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Imipramine demethylation and norepinephrine storage in brain
EUROPEAN JOURNAL OF PHARMACOLOGY 10 (1970) 355-359. NORTH-HOLLAND PUBLISHING COMPANY
IMIPRAMINE
DEMETHYLATION
AND NOREPINEPHRINE
STORAGE
IN BRAIN
G. STILLE and W. MICHAELIS Research Institute Dr. A. Wander S.A., Berne, Switzerland
Accepted 13 February 1970
Received 13 January 1970
G. STILLE and W. MICHAELIS, hnipramine demethylation and norepinephrine storage in brain, European J. Pharmacol. 10 (1970) 355-359. Imipramine administered i.p. in rats following intraventricular administration of DL-NE-73H decreases the amount of unchanged NE in the brain and leads to a significant increase of normetanephrine, methylated acid and methylated glycol. The results are the same if demethylation of imipramine is inhibited by pretreatment with SKF 525-A. According to current findings, imipramine itself has an effect on the storage capacity of brain tissue for NE. Imipramine Intraventricular administration
1. INTRODUCTION Brodie et al. (1961a, 1962) have shown that the compulsive behaviour seen in rats injected with reserpine-like substances following pretreatment with imiprmnine is effected by the desmethylimipramine * formed in the body. However, there are several other effects o f imipramine caused, not by the demethylation product, but by the substance itself. Firstly there is the general sedative effect which, in animals as well as in man, is more pronounced with imipramine than with DMI (Brodie et al., 1961b; Stifle et al., 1965; Mety~ovfi and Metyg, 1965). This correlates with the fact that imipramine, as opposed to DMI, raises the threshold
* The following abbreviations are used: DMI = Desmethylimipramine NE = Norepinephrine NM = Normetanephrinc DHMA = 3,4-Dihydroxymandelic acid DHPG = 3,4-Dihydroxyphenylglycol MHMA = 3-Methoxy-4-hydroxymandelic acid MHPG = 3-Methoxy-4-hydroxyphenylglycol MHPG-SO4 = 3-Methoxy-4-hydroxyphenylglycol-4-O-sulfate DHPH-SO4 = 3,4-Dihydroxyphenylglycol-sulfate 5-HT = 5-Hydroxytryptamine
NE-storage
Demethylation SKF-525 A
for the afterdischarge following cortical stimulation in rabbits (Stille and Sayers, 1964). The same applies for the anticonvulsive effect in mice (Garattini et al., 1962). The anticholinergic effect of imipramine is also stronger than that o f DMI (Loew and Taeschler, 1964). In man, the effect of DMI on the psychomotoric parameters chosen for evaluation was weaker than that o f imipramine (Di Mascio et al., 1964; Ban and Lehman, 1962). A notable difference between imipramine and DMI in regard to their action on the biogenic amines was discovered by Fuxe and Ungerstedt (1968) during histochemical investigations. Imipramine blocked the amine-uptake on the nervecell membranes o f the 5-HT neurones, whereas DMI had a corresponding effect on the NE neurones. In a previous publication we have shown that formation o f DMI is not necessary for the antagonism by imipramine of the catalepsy and ptosis induced by tetrabenazine (Michaelis and Stille, 1968). Under our experimental conditions the tetrabenazine catalepsy and ptosis were completely inhibited although hardly a trace of DM1 appeared in the brain. Considerable inhibition of the imipramine demethylation, as measured b y the appearance of DMI in the liver, occurred after administration o f 80 mg/ kg SKF 525-A p.o. Inhibition o f the breakdown of
356
G.Stille, W.Michaelis,Imipramine demethylation and norepinephrine storage in brain
imipramine by SKF 525-A, in vivo as well as in vitro, has been confirmed by Bickel and Weder (1968) and Jori and Bernardi (1968). In the experiments of Jori and Bernardi (1968), SKF 525-A completely inhibited the antagonism of imipramine toward the reserpine hypothermia "although traces of DMI could be detected in the brain. In our experiments with animals pretreated with SKF 525-A, containing no DMI in their brain, imipramine produced the usual antagonism of the tetrabenazine effects. The two tests thus give contradictory statements concerning the significance of the imipramine demethylation for the antagonism of reserpine and reserpine-like substances. The results of Jori and Bernardi (1968) suggest that in our experiments the antagonism toward the tetrabenazine catalepsy and ptosis is due to the anticholinergic action of imipramine itself, whereas the formation of DMI is necessary for the NE potentiation and related effects. In order to throw more light on the problem the extent to which SKF 525-A influenced the effect of imipramine on the storage of NE and the distribution of NE metabolites in the brain in vivo (Glowinski and Axelrod, 1964) was investigated.
2. METHODS NE was administered according to the method originally described by Milhaud and Glowinski (1962, 1963); preparation of the brains was based on the method of Giese, Ruether and Matussek (1967). 2.1. Treatment o f the animals Groups of 6 female Holtzmann rats ( 2 0 0 - 2 5 0 g) were used for all experiments. Imipramine (40 mg/kg i.p.) was administered 1 hr, and SKF 525-A (80 rag/ kg p.o.) 2 hr before NE. The animals were anaesthetized with sodium pentobarbital ( 3 0 - 3 5 mg]kg i.p.) and were placed in a stereotaxic instrument (KriegStoelting). After the skull had been exposed, fine holes were bored 1 mm anterior to the bregma and 1.3 mm to the side of the saggital suture. The glass capillary used for injection was lowered 4 mm into the brain, and by means of a microsyringe 0.33/ag 3H-NE in 20/al (DL-NE-7-3H, 1 mCi/ml; New England Corp., USA) was injected into a lateral ventricle. The experimental solution was investigated chromato-
graphically before and after its use and found to be over 98% pure. After 1 hr the animals were decapitated. The brains were removed, frozen on dry ice, and stored at -30°C until extraction. It is important that all traces of blood are removed from the brains, as this can interfere with the further treatment. 2.2. Extraction o f the brains The work was carried out in an ice-bath at 0°C and all of the extracts were stored at 0°C. The frozen brains were treated singly. Each brain was weighed and homogenized with 1.0 ml ice-cold 0.4 N perchloric acid (Polytron homogenizer). The resulting homogenate was centrifuged for 15 rain at 10,000 g and 0°C. The supernatant liquid was removed and stored at 0°C (extract 1). The deposit was further extracted with two 1 ml portions of perchloric acid as above (extracts 2 and 3). 2.3. Measurem en t o f activity The radioactivity of all the extracts and residues was determined by liquid-scintillation counting (Tri Carb 314 EX, Packard Instr. Comp., USA). Evaluation of the radiochromatograms was carried out using a flow counter with impulse amplifier VP 200 spec. (Laboratory Prof. Berthold, West Germany) and a "Precision Ratemeter", series 260 (Packard Instr. Comp.). The brains contained between 27 and 48% of the NE administered, and of this amount between 80 and 85% was extracted and investigated chromatographically.
2.4. Thin-layer chromatography 4 N KOH was added to the extracts until pH 2.5 (controlled by a pH-meter), and the precipitated KC104 removed by centrifuging. The supernatant liquid was divided into aliquots of 100-300 ~tl (according to activity) and 25/~g of each of the following substances was added to each ,aliquot: NE, NM, DHMA, DHPG, MHPG (Calbiochem., USA). The solutions were applied to the plates in the form of a 5 - 6 cm line, dried with nitrogen, and immediately chromatographed (Antec plates, Cellulose MN 300, butan-l-ol 5 N acetic acid (100: 35)). The activity lying between the start and NE according to Schanberg et at. (1968) and Good'all and Alton (1968), is due to sulphuric acid conjugates of DHPG and MHPG. This was confirmed by enzymatic splitting
G.Stille, W.Michaelis, Imipramine demethylation and norepinephrine storage in brain
with phenolsulfatase (Sigma Chem. Corp., USA) and rechromatography. 2.5. Evaluation Following measurement of the radiochromatograms with the flow counter, the reference compounds were stained with Gibbs' reagent and the chromatograms were divided into the zones shown in table 1. The proportional distribution o f the metabolites was calculated from the peak-areas. In several control experiments the radioactive zones were carefully removed, suspended in thixotropic gel (Cab-osil), and counted in the liquid-scintillation counter. The results so obtained were in good agreement with those obtained b y peak-area measurement.
3. RESULTS The amounts o f NE and the NE metabolites in the
357
radiochromatograms from the various treatment groups, expressed as percentages o f the total activity, are summarized in table 1. The effect o f imipramine (40 mg/kg i.p.) is clear-cut and the values for NE and its unconjugated metabolites differ significantly (ttest) from those o f the controls ( p < 0 . 0 1 < 0.005). The conjugate fraction showed no variation between the groups. Apart from decreasing the amount of unchanged NE, imipramine led to a significant increase in normetanephrine, methylated acid and methylated glycol. The non-methylated metabolites were significantly decreased. Following treatment with SKF 525-A (80 mg/kg p.o.) and imipramine (40 mg/kg i.p.) the results were as above, and there was no significant difference between the imipramine-treated animals with and without pretreatment with SKF 525-A. Treatment with SKF 525-A alone also caused no significant difference in the proportions o f NE and its metabolites in comparison to the controls.
Table 1 Percentages of 3H-NE and its metabolites in the radiochromatograms of the brain extracts (average values from 6 rats) 1 hr after intraventricular injection of 0.33 ~tg 3H-NE. Treatment group
Imipramine SKF 525-A mg/kg ip. mg/kg po. %
Start p*
p**
%
2.8
-
n.s.
26.8
-
2.0
tl.s.
29.1
n.s.
80
1.8
n.s.
n.s.
25.6
n.s.
n.s.
38.1 <0.005
= 0.05
80
1.8
n.s.
n.s.
27.5
n.s.
n.s.
44.1 n.s.
<0.01
1
-
2
40
3
40
-
4
Radiochromatogram DHPG-SO4/MHPG-SO 4 p. ~**
NM group
Imipramine SKF .525-A mg/kg ip. mg/kg po. %
1
p**
46.6
-
<0.005
34.5
<0.005
-
DHMA/DHPG
p*
p**
% 4.0
p*
MHMA/MHPG p**
%
p*
p**
<0.005
11.0
-
<0.01
-
<0.005
16.5
< 0.005
-
1.3
16.1 <0.01
-
80
16.5
<0.005
n.s.
1.3
n.s.
16.5 <0.01
n.s.
80
8.8
n.s.
<0.005
3.7
n.s.
14.1 n.s.
n.s.
-
2
40
-
3
40
4
8.7
n.s.
NE p,
%
* p compared with group 1 ** p compared with group 2 *** n.s. = not significant.
358
G.Stille, W.Michaelis, Irnipramine demethylation and norepinephrine storage in brain
4. DISCUSSION The proportions of NE and its metabolites in rat brain 1 hr after intraventricular application of 3 H-NE give a good indication of the effect of previously administered drugs on the kinetics o f NE and particularly o f the storage capacity of the tissue for this amine (Giese, Ruether and Matussek, 1967). One objection to this method is the fact that NE, when injected into tire ventricle, hardly penetrates into the brain tissue, as shown by the histochemical studies o f Fuxe and Ungerstedt (1968). In spite o f this, investigations with various drugs have given results of good reproducibility, so that the method can be used at least for obtaining general information. This method for the detection of cerebral storage inhibition has previously been used by Glowinski and Axelrod (1964), Glowinski et al. (1966), Giese et al. (1967), Stille (1968), Stille et al. (1968), and has been used for these current studies. Imipramine (40 mg/kg i.p.) caused a significant decrease in the storage capacity for NE in comparison to the control values, and, as an expression of an increased extr~meuronal metabolism with increased O-methylation, also caused a considerable increase o f NM, MttMA and MHPG. The decisive point for our investigations is, however, the fact that pretreatment with SKF 525-A is without influence on the changes in the spectrum of NE and its metabolites caused by imipramine, i.e. it is apparently without effect on the NE storage inhibition caused by imipramine. However, as our previous experiments have shown (Michaelis and Stille, 1968), and as has been demonstrated by other workers (Bickel and Weder, 1968: Jori and Bernardi, 1968), SKF 525-A considerably inhibits the demethylation o f imipramine, so that the demethylation product is not to be found in the brain, or at most only in traces. In consideration of the above findings it must be assumed that demethylation of imipramine has little or no bearing on the NE storage inhibition. This is in agreement with our previous findings (Michaelis and Stille, 1968) showing that the antagonism o f imipramine toward the tetrabenazine catalepsy and ptosis is independent o f the appearance of DMI in the brain. We have no explanation for the contradictory findings of Jori and Bernardi (1968) in respect to the reserpine hypothermia. It must be said, however, that imipramine itself, without formation of the deme-
thylation product, can also raise the temperature of reserpinized rats, as Bernardi et al. (1966) have shown by intraventricular injection of the substance. Dingell et al. (1964) have shown that no demethylation of imipramine worth mentioning takes place in the brain. This makes it all the more surprising that an inhibition of demethylation by SKF 525-A blocks the effect of imipramine on the reserpine hypothermia. However, according to the current findings imipramine itself has an effect on the storage capacity o f the tissue for NE, as indeed indicated by the earlier experiments of Dengler and Titus (1961) using brain slices. As Dingell et al. (1964) have shown, no demethylation can be expected to take place in brain slices.
ACKNOWLEDGEMENTS The authors wish to acknowledge the technical assistance of Mr. C. Niemitz and Mr. A. Sayers.
REFERENCES Ban, T.A. and H.E. Lehmann, 1962, Clinical trial with desmethylimipramine, a new antidepressant compound, Can. Med. Assoc. J. 86, 1030 1031. Bernardi, D., A. Jori, P. Morselli, L. Valzelli and S. Garattini, 1966, Further effects of imipramine and its desmethyl derivatives on the hypothermia induced by reserpine, J. Pharm. Pharmacol. 18,278 282. Bickel, M.tt. and lt.J. Weder, 1968, Demethylation of imipramine in the rat as influenced by SKF 525-A and by different routes of administration, Life Sci. 7. 12231230. Brodie, B.B., M.H. Bickel and F. Sulser, 1961a, Desmethylimipramine, a new type of antidepressant drug, Med. Exptl. 5,454 458. Brodie, B.B., P. Dick, P. Kielholz, W. Poeldinger and W. Theobald, 1961b, Preliminary pharmacological and clinical results with desmethylimipramine, a metabolite of imipramine, Psychopharmacologia 2,467 474. Dengler, N.J. and E.O. Titus, 1961, The effect of drugs on the uptake of isotopic norepinephrine in various tissues, Biochem. Pharmacol. 8, 64. Di Mascio, A., G. Heninger and G.L. Klerman, 1964, Psychopharmacology of imipramine and desipramine: A comparative study of their effects in normal males, Psychopharmacologia 5,367 371. Dingell, J.V., F. Sulser and J.R. Gillette, 1964, Species differences in the metabolism of imipramine and desme-
G.Stille, W.Michaelis, Imipramine demethylation and norepinephrine storage in brain thylimipramine (DMI), J. Pharmacol. Exptl. Therap. 143, 14-22. Fuxe, K. and U. Ungerstedt, 1968, Histochemical studies on the effect of (+)-amphetamine, drugs of the imipramine group and tryptamine on central catecholamine and 5-hydroxytryptamine neurons after intraventricular injection of catecholamines and 5-hydroxytryptamine, European J. Pharmacol. 4, 135-144. Garattini, S., A. Giachetti, A. Jori, L. Pieri and L. Valzelli, 1962, Effect of imipramine, antitriptyline and their monomethyl derivatives on reserpine activity, J. Pharm. Pharmacol. 1 4 , 5 0 9 - 5 1 4 . Giese, J., E. Ruethor and M. Matussek, 1967, Quantitative estimation of H3-norepinephrine and its metabolites by thin-layer chromatography, Life Sci. 6, 1975. Glowinski, J. and J. Axelrod, 1964, Inhibition of uptake of tritiated noradrenaline in the intact rat brain by imipramine and structurally related compounds, Nature 204, 1318-1319. Glowinski, J., J. Axelrod and L.L. Iversen, 1966, Effects of drugs on the disposition and metabolism of H3-norepine phrine and H3-dopamine, J. Pharmacol. 153, 3 0 - 4 1 . Goodall, M. and H. Alton, 1968, Metabolism of 3-hydroxytyramine (dopamine) in human subjects, Biochem. Pharmacol. 17,905. Jori, A. and D. Bernardi, 1968, Brain desipramine level in relation to the antireserpine activity of imipramine, J. Pharm. Pharmacol. 20, 9 5 5 - 9 5 6 . Loew, D. and M. Taeschler, 1964, Untersuchungen uber anticholinergische Wirkungen yon antidepressiv wirkenden Substanzen am Kaninchen, Helv. Physiol. Acta 22, C 133-136. Merck, E. AG, 1964, Anf'/ffbereagentien fiir Diinnschichtund Papierchromatographie (Darmstadt, 1964) p. 14. Mety~ov~, J. and J. Mety~, 1965, Pharmacological properties of the desmethyl derivatives of some antidepressants of imipramine type, Intern. J. Neuropharmacol. 4, 111-124.
359
Michaelis, W. and G. Stille, 1968, Demethylation of imipramine and antitetrabenazine effect in the catalepsy and ptosis test, Life Sci. 7, 99 106. Milhaud, G. and J. Glowinski, 1962, MOtabolisme de la dopamine-14C dans le cerveau du rat. F~tude du mode d'administration, Compt. Rend. Acad. Sci. 255, 203-205. Milhaud, G. and J. Glowinski, 1963, Metabolisme de la noradr~naline-laC dans le cerveau du rat, Compt. Rend. Acad. Sci. 256, 1033-1035. Schandberg, S.M. et al., 1968, Metabolism of normetanephrine-H 3 in rat brain. Identification of conjugated 3-methoxy-4-hydrophenylglycol as the major metabolite, Biochem. Pharmacol. 17,247. Stille, G. and A. Sayers, 1964, The effect of antidepressant drugs on the convulsive excitability of brain structures, Intern. J. Neuropharmacol. 3 , 6 0 5 - 6 0 9 . Stille, G., H. Lauener and E. Eichenberger, 1965, Ein pharmakologischer Vergleich klinisch gebr~iuchlicher Antidepressiva unter besonderer Berficksichtigung von Noveril (HF-1927 Wander), Schweiz Med. Wochschr. 95, 3 6 6 372. Stille, G., 1968, Pharmacological investigation of antidepressant compounds, Pharmacopsychiat. Neuro-Psychopharmacol. 1, 9 2 - 1 0 6 . Stille, G., H. Lauener, E. Eichenberger, N. Matussek and W. Poeldinger, 1968, Observations concerning adrenergic functions and antidepressant activity. Pharmacological, biochemical and clinical results with a new imidazoquinazoline derivative, Pharmacopsychiat. Neuro-Psychopharmacol. 1, 123-135. Sulser, F., J. Watts and B.B. Brodie, 1962, On the mechanisms of antidepressant action of imipraminelike drugs, Ann. N.Y. Acad. Sci. 9 6 , 2 7 9 - 2 8 6 .
IMIPRAMINE
DEMETHYLATION
AND NOREPINEPHRINE
STORAGE
IN BRAIN
G. STILLE and W. MICHAELIS Research Institute Dr. A. Wander S.A., Berne, Switzerland
Accepted 13 February 1970
Received 13 January 1970
G. STILLE and W. MICHAELIS, hnipramine demethylation and norepinephrine storage in brain, European J. Pharmacol. 10 (1970) 355-359. Imipramine administered i.p. in rats following intraventricular administration of DL-NE-73H decreases the amount of unchanged NE in the brain and leads to a significant increase of normetanephrine, methylated acid and methylated glycol. The results are the same if demethylation of imipramine is inhibited by pretreatment with SKF 525-A. According to current findings, imipramine itself has an effect on the storage capacity of brain tissue for NE. Imipramine Intraventricular administration
1. INTRODUCTION Brodie et al. (1961a, 1962) have shown that the compulsive behaviour seen in rats injected with reserpine-like substances following pretreatment with imiprmnine is effected by the desmethylimipramine * formed in the body. However, there are several other effects o f imipramine caused, not by the demethylation product, but by the substance itself. Firstly there is the general sedative effect which, in animals as well as in man, is more pronounced with imipramine than with DMI (Brodie et al., 1961b; Stifle et al., 1965; Mety~ovfi and Metyg, 1965). This correlates with the fact that imipramine, as opposed to DMI, raises the threshold
* The following abbreviations are used: DMI = Desmethylimipramine NE = Norepinephrine NM = Normetanephrinc DHMA = 3,4-Dihydroxymandelic acid DHPG = 3,4-Dihydroxyphenylglycol MHMA = 3-Methoxy-4-hydroxymandelic acid MHPG = 3-Methoxy-4-hydroxyphenylglycol MHPG-SO4 = 3-Methoxy-4-hydroxyphenylglycol-4-O-sulfate DHPH-SO4 = 3,4-Dihydroxyphenylglycol-sulfate 5-HT = 5-Hydroxytryptamine
NE-storage
Demethylation SKF-525 A
for the afterdischarge following cortical stimulation in rabbits (Stille and Sayers, 1964). The same applies for the anticonvulsive effect in mice (Garattini et al., 1962). The anticholinergic effect of imipramine is also stronger than that o f DMI (Loew and Taeschler, 1964). In man, the effect of DMI on the psychomotoric parameters chosen for evaluation was weaker than that o f imipramine (Di Mascio et al., 1964; Ban and Lehman, 1962). A notable difference between imipramine and DMI in regard to their action on the biogenic amines was discovered by Fuxe and Ungerstedt (1968) during histochemical investigations. Imipramine blocked the amine-uptake on the nervecell membranes o f the 5-HT neurones, whereas DMI had a corresponding effect on the NE neurones. In a previous publication we have shown that formation o f DMI is not necessary for the antagonism by imipramine of the catalepsy and ptosis induced by tetrabenazine (Michaelis and Stille, 1968). Under our experimental conditions the tetrabenazine catalepsy and ptosis were completely inhibited although hardly a trace of DM1 appeared in the brain. Considerable inhibition of the imipramine demethylation, as measured b y the appearance of DMI in the liver, occurred after administration o f 80 mg/ kg SKF 525-A p.o. Inhibition o f the breakdown of
356
G.Stille, W.Michaelis,Imipramine demethylation and norepinephrine storage in brain
imipramine by SKF 525-A, in vivo as well as in vitro, has been confirmed by Bickel and Weder (1968) and Jori and Bernardi (1968). In the experiments of Jori and Bernardi (1968), SKF 525-A completely inhibited the antagonism of imipramine toward the reserpine hypothermia "although traces of DMI could be detected in the brain. In our experiments with animals pretreated with SKF 525-A, containing no DMI in their brain, imipramine produced the usual antagonism of the tetrabenazine effects. The two tests thus give contradictory statements concerning the significance of the imipramine demethylation for the antagonism of reserpine and reserpine-like substances. The results of Jori and Bernardi (1968) suggest that in our experiments the antagonism toward the tetrabenazine catalepsy and ptosis is due to the anticholinergic action of imipramine itself, whereas the formation of DMI is necessary for the NE potentiation and related effects. In order to throw more light on the problem the extent to which SKF 525-A influenced the effect of imipramine on the storage of NE and the distribution of NE metabolites in the brain in vivo (Glowinski and Axelrod, 1964) was investigated.
2. METHODS NE was administered according to the method originally described by Milhaud and Glowinski (1962, 1963); preparation of the brains was based on the method of Giese, Ruether and Matussek (1967). 2.1. Treatment o f the animals Groups of 6 female Holtzmann rats ( 2 0 0 - 2 5 0 g) were used for all experiments. Imipramine (40 mg/kg i.p.) was administered 1 hr, and SKF 525-A (80 rag/ kg p.o.) 2 hr before NE. The animals were anaesthetized with sodium pentobarbital ( 3 0 - 3 5 mg]kg i.p.) and were placed in a stereotaxic instrument (KriegStoelting). After the skull had been exposed, fine holes were bored 1 mm anterior to the bregma and 1.3 mm to the side of the saggital suture. The glass capillary used for injection was lowered 4 mm into the brain, and by means of a microsyringe 0.33/ag 3H-NE in 20/al (DL-NE-7-3H, 1 mCi/ml; New England Corp., USA) was injected into a lateral ventricle. The experimental solution was investigated chromato-
graphically before and after its use and found to be over 98% pure. After 1 hr the animals were decapitated. The brains were removed, frozen on dry ice, and stored at -30°C until extraction. It is important that all traces of blood are removed from the brains, as this can interfere with the further treatment. 2.2. Extraction o f the brains The work was carried out in an ice-bath at 0°C and all of the extracts were stored at 0°C. The frozen brains were treated singly. Each brain was weighed and homogenized with 1.0 ml ice-cold 0.4 N perchloric acid (Polytron homogenizer). The resulting homogenate was centrifuged for 15 rain at 10,000 g and 0°C. The supernatant liquid was removed and stored at 0°C (extract 1). The deposit was further extracted with two 1 ml portions of perchloric acid as above (extracts 2 and 3). 2.3. Measurem en t o f activity The radioactivity of all the extracts and residues was determined by liquid-scintillation counting (Tri Carb 314 EX, Packard Instr. Comp., USA). Evaluation of the radiochromatograms was carried out using a flow counter with impulse amplifier VP 200 spec. (Laboratory Prof. Berthold, West Germany) and a "Precision Ratemeter", series 260 (Packard Instr. Comp.). The brains contained between 27 and 48% of the NE administered, and of this amount between 80 and 85% was extracted and investigated chromatographically.
2.4. Thin-layer chromatography 4 N KOH was added to the extracts until pH 2.5 (controlled by a pH-meter), and the precipitated KC104 removed by centrifuging. The supernatant liquid was divided into aliquots of 100-300 ~tl (according to activity) and 25/~g of each of the following substances was added to each ,aliquot: NE, NM, DHMA, DHPG, MHPG (Calbiochem., USA). The solutions were applied to the plates in the form of a 5 - 6 cm line, dried with nitrogen, and immediately chromatographed (Antec plates, Cellulose MN 300, butan-l-ol 5 N acetic acid (100: 35)). The activity lying between the start and NE according to Schanberg et at. (1968) and Good'all and Alton (1968), is due to sulphuric acid conjugates of DHPG and MHPG. This was confirmed by enzymatic splitting
G.Stille, W.Michaelis, Imipramine demethylation and norepinephrine storage in brain
with phenolsulfatase (Sigma Chem. Corp., USA) and rechromatography. 2.5. Evaluation Following measurement of the radiochromatograms with the flow counter, the reference compounds were stained with Gibbs' reagent and the chromatograms were divided into the zones shown in table 1. The proportional distribution o f the metabolites was calculated from the peak-areas. In several control experiments the radioactive zones were carefully removed, suspended in thixotropic gel (Cab-osil), and counted in the liquid-scintillation counter. The results so obtained were in good agreement with those obtained b y peak-area measurement.
3. RESULTS The amounts o f NE and the NE metabolites in the
357
radiochromatograms from the various treatment groups, expressed as percentages o f the total activity, are summarized in table 1. The effect o f imipramine (40 mg/kg i.p.) is clear-cut and the values for NE and its unconjugated metabolites differ significantly (ttest) from those o f the controls ( p < 0 . 0 1 < 0.005). The conjugate fraction showed no variation between the groups. Apart from decreasing the amount of unchanged NE, imipramine led to a significant increase in normetanephrine, methylated acid and methylated glycol. The non-methylated metabolites were significantly decreased. Following treatment with SKF 525-A (80 mg/kg p.o.) and imipramine (40 mg/kg i.p.) the results were as above, and there was no significant difference between the imipramine-treated animals with and without pretreatment with SKF 525-A. Treatment with SKF 525-A alone also caused no significant difference in the proportions o f NE and its metabolites in comparison to the controls.
Table 1 Percentages of 3H-NE and its metabolites in the radiochromatograms of the brain extracts (average values from 6 rats) 1 hr after intraventricular injection of 0.33 ~tg 3H-NE. Treatment group
Imipramine SKF 525-A mg/kg ip. mg/kg po. %
Start p*
p**
%
2.8
-
n.s.
26.8
-
2.0
tl.s.
29.1
n.s.
80
1.8
n.s.
n.s.
25.6
n.s.
n.s.
38.1 <0.005
= 0.05
80
1.8
n.s.
n.s.
27.5
n.s.
n.s.
44.1 n.s.
<0.01
1
-
2
40
3
40
-
4
Radiochromatogram DHPG-SO4/MHPG-SO 4 p. ~**
NM group
Imipramine SKF .525-A mg/kg ip. mg/kg po. %
1
p**
46.6
-
<0.005
34.5
<0.005
-
DHMA/DHPG
p*
p**
% 4.0
p*
MHMA/MHPG p**
%
p*
p**
<0.005
11.0
-
<0.01
-
<0.005
16.5
< 0.005
-
1.3
16.1 <0.01
-
80
16.5
<0.005
n.s.
1.3
n.s.
16.5 <0.01
n.s.
80
8.8
n.s.
<0.005
3.7
n.s.
14.1 n.s.
n.s.
-
2
40
-
3
40
4
8.7
n.s.
NE p,
%
* p compared with group 1 ** p compared with group 2 *** n.s. = not significant.
358
G.Stille, W.Michaelis, Irnipramine demethylation and norepinephrine storage in brain
4. DISCUSSION The proportions of NE and its metabolites in rat brain 1 hr after intraventricular application of 3 H-NE give a good indication of the effect of previously administered drugs on the kinetics o f NE and particularly o f the storage capacity of the tissue for this amine (Giese, Ruether and Matussek, 1967). One objection to this method is the fact that NE, when injected into tire ventricle, hardly penetrates into the brain tissue, as shown by the histochemical studies o f Fuxe and Ungerstedt (1968). In spite o f this, investigations with various drugs have given results of good reproducibility, so that the method can be used at least for obtaining general information. This method for the detection of cerebral storage inhibition has previously been used by Glowinski and Axelrod (1964), Glowinski et al. (1966), Giese et al. (1967), Stille (1968), Stille et al. (1968), and has been used for these current studies. Imipramine (40 mg/kg i.p.) caused a significant decrease in the storage capacity for NE in comparison to the control values, and, as an expression of an increased extr~meuronal metabolism with increased O-methylation, also caused a considerable increase o f NM, MttMA and MHPG. The decisive point for our investigations is, however, the fact that pretreatment with SKF 525-A is without influence on the changes in the spectrum of NE and its metabolites caused by imipramine, i.e. it is apparently without effect on the NE storage inhibition caused by imipramine. However, as our previous experiments have shown (Michaelis and Stille, 1968), and as has been demonstrated by other workers (Bickel and Weder, 1968: Jori and Bernardi, 1968), SKF 525-A considerably inhibits the demethylation o f imipramine, so that the demethylation product is not to be found in the brain, or at most only in traces. In consideration of the above findings it must be assumed that demethylation of imipramine has little or no bearing on the NE storage inhibition. This is in agreement with our previous findings (Michaelis and Stille, 1968) showing that the antagonism o f imipramine toward the tetrabenazine catalepsy and ptosis is independent o f the appearance of DMI in the brain. We have no explanation for the contradictory findings of Jori and Bernardi (1968) in respect to the reserpine hypothermia. It must be said, however, that imipramine itself, without formation of the deme-
thylation product, can also raise the temperature of reserpinized rats, as Bernardi et al. (1966) have shown by intraventricular injection of the substance. Dingell et al. (1964) have shown that no demethylation of imipramine worth mentioning takes place in the brain. This makes it all the more surprising that an inhibition of demethylation by SKF 525-A blocks the effect of imipramine on the reserpine hypothermia. However, according to the current findings imipramine itself has an effect on the storage capacity o f the tissue for NE, as indeed indicated by the earlier experiments of Dengler and Titus (1961) using brain slices. As Dingell et al. (1964) have shown, no demethylation can be expected to take place in brain slices.
ACKNOWLEDGEMENTS The authors wish to acknowledge the technical assistance of Mr. C. Niemitz and Mr. A. Sayers.
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