Demonstration of endogenous “imipramine like” material in rat brain

Life Sciences, Vol. 36, pp. 687-693 Printed in the U.S.A.

Pergamon Press

DEMONSIRAIION OF ENDOGENOUS "IMIPRAMINE LIKE" MAIERIAL IN RAT BRAIN Moshe Rehav%, Ilana Ventura and Yosef Sarne Department of Physiology and Pharmacology, Sackler School of Medlc%ne, l e l Av%v Un%verslty, Ramat Av%v 69978, Israel (Received in final form December 7, 1984)

SUMMARY lhe extract%on and partial pur%flcat%on of an endogenous "%m%pramlne-l%ke" material from rat bra%n %s described. The endogenous factor obtained after gel f i l t r a t i o n and s%l%ca chromatography %nhlb%ts [3H] imlpramlne specific blnd%ng and mimics the %nhlb%tory effect of %mlpramlne on [3H] serotonln uptake in both brain and platelet preparations. The effects of the endogenous material are dose-dependent and i t %nhlblts [3H] %mlpramlne binding %n a compet%t%ve fashion. The factor is unevenly distributed %n the brain with high concentration %n the hypothalamus and low concentration In the cerebellum. Tr%cycllc antidepressants are the most widely prescribed drugs for treatment of depression. In studying the blnd%ng of rad%oactlvely labeled %m%pramlne in v i t r o , specific and saturable high a f f i n i t y b%ndlng sites have been demonstrated %n membrane preparations from rat brain (1) human platelets (2) and human brain (3). Recent studies have demonstrated that the b%ndlng site for [3H] Imlpram%ne is closely associated wlth the presynaptlc neuronal and platelet uptake site for serotonln (4,5). An Ident%cal pharmacological p r o f i l e for [3H] %mlpramlne binding in human brain and platelets was demonstrated by competition studies with various antidepressant agents (2, 3, 6), thus, %t was suggested that the platelet may serve as a model for studying [3H] %mlpramlne b%nd%ng sites in human brain. A decrease %n[3H] Imipramine b~ndlng sites in platelets of depressed patients (7, 8) as well as %n the serotonln uptake (9) suggests that a defect in the seroton%n transport system may be involved %n the pathophys%ology of depression. The existence of spec%flc and functional hlgh a f f i n i t y Imlpramlne binding sites suggested the posslb%l%ty that an unldent%f%ed endogenous l%gand may exist (lO). Conf%rm%ng a recent study of Barbaccla et al ( l l ) , we demonstrate in this report the existence of an endogenous factor in rat braln extract wh%ch is a potent Inh%bltor of the In v i t r o [3H] imlpramlne bindlng and [3H] serotonln uptake in rat brain Furthermore, the endogenous factor is effective in Inh%bltlng [3H] imlpram%ne binding and [aH] seroton%n uptake in human platelets. Our study also shows the competitive nature of the interact%on between the endogenous factor and Im%pram%ne and the uneven d%str%butlon of the factor over the brain. Materials and Methods Endogenous "lmlpram%ne-llke" materlal was extracted from the brains of Charles River descendent albino rats (300-400 g). Brains were homogen%zed by an Ultra Turrax tlssue homogenizer In 5 volumes Ice-cold 50/0 trlchloracetlc 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.

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acid (TCA). The homogenate was centrifuged for lO mln at go00 g and the pellet was resuspended in 50/0 ICA and centrifuged again. The pooled supernatants were extracted with ether to remove the acid. The residual ether was evaporated from the aqueous phase and the protein-free, l l p l d - f r e e solution was freeze-drled, dissolved in O.2M acetic acid and applied to a Biogel P-2 gel f l l t r a t l o n column (5 x 80 cm). The column was eluted with 0.2M acetic acld at a rate of 3.5 ml per minute and fractions of 14.5 ml were collected. Following lyophillzatlon and reconstitutlon in d i s t i l l e d water, each of these fractions was tested for i t s a b i l i t y to i n h i b i t specific [3H] imlpramlne binding and [3H] serotonin uptake in platelets. The potent fractions from the Biogel P-2 column (41-58) were pooled, lyophilized and applied in l ml water to a s i l i c a column ( I . 5 x 18 cm). The column was eluted stepwlse by acetonitrile, 15°/o water in acetonitrile, 300/0 water in acetonltrile and pure water. Following freeze-drylng and reconstltution in water, fractions were tested for their a b i l i t y to i n h i b i t specif~c [3H] Imlpramlne binding and [3H] serotonln uptake in platelets. [JH] Imlpramine binding to human platelet and rat brain membrane preparations was measured as described before ( l , 2). [3H] Serotonln (5HI) uptake into platelets and brain synaptosomes and [3H] noreplnephrlne (NE) uptake into brain synaptosomes were performed by previously described methods (6, 1 2 ) . [3H] Dihydroalprenolol (DHA) binding to the B-adrenerglc receptor was measured according to Bylund and Snyder (13) and [3H] quinuclldlnyl benzilate (QNB) binding to the muscarinlc-chollnerglc receptor was assessed by the method of Yamamura and Snyder (14). [3H] Imipramine ~55.4 Ci/mmole), [3H] 5HI (26.7 Cl/mmole), [3H] NE ( l l . B Ci/mmole) and [JH],QNB (33.1 Ci/mmole) were purchased from New England Nuclear (Boston, MA). [ J H ] DHA (55 Cl/mmole) was purchased from Amersham (England). Results Following elutlon on Biogel P-2 column, fractions 41-58 were found to inhibit [3H] Imlpramlne binding in platelet membranes. These fractions were

pooled, freeze-drled and analyzed for their a b i l i t y to i n h i b i t [3H] Imlpramlne binding and [3H] serotonln uptake as compared to Imlpramine in both platelets and brain. The endogenous compound (Biogel) inhibited [3H] %mlpramlne (2nM) binding in platelets with IC50 values of 12 units/ml (l unit = lO/o content of whole brain extract) as compared to an ICso value of 1.6 nM for unlabeled Imlpramlne. The log dose-response curves for the endogenous compound and for Imlpramlne were parallel (Fig. l) suggesting a common site of action for Imlpramlne and the endogenous factor. The inhibition of [3H] serotonin uptake into platelets by the endogenous compound (Biogel) was dose dependent with an IC50 values of 30 unlts/ml as compared to 3.5 nM Imipramlne. Similar results were also found in brain where the endogenous compound inhibited both [3H] Imlpramlne binding (IC50 = 24 unlts/ml) and [3HI serotonln uptake (IC50 = 3 unlts/ml). Interestingly, the endogenous factor was 7 times less potent (IC50 = 20 unlts/ml) in inhibiting noreplnephrlne uptake into brain synaptosomes as has been also reported for imlpramlne (15) which is a better inhibitor of serotonin uptake than of noreplnephrine uptake in braln.Lineweaver-Burk analysis of serotonln uptake to platelets(Fig.2) revealed that the endogenous compound increased the Km of the serotonln uptake without any effect on Vmax. lhe specificity of the endogenous factor is manifested by our finding that extracts purified bX either Bio-gel gel f i l t r a t i o n or s l l l c a chrmatography failed to i n h i b i t [OH] QNB binding to the muscarlnic-chollnerglc receptor (ICso > lO0 units/ml) and [3H] DHA binding to the B-adrenerglc receptor (ICso > lO0 unlts/ml) in rat brain.

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The competitive nature of [3H] Imipramlne binding I n h i b i t i o n by the endogenous materlal ( s i l l c a chromatography) is demonstrated In Ftg. 3. Scatchard analysis of [3H] lmlpramtne binding to p l a t e l e t membranes In the absence and in the presence of the endogenous factor revealed an apparant decrease in a f f i n i t y (Kd) with no change In the number of binding sites (Bmax) suggesting a competitive reversible type of i n h i b i t i o n . The active fractions obtained from the BlogelP-2 column were f u r t h e r p u r i f i e d by using a s i l l c a column. The column was eluted with increasing proportions of water in a c e t o n i t r l l e , the elution p r o f i l e is demonstrated in Ftg. 4. A peak which contained i n h i b i t o r y a c t i v i t i e s against [3H] Imlpramlne binding and [3H] serotonin uptake In p l a t e l e t s was eluted by 300/0 water in a c e t o n l t r l l e ( f r a c t i o n s 9-15).The a c t i v i t y counted for 800/0 of the orlgtnal material subjected to the s i l i c a column. A marker of radioactive serotonln was eluted from the s i l i c a column by 15O/o water in a c e t o n l t r i l e ( f r a c t i o n s 4-6). Regional d i s t r i b u t i o n of the endogenous compound in rat brain was studied by extracting the compound from f l v e d i f f e r e n t regions of the brain. Extracts of these regions were tested for t h e i r potency In i n h i b i t i n g [3H] tmipramtne binding and compared to a standard curve of whole brain e x t r a c t . The hypothalamus was the richest region tested (49 unit equivalents per gram wet tissue) which is almost twice the concentration found in the cerebellum (28 units per gram wet t i s s u e ) . High concentration was also found in the strtatum (47 unlts/gram) while the medula-pons and the mldbraln had 37 units/gram and 24 units/gram respectively. Incubation of the endogenous extract with d i f f e r e n t p r o t e o l y t l c enzymes ( trypsin, ~-chemotrypsln, protease, carboxypeptidase A) had no e f f e c t upon i t s i n h i b i t o r y a c t i v i t y . . Endogenous Factor /umts/ml lO

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FIG. l Log dose-response curves for the Inhlbltlon of [3H] Imlpramlne blndlng by unlabeled Imlpramlne (e) and the endogenous materla1(A). Platelet membranes were incubated wlth [3H] Imlpramlne (2 nM) for 60 mln at 0-4°C. Bound [3H] Imlpramine was separated from the free llgand on GF/C f i l t e r s . Nonspeclflc binding was measured In the presence of l ~M chlorImlpramlne.

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Imlpramine Endocold in Rat Brain

Vol.

36, No.

7, 1985

The Identlflcat~on of h~gh-afflnity [3H] Im~pram~ne b~ndlng s~tes in the central nervous system and in platelets suggests the presence of an endogenous substance capable of regulating the physiological events mediated by these sites. The physlolog~cal functions of Imlpram~ne b~ndlng sites in the brain are unknown; nevertheless, there is a great deal of evidence suggestlng a close relatlonsh~p between Imlpramlne binding sites and serotonln uptake sites in both brain and platelets (4, 5). Moreover, the decreased

160

120

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FIG. 2 Lineweaver - Burk analysis of [3H] serotonln uptake to platelets in the absence ( • ) and in the presence (m) of 5 units /ml of the endogenous material ( s i l i c a chromatography) levels of Imlpramlne binding and serotonln uptake found in depressed patients (7, B, 9) suggest that there Is some physiological function for the Imlpramlne binding sites in the pathophyslology of depression. In the present study an endogenous "Imlpramlne-llke" material has been demonstrated In rat brain. The endogenous factor not only i n h i b i t s the binding of Imlpramlne to i t s specific site but also mimics i t s effect on the uptake of serotonln In both brain and platelets. Serotonln is an endogenous

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Imipramine Endocoid in Rat Brain

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neurotransmltter known to i n h i b i t [3H] Imipramine binding (2, 3). The endogenous factor demonstrated ~n the present study is not serotonln s~nce both P-2 and sillca columns completely separated between the endogenous factor and exogenous [3HI serotontn added to the columns as a marker, The ~nabillty of the endogenous materlal to Inhlblt radlollgand binding to the muscarln~c-cho1~nerglc and to the 8-adrenerglc receptors in rat brain membranes suggests that the effect of the endogenous materlal on the ~mlpramlne blnd~ng site is not due to nonspeclflc Interactions. Moreover, the ~mlpramlne-llke factor was found to be a more potent Inhlbltor of [3H] SHT uptake than [3H] NE uptake into rat brain synaptosomes and th~s s e l e c t i v i t y for the blogenic amine transporters has been also reported for Imtpramtne (15). Gel f l l t r a t l o n (Biogel P-2) gives also a preltmlnary estimation of the size of the endogenous compound which Is probably less than

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3000

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FIG 3 Scatchard analysls of [3HI lmlpramlne binding with (A) or without (e) 18 untts/ml of the endogenous f a c t o r . ( S l l l c a chromatography) [3H]Imtpramtne was incubated at d i f f e r e n t concentratlons(0.2-6 nN) with p l a t e l e t membranes (0.3 mg protein per tube)for 60 min at O°C. The incubation was carried out In a t o t a l volume of 350 pl of a 50 mN Trls-HC1 buffer, pH 7.4 containing 120 mN NaCl and S mR KC1. Non-speclflc binding was defined In the presence of 1 ~M chlorimlpramine. The values are the means of t r i p l i c a t e determinations with S.E.R. less than 5O/o

1000 daltons,

The log dose-response curves for [3H] tmlpramine binding I n h l b l t l o n by unlabelled tmipramtne and the endogenous material were found to be p a r a l l e l which suggests a common s l t e of action. The competitive nature of the i n h i b i t i o n was also supported by the Scatchard analysis and by Ltneweaver-Burk analysis In which there was no change in Bmax for [3H] Imipramlne binding or in Vmax for [3H] serotonin uptake in the presence of the endogenous f a c t o r . The broad peaks of a c t i v i t y that were obtained following 81o-gel and s t l l c a chromatography do not exclude the p o s s i b t l l t y of the exslstance of more than one active substance. The simple competitive k i n e t i c s observed for the i n h i b i t i o n of both serotontn uptake and tmipramine binding suggest that the p a r t l a l l y p u r i f i e d extract contains one I n h i b i t o r y substance. I f the extract contains two or more i n h i b i t o r y substances with d i f f e r e n t potencies I t would r e s u l t in a complex type of I n h l b l t l o n . On the

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other hand, a mixture of several compounds having the same i n h i b i t o r y potencies w i l l s t i l l reveal simple kinetics similar to those described. The regional d i s t r i b u t i o n of the endogenous compound in rat brain supports the physiological importance of the substance. As was found for [3H] Imlpramlne binding sites (3), the highest concentration of the new compound was found in the hypothalamus while the cerebellum had the lowest concentration. While this investigation was carried out, a study has been published ( l l ) in which the existence of an endogenous llgand of imlpramlne recognition sites

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Silica chromatography of the endogenous Imlpramlne-llke substance. Pooled fractions (41-5B) of Biogel-P-2 column were applied in l ml water to a s i l i c a column ( I . 5 x 18 cm). The column was eluted stepwise by a c e t o n l t r i l e (fractions I-2), 15O/o water in a c e t o n l t r i l e (3-7), 300/0 water in a c e t o n l t r i l e (8-17) and pure water (18-29). Fractions of 15 ml were collected. [ 3 H ] Imlpramlne binding (2 riM) in platelets (0) was measured as described in Fig. I. Uptake of serotonln into platelets (A) was measured using l x 10-8 M [3H] serotonln at 37QC for 2 minutes. The free [3H] serotonln was separated by centrlfugatlon and the radioactivity of the p e l l e t was measured after being extracted with 0.4 N HCI04. Non specific uptake was defined at 0-4°C. was suggested. I t is not clear yet whether we are demonstrating the existence of the same factor since our procedure is a l i t t l e d i f f e r e n t . Furthermore, in the previous report of Barbaccla et al. ( l l ) , there was no information concerning the nature of the interaction between the endogenous llgand and the Imlpramlne binding slte or i t s regional d i s t r i b u t i o n over the brain. Our prellnlnary studies confirm Barbaccla's observation that the endogenous compound is not a peptlde, thus, in our experiments, the Imlpramlne-llke a c t i v i t y was not affected by protease, trypsin, chemotrypsln

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Imipramine Endocoid in Rat Brain

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and carboxypeptldase A. In order to understand the physlologlcal role of the endogenous Imlpramlne-llke factor, the effect of various antldepresslve treatments (e.g. chronic electroconvulslve treatment, llthlum and MAO Inhlbltors) on the level of the endogenous factor In different brain regions is currently under Investlgatlon In our laboratory. Acknowledgement Thls research was supported by grants from the Basic Research Foundation of Tel Avlv University to M.R. and from the Schrelber Foundation to Y.S. References I. 2. 3. 4. 5. 6. 7. 8. .9. TO. IT. 12. 13. 14. 15.

R. RAISMAN, M. BRILEY and S.Z. LANGER, Nature 281, 148-150 (1979). S.M. PAUL, M. REHAVI, P. SKOLNICK and F.K. GOODWIN, Llfe Scl. 26, 953-959 (1980). M. REHAVI, S.M. PAUL, P. SKOLNICK and F.K. GOODWIN, Llfe Scl. 26, 2273-2279 (1980). S.Z. LANGER, C. MOREl, R. RAISMAN, M.L. DUBOCOVICH and M. BRILEY, Science 210, 1133-1135 (1980). S.M. PAUL, M. REHAVI, K.C. RICE, Y. ITTAH and P. SKOLNICK, Life Scl. 28, 2753-2760 (1981). M. BRILEY, R. RAISMAN and S.Z. LANGER, Eur. J. Pharmacol. 58, 347-348 (]979). M.S. BRILEY, S.Z. LANGER, R. RAISMAN, D. SECHTER and E. ZARIFIAN, Science 209, 303-305 (1980). S.M. PAUL, M. REHAVI, P. SKOLNICK, 3.C. BALLENGER and F.K. GOODWIN, Arch. Gen. Psychiatry 38__8~ 1315-1317 (1981). 3. TUOMISTO AND E. TUKIANIEN, NATURE 262., 596-598 (1976). M.L. BARBACCIA, N. BRUNELLO, D.M. CHUANGand E. COSTA, Neuropharmacology 22, 373-383, (1982) M.L. BARBACCIA, O. GANDOLFI, D. CHUANGand E. COSTA, Proc. Natl. Acad. Scl. 80, 5134-5138 (1983). M.J. KUHAR, R.H. ROTHand G.K. AGHAJANIAN, 3. Pharmacol. Exp. Ther. ]81, 36-45 (1972). D.B. BYLUNDand S.H. SNYDER, MoT. Pharmacol. 12, 568-580 (1976). H.I. YAMAMURAand S.H. SNYDER, Proc. Natl. Acad. Scl. 71, 1725-1729 (1974). B.K. KOE, J. Pharmacol. Exp. Ther. 199, 649-661 (1976).