- Email: [email protected]
Changes in rat hypothalamic and brain stem catecholamine concentrations following acute administration of phenylethanolamine-N-methyltransferase inhibitors
Neuroscience Letters, 20 (1980) 67-71
67
© Elsevier/North-Holland Scientific Publishers Ltd.
C H A N G E S IN R A T H Y P O T H A L A M I C A N D B R A I N STEM C A T E C H O L A M I N E C O N C E N T R A T I O N S F O L L O W I N G A C U T E A D M I N I S T R A T I O N OF PHENYLETHANOLAMINE-N-METHYLTRANSFERASE INHIBITORS
IVAN N. M E F F O R D , KEVIN A. ROTH and JACK D. B A R C H A S
Nancy Pritzker Laboratory of Behavioral Neurochernistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) (Received April 9th, 1980; Accepted July 3rd, 1980)
The effect of acute phenylethanolamine-N-methyltransferase (PNMT) inhibition on rat brain catecholamine concentrations was studied. All P N M T inhibitors tested were effective in depleting hypothalamic and brain stem epinephrine. Norepinephrine concentrations were unaffected; however, dopamine levels were increased in all areas studied. While several possible explanations are offered for this p h e n o m e n o n , the mechanism is not yet understood.
A number of compounds have been shown to be potent inhibitors of brain or adrenal phenylethanolamine-N-methyltransferase (PNMT) (EC 2.1.1.28) in vitro. SKF 64139 (7,8-dichloro-l,2,3,4-tetrahydroisoquinoline) has been demonstrated to be a potent inhibitor of P N M T [8-10] and to deplete brain epinephrine [11]. Both the 2,3-dichloro- (DCMB) and 2-chloro-3-trifluoromethyl-c~-methylbenzylamine (CTFMB) compounds have been shown to be potent in vitro and in vivo P N M T inhibitors [1, 3] and to deplete hypothalamic epinephrine [2]. These compounds appear to be the most frequently used 'in vivo' inhibitors of PNMT. The effect of acute administration of these three compounds on catecholamine concentrations in the hypothalamus and the cell body areas of CI and C2 in the brain stem was studied. Male Sprague-Dawley rats, 200-250 g, were used throughout. Each PNMT inhibitor was dissolved in saline and animals were injected i.p. at 50 mg/kg body weight. Controls received a 1 ml injection of saline. After 2 h, animals were decapitated, brains removed and dissected and brain parts stored on dry ice. Catecholamine determinations were accomplished using high performance liquid chromatography (HPLC) with electrochemical detection as previously described [6]. Statistical analysis was performed using a two-way analysis of variance followed by post-hoc comparisons using Student's t-test.
68
The effects on norepinephrine levels seen after treatment with these three drugs are seen in Fig. 1. Only one region showed any effect. C T F M B lowered norepinephrine significantly in hypothalamus. As expected, all three compounds were generally effective in lowering epinephrine in various regions, as shown in Fig. 2. An exception was the effect of SKF 64139, which was ineffective in depleting 140
r--i SALINE SKF 64139
~ DCMB roll CTFMB
120
100
80
60
40
20
0 C2
C1
HYPOTHALAMUS
Fig. 1. Effects o f acute P N M T i n h i b i t i o n on rat brain n o r e p i n e p h r i n e levels, n = 8, control values were: C I , 717 _+ 30; C2, 2137 + 74; h y p o t h a l a m u s , 1368 _+ 77 ( n g / g wet wt). * P < 0.05; ** P < 0.01; *** P < 0.001.
140 r
/
i..-..1 SALINE IrA SKF 64139
1~3 DCMB lIB CTFMB
120 I
~,~
,..
~
60
a_
40
~
*
*
C2
C1
*
**
HYPOTHALAMUS
Fig. 2. Effects of acute P N M T i n h i b i t i o n on rat brain e p i n e p h r i n e levels, n = 8, control values were: C I , 21.7 _+ 3.2; C2, 28.5 _+ 3.5; h y p o t h a l a m u s , 10.2 +_ 2.3 ( n g / g wet wt). * P < 0.05; +* P < 0.01; *** P < 0.001.
69
epinephrine in the C2 area. This effect may be related to the potent monoamine oxidase inhibitory properties of the drug [7]. Perhaps the most intriguing results can be seen in Fig. 3. All three compounds were effective in increasing dopamine levels significantly in all the regions examined. It has previously been shown that acute administration of P N M T inhibitors does not lead to an increase in brain norepinephrine, even though epinephrine is decreased [1]. Typically, enzyme inhibition leads to an increase of substrate which does not appear to be the case in this instance. It is possible that due to the small fraction of total norepinephrine, which is transformed to epinephrine, P N M T inhibition might lead to an insignificant increase in norepinephrine. The significant elevation in the concentration of dopamine in all areas following acute P N M T inhibition is striking. There are several possible explanations for the results obtained. We have previously obtained evidence that one of the PNMT inhibitors, SKF 64139, is a potent monoamine oxidase (MAO) inhibitor in vivo, causing within 2 h of administration a 70% depletion of the dopamine metabolite, 3,4dihydroxyphenylacetic acid (DOPAC) [7]. One might speculate that the concomitant increase in dopamine was a result of MAO inhibition. However, only SKF 64139 appears to have MAO inhibition properties. The other two PNMT inhibitors used in this study, DCMB and CTFMB, do not cause depletion of D O P A C and, in fact, preliminary results suggest that CTFMB causes a slight increase in DOPAC concentrations. In spite of the MAO inhibition accompanying administration of SKF 64139, dopamine levels are not enhanced to a greater degree
16018°tosAi SK 4139 NE O" CTFM OCMB ~ ~4Ol-
j.
/
:
.
-
~ 100I FT"il [ 80 I-
° 1"
//l;~
/4I~lm C2
C1 HYPOTHALAMUS
Fig. 3. E f f e c t s o f a c u t e P N M T i n h i b i t i o n o n rat b r a i n d o p a m i n e levels, n = 8, c o n t r o l values were: C I , 61.8 _+ 3.6; ;C2, 167 _+ 6; h y p o t h a l a m u s , 251 _+ 8 . * P < 0 . 0 5 ; * * P < 0 . 0 1 ; * * * P < 0 . 0 0 1 .
70 than either of the other two drugs. Thus, there would appear to be evidence that the mechanism does not generally involve MAO inhibition. A decreased firing rate in dopamine neurons could also be responsible for an increase in dopamine levels; however, this would also be reflected by a decrease in D O P A C concentration, which does not occur. The evidence on this point is indirect but suggestive of a lack of effect through such a mechanism. Still a third possibility is that these compounds are weak dopamine-~-hydroxylase (DBH) inhibitors. If this were the case one might expect a decrease in norepinephrine concentrations associated with an increase in the concentration of dopamine and an increase in D O P A C concentrations. Only CTFMB caused depletion of norepinephrine. This was accompanied by the slightest amount of dopamine increase associated with any of the three compounds. DBH inhibition, if it occurred with CTFMB, is not a property associated with DCMB and SKF 64139. Another possible explanation for the results centers about the suggestion that epinephrine neurons exert a tonic inhibitory effect on dopamine neurons in the substantia nigra [4]. Depletion of epinephrine would lead to a decrease in this inhibition and perhaps an increase in dopamine turnover. This would be reflected in increased D O P A C levels and perhaps a decrease in dopamine concentrations, under acute conditions. This was not observed in the regions examined; however, such an effect might be very subtle. Another alternative, for which there is no evidence, would center about the speculation that N-methylation of dopamine has been inhibited, therefore causing a buildup of dopamine. This infers that epinephrine may be formed by an alternate pathway in brain instead of the synthetic pathway clearly demonstrated in adrenals (dopamine/norepinephrine/epinephrine). This alternate pathway has been suggested earlier [5] involving N-methylation of dopamine to epinine followed by/3hydroxylation to epinephrine, however the supporting data for this hypothesis was at that time found to be in error. The presence of epinine in brain has not been demonstrated and it is generally assumed n o t to be present. The mechanism underlying the increase in dopamine following acute P N M T inhibition is not presently known. Perhaps the consistent elevation of dopamine observed after acute administration of these P N M T inhibitors is coincidence which may be attributed to different properties of these three drugs. Nevertheless, we are assuming that a mechanism c o m m o n to all three of the P N M T inhibitors is involved. Perhaps the most likely explanation involves feed-back inhibition of tyrosine hydroxylase. With the inhibition of P N M T and resultant decline in concentration of epinephrine, there would be a release of feed-back inhibition of tyrosine hydroxylase. The increased tyrosine hydroxylase activity would lead to an increased concentration of dopamine. We are critically evaluating this and other alternative explanations. This information about P N M T inhibition may prove to be relevant in both pharmacological and behavioral studies using these drugs. Whether the dopamine is
71
neuroactive or released under these conditions is unclear. Whether the formed dopamine can act as a transmitter in epinephrine cells will also have to be evaluated. Nevertheless, it is clear that P N M T inhibition sets in motion changes which would have to be considered in evaluation of behavioral and pharmacological effects of such inhibition.
SKF 64139 was kindly provided by Dr. Robert Pendleton, Smith Kline and French. D C M B and CTFMB were provided by Dr. Ray Fuller, Eli Lilly. This work was supported by the O N R and MH 23861.
1
Fuller, R.W., Inhibitors of norepinephrine-N-methyltransferase. In E. Usdin, C. Creveling and R. Borchardt (Eds.), Transmethylation, Elsevier, Amsterdam, 1979, pp. 251-259. 2 Fuller, R.W. and Perry, K.W., Lowering of epinephrine concentration in rat brain by 2,3-dichloro-(,methylbenzylamine, an inhibitor of norepinephrine N-methyltransferase, Biochem. Pharmacol., 26 (1977) 2087 2090. 3 Fuller, R., Roush, B.W., Snoddy, H.D. and Molloy, B.B., Inhibition of phenylethanolamine-Nmethyltransferase by benzylamines. 2: In vitro and in vivo studies with 2,3-dichloro-c~-methyl benzylamine, J. med. Chem., 16(1973) 106 109. 4 Katz, R.J., Turner, B.B., Roth, K.A. and Carroll, B.J., Central adrenergic neurons as mediators of motivation and behavior - evidence from the specific inhibition of PNMT. In E. Usdin, 1. Kopin and J. Barchas (Eds.), Catecholamines: Basic and Clinical Frontiers, Vol. 11, Pergamon Press, 1979, pp. 1627-1689. 5 Laduron, P., N-methylation of dopamine to epinine in brain tissue using N-methyltetrahydrofolic acid as the methyl donor, Nature New Biol., 238 (1972) 212 213. 6 Mefford, I.N., Gilberg, M. and Barchas, J.D., Simultaneous determination of catecholamiues and 3,4-dihydroxyphenylacetic acid (DOPAC) in rat brain by HPLC with electrochemical detection, Analyt. Biochem., in press. 7 Mefford, I.N., Roth, K.A., Gilberg, M. and Barchas, J.D., In vivo monoamine oxidase inhibition in rat brain by SKF 64139 - comparison to other potent PNMT inhibitors, submitted. 8 Pendleton, R.G., Studies defining new pharmacological tools, inhibitors of phenylethanolamine-Nmethyltransferase (PNMT). In E. Usdin, C. Creveling and R. Borchardt (Eds.), Transmethylation, Elsevier, Amsterdam, 1979, pp. 261-268. 9 Pendleton, R.G., Kaiser, C. and Gessner, G., Studies on adrenal phenylethanotamine-Nmethyltransferase (PNMT) with SKF 64139, a selective inhibitor, J. Pharmacol. exp. Ther., 197 (1976) 623-632. 10 Pendleton, R.G., Khalsa, J., Gessver, G. and Sawyer, J., Studies on the characterization and inhibition of rat brain phenylethanolamine-N-methyltransferase,Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 299 (1977) 219 224. 11 Sauter, A.M., Lew, J.Y., Baba, Y. and Goldslein, M., Effect of phenylethanol N-methyltransferase and dopamine-t3-hydroxylase inhibition on epinephrine levels in the brain, Life Sci., 21 (1977) 261 266.
67
© Elsevier/North-Holland Scientific Publishers Ltd.
C H A N G E S IN R A T H Y P O T H A L A M I C A N D B R A I N STEM C A T E C H O L A M I N E C O N C E N T R A T I O N S F O L L O W I N G A C U T E A D M I N I S T R A T I O N OF PHENYLETHANOLAMINE-N-METHYLTRANSFERASE INHIBITORS
IVAN N. M E F F O R D , KEVIN A. ROTH and JACK D. B A R C H A S
Nancy Pritzker Laboratory of Behavioral Neurochernistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) (Received April 9th, 1980; Accepted July 3rd, 1980)
The effect of acute phenylethanolamine-N-methyltransferase (PNMT) inhibition on rat brain catecholamine concentrations was studied. All P N M T inhibitors tested were effective in depleting hypothalamic and brain stem epinephrine. Norepinephrine concentrations were unaffected; however, dopamine levels were increased in all areas studied. While several possible explanations are offered for this p h e n o m e n o n , the mechanism is not yet understood.
A number of compounds have been shown to be potent inhibitors of brain or adrenal phenylethanolamine-N-methyltransferase (PNMT) (EC 2.1.1.28) in vitro. SKF 64139 (7,8-dichloro-l,2,3,4-tetrahydroisoquinoline) has been demonstrated to be a potent inhibitor of P N M T [8-10] and to deplete brain epinephrine [11]. Both the 2,3-dichloro- (DCMB) and 2-chloro-3-trifluoromethyl-c~-methylbenzylamine (CTFMB) compounds have been shown to be potent in vitro and in vivo P N M T inhibitors [1, 3] and to deplete hypothalamic epinephrine [2]. These compounds appear to be the most frequently used 'in vivo' inhibitors of PNMT. The effect of acute administration of these three compounds on catecholamine concentrations in the hypothalamus and the cell body areas of CI and C2 in the brain stem was studied. Male Sprague-Dawley rats, 200-250 g, were used throughout. Each PNMT inhibitor was dissolved in saline and animals were injected i.p. at 50 mg/kg body weight. Controls received a 1 ml injection of saline. After 2 h, animals were decapitated, brains removed and dissected and brain parts stored on dry ice. Catecholamine determinations were accomplished using high performance liquid chromatography (HPLC) with electrochemical detection as previously described [6]. Statistical analysis was performed using a two-way analysis of variance followed by post-hoc comparisons using Student's t-test.
68
The effects on norepinephrine levels seen after treatment with these three drugs are seen in Fig. 1. Only one region showed any effect. C T F M B lowered norepinephrine significantly in hypothalamus. As expected, all three compounds were generally effective in lowering epinephrine in various regions, as shown in Fig. 2. An exception was the effect of SKF 64139, which was ineffective in depleting 140
r--i SALINE SKF 64139
~ DCMB roll CTFMB
120
100
80
60
40
20
0 C2
C1
HYPOTHALAMUS
Fig. 1. Effects o f acute P N M T i n h i b i t i o n on rat brain n o r e p i n e p h r i n e levels, n = 8, control values were: C I , 717 _+ 30; C2, 2137 + 74; h y p o t h a l a m u s , 1368 _+ 77 ( n g / g wet wt). * P < 0.05; ** P < 0.01; *** P < 0.001.
140 r
/
i..-..1 SALINE IrA SKF 64139
1~3 DCMB lIB CTFMB
120 I
~,~
,..
~
60
a_
40
~
*
*
C2
C1
*
**
HYPOTHALAMUS
Fig. 2. Effects of acute P N M T i n h i b i t i o n on rat brain e p i n e p h r i n e levels, n = 8, control values were: C I , 21.7 _+ 3.2; C2, 28.5 _+ 3.5; h y p o t h a l a m u s , 10.2 +_ 2.3 ( n g / g wet wt). * P < 0.05; +* P < 0.01; *** P < 0.001.
69
epinephrine in the C2 area. This effect may be related to the potent monoamine oxidase inhibitory properties of the drug [7]. Perhaps the most intriguing results can be seen in Fig. 3. All three compounds were effective in increasing dopamine levels significantly in all the regions examined. It has previously been shown that acute administration of P N M T inhibitors does not lead to an increase in brain norepinephrine, even though epinephrine is decreased [1]. Typically, enzyme inhibition leads to an increase of substrate which does not appear to be the case in this instance. It is possible that due to the small fraction of total norepinephrine, which is transformed to epinephrine, P N M T inhibition might lead to an insignificant increase in norepinephrine. The significant elevation in the concentration of dopamine in all areas following acute P N M T inhibition is striking. There are several possible explanations for the results obtained. We have previously obtained evidence that one of the PNMT inhibitors, SKF 64139, is a potent monoamine oxidase (MAO) inhibitor in vivo, causing within 2 h of administration a 70% depletion of the dopamine metabolite, 3,4dihydroxyphenylacetic acid (DOPAC) [7]. One might speculate that the concomitant increase in dopamine was a result of MAO inhibition. However, only SKF 64139 appears to have MAO inhibition properties. The other two PNMT inhibitors used in this study, DCMB and CTFMB, do not cause depletion of D O P A C and, in fact, preliminary results suggest that CTFMB causes a slight increase in DOPAC concentrations. In spite of the MAO inhibition accompanying administration of SKF 64139, dopamine levels are not enhanced to a greater degree
16018°tosAi SK 4139 NE O" CTFM OCMB ~ ~4Ol-
j.
/
:
.
-
~ 100I FT"il [ 80 I-
° 1"
//l;~
/4I~lm C2
C1 HYPOTHALAMUS
Fig. 3. E f f e c t s o f a c u t e P N M T i n h i b i t i o n o n rat b r a i n d o p a m i n e levels, n = 8, c o n t r o l values were: C I , 61.8 _+ 3.6; ;C2, 167 _+ 6; h y p o t h a l a m u s , 251 _+ 8 . * P < 0 . 0 5 ; * * P < 0 . 0 1 ; * * * P < 0 . 0 0 1 .
70 than either of the other two drugs. Thus, there would appear to be evidence that the mechanism does not generally involve MAO inhibition. A decreased firing rate in dopamine neurons could also be responsible for an increase in dopamine levels; however, this would also be reflected by a decrease in D O P A C concentration, which does not occur. The evidence on this point is indirect but suggestive of a lack of effect through such a mechanism. Still a third possibility is that these compounds are weak dopamine-~-hydroxylase (DBH) inhibitors. If this were the case one might expect a decrease in norepinephrine concentrations associated with an increase in the concentration of dopamine and an increase in D O P A C concentrations. Only CTFMB caused depletion of norepinephrine. This was accompanied by the slightest amount of dopamine increase associated with any of the three compounds. DBH inhibition, if it occurred with CTFMB, is not a property associated with DCMB and SKF 64139. Another possible explanation for the results centers about the suggestion that epinephrine neurons exert a tonic inhibitory effect on dopamine neurons in the substantia nigra [4]. Depletion of epinephrine would lead to a decrease in this inhibition and perhaps an increase in dopamine turnover. This would be reflected in increased D O P A C levels and perhaps a decrease in dopamine concentrations, under acute conditions. This was not observed in the regions examined; however, such an effect might be very subtle. Another alternative, for which there is no evidence, would center about the speculation that N-methylation of dopamine has been inhibited, therefore causing a buildup of dopamine. This infers that epinephrine may be formed by an alternate pathway in brain instead of the synthetic pathway clearly demonstrated in adrenals (dopamine/norepinephrine/epinephrine). This alternate pathway has been suggested earlier [5] involving N-methylation of dopamine to epinine followed by/3hydroxylation to epinephrine, however the supporting data for this hypothesis was at that time found to be in error. The presence of epinine in brain has not been demonstrated and it is generally assumed n o t to be present. The mechanism underlying the increase in dopamine following acute P N M T inhibition is not presently known. Perhaps the consistent elevation of dopamine observed after acute administration of these P N M T inhibitors is coincidence which may be attributed to different properties of these three drugs. Nevertheless, we are assuming that a mechanism c o m m o n to all three of the P N M T inhibitors is involved. Perhaps the most likely explanation involves feed-back inhibition of tyrosine hydroxylase. With the inhibition of P N M T and resultant decline in concentration of epinephrine, there would be a release of feed-back inhibition of tyrosine hydroxylase. The increased tyrosine hydroxylase activity would lead to an increased concentration of dopamine. We are critically evaluating this and other alternative explanations. This information about P N M T inhibition may prove to be relevant in both pharmacological and behavioral studies using these drugs. Whether the dopamine is
71
neuroactive or released under these conditions is unclear. Whether the formed dopamine can act as a transmitter in epinephrine cells will also have to be evaluated. Nevertheless, it is clear that P N M T inhibition sets in motion changes which would have to be considered in evaluation of behavioral and pharmacological effects of such inhibition.
SKF 64139 was kindly provided by Dr. Robert Pendleton, Smith Kline and French. D C M B and CTFMB were provided by Dr. Ray Fuller, Eli Lilly. This work was supported by the O N R and MH 23861.
1
Fuller, R.W., Inhibitors of norepinephrine-N-methyltransferase. In E. Usdin, C. Creveling and R. Borchardt (Eds.), Transmethylation, Elsevier, Amsterdam, 1979, pp. 251-259. 2 Fuller, R.W. and Perry, K.W., Lowering of epinephrine concentration in rat brain by 2,3-dichloro-(,methylbenzylamine, an inhibitor of norepinephrine N-methyltransferase, Biochem. Pharmacol., 26 (1977) 2087 2090. 3 Fuller, R., Roush, B.W., Snoddy, H.D. and Molloy, B.B., Inhibition of phenylethanolamine-Nmethyltransferase by benzylamines. 2: In vitro and in vivo studies with 2,3-dichloro-c~-methyl benzylamine, J. med. Chem., 16(1973) 106 109. 4 Katz, R.J., Turner, B.B., Roth, K.A. and Carroll, B.J., Central adrenergic neurons as mediators of motivation and behavior - evidence from the specific inhibition of PNMT. In E. Usdin, 1. Kopin and J. Barchas (Eds.), Catecholamines: Basic and Clinical Frontiers, Vol. 11, Pergamon Press, 1979, pp. 1627-1689. 5 Laduron, P., N-methylation of dopamine to epinine in brain tissue using N-methyltetrahydrofolic acid as the methyl donor, Nature New Biol., 238 (1972) 212 213. 6 Mefford, I.N., Gilberg, M. and Barchas, J.D., Simultaneous determination of catecholamiues and 3,4-dihydroxyphenylacetic acid (DOPAC) in rat brain by HPLC with electrochemical detection, Analyt. Biochem., in press. 7 Mefford, I.N., Roth, K.A., Gilberg, M. and Barchas, J.D., In vivo monoamine oxidase inhibition in rat brain by SKF 64139 - comparison to other potent PNMT inhibitors, submitted. 8 Pendleton, R.G., Studies defining new pharmacological tools, inhibitors of phenylethanolamine-Nmethyltransferase (PNMT). In E. Usdin, C. Creveling and R. Borchardt (Eds.), Transmethylation, Elsevier, Amsterdam, 1979, pp. 261-268. 9 Pendleton, R.G., Kaiser, C. and Gessner, G., Studies on adrenal phenylethanotamine-Nmethyltransferase (PNMT) with SKF 64139, a selective inhibitor, J. Pharmacol. exp. Ther., 197 (1976) 623-632. 10 Pendleton, R.G., Khalsa, J., Gessver, G. and Sawyer, J., Studies on the characterization and inhibition of rat brain phenylethanolamine-N-methyltransferase,Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 299 (1977) 219 224. 11 Sauter, A.M., Lew, J.Y., Baba, Y. and Goldslein, M., Effect of phenylethanol N-methyltransferase and dopamine-t3-hydroxylase inhibition on epinephrine levels in the brain, Life Sci., 21 (1977) 261 266.