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Biochimica et Biophysica Acta, 584 (1979) 242--245 ~) Elsevier/North-Holland Biomedical Press

BBA 28864

ENDOCYTOSIS IN ADENOSINE TRIPHOSPHATE-DEPLETED ERYTHROCYTES

HAROLD ZARKOWSKY and JOHN RINEHART

Edward Mallinckrodt Department o f Pediatrics, Washington University School o f Medicine, and the Division o f Hematology-Oncology, St, Louis Children's Hospital, P.O. Box 148 71 St. Louis, MO 63110 (U.S.A.)

(Received September 5th, 1978)

Key words: Endocytosis; A TP depletion; Membrane invagination; Chlorpromazine; Vinblastine ; (Ery throcyte )

Summary

The extent of membrane invagination or endocytosis in intact erythrocytes was quantified by measuring the loss of acetylcholinesterase activity. Primaquine-induced endocytosis was completely inhibited in ATP-depleted cells. However, chlorpromazine and vinblastine were capable of inducing mem- brane invagination in depleted cells. With both drugs, the loss of enzyme activ- ity was less than that measured in fresh cells. We conclude that drug-induced endocytosis is not necessarily an energy-dependent process.

Introduct ion

A variety of agents can induce invagination of the erythrocyte membrane with formation of intracellular vacuoles [ 1 ]. When this process was quantified by a radioactive membrane-bound marker, Ben-Basset et al. [1] found markedly reduced to absent membrane invagination in ATP-depleted erythro- cytes. They concluded that membrane invagination or endocytosis was an energy-dependent process.

Endocytosis is also the final stage in the stomatocytic transformation of red cells from discocyte to bowl-shape to sphere [2]. Morphologic studies demonstrated that ATP-depleted red cells responded to the stomatocytic agent, chlorpromazine, and at high concentration invagination was observed [3]. Since these morphologic observations were not consistent with the quantative data, we measured the extent of membrane invagination in ATP-depleted cells exposed to several membrane-active drugs over a range of concentrations.

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Materials and Methods

Blood was obtained from normal volunteers and collected in heparin. The erythrocytes were washed three times and then suspended in phosphate buffered saline, pH 7.4 (PBS). ATP-depletion was accomplished by incubating the red cells suspension with 100 p/ml penicillin and 0.1 mg/ml s treptomycin for 20--24 h at 37°C. To restore ATP, depleted cells were washed once with PBS, resuspended at a 30% hematocrit in PBS with 20 mM adenosine and incubated at 37°C for 3 h.

Incubations with chlorpromazine were performed on 2% erythrocyte sus- pensions for 30 min at room temperature. A 30% erythrocyte suspension con- taining 0.5 mg glucose/ml was used for incubation with primaquine, and the incubations were for 1 h at 37°C. Vinblastine was added to 30% erythrocyte suspensions and incubations were performed at room temperature for 30 min. Preincubation of fresh cells with lysolecithin was performed at room temperature for 20 min. The cells were washed in PBS before incubation with chlorpromazine.

Membrane invagination was quantified by measuring acetylcholinesterase activity [4]. ATP was determined by NADP-coupled reaction on neutralized perchloric acid extracts of erythrocytes [5]. Erythrocytes were fixed in 1% gluteraldehyde for examination by light microscopy.

Results

The maximum concentrations of chlorpromazine and primaquine used in these studies were 0.25 mM and 2.0 mM, respectively. At these concentrations, the average decrease in acetylcholinesterase activity was 34% for chlor- promazine and 27% for primaquine. Studies on primaquine-induced endo- cytosis in human erythrocyte ghosts by Jarrett and Penniston [4] showed an approximately 25% reduction in acetylcholinesterase activity. As the concen- tration of chlorpromazine was decreased, less enzyme activity was lost (Table I). Although only bowl shapes with deep central depression were observed in cells exposed to 0.1 mM chlorpromazine, the loss of enzyme activ- ity indicates that some membrane internalization had occurred.

The ATP content of depleted cells was 0--10% of the initial level and incuba- tion with adenosine increased to approximately 80% of the initial value. In the depleted state, membrane invagination was minimal at the lower chlor- promazine concentrations. At 0.25 mM chlorpromazine, the loss of acetyl- cholinesterase activity in the ATP-depleted cells was 74% of that seen in the fresh erythrocytes. Upon repletion of ATP the response to all concentrations of chlorpromazine was comparable to that of the fresh cells.

Fresh cells were incubated with a glycolytic inhibitor 5.0 mM NaF for 30 min at 37°C before the addition of chlorpromazine. At the end of the pre- incubation the ATP level was reduced 60%. The loss of acetylcholinesterase activity was not affected by the lower ATP concentration and presence of NaF.

There was no endocytosis, i.e., loss of acetylcholinesterase activity, after ATP-depleted cells were incubated with primaquine. The loss of enzyme activ- ity in ATP-depleted cells treated with vinblastine was 25% of the value of fresh

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T A B L E I

A C E T Y L C H O L I N E S T E R A S E A C T I V I T Y OF I N T A C T E R Y T H R O C Y T E S

A c e t y l c h o l i n e s t e r a s e a c t i v i t y w a s m e a s u r e d p r i o r to exposu re to c h l o r p r o m a z i n e in fresh, ATP-dep l e t ed a n d ATP-res to red e r y t h r o c y t e s . T h e p e r c e n t of e n z y m e ac t iv i ty a f t e r i ncuba t ion wi th c h l o r p r o m a z i n e is p r e s e n t e d as t h e m e a n ± t h e s t a n d a r d d e v i a t i o n (n = 5).

C h l o ~ r o m a z i n e Fresh A T P - D e p l e t e d A T P - R e s t o r e d (mM~)

0 .10 8 7 . 6 ÷ 5 . 8 9 3 . 0 ± 3 . 8 8 0 . 4 ± 8 , 8 0 .15 7 5 . 0 + 4 . 5 8 9 . 3 + 6 . 0 7 6 . 8 ± 7 . 4 0 .25 6 6 . 4 ~ 5.2 7 5 . 1 ± 6 . 6 7 0 . 2 ± 4 . 6

cells similarly treated. After restoration of ATP with adenosine, the response to both vinblastine and primaquine was comparable to that seen with fresh cells.

ATP-depleted cells were a mixture of echinocytes and spheroechinocytes. In order to produce a more homogeneous population of echinocytes and smooth spherocytes, red cells were treated with lysolecithin. Smooth spheres were produced with 100 pg of lysolecithin/ml of red cells. When these cells were incubated with 0.25 mM chlorpromazine, no enzyme loss occurred. Endo- cytosis, as measured by loss of enzyme activity, was not inhibited by prior t reatment with concentrations of lysolecithin between 20 and 50 pg/ml cells.

Discussion

In drug-induced shape transformation, echinocytic and s tomatocyt ic agents are antagonistic [2]. At an appropriate concentrat ion a s tomatocyt ic agent can neutralize the effect of an echinocytic drug and restore the red cell's discoidal shape. As the concentration is increased, bowl shapes and other s tomatocyt ic shape changes develop [6]. ATP-depleted cells are echinocytes. Therefore, two factors might effect the response of ATP-depleted cells to s tomatocyt ic agents: 1, lack of a necessary source of energy; and 2, the biophysical perturbations of the membrane. If the latter were a factor in retarding endocytosis, the inhibi- tion might be overcome by increasing the concentrat ion of the s tomatocyt ic agent. The results of the studies with chlorpromazine illustrate this point. The extent of membrane invagination that occurred in ATP-depleted cells at 0.25 mM chlorpromazine was comparable to that attained by fresh cells incubated in 0.15 mM chlorpromazine. Although ATP-depleted cells did not invaginate as extensively as did fresh cells, it is clear that ATP is not a requirement for endo- cytosis induced by chlorpromazine. The presence of NaF also failed to inhibit endocytosis. The response of lysolecithin-induced ATP-replete spheres to chlor- promazine also suggests that the physical constraints of cell shape affect endo- cytosis.

Ben-Basset et al. [1] reported that the extent of chlorpromazine-induced membrane invagination in ATP-depleted cells was only 3% of that measured in fresh cells. More extensive studies on drug-induced endocytosis were performed using primaquine, hydrocort isone and vinblastine. In all cases ATP-depleted cells had markedly impaired membrane invagination. Our results confirm the

2 4 5

adverse effect of ATP-depletion on primaquine-induced endocytosis. The mechanism of endocytosis is unknown and might vary with the inducing

agent. Schrier et al. [ 7] demonstrated that the requisites for maximum mem- brane invagination varied with the agent under study. Penetrating anions in the medium were necessary for the action of primaquine, whereas hydrocortisone was most effective in low ionic strength medium containing sucrose. Our studies show that the effect of ATP-depletion also varies with the agent. We suggest that endocytosis is not invariably an energy dependent process. Perhaps, the biophysical perturbations induced by ATP-depletion, rather than the absence of a source of energy, account for the apparent requirement for ATP.

Acknowledgement

This study was supported by the National Institutes of Health Grant HL-20241-02.

References

1 Ben-Bassat, I., Bensch, K. and Schrier, S? (1971) J. Clin. Invest. 51, 1833--1844 2 Deutickc, B. (1968) Bioch im. Biophys. Acta 163 ,494 - -500 3 Zarkowsky, H., Mohandas, N. and Speaker, C. (1973) Proc. Am. Soc. Hematol. p. 97 4 Jarret t , H. and Penniston, J. (1976) Biochim. Biophys. Acta 448, 314--324 5 Adam, H. (1963) in Methods of Enzymatic Analysis (Bergmcyer, H., ed.), p. 539, Academic Press,

New York 6 ChalUey, B., Weed, R., Lcblond. P. and Maigne, J. (1973) Nouv. Rev. ft. Hematol . 13, 71--88 7 Schrier, S., Junga, I. and Secger, M. (1974) J. Lab. Clin. Med. 83 ,215- -227