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The characteristics and distribution of monoamine oxidase (mao) activity in different tissues of the rainbow trout, salmo gairdneri
Camp. Biochem.
Physiol. Vol. 84C,No. I,pp.73-77,1986
0306-4492/86 $3.00+0.00 Pergamon Journals Ltd
Printed in Great Britain
THE CHARACTERISTICS AND DISTRIBUTION OF MONOAMINE OXIDASE (MAO) ACTIVITY IN DIFFERENT TISSUES OF THE RAINBOW TROUT, SALMO GAIRDNERI TERENCE R. HALL? and J. ANNE BROWN* of Zoology and tWolfson Institute, University of Hull, Hull HU6 7RX, UK. Telephone: 0482 46311
DENISE EDWARDS,*
*Department
(Received
16 September
1985)
Abstract-l. Monoamine oxidase (MAO) activity was determined fluorometrically in brain, intestine, kidney and liver tissues of the rainbow trout, S&no gairdneri. 2. MAO activity was inhibited by various drugs in a concentration-related manner, with single sigmoid inhibition curves, the inhibitors of type A MAO, harmaline and clorgyline being more effective than deprenyl, an inhibitor of type B MAO.
3. Intestine exhibited greatest MAO activity followed by liver and brain with kidney showing least activity. The Michaelis constants (K,,,) also showed variability between tissues. 4. Inhibition of MAO by harmaline was non-competitive and dependent on the concentration of substrate present.
INTRODUCTION
MATERIALSAND Experimental
Monoamine oxidase (monoamine:oxygen oxidoreductase, deaminating; EC 1.4.3.4; MAO) is present in a variety of tissues of many species (Blaschko, 1952; Squires, 1972) where it is involved in the metabolic inactivation of monoamines. Much of our present knowledge of the characteristics of MAO stems from studies of mammalian species using specific MAO inhibitors and various substrates (Youdim, 1975; Murphy, 1978; Hall and Urueiia, 1983a). As a result two enzyme forms have been distinguished. MAO A is more sensitive to inhibition by clorgyline and harmaline and preferentially deaminates serotonin and noradrenaline (Burkard and Kettler, 1977; Mitra and Guha, 1978) whereas MAO B is more sensitive to inhibition by deprenyl and preferentially deaminates phenylethylamine and benzylamine (Knoll et al., 1978). Dopamine is readily deaminated by both forms of the enzyme in most species (Youdim, 1975; Achee et al., 1977). The B form of MAO appears to be absent from aquatic vertebrates. Only MAO A appears to be present in the aquatic urodeles and freshwater teleosts which have been investigated, whereas terrestrial amphibians and birds have both MAO A and B (Hall and Urueiia, 1983a). This led Hall and Uruefia (1983a) to suggest that MAO B confers an advantage to a successful terrestrial lifestyle. However, since MAO A can metabolize type B substrates to some extent (Ekstedt, 1976), MAO B may be a nonessential component of MAO activity (Hall and Urueiia, 1983b). The characteristics of MAO have only been investigated in a small number of teleostean species. The present study aimed to characterize MAO activity in tissues of the rainbow trout by the use of the specific MAO inhibitors harmaline, clorgyline and deprenyl.
METHODS
animals
Five rainbow trout, Sulmo gairdneri, obtained commercially were maintained in well aerated tap water (osmolarity approximately lOmOsm/l) at 10 + 2°C on a 12 hr light: 12 hr d&k photoperiod and fed with pelleted trout food (Mainstream, BP). Fish were killed by a blow to the head, and liver, caudal kidney, small intestine and brain, except for hypothalamus, were removed and rinsed in trout Ringer solution (Brown et nl., 1983) following which all tissues were kept at -20°C prior to MAO characterization. MAO
assay
Tissues were homogenized in 0.2 M sodium glycine buffer, pH 9.2, at a concentration of 30 mg/ml for brain, intestine and kidney, and 50 mg/ml for liver. MAO was determined fluorometrically using kynuramine as a substrate (Hall and Figueroa, 1982; Hall et al., 1984). Liver homogenate (25 ~1) was incubated in 0.5 ml buffer containing 1O-4 M kynuramine and 100 pl of intestine, kidney or brain homogenate were incubated in 1 ml buffer containing 5 x lo-’ M kynuramine. After 30min incubation at 37°C the reaction was stopped by placing on ice and addition of 100~1 10% (wt/vol) ZnSO,. On mixing with 1 ml of 1 M NaOH the fluorescence of the product, 4-hydroxyquinolene (4-HQ), was determined in a Perkin-Elmer luminescence spectrometer (LS5) at 3 15 nm excitation, 380 nm emission. Blanks and 4-HQ standards were run in parallel with each assay. MAO activity was expressed as pmol 4-HQ produced/g tissue/hr. Inhibitor
studies
Trout liver homogenate was preincubated for 30min in buffer containing the MAO inhibitors harmaline. clorevline or deprenyl at concentrations of from IO-” to 10e5 MyThe incubation was then started by the addition of the substrate kynuramine. The action of the MAO inhibitors harmaline and deprenyl (10~‘0~10-5 M) on kidney, intestine and brain homogenates was also investigated. Production of 4-HQ was determined and MAO activity expressed as percentage control activity without inhibitor. 73
14 MAO
DENSE EDWARDS et (11. kinetics
studies
The kinetics of MAO activity in kidney, intestine and brain was studied by incubation of tissues with a range of kynuramine concentrations (2 x 10m6-5 x 1O-5 M). Liver homogenate was incubated with 5 x 10m6-5 x IOmSM kynuramine alone and in the presence of various concentrations of harmaline (10m9, 3 x 10m9 and 10--R M).
Clorgyline was kindly provided by May and Baker, Dagenham, Essex (UK) and deprenyl was donated by Professor J. Knoll, Semmelweis University, Budapest (Hungary). All other drugs were purchased from Sigma Chemicals Co., Poole, Dorset (UK). Datu analysis All results were expressed as mean &-standard error of the mean (SEM). The apparent K, and V,,,,, were determined by a least squares regression analysis. Differences between groups were compared using Student’s t test and differences between slopes were tested by comparison of regression coefficients, with the level of significance as P < 0.05. Regression analysis was further employed to determine the inhibitor constants on replotting the original data. A replot of slope (V,,,.,,/X;.) against harmaline concentration determines the slope inhibition constant (K,,), while a replot of intercept (l/V,,,,,) against harmaline concentration determines the intercept inhibition constant (K,,), 10-5
10-10
RESULTS
Liver MAO activity was inhibited by harmaline, clorgyline and deprenyl in a concentration dependent fashion producing single-phase sigmoid inhibition
lnhlbltor
concmtratlon
(MI
Fig. 2. Effects of harmaline (closed symbols) and deprenyl (open symbols) on monoamine oxidase activity in rainbow trout tissues. Activity is expressed as percentage of uninhibited value. Intestine, triangles; kidney, squares; brain, circles. Vertical bars represent SEM of determinations from five fish.
(Fig. 1). However, the potencies of the three inhibitors were different. Harmaline was the most potent, producing 50% inhibition of activity (I,,) at a concentration of 2.5 x lOma M, followed by clorgyline (I,, = 2.0 x lo-‘M) and deprenyl (IS,, = 3.6 x lo-’ M). Monoamine oxidase activity of intestine, brain and kidney was also inhibited by harmaline and deprenyl in a concentration-related fashion with single-phase sigmoid inhibition curves (Fig. 2). In these three tissues, MAO activity was approximately 30-fold more sensitive to inhibition by harmaline than by deprenyl. Homogenates of intestine, brain, liver and kidney, incubated with different kynuramine concentrations, yielded the maximum reaction velocities (V,,,,,) and apparent Michaelis constants (I&) presented in Table 1. Comparison of V,,, of brain and liver showed no significant difference, however all other differences in curves
Table
,
I. Characteristics of monoamine oxidase in different tissues of rainbow trout
,
I
10-10
1 o-5 Inhibitor
concentration
CM)
Fig. 1. Effects of harmaline (closed circle), clorgyline (open circle) and deprenyl (closed triangle) on monoamine oxidase activity in homogenates of trout liver. Activity is expressed as percentage yield of 4-hydroxyquinolene (4-HQ) compared with control incubations without inhibitor. Vertical bars represent SEM of determinations from five fish.
Intestine Liver Brain Kidnev
23.51 & 3.21
1.89 i_ 0.25 I.31 f 0.24 0.40 + 0.08
31.19&4.0X
3.00 f 0.33 5.44 + 0.35 9.45 + 1.24
K, is the apparent Michaelis constant and V,,,,, the maximal velocity. Results show means k SEM of determinations on four (liver) or five (other tissues) fish.
Activity and distribution of MAO in trout 10
1
(b)
/
(a)
-4
-2
0
Kynuramine
2
(10-5h4(-1)
01 0 Harmaline
10 (nM)
Fig. 3. Kinetics of MAO activity. (a) Lineweaver-Burk plot of 4-hydroxyquinolene (4-HQ) produced by incubation of liver homogenates with various amounts of kynuramine and harmaline. All regression coefficients were significantly different from each other (P < 0.05). (b) Replot of l/V,, against harmaline concentration. (c) Replot of K,,,/V,,,,, against hannaline concentration. Results are the mean determination
from livers from four fish.
V,,,,, and K,,, between tissues were significant at the
P ~0.02
level. The small intestine exhibited a markedly higher activity than the other types of tissues tested (P < O.OOl), with the liver and brain
having lower activity and the kidney least activity
(P c 0.01). The apparent Km of intestine MAO was also considerably higher than the other tissues assayed (P < 0.001) and lowest in liver (P c 0.001). Figure 3a shows Lineweaver-Burk plots of the liver yield of 4-HQ-’ against kynuramine concentration-’ in the presence of various concentrations of the inhibitor harmaline. The lines for various harmaline concentrations intersect at a point to the left of the l/velocity axis indicating that harmaline apparently increases both the slope and y intercept, suggesting non-competitive inhibition. Inhibition constants were determined by replotting the data shown in Fig. 3a. A replot of slope against harmaline concentration (Fig. 3b) provides the slope inhibition constant, (Ki, = 2.12 nM). A replot of the intercept (l/V,,,,,) against harmaline concentration (Fig. 3c) provides the intercept inhibition constant (K,, = 25.68 nM). DISCUSSION
The present study provides further information on some of the characteristics of MAO activity in aquatic vertebrates. The production of single sigmoid inhibition curves
on incubation with harmaline, clorgyline and deprenyl is indicative of the presence of only one form of MAO. When both forms are present distinct plateau regions are apparent (Youdim, 1975; Das and Guha, 1980) as MAO type A and B exhibit differing sensitivities to various substrates and inhibitors. The use of the non-physiological substrate, kynuramine, allowed the determination of the sensitivity of whichever form of MAO was present since kynuramine, like the trace amine tyramine, can be deaminated by either form of MAO (Youdim, 1975; Murphy, 1978). The MAO activity of rainbow trout liver was most sensitive to harmaline, less sensitive to clorgyline and least sensitive to deprenyl inhibition indicating the presence of an MAO form resembling mammalian MAO type A. Large differences exist in the relative proportions of type A: type B in different tissues of a given species (Hall and Urueiia, 1983a) with some tissues composed almost exclusively of one form while two forms exist in other tissues (Tipton et al., 1976). In the rainbow trout, the greater sensitivity of MAO from brain, intestine and kidney to harmaline and the single inhibition curves suggest that MAO A activity is solely responsible for the inactivation of monoamines in rainbow trout tissues. This is in agreement with work on other aquatic vertebrates (Figueroa et al., 1981; Hall et al., 1982a; Hall and Urueiia, 1983b). There were large differences in V,,,,, of MAO between the four tissues tested. Intestine showed the
16
DENISEEDWARDS et al.
greatest, and kidney least, MAO activity. The level of activity in the kidney was surprisingly low compared to that measured in the goldfish (Hall and Urueiia, 1982) where activity was greater than that seen in the intestine which was also high. Liver and brain MAO levels were not significantly different in the present study, a trend which is also seen in goldfish (Hall and Urueria, 1982), tiger salamander (Hall and Uruena, 1983b) and grassfrog (Urueiia and Hall, 1982). High MAO activity would be expected in the four tissues studied as brain MAO is important in the deactivation of monoamine neurotransmitters (Achee et al., 1977) while the other three tissues are important detoxifying organs responsible for the breakdown of exogenous and endogenous monoamines. High levels of intestinal MAO activity have been noted in goldfish (Hall and Uruena, 1982), mudpuppy (Hall and Uruefia, 1983b) and ring dove (Hall et al., 1985b) as well as the present study. This may be due to MAO providing the primary route of inactivation of serotonin (Curzon, 1981) which may be important in intestinal motility (Huidobro-Toro and Foree, 1980). Rainbow trout intestine also exhibited a high apparent K,,, (Michaelis constant), a measure of the affinity of the enzyme and substrate. The K,,, for kidney was also significantly higher than that of liver and brain. High K,,, in conjunction with low V,,,,, suggests that kidney MAO is less efficient at deamination of the substrate, kynuramine, than the other tissues. Differences in MAO activity, primary enzyme structure, substrate specificity or lipid environment between the tissues could equally be responsible for the differences observed (Achee et al., 1977; Huang, 1981). Further information on the action of MAO in rainbow trout was obtained from analysis of the effects of harmaline on MAO kinetics. These show harmaline to be a non-competitive inhibitor (Spector and Cleland, 1981). Non-competitive inhibition of MAO by harmaline was also observed with mouse brain (Hall et al., 1982b) and ring dove kidney (Hall et al., 1985a) incubated with kynuramine. The requirement for oxygen for MAO activity, on the other hand, has been reported to display uncompetitive kinetics (Fowler and Oreland, 1980; Hall et al., 1985a). Oxygen was not monitored in the present study. Transformation of data allowed the determination of the slope and intercept inhibitor constants (K,, and K,,), but the dissociation constant, KI, could not be determined. KiS is the inhibition constant at zero substrate concentration and K,, is the inhibition constant at infinite substrate concentration (Spector and Cleland, 1981). In the present study, the constants varied markedly, suggesting that the inhibition of trout MAO is dependent on the concentration of kynuramine. This is in contrast to ring dove MAO where similar values for the two constants were observed leading Hall et al. (1985a) to suggest that harmaline inhibition of dove MAO is independent of kynuramine substrate. In conclusion, the apparent absence of MAO type B from various tissues of the rainbow trout supports the concept that this isoenzyme may not be necessary in aquatic animals. The variations in K,,, and V,,, between the different tissues studied indicate that
MAO is present in different amounts and exhibits different binding affinities in these tissues, which may be related to variations in its role in different tissues. Finally the study of kinetics of the MAO reaction indicates that inhibition by harmaline is noncompetitive with harmaline interacting at a separate site to that occupied by kynuramine, and that inhibition is dependent on the concentration of kynuramine present. REFERENCES
Achee F. M., Gabay S. and Tipton K. F. (1977) Some aspects of monoamine oxidase activity in brain. Prog. Neurobiol.
Blaschko
8, 325-348.
H. (1952) Amine
Pharmac.
oxidase
and amine metabolism.
Rev. 4, 415453.
Brown J. A., Taylor S. M. and Gray C. J. (1983) Glomerular ultrastructure of the trout, Salmo gairdneri. Cell Tissue Res. 230,
205-218.
Burkard W. P. and Kettler R. (1977) Harmaline and its sulfur analogue: increase of cerebellar cyclic GMP and inhibition of monoamine oxidase. Biochem. Pharmac. 26, 1303-l 306. Curzon G. (1981) The turnover of 5-hydroxytryptamine. In Central Neurotransmitter Turnover (Edited by Pycock C. J. and Taberner P. V.), pp. 59-80. University Park Press, Baltimore. Das P. K. and Guha S. R. (1980) MAO types in guinea-pig liver mitochondria. Biochem. Pharmac. 29, 2049-2053. Ekstedt B. (1976) Substrate specificity of the different forms of monoamine oxidase in rat liver mitochondria. Biothem. Pharmac. 25, 113331138. Figueroa H. R., Hall T. R., Olcese J. M. and De Vlaming V. L. (1981) Effects of inhibitors of monoamine oxidase activity in goldfish (Carassius auratus) brain in vitro. Comp.
Biochem.
Physiol.
7OC, 281-284.
Fowler C. J. and Oreland L. (1980) The nature of the substrate-selective interaction between rat liver mitochondrial monoamine oxidase and oxygen. Biochem. Pharmac.
29, 2225-2233.
Hall T. R. and Figueroa H. R. (1982) Characteristics of monoamine oxidase activity in mouse brain. Pharmac. Res. Commun.
14, 431441.
Hail T. R. and Uruetia G. (1982) Monoamine activity in several tissues of the goldfish, auratus.
Camp.
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oxidase Carassius
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Hall T. R. and Uruefia G. (1983a) Pharmacology and physiology of monoamine oxidase activity in vertebrates -a comparative study. Comp. Biochem. Physiol. 76B, 393-397.
Hall T. R. and Urueiia G. (1983b) Distribution of monoamine oxidase activity in tissues of the urodeles Ambystoma tigrinum (tiger salamander) and Necturus maculosus (mudpuppy). _ _.. Comp. Biochem. Physiol. 74C, 35-39. Hall T. R., Olcese J: M., Figueroa H. R. and De Vlaming V. L. (1982a) Effects of inhibitors on monoamine oxidase activity in perch (Perca,flavescens) brain in vitro. Comp. Biochem.
Physiol.
71C,
Hall T. R., Figueroa
141L144.
H. R. and Yurgens P. B. (1982b) on monoamine-oxidase activity in
Effects of inhibitors mouse brain. Pharmac. Res, Commun. 14, 443-453. Hall T. R., Yurgens P. B. and Figueroa H. R. (1984) Effects of reserpine on recovery rates after inhibition of monoamine oxidase in different regions of mouse brain. Gen. Phnrmac.
15, 485-489.
Hall T. R., Lea R. W., Vowles D. M. and Harvey S. (1985a) Biochemistry and physiology of monoamine oxidase (MAO) activity in the ring dove (Streptopelia risoria)_l. Characteristics of MAO activity in vitro. Comp. Biochem. Physiol.
82C,
417422.
Hall T. R., Lea R. W., Harvey
S. and Vowles D. M. (1985b)
Activity
and distribution
Biochemistry and physiology of monoamine oxidase (MAO) activity in the ring dove (Streptopeka risoriut_II. Distribution of MAO in different tissues. Comp. Biochem. Physiol. 82C, 423426. Huang R. H. (1981) Topology and lipid protein association of MAO-A and MAO-B. In Function and Re,qdation of Monoamine Enzymes: Basic and Clinical Aspects (Edited by Usdin E., Weiner N. and Youdim M. B. H.), pp. 4899501. Macmillan, London. Huidobro-Toro J. P. and Foree B. (1980) Dual agonistantagonist effects of 5hydroxytryptamine (5HT) in the guinea-pig ileum: evidence for a selective receptor desensitization effect. Eur. J. Pharmac. 61, 335-345. Knoll J., Escery Z., Magyar K. and Satory E. (1978) Novel (-)deprenyl-derived selective inhibitors of B-type monoamine oxidase. The relation of structure to their action. Biochem. Pharmac. 27, 1739-1749. Mitra C. and Guha S. R. (1978) Inhibition of monoamine oxidation in subfractions of crude mitochondria of rat brain by clorgyline and Lilly 51641. Biochem. Pharmac. 27, 2455-2457.
of MAO
in trout
77
Murphy D. L. (1978) Substrate-selective monoamine oxidases. Inhibitor, tissues, species and functional differences. Biochem. Pharmac. 27, 188991893. Spector T. and Cleland W. W. (1981) Meanings of K, for conventional and alternate-substrate inhibitors. Biochem. Pharmac. 30, 1-7. Squires R. F. (1972) Multiple forms of monoamine oxidase in intact mitochondria as characterized by selective inhibitors and thermal stability: a comparison of eight mammalian species. Adu. Biochem. Pharmac. 5, 355-370. Tipton K. F., Houslay M. D. and Mantle T. J. (1976) The nature and locations of multiple forms of monoamine oxidase. In Monoamine Oxidase and its Inhibition. CIBA Foundation Symposium 39, pp. 5-16. Elsevier, New York. Urueiia G. and Hall T. R. (1982) Some characteristics of monoamine oxidase activity in tissues of the adult female grassfrog (Rana pipiens). Camp. Biochem. Physiol. 73C, 67-70. Youdim M. B. H. (1975) Monoamine oxidase: its inhibition. Mod. Probl. Pharmaco-psych. 10, 65-88.
Physiol. Vol. 84C,No. I,pp.73-77,1986
0306-4492/86 $3.00+0.00 Pergamon Journals Ltd
Printed in Great Britain
THE CHARACTERISTICS AND DISTRIBUTION OF MONOAMINE OXIDASE (MAO) ACTIVITY IN DIFFERENT TISSUES OF THE RAINBOW TROUT, SALMO GAIRDNERI TERENCE R. HALL? and J. ANNE BROWN* of Zoology and tWolfson Institute, University of Hull, Hull HU6 7RX, UK. Telephone: 0482 46311
DENISE EDWARDS,*
*Department
(Received
16 September
1985)
Abstract-l. Monoamine oxidase (MAO) activity was determined fluorometrically in brain, intestine, kidney and liver tissues of the rainbow trout, S&no gairdneri. 2. MAO activity was inhibited by various drugs in a concentration-related manner, with single sigmoid inhibition curves, the inhibitors of type A MAO, harmaline and clorgyline being more effective than deprenyl, an inhibitor of type B MAO.
3. Intestine exhibited greatest MAO activity followed by liver and brain with kidney showing least activity. The Michaelis constants (K,,,) also showed variability between tissues. 4. Inhibition of MAO by harmaline was non-competitive and dependent on the concentration of substrate present.
INTRODUCTION
MATERIALSAND Experimental
Monoamine oxidase (monoamine:oxygen oxidoreductase, deaminating; EC 1.4.3.4; MAO) is present in a variety of tissues of many species (Blaschko, 1952; Squires, 1972) where it is involved in the metabolic inactivation of monoamines. Much of our present knowledge of the characteristics of MAO stems from studies of mammalian species using specific MAO inhibitors and various substrates (Youdim, 1975; Murphy, 1978; Hall and Urueiia, 1983a). As a result two enzyme forms have been distinguished. MAO A is more sensitive to inhibition by clorgyline and harmaline and preferentially deaminates serotonin and noradrenaline (Burkard and Kettler, 1977; Mitra and Guha, 1978) whereas MAO B is more sensitive to inhibition by deprenyl and preferentially deaminates phenylethylamine and benzylamine (Knoll et al., 1978). Dopamine is readily deaminated by both forms of the enzyme in most species (Youdim, 1975; Achee et al., 1977). The B form of MAO appears to be absent from aquatic vertebrates. Only MAO A appears to be present in the aquatic urodeles and freshwater teleosts which have been investigated, whereas terrestrial amphibians and birds have both MAO A and B (Hall and Urueiia, 1983a). This led Hall and Uruefia (1983a) to suggest that MAO B confers an advantage to a successful terrestrial lifestyle. However, since MAO A can metabolize type B substrates to some extent (Ekstedt, 1976), MAO B may be a nonessential component of MAO activity (Hall and Urueiia, 1983b). The characteristics of MAO have only been investigated in a small number of teleostean species. The present study aimed to characterize MAO activity in tissues of the rainbow trout by the use of the specific MAO inhibitors harmaline, clorgyline and deprenyl.
METHODS
animals
Five rainbow trout, Sulmo gairdneri, obtained commercially were maintained in well aerated tap water (osmolarity approximately lOmOsm/l) at 10 + 2°C on a 12 hr light: 12 hr d&k photoperiod and fed with pelleted trout food (Mainstream, BP). Fish were killed by a blow to the head, and liver, caudal kidney, small intestine and brain, except for hypothalamus, were removed and rinsed in trout Ringer solution (Brown et nl., 1983) following which all tissues were kept at -20°C prior to MAO characterization. MAO
assay
Tissues were homogenized in 0.2 M sodium glycine buffer, pH 9.2, at a concentration of 30 mg/ml for brain, intestine and kidney, and 50 mg/ml for liver. MAO was determined fluorometrically using kynuramine as a substrate (Hall and Figueroa, 1982; Hall et al., 1984). Liver homogenate (25 ~1) was incubated in 0.5 ml buffer containing 1O-4 M kynuramine and 100 pl of intestine, kidney or brain homogenate were incubated in 1 ml buffer containing 5 x lo-’ M kynuramine. After 30min incubation at 37°C the reaction was stopped by placing on ice and addition of 100~1 10% (wt/vol) ZnSO,. On mixing with 1 ml of 1 M NaOH the fluorescence of the product, 4-hydroxyquinolene (4-HQ), was determined in a Perkin-Elmer luminescence spectrometer (LS5) at 3 15 nm excitation, 380 nm emission. Blanks and 4-HQ standards were run in parallel with each assay. MAO activity was expressed as pmol 4-HQ produced/g tissue/hr. Inhibitor
studies
Trout liver homogenate was preincubated for 30min in buffer containing the MAO inhibitors harmaline. clorevline or deprenyl at concentrations of from IO-” to 10e5 MyThe incubation was then started by the addition of the substrate kynuramine. The action of the MAO inhibitors harmaline and deprenyl (10~‘0~10-5 M) on kidney, intestine and brain homogenates was also investigated. Production of 4-HQ was determined and MAO activity expressed as percentage control activity without inhibitor. 73
14 MAO
DENSE EDWARDS et (11. kinetics
studies
The kinetics of MAO activity in kidney, intestine and brain was studied by incubation of tissues with a range of kynuramine concentrations (2 x 10m6-5 x 1O-5 M). Liver homogenate was incubated with 5 x 10m6-5 x IOmSM kynuramine alone and in the presence of various concentrations of harmaline (10m9, 3 x 10m9 and 10--R M).
Clorgyline was kindly provided by May and Baker, Dagenham, Essex (UK) and deprenyl was donated by Professor J. Knoll, Semmelweis University, Budapest (Hungary). All other drugs were purchased from Sigma Chemicals Co., Poole, Dorset (UK). Datu analysis All results were expressed as mean &-standard error of the mean (SEM). The apparent K, and V,,,,, were determined by a least squares regression analysis. Differences between groups were compared using Student’s t test and differences between slopes were tested by comparison of regression coefficients, with the level of significance as P < 0.05. Regression analysis was further employed to determine the inhibitor constants on replotting the original data. A replot of slope (V,,,.,,/X;.) against harmaline concentration determines the slope inhibition constant (K,,), while a replot of intercept (l/V,,,,,) against harmaline concentration determines the intercept inhibition constant (K,,), 10-5
10-10
RESULTS
Liver MAO activity was inhibited by harmaline, clorgyline and deprenyl in a concentration dependent fashion producing single-phase sigmoid inhibition
lnhlbltor
concmtratlon
(MI
Fig. 2. Effects of harmaline (closed symbols) and deprenyl (open symbols) on monoamine oxidase activity in rainbow trout tissues. Activity is expressed as percentage of uninhibited value. Intestine, triangles; kidney, squares; brain, circles. Vertical bars represent SEM of determinations from five fish.
(Fig. 1). However, the potencies of the three inhibitors were different. Harmaline was the most potent, producing 50% inhibition of activity (I,,) at a concentration of 2.5 x lOma M, followed by clorgyline (I,, = 2.0 x lo-‘M) and deprenyl (IS,, = 3.6 x lo-’ M). Monoamine oxidase activity of intestine, brain and kidney was also inhibited by harmaline and deprenyl in a concentration-related fashion with single-phase sigmoid inhibition curves (Fig. 2). In these three tissues, MAO activity was approximately 30-fold more sensitive to inhibition by harmaline than by deprenyl. Homogenates of intestine, brain, liver and kidney, incubated with different kynuramine concentrations, yielded the maximum reaction velocities (V,,,,,) and apparent Michaelis constants (I&) presented in Table 1. Comparison of V,,, of brain and liver showed no significant difference, however all other differences in curves
Table
,
I. Characteristics of monoamine oxidase in different tissues of rainbow trout
,
I
10-10
1 o-5 Inhibitor
concentration
CM)
Fig. 1. Effects of harmaline (closed circle), clorgyline (open circle) and deprenyl (closed triangle) on monoamine oxidase activity in homogenates of trout liver. Activity is expressed as percentage yield of 4-hydroxyquinolene (4-HQ) compared with control incubations without inhibitor. Vertical bars represent SEM of determinations from five fish.
Intestine Liver Brain Kidnev
23.51 & 3.21
1.89 i_ 0.25 I.31 f 0.24 0.40 + 0.08
31.19&4.0X
3.00 f 0.33 5.44 + 0.35 9.45 + 1.24
K, is the apparent Michaelis constant and V,,,,, the maximal velocity. Results show means k SEM of determinations on four (liver) or five (other tissues) fish.
Activity and distribution of MAO in trout 10
1
(b)
/
(a)
-4
-2
0
Kynuramine
2
(10-5h4(-1)
01 0 Harmaline
10 (nM)
Fig. 3. Kinetics of MAO activity. (a) Lineweaver-Burk plot of 4-hydroxyquinolene (4-HQ) produced by incubation of liver homogenates with various amounts of kynuramine and harmaline. All regression coefficients were significantly different from each other (P < 0.05). (b) Replot of l/V,, against harmaline concentration. (c) Replot of K,,,/V,,,,, against hannaline concentration. Results are the mean determination
from livers from four fish.
V,,,,, and K,,, between tissues were significant at the
P ~0.02
level. The small intestine exhibited a markedly higher activity than the other types of tissues tested (P < O.OOl), with the liver and brain
having lower activity and the kidney least activity
(P c 0.01). The apparent Km of intestine MAO was also considerably higher than the other tissues assayed (P < 0.001) and lowest in liver (P c 0.001). Figure 3a shows Lineweaver-Burk plots of the liver yield of 4-HQ-’ against kynuramine concentration-’ in the presence of various concentrations of the inhibitor harmaline. The lines for various harmaline concentrations intersect at a point to the left of the l/velocity axis indicating that harmaline apparently increases both the slope and y intercept, suggesting non-competitive inhibition. Inhibition constants were determined by replotting the data shown in Fig. 3a. A replot of slope against harmaline concentration (Fig. 3b) provides the slope inhibition constant, (Ki, = 2.12 nM). A replot of the intercept (l/V,,,,,) against harmaline concentration (Fig. 3c) provides the intercept inhibition constant (K,, = 25.68 nM). DISCUSSION
The present study provides further information on some of the characteristics of MAO activity in aquatic vertebrates. The production of single sigmoid inhibition curves
on incubation with harmaline, clorgyline and deprenyl is indicative of the presence of only one form of MAO. When both forms are present distinct plateau regions are apparent (Youdim, 1975; Das and Guha, 1980) as MAO type A and B exhibit differing sensitivities to various substrates and inhibitors. The use of the non-physiological substrate, kynuramine, allowed the determination of the sensitivity of whichever form of MAO was present since kynuramine, like the trace amine tyramine, can be deaminated by either form of MAO (Youdim, 1975; Murphy, 1978). The MAO activity of rainbow trout liver was most sensitive to harmaline, less sensitive to clorgyline and least sensitive to deprenyl inhibition indicating the presence of an MAO form resembling mammalian MAO type A. Large differences exist in the relative proportions of type A: type B in different tissues of a given species (Hall and Urueiia, 1983a) with some tissues composed almost exclusively of one form while two forms exist in other tissues (Tipton et al., 1976). In the rainbow trout, the greater sensitivity of MAO from brain, intestine and kidney to harmaline and the single inhibition curves suggest that MAO A activity is solely responsible for the inactivation of monoamines in rainbow trout tissues. This is in agreement with work on other aquatic vertebrates (Figueroa et al., 1981; Hall et al., 1982a; Hall and Urueiia, 1983b). There were large differences in V,,,,, of MAO between the four tissues tested. Intestine showed the
16
DENISEEDWARDS et al.
greatest, and kidney least, MAO activity. The level of activity in the kidney was surprisingly low compared to that measured in the goldfish (Hall and Urueiia, 1982) where activity was greater than that seen in the intestine which was also high. Liver and brain MAO levels were not significantly different in the present study, a trend which is also seen in goldfish (Hall and Urueria, 1982), tiger salamander (Hall and Uruena, 1983b) and grassfrog (Urueiia and Hall, 1982). High MAO activity would be expected in the four tissues studied as brain MAO is important in the deactivation of monoamine neurotransmitters (Achee et al., 1977) while the other three tissues are important detoxifying organs responsible for the breakdown of exogenous and endogenous monoamines. High levels of intestinal MAO activity have been noted in goldfish (Hall and Uruena, 1982), mudpuppy (Hall and Uruefia, 1983b) and ring dove (Hall et al., 1985b) as well as the present study. This may be due to MAO providing the primary route of inactivation of serotonin (Curzon, 1981) which may be important in intestinal motility (Huidobro-Toro and Foree, 1980). Rainbow trout intestine also exhibited a high apparent K,,, (Michaelis constant), a measure of the affinity of the enzyme and substrate. The K,,, for kidney was also significantly higher than that of liver and brain. High K,,, in conjunction with low V,,,,, suggests that kidney MAO is less efficient at deamination of the substrate, kynuramine, than the other tissues. Differences in MAO activity, primary enzyme structure, substrate specificity or lipid environment between the tissues could equally be responsible for the differences observed (Achee et al., 1977; Huang, 1981). Further information on the action of MAO in rainbow trout was obtained from analysis of the effects of harmaline on MAO kinetics. These show harmaline to be a non-competitive inhibitor (Spector and Cleland, 1981). Non-competitive inhibition of MAO by harmaline was also observed with mouse brain (Hall et al., 1982b) and ring dove kidney (Hall et al., 1985a) incubated with kynuramine. The requirement for oxygen for MAO activity, on the other hand, has been reported to display uncompetitive kinetics (Fowler and Oreland, 1980; Hall et al., 1985a). Oxygen was not monitored in the present study. Transformation of data allowed the determination of the slope and intercept inhibitor constants (K,, and K,,), but the dissociation constant, KI, could not be determined. KiS is the inhibition constant at zero substrate concentration and K,, is the inhibition constant at infinite substrate concentration (Spector and Cleland, 1981). In the present study, the constants varied markedly, suggesting that the inhibition of trout MAO is dependent on the concentration of kynuramine. This is in contrast to ring dove MAO where similar values for the two constants were observed leading Hall et al. (1985a) to suggest that harmaline inhibition of dove MAO is independent of kynuramine substrate. In conclusion, the apparent absence of MAO type B from various tissues of the rainbow trout supports the concept that this isoenzyme may not be necessary in aquatic animals. The variations in K,,, and V,,, between the different tissues studied indicate that
MAO is present in different amounts and exhibits different binding affinities in these tissues, which may be related to variations in its role in different tissues. Finally the study of kinetics of the MAO reaction indicates that inhibition by harmaline is noncompetitive with harmaline interacting at a separate site to that occupied by kynuramine, and that inhibition is dependent on the concentration of kynuramine present. REFERENCES
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