J o i m t d 01N ~ , i , r , ~ ' l i , , i , , , . ~ iVal. ~ , . 32. pp. I I I 119 Pcrgamon Press Ltd. 1979. Printed in Great Britain. 0 International Society for Neoiochemistry Lfd
0022-3042/79/0lOl-01 II S02.00/0
I N V I T R O STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5.7-DIHYDROXYTRYPTAMINE1,' BAUMGARTENj and H.G. SCHLOSSBERCER4 3Depdrtment of Functional Anatomy, University of Hamburg, 2000 Hamburg 20, GFR and 4Max-Planck-Institute for Biochemistry, Department of Organic Chemistry and Spectroscopy, 8033 Martinsried, GFR
H.-P. KLEMM,j H. G.
(Receioed 2 March 1978. Revised 15 M a y 1978. Accepted 6 June 1978)
Ab~tract-['~C]5.6-Dihydroxytryptamine ([I4C]5,6-DHT) and rL4C]5,7-dihydroxytryptamine ([I4C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [14C]5,6-Dihydroxyindoleacetic acid ([14C]5,6-DHIAA) and ['4C]5,7-dihydroxyindole acetic acid ([I4C]5,7-DHIAA) were detected as M A 0 metabolites by TLC besides non-identified components. The conversion of [I4C]5,6-DHT and [14C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics ( K , 5,7-DHT: 0.5 x ~O-'M;K , 5,6-DHT: 1.25 x I ~ - ' M ) . Inhibition of the reaction by the M A 0 A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of M A 0 (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of M A 0 A. Incubation of rat brain homogenates with [I4C]5,6-DHT and [l4C]5,7-DHT or with the M A 0 metabolites [14C]5,6-DHIAA and [14C]5:7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5.6-DHT inactivated M A 0 in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of M A 0 by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O,] in the incubation medium slightly attenuated the inactivation of M A 0 by 5,6-DHT. Catalase or superoxide dismutase failed to prevent M A 0 from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT. e.g. its corresponding o-quinone, arc involved in the inactivation of M A 0 in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in uivo neurotoxicity of 5,6-DHT. The lack of inactivation of M A 0 by 5,7-DHT in uitro correlated with a low degree of radioactivity binding (from [I4C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and L-ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in uivo neurotoxicity of 5.7-DHT.
5,7-DIHYDROXYTRYPTAMINE (5,7-DHT), a neurotoxic congener of serotonin, has recently become a n important tool for producing rather selective lesions in central serotonergic fibre systems (for review, cf. BAUMGARTEN & BJORKLIJND,1976; BAUMGARTEN et a/., 1977, 1978). The mechanism of action of this drug
This investigation was supported by grants from the Deutsche Forschungsgerneinschaft Dedicated to Prof. Dr. A. BUTENANDTon the occasion of his 75th anniversary Abbreviations used: MAO, monoamine oxidase; 5,6-DHT. 5.6-dihydroxytryptamine; 5,7-DHT, 5.7-dihydroxytryptamine; 5,6-DHIAA. 5,6-dihydroxyindole acetic acid; 5,7-DHIAA. 5,7-dihydroxyindole acetic acid; 5-HT, 5-hydroxytryptamine (serotonin); 6-hydroxydopamine, 6-OH-DA; dithiothreitol, DTT.
is not known at present. Recent findings by DALY et al. (1974) and CREVELING et al. (1975) on the prevention of the toxicity of 5.7-DHT in peripheral noradrenergic (NA) axons by monoamine oxidase inhibitors suggest that metabolism of 5,7-DHT by M A 0 might be a n essential step in the events that result in its neurodegenerative actions. The prescnt study was initiated t o find out whether 5.7-DHT and its positional isomer, 5,6-dihydroxytryptamine (5,6-DHT), are substrates for monoamine oxidase, the most important enzyme in serotonin catabolism, t o establish whether both compounds interact with both forms of M A 0 (A and B), and whether oxidation products of either drug are capable of inactivating MAO, analogous t o the inactivation of catechol-0-methyltransferase by 5,6-indolequinone, formed from 6-hydroxydopamine during its oxidation (BORCHARDT, 1975).
111
112
and H. G. SCHLOSBERGER H.-P. KLEMM,H. G. BAUMGARTEN
MATERIALS AND METHODS ger, Mannheim, GFR) and ascorbate, 0.125 mg superoxide dismutase (Sigma, Munich, G F R ) or 0.125 mg catalase Female albino rats, Sprague~Dawley strain (Wiga, Sulz(Boehringer, Mannheim, GFR). feld, GFR), 180-220 g body weight, were used throughout In vitro binding of [I4C]5,6-DHT, [I4C]5,7-DHT, the present study. Animals were killed by decapitation and [I4C]5,6-DHIAA and [14C]5,7-DHIAA to homogenates of the brains quickly removed from the skull. The dissected rat hypothala~nus. or M-['~C]~,~-DH o rT brains or brain regions (hypothalamus, pons plus medulla 5,7-DHT, and lo-' or 10-4~-[14C]5,6-or 5,7-DHIAA oblongata) were stored in liquid nitrogen until use. were dissolved in 0.25 ml phosphate buffer at p H 7.4 and 5,6-Dihydroxytryptamine creatininesulphate-2 H,O or incubated with 50 pl homogenate of rat hypothalamus 5,7-dihydroxytryptamine creatininesulphate-l/2 H 2 0 were (1 :SO w/v) at 37°C. The incubation was terminated by the purchased from Regis Chemical Co. (Morton Grove, IL, addition of 2 ml 0.4 N-perchloric acid and by cooling the U.S.A.) and dissolved in sterile physiologic saline, gassed sample down to 0°C. The samples were then ccntrifugcd with nitrogen and contained I mg/ml L-ascorbic acid. at 10,000rev.jmin for lOmin. One millilitre of the per[1-I4C]5,6-DHT- and [I4C]S,7-DHT creatininesulphatc chloric acid supernatant was diluted in 10ml of Triton(5.5, 10 or 25mCi/mmol) were synthesized from X-100 cocktail and counted by liquid scintillation spec14C-labelled cyanide following the method of SCHLOSStrometry. Binding of radioactivity to the acid-insoluble pelBERGER & KUCH (1963) (for details, cf. SCHLOSSBERGER, let was calculated by measuring the decline of radioactivity 1978). and dissolved as described above. in the supernatant fraction. In separate experiments it was Monoornine oxidase assays. A modification of the ascertained that the decline of radioactivity from the super& AXELROD (1963) was used to method by WUKTMAN natant corresponded to an equivalent accumulation of determine the kinetics of M A 0 with 5,6- and 5,7-DHT radioactivity in the pellet fraction (by counting thc radioacas substrates. Hypothalami of rats wcrc homogenized in tivity in thc pellet washed several times with perchloric cold physiologic KCI (1 :50 w/v); an aliquot of this homoacid and dissolved in Soluene-350, Packard). genate (Sop1 = 1 mg tissue) was incubated with labelled TLC qf M A 0 catalysed metabolites. The following refer5 6 - or 5,7-DHT. The incubation time was 1&20min for ence substances were available: S,6-DHT. 5,7-DHT. [I4C]5,6-DHT but 30-60 min for CL4C]5,7-DHT. Thc dc- 5,6-DHIAA, and 5,7-DHIAA. About 3 x 106d.p.m. of aminated radioactive material was extracted with 6 ml ['4C]S,6-DHT or 3 x 10-6d.p.m. of CL4C]S,7-DHT toluene-isoamylalcohol (5: 1 v/v) by vigorous shaking for (LO mCi/mmol) were incubated in homogenates of rat 10 min. The recovery of the extraction step was evaluated hypothalamus (1 : 10, w/v) for 10 min (5.6-DHT) or 30 min by internal standards ([1-I4C]5,6-DHIAA (0.5 mCi/mmol) (5.7-DHT) and extracted as described above. Another and [I-I4C]5,7-DHIAA (1.3 mCi/mmol)), both acids being series of incubations was run in the presence of 2 U of available as diethylammonium salts. Four moi of the aldehyde dchydrogenase (Boehringer, Mannheim) and organic layer were counted in 10 ml Triton X-100 cocktail M-NAD (Boehringer, Mannheim). To the extract, in a Packard liquid scintillation counter (C 2405). pooled from 10 tubes, 0.1 mg of 5,6-DHT and 0.1 mg of Five diflerent substrate concentrations (ranging from S,6-DHIAA or 0.1 mg of 5,7-DHT and 0.1 mg of lo-' M to M) were employed, and the linearity of the 5,7-DHIAA were added, and the extract was evaporated reaction was tested in time-course experiments. In case of to dryness in a nitrogen atmosphere. The residue was takcn 5-HT or tryptamine as substrates, 5-1 1,2-3H(N)]hydroxy- up in butanol-glacial acetic acid-water (12:3:5, by vol) and tryptamine binoxalate (21.4 Ci/mmol) or [2-I4C:]trypta(Merck, chromatographed on Cellulose-Fertigplatten mine bisuccinate (48 mCi/mmol) (NEN. Dreieichenhain, Darmstadt). The reference compounds were identified by G F R ) were incubated with homogenates for 20min. and spraying p-dimethylaminocinnamaldehydc onto the chro6 ml of toluene-isoamylalcohol ( I 0: 1 v/v) or 6 ml of matograms. One centimetre strips were scraped off the toluene, respectively. were used for extraction. plates and suspended in 3 ml of H 2 0 and 10 rnl of lnstagel Clorgylinel- and depreny1'-dependent inhibition of (Packard, Frankfurt, G F R ) and counted by LSC. Counting M A 0 activity with [14C]S,6-DHT, [I4C]S,7-DHT, efficiency was monitored by internal standards that con['4C]tryptamine, [3H]5-HT, and [14C]phenylethylamine tained ['4C]toluene (Packard, Frankfurt). (50.9 mCi/mmol, NEN) as substrates were performed in radiometric experiments according to CHRisrMAs et al. (1972). Following a IOmin incubation of each substrate, RESULTS the deaminated radioactive material was extracted into Kinetics OJ conversion of [I4C]5,6-DHT and toluene o r toluene-isoamylalcohol mixturcs as dcscribed ['4C]5.7-DHTOy M A 0 above. Iuactiuatiorz qf M A 0 in vitro. Homogenates of rat ponsWhen homogenates of r a t hypothalamus were incumedulla oblongata (I: 10Ow/v) were incubated with bated in t h e presence of low concentrations of M-S,~-DHT or 5,7-DHT for 15, 30, 45 or 60 min 2.5 x ['4C]5,6- a n d [14C]5,7-DHT (lO-'-IO M), there at 37°C; at the end of each incubation, M A 0 activity was was a time-dependent, linear accumulation of deassayed with 3.4 x I O - ' ~ - [ ~ ~ C ] t r y p t a m i nusing e Sop1 of aminatcd radioactive material in the organic extracthe preincubated homogenate. In 2 series of cxperimcnts, tion medium (toluene-isoamylalcohol). Doubling of all solutions were gassed with N 2 and the preincubation t h e a m o u n t of homogenate used for incubation carried out with air sealed off as well as possiblc. Further resulted in a doubling of the substrate consumption. series of incubations were run in the presence of 4 mM-djthiothreitol (Sigma, Munich. GFR), glutathione (BoehrinRaising the concentrations of b o t h D H T s t o 10-5-10-4 M, however, n o longer yielded proportional increases i n the a m o u n t of deaminated product. With 5,6-DHT, t h e conversion was linear only up to Clorgyline and deprenyl were a gift from CIBAGEIGY. Basle. 10- 15 min but with 5 , 7 - D H T a t least u p to 3Omin.
'
113
Interaction of 5.6- and 5,7-DHT with M A 0
400 1 -
”
300
a00
100
FIG. 1. Lineweaver-Burk representation of MAO-catalysed conversion of [14C]5.6-DHT, [I4C]5,7-DHT, C3H]5-HT and [I4C]tryptamine. Aliquots of rat hypothalamus homogenized in isotonic KCl ( 5 0 ~ 1= I mg tissue) were added to the labelled substrates dissolved in 0.25 ml 0.5 M-phosphate buffer, pH 7.4 at 37°C. Due to graphical reasons. the intcrsection of the [I4C]tryptamine characteristic with the abscissa is omitted from the diagram.
Figure 1 demonstrates the Lineweaver-Burk repre-, sentation of the initial reaction velocities of M A 0 in hypothalamic homogenates plotted against different concentrations of four substrates, [I4C]5,6-DHT. [I4C]5,7-DHT, [3H]5-HT and [‘“Cltryptamine. The K , (app.) and V,,, for the different substrates arc given in Table I . It is obvious that the K , for the less polar substrate tryptamine is much lower than for the more polar ones 5-HT, 5,7-DHT and 5,6-DHT.
in triplication of the [14C]5,6-DHIAA spot but little or no increase in the radioactivity accumulating at the R , of 5,7-DHIAA. Though the background activity on the chromatograms was considerable, the aniines and acids showed up as distinct radioactivity pcaks. A considerable fraction of the radioactivity moved with the solvent front and was not identified.
Thin-layer chromatography
The question which of the two types of MAO, A and B, are mainly responsible for the conversion of 5,6- and 5,7-DHT, was evaluated by studying the effects of inhibitors, said to be selective for the A form (clorgyline) and the B form (deprenyl), on the deamination of both substrates. Figures 2a and 2b depict plots of the percentage inhibition of M A 0 activity by clorgyline and [‘“C]5,6-DHT, deprenyl, respectively, with [I4C]5,7-DHT. C3H]5-HT. [14C]tryptamine and [‘4C]P-phenylethylamine as substrates. Whereas the
Besides non-metabolized [I4C]5,6- or [‘“C]5,7DHT, [‘“C]5,6-DHIAA and [14C]5,7-DHIAA were identified in the organic extract of M A 0 reaction mixtures. The postulated primary products of the cnzyme reaction, the aldehydes, were not detectable because of their presumed instability during the isolation procedure from tissue medium and thin-layer chromatographic procedures. The addition of aldehyde-dehydrogenase and NAD to the reaction mixtures resulted TABLE1. MICHAELIS
Diff~errritial inhibition of deprenyl
CONSTANTS A N D MAXIMAL VELOClTltS FOR THE CONVERSION OF
L3H]5-HT A N D [‘4C]TKYPTAMINE [I
4C]5,6-DHT
app).
[I4C]5.6-DHT, [I4C]5,7-DHT,
I N H A T HYPOTHALAMIC HOMOGENATES
[ 14C]5,7-DHT
[3H]5-HT
[14C]tryptamine
500
200 250
80
70 (K,
M A 0 by clorgyline and
Michaelis constants; (V,,,;,,),maximal velocities.
7
114
H.-P. KLEMM. H. G. BAUMGARTEN and H. G .
(a) 100
90
80 70
SCHLOSSaERGER
clorgyline-dependent inhibition of the serotonin oxidation is represented by a single sigmoidal pattern, the corresponding inhibition of the conversion of [14C]5,6- and 5,7-DHT obeys double sigmoidal characteristics with a broad intermediate plateau. This pattern of enzyme inhibition resembles that observed with ambivalent substrates, such as dopamine or tryptamine, that are metabolized by both forms of MAO. The first part of the double sigmoidal clorgyline inhibition curve appears to represent the inhibition of M A 0 A and the second portion of the curve may be referred to the inhibition of M A 0 B. Thc simple sigmoidal shape of the clorgyline inhibition with serotonin as substrate reflects the fact that serotonin is preferentially oxidized by M A 0 A (JOHNSTON, 1968). The dose-response curves for the deprenyl-dependent inhibition of M A 0 yielded unisigmoidal characteristics for the five substrates tested (Fig. 2b). The substrate-dependent preference of this selective inhibitor of M A 0 B is clarified by the fact that 8-phenylethylamine-a typical substrate for M A 0 B (YANG& NEFF,1973)-has a lower I,, value [-log ~ ~ o r g y ~ i n e ] (molar concentration to inhibit enzyme activity by 5079 in the overall deamination reaction than serotonin, a typical substrate for M A 0 A (2.0 x IO-'M versus 4.5 x M for 8-phenylethylamine and 5-HT, respectively). Deamination of the ambivalent substrate tryptamine was found to be intermediate in its sensitivity to the inhibitor deprenyl (I,, 2.5 x M) whereas 5,6-DHT (I5o 3.2 x M) and 5,7-DHT (I,* 1.1 x 1 0 - ' ~ ) proved to be even less sensitive to inhibition by deprenyl than serotonin.
60
% of control
50 100
40
90
30
80
20
70
10
60 50 [-log Deprenyl]
(b)
40
30
20 FIG.2a, b. EfTects of clorgyline (Fig. 2a) and deprenyl (Fig. 2b) on rat brain M A 0 activity with 0.5 ~ M - [ ' ~ C ] ~ , ~ - D H T , [I4C]5,7-DHT, ['4C]tryptamine, Li4C]P-phenylethyl-
amine and C3H]5-HT as substrates. Homogenates of rat brain (0.2 ml = 20mg tissue) were incubated with various amounts of inhibitor in 0.1 M-sodium phosphate buffer, pH 7.4 at 37°C. At the beginning of the reaction. labelled substrate was added to make a final volume of 1 ml. % inhibition was determined by comparison with control samples containing no inhibitor. The values obtained were plotted against the negative logarithm of the inhibitor molar concentration.
0
15
30
45
60 min preincubation time
FIG. 3. I n uitro inactivation of M A 0 in homogenates of rat pons-medulla oblongata by 5,6-DHT or 5,7-DHT. Preincubation of homogenates with 2.5 x M-~,~-DHT or 5,7-DHT. n = 4; mean f S.D. loo%, = 53.6 x lo-'' mol ['4C]tryptamine/min x mg (control activity in the 5,6-DHT incubations) and 60.0 x lo-'' mol ['4C]tryptamine/min x mg (control activity in the 5,7-DHT incubations).
Interaction of 5.6- and 5,7-DHT with M A 0
In vitro inactivation of MA0 by 5,6- and 5,7-DHT Preincubation of pons-medulla oblongata homogenates with 5,6- or 5,7-DHT and subsequent incubation of an aliquot of the preincubated homogenate with ['4C]tryptamine (generally 6 min) allowed a distinction between reversible enzyme inhibition and time-dependent irreversible M A 0 inactivation. Figure 3 shows the time-dependent loss of enzyme activity due to 5,6-DHT. After 30 min preincubation in the homogenates, only 40% of the initial activity is detected in the homogenates. In prolonged incubations, there was but little increase in the time-dependent enzyme inactivation. The extent of this late M A 0 inactivation by 5,6-DHT roughly corresponds to the time-dependent loss of M A 0 activity brought about by the incubation procedure itself. The inactivation of M A 0 by 5,6-DHT could be partially prevented by the presence of dithiothreitol. glutathione or reduced oxygen concentration in the preincubation medium. There was n o effect, whatsoever, on M A 0 activity by catalase or superoxide dismutase (Table 2). By contrast, there was no clear effect of equimolar amounts of 5,7-DHT on M A 0 activity. The loss of activity bya60 min preincubation with 5,7-DHT(Fig. 3) exceeded that seen in control incubations by only 10%. The initial fall of M A 0 activity by 5,7-DHT is readily explained by the transfer from the preincubate of small amounts of 5,7-DHT into the reaction mixture. The addition of ascorbate, glutathione or dithiothreitol had no effect on the enzyme activity (Table 2).
In vitro binding of [I4C]5,6- arid [I4C]5,7-DffT, ['4C]5.6-DHIA A and [ L4C]5,7-DHlA A to honzogenates of rat hypothalanius A time-dependent binding of all four dihydroxyindoles to components of hypothalamic homogenates was disclosed in in uitro studies (Figs. 4 and 5).
115
Homogenates of rat hypothalamus were incubated in 0.5~-phosphatebuffer at pH 7.4 with different concentrations of the ''C-labelled amines and acids. The incubation conditions were the same as those used for the in vitro assay of MAO. The amount of radioactivity removed from the supernatant fractions containing either [I4C]5,6-DHT or [l4C]5,7-DHT (Fig. 4) corresponded to the retention of radioactivity in the pcrchloric acid insoluble pellet fraction. However, part of the radioactivity in the homogenates containing [14C]5,6-DHT precipitated as an insoluble black product (probably a melanin-like polymer). The precipitation of this coloured product occurred in a concentration-dependcnt manner at different times: with M - [ ' ~ C ] ~ , ~ - D Hprecipitation T, was evident by 30 min, with M-['~C]~,~-DH byT 60 min after onset of incubation. Formation of this melanin-like polymer was noted even in the absence of homogenates, i s . when [I4C]5,6-DHT was dissolved in 0.5 M-phosphate buffer at pH 7.4 at 37°C. The removal of radioactivity from [ I4C]5,7-DHTcontaining homogcnate supernatants (paralleled by a corresponding accumulation of radioactivity in the protein pellet) progressed slowly with time and, after 90min of incubation, did not exceed 10% of the amount of radioactivity added to the homogenates. There was no indication of formation of an insolublc coloured polymer by 5.7-DHT. It is important to note that there was a n initial lag phase of about 10min after onset of incubation during which little, if any. radioactivity was found in the acid insoluble homogenate pellet fraction. The ''C-labelled dihydroxyindole acetic acids reacted similarly to the labelled amines (Fig. 5), and the difference in % removal from the homogcnate supernatants of [I4C]5.6-DHIAA and ['4C]5,7DHIAA was also similar to the difference noted with the amines (see above), and part of the 5,6-DHIAA precipitated from the solutions in the form of coloured, insoluble products. As shown in Tablc 3, thc
2. EFFECTSOF PROTECTIVE AGENTS, REDUCED [O,] A N D CAI.ALASE OK SUPEKOXIIIE TABLE DISMUTASE ON THE INACTIVATION OF MA0 BY 5.6- OR 5.7-DHT FOLLOWING A 60 min PKEINCURATION PERIOD
M A 0 activity in control samples preincubated for 60 min
MA0 activity with 5.6or 5.7-DHT added (2.5 DTT (4 mM) GSH (4 m ~ ) L-Ascorbate (4 mM) Reduced [O,] Catalase (0.125 mg/ml) Superoxide dismutase (0.125 mg/ml)
x
M)
88.6 k 5.2%
88.3 k 2.0%
5:6-DHT
5,7-DHT
+ 1.5 70.9 + 1.7 35.8
60.4 k 2.0 not assayed 43.4 0.8 36.9 & 7.2 35.4 1.1
*
79.3
+ 2.2
66.5 & 2.2 76.7 3.8
+
81.7 Ifr 1.9 86.1 4.0 not assaycd not assayed
(Means S.E.M.of 4 determinations, expressed as % of control. For 0 h control activity, cf. Legend to Fig. 3.)
H.-P. KLEMM, H. G. BAUMGARTEN and H. G. SCHLOSSBERCER
116 % radloacti vity
LO
30
20
10
0 15
1
15 20
L5
30
75
60
90
2.5 10
120
min
incubation time
. . .. " . . " . . . FIG.1 In uitro binding ot L"CJ5,b-UHI and L"CJ>./-UH 1 to homogenates 01 rat hypothalamus. [14C]5,6-DHT or [I4C]5,7-DHT lo-' or 1 0 - 6 ~ were ) dissolved in 2 5 0 ~ 10.5 M phosphate buffer, pH 7.4 at 37°C. 50pl (= 1 mg tissue) homogenate of rat hypothalamus were added to the S.D. % radioactivity in Fig. 4 denotes percentage of radioacamine-containing solutions. n = 4; mean tivity removed from the supernatant fraction, i.e. bound to the pellet. 100% radioactivity = amount of radioactivity recovered in the supernatant immediately after addition of [I4C]5,6-DHT or [I4C]5.7-DHT to a perchloric acid treated homogenate (= 0 min value in Fig. 4). - 1 1 - _ _ ,
LO
',
polymerization
T I
30
-
20
-
10
-
_ I "
14C-5,6- DHIAA (1O-CM)
:
W-5.7-DHIAA (10 -&MI 14C -5,7-DHIAA (10-5~)
0 15
I
2,5 10
15 20
30
45
60
75
120
90
rnin
Incubation time
FIG.5. I n uitro binding of [l4C]5,6-DHIAA and CL4C]5,7-DHIAAto homogenates of rat hypothalamus. [14C]5.6-DHIAA or [I4C]5,7-DHIAA M or lo-' M ) were dissolved in 2 5 0 ~ 10.5 M-phosphate buffer, pH 7.4 at 37°C. 50pl (= 1 mg tissue) homogenate of rat hypothalamus were added to the dihydroxyindole acetic acid-containing solutions. n = 4; mean i S.D. % radioactivity in Fig. 6 denotes percentage of radioactivity removed from the supernatant fraction, i.e. bound t o the pellet. 100% radioactivity = amount of radioactivity recovered in the supernatant immediately after addition of [ 14C]5,6-DH1AA or [ 14C]5,7-DH1AA to a pcrchloric acid treated homogenate ( = 0 min value in Fig. 5).
Interaction of 5,6- and 5.7-DHT with M A 0
117
TABLE3. EFFECTSOF
P R O T ~ C T I V EA ~ ~ E N TON S THE BINDING OF RADIOACTIVITY TO HYPOTHALAMIC PROTEIN PELLETS (% OF CONTROL) FOLLOWING 111 ~ i t r oINCUBATION WITH [I4C]5,6- OR [14C]5.7-DHT
Protective agent added to incubation
.I.
GSH DTT Cysteine L-ascorbic acid
[I4C]5.6-DHT
[' 4C]5,7-DHT
100 f 11.7 8.3 k 0.2
100 k 10.5 86.5 6.8 71.6 & 6.8 91.8 & 12.3 92.8 1.0
10.3
0.4
6.6 0.1 10.8 & 0.2
Retention of radioactivity in the protein pellet of hypothalamic hornogenatcs incubated for 20min at 37"C, pH 7.4 with 10-4~-[14C]5,6-DHTor [14C]5,7-DHT, with or without 4 mM-GSH, DTT. cysteine or L-ascorbic acid added to the incubation medium. The incubation conditions were the same as employed in the measurements of the inactivation of M A 0 irt vitro (cf. Material and Methods). The proteins were precipitated with 0 . 4 ~cold pcrchloric acid. The data represent means S.E.M. of 4 determinations and are expressed as :( radioactivity (d.p.m./pellet)recovered from the protein pellet following incubation of homogenates with radioactive 5,h- or 5,7-DHT (lo@% control in Table 3). Blank values were obtained by running incubations without tissue added.
sulphydryl reagents GSH, DTT and cysteine as well as ascorbic acid-previously found to counteract the inactivation of M A 0 in uitro (Table 2), antagonized the binding of [I4C]5,6-DHT-derived radioactivity to protein pellets. By contrast, there was little effect of these protective agents on the binding of radioactivity from [14C]5,7-DHT to protein pellets. DISCUSSION The results of the present radiometric study show that both 5.6- and 5,7-DHT are metabolized by M A 0 in homogenates of rat brain. The conversion of both drugs by M A 0 was inhibited by the M A 0 inhibitors clorgyline and deprenyl and obeyed, at least initially, Michaelis--Menten kinetics. 5,6-DHIAA and 5,7-DHIAA were identified as part of the deamination products in thin-layer chromatography. A comparison of the kinetic data of 5,6-DHT and 5,7-DHT with those of 5-HT obtained from Lineweaver-Burk diagrams permits the following conclusions as to the enzyme-substrate interaction: (1) The K , of $6- and 5,7-DHT is in the order of 1 0 - 4 ~and thus in a concentration range corresponding to the postulated concentration of extravesicular, cytoplasmic monoamines (EULER,I967 ; cited 1973). The affinity of M A 0 to 5,6-DHT from TIPTON, and 5,7-DHT is thus high enough to ensure metabolism of these false neurochemicals following accumulation in the neuron mediated by the membranebound amine uptake mechanism. (2) The introduction of a second hydroxyl function into the indole nucleus of the serotonin moiety, as in 5,6- and 5,7-DHT, diminishes the affinity of the substrate to the catalytic binding site. The K , of 5,6-DHT (1.3 x 10- M) is about 6 times that of 5-HT (0.2 x lo-' M); the K , of 5,7-DHT (0.5 x M) is about 2.5 times that of 5-HT. Since a hydroxyl funcN.C.
32 I
11
tion in p-position seems to be required for fixation of the substrate in the active center of M A 0 A (SEVERINA: 1973), the additional hydroxyl in the indole nucleus could interfere with the binding by steric hindrance or by clcctrostatic repulsion. This appears to be more important for o-dihydroxylated derivatives, such as 5,6-DHT. In the presence of thc specific M A 0 A type inhibitor, clorgylinc, M A 0 activity with 5,6-DHT and 5,7-DHT as substrates was depressed in a typical manner, indicating that both amines are metabolized by both types of the enzymes, M A 0 A and B, or that both amines are interacting with two active centres in a single enzyme molecule (for details, cf. TIPTONet a/.; 1976). On the other hand, the analysis of the deprenyl inhibition curves (an inhibitor which is known to preferentially inhibit M A 0 B) does not allow a clear distinction between the two enzyme activities, A or B, that interact with 5.6- or 5,7-DHT. In this case, the discussion of the I,, values of both amines leads to the conclusion that 5,6-DHT and 5.7-DHT are mainly metabolized by M A 0 A rather than M A 0 B. However, it has been found that deprenyl is not entirely specific in cases where clorgyline has proved to be a selective inhibitor (SQUIRES, 1972). Therefore. clorgyline seems to be a more reliable standard inhibitor in a binary M A 0 classification than dcprenyl (FOWLERer al., 1977). UV-absorption spectrographical analysis and oxygen consumption measurements rcveal a quick, time-dependent break-down of 5.6-DHT at biological pH and 37°C undcr aerobic conditions (SCHLOSSBERGER,1978 and KLEMM& BAUMGARTEN, unpublished observations), finally resulting in the formation of an insoluble black product. This resembles the air oxidation of 5,6-dihydroxyindole to a melanin species via the quinone intermediate aminochrome (CROMARTIE & HARLEY-MASON, 1957; review by SWAN.
118
H.-P. KLLMM,H. G . BAUMGARTEN and H. G. SCHLOSSBERGFR
FIG. 6. Proposed oxidation products of 5,6-DHT and their reaction with SH-groups in proteins or peptides (R-SH). a = 5,6-DHT; b = o-indolequinone of 5,6-DHT; c = addition product of 'b' and R-SH. The oxidation of (c) is supposed to result in addition of further molecules of 5,6-DHT '(a) thus forming melanin-like high molecular weight products and/or addition of another peptide/protcinlinked indoleamine species (c). The latter reaction would lead to crosslinkage of peptides or proteins as proposed by ROTMANet nl. (1976).
1974). The aminochrome intermediates or related quinones such as 5,6-indolequinone formed from 5,6-DHT are highly reactive structures which rapidly undergo addition reactions especially with nuclcophiles in proteins or peptides (ix. SH- or NH2groups) as demonstrated by ROTMANet al. (1976) in model proteins. A similar mechanism may be responsible for (a) the binding of radioactivity from ['4C]5:6-DHT to perchloric acid insoluble protein pellets and (b) for the inactivation of M A 0 by 5,6-DHT which closely resembles the inactivation of catechol-0-methyltransferase by 5,6-dihydroxyindole (BORCHARDT, 1975). Both, the inactivation of M A 0 by 5,6-DHT and the binding of radioactivity from [l4C)5,6-DHT to protein pellets wcrc counteracted by ascorbic acid, GSH, DTT and cysteine suggesting that the binding of radioactivity may correlate with and possibly reflect the degree of the inactivation of M A 0 by a reactive intermediate of 5,6-DHT, such as its corresponding o-indolequinone (cf. Fig. 6). The mechanism by which e.g. GSH antagonized the protein binding of radioactivity in incubations of homogenates with ['4C]5,6DHT was probably not ,simply to prevent its oxidation by maintaining a reductive milieu since a time-dependent loss of GSH occurred irz uitro duiing incubation with 5,6-DHT which was not followed by a parallel increase in the concentration of GSSG
(Baumgarten, Gercken, Junghanns. unpublished observations). This implies that part of the quinone formed from 5,6-DHT might have undergone covalent binding to glutathione (cf. Fig. 6). Our concept of the formation of a thioether compound of 56indolequinone and GSH receives support from recent studies of L ~ A Net G al. (1977) who isolated and identified 2-(2-S-glutathionyl-3,4,6-trihydroxyphenyl)ethylamine, formed from 6-OH-DA with GSH in uiao in rat brain, a precursor of the intracyclization product 5,6-dihydroxy-4-S-glutathionylindole (cf. Fig. 6). [14C]5,6-DHIAA, the ultimate metabolite of the deamination pathway of S,6-DHT, exhibited similar patterns of protein binding in uitro as [I4C]5,6-DHT indicating that the side chain is not significantly involved in mcdiating the observed binding process. There was no effect, whatsoever, of catalase or superoxide dismutase on the inactivation of M A 0 by 5,6-DHT, an observation which parallels findings by BORCHARDT (1975) using 6-OH-DA and 5,6-dihydroxyindole as substrates for catechol-0-methyltransfcrase. This casts doubt on the potential role of H,Oz. oxygen or hydroxyl radical in the mechanism of M A 0 inactivation by 5,6-DHT. 5,7-DHT, in contrast to 5,6-DHT, did not inactivate M A 0 in uitro and did not bind to a substantial degree to homogenatc pellets irr vitro, though it consumed oxygen accompanied by time-dependent
1 -CH2-NHZ
FIG. 7. Keto-enol tautomeric forms of 5,7-DHT (a-d) and proposed oxidation product of 5,7-DHT (e) and its reaction with SH-groups in proteins or peptides (R-SH) (0. e = p-quinoneimine of' 5,7-DHT.
Interaction of 5,6- and 5.7-DHT with M A 0
1978; changes in UV-absorption (SCHLOSSIIEKGER, KLEMM& BALMGARTENunpublished observations). However, t h e time-dependent air oxidation of S.7-DHT d i d n o t result in the formation of insolublc, coloured precipitates as in case of 5,6-DHT. Furthermore, in buffer solutions a t physiological pH, there is an equilibrium between different keto-cnol tautomeric forms of 5.7-DHT (SCHLOSSHEKGER, 1978; cf. Fig. 7). T h e degree of binding of ['4C]5,7-DHT-derived radioactivity t o proteins in uitro was low when compared to that found after Ci4C]5,6-DHT; may b e mediated by nuclcophilic reactions of the tautomeric j-diketoanion of 5.7-DHT (Fig. 7), by reaction of nuclcophiles in proteins with t h e postulated oxid a t i o n product of 5,7-DHT, i.e. its p-quinoneimine (Fig. 7), by covalent binding of t h e primary metabolite of the deamination pathway of S,7-DHT (its corresponding aldehyde), or by other as yet u n k n o w n mechanism. Finally. it may be asked whether t h e present in uitro findings have s o m e relevance t o the iri uiuo fate of 5,6- and 5,7-DHT. While t h e binding of radioactivity t o protein pellets of rat brain derived from [I4C]5,6-DHT is similar in vitro a n d in viuo (BAUMGAKTEN et a/., 1978) there is a discrepancy between t h e negligible extent of protein binding (from ['4C]5.7-DHT) traced in vitro when c o m p a r e d t o t h e extensive, time-dependent increase in covalcntly bound radioactivity seen in uiuo. This discrepancy suggcsts that there exists a toxicity-reinforcing catalytic mechanism in aivo which renders S,7-DHT o r o n e of its metabolites and/or oxidation products more reactive which d o e s n o t operatc in vitro. 11 is as yet u n k n o w n whether inactivation of brain M A 0 by S,6-DHT occurs also in vivo a n d whether it has a n y dclcterious conscqucnccs for the neurons affected.
119
CREVEL~N C.CR., LUNDSTKOM J., MCNEALE. T., TICFL. & DALYJ. W. (1975) Dihydroxytryptamines: effects on noradrenergic function in mouse heart in uiuo. Molec. Pkarinac. 11, 21 1-227. DALYJ . W., LUNDSTKOM J. & CREVELING C. R. (1974) Structure-activity correlations in trihydroxyphenylethylamines and dihydroxytryptamines. Relationship to cytotoxicity in adrenergic and serotonergic neurons, in DynaK., mics of Degeneration atid Grow~thin Neurons (FUXE OLSON L. & ZOTTERMAN Y.. eds.), pp. 29-42. Pergamon Press. Oxford. K. F. (1973) EULEKU . S. VON (1967) cited from TIPTOW Biochemical aspects of monoamine oxidase. Br. ined. E L / / / 29. . 116-1 19. FOWLER C. J., CALLINGHAM B. A., MANTLE T. J. & TIPTON K. F. (1978) Monoamine oxidase A and B: A useful concept? Biuchem. Pharinac. 27, 97-101. HEIKKILA R. & COHENG. (1971) Inhibition of hiogenic amine uptake by hydrogen peroxide: A mechanism for toxic effects of 6-hydroxydopamine. Science 72, 1257-1 258. JOHNSTOW J. P. (1968) Some observations upon a new inhibitor of monoamine oxidase in brain tissue. BiOChEL WURTMANR. J. & AXELROD J . (1963) A sensitive and CHKISTMAS D. (1972) A cornparison of the pharmacological and biospecific assay for the estimation of monoamine oxidase. chemical properties of substrate-sclcctive monoaminc BiocAein. Pliarinac. 12, 1439-1 440. oxidasc inhibitors. Br. J . Pharniuc. 45, 49&503. YANGH. Y. T. &. NEFFN. H. (1973) P-Phenylethylamine: CROMARTIE R. I. T. & HARLEY-MASON J . (1957) Melanin a specific substrate for type B monoamine oxidase of and its precursors. Biocheni. J . 66, 713-720. brain. J . Pharmac. exp. Ther. 187. 365-371.
0022-3042/79/0lOl-01 II S02.00/0
I N V I T R O STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5.7-DIHYDROXYTRYPTAMINE1,' BAUMGARTENj and H.G. SCHLOSSBERCER4 3Depdrtment of Functional Anatomy, University of Hamburg, 2000 Hamburg 20, GFR and 4Max-Planck-Institute for Biochemistry, Department of Organic Chemistry and Spectroscopy, 8033 Martinsried, GFR
H.-P. KLEMM,j H. G.
(Receioed 2 March 1978. Revised 15 M a y 1978. Accepted 6 June 1978)
Ab~tract-['~C]5.6-Dihydroxytryptamine ([I4C]5,6-DHT) and rL4C]5,7-dihydroxytryptamine ([I4C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [14C]5,6-Dihydroxyindoleacetic acid ([14C]5,6-DHIAA) and ['4C]5,7-dihydroxyindole acetic acid ([I4C]5,7-DHIAA) were detected as M A 0 metabolites by TLC besides non-identified components. The conversion of [I4C]5,6-DHT and [14C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics ( K , 5,7-DHT: 0.5 x ~O-'M;K , 5,6-DHT: 1.25 x I ~ - ' M ) . Inhibition of the reaction by the M A 0 A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of M A 0 (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of M A 0 A. Incubation of rat brain homogenates with [I4C]5,6-DHT and [l4C]5,7-DHT or with the M A 0 metabolites [14C]5,6-DHIAA and [14C]5:7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5.6-DHT inactivated M A 0 in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of M A 0 by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O,] in the incubation medium slightly attenuated the inactivation of M A 0 by 5,6-DHT. Catalase or superoxide dismutase failed to prevent M A 0 from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT. e.g. its corresponding o-quinone, arc involved in the inactivation of M A 0 in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in uivo neurotoxicity of 5,6-DHT. The lack of inactivation of M A 0 by 5,7-DHT in uitro correlated with a low degree of radioactivity binding (from [I4C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and L-ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in uivo neurotoxicity of 5.7-DHT.
5,7-DIHYDROXYTRYPTAMINE (5,7-DHT), a neurotoxic congener of serotonin, has recently become a n important tool for producing rather selective lesions in central serotonergic fibre systems (for review, cf. BAUMGARTEN & BJORKLIJND,1976; BAUMGARTEN et a/., 1977, 1978). The mechanism of action of this drug
This investigation was supported by grants from the Deutsche Forschungsgerneinschaft Dedicated to Prof. Dr. A. BUTENANDTon the occasion of his 75th anniversary Abbreviations used: MAO, monoamine oxidase; 5,6-DHT. 5.6-dihydroxytryptamine; 5,7-DHT, 5.7-dihydroxytryptamine; 5,6-DHIAA. 5,6-dihydroxyindole acetic acid; 5,7-DHIAA. 5,7-dihydroxyindole acetic acid; 5-HT, 5-hydroxytryptamine (serotonin); 6-hydroxydopamine, 6-OH-DA; dithiothreitol, DTT.
is not known at present. Recent findings by DALY et al. (1974) and CREVELING et al. (1975) on the prevention of the toxicity of 5.7-DHT in peripheral noradrenergic (NA) axons by monoamine oxidase inhibitors suggest that metabolism of 5,7-DHT by M A 0 might be a n essential step in the events that result in its neurodegenerative actions. The prescnt study was initiated t o find out whether 5.7-DHT and its positional isomer, 5,6-dihydroxytryptamine (5,6-DHT), are substrates for monoamine oxidase, the most important enzyme in serotonin catabolism, t o establish whether both compounds interact with both forms of M A 0 (A and B), and whether oxidation products of either drug are capable of inactivating MAO, analogous t o the inactivation of catechol-0-methyltransferase by 5,6-indolequinone, formed from 6-hydroxydopamine during its oxidation (BORCHARDT, 1975).
111
112
and H. G. SCHLOSBERGER H.-P. KLEMM,H. G. BAUMGARTEN
MATERIALS AND METHODS ger, Mannheim, GFR) and ascorbate, 0.125 mg superoxide dismutase (Sigma, Munich, G F R ) or 0.125 mg catalase Female albino rats, Sprague~Dawley strain (Wiga, Sulz(Boehringer, Mannheim, GFR). feld, GFR), 180-220 g body weight, were used throughout In vitro binding of [I4C]5,6-DHT, [I4C]5,7-DHT, the present study. Animals were killed by decapitation and [I4C]5,6-DHIAA and [14C]5,7-DHIAA to homogenates of the brains quickly removed from the skull. The dissected rat hypothala~nus. or M-['~C]~,~-DH o rT brains or brain regions (hypothalamus, pons plus medulla 5,7-DHT, and lo-' or 10-4~-[14C]5,6-or 5,7-DHIAA oblongata) were stored in liquid nitrogen until use. were dissolved in 0.25 ml phosphate buffer at p H 7.4 and 5,6-Dihydroxytryptamine creatininesulphate-2 H,O or incubated with 50 pl homogenate of rat hypothalamus 5,7-dihydroxytryptamine creatininesulphate-l/2 H 2 0 were (1 :SO w/v) at 37°C. The incubation was terminated by the purchased from Regis Chemical Co. (Morton Grove, IL, addition of 2 ml 0.4 N-perchloric acid and by cooling the U.S.A.) and dissolved in sterile physiologic saline, gassed sample down to 0°C. The samples were then ccntrifugcd with nitrogen and contained I mg/ml L-ascorbic acid. at 10,000rev.jmin for lOmin. One millilitre of the per[1-I4C]5,6-DHT- and [I4C]S,7-DHT creatininesulphatc chloric acid supernatant was diluted in 10ml of Triton(5.5, 10 or 25mCi/mmol) were synthesized from X-100 cocktail and counted by liquid scintillation spec14C-labelled cyanide following the method of SCHLOSStrometry. Binding of radioactivity to the acid-insoluble pelBERGER & KUCH (1963) (for details, cf. SCHLOSSBERGER, let was calculated by measuring the decline of radioactivity 1978). and dissolved as described above. in the supernatant fraction. In separate experiments it was Monoornine oxidase assays. A modification of the ascertained that the decline of radioactivity from the super& AXELROD (1963) was used to method by WUKTMAN natant corresponded to an equivalent accumulation of determine the kinetics of M A 0 with 5,6- and 5,7-DHT radioactivity in the pellet fraction (by counting thc radioacas substrates. Hypothalami of rats wcrc homogenized in tivity in thc pellet washed several times with perchloric cold physiologic KCI (1 :50 w/v); an aliquot of this homoacid and dissolved in Soluene-350, Packard). genate (Sop1 = 1 mg tissue) was incubated with labelled TLC qf M A 0 catalysed metabolites. The following refer5 6 - or 5,7-DHT. The incubation time was 1&20min for ence substances were available: S,6-DHT. 5,7-DHT. [I4C]5,6-DHT but 30-60 min for CL4C]5,7-DHT. Thc dc- 5,6-DHIAA, and 5,7-DHIAA. About 3 x 106d.p.m. of aminated radioactive material was extracted with 6 ml ['4C]S,6-DHT or 3 x 10-6d.p.m. of CL4C]S,7-DHT toluene-isoamylalcohol (5: 1 v/v) by vigorous shaking for (LO mCi/mmol) were incubated in homogenates of rat 10 min. The recovery of the extraction step was evaluated hypothalamus (1 : 10, w/v) for 10 min (5.6-DHT) or 30 min by internal standards ([1-I4C]5,6-DHIAA (0.5 mCi/mmol) (5.7-DHT) and extracted as described above. Another and [I-I4C]5,7-DHIAA (1.3 mCi/mmol)), both acids being series of incubations was run in the presence of 2 U of available as diethylammonium salts. Four moi of the aldehyde dchydrogenase (Boehringer, Mannheim) and organic layer were counted in 10 ml Triton X-100 cocktail M-NAD (Boehringer, Mannheim). To the extract, in a Packard liquid scintillation counter (C 2405). pooled from 10 tubes, 0.1 mg of 5,6-DHT and 0.1 mg of Five diflerent substrate concentrations (ranging from S,6-DHIAA or 0.1 mg of 5,7-DHT and 0.1 mg of lo-' M to M) were employed, and the linearity of the 5,7-DHIAA were added, and the extract was evaporated reaction was tested in time-course experiments. In case of to dryness in a nitrogen atmosphere. The residue was takcn 5-HT or tryptamine as substrates, 5-1 1,2-3H(N)]hydroxy- up in butanol-glacial acetic acid-water (12:3:5, by vol) and tryptamine binoxalate (21.4 Ci/mmol) or [2-I4C:]trypta(Merck, chromatographed on Cellulose-Fertigplatten mine bisuccinate (48 mCi/mmol) (NEN. Dreieichenhain, Darmstadt). The reference compounds were identified by G F R ) were incubated with homogenates for 20min. and spraying p-dimethylaminocinnamaldehydc onto the chro6 ml of toluene-isoamylalcohol ( I 0: 1 v/v) or 6 ml of matograms. One centimetre strips were scraped off the toluene, respectively. were used for extraction. plates and suspended in 3 ml of H 2 0 and 10 rnl of lnstagel Clorgylinel- and depreny1'-dependent inhibition of (Packard, Frankfurt, G F R ) and counted by LSC. Counting M A 0 activity with [14C]S,6-DHT, [I4C]S,7-DHT, efficiency was monitored by internal standards that con['4C]tryptamine, [3H]5-HT, and [14C]phenylethylamine tained ['4C]toluene (Packard, Frankfurt). (50.9 mCi/mmol, NEN) as substrates were performed in radiometric experiments according to CHRisrMAs et al. (1972). Following a IOmin incubation of each substrate, RESULTS the deaminated radioactive material was extracted into Kinetics OJ conversion of [I4C]5,6-DHT and toluene o r toluene-isoamylalcohol mixturcs as dcscribed ['4C]5.7-DHTOy M A 0 above. Iuactiuatiorz qf M A 0 in vitro. Homogenates of rat ponsWhen homogenates of r a t hypothalamus were incumedulla oblongata (I: 10Ow/v) were incubated with bated in t h e presence of low concentrations of M-S,~-DHT or 5,7-DHT for 15, 30, 45 or 60 min 2.5 x ['4C]5,6- a n d [14C]5,7-DHT (lO-'-IO M), there at 37°C; at the end of each incubation, M A 0 activity was was a time-dependent, linear accumulation of deassayed with 3.4 x I O - ' ~ - [ ~ ~ C ] t r y p t a m i nusing e Sop1 of aminatcd radioactive material in the organic extracthe preincubated homogenate. In 2 series of cxperimcnts, tion medium (toluene-isoamylalcohol). Doubling of all solutions were gassed with N 2 and the preincubation t h e a m o u n t of homogenate used for incubation carried out with air sealed off as well as possiblc. Further resulted in a doubling of the substrate consumption. series of incubations were run in the presence of 4 mM-djthiothreitol (Sigma, Munich. GFR), glutathione (BoehrinRaising the concentrations of b o t h D H T s t o 10-5-10-4 M, however, n o longer yielded proportional increases i n the a m o u n t of deaminated product. With 5,6-DHT, t h e conversion was linear only up to Clorgyline and deprenyl were a gift from CIBAGEIGY. Basle. 10- 15 min but with 5 , 7 - D H T a t least u p to 3Omin.
'
113
Interaction of 5.6- and 5,7-DHT with M A 0
400 1 -
”
300
a00
100
FIG. 1. Lineweaver-Burk representation of MAO-catalysed conversion of [14C]5.6-DHT, [I4C]5,7-DHT, C3H]5-HT and [I4C]tryptamine. Aliquots of rat hypothalamus homogenized in isotonic KCl ( 5 0 ~ 1= I mg tissue) were added to the labelled substrates dissolved in 0.25 ml 0.5 M-phosphate buffer, pH 7.4 at 37°C. Due to graphical reasons. the intcrsection of the [I4C]tryptamine characteristic with the abscissa is omitted from the diagram.
Figure 1 demonstrates the Lineweaver-Burk repre-, sentation of the initial reaction velocities of M A 0 in hypothalamic homogenates plotted against different concentrations of four substrates, [I4C]5,6-DHT. [I4C]5,7-DHT, [3H]5-HT and [‘“Cltryptamine. The K , (app.) and V,,, for the different substrates arc given in Table I . It is obvious that the K , for the less polar substrate tryptamine is much lower than for the more polar ones 5-HT, 5,7-DHT and 5,6-DHT.
in triplication of the [14C]5,6-DHIAA spot but little or no increase in the radioactivity accumulating at the R , of 5,7-DHIAA. Though the background activity on the chromatograms was considerable, the aniines and acids showed up as distinct radioactivity pcaks. A considerable fraction of the radioactivity moved with the solvent front and was not identified.
Thin-layer chromatography
The question which of the two types of MAO, A and B, are mainly responsible for the conversion of 5,6- and 5,7-DHT, was evaluated by studying the effects of inhibitors, said to be selective for the A form (clorgyline) and the B form (deprenyl), on the deamination of both substrates. Figures 2a and 2b depict plots of the percentage inhibition of M A 0 activity by clorgyline and [‘“C]5,6-DHT, deprenyl, respectively, with [I4C]5,7-DHT. C3H]5-HT. [14C]tryptamine and [‘4C]P-phenylethylamine as substrates. Whereas the
Besides non-metabolized [I4C]5,6- or [‘“C]5,7DHT, [‘“C]5,6-DHIAA and [14C]5,7-DHIAA were identified in the organic extract of M A 0 reaction mixtures. The postulated primary products of the cnzyme reaction, the aldehydes, were not detectable because of their presumed instability during the isolation procedure from tissue medium and thin-layer chromatographic procedures. The addition of aldehyde-dehydrogenase and NAD to the reaction mixtures resulted TABLE1. MICHAELIS
Diff~errritial inhibition of deprenyl
CONSTANTS A N D MAXIMAL VELOClTltS FOR THE CONVERSION OF
L3H]5-HT A N D [‘4C]TKYPTAMINE [I
4C]5,6-DHT
app).
[I4C]5.6-DHT, [I4C]5,7-DHT,
I N H A T HYPOTHALAMIC HOMOGENATES
[ 14C]5,7-DHT
[3H]5-HT
[14C]tryptamine
500
200 250
80
70 (K,
M A 0 by clorgyline and
Michaelis constants; (V,,,;,,),maximal velocities.
7
114
H.-P. KLEMM. H. G. BAUMGARTEN and H. G .
(a) 100
90
80 70
SCHLOSSaERGER
clorgyline-dependent inhibition of the serotonin oxidation is represented by a single sigmoidal pattern, the corresponding inhibition of the conversion of [14C]5,6- and 5,7-DHT obeys double sigmoidal characteristics with a broad intermediate plateau. This pattern of enzyme inhibition resembles that observed with ambivalent substrates, such as dopamine or tryptamine, that are metabolized by both forms of MAO. The first part of the double sigmoidal clorgyline inhibition curve appears to represent the inhibition of M A 0 A and the second portion of the curve may be referred to the inhibition of M A 0 B. Thc simple sigmoidal shape of the clorgyline inhibition with serotonin as substrate reflects the fact that serotonin is preferentially oxidized by M A 0 A (JOHNSTON, 1968). The dose-response curves for the deprenyl-dependent inhibition of M A 0 yielded unisigmoidal characteristics for the five substrates tested (Fig. 2b). The substrate-dependent preference of this selective inhibitor of M A 0 B is clarified by the fact that 8-phenylethylamine-a typical substrate for M A 0 B (YANG& NEFF,1973)-has a lower I,, value [-log ~ ~ o r g y ~ i n e ] (molar concentration to inhibit enzyme activity by 5079 in the overall deamination reaction than serotonin, a typical substrate for M A 0 A (2.0 x IO-'M versus 4.5 x M for 8-phenylethylamine and 5-HT, respectively). Deamination of the ambivalent substrate tryptamine was found to be intermediate in its sensitivity to the inhibitor deprenyl (I,, 2.5 x M) whereas 5,6-DHT (I5o 3.2 x M) and 5,7-DHT (I,* 1.1 x 1 0 - ' ~ ) proved to be even less sensitive to inhibition by deprenyl than serotonin.
60
% of control
50 100
40
90
30
80
20
70
10
60 50 [-log Deprenyl]
(b)
40
30
20 FIG.2a, b. EfTects of clorgyline (Fig. 2a) and deprenyl (Fig. 2b) on rat brain M A 0 activity with 0.5 ~ M - [ ' ~ C ] ~ , ~ - D H T , [I4C]5,7-DHT, ['4C]tryptamine, Li4C]P-phenylethyl-
amine and C3H]5-HT as substrates. Homogenates of rat brain (0.2 ml = 20mg tissue) were incubated with various amounts of inhibitor in 0.1 M-sodium phosphate buffer, pH 7.4 at 37°C. At the beginning of the reaction. labelled substrate was added to make a final volume of 1 ml. % inhibition was determined by comparison with control samples containing no inhibitor. The values obtained were plotted against the negative logarithm of the inhibitor molar concentration.
0
15
30
45
60 min preincubation time
FIG. 3. I n uitro inactivation of M A 0 in homogenates of rat pons-medulla oblongata by 5,6-DHT or 5,7-DHT. Preincubation of homogenates with 2.5 x M-~,~-DHT or 5,7-DHT. n = 4; mean f S.D. loo%, = 53.6 x lo-'' mol ['4C]tryptamine/min x mg (control activity in the 5,6-DHT incubations) and 60.0 x lo-'' mol ['4C]tryptamine/min x mg (control activity in the 5,7-DHT incubations).
Interaction of 5.6- and 5,7-DHT with M A 0
In vitro inactivation of MA0 by 5,6- and 5,7-DHT Preincubation of pons-medulla oblongata homogenates with 5,6- or 5,7-DHT and subsequent incubation of an aliquot of the preincubated homogenate with ['4C]tryptamine (generally 6 min) allowed a distinction between reversible enzyme inhibition and time-dependent irreversible M A 0 inactivation. Figure 3 shows the time-dependent loss of enzyme activity due to 5,6-DHT. After 30 min preincubation in the homogenates, only 40% of the initial activity is detected in the homogenates. In prolonged incubations, there was but little increase in the time-dependent enzyme inactivation. The extent of this late M A 0 inactivation by 5,6-DHT roughly corresponds to the time-dependent loss of M A 0 activity brought about by the incubation procedure itself. The inactivation of M A 0 by 5,6-DHT could be partially prevented by the presence of dithiothreitol. glutathione or reduced oxygen concentration in the preincubation medium. There was n o effect, whatsoever, on M A 0 activity by catalase or superoxide dismutase (Table 2). By contrast, there was no clear effect of equimolar amounts of 5,7-DHT on M A 0 activity. The loss of activity bya60 min preincubation with 5,7-DHT(Fig. 3) exceeded that seen in control incubations by only 10%. The initial fall of M A 0 activity by 5,7-DHT is readily explained by the transfer from the preincubate of small amounts of 5,7-DHT into the reaction mixture. The addition of ascorbate, glutathione or dithiothreitol had no effect on the enzyme activity (Table 2).
In vitro binding of [I4C]5,6- arid [I4C]5,7-DffT, ['4C]5.6-DHIA A and [ L4C]5,7-DHlA A to honzogenates of rat hypothalanius A time-dependent binding of all four dihydroxyindoles to components of hypothalamic homogenates was disclosed in in uitro studies (Figs. 4 and 5).
115
Homogenates of rat hypothalamus were incubated in 0.5~-phosphatebuffer at pH 7.4 with different concentrations of the ''C-labelled amines and acids. The incubation conditions were the same as those used for the in vitro assay of MAO. The amount of radioactivity removed from the supernatant fractions containing either [I4C]5,6-DHT or [l4C]5,7-DHT (Fig. 4) corresponded to the retention of radioactivity in the pcrchloric acid insoluble pellet fraction. However, part of the radioactivity in the homogenates containing [14C]5,6-DHT precipitated as an insoluble black product (probably a melanin-like polymer). The precipitation of this coloured product occurred in a concentration-dependcnt manner at different times: with M - [ ' ~ C ] ~ , ~ - D Hprecipitation T, was evident by 30 min, with M-['~C]~,~-DH byT 60 min after onset of incubation. Formation of this melanin-like polymer was noted even in the absence of homogenates, i s . when [I4C]5,6-DHT was dissolved in 0.5 M-phosphate buffer at pH 7.4 at 37°C. The removal of radioactivity from [ I4C]5,7-DHTcontaining homogcnate supernatants (paralleled by a corresponding accumulation of radioactivity in the protein pellet) progressed slowly with time and, after 90min of incubation, did not exceed 10% of the amount of radioactivity added to the homogenates. There was no indication of formation of an insolublc coloured polymer by 5.7-DHT. It is important to note that there was a n initial lag phase of about 10min after onset of incubation during which little, if any. radioactivity was found in the acid insoluble homogenate pellet fraction. The ''C-labelled dihydroxyindole acetic acids reacted similarly to the labelled amines (Fig. 5), and the difference in % removal from the homogcnate supernatants of [I4C]5.6-DHIAA and ['4C]5,7DHIAA was also similar to the difference noted with the amines (see above), and part of the 5,6-DHIAA precipitated from the solutions in the form of coloured, insoluble products. As shown in Tablc 3, thc
2. EFFECTSOF PROTECTIVE AGENTS, REDUCED [O,] A N D CAI.ALASE OK SUPEKOXIIIE TABLE DISMUTASE ON THE INACTIVATION OF MA0 BY 5.6- OR 5.7-DHT FOLLOWING A 60 min PKEINCURATION PERIOD
M A 0 activity in control samples preincubated for 60 min
MA0 activity with 5.6or 5.7-DHT added (2.5 DTT (4 mM) GSH (4 m ~ ) L-Ascorbate (4 mM) Reduced [O,] Catalase (0.125 mg/ml) Superoxide dismutase (0.125 mg/ml)
x
M)
88.6 k 5.2%
88.3 k 2.0%
5:6-DHT
5,7-DHT
+ 1.5 70.9 + 1.7 35.8
60.4 k 2.0 not assayed 43.4 0.8 36.9 & 7.2 35.4 1.1
*
79.3
+ 2.2
66.5 & 2.2 76.7 3.8
+
81.7 Ifr 1.9 86.1 4.0 not assaycd not assayed
(Means S.E.M.of 4 determinations, expressed as % of control. For 0 h control activity, cf. Legend to Fig. 3.)
H.-P. KLEMM, H. G. BAUMGARTEN and H. G. SCHLOSSBERCER
116 % radloacti vity
LO
30
20
10
0 15
1
15 20
L5
30
75
60
90
2.5 10
120
min
incubation time
. . .. " . . " . . . FIG.1 In uitro binding ot L"CJ5,b-UHI and L"CJ>./-UH 1 to homogenates 01 rat hypothalamus. [14C]5,6-DHT or [I4C]5,7-DHT lo-' or 1 0 - 6 ~ were ) dissolved in 2 5 0 ~ 10.5 M phosphate buffer, pH 7.4 at 37°C. 50pl (= 1 mg tissue) homogenate of rat hypothalamus were added to the S.D. % radioactivity in Fig. 4 denotes percentage of radioacamine-containing solutions. n = 4; mean tivity removed from the supernatant fraction, i.e. bound to the pellet. 100% radioactivity = amount of radioactivity recovered in the supernatant immediately after addition of [I4C]5,6-DHT or [I4C]5.7-DHT to a perchloric acid treated homogenate (= 0 min value in Fig. 4). - 1 1 - _ _ ,
LO
',
polymerization
T I
30
-
20
-
10
-
_ I "
14C-5,6- DHIAA (1O-CM)
:
W-5.7-DHIAA (10 -&MI 14C -5,7-DHIAA (10-5~)
0 15
I
2,5 10
15 20
30
45
60
75
120
90
rnin
Incubation time
FIG.5. I n uitro binding of [l4C]5,6-DHIAA and CL4C]5,7-DHIAAto homogenates of rat hypothalamus. [14C]5.6-DHIAA or [I4C]5,7-DHIAA M or lo-' M ) were dissolved in 2 5 0 ~ 10.5 M-phosphate buffer, pH 7.4 at 37°C. 50pl (= 1 mg tissue) homogenate of rat hypothalamus were added to the dihydroxyindole acetic acid-containing solutions. n = 4; mean i S.D. % radioactivity in Fig. 6 denotes percentage of radioactivity removed from the supernatant fraction, i.e. bound t o the pellet. 100% radioactivity = amount of radioactivity recovered in the supernatant immediately after addition of [ 14C]5,6-DH1AA or [ 14C]5,7-DH1AA to a pcrchloric acid treated homogenate ( = 0 min value in Fig. 5).
Interaction of 5,6- and 5.7-DHT with M A 0
117
TABLE3. EFFECTSOF
P R O T ~ C T I V EA ~ ~ E N TON S THE BINDING OF RADIOACTIVITY TO HYPOTHALAMIC PROTEIN PELLETS (% OF CONTROL) FOLLOWING 111 ~ i t r oINCUBATION WITH [I4C]5,6- OR [14C]5.7-DHT
Protective agent added to incubation
.I.
GSH DTT Cysteine L-ascorbic acid
[I4C]5.6-DHT
[' 4C]5,7-DHT
100 f 11.7 8.3 k 0.2
100 k 10.5 86.5 6.8 71.6 & 6.8 91.8 & 12.3 92.8 1.0
10.3
0.4
6.6 0.1 10.8 & 0.2
Retention of radioactivity in the protein pellet of hypothalamic hornogenatcs incubated for 20min at 37"C, pH 7.4 with 10-4~-[14C]5,6-DHTor [14C]5,7-DHT, with or without 4 mM-GSH, DTT. cysteine or L-ascorbic acid added to the incubation medium. The incubation conditions were the same as employed in the measurements of the inactivation of M A 0 irt vitro (cf. Material and Methods). The proteins were precipitated with 0 . 4 ~cold pcrchloric acid. The data represent means S.E.M. of 4 determinations and are expressed as :( radioactivity (d.p.m./pellet)recovered from the protein pellet following incubation of homogenates with radioactive 5,h- or 5,7-DHT (lo@% control in Table 3). Blank values were obtained by running incubations without tissue added.
sulphydryl reagents GSH, DTT and cysteine as well as ascorbic acid-previously found to counteract the inactivation of M A 0 in uitro (Table 2), antagonized the binding of [I4C]5,6-DHT-derived radioactivity to protein pellets. By contrast, there was little effect of these protective agents on the binding of radioactivity from [14C]5,7-DHT to protein pellets. DISCUSSION The results of the present radiometric study show that both 5.6- and 5,7-DHT are metabolized by M A 0 in homogenates of rat brain. The conversion of both drugs by M A 0 was inhibited by the M A 0 inhibitors clorgyline and deprenyl and obeyed, at least initially, Michaelis--Menten kinetics. 5,6-DHIAA and 5,7-DHIAA were identified as part of the deamination products in thin-layer chromatography. A comparison of the kinetic data of 5,6-DHT and 5,7-DHT with those of 5-HT obtained from Lineweaver-Burk diagrams permits the following conclusions as to the enzyme-substrate interaction: (1) The K , of $6- and 5,7-DHT is in the order of 1 0 - 4 ~and thus in a concentration range corresponding to the postulated concentration of extravesicular, cytoplasmic monoamines (EULER,I967 ; cited 1973). The affinity of M A 0 to 5,6-DHT from TIPTON, and 5,7-DHT is thus high enough to ensure metabolism of these false neurochemicals following accumulation in the neuron mediated by the membranebound amine uptake mechanism. (2) The introduction of a second hydroxyl function into the indole nucleus of the serotonin moiety, as in 5,6- and 5,7-DHT, diminishes the affinity of the substrate to the catalytic binding site. The K , of 5,6-DHT (1.3 x 10- M) is about 6 times that of 5-HT (0.2 x lo-' M); the K , of 5,7-DHT (0.5 x M) is about 2.5 times that of 5-HT. Since a hydroxyl funcN.C.
32 I
11
tion in p-position seems to be required for fixation of the substrate in the active center of M A 0 A (SEVERINA: 1973), the additional hydroxyl in the indole nucleus could interfere with the binding by steric hindrance or by clcctrostatic repulsion. This appears to be more important for o-dihydroxylated derivatives, such as 5,6-DHT. In the presence of thc specific M A 0 A type inhibitor, clorgylinc, M A 0 activity with 5,6-DHT and 5,7-DHT as substrates was depressed in a typical manner, indicating that both amines are metabolized by both types of the enzymes, M A 0 A and B, or that both amines are interacting with two active centres in a single enzyme molecule (for details, cf. TIPTONet a/.; 1976). On the other hand, the analysis of the deprenyl inhibition curves (an inhibitor which is known to preferentially inhibit M A 0 B) does not allow a clear distinction between the two enzyme activities, A or B, that interact with 5.6- or 5,7-DHT. In this case, the discussion of the I,, values of both amines leads to the conclusion that 5,6-DHT and 5.7-DHT are mainly metabolized by M A 0 A rather than M A 0 B. However, it has been found that deprenyl is not entirely specific in cases where clorgyline has proved to be a selective inhibitor (SQUIRES, 1972). Therefore. clorgyline seems to be a more reliable standard inhibitor in a binary M A 0 classification than dcprenyl (FOWLERer al., 1977). UV-absorption spectrographical analysis and oxygen consumption measurements rcveal a quick, time-dependent break-down of 5.6-DHT at biological pH and 37°C undcr aerobic conditions (SCHLOSSBERGER,1978 and KLEMM& BAUMGARTEN, unpublished observations), finally resulting in the formation of an insoluble black product. This resembles the air oxidation of 5,6-dihydroxyindole to a melanin species via the quinone intermediate aminochrome (CROMARTIE & HARLEY-MASON, 1957; review by SWAN.
118
H.-P. KLLMM,H. G . BAUMGARTEN and H. G. SCHLOSSBERGFR
FIG. 6. Proposed oxidation products of 5,6-DHT and their reaction with SH-groups in proteins or peptides (R-SH). a = 5,6-DHT; b = o-indolequinone of 5,6-DHT; c = addition product of 'b' and R-SH. The oxidation of (c) is supposed to result in addition of further molecules of 5,6-DHT '(a) thus forming melanin-like high molecular weight products and/or addition of another peptide/protcinlinked indoleamine species (c). The latter reaction would lead to crosslinkage of peptides or proteins as proposed by ROTMANet nl. (1976).
1974). The aminochrome intermediates or related quinones such as 5,6-indolequinone formed from 5,6-DHT are highly reactive structures which rapidly undergo addition reactions especially with nuclcophiles in proteins or peptides (ix. SH- or NH2groups) as demonstrated by ROTMANet al. (1976) in model proteins. A similar mechanism may be responsible for (a) the binding of radioactivity from ['4C]5:6-DHT to perchloric acid insoluble protein pellets and (b) for the inactivation of M A 0 by 5,6-DHT which closely resembles the inactivation of catechol-0-methyltransferase by 5,6-dihydroxyindole (BORCHARDT, 1975). Both, the inactivation of M A 0 by 5,6-DHT and the binding of radioactivity from [l4C)5,6-DHT to protein pellets wcrc counteracted by ascorbic acid, GSH, DTT and cysteine suggesting that the binding of radioactivity may correlate with and possibly reflect the degree of the inactivation of M A 0 by a reactive intermediate of 5,6-DHT, such as its corresponding o-indolequinone (cf. Fig. 6). The mechanism by which e.g. GSH antagonized the protein binding of radioactivity in incubations of homogenates with ['4C]5,6DHT was probably not ,simply to prevent its oxidation by maintaining a reductive milieu since a time-dependent loss of GSH occurred irz uitro duiing incubation with 5,6-DHT which was not followed by a parallel increase in the concentration of GSSG
(Baumgarten, Gercken, Junghanns. unpublished observations). This implies that part of the quinone formed from 5,6-DHT might have undergone covalent binding to glutathione (cf. Fig. 6). Our concept of the formation of a thioether compound of 56indolequinone and GSH receives support from recent studies of L ~ A Net G al. (1977) who isolated and identified 2-(2-S-glutathionyl-3,4,6-trihydroxyphenyl)ethylamine, formed from 6-OH-DA with GSH in uiao in rat brain, a precursor of the intracyclization product 5,6-dihydroxy-4-S-glutathionylindole (cf. Fig. 6). [14C]5,6-DHIAA, the ultimate metabolite of the deamination pathway of S,6-DHT, exhibited similar patterns of protein binding in uitro as [I4C]5,6-DHT indicating that the side chain is not significantly involved in mcdiating the observed binding process. There was no effect, whatsoever, of catalase or superoxide dismutase on the inactivation of M A 0 by 5,6-DHT, an observation which parallels findings by BORCHARDT (1975) using 6-OH-DA and 5,6-dihydroxyindole as substrates for catechol-0-methyltransfcrase. This casts doubt on the potential role of H,Oz. oxygen or hydroxyl radical in the mechanism of M A 0 inactivation by 5,6-DHT. 5,7-DHT, in contrast to 5,6-DHT, did not inactivate M A 0 in uitro and did not bind to a substantial degree to homogenatc pellets irr vitro, though it consumed oxygen accompanied by time-dependent
1 -CH2-NHZ
FIG. 7. Keto-enol tautomeric forms of 5,7-DHT (a-d) and proposed oxidation product of 5,7-DHT (e) and its reaction with SH-groups in proteins or peptides (R-SH) (0. e = p-quinoneimine of' 5,7-DHT.
Interaction of 5,6- and 5.7-DHT with M A 0
1978; changes in UV-absorption (SCHLOSSIIEKGER, KLEMM& BALMGARTENunpublished observations). However, t h e time-dependent air oxidation of S.7-DHT d i d n o t result in the formation of insolublc, coloured precipitates as in case of 5,6-DHT. Furthermore, in buffer solutions a t physiological pH, there is an equilibrium between different keto-cnol tautomeric forms of 5.7-DHT (SCHLOSSHEKGER, 1978; cf. Fig. 7). T h e degree of binding of ['4C]5,7-DHT-derived radioactivity t o proteins in uitro was low when compared to that found after Ci4C]5,6-DHT; may b e mediated by nuclcophilic reactions of the tautomeric j-diketoanion of 5.7-DHT (Fig. 7), by reaction of nuclcophiles in proteins with t h e postulated oxid a t i o n product of 5,7-DHT, i.e. its p-quinoneimine (Fig. 7), by covalent binding of t h e primary metabolite of the deamination pathway of S,7-DHT (its corresponding aldehyde), or by other as yet u n k n o w n mechanism. Finally. it may be asked whether t h e present in uitro findings have s o m e relevance t o the iri uiuo fate of 5,6- and 5,7-DHT. While t h e binding of radioactivity t o protein pellets of rat brain derived from [I4C]5,6-DHT is similar in vitro a n d in viuo (BAUMGAKTEN et a/., 1978) there is a discrepancy between t h e negligible extent of protein binding (from ['4C]5.7-DHT) traced in vitro when c o m p a r e d t o t h e extensive, time-dependent increase in covalcntly bound radioactivity seen in uiuo. This discrepancy suggcsts that there exists a toxicity-reinforcing catalytic mechanism in aivo which renders S,7-DHT o r o n e of its metabolites and/or oxidation products more reactive which d o e s n o t operatc in vitro. 11 is as yet u n k n o w n whether inactivation of brain M A 0 by S,6-DHT occurs also in vivo a n d whether it has a n y dclcterious conscqucnccs for the neurons affected.
119
CREVEL~N C.CR., LUNDSTKOM J., MCNEALE. T., TICFL. & DALYJ. W. (1975) Dihydroxytryptamines: effects on noradrenergic function in mouse heart in uiuo. Molec. Pkarinac. 11, 21 1-227. DALYJ . W., LUNDSTKOM J. & CREVELING C. R. (1974) Structure-activity correlations in trihydroxyphenylethylamines and dihydroxytryptamines. Relationship to cytotoxicity in adrenergic and serotonergic neurons, in DynaK., mics of Degeneration atid Grow~thin Neurons (FUXE OLSON L. & ZOTTERMAN Y.. eds.), pp. 29-42. Pergamon Press. Oxford. K. F. (1973) EULEKU . S. VON (1967) cited from TIPTOW Biochemical aspects of monoamine oxidase. Br. ined. E L / / / 29. . 116-1 19. FOWLER C. J., CALLINGHAM B. A., MANTLE T. J. & TIPTON K. F. (1978) Monoamine oxidase A and B: A useful concept? Biuchem. Pharinac. 27, 97-101. HEIKKILA R. & COHENG. (1971) Inhibition of hiogenic amine uptake by hydrogen peroxide: A mechanism for toxic effects of 6-hydroxydopamine. Science 72, 1257-1 258. JOHNSTOW J. P. (1968) Some observations upon a new inhibitor of monoamine oxidase in brain tissue. BiOChEL WURTMANR. J. & AXELROD J . (1963) A sensitive and CHKISTMAS D. (1972) A cornparison of the pharmacological and biospecific assay for the estimation of monoamine oxidase. chemical properties of substrate-sclcctive monoaminc BiocAein. Pliarinac. 12, 1439-1 440. oxidasc inhibitors. Br. J . Pharniuc. 45, 49&503. YANGH. Y. T. &. NEFFN. H. (1973) P-Phenylethylamine: CROMARTIE R. I. T. & HARLEY-MASON J . (1957) Melanin a specific substrate for type B monoamine oxidase of and its precursors. Biocheni. J . 66, 713-720. brain. J . Pharmac. exp. Ther. 187. 365-371.
