Dopamine uptake in striatal and hypothalamic synaptosomes: Conformational selectivity of the inhibition

EUROPEAN JOURNAL OF PHARMACOLOGY 25 (1974) 351-361. NORTH-HOLLAND PUBLISHING COMPANY

DOPAMINE UPTAKE IN STRIATAL AND HYPOTHALAMIC SYNAPTOSOMES: CONFORMATIONAL SELECTIVITY OF THE INHIBITION Leena TUOMISTO, Jouko TUOMISTO and Edward E. SMISSMAN Department of Pharmacology, University of Helsinki, Helsinki, Finland, and Department of Medicinal Chemistry, University of Kansas, Lawrence, Ks., U.S.A. Received 2 July 1973

Accepted 3 October 1973

L. TUOMISTO, J. TUOMISTO and E.E. SMISSMAN,Dopamine uptake in strlatal and hypothalamic synaptosomes: conformational selectivity of the inhibition, European J. Pharmaeol. 25 (1974) 351-361. The eonformational selectivity of dopamine uptake mechanism in rat striatum and hypothalamus was studied by utilizing rigid analogs of noradrenaline as uptake inhibitors. Noradrenaline was rendered sterically rigid by incorporating the essential groups into the trans-deealin structure. Four isomers were obtained. In three of them, the relation of the cateehol ring and the amino function was gauche (i.e. the dihedral angle created by those groups was 60°), in one it was anti (the angle was 180° , resp.). In striatal synaptosomes one of the gauche isomers was the most potent inhibitor of dopamine uptake. It was 22 times as potent as the anti-isomer. In the hypothalamie synaptosomes the difference was less marked, but the ganehe form was still 5 times as potent as the anti-isomer. In both cases the inhibition was competitive. It is suggested that the differences in potency are due to conformational selectivity of the dopamine-uptake mechanism, and that the difference between the striatum and the hypothalamus is due to different distribution of dopamine neurons. Decalin derivatives Uptake mechanisms

1. Introduction The configurational selectivity o f amine uptake processes is well known (for ref. see Patil et al., 1970). One o f the intriguing findings in the brain is that noradreline uptake in the hypothalamus, or cerebral cortex, exhibited configurational selectivity with a preference for (-).noradrenaline, but no such selectivity was seen in the striatum (Coyle and Snyder, 1969). Configurational selectivity was also demonstrated for inhibition o f uptake. (+)-Amphetamine and (-)-noradrenaline were more potent inhibitors o f amine uptake than their enantiomers in the cortex or cortex and hypothalamus, respectively, but not in the striatum (Coyle and Snyder, 1969). The finding with amphetamine has been challenged by Ferris et al. (1972) as well as Thornburg and Moore (1973). They found (+)-amphetamine more potent in the striatum, whereas both enantiomers were equipotent in other brain areas studied.

Brain chemistry Synaptosomes

Receptors Dopamine

Relatively few studies have been done on the possible conformational requirements o f amine transport mechanism. They suggest that in areas where the amine uptake is not considered stereoselective in the configurational sense, the mechanism may still be sensitive to conformational differences. H e m and Snyder (1972) demonstrated that tranylcypromine was 300 times more potent than its cisoid isomer, cis-2phenylcyclopropylamine, on dopamine uptake by striatum. 2-Aminoindane, which was viewed as an anti-derivative of phenylethylamine, was also 300 times more potent than 1-aminoindane, which is closer to the gauche conformer ofphenylethylamine. Our recent data (Tuomisto et al., 1973) on decalin derivatives of noradrenaline or amphetamine, suggested that the conformational requirements o f the dopamine uptake may actually be clearer than those o f the noradrenaline or o f the 5-HT uptake. These findings might indicate that configurational and conformational requirements are quite independent of one another.

352

L. Tuomisto et aL, Conformational selectivity of dopamine uptake

The present study was performed to eludicate the conformational requirements of dae inhibition of dopamine uptake in synaptosomes. Noradrenaline analogs were used as inhibitors. They were rendered conformationally rigid by incorporating the catechol ring, the hydroxyl group and the amino function into the trans-decalin structure. Four different racemic isomers were studied. In three of them the relation between the catechol ring and the amino function was gauche and in one isomer it was anti. Their potency was also compared with that of the parent amine, noradrenaline. Both striatal and hypothalamic synaptosomes were used.

2. Materials and methods

Dopamine uptake by crude synaptosomal fractions of rat striatum and hypothalamus was determined, slightly modifying the procedure of Snyder and Coyle (1969) as described previously (Tuomisto et al., 1973). Male Sprague-Dawley rats, weighing 200-250 g, were killed in a cold room (+4°C) by decapitation. The brains were rapidly removed and the striatum and the hypothalamus dissected on a cold surface as described by Glowinski and Iversen (1966), and homogenized in a glass-teflon homogenizer in 19 volumes of 0.32 M sucrose made in 0.01 M phosphate buffer pH 7.4. The homogenate was centrifuged at 750Xg (maximum radius) for 10 min in a refrigerated Sorvall centrifuge. The supernatant was stirred and aliquots of 100/al were added with an automatic pipettor (Finnpipette) to 3.9 ml of KrebsHenseleit bicarbonate buffer (Krebs, 1950), containing the radioactive substrate, the inhibitor to be studied or an equivalent amount of solvent, 0.2 mg/ ml ascorbic acid and 1.25 X I0-s M nialamide. Tritiated dopamine was stored at -18°C as 10-s M water solution containing ascorbid acid, to give 0.1 mg/ml in the final incubation medium. It was diluted with ice-cold buffer just before use. The inhibitors were dissolved in Krebs-Henseleit buffer also containing ascorbate to give 0.1 mg/ml of ascorbid acid in the final incubation medium. The incubation was performed in disposable polypropylene vials, which were gassed with 95% 0 2 - 5 % COs after pipetting the buffer and the reagents, and then closed with plastic film (Parafiim ®). The vials were kept on ice through-

out this procedure. At the beginning of the incubation, a small opening was made with a scalpel and the tissue homogenate pipetted into the vial, which was then transferred to a metabolic shaker and shaken at 37°C for 5 min -+ 2 sec. The incubation was terminated by rapid filtration ( 5 - 1 0 sec) through a membrane filter (Schleicher & Schull, Selectron BA 85 cellulose nitrate filter with 0.45 /am pore size). The filter discs were premoistened with 0.9% saline, and the sample was washed with 10 ml of ice-cold saline (15-20 sec). No preincubation with the inhibitor was used. About 40 samples were included in each experiment and they were incubated at one min intervals. By using several control samples, it was checked that the rate of uptake did not change during the experiment and, in addition, the samples were randomized. The whole procedure was preformed within 1 hr from the decapitation of the animal. The presence and condition of synaptosomes were checked electron microscopically. After the filtration procedure, the filter discs were transferred to scintillation vials, and I0 ml of scintillation fluid was added (0.4% PPO and 0.01% dimethyl-POPOP in equal parts of toluene and ethylenglycol monoethyl ether, Bruno and Christian, 1961). The scintillation fluid completely dissolved the moist filters. The samples were counted with a Wallac Decem 314 scintillation counter. The radioactivity in samples incubated at 0°C in otherwise identical conditions, was used as a blank value and subtracted from the results except where stated otherwise. These 0°C blanks were very close to the 'filter blanks' obtained by omission of tissue, and were 4.5 + 0.9 (S.D.)% of the controls incubated at 37°C in experiments with striatal synaptosomes, and 15.5 -+ 4.0 (S.D.)% in experiments with hypothalamic synaptosomes. Hence, the activity in the 0°C samples appeared mainly to be due to the adsorption of dopamine onto the filter. The inhibition of uptake was expressed as the % of controls incubated without an inhibitor. Duplicate samples were always used and 4 - 6 controls were included in each experiment. The duplicate samples deviated from their mean less than 5% on an average. The IDs o values were calculated by converting the % inhibition of uptake to probit and by plotting this on the semilogarithmic paper against the concentration of the inhibitor. For kinetic analysis, the samples were incubated with different concentrations of 3 H-dopamine. For

L. Tuomisto et al., Conformational selectivity of dopamine uptake concentrations higher than 10-7 M, 10- 7 M 3H-dopamine and a necessary amount of cold carrier was used. In these experiments no 0°C blanks were subtracted. Tissue/medium ratios were calculated as cpm per original wet tissue weight to initial cpm per 1 #1 medium. For calculating linear correlation coefficients as well as for other statistical calculations, the Olivetti Programma 101 desk computer was used. Statistical significances between means were determined by using Student's t-test. Chemicals: 3H-Dopamine hydrochloride (500 mCi/mmol) was purchased from the Radiochemical Centre, Dopamine hydrochloride and (-)-noradrenaline hydrogen tartrate from Fluka AG, PPO (2,5diphenyloxazole) and dimethyl-POPOP (1,4-bis-2(4-methyl-5-phenyloxazolyl)-benzene) from Packard Instrument Co, the racemic decalin compounds of the noradrenaline type were synthesized by Dr. R.T. Borchardt (Smissman and Borchardt, 1971)and the others by Dr. T.L. Pazdernik. Nialamide was donated by Pfizer.

3. Results

3.1. Active uptake of dopamine When incubated at 37°C for 5 min, the striatal synaptosomes took up a H-dopamine against a concentration gradient. As calculatod for the original wet weight of the tissue, the tissue/medium (T/M) ratio of 59.5 was formed when the initial concentration in the medium was 10-7 M. This uptake corresponds to 7.5% of the total amount of tritiated amine added to the medium, whereas the amount of tissue (actually its original weight) was only 0.125% of the total volume of the incubate. At 0°C a ratio of only 2.0 was formed, which in this case includes the amine adsorbed onto the filter. Previously, Snyder and Coyle (1969) found a T/M ratio of more than 200. However, they calculated it to the weighed particulate fraction rather than to the original weight of the tissue. This causes a 2.5-fold difference. Their technique also differed from the present one inasmuch that the sampies were preincubated for 5 min, the homogenate added to the prewarmed buffer, and the synaptosomes separated by centrifugation. These factors ren-

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Fig. 1. Uptake of dopamine by striatal synaptosomes when the dopamine concentration was varied from 5 × 10 8 M to 2 X 10-6 M. The synaptosomes were incubated for 5 rain in Krebs-Henseleit bicarbonate buffer at pH 7.4 and separated by membrane t'titration. The medium contained 0.2 mg/ml aseorbic acid and 1.25 × l 0 s M nialamide, to prevent oxidation of dopamine. The upper curve is the total uptake and the lower curve indicates the remaining portion after the linear process was subtracted (see text and fig. 3). Mean ± S.E.M. of 5 - 1 7 (average 9) duplicate experiments.

der the effective incubation time longer than that used in the present experiments. Taking these corrections into consideration, the results are quite close to one another. The hypothalamic uptake was less active. A T/M ratio of 9.2 was found (about 25 according to Snyder and Coyle as calculated to the particulate fraction). The apparent T/M ratio at 0°C was 0.8 in the hypotalamus, including the amine adsorbed onto the filter. The uptake exhibited a saturable type of kinetics at low concentrations, both in the striatum and in the hypothalamus (figs. 1 and 2). However, at high concentrations no apparent Vma x was seen, and the uptake increased linearly to the highest concentrations used (figs. 3 and 4). This indicates that at least two processes were involved, a saturable one and an additional linear process which was assumed to be due to passive processes like adsorption as well as diffusion aided by granular storage (the animals were not reserpinized). The linear portion was extrapolated to the zero concentration and used to calculate the pas-

L. Tuomisto et al., Conformational selectivity of dopamine uptake

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10- 7 M, and the other from 5 X 10-7 M to 10-s M (fig. 2). The corrected data were used for double-reciprocal plots (Lineweaver and Burk, 1934)after subtracting the passive process. In the striatum, the plot was linear (fig. 5) and gave a K m of 1.09 X 10- ~ M, and a Vma x of 12.7 nmol/g/5 rain. In the hypothalamus, the plot was linear at low concentrations (5 X 10-s M to 2 X 10-7 M) and a K m o f l . 0 3 X 10- ~ M a n d a Vma x of 1.5 nmol/g/5 min was calculated. At higher concentrations a higher K m o f 9.7 × 10-~ M was estimated, and the uptake approached another Vma x of about 4.4 nmol/g/5 ndn.

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Fig. 3. Uptake of dopamine by striatal synaptosomes at high dopamine concentrations. Two separate processes ate soon, one saturable and one linear. The lower curve was obtained by extrapolating the linear process to zero concentration and subtracting it from the total uptake. Other conditions as in fig. 1.

(-)-Noradrenaline was the most effective o f the compounds studied in inhibiting 3 H-dopamine uptake by striatal synaptosomes (table 1). Its IDs 0 (the concentration causing an apparent 50% inhibition o f uptake) was 1.2 X 10"~ M when the aH-dopamine concentration was 10-7 M. This is slightly lower than the K i o f noradrenaline in the striatum, as given by Coyle and Snyder (1969). The most potent of the decalin analogs o f noradrenaline was the gauche isomer with an equatorial catechol ring and an axial amino group (2(a)-amino-3(e)3,4-dihydroxyphenyl.3-trans-decalol). Its potency was one half o f that o f (-)-noradrenaline, and 22

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Fig..5. a. Double-reciprocal plots of dopamine uptake by striatal synaptosomes. The plots with inhibitor, 2(a)-amino-3(e)-3,4dihydroxyphenyl-3-trans-decalol, demonstrate that the inhibition is competitive with an unchanged Vmax. The positions of the lines, as well as the Km and Vma x values, were calculated by computer. • •, without an inhibitor, Km = 1.09 X 10-7 M, Vma x = 12.7 nmol/g/5 rain, r 0in•at correlation coefficient o f the double reciprocal plot) = 0.992; x x, inhibitor 2 X 10-6 M, Km ffi 3.51 X 10-7 M, Vma x = 14.8 nmot/g/5 rain, r = 0.998; o o, inhibitor 5 X 10"~ M, Km = 8.55 X 10- 7 M, Vmax = 18.5 nmol/g/5 rain, r = 0.999. The results were also checked by using Eadie-Hofstee plot (Eadie, 1952; Hofstee, 1952), and essentially identical results were obtained. Mean of 5 - 6 duplicate experiments on an average. Fig. 5. b. Double-reciprocal plots of dopamine uptake by hypothalamic synaptosomes. Two different uptake processes are seen, with different Km and Vmax values. Both of these were inhibited competitively by 2(a)-amino-3(e)-3,4-dihydroxyphenyl-3-transdecalol. Low-concentration area; • e, without an inhibitor, Kin = 1.03 × 10-7 M, Vmax = 1.5 nmol/g/5 rain, r = 0.978; inhibitor 2 × 10-.6 M (for clarity not shown in the figure), K m = 2.48 X 10-7 M, Vma x = 1.5 nmol/g/5 rain, r = 0.995; o o, inhibitor 5 X 10"~ M, Kin 3.01 X 10 -7 M, Vmax ffi 1.5 nmol/g/5 min, r ffi 1.000. High-concentration area; • •, without an inhibitor, Km = 9.66 X 10-7 M, Vmax = 4.4 nmol/g/5 rain, r ffi 0.999; 2 X 10-6 M inhibitor (not shogn), K m = 12.2 X 10- 7 M, Vmax ffi 2.9 nmol/g/5 min, r = 0.954; o o, 5 X 10-6 M inhibitor, Km = 13.5 X 10-7 M, Vmax = 4.1 nmol/g/5 rain, r ffi 0.962. The results were also checked by Eadie-Hofstee plot with similar results. Mean of 5 - 6 duplicate experiments on an average.

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times higher than that of the anti-isomer. The IDs 0 of the gauche and the anti-isomer were 2.4 × 10-~ M and 5.2 X 10-s M, resp. The other two gauche isomers were of a low potency.

3.3. Inhibition of dopamine uptake in the hypothalamu$

• (-)-Noradrenaline inhibited 50% of dopamine uptake in the hypothalamus at a similar concentration as in the striatum (table 2). However, even the most active of the decalin analogs was only one fourth as potent as (-)-noradrenaline. The active isomer was the same as in the striatum, but it was only four times more potent than the anti4somer. The other two isomers were about as active in the hypothalamus as in the striatum. Thus the main difference between these two brain regions was that the most potent gauche isomer and the anti-isomer were closer to one another in potency in the hypothalamus than in the striatum.

3.4. Kinetics of the inhibition According to the double reciprocal plot the K m for dopamine uptake in the striatum was 1.09 X 10-7

M (fig. 5). This is somewhat lower than previously reported by Coyle and Snyder (1969). The most potent decalin analog, 2(a)-amino-3(e)-3,4-dihydroxyphenyl-3-trans-decalol, was a competitive inhibitor of this uptake, increasing the apparent Kin, whereas the Vma x remained the same. In the hypothalamus, the picture was more complicated, as discussed above. At low concentrations, a typical plot of a competitive inhibition was obtained (fig. 5). Also at high concentrations, the plot approached that of competitive inhibition although at the concentrations used the plot was slightly hyperbolic rather than completely linear. The correlation was, however, very good. The form is possibly a result of the overlapping of the high K m and low K m areas and is not necessarily due to a truly hyperbolic curve.

3.5. Inhibition of dopamine uptake by decalin compounds devoid of the noradrenaline structure To rule out the possibility that the inhibition might be unspecific and due to the aminodecalin moiety, some decalin compounds without a complete noradrenaline structure were tested. 3(e)-Aminotrans-decalin inhibited dopamine uptake both in stria-

L. Tuomisto et al., Conformational selectivity of dopamine uptake

tal and in hypothalamic synaptosomes to an appreciable extent (table 3). Its potency in the striatum was within the same range as that of the less active noradrenaline analogs. The inhibition was competitive (fig. 6). The other compounds tested were of low activity.

4. Discussion

The present results revealed a most interesting difference between different stereoisomers of noradrenaline-like decalin analogs in their potency to inhibit dopamine uptake in synaptosomes. A summary of the results is shown in fig. 7. Since the most potent analog was close in potency to its parent amine, noradrenaline, and since the inhibition was competitive, it is conceivable that it inhibited dopamine uptake by competing with the substrate of the receptor of the amine carrier through its dopamine-like moiety. This does not necessarily mean that this compound itself would be transported. In any case, the differences in potency between different stereoisomers may reflect

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the true conformational selectivity of the uptake receptor. If this assumption is correct, it seems as though the most favourable conformation for attachment to the carrier receptor was gauche. That the other gauche isomers were not more active, may easily be explained by the possibility of a steric hindrance caused by the decalin structure. In the hypothalamus, the preference was much less marked: the gauche isomer was relatively less active and the anti-isomer more active than in the striatum. This is in accordance with our recent results on noradrenaline uptake in the hypothalamus (Tuomisto et al., 1973). The same isomer which was the most active in the present study, was also the most active noradrenaline uptake inhibitor in the hypothalamus, but the difference to the anti-isomer was only twofold (IDso 4.5 × 10-6 M and 9 X 10-~ M, respectively). One possibility to rationalize these findings would be as follows: the striatum is assumed to contain mainly dopamine neurons (Fuxe, 1965). These are responsible for the highly active uptake in this area, and they are stereoselective with a preference for the gauche form. In the hypothalamus there are mainly noradrenaline neurons but also some dopamine neurons. When noradrenaline uptake is tested, the uptake is mainly to the noradrenaline neurons with a low Km and high affinity for (-)-noradrenaline (Snyder and Coyle, 1969). These apparently are quite flexible in their conformational requirements. If dopamine uptake is measured in the hypothalamus, the presence of the few dopamine neurons becomes important since the Km for dopamine in them is lower than in the NA neurons. Therefore the difference of potencies is about 5 if the substrate is DA but only 2 if the substrate is NA in a similar preparation. This explanation is only one attempt to explain the differences in stereoselectivity and may be an oversimplification, but it might help in understanding these processes when studying them further. These assumptions do not agree with some kinetic results of Snyder and Coyle (1969). They found a Km for dopamine of 4.0 X 10-7 M in the striatum. In the hypothalamus the Km was 0.8 X 10-7 M, when low concentrations were used, and 1.4 X 10-~ M in the high concentration area. This suggested that at low concentrations the affinity for dopamine is actually higher in the hypothalamus than in the stria-

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L. Tuomisto et al., Conformational selectivity of dopamine uptake

tum. However, according to the present data the Km is the same in both the striatum and in the hypothalamus (1.09 × 10-7 M and 1.03 × 10-7 M, resp.). Assuming that the present data are correct, and that the Km for dopamine in the striatum and in the hypothalamus are identical at low concentrations, the rationalization given above becomes reasonable. At low concentrations the identical Km's but differing Vma x values suggest a presence of similar receptors, but that their number would be different. This again, suggests the presence of many neurones, conceivably dopamine neurones, in the striatum and relatively few, perhaps only one tenth, in the hypothalamus. At high concentrations, remarkable amounts of dopamine would also be taken up by other hypothalamic neurons as well, but the affinity would be lower, reflecting in high Km . It is intriguing that according to molecular orbital calculations (Kier and Truitt, 1970) the preferred conformation of dopamine is gauche. Noradrenaline has its energy minimum at 180 ° or anti-conformation, but the difference to the gauche form is not great (Kier, 1969). The recent results of Katz et al. (1973) also suggest that dopamine exists predominantly as the gauche conformer. Although the energy minimum of noradrenaline also exhibited a gauche conformation according to this study, both anti and gauche conformers were interpreted to exist in solution and to be biologically important. Therefore it would seem logical that the uptake mechanism for dopamine would prefer the gauche conformer: this would be in favour of dopamine rather than noradrenaline as the substrate. In noradrenaline neurons dopamine is present as the precursor of noradrenaline. Therefore it is perhaps not advantageous that the uptake into these ceils would discriminate dopamine. Consequently the carrier should accept the two conformations of noradrenaline, one of which is the preferred conformation of dopamine as well. The finding that aminodecalin, without a complete catecholamine structure, is also able to inhibit dopamine uptake, undoubtedly warns against drawing too definite conclusions. The amine uptake mechanisms appear to be fairly unspecific, and competitive inhibition is achieved by substances not too closely related to the catecholamines. These problems were discussed at length previously (Tuomisto, 1972). However, this unspecificity may also help in under-

standing the effectiveness of the substances with a 'wrong conformation'. 3(e)Amino-trans-decalin was actually more potent than the least active norepinephrine analogs. Hence there exists a possibility that the weaker isomers attach to the receptor by using the aminodecalin moiety and not the catechol ring at all. On the other hand, this explanation does not appear likely as far as the most active gauche isomer and the anti-isomer are concerned. In these isomers, the amino group is axial, and the aminodecalin with an axial amino group was of very low potency. Certainly the aminodecalin moiety does not explain the difference between the most active gauche isomer and the anti-isomer, since that moiety was identical in both of them. Another disadvantage with the large decalin structure is that it causes steric hindrance. These problems of having one or more of the angles of attachment excluded were discussed previously (Tuomisto et al., 1973). It could be argued that the apparent uptake inhibition might be due to the amine releasing effect of the decalin derivatives, since the animals were not reserpinized. The endogenous dopamine released, would compete for uptake sites with the substrate added. However, the amount of tissue used for one sample was only 5 mg. Assuming 6/ag/g of dopamine in the striatum (I-Iolzbauer and Sharman, 1972), a maximal dopamine concentration of 0.5 × 10-7 M could be achieved in the incubation medium after the release of all endogenous amine. The resultant theoretical and most unlikely rise of substrate concentration from 1 X 10-7 M to 1.5 X 10-7 M could cause only a trivial decrease in the uptake of radioactive amine (cf. fig. 1). Hence the influence of release on the differences in potency of the decalin derivatives, if any, is likely to be negligible. In conclusion, the present results suggest that the amine uptake mechanism at least in the dopamine neurons in the striatum is conformationally selective. It appears that the preferred conformation for the inhibitors of uptake is gauche, and since the inhibition is competitive, it is conceivable (although not certain) that the uptake mechanism would also favour the gauche conformer of the substrate. These findings might have important implications in efforts to develop specific inhibitors of different kinds of uptake.

L. Tuomisto et al., Conformational selectivity of dopamine uptake Note added in proof Subsequently some of the results were repeated by using animals given 1 mg/kg of reserpine 18 hr before sacrifice. In the striatum, the IDs o of noradrenaline, the most potent gauche derivative and the anti derivative, was 9 X l 0 -7 M, 2.2 X l 0 -6 M, and 3.8 X 10-s M, respectively. The inhibition was competitive. Hence the results support those on unreserpinized animals. In the hypothalamus the gauche isomer was somewhat less potent after reserpinization, but it was still about 3 times as potent as the anti-isomer.

Acknowledgements The authors are indebted to Drs. R.T. Borchardt and T.L. Pazdernik for the synthesis of the decalin compounds, and to Dr. E. Solatunturi for the electron microscopy of the brain homogenates. For skilful technical assistance we wish to thank Mrs. Anneli v. Behr and Miss Aita Klemela. This study was supported in part by the National Research Council for Medical Sciences, Helsinki, Finland, and the Yrjo Jahnsson Foundation, Helsinki, Finland.

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