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Choline Uptake and Metabolism by Nerve Cell Cultures
Choline Uptake and Metabolism by Nerve Cell Cultures R. MASSARELLI, TUEN YEE WONG, CHANTAL FROISSART and
I. ROBERT
Centre d e Neurochimie d u CNRS and Institute of Biological Chemistry, Faculty of Medicine, Universite Louis Pasteur, I 1 R u e Humann, 67085 Strasbourg (France)
INTRODUCTION The transport of choline (Ch) into synaptosomes has been suggested t o be mediated by two mechanisms which differ in their affinity towards the substrate. Initial studies showed only one Km (about M) for the uptake of choline into synaptosomes (Diamond and Milfay, 1972; Hemsworth e t al., 1971 ;Marchbanks, 1968; Diamond and Kennedy, 1969). However, o n lowering Ch concentrations and using a larger range (from 0.5 t o 100 pM), it was shown by a Lineweaver-Burk plot that the experimental points did not fit a straight line, but could be resolved into two straight lines which correspond t o the high and low affinity mechanisms (Yamamura and Snyder, 1973). A correlation was shown between cholinergic markers and high affinity uptake (HAU) of Ch in various areas of rat brain, and HAU of Ch was lost after degeneration of cholinergic terminals (Kuhar et al., 1975a, b); thus, it was suggested that HAU was specific for cholinergic neurons, and there is considerable evidence supporting the concept that Ch transported by this mechanism is necessary for, and may even limit, ACh synthesis (see Kuhar and Murrin, 1978). However, using nerve cell. cultures as a model of the nervous system, several laboratories have shown that HAU of Ch can be demonstrated in non-cholinergic neurons as well as in glial cells (Haber and Hutchison, 1976; Massarelli and Mandel, 1976). Why should a non-cholinergic cell show a mechanism which has so clearly been demonstrated as characteristic of and necessary for the synthesis of ACh? We have previously shown (Massarelli, 1978) that the incubation of nerve cell cultures in Krebs-Ringer phosphate containing no Ch can create a non-steadystate in the endogenous concentration of Ch; the steady-state appeared only to be maintained by incubating cells with the normal Ch concentration of the growth medium (30 pM). Thus incubation with Ch at concentrations necessary t o measure high affinity uptake (considerably less than 30 pM) appears t o disturb the equilibrium of the endogenous Ch pool. We suggest that, under these conditions, the cell may regulate the transport of choline depending on its needs. The high and low affinities shown in a Lineweaver Burk plot may just be the graphical representation of conforrnational changes brought t o a single transport mechanism by varying the exo/endocellular concentrations of choline.
90
The present experiments were made in the hope that they might provide evidence for o r against this postulate. MATERIALS AND METHODS
Culture Cultures from mouse neuroblastoma C1300, clone M I cells, were grown in Falcon plastic Petri dishes and used at confluency. The cells did not show any choline acetyltransferase (EC 2.3.1.6, ChAT) activity. The growth medium was Dulbecco’s modified Eagle’s containing 10%foetal calf serum.
Uptake experiments Cells attached at the bottom of Petri dishes were washed 3 times with 0.147 M NaCl at 37°C and the preincubation was performed in Krebs-Ringer phosphate pH 7.4 (137 mM NaCl, 2.6 mM KCI, 0.7 mM CaCl,, 0.5 mM MgCl,, 3.2 mM Na,HP04, 1.4 mM KH2PO4 and 10 mM glucose). Various concentrations of [ 14C]choline (Amersham), kept at the same specific radio activity, were added for various periods of incubation, after which the Petri dishes were washed 3 times with 0.147 mM NaCl, and the cells were digested with concentrated formic acid. Aliquots of the acid extract were used for the determination of total radioactivity; this represented only 2-3% of medium radioactivity at the longest time of incubation. In the studies that measured Ch and its metabolites, a mixture of ice-cold 1 N formic acid/acetone (15/85) was added after washing the cells, the cells were then scraped off, and homogenized. After centrifugation at 3000 X g for 20 min, the supernatant was collected, frozen with liquid N,, and lyophilized. 50 p1 of 0.04 N HCl containing 10 mM Ch-chloride and phosphorylcholine (PCh) chloride were added to the dry material, 10 pl were counted (acid total extract), and 10 pl were spotted on TLC cellulose plates. Chromatography used the system of Marchbanks and Israel (1971) (Butanol/ethanol/acetic acid/ water, 100 : 20 : 17 : 33); the areas containing Ch and PCh (identified from the migration of standards) were scraped and the radioactivity determined. Lipids from the pellet remaining after acid homogenization were extracted in chloroform/methanol 1 : 1 and aliquots were taken for scintillation counting. The tissue residue was dissolved in NaOH (1 M) and aliquots were taken for assay of protein by the method of Lowry et al. (1 95 1). RESULTS AND DISCUSSION
Uptake and metabolism Initial experiments characterized the uptake and metabolism of Ch by the M, cells. The accumulation of radioactivity into the total acid extract of M, cells incubated with varying concentrations of [ 14C]Ch was approximately linear
91 A. TOTAL (acid molubhl
/
B. CHOLINE
[-:
80
I A
I 1;.
C. PHOSPHORYLCHOLINE
D. LIPID TOTAL
I EI P O '
4i
i
t 0
10
I I t
20
40 Ty (nln)
f
A -.
Al
/
10
0
10
40 T l M l (mln)
Fig. I . Distribution of radioactivity originating from [14Clcholine in the incubation meaium. Cells were incubated with various concentrations of [I4C]Ch kept at the same specific activity and the various compartments extracted and separated as described in the Materials and Methods section. The data are expressed as pmoles of [ 14C]Ch incorporated into each compartment/mg protein. Each point represents the average of two experiments. 0, 0.38 pM; 0.0.64 pM;n, 1.28 pM;A, 6.41 pM;A, 19.23 pM.
92 with time (Fig. IA). However, when the free Ch compartment was measured, it appeared that a saturation plateau was already present after 5 min of incubation (Fig. 1B). The incorporation of Ch into PCh increased linearly with time (Fig. IC), so that the ratio PCh/Ch varied between 5 and 50. Thus, choline enters these cells, rapidly saturates the free Ch compartment, and is essentially directed towards the synthesis of PCh; the lipid incorporated much less radioactivity, as is shown in Fig. 1D.These experimental results are interpreted as supporting the finding (Massarelli, 1978) that endogenous Ch is in a non steadystate when cells are exposed to low exogenous Ch; The radioactivity in the free Ch compartment reaches different saturation plateaus at different exogenous Ch concentrations (Fig. 1B). The kinetic parameters of the uptake process could be calculated, but we d o not consider them to be valid. When tissue is incubated with labelled Ch, the amount of total tissue radioactivity can only be analysed as Ch transport if it is clearly demonstrated that uptake of Ch into its free endocellular compartment is linear with time, and that the production of metabolites (i.e. the activity of the enzymes involved in Ch metabolism) is unimportant. If the latter requirement is not fulfilled, a Michaelis-Menten treatment of total uptake of radioactivity would represent several enzymatic activities and not uptake alone. This was clearly so for the uptake of choline in our cell cultures.
Effect of temperature on choline uptake One possible way t o characterize the uptake mechanism is to study its behaviour at different temperatures. Such a study made on cultured cells incubated with 1 pM Ch is shown in Fig. 2. The accumulation of radioactivity in the total acid extract showed a break in the Arrhenius plot a t 17°C. The two resulting energies of activation would suggest that the uptake may change its energy requirement with temperature and that, at physiological temperature, this requirement is rather low. Similar results have already been published (Massarelli e t al., 1976) and it was suggested that, in nerve cell cultures, the HAU of Ch may be a facilitated diffusion mechanism. However, when the effect of temperature was studied on the incorporation of Ch into the endocellular free Ch compartment (representing thus the transport of Ch) a rather strange Arrhenius plot was obtained (Fig. 2B). One possible explanation for this bell shaped curve is that, when cells are incubated with 1 p M [I4C]Ch, there are great differences in the metabolism of the endogenous Ch pool at different temperatures. An anomalous Arrhenius plot was also observed for Ch incorporation into the lipid fraction (Fig. 2D),while PCh synthesis gave only one energy component (Fig. 2C). These data may reflect the non steady-state conditions referred to earlier, and it is possible that different metabolic pathways intervene at different temperatures. If this were so, at a concentration of external Ch that would not change the endogenous size of the pool, the temperature dependency should not show anomalies. When cells were incubated with 30 p M choline (normal concentrations in the medium) the Arrhenius plots showed only one energy of activation for all parameters measured (Fig. 3).
93
1.
CHOLlNE
C.
PHOSPHORYLCHOLINE
D. UPlD TOTAL O k
o\o
\
\
0
>
-B 1.6
O O \
Fig. 2. Arrhenius plot of cellular 14C-distributionafter incubation with 1 p M [ 14C]Ch. (A) The values of E, are: 8.6 kcal mol-l above 17OC and 19.1 kcal mol-1 below 17'C. (C) 14C-incorporation in PCh showed an E, of 18.1 kcal . mol-'. Each point is the average of three independent determinations. The points were fitted by regression analysis.
-
94 A. TOTAL ( a d d &bk
I E. CHOLINL
a
#
1 4
,PnObPHORYLCHOUNL
L LIPID
TOTAL
a 9.C
>
1 a
>
-t
au
1
Fig. 3 . Arrhenius plot of cellular choline distribution after incubation with 30 I.~M[ l4C1Ch. (A) E, = 12.7 kcal . mol-I; (B) E, = 6.7 kcal . mol-l; (C) E, = 29.0 kcal . mol-I; (D)E, = 20.2 kcal . mol-l. Each point is the average of three independent determinations.
95 CONCLUSIONS These results and other arguments which have been developed elsewhere (Massarelli, 1978) suggest that we can not exclude the possibility that a single uptake mechanism exists; it is energy dependent (not as much, though, as the other processes involved in Ch metabolism as can be judged by their energies of activation (Fig. 3)), and it may change in conformation depending on the concentration of external Ch and/or on endocellular steady-state conditions. It should be emphasized that these suggestions are based upon experiments on cell cultures, and it would be inappropriate to extrapolate the suggestions to intact tissues or to synaptosomes. It would, however be interesting to test whether synaptosomes under HAU conditions can maintain steady-state concentrations of c h , and if these may influence Ch uptake as we have shown in nerve cell structures. SUMMARY Uptake and metaboIic distribution of Me[*4C]cholinewas studied in clone
M 1 of mouse neuroblastoma 1300. Total incorporation of I4C and labelling of
phosphorylcholine were linear with time, while the free Ch compartment was rapidly saturated, Incubation of cells at different temperature, and under high affinity uptake conditions (1 pM of exogenous Ch), resulted in abnormal Arrhenius plots. This anomaly was not shown when cells were incubated with 30 pM choline (concentration used normally in the growth medium). The results support previously published data suggesting that conditions used to study high affinity Ch uptake produce a non steady-state in the endocellular pool of choline. It is postulated that the uptake of Ch may be regulated by the exo-endocellular concentrations of choline, and that the high and low affinity uptake systems may represent a single mechanism which changes in conformation with different concentrations of substrate. REFERENCES Diamond, I. and Kennedy, E.P. (1 969) Carrier-mediated transport of choline into synaptic nerve endings. J. Biol. Chem., 244,3258-3263. Diamond, 1. and Milfay, D. (1 972) Uptake of [3H-methyl]choline by microsomal, synaptosomal, mitochondria1 and synaptic vesicle fractions of rat brain. J. Neurochem., 19, 1899-1909. Haber, B. and Hutchison, H.T. ( 1 976) Uptake of neurotransmitters and precursors by clonal cell lines of neural origin. In Transport Phenomena in the Nervous System, C . Levi, L. Battistin and A. Lajtha (Eds.), Plenum Press, New York, pp. 179-198. Hemsworth, B.A., Darmer, K.I. and Bosmann, H.B. (1971) The incorporation of choline into isolated synaptosomal and synaptic vesicles fractions in the presence of quaternary ammonium compounds. Neuropharmacol.. 10,109- 120. Kuhar, M.J., Dehaven, R.M., Yamamura, H.I.,Rommelspacher, H.and Simon, J.R. (197Sa) Further evidence for cholinergic habenulo-inter-peduncular neurons: pharmacologic and functional characteristics. Brain Res., 97,265-275. Kuhar, M.J., Sethy, V.H., Roth, R.H. and Aghajanian, G.K. (1975b) Choline: selective accumulation by central cholinergic neurons. J. Neurochem., 20, 581 -593.
96 Kuhar, M.J. and Murrin, L.C. (1978) Sodium-dependent, high affinity choline uptake. J. Neurochem., 30,15-21. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin Phenol Reagent. J. Bioi. Chem., 193,265-275. Marchbanks, R.M. (1968) The uptake of ['4C]choline into synaptosomes in vitro. Biochem. J., 110, 533-541. Marchbanks, R.M. and Israel, M. (1971) Aspects of acetylcholine metabolism in the electric organ of Torpedo marmorata. J. Neurochem., 1 8 , 4 3 9 4 4 8 , Massarelli, R. (1978) Uptake of choline in nerve cell cultures: correlation with the endogenous pool of choline. In Cholinergic Mechanisms and Psychopharmacology, D.J. Jenden (Ed.), Plenum Press, New York,pp. 539-550. Massarelli, R. and Mandel, P. (1976) On the uptake mechanism of choline in nerve cell cultures. In Transport phenomena in the neruous system (Levi, G . , Battistin, L. and Lajtha, A., eds.), pp. 199-209, Plenum Press, New York. Massarelli, R., Stefanovic, V. and Mandel, P. (1976) Cholinesterase activity and choline uptake in intact nerve cell cultures. Brain Res., 112, 103-1 12. Yamamura, H.I. and Snyder, S.H. (1973) High affinity transport of choline into synaptosomes of rat brain.J. Neurochem., 2 1 , 1355-1374.
I. ROBERT
Centre d e Neurochimie d u CNRS and Institute of Biological Chemistry, Faculty of Medicine, Universite Louis Pasteur, I 1 R u e Humann, 67085 Strasbourg (France)
INTRODUCTION The transport of choline (Ch) into synaptosomes has been suggested t o be mediated by two mechanisms which differ in their affinity towards the substrate. Initial studies showed only one Km (about M) for the uptake of choline into synaptosomes (Diamond and Milfay, 1972; Hemsworth e t al., 1971 ;Marchbanks, 1968; Diamond and Kennedy, 1969). However, o n lowering Ch concentrations and using a larger range (from 0.5 t o 100 pM), it was shown by a Lineweaver-Burk plot that the experimental points did not fit a straight line, but could be resolved into two straight lines which correspond t o the high and low affinity mechanisms (Yamamura and Snyder, 1973). A correlation was shown between cholinergic markers and high affinity uptake (HAU) of Ch in various areas of rat brain, and HAU of Ch was lost after degeneration of cholinergic terminals (Kuhar et al., 1975a, b); thus, it was suggested that HAU was specific for cholinergic neurons, and there is considerable evidence supporting the concept that Ch transported by this mechanism is necessary for, and may even limit, ACh synthesis (see Kuhar and Murrin, 1978). However, using nerve cell. cultures as a model of the nervous system, several laboratories have shown that HAU of Ch can be demonstrated in non-cholinergic neurons as well as in glial cells (Haber and Hutchison, 1976; Massarelli and Mandel, 1976). Why should a non-cholinergic cell show a mechanism which has so clearly been demonstrated as characteristic of and necessary for the synthesis of ACh? We have previously shown (Massarelli, 1978) that the incubation of nerve cell cultures in Krebs-Ringer phosphate containing no Ch can create a non-steadystate in the endogenous concentration of Ch; the steady-state appeared only to be maintained by incubating cells with the normal Ch concentration of the growth medium (30 pM). Thus incubation with Ch at concentrations necessary t o measure high affinity uptake (considerably less than 30 pM) appears t o disturb the equilibrium of the endogenous Ch pool. We suggest that, under these conditions, the cell may regulate the transport of choline depending on its needs. The high and low affinities shown in a Lineweaver Burk plot may just be the graphical representation of conforrnational changes brought t o a single transport mechanism by varying the exo/endocellular concentrations of choline.
90
The present experiments were made in the hope that they might provide evidence for o r against this postulate. MATERIALS AND METHODS
Culture Cultures from mouse neuroblastoma C1300, clone M I cells, were grown in Falcon plastic Petri dishes and used at confluency. The cells did not show any choline acetyltransferase (EC 2.3.1.6, ChAT) activity. The growth medium was Dulbecco’s modified Eagle’s containing 10%foetal calf serum.
Uptake experiments Cells attached at the bottom of Petri dishes were washed 3 times with 0.147 M NaCl at 37°C and the preincubation was performed in Krebs-Ringer phosphate pH 7.4 (137 mM NaCl, 2.6 mM KCI, 0.7 mM CaCl,, 0.5 mM MgCl,, 3.2 mM Na,HP04, 1.4 mM KH2PO4 and 10 mM glucose). Various concentrations of [ 14C]choline (Amersham), kept at the same specific radio activity, were added for various periods of incubation, after which the Petri dishes were washed 3 times with 0.147 mM NaCl, and the cells were digested with concentrated formic acid. Aliquots of the acid extract were used for the determination of total radioactivity; this represented only 2-3% of medium radioactivity at the longest time of incubation. In the studies that measured Ch and its metabolites, a mixture of ice-cold 1 N formic acid/acetone (15/85) was added after washing the cells, the cells were then scraped off, and homogenized. After centrifugation at 3000 X g for 20 min, the supernatant was collected, frozen with liquid N,, and lyophilized. 50 p1 of 0.04 N HCl containing 10 mM Ch-chloride and phosphorylcholine (PCh) chloride were added to the dry material, 10 pl were counted (acid total extract), and 10 pl were spotted on TLC cellulose plates. Chromatography used the system of Marchbanks and Israel (1971) (Butanol/ethanol/acetic acid/ water, 100 : 20 : 17 : 33); the areas containing Ch and PCh (identified from the migration of standards) were scraped and the radioactivity determined. Lipids from the pellet remaining after acid homogenization were extracted in chloroform/methanol 1 : 1 and aliquots were taken for scintillation counting. The tissue residue was dissolved in NaOH (1 M) and aliquots were taken for assay of protein by the method of Lowry et al. (1 95 1). RESULTS AND DISCUSSION
Uptake and metabolism Initial experiments characterized the uptake and metabolism of Ch by the M, cells. The accumulation of radioactivity into the total acid extract of M, cells incubated with varying concentrations of [ 14C]Ch was approximately linear
91 A. TOTAL (acid molubhl
/
B. CHOLINE
[-:
80
I A
I 1;.
C. PHOSPHORYLCHOLINE
D. LIPID TOTAL
I EI P O '
4i
i
t 0
10
I I t
20
40 Ty (nln)
f
A -.
Al
/
10
0
10
40 T l M l (mln)
Fig. I . Distribution of radioactivity originating from [14Clcholine in the incubation meaium. Cells were incubated with various concentrations of [I4C]Ch kept at the same specific activity and the various compartments extracted and separated as described in the Materials and Methods section. The data are expressed as pmoles of [ 14C]Ch incorporated into each compartment/mg protein. Each point represents the average of two experiments. 0, 0.38 pM; 0.0.64 pM;n, 1.28 pM;A, 6.41 pM;A, 19.23 pM.
92 with time (Fig. IA). However, when the free Ch compartment was measured, it appeared that a saturation plateau was already present after 5 min of incubation (Fig. 1B). The incorporation of Ch into PCh increased linearly with time (Fig. IC), so that the ratio PCh/Ch varied between 5 and 50. Thus, choline enters these cells, rapidly saturates the free Ch compartment, and is essentially directed towards the synthesis of PCh; the lipid incorporated much less radioactivity, as is shown in Fig. 1D.These experimental results are interpreted as supporting the finding (Massarelli, 1978) that endogenous Ch is in a non steadystate when cells are exposed to low exogenous Ch; The radioactivity in the free Ch compartment reaches different saturation plateaus at different exogenous Ch concentrations (Fig. 1B). The kinetic parameters of the uptake process could be calculated, but we d o not consider them to be valid. When tissue is incubated with labelled Ch, the amount of total tissue radioactivity can only be analysed as Ch transport if it is clearly demonstrated that uptake of Ch into its free endocellular compartment is linear with time, and that the production of metabolites (i.e. the activity of the enzymes involved in Ch metabolism) is unimportant. If the latter requirement is not fulfilled, a Michaelis-Menten treatment of total uptake of radioactivity would represent several enzymatic activities and not uptake alone. This was clearly so for the uptake of choline in our cell cultures.
Effect of temperature on choline uptake One possible way t o characterize the uptake mechanism is to study its behaviour at different temperatures. Such a study made on cultured cells incubated with 1 pM Ch is shown in Fig. 2. The accumulation of radioactivity in the total acid extract showed a break in the Arrhenius plot a t 17°C. The two resulting energies of activation would suggest that the uptake may change its energy requirement with temperature and that, at physiological temperature, this requirement is rather low. Similar results have already been published (Massarelli e t al., 1976) and it was suggested that, in nerve cell cultures, the HAU of Ch may be a facilitated diffusion mechanism. However, when the effect of temperature was studied on the incorporation of Ch into the endocellular free Ch compartment (representing thus the transport of Ch) a rather strange Arrhenius plot was obtained (Fig. 2B). One possible explanation for this bell shaped curve is that, when cells are incubated with 1 p M [I4C]Ch, there are great differences in the metabolism of the endogenous Ch pool at different temperatures. An anomalous Arrhenius plot was also observed for Ch incorporation into the lipid fraction (Fig. 2D),while PCh synthesis gave only one energy component (Fig. 2C). These data may reflect the non steady-state conditions referred to earlier, and it is possible that different metabolic pathways intervene at different temperatures. If this were so, at a concentration of external Ch that would not change the endogenous size of the pool, the temperature dependency should not show anomalies. When cells were incubated with 30 p M choline (normal concentrations in the medium) the Arrhenius plots showed only one energy of activation for all parameters measured (Fig. 3).
93
1.
CHOLlNE
C.
PHOSPHORYLCHOLINE
D. UPlD TOTAL O k
o\o
\
\
0
>
-B 1.6
O O \
Fig. 2. Arrhenius plot of cellular 14C-distributionafter incubation with 1 p M [ 14C]Ch. (A) The values of E, are: 8.6 kcal mol-l above 17OC and 19.1 kcal mol-1 below 17'C. (C) 14C-incorporation in PCh showed an E, of 18.1 kcal . mol-'. Each point is the average of three independent determinations. The points were fitted by regression analysis.
-
94 A. TOTAL ( a d d &bk
I E. CHOLINL
a
#
1 4
,PnObPHORYLCHOUNL
L LIPID
TOTAL
a 9.C
>
1 a
>
-t
au
1
Fig. 3 . Arrhenius plot of cellular choline distribution after incubation with 30 I.~M[ l4C1Ch. (A) E, = 12.7 kcal . mol-I; (B) E, = 6.7 kcal . mol-l; (C) E, = 29.0 kcal . mol-I; (D)E, = 20.2 kcal . mol-l. Each point is the average of three independent determinations.
95 CONCLUSIONS These results and other arguments which have been developed elsewhere (Massarelli, 1978) suggest that we can not exclude the possibility that a single uptake mechanism exists; it is energy dependent (not as much, though, as the other processes involved in Ch metabolism as can be judged by their energies of activation (Fig. 3)), and it may change in conformation depending on the concentration of external Ch and/or on endocellular steady-state conditions. It should be emphasized that these suggestions are based upon experiments on cell cultures, and it would be inappropriate to extrapolate the suggestions to intact tissues or to synaptosomes. It would, however be interesting to test whether synaptosomes under HAU conditions can maintain steady-state concentrations of c h , and if these may influence Ch uptake as we have shown in nerve cell structures. SUMMARY Uptake and metaboIic distribution of Me[*4C]cholinewas studied in clone
M 1 of mouse neuroblastoma 1300. Total incorporation of I4C and labelling of
phosphorylcholine were linear with time, while the free Ch compartment was rapidly saturated, Incubation of cells at different temperature, and under high affinity uptake conditions (1 pM of exogenous Ch), resulted in abnormal Arrhenius plots. This anomaly was not shown when cells were incubated with 30 pM choline (concentration used normally in the growth medium). The results support previously published data suggesting that conditions used to study high affinity Ch uptake produce a non steady-state in the endocellular pool of choline. It is postulated that the uptake of Ch may be regulated by the exo-endocellular concentrations of choline, and that the high and low affinity uptake systems may represent a single mechanism which changes in conformation with different concentrations of substrate. REFERENCES Diamond, I. and Kennedy, E.P. (1 969) Carrier-mediated transport of choline into synaptic nerve endings. J. Biol. Chem., 244,3258-3263. Diamond, 1. and Milfay, D. (1 972) Uptake of [3H-methyl]choline by microsomal, synaptosomal, mitochondria1 and synaptic vesicle fractions of rat brain. J. Neurochem., 19, 1899-1909. Haber, B. and Hutchison, H.T. ( 1 976) Uptake of neurotransmitters and precursors by clonal cell lines of neural origin. In Transport Phenomena in the Nervous System, C . Levi, L. Battistin and A. Lajtha (Eds.), Plenum Press, New York, pp. 179-198. Hemsworth, B.A., Darmer, K.I. and Bosmann, H.B. (1971) The incorporation of choline into isolated synaptosomal and synaptic vesicles fractions in the presence of quaternary ammonium compounds. Neuropharmacol.. 10,109- 120. Kuhar, M.J., Dehaven, R.M., Yamamura, H.I.,Rommelspacher, H.and Simon, J.R. (197Sa) Further evidence for cholinergic habenulo-inter-peduncular neurons: pharmacologic and functional characteristics. Brain Res., 97,265-275. Kuhar, M.J., Sethy, V.H., Roth, R.H. and Aghajanian, G.K. (1975b) Choline: selective accumulation by central cholinergic neurons. J. Neurochem., 20, 581 -593.
96 Kuhar, M.J. and Murrin, L.C. (1978) Sodium-dependent, high affinity choline uptake. J. Neurochem., 30,15-21. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin Phenol Reagent. J. Bioi. Chem., 193,265-275. Marchbanks, R.M. (1968) The uptake of ['4C]choline into synaptosomes in vitro. Biochem. J., 110, 533-541. Marchbanks, R.M. and Israel, M. (1971) Aspects of acetylcholine metabolism in the electric organ of Torpedo marmorata. J. Neurochem., 1 8 , 4 3 9 4 4 8 , Massarelli, R. (1978) Uptake of choline in nerve cell cultures: correlation with the endogenous pool of choline. In Cholinergic Mechanisms and Psychopharmacology, D.J. Jenden (Ed.), Plenum Press, New York,pp. 539-550. Massarelli, R. and Mandel, P. (1976) On the uptake mechanism of choline in nerve cell cultures. In Transport phenomena in the neruous system (Levi, G . , Battistin, L. and Lajtha, A., eds.), pp. 199-209, Plenum Press, New York. Massarelli, R., Stefanovic, V. and Mandel, P. (1976) Cholinesterase activity and choline uptake in intact nerve cell cultures. Brain Res., 112, 103-1 12. Yamamura, H.I. and Snyder, S.H. (1973) High affinity transport of choline into synaptosomes of rat brain.J. Neurochem., 2 1 , 1355-1374.