(CANCER RESEARCH 47, 4830-4834, September 15, 1987]

Differences between Human and Goose Erythrocytes in Response to Phorbol Estersand Expression of Phorbol Ester Receptors1

Louis A. Speizer,2 Sue E. Atherton, and Julianne J. Sando3

Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908

ABSTRACT

Phorbol esters inhibited the uptake of a fluorescent glucose analoguein goose but not in human erythrocytes. Specific phorbol-12,13-dibutyrate(PDB) binding sites were identified in both goose and human erythrocytes.In the absence of Ca2* and phospholipid, PDB binding in whole celllysates was similar to that in intact cells, but addition of Or* (0.5 m\i)

and phosphatidyl serine (96 Mg/ml)caused a 4-fold increase in the bindingdetected in lysates. Nonlinear least-squares analysis of the PDB bindingisotherm revealed that the data for lysates from both goose and humancells were best fit by a two-site model, with goose erythrocytes havingapproximately 3 times as many sites per class of receptors. Subcellularfractionation of human lysates indicated that the high (K,, = 3.6 ±2.2MM)and low (A,, = 20 ±5 nM) affinity sites could be accounted for bythe contributions from cytosol and crude membrane, respectively. Separation of the high and low affinity sites was not achieved in goose lysates.PDB binding to intact goose erythrocytes exhibited the lower affinity (Kt-30 UM)and was enhanced ~2-fold by incubation at 37°Crelative toincubation at 4°C.This was due to an increased Bni,v,with no change inA'j of the whole cell binding. Human erythrocytes did not demonstrate

this temperature-enhanced binding of PDB to intact cells. These data areconsistent with a temperature-induced translocation of PDB receptorsfrom cytosol to membrane in goose erythrocytes. The failure of humanerythrocytes to respond to PDB is not due to an absence of PDB receptorsbut may be related to the diminished number of receptors or to the lackof a temperature-induced increase in whole cell receptor number.

INTRODUCTION

Many effects of PE4 tumor promoters are exerted at the level

of the plasma membrane. PEs influence various cell surfacereceptors including those for insulin (1), epidermal growthfactor (2, 3), transferrin (4, 5), and a- and /3-adrenergic agonists(6-8). They also affect transport of a number of ions and smallmolecules such as Ca2+ (9), lactate (10), and glucose (11-13).These effects are consistent with the presence of PK-C, the PEreceptor (14-21), in membrane and the PE-induced translocation of PK-C from cytosol to membrane (22, 23).

In this report, we show that PEs inhibit the uptake of afluorescent glucose analogue (NBDG) in goose but not inhuman erythrocytes. The lack of PE effect in the human cellswas consistent with earlier reports (24, 25) that human erythrocytes lacked PE receptors. However, we were able to detectspecific PE binding in both human and goose erythrocytes.Several differences between the PE binding of human and gooseerythrocytes were noted, though, and these differences mightexplain differences in the functional response to PE.

Received 10/22/86; revised 6/1/87; accepted 6/23/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by NIH Grant GM31184.2Initially supported by NIH Training Grant HL07284. Present address: Di

vision of Pharmacology, M-013H, Dept. of Medicine, University of California,San Diego, San Diego, CA 92093. To whom requests for reprints should beaddressed.

3 Recipient of American Cancer Society Grant FRA-306.4The abbreviations used are: PE, phorbol ester; PK-C, protein kinase C;

NBDG, 6-deoxy-iV-(7-nitrobenz-2-oxa-I,3-diazol-4-yl)amino glucose; PDB,phorbol-12,13-dibutyrate; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidyl serine; PBS, phosphate-buffered saline, 5 min Na2HPO4/150 mM NaCI, pH7.4.

MATERIALS AND METHODS

Materials. [20-3H]PDB (13.4 or 12.5 Ci/mmol in ethanol; 1 Ci =3.7 x IO10Bq) was purchased from New England Nuclear. Unlabeled

phorbol esters, DNase I (type II from bovine pancreas), and RNase A(type I-AS from bovine pancreas) were obtained from Sigma. PS wasobtained from Avanti Polar Lipids or Sigma. Freshly drawn gooseblood was obtained from Colorado Serum, and isolated erythrocyteswere stored in a modified Alsever's solution. The cells were used within

1 to 2 days after the blood was drawn. Human RBC were freshlyisolated from blood drawn on the day of the experiment. NBDG wasobtained from Dr. Richard Haugland of Molecular Probes. Ficoll-Pacque was from Pharmacia.

Erythrocyte Isolation and Fractionation. Both human and goose bloodcells were washed, layered onto Ficoll-Pacque (0.1425 mM Ficoll 400/141.5 mM diatrizoate sodium), and spun at 1600 rpm for 20 min. Thepelleted erythrocytes were subjected to a second centrifugation throughFicoll and washed 2 additional times with PBS (5 mM Na2HPO4/150mM NaCI, pH 7.4). In one experiment the cells were washed 3 times inPBS and then passed through an a-cellulose:microcrystalline cellulose(1:1) column which permits erythrocytes to pass through while retainingapplied leukocytes and platelets (26). For whole cell experiments,erythrocytes were diluted with PBS. Whole cell lysates were preparedin water. Cytosols (supernatant) and crude membrane (pellet) fractionswere prepared by centrifugation of lysates at 100,000 x g. Humanmembrane was washed once by resuspension in water and centrifugationat 27,000 x g for 15 min and ultimately resuspended in a volume ofwater equal to one-half to 1 times that of the cytosol. For gooseerythrocytes, which are nucleated and thus contain a lot of DNA,membrane fractions were treated at 4°Cfor 15 min with 250 /ig/ml

DNase I, 75 Mg/ml RNase, and 5 mM magnesium acetate in 49 mMTris, pH 7-8. Although DNase did not affect the affinity of PDBbinding when added to highly purified PK-C from rat brain, the DNasepreparation used in many of the experiments was subsequently foundto decrease the number of sites by up to 50%. One possible explanationfor this effect is contamination of the enzyme with proteases.

PDB Binding Assay. Binding of [3H]PDB was carried out as described(16). Briefly, intact or lysed cells or subcellular fractions (10* to 10'cells or cell equivalents in 0.33 to 0.36 ml) were incubated with [3H]-

PDB (0.1 to 120 nM for Scatchard analysis, 40 nM elsewhere), 0.5 HIMCaCI2, 75 mM magnesium acetate, bovine serum albumin (1.2 mg/ml),PS (96 Mg/ml), and either vehicle (1 to 2% ethanol) or 3 n\i unlabeledPDB or PMA in a total volume of 0.5 ml. Incubations were carried outat 4°C,pH 7.4, for 2 to 18 h or, in the experiments of Figs. 4 and 5, at37°Cfor up to 8 h. Bound [3H]PDB was separated from free by rapid

filtration through glass fiber filters. The filters were decolorized with30% H2O2 (0.4 ml) prior to addition of scintillation fluor (ACS II;Amersham) and counting. Specific binding represents the differencebetween the binding in the absence and that in the presence of excesscompetitor.

NBDG and 3-O-Methylglucose Uptake. NBDG uptake was determined spectrofluorometrically as previously described (27). Briefly,goose or human erythrocytes were washed 3 times in glucose-freeHanks' balanced salt solution (142 mM NaCl/5.4 mM KC1/0.57 mM

Na2HPO4/0.74 mM KH2PO4/1.8 mM CaCl2/0.83 mM MgSO4/16.7mM NaHCO3, pH 7.4), suspended to a 10% hematocrit, and incubatedat 37°Cfor l h in the presence or absence of 100 nM PMA. They were

then incubated with 108 MMNBDG, and uptake was monitored atvarious times by stopping the transport with 3 washes in stoppingsolution (1% NaCl/1 mM HgCl2/1.25 mM KI/100 MMphloretin) (28)at 4°C.Cells were lysed in 2.5 ml of Tris-HCl/5 HIMNaCI, pH 7.4,

hemoglobin was precipitated with 0.38 ml of 20% trichloroacetic acid,

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PHORBOL ESTER RECEPTORS IN HUMAN AND GOOSE RBC

and NBDG content of the soluble fraction was assayed spectrofluoro-metrically. In one experiment, PMA-treated cells were incubated with108 MMNBDG and 10 nM 3-0-[3H]methylglucose. In this case, twoaliquots of the trichloroacetic acid-soluble supernatant were assayed,one for NBDG and one for 3-0-[3H]methylglucose by scintillation

counting. The intracellular water available for NBDG movement wasassumed to be equal to the 0.525 ml water/ml goose erythrocyte asdetermined by Wood and Morgan (29).

Data Analysis. All experiments were performed at least twice withsimilar results. Nonlinear least-squares analysis of the data was performed on a PDP-11/23 minicomputer utilizing the program NONLIN

(30).

RESULTS

Effect of PMA on NBDG Uptake in Human and GooseErythrocytes. NBDG has been shown to interact with the hex-ose transport system of the human erythrocyte (27). Since thetransporter in human erythrocytes is not hormonally regulated,goose erythrocytes were examined to determine whether NBDGuptake could be affected by agents which regulate glucosetransport. Since PMA regulates glucose uptake in numeroussystems, the effects of PMA on NBDG uptake were investigated. As shown in Fig. 1, 100 nM PMA inhibited NBDGuptake by 37% (K, = 5 nM), but did not inhibit uptake of 3-0-methylglucose. This failure of maximally effective concentrations of PMA to inhibit uptake of 3-O-methylglucose suggeststhat the PMA effect is exerted at a site other than the hexosetransporter. No effect of PMA was observed on NBGD or 3-O-methylglucose uptake in human erythrocytes (data notshown).

Identification of PE Receptors in Goose and Human Erythrocytes. The inhibition by PMA of a component of NBDG uptakein goose erythrocytes suggested the presence of PE receptors inthe goose cells. To test this possibility, we examined [3H]PDB

binding in both lysed and intact goose erythrocytes. Table 1shows that lysed cells without PS exhibit about the sameamount of PDB binding as do intact cells with PS. Addition ofPS with CaCl2 enhanced the binding in lysed cells approximately 4-fold. Since more binding could be detected in lysatesthan in whole cells, these preparations were used for optimizing

9060

ó i

•0 200 300

TIME (mimjlesl

Fig. 1. Effect of PMA on uptake of NBDG and 3-O-methylglucose in gooseerythrocytes. Goose erythrocytes were preincubated with vehicle (O) or 100 nMPMA (•)for I h at 37°C.They were then transferred to medium containing 108ii\i NBDG (. I ) or l o n\i 3-O-methylglucose (B), and uptake was measured at theindicated times as described in "Materials and Methods." Points, mean; bars, SD.

Table 1 Conditions for identification ofPE receptors in goose erythrocytes

ConditionsSpecifically bound PDB

Cells Method of Isolation CaCl2° PS* (cpm/10" cells)

IntactLysedLysedLysedLysedFicoll ++Ficoll+Ficoll

-+Ficoll++Ficoll,

a-cellulose + +190

±60*280

±80520±60930±120950±130

' +, added CaCI2 (500 ¿IM);-, no added CaCl2. Endogenous CaCI2 ~ 1 I¡M.* +, added PS (96 fig/ml); -, no added PS.c Mean ±SD.

0 S

¡ly ~E

\2O

80

40

20 40 60 80SPECIFICALLY BOUND PDB (fmol/IO8 calls)

00

Fig. 2. SLali-hard analysis of [3H]PDB binding to whole cell lysates of goose(•)or human (A) erythrocytes. Binding was performed at 4"C as described in"Materials and Methods." The data have been fit to a 2-site model.

the binding assay. The concentrations of CaCl2 (0.5 HIM),magnesium acetate (75 HIM),and PS (96 ng/m\) were demonstrated to be optimal for detection of binding in these lysates,and binding was observed to reach equilibrium within 1 to 2 hat 4°C,to be stable for at least 20 h, and to be reversible (data

not shown). Nonspecific binding was approximately 500 cpmat 40 nM PDB. This represented ~30% of the total binding inlysates and often more than 60% of the total binding in intactcells.

Although leukocytes and platelets are only a few percentagesas numerous as erythrocytes in blood, the number of phorbolester binding sites on leukocytes and platelets may be highenough to influence the apparent binding of [3H]PDB to eryth

rocytes. Microscopic examination of our cells (purified throughFicoll and washed with PBS) did not reveal the presence of anycells other than erythrocytes. However, to further rule outcontamination by other blood elements, we also passed the cellsthrough an a-cellulose:microcrystalline cellulose (1:1) columnwhich removes greater than 99.8% of the applied leukocytesfrom whole blood (26). These highly purified erythrocytesshowed PDB binding that was identical to that in the standardpreparation (Table 1), suggesting that the standard preparationyields pure erythrocytes. Since the standard procedure gavemuch better cell yields, we used that purification procedure forother experiments.

Fig. 2 shows the Scatchard analysis for binding of [3H]PDB

to lysed preparations of both human and goose erythrocytes. Itis readily apparent that goose cell lysates have many morereceptors than do human cells. However, the human cells clearlydo have detectable PE binding capacity. Curvilinear Scatchardplots suggest the existence of more than one class of bindingsites in lysates from erythrocytes of both species. Analysis byleast-squares shows the data to be consistent with two classesof receptors in each cell type with similar affinities in humanand goose. Human erythrocyte lysates have approximately 100sites/cell of each affinity class, whereas the goose cell lysateshave ~300 sites/cell of each class.

In an attempt to separate the two classes of receptors, we4831

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PHORBOL ESTER RECEPTORS IN HUMAN AND GOOSE RBC

prepared cytosolic (100,000 x g supernatant) and crude membrane (100,000 x g pellet) fractions from the lysates. Fig. 3shows the Scatchard analysis for data from human cells. Asingle class of PDB binding sites is present in each fractionwith the cytosolic site exhibiting a higher affinity than themembrane site. Fractionation of goose lysates did not provideseparation of the two affinity sites. This may have been due tothe technical difficulty of having to use such a large number ofnucleated cells. The 100,000 x g centrifugation ruptures nuclei.While constituents released into the cytosol may contribute tothe inability to observe a high affinity site in that fraction, thelarge amount of released DNA contaminates the membraneand makes pipetting impossible. Attempts to remove nucleiprior to centrifugation resulted in large decreases in yields and,because of the high cell density required, significant cross-contamination of the various subcellular fractions. We thereforedid not pursue the subcellular fractionation with the goose cellsbut treated the membrane preparation with DNase to permitpipetting of the samples.

Quantitäten of the equilibrium binding data from severalexperiments with human and goose subcellular fractions ispresented in Table 2. The low affinity site in lysates from bothgoose and human cells is of comparable affinity to the site inmembrane. The high affinity site in lysates is of somewhathigher affinity than the site in cytosol, especially in the case ofthe goose cells. Whether this might reflect dissociation of thereceptor from constituents separated into the pellet fraction orassociation of the receptor with constituents released fromnuclei or other subcellular organelles is unknown. The totalnumber of sites in whole cell lysates of human erythrocytes canbe accounted for by the sum of the sites in cytosol plus membrane. The discrepancy in goose erythrocytes between the sumof cytosolic and membrane sites and the total number of sites

I

\\

an

20 30 O I 2

SPECIFICALLY BOUND POB (fmol/IOB cdli)

Fig. 3. Scatchard analysis of [3H]PDB binding to subcellular fractions ofhuman erythrocytes. Cytosol (A, A) or membrane (B, A) fractions of humanerythrocytes were incubated with the indicated concentrations of [3H]PDB for 2h at 4°C.and binding was assayed as described.

Table 2 PDB binding in subcellular fractions of goose and human erythrocytes

SpeciesHumanGooseCellfractionWhole

celllysateCytosolMembraneWhole

celllysateCytosolMembrane«,,0.33.60.412(nM)±0.1*±2.2±0.2±3Bm„,

(sites/cell)110±27178

±68340

±30383±57(HM)17±

1120

±528

+2015±4Bmu2

(sites/cell)101

±1831

±12330

±61102

±34n"232143" Number of experiments.* Means ±SD.

in whole lysates is probably accounted for by the decrease innumber of membrane sites due to treatment with some preparations of DNase.

Effect of Temperature on PDB Binding to Intact Cells. Incubation of cell lysates at 37°Cas opposed to 4"C had no effect

on the equilibrium binding of PDB in human or goose erythrocytes. However, incubation of intact goose cells at 37°Cinstead of 4°Crevealed an increase in the amount of PDB

bound at equilibrium of 3.3 ±0.4-fold, n = 3 (Fig. 4). Thistemperature-induced increase in whole cell PDB binding wasnot observed in intact human erythrocytes (ratio of binding at37°Cto 4°C= 0.9 ±0.2, «= 2). Since PDB binding to intact

goose erythrocytes did not decay after reaching equilibrium at37°C,we were able to analyze the effect of temperature on

receptor number and affinity. Fig. 5 shows that the increasedtemperature caused an increase in the number of detectable PEreceptors (from 121 ±3 sites/cell to 280 ±50 sites/cell) withoutcausing a significant change in the affinity (Kd at 4°C= 31 ±13 HM, Kt at 37°C= 36 ±12 nM, n = 3 experiments). This

increase in receptor number was reversed when cells which hadbeen incubated at 37°Cwere returned to 4°C(data not shown).

Thus, it was not possible to isolate cytosol and membranefractions after incubation of cells at 37°Cto determine directly

whether increased temperature could cause a shift in subcellularlocalization of PE receptors. While this effect is clearly notinduced by PE at 4°C,it is possible that the presence of PE isrequired to see this effect at 37°C.

DISCUSSION

Our observation that PMA inhibits a component of NBDGuptake in goose erythrocytes is consistent with reports of otherPE effects in other nucleated erythrocytes. PEs have been shown

100 300200TIME (minutes)

Fig. 4. Time course for the effect of temperature on binding of (3H)PDB tointact goose erythrocytes. Intact goose cells were incubated with 40 nM | '11|I'I)Hat 4'C (O) or 37'C (•),and binding was assayed at the indicated times.

20 40 60 80 100

FREE PDB CONCENTRATION (nM)

Fig. 5. Concentration dependence for the effect of temperature on binding of[3H]PDB to intact goose erythrocytes. Intact goose cells were incubated with theindicated concentrations of [3HJPDB at 4'C (O) or 37'C (•)for 2 h. Binding was

assayed as described.

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PHORBOL ESTER RECEPTORS IN HUMAN AND GOOSE RBC

to inhibit isoproterenol-stimulated adenylate cyclase activity inturkey (6) and duck (8) erythrocytes and to potentiate adenylatecyclase activity in frog erythrocytes (31). The identification ofPE receptors in goose erythrocytes was thus not surprising.

Effects of PEs on hormone-stimulated adenylate cyclase hadnot been studied in human erythrocytes because isoproterenoldoes not stimulate adenylate cyclase in the human cells (32).Our finding that PEs failed to inhibit NBDG uptake in humanerythrocytes was compatible with earlier reports (24, 25) thathuman erythrocytes lacked PE receptors. However, we didobserve specific PE binding in highly purified human erythrocytes. It is likely that these receptors were missed in earlierstudies for two reasons, (a) The number of receptors/cell isquite low, and thus large numbers of cells and (3H]PDB of high

specific activity must be used to allow detection, (b) The hemoglobin present in erythrocytes effectively quenches the detectionby scintillation counting of radioactivity associated with thecells. Use of large numbers of cells, high specific activity [3H]-

PDB, and decolorization of cell samples with H2O2 alloweddetection of the PDB binding. Recently. Palfrey and Waseem(33) demonstrated PK-C activity and existence of PK-C substrates in human erythrocytes. Based on their assumption ofthe specific activity of cytosolic PK-C, they predicted 300molecules/cell. This value is in excellent agreement with ourfinding of 211 ±45 sites/cell as calculated from PE binding inlysates.

The PE binding in both goose and human erythrocytes issimilar to that in other cells. Cytosolic binding is dependent onCa2+, Mg2*, and PS at concentrations similar to those required

in other cells, such as the EL4 thymoma line (16). As with EL4cells, Scatchard analysis of PE binding in whole cell lysates oferythrocytes indicated more than one class of binding sites. Inthe case of the erythrocytes, it was possible, by least-squaresanalysis of the data (30), to separate the binding into twocomponents. Separation of the human whole cell lysate intocytosolic and crude membrane fractions effectively separatedthe two components, yielding the high affinity site in cytosoland the low affinity site in membrane. Sharkey et al. haveshown that binding sites of different affinities can be accountedfor by association of a single aporeceptor with phospholipidsof different composition (34), and it is likely that this effectaccounts for the two affinity sites we detect. The only phospho-lipid present in the assay of cytosolic binding is the added PSwhich is the most effective phospholipid for supporting PEbinding (16, 17). The membrane preparations also containendogenous lipids, including phosphatidyl choline, which candiminish PE binding affinity.

In EL4 cells, whole cell binding was comparable to that inlysates, but in these cells, the majority of the PE binding wasrecovered in the membrane fraction (16). In both goose andhuman erythrocytes the majority of the PE binding (75 to 85%)was recovered in the cytosolic fraction. The facts that fewer PEbinding sites were detected in intact as opposed to lysed erythrocytes and that the A',,for binding to intact cells was similar

to that for the membrane site suggest that only those receptorspresent in the membrane can bind PE. Since PE binding tointact erythrocytes at 37°Cwas stable after reaching equilibrium

(unlike that in EL4 cells) (35), it was possible to analyze theeffect of temperature on whole cell binding. Human erythrocytes showed identical binding at 4°Cand at 37°C;however,incubation at 37°Ccaused a 2- to 3-fold increase in the PE

binding detected in intact goose cells. This did not appear to bedue to a toxic effect, since no hemolysis was observed and theeffect was reversible. Analysis of the binding isotherms indi

cated that the increase was due to an increased number ofreceptors rather than to the ability to detect a receptor of higheraffinity at 37°C.This result is compatible with the interpreta

tion that a temperature-dependent translocation of PE receptorfrom cytosol to membrane occurs.

Several explanations are possible for the failure of humanerythrocytes to respond to PE with an inhibition of NBDGuptake. The previous conclusion that these cells lack PE receptors (24, 25) is no longer tenable since we can now detectspecific PE binding, and Palfrey and Waseem (33) have detectedPK-C activity in human erythrocytes. One possibility is that thehuman cells lack the component of NBDG uptake that isinhibitable by PE, and thus the PK-C target is missing. It wasshown in human cells that NBDG accumulates due to itsbinding to hemoglobin (27). It seems unlikely that PK-C isdirectly affecting this binding component in goose cells becausepurified hemoglobin did not serve as a PK-C substrate.5 Other

possibilities are that the diminished number of PE binding siteson human as compared with goose erythrocytes or, perhapsmore importantly, the failure of the human cells to exhibit atemperature-dependent increase in whole cell binding may contribute to the lack of response.

ACKNOWLEDGMENTS

We would like to thank Dr. Howard Kutchai and Dr. CarolineKramer for helpful discussions during this work and Dr. B. W. Turnerfor providing purified human hemoglobin.

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