Neurochemical Research (1) 201-215 (1976)

U P T A K E OF N E U R O T R A N S M I T T E R S A N D P R E C U R S O R S BY C L O N A L L I N E S OF A S T R O C Y T O M A A N D NEUROBLASTOMA: III. T R A N S P O R T OF C H O L I N E H.T. HUTCHISON, R.L. SUDDITH, M. RISK, AND B. HABER Department of Human Biological Chemistry and Genetics The Division of Comparative Neurobiology The Marine Biomedical Institute and Department of Neurology The University of Texas Medical Branch Galveston, Texas 77550

Accepted February 9, 1976

Clonal lines of glial, neuronal, and nonneural origin accumulate choline via a high-affinity carrier-mediated transport system with K,, in the range of 10-14 txM. These cell lines also accumulate choline by a second system that is not saturable at 10 mM choline, and that may represent diffusion. The transport of choline in glial cells differs from that seen in neuronal ceils with respect to its Na + requirement. The omission of Na + from the incubation medium reduces high-affinity choline transport in neuronal cells and enhances it in glial cells. Kinetic analysis of the data indicates that reversible cholinesterase inhibitors and hemicholinium-3 (HC-3) inhibit the high-affinity transport system for choline. On the other hand, the diffusional or low-affinity component of choline transport in either cell type appears to have no Na + requirement and is unaffected by either cholinesterase inhibitors or 10-4 M HC-3. The neuronal-glial differences in the Na + requirement of choline transport may be related to the coupling of transport to choline metabolism, which differs in the two cell types. The presence of a high-affinity transport system for choline in clonal glial lines used as models of normal glia suggest that glia may modulate the availability of choline for acetylcholine synthesis at cholinergic synapses.

INTRODUCTION Acetylcholine inactivated

by

(ACh)

differs from

extracellular

other

hydrolysis,

neurotransmitters rather

than

by

i n t h a t it i~s reuptake

into

201 @ 1976 Plenum Publishing Corporation., 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.

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presynaptic nerve endings and into glia. F u r t h e r m o r e , the central nervous system is incapable of de novo synthesis o f choline (1,2). Thus, for the synthesis of ACh, cholinergic synapses must depend on the uptake either of exogenous choline or of choline formed by hydrolysis of ACh. Evidence that choline may be rate-limiting for ACh synthesis was first shown in frog sympathetic ganglia (1), in which repetitive stimulation fails in the absence of exogenous choline. The suggestion that reuptake of choline is important to cholinergic function has led to a number of investigations of the transport of choline into brain slices (35) and synaptosomes (6,7). These studies have shown that both slices and synaptosomes accumulate choline via a high-affinity, saturable mechanism and by a second, less well characterized c o m p o n e n t of lower affinity. It has been suggested that, at cholinergic synapses, the reuptake of choline by the high-affinity system is tightly coupled to the synthesis of ACh (6,3). The understanding of the role of the various cellular c o m p o n e n t s of the nervous system has been accelerated by using clonal cell lines derived from tumors of neural origin as models of the individual cell types within the nervous system. In particular, the use of these cell lines to assess the role of the corresponding cell types in neurotransmitter reuptake in vivo is well established (8-15). We have chosen to study the transport of choline by clonal cell lines of neuronal (NB41 and N 2 A G ) and glial (C6 and RN22) origin. The presence of a high-affinity transport system for choline has been shown in astrocytoma cells by Richelson and T h o m p s o n (13), by ourselves (8), and subsequently in neuroblastoma cells by others (10,11). In this study, we have characterized the kinetic characteristics of choline transport by both cell types with respect to ionic requirements and inhibition by cholinergic drugs. Furthermore, we have found that glial and neuronal cell lines respond in opposite directions to removal of sodium from the incubation medium.

EXPERIMENTAL

PROCEDURE

The NB41 neuroblastoma, a subclone of the C13013murine neuroblastoma and the C6 rat astrocytoma, which were originally described by Benda (16), were kindly supplied by Dr. Jean de VeUis. The N2AG neuroblastoma, the B16 melanoma, the RAG mouse renal adenocarcinoma, and the HTC rat hepatoma cell lines were supplied by Dr. R. Klebe at UTMB. The B50 line is an N-nitrosomethylurea-induced rat tumor cell line (17) described as neuronal, and provided by Dr. D. Shubert, Salk Institute. The RN22 has been described as a peripheral neurinoma (18), and was supplied by Dr. S. Pfeiffer. The human

N E U R O N A L - G L I A L CHOLINE TRANSPORT

203

meningioma was cultured from a biopsy specimen supplied by the Division of Neurosurgery at UTMB. Human fibroblasts were derived from a biopsy scar. Growth of these cell lines and measurement of choline uptake was as described by Hutchison et al. (9), except that unmodified Hanks' Balanced Salt Solution (containing 141 mM sodium) (19) was used as the incubation medium. Hanks BSS was modified to contain an isoosmotic amount of sucrose in experiments where the external Na + concentration was varied. Sodium phosphate and sodium bicarbonate, however, remain in the medium; thus, the sodium concentration in "sodium-free" Hanks' BSS is reduced to 4.5 raM. Protein was determined by a modification of the method of Lowry et al. (20). The [3HI-choline (sp act >2 Ci/mmol) was purchased from New England Nuclear, and all other reagents were analytical grade. Hemicholinium-3 (HC-3), neostigmine, and physostigmine were made up fresh, and added to the incubation medium at the stated concentrations. Unless stated otherwise, the incubation times were 5 rain. The washing procedure used to stop the uptake was as follows: It was found that 5 washes with ice-cold saline left a large and variable amount of radioactivity bound nonspecifically to the cells. This material was not removed by washing with 1 mM choline in saline or with 0.15 M NH4CI at 0~ Following the incubation with labeled choline,

,! o InitialIncubation Time

~

,b

Post Incubation Time (Mln)

FIG. 1. Time course of removal of nonspecifically bound radioactive choline from NB41 neuroblastoma. The inverted triangles ( , ) in curve A represent the radioactivity present in cells incubated with 3H-choline (0.21/xM) for the indicated times and washed immediately 5 times with ice-cold NH4C1. The erect triangles ( A ) in curve A represent the radioactivity present in cells incubated with all-choline for 5 rain, postincubated at 37~ with Hanks' BSS containing 1 mM choline for the indicated times, and then washed with ice-cold NG4CI. The points in curve B represent the radioactivity present in cells incubated with all-choline for the indicated times, postincubated in Hanks' 1 mM choline for 2 rain, and then washed with ice-cold NH4Cl. Each point represents the mean of 4 determinations.

204

H U T C H I S O N ET AL.

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Post Incubation Time (Min)

Fzo. 2. Time course of removal of nonspecifically bound radioactive choline from human fibroblasts in culture. The inverted triangles (!?) in curve A represent the radioactivity present in cells incubated with all-choline (0.21 /zM) for the indicated times and washed immediately 5 times with ice-cold NH~C1. The erect triangles (&) in curve A represent the radioactivity present in ceils incubated with 3H-choline for 10 min, postincubated at 37~ with Hanks' BSS containing 1 mM choline for the indicated times, and then washed with ice-cold NH4CI. The points in curve B represent the radioactivity present in cells incubated with 3H-choline for the indicated times, postincubated in Hanks' 1 mM choline for 10 min, and then washed with ice-cold NH4CI. Each point represents the mean of 4 determinations.

therefore, the ceils were washed 3 times with Hanks' BSS containing 1 mM choline at 37~ the third wash was left on the cells for the specified time period (usually 2 min). The cells were then washed rapidly 5 times with ice-cold 0.15 M NH4CI. The remaining radioactivity, which is stable for 10 min, represents intracellular chofine and choline metabolites. Figure 1 shows that for the NB41 neuroblastoma, the nonspecifically bound radioactivity is completely removed by a 2-min incubation in Hanks' BSS containing 1 mM choline, and that the remaining radioactivity is stable over a 10-min period under these conditions. Similar curves were obtained for the N 2 A G neuroblastoma and for the C6 and RN22 glial cell lines used in this study. In contrast, the choline bound nonspecifically to human fibroblasts is removed only after 10 min (Fig. 2) of a postincubation in Hanks' BSS containing 1 mM choline. These additional washing procedures improve the accuracy, but do not materially change the estimates, of the Km values of the high-affinity transport system. The kinetics of choline transport were analyzed by hyperbolic least-squares regression techniques, as previously described (21). The fitted equation for a two-carrier mediated transport system is V

Vmax~

S

Kml+S

_t-

Vmax~

K,,.,~+S

205

NEURONAL-GLIAL CHOLINE TRANSPORT

and for a one-carrier mediated transport system and diffusion it is

V_

S

Vmax~ + D

Kml +S

Where K~ and Vmaxare Michaelis constants and maximum velocities, respectively, and D is a diffusion constant. The velocities, V, have units of pmol choline taken up/rag cell protein per rain incubation; the substrate concentrations, S, are expressed in tzM units. The data are plotted as V/S vs. V, a modified Hofstee (22) plot.

RESULTS

Sodium Dependence of Choline Uptake in NB41 Neuroblastoma Cells The substrate dependence of choline uptake in NB41 neuroblastoma cells is shown in Fig. 3. The curved lines in this figure indicate that more than one mechanism for choline uptake is operative in these cells. The solid line (A) represents a computer fit of these points by a model

50A

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Vmax

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Vmax

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v FIG. 3. Kinetic analysis of choline transport by NB41 neuroblastoma cells in culture. Curve A: graphic description of a single high-affinity transport system plus a diffusional component; Curve B: graphic description of a carrier-mediated transport system, consisting of two components of high and low affinities. Legend gives numerical values for the kinetic parameters described by curves A and B. Each point represents the mean of 5 determinations; the bar, the sE.

206

H U T C H I S O N ET AL.

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FIG. 4. Kinetic analysis of choline transport by NB41 neuroblastoma in the presence and absence of Na--. Legend gives values for the kinetic parameters described by each curve. The solid line describes the kinetics of choline transport in the presence of sodium. Since a K,, cannot be estimated from the points obtained in the absence of sodium, the value of K,~ was set constant at 11.7/xM, and the values of Vm~, and D estimated accordingly (broken line). Each point represents the mean of 4 determinations; the bar, the SE.

consisting of a single carrier-mediated transport system and a second component, which is diffusion. The dashed line (B) represents a double carrier-mediated transport system model fit to the same data points. Either curve fits the data well, and the kinetic parameters obtained from these fits are shown in the legend in Fig. 3. Either fit yields an estimated Km for the high-affinity choline transport system in the range of 10-14 /zM. The low-affinity component is less well characterized by these data, but if it is a saturable system, it has a K,, greater than 1 mM and is not saturated at 10 mM, the highest concentration used in this series of experiments. Since we are interested primarily in the high-affinity transport of choline, we have assumed, in the remaining kinetic analyses in this paper, that the low-affinity component represents diffusion. The sodium dependence of choline transport in NB41 cells is shown in Fig. 4. The solid line (A) represents the computer fit to the observed choline uptake in the presence of sodium. The kinetic constants described by this curve are shown in the legend in Fig. 4. The Km of 11.7 /zM obtained from these points agrees well with the K,, of 14.2 /zM derived from the experiments displayed in Fig. 3. Choline uptake in the absence of sodium is shown by the dashed line (B) in Fig. 4. At low substrate concentrations, the uptake of choline is drastically reduced in

NEURONAL--GLIAL

CHOLINE

TRANSPORT

207

the absence of sodium. This effect is not seen at high substrate concentrations, however, because the low-affinity system is predominant here. These results agree with similar studies in synaptosomes reported earlier by Haga and Noda (6) and in brain slices by Kuhar et al. (3), but differs from the recent report by Lanks et al. (10) that choline transport in similar neuroblastoma cell lines is sodium-independent. We have found a partial sodium dependence of choline transport at low substrate concentrations in several neuronal (N2AG and B50) and in some, but not all, of the nonneural cell lines we have examined (see Table I).

Stimulation of Choline Transport in the C6 Rat Astrocytoma Cell Line in the Absence of Sodium Figure 5 illustrates the substrate dependence of choline transport in the C6 astrocytoma cell line; the kinetic parameters of choline transport

TABLE I EFFECTS OF N A + ON CHOLINE TRANSPORT IN CELL LINES OF N E U R A L AND NONNEURAL ORIGIN a V

Cell line Neuronal NB41 N2AG B50 Glial C6 RN22 Other RAG Fibroblasts HTC B16 Meningioma

+ Na +

- Na +

%

16.90 2.64 6.61

8.60 0.75 3.75

51 28 57

2.85 2.84

7.08 6:75

248 237

3,16 13.00 10.27 5.05 13.30

1.27 10.90 28.77 15.41 21.20

40 84 280 305 159

Choline uptake in the absence of Na + is expressed as a velocity and as a percentage of the velocity observed in the presence of Na +. The table includes representative values obtained from at least three separate determinations on each cell ine. The concentration of ~H-choline in the incubation medium was 0.42 tzM for the meningioma cells, 0.16 p.M for the B50 and B16 cells, and 0.21 /zM for all others.

208

H U T C H I S O N ET AL.

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15-

Km Vmox D ~Naf 1 3 ~ 0 I10_~13 089• rl -Na~ 47_~0.47 115_+Z3 1.23• 15 I

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V

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A

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F~G. 5. Kinetics of choline transport in the C6 astrocytoma cell line: stimulation in the absence of NA +. Curves A and B describe the kinetics of choline transport in the presence and absence of Na +, respectively. Legend gives numerical values for the kinetic parameters described by curves A and B. Each point represents the mean of 8 determinations; the bar, the SE.

in the presence, and in the absence, of sodium in these cells are listed in the legend in Fig. 5. The Km value of 13.8 k~M for the high-affinity system is quite similar to that shown for the NB41 neuroblastoma cells in Fig. 3. Choline uptake at low substrate concentrations in the absence of sodium is shown by the dashed line (B) in Fig. 5. The striking acceleration of choline transport is seen only at low substrate concentrations. The stimulation of choline transport in the absence of sodium apparently results from a decrease in the Km of the high-affinity choline transport system. It should be pointed out, however, that a variety of closely related curves may be fitted to these points; thus, we do not reject the possibility that the difference may be a result of a higher Vmax rather than a lower Km (21,23). Nonetheless, it is quite apparent that choline transport is stimulated in the absence of sodium, and that this stimulation is essentially a stimulation of the high-affinity transport system exclusive of the diffusion or low-affinity component. This stimulation of choline uptake decreases with increasing sodium concen-

NEURONAL-GLIAL

CHOLINE TRANSPORT

209

tration (data not shown); at 50 mM sodium, the stimulation is reduced to less than half that seen in the absence of sodium. Table I shows a similar stimulation of choline transport in the absence of sodium in the RN22 neurinoma (glial) cell line and in several cell lines of nonneural origin.

Similar Effect of Chotinergic Drugs on Choline Transport in Neuronal and Glial Cell Lines The pharmacology of many cholinergic drugs indicates that their actions are not simple, but that they probably influence more than a single aspect of cholinergic transmission. These secondary effects may become important in the long-term management of diseases such as myasthenia gravis. In this disease, neostigmine, a quaternary amine cholinesterase inhibitor, is used to increase the effective concentration of acetylcholine at the neuromuscular junction. Neostigmine may also function directly, however, as a cholinomimetic agent (24). Indeed, we find that neostigmine (10 .2 M) does inhibit the active transport of exogenous choline (Table II). Similarly, physostigmine, a tertiary amine cholinesterase inhibitor, also interferes with choline transport (Table I).

TABLE II EFFECTS OF HEMICHOLINIUM-3

(HC-3) AND

CHOLINESTERASE ~NHIBITORS ON CHOLINE TRANSPORT BY C6 ASTROCYTOMA CELLS IN CULTURE a

Additions

None Hemicholinium-3

Neostigmine

Physostigmine

Concentration [M]

10.4 10.3 10-z 10.4 10-3 I0 -~ 10.4 10.3 10 - z

V

Percentage of control

4.44 1.63 0.90 0.20 4.19 2.78 1.56 4.06 3.22 1.17

100 37 22 4 95 70 35 92 80 26

a The experiments were done on C6 astrocytoma cells as described in "Materials and M e t h o d s . " The inhibitors were added at the stated concentration; 3H-choline was present at 0.21 /xM.

210

HUTCHISON

E T AL.

This compound has long been known to have a number of effects on cholinergic transmission other than its action as a cholinesterase inhibitor (4,5). Thus, physostigmine and neostigmine both interfere with choline transport, probably by functioning as choline analogues. In results not shown here, the cholinergic antagonist atropine was found to inhibit choline transport in cell lines of both neural and glial origin, but only at very high concentrations. Atropine at 10-2 M reduced choline uptake to 40% of control values. Equivalent concentrations of Dtubocurarine did not block the accumulation of choline. In contrast to the agents discussed above, which act at only rather high concentrations, hemicholinium-3 (HC-3) inhibits choline uptake at lower concentrations (Table II). This drug is thought to be a specific inhibitor of choline uptake at nerve endings (25). Thus, HC-3 at 10-4 M is an effective inhibitor of choline uptake, a concentration at which neostigmine, physostigmine, and atropine are ineffective. At this concentration of HC-3, choline uptake by the high-affinity system is reduced about ninefold (data not shown), whereas the low-affinity system is hardly affected. Thus, the high-affinity choline transport system is selectively inhibited by HC-3 at 10-4 M concentration. Table II shows further that even the nonsaturable uptake of choline is inhibited by 10-2 M HC-3. With the exception of the Na + dependence, the high-affinity choline transport systems in neuroblastoma and astrocytoma cells appear to be identical; they are similar in their kinetic parameters and in their response to all the drugs we tested.

DISCUSSION We present data supporting the following conclusions: (1) Cells of neural and nonneural origin possess a high-affinity transport system for exogenous choline with Km in the 10-15 /zM range, plus a second lowaffinity system that may represent diffusion; (2) external sodium modulates high-affinity choline transport systems differentially in neuronal and glial cell lines; and (3) the high-affinity uptake process is preferentially inhibited by reversible cholinesterase inhibitors and by HC-3. The kinetic parameters for the sodium-dependent high-affinity transport of choline by neuroblastoma cells described herein is in general agreement with previous studies on rat brain synaptosomes (6,7) and on clones of the C1300 neuroblastoma (10,11,13). In contrast to the findings of Lanks et al. (10), we find that choline transport in neuroblastoma is

NEURONAL-GLIAL CHOLINE TRANSPORT

211

sodium-dependent and sensitive to HC-3. It must be noted that our experimental procedure differs somewhat from that of Lanks et al. (10), in that we use a 1 mM choline postincubation wash; furthermore, the cells in our experiments were never starved for choline. Our data provide no evidence for an intermediate-affinity choline transport system (Kin in the 200/zM range). The stimulation of the glial cell high-affinity choline transport system in the absence of sodium is a striking new finding. It is not, however, an exclusive glial trait, because such diverse cell types as hepatoma and melanoma cell lines exhibit a similar response to low sodium. This differential response to low sodium may provide a new biochemical marker to distinguish between glial and neuronal cells in culture. The physiological significance of the differential modulation by sodium of choline transport in glial and neuronal cells remains to be elucidated. Kuffler and Nicholls (26) have suggested that the ionic fluxes generated in the process of neurotransmission may be a mechanism whereby neurons can communicate with glia. In invertebrates, the accumulation of K + by glia is thought to be passive (26), whereas in clonal mammalian astrocytoma cells, the Na+-K + exchange appears to be an active and coupled process (27,28). Thus, glia may modulate the extracellular concentration of monovalent cations, which, in turn, may differentially affect the transport of neurotransmitters or precursors into neurons and glia. It has been suggested that the influx of Na + during the depolarization of presynaptic terminals results in a transitory decrease in extracellular Na +, and an increase in K + (29,30,26,28). Such transitory changes in extracellular monovalent cations might trigger the influx of choline into glial cells, whereas during repolarization the external Na + would increase, and thus promote neuronal reuptake of choline for the resynthesis of ACh. The role of sodium on glial choline transport and its relationship to cholinergic function in vivo is not presently understood. Moreover, the extracellular concentration of divalent cations may influence choline transport; experiments to elucidate the modulation of choline transport by monovalent and divalent cations are under way. The transport of choline may be influenced by its intracellular conversion into ACh, or into phosphorylated intermediates. We find that the majority (about 80%) of the transported choline in C6 glial cells is converted to phosphorylcholine during the 5-min incubation, whereas in the neuronal NB41 cells only about 15% of the choline is so converted during the same time interval. About 80% of the choline remains as free choline in these neuronal cells. This finding is in contrast to the findings

212

HUTCHISON

E T AL.

of Lanks et al. (10) that nearly 95% of the choline taken up by neuroblastoma cells during a 4-min incubation is converted to phosphorylcholine. Hemicholinium-3 (HC-3) at concentrations as low as 10-4 M has a strong inhibitory effect on choline transport in the glial and in the neuronal cells similar to that reported for rat synaptosomes (6). This low concentration of HC-3 preferentially blocks the high-affinity component of choline transport, while the low-affinity or diffusional component is affected only at higher concentrations (10 -2 M). On the other hand, drugs that are not primarily inhibitors of transport may inhibit choline transport at high concentrations. The cholinesterase inhibitors neostigmine and physostigmine effectively inhibit the high-affinity transport of choline, probably by acting as choline analogues (24). This is a new observation in clonal cell lines and is in agreement with earlier findings that physostigmine inhibits choline uptake in synaptosomes (31). Thus, these findings suggest the possibility that the clinical effectiveness of these drugs in the treatment of such disorders as myasthenia gravis may be limited by their inhibition of choline transport. The experiments using atropine and D-tubocurarine were suggested by the observations that cholinergic antagonists influence the influx of Na § in muscle (30). Since neuronal and glial cell lines differ in their sodium requirements for choline transport, it was expected that the influx of choline into these cell lines might be differentially affected by atropine and D-tubocurarine. We observed, however, that atropine at high doses inhibits choline transport in both cell types, and that D-tubocurarine is ineffective. The findings of a high-affinity transport system for choline in a variety of clonal cell lines of glial origin suggests a glial role in the modulation of cholinergic transmission. The precise fate of 3H choline taken up by glia, however, remains to be determined. The presence of a high-affinity transport system for choline in nonneural cell types is not surprising, and may be coupled to choline kinase and membrane biosynthesis (32,33). Likewise, neuronal cell types possess high-affinity transport systems for choline that may or may not be coupled to the synthesis of ACh. The degree of such coupling to ACh synthesis will depend on the degree of differentiated cholinergic function. The radioautographic evidence of glial accumulation of GABA and glycine in vivo (34,35) and in vitro (36) strongly supports the data obtained with clonal cell lines, suggesting a possible glial role in the modulation of synaptic transmission. The observations of Denis and Miledi (37) on the quantal release of ACh from Schwann cells at the denervated neuromuscular junction is suggestive of a glial role in the modulation of cholinergic transmission.

NEURONAL-GLIAL CHOLINE TRANSPORT

213

Taken together, the results presented here suggest that clonal lines of glial origin may be useful in the further elucidation of the possible glial role in cholinergic transmission.

ACKNOWLED GMENTS Supported by D H E W grants NS 11354, NS 11255, Welch Grant H504, a grant from the Muscular Dystrophy Association of America, and supporting funds from the Multidisciplinary Research Program in Mental Health Group, UTMB. R.L.S. is a recipient of a postdoctoral fellowship from the Muscular Dystrophy Association of America. M.R. is a Welch Foundation predoctoral fellow. The skillful assistance of Ms. Karin Werrbach, Ms. Tina Colmore, and Ms. E.A. Murray is gratefully acknowledged.

REFERENCES 1. BIRKS, R.I., and MACINTOSH, F.S. (1961) Acetylcholine metabolism of a sympathetic ganglion. Can. J. Biochem. Physiol. 39, 787. 2. BROWNING, E.T., and SCHULMAN, M.P. (1968) (14C) Acetylcholine synthesis by cortex slices of rat brain. J. Neurochem. 15, 1391-1405. 3. KUHAR, M.J., SETHY, V.H., ROTH, R.H., and AGHAJANIAN, G.K. (1973) Choline: selective accumulation by central cholinergic neurons. J. Neurochem. 20, 581-593. 4. LIANG, C.C., and QUASTEL, J.H. (1969) Effects of drugs on the uptake of acetylcholine in rat brain cortex slices. Biochem. Pharmacol. 18, 1187-1194. 5. POLAK, R.L. (1969) The influence of drugs on the uptake of acetylcholine by slices of rat cerebral cortex. Brit. J. Pharmacol. 36, 144-152. 6. HAGA, T., and NODA, H. (1973) Choline uptake systems of rat brain synaptosomes, Biochim. Biophys. Acta. 291,564-575. 7. YAMAMURA, H., and SNYDER, S. (1972) Choline: high affinity uptake by rat brain synaptosomes. Science 178, 626-628. 8. HABER, B., COLMORE, T., WERRBACH, K., and HUTCHISON, H.T. (1973) Uptake of choline by clonal lines of astrocytoma and neuroblastoma in culture. Proc. Soc. Neuroscience, 3rd Annual Meeting 75, 399. 9. HUTCHISON, H.T., WERRBACH, K., VANCE, C., and HABER, B. (1974) Uptake of neurotransmitters by clonal lines of astrocytoma and neuroblastoma in culture. I. Transport of gamma-aminobutyric acid. Brain Res. 66, 265-274. 10. LANKS, K., SOMERS, L., PAPERMEISTER, B., and YAMAMURA, H. (1974) Choline transport by neuroblastoma cells in tissue culture. Nature 252, 476-478. 11. MASSARELLI,R., CIESIELSKI-TRESKA,J., EBEL, A., and MANDEL, P., (1974) Kinetics of choline uptake in neuroblastoma clones. Biochem. Pharm. 23, 2857-2865. 12. MASSARELLI,R., CIESIELSKI-TRESKA,J., EBEL, A., and MANOEL, P. (1974) Choline uptake in glial cell cultures. Brain Res. 81,361-363.

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Uptake of neurotransmitters and precursors by clonal lines of astrocytoma and neuroblastoma: III. Transport of choline.

Clonal lines of glial, neuronal, and nonneural origin accumulate choline via a high-affinity carrier-mediated transport system withK m in the range of...
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