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A comparison of the inhibitory effects of new non-tricyclic amine uptake inhibitors on the uptake of norepinephrine and 5-hydroxytryptamine into synaptosomes of the rat brain
Neurophormmoloy~. Vol. 19. pp. 349 to 354 Pergamon Press Ltd 1980. PrInted m Great Britain
A COMPARISON OF THE INHIBITORY EFFECTS OF NEW NON-TRICYCLIC AMINE UPTAKE INHIBITORS ON THE UPTAKE OF NOREPINEPHRINE AND 5HYDROXYTRYPTAMINE INTO SYNAPTOSOMES OF THE RAT BRAIN T. KOIDE* and K. UYEMURA Department of Physiology, Saitama Medical School, Moroyama, Irumagun, Saitama, 350-04, Japan (Accepted 15 October 1979) Summary-The effects of new non-tricyclic amine uptake inhibitors, FS32 and FS97, on the uptake of [3H]-norepinephrine (NE) into the hypothalamic synaptosomes and [3H]-5-hydroxytryptamine (5-HT) into whole brain synaptosomes were studied. Their effects were compared with those of tricyclic antidepressants, The uptake of C3H]-NE was inhibited competitively by FS32 and FS97 with a respective Ki value of 6.5 x lo-’ M and 3.8 x lo-’ M. The potency of FS32 and FS97 to inhibit this uptake was almost comparable to that of clomipramine and imipramine, respectively. In the case of C3H]-5-HT uptake, FS32 and FS97 also showed competitive inhibition with a respective Ki value of 2.9 x 1O-6 M and 5.9 x 1O-6 M. The ability of FS32 to inhibit C3H]-5-HT uptake was almost equal to that of nortriptyline, while FS97 was two times more potent than iprindole in inhibiting this uptake.
During recent years growing support has been accumulating for the view that a decreased function of the neurotransmitter systems for norepinephrine (NE) and 5-hydroxytryptamine (5-HT) is a common feature of depressive illness (Schildkraut, 1965; Lapin and Oxenkrug, 1969; Van Praag and Korf, 1971; Asberg, Bertilsson, Tuck, Cronholm and SjBqvist, 1973; Schildkraut, 1973) and that the facilitation of NE and of 5-HT transmission is related to an increase in drive and in mood elevation, respectively. It was also reported that the therapeutic efficacy of tricyclic antidepressants, such as imipramine and desipramine, depends on the inhibition of the uptake of each amine into nerve endings, which allows the transmitters to remain at the receptor sites for a longer period (Carlsson, Corrodi, Fuxe and Hokfelt, 1969a; Carlsson, Corrodi, Fuxe and Hiikfelt, 1969b; Carlsson, 1970; Lidbrink. Jonsson and Fuxe, 1971). In the course of studying the pharmacological actions of a series of indazole derivatives, FS32 (l-[3-(Dimethylamino)propyl]-S-methyl-3-phenyl-lHindazole) and its N-desmethylated compound, FS97 (Fig. 1) were selected as a new group of antidepressive drugs, because they revealed a favourable therapeutic index in experimental animals and showed a uniform effect after oral administration (Ikeda, Takano, Matsushita, Shiraki. Koide, Nagashima, Fujimura,
Shindo. Suzuki and Iwasaki, 1979). As the nontricyclic structure in their chemical formula differs from the conventional tricyclic antidepressants, it is of interest to compare the neurochemical effects with those of common tricyclic antidepressants. In the present study, the effects of such drugs on C3H]-NE and C3H]-5-HT uptake into synaptosomes were examined comparatively. METHODS Tissue preparations Purtjied whole brain synaptosomes. Synaptosomes were prepared from rat whole brain essentially according to the method described by White (1975) with some modifications. All steps in the procedure were carried out in the cold (@4”C). A male SpragueDawley rat (weighing about 250 g) was killed by cervical dislocation and the whole brain was removed, chilled in 0.32 M-sucrose and homogenized by 15 strokes of a Teflon-glass homogenizer at 800 rpm. The
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Department of Pharmacology, * Present address: Research Laboratories of Chugai Pharmaceutical Company Ltd, No. 41-8, 3-chome, Takada, Toshima-ku, Tokyo, Japan. Key words: synaptosomes, tricyclic non-tricyclic amine uptake inhibitors.
antidepressants,
new
Fig. 1. Chemical 349
I
I
FS32 : R=CH3 697 : R=H
CH3 structure
of FS32 and FS97.
T. KOIDE and K. UYEMURA
350
homogenate was divided into two equal portions and centrifuged at 1000 g for 10 min, and the resulting supernatants were then centrifuged at 12,000g for 20 min. The final pellets were suspended in 0.32 Msucrose to give a total volume of 6 ml, and each 2 ml was layered on a discontinuous density gradient consisting of 5 ml of 0.8 M-sucrose on 5 ml of 1.2 Msucrose. The gradients were centrifuged at 150,000 g for 60 min and the interfaces between 0.8 M- and 1.2 M-sucrose were collected. The pooled fraction was diluted to 40 ml with 0.32 M-sucrose. Then the fraction was centrifuged at 20,000 g for 30 min. The resulting pellets were finally resuspended with a total volume of 3.0 ml of 0.32 M-sucrose and were used immediately as a synaptosomal fraction for the C3H]S-HT uptake study. This fraction contained a large number of intact synaptosomes as revealed by electron microscopy. Hypothalamic synaptosomes. The brain was removed as described above and the hypothalamus was dissected out in the cold according to the method of Glowinski and Iversen (1966). The tissue was gently homogenized in 30 vol of ice-coid 0.32 M-sucrose by 10 strokes of a Teflon-glass homogenizer at 800 rpm. The homogenate was centrifuged at 1000 g for 10 min, and the resulting supernatants were then centrifuged at 12,000 g for 20 min. The final pellets were suspended in 0.32 M-sucrose and were used immediately as the hypothalamic synaptosomes for C3H]-NE uptake study and releasing study. Uptake of amines Fifty pl of synaptosomal suspension was added to 450~1 of incubation medium containing various amounts of drugs and preincubated for 5 min at 37’C. Then 50 ~1 of C3H]-NE or C3H]-5-HT was added and the incubation was continued for another 5 min or 3 min for the uptake of c3H]-NE and C3H]-5-HT, respectively. Preliminary experiments indicated that the reaction proceeds linearly within the incubation times. The incubation medium contained; 130 mMNaCl, 3 mM-KCl, 2 mM-CaCl,, 2 mM--MgCl, and 20mM-Tris-HCI buffer (pH 7.4). Incubation was terminated by filtering 500 ~1 of the incubation mixture through a 0.45 pm pore-size membrane filter Table 1. Inhibition
of 13H]-NE
uptake
(TM-2, Toyo Roshi) by suction. The filter was washed with 10 ml of washing solution containing 260 mM---sucrose, 2 mM-CaCI,, 2 mM_MgCl, and 20 mM ~ Tris-HCI buffer (pH 7.4). Each membrane filter was dissolved in 10 ml of scintillating fluid containing lo”,, (v/v)-ethylene glycol monoethyl ether, 204; (v!v)-Triton X-100, 0.5% (w/v)-DPO and 0.03”/;, (w/v)-POPOP in xylene. The radioactivity was determined by liquid scintillation counter (LSC-653, Aloka). The actual moles of NE or 5-HT taken up/mg synaptosomal protein was obtained by subtracting the mol of each amine/mg protein at O’C (= uptake at 37 C minus uptake at 0°C). Any significant decrease in the uptake activity was not shown within the experimental periods in this study (about 2 hr after preparation). Release of [3H]-norepinephrine One volume of the hypothalamic synaptosomal suspension was incubated with ten volumes of the incubation medium containing C3H]-NE at 37°C for 20 min. After incubation, the mixture was centrifuged at 12,000 g for 20 min. The pellet was washed gently three times by resuspending in the incubation medium (omitting [‘HI-NE) and was centrifuged. After final washing, the radioactive synaptosomes were resuspended in the same volume of the incubation medium. Aliquots (500 ~1) of the final suspension were incubated with 50 ~1 of drug solution for 5, 10, 20 and 30 min at 37°C. After incubation, the mixture was passed through a 0.45 pm pore-size membrane filter and washed with 10ml of the washing solution. The amount of C3H]-NE remaining on the filter was counted as described above. Protein estimution The protein concentration of the synaptosomal suspension was determined according to the procedure described by Lowry, Rosebrough, Farr and Randall (1951). Chemicals The laboratories of Chugai Pharmaceutical Company Ltd synthesized FS32 and FS97 and these were used as the HCI salt in this study. DL-Norepinephrine-
into the hypothalamic
synaptosomes
by FS32, FS97 and tricyclic
Concentrations Drugs FS32 FS97 Imipramine Desipramine Amitriptyline Nortriptyline Clomipramine Iprindole
1 X lo-’ 28.8 40.0 35.0 43.8 42.3 38.0 41.7 17.8
k * + 2 + i + +
2.3 3.8 2.3 1.2 4.3 2.0 2.4 4.7
5 x lo-’ 43.5 49.8 49.5 57.3 49.0 51.4 47.7 22.5
f f * + + f & +
2.5 2.6 1.9 2.1 2.6 2.1 0.8 3.5
I x 1om6 52.3 54.5 56.3 61.8 52.0 55.1 51.0 28.3
+ + + * + + + +
3.1 1.8 1.5 3.3 1.5 2.3 2.2 4.4
(M)
5 x 10-h 66.0 64.3 69.3 72.8 58.3 62.3 56.7 32.3
+ _I + + k + + +
antidepressants
3.6 2.0 1.6 3.0 I.4 1.8 2.0 3.7
1 X lo-5 71.8 68.0 72.5 16.5 62.2 64.8 60.0 36.3
+ & ) ) + + + +
3.3 2.4 I.8 3.2 I.5 0.6 2.8 3.1
Inhibition is expressed as the mean percentage inhibition of control + SEM of 5 independent tion was performed at 37’C for 5 min with 2.9 x lo-* M of C3H]-NE.
I X 1om4
5 x IO_’ 81.5 75.0 81.0 83.0 67.0 73.3 64.4 46.5
+ ) * * + f + k
2.5 2.1 2.1 2.3 2.4 2.5 I .7 I.5
84.5 71.5 83.8 85.0 69.0 15.3 66.8 51.5
determinations.
+ * * i + ) f &
1.8 2.8 2.0 2.6 I.8 2.9 2.9 1.4
Incuba-
Non-tricyclic amine uptake inhibitors
351
L-bitartrate (7-3H(N)) (specific activity 8.72 Ci/mmol) and S-hydroxytryptamine creatinine sulphate (5-(1,2)3H(N)) (s.a. 27.8 Ci/mmol) were obtained from New England Nuclear. All other chemicals were reagent grade. RESULTS
Inhibition oj C3H]-NE uptake Inhibitory effects of C3H]-NE uptake by FS32, FS97 and a number of tricyciic antidepressants were compared (Table 1). Among tricychc antidepressants examined, desipramine was the most potent inhibitor. On the other hand, the weakest inhibitor was iprindole. The uptake was inhibited by FS32 and FS97 to the same extent as by clomipramine and imipramine, respectively. The results shown in Table 1 indicate that secondary amines (such as FS97, desipramine and nortriptyline) were more potent than their respective tertiary amines (i.e. FS32, imipramine and amitriptyline).
0.5
0 i/C3H]-NE
Kinetics of [3H]-NE uptake inhibition The kinetics of [‘HI-NE uptake inhibition by FS32 and FS97 were studied according to the method of Lineweaver-Burk (1934) (Fig. 2). According to this plot, the K, value for [“HI-NE uptake in the absence of drugs was 1.5 x 1K’ M and the V,,, value was 15 pmol/mg protein/S min. lnhibjtion of the uptake by ES32 and FS97 was considered to be competitive because they increased the apparent K, values without changing the V,,, value.
x10-8(~)
Fig. 2. Kinetic analysis of [3H]-NE uptake inhibition by FS32 and FS97. The method of graphical analysis was that of Lineweaver-Burk (1934). Incubation was performed at 37°C for 5 min with concentrations of CJH]-NE ranging from 2.0 to 12.5 x 10-s M and a constant drug concentration (1 x lo-’ M). Each point represents the mean li; SEM of 5 independent determinations. The hy~thalamic synaptosomes were used in this study. Control-O; FS32- x : FS97-0.
Inhibition of [3H]-5-HT uptake
Kinetics of [‘HI-.5-HT uptake inhibition
The potency of drugs to inhibit [3H]-5-HT uptake is summarized in Table 2. Clomipramine was the most potent inhibitor while iprindole was the weakest. The uptake was inhibited by FS32 to the same extent as nortriptyline, while FS97 showed low activity. In addition, the results shown in this Table also indicated that tertiary amines were, in contrast to C3H]-NE uptake inhibition, more effective than their respective secondary amines in inhibiting the uptake of [3H]-5-HT.
The nature of E3H]-5-HT uptake inhibition by FS32 and FS97 was studied by Lineweaver-Burk plot (Fig. 3). It can be seen from Figure 3 that when drugs were absent (refered to as control in the Figure), the K, value was 6.7 x 10T8 M and the V,,, value was 25 pmol/mg protein/3 min. In the presence of drugs, the K, values increased with more or less no alteration in the V,,, values. This indicates that FS32 and FS97 were competitive inhibitors of C3H]-5-HT uptake.
Table 2. Inhibition of [‘HI-S-HT uptake into whole brain synaptosomes by FS32, FS97 and tricyciic antidepressants Concentrations Drugs
1 x lo-’
FS32 FS97 Imipramine Desipramine Amitriptyline Nortriptyline Clomipramine Iprindole
20.0 22.8 38.3 28.3 39.2 32.8 47.7 23.8
+ 1.2 + 3.3 + 3.7 f 2.6 f 2.7 k 2.4 & 2.8 & 3.4
5 x lo-’ 28.5 10.9 29.2 i 3.0 47.5 5 2.0 35.5 + 2.2 49.3 5 1.9 40.3 + 2.3 55.3 i 1.5 29.2 f 3.3
1 x 10-6 33.2 + 32.5 i 51.7 It 39.3 + 54.3 * 43.8 k 58.8 It 31.4 +
0.9 2.7 1.6 1.9 1.7 2.4 1.3 3.3
(M)
5 x lo-6
1 X 10-s
5 x 10-5
1 x lo-4
47.5 + 1.0 41.8 i 2.2 60.5 + 2.3 49.0 f 1.4 63.2 + 1.8 52.5 + 2.7 65.7 + 2.0 39.2 &-3.2
54.3 + 1.2 47.2 _t 2.0 63.8 & 2.8 54.3 + 1.9 66.7 f 2.0 55.7 1 3.0 68.0 I2.1 43.6 + 3.2
68.5 + 1.8 59.0 L?:3.4 70.0 rt 3.3 62.1 rt 1.4 13.3 It 2.3 62.5 rf: 3.3 72.8 + 2.8 52.8 + 2.6
72.3 f 63.3 + 72.5 + 66.0 + 75.7 i 64.8 k 75.0 k 57.2 +
Inhibition is expressed as the mean percentage inhibition of control f SEM of 6 independent determinations. tion was performed at 37°C for 3 min with 5.4 x lo-* M of [3H]-5-HT.
1.7 2.8 3.4 1.6 2.2 3.3 2.9 2.5
Incuba-
T. KOIDE and K. UYEMURA
352
0
._
a5 1 /[3H1-5-HT
0
x lO-e(M)
Fig. 3. Kinetic analysis of C3H]-5-HT uptake inhibition by FS32 and FS97. The method of graphical analysis was that of Lineweaver-Burk (1934). Incubation was performed at 37°C for 3 min with concentrations of c3H]-5-HT ranging from 1.45 to 7.69 x 10e8 M and a constant drug concentration (1 x 10m5 M). Each point represents the mean +SEM of 5 independent determinations. The whole brain synaptosomes were used in this study. ControlPO; FS32p
Releasing activities synaptosomes
x
; FS97-0.
of C3H]-NE from
the preloaded
When the hypothalamic synaptosomes, preloaded with c3H]-NE (2.9 x lo-’ M), were resuspended and incubated at 37”C, a spontaneous release of radioactivity into the medium occurred. The addition of d-amphetamine (1 x lo- 5 M) strongly increased its releasing rate from the preparation. When either FS32 Table 3. The K, values of FS32, FS97 and tricyclic antidepressants for the uptake of C3H]-NE and of C3H]-5-HT K, values (M) Drugs FS32 FS97 Imipramine Desipramine Amitriptyline Nortriptyline Clomipramine Iprindole
C3H]-NE 6.5 3.8 4.2 7.0 5.0 2.7 5.4 6.5
x x x x X x X x
lo-’ lo-’ IO-’ 1om8 lo-’ lo-’ lo-’ 1O-5
10
incubation
13H]-5-HT 2.9 5.9 1.3 5.5 6.0 2.0 1.5 1.0
x X X x x X X X
10mh 1o-6 10-b lomh lo-’ 1om6 IO_’ 10-s
Synaptosomes were incubated at 37°C for 5 and 3 min in the presence of C3H]-NE and C3H]-5-HT. respectively. The Ki values were obtained by the method of Dixon (1953). For the Ki values of C3H]-NE uptake, either 2.9 x lo- ’ M or 5.4 x 10m8 M of C3H]-NE was used and for that of C3H]-5-HT uptake, 2.7 x lO-‘M or 5.4 x 10e8 M of [‘HI-5-HT was used. The hypothalamic synaptosomes were used for C3H]-NE uptake and whole brain synaptosomes were used for C3H]-5-HT uptake
20 timetmin)
30
Fig. 4. Release of C3H]-NE from the preloaded hypothalamic synaptosomes. Synaptosomal suspension were preloaded by incubating with [3H]-NE (2.9 x 10m8 M) for 20 min at 37°C. After cooling in ice, the synaptosomes were separated by centrifugation, and the pellet was rinsed and resuspended in fresh buffer (see details in the Methods).
The drugs were added at zero time, and incubation was performed for different periods as indicated in the Figure. Control-o; FS32 (1 x 10m4 Mb x ; FS97 (1 x 10m4 MtO; d-amphetamine (1 x 10-s M) --A. The percentage of the radioactivity remaining on the filter was evaluated from 5 to 6 independent determinations after various incubation times. The results were given as the mean k SEM.
or FS97 was added
to the medium at a concentration of 1 x 10e4 M, a dose level exceeding 50% inhibition of C3H]-NE uptake, no marked changes of NE-releasing rate were observed (Fig. 4). Comparisons of the Ki values The Ki values of FS32, FS97 and tricyclic antidepressants for the uptake of C3H]-NE and C3H]-5-HT were obtained by the method of Dixon plot (1953) and are summarized in Table 3. Among the tricychc antidepressants studied, desipramine showed the highest affinity for C3H]-NE uptake sites while clomipramine showed it for C3H]-5-HT uptake sites. In both uptake systems, all points of intersection of the two lines (which gives the Ki values) occurred at a height equivalent to l/V,,,. This suggests that all the drugs examined were competitive inhibitors of uptake. DtSCUSSlOlV
Despite the widespread use of antidepressants, the precise mechanisms of their antidepressive actions has been unknown for some time. In recent years, however, it has been generally accepted that the thera-
353
Non-tricyclic amine uptake inhibitors peutic efficiency of antidepressants may be the consequence of an increased availability of NE and/or 5-HT at the postsynaptic receptor sites by inhibiting the uptake of each amine into the presynaptic nerve terminals. In this paper, an examination was made of the drug effects of new non-tricyclic compounds, FS32 and FS97, on the uptake of each amine into rat hypothalamic and whole brain synaptosomes. It was observed that secondary tricyclic antidepressants (i.e. desipramine and nortriptyline) were more potent than their corresponding tertiary amines (i.e. imipramine and amitriptyline) in inhibiting C3H]-NE uptake. On the contrary, tertiary amines inhibited the uptake of E3H)-5-HI’ more effectively than their respective secondary amines. This is consistent with previous findings (Shaskan and Snyder, 1970; Koe, 1976; Goodlet, Mireylees and Sugrue, 1977). In the case of FS32 and FS97, the results also support the above hypothesis: FS32 inhibited C3H]-5-HT uptake more effectively than C3H]-NE uptake. On the other hand, FS97 showed a comparable inhibiting activity of C3H]-NE uptake to that of imipramine, although it showed a relatively weak inhibition of [‘HI-5-HT. These results suggest that the presence of a methyl group at the side chain of the drugs may be the main contribution to the differences between NE and 5-HT uptake inhibition. In studying the actions of drugs on neural transmission, effects of both the uptake inhibition and releasing activity by the drugs should be considered. AS both FS32 and FS97 showed no significant releasing activities of C3H]-NE from the hypothalamic synaptosomes, the apparent inhibitory effects could mainly be due to the inhibition of uptake mechanisms. The K, value for C3H]-NE and E3H]-.5-HT uptake into the synaptosomes was 1.5 x 10m7 M and 6.7 x 10e8 M, respectively. These values accorded well with the values previously reported (Snyder and Coyfe, 1969; Shaskan and Snyder, 1970; White and Paton, 1972; Tuomisto and Tuomisto, 1973). Under these conditions, both FS32 and FS97 inhibited the uptake of each amine in a competitive manner. Tricyclic antidepressants also inhibited the uptake competitively, which confirms previous reports (Wong, Horng, Bymaster, Hauser and Molloy, 1974; Classen, Davies, Hertting and Placheta, 1977; Goodlet er al., 1977). Among the drugs studied, desipramine showed the highest affinity for C3H]-NE uptake sites, while clomipramine showed it for C3H]-.5-HT uptake sites. The lowest affinity was shown by iprindole for both uptake sites. These results are in agreement with the previous studies by several authors (Todrick and Tait, 1969; Ross, Renyi and Ogren, 1971; RosIofT and Davis, 1974; Nyback, Walters, Aghajanian and Roth, 1975; Koe, 1976; Blackburn, Foster, Greenwood and Howe, 1978). A similar affinity as clomipramine and imipramine respectively for c3H]-NE uptake sites was shown by FS32 and FS97. On the other hand, the aftinity of both drugs for C3H]-5-HT uptake sites,
FS32 and FS97 showed a similar affinity to nortriptyline and desipramine, respectively. These results correspond well with the inhibiting potencies of the uptake systems. In conclusion, the present study indicates that new non-tricyclic compounds, FS32 and FS97, inhibit the uptake of NE or 5-HT competitively and this may be of pharmacological significance for their antidepressant actions. REFERENCES
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White, T. D. and Paton, D. M. (1972). Effects of external NA+ and K+ on the initial rates of noradrenaline uptake by synaptosomes prepared from rat brain. Biochim. biophys. AC~A 206: 116-127. Wang, D. T., Horng, J. S., Bymaster, F. P.. Hauser, K. L. and Molloy, B. B. (1974). A selective inhibitor of serotonin uptake: Lilly 110140, 3-(p-trifluoromethylphenoxy)N-methyl-3-phenylpropylamine. Life Sci. 15: 471-479.
A COMPARISON OF THE INHIBITORY EFFECTS OF NEW NON-TRICYCLIC AMINE UPTAKE INHIBITORS ON THE UPTAKE OF NOREPINEPHRINE AND 5HYDROXYTRYPTAMINE INTO SYNAPTOSOMES OF THE RAT BRAIN T. KOIDE* and K. UYEMURA Department of Physiology, Saitama Medical School, Moroyama, Irumagun, Saitama, 350-04, Japan (Accepted 15 October 1979) Summary-The effects of new non-tricyclic amine uptake inhibitors, FS32 and FS97, on the uptake of [3H]-norepinephrine (NE) into the hypothalamic synaptosomes and [3H]-5-hydroxytryptamine (5-HT) into whole brain synaptosomes were studied. Their effects were compared with those of tricyclic antidepressants, The uptake of C3H]-NE was inhibited competitively by FS32 and FS97 with a respective Ki value of 6.5 x lo-’ M and 3.8 x lo-’ M. The potency of FS32 and FS97 to inhibit this uptake was almost comparable to that of clomipramine and imipramine, respectively. In the case of C3H]-5-HT uptake, FS32 and FS97 also showed competitive inhibition with a respective Ki value of 2.9 x 1O-6 M and 5.9 x 1O-6 M. The ability of FS32 to inhibit C3H]-5-HT uptake was almost equal to that of nortriptyline, while FS97 was two times more potent than iprindole in inhibiting this uptake.
During recent years growing support has been accumulating for the view that a decreased function of the neurotransmitter systems for norepinephrine (NE) and 5-hydroxytryptamine (5-HT) is a common feature of depressive illness (Schildkraut, 1965; Lapin and Oxenkrug, 1969; Van Praag and Korf, 1971; Asberg, Bertilsson, Tuck, Cronholm and SjBqvist, 1973; Schildkraut, 1973) and that the facilitation of NE and of 5-HT transmission is related to an increase in drive and in mood elevation, respectively. It was also reported that the therapeutic efficacy of tricyclic antidepressants, such as imipramine and desipramine, depends on the inhibition of the uptake of each amine into nerve endings, which allows the transmitters to remain at the receptor sites for a longer period (Carlsson, Corrodi, Fuxe and Hokfelt, 1969a; Carlsson, Corrodi, Fuxe and Hiikfelt, 1969b; Carlsson, 1970; Lidbrink. Jonsson and Fuxe, 1971). In the course of studying the pharmacological actions of a series of indazole derivatives, FS32 (l-[3-(Dimethylamino)propyl]-S-methyl-3-phenyl-lHindazole) and its N-desmethylated compound, FS97 (Fig. 1) were selected as a new group of antidepressive drugs, because they revealed a favourable therapeutic index in experimental animals and showed a uniform effect after oral administration (Ikeda, Takano, Matsushita, Shiraki. Koide, Nagashima, Fujimura,
Shindo. Suzuki and Iwasaki, 1979). As the nontricyclic structure in their chemical formula differs from the conventional tricyclic antidepressants, it is of interest to compare the neurochemical effects with those of common tricyclic antidepressants. In the present study, the effects of such drugs on C3H]-NE and C3H]-5-HT uptake into synaptosomes were examined comparatively. METHODS Tissue preparations Purtjied whole brain synaptosomes. Synaptosomes were prepared from rat whole brain essentially according to the method described by White (1975) with some modifications. All steps in the procedure were carried out in the cold (@4”C). A male SpragueDawley rat (weighing about 250 g) was killed by cervical dislocation and the whole brain was removed, chilled in 0.32 M-sucrose and homogenized by 15 strokes of a Teflon-glass homogenizer at 800 rpm. The
9
o? I
0
0
t/ I
-N-I3
Department of Pharmacology, * Present address: Research Laboratories of Chugai Pharmaceutical Company Ltd, No. 41-8, 3-chome, Takada, Toshima-ku, Tokyo, Japan. Key words: synaptosomes, tricyclic non-tricyclic amine uptake inhibitors.
antidepressants,
new
Fig. 1. Chemical 349
I
I
FS32 : R=CH3 697 : R=H
CH3 structure
of FS32 and FS97.
T. KOIDE and K. UYEMURA
350
homogenate was divided into two equal portions and centrifuged at 1000 g for 10 min, and the resulting supernatants were then centrifuged at 12,000g for 20 min. The final pellets were suspended in 0.32 Msucrose to give a total volume of 6 ml, and each 2 ml was layered on a discontinuous density gradient consisting of 5 ml of 0.8 M-sucrose on 5 ml of 1.2 Msucrose. The gradients were centrifuged at 150,000 g for 60 min and the interfaces between 0.8 M- and 1.2 M-sucrose were collected. The pooled fraction was diluted to 40 ml with 0.32 M-sucrose. Then the fraction was centrifuged at 20,000 g for 30 min. The resulting pellets were finally resuspended with a total volume of 3.0 ml of 0.32 M-sucrose and were used immediately as a synaptosomal fraction for the C3H]S-HT uptake study. This fraction contained a large number of intact synaptosomes as revealed by electron microscopy. Hypothalamic synaptosomes. The brain was removed as described above and the hypothalamus was dissected out in the cold according to the method of Glowinski and Iversen (1966). The tissue was gently homogenized in 30 vol of ice-coid 0.32 M-sucrose by 10 strokes of a Teflon-glass homogenizer at 800 rpm. The homogenate was centrifuged at 1000 g for 10 min, and the resulting supernatants were then centrifuged at 12,000 g for 20 min. The final pellets were suspended in 0.32 M-sucrose and were used immediately as the hypothalamic synaptosomes for C3H]-NE uptake study and releasing study. Uptake of amines Fifty pl of synaptosomal suspension was added to 450~1 of incubation medium containing various amounts of drugs and preincubated for 5 min at 37’C. Then 50 ~1 of C3H]-NE or C3H]-5-HT was added and the incubation was continued for another 5 min or 3 min for the uptake of c3H]-NE and C3H]-5-HT, respectively. Preliminary experiments indicated that the reaction proceeds linearly within the incubation times. The incubation medium contained; 130 mMNaCl, 3 mM-KCl, 2 mM-CaCl,, 2 mM--MgCl, and 20mM-Tris-HCI buffer (pH 7.4). Incubation was terminated by filtering 500 ~1 of the incubation mixture through a 0.45 pm pore-size membrane filter Table 1. Inhibition
of 13H]-NE
uptake
(TM-2, Toyo Roshi) by suction. The filter was washed with 10 ml of washing solution containing 260 mM---sucrose, 2 mM-CaCI,, 2 mM_MgCl, and 20 mM ~ Tris-HCI buffer (pH 7.4). Each membrane filter was dissolved in 10 ml of scintillating fluid containing lo”,, (v/v)-ethylene glycol monoethyl ether, 204; (v!v)-Triton X-100, 0.5% (w/v)-DPO and 0.03”/;, (w/v)-POPOP in xylene. The radioactivity was determined by liquid scintillation counter (LSC-653, Aloka). The actual moles of NE or 5-HT taken up/mg synaptosomal protein was obtained by subtracting the mol of each amine/mg protein at O’C (= uptake at 37 C minus uptake at 0°C). Any significant decrease in the uptake activity was not shown within the experimental periods in this study (about 2 hr after preparation). Release of [3H]-norepinephrine One volume of the hypothalamic synaptosomal suspension was incubated with ten volumes of the incubation medium containing C3H]-NE at 37°C for 20 min. After incubation, the mixture was centrifuged at 12,000 g for 20 min. The pellet was washed gently three times by resuspending in the incubation medium (omitting [‘HI-NE) and was centrifuged. After final washing, the radioactive synaptosomes were resuspended in the same volume of the incubation medium. Aliquots (500 ~1) of the final suspension were incubated with 50 ~1 of drug solution for 5, 10, 20 and 30 min at 37°C. After incubation, the mixture was passed through a 0.45 pm pore-size membrane filter and washed with 10ml of the washing solution. The amount of C3H]-NE remaining on the filter was counted as described above. Protein estimution The protein concentration of the synaptosomal suspension was determined according to the procedure described by Lowry, Rosebrough, Farr and Randall (1951). Chemicals The laboratories of Chugai Pharmaceutical Company Ltd synthesized FS32 and FS97 and these were used as the HCI salt in this study. DL-Norepinephrine-
into the hypothalamic
synaptosomes
by FS32, FS97 and tricyclic
Concentrations Drugs FS32 FS97 Imipramine Desipramine Amitriptyline Nortriptyline Clomipramine Iprindole
1 X lo-’ 28.8 40.0 35.0 43.8 42.3 38.0 41.7 17.8
k * + 2 + i + +
2.3 3.8 2.3 1.2 4.3 2.0 2.4 4.7
5 x lo-’ 43.5 49.8 49.5 57.3 49.0 51.4 47.7 22.5
f f * + + f & +
2.5 2.6 1.9 2.1 2.6 2.1 0.8 3.5
I x 1om6 52.3 54.5 56.3 61.8 52.0 55.1 51.0 28.3
+ + + * + + + +
3.1 1.8 1.5 3.3 1.5 2.3 2.2 4.4
(M)
5 x 10-h 66.0 64.3 69.3 72.8 58.3 62.3 56.7 32.3
+ _I + + k + + +
antidepressants
3.6 2.0 1.6 3.0 I.4 1.8 2.0 3.7
1 X lo-5 71.8 68.0 72.5 16.5 62.2 64.8 60.0 36.3
+ & ) ) + + + +
3.3 2.4 I.8 3.2 I.5 0.6 2.8 3.1
Inhibition is expressed as the mean percentage inhibition of control + SEM of 5 independent tion was performed at 37’C for 5 min with 2.9 x lo-* M of C3H]-NE.
I X 1om4
5 x IO_’ 81.5 75.0 81.0 83.0 67.0 73.3 64.4 46.5
+ ) * * + f + k
2.5 2.1 2.1 2.3 2.4 2.5 I .7 I.5
84.5 71.5 83.8 85.0 69.0 15.3 66.8 51.5
determinations.
+ * * i + ) f &
1.8 2.8 2.0 2.6 I.8 2.9 2.9 1.4
Incuba-
Non-tricyclic amine uptake inhibitors
351
L-bitartrate (7-3H(N)) (specific activity 8.72 Ci/mmol) and S-hydroxytryptamine creatinine sulphate (5-(1,2)3H(N)) (s.a. 27.8 Ci/mmol) were obtained from New England Nuclear. All other chemicals were reagent grade. RESULTS
Inhibition oj C3H]-NE uptake Inhibitory effects of C3H]-NE uptake by FS32, FS97 and a number of tricyciic antidepressants were compared (Table 1). Among tricychc antidepressants examined, desipramine was the most potent inhibitor. On the other hand, the weakest inhibitor was iprindole. The uptake was inhibited by FS32 and FS97 to the same extent as by clomipramine and imipramine, respectively. The results shown in Table 1 indicate that secondary amines (such as FS97, desipramine and nortriptyline) were more potent than their respective tertiary amines (i.e. FS32, imipramine and amitriptyline).
0.5
0 i/C3H]-NE
Kinetics of [3H]-NE uptake inhibition The kinetics of [‘HI-NE uptake inhibition by FS32 and FS97 were studied according to the method of Lineweaver-Burk (1934) (Fig. 2). According to this plot, the K, value for [“HI-NE uptake in the absence of drugs was 1.5 x 1K’ M and the V,,, value was 15 pmol/mg protein/S min. lnhibjtion of the uptake by ES32 and FS97 was considered to be competitive because they increased the apparent K, values without changing the V,,, value.
x10-8(~)
Fig. 2. Kinetic analysis of [3H]-NE uptake inhibition by FS32 and FS97. The method of graphical analysis was that of Lineweaver-Burk (1934). Incubation was performed at 37°C for 5 min with concentrations of CJH]-NE ranging from 2.0 to 12.5 x 10-s M and a constant drug concentration (1 x lo-’ M). Each point represents the mean li; SEM of 5 independent determinations. The hy~thalamic synaptosomes were used in this study. Control-O; FS32- x : FS97-0.
Inhibition of [3H]-5-HT uptake
Kinetics of [‘HI-.5-HT uptake inhibition
The potency of drugs to inhibit [3H]-5-HT uptake is summarized in Table 2. Clomipramine was the most potent inhibitor while iprindole was the weakest. The uptake was inhibited by FS32 to the same extent as nortriptyline, while FS97 showed low activity. In addition, the results shown in this Table also indicated that tertiary amines were, in contrast to C3H]-NE uptake inhibition, more effective than their respective secondary amines in inhibiting the uptake of [3H]-5-HT.
The nature of E3H]-5-HT uptake inhibition by FS32 and FS97 was studied by Lineweaver-Burk plot (Fig. 3). It can be seen from Figure 3 that when drugs were absent (refered to as control in the Figure), the K, value was 6.7 x 10T8 M and the V,,, value was 25 pmol/mg protein/3 min. In the presence of drugs, the K, values increased with more or less no alteration in the V,,, values. This indicates that FS32 and FS97 were competitive inhibitors of C3H]-5-HT uptake.
Table 2. Inhibition of [‘HI-S-HT uptake into whole brain synaptosomes by FS32, FS97 and tricyciic antidepressants Concentrations Drugs
1 x lo-’
FS32 FS97 Imipramine Desipramine Amitriptyline Nortriptyline Clomipramine Iprindole
20.0 22.8 38.3 28.3 39.2 32.8 47.7 23.8
+ 1.2 + 3.3 + 3.7 f 2.6 f 2.7 k 2.4 & 2.8 & 3.4
5 x lo-’ 28.5 10.9 29.2 i 3.0 47.5 5 2.0 35.5 + 2.2 49.3 5 1.9 40.3 + 2.3 55.3 i 1.5 29.2 f 3.3
1 x 10-6 33.2 + 32.5 i 51.7 It 39.3 + 54.3 * 43.8 k 58.8 It 31.4 +
0.9 2.7 1.6 1.9 1.7 2.4 1.3 3.3
(M)
5 x lo-6
1 X 10-s
5 x 10-5
1 x lo-4
47.5 + 1.0 41.8 i 2.2 60.5 + 2.3 49.0 f 1.4 63.2 + 1.8 52.5 + 2.7 65.7 + 2.0 39.2 &-3.2
54.3 + 1.2 47.2 _t 2.0 63.8 & 2.8 54.3 + 1.9 66.7 f 2.0 55.7 1 3.0 68.0 I2.1 43.6 + 3.2
68.5 + 1.8 59.0 L?:3.4 70.0 rt 3.3 62.1 rt 1.4 13.3 It 2.3 62.5 rf: 3.3 72.8 + 2.8 52.8 + 2.6
72.3 f 63.3 + 72.5 + 66.0 + 75.7 i 64.8 k 75.0 k 57.2 +
Inhibition is expressed as the mean percentage inhibition of control f SEM of 6 independent determinations. tion was performed at 37°C for 3 min with 5.4 x lo-* M of [3H]-5-HT.
1.7 2.8 3.4 1.6 2.2 3.3 2.9 2.5
Incuba-
T. KOIDE and K. UYEMURA
352
0
._
a5 1 /[3H1-5-HT
0
x lO-e(M)
Fig. 3. Kinetic analysis of C3H]-5-HT uptake inhibition by FS32 and FS97. The method of graphical analysis was that of Lineweaver-Burk (1934). Incubation was performed at 37°C for 3 min with concentrations of c3H]-5-HT ranging from 1.45 to 7.69 x 10e8 M and a constant drug concentration (1 x 10m5 M). Each point represents the mean +SEM of 5 independent determinations. The whole brain synaptosomes were used in this study. ControlPO; FS32p
Releasing activities synaptosomes
x
; FS97-0.
of C3H]-NE from
the preloaded
When the hypothalamic synaptosomes, preloaded with c3H]-NE (2.9 x lo-’ M), were resuspended and incubated at 37”C, a spontaneous release of radioactivity into the medium occurred. The addition of d-amphetamine (1 x lo- 5 M) strongly increased its releasing rate from the preparation. When either FS32 Table 3. The K, values of FS32, FS97 and tricyclic antidepressants for the uptake of C3H]-NE and of C3H]-5-HT K, values (M) Drugs FS32 FS97 Imipramine Desipramine Amitriptyline Nortriptyline Clomipramine Iprindole
C3H]-NE 6.5 3.8 4.2 7.0 5.0 2.7 5.4 6.5
x x x x X x X x
lo-’ lo-’ IO-’ 1om8 lo-’ lo-’ lo-’ 1O-5
10
incubation
13H]-5-HT 2.9 5.9 1.3 5.5 6.0 2.0 1.5 1.0
x X X x x X X X
10mh 1o-6 10-b lomh lo-’ 1om6 IO_’ 10-s
Synaptosomes were incubated at 37°C for 5 and 3 min in the presence of C3H]-NE and C3H]-5-HT. respectively. The Ki values were obtained by the method of Dixon (1953). For the Ki values of C3H]-NE uptake, either 2.9 x lo- ’ M or 5.4 x 10m8 M of C3H]-NE was used and for that of C3H]-5-HT uptake, 2.7 x lO-‘M or 5.4 x 10e8 M of [‘HI-5-HT was used. The hypothalamic synaptosomes were used for C3H]-NE uptake and whole brain synaptosomes were used for C3H]-5-HT uptake
20 timetmin)
30
Fig. 4. Release of C3H]-NE from the preloaded hypothalamic synaptosomes. Synaptosomal suspension were preloaded by incubating with [3H]-NE (2.9 x 10m8 M) for 20 min at 37°C. After cooling in ice, the synaptosomes were separated by centrifugation, and the pellet was rinsed and resuspended in fresh buffer (see details in the Methods).
The drugs were added at zero time, and incubation was performed for different periods as indicated in the Figure. Control-o; FS32 (1 x 10m4 Mb x ; FS97 (1 x 10m4 MtO; d-amphetamine (1 x 10-s M) --A. The percentage of the radioactivity remaining on the filter was evaluated from 5 to 6 independent determinations after various incubation times. The results were given as the mean k SEM.
or FS97 was added
to the medium at a concentration of 1 x 10e4 M, a dose level exceeding 50% inhibition of C3H]-NE uptake, no marked changes of NE-releasing rate were observed (Fig. 4). Comparisons of the Ki values The Ki values of FS32, FS97 and tricyclic antidepressants for the uptake of C3H]-NE and C3H]-5-HT were obtained by the method of Dixon plot (1953) and are summarized in Table 3. Among the tricychc antidepressants studied, desipramine showed the highest affinity for C3H]-NE uptake sites while clomipramine showed it for C3H]-5-HT uptake sites. In both uptake systems, all points of intersection of the two lines (which gives the Ki values) occurred at a height equivalent to l/V,,,. This suggests that all the drugs examined were competitive inhibitors of uptake. DtSCUSSlOlV
Despite the widespread use of antidepressants, the precise mechanisms of their antidepressive actions has been unknown for some time. In recent years, however, it has been generally accepted that the thera-
353
Non-tricyclic amine uptake inhibitors peutic efficiency of antidepressants may be the consequence of an increased availability of NE and/or 5-HT at the postsynaptic receptor sites by inhibiting the uptake of each amine into the presynaptic nerve terminals. In this paper, an examination was made of the drug effects of new non-tricyclic compounds, FS32 and FS97, on the uptake of each amine into rat hypothalamic and whole brain synaptosomes. It was observed that secondary tricyclic antidepressants (i.e. desipramine and nortriptyline) were more potent than their corresponding tertiary amines (i.e. imipramine and amitriptyline) in inhibiting C3H]-NE uptake. On the contrary, tertiary amines inhibited the uptake of E3H)-5-HI’ more effectively than their respective secondary amines. This is consistent with previous findings (Shaskan and Snyder, 1970; Koe, 1976; Goodlet, Mireylees and Sugrue, 1977). In the case of FS32 and FS97, the results also support the above hypothesis: FS32 inhibited C3H]-5-HT uptake more effectively than C3H]-NE uptake. On the other hand, FS97 showed a comparable inhibiting activity of C3H]-NE uptake to that of imipramine, although it showed a relatively weak inhibition of [‘HI-5-HT. These results suggest that the presence of a methyl group at the side chain of the drugs may be the main contribution to the differences between NE and 5-HT uptake inhibition. In studying the actions of drugs on neural transmission, effects of both the uptake inhibition and releasing activity by the drugs should be considered. AS both FS32 and FS97 showed no significant releasing activities of C3H]-NE from the hypothalamic synaptosomes, the apparent inhibitory effects could mainly be due to the inhibition of uptake mechanisms. The K, value for C3H]-NE and E3H]-.5-HT uptake into the synaptosomes was 1.5 x 10m7 M and 6.7 x 10e8 M, respectively. These values accorded well with the values previously reported (Snyder and Coyfe, 1969; Shaskan and Snyder, 1970; White and Paton, 1972; Tuomisto and Tuomisto, 1973). Under these conditions, both FS32 and FS97 inhibited the uptake of each amine in a competitive manner. Tricyclic antidepressants also inhibited the uptake competitively, which confirms previous reports (Wong, Horng, Bymaster, Hauser and Molloy, 1974; Classen, Davies, Hertting and Placheta, 1977; Goodlet er al., 1977). Among the drugs studied, desipramine showed the highest affinity for C3H]-NE uptake sites, while clomipramine showed it for C3H]-.5-HT uptake sites. The lowest affinity was shown by iprindole for both uptake sites. These results are in agreement with the previous studies by several authors (Todrick and Tait, 1969; Ross, Renyi and Ogren, 1971; RosIofT and Davis, 1974; Nyback, Walters, Aghajanian and Roth, 1975; Koe, 1976; Blackburn, Foster, Greenwood and Howe, 1978). A similar affinity as clomipramine and imipramine respectively for c3H]-NE uptake sites was shown by FS32 and FS97. On the other hand, the aftinity of both drugs for C3H]-5-HT uptake sites,
FS32 and FS97 showed a similar affinity to nortriptyline and desipramine, respectively. These results correspond well with the inhibiting potencies of the uptake systems. In conclusion, the present study indicates that new non-tricyclic compounds, FS32 and FS97, inhibit the uptake of NE or 5-HT competitively and this may be of pharmacological significance for their antidepressant actions. REFERENCES
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