Journal oJ Nuurochrwisrry Vol. 30, pp. 1391-1397
0022-3042/78/0601- I391 SOZ.00/0
Pergarnon Press Ltd. 1978. Printed in Great Britain 0 International Society lor Neurochemistry Ltd.
MONOAMINE OXIDASE A AND B IN CULTURED CELLS MORRISHAWKINS, JR. and XANDRA0. BREAKEFIELD Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510, U.S.A. (Received 5 M a y 1977. Accepted 6 September 1977)
Abstract-Monoamine oxidase (MAO) activity against tryptamine was compared in a number of continuous rodent lines, including neuroblastoma, hepatoma, melanoma, nephroma, sarcoma and L cells. Activities against tryptamine varied over 300-fold in homogenates of different lines, being highest in hepatoma line MHICl and lowest in a neuroblastoma line lacking hypoxanthine phosphoribosyltransferase (HPRT) activity. The amount, but not the type, of MA0 activity varied with the stage of growth in homogenates of neuroblastoma and hepatoma cells. Measurements of succinatexytochrome c reductase (SCCR), another mitochondria1 enzyme, also showed 20-fold variations between lines, being highest in neuroblastoma line N1E-115 and lowest in hepatoma line MH,C,; SCCR and M A 0 activities appeared to be regulated independently. The relative proportions of the A and B types of MA0 activity were determined in homogenates and living cultures. Clorgyline inhibition of tryptamine deamination in homogenates indicated that in all lines except MHIC,, greater than 95% of the MA0 activity was of the A type. In MHICl homogenates, using clorgyline or deprenyl, 40-70% of the activity appeared to be of the A type and 3MO% of the B type. In cultures of neuroblastoma N1E-115 cells, deamination of tryptamine and dopamine was sensitive to inhibition by low concentrations of clorgyline, indicating that the A type of activity is present intracellularly. as in homogenates. In MHIC, hepatoma cultures, tryptamine deamination showed a biphasic sensitivity to clorgyline. We interpret this to mean that A and B types of MA0 activity occur together in living hepatoma cells.
Because of the potential usefulness of cell culture MONOAMINE oxidase (MAO, monoamine :O2 oxidoreductase EC 1.4.3.4) is important in the degradative for studying the regulation of M A 0 activity, we deterdeamination of biogenic amines throughout the body. mined the relative proportions of A and B activity Studies using homogenates prepared from various in a number of continuous cell lines arising from diftissues have suggested the presence of two types of ferent tissue types. Proportions of A and B activity M A 0 activity which differ in their drug sensitivities were studied in homogenates prepared from cells at and substrate preferences (JOHNSTON,1968; NEFF & different stages of growth and under different condiYANG,1974; HOUSLAY et a/., 1976; HUANG& EIDU- tions of culture. We also examined another mitochondrial enzyme, succinate-cytochrome c reductase SON,1977). Classically, the A activity is preferentially & RACKER,1969) t o determine inhibited by low concentrations of clorgyline and acts (SCCR) (YAMASHITA against serotonin, whereas the B form is more sensi- whether there was co-ordinate expression with M A 0 tive t o deprenyl and acts against phenethylamine and activity. Further, we examined the metabolites formed from labeled amines in culture in the presence of benzy lamine. Monoamine oxidase activity has been described in varying extracellular concentrations of clorgyline t o a variety of mammalian cell cultures (JACOBOWITZ, determine whether the A and B types of M A 0 could et al., 1972; SEEDS,1975; BREAKE- be distinguished in homogeneous populations of 1972; SILBERSTEIN FIELD et al., 1976; HONNEGGER & RICHELSON,1976; living cells. SKMERet al., 1976; SMALLet al., 1977). In only three studies have the relative activities of the A and B types of M A 0 been determined. Homogenates of MATERIALS AND METHODS et al., 1976) and mouse neuroblastoma (DONNELLY Materials. Tissue culture dishes and flasks were obtained rat glioma cells (MURPHYet al., 1976) contain exclusfrom Falcon, Costar or Corning; the Dulbecco-Vogt modiively the A type of activity, while normal human skin fication of Eagle’s medium (DMEM, No. H-21), 1OX Viokfibroblasts contain predominately the A, but also ase and 0.4% Trypan Blue from Grand Island Biological some B activity (ROTH et al., 1976). Co. (Santa Clara, CA); and serum from Flow Laboratories Inc. (Rockville, MD). F-14 (VOGEL et al., 1972) was preAbbreviations used: MAO, monoamine oxidase; HPRT, pared from component chemicals in our laboratory. All hypoxanthine phosphoribosyltransferase ; SCCR, succinate- chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), except clorgyline (Dr. SABATGABAY, cytochrome c reductase. V.A. 1391
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JR. and XANDRA0.BREAKEFIELD MORRISHAWKINS,
Hospital, Boston, MA) and deprenyl (Professor J. KNOLL, 37°C. A zero time blank was used in which 200 pl of 2 N Sammelweis University of Medicine, Budapest, Hungary). HCI was added prior to the labeled substrate. In cases Cell culfure. Mouse neuroblastoma clones NIE-115, where inhibitors were used, the homogenate preparations N-18, and N-l8TG2, derived from the C1300 tumor, were were preincubated for 15 min at 37°C in presence of either laboratory clorgyline or deprenyl. Monoamine oxidase activities were obtained from Dr. MARSHALLNIRENBERG'S (Natl. Inst. Health, Bethesda, MD). Lines designated as assayed within the range of linearity for protein and time. TG were selected from the parent clone by their resistance Succinate-cytochrome c reductase activity was measured to M-Cthioguanine. Line NlE-115TG2 was obtained by a modification of the method of YAMASHITA& RACKER in our laboratory; both NlE-115TG2 and N-18TG2 lack (1969), provided by Anne Hubbard (Yale Univ. Sch. Med., hypoxanthine phosphoribosyltransferase activity (HPRT) New Haven, CT). Homogenates were assayed in duplicate (BREAKEFIELD et al., 1976). Rat hepatoma line HTC and over 1-5 min at 23-25°C in a Gilford spectrophotometer mouse L-cell line LEA-2a line were obtained from Dr. JER- (model 250) with an automatic cuvette positioner (model OME EISENSTADT; rat hepatoma line HTCPRT- and mouse 2451-A) and recorder (model 2530). Each cuvette received L-cell line LMTK- from Dr. EDWARDADELBERG; rat 0.91111 of a reaction mixture consisting of 40mw-sodium hepatoma line MHIC, (No. CCL 114) from the American phosphate buffer, pH 7.4, 20 mM-Na succinate, 1 mM-KCN Type Culture Collection; mouse melanoma lines PSI and and 0.12 pM-cytochrome c. Mixtures were allowed t o equiand RUTHHALA- librate for 1-2min and then 100pl of homogenate was PClA-HPRT2 from Drs. JOHNPAWELEK BAN;and baby hamster kidney line BHK (Cat. No. 026200) added. Samples were read at 550nm. Blanks contained and mouse sarcoma line S-180 (Cat. No. 0-27700) from antimycin, an inhibitor of SCCR, at a final concentration Flow Laboratories. (The preceding investigators are all at of 2.4 x 1 0 - ' ~ . Enzyme activities were calculated using Yale Univ. Med. School, New Haven, CT) Cells were an extinction coefficient of 18.5 x lo6 cmz mol reduced grown as monolayers on plastic tissue culture dishes cytochrome c. (100 mm) or flasks (75 cmZ)in medium supplemented with Protein was determined by a modification of the method 5-10% (v/v) fetal calf serum, without antibiotics, at 37°C of LOWRYet al. (1951) using bovine serum albumin as in a humidified atmosphere of 5% C 0 2 and 95% air. a standard. Neuroblastoma cells, hepatoma HTC cells and LMTKChromatographic examination of ['Hlamine metabolites. cells were routinely grown in DMEM, other lines were The metabolism of [G-3H]3,4-dihydroxyphenylethylamine grown in F-14. Cells were fed every 2-3 days and subcul- (dopamine, 6.5 Ci/mmol, New England Nuclear) and tured every 7-14 days. Neuroblastoma lines were resus- [G-3H]tryptamine HC1 (1 Ci/mmol, Amersham/Searle) in pended by gentle trituration and subcultured in a ratio neuroblastoma cells was determined by adding 30pCi of of 1:lo. Other cell lines required 3-10 min treatment with each t o confluent monolayers grown on l00mm dishes 1X Viokase prior to resuspension, and were subcultured in 3 ml of either medium with 5% serum or isotonic phosat a ratio of 1: 10, except for MHICl which was subcul- phate buffered saline with 0.1 mM-ascorbic acid. For hepatured at 1:3. Growth curves for N1E-115 and MHIC, were toma cells, 2pCi of [3H]tryptamine was added to cells established by inoculating 100mm dishes in parallel. For resuspended in 0.2ml of medium with 10% serum. (Cells neuroblastoma cells, we determined cell numbers using a were grown to confluency on 150mm dishes and resushemacytometer and per cent viability was determined by pended in 4ml of medium per dish.) Cultures were incutrypan blue exclusion. For hepatoma cells, which are diffi- bated at 37°C either in a humidified atmosphere of 5% cult to resuspend, confluency of cultures was monitored C 0 2 and 95% air (medium) or in room atmosphere by microscopic examination. (buffer). When present, clorgyline was preincubated with Preparation of homogenates. In our standard procedure, the cells for 15min prior to the addition of isotope. The monolayer cultures of stationary phase cells were rinsed incubation was terminated by removal of medium (or quickly three times with an isotonic phosphate' buffered buffer), monolayers were rinsed once with isotonic buffer saline at room temperature. This buffer, used routinely in and the rinse was pooled with the medium (or buffer). Cells our laboratory, consists of 129 mM-NaC1, 2.5 mM-KCI, were scraped off the dish in a small volume of buffer 7.4 mM-NaZHPO.,, 1.3 mM-KH,PO,, 0.63 m~-CaCl,, (0.2-0.3 ml) and extracted in 0.1 N-acetic acid in the pres0.74 m~-MgSo,, 5.3 mM-glucose and 46 mM-sucrose, pH ence of lo-" M-ascorbic acid. Cell extracts and medium 7.2, 330 mOSM. Cells grown on flasks were resuspended by were chromatographed on a thin layer cellulose (Eastman trituration; in the case of all except the neuroblastoma 6064) in a solvent system consisting of 1-butanol-1 N-acetic lines, this entailed 2-5 min of pre-treatment with 1X Viok- acid-95% ethanol (3.5:1:1, by vol). The distribution of ase to dissociate cells. Resuspended cells were pelleted by radioactivity was determined, as previously described low speed centrifugation. Cells grown on dishes were (BREAKEFIELD, 1975). For both cell types, >95% of the dedrained and scraped off with a Teflon-coated straight edge. aminated metabolites were found in the medium with only Following either procedure, cells were concentrated in a a few per cent in the cell extracts. There was no breakdown small volume of cold 0.1 M-potassium phosphate buffer, pH or metabolism of these amines under the same incubation 7.4, at 5 1 0 m g protein/ml, frozen on dry ice and stored conditions in the absence of cells. in the vapor phase of liquid nitrogen (-70°C). Prior to RESULTS assay homogenates were thawed and sonicated for 30 s on ice using a microprobe (Biosonik IV.). Monoamine oxidase activity against tryptamine, a Enzyme assays. Monoamine oxidase activity in cell substrate for both A and B types varied widely among homogenates was measured as described by NAGATSU the cell lines tested (Table 1). The highest activity, (1973). Homogenate preparations were assayed in triplicate by incubating 50 pl aliquots with 50 pl of 0.1 M-potassium 1043 pmol/min/mg protein, was observed in rat hepatoma line MH,C1.The other rat hepatoma line HTC, phosphate buffer, pH 7.4, containing 0.1 pCi [2-14C]tryptamine bisuccinate (48.5 mCi/mmol, New England Nuclear) as well as mouse melanoma PS1, mouse sarcoma and 0.17 mht-tryptamine. Reactions were run for 20min at S-180, and mouse L cell LEA-2a showed about half
Monoamine oxidase A and B in cultured cells TABLE1. M A 0
AND
SCCR
Cell line
Cell type
Species
NlE-115 N1 E-115TG2 N-18 N-18TG2 HTC HTCPRTMHlCl PS 1 PCIA-HPRT; BHK S- 180 LMTKLEA-2a
Neuroblastoma Neuroblastoma Neuroblastoma Neuroblastoma Hepatoma Hepatoma Hepatoma Melanoma Melanoma Nephroma Sarcoma L-cell L-cell
Mouse Mouse Mouse Mouse Rat Rat Rat Mouse Mouse Hamster Mouse Mouse Mouse
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ACTIVITIES IN CELL HOMOGENATES
M A 0 activity* SCCR activity? pmol/min/mg nmol/min/mg protein Cytochrome c reduced 125 f 76 3 f 1 196 f 45 67 & 1 1 502 f 77 478 f 41 1043 f 148 492 f 158 175 8 49 & 8 385 f 99 94 f 5 614 & 62
49 f 2 8f1 15 f 4 11 + 5 15 f 3 17 & 4 2 f 0.1 10 f 3 9+3 10 f 2 14 & 3 25 * 4
9f3
MA0:SCCR ratio 0.003 0.0004 0.013 0.006 0.034 0.027 0.603 0.051 0.019 0.005 0.027 0.0003 0.068
* Monoamine oxidase activity was measured in homogenates of stationary phase cells over 20 min at 37°C in 0.1 M-potassium phosphate buffer, pH 7.4 with [14C]tryptamine (1.7 x l o T4M, 48.5 mCi/mmol) (NAGATSU,1973). Deaminated products were extracted into toluene and counted using a liquid scintillation spectrometer. Values represent the mean of 3-5 determinations f S.E.M. t Succinate cytochrome c reductase was measured in homogenates of stationary phase cells over 3-5 min at 23-25°C using 0.12 mM-cytochrome c, 40 mM-sodium phosphate buffer, pH 7.4, 20 mM-Na succinate and 1 mM-KCN and measuring absorbance changes - at O.D. 550nm with a spectrophotometer (YAMASHITA& RACKER, 1969). Values represent the mean of 3-5 determinations f S.E.M. I
this activity. Even lower activity was observed in mouse neuroblastoma lines N1E-115 and N-18, baby hamster BHK and mouse L ceH LMTK-. Monoamine Gxidase activity was lower in 3 lines lacking HPRT activity as compared to the parental lines from which they were derived; activities were 100-fold lower in NlE-115TG2, and 3-fold lower in both N-18TG2 and PClA-HPRT;. However, no difference was found between MA0 activities in HTC and HTCPRT- cells. Succinate-cytochrome c reductase activity also showed wide variation in the cell lines tested (Table 1). The highest activity, 49nmol cytochrome c reduced/min/mg protein, was seen in mouse neuroblastoma NlE-115. All other lines showed one-half to one-third less activity than NIE-115, with rat hepatoma MH,C, showing the lowest activity, 2nmol/ min/mg protein. There appears to be no consistent relationship between the levels of these two mitochondrial enzyme activities in the lines tested, as shown by the MA0:SCCR ratios in Table 1. Rat hepatomas MHICl and HTC have MA0:SCCR ratios of 0.603 and 0.034, respectively, while mouse neuroblastomas N-18 and N1E-115 have ratios of 0.013 and 0.003, respectively. The lowest ratio, 0.0003, was observed in mouse L cell line LMTK-. The relative proportion of M A 0 A and B activities in cell homogenates was determined by measuring the sensitivity of tryptamine deamination to varying concentrations of clorgyline, 1 0 - ~ - 1 0 - ' ~M. In all lines except the rat hepatoma MHIC1, M A 0 activity was completely inhibited by 10- M-clorgyline indicating the exclusive presence of the A type of M A 0 activity. Considering the lower range of sensitivity of our MA0 assay (0.2 pmol/min/mg protein), we would not have been able to detect up to 1% of B activity in
lines such as BHK and LMTK- with low M A 0 values. In fine MH,C,, 70% of the activity appeared to be type A, by its sensitivity to lo-' M-clorgyline, while the remaining 30% required M-dOrgyhe for complete inhibition, as expected for type B (Fig. 1A). Essentially the same biphasic curve of clorgyline sensitivity was observed when the concentration of tryptamine in the assay was reduced from 1.7 x 1 0 - 4 ~ to 1.7 x 1 0 - 6 ~ The . sensitivity of tryptamine deamination to deprenyl in these cells also showed a biphasic pattern with 60% of activity being inhibited at 10T5wand the remaining 40% of (Fig. 1B). This finding suggests somewhat different proportions of A and B activities in these cells. Monoamine oxidase activity in homogenates was blocked only 8% by 1 0 - 4 ~semicarbazide, a drug which blocks another amine oxidase (BLASCHKO, 1963). Changes in M A 0 activity with the stage of cell growth for line MH,CI is shown in Fig. 2. Monoamine oxidase activity is low in homogenates prepared from cells within a few days after subculture, rises during the most rapid stage of growth and decreases again as the cultures become confluent. The proportion of the A and B types of M A 0 activity in these cells, as measured by clorgyline sensitivity, is the same whether homogenates are prepared from cells in logarithmic or stationary phases of growth. The specific activity of M A 0 and the relative proportion of the A and B types seen in MHiCl h o m e genates are not affected by growth in different media (DMEM and F-14) or with different amounts or types of sera (5 and 1004; fetal bovine and human); specific activity did not vary with intervals after feeding (1, 3 and 5 days). Further, homogenates of sarcoma or MHICl cells, prepared from cultures grown fot 7
MORRIS HAWKINS, JR. and XANDRA0. BREAKEFIELD
1394
days in medium (DMEM with 10% fetal calf serum) supplemented with that conditioned by M H I C l or sarcoma cells, respectively, showed no change in the specific activity of M A 0 or its sensitivity to clorgyline. The relation between M A 0 activity and stage of growth in line N1E-115 is shown in Fig. 3. Although M A 0 activity is high one day after plating, the level drops three fold during rapid cell division and returns to its higher level as the cells enter stationary phase. The A type of M A 0 is the only form present during logarithmic and stationary phases, as measured by the sensitivity of homogenate activity to clorgyline. To determine whether A and B types of M A 0 activity could be distinguished in living cells, cultures were incubated with c3H]amines in the presence of varying concentrations of clorgyline. [3H]Tryptamine is metabolized almost exclusively by deamination in both neuroblastoma line N1E-115 and hepatoma line M H I C , . In N1E-115 cells during a 2 h incubation at 37"C, 74% of the tryptamine was converted to indoleacetic acid. Assuming no endogenous tryptamine and linearity with time and protein, the conversion to indoleacetic acid occurs at a rate of 3.6 nmol/h/mg 12 10 8 6 4 (-log concantration, M I protein when tryptamine is present a t an extracellular concentration of low5M. In M H I C , cells, 75% conFIG. 1. Drug inhibition of tryptamine deamination in version to indoleacetic acid was observed over a MH,C, cell homogenates. Deamination of [14C]trypta- 20min incubation at 37°C corresponding to a rate mine was measured as described in the footnote to Table of 74 nmol/h/mg protein at the same tryptamine con1. Clorgyline (A) and deprenyl (B) were present at varying centration. In line N1E-115 complete inhibition of concentrations for l5min prior to and during M A 0 assays. Curves represent typical results from 2 or 3 experi- tryptamine deamination occurred with approx M-ClOrgyline, and the dose response curve was ments. Each point is the mean of triplicate assays which differed by less than 15%. Percent inhibition is expressed monophasic, indicating that the A type of activity is present exclusively in cells, as in homogenates (Fig, relative to the control activity in each experiment. I200
-
100
1000
0 .-C
0 c
en LL
50 V
3
I
5
9
13
17
21
24
D A Y S AFTER PLATING
FIG.2. Variation in M A 0 activity with stage of growth for MH,C1 cells. M A 0 activity against ['*C]tryptamine was measured in cell homogenates as described in the footnote to Table 1. Homogenates were prepared from parallel cultures at sequential stages of growth. Percent confluency was estimated by microscopic examination of cultures. Results are pooled from 2 experiments. Each point is the mean value of one homogenate assayed in triplicate; triplicate values differed by less than 15%.
Monoamine oxidase A and B in cultured cells
oLI
I
I
2
I
I
4
3
I
5
I
6
7
1
8
4
9
1395
4A). In MH,C, cells, clorgyline sensitivity showed a biphasic curve, as expected for A and B type activity (Fig. 4B). Freezing and thawing MHICl cells in medium prior t o incubation with tryptamine gave an identical curve t o that in living cells. [3H]Dopamine metabolism in murine neuroblastoma cells has been well characterized (BREAKEFIELD, 1975; STOUTet al., 1976; WEXLER& KATZMAN, 1975). In these experiments N1E-115 cells converted 13% of the dopamine to 3,4-dihydroxyphenylacetic acid and homovanillic acid during a 2 h incubation a t 37°C. This would correspond to a rate of 120
I f
10
DAYS AFTER PLATING
FIG. 3. Variation in M A 0 activity with stage of growth for N1E-115 cells. M A 0 activity against [14C]tryptamine was measured in cell homogenates as described in the footnote to Table 1. Homogenates were prepared from parallel cultures at sequential stages of growth. Viable cell numbers were determined by microscopic examination in the presence of 0.05% trypan blue using a hemacytometer. The per cent viable cells in the population decreased from 99% to 73% from day 1 to 10. Results shown are from one of two similar experiments. Each point is the mean value of one homogenate assayed in triplicate; triplicate values differed by less than 15%.
g 100 1 2 80 0 2 60
pmol/h/mg protein assuming that endogenous levels of dopamine are negligible. This M A 0 activity appears t o be all of the A type, as it is completely inhibited by l o - * M-clorgyline (Fig. 5).
DISCUSSION These studies were undertaken t o explore the usefulness of cell culture in studying the expression of A and B types of M A 0 activity. Continuous lines were used which derived from a number of differentiated peripheral cell types: neuroblasts of sympathetic ganglia, hepatocytes, kidney cells, melanocytes and
I-
't
0
a
z
40
0 c
E
z=
20
8 6 4 1 2 1 0 8 C L O R G Y L I N E (-log concentrotion, M )
1 2 1 0 8
6
4
FIG.4. drug sensitivity of tryptamine metabolism in living cells. Stationary phase cultures were incubated with C3H]tryptamine (1 x lo5M, 1 mCi/mmol) in growth medium at 37°C in a humidified atmosphere of 5% CO, and 95% air. Clorgyline was present at varying extracellular concentrations for 15 min prior to and during incubation with tryptamine. At the end of the incubation period, the medium (containing 2 9 5 % of the metabolites) was removed, stored frozen and used for TLC analysis. The formation of deaminated products was determined as the percent radioactivity recovered from the chromatogram which co-migrated with indoleacetic acid. Each point represents chromatographic examination of medium from a single culture. Results are from 1 of 2 essentially identical experiments. (A) N1E-115 cells were grown on lOOmm dishes and incubated with tryptamine for 2 h. An uninhibited culture showed a 74% conversion of tryptamine to indoleacetic acid. (B) MHICl cells were resuspended and incubated with tryptamine for 20 min. Two uninhibited cultures showed 74% and 76% conversion of tryptamine to indoleacetic acid.
CLORGY LI N E (-log concentrotion, M )
FIG.5. Drug sensitivity of dopamine metabolism in living N1E-115 cells. Stationary phase cultures were incubated M, 6.45 mCi/mmol) for 2 h with C3H]dopamine (1.5 x at 37°C in an isotonic phosphate buffered saline with 0.1 mM-ascorbic acid. Clorgyline was present a t varying extracellular concentrations for 15 min prior to and during incubation with dopamine. At the end of the incubation period, the buffer (containing >95% of the metabolites) was removed, stored frozen and used for TLC analysis. The formation of deaminated products was determined as the per cent radioactivity recovered from the chromatogram which co-migrated with 3,4-dihydroxyphenylacetic acid and homovanillic acid. Each point represents chromatographic examination of medium from a single culture. Results are from 1 of 2 essentially identical experiments. Cells were grown on lOOmm dishes. Control cultures showed a 13% conversion of dopamine to deaminated metabolites.
1396
MORRIS HAWKINS, JR. and XANDRA0. BREAKEFIELD
fibroblasts. Levels of M A 0 activity against trypt- cells were broken by vigorous douncing, there was amine measured in cell homogenates varied widely a preferential loss of A type activity, so that the total between these lines, but was exclusively of the A type, M A 0 activity decreased and the remaining activity as measured by clorgyline sensitivity, in all but one showed a sensitivity to clorgyline characteristic of the hepatoma line, MH,C,. The preponderance of A ac- B type (data not shown). tivity in these lines could be explained in several In view of the effects of homogenization procedure ways: (1) These cells, which are ab!e to grow in cul- on the proportion of A and B activity seen in vitro, ture, may have characteristics of developmentally im- we were anxious to determine the expression of these mature cells. In rat brain (MANTLEet al., 1976), as types of activity within living cells. In N1E-115 neurowell as mouse brain (JOURDIKIAN et al., 1975), the blastoma cells, deamination of C3H]tryptamine and B type of M A 0 activity develops later than the A [3H]dopamine was inhibited by low concentrations type. (2) The conditions of culture may affect the of clorgyline as expected for the A type of monoamine expression of M A 0 activity. Lines were grown rou- oxidase activity. Thus the same type of M A 0 activity tinely in medium supplemented with fetal calf serum. occurs in living neuroblastoma cells, as observed in If this serum serves as a common source of lipids homogenates (DONNELLYet al., 1975). In living for the cells, and if the nature of M A 0 activity MH,C, cells, deamination of tryptamine appears to depends on the phospholipid environment (HOUSLAY be mediated by both A and B type activity as in eta!., 1976), then the nature of the lipids in the serum homogenates. The similarity of doseresponse curves may affect the type of M A 0 activity observed. (3) in vitro and in uivo suggests that clorgyline fully penMany of the cells which grow out of tumors from etrates cell membranes. different tissues may represent ‘fibroblastic’ cell types, We conclude that: (1) homogenous populations of and the A type of activity may be found in these a number of peripheral, rodent cell types possess cells in uiuo, as in vitro. almost exclusively the A type of M A 0 activity against In previous studies, we and others have observed tryptamine, as measured in homogenates by sensia decrease in M A 0 activity associated with decreased tivity t o clorgyline; (2) total M A 0 activity is reguHPRT activity in mouse neuroblastoma cells lated in culture with the stage of growth and varies (BREAKEFIELD et al., 1976), rat glioma cells (SKAPER independently of at least one other mitochondrial & SEEGMILLER, 1976) and normal human fibroblasts enzyme, SCCR; (3) in mouse neuroblastoma clone (BREAKEFIELD et al., 1976; R o w et al., 1976). Here N1E-115, M A 0 activity of the A type against tryptwe compared M A 0 activities in two additional par- amine and dopamine occurs in living cells, as in ental lines with that in the HPRT deficient lines de- homogenates (DONNELLY et al., 1976); and (4) in rat rived from them. Three-fold lower M A 0 activity was hepatoma line MH,C, cells both A and B activity observed in an HPRT deficient mouse melanoma line, are present in living cells and homogenates. but no change was observed in an HPRT deficient rat hepatoma line. Further, low M A 0 activity did Acknowledgements-We thank Drs. JAMESK. COWARD, and JEROME A. ROTH for their advice; Mr. not correlate with low SCCR activity, suggesting no PETERCROOKS GRANTfor technical assistance and Ms. ELIZAdramatic changes in mitochondria1 number or func- STEPHEN tion. The lowering of M A 0 activity seen with HPRT BETH AMMANNfor preparation of the manuscript. This deficiency may depend on factors of intermediary work was supported by USPHS Grant NS12105 and a metabolism which vary between lines, or may reflect grant from the National Foundation-March of Dimes. Dr. HAWKINS has been supported by National Science Foundifferent mutational or epigenetic events leading to dation Faculty Fellowship (32-3723 (1975-76) and HPRT deficiency. NIGMS-MRC Fellowship GM 05913 (1976-77). In both neuroblastoma and hepatoma cells the level of M A 0 activity observed in homogenates REFERENCES varied with the stage of growth. In neuroblastoma line N1E-115, M A 0 activity was higher during BLMCHKO H. (1963) in T h e Enzymes, (BOYER P. D., LARDY stationary, as compared to logarithmic phases of H. & MYRBACK K., eds.) Vol. 8, 2nd edn, pp. 337-351. growth, whereas in hepatoma line MHIC, the inverse Academic Press, New York. pattern was observed. No variation in the relative BREAKEFIELD X. 0. (1975) J. Neurochem. 25, 877-882. amounts of A and B activity occurred in homogenates BREAKEFIELD X. O., CASTIGLIONE C. M. & EDELSTEIN S. B. (1976) Science, N.Y. 192, 1018-1020. prepared from either line in logarithmic or stationary C. H., RICHELSONE. & MURPHYD. L. (1976) phases. Other variations in culture conditions, such DONNELLY Biochem. Pharmac. 25, 1639-1643. as the type of medium and serum used for growth, P. & RICHELSON E. (1976) Brain Res. 109, also did not affect the amount or types of M A 0 ac- HONNECCER 335-354. tivity in cell homogenates. Preliminary experiments HOUSLAY M. D., TIPTONK. F. & YOUDIMM. G. H. (1976) suggested that the method of homogenization can Lge Sci. 19, 467-478. change the clorgyline sensitivity of tryptamine dea- HUANGR. H. & EIDUSONS. (1977) J . bid. Chem. 252, mination in MH,C, cells. However, when homo284-290. genates were prepared by our standard procedure JACOBOWITZ D. M. (1972) Life Sci. 11, 965-974. J. P. (1968) Biochem. Pharmac. 17, 1285-1297. both A and B types of M A 0 activity were seen. When JOHNSTON
Monoamine oxidase A and B in cultured cells JOURDIKIAN F., TABAKOFF B. & ALIVISATIOS S. G. A. (1975) Brain Res. 93, 301-308. KNOLLJ. & MAGYARK. (1972) Ado. Biochem. Psychopharmac. 5, 393-408. LOWRY0. H., ROSEBROUGH N. J., FARRA. L. & RANDALL R. J. (1951) J . biol. Chem. 193, 265275. MANTLET. J., GARRETT N. J. & TIPTONK. F. (1976) FEBS Lett. 64,227-230. MURPHYD. L., DONNELLY C. H. & R~CHELSON E. (1976) J . Neurochem. 26, 1231-1235. T. (1973) in Biochemistry of Catecholamines, pp. NAGATSU 203-205. University Park Press. Baltimore. NEFF N. H. & YANG H.-Y. T. (1974) Life Sci. 14, 2061-2074. ROTH J. A., BREAKEFIELD X. 0. & CASTIGLIONEC. M. (1976) Life Sci. 19. 1701-1710. SCHNAITMAN C., ERWINV. G. & GREENAWALT J. W. (1967) J . cell Biol. 32. 719-735.
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SEEDSN. W. (1975) J . biol. Chem. 250, 54555458. S. D., SHEINH. M. & BERVK. R. (1972) Brain SILBERSTEIN Res. 41, 245-248. SKAPERS . D., ADELSONG. L. & SEEGMILLER J. E. (1976) J . Neurochem. 27, 1065-1070. J. E. (1976) Science, N . Y 194, SKAPERS. D. & SEEGMILLER 1171-1 173. SMALLR.. MACARAK E. & FISHER A. B. (1977) J . cell Physiot. 90, 22S232. STOUTR. W., MICHELOTR. J., MOLNARI., HORVATHC. & COWARDJ. C. (1976) Analyt. Biochem. 76, 330-341. VOGEL Z., SYTKOWSKI A. J. & NIRENBERG M. (1972) Proc. natn. Acad. Sci., U.S.A. 69, 318&3 184. WEXLERB. & KATZMANR. (1975) Expl. Cell Res. 92, 291-298. YAMASHITA S. & RACKERE. (1969) J . biol. Chem. 244, 1220-1227.
0022-3042/78/0601- I391 SOZ.00/0
Pergarnon Press Ltd. 1978. Printed in Great Britain 0 International Society lor Neurochemistry Ltd.
MONOAMINE OXIDASE A AND B IN CULTURED CELLS MORRISHAWKINS, JR. and XANDRA0. BREAKEFIELD Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510, U.S.A. (Received 5 M a y 1977. Accepted 6 September 1977)
Abstract-Monoamine oxidase (MAO) activity against tryptamine was compared in a number of continuous rodent lines, including neuroblastoma, hepatoma, melanoma, nephroma, sarcoma and L cells. Activities against tryptamine varied over 300-fold in homogenates of different lines, being highest in hepatoma line MHICl and lowest in a neuroblastoma line lacking hypoxanthine phosphoribosyltransferase (HPRT) activity. The amount, but not the type, of MA0 activity varied with the stage of growth in homogenates of neuroblastoma and hepatoma cells. Measurements of succinatexytochrome c reductase (SCCR), another mitochondria1 enzyme, also showed 20-fold variations between lines, being highest in neuroblastoma line N1E-115 and lowest in hepatoma line MH,C,; SCCR and M A 0 activities appeared to be regulated independently. The relative proportions of the A and B types of MA0 activity were determined in homogenates and living cultures. Clorgyline inhibition of tryptamine deamination in homogenates indicated that in all lines except MHIC,, greater than 95% of the MA0 activity was of the A type. In MHICl homogenates, using clorgyline or deprenyl, 40-70% of the activity appeared to be of the A type and 3MO% of the B type. In cultures of neuroblastoma N1E-115 cells, deamination of tryptamine and dopamine was sensitive to inhibition by low concentrations of clorgyline, indicating that the A type of activity is present intracellularly. as in homogenates. In MHIC, hepatoma cultures, tryptamine deamination showed a biphasic sensitivity to clorgyline. We interpret this to mean that A and B types of MA0 activity occur together in living hepatoma cells.
Because of the potential usefulness of cell culture MONOAMINE oxidase (MAO, monoamine :O2 oxidoreductase EC 1.4.3.4) is important in the degradative for studying the regulation of M A 0 activity, we deterdeamination of biogenic amines throughout the body. mined the relative proportions of A and B activity Studies using homogenates prepared from various in a number of continuous cell lines arising from diftissues have suggested the presence of two types of ferent tissue types. Proportions of A and B activity M A 0 activity which differ in their drug sensitivities were studied in homogenates prepared from cells at and substrate preferences (JOHNSTON,1968; NEFF & different stages of growth and under different condiYANG,1974; HOUSLAY et a/., 1976; HUANG& EIDU- tions of culture. We also examined another mitochondrial enzyme, succinate-cytochrome c reductase SON,1977). Classically, the A activity is preferentially & RACKER,1969) t o determine inhibited by low concentrations of clorgyline and acts (SCCR) (YAMASHITA against serotonin, whereas the B form is more sensi- whether there was co-ordinate expression with M A 0 tive t o deprenyl and acts against phenethylamine and activity. Further, we examined the metabolites formed from labeled amines in culture in the presence of benzy lamine. Monoamine oxidase activity has been described in varying extracellular concentrations of clorgyline t o a variety of mammalian cell cultures (JACOBOWITZ, determine whether the A and B types of M A 0 could et al., 1972; SEEDS,1975; BREAKE- be distinguished in homogeneous populations of 1972; SILBERSTEIN FIELD et al., 1976; HONNEGGER & RICHELSON,1976; living cells. SKMERet al., 1976; SMALLet al., 1977). In only three studies have the relative activities of the A and B types of M A 0 been determined. Homogenates of MATERIALS AND METHODS et al., 1976) and mouse neuroblastoma (DONNELLY Materials. Tissue culture dishes and flasks were obtained rat glioma cells (MURPHYet al., 1976) contain exclusfrom Falcon, Costar or Corning; the Dulbecco-Vogt modiively the A type of activity, while normal human skin fication of Eagle’s medium (DMEM, No. H-21), 1OX Viokfibroblasts contain predominately the A, but also ase and 0.4% Trypan Blue from Grand Island Biological some B activity (ROTH et al., 1976). Co. (Santa Clara, CA); and serum from Flow Laboratories Inc. (Rockville, MD). F-14 (VOGEL et al., 1972) was preAbbreviations used: MAO, monoamine oxidase; HPRT, pared from component chemicals in our laboratory. All hypoxanthine phosphoribosyltransferase ; SCCR, succinate- chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), except clorgyline (Dr. SABATGABAY, cytochrome c reductase. V.A. 1391
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JR. and XANDRA0.BREAKEFIELD MORRISHAWKINS,
Hospital, Boston, MA) and deprenyl (Professor J. KNOLL, 37°C. A zero time blank was used in which 200 pl of 2 N Sammelweis University of Medicine, Budapest, Hungary). HCI was added prior to the labeled substrate. In cases Cell culfure. Mouse neuroblastoma clones NIE-115, where inhibitors were used, the homogenate preparations N-18, and N-l8TG2, derived from the C1300 tumor, were were preincubated for 15 min at 37°C in presence of either laboratory clorgyline or deprenyl. Monoamine oxidase activities were obtained from Dr. MARSHALLNIRENBERG'S (Natl. Inst. Health, Bethesda, MD). Lines designated as assayed within the range of linearity for protein and time. TG were selected from the parent clone by their resistance Succinate-cytochrome c reductase activity was measured to M-Cthioguanine. Line NlE-115TG2 was obtained by a modification of the method of YAMASHITA& RACKER in our laboratory; both NlE-115TG2 and N-18TG2 lack (1969), provided by Anne Hubbard (Yale Univ. Sch. Med., hypoxanthine phosphoribosyltransferase activity (HPRT) New Haven, CT). Homogenates were assayed in duplicate (BREAKEFIELD et al., 1976). Rat hepatoma line HTC and over 1-5 min at 23-25°C in a Gilford spectrophotometer mouse L-cell line LEA-2a line were obtained from Dr. JER- (model 250) with an automatic cuvette positioner (model OME EISENSTADT; rat hepatoma line HTCPRT- and mouse 2451-A) and recorder (model 2530). Each cuvette received L-cell line LMTK- from Dr. EDWARDADELBERG; rat 0.91111 of a reaction mixture consisting of 40mw-sodium hepatoma line MHIC, (No. CCL 114) from the American phosphate buffer, pH 7.4, 20 mM-Na succinate, 1 mM-KCN Type Culture Collection; mouse melanoma lines PSI and and 0.12 pM-cytochrome c. Mixtures were allowed t o equiand RUTHHALA- librate for 1-2min and then 100pl of homogenate was PClA-HPRT2 from Drs. JOHNPAWELEK BAN;and baby hamster kidney line BHK (Cat. No. 026200) added. Samples were read at 550nm. Blanks contained and mouse sarcoma line S-180 (Cat. No. 0-27700) from antimycin, an inhibitor of SCCR, at a final concentration Flow Laboratories. (The preceding investigators are all at of 2.4 x 1 0 - ' ~ . Enzyme activities were calculated using Yale Univ. Med. School, New Haven, CT) Cells were an extinction coefficient of 18.5 x lo6 cmz mol reduced grown as monolayers on plastic tissue culture dishes cytochrome c. (100 mm) or flasks (75 cmZ)in medium supplemented with Protein was determined by a modification of the method 5-10% (v/v) fetal calf serum, without antibiotics, at 37°C of LOWRYet al. (1951) using bovine serum albumin as in a humidified atmosphere of 5% C 0 2 and 95% air. a standard. Neuroblastoma cells, hepatoma HTC cells and LMTKChromatographic examination of ['Hlamine metabolites. cells were routinely grown in DMEM, other lines were The metabolism of [G-3H]3,4-dihydroxyphenylethylamine grown in F-14. Cells were fed every 2-3 days and subcul- (dopamine, 6.5 Ci/mmol, New England Nuclear) and tured every 7-14 days. Neuroblastoma lines were resus- [G-3H]tryptamine HC1 (1 Ci/mmol, Amersham/Searle) in pended by gentle trituration and subcultured in a ratio neuroblastoma cells was determined by adding 30pCi of of 1:lo. Other cell lines required 3-10 min treatment with each t o confluent monolayers grown on l00mm dishes 1X Viokase prior to resuspension, and were subcultured in 3 ml of either medium with 5% serum or isotonic phosat a ratio of 1: 10, except for MHICl which was subcul- phate buffered saline with 0.1 mM-ascorbic acid. For hepatured at 1:3. Growth curves for N1E-115 and MHIC, were toma cells, 2pCi of [3H]tryptamine was added to cells established by inoculating 100mm dishes in parallel. For resuspended in 0.2ml of medium with 10% serum. (Cells neuroblastoma cells, we determined cell numbers using a were grown to confluency on 150mm dishes and resushemacytometer and per cent viability was determined by pended in 4ml of medium per dish.) Cultures were incutrypan blue exclusion. For hepatoma cells, which are diffi- bated at 37°C either in a humidified atmosphere of 5% cult to resuspend, confluency of cultures was monitored C 0 2 and 95% air (medium) or in room atmosphere by microscopic examination. (buffer). When present, clorgyline was preincubated with Preparation of homogenates. In our standard procedure, the cells for 15min prior to the addition of isotope. The monolayer cultures of stationary phase cells were rinsed incubation was terminated by removal of medium (or quickly three times with an isotonic phosphate' buffered buffer), monolayers were rinsed once with isotonic buffer saline at room temperature. This buffer, used routinely in and the rinse was pooled with the medium (or buffer). Cells our laboratory, consists of 129 mM-NaC1, 2.5 mM-KCI, were scraped off the dish in a small volume of buffer 7.4 mM-NaZHPO.,, 1.3 mM-KH,PO,, 0.63 m~-CaCl,, (0.2-0.3 ml) and extracted in 0.1 N-acetic acid in the pres0.74 m~-MgSo,, 5.3 mM-glucose and 46 mM-sucrose, pH ence of lo-" M-ascorbic acid. Cell extracts and medium 7.2, 330 mOSM. Cells grown on flasks were resuspended by were chromatographed on a thin layer cellulose (Eastman trituration; in the case of all except the neuroblastoma 6064) in a solvent system consisting of 1-butanol-1 N-acetic lines, this entailed 2-5 min of pre-treatment with 1X Viok- acid-95% ethanol (3.5:1:1, by vol). The distribution of ase to dissociate cells. Resuspended cells were pelleted by radioactivity was determined, as previously described low speed centrifugation. Cells grown on dishes were (BREAKEFIELD, 1975). For both cell types, >95% of the dedrained and scraped off with a Teflon-coated straight edge. aminated metabolites were found in the medium with only Following either procedure, cells were concentrated in a a few per cent in the cell extracts. There was no breakdown small volume of cold 0.1 M-potassium phosphate buffer, pH or metabolism of these amines under the same incubation 7.4, at 5 1 0 m g protein/ml, frozen on dry ice and stored conditions in the absence of cells. in the vapor phase of liquid nitrogen (-70°C). Prior to RESULTS assay homogenates were thawed and sonicated for 30 s on ice using a microprobe (Biosonik IV.). Monoamine oxidase activity against tryptamine, a Enzyme assays. Monoamine oxidase activity in cell substrate for both A and B types varied widely among homogenates was measured as described by NAGATSU the cell lines tested (Table 1). The highest activity, (1973). Homogenate preparations were assayed in triplicate by incubating 50 pl aliquots with 50 pl of 0.1 M-potassium 1043 pmol/min/mg protein, was observed in rat hepatoma line MH,C1.The other rat hepatoma line HTC, phosphate buffer, pH 7.4, containing 0.1 pCi [2-14C]tryptamine bisuccinate (48.5 mCi/mmol, New England Nuclear) as well as mouse melanoma PS1, mouse sarcoma and 0.17 mht-tryptamine. Reactions were run for 20min at S-180, and mouse L cell LEA-2a showed about half
Monoamine oxidase A and B in cultured cells TABLE1. M A 0
AND
SCCR
Cell line
Cell type
Species
NlE-115 N1 E-115TG2 N-18 N-18TG2 HTC HTCPRTMHlCl PS 1 PCIA-HPRT; BHK S- 180 LMTKLEA-2a
Neuroblastoma Neuroblastoma Neuroblastoma Neuroblastoma Hepatoma Hepatoma Hepatoma Melanoma Melanoma Nephroma Sarcoma L-cell L-cell
Mouse Mouse Mouse Mouse Rat Rat Rat Mouse Mouse Hamster Mouse Mouse Mouse
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ACTIVITIES IN CELL HOMOGENATES
M A 0 activity* SCCR activity? pmol/min/mg nmol/min/mg protein Cytochrome c reduced 125 f 76 3 f 1 196 f 45 67 & 1 1 502 f 77 478 f 41 1043 f 148 492 f 158 175 8 49 & 8 385 f 99 94 f 5 614 & 62
49 f 2 8f1 15 f 4 11 + 5 15 f 3 17 & 4 2 f 0.1 10 f 3 9+3 10 f 2 14 & 3 25 * 4
9f3
MA0:SCCR ratio 0.003 0.0004 0.013 0.006 0.034 0.027 0.603 0.051 0.019 0.005 0.027 0.0003 0.068
* Monoamine oxidase activity was measured in homogenates of stationary phase cells over 20 min at 37°C in 0.1 M-potassium phosphate buffer, pH 7.4 with [14C]tryptamine (1.7 x l o T4M, 48.5 mCi/mmol) (NAGATSU,1973). Deaminated products were extracted into toluene and counted using a liquid scintillation spectrometer. Values represent the mean of 3-5 determinations f S.E.M. t Succinate cytochrome c reductase was measured in homogenates of stationary phase cells over 3-5 min at 23-25°C using 0.12 mM-cytochrome c, 40 mM-sodium phosphate buffer, pH 7.4, 20 mM-Na succinate and 1 mM-KCN and measuring absorbance changes - at O.D. 550nm with a spectrophotometer (YAMASHITA& RACKER, 1969). Values represent the mean of 3-5 determinations f S.E.M. I
this activity. Even lower activity was observed in mouse neuroblastoma lines N1E-115 and N-18, baby hamster BHK and mouse L ceH LMTK-. Monoamine Gxidase activity was lower in 3 lines lacking HPRT activity as compared to the parental lines from which they were derived; activities were 100-fold lower in NlE-115TG2, and 3-fold lower in both N-18TG2 and PClA-HPRT;. However, no difference was found between MA0 activities in HTC and HTCPRT- cells. Succinate-cytochrome c reductase activity also showed wide variation in the cell lines tested (Table 1). The highest activity, 49nmol cytochrome c reduced/min/mg protein, was seen in mouse neuroblastoma NlE-115. All other lines showed one-half to one-third less activity than NIE-115, with rat hepatoma MH,C, showing the lowest activity, 2nmol/ min/mg protein. There appears to be no consistent relationship between the levels of these two mitochondrial enzyme activities in the lines tested, as shown by the MA0:SCCR ratios in Table 1. Rat hepatomas MHICl and HTC have MA0:SCCR ratios of 0.603 and 0.034, respectively, while mouse neuroblastomas N-18 and N1E-115 have ratios of 0.013 and 0.003, respectively. The lowest ratio, 0.0003, was observed in mouse L cell line LMTK-. The relative proportion of M A 0 A and B activities in cell homogenates was determined by measuring the sensitivity of tryptamine deamination to varying concentrations of clorgyline, 1 0 - ~ - 1 0 - ' ~M. In all lines except the rat hepatoma MHIC1, M A 0 activity was completely inhibited by 10- M-clorgyline indicating the exclusive presence of the A type of M A 0 activity. Considering the lower range of sensitivity of our MA0 assay (0.2 pmol/min/mg protein), we would not have been able to detect up to 1% of B activity in
lines such as BHK and LMTK- with low M A 0 values. In fine MH,C,, 70% of the activity appeared to be type A, by its sensitivity to lo-' M-clorgyline, while the remaining 30% required M-dOrgyhe for complete inhibition, as expected for type B (Fig. 1A). Essentially the same biphasic curve of clorgyline sensitivity was observed when the concentration of tryptamine in the assay was reduced from 1.7 x 1 0 - 4 ~ to 1.7 x 1 0 - 6 ~ The . sensitivity of tryptamine deamination to deprenyl in these cells also showed a biphasic pattern with 60% of activity being inhibited at 10T5wand the remaining 40% of (Fig. 1B). This finding suggests somewhat different proportions of A and B activities in these cells. Monoamine oxidase activity in homogenates was blocked only 8% by 1 0 - 4 ~semicarbazide, a drug which blocks another amine oxidase (BLASCHKO, 1963). Changes in M A 0 activity with the stage of cell growth for line MH,CI is shown in Fig. 2. Monoamine oxidase activity is low in homogenates prepared from cells within a few days after subculture, rises during the most rapid stage of growth and decreases again as the cultures become confluent. The proportion of the A and B types of M A 0 activity in these cells, as measured by clorgyline sensitivity, is the same whether homogenates are prepared from cells in logarithmic or stationary phases of growth. The specific activity of M A 0 and the relative proportion of the A and B types seen in MHiCl h o m e genates are not affected by growth in different media (DMEM and F-14) or with different amounts or types of sera (5 and 1004; fetal bovine and human); specific activity did not vary with intervals after feeding (1, 3 and 5 days). Further, homogenates of sarcoma or MHICl cells, prepared from cultures grown fot 7
MORRIS HAWKINS, JR. and XANDRA0. BREAKEFIELD
1394
days in medium (DMEM with 10% fetal calf serum) supplemented with that conditioned by M H I C l or sarcoma cells, respectively, showed no change in the specific activity of M A 0 or its sensitivity to clorgyline. The relation between M A 0 activity and stage of growth in line N1E-115 is shown in Fig. 3. Although M A 0 activity is high one day after plating, the level drops three fold during rapid cell division and returns to its higher level as the cells enter stationary phase. The A type of M A 0 is the only form present during logarithmic and stationary phases, as measured by the sensitivity of homogenate activity to clorgyline. To determine whether A and B types of M A 0 activity could be distinguished in living cells, cultures were incubated with c3H]amines in the presence of varying concentrations of clorgyline. [3H]Tryptamine is metabolized almost exclusively by deamination in both neuroblastoma line N1E-115 and hepatoma line M H I C , . In N1E-115 cells during a 2 h incubation at 37"C, 74% of the tryptamine was converted to indoleacetic acid. Assuming no endogenous tryptamine and linearity with time and protein, the conversion to indoleacetic acid occurs at a rate of 3.6 nmol/h/mg 12 10 8 6 4 (-log concantration, M I protein when tryptamine is present a t an extracellular concentration of low5M. In M H I C , cells, 75% conFIG. 1. Drug inhibition of tryptamine deamination in version to indoleacetic acid was observed over a MH,C, cell homogenates. Deamination of [14C]trypta- 20min incubation at 37°C corresponding to a rate mine was measured as described in the footnote to Table of 74 nmol/h/mg protein at the same tryptamine con1. Clorgyline (A) and deprenyl (B) were present at varying centration. In line N1E-115 complete inhibition of concentrations for l5min prior to and during M A 0 assays. Curves represent typical results from 2 or 3 experi- tryptamine deamination occurred with approx M-ClOrgyline, and the dose response curve was ments. Each point is the mean of triplicate assays which differed by less than 15%. Percent inhibition is expressed monophasic, indicating that the A type of activity is present exclusively in cells, as in homogenates (Fig, relative to the control activity in each experiment. I200
-
100
1000
0 .-C
0 c
en LL
50 V
3
I
5
9
13
17
21
24
D A Y S AFTER PLATING
FIG.2. Variation in M A 0 activity with stage of growth for MH,C1 cells. M A 0 activity against ['*C]tryptamine was measured in cell homogenates as described in the footnote to Table 1. Homogenates were prepared from parallel cultures at sequential stages of growth. Percent confluency was estimated by microscopic examination of cultures. Results are pooled from 2 experiments. Each point is the mean value of one homogenate assayed in triplicate; triplicate values differed by less than 15%.
Monoamine oxidase A and B in cultured cells
oLI
I
I
2
I
I
4
3
I
5
I
6
7
1
8
4
9
1395
4A). In MH,C, cells, clorgyline sensitivity showed a biphasic curve, as expected for A and B type activity (Fig. 4B). Freezing and thawing MHICl cells in medium prior t o incubation with tryptamine gave an identical curve t o that in living cells. [3H]Dopamine metabolism in murine neuroblastoma cells has been well characterized (BREAKEFIELD, 1975; STOUTet al., 1976; WEXLER& KATZMAN, 1975). In these experiments N1E-115 cells converted 13% of the dopamine to 3,4-dihydroxyphenylacetic acid and homovanillic acid during a 2 h incubation a t 37°C. This would correspond to a rate of 120
I f
10
DAYS AFTER PLATING
FIG. 3. Variation in M A 0 activity with stage of growth for N1E-115 cells. M A 0 activity against [14C]tryptamine was measured in cell homogenates as described in the footnote to Table 1. Homogenates were prepared from parallel cultures at sequential stages of growth. Viable cell numbers were determined by microscopic examination in the presence of 0.05% trypan blue using a hemacytometer. The per cent viable cells in the population decreased from 99% to 73% from day 1 to 10. Results shown are from one of two similar experiments. Each point is the mean value of one homogenate assayed in triplicate; triplicate values differed by less than 15%.
g 100 1 2 80 0 2 60
pmol/h/mg protein assuming that endogenous levels of dopamine are negligible. This M A 0 activity appears t o be all of the A type, as it is completely inhibited by l o - * M-clorgyline (Fig. 5).
DISCUSSION These studies were undertaken t o explore the usefulness of cell culture in studying the expression of A and B types of M A 0 activity. Continuous lines were used which derived from a number of differentiated peripheral cell types: neuroblasts of sympathetic ganglia, hepatocytes, kidney cells, melanocytes and
I-
't
0
a
z
40
0 c
E
z=
20
8 6 4 1 2 1 0 8 C L O R G Y L I N E (-log concentrotion, M )
1 2 1 0 8
6
4
FIG.4. drug sensitivity of tryptamine metabolism in living cells. Stationary phase cultures were incubated with C3H]tryptamine (1 x lo5M, 1 mCi/mmol) in growth medium at 37°C in a humidified atmosphere of 5% CO, and 95% air. Clorgyline was present at varying extracellular concentrations for 15 min prior to and during incubation with tryptamine. At the end of the incubation period, the medium (containing 2 9 5 % of the metabolites) was removed, stored frozen and used for TLC analysis. The formation of deaminated products was determined as the percent radioactivity recovered from the chromatogram which co-migrated with indoleacetic acid. Each point represents chromatographic examination of medium from a single culture. Results are from 1 of 2 essentially identical experiments. (A) N1E-115 cells were grown on lOOmm dishes and incubated with tryptamine for 2 h. An uninhibited culture showed a 74% conversion of tryptamine to indoleacetic acid. (B) MHICl cells were resuspended and incubated with tryptamine for 20 min. Two uninhibited cultures showed 74% and 76% conversion of tryptamine to indoleacetic acid.
CLORGY LI N E (-log concentrotion, M )
FIG.5. Drug sensitivity of dopamine metabolism in living N1E-115 cells. Stationary phase cultures were incubated M, 6.45 mCi/mmol) for 2 h with C3H]dopamine (1.5 x at 37°C in an isotonic phosphate buffered saline with 0.1 mM-ascorbic acid. Clorgyline was present a t varying extracellular concentrations for 15 min prior to and during incubation with dopamine. At the end of the incubation period, the buffer (containing >95% of the metabolites) was removed, stored frozen and used for TLC analysis. The formation of deaminated products was determined as the per cent radioactivity recovered from the chromatogram which co-migrated with 3,4-dihydroxyphenylacetic acid and homovanillic acid. Each point represents chromatographic examination of medium from a single culture. Results are from 1 of 2 essentially identical experiments. Cells were grown on lOOmm dishes. Control cultures showed a 13% conversion of dopamine to deaminated metabolites.
1396
MORRIS HAWKINS, JR. and XANDRA0. BREAKEFIELD
fibroblasts. Levels of M A 0 activity against trypt- cells were broken by vigorous douncing, there was amine measured in cell homogenates varied widely a preferential loss of A type activity, so that the total between these lines, but was exclusively of the A type, M A 0 activity decreased and the remaining activity as measured by clorgyline sensitivity, in all but one showed a sensitivity to clorgyline characteristic of the hepatoma line, MH,C,. The preponderance of A ac- B type (data not shown). tivity in these lines could be explained in several In view of the effects of homogenization procedure ways: (1) These cells, which are ab!e to grow in cul- on the proportion of A and B activity seen in vitro, ture, may have characteristics of developmentally im- we were anxious to determine the expression of these mature cells. In rat brain (MANTLEet al., 1976), as types of activity within living cells. In N1E-115 neurowell as mouse brain (JOURDIKIAN et al., 1975), the blastoma cells, deamination of C3H]tryptamine and B type of M A 0 activity develops later than the A [3H]dopamine was inhibited by low concentrations type. (2) The conditions of culture may affect the of clorgyline as expected for the A type of monoamine expression of M A 0 activity. Lines were grown rou- oxidase activity. Thus the same type of M A 0 activity tinely in medium supplemented with fetal calf serum. occurs in living neuroblastoma cells, as observed in If this serum serves as a common source of lipids homogenates (DONNELLYet al., 1975). In living for the cells, and if the nature of M A 0 activity MH,C, cells, deamination of tryptamine appears to depends on the phospholipid environment (HOUSLAY be mediated by both A and B type activity as in eta!., 1976), then the nature of the lipids in the serum homogenates. The similarity of doseresponse curves may affect the type of M A 0 activity observed. (3) in vitro and in uivo suggests that clorgyline fully penMany of the cells which grow out of tumors from etrates cell membranes. different tissues may represent ‘fibroblastic’ cell types, We conclude that: (1) homogenous populations of and the A type of activity may be found in these a number of peripheral, rodent cell types possess cells in uiuo, as in vitro. almost exclusively the A type of M A 0 activity against In previous studies, we and others have observed tryptamine, as measured in homogenates by sensia decrease in M A 0 activity associated with decreased tivity t o clorgyline; (2) total M A 0 activity is reguHPRT activity in mouse neuroblastoma cells lated in culture with the stage of growth and varies (BREAKEFIELD et al., 1976), rat glioma cells (SKAPER independently of at least one other mitochondrial & SEEGMILLER, 1976) and normal human fibroblasts enzyme, SCCR; (3) in mouse neuroblastoma clone (BREAKEFIELD et al., 1976; R o w et al., 1976). Here N1E-115, M A 0 activity of the A type against tryptwe compared M A 0 activities in two additional par- amine and dopamine occurs in living cells, as in ental lines with that in the HPRT deficient lines de- homogenates (DONNELLY et al., 1976); and (4) in rat rived from them. Three-fold lower M A 0 activity was hepatoma line MH,C, cells both A and B activity observed in an HPRT deficient mouse melanoma line, are present in living cells and homogenates. but no change was observed in an HPRT deficient rat hepatoma line. Further, low M A 0 activity did Acknowledgements-We thank Drs. JAMESK. COWARD, and JEROME A. ROTH for their advice; Mr. not correlate with low SCCR activity, suggesting no PETERCROOKS GRANTfor technical assistance and Ms. ELIZAdramatic changes in mitochondria1 number or func- STEPHEN tion. The lowering of M A 0 activity seen with HPRT BETH AMMANNfor preparation of the manuscript. This deficiency may depend on factors of intermediary work was supported by USPHS Grant NS12105 and a metabolism which vary between lines, or may reflect grant from the National Foundation-March of Dimes. Dr. HAWKINS has been supported by National Science Foundifferent mutational or epigenetic events leading to dation Faculty Fellowship (32-3723 (1975-76) and HPRT deficiency. NIGMS-MRC Fellowship GM 05913 (1976-77). In both neuroblastoma and hepatoma cells the level of M A 0 activity observed in homogenates REFERENCES varied with the stage of growth. In neuroblastoma line N1E-115, M A 0 activity was higher during BLMCHKO H. (1963) in T h e Enzymes, (BOYER P. D., LARDY stationary, as compared to logarithmic phases of H. & MYRBACK K., eds.) Vol. 8, 2nd edn, pp. 337-351. growth, whereas in hepatoma line MHIC, the inverse Academic Press, New York. pattern was observed. No variation in the relative BREAKEFIELD X. 0. (1975) J. 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