Proc. NatL Acad. Sci. USA Vol. 80, pp. 5134-5138, August 1983 Neurobiology

Modulation of neuronal serotonin uptake by a putative endogenous ligand of imipramine recognition sites (serotonergic axons/adenylate cyclase/p-adrenergic receptor function/antidepressants) MARIA LUISA BARBACCIA, OTTAVIo GANDOLFI, DE-MAW CHUANG, AND ERMINIO COSTA Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 Contributed by Erminio Costa, May 16, 1983

ABSTRACT Imipramine inhibits the serotonin uptake by [3H]imipramine recognition sites, prevented the down-regu- binding with high affinity to regulatory sites of this uptake located lation of f3-adrenergic recognition sites elicited by two daily in- on axons that release serotonin. The number of imipramine rec- jections of DMI repeated for 3 weeks (18). These data sug- ognition sites located on crude synaptic membrane preparations gested to us that a neuronal regulatory loop might connect 5HT is reduced by two daily injections of imipramine or desmethyl- terminals with the neuron where ,B-adrenergic receptors are imipramine for 3 weeks. When the binding sites for [3H]imipra- located and that this link participates in the attenuation of NE mine are down-regulated the Vma of the neuronal uptake of sero- receptor function and, perhaps, in the antidepressant action of tonin is increased. Moreover, in minces prepared from the brain imipramine and drugs (17, 18). hippocampus of rats receiving imipramine in a dose regimen that related These findings have been reduces the number of [3H]imipramine recognition sites, the ef- replicated by us with repeated daily injections of imipramine ficiency of imipramine as a blocker of the serotonin uptake is di- (19) and confirmed by other laboratories (20). Therefore we de- minished. Hence the high-affinity binding sites for [3H]imipra- cided to study the effect of repeated imipramine injections on mine may have a physiological role in modulation of serotonin the 5HT uptake as a departure point to elucidate the neuronal reuptake. Probably this is mediated by an endogenous effector of circuitry and identify the molecular nature of the modulator(s) these regulatory sites. A nonpeptidic constituent of rat brain ca- that may physiologically operate at the sites where imipramine pable of displacing [3H]imipramine from its high-affinity binding binds, thereby triggering a modulation of the 5HT uptake site and of inhibiting the serotonin uptake in a dose-related man- mechanism. ner has been extracted and its partial purification is described. METHODS Imipramine and desmethylimipramine (DMI) have been used Animals. Male Sprague-Dawley rats (150-250 g) from Zivic- for longer than two decades to relieve the symptoms of depres- Miller were used for all the experiments. The animals were sion (1, 2). Their capacity to block norepinephrine (NE) and housed under standard laboratory conditions with a 12-hr light/ serotonin (5-hydroxytryptamine; 5HT) uptake has been consid- dark cycle and food and water ad lib. The rats were injected ered to be important for their therapeutic action, but when it with imipramine or DMI (10 mg/kg intraperitoneally, twice daily was noted that imipramine and DMI block amine uptake almost for 21 days) and were decapitated 72 hr after the last drug in- instantaneously, whereas their antidepressant action occurs with jection. The control rats received saline intraperitoneally with a latency time of 1-2 weeks (1), the importance of the uptake the same injection schedule. inhibition in explaining their therapeutic action was deem- Materials. Imipramine hydrochloride either was a gift from phasized. Upon repeated daily administration to rats imipra- CIBA-Geigy or was purchased from Sigma. DMI as the hy- mine and a number of its congeners down-regulate the function drochloride was a gift from Merrel Laboratories (Cincinnati, of NE recognition sites in several brain structures (3-6); be- OH). Fluoxetine hydrochloride (LY 110140) and nisoxetine hy- cause the onset of this down-regulation and that of the anti- drochloride (LY 94939) were gifts from Eli Lilly. 5HT as the depressant action are delayed with respect to the beginning of creatine sulfate and pargyline hydrochloride were purchased the treatment, it has been suggested that the NE response down- from Sigma; 5,7-DHT as the creatine sulfate was purchased from regulation and the antidepressant action may be related (6). An Calbiochem-Behring; Pronase was purchased from Boehringer additional tool to study the mechanism of the down-regulation Mannheim; [3H]5HT creatine sulfate (=20 Ci/mmol) and [3H]- of /3-adrenergic receptors became available when Langer and imipramine (-24 Ci/mmol) were from New England Nuclear his colleagues reported that specific Na+-dependent and high- (1 Ci = 3.7 X 1010 Bq). Bio-Gel P-10 (200-400 mesh) and AG affinity binding sites for [3H]imipramine are located in crude 50W-X8 (-SO3 strong cation exchanger) resin were from Bio- synaptic membranes prepared from various structures of mam- Rad. malian brains (7, 8). The maximal number, Bmax, of [3H]imip- 5HT Uptake Assay. 5HT uptake was measured either in syn- ramine binding sites is down-regulated by repeated injections aptosomes or in minces freshly prepared from hippocampi of of imipramine or DMI (9-11). These recognition sites are lo- rats receiving saline or imipramine. The animals were decap- cated on 5HT axon terminals (12-14) and appear to be anatom- itated and the hippocampi were dissected on ice within 45 sec ically and functionally associated with the 5HT uptake mech- and placed in ice-cold Krebs bicarbonate buffer (NaCl, 114 mM; anism (15-17). KCI, 4.7 mM; CaCl2, 2.0 mM; KH2PO4, 1.2 mM; MgSO4, 1.2 In 1981 at a symposium on the mode of action of antide- mM; NaHCO3, 25 mM; dextrose, 2 mg/ml; ascorbic acid, 0.2 pressants we reported that the selective destruction of 5HT ax- mg/ml; Na2EDTA, 0.05 mg/ml; pargyline, 10 AM) until minces ons by 5,7-dihydroxytryptamine (5,7-DHT), which eliminates or synaptosomes were prepared. Minces from rat hippocampi were prepared according to the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: DMI, desmethylimipramine; NE, norepinephrine; 5HT, ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. serotonin (5-hydroxytryptamine); 5,7-DHT, 5,7-dihydroxytryptamine. 5134 Downloaded by guest on October 3, 2021 Neurobiology: Barbaccia et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5135 procedure described by Shaskan and Snyder (21) with minor modifications. The hippocampi were cross cut (300 x 300 tim) 0~ by using a tissue chopper (McIlwain and Rodnight). These tis- E Total uptake sue prisms (two hippocampi per 10 ml of buffer) were gently 04 dispersed in Krebs bicarbonate buffer, saturated with O2/CO2 $0 (95%:5%, vol/vol). Aliquots of the mince dispersion (800 .ld b1-3 Fluoxetine blank 0.5 mg of protein) were transferred into test tubes and incu- 0 bated for 20 min at 370C in a Dubnoff metabolic shaker under CS- 02/CO2 atmosphere in the presence or in the absence of 50 a, tkM fluoxetine. The net 5HT uptake was determined as the dif- bf Net uptake ference in the [3H]5HT taken up in the absence and in the pres- LO ence of fluoxetine. In some experiments, blank values were determined in parallel at 00C, and, in these cases, the samples were incubated for 20 min in ice. Accumulation of 5HT was 0 100 300 500 700 proportional to the amount of-tissue present in each tube. The 1,000 incubation was started by adding to each tube (final volume 1 5HT, nM ml) 100-jul aliquots of [3H]5HT (to attain final concentrations FIG. 1. Saturation isotherm of [3H]5HT uptake in minces pre- ranging between 0.01 and 1 ,uM) dissolved in Krebs bicarbon- pared from rat hippocampus. The net uptake was determined from the ate buffer. The uptake was allowed to continue for 4 min and difference between the total uptake and the uptake in the presence of the reaction was stopped by dilution with 4 ml of ice-cold saline 50 ,uM fluoxetine. The incubation was at 370C for 4 min. Tissue minces and by rapid filtration under reduced pressure through Milli- were preincubated with fluoxetine for 20 min at 37°C under 02/CO2 pore filters (type HA; 0.45-,um pores). Each filter was washed atmosphere. three times with 3 ml of ice-cold saline. The minces were then collected from each filter and placed in glass vials, where they between 0.01 and 1.0 ,uM a monophasic 5HT uptake was de- were allowed to dissolve in 1 ml of 1 M NaOH. Protein content tectable. This, like the high-affinity 5HT uptake previously de- was determined in 25- or 50-1LI aliquots of this solution. The scribed (21), showed an apparent Km value for 5HT of -0.4- [3H]5HT taken up by the tissue was determined by scintillation 0.5 ,M and approached saturation at a 5HT concentration of spectrometry after adding 10 ml of scintillation fluid (Atom- about 1 ,uM. When higher 5HT concentrations (up to 10 tLM) light, New England Nuclear) to each vial. When the synap- were used a biphasic 5HT uptake was detected due to a second, tosomal uptake of 5HT was studied the synaptosomes were pre- low-affinity, uptake component (data not shown). In order to pared as described (22) and aliquots (50 ,ul) of the synaptosome measure the blank values the samples were preincubated with preparation were incubated with [3H]5HT in concentrations a maximally active concentration (50 uM) of fluoxetine, a se- ranging between 0.01 and 0.5 /iM. After 4 min the reaction was lective inhibitor of 5HT uptake. The net neuronal uptake was terminated by dilution with 4 ml of ice-cold saline and rapid calculated as the difference between the radioactivity re- filtration under reduced pressure through Whatman GF/B glass covered in the minces preincubated without and with fluox- fiber filters. Each filter was washed three times with ice-cold etine. The blank values obtained by incubating the samples at saline and the radioactivity present was counted by scintillation 37°C in the presence of fluoxetine were significantly higher spectrometry after adding 10 ml of scintillation fluid (Atom- than those obtained by incubating the samples without fluox- light or Aquassure, New England Nuclear). etine at 2-4°C (data not shown). This difference probably ac- [3H]Imipramine Binding. [3H]Imipramine binding was as- counts for a number of processes that are not corrected by low- sayed in crude synaptic membranes prepared from hippocampi temperature blanks: nonneuronal uptake, transport into sub- and cortices of rats treated daily for 21 days with saline, imipra- cellular organelles, or temperature-dependent passive diffu- mine, or DMI, according to the procedures previously de- sion. To validate the use of blanks with fluoxetine, experiments scribed (23). The protein content was determined colorimet- were carried out with hippocampal minces prepared either from rically as described by Lowry et al. (24), using bovine serum control sham-operated rats or from rats having a selective lesion albumin as standard. of the 5HT terminals produced by 5,7-DHT. The selectivity of 5HT Axon Terminal Lesions. Selective lesions of the 5HT the lesion was assessed by measuring the hippocampal content axon terminals were produced by injecting 200 ,ug (free base) of 5HT (25) and NE (26) in five rats taken at random from 40 of 5,7-DHT into the lateral ventricle of rats previously anes- that were injected with the neurotoxin. The brain 5HT content thetized with pentobarbital (35 mg/kg, intraperitoneally). 5,7- was decreased by about 80-85% in the rats with lesions, whereas DHT was injected in a volume of 10 ,ul (saline containing 0.01% the NE content was virtually unchanged in comparison to that ascorbic acid) at a rate of 2.5 ,ul/min. At 40-45 min prior to the of sham-operated rats. Fig. 2 shows that the net 5HT uptake neurotoxin injection the rats were treated with nisoxetine (20 measured in hippocampal minces from sham-operated rats against mg/kg, intraperitoneally), which blocks NE uptake and thereby blanks with fluoxetine is significantly lower than that measured enhances the neurotoxin selectivity of the 5HT neuron lesions. against blanks at 2-4°C. However, uptake calculations using The sham-operated rats were injected intracerebroventricu- fluoxetine blank values appear to be more reliable than those larly with the same amount of saline containing 0.01% ascorbic made by using blanks at 2°C: in fact, a 5,7-DHT lesion that re- acid. duces the 5HT content by 85% reduces the uptake calculated from the fluoxetine blanks by about 80-85%, whereas it de- creases only by about 40-45% the 5HT uptake calculated from RESULTS the blanks at 2-40C without fluoxetine. With fluoxetine as a Characterization of 5HT Uptake by Rat Hippocampus blank the Ki values for the inhibition of SHT uptake by fluox- Minces. The kinetic characteristics of the [3H]5HT uptake into etine, imipramine, and DMI were 0.6, 1.1, and 5.0 tiM, re- hippocampal minces prepared from rats receiving saline are spectively. Moreover, in hippocampal minces the net uptake of shown in Fig. 1. With an incubation time of 4 min, 0.4-0.6 SHT was inhibited by about 40-50% 40 min after a single in- mg of protein per tube, and concentrations of [3H]5HT ranging jection of imipramine (10 or 20 mg/kg, intraperitoneally). Downloaded by guest on October 3, 2021 5136 Neurobiology: Barbaccia et alPProc. Natl. Acad. Sci. USA 80 (1983) Table 1. Increase of the 5HT uptake in minces from rat hippocampi with a reduced number of imipramine recognition sites 2-v [3H]Imipramine [3H]5HT uptake _~= r binding B,,,., pmol/mg Treatment n fmol/mg protein protein per 4 min % ,1 ?cX ..... Saline 4 500 ± 25 6.9 ± 0.60 100 -. ..f DMI 4 390 ± 30* 11 ± 0.90* 160* n- The concentration of [3HI5HT used in uptake studies (run in tripli- S h i 5.7-1)11IT Sham- 5. i -D1)T cate) was 400 nM. [3H]Imipramine binding was assayed as described P at;lt (Ied epIrI11(, t, t es I 11 (23) over a range of [3Hlimipramine concentrations between 0.5 and 10 nM. The rats were given either saline or DMI (10 mg/kg, intraperi- FIG. 2. Net uptake of 5HT by hippocampal minces prepared from toneally) twice daily for 21 days and were killed 72 hr after the last in- sham-operated rats and rats with 5,7-DHT lesions. The net uptake was jection. Results are presented as mean ± SEM. calculated by subtracting blank values determined at 2-4°C (open bars), *fP < 0.05 comparedto values obtained in saline-treated rats (Student's or in the presence of 50 MM fluoxetine at 370C (shadowed bars). Each t test). bar represents the mean SEM ofthree different determinations run in triplicate. The [3H]5HT concentration used in these experiments was 200 nM. Student's t test: *, P < 0.005 compared to the values ob- efficiency of 5HT uptake, selectively; in fact, neither the up- tained in sham-operated rats; t, P < 0.005 compared to the net uptake take of NE nor that of DA was changed in hippocampi, cor- values obtained after subtraction ofblank values determined at 2-4°C. tices, and striata of rats receiving imipramine or DMI (data not The extent of 5HT uptake reduction due to the lesion of 5HT axon ter- minals was similar in the two conditions, but the percent of decrease shown). (-44% and -88%, respectively) was greater when the net uptake was Relationship Between the fH]Imipramine Binding Sites and calculated by considering the blank values at 37°C in the presence of the 5HT Uptake Mechanism. The Bma. of [3H]imipramine fluoxetine. The lesion was produced by injecting 5,7-DHT (200 Mg of binding to crude synaptic membranes prepared from hippo- free base per 10 ul of saline, containing 0.01% ascorbic acid) into the campi of rats receiving imipramine (twice daily for 3 weeks) is lateral ventricle. reduced, whereas the net 5HT uptake (Vm.) is increased (Table 2). Furthermore, Table 2 shows that the inhibitory effect on the 5HT Uptake by Minces of Hippocampi with [3H]Imipra- 5HT uptake by various imipramine concentrations added in vi- mine Recognition Sites Down-Regulated by Daily Injections tro to the hippocampal minces is attenuated when the number of Imipramine or DMI Repeated for 21 Days. Daily injections of [3H]imipramine binding sites is decreased by repeated imip- of imipramine or DMI (10 mg/kg, twice daily) for 3 weeks re- ramine injections. In fact, 1 /iM imipramine inhibited by 33% duced the Bma. of [3H]imipramine specific binding to crude the 5HT uptake in saline-treated rats, whereas it was ineffective synaptic membranes prepared from several structures of rat (11% inhibition) in imipramine-treated rats. A higher concen- brain, including the hippocampus (Tables 1 and 2). Fig. 3 shows tration of imipramine (5 ,uM) inhibited by 48% (P < 0.05) the that such a dose regimen of imipramine (10 mg/kg twice daily 5HT uptake in hippocampal minces prepared from imipramine- for 21 days) increases the net uptake of 5HT by hippocampal treated rats and by 60% (P < 0.005) the 5HT uptake in minces minces. The saturation isotherm and the transformation of the prepared from rats receiving saline. data into a double-reciprocal plot clearly show that the en- Extraction and Partial Purification from Rat Brain of an hancement of the 5HT uptake is due to an increase in Vma, Endogenous Factor Endowed with Imipramine-Like Activity. (+40%), while the apparent Km value for 5HT is virtually un- Typically, four to six rat brains were used for each preparation. affected. A qualitatively similar result was obtained when the The animals were decapitated and each brain was rapidly ex- 5HT uptake was measured in unwashed crude synaptosome cised from the skull, cut in small prisms, and dumped in hot preparations. However, the extent of the uptake increase was (90°C) 1 M acetic acid (1/10, wt/vol) within 30 sec from de- found to be somewhat lower than in minces prepared from hip- capitation. After 10-15 min the brain prisms were homoge- pocampi of rats receiving daily injections of imipramine. Table nized with an Ultra-Turrax tissue homogenizer for 30-40 sec 1 shows that daily injections of DMI, for 3 weeks, decrease the and heated again (90°C). After 5-10 min, the homogenate was number of [3H]imipramine binding sites and enhance the 5HT centrifuged (48,000 X g for 10 min). The first supernatant was uptake by hippocampal minces. Moreover, daily injections of combined with a 1 M hot acetic acid extract (1:5, wt/vol) of the imipramine or DMI, given for 3 weeks, appear to increase the first pellet and these two extracts were recentrifuged (48,000

Table 2. Attenuation of the imipramine-induced V,,, inhibition of 5HT uptake in hippocampal minces prepared from rats showing a decrease of [3Hlimipramine binding sites after repeated imipramine injections [3H]5HT uptake No additions 1 MuM imipramine 5 ,uM imipramine [3HlImipramine specific binding pmol/mg protein pmol/mg protein pmol/mg protein Kd, Treatment n per 4 min % per 4 min % per 4 min % fmol/mg protein % nM Saline 6 3.9 ± 0.26 100 2.6 ± 0.47* -33 1.5 ± 0.12t -60 520 ± 41 100 5.7 ± 0.5 Imipramine 6 5.4 ± 0.42* 140 4.8 ± 0.57 -11 2.8 ± 0.40* -48 370 ± 25* 71 7.2 ± 0.9 The rats received saline or imipramine (10 mg/kg, intraperitoneally) twice daily for 21 days and were killed 72 hr after the last injection. The concentration of [3H]5HT for the 5HT uptake assay was 300 nM. [3H]Imipramine binding was assayed as described (23) in a range ofconcentrations between 0.5 and 10 nM. Each value is the mean ± SEM of three different determinations, each one done in triplicate. *P < 0.05 compared to no-addition control values (Student's t test). tP < 0.005 compared to no-addition control values (Student's t test). tP < 0.05 compared to values for saline-treated rats (Student's t test). Downloaded by guest on October 3, 2021 Neurobiology: Barba-ccia et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5137 FIG. 3. Net uptake of 5HT Table 3. Pronase fails to reduce the [3H]imipramine binding by minces prepared from hip- inhibition by an endogenous effector lo°-10.0£ pocampi of saline-treated (i) and X *¢ 8.0 , imipramine-treated (i) rats. The [3H]Imipramine z 6.0 rats received either saline or Addition specific binding Sz E- o. 4.0 ,/ imipramine (10 mg/kg, twice (30 1l) fmol/mg protein % E - 2.0 daily for 21 days) and were killed t ' . .. . Buffer 244 ± 13 100 ~0 72 hr after the last injection. The E 200 600 1,000 saturation curve (Inset) repre- Boiled Pronase 253 ± 3 103

5HT, nM sents a typical experiment in Rat brain extract 158 ± 7* 62 * 1.0 which both hippocampi from five Rat brain extract + Pronase 168 ± 6* 69 o rats were pooled. Each point ", 0.8- represents the mean of quad- The [3H]imipramine binding was assayed on crude synaptic mem- C_ X ruplicate determinations. The branes prepared from frontal cortex and hippocampus of rat brain. -0.6o SEM was always

a - It$ 80 - 80 - DISCUSSION O In rats receiving repeated daily injections of imipramine (19) or CD .E 60~~~~~~~~~~~~~~~~~06 DMI (18, 27) the integrity of the brain serotonergic axon ter- 60- - E-- is a necessary requirement to obtain the attenuation of zT minals FIs ,B-adrenergic receptor responsiveness to specific agonists. We interpret this finding to indicate that the occupancy of [3H]- 4 8 12 16 20 Fraction imipramine binding sites by this drug can trigger a chain of bi- ological responses leading to the down-regulation of f3-adren- FIG. 4. AG 50W-X8 column (0.7 x 4 cm) chromatography of the ergic receptors. This finding suggests that a site located on 5HT- rat brain extract endowed with inhibitory activity on [3H]imipramine releasing axons could be the primary site whereby imipramine binding and [3H]5HT uptake. The resin was previously regenerated with 5 ml of 1 M HCl and then extensively washed with H20 (20-30 ml). A and related drugs act in the central nervous system. Perhaps 1-ml sample in H20 (corresponding to the extract from one rat brain) the synaptic signal(s) generated by 5HT axon terminals is mod- was applied and was eluted with 3 ml of H20 (1); 4 ml of 0.2 M HCl (2); ulated by repeated imipramine injections and as result of this 4 ml of 1 M HCl (3); 4 ml of 2 M HCl (4); 4 ml of 2 M HCl/30% (vol/ modulation a P3-adrenergic receptor down-regulation ensues. It vol) methanol (5). Fractions (1 ml) were collected, lyophilized, and re- is interesting now to test whether this down-regulation has be- suspended in 200 of H20. Aliquots (30 Dl) from each fraction were havioral and therapeutic significance. This evaluation requires tested for their effects on [3H]imipramine binding (.) and on [3H]5HT we know the functional significance of [3H]imipramine uptake in rat brain synaptosomes (o). [3H]5HT (-130,000 cpm) added that to the extract could not be eluted under any of the elution conditions recognition sites. Several lines of independent investigation (28- listed above (A). 30) led us to believe that the [3H]imipramine binding site could Downloaded by guest on October 3, 2021 5138 Neurobiology: Barbaccia et aL Proc. Natl. Acad. Sci. USA 80 (1983) not be identified with the 5HT recognition site located on the imipramine recognition sites. Such a molecule may be of im- uptake carrier. The present results further support this view by portance in studying the endogenous factors that are operative showing that the decrease in the Bm,,a of [3H]imipramine bind- in the affective disorders of man. ing sites is associated with an increase in the Vma. of 5HT up- take (Fig. 3). In addition, these data support the possibility that 1. Klein, D. F. & Davis, J. M. (1969) Diagnosis and Drug Treat- the sites where [3H]imipramine binds are physiological regu- ment of Psychiatric Disorders (Williams & Wilkins, Baltimore). latory sites for the uptake of 5HT (17, 30). Hence one might 2. Kuhn, R. (1958) Am. J. Psychiatry 115, 459-464. infer that the 5HT uptake functions as a supramolecular or- 3. Banerjee, S. P., Kung, L. S., Riggi, S. J. & Chanda, S. K. (1977) Nature (London) 268, 455-456. ganization in which various subunits are involved in the fine 4. Sarai, K., Frazer, A., Brunswick, D. & Mendels, J. (1978) Biochem. tuning of the uptake, which therefore can be viewed as an en- Pharmacol 27, 2179-2181. ergy-dependent, receptor-modulated process. So far, two func- 5. Vetulani, J. & Sulser, F. (1975) Nature (London) 257, 495-496. tional components of the supramolecular system that takes up 6. Sulser, F., Vetulani, J. & Mobley, P. L. (1978) Biochem. Phar- 5HT in serotonergic axons have been unveiled. The recognition macot 27, 257-261. site for 5HT located on the uptake carrier and the uptake mod- 7. Raisman, R., Briley, M. S. & Langer, S. Z. (1979) Nature (Lon- ulatory site where imipramine binds are revealed by the pres- don) 281, 148-150. 8. Langer, S. Z., Raisman, R. & Briley, M. S. (1980) Eur. J. Phar- ent experiments. Studies on porcine and human platelets have macot 64, 89-90. shown that the 5HT uptake is also regulated by the flux of Na' 9. Kinnier, W. J., Chuang, D. M. & Costa, E. (1980) Eur. J. Phar- and K+ (31, 32). If such an interaction is operative in neuronal macol 67, 289-294. membranes, the complexity of the 5HT uptake system in- 10. Brunello, N., Chuang, D. M. & Costa, E. (1982) in Typical and creases, resembling the organization of an ion channel linked Atypical Antidepressantsfrom Molecular Mechanisms to Clinical Practice, Advances in Biochemical Psychopharmacology, eds. to transmitter postsynaptic receptors such as a chemically reg- Costa, E. & Racagni, G. (Raven, New York), pp. 179-184. ulated Na' channel. The supramolecular complexity of the 5HT 11. Briley, M., Raisman, R., Arbilla, S., Casadamont, M. & Langer, uptake system could explain why we were able to obtain more S. Z. (1982) Eur. J. Pharmacol 81, 309-314. consistent results in minces than in synaptosomes; probably when 12. Sette, M., Raisman, R., Briley, M. & Langer, S. Z. (1981)J. Neu- the preparation procedures are too lengthy, the function of im- rochem. 37, 40-42. portant subunits that regulate the fine tuning of 5HT uptake is 13. Brunello, N., Chuang, D. M. & Costa, E. (1982) Science 215, 1112- 1115. lost. Repeated daily injections of DMI elicit changes on the 5HT 14. Grob, G., Gothert, M., Ender, M.-P. & Schumann, H.-J. (1981) uptake that are similar to those elicited by imipramine. This Naunyn-Schmiedeberg's Arch. Pharmacol 317, 310-314. similarity was unexpected because the specific binding sites for 15. Langer, S. Z., Moret, C., Raisman, R., Dubocovich, M. L. & [3H]DMI are located on NE axon terminals of various struc- Briley, M. (1980) Science 210, 1133-1135. tures of rat brain (33-36). However, that DMI acts also on 5HT 16. Paul, S. M., Rehavi, M., Rice, K. C., Ittah, Y. & Skolnick, P. (1981) axons, similarly to imipramine, can be inferred from the in- Life Sci. 28, 2753-2760. 17. Barbaccia, M. L., Brunello, N., Chuang, D. M. & Costa, E. (1982) hibition'by 5HT axon lesions (17, 18) of the long-term action of Neuropharmacology 22, 373-383. DMI on the accumulation of cyclic AMP elicited by NE; more- 18. Brunello, N., Chuang, D. M. & Costa, E. (1982) in New Vistas in over, a long-term treatment with DMI down-regulates the Bma. Depression, Advances in Bioscience, eds. Langer, S.].-.& Briley, of [3H]imipramine binding sites (10, 17). Although DMI is less M. S. (Pergamon, Oxford), Vol. 40, pp. 141-145. selective-than imipramine as an inhibitor of the 5HT uptake, 19. Barbaccia, M. L., Chuang, D. M., Gandolfi, 0. & Costa, E. (1983) it can displace the [3H]imipramine specifically bound to crude in Frontiers in Neuropsychiatric Research, ed. Usdin, E. (Mac- millan, Houndmills, UK), pp. 19-31. synaptic membranes (8, 15, 30). At the present level of knowl- 20. Janowski, A., Okada, F., Manier, D. H., Applegate, C. D., Sul- edge the down-Tegulation of brain A-adrenergic receptors elic- ser, F. & Steranka, L. (1982) Science 218, 900-901. ited by DMI could be explained by its binding to 5HT axons 21. Shaskan, E. G. & Snyder, S. H. (1970)J. Pharmacol Exp. Ther. and could be considered to be unrelated to the specific 3H-DMI 175, 404-418. binding to noradrenergic axons. 22. Baumann, P. A. & Maitre, L. (1977) Naunyn-Schmiedeberg's Arch. All these data taken together give credit to the view that the Pharmacol 300, 31-37. 23. Kinnier, W. J., Chuang, D. M., Gwynn, G. & Costa, E. (1981) [3H]imipramine recognition sites in the central nervous system Neuropharmacology 20, 411-419. are not merely acceptor sites for a drug but function as receptor 24. Lowry, 0. H., Rosebrough, M. J., Farr, A. L. & Randall, R. J. sites where endogenous effector(s) can bind in order to regulate (1951)J. Biol Chem. 193, 265-275. the efficiency of 5HT reuptake by'5HT neurons. In support of 25. Lackovic, Z., Parenti, M. & Neff, N. H. (1981) Eur.J. Pharmacol this inference we have been successful in extracting and- par- 69, 347-352. a rat brain factor that appears to 26. Saiani, L. & Guidotti, A. (1982)J. Neurochem. 39, 1669-1676. tially purifying endogenous 27. Brunello, N., Barbaccia, M. L., Chuang, D. M. & Costa, E. (1982) have the profile of imipramine action; i.e., it inhibits the 5HT Neuropharmacology 21, 1145-1149. uptake and displaces [3H]imipramine specifically bound to its 28. Ahtee, L., Briley, M., Raisman, R., Lebrec, D. & Langer, S. Z. recognition sites. We have tested this endogenous factor also on (1981) Life Sci. 29, 2323-2329. binding of [3H]flunitrazepam, [3H]mianserin, and [3H]dihy- 29. Abbott, W. M., Briley, M. S., Langer, S. Z. & Sette, M. (1982) droalprenolol, and none of these ligands could be displaced by Br. J. Pharmacol 76, 295P (abstr.). This activity is not 30. Sette, M., Briley, M. S. & Langer, S. Z. (1983)J. Neurochem. 40, the inhibitor of [3H]imipramine binding. 622-628. due to 5HT because chromatography on a strong cation-ex- 31. Rudnick, G. (1977)J. BioL Chem. 252, 2170-2174. change resin separates the endogenous ligand of imipramine 32. Talvenheimo, J., Nelson, P. J. & Rudnick, G. (1979)J. Biol. Chem. binding sites from [3H]5HT. The data in Table 3 indicate that 254, 4631-4635. the inhibition of [3H]imipramine binding by the extract is vir- 33. Rehavi, M., Skolnick, P., Hulihan, B. & Paul, S. M. (1981) Eur. tually unchanged by a Pronase digestion that could abolish the J. Pharmacol. 70, 597-599. in These observations 34. Langer, S.iZ., Raisman, R. & Briley, M. (1981) Eur.J. Pharmacol. action of other proteins treated parallel. 72, 423-424. suggest that the endogenous effector of the [3H]imipramine 35. Lee, C.-M. & Snyder, S. H. (1981) Proc. NatL Acad. Sci. USA 78, recognition site may not be a neuropeptide; further experi- 5250-5254. ments are needed to determine the chemical nature of this en- 36. Hrdina, P. D., Elson-Hartman, K., Roberts, D. C. S. & Pappas, dogenous modulator of 5HT uptake, which is a ligand of [3H]- B. A. (1981) Eur. J. PharmacoL 73, 375-376. Downloaded by guest on October 3, 2021