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m -Octopamine as a substrate for monoamine oxidase
Pergamon Press
Life Sciences, Vol . 25, pp . 1231-1236 Printed in the U .S .A .
m-OCTOPAMINE AS A SUBSTRATE FOR MONOAMINE OXIDASE Osamu Suzuki and Hideki Hattori' Yoshinao Katsumata and Masakazu Oya2 'Department of Legal Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan 2 Department of Legal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan (Received in final form August 21, 1979) Summary m-Octopamine was characterized as substrate for monoamine oxidase (MAO) in rat brain and liver mitochondria . The ICm and V~ ax values of the brain enzyme were 735 uM and 32 .5 nmoTes/mg protein/30 min, and those of the liver enzyme 351 pM and 125 nmoles/mg protein/30 min, respectively . The inhibition experiments with clorgyline and deprenyl showed that m-octopamine was a common substrate for type A and type B MAU, though a major part of the activity was due to type A enzyme . The functional forms of mitochondrial monoamine oxidase [amine : oxygen oxidoreductase (deaminating, flavin-containing) ; EC 1 .4 .3 .4] (MAO) have generally been classified as type A and type B, depending on inhibitor sensitivity and substrate specificity (1) . Recently, various biogenic monoamines have been characterized as substrates for type A and type B MAO : 5-hydroxytry tamine (2), 5-methoxytryptamine (3), norepinephrine and epinephrine (2) are specific for type A MAO, while 0-phenylethylamine ~4,5), phenylethanolamine (6,7) at low substrate concentrations, o-tyramine 8) and 1,4-methylhistamine (9) are specific for type B MAO . Some substrates, such as p tyramine (1), m-tyramine (8), tryptamine (1), dopamine (2), p7octopamine (7,10) and p,-synephrine (11), are oxidized by either type of MAO . Recently, m-octopamine was identified and quantified in rat brain after the administration of a MAO inhibitor pargyline (12), showing that this amine is actively metabolized in vivo by MAO . Therefore, in the present paper, we characterized _m-octopamTne as substrate for type A and type B MAO in vit ro . Materials and Methods Mitochondrial fractions were isolated from the whole brains and the livers of male Sprague-Dawley rats weighing 150-200 g as described previously (13) . m-Octopamine-HC1 (DL-norphenylephrine-HC1) was obtained as a gift from Pharmacia, Uppsala, Sweden . Clorgyline, a selective inhibitor of type A MAO (1), was generously supplied by May & Baker Ltd ., Dagenham,
0024-3205/79/141231-05$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1232
Vol . 25, No . 14, 1979
m-Octopamine as a MAO Substrate
12 .0 10 .0 8 .0
4 .0 2 .0 0
-3 .0
-2 .0
-1 .0
0
1/[S]
1 .0 X
2 .0
3 .0
4 .0
10-1 (m -, )
FIG . 1 Lineweaver-Burk plots for MAO in rat brain (0--0) and liver (" mitochondria with m-octopamine as substrate . Each point represents the mean obtained from duplicate determinations ; v : moles H202 formed/mg protein/30 min . England . Deprenyl, a selective inhibitor of type B MAO (14), was kindly donated by Prof . J . Knoll, Department of Pharmacology, Semmelweis Univerisity of Medicine, Budapest, Hungary . MAO activities were determined fluorometrically by the method of Guilbault et al . (15) and Snyder and Hendley (16) . The details of the procedure are described in our previous paper (7) . For each assay (final volume, 3 .0 ml) 0 .225-2 .26 mg of mitochondrial protein was used . The assays were carried out at 37°C for 30 or 60 min . Under the conditions used, the assays were linear during incubation for at least 60 min . For kinetic analyses, the concentrations of _m-octopamine over the range of 300-3000 1iM were used . For inhibition experiments with clorgyline and deprenyl, the assay mixtures were preincubated with each inhibitor at 37°C for 10 min to ensure maximal enzyme inhibition . It was confirmed that the inhibitors neither interfered with the formation of the fluorescent compound nor quenched its fluorescence when hydrogen peroxide was added directly . Protein was measured by a slight modification (17) of the conventional biuret method . Results m-Octopamine was found to be an active substrate for MAO in rat brain and liver mitochondria . The Michaelis-Menten kinetic constants for moctopamine were determined from the Lineweaver-Burk plots as shown Fig . 1 . The Ksn and VMax values of the brain enzyme were 735 uM and 32 .5 nmoles/
in
m-Octopamine as a MAO Substrate
Vol . 25, No . 14, 1979
1233
100 ó 80 â
60
41
w u 40 s v a 20 0
10
9
8
7
6
5
4
Clorgyline (-log M)
Deprenyl
(-log M)
FIG . 2
Inhibition of MAO in rat brain mitochondria by clorgyline and deprenyl using various concentrations of m-octopamine as substrate . The substrate concentrations were 73 .5 (C)--O), 735 (ate) and 7350 uM Each point represents the mean obtained from duplicate determinations upon a single enzyme source prepared from the pooled brains of 7 rats . mg protein/30 min, and those of the liver enzyme were 351 uM and 125 nmoles/ mg protein/30 min, respectively . To determine the specific type of MAO involved in the metabolism of moctopamine, the sensitivity of MAO to clorgyline and deprenyl was studied Since we reported that the inhibition pattern may be affected by substrate concentration (5,7), the inhibition experiments were carried out at three different concentrations, viz ., at the K value, one-tenth and ten-fold of the KSm value . In Fig . Y, the inhibition patterns with the brain enzyme are illustrated . In the curves with clorgyline, clear plateaus appeared at 10-8_10-7 M . The sensitivity to clorgyline with 73 .5 uM m-octopamine In was slightly lower than that at the higher substrate concentrations . the curves with deprenyl, plateaus were not clear . The deamination of 73 .5 yM m-octopamine was somewhat more susceptible to deprenyl than that at the higher substrate concentrations . The inhibition of MAO in rat liver mitochondria by both inhibitors using three concentrations of m-octopamine is presentedin Fiq . 3 . In the curves with clorgyline, there were clear plateaus at 10 -8 -10- M as in the brain enzyme . The sensitivity to clorgyline with 35 .1 uM m-octopamine was lower than that at the higher concentrations . In the inhibition patterns with deprenyl, plateaus were also not clear . Discussion In the present study, we have characterized m-octopamine as substrate In both for type A and type B MAO using rat brain and livér mitochondria . brain and liver enzymes, clear plateaus were observed in all the curves
1234
m-octopamine as a MAO Substrate
Vol . 25, No . 14, 1979
100
c0 â
rc
80 60
cd
+1
U 40 L C.-
20 0
10
9
8
7
6
5
Clorgyline (-log M)
Deprenyl
(-l09 M)
FIG . 3 Inhibition of MAO in rat liver mitochondria by clorgyline and deprenyl using various concentrations of m-octopamine as substrate . The substrate concentrations were 35 .1 (o-O), 351 (*--a) and 3510 uM Each point represents the mean obtained from duplicate determinations upon a single enzyme source prepared from the pooled livers of 7 rats . with clorgyline, but not in the curves with deprenyl (Figs . 2 and 3) . However, since it has been found that deprenyl is not entirely specific in cases where clorgyline has proved to be a selective inhibitor (18), clorgyline seems to be a more reliable inhibitor for the type A and type B classification than deprenyl . Therefore, it can be concluded that moctopamine is deaminated by both types of MAO, though a major part of the activity is due to type A MAO . The susceptibility to clorgyline of m-octopamine deamination with the brain MAO was higher than that with the liver enzyme, while the susceptibility to deprenyl with the brain MAO was generally lower than that with the liver enzyme (Figs . 2 and 3) . These results are consistent with the report that the ratio of type A to type B in rat brain is higher than that in rat liver (18) . We reported that the inhibition curves were affected by substrate concentration especially when 0-phenylethylamine and phenylethanolamine were used as substrates (5,7) . This was also true for m-octopamine although the change in the pattern was not so dramatical as thatfor B-phenylethylamine and phenylethanolamine : the deamination of m-octopamine at the concentrations of one-tenth of the K values was more resistant to clorgyline than that at the higher substrate concentrations for both brain and liver enzymes (Figs . 2 and 3) . Therefore, our results with m-octopamine lend further support to the suggestion that, in the inhibition studies with clorgyline and deprenyl, we should check the effect of substrate concentration for each substrate and enzyme preparation, suspecting the different affinities of the substrate for type A and type B MAO (7) .
Vol . 25, No . 14, 1979
m-Octopamine as a MAO Substrate
1235
The present results with m-octopamine should be compared with those with poctopamine (7) . The inFibition patterns by clorgyline with moctopamine are generally similar to those with the pisomer . In this connection, it should be recalled that p and m-tyramine revealed extremely similar inhibition patterns (8) . In the inhibition by deprenyl, however, octopamine showed clear plateaus at 10-8_10-7 M of the inhibitor (7), while m-octopamine did not (Figs, 2 and 3) . Although physiological roles of m-octopamine have not been investigated, this amine is indeed present rat brain in extremely small quantities (12) . It was also reported that _m-octopamine can be formed from injected m-tyramine in rat heart (19) . Therefore, our present results may serve forunderstanding the in vivo dynamics of _m-octopamine in mammalian tissues in future .
in
References l. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 .
J . P . JOHNSTON, Biochem. Pharmacol . 17 1285-1297 (1968) . M. D. HOUSLAY an K. . ocFem. J . 139 645-652 (1974) . W . C . PROZIALECK and W. H. VOGEL, LET. 2561-569 (1978) H .-Y . T. YANG and N. H . NEFF, J . P i rmacoT. exp. Ther . 187 365-371 (1973) . 0 . SUZUKI, Y. KATSUMATA, M. OYA and T . MATSUMOTO, Biochem. Pharmacol . 28 953-956 (1979) . 157 . J . EDWARDS, Life Sci . 23 1201-1207 (1978) . 0 . SUZUKI, Y. KAT9UFPT, M . OYA and T . MATSUMOTO, Biochem. Pharmacol . i n press . 0 . SUZUKI, M. OYA and Y . KATSUMATA, Biochem . Pharmacol . i n press . 0 . SUZUKI, Y. KATSUMATA and M. OYA, e Sc i . _ -2230 (1979) . G . A. LYLES, Life Sci . 23 223-230 0 . SUZUKI, T._RATWTO,M . OYA and Y . KATSUMATA, Ex rientia in press . T . J . DANIELSON, A . A . BOULTON and H . A. ROBERTSON, . uroc em . 29 1131-1135 (1977) . 0 . SUZUKI, Y. KATSUMATA, M. OYA, V. M . CHARI, R . KLAPFENBERGER, H . WAGNER and K. HOSTETTMANN, Biochem. Pharmacol . 27 2075-2078 (1978) . J . KNOLL and K . MAGYAR, Advances n B oc em cal Ps cho harmacolo , vol . 5, pp . 393-408, Raven Press, Ne rk (1972) . G . G . GUILBAULT, P . J . BRIGNAC, JR . and M. JUNEAU, Anal . Chem . 40 1256-1263 (1968) . S . H . SNYDER and E . D . HENDLEY, J . Pharmacol . exp. Ther . 16 3 386392 (1968) . 0 . SUZUKI, E . NOGUCHI and K . YAGI, Brain Res . 135 305-313 (1977) . R . F . SQUIRES, Advances in Biochemica cFo írmacol , vol . 5, pp . 355-370, Raven ress, ew .Yor J . M . MUSACCHIO, I . J . KOPIN and V. K . WEISE, J . Pharmacol . exp. Ther . 148 22-28 (1965) .
Life Sciences, Vol . 25, pp . 1231-1236 Printed in the U .S .A .
m-OCTOPAMINE AS A SUBSTRATE FOR MONOAMINE OXIDASE Osamu Suzuki and Hideki Hattori' Yoshinao Katsumata and Masakazu Oya2 'Department of Legal Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan 2 Department of Legal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan (Received in final form August 21, 1979) Summary m-Octopamine was characterized as substrate for monoamine oxidase (MAO) in rat brain and liver mitochondria . The ICm and V~ ax values of the brain enzyme were 735 uM and 32 .5 nmoTes/mg protein/30 min, and those of the liver enzyme 351 pM and 125 nmoles/mg protein/30 min, respectively . The inhibition experiments with clorgyline and deprenyl showed that m-octopamine was a common substrate for type A and type B MAU, though a major part of the activity was due to type A enzyme . The functional forms of mitochondrial monoamine oxidase [amine : oxygen oxidoreductase (deaminating, flavin-containing) ; EC 1 .4 .3 .4] (MAO) have generally been classified as type A and type B, depending on inhibitor sensitivity and substrate specificity (1) . Recently, various biogenic monoamines have been characterized as substrates for type A and type B MAO : 5-hydroxytry tamine (2), 5-methoxytryptamine (3), norepinephrine and epinephrine (2) are specific for type A MAO, while 0-phenylethylamine ~4,5), phenylethanolamine (6,7) at low substrate concentrations, o-tyramine 8) and 1,4-methylhistamine (9) are specific for type B MAO . Some substrates, such as p tyramine (1), m-tyramine (8), tryptamine (1), dopamine (2), p7octopamine (7,10) and p,-synephrine (11), are oxidized by either type of MAO . Recently, m-octopamine was identified and quantified in rat brain after the administration of a MAO inhibitor pargyline (12), showing that this amine is actively metabolized in vivo by MAO . Therefore, in the present paper, we characterized _m-octopamTne as substrate for type A and type B MAO in vit ro . Materials and Methods Mitochondrial fractions were isolated from the whole brains and the livers of male Sprague-Dawley rats weighing 150-200 g as described previously (13) . m-Octopamine-HC1 (DL-norphenylephrine-HC1) was obtained as a gift from Pharmacia, Uppsala, Sweden . Clorgyline, a selective inhibitor of type A MAO (1), was generously supplied by May & Baker Ltd ., Dagenham,
0024-3205/79/141231-05$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1232
Vol . 25, No . 14, 1979
m-Octopamine as a MAO Substrate
12 .0 10 .0 8 .0
4 .0 2 .0 0
-3 .0
-2 .0
-1 .0
0
1/[S]
1 .0 X
2 .0
3 .0
4 .0
10-1 (m -, )
FIG . 1 Lineweaver-Burk plots for MAO in rat brain (0--0) and liver (" mitochondria with m-octopamine as substrate . Each point represents the mean obtained from duplicate determinations ; v : moles H202 formed/mg protein/30 min . England . Deprenyl, a selective inhibitor of type B MAO (14), was kindly donated by Prof . J . Knoll, Department of Pharmacology, Semmelweis Univerisity of Medicine, Budapest, Hungary . MAO activities were determined fluorometrically by the method of Guilbault et al . (15) and Snyder and Hendley (16) . The details of the procedure are described in our previous paper (7) . For each assay (final volume, 3 .0 ml) 0 .225-2 .26 mg of mitochondrial protein was used . The assays were carried out at 37°C for 30 or 60 min . Under the conditions used, the assays were linear during incubation for at least 60 min . For kinetic analyses, the concentrations of _m-octopamine over the range of 300-3000 1iM were used . For inhibition experiments with clorgyline and deprenyl, the assay mixtures were preincubated with each inhibitor at 37°C for 10 min to ensure maximal enzyme inhibition . It was confirmed that the inhibitors neither interfered with the formation of the fluorescent compound nor quenched its fluorescence when hydrogen peroxide was added directly . Protein was measured by a slight modification (17) of the conventional biuret method . Results m-Octopamine was found to be an active substrate for MAO in rat brain and liver mitochondria . The Michaelis-Menten kinetic constants for moctopamine were determined from the Lineweaver-Burk plots as shown Fig . 1 . The Ksn and VMax values of the brain enzyme were 735 uM and 32 .5 nmoles/
in
m-Octopamine as a MAO Substrate
Vol . 25, No . 14, 1979
1233
100 ó 80 â
60
41
w u 40 s v a 20 0
10
9
8
7
6
5
4
Clorgyline (-log M)
Deprenyl
(-log M)
FIG . 2
Inhibition of MAO in rat brain mitochondria by clorgyline and deprenyl using various concentrations of m-octopamine as substrate . The substrate concentrations were 73 .5 (C)--O), 735 (ate) and 7350 uM Each point represents the mean obtained from duplicate determinations upon a single enzyme source prepared from the pooled brains of 7 rats . mg protein/30 min, and those of the liver enzyme were 351 uM and 125 nmoles/ mg protein/30 min, respectively . To determine the specific type of MAO involved in the metabolism of moctopamine, the sensitivity of MAO to clorgyline and deprenyl was studied Since we reported that the inhibition pattern may be affected by substrate concentration (5,7), the inhibition experiments were carried out at three different concentrations, viz ., at the K value, one-tenth and ten-fold of the KSm value . In Fig . Y, the inhibition patterns with the brain enzyme are illustrated . In the curves with clorgyline, clear plateaus appeared at 10-8_10-7 M . The sensitivity to clorgyline with 73 .5 uM m-octopamine In was slightly lower than that at the higher substrate concentrations . the curves with deprenyl, plateaus were not clear . The deamination of 73 .5 yM m-octopamine was somewhat more susceptible to deprenyl than that at the higher substrate concentrations . The inhibition of MAO in rat liver mitochondria by both inhibitors using three concentrations of m-octopamine is presentedin Fiq . 3 . In the curves with clorgyline, there were clear plateaus at 10 -8 -10- M as in the brain enzyme . The sensitivity to clorgyline with 35 .1 uM m-octopamine was lower than that at the higher concentrations . In the inhibition patterns with deprenyl, plateaus were also not clear . Discussion In the present study, we have characterized m-octopamine as substrate In both for type A and type B MAO using rat brain and livér mitochondria . brain and liver enzymes, clear plateaus were observed in all the curves
1234
m-octopamine as a MAO Substrate
Vol . 25, No . 14, 1979
100
c0 â
rc
80 60
cd
+1
U 40 L C.-
20 0
10
9
8
7
6
5
Clorgyline (-log M)
Deprenyl
(-l09 M)
FIG . 3 Inhibition of MAO in rat liver mitochondria by clorgyline and deprenyl using various concentrations of m-octopamine as substrate . The substrate concentrations were 35 .1 (o-O), 351 (*--a) and 3510 uM Each point represents the mean obtained from duplicate determinations upon a single enzyme source prepared from the pooled livers of 7 rats . with clorgyline, but not in the curves with deprenyl (Figs . 2 and 3) . However, since it has been found that deprenyl is not entirely specific in cases where clorgyline has proved to be a selective inhibitor (18), clorgyline seems to be a more reliable inhibitor for the type A and type B classification than deprenyl . Therefore, it can be concluded that moctopamine is deaminated by both types of MAO, though a major part of the activity is due to type A MAO . The susceptibility to clorgyline of m-octopamine deamination with the brain MAO was higher than that with the liver enzyme, while the susceptibility to deprenyl with the brain MAO was generally lower than that with the liver enzyme (Figs . 2 and 3) . These results are consistent with the report that the ratio of type A to type B in rat brain is higher than that in rat liver (18) . We reported that the inhibition curves were affected by substrate concentration especially when 0-phenylethylamine and phenylethanolamine were used as substrates (5,7) . This was also true for m-octopamine although the change in the pattern was not so dramatical as thatfor B-phenylethylamine and phenylethanolamine : the deamination of m-octopamine at the concentrations of one-tenth of the K values was more resistant to clorgyline than that at the higher substrate concentrations for both brain and liver enzymes (Figs . 2 and 3) . Therefore, our results with m-octopamine lend further support to the suggestion that, in the inhibition studies with clorgyline and deprenyl, we should check the effect of substrate concentration for each substrate and enzyme preparation, suspecting the different affinities of the substrate for type A and type B MAO (7) .
Vol . 25, No . 14, 1979
m-Octopamine as a MAO Substrate
1235
The present results with m-octopamine should be compared with those with poctopamine (7) . The inFibition patterns by clorgyline with moctopamine are generally similar to those with the pisomer . In this connection, it should be recalled that p and m-tyramine revealed extremely similar inhibition patterns (8) . In the inhibition by deprenyl, however, octopamine showed clear plateaus at 10-8_10-7 M of the inhibitor (7), while m-octopamine did not (Figs, 2 and 3) . Although physiological roles of m-octopamine have not been investigated, this amine is indeed present rat brain in extremely small quantities (12) . It was also reported that _m-octopamine can be formed from injected m-tyramine in rat heart (19) . Therefore, our present results may serve forunderstanding the in vivo dynamics of _m-octopamine in mammalian tissues in future .
in
References l. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 .
J . P . JOHNSTON, Biochem. Pharmacol . 17 1285-1297 (1968) . M. D. HOUSLAY an K. . ocFem. J . 139 645-652 (1974) . W . C . PROZIALECK and W. H. VOGEL, LET. 2561-569 (1978) H .-Y . T. YANG and N. H . NEFF, J . P i rmacoT. exp. Ther . 187 365-371 (1973) . 0 . SUZUKI, Y. KATSUMATA, M. OYA and T . MATSUMOTO, Biochem. Pharmacol . 28 953-956 (1979) . 157 . J . EDWARDS, Life Sci . 23 1201-1207 (1978) . 0 . SUZUKI, Y. KAT9UFPT, M . OYA and T . MATSUMOTO, Biochem. Pharmacol . i n press . 0 . SUZUKI, M. OYA and Y . KATSUMATA, Biochem . Pharmacol . i n press . 0 . SUZUKI, Y. KATSUMATA and M. OYA, e Sc i . _ -2230 (1979) . G . A. LYLES, Life Sci . 23 223-230 0 . SUZUKI, T._RATWTO,M . OYA and Y . KATSUMATA, Ex rientia in press . T . J . DANIELSON, A . A . BOULTON and H . A. ROBERTSON, . uroc em . 29 1131-1135 (1977) . 0 . SUZUKI, Y. KATSUMATA, M. OYA, V. M . CHARI, R . KLAPFENBERGER, H . WAGNER and K. HOSTETTMANN, Biochem. Pharmacol . 27 2075-2078 (1978) . J . KNOLL and K . MAGYAR, Advances n B oc em cal Ps cho harmacolo , vol . 5, pp . 393-408, Raven Press, Ne rk (1972) . G . G . GUILBAULT, P . J . BRIGNAC, JR . and M. JUNEAU, Anal . Chem . 40 1256-1263 (1968) . S . H . SNYDER and E . D . HENDLEY, J . Pharmacol . exp. Ther . 16 3 386392 (1968) . 0 . SUZUKI, E . NOGUCHI and K . YAGI, Brain Res . 135 305-313 (1977) . R . F . SQUIRES, Advances in Biochemica cFo írmacol , vol . 5, pp . 355-370, Raven ress, ew .Yor J . M . MUSACCHIO, I . J . KOPIN and V. K . WEISE, J . Pharmacol . exp. Ther . 148 22-28 (1965) .