Acetylcholine and carnitine sensitive growth in a Drosophila cell line

Camp. Biochem. Physiol.

030~4492/U s3.00 + 0.00 0 1985Pergamon Press Ltd

Vol. 82C, No, 1, pp. 235.-241,1985

Printed in Great Britain

ACETYLCHOLINE AND CARNITINE SENSITIVE GROWTH IN A DROSOPHILA CELL LINE ALAN R.

GINGLE

College of Pharmacy, University of Georgia,

Athens, GA 30602, USA

(Received 4 Februar}~ 1985)

Abstract-l. Growth yield in cultures of Schneider’s line 2 cells was studied as a function of plating cell density. 2. It was measured in the presence of various neurotransmitters, hormones and related chemicals. Of these, acetylcholine and carnitine were studied in detail. Both significantly increased growth yields at low plating cell densities. 3. Atropine and tubocurarine significantly reduced growth yield, suggesting the presence of a cholinergic-like receptor. 4. Cell populations were incubated with [‘4CJcholine and uptake occurred. 5. The [‘4C]choline was incorporated into the production of secreted molecules which comigrated with carnitine offering evidence for carnitine production and secretion.

INTRODUCTION

Much of insect development is under hormonal control and the actions of some of these hormones can be observed in vitro. For example, cell division in cultures of many Drosophila cell lines stops after addition of the developmental hormone, @-ecdysone (Rosset, 1978; Berger et al., 1978). Also, it stimulates acetylcholinesterase production by a Drosophila cell line (Cherbas et ai., 1977). In the study reported here, the growth of a Drosophila cell line, Schneider’s line 2, was tested for sensitivity to some neural hormones and other related chemicals. A chemical which is at least a neural humoral factor in insects is acetylcholine. When the results of preliminary tests suggested that it enhanced growth, a detailed study of its effects followed as well as tests for its secretion. Its effects were measured over a range of cell densities, a variable which amongst other things is important to growth regulation both in uiuo and in vitro. The actions of carnitine, a similar molecule, were also studied in detail. Carnitine and acetylcholine are not unrelated to insect growth and development. Carnitine is an essential nutritional requirement of the meal worm, Tenebrie nt~~itor (Fraenkel and Friedman, 19.57). Also it is an effective substitute for choline in the diet of the blowfly, Phor~ia regina (Newburgh, 1965). In addition to its role as a neural transmitter, ~cetylcholine has been observed in large amounts in some nonnervous tissues of insects. For example, it has been found in high concentrations in the silk gland of larvae, the reproductive tract of the adults, and in eggs of Arotia cuja (Morley and Schachter, 1963). Also it is present in royal jelly of honey bees in large amounts (Colhoun and Smith, 1960). MATERIALS

AND METHODS

Culturing

Schneider’s line 2 was maintained in the usual manner {Schneider, 1972). stock cultures were grown (24-25”Cf in

plastic T-25 flasks containing peptone-supplemented (500 mg/lOO ml) Scheider’s medium (GIBCO) and 10% heat inactivated fetal bovine serum, iFBS (GIBCO). No antibiotics were added to these stock cultures. Culturing for experiments was similar, in general peptone-supplemented Schneider’s medium plus 10% iFBS was used with the of antibiotics (penicillin-streptomycin, 50 addition units/ml). Where deviations occurred, such as changes in percentage of iFBS or the absence of antibiotics, they are noted. Also, plastic 24-well culture trays (Falcon No. 3008) were employed for these experiments. Haruesting and planting Stock cultures were harvested by resuspending the cells, cent~fuging the suspension at 300g (Son/al RCZ-B) for 3 min, pouring off the old and resuspending in fresh medium. The resulting cell concentration was measured by counting in a hemocytometer grid. Plating cell density (cells/cm2) was an important variable in these experiments and the cell concentration was used in calculating the volume of cell suspension to be added to each well (area = 2 cm2) for its required cell density. Prior to cell plating, a fixed volume of culture medium, V, (usually 0.5 ml), was added to each of the wells. Then the appropriate volumes of cell suspension were added to the wells and the mixtures were gently stirred to ensure even settling of the cetls. Cell concentration was kept large and only small volumes of cell suspension were added to achieve the required cell densities. As a result, total m~ium volume varied by only a few per cent. Once plated, the cultures were incubated at constant temperature for times ranging from 20 to SOhr. During the incubations, the 24-well trays were contained in sealed metal boxes to prevent differential growth due to temperature gradients.

Growth yield measurements

In most experiments, the growth yield was measured for each well culture. This was done by gently resuspending the cells and counting them in a hemocytometer grid to determine the final number of cells per well, n,. This was divided by the initial number of cells per well, n,, determined earlier and gave a growth yield, nr/ni, quantifying the average growth in ceil number during the incubation period. Each well culture yielded a value of n&r,. In some experiments chemicals were added to one set of cultures prior to plating

235

236

ALAN R. GINGLE

and in these, growth yield was measured relative to that of the control cultures, without added chemicals. This was quantified in terms of a relative growth yield, nr/nfO. Conditioned medium As part of this study, the effect of conditioned medium on the growth of these cells was measured. The usual mixture of Schneider’s plus peptone, and iFBS, was conditioned by allowing line 2 cells to grow in it for 4 days, reaching final cell densities of about 10” cells/cm’. Afterward, the cells were centrifuged free of the medium (3000g for 5 min) which was decanted for immediate use. [‘4C]Choline uptake Choline uptake was measured in some experiments. It was done by incubating Schneider’s line 2 cells in medium containing [‘%]choline (New England Nuclear). First the cells were plated and allowed 30 min to settle. Next, 10~1 of the labelled choline stock was added to the 500~1 of medium in each well. The total choline concentration ranged from 0.19 to 0.56 mM (0.4-1.2 pCi/ml) and the incubations from 0.5 to 24 hr. After the incubations, the cells were resuspended, transferred to centrifuge tubes and washed three times. Washing was done by centrifuging at 3000g (Sorvall RCZ-B) for 3 mitt, decanting the medium and resuspending in fresh Schneider’s medium (4C). The decanted culture medium was saved for electrophoretic separation, described below. After the last centrifugation and decanting, scintillant (a mixture of toluene, Triton X-100 and PPO-POPOP) was poured over each cell pellet, mixed thoroughly, and then transferred to scintillation vials for “counting”. Also, vials were prepared with known amounts of [%]choline for calibration. Each vial was “counted” for IOmin in a Searle-Delta 300. The radioactivities were recorded and the uptakes computed.

RESULTS

The cell density dependence

of growth

The growth yield vs plating cell density data for Schneider’s line 2 exhibited a characteristic form. The growth yield increased from near unity to a plateau beginning at about 9 x lo4 cells/cm*. Providing that the cultures were not allowed to grow to confluence, the basic form of the growth yield curves was independent of the incubation durations (20-80 hr), temperatures (22-25(C), and serum contents (lo-20% iFBS) tried. Three typical curves are shown in Fig. 1. All exhibit the characteristic rise in growth yield; however, not all of the curves plateau. The uppermost curve shows a decrease, characteristic of incubations which lead to large final cell densities. Density dependence of ecdysterone

sensitivity

Schneider’s line 2 cells were incubated in the presence of ecdysterone with a resulting reduction in growth yield at most cell densities tried. The threshold concentration for this reduction is about lo-’ M and at 5 x 10m8 M there is about a 50% reduction in growth yield. At ecdysterone concentrations of lo-’ M and greater, the growth yield is independent of plating cell density and near unity. Typical curves are shown in Fig. 2, the uppermost being a control curve.

12

r

Paper electrophoresis The culture media, saved after incubation with [‘%]choline (described above), were analyzed via paper electrophoresis according to the procedure of Potter and Murphy (1967). A 10 ~1 sample of each culture medium was applied to a 4 x 30cm strip of Whatman No. 1 paper and allowed to air dry. The culture media samples were either taken immediately after incubation or after storage at -20°C and in some cases TCA (10%) or butyrylcholinesterase were added prior to applying. The strips were mounted horizontally in the electrophoresis cell and an acetic acid-formic acid buffer was used. Electrophoresis was carried out at a constant field of 28.8 V/cm for 45 min. Afterwards, the paper strips were dried and either stained in iodine vapor or cut into 5 mm pieces. Each piece was placed in a Scintillation vial with scintillant (toluene and PPOPOPOP) and “counted” for 10 min in a liquid scintillation counter (Searle-Delta 300). Samples containing known DL-carnitine-HCl and succhemicals (acetylcholine, cinylcholine) were electrophorezed for calibration.

Fig. 1. Growth yield, nr/ni, vs N, for Schneider’s line 2 cells. The incubation times and temperatures were: 48 hr and 26°C (O), 46 hr and 24°C (0) and 29 hr and 23°C (A),

Measurement of transformed growth characteristics The growth characteristics of these cells sometimes transformed under abnormal conditions and experiments were designed to quantify the growth properties of these cultures. Stock cultures were allowed to grow to a range of final cell densities (5.5 x 104-2.8 x lo6 cells/cm*). The cells were then harvested and tested for sensitivities to acetylcholine and plating cell density. Sensitivity to acetylcholine was tested at a concentration of 10m6M and quantified by the relative growth yield at 2 x lo4 cells/cm’. Sensitivity to cell density was tested for by comparing the growth yield at 6 x lo4 cells/cm2 with that at 2 x lo4 cells/cm* and quantified by their ratio.

$!! 0

2

4

6

8

10

12

14

16

18

Ni (104~~11~/~~*) Fig. 2. Growth yield vs N, for Schneider’s line 2 cells in the presence of ecdysterone. The incubation time and temperature were 48 hr and 24°C respectively. The ecdysterone concentrations were: 1 x IO-’ M (0) 5 x lOm8 M (0) and 0 M (A).

ACh and carnitine sensitive growth Enhunced growth with ocetylchol~ne or cczrnitine

231 A

3.0 2.6

Acetylcholine and carnitine enhanced growth in cultures of these cells. The enhancements were in

terms of relative growth measured over a range of (2 x lo-“-5 x 10-5M) chemical concentrations (1.0x 104
Enhanced growth was observed when cultures of line 2 cells contained conditioned medium, medium taken from previously incubated cultures and centrifuged free of cells. The growth enhancement was dependent upon plating cell density in a fashion very similar to that observed with acetylcholine and carnitine. The data from two of these experiments are plotted in Fig. 4c. Growth enhancements as large as 2.00 were measured with conditioned medium diluted to one-half strength with Fresh medium.

2.2 1.6

0

*

4

6

Ni

e 10 (104cel16/cm2

12

14

16

1s

)

Fig, 4. Growth yield vs N, for Schneider’s line 2 cells in the presence of carnitine (A), acetylcholine (B) or conditioned medium (C). Three values of [carnitine] are plotted: t.5 x 10e9 M (a), 1.5 x 10-8M (O), and 5 x lo-‘M (A). Six values of [acetylcholine] are plotted: 3 x lo-‘M (W), 1.25 x 10..*M (O), 3 x lo-” M (O), 1 x 10-7M (a), 3 x lo-’ M (A) and 1 x 10e6M (0). Two dilutions of conditioned medium by fresh medium were studied l/2 (@) and l/l00 (0).

The effects of acefylcholimesterase, atropine, tubocurarine and decamethoniun?

A~iylcholinesterase and the cho~inergic blocking agents, atropine and tubocurarine, each decreased growth of line 2 cells when added to the culture medium. The cell density dependences of their effects were measured and are plotted in Fig. 5. There is a plot for acetylcholinesterase (specific activity = 235 units/mg solid at 37°C and pH 8; Sigma) at a concentration of 116 pg/ml which corresponds to a very high specific activity [27 (enzyme units/ml)] for this enzyme. Also, the control is included for comparison.

54< c’

3 e-

lo-‘0

lo-*

10-e CHEMICAL

10” CONCENTRATION

10.6

lo-’

10.'

I-

(M)

Fig. 3. Relative growth yield, n&z,,, vs chemica1 concentration for Schneider’s line 2 cells in the presence of acctylcholine or carnitine. Typical plots are shown for carnitine (A) and acetylcholine (B) as well as the average plot of all 26 data sets for acetylcholine (C). The incubation times and temperatures were: 29 hr and 24°C (A), 48 hr and 25°C (B), and 47 + 3 hr and 25°C (C). The bars either equal errors (A and B) or SD (C).

Fig. 5. Growth yield vs N, for Schneider’s line 2 cells in the presence of 116 pg/ml of acetylcholinesterase (a), tom4 M atropine (O), or 10e4M tubocurarine (A). A plot for untreated cuhures (0) is shown for comparison. The incubation time and temperature were 46 hr and 24°C respectively.

238

ALAN R. GINGLE

The overall inhibition due to each chemical was quantified by the mean of the relative growth yields. The mean for tubocurarine (0.96) was larger than that for atropine (0.73) which was roughly equal to that for acetylcholinesterase (0.72). Thus, of the two blocking agents, atropine was the more potent growth inhibitor. The depolarizing blocker, decamethonium, was also tested (10m4 M) over a range of plating cell densities (5 x 103-9 x IO4cells/cm2) and it generally enhanced growth (1.41). Among the treatments which inhibited growth, there were differences in the cell density dependences. Tubocurarine ( 10m4 M) only inhibited growth in cultures with initial cell densities less than 1.4 x lo5 cells/cm2 whereas atropine and acetylcholinesterase inhibited growth at all plating cell densities. One prominent feature of all three curves was a peak in growth yield near 8 x lo4 cells/cm2. However, the details of the basis for this feature are at present unknown. Transformation

of growth characteristics

The growth characteristics sometimes transformed when cultures were allowed to grow to abnormally high cell densities. The sensitivities to acetylcholine and carnitine vanished as well as the characteristic form of the growth yield data. The growth yield became insensitive to cell density and was depressed to levels normally characteristic of low initial cell density control cultures. The cells returned to normalcy after about 1 week of culturing at normal cell density. As the effect of carnitine was discovered late in this study, most of the measured transformed properties involved acetylcholine rather than carnitine sensitivity. An experiment designed to study the transformation was performed, and sensitivity to plating cell density was found to be correlated to acetylcholine sensitivity as the correlation coefficients between their respective growth yield enhancement ratios indicates (r = 0.48 and P < 0.05). Thus, the loss of sensitivity to cell density is significantly correlated with the loss of sensitivity to acetylcholine.

11.3 cm/45 min at 28.8 V/cm). In these experiments, media were taken from cultures after incubations ranging from 3 to 24 hr, where the populations grew to final cell densities ranging from 1.8 x lo5 to 9 x 105cells/cm2 respectively. The data from two of these experiments are plotted in Fig. 6. The [‘“Cl label (counts per min) in each 5 mm piece of the paper strip is plotted with respect to its distance from the origin. The [‘“Cl label near the origin in Fig. 6A was removed in the experiment of Fig. 6B by adding TCA and centrifuging the precipitate. The ratio of the [‘“Cl label in Band I to that in Band II varied between 0.13 and 7.28, and was correlated with final cell density (r = 0.74). The chemical associated with Band I, electrophoretically identical to carnitine, was very stable at room temperature. When the cell free media were maintained at room temperature for times up to 23 hr, no significant differences in the radioactive label of Band I and Band II occurred. For example, the ratio of the radioactivity in Band I of the 23 hr sample to that of the zero time control was 1.05. The cell free medium was also unaffected by the enzyme, butyrylcholinesterase, at room temperature. Cell free medium, to which 0.71 mg/ml of butylcholinesterase (14 enzyme units/mg solid at pH 8 and 37°C) was added 100min prior to electrophoresis, was not significantly different from that without. The ratio of radioactive label in Band I with, to that without, butyrylcholinesterase was 0.93. The effects of various chemicals on growth A variety of chemicals have been tested for their effects on the growth properties of line 2 cells. The effects on growth yield were measured over a range of chemical concentrations and the results are listed in Table 1. Various parameters and results are listed for each chemical, the chemical concentration range and plating cell density tested, the mean of the

[‘4C]Choline uptake The uptake of [‘4C]choline by line 2 cells was measured for a range of plating cell densities. Uptake generally decreased as cell density was increased up to about 8 x IO4 cells/cm*. Above this, [‘4C]choline uptake per cell was insensitive to plating cell density. In a particular experiment [‘4C]choline uptake was measured at a range of cell densities. The incubation parameters were 25°C 10% iFBS and 3 hr. Also 10e4 M eserine, an acetylcholinesterase inhibitor was present. The [‘4C]choline uptake per cell decreased from 4 x 10-‘5moles/cell/3 hr at 1 x lo4 cells/cm2 to about 5 x lo-l6 moles/cell/3 hr for 8 x lo4 cells/cm2 or greater. Electrophoretic

separation

Media taken from line 2 cultures after incubation via with [‘4C]choline were analyzed paper electrophoresis. The electrophoresis revealed two [‘“Cl labelled bands. Band I comigrated with DL-carnitine (approx. 8.3 cm/45 min at 28.8 V/cm) and Band II comigrated with choline (approximately

Z

600

%o 200 0

1

2

3

4

6

6

DISTANCE

7

6

9

10

FROM

I,

12 13

ORIGIN

14

IS

16

‘7

16

(cm)

Fig. 6. [‘“Cl label vs distance from the origins on paper electronhoresis strius made from the extracellular medium of Schneider’s line 2 cell cultures incubated with V4Clcholine. The cultures were incubated for 24 hr at 25°C iA)‘or 23°C (B), and in the presence of 9.4 x 10-‘M [‘4C]cho1ine. During that period they grew to final cell densities of 1 x lo6 cells/cm* (A) and 7 x lo5 cells/cm2 (B). In the experiment described in B, TCA was added to the cell free culture medium prior to electrophoresis and the electric field was 90% of that used in A.

ACh and carnitine sensitive growth

Table I, Efkts

Chemical ____~~ Adrenaline CAMP CAMP

of chemicals

on Schneider’s

line 2 growth

with constant

239 plating

cell

density

Plating cell density (104 cells/cm~)

Mean relative growth yield

10-9-10-5 2 x 10-10-3 x 10-s IO-~-IO-~

2.5 2.0 2.5

1.15 1.50 1.28

3.98 1.60 I .32

<0.005 O.l0.25

1O-5 10-v-10-5 10-L10-5

1.0 2.5

0.91 0.72

10.58 3.90


5 x 10-S 4 x IO_‘“-5 x 10-5 IO‘‘-10-S

2.0 2.0

1.44 1.15

3.69 3.18


2.0

1.06

1.60

O.I
2.0 2.0

1.33 0.84

2.49 1.54


Concentration range (M)

+ IBMX Serotonin Serotonin

F-Value

Significance level

+ Iproniazid Acetylcholine Acetylcholine

+ lO-5 lo-9-10-s

Eserine Acetylcholine J

10-l 2 x lo-‘“-4 x 10-S 10-9-10-5

Ecdysterone m-Carnitine-HCI rx-Carnitine-HCI

+ Atropine

relative growth yields, the F-ratio of the treatments mean square to the error mean square (each chemical concentration is considered to be a different treatment in this analysis), and the corresponding significance level based on an F-test. With respect to the second to the last combination, acetylcholinesterase (0.036 mg/ml) added to was 100 x stock solutions of acetylcholine 20 hr prior to use and the solutions were kept at 22°C. Thus the final acetylcholinesterase concentration was 3.6 pgg/ml. The effects of DL-carnitine-HCI, acetylcholine, adrenaline, serotonin and the combinations acetylcholine with eserine and serotonin with iproniazid were highly significant (P < 0.01); while those for the other chemicals were not significant (P > 0.05). Only the serotonin and serotonin with iproniazid significantly inhibited growth. DISCUSSION

Responses to acetylcholine, carnitine and conditioned medium As the data have shown, acetylcholine, carnitine and conditioned medium are capable of enhancing the growth of line 2 cells. The relative growth yield vs [chemical] plots for acetylcholine and carnitine (Fig. 3) exhibit four peaks, spaced approximately an order of magnitude apart. Thus, the active concentrations range over several orders of magnitude. This line was established from cells of Drosophila melanogaster embryos and perhaps they shared its sensitivity to acetylcholine and carnitine. If so, the nature of these sensitivities could have significant implications for pattern’formation in the development of this organism. For example, if its sensitivity is shared by at least a portion of Drosophila cells in uivo, simple concentration profiles of acetylcholine or carnitine can initiate the formation of periodic or segmented structures by differential cell growth. The cell density dependence data (Fig. 4) offer an internal consistency check on the chemical concentration data. At lower cell densities, the curves corresponding to maximal effect concentrations are

consistently larger than those corresponding to minimal effect concentrations. This rules out the possibility of differential growth due to temperature variations as this would depend on the well location within the culture tray. In fact, the basic form of the cell density dependence curves does not change significantly. However, this data does show that the growth enhancement decreases with increasing cell density. These data are consistent with cells secreting a humoral chemical, like acethylcholine or carnitine. Its concentration in the culture medium would then be an increasing function of cell density. The activity of conditioned medium and its similar cell density dependence offer support for this interpretation in which the addition of acetylcholine, carnitine or conditioned medium can in some measure supplement the lower, less effective, concentration of active chemical present at lower cell densities and thereby enhance growth. [‘4C]choline uptake and evidence for carnitine secretion Two labelled bands were detected after the electrophoresis of medium taken from cultures incubated with [‘4C]choline. One band (II) comigrated with choline; the other (I) comigrated with carnitine. This does not conclusively identify the first band as carnitine because other chemicals may comigrate with it as does butyrylcholine (Potter and Murphy, 1967). However, conditioned medium’s stability in the presence of butyrylcholinesterase all but ruled out butyrylcholine. In addition to the two labelled bands, some label appeared near the orign after electrophoresis (Fig. 6A); however, this was probably label bound to protein since it was completely removed by treatment with trichloroacetic acid prior to electrophoresis (Fig. 6B). Also, the bands (I and II) were unaffected by the TCA treatment. While the evidence for carnitine is not complete, it is quite substantial. Its growth enhancing activity, as well as that of conditioned medium, and the secreted chemical comigrating with it imply a role for carnitine as a humoral mediator affecting growth.

240

ALAN R. GINGLE

Measured [‘4C]choline uptake, a decreasing function of cell density, varied from 1.34 x 10’ to However, these 1.12 x 10-6 molecules/~ell/min. measured values are underestimates of the actual [‘4C]choline uptakes because a portion of the radioactive label, taken up as [‘4C]choline, was incorporated into the secreted molecules (e.g. Band I). This component of the uptake was not detected by measurements of the radioactive content of the cells, but it could be determined from the electrophoresis data. Its value was 1.33 x 10~‘5moles~cell/hr for 1.7 x lo5 cells/cm2. When it is added to the measured [‘4C]choline uptake at 1.7 x lo5 cells/cm* it yields the actual [‘4C]choline uptake at this cell density, 1.44 x lo-” moles/cell/hr. This is only slightly greater than the measured [14C]choline uptake at lo4 cells/cm’. Thus the actual [‘4C]choline uptake appears to be independent of cell density. Responses to drugs which affect cholinergic transmission

The responses to acetylcholine and carnitine are quite possibly mediated by receptors which are similar to those found in cholinergic synapses. Drosophila melanogaster contains cholinergic receptors, both nicotinic (Dudai, 1977) and muscarinic (Dudai and Ben-Barak, 1977). Of course, these cell line 2 receptors would not be as specific as those of D. mefanogaster since they respond equally well to carnitine. However, a~etyl~holine and carnitine are quite similar; for example, they both have a quaternized nitrogen atom forming a cationic head. Atropine which blocks muscarinic cholinergic receptors can completely eliminate carnitine’s growth yield enhancing effect (Table 1). Also it had a greater inhibitory effect on growth than tubo~urarine which blocks nicotinic cholinergic receptors (Fig. 5). All of this data is consistent with the presence of cholinergic-like receptors on the surfaces of these cells which have a muscarinic nature while still displaying some nicotonic properties. It should be noted that in the housefly, Musca domestica, cholinergic receptors with a mixture of nicotinic and muscarinic properties have been found (Donnellan et al., 1975). These receptors have a high affinity for decamethonium (Donnellan et al., 1975) an affinity which is not shared by the nicotinic or muscarinic receptors of Drosophila (Dudai, 1977; Dudai and Ben-Barak, 1977). Decamethonium did enhance the growth of line 2 cells. So, perhaps cholinergic receptors are mediating acetylcholine and carnitine’s effects on the growth of these cells. The effects of acetylcholinesterase were not as interpretable as those of atropine or tubocurarine. It appears that no acetylcholinesterase was present in the cultures of these cells since eserine had no si~i~~ant effect when added alone and did not alter the growth enhancements due to acetylcholine. However, acetylcholinesterase, when added to cultures, inhibited growth as did atropine and tubocurarine (Fig. 5). The basis for its effect here is unknown, but a very large acetylcholinesterase activity (>27 enzyme units/ml) was required to have a sizeable effect. Therefore, it is unlikely that its effect was only due to acetylcholine hydrolysis.

Responses to other drugs and hormones

Of these chemicals, only adrenaline significantly enhanced the growth of line 2 cells (Table I). This monoamine has been identified in insects such as Tenehrio molitor and has been found to stimulate the central nervous system of the cockroach, Periplaneta americana (Novak, 197.5). Also, substances which block the uptake of adrenaline at c~and p activity sites reduced the incidence of melanotic tumors in a mutant of DrosophiZu ~ne~a~ogaster; though, adrenaline was thought to influence the balance between the juvenile and molting hormones rather than directly affecting tumor incidence (Sang, 1969). So adrenaline is certainly present and active in some insects, thus the sensitivity which line 2 cells display is not without precedent. Another monoamine, serotonin, significantly inhibited the growth of line 2 cells (Table 1). It is present in considerable amounts in insect ganglia and in the corpora cardiaca (Novak, 1975), and it inhibits phosphorylase activity in the nerve cord of P. americana (Hart and Steele, 1969). Perhaps a similar inhibitory effect on metabolism is responsible for its action on these cells. When added in the presence of iproniazid, a monoamine oxidase inhibitor, serotonin again significantly inhibited growth (Table I), but at much lower concentrations. (The threshold with iproniazid was 5 x 10m9M as compared to 2.5 x 10m6M without.) This is consistent with the presence of a monoamine oxidase in these cultures; however, in addition to its monoamine oxidase inhibiting activity, iproniazid is known to induce hepatocellular damage due to covalent bonding of its major metabolites to proteins (Doull et al., 1980). Its toxicity may be a factor in its effect on line 2 cells. Finally, ecdysterone significantly inhibited growth. This was not surprising since a similar growth inhibition has been reported for this cell line in its presence (Berger et al., 1978). However, it is interesting that ecdysterone’s growth inhibiting effect on this line blocked the expression of its acetylcholine sensitivity. Tra~.~~~rmationC$ growth characteristics

The transformation which sometimes occurred after overcrowding almost always resulted in the loss of sensitivity to cell density and to acetylcholine and carnitine. And, while there were transformed cells exhibiting some acetylcholine sensitivity without cell density sensitivity, there were no transformed cells exhibiting cell density sensitivity without acetylcholine and carnitine sensitivities. Thus, when the acetylcholine and carnitine sensitivities were lost, the cell density sensitivity was lost also, and upon its return, the acetylcholine and carnitine sensitivities returned as well. This correlation is consistent with a causal relationship between carnitine as a humoral mediator affecting growth and the sensitivity of growth rate to cell density. Finally, the conditions which led to this transformation suggest that, while growth to large cell densities can lead to transformation, it is not the only factor involved. For example, there was not a significant correlation between the loss of sensitivities and the final cell densities to which stock cultures were allowed to grow (Y = -0.21 and P ~0.53).

ACh and camitine sensitive growth SUMMARY

241

Donnellan J. F., Jewess P. J. and Cattell K. J. (1975)

Subcellular localization and properties of a cholinergic There is a substantial amount of evidence for receptor isolated from housefly heads. J. Neurochem. 25, carnitine as a humoral mediator which affects growth 623-629. by acting at cholinergic-like receptors on these cetls. Doull J., Klaassen C. D. and Amdur M. 0. (Editors) (1980) The responses to acetylcholine, carnitine and conCasarett and Doull’s Toxicology/The Basic Scienre qf ditioned medium are very similar. Drugs like atroPoison. MacMillan, New York. pine, tubocurarine and decamethonium which affect Dudai Y. (1977) Demonstration of an a-bungarotoxin binding nicotinic receptor in flies. FEBS Left. 76, chohnergic transmission have corresponding growth 21 I-213. yield inhibiting or enhancing activity on these cells. Dudai Y. and Ben-Barak J. (1977) Muscarinic receptor in The choline uptake and electrophoresis data suggest Drosophila melanogaster demonstrated by binding of carnitine production and secretion. Also, the trans13Hlauinuclidinvl benzilate. FEBS Lett. 81. 134-l 36. formation which ied to the loss of acetylcholine and Fraenxel G. and Friedman S. (1957) Carnitink. In Vitamins carnitine sensitivity always resulted in reduced and hormones (Edited by Harris R. S., Marrian G. F. and growth yield and insensitivity to cell density. These Thimann K. V.), Vol. 15, pp. 73-l 18. Academic Press, data along with the periodicities in the chemical New York. concentration dependences may be significant to Hart D. E. and Steele J. E. (1969) Inhibition of insect nerve growth and pattern formation in Drosophila rrzelano- cord phosphorylase activity by 5-hydroxytryptamine. Exgas&r. Acknowledgements--I wish to thank those who made equipment and facilities available: W. B. Iturrian, U. E. Brady and R. L. Anderson at the University of Georgia and A. Robertson and H. C. Friedman at the University of Chicaeo. Also. soecial thanks to W. R. Iturrian and U. E. Brady forreading the manuscript and to M. Miranda, upon whose suggestion carnitine was tested.

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