DEVELOI’MENTAL
High
RIOLOGY
Affinity
65,
w-99
Choline
(1978)
Uptake
K. F. Department
of Biology, Received
by Spinal Cord Culture
BARALD
University
November
DARWIN
AND of California,
8, 1977; accepted
Neurons
Cell
K. BERG
San Diego, in revised
in Dissociated
form
La Jolla, February
California
92093
17, 1978
Spinal cord-myotube cultures prepared with dissociated embryonic chick spinal cord cells and myoblasts exhibit a high affinity mechanism for accumulating choline. The uptake mechanism has a K,,, of 3.4 + 0.5 (.&f (7) and a V,,, of 40.0 f 0.1 (7) pmoles/min/mg of protein (mean f SEM; number of determinations in parentheses). It is inhibited 90-95% by 10 $4 hemichohnium-3 or by replacement of Na’ in the incubation solution with Li+. Part of the choline (10-20s) accumulated by the high affinity system is converted to acetylcholine (ACh). Uptake studies on spinal cord cells and myotubes grown separately demonstrate that the spinal cord cells can account for virtually all of the choline uptake observed in the mixed cultures. Myotubes are unnecessary under these conditions for the expression of the high affinity uptake mechanism by spinal cord cells. Neurons are not the only cell type in culture to exhibit high affinity choline uptake. Chick fibroblasts in both rapidly growing and stationary phase can accumulate choline with kinetics similar to those observed for the high affinity uptake by spinal cord cells. Little if any of the choline accumulated by fibroblasts, however, is converted to ACh. In most uptake studies with spinal cord cells, contributions from fibroblasts were minimized by carrying out the analysis at a time when few non-neuronal cells were present in the spinal cord cultures. These observations suggest that a population of chick central nervous system (CNS) neurons develop a high affinity choline uptake mechanism in cell culture that has many of the properties described for uptake by cholinergic neurons in vivo and that at least part of the choline accumulated by the system can be used for neurotransmitter synthesis. INTRODUCTION
Dissociated cell cultures prepared with embryonic chick spinal cord cells and myotubes contain a population of cholinergic neurons. This is indicated by the fact that some of the surviving neurons form functional cholinergic synapses on the myotubes, and high levels of the enzyme choline acetyltransferase appear in the cultures (Fischbach, 1972; Berg and Fischbach, 1978). The experiments reported here were undertaken to assay another property of cholinergic neurons, the ability to accumulate choline by a high affinity uptake mechanism. We were interested in determining (1) if high affinity choline uptake could be expressed by CNS neurons in dissociated ceII culture, (2) if the uptake could provide choline for ACh synthesis and resemble in other respects uptake by choline& neu-
rons in vivo, and (3) if synaptic connections with muscle would play a role in development of the uptake mechanism. Neurons of the central nervous system appear unable to synthesize choline de novo (Bremer and Greenberg, 1961; Ansell and Spanner, 1967; Browning and Schuhnan, 1968). It has been proposed that choline needed for neuronal metabolism is derived from external sources either as free choline (Groth et al., 1958) or as a lipid-bound form (Illingworth and Portman, 1972). Much evidence has been presented that cholinergic neurons have a high affinity uptake mechanism for accumulating choline from external sources and that choline accumulated by the uptake mechanism can serve as substrate for ACh synthesis. Studies on tissue fragments and synaptosome preparations containing cholinergic nerve terminals from 90
0012-X06/78/0651-0090$02.00/0 Copyright AU rights
0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
BARALD
AND BERG
Choline Uptake by Spinal Cord Cells
a variety of vertebrate and invertebrate postembryonic sources have revealed an uptake mechanism for choline with K,,, of l-4 fl (Haga and Noda, 1973; Yamamura and Snyder, 1973; Pert and Snyder, 1974; Schwartz et al., 1975; Suszkiw and Pilar, 1976). The high affinity choline uptake mechanism is dependent on the presence of external Na+ and can be inhibited by low concentrations of hemicholinium-3 (10 $kf); much of the accumulated choline (ca. 70%) is converted to ACh. Tissue though!, to lack cholinergic nerve terminals or tissue in which cholinergic terminals have been destroyed contains only a low affinity, Na+independent mechanism for accumulating choline (Yamamura and Snyder, 1973; Kuhar et al., 1973; Pert and Snyder, 1974; Mulder et al., 1974; Carroll and Buterbaugh, 1975; Suszkiw and Pilar, 1976). Choline uptake studies on different cell types grown in culture have yielded mixed results. Dissociated cerebral cells from chick display a high affinity choline uptake mechanism in culture (K, = 2-3 $V) with a partial dependence of Na+ (Massarelli et al., 1974a), while cultured cerebral cells from rat exhibit uptake mechanisms of only intermediate and low affinities (K,,, = 16 and 960 @f, respectively) and convert little of the accumulated choline to ACh (Yavin, 1976). High affinity choline uptake is found in several neuroblastoma cell lines, but the uptake is not inhibited by hemicholinium3 and is not restricted to cell lines capable of synthesizing ACh (Richelson and Thompson, 1973; Massarelli et al., 1974b; Lanks et al., 1974). High affinity choline uptake has also been demonstrated for two non-neuronal cell lines: glioma cells (Massarelli et al., 1974c) and hepatoma cells (Plagemann, 1971). It remained unclear whether cholinergic neurons grown in cell culture would develop a high affinity choline uptake mechanism and whether the uptake would have properties distinguishing it from uptake mechanisms in other cell types. We have found that chick spinal
91
cord-myotube cultures have a high affinity uptake mechanism for choline. The uptake is Na+-dependent and can be blocked by hemicholinium-3. Part of the accumulated choline is converted to ACh. Spinal cord neurons grown with and without myotubes have high affinity uptake mechanisms of equivalent magnitude, indicating that nerve-muscle synapse formation is not necessary for development of the uptake mechanism. A preliminary account of some of these results has appeared (Barald and Berg, 1977). MATERIALS
AND METHODS
Preparation of cell cultures. Myotube cultures were prepared by mechanically dissociating pectoral muscle from 1l-dayold chick embryos into a suspension of single cells as previously described (Berg and Fischbach, 1978). Cells were added to collagen-coated dishes at a concentration of 2-3 X lo5 tells/35-mm dish. After an initial 48-h period in culture medium (see below), cultures were incubated for an additional 48 hr in medium containing 10e5M cytosine arabinoside to kill rapidly dividing fibroblasts (Fischbach, 1972). Dissociated spinal cord cells were prepared from 4-day-old chick embryos by incubating cord fragments in Ca’+, Mg”+-free salt solution and using gentle trituration to separate the cells. Previously described techniques were used (Berg and Fischbach, 1978) except that the dissected spinal cords were first carefully stripped free of other tissue and then were dissociated in the absence of trypsin. Cell yields (ca. 1.4 x 10” cells/cord) were similar to those reported previously for cell preparations obtained by trypsin treatment (Berg and Fischbach, 1978). These modifications resulted in fewer non-neuronal cells present in spinal cord cultures during the first week after plating. Spinal cord cells were grown in culture by adding them at a concentration of 4.0 X 10”/35-mm dish either in 4-day-old myotube cultures or on collagen-coated dishes in the absence of myotubes. Unless
92
DEVELOPMENTAL BIOLOGY
otherwise indicated, all cultures were taken for analysis 7-8 days after plating the spinal cord cells. Fibroblast cultures were prepared by incubating the initial muscle cell suspension, obtained as described above, in 60-mm tissue culture plastic dishes for 20 min at 37°C. Fibroblasts preferentially attached to the plastic surface during this time. The cell suspension was then replaced with fibroblast culture medium (see below), and the attached cells were allowed to grow almost to confluency. The attached cells were then resuspended by treatment with crude trypsin (0.33% for 10 min), collected by centrifugation at 50g for 5 min, filtered through a double layer of lens paper to remove cell clumps, and added to tissue culture dishes at 3.0 x lo5 cells/35mm dish. To minimize residual contamination by myoblasts, the procedure was repeated twice, each time allowing the cells to grow almost to confluency. The hnal round of cultures was used for experiments when they were approximately half confluent (rapidly growing cultures) or when they were 2-3 days postconfluent (stationary cultures). Choline uptake. Choline uptake was examined by incubating cultures with r3H]choline. Cultures were rinsed twice with PBSS (2 ml) and then incubated at room temperature in 1.0 ml of PBSS containing 0.1-100 fi r3H]choline at a specific activity of 0.1-10 Ci/mmole (obtained by diluting 10 Ci/mmole [3H]choline chloride with unlabeled choline chloride). Uptake was terminated by the addition of 20 ~1 of 0.5 M choline; the cultures were then rinsed three times with PBSS (2 ml). Cell extracts were prepared by incubating the cultures in 0.5 ml of 0.6 N NaOH overnight and then scraping the dishes with a Teflon pad. Accumulated radioactivity was determined by liquid scintillation counting of acidified aliquots in Aquasol-2. Background levels were determined by incubating cultures under the same conditions with the addition of 10 mit4 unlabeled choline to saturate the uptake mechanisms; blanks determined in
VOLUME 65.1978
this manner did not exceed 5% of the uptake observed at 0.1 fl [3H]choline. Unless otherwise indicated, values for uptake have been normalized for the total amount of cellular protein present. In cases where uptake was examined in the presence of a drug or in the absence of Na+, experimental and control cultures were preincubated for 10 min in the appropriate solutions to allow equilibration prior to the initiation of uptake. Acetylcholine synthesis. Acetylcholine synthesis was measured by high voltage paper electrophoresis (Hildebrand et al., 1971). Cultures were incubated with 1.0 fl [3H]choline and rinsed as described above except that either PBSS or Earle’s BSS were used, and both were supplemented with 5 gm/liter of glucose, 50 units/ml of penicillin, and 50 pg/ml of streptomycin. Incubations were carried out at 37°C. Cell extracts were prepared by scraping the dishes in 0.20 ml of 0.05 M sodium phosphate buffer, pH 7.5, containing 0.3% sodium dodecyl sulfate, 0.7 mM ACh, 2 mM choline, and 0.1 mil4 neostigmine methylsulfate. The extracts were homogenized in a glass tissue grinder equipped with a Teflon pestle, and stored frozen. r3H]ACh was assayed by applying 20-~1 aliquots to strips of Whatman 3 MM chromatography paper and submitting the sample to electrophoresis for 45 min at 2500 V (ca. 50 V/cm). Electrophoresis buffer contained 0.47 M formic acid and 1.4 M acetic acid (pH 1.9). The positions of [3H]ACh and [3H]choline were determined by iodine vapor staining of unlabeled carrier ACh and choline. After cutting the strips into segments (1.5 x 0.5 in), the radioactivity was eluted with 0.5 ml of 0.1 N HCl, mixed with 5 ml of Aquasol2, and counted by liquid scintillation counting. Under the conditions used, ACh synthesis was linear with time for periods up to 1 br and was proportional to the number of spinal cord cells initially added to the cultures (Berg, 1978). Protein determinations. The amount of protein in cell extracts was determined by
BARALD
AND BERG
Choline
the method of Lowry et al. (1951). Bovine serum albumin was used as a standard. Background values were determined by assaying extracts prepared from collagencoated dishes carried through the same feeding regimen as the cell cultures and were equivalent to lo-20% of the values obtained for spinal cord-myotubes cultures. Media and chemicals. Culture media and biological materials were obtained and prepared as previously described (Nishi and Berg, 1977). All media contained 5% (v/v) chick embryo extract except medium containing low5 M cytosine arabinoside which had 2% embryo extract. Fibroblast culture medium contained 10% (v/v) fetal calf serum (Microbiological Associates, Inc.) instead of horse serum and lacked embryo extract. Culture feeding regimens were as previously described (Nishi and Berg, 1977). Earle’s BSS was purchased from Microbiological Associates, Inc. PBSS con-
Uptake
by Spinal
Cord
Cells
93
tained 123 n&f NaCl, 5.4 m&f KCl, 11 mM NaP04 (pH 7.3), 0.9 n&f CaC12, 0.4 rniV MgS04, and 1 g/liter of glucose. r3H]Choline chloride (10.1 Ci/mmole) was purchased from Amersham/Searle, Aquasolfrom New EngIand Nuclear, hemicholinium-3 from Eastman, and acetylcholine chloride, choline chloride, ouabain, and neostigmine methylsulfate from Sigma. RESULTS
Choline uptake by spinal cord-myotube cultures. Myotube cultures seeded with spinal cord cells prepared from 4-day-old chick embryos by mechanical means contained two major cell types at early times: neurons and myotubes (Fig. 1A). Usually few fibroblast-like cells were apparent under these conditions during the first week. We examined choline uptake in spinal cord-myotube cultures at 1 week by incubating them with [3H]choline. Uptake was linear with time for at least 10 min at all
FIG 1. Micrographs of dissociated cell cultures. Cultures were prepared and maintained as describe :d in M aterials and Methods. Micrographs were taken with phase contrast optics; bar = 100 pm. (A) Spinal cord cells myotubes. (B) Spinal cord cells grown alone for 7 days. (C) Myotubes gr -own IT own for 7 days on 4-day-old al one for 11 days.
94
DEVF,I.OPMENTAI,
BIOLOGY
choline concentrations examined. Results obtained at 0.1 and 100 $V are shown in Fig. 2. Double reciprocal analysis of the data for choline concentrations of 0.1-1.0 fl revealed a high affinity uptake component. The uptake was characterized by a K,, of 3.4 + 0.5 fl and a V,,, of 40.0 + 0.1 pmoles/min/mg of protein (mean f SEM, seven separate determinations). A typical experiment is shown in Fig. 3. Uptake at 0.1 fl choline was examined in the presence of inhibitors to determine whether the uptake mechanism had properties similar to those reported for high
A 2/
I/
Y 0
5 TIME (mid
1
IO
FIG. 2. Time dependence of choline uptake in spinal cord-myotube cultures. Spinal cord-myotube cultures were examined for choline uptake by incubating the cultures in [“HIcholine for the indicated periods of time and determining the amount of radioactivity retained as described in Materials and Methods. Values represent the means of triplicate cultures; vertical lines indicate the SEM. (a) Incubation in 0.1 fl choline. (b) Incubation in 100 fl choline.
VOLIJME
65, 1978
‘1s IpM-‘I FIG. 3. High affinity choline uptake in spinal cord-myotube cultures. Spinalcord-myotube cultures were incubated in 0.1-1.0 aA choline, and the amount of choline accumulated in the fist 5 min was used to calculate the initial rates of uptake. The reciprocal of the initial velocity, l/V, is shown as a function of the reciprocal of the choline concentration, l/S. Values represent the means of triplicate cultures; vertical lines indicate the SEM. The line was fitted by hand. Values for K,, and V,,, were calculated to be 3.4 fl and 40 pmoles/min/mg of protein, respectively, for this experiment.
affinity choline uptake in cholinergic tissue and synaptosome preparations (Haga and Noda, 1973; Yamamura and Synder, 1973; Pert and Snyder, 1974; Suszkiw and Pilar, 1976). Consistent with those findings, uptake in spinal cord-myotube cultures was almost completely blocked by hemicholinium-3 (10 @fl and was significantly reduced when Na’ in the medium was replaced with Li’. ,Ouabain, an inhibitor of the Nat, K+-dependent ATPase, and KCN, a metabolic inhibitor, each inhibited uptake slightly (Table 1). Uptake at 100 fl choline was inhibited much less by replacement of Na’ with Li’ (Table 1). This raised the possibility that spinal cord-myotube cultures had a second choline uptake mechanism with a lower affinity for choline. To test for the presence of such a system, choline uptake was examined under conditions that minimized contributions from high affinity uptake. Na+ in the medium was replaced with Li’, and uptake was examined over the range of lo-100 fl choline. In four experiments double reciprocal analysis of the data yielded values for Km of 70 f 25 fl and V,
BAIIALD
TABLE INHIBITION
OF CHOLINE
Choline
IN SPINAL
C~lI.T~lH~S”
Choline
uptake
0.1 LLM
Li’ (low Na’) Hemicholinium-3 Ouabain KCN
BERG
1 UPTAKE
CORD-MYCW~IFIE
Condition
AND
34 f 3 (9) 9 -c 1 (9)
(9, of control) lC@lLM
71 -c 8 (8)
60 -t 14 (4) 64 + 12 (4) 70 f 5 (4)
87 + 3 (7)
69 k 13 (4)
” Inhibition of choline uptake was examined in spinal cord-myotube cultures at 0.1 and 100 fl choline under four conditions. Uptake in experimental cultures is expressed as a percentage of that observed for controls incubated in normal PBSS. Values represent the mean & SEM; the number in parentheses indicates the number of separate determinations. Each determination for a given condition was taken from a separate cell plating and represents the mean of three separate cultures. The conditions examined were (a) low Na’, in which 137 mJ4 LiCl was used to replace the NaCl normally present in PBSS, (b) 10 N hemicholinium-3, (c) 0.1 mM ouabain, and (d) 1 mM KCN.
of 40 f 14 pmoles/min/mg of protein (mean 4 SEM). One such experiment is shown in Fig. 4. While these results indicate that spinal cord-myotube cultures can also display a low affinity choline uptake mechanism, it is not clear whether the low affinity mechanism is present under normal conditions. Uptake studies in the presence of Na’ yielded ambiguous results in the range of 10-100 @II choline, both because of the large contribution from the high affinity mechanism and because of the small radioactive signal under these conditions. Conceivably the absence of Na’ could alter both the K,,, and the V, of the high affinity system. High affinity uptake by individual cell types. To distinguish whether spinal cord cells or myotubes were responsible for the high affinity choline uptake observed in the mixed cultures, the cell types were grown separately (Figs. 1B and 1C) and analyzed for choline uptake as described above. For both culture types choline uptake was linear with time for at least 8 min at all choline concentrations examined. Double reciprocal analysis of the data revealed a major high affinity uptake component in spinal
Uptake
by Spinal
Cord
95
Cells
cord cultures that quantitatively accounted observed in spinal for the uptake cord-myotube cultures. A typical experiment is illustrated in Fig. 5A; compiled results from several experiments are listed in Table 2. Thus, at 1 week, myotubes contribute very little to the choline uptake observed in mixed cultures, either directly by accumulating choline, or indirectly by influencing the development of spinal cord cells that express the high affinity system. Cultures of myotubes lacking spinal cord cells also exhibited a high affinity choline uptake mechanism, but the capacity of the system was very small (Fig. 5B, Table 2). The V, was about 1% of that observed for spinal cord cells. Since fibroblasts often contaminate myotube cultures in small amounts, choline uptake by chick fibroblast cultures was examined in a similar manner. Both rapidly growing fibroblasts and fibroblasts in stationary phase displayed a major high affinity uptake component (Fig. 5C,
/
-02
I
0
02
04
06
08
I
IO
I/s (/API
4. Low affinity choline uptake by spinal cord-myotube cultures in the absence of Na’. Initial rates of choline uptake by spinal cord-myotube cultures in the absence of Na+ were determined over the range of lo-100 $lJ choline as described for Fig. 3. (NaCl in the incubation solution was replaced with LiCl.1 The reciprocal of the initial rate is shown as a function of the reciprocal of the substrate concentration. Values represent the means of triplicate cultures; vertical lines indicate the SEM. The line was fitted by hand. Values of K,,, and V,,, were calculated to be 63 $U and 29 pmoles/min/mg of protein, respectively, for this experiment. FIG.
96
DEVELOPMENTAL
BIOLOGY
VOLUME
65. 1978
bain (0.1 m.il4) had little effect on uptake by spinal cord cells or myotubes, but did partially inhibit uptake by fibroblasts (ca.
50%). Acetylcholine
synthesis. Acetylcholine (ACh) synthesis was measured to determine the relation between high affinity choline uptake and ACh synthesis. Spinal cord, spinal cord-myotube, myotube, and fibroblast cultures were incubated for 1 hr in a low concentration of [3H]choline (1 $V) so that uptake would occur primarily by the IO
TABLE
t
2
KINETIC PARAMETERS FOR HIGH AFFINITY CHOLINE UPTAKE IN CELL CULTURE” Cultures V, (pmoles/min) Km (p.M x -2
0
2
4
6
8
IO
I2
‘5 (j&P) FIG. 5. High affinity choline uptake in cultures of different cell types. Initial rates of choline uptake were determined in 0.1-1.0 aikf choline as described in Fig. 3, and double reciprocal plots of l/V vs l/S were constructed. Values represent means of triplicate cultures; vertical bars indicate the SEM. Lines were fitted by hand. (A) Spinal cord cells grown for 1 week in the absence of myotubes. Values of 2.5 @!4 and 67 pmoles/min/mg of protein were obtained for K,,, and V,,,, respectively, in this determination. (B) Myotube cultures 11 days old (i.e., equivalent in age to myotubes present in spinal cord-myotube cultures at the time of analysis). Value of 0.5 $W and 0.63 pmoles/min/mg of protein were obtained for K,,, and V, in this determination. (C) Rapidly dividing fibroblast cultures. Values of 3.3 fl and 50 pmoles/min/mg of protein were obtained for K,,, and V, in this determination.
Table 2). The specific activity of uptake (picomoles per minute per milligram of protein) was comparable to that observed with spinal cord cells. Thus some non-neuronal cell types in vitro can express a high affinity uptake mechanism for choline. Uptake at 0.1 $t4 choline was examined in the presence of inhibitors to determine whether the high affinity uptake mechanism in spinal cord cells could be distinguished from that observed with myotubes and fibroblasts. In all cases uptake was substantially inhibited by hemicholinium-3 (10 CLM)or by replacing Na+ in the medium with Li+ (Table 3). KCN (1 mM) and oua-
Spinal cordmyotube Spinal cord Myotube Fibroblast
3.4 f (n = 3.5 f (n = 0.63 f (n = 2.9 f (n=9)
0.5 7) 0.6 7) .04 3) 0.4
(per culture)
(per mg of protein)
13.0 k (n=6) 13.0 f (n = 0.18 f (n=3)
40.0 f (n = 67.0 + (n = 0.61 f (n=3) 71.0 f (n=9)
1.7 2.0 7) 0.08
0.1 7) 1.3 7) 0.05 9.6
” Spinal cord-myotube, spinal cord, myotube, and fibroblast cultures were examined for high affinity choline uptake, and the kinetic parameters of uptake were determined by double reciprocal analysis of the data as shown in Figs. 3-5. Results are expressed as the mean f SEM. The number in parentheses indicates the number of determinations; each determination represents a separate cell plating and corresponding double reciprocal plot. Cultures were processed in triplicate for each choline concentration used to generate each plot. TABLE INHIBITION Condition
Choline W;J
Li+ (low Na’) Hemicholinium-3 Ouahain KCN
3
OF CHOLINE UPTAKE CELL TYPES”
33 f 2 12 f 4 89 f 5 lOOf
FOR DIFFERENT
uptake
(W of control) Fibroblast
Myotube 23 16 93 82
+ f f f
4 5 7 15
12 -e 1 11f2 5423 43 + 5
n Choline uptake in spinal cord, myotube, and libroblast cultures was examined at 0.1 fl under four different conditions as described in Table 1. Uptake in experimental cultures is expressed as a percentage of that observed for control cultures incubated in normal PBSS. Values represent the mean + SEM for five separate determinations. Each determination for a given condition was taken from a separate cell plating and represents the mean of three cultures.
BARALD
high affinity uptake system. Cell extracts were then prepared and assayed for [3H]ACh by high voltage paper electrophoresis. Large amounts of C3H]ACh were present in extracts of both spinal cord and spinal cord-myotube cultures; levels in myotube and fibroblast cultures were at the limits of detection (Table 4). Thus significant amounts of [3H]ACh accumulated only in cultures containing spinal cord cells, and the amount was not significantly dependent on myotube influence under the conditions examined. High affinity choline uptake can apparently exist in some cell types (e.g., TABLE ACETYLCHOLINE
4
SYNTHESIS
Cell culture
IN CELL CULTURE”
ACh
synthesis
(pmoles/hr/mg protein) Spiral cordmyotube Spinal cord Myotube Fibroblaat
of
(% of total uptake)
51.1 f 5.3 (6)
15 + 2
67.1 + 3.4 (6) 0.9 2 0.2 (6) 0.9 + 0.3 (5)
12 f 2
High
RIOLOGY
Affinity
65,
w-99
Choline
(1978)
Uptake
K. F. Department
of Biology, Received
by Spinal Cord Culture
BARALD
University
November
DARWIN
AND of California,
8, 1977; accepted
Neurons
Cell
K. BERG
San Diego, in revised
in Dissociated
form
La Jolla, February
California
92093
17, 1978
Spinal cord-myotube cultures prepared with dissociated embryonic chick spinal cord cells and myoblasts exhibit a high affinity mechanism for accumulating choline. The uptake mechanism has a K,,, of 3.4 + 0.5 (.&f (7) and a V,,, of 40.0 f 0.1 (7) pmoles/min/mg of protein (mean f SEM; number of determinations in parentheses). It is inhibited 90-95% by 10 $4 hemichohnium-3 or by replacement of Na’ in the incubation solution with Li+. Part of the choline (10-20s) accumulated by the high affinity system is converted to acetylcholine (ACh). Uptake studies on spinal cord cells and myotubes grown separately demonstrate that the spinal cord cells can account for virtually all of the choline uptake observed in the mixed cultures. Myotubes are unnecessary under these conditions for the expression of the high affinity uptake mechanism by spinal cord cells. Neurons are not the only cell type in culture to exhibit high affinity choline uptake. Chick fibroblasts in both rapidly growing and stationary phase can accumulate choline with kinetics similar to those observed for the high affinity uptake by spinal cord cells. Little if any of the choline accumulated by fibroblasts, however, is converted to ACh. In most uptake studies with spinal cord cells, contributions from fibroblasts were minimized by carrying out the analysis at a time when few non-neuronal cells were present in the spinal cord cultures. These observations suggest that a population of chick central nervous system (CNS) neurons develop a high affinity choline uptake mechanism in cell culture that has many of the properties described for uptake by cholinergic neurons in vivo and that at least part of the choline accumulated by the system can be used for neurotransmitter synthesis. INTRODUCTION
Dissociated cell cultures prepared with embryonic chick spinal cord cells and myotubes contain a population of cholinergic neurons. This is indicated by the fact that some of the surviving neurons form functional cholinergic synapses on the myotubes, and high levels of the enzyme choline acetyltransferase appear in the cultures (Fischbach, 1972; Berg and Fischbach, 1978). The experiments reported here were undertaken to assay another property of cholinergic neurons, the ability to accumulate choline by a high affinity uptake mechanism. We were interested in determining (1) if high affinity choline uptake could be expressed by CNS neurons in dissociated ceII culture, (2) if the uptake could provide choline for ACh synthesis and resemble in other respects uptake by choline& neu-
rons in vivo, and (3) if synaptic connections with muscle would play a role in development of the uptake mechanism. Neurons of the central nervous system appear unable to synthesize choline de novo (Bremer and Greenberg, 1961; Ansell and Spanner, 1967; Browning and Schuhnan, 1968). It has been proposed that choline needed for neuronal metabolism is derived from external sources either as free choline (Groth et al., 1958) or as a lipid-bound form (Illingworth and Portman, 1972). Much evidence has been presented that cholinergic neurons have a high affinity uptake mechanism for accumulating choline from external sources and that choline accumulated by the uptake mechanism can serve as substrate for ACh synthesis. Studies on tissue fragments and synaptosome preparations containing cholinergic nerve terminals from 90
0012-X06/78/0651-0090$02.00/0 Copyright AU rights
0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
BARALD
AND BERG
Choline Uptake by Spinal Cord Cells
a variety of vertebrate and invertebrate postembryonic sources have revealed an uptake mechanism for choline with K,,, of l-4 fl (Haga and Noda, 1973; Yamamura and Snyder, 1973; Pert and Snyder, 1974; Schwartz et al., 1975; Suszkiw and Pilar, 1976). The high affinity choline uptake mechanism is dependent on the presence of external Na+ and can be inhibited by low concentrations of hemicholinium-3 (10 $kf); much of the accumulated choline (ca. 70%) is converted to ACh. Tissue though!, to lack cholinergic nerve terminals or tissue in which cholinergic terminals have been destroyed contains only a low affinity, Na+independent mechanism for accumulating choline (Yamamura and Snyder, 1973; Kuhar et al., 1973; Pert and Snyder, 1974; Mulder et al., 1974; Carroll and Buterbaugh, 1975; Suszkiw and Pilar, 1976). Choline uptake studies on different cell types grown in culture have yielded mixed results. Dissociated cerebral cells from chick display a high affinity choline uptake mechanism in culture (K, = 2-3 $V) with a partial dependence of Na+ (Massarelli et al., 1974a), while cultured cerebral cells from rat exhibit uptake mechanisms of only intermediate and low affinities (K,,, = 16 and 960 @f, respectively) and convert little of the accumulated choline to ACh (Yavin, 1976). High affinity choline uptake is found in several neuroblastoma cell lines, but the uptake is not inhibited by hemicholinium3 and is not restricted to cell lines capable of synthesizing ACh (Richelson and Thompson, 1973; Massarelli et al., 1974b; Lanks et al., 1974). High affinity choline uptake has also been demonstrated for two non-neuronal cell lines: glioma cells (Massarelli et al., 1974c) and hepatoma cells (Plagemann, 1971). It remained unclear whether cholinergic neurons grown in cell culture would develop a high affinity choline uptake mechanism and whether the uptake would have properties distinguishing it from uptake mechanisms in other cell types. We have found that chick spinal
91
cord-myotube cultures have a high affinity uptake mechanism for choline. The uptake is Na+-dependent and can be blocked by hemicholinium-3. Part of the accumulated choline is converted to ACh. Spinal cord neurons grown with and without myotubes have high affinity uptake mechanisms of equivalent magnitude, indicating that nerve-muscle synapse formation is not necessary for development of the uptake mechanism. A preliminary account of some of these results has appeared (Barald and Berg, 1977). MATERIALS
AND METHODS
Preparation of cell cultures. Myotube cultures were prepared by mechanically dissociating pectoral muscle from 1l-dayold chick embryos into a suspension of single cells as previously described (Berg and Fischbach, 1978). Cells were added to collagen-coated dishes at a concentration of 2-3 X lo5 tells/35-mm dish. After an initial 48-h period in culture medium (see below), cultures were incubated for an additional 48 hr in medium containing 10e5M cytosine arabinoside to kill rapidly dividing fibroblasts (Fischbach, 1972). Dissociated spinal cord cells were prepared from 4-day-old chick embryos by incubating cord fragments in Ca’+, Mg”+-free salt solution and using gentle trituration to separate the cells. Previously described techniques were used (Berg and Fischbach, 1978) except that the dissected spinal cords were first carefully stripped free of other tissue and then were dissociated in the absence of trypsin. Cell yields (ca. 1.4 x 10” cells/cord) were similar to those reported previously for cell preparations obtained by trypsin treatment (Berg and Fischbach, 1978). These modifications resulted in fewer non-neuronal cells present in spinal cord cultures during the first week after plating. Spinal cord cells were grown in culture by adding them at a concentration of 4.0 X 10”/35-mm dish either in 4-day-old myotube cultures or on collagen-coated dishes in the absence of myotubes. Unless
92
DEVELOPMENTAL BIOLOGY
otherwise indicated, all cultures were taken for analysis 7-8 days after plating the spinal cord cells. Fibroblast cultures were prepared by incubating the initial muscle cell suspension, obtained as described above, in 60-mm tissue culture plastic dishes for 20 min at 37°C. Fibroblasts preferentially attached to the plastic surface during this time. The cell suspension was then replaced with fibroblast culture medium (see below), and the attached cells were allowed to grow almost to confluency. The attached cells were then resuspended by treatment with crude trypsin (0.33% for 10 min), collected by centrifugation at 50g for 5 min, filtered through a double layer of lens paper to remove cell clumps, and added to tissue culture dishes at 3.0 x lo5 cells/35mm dish. To minimize residual contamination by myoblasts, the procedure was repeated twice, each time allowing the cells to grow almost to confluency. The hnal round of cultures was used for experiments when they were approximately half confluent (rapidly growing cultures) or when they were 2-3 days postconfluent (stationary cultures). Choline uptake. Choline uptake was examined by incubating cultures with r3H]choline. Cultures were rinsed twice with PBSS (2 ml) and then incubated at room temperature in 1.0 ml of PBSS containing 0.1-100 fi r3H]choline at a specific activity of 0.1-10 Ci/mmole (obtained by diluting 10 Ci/mmole [3H]choline chloride with unlabeled choline chloride). Uptake was terminated by the addition of 20 ~1 of 0.5 M choline; the cultures were then rinsed three times with PBSS (2 ml). Cell extracts were prepared by incubating the cultures in 0.5 ml of 0.6 N NaOH overnight and then scraping the dishes with a Teflon pad. Accumulated radioactivity was determined by liquid scintillation counting of acidified aliquots in Aquasol-2. Background levels were determined by incubating cultures under the same conditions with the addition of 10 mit4 unlabeled choline to saturate the uptake mechanisms; blanks determined in
VOLUME 65.1978
this manner did not exceed 5% of the uptake observed at 0.1 fl [3H]choline. Unless otherwise indicated, values for uptake have been normalized for the total amount of cellular protein present. In cases where uptake was examined in the presence of a drug or in the absence of Na+, experimental and control cultures were preincubated for 10 min in the appropriate solutions to allow equilibration prior to the initiation of uptake. Acetylcholine synthesis. Acetylcholine synthesis was measured by high voltage paper electrophoresis (Hildebrand et al., 1971). Cultures were incubated with 1.0 fl [3H]choline and rinsed as described above except that either PBSS or Earle’s BSS were used, and both were supplemented with 5 gm/liter of glucose, 50 units/ml of penicillin, and 50 pg/ml of streptomycin. Incubations were carried out at 37°C. Cell extracts were prepared by scraping the dishes in 0.20 ml of 0.05 M sodium phosphate buffer, pH 7.5, containing 0.3% sodium dodecyl sulfate, 0.7 mM ACh, 2 mM choline, and 0.1 mil4 neostigmine methylsulfate. The extracts were homogenized in a glass tissue grinder equipped with a Teflon pestle, and stored frozen. r3H]ACh was assayed by applying 20-~1 aliquots to strips of Whatman 3 MM chromatography paper and submitting the sample to electrophoresis for 45 min at 2500 V (ca. 50 V/cm). Electrophoresis buffer contained 0.47 M formic acid and 1.4 M acetic acid (pH 1.9). The positions of [3H]ACh and [3H]choline were determined by iodine vapor staining of unlabeled carrier ACh and choline. After cutting the strips into segments (1.5 x 0.5 in), the radioactivity was eluted with 0.5 ml of 0.1 N HCl, mixed with 5 ml of Aquasol2, and counted by liquid scintillation counting. Under the conditions used, ACh synthesis was linear with time for periods up to 1 br and was proportional to the number of spinal cord cells initially added to the cultures (Berg, 1978). Protein determinations. The amount of protein in cell extracts was determined by
BARALD
AND BERG
Choline
the method of Lowry et al. (1951). Bovine serum albumin was used as a standard. Background values were determined by assaying extracts prepared from collagencoated dishes carried through the same feeding regimen as the cell cultures and were equivalent to lo-20% of the values obtained for spinal cord-myotubes cultures. Media and chemicals. Culture media and biological materials were obtained and prepared as previously described (Nishi and Berg, 1977). All media contained 5% (v/v) chick embryo extract except medium containing low5 M cytosine arabinoside which had 2% embryo extract. Fibroblast culture medium contained 10% (v/v) fetal calf serum (Microbiological Associates, Inc.) instead of horse serum and lacked embryo extract. Culture feeding regimens were as previously described (Nishi and Berg, 1977). Earle’s BSS was purchased from Microbiological Associates, Inc. PBSS con-
Uptake
by Spinal
Cord
Cells
93
tained 123 n&f NaCl, 5.4 m&f KCl, 11 mM NaP04 (pH 7.3), 0.9 n&f CaC12, 0.4 rniV MgS04, and 1 g/liter of glucose. r3H]Choline chloride (10.1 Ci/mmole) was purchased from Amersham/Searle, Aquasolfrom New EngIand Nuclear, hemicholinium-3 from Eastman, and acetylcholine chloride, choline chloride, ouabain, and neostigmine methylsulfate from Sigma. RESULTS
Choline uptake by spinal cord-myotube cultures. Myotube cultures seeded with spinal cord cells prepared from 4-day-old chick embryos by mechanical means contained two major cell types at early times: neurons and myotubes (Fig. 1A). Usually few fibroblast-like cells were apparent under these conditions during the first week. We examined choline uptake in spinal cord-myotube cultures at 1 week by incubating them with [3H]choline. Uptake was linear with time for at least 10 min at all
FIG 1. Micrographs of dissociated cell cultures. Cultures were prepared and maintained as describe :d in M aterials and Methods. Micrographs were taken with phase contrast optics; bar = 100 pm. (A) Spinal cord cells myotubes. (B) Spinal cord cells grown alone for 7 days. (C) Myotubes gr -own IT own for 7 days on 4-day-old al one for 11 days.
94
DEVF,I.OPMENTAI,
BIOLOGY
choline concentrations examined. Results obtained at 0.1 and 100 $V are shown in Fig. 2. Double reciprocal analysis of the data for choline concentrations of 0.1-1.0 fl revealed a high affinity uptake component. The uptake was characterized by a K,, of 3.4 + 0.5 fl and a V,,, of 40.0 + 0.1 pmoles/min/mg of protein (mean f SEM, seven separate determinations). A typical experiment is shown in Fig. 3. Uptake at 0.1 fl choline was examined in the presence of inhibitors to determine whether the uptake mechanism had properties similar to those reported for high
A 2/
I/
Y 0
5 TIME (mid
1
IO
FIG. 2. Time dependence of choline uptake in spinal cord-myotube cultures. Spinal cord-myotube cultures were examined for choline uptake by incubating the cultures in [“HIcholine for the indicated periods of time and determining the amount of radioactivity retained as described in Materials and Methods. Values represent the means of triplicate cultures; vertical lines indicate the SEM. (a) Incubation in 0.1 fl choline. (b) Incubation in 100 fl choline.
VOLIJME
65, 1978
‘1s IpM-‘I FIG. 3. High affinity choline uptake in spinal cord-myotube cultures. Spinalcord-myotube cultures were incubated in 0.1-1.0 aA choline, and the amount of choline accumulated in the fist 5 min was used to calculate the initial rates of uptake. The reciprocal of the initial velocity, l/V, is shown as a function of the reciprocal of the choline concentration, l/S. Values represent the means of triplicate cultures; vertical lines indicate the SEM. The line was fitted by hand. Values for K,, and V,,, were calculated to be 3.4 fl and 40 pmoles/min/mg of protein, respectively, for this experiment.
affinity choline uptake in cholinergic tissue and synaptosome preparations (Haga and Noda, 1973; Yamamura and Synder, 1973; Pert and Snyder, 1974; Suszkiw and Pilar, 1976). Consistent with those findings, uptake in spinal cord-myotube cultures was almost completely blocked by hemicholinium-3 (10 @fl and was significantly reduced when Na’ in the medium was replaced with Li’. ,Ouabain, an inhibitor of the Nat, K+-dependent ATPase, and KCN, a metabolic inhibitor, each inhibited uptake slightly (Table 1). Uptake at 100 fl choline was inhibited much less by replacement of Na’ with Li’ (Table 1). This raised the possibility that spinal cord-myotube cultures had a second choline uptake mechanism with a lower affinity for choline. To test for the presence of such a system, choline uptake was examined under conditions that minimized contributions from high affinity uptake. Na+ in the medium was replaced with Li’, and uptake was examined over the range of lo-100 fl choline. In four experiments double reciprocal analysis of the data yielded values for Km of 70 f 25 fl and V,
BAIIALD
TABLE INHIBITION
OF CHOLINE
Choline
IN SPINAL
C~lI.T~lH~S”
Choline
uptake
0.1 LLM
Li’ (low Na’) Hemicholinium-3 Ouabain KCN
BERG
1 UPTAKE
CORD-MYCW~IFIE
Condition
AND
34 f 3 (9) 9 -c 1 (9)
(9, of control) lC@lLM
71 -c 8 (8)
60 -t 14 (4) 64 + 12 (4) 70 f 5 (4)
87 + 3 (7)
69 k 13 (4)
” Inhibition of choline uptake was examined in spinal cord-myotube cultures at 0.1 and 100 fl choline under four conditions. Uptake in experimental cultures is expressed as a percentage of that observed for controls incubated in normal PBSS. Values represent the mean & SEM; the number in parentheses indicates the number of separate determinations. Each determination for a given condition was taken from a separate cell plating and represents the mean of three separate cultures. The conditions examined were (a) low Na’, in which 137 mJ4 LiCl was used to replace the NaCl normally present in PBSS, (b) 10 N hemicholinium-3, (c) 0.1 mM ouabain, and (d) 1 mM KCN.
of 40 f 14 pmoles/min/mg of protein (mean 4 SEM). One such experiment is shown in Fig. 4. While these results indicate that spinal cord-myotube cultures can also display a low affinity choline uptake mechanism, it is not clear whether the low affinity mechanism is present under normal conditions. Uptake studies in the presence of Na’ yielded ambiguous results in the range of 10-100 @II choline, both because of the large contribution from the high affinity mechanism and because of the small radioactive signal under these conditions. Conceivably the absence of Na’ could alter both the K,,, and the V, of the high affinity system. High affinity uptake by individual cell types. To distinguish whether spinal cord cells or myotubes were responsible for the high affinity choline uptake observed in the mixed cultures, the cell types were grown separately (Figs. 1B and 1C) and analyzed for choline uptake as described above. For both culture types choline uptake was linear with time for at least 8 min at all choline concentrations examined. Double reciprocal analysis of the data revealed a major high affinity uptake component in spinal
Uptake
by Spinal
Cord
95
Cells
cord cultures that quantitatively accounted observed in spinal for the uptake cord-myotube cultures. A typical experiment is illustrated in Fig. 5A; compiled results from several experiments are listed in Table 2. Thus, at 1 week, myotubes contribute very little to the choline uptake observed in mixed cultures, either directly by accumulating choline, or indirectly by influencing the development of spinal cord cells that express the high affinity system. Cultures of myotubes lacking spinal cord cells also exhibited a high affinity choline uptake mechanism, but the capacity of the system was very small (Fig. 5B, Table 2). The V, was about 1% of that observed for spinal cord cells. Since fibroblasts often contaminate myotube cultures in small amounts, choline uptake by chick fibroblast cultures was examined in a similar manner. Both rapidly growing fibroblasts and fibroblasts in stationary phase displayed a major high affinity uptake component (Fig. 5C,
/
-02
I
0
02
04
06
08
I
IO
I/s (/API
4. Low affinity choline uptake by spinal cord-myotube cultures in the absence of Na’. Initial rates of choline uptake by spinal cord-myotube cultures in the absence of Na+ were determined over the range of lo-100 $lJ choline as described for Fig. 3. (NaCl in the incubation solution was replaced with LiCl.1 The reciprocal of the initial rate is shown as a function of the reciprocal of the substrate concentration. Values represent the means of triplicate cultures; vertical lines indicate the SEM. The line was fitted by hand. Values of K,,, and V,,, were calculated to be 63 $U and 29 pmoles/min/mg of protein, respectively, for this experiment. FIG.
96
DEVELOPMENTAL
BIOLOGY
VOLUME
65. 1978
bain (0.1 m.il4) had little effect on uptake by spinal cord cells or myotubes, but did partially inhibit uptake by fibroblasts (ca.
50%). Acetylcholine
synthesis. Acetylcholine (ACh) synthesis was measured to determine the relation between high affinity choline uptake and ACh synthesis. Spinal cord, spinal cord-myotube, myotube, and fibroblast cultures were incubated for 1 hr in a low concentration of [3H]choline (1 $V) so that uptake would occur primarily by the IO
TABLE
t
2
KINETIC PARAMETERS FOR HIGH AFFINITY CHOLINE UPTAKE IN CELL CULTURE” Cultures V, (pmoles/min) Km (p.M x -2
0
2
4
6
8
IO
I2
‘5 (j&P) FIG. 5. High affinity choline uptake in cultures of different cell types. Initial rates of choline uptake were determined in 0.1-1.0 aikf choline as described in Fig. 3, and double reciprocal plots of l/V vs l/S were constructed. Values represent means of triplicate cultures; vertical bars indicate the SEM. Lines were fitted by hand. (A) Spinal cord cells grown for 1 week in the absence of myotubes. Values of 2.5 @!4 and 67 pmoles/min/mg of protein were obtained for K,,, and V,,,, respectively, in this determination. (B) Myotube cultures 11 days old (i.e., equivalent in age to myotubes present in spinal cord-myotube cultures at the time of analysis). Value of 0.5 $W and 0.63 pmoles/min/mg of protein were obtained for K,,, and V, in this determination. (C) Rapidly dividing fibroblast cultures. Values of 3.3 fl and 50 pmoles/min/mg of protein were obtained for K,,, and V, in this determination.
Table 2). The specific activity of uptake (picomoles per minute per milligram of protein) was comparable to that observed with spinal cord cells. Thus some non-neuronal cell types in vitro can express a high affinity uptake mechanism for choline. Uptake at 0.1 $t4 choline was examined in the presence of inhibitors to determine whether the high affinity uptake mechanism in spinal cord cells could be distinguished from that observed with myotubes and fibroblasts. In all cases uptake was substantially inhibited by hemicholinium-3 (10 CLM)or by replacing Na+ in the medium with Li+ (Table 3). KCN (1 mM) and oua-
Spinal cordmyotube Spinal cord Myotube Fibroblast
3.4 f (n = 3.5 f (n = 0.63 f (n = 2.9 f (n=9)
0.5 7) 0.6 7) .04 3) 0.4
(per culture)
(per mg of protein)
13.0 k (n=6) 13.0 f (n = 0.18 f (n=3)
40.0 f (n = 67.0 + (n = 0.61 f (n=3) 71.0 f (n=9)
1.7 2.0 7) 0.08
0.1 7) 1.3 7) 0.05 9.6
” Spinal cord-myotube, spinal cord, myotube, and fibroblast cultures were examined for high affinity choline uptake, and the kinetic parameters of uptake were determined by double reciprocal analysis of the data as shown in Figs. 3-5. Results are expressed as the mean f SEM. The number in parentheses indicates the number of determinations; each determination represents a separate cell plating and corresponding double reciprocal plot. Cultures were processed in triplicate for each choline concentration used to generate each plot. TABLE INHIBITION Condition
Choline W;J
Li+ (low Na’) Hemicholinium-3 Ouahain KCN
3
OF CHOLINE UPTAKE CELL TYPES”
33 f 2 12 f 4 89 f 5 lOOf
FOR DIFFERENT
uptake
(W of control) Fibroblast
Myotube 23 16 93 82
+ f f f
4 5 7 15
12 -e 1 11f2 5423 43 + 5
n Choline uptake in spinal cord, myotube, and libroblast cultures was examined at 0.1 fl under four different conditions as described in Table 1. Uptake in experimental cultures is expressed as a percentage of that observed for control cultures incubated in normal PBSS. Values represent the mean + SEM for five separate determinations. Each determination for a given condition was taken from a separate cell plating and represents the mean of three cultures.
BARALD
high affinity uptake system. Cell extracts were then prepared and assayed for [3H]ACh by high voltage paper electrophoresis. Large amounts of C3H]ACh were present in extracts of both spinal cord and spinal cord-myotube cultures; levels in myotube and fibroblast cultures were at the limits of detection (Table 4). Thus significant amounts of [3H]ACh accumulated only in cultures containing spinal cord cells, and the amount was not significantly dependent on myotube influence under the conditions examined. High affinity choline uptake can apparently exist in some cell types (e.g., TABLE ACETYLCHOLINE
4
SYNTHESIS
Cell culture
IN CELL CULTURE”
ACh
synthesis
(pmoles/hr/mg protein) Spiral cordmyotube Spinal cord Myotube Fibroblaat
of
(% of total uptake)
51.1 f 5.3 (6)
15 + 2
67.1 + 3.4 (6) 0.9 2 0.2 (6) 0.9 + 0.3 (5)
12 f 2
