4 nmnnol/mIil cellsper 4 h) assuming a surface area of' 134 inn2 andmean cell volume of' 85 fl (22). For matiure cellsthe flux is 2 x 10-5 pmol/cm112 per s and for reticuilo-cytes about 1 x 10-3 pinol/c'm2 per s, although thelatter value is approximate and depends onl the as-suimption that reticulocytes and matuire cells have thesame membrane area per tuinit volume. This assiump-tioIn is only approximate since retictulocytes are moreosmotically resistant than matture cells which in(licatesa slightly greater surface area-to-volume ratio of' thesemore immatuire cells (3). Whatever the exact valtues,it is clear that erythrocyte Ca21 permeability is manyorders of magnitude less than for squiid axon, HeLacells, or ascites tumor cells where fluix aluies about0(.1 pmol/cm2 per s have been foOund (23-25).

The concept of a "normal" cell Ca2+ -oncentra-tion is imprecise since cell Ca2+ depends upon severalfactors such as ambient Ca2+ levels and the tumberof saline washes before dry ashing of erythrocytes.Previous stuidies have reported mean valuies for eryth-rocyte Ca21 of 16 and 15 nmol/ml cells (26, 27). Awide range of individual values from 5 to 22 nmol/mlcells was observed by Harrison and Long 126) who

1118 J. S. Wiley and C. C. Shaller

found that incltusion of EDTA in the Walilng mide(lilno-redutced mean ervthri-ocvte Ca2+ to onio. 2 nimol/unlcells. The present i-esiilts gave a meanIm erthro-flir-vrtcCa2+ of 5.9±+1.8 nmol/iml cells whether E(GTA w,vfaspresent in the Ca2tf'ree washing mediuimii o)r not.The discrepancies between these sttudies mray in p)artrelate to the small Ia2 contamination iabl)out 1-2,u M) always present in saline washing miedia if'previous studies. This Ca2' may associate with phos-phate anions leaking from the cells durinlyg %v'ashllinlgso that insoluble calciiumn phosphate formns oni tle cewllsturface and is incliide(l in the cell ( Ja24anal vsWhatever valuie is accepted for ervthrocvte Ca'2the analyses in this study employed conisisteint lilethio(dsso it is valid to concluide that reticulocvte-rich (5-25%) erythrocytes have the same total ,eil Ca(.concentration as matuire cells. However, C(a 2- Of retiCe i-locytes can exchange more completely with isotop1ic45Ca21 in the externacl imeditum, presunnablv becauseof the greater Ca2+ perineability of' reticuilocvte oiemI-branes.

Reticulocytes possess an increased nmimh)er of i:ationptimp sites wlicih have been (iltantitate(l in this studyby the [3H]ouiabain binding techniq(utie. Putmip siteswere increased abouit three-fold in cells with 20'%reticuilocytosis which extrapolates to aI valie of about4,000 sites for each reticulocyte. This value is 11-fold higher than the p)ump site numrbers touind formiatture erythrocytes of :362 ±69 sites/cell. Estti mates forthe ouabain-binding sites in matture huiman erythro-cytes range between 228 and 1,200 sites/cells 2b.29), with most stuidies finding between 250 and 500sites/cell (30-32). Active K+ influix into reticuilocyte-rich cells is also increased above normial althouighthe stimuilation of' fluix is less than expected fromi theincrement in pLump sites. This may be explained bythe low normal or suibnormal concentrations of' Na'in the retictilocyte (6).

Control of cytoplasmic Ca2+ concentration is an im-portant function of all cell types. Low cytoplasmicCa2+ concentrations rnay be achieved by acciunimula-tion of' this cation within organelles such as the sarco-plasmic reticuluim of muscle, the dense granuiles *ofblood platelets, or mitochondria of variouis cells. `I'tchuman erythrocyte lacks organelles and the low Ca'content of this cell is maintained by a calciuim pumpwhich extrudes this ion from the cytoplasrn to the cellexterior (18). This calcium pump is an ATPase, whichis active at the micromolar Ca2+ concentrations nor-mally present in human erythrocytes (33, 34). Energyrequirements by this calcium pump normnally are oniya negligible fraction of ATPproduction from glycolysis.The 43-fold decrease in Ca2+ permeability whichoccurs on maturation of'the retictilocyte thus effective v

limits the energy needed by the mature cell for Ca+homeostasis. Any failure by the mature cell to acquire

this low Ca" permeability will lead to cell destruc-tion, which is well illustrated bx the association oflhemolytic anemia with a congenital erythrocyte Ca2+leak (35).

A CKNOWLEDGMENTSWe thank Dr. Richard A. Cooper for helpful diseussionsand Dr. Toshio Asakura for measurements of ATP.

This work was supported by grant HL 16201 from the Na-tional Institutes of Health.

REFERENCES

. Stryckmans, P. A., E. P. Cronkite, G. Giacomelli, L. M.Schiffer, and H. Schnappauf. 1968. The maturation andf;ate of reticulocytes after in vitro labeling with tritiatedamino acids. Blood. 31: 33-43.

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