Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. 13814-1318, March 1978 Biochemistry Regulation of acetylcholine release from neuroblastoma X glioma hybrid cells (synapse formation/dibutyryl cyclic AMP/neuron development) RICHARD MCGEE®®, PAUL SIMPSON®°, CLIFFORD CHRISTIAN’, MARINA MATA‘, PHILLIP NELSON?, AND MARSHALL NIRENBERG? * Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, and 4Behavioral Biology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, January 10, 1978 ABSTRACT Neuroblastoma X glioma NG108-15 hybrid cells exposed to N®,O0?'-dibutyryladenosine 3':5'-cyclic mono- phosphate for several days release [SH]acetylcholine in response to serotonin, prostaglandin F2,,, KCI, or veratridine. NG108-15 cells grown in the absence of dibutyryl cyclic AMP do not re- spond to an excitatory stimulus by releasing [2H]acetylcholine but can be shifted to a responsive state by treatment with di- butyryl cyclic AMP. Thus, the reactions that are required for acetylcholine release can be regulated in NG108-15 cells, thereby regulating the ability of cells to form synapses and the efficiency of synaptic communication. Neuroblastoma X glioma NG108-15 hybrid cells form synapses with cultured striated muscle cells (1-4). Synaptogenesis by NG108-15 cells is greatly increased when hybrid cells are treated for a period of days with N®,O?-dibutyryladenosine 3’:5’-cyclic monophosphate (BtpcAMP). Culture of NG108-15 cells with BtgcAMP also results in increases in cell body diam- eter, neurite extension, abundance of clear vesicles 600 A in diameter (5), membrane excitability, and specific activities of choline acetyltransferase (acetyl-CoA:choline O-acetyltrans- ferase, EC 2.3.1.6) and acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7).¢ The cells generate both Nat and Ca?* action potentials and have endorphin receptors, excitatory muscarinic acetylcholine {ACh) receptors, a-adrenergic receptors, prostaglandin E) (PGE) receptors, PGF2, receptors, and adenosine receptors that are coupled to shifts in cyclic AMP and/or cyclic GMP levels of the cells (6-14).¢£8 Serotonin (13), ACh (13), PGF, (13),8 or dopamine (14) can depolarize the cells and initiate action potentials. In this report, we describe the properties of stimulus-de- pendent release of [7H]ACh from NG108-15 cells, a response required for synaptic communication, and show that the ability of the cells to release [[H]ACh in response to stimulation can be regulated by BtgcAMP. A preliminary report of this work has appeared (15). MATERIALS AND METHODS Cell Culture. The culturing of NG108-15 hybrid cells has been described (1). To switch cells to a more differentiated state, growth medium [90% Dulbecco's modified Eagle’s minimal essential medium (DMEM) (GIBCO H-21)/10% fetal bovine serum/100 »#M hypoxanthine/] uM aminopterine/16 uM thymidine] was supplemented with 1 mM BtecAMP, purified The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S. C. §1734 solely to indicate this fact. 1314 as described (1), and the fetal bovine serum concentration was decreased from 10% to 5%. Cells to be used for experiments were dissociated and transferred to 200-ul disposable glass capillary pipettes (total volume, 300 ul) bent in the form of a “U” and connected in series (1.5-2.0 X 10° cells per capillary) and incubated for 3 hr at 37° to promote cell attachment to the glass. The tubes then were perfused with 2 ml of medium per hr for 2 days unless indicated otherwise." Each U tube then contained 200-400 yg of cell protein. Measurement of [3H|Choline Uptake and [7HJACh Re- lease. Cells in capillaries were washed for 3 min by perfusion (0.4 ml/min) with medium A [DMEM without choline, NaHCOs, phenol red, and fetal bovine serum but with 15 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) and 125 mM NaCl, adjusted to pH 7.4 and 340 mOsm/liter]. Cells then were incubated without perfusion in medium A supplemented with [methyl-°H]choline (10.1 Ci/mmol, Am- ersham/Searle) as indicated in figure legends. For cells grown in the presence of BtgpcAMP, 1 mM BtgcAMP also was added to the [2H]choline uptake medium but not to the wash medium. After incubation, most of the [2H ]choline in the medium was removed by perfusion with medium B (medium A with 20 uM eserine sulfate), When only intracellular [SHjcholine, (8H]ACh, and other 3H-labeled metabolites of choline were to be deter- mined, the tubes were perfused for 1.5 min (0.7 ml/min) with medium A, a small air bubble was introduced into the line, and the capillary was filled with acetone/1 M formic acid, 85:15 (vol/vol), to precipitate protein and extract SH-labeled com- pounds from cells (16). The acetone/formic acid extract was collected and each tube was washed twice with 100 yl of the acetone /formic acid solution; the extracts and washes then were combined. Greater than 98% of the intracellular 9H-labeled compounds were recovered. Cell protein in each capillary then was dissolved in 0.4 M NaOH and assayed by a modification of the method of Lowry et al. (17) with crystalline bovine serum albumin as the standard. The 2H-labeled compounds extracted from cells were frac- tionated by high-voltage paper electrophoresis with 1.5 M acetic Abbreviations: BtgcAMP, N®,O9-dibutyryladenosine 3':5’-cyclic monophosphate; ACh, acetylcholine; PG, prostaglandin; DMEM. Dulbecco's modification of Eagle’s minimum essential medium; 5-HT. 5-hydroxytrypamine (serotonin). > Present address: Department of Pharmacology, Georgetown Uni- versity, Washington, DC 20007. © Present address: Cardiology 111C, Veterans Administration Hospital. San Francisco, CA 9412). ¢ B. Hamprecht, T. Amano, and M. Nirenberg, unpublished data. f S, Sharma and M. Nirenberg, unpublished data. & H. Matsuzawa and M. Nirenberg, unpublished data. h Details of the capillary culture tube system will appear elsewhere. Biochemistry: McGee et al. T T T T T t 4 (HI ACh [°H} Choline a) " 3 50 3 L 450 & 2 £5 s £ % (—) Enzyme x B40} & . 1 2 x a9 5 10 ( | = ® O18 fmt X 10° | 00's gE 2 q |* x 207 4 any a (+) or (—) enzymef 450 3 oo IOF 1 £ E (+) Enzyme 1 1 4 L 1 1 i Oo 16 20736 40 560 50 io 150° fmot [9H] ACh or [3H] choline Fic. 1. [2H]ACh was separated from [?H]choline by conversion of [3H] choline to [7H}phosphorylcholine with choline kinase and ex- traction of [SHJACh into 3-heptanone/30 mM tetraphenylboron. Indicated amounts of [7H]ACh (Left) or [?H]choline (Right) (both 10.1 Ci/mmol) were incubated in the absence or presence of 2.5 munits of choline kinase. [7H]ACh or unreacted {*H]choline then were ex- tracted into the organic phase and radioactivity was determined. Each point represents the mean of four values; SEM <4%. acid/0.75 M formic acid (18). ACh, choline, phospholipids, and a fraction containing phosphorylcholine and CDP-choline were identified by coelectrophoresis with authentic standards. The paper was treated with Ip vapor to visualize spots, 9H-labeled compounds in each fraction were eluted from the paper with 1 ml of water, the eluate was mixed with 10 ml of Hydromix (Yorktown Research), and radioactivity was determined by using a liquid scintillation spectrometer. When [8H]ACh release from cells was measured, cells that had been incubated with [°H]choline were washed by perfusion with medium B at 0.4 ml/min for 8-14 min as indicated in the figure legends and fractions then were collected at 2-min in- tervals unless indicated otherwise. Cells were exposed to dif- ferent compounds by stopping the peristaltic pump for a few seconds to switch from one perfusion medium to another. To separate (7H]ACh and [°H]choline in the perfusate, a modification of the method of Goldberg and McCaman (19) was used in which (8H|choline was converted to (7H |phospho- rylcholine during incubation with choline kinase, and then (SHJACh and remaining traces of [3H]choline, but not (3H]- phosphorylcholine, were extracted with 3-heptanone/30 mM tetraphenylboron (Sigma). Choline was converted to phos- Phorylcholine in a reaction mixture containing, in a final vol- ume of 0.275 ml, the following: 0.25 ml of perfusate, 10 mM NaATP, 10 mM MgCl, and 2.5 munits of choline kinase (ap- proximately 10 ug of protein, Sigma). Reaction mixtures were incubated for 15 min at 36° and then extracted with 2 ml of 3-heptanone/30 mM tetraphenylboron. The organic phase was removed and part (1.7 ml) was evaporated at 60° under a stream of air, the residue was dissolved in Hydromix, and ra- dioactivity was determined with a liquid scintillation spec- trometer. The amount of [3H]JACh or [*H]choline released by cells was calculated with the assumption that the specific ra- dioactivity of [SH]choline was not diluted. Because the (°H]- choline probably is diluted by unlabeled choline of cells, the values reported are lower than true values. As shown in Fig. 1, >97% of authentic [3H}]ACh or [2H |cho- line is extracted into the organic phase over the wide range of concentrations tested. However, after incubation with choline kinase, 97% of the [SH]ACh, but only 3-5% of the [3H |choline (or other 3H-labeled compounds present), is extracted into the organic phase. Purification of the [methyl-2H|choline by paper electrophoresis prior to use decreased the amount of 3H-labeled Proc. Natl. Acad. Sci. USA 75 (1978) 1315 material extracted after incubation with choline kinase to <1% of the (8H]choline present initially. The amount of 3H-labeled material extracted into the organic phase after incubation of [H]choline with choline kinase was determined for each ex- periment and the amount, <25% of the total 3H-labeled com- pounds extracted was subtracted from the total to calculate the amount of [SHJACh. The sensitivity of the assay is limited by the specific activity of the [SH]ACh and the quantity of other 3H-labeled compounds which are extracted with [SH]ACh. The maximum sensitivity in our experiments was 2 fmol of (SH|ACh per sample. The assay also is rapid, and as many as 300 samples were analyzed in a single day by one person. Characterization of Released [*H]JACh. Perfusate fractions from three tubes were pooled and eserine was removed by ex- traction with equal volumes of CHCls. The aqueous phase then was warmed to 36° to remove residual CHCl, and the solution was incubated with choline kinase as described above in the presence or absence of 0.5 unit of acetylcholinesterase (0.5 yg of protein, electric eel, Sigma) in a 0.25-ml! reaction mixture. Greater than 95% of authentic [SH]ACh was hydrolyzed under these conditions. RESULTS Release of [2HJACh from Cells Grown with or without BtgcAMP. NG108-15 cells incubated with (3H choline release both ([SHJACh and [H]choline into the medium. The release of [7H]ACh and [°H]choline into the medium from NG108-15 cells grown with or without BtgcAMP is shown in Fig. 2. NG108-15 cells grown without BtgcAMP released (SH|ACh into the medium but 80 mM K* had little or no effect on the rate of release. In contrast, the rates of [SH]ACh release from cells grown in the presence of 1 mM BtgcAMP increased in response to KCI. The basal unstimulated rates of [SH]ACh release from cells treated with BtseAMP were twice those of control cells, Both basal and KC]-stimulated rates of (3HJACh release in- creased throughout the 5 days of treatment with BtgcAMP. These results show that populations of NG108-15 cells can be shifted from an unresponsive to a responsive state with respect to KCl-dependent ACh release by exposure of cells to BtocAMP. KC] also stimulated the release of [8H]choline from both control and BtgcAMP-treated cells to approximately the same extent. The rates of basal and KCl-stimulated [2H]choline re- lease decreased between the 1st and 5th days of culture. These results show that BtgcAMP has little or no effect on basal or KCl-stimulated release of [SH choline. The (SHJACh released from cells was characterized further by determining the sensitivity of the 3H-labeled material re- leased by hydrolysis catalyzed by acetylcholinesterase (Table 1). After incubation in the presence of acetylcholinesterase, the 3H-labeled material recovered in the organic phase was <5% of the amount obtained in the absence of acetylcholinesterase. In experiments not shown here, >97% of the $H-labeled com- pounds released from cells exhibited the electrophoretic mobilities of ACh or choline. Veratridine, which activates action potential Na* ionophores, also stimulated (7H]ACh release from NG108-15 cells grown in the presence of BtgcAMP but had little or no effect on cells grown without BtgcAMP (Fig. 3A). The response to veratridine decreased with time. Veratridine-stimulated ACh release was abolished in the presence of 1 uM tetrodotoxin, a specific in- hibitor of action potential Na+ ionophore activation, but te- trodotoxin had no effect on the basal, unstimulated rate of [SHJACh release. Veratridine increased the rate of [SH ]choline release, after a delay, from cells grown with or without 1316 Biochemistry: McGee et al. eo T r rr r : 7 50k A Control cells I B Bt,cAMP-treated cells Acetylcholine c g 2 4 (OOF aD E ~ , e KCI 5 days £ 3 50r 4 o 3 days s 3 days K 3 c 1 day g Ow + + ; oy + +t + t 3 900F c yD | £ = Choline a 3 KL = 600f / 1 day, 5 / NOR - bt—H 3 days 1 ‘ KCI KCl 5 days © 10 \6 22 28 10 6 22 28 Minutes Fic. 2. Effect of culturing NG108-15 cells without or with BtscAMP on KCl-stimulated (solid symbols) release of (SHJACh (A and B) and of [8H] choline release (C and D). A cell suspension (ap- proximately 200 ug of cell protein) was added to each capillary and the tubes then were perfused with DMEM supplemented with 5% fetal bovine serum for 24 hr. Some tubes then were perfused with the above medium supplemented with 1 mM BtocAMP. One (0), 3 (0), or 5 (A) days later, the release of (7H]ACh and {?H] choline from cells was determined. Cells were incubated with 10 uM [*H]choline for 45 min and washed for 10 min with medium without choline but with 20 uM eserine sulfate (wash discarded), and then fractions were collected at 2-min intervals (0.8 ml per fraction). Stimulation was with 80 mM KCI for 6 min as indicated. Each point represents the mean of four values obtained with separate cultures. Protein ranged from 200 yg per culture on the first day to 550 wg on the fifth day with or without BtocAMP. BtpcAMP but tetrodotoxin did not inhibit veratridine-stimu- lated [2H]choline release. This suggests that the increase in choline release due to veratridine is not mediated by the acti- vation of action potential Nat ionophores. Choline Metabolism. The effect of growing cells in the presence of BtgcAMP for 0, 9, or 16 days on [°H]choline me- tabolism is shown in Table 2. The intracellular concentrations of (SHJACh and [H]choline increased and those of phospho- Table 1. Effect of acetylcholinesterase on released [>H]ACh [SHJACh released, Stimulation fmol/mg protein of cells ~AChE +AChE None (before stimulation) 126 6 80 mM KCl] 211 3 None (after stimulation) 136 0 NG108-15 cells grown for 9 days with 1 mM BtgcAMP were incu- bated with 10 uM [H]choline for 45 min and washed by perfusion for 10 min. Six-minute fractions were collected before, during, and after stimulation with 80 mM KCl and perfusates from three cultures were then combined. Eserine was then extracted with CHCl, and the [2H]ACh content of the perfusates was determined after incubation in the presence or absence of acetylcholinesterase (AChE). Each value is the mean of duplicate determinations. Proc. Natl. Acad. Sci. USA 75 (1978) 3 — oO | Acetylcholine A |. Choline B | o c 2100) > fA 500 2 \. Treated 3 E 3 re ; ® € 490 o£ £* Jee Treated|“” £3 o 3 z° 3 =a © 0 TTX Control | o> 3 SO CE C = £ as g Control UE — 8 H—H 150 6 = Veratridine £ = Veratridine 2 Olu 1 1. 1. lye po 1 0 E 6 0 4 2&8 2 66 M0 4 BW RW 3 Minutes Fic. 3. Effect of culturing NG108-15 cells with or without BtgcAMP on veratridine-stimulated release of [PHJACh (A) or [3H] choline (B). Cells cultured with or without 1 mM BtgcAMP for 7 days were dissociated and transferred to U tubes (about 300 ug of protein per tube) and then perfused with the same growth medium with or without BtecAMP for 2 additional days. Cells were incubated with 10 uM [H]choline for 45 min and washed for 16 min as described in the legend to Fig. 2. Cells were exposed to 0.2 mM veratridine for 6 min as indicated. @,0, Cells treated with BtecAMP for 9 days; 4,4, cells cultured without BtscAMP for 9 days. 0,4, 1 uM tetrodotoxin (TTX) added at the start of the 16-min wash. Each point is the mean of four values obtained with separate cultures. rylated 3H-labeled compounds derived from [2H]choline de- creased as the time of exposure of cells to BtpeAMP was in- creased. After 16 days of exposure to BtgecAMP, NG108-15 cells had 2- and 5-fold higher levels of (SH|ACh and [°H]choline, respectively, than cells not exposed to BtgeAMP. The results shown in Table 2 were obtained with NG108-15 cells that had been incubated with [8H ]choline and then washed in the absence of extracellular choline for 36 min. The results shown in Fig. 4 were obtained with cells that had been incu- bated with [2H]choline and washed only 1.5 min prior to the extraction of 3H-labeled compounds from cells. The intracel- lular level of [2H]choline increased rapidly during the first 10 min of incubation and then plateaued, whereas the amount of (SHIACh in cells increased throughout the 60-min incubation period. (8HJPhosphorylcholine accumulated at a rapid linear rate for 60 min. After a short lag, [SH ]phospholipids also ac- cumulated rapidly, which suggests a precursor—product rela- tionship between [*H|phosphorylcholine and [8H]phospho- lipids. The relationship between extracellular choline concentration and uptake and metabolism of [*H]choline by NG108-15 cells is shown in Fig. 4 right. The accumulation of 3H-labeled phosphorylated compounds derived from [3H|choline and of Table 2. [H]Choline metabolism by NG108-15 cells grown with or without BtgcAMP Intracellular levels, pmol/mg protein Days Phosphorylated Total with {H]- [H]- [3H] choline (SH] BtacAMP ACh Choline metabolites compounds 0 19.3 3.3 685 708 9 33.8 11.3 704 750 16 40.0 16.2 519 575 NG108-15 cells grown with BtgcAMP for 0, 7, or 14 days were transferred to U tubes and perfused with medium with or without BtocAMP for 2 days. Cells then were incubated with 10 2M [3H]cho- line for 45 min and washed for 36 min with medium B. 3H-Labeled metabolites in the cells were extracted and subjected to paper elec- trophoresis, Each value represents the mean of three values obtained with separate cultures. Biochemistry: McGee et al. 900; + PL, Pch L Choline; 300 Choline + 4 pmol [*H] choline incorporated/mg protein ACh L i L i 10 2 30 40 50 60051 5 10 50 100 500 Choline, uM Fic. 4. Choline uptake and metabolism by BtecAMP-treated NG108-15 cells. (Left) Cells that had been grown with 1 mM BtecAMP for 16 days were incubated with 10 4M [8H] choline for the times indicated and then washed for 1.5 min at a perfusion rate of 0.7 ml/min. The cells then were extracted with acetone/1 M formic acid, 85:15 (vol/vol), the 3H-labeled compounds extracted were separated by high-voltage paper-electrophoresis, and the protein content of each culture was determined. Each point represents the mean of three values from separate cultures. PCh, (?H]phosphorylcholine and [SH]CDP-choline; PL, [H] phospholipids; choline, [2H] choline; ACh, (83H}ACh. (Right) Cells that had been treated with BtzcAMP for 11 days were incubated with [*H]choline at the concentrations indicated for 15 min and then washed; then, [H]choline and metabolites were extracted as for Left. PCh + PL, phosphorylcholine, CDP-choline, and phospholipid; choline, [3H]choline; ACh, [SHJACh. (Inset) Total uptake of ['H]choline by cells. Minutes total 3H-labeled compounds increased as the extracellular choline concentration was increased and plateaued at 250 uM (2H |choline. However, [2H]choline and [SH]ACh levels in cells increased over the entire range of extracellular [SH|choline concentrations tested (0.5-500 uM). Data analysis by the method of Lineweaver and Burk suggests that NG108-15 cells have both high- and low-affinity choline uptake mechanisms (not shown); however, further work is needed to define more accurately the properties of the choline uptake systems. The results also show that, as the extracellular choline con- centration is increased, the levels of intracellular (H|choline and (3H]ACh increase at approximately the same rate. The distribution of 3H-labeled compounds accumulated by the cell remained constant between 0.5 and 250 uM extracellular (3H choline. These results suggest that the synthesis of (7HJACh is not preferentially coupled to the high- or low-affinity choline uptake systems. Properties of [7HJACh and {3H]Choline Release. KCl repetitively stimulated [7H]ACh release from NG108-15 cells perfused with control medium containing 1.8 mM Ca?* and 0.8 mM Mg?*; but the second response to KC] was smaller than the first response (Fig. 5). Omission of Ca?* and increasing Mg?+ to 4 mM abolished the KC]-dependent (9H]ACh release and also decreased the unstimulated rate of [7H]JACh release. Omission of Ca?+ and increasing Mg?* partially decreased the KCl-stimulated release of [2H |choline from cells. The relationship between KCI concentration and [SH]ACh release from cells is shown in Fig. 6 left. KCI at 30 mM stimu- lated the release of (2HJACh from cells but not as effectively as 40 or 80 mM KCI. A second addition of KCl resulted in the release of approximately half the amount of [H]ACh compared to the first response to KC] at each concentration of KC] tested. These results suggest that the decreased (8H|ACh release evoked by the second application of KCl may not be due to depletion of an intracellular pool of releasable [SHJACh. As shown in Fig. 6 right, 10 uM 5-hydroxytryptamine (5-HT) Proc. Natl. Acad. Sci. USA 75 (1978) 1317 £ aan 7 7 ph r + ; r . 120+ + = A Acetylcholine 8 Choline aad a ~ & 100} Contrat 4+ 4750 3 & Control 2 a & 3s — 80 4600 £3 ~ ® 5 3 ex oe aa ® 60 4450 2a 3 SE 2 No Ca” - re 6 4mM Mg 490 CLE s KH 4 8 = 2 KCl KCI 4 KCI KCI digo E 6 &€ oY 1 i 1 1 4 4 4 i 1 1 1 0 - 4 #18 2 26 20 4 4 8 2B MW HU Minutes Fic. 5. Effects of omission of Ca2+ and increasing Mg?* on KCl-stimulated release of [7H]ACh (A) and [8H]choline (B) from NG108-15 cells. Cells grown with 1 mM BtecAMP for 35 days were incubated with 13 »M [H]choline for 45 min and washed, as described in the legend to Fig. 2. Cells then were perfused with control medium (0,@) (1.8 mM Ca2*+, 0.8 mM Mg?*) or medium without Ca2+ and adjusted to 4 mM MgCl (4,4). Cells were stimulated with 80 mM KC] for 4 min as indicated (solid symbols). Perfusate fractions were assayed for [7H]ACh and [*H]choline. Each point represents the mean of three values from separate cultures. or PGF, stimulated (SHJACh release from NG108-15 cells; whereas, PGE, stimulated [SH]ACh release only slightly. Per- fusate fractions were collected at 1-min intervals since cell re- sponses to 5-HT at PGF, rapidly desensitized. The addition of 5-HT, PGF», or PGE; had little or no effect on the rate of [3H]choline release from cells (not shown). DISCUSSION The results show that the ability of NG108-15 cells to respond to excitatory stimuli by releasing ACh into the medium can be f 8 | | | | yw 8. 200 fmol [7H] ACh released/min/mg protein | 3a? | fmol {°7H] ACh released/min/mg protein wage! ow. 100 Ma : KCI KCI gl s00l-- es Se 3 10 16 #22 28 3M 40 14 «#16 18 20 22 Minutes Minutes Fic. 6. (Left) Effect of KC] concentration on the amount of (PHJACh released from NG108-15 cells. Cells cultured with BtecAMP for 11 days were incubated with 20 uM (#H]choline for 45 min. The cells then were washed and perfusate fractions were collected and assayed for [3H]ACh as in the legend to Fig. 2. Each point represents the mean of three values from separate cultures. Solid symbols cor- respond to fractions collected during stimulation with KCl, open symbols correspond to fractions collected from cells before and after the stimulus. A, 80 mM KCI; 0, 40 mM KCi, 5, 30 mM KCl. (Right) Effects of serotonin (5-HT), PGF 2q, or PGE, on the release of (°H]- ACh from NG108-15 cells. Cells grown in BtpcAMP for 9 days were incubated with 35 »M [°H|choline for 45 min and then washed for 14 min as in the legend to Fig. 2; fractions then were collected at 1-min intervals. Cells were stimulated for 3 min (solid symbols) with 10 «M serotonin creatinine sulfate (A), 10 uM PGF, (@), or 10 .M PGE; plus 0.1% ethanol (0). Perfusate fractions were assayed for [H|ACh. Each point represents the mean of three values from separate cul- tures. 1818 Biochemistry: McGee et al. regulated. Cells grown without BtgcAMP do not respond to excitatory stimuli by releasing [2H]ACh but they can be shifted toa responsive state by treatment with BtgcAMP. The response to stimulation slowly increases over a period of at least 5 days while cells are cultured with BtgcAMP. Because ACh release in response to stimulation is required for synaptic communi- cation, the ability of the cells to form synapses and the efficiency of synaptic communication can be regulated by factors that control stimulus-dependent ACh release. Exposure of NG108-15 cells to BtpeeAMP also results in an increase in cell body diameter, neurite length, number of clear vesicles, membrane excitability, and the specific activities of acetyl- cholinesterase and choline acetyltransferase (5-14). Thus, many reactions required for synapse formation are regulated, directly or indirectly, by BtgcAMP. The properties of evoked ACh release from NG108-15 cells treated with BtgcAMP are similar to those of neurons. The amount of ACh release due to exposure of cells to KCI is a function of KCI concentration, 40-80 mM KCl evokes maximal ACh release. The response to KCI is abolished by removal of extracellular Ca?+ and elevation of Mg?* from 0.8 to 4 mM. Veratridine-stimulated ACh release is completely inhibited by 1 uM tetrodotoxin, a specific inhibitor of action potential Nat ionophore activation. Serotonin or PGF2,-stimulated ACh re- lease ceases rapidly, possibly due to receptor desensitization, whereas veratridine- or KCl-dependent ACh release can be maintained for longer periods (4-8 min). In each case, however, only 1% or Jess of the intracellular [7H]ACh is released before cell responsiveness to the stimulating agent decreases or dis- appears. If [SH]ACh is released into the medium from vesicles that contain approximately 10,000 molecules of ACh per vesicle, then one can estimate that ACh may be released from 10 ves- icles per min per cell without stimulation and 15 vesicles per min per cell when stimulated by serotonin. Both basal and serotonin-stimulated rates of ACh release are compatible with muscle responses observed at synapses between NG108-15 cells and striated muscle cells (1-4, 13), but response rates at different synapses vary greatly. Such data suggest that the NG108-15 cell population is heterogeneous with respect to the amount of ACh released and the responsiveness of cells to stimulation. Bio- chemical measurements of [7H]ACh represent the mean values for the population of cells. The results suggest that ACh is released from NG108-15 by two mechanisms because, Ca?* is required for KC]-stimulated release of ACh but not for unstimulated release of ACh. KC] stimulates the release of both [SH]ACh and (*H]choline; how- ever, these compounds seem to be released by different mechanisms. About 15% of the [H]choline taken up by NG108-15 cells is converted to [SH]ACh at each choline concentration tested between 0.5 and 250 uM. Thus, the synthesis of [7H]ACh is not preferentially coupled to high- or low-affinity choline uptake systems. In contrast, Yavin (20) has observed that an increased percentage of accumulated [H]choline is converted to [SHJACh by cultured rat embryo brain cells as the external concentration of (8H]choline is increased. The reasons for the differences between these two systems is unknown at present but both differ Proc. Natl. Acad. Sci. USA 75 (1978) substantially from what has been observed with synaptosomes, in which 50-80% of the {[*H]choline accumulated is converted to [SHJACh (21). One major difference between choline me- tabolism in cultured cells and synaptosomes is that much of the choline probably is taken up by cell bodies rather than nerve terminals and is thus exposed to both acetylation and phos- phorylation pathways; little if any phosphorylation of choline is observed with synaptosomes (21). The results show that stimulus-dependent [SH]ACh release from NG108-15 cells is acquired slowly by cells over a period of at least 5 days while cells are exposed to BtpcAMP. The slow increase in cell responsiveness to stimuli suggests that one or more components required for ACh release may be formed during this time. Whether regulation of stimulus-dependent ACh release by BtecAMP is mediated by cyclic AMP is not known; however, if the process is regulated by cyclic AMP, neurotransmitters, hormones, or other molecules coupled to the activation or inhibition of adenylate cyclase might then regulate both the ability of a neuron to form functional synapses and the efficiency of communication across the synapse. 1. Nelson, P., Christian, C. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 123-127. 2. Puro, D. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 3544-3548. 3. Christian, C., Nelson, P., Peacock, J. & Nirenberg, M. (1977) Science 196, 995-998. 4. Nelson, P. G., Christian, C. N., Daniels, M. P., Henkart, M., Bullock, P., Mullinax, D. & Nirenberg, M. (1978) Brain Res., in press. 5. Daniels, M. P. & Hamprecht, B. (1974) J. Cell Biol. 63, 691- 699. 6. Traber, J., Fischer, K., Latzin, S. & Hamprecht, B. (1975) Nature 253, 120-122. 7. Sharma, S. K., Klee, W. & Nirenberg, M. (1975) Proc. Natl. Acad. Sci. USA 73, 3092-3096. 8. Traber, J., Fischer, K., Buchen, C. & Hamprecht, B. (1975) Na- ture 255, 558-560. 9. Klein, W. L., Nathanson, N. M. & Nirenberg, M. (1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1576 (Abstr.). 10. Traber, J., Reiser, G., Fischer, K. & Hamprecht, B. (1975) FEBS Lett. 52, 327-332. . ll. Sabol, S. L. & Nirenberg, M. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, 736 (Abstr. ). 12. Traber, J., Fischer, K., Latzin, S. & Hamprecht, B. (1974) FEBS Lett. 49, 260-263. 13. Christian, C. N., Nelson, P. G., Bullock, P., Mullinax, D. & Nirenberg, M. (1978) Brain Res., in press. 14. Myers, P. R. & Livengood, D. R. (1975) Nature 255, 235-237. 15. McGee, R., Simpson, P., Christian, C., Mata, M., Nelson, P. & Nirenberg, M. (1977) Trans. Am. Soc. Neurochem. 8, 108. 16. Toru, M. & Aprison, M. H. (1966) J. Neurochem. 13, 1533- 1544. 17. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. Potter, L. T. & Murphy, W. (1967) Biochem. Pharmacol. 16, 1386-1388. 19. Goldberg, A.M. & McCaman, R. E. (1973) J. Neurochem. 20, 1-8. 20. Yavin, E. (1976) J. Biol. Chem. 251, 1392-1397. 21. Yamamura, H. I. & Snyder, S. H. (1973) J. Neurochem. 21, 1355-1374.