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Evidence for a specific monoamine oxidase associated with sympathetic nerves
Neuropharmacology,
1971,10,557-564
Pergamon
Press.
Printed
in Gt. Britain.
EVIDENCE FOR A SPECIFIC MONOAMINE OXIDASE ASSOCIATED WITH SYMPATHETIC NERVES* Laboratory
C. GORID@ and N. H. NEFF of Preclinical Pharmacology, National Institute of Mental Health, St. Elizabeth’s Hospital, Washington, D.C. 20032 (Accepted 30 March 1971)
Summary-Rat brain has been found to contain two forms of monoamine oxidase: an enzyme highly sensitive to the inhibitor clorgyline that acted on tyramine and serotonin (Type A); and an enzyme that was relatively insensitive to clorgyline and acted on tyramine but not serotonin (Type B). The superior cervical ganglion was found to contain about 90% Type A and 10% Type B enzyme. In contrast, the rat pineal gland contained 8.5% Type B and 15 % Type A enzyme. After bilateral superior cervical ganglionectomy, Type A enzyme was barely detectable in the pineal gland. It is concluded that sympathetic nerves contain, almost exclusively, Type A monoamine oxidase.
J~WNSTON (1968) reported that a plot of per cent inhibition of rat brain monoamine oxidase (MAO) versus the logarithm of ciorgyline [M+B 9302, N-methyl-N-propar~l-3-(2,4 dichlorophenoxy) propylamine hydrochloride] concentration did not show a normal sigmoid curve, but a pair of sigmoid curves joined by a horizontal region or plateau where inhibition remained essentially constant. He concluded that rat brain MAO couId be described as a binary system of enzymes, each with a different sensitivity to the inhibitor clorgyline. Johnston designated the enzymes as A and B, where enzyme A is highly sensitive to clorgyline and enzyme B is relatively insensitive. Evidence was provided that only enzyme A acted on serotonin, whereas both enzymes acted on tyramine. Rat pineal gland is innervated solely by sympathetic nerves that originate in the superior cervical ganglion (RAPPER, 1960). Bilateral superior cervical ganglionectomy results in a significant fall of pineal MAO activity when tryptamine is used as a substrate presumably as a result of the loss of MAO from sympathetic nerve endings or from structures within pineal dependent on the nerves (SNYDERet al., 1965). In the present experiments, the effect of increasing concentrations of clorgyline on MAO activity in the superior cervical ganglion, in the pineal gland of normal animals, and in the pineal gland after bilateral superior cervica1 ganglionectomy has been studied to determine whether specific enzyme types are associated with sympathetic nerves. METHODS
Normal male Sprague-Dawley rats and rats with bilateral superior cervical ganglionectomy (180-220 g) were obtained from Zivic Miller Labs. (Pittsburgh, Penna.) and kept in our animal quarters for 2 weeks before experimentation. Pineal glands were pooled *Presented in part at the National Meeting of the American Society for Neurochemistry, 15-19 March 1971, Hershey, Penna. tThe work described in this paper has been submitted by C. G. in partial ful~llment of the requirements for the degree D.Sc., University of Strasbourg, France, 557
558
C. GORIDIS and N.
H. NEFF
and homogenized in 0.25 M sucrose (20 pl/pineal) in a 1 ml homogenizer fitted with a Teflon pestle. The homogenate was diluted 1 : 50 assuming a weight of 1 mg per pineal and the sample was centrifuged at 7 x lo2 g for 10 min. The sediment was discarded and the supernatant was subjected to 1.2~ IO4 g for 20 min to prepare a mitochondrial fraction. The mitochondria was washed once with 0.25 M sucrose. The pellet was suspended in phosphate buffer (25 pl/pineal) 0,067 M, pH 7.2. Mitochondria were prepared from cerebral hemispheres in a similar manner. A 4-10 superior cervical ganglia was homogenized in 0.25 M sucrose (75 pi/ganglion) in a 1 ml homogenizer fitted with a Teflon pestle and proceeded as described above. Greater enzyme activity was found following homogenization in a glass-Teflon homogenizer than in an all-glass homogenizer. Rat brain mitochondrial MAO was solubilized by a method similar to that reported by YOUDIM et al. (1969). Benzylamine was omitted from the I.5 % Triton x 100 buffer solution and the enzyme activity in the crude soluble mitochondrial fraction was determined after centrifugation at lo5 g for 1 hr. MAO activity, using tyramine as substrate, was determined as described previously (GORIDIS and NEFF, 1971). Increasing concentrations of clorgyline were preincubated with either a 7 x IO2 g supernatant or with a mitochondrial preparation at 22°C for 15 min before tyramine-l-Cl4 (New England Nuclear Corp., Boston, Mass., 5 mCi/mmol) in a final concentration of 2.1 mM (about 6 x IO6 dpm) was added. The preparations were then incubated for 30 min at 37°C and the reaction was stopped by adding 50 ~1 of 60 % perchloric acid. After centrifuging the samples to remove protein, the supernatant and a 200 ~1 water wash were placed on a Rexyn 101 column (Fisher Scientific Co., Springfield, N.J.) H+ form, 200-400 mesh, 2.5 x 0.5 cm inner diameter. The effluent was discarded and the tyramine metabolites were eluted with 5 ml of water into glass scintillation counting vials containing 15 ml of Aquasol (New England Nuclear Co.). Radioactivity was determined in a Beckman LS 250 liquid scintillation system with automatic quench correction. The preincubation mixture, when serotonin was used as substrate, consisted of: 2 ~1 of mitochondrial preparation (0.02-0.04 mg protein); nicotinamide, 5 pmol; NAD+, 0.12 pmol; aldehyde dehydrogenase preparation (WEISSBACHet al., 1957) lo-20 ~1 containing 5-8 units of enzyme activity; and appropriate concentrations of clorgyline in a total volume of 130 ~1. The reagents were prepared in phosphate buffer 0.067 M, pH 7.2. After 15 min of preincubation at 22°C 30 ~1 of serotonin-2-14C (New England Nuclear, 15 mCi/ mmol, about 6 x lo5 dpm) were added resulting in a final concentration of I.2 mM. The mixture was then incubated for 20 min at 37°C. The reaction was stopped by adding 25 ~1 each of 0.25 M zinc sulfate and 0.20 M barium hydroxide. The precipitate was removed by centrifugation and the supernatant and a 200 ~1 water wash were transferred to a Rexyn 101 column, H+ form, 20&400 mesh, 1.5 x0.5 cm inner diameter. The effluent was discarded and 6 ml of water were passed through the column. The water effluent was mixed with Aquasol counting mixture and the radioactivity was determined. Boiled homogenate and reagents gave identical blank values. The recovery of the reaction end product, 5-hydroxyindole-acetic acid, was determined by adding known quantities of 5-hydroxyindoleacetic-l-14C acid (New England Nuclear Co., 5 mCi/mmol, about 5 x lo4 dpm) to a sample that was processed in the usual manner. The recovery of the acid averaged 60 %. This assay was linear with protein and time of incubation. Enzyme activity was not linear with protein in the absence of aldehyde dehydrogenase, probably because 5-hydroxyindolyl-3-acetaldehyde binds to protein (ALIVISATOS et al., 1966). Addition of aldehyde dehydrogenase and cofactors to the incubation mixture, when tyramine was the substrate, had no effect on
Sympatheticnerve monoamineoxidase
559
enzyme activity or on the inhibitory action of clorgyline. The concentrations of tyramine and serotonin used gave maximal activities in the present test system. Aldehyde dehydrogenase was prepared from guinea pig kidney as described by WEISSBACH et al. (1957). The supernatant fraction following acid precipitation was dialyzed for 12 hr against 0.01 M potassium phosphate buffer, pH 7.4, containing O-6 mg disodium edetate per ml. We found no detectable MAO after dialysis. Aldehyde dehydrogenase was assayed using acetaldehyde as substrate, by following the appearance of NADH as described by RACKER(1955). One enzyme unit was equivalent to AE’,,,=O.OOl at 22°C. The preparation gradually lost activity and therefore the volume added to the MAO test system varied from 10 to 20 ~1. RESULTS The effect of increasing concentrations of clorgyline on rat brain MAO activity when tyramine or serotonin was used as substrate are given in Fig. 1. A sigmoid curve was observed when serotonin was used as substrate. When tyramine was substrate, a pair of sigmoid
CLORGYLINE (-log
FIG. 1. Inhibition of rat brain monoamine Figs., values are presented as mean and homogenates. At some concentrations of single homogenate;
cone
Ml
oxidase by clorglyine. In this Fig. and subsequent range for double determinations from 3 or more clorgyline, a double determination was made on a therefore, no range is shown.
curves were observed. These were joined by a horizontal region or plateau where a lo3 concentration change of clorgyline had no effect on MAO activity. Significantly higher concentrations of clorgyline were required to block the oxidative deamination of tyramine completely than those required for serotonin. The plateau observed with the substrate tyramine occurred at about 55 % inhibition of enzyme activity. Using the terminology of JOHNSTON(1968), about 55 % of the total MAO activity could be attributed to enzyme A. A pair of sigmoid curves were also observed when the experiment was repeated with a solubilized MAO preparation from rat brain mitochondria (Fig. 2). The plateau again occurred at about 55 % inhibition of enzyme activity. In contrast to brain, a plateau occurred with pineal gland MAO at about 15 % of the total MAO activity when increasing concentrations of clorgyline were incubated together with tyramine, e.g. about 15 % of the total activity was attributable to enzyme A (Fig. 3).
C. GORIDISand N. I-I. NEFF
CLORGYLlNE
(-log cmc Ml
FXG. 2. Inhibition of solubilized rat brain mitochondrial monoamine axidase by ciorgyline. Values are presented as the mean far a double determination on a single enzyme preparation.
CLORFYLINE Gq FIG. 3.
fnhibition
cone M:f
of rat pineal gland monoamine
oxidase by clorgyiine.
The ~~~~e~~rations of clorgyfine required to inhibit the oxidation of serotonin by the pineal gland preparation were similar to those required for brain. After bilateral superior cervical ganglionectomy (Fig. 4), the inhibition of tyramine metabolism by clorgyline followed a simple sigmoid curve in the pineal gland. Denervation appeared to have almost completely eliminated enzyme A from the pineal. Serotonin was still deaminated by this preparation, but the total activity was reduced by 70% (Table 1). There was no significant change in the metabolism of tyramine (Table 1). The superior cervical ganglion showed a plateau at about 90% inhibition of MAO activity with the substrate tyramine in the presence of cforgyline (Fig. 5). In contrast to the restits for the pineal gland and brain, enzyme A activity adopted for about 90% of the t~~~ne-~~e~boli~ing activity. As in the brain (Fig. 1) and pineal gland (Fig. 3)>serotonin
Sympathetic nerve monoamine
CLORGYLINE FIG.
4, Inhibition
TABLE
1,
561
oxidase
(-log cone M)
of rat pineal gland monoamine oxidase by clorgyhne 14 days after bilateral superior cervical ganglionectomy.
MONOAMINE
OXIDhSE
ACTMTY
IN PINEAL
GLAND
AFTER
SUPERIOR
CERVICAL
GANGLIONECTGMY*
Substrate
nrno~~~in~l/~~~.~.M. (N) Denervated
Innervated
Activity Ioss
Tyramine Serotonin *Pineal mitochondria were isolated as described in methods 2.1 mm01 tyramine or 1.2 mm01 serotonin. tP> 0.05 when compared with innervated preparation. $P< 0.01 when compared with innervated preparation.
and incubated
FIG. 5. Inhibition
oxidase by clorgyhne using
of rat superior cervical ganglion monoamine tyramine as substrate,
with either
562
C. GORIDIS andN.H.
NEFF
was deaminated exclusively in our test system by enzyme A (Fig. 6) which appears to be the primary enzyme in the ganglion. The steady-state MAO activity in the 3 tissues studied, with tyramine and serotonin as substrate, and the ratios of tyramine to serotonin metabolism among the tissues are shown in Table 2. The ability of enzyme A to metabolize tyramine and serotonin in the same tissue was calculated from the relative proportions of the enzyme in the tissue (Figs. 1, 3 and 5) and the steady-state MAO activity toward tyramine (Table 2). Ratios of the activities are shown in Table 3. lOOr ? % I 2
:
25 80Y E 2
I
_
/
60-
z
r;
-
k $ I= B
40-
z
20-
./I ,/
8
0
/.
1
!2
II
/ /
/
9 I
I
I
I
I
IO
9
8
7
6
CLORGYLINE
FIG. 6. Inhibition
/
(-LOG
CONC
M)
of rat superior cervical ganglion monoamine serotonin as substrate.
oxidase by clorgyline using
TABLET. COMPARISONOFMITOCHONDRIALMONOAMINEOXIDASEAC~YINTISSUESUSING TYRAMINEORSEROTONINASSUBSTRATE*
Serotonin
Tyramine Tissue
-nmol/mg
Superior cervical ganglion? Pineal gland Cerebral hemispheres
8.5kO.5 9.440.5 11 f0.9
Ratio of activity serotonin/tyramine
tissue/hr & S.E.M. (N) (4) (4) (4)
13 zt3 3.4f0.8 6.7&l
1.5 0.36 0.61
(4) (7) (6)
*Mitochondria were isolated from tissues and incubated with either 2.1 mm01 tyrarnine or 1.2 mm01 serotonin as described in Methods. tActivity expressed as nmol/ganglion/hrfS.E.M. (N). TABLET. COMPARISONOFTYRAMINEANDSEROTONINMETABOLISMBYENZYMEAINTISSUE
Tyramine: enzyme A
Serotonin §
Ratio of activity -Serotonin enzyme A Tyramine
11 9.4
6.1 1.4
6.7 3.4
1.1 2.4
8.5
7.7
nmol/mg tissue/hr Tissue
A: B*
Cerebral hemisphere Pineal gland Superior cervical ganglion
55.45 15.85 90.10
*By extrapolation from Figs. 1, 3 and 5. $By calculation from preceding columns.
Tyraminet enzyme A+B
tFrom Table 2. IFrom Table 2.
13
1.7
Sympatneticnerve monoamineoxidase
563
DISCUSSION Clorgyline is one of the most potent MAO inhibitors available (JOHNSTON,1968). HALL et al. (1969) demonstrated the existence of what appeared to be a double enzyme system in the brain and liver of man and rat with this inhibitor. Some species examined showed only a simple sigmoid curve for the liver, but a double enzyme system in the brain. These species are cat, dog, rabbit and ox. The liver of these animals deaminated both tyramine and serotonin as with the A enzyme in the rat. Only a single enzyme system has been found in the brain and liver of the pig. These studies are compatible with the multiple enzyme forms of MAO isolated by polyacrylamide gel (YOUDIMet al., 1969; SIERENSand D’IORIO, 1970), cellulosepolyacetate (KIM and D’IORIO, 1968) and density gradient column electrophoresis techniques (GORKIN, 1969) from the brain and liver of various species. For discussion we have adopted Johnston’s functional definition for enzymes A and B, i.e. A acts on both serotonin and tyramine and is most sensitive to clorgyline, while B acts on tyramine but not on serotonin (JOHNSTON,1968). Based on substrate affinity and polyacrylamide gel studies, SIERENSand D’IORIO(1970) concluded that 2 distinct forms of MAO are present in the rat liver. These forms may be related to enzymes A and B. The original studies using clorgyline were performed with isolated mitochondria (JOHNSTON,1968). The location of MAO on mitochondria is unclear at present. MAO has been associated with outer (BEATTIE,1968; SCHNAITMAN and GREENAWALT,1968; MARCO et al., 1969) and inner membranes (GREEN et al., 1968), cristae (BYINGTONet al., 1968) and matrix (BRDICZKAet al., 1968). The inability of either the substrates or clorgyline to readily penetrate through mitochondrial membranes to the active site of the enzyme could have resulted in apparent enzyme types. However, as shown in Fig. 2, a double enzyme system was found with solubilized MAO too. Enzyme A is responsible for about half of the metabolism of tyramine in a rat brain homogenate (Fig. 1). The PI,, values using the inhibitor clorgyline and the substrate tyramine are about 8.5 and 5 for enzyme A and B, respectively. Similar values were reported by JOHNSTON(1968). In contrast to the brain, only about 15 % of the total tyramine metabolized by the pineal is associated with enzyme A (Fig. 3). The rat pineal gland is innervated solely by sympathetic nerves that originate in the superior cervical ganglion (KAPPERS,1960). After ganglionectomy, enzyme A was no longer detectable in the pineal (Fig. 4) and there was a 70% reduction of serotonin metabolism by the pineal gland (Table 1). No significant change of tyramine metabolism was observed suggesting that there was little change of enzyme B in the pineal. Apparently, enzyme B is almost exclusively associated with pineal gland cells whereas enzyme A is found within sympathetic nerve endings or in structures dependent upon the nerves. MAO of the superior cervical ganglia is associated with sympathetic nerve cell bodies and not with preganglionic cholinergic nerves (CONSOLOet al., 1968; GIACOBINIet al., 1970). As shown in Figs. 5 and 6, enzyme A, the form of MAO most sensitive to clorgyline, is responsible for about 90 % of the metabolism of tyramine by the ganglia. This observation is consistent with the denervation experiments and suggests that sympathetic nerves contain a specific MAO, enzyme A. Enzymes A and B may be analogous to specific and nonspecific cholinesterases. The marked differences of the ratio of tyramine to serotonin metabolized by the three tissues studied supports the existence of several MAO forms (Table 2). The ratio of the ability of enzyme A to metabolize tyramine and serotonin should be similar in tissues if enzyme A is a single entity. Apparently, enzyme A is not a single entity as the ratio of activities varied among the tissues (Table 3). Our observations are consistent with reports
C. GOR~XSand N. H. NEFF
564
that more than 2 isoenzymes can be identified in rat tissue ~YOUDIM et al., 1969; SIERENS and D’IORKI, 1970; Kr?/r and I)%RIo, 1968 ; GORKIN, 1969) and they suggest that enzymes A and B represent cIasses of enzymes rather than single enzymes. The superior cervical ganglion and denervated pineai gland of the rat might be valuable tissues for isolating and characterizing these enzymes. The present experiments suggest that new MAO inhibitors might be developed which could selectively inactivate sympathetic nerve MAO, Type A enzymes. Such drugs might be valuable for treating hypertension as they should not cause the adverse circulatory effects associated with the ingestion of foods containing tyramine. Furthermore, these drugs might be useful for treating depression as Type A enzyme appears to inactivate biogenic amines in brain. Aek~o~le~gerneni-We
gratefully acknowledge the expert technicai assistance of Mr. J. ~UEE~STEINand the generous supply of clorgyline provided by Dr. GMMSTERof May and Baker, Ltd.
RE.FERENCES AL~XX~ATOS, S, G. A., UNGAR, F. and PARMAR,S. S. (1966). Effect of monoamine oxidase inhibitors on the labeling of subcellular fractions of brain and liver by “C serotonin. Biochem. Bio&vs. Res. Commun. 25: 495-500. BEATTIE,D. S. (1968). Enzyme localization in the inner and outer membranes of rat liver mitochondtia. Biochem. Biophys. Res. Commun. 31: 901-907. BRDICZKA,D., PETTE,D., BRUNNER,G. and MILLER,I?. (1968). Kompartimentierte Verteilung von Enzymen in Ratte~e~~itocbo~dr~en. Eur. J. B~u~kern* 5: 294-304. Bn~~-ror+, K. H., SMOLY,3. M., MOSEY, A. V. and GREEN,D. E. (IP@). On the fragmentation of mitochondria by diethylst~~bestero~. I. Conditions for maximizing fragmentation. Arch Biochem. Biq&~s.
128:762-773. CONSOLO,S., GLACOBINI,E. and KARJALA~NEN, K. (1968). Monoamine oxidase in syn~pat~letic ganglia of the cat. Acta pkysiof. scand. 74 : 513-520. CIACOBINI,I?,, KARJALAMEN,K., KERPEL-FR~NIUS,S. and RITZEX, M. (1970). Monoamines and monoamine oxidase in denervated sympathetic ganglia of the cat. ~euruph~rma~~~ugy 9: 59-66. GORXLXS,C. and NEFF, N. H. (I97 I). Monoamine oxidase: an approximation of turnover rates. J. ~e~ruchef?~. In press. GORKIN, V. 2. (1969). Separation of rat liver mitochondrial amine oxidase. Experientia 25: 1142. GREEN, D. E., ALLMANN,D. W., HARRIS, R. A. and TAN, W. C. (1968). Enzyme localization in the inner and outer mitochondrial membranes. Biochem. Biophys. Res. Commun. 31: 368-378. HALL, D. W. R., LOGAN,B. W. and PARSONS,G. H. (1969). Further studies on the inhibition of monoamine oxidase by M&B 9302 (clorgyline). I. Substrate specificity in various mammalian species. Biochem. &armac. 18: 1447-1454. JOHNSTON, J. P. f1968). Some observations upon a new inhibitor of monoamine oxidase in brain tissue, Biochem. Rharmac. 17: 1285-1297.
KAPPER, .I. A. (1960). The develnpm~t, topo~aphicai retations and i~ervation of the epiphysis cerebri in the albino rat. Z. ~e~lfo~~ehrnikro~k. Aprat. 52: 163-215. KIM, II. C. and D’IORIO, A. (1968). Possible isoenzymes of monoamine oxidase in rat tissue, Can. J. ~~~che~~. 46: 295-297.
MARCO, R., SEBASTIAN, J. and SC&S,A. (1969). Location of the enzymes of the oxalacetate metabolic cross roads in rat Iiver mitochondria. Biockent. Biopl~~s. Res. Commrm. 34: 725-730. RACKER, E, (1955). Liver aldehyde dehydrogenase. In: Methods in Enzq’mology (COLOWICK,S. P. and KAPLAN, N. O., Eds.), Vol. 1, pp. 514-517. Academic Press, New York. SCHNAITMAN,C. and GREENAWALT,J. W. (1968). Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J. Cell Biol. 38: 158-17.5. SIER~NS,L. and D’IORIO,A. (1970). Multiple monoamine oxidases in rat liver mitochondria. Can. J. Biochenr. 48: 659-663, SNYDER,S. H., FISCHER,J. and AXELROR,J. (1965). Evidence for the presence of monoamine oxidase in sympathetic nerve endings. Biockem. Pkurmnc. 14: 363-365. WEISSBACX,H,, REDF~~LD,B. G. and UDENFRXEND, S. (1957). SofubIe monoamine oxidase: Its properties and actions on serotonin. J. 6iol. Ckem. 229: 953-963. YOUDIM,M. B. H., COLL~S, G. G. S. and SANDLER,M. (1969). ~ILlitipIe forms of rat brain mono~ine oxidase, N~~~~re,Lo&. 223: 626628.
1971,10,557-564
Pergamon
Press.
Printed
in Gt. Britain.
EVIDENCE FOR A SPECIFIC MONOAMINE OXIDASE ASSOCIATED WITH SYMPATHETIC NERVES* Laboratory
C. GORID@ and N. H. NEFF of Preclinical Pharmacology, National Institute of Mental Health, St. Elizabeth’s Hospital, Washington, D.C. 20032 (Accepted 30 March 1971)
Summary-Rat brain has been found to contain two forms of monoamine oxidase: an enzyme highly sensitive to the inhibitor clorgyline that acted on tyramine and serotonin (Type A); and an enzyme that was relatively insensitive to clorgyline and acted on tyramine but not serotonin (Type B). The superior cervical ganglion was found to contain about 90% Type A and 10% Type B enzyme. In contrast, the rat pineal gland contained 8.5% Type B and 15 % Type A enzyme. After bilateral superior cervical ganglionectomy, Type A enzyme was barely detectable in the pineal gland. It is concluded that sympathetic nerves contain, almost exclusively, Type A monoamine oxidase.
J~WNSTON (1968) reported that a plot of per cent inhibition of rat brain monoamine oxidase (MAO) versus the logarithm of ciorgyline [M+B 9302, N-methyl-N-propar~l-3-(2,4 dichlorophenoxy) propylamine hydrochloride] concentration did not show a normal sigmoid curve, but a pair of sigmoid curves joined by a horizontal region or plateau where inhibition remained essentially constant. He concluded that rat brain MAO couId be described as a binary system of enzymes, each with a different sensitivity to the inhibitor clorgyline. Johnston designated the enzymes as A and B, where enzyme A is highly sensitive to clorgyline and enzyme B is relatively insensitive. Evidence was provided that only enzyme A acted on serotonin, whereas both enzymes acted on tyramine. Rat pineal gland is innervated solely by sympathetic nerves that originate in the superior cervical ganglion (RAPPER, 1960). Bilateral superior cervical ganglionectomy results in a significant fall of pineal MAO activity when tryptamine is used as a substrate presumably as a result of the loss of MAO from sympathetic nerve endings or from structures within pineal dependent on the nerves (SNYDERet al., 1965). In the present experiments, the effect of increasing concentrations of clorgyline on MAO activity in the superior cervical ganglion, in the pineal gland of normal animals, and in the pineal gland after bilateral superior cervica1 ganglionectomy has been studied to determine whether specific enzyme types are associated with sympathetic nerves. METHODS
Normal male Sprague-Dawley rats and rats with bilateral superior cervical ganglionectomy (180-220 g) were obtained from Zivic Miller Labs. (Pittsburgh, Penna.) and kept in our animal quarters for 2 weeks before experimentation. Pineal glands were pooled *Presented in part at the National Meeting of the American Society for Neurochemistry, 15-19 March 1971, Hershey, Penna. tThe work described in this paper has been submitted by C. G. in partial ful~llment of the requirements for the degree D.Sc., University of Strasbourg, France, 557
558
C. GORIDIS and N.
H. NEFF
and homogenized in 0.25 M sucrose (20 pl/pineal) in a 1 ml homogenizer fitted with a Teflon pestle. The homogenate was diluted 1 : 50 assuming a weight of 1 mg per pineal and the sample was centrifuged at 7 x lo2 g for 10 min. The sediment was discarded and the supernatant was subjected to 1.2~ IO4 g for 20 min to prepare a mitochondrial fraction. The mitochondria was washed once with 0.25 M sucrose. The pellet was suspended in phosphate buffer (25 pl/pineal) 0,067 M, pH 7.2. Mitochondria were prepared from cerebral hemispheres in a similar manner. A 4-10 superior cervical ganglia was homogenized in 0.25 M sucrose (75 pi/ganglion) in a 1 ml homogenizer fitted with a Teflon pestle and proceeded as described above. Greater enzyme activity was found following homogenization in a glass-Teflon homogenizer than in an all-glass homogenizer. Rat brain mitochondrial MAO was solubilized by a method similar to that reported by YOUDIM et al. (1969). Benzylamine was omitted from the I.5 % Triton x 100 buffer solution and the enzyme activity in the crude soluble mitochondrial fraction was determined after centrifugation at lo5 g for 1 hr. MAO activity, using tyramine as substrate, was determined as described previously (GORIDIS and NEFF, 1971). Increasing concentrations of clorgyline were preincubated with either a 7 x IO2 g supernatant or with a mitochondrial preparation at 22°C for 15 min before tyramine-l-Cl4 (New England Nuclear Corp., Boston, Mass., 5 mCi/mmol) in a final concentration of 2.1 mM (about 6 x IO6 dpm) was added. The preparations were then incubated for 30 min at 37°C and the reaction was stopped by adding 50 ~1 of 60 % perchloric acid. After centrifuging the samples to remove protein, the supernatant and a 200 ~1 water wash were placed on a Rexyn 101 column (Fisher Scientific Co., Springfield, N.J.) H+ form, 200-400 mesh, 2.5 x 0.5 cm inner diameter. The effluent was discarded and the tyramine metabolites were eluted with 5 ml of water into glass scintillation counting vials containing 15 ml of Aquasol (New England Nuclear Co.). Radioactivity was determined in a Beckman LS 250 liquid scintillation system with automatic quench correction. The preincubation mixture, when serotonin was used as substrate, consisted of: 2 ~1 of mitochondrial preparation (0.02-0.04 mg protein); nicotinamide, 5 pmol; NAD+, 0.12 pmol; aldehyde dehydrogenase preparation (WEISSBACHet al., 1957) lo-20 ~1 containing 5-8 units of enzyme activity; and appropriate concentrations of clorgyline in a total volume of 130 ~1. The reagents were prepared in phosphate buffer 0.067 M, pH 7.2. After 15 min of preincubation at 22°C 30 ~1 of serotonin-2-14C (New England Nuclear, 15 mCi/ mmol, about 6 x lo5 dpm) were added resulting in a final concentration of I.2 mM. The mixture was then incubated for 20 min at 37°C. The reaction was stopped by adding 25 ~1 each of 0.25 M zinc sulfate and 0.20 M barium hydroxide. The precipitate was removed by centrifugation and the supernatant and a 200 ~1 water wash were transferred to a Rexyn 101 column, H+ form, 20&400 mesh, 1.5 x0.5 cm inner diameter. The effluent was discarded and 6 ml of water were passed through the column. The water effluent was mixed with Aquasol counting mixture and the radioactivity was determined. Boiled homogenate and reagents gave identical blank values. The recovery of the reaction end product, 5-hydroxyindole-acetic acid, was determined by adding known quantities of 5-hydroxyindoleacetic-l-14C acid (New England Nuclear Co., 5 mCi/mmol, about 5 x lo4 dpm) to a sample that was processed in the usual manner. The recovery of the acid averaged 60 %. This assay was linear with protein and time of incubation. Enzyme activity was not linear with protein in the absence of aldehyde dehydrogenase, probably because 5-hydroxyindolyl-3-acetaldehyde binds to protein (ALIVISATOS et al., 1966). Addition of aldehyde dehydrogenase and cofactors to the incubation mixture, when tyramine was the substrate, had no effect on
Sympatheticnerve monoamineoxidase
559
enzyme activity or on the inhibitory action of clorgyline. The concentrations of tyramine and serotonin used gave maximal activities in the present test system. Aldehyde dehydrogenase was prepared from guinea pig kidney as described by WEISSBACH et al. (1957). The supernatant fraction following acid precipitation was dialyzed for 12 hr against 0.01 M potassium phosphate buffer, pH 7.4, containing O-6 mg disodium edetate per ml. We found no detectable MAO after dialysis. Aldehyde dehydrogenase was assayed using acetaldehyde as substrate, by following the appearance of NADH as described by RACKER(1955). One enzyme unit was equivalent to AE’,,,=O.OOl at 22°C. The preparation gradually lost activity and therefore the volume added to the MAO test system varied from 10 to 20 ~1. RESULTS The effect of increasing concentrations of clorgyline on rat brain MAO activity when tyramine or serotonin was used as substrate are given in Fig. 1. A sigmoid curve was observed when serotonin was used as substrate. When tyramine was substrate, a pair of sigmoid
CLORGYLINE (-log
FIG. 1. Inhibition of rat brain monoamine Figs., values are presented as mean and homogenates. At some concentrations of single homogenate;
cone
Ml
oxidase by clorglyine. In this Fig. and subsequent range for double determinations from 3 or more clorgyline, a double determination was made on a therefore, no range is shown.
curves were observed. These were joined by a horizontal region or plateau where a lo3 concentration change of clorgyline had no effect on MAO activity. Significantly higher concentrations of clorgyline were required to block the oxidative deamination of tyramine completely than those required for serotonin. The plateau observed with the substrate tyramine occurred at about 55 % inhibition of enzyme activity. Using the terminology of JOHNSTON(1968), about 55 % of the total MAO activity could be attributed to enzyme A. A pair of sigmoid curves were also observed when the experiment was repeated with a solubilized MAO preparation from rat brain mitochondria (Fig. 2). The plateau again occurred at about 55 % inhibition of enzyme activity. In contrast to brain, a plateau occurred with pineal gland MAO at about 15 % of the total MAO activity when increasing concentrations of clorgyline were incubated together with tyramine, e.g. about 15 % of the total activity was attributable to enzyme A (Fig. 3).
C. GORIDISand N. I-I. NEFF
CLORGYLlNE
(-log cmc Ml
FXG. 2. Inhibition of solubilized rat brain mitochondrial monoamine axidase by ciorgyline. Values are presented as the mean far a double determination on a single enzyme preparation.
CLORFYLINE Gq FIG. 3.
fnhibition
cone M:f
of rat pineal gland monoamine
oxidase by clorgyiine.
The ~~~~e~~rations of clorgyfine required to inhibit the oxidation of serotonin by the pineal gland preparation were similar to those required for brain. After bilateral superior cervical ganglionectomy (Fig. 4), the inhibition of tyramine metabolism by clorgyline followed a simple sigmoid curve in the pineal gland. Denervation appeared to have almost completely eliminated enzyme A from the pineal. Serotonin was still deaminated by this preparation, but the total activity was reduced by 70% (Table 1). There was no significant change in the metabolism of tyramine (Table 1). The superior cervical ganglion showed a plateau at about 90% inhibition of MAO activity with the substrate tyramine in the presence of cforgyline (Fig. 5). In contrast to the restits for the pineal gland and brain, enzyme A activity adopted for about 90% of the t~~~ne-~~e~boli~ing activity. As in the brain (Fig. 1) and pineal gland (Fig. 3)>serotonin
Sympathetic nerve monoamine
CLORGYLINE FIG.
4, Inhibition
TABLE
1,
561
oxidase
(-log cone M)
of rat pineal gland monoamine oxidase by clorgyhne 14 days after bilateral superior cervical ganglionectomy.
MONOAMINE
OXIDhSE
ACTMTY
IN PINEAL
GLAND
AFTER
SUPERIOR
CERVICAL
GANGLIONECTGMY*
Substrate
nrno~~~in~l/~~~.~.M. (N) Denervated
Innervated
Activity Ioss
Tyramine Serotonin *Pineal mitochondria were isolated as described in methods 2.1 mm01 tyramine or 1.2 mm01 serotonin. tP> 0.05 when compared with innervated preparation. $P< 0.01 when compared with innervated preparation.
and incubated
FIG. 5. Inhibition
oxidase by clorgyhne using
of rat superior cervical ganglion monoamine tyramine as substrate,
with either
562
C. GORIDIS andN.H.
NEFF
was deaminated exclusively in our test system by enzyme A (Fig. 6) which appears to be the primary enzyme in the ganglion. The steady-state MAO activity in the 3 tissues studied, with tyramine and serotonin as substrate, and the ratios of tyramine to serotonin metabolism among the tissues are shown in Table 2. The ability of enzyme A to metabolize tyramine and serotonin in the same tissue was calculated from the relative proportions of the enzyme in the tissue (Figs. 1, 3 and 5) and the steady-state MAO activity toward tyramine (Table 2). Ratios of the activities are shown in Table 3. lOOr ? % I 2
:
25 80Y E 2
I
_
/
60-
z
r;
-
k $ I= B
40-
z
20-
./I ,/
8
0
/.
1
!2
II
/ /
/
9 I
I
I
I
I
IO
9
8
7
6
CLORGYLINE
FIG. 6. Inhibition
/
(-LOG
CONC
M)
of rat superior cervical ganglion monoamine serotonin as substrate.
oxidase by clorgyline using
TABLET. COMPARISONOFMITOCHONDRIALMONOAMINEOXIDASEAC~YINTISSUESUSING TYRAMINEORSEROTONINASSUBSTRATE*
Serotonin
Tyramine Tissue
-nmol/mg
Superior cervical ganglion? Pineal gland Cerebral hemispheres
8.5kO.5 9.440.5 11 f0.9
Ratio of activity serotonin/tyramine
tissue/hr & S.E.M. (N) (4) (4) (4)
13 zt3 3.4f0.8 6.7&l
1.5 0.36 0.61
(4) (7) (6)
*Mitochondria were isolated from tissues and incubated with either 2.1 mm01 tyrarnine or 1.2 mm01 serotonin as described in Methods. tActivity expressed as nmol/ganglion/hrfS.E.M. (N). TABLET. COMPARISONOFTYRAMINEANDSEROTONINMETABOLISMBYENZYMEAINTISSUE
Tyramine: enzyme A
Serotonin §
Ratio of activity -Serotonin enzyme A Tyramine
11 9.4
6.1 1.4
6.7 3.4
1.1 2.4
8.5
7.7
nmol/mg tissue/hr Tissue
A: B*
Cerebral hemisphere Pineal gland Superior cervical ganglion
55.45 15.85 90.10
*By extrapolation from Figs. 1, 3 and 5. $By calculation from preceding columns.
Tyraminet enzyme A+B
tFrom Table 2. IFrom Table 2.
13
1.7
Sympatneticnerve monoamineoxidase
563
DISCUSSION Clorgyline is one of the most potent MAO inhibitors available (JOHNSTON,1968). HALL et al. (1969) demonstrated the existence of what appeared to be a double enzyme system in the brain and liver of man and rat with this inhibitor. Some species examined showed only a simple sigmoid curve for the liver, but a double enzyme system in the brain. These species are cat, dog, rabbit and ox. The liver of these animals deaminated both tyramine and serotonin as with the A enzyme in the rat. Only a single enzyme system has been found in the brain and liver of the pig. These studies are compatible with the multiple enzyme forms of MAO isolated by polyacrylamide gel (YOUDIMet al., 1969; SIERENSand D’IORIO, 1970), cellulosepolyacetate (KIM and D’IORIO, 1968) and density gradient column electrophoresis techniques (GORKIN, 1969) from the brain and liver of various species. For discussion we have adopted Johnston’s functional definition for enzymes A and B, i.e. A acts on both serotonin and tyramine and is most sensitive to clorgyline, while B acts on tyramine but not on serotonin (JOHNSTON,1968). Based on substrate affinity and polyacrylamide gel studies, SIERENSand D’IORIO(1970) concluded that 2 distinct forms of MAO are present in the rat liver. These forms may be related to enzymes A and B. The original studies using clorgyline were performed with isolated mitochondria (JOHNSTON,1968). The location of MAO on mitochondria is unclear at present. MAO has been associated with outer (BEATTIE,1968; SCHNAITMAN and GREENAWALT,1968; MARCO et al., 1969) and inner membranes (GREEN et al., 1968), cristae (BYINGTONet al., 1968) and matrix (BRDICZKAet al., 1968). The inability of either the substrates or clorgyline to readily penetrate through mitochondrial membranes to the active site of the enzyme could have resulted in apparent enzyme types. However, as shown in Fig. 2, a double enzyme system was found with solubilized MAO too. Enzyme A is responsible for about half of the metabolism of tyramine in a rat brain homogenate (Fig. 1). The PI,, values using the inhibitor clorgyline and the substrate tyramine are about 8.5 and 5 for enzyme A and B, respectively. Similar values were reported by JOHNSTON(1968). In contrast to the brain, only about 15 % of the total tyramine metabolized by the pineal is associated with enzyme A (Fig. 3). The rat pineal gland is innervated solely by sympathetic nerves that originate in the superior cervical ganglion (KAPPERS,1960). After ganglionectomy, enzyme A was no longer detectable in the pineal (Fig. 4) and there was a 70% reduction of serotonin metabolism by the pineal gland (Table 1). No significant change of tyramine metabolism was observed suggesting that there was little change of enzyme B in the pineal. Apparently, enzyme B is almost exclusively associated with pineal gland cells whereas enzyme A is found within sympathetic nerve endings or in structures dependent upon the nerves. MAO of the superior cervical ganglia is associated with sympathetic nerve cell bodies and not with preganglionic cholinergic nerves (CONSOLOet al., 1968; GIACOBINIet al., 1970). As shown in Figs. 5 and 6, enzyme A, the form of MAO most sensitive to clorgyline, is responsible for about 90 % of the metabolism of tyramine by the ganglia. This observation is consistent with the denervation experiments and suggests that sympathetic nerves contain a specific MAO, enzyme A. Enzymes A and B may be analogous to specific and nonspecific cholinesterases. The marked differences of the ratio of tyramine to serotonin metabolized by the three tissues studied supports the existence of several MAO forms (Table 2). The ratio of the ability of enzyme A to metabolize tyramine and serotonin should be similar in tissues if enzyme A is a single entity. Apparently, enzyme A is not a single entity as the ratio of activities varied among the tissues (Table 3). Our observations are consistent with reports
C. GOR~XSand N. H. NEFF
564
that more than 2 isoenzymes can be identified in rat tissue ~YOUDIM et al., 1969; SIERENS and D’IORKI, 1970; Kr?/r and I)%RIo, 1968 ; GORKIN, 1969) and they suggest that enzymes A and B represent cIasses of enzymes rather than single enzymes. The superior cervical ganglion and denervated pineai gland of the rat might be valuable tissues for isolating and characterizing these enzymes. The present experiments suggest that new MAO inhibitors might be developed which could selectively inactivate sympathetic nerve MAO, Type A enzymes. Such drugs might be valuable for treating hypertension as they should not cause the adverse circulatory effects associated with the ingestion of foods containing tyramine. Furthermore, these drugs might be useful for treating depression as Type A enzyme appears to inactivate biogenic amines in brain. Aek~o~le~gerneni-We
gratefully acknowledge the expert technicai assistance of Mr. J. ~UEE~STEINand the generous supply of clorgyline provided by Dr. GMMSTERof May and Baker, Ltd.
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128:762-773. CONSOLO,S., GLACOBINI,E. and KARJALA~NEN, K. (1968). Monoamine oxidase in syn~pat~letic ganglia of the cat. Acta pkysiof. scand. 74 : 513-520. CIACOBINI,I?,, KARJALAMEN,K., KERPEL-FR~NIUS,S. and RITZEX, M. (1970). Monoamines and monoamine oxidase in denervated sympathetic ganglia of the cat. ~euruph~rma~~~ugy 9: 59-66. GORXLXS,C. and NEFF, N. H. (I97 I). Monoamine oxidase: an approximation of turnover rates. J. ~e~ruchef?~. In press. GORKIN, V. 2. (1969). Separation of rat liver mitochondrial amine oxidase. Experientia 25: 1142. GREEN, D. E., ALLMANN,D. W., HARRIS, R. A. and TAN, W. C. (1968). Enzyme localization in the inner and outer mitochondrial membranes. Biochem. Biophys. Res. Commun. 31: 368-378. HALL, D. W. R., LOGAN,B. W. and PARSONS,G. H. (1969). Further studies on the inhibition of monoamine oxidase by M&B 9302 (clorgyline). I. Substrate specificity in various mammalian species. Biochem. &armac. 18: 1447-1454. JOHNSTON, J. P. f1968). Some observations upon a new inhibitor of monoamine oxidase in brain tissue, Biochem. Rharmac. 17: 1285-1297.
KAPPER, .I. A. (1960). The develnpm~t, topo~aphicai retations and i~ervation of the epiphysis cerebri in the albino rat. Z. ~e~lfo~~ehrnikro~k. Aprat. 52: 163-215. KIM, II. C. and D’IORIO, A. (1968). Possible isoenzymes of monoamine oxidase in rat tissue, Can. J. ~~~che~~. 46: 295-297.
MARCO, R., SEBASTIAN, J. and SC&S,A. (1969). Location of the enzymes of the oxalacetate metabolic cross roads in rat Iiver mitochondria. Biockent. Biopl~~s. Res. Commrm. 34: 725-730. RACKER, E, (1955). Liver aldehyde dehydrogenase. In: Methods in Enzq’mology (COLOWICK,S. P. and KAPLAN, N. O., Eds.), Vol. 1, pp. 514-517. Academic Press, New York. SCHNAITMAN,C. and GREENAWALT,J. W. (1968). Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J. Cell Biol. 38: 158-17.5. SIER~NS,L. and D’IORIO,A. (1970). Multiple monoamine oxidases in rat liver mitochondria. Can. J. Biochenr. 48: 659-663, SNYDER,S. H., FISCHER,J. and AXELROR,J. (1965). Evidence for the presence of monoamine oxidase in sympathetic nerve endings. Biockem. Pkurmnc. 14: 363-365. WEISSBACX,H,, REDF~~LD,B. G. and UDENFRXEND, S. (1957). SofubIe monoamine oxidase: Its properties and actions on serotonin. J. 6iol. Ckem. 229: 953-963. YOUDIM,M. B. H., COLL~S, G. G. S. and SANDLER,M. (1969). ~ILlitipIe forms of rat brain mono~ine oxidase, N~~~~re,Lo&. 223: 626628.