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The irreversible inhibition of monoamine oxidase in vivo
ARCHIVES
OF
BIOCHEMISTRY
The Irreversible
AND
BIOPHYSICS
Inhibition
77, 363-371 (1958)
of Monoamine Oxidase in Viva
A. N. Davison From
the
Department
of Pathology, London,
Guy’s England
Hospital
Medical
School,
Received January 29, 1953 INTRODUCTION
Recent work on the role of monoamine oxidase in the biological inactivation of 5-hydroxytryptamine, adrenaline, and the catechol amines has been facilitated by the use of enzyme inhibitors in tivo. Cholinep-tolyl ether bromide (TM6) and I-isonicotinoyl-2-isopropyl hydrazine phosphate (iproniazid or Marsilid) have been widely used for this purpose (1). It seemed interesting to compare the mechanisms of the inhibition reactions of these two compounds together with that of pentamidine, shown by Blaschko and Duthie (2) to inhibit monoamine oxidase irreversibly. METHODS
In vitro Studies Rat liver mitochondria were used as a source of monoamine oxidase in vitro. The methods employed for enzyme estimations and kinetic studies were as described previously (3).
In vivo Studies White mice were given intraperitoneal injections of inhibitor and killed by breaking their necks. Tissues were dissected out, washed, and dried on filter paper. The preparation from mouse intestine consisted of about 10 cm. of ileum washed free from contents and scraped on filter paper. The tissues were then chilled and homogenized in cold distilled water (about 10% w/v). The homogenates were centrifuged down at 8000 X g at 0”, and the particulate matter was resuspended in Sorensen’s 0.067 M phosphate buffer pH 7.2, so that 1 ml. contained the equivalent of 400 mg. brain, 250 mg. liver, or 250 mg. intestine, as wet tissue. Each flask contained 1 ml. of homogenate, 1.8 ml. of phosphate buffer with 0.2 ml. of 0.032 M KCN, and 0.2 ml. of 0.16 M tyramine in the side arm. After flushing with oxygen (2 min.), the flasks were allowed to equilibrate on the bath for 15 368
INHIBITION
OF MONOAMINE
369
OXIDASE
min. before adding cyanide and substrate. The oxygen uptake wasread every 5 min. for at least 35 min., and results were expressed as ~1. OJhr./g. of original wet tissue, corrected for a blank oxygen uptake. Choline-p-tolyl ether bromide was kindly given by Professor W. A. Bain and pentamidine and Baker Ltd., Dagenham, England.
isethionate
by May
RESULTS
Iproniazid is a competitive inhibitor of monoamine oxidase activity. In. vitro the inhibition is progressive and cannot easily be reversed (3). Table I shows that iproniazid also inhibits tissue monoamine oxidase activity in viva. Reactivation of the enzyme in vivo is slow (4). The method of Lineweaver and Burk (5) was used to determine the nature of the inhibition by pentamidine. The effect of addition of pentamidine (1O-4-1O-6 M) on the enzymic oxidation of different concentrations of tyramine clearly showed that inhibition was noncompetitive (Fig. 1). Inhibition of monoamine oxidase activity was immediate and showed no dependence on the length of incubation of pentamidine with the enzyme. Furthermore, the inhibition was irreversible, and sensitivity to the inhibitor was the same for monoamine oxidase of whole liver TABLE Znhibition
of Mouse
I
Monoamine
Ozidase
Mice are given drugs by intraperitoneal injection. tissues are dissected out and washed, and monoamine mined as described. Monoamine
__ Brain Liver Intestine
oxidase
--.~-
Animals are killed, the oxidase activity is deter-
activity,
pl. Oz/g./hr.
~- ~___.~ 156 f 1194 f 737 f
12 63 15
107 125 260
0 0 0
197 1140 380
4 hr. after 25 mg./kg.
Brain Liver Intestine
pentamidine
114 455 560
75 mg./kg.
pentamidine
100 12 -
100 mg./kg.
pentmidine
98 0 400
370
DAVISON
suspensions or mitochondria, in the presence or absence of cyanide. It was therefore concluded that pentamidine acts by an entirely different mechanism from that of iproniazid. The dissociation constant of the enzyme-inhibitor complex for pentamidine is 4 X 10e6 and for iproniazid 2 X 10e4. Thus the affinity of pentamidine for the enzyme is some 75 times as great as that of the tyramine (K,/Ki = 75). The affinity of iproniazid for monoamine oxidase is about 15 times as great as that of the substrate.
1.6
.8
0
250
500
750
1000
‘/cs,
FIG. 1. Noncompetitive inhibition of monoamine oxidase by pentamidine. In these experiments rat liver mitochondria (equivalent to 300 mg. of wet liver) were suspended in 0.067 M Sorensen’s phosphate pH 7.2. Suitable concentrations of tyramine and pentamidine were placed in the side arm of Warburg flasks with 1 ml. of mitochondria in the center. The volume in each flask was 3.2 ml. Bath temperature was 37”. Ten minutes after flushing with oxygen, substrate and inhibitor were added together and oxygen uptake was determined.
INHIBITION
OF
MONOAMINE
OXIDASE
371
Following injection of pentamidine into mice, liver monoamine oxidase is strongly inhibited but brain monoamine oxidase activity is only slightfly reduced. Recovery of enzyme activity is slow. On the other hand choline-p-tolyl ether is a competitive inhibitor of monoamine oxidase (6). Since it is active in vioo it was thought likely t’hat the inhibition was irreversible. However, when choline-p-tolyl ether was injected into mice (100 mg./kg.) and monoamine oxidase activity determined after 90 min., the slight inhibition found in a 25 % w/v liver suspension was easily reversed by dilution. Similarly, when mitochondria were completely inhibited in vitro (250 pg./ml.), a practically complete reversal of inhibition could be obtained by diluting tenfold with buffer, followed by sedimentation of the mitochondrin and redetermination of the enzyme activity. SUMMARY
These results confirm the conclusion of Corne and Graham (1) that iproniazid is superior to choline-p-tolyl ether as an inhibitor of monoamine oxidase in tivo. Furthermore, inhibition by pentamidine is noncompetitive, and in vivo brain monoamine oxidase activity is not easily inhibited. REFERENCES 1. 2. 3. 4. 5. 6.
CORNE, S. J., AND GRAHAM, J. D. P., J. Physiol. (London) 136, 339, (1957). BLASCHKO, H., AND DUTHIE, R., BiochenL. J. 39, 347 (1945). DAVISON, A. N., Biochem. J. 67, 316 (1957). DAVISON, A. N., LESSIN, A. W., AND PARKES, M. W., Ezperientia 13, 329 (1957). LINEWEAVER, H., AND BURK, D., J. Am. Chern. Sot. 66, 658 (1934). BROWN, B. G., AND HEY, P., &-it. J. PharmacoZ. 11, 58 (1956).
OF
BIOCHEMISTRY
The Irreversible
AND
BIOPHYSICS
Inhibition
77, 363-371 (1958)
of Monoamine Oxidase in Viva
A. N. Davison From
the
Department
of Pathology, London,
Guy’s England
Hospital
Medical
School,
Received January 29, 1953 INTRODUCTION
Recent work on the role of monoamine oxidase in the biological inactivation of 5-hydroxytryptamine, adrenaline, and the catechol amines has been facilitated by the use of enzyme inhibitors in tivo. Cholinep-tolyl ether bromide (TM6) and I-isonicotinoyl-2-isopropyl hydrazine phosphate (iproniazid or Marsilid) have been widely used for this purpose (1). It seemed interesting to compare the mechanisms of the inhibition reactions of these two compounds together with that of pentamidine, shown by Blaschko and Duthie (2) to inhibit monoamine oxidase irreversibly. METHODS
In vitro Studies Rat liver mitochondria were used as a source of monoamine oxidase in vitro. The methods employed for enzyme estimations and kinetic studies were as described previously (3).
In vivo Studies White mice were given intraperitoneal injections of inhibitor and killed by breaking their necks. Tissues were dissected out, washed, and dried on filter paper. The preparation from mouse intestine consisted of about 10 cm. of ileum washed free from contents and scraped on filter paper. The tissues were then chilled and homogenized in cold distilled water (about 10% w/v). The homogenates were centrifuged down at 8000 X g at 0”, and the particulate matter was resuspended in Sorensen’s 0.067 M phosphate buffer pH 7.2, so that 1 ml. contained the equivalent of 400 mg. brain, 250 mg. liver, or 250 mg. intestine, as wet tissue. Each flask contained 1 ml. of homogenate, 1.8 ml. of phosphate buffer with 0.2 ml. of 0.032 M KCN, and 0.2 ml. of 0.16 M tyramine in the side arm. After flushing with oxygen (2 min.), the flasks were allowed to equilibrate on the bath for 15 368
INHIBITION
OF MONOAMINE
369
OXIDASE
min. before adding cyanide and substrate. The oxygen uptake wasread every 5 min. for at least 35 min., and results were expressed as ~1. OJhr./g. of original wet tissue, corrected for a blank oxygen uptake. Choline-p-tolyl ether bromide was kindly given by Professor W. A. Bain and pentamidine and Baker Ltd., Dagenham, England.
isethionate
by May
RESULTS
Iproniazid is a competitive inhibitor of monoamine oxidase activity. In. vitro the inhibition is progressive and cannot easily be reversed (3). Table I shows that iproniazid also inhibits tissue monoamine oxidase activity in viva. Reactivation of the enzyme in vivo is slow (4). The method of Lineweaver and Burk (5) was used to determine the nature of the inhibition by pentamidine. The effect of addition of pentamidine (1O-4-1O-6 M) on the enzymic oxidation of different concentrations of tyramine clearly showed that inhibition was noncompetitive (Fig. 1). Inhibition of monoamine oxidase activity was immediate and showed no dependence on the length of incubation of pentamidine with the enzyme. Furthermore, the inhibition was irreversible, and sensitivity to the inhibitor was the same for monoamine oxidase of whole liver TABLE Znhibition
of Mouse
I
Monoamine
Ozidase
Mice are given drugs by intraperitoneal injection. tissues are dissected out and washed, and monoamine mined as described. Monoamine
__ Brain Liver Intestine
oxidase
--.~-
Animals are killed, the oxidase activity is deter-
activity,
pl. Oz/g./hr.
~- ~___.~ 156 f 1194 f 737 f
12 63 15
107 125 260
0 0 0
197 1140 380
4 hr. after 25 mg./kg.
Brain Liver Intestine
pentamidine
114 455 560
75 mg./kg.
pentamidine
100 12 -
100 mg./kg.
pentmidine
98 0 400
370
DAVISON
suspensions or mitochondria, in the presence or absence of cyanide. It was therefore concluded that pentamidine acts by an entirely different mechanism from that of iproniazid. The dissociation constant of the enzyme-inhibitor complex for pentamidine is 4 X 10e6 and for iproniazid 2 X 10e4. Thus the affinity of pentamidine for the enzyme is some 75 times as great as that of the tyramine (K,/Ki = 75). The affinity of iproniazid for monoamine oxidase is about 15 times as great as that of the substrate.
1.6
.8
0
250
500
750
1000
‘/cs,
FIG. 1. Noncompetitive inhibition of monoamine oxidase by pentamidine. In these experiments rat liver mitochondria (equivalent to 300 mg. of wet liver) were suspended in 0.067 M Sorensen’s phosphate pH 7.2. Suitable concentrations of tyramine and pentamidine were placed in the side arm of Warburg flasks with 1 ml. of mitochondria in the center. The volume in each flask was 3.2 ml. Bath temperature was 37”. Ten minutes after flushing with oxygen, substrate and inhibitor were added together and oxygen uptake was determined.
INHIBITION
OF
MONOAMINE
OXIDASE
371
Following injection of pentamidine into mice, liver monoamine oxidase is strongly inhibited but brain monoamine oxidase activity is only slightfly reduced. Recovery of enzyme activity is slow. On the other hand choline-p-tolyl ether is a competitive inhibitor of monoamine oxidase (6). Since it is active in vioo it was thought likely t’hat the inhibition was irreversible. However, when choline-p-tolyl ether was injected into mice (100 mg./kg.) and monoamine oxidase activity determined after 90 min., the slight inhibition found in a 25 % w/v liver suspension was easily reversed by dilution. Similarly, when mitochondria were completely inhibited in vitro (250 pg./ml.), a practically complete reversal of inhibition could be obtained by diluting tenfold with buffer, followed by sedimentation of the mitochondrin and redetermination of the enzyme activity. SUMMARY
These results confirm the conclusion of Corne and Graham (1) that iproniazid is superior to choline-p-tolyl ether as an inhibitor of monoamine oxidase in tivo. Furthermore, inhibition by pentamidine is noncompetitive, and in vivo brain monoamine oxidase activity is not easily inhibited. REFERENCES 1. 2. 3. 4. 5. 6.
CORNE, S. J., AND GRAHAM, J. D. P., J. Physiol. (London) 136, 339, (1957). BLASCHKO, H., AND DUTHIE, R., BiochenL. J. 39, 347 (1945). DAVISON, A. N., Biochem. J. 67, 316 (1957). DAVISON, A. N., LESSIN, A. W., AND PARKES, M. W., Ezperientia 13, 329 (1957). LINEWEAVER, H., AND BURK, D., J. Am. Chern. Sot. 66, 658 (1934). BROWN, B. G., AND HEY, P., &-it. J. PharmacoZ. 11, 58 (1956).