Ligand binding studies in the mouse olfactory bulb: Identification and characterization of al -[3H]carnosine binding site

Brain Research, 158 (1978) 407422 © Elsevier/North-Holland Biomedical Press

407

L I G A N D B I N D I N G STUDIES IN T H E MOUSE O L F A C T O R Y BULB: IDENTIFICATION A N D C H A R A C T E R I Z A T I O N OF A L-[3H]CARNOSINE B I N D I N G SITE

JAMES D. HIRSCH, MARY GRILLO and FRANK L. MARGOLIS* Department of Physiological Chemistry andPharmacology, Roche Institute of Molecular Biology, Nutley, N.J. 07110 (U.S.A.)

(Accepted March 23rd, 1978)

SUMMARY Binding sites for the dipeptide L-carnosine (fl-alanyl-L-histidine) have been detected in membranes prepared from mouse olfactory bulbs. The binding of L-[aH]carnosine was saturable, reversible and stereospecific and had a Kd of about 770 nM. The stereospecific binding of L-carnosine represented about 30 ~o of the total binding at pH 6.8, and decreased markedly with increasing pH. Binding was stimulated by calcium, unaffected by zinc, magnesium or manganese and inhibited by sodium and potassium. Carnosine binding was sensitive to trypsin and phospholipases A and C, but not to neuraminidase. Nystatin and filipin, which interact with membrane lipids, also interfered with binding. Some peptide analogues of carnosine were potent inhibitors of binding, but a variety of drugs serving as potent inhibitors in other binding systems had no effect on carnosine binding. Carnosine binding to mouse olfactory bulb membranes was 15-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than in cerebellum membranes and 3-fold higher than in membranes from spinal medulla and the olfactory tubercle-lateral olfactory tract area. Binding sites for 6 other radiolabeled receptor ligands were also detected in bulb membranes. Peripheral deafferentation of the olfactory bulbs by intranasal irrigation with ZnSO4 led to a loss of > 90 ~o of the L-[3H]carnosine binding in 4-5 days with much smaller losses in binding of the other 6 ligands over a 180-day observation period. This initial loss ofcarnosine binding after denervation was due to a loss of binding site stereospecificity followed by a loss of binding sites. The characteristics of the carnosine binding site in olfactory bulb fulfil 6 of the 7 criteria considered relevant for a functional receptor.

* To whom all correspondence should be sent.

408 INTRODUCTION The neurotransmitter utilized in the synapses made by afferent olfactory axons on the dendrites of the mitral cells and on the periglomerular cells in the glomerular layer of the olfactory bulb is not known. The available evidence derived from denervation, uptake, histochemical and electrophysiological studies does not support catecholamines, indoleamines, acetylcholine, ~,-aminobutyric acid or a variety of amino acids as the neurotransmitter active in these particular synapses in the mammalian olfactory bulb 1,4,5,11A5,17,24,27,31. In contrast, the dipeptide L-carnosine (fl-alanyl-L-histidine) has recently become a neurotransmitter candidate in these synapses. In the mammalian brain, carnosine is found at high concentration in the primary olfactory pathway ; the olfactory bulbs and epithelium of rodents contain >_ l nmole of carnosine/mg tissue, while the rest of the brain contains only 0.01-0.2 nmole of the compound/mg tissue2L The enzymes responsible for the metabolism of carnosine are found to be both highly active and generally localized in olfactory tissue. Significantly, carnosine levels and the activity of the enzyme, carnosine synthetase, decrease markedly in the olfactory bulb after olfactory nerve section or other means of olfactory neuron destruction, while carnosinase activity is almost unaffected19,2L in addition, a previous report from this laboratory (Brown et al. 6) based on proton magnetic resonance spectral studies has shown the existence of a low-affinity carnosine binding site in membranes prepared from olfactory epithelium. If carnosine has a neurotransmitter or modulator role in the olfactory pathway, one might expect to find carnosine binding sites in the olfactory bulb where the olfactory axons form synapses. Efforts were made to identify and characterize a carnosine binding site in olfactory bulb by the 7 criteria considered characteristic of receptors 1°. The data in this paper show that a binding site for L-carnosine is present in membranes derived from mouse olfactory bulbs which satisfies 6 of the 7 criteria. In addition, binding of several other radiolabeled receptor ligands was studied in bulb membranes in an effort to identify other receptors found in this brain region. MATERIALS AND METHODS

Animals and tissues Retired female breeder CD-1 albino mice were obtained from Charles River Breeding Laboratories (Wilmington, Mass.) and maintained on Purina Chow and water ad libitum. Mice were killed by CO2 asphyxiation followed by exsanguination, and both olfactory bulbs were removed and placed on ice or frozen at --70 °C. No differences in binding between fresh or frozen tissue were detected. Destruction of the olfactory nasal mucosa was performed as previously described by intranasal irrigation with a 0.17 M ZnSOa solution 29. Preparation of membranes For the carnosine binding studies, fresh or frozen olfactory bulbs, other brain regions or tissues were homogenized in 20 vols of ice-cold 20 m M potassium phosphate

409 buffer (pH 6.8 at 23 °C) using a Brinkman Polytron (setting 5.5, 20 sec). The homogenate was centrifuged at 49,000 × g for 10 min and the membrane pellet was rehomogenized in 20 vols of fresh buffer as above and recovered by centrifugation. The pellet was resuspended by homogenization in 10 vols of buffer and stirred gently at 23 °C with a small magnetic stirring bar. Membranes for use in [ZH]-7-aminobutyric acid binding assays were prepared and treated with Triton X-100 by the method of Enna and Snyder 14. For binding of other ligands, olfactory bulb membranes were prepared by homogenization in 20 m M Tris • HC1 buffer (pH 7.4) as described by Bylund and Snyder s.

Binding assays e-[3H]carnosine binding was assayed in triplicate in 100/A reaction mixtures containing 50/tl of membranes (300-400 #g of protein), 1 m M unlabeled L- or D-carnosine or other competitors at the desired concentration, L-[ZH]carnosine at the specified concentrations (usually 188 nM) and 20 m M potassium phosphate buffer (pH 6.8, 23 °C). The e-[3H]carnosine in 50 ~o ethanol was added to each 12 × 75 mm plastic reaction tube and evaporated to dryness in a stream of air just prior to the assay. The remaining reagents were added and the reaction begun by the addition of the membrane suspension. The tubes were mixed with a vortex mixer and incubation took place at 23 °C for 40 rain. Carnosine binding was terminated by rapidly diluting the reaction mixtures with 4 ml of ice-cold 20 m M potassium phosphate buffer (pH 6.8) and the membranes were removed from suspension by vacuum filtration through presoaked Whatman GF/B glass fiber filters. The reaction tubes were washed twice more with 4 ml of buffer which was poured over the filters. Total filtration time was less than 15 sec. The filters were dried at 100 °C for 20 min, placed in glass scintillation vials, covered with 5 ml of ACS counting solution (Amersham-Searle, Arlington Heights, Ill.), allowed to equilibrate at 4 °C until the filters were uniformly translucent and counted in a Beckman LS-100 liquid scintillation spectrometer. For each experiment, blanks consisted of unincubated complete reaction mixtures that were diluted and filtered immediately. Using this method, less than 0.1 ~ of the total input radioactivity was bound to the filters even in the presence of membranes. Repeated washing of the filters with from 4 to 60 ml of buffer reduced both the total and specific binding. However, the fraction of the total binding represented by the specific binding remained the same regardless of the wash volume. Comparison of the centrifugation and filter binding assays indicated that the latter was far superior and was therefore used routinely. In all of these experiments, stereospecific binding of L-[SH]carnosine was determined by subtracting the radioactivity displaced by 1 m M unlabeled D-carnosine from that displaced by 1 m M unlabeled e-carnosine. In control animals, the amount of radioactivity bound in the presence of D-carnosine was always 90-100 ~ of that bound in the absence of any added cold ligand. Triplicate determinations varied < 10 ~. Those experiments performed to test the effect of ions were carried out in 20 m M Tris • maleate buffer (pH 6.8) rather than the usual phosphate buffer to avoid the formation of insoluble phosphate salts. Tris • maleate buffer was shown in separate experiments to have no effect on binding. For some experiments, membranes were treated with enzymes or lipid-perturbing

410 drugs before use. After the second membrane washing step in 20 m M potassium phosphate buffer (pH 6.8), the pellets were resuspended in 20 vols of the buffer. One ml aliquots of membranes were incubated at 37 °C for 30 min with 5 #g (in 5/~1 buffer) each of either trypsin, neuraminidase, phospholipases A or C or 500 #g of amphotericin B, nystatin or filipin and chilled. The membranes were recovered by centrifugation and resuspended in 0.5 ml buffer. The lipid-perturbing drugs were prepared for use and solubilized in the membranes by the methods of Limbird and Lefkowitz '25. Binding of [3H]quinuclidinyl benzilate*, [3H]dihydroergocryptine, [3H]dihydroalprenolol, [3H]haloperidol and [aH]etorphine was performed in triplicate in 1 ml reaction mixtures in plastic 12 × 75 mm tubes containing 0.2 ml of membranes (250350 #g of protein) prepared as described above, 5 nM [3H]ligand, 20 mM Tris • HCI buffer (pH 7.4) and the following unlabeled competing compounds: 2/~M unlabeled QNB (QNB); 10/~M phentolamine (DHE); 10 # M (--)-propranolol (DHA); 100 # M dopamine (HAL) and 3 # M naloxone (ETO). For measurement of [3H]HAL binding in the presence of unlabeled dopamine, an aliquot of membranes was treated with ascorbic acid and pargyline by the method of Burt et al. 7 immediately before use. After incubation at 37 °C for 15 min the reactions were terminated by dilution with ice-cold buffer and filtration through GF/B filters. Radioactivity was determined as above. Specific binding was defined as the difference between the radioactivity bound to the filter in the absence and presence of unlabeled competitors. Blanks consisted of complete unincubated reaction mixtures that were prepared, diluted, filtered and washed immediately. Triplicate determinations varied ~ l0 ~o. Binding of [3H]GABA was performed in triplicate in conical 1 ml polypropylene centrifuge tubes as described by Enna and Snyder 14. After incubation for 10 min at 4 °C, the membranes were recovered by centrifugation at 49,000 × g for 10 min. The pellets were washed once with 0.7 ml of ice-cold distilled water, resuspended in 1 ml of 1 ~o Triton X-100 and mixed with 10 ml of ACS counting solution. Blanks prepared in the absence of membranes were carried through the incubation and centrifugation steps. Protein was determined by the method of Lowry et al. 26 using bovine albumin as the standard. Materials [3-3H]QNB (13 Ci/mmole) and [15,16(N)-3H]ETO (40 Ci/mmole) were obtained from Amersham-Searle, Arlington Heights, Ill. and stored in ethanol at --20 °C, 9,10[9,10-~H(N)]DHE (24.1 Ci/mmole), [SH(G)]HAL (13.2 Ci/mmole), [2,3-3H(N)] GABA (35.1 Ci/mmole) and levo-[propyl-2,3-3H]DHA (48.6 Ci/mmole) were obtained from New England Nuclear, Boston, Mass. [3H]DHE was stored in ethanol in the dark at --20 °C and [3H]HAL was stored in 0.01 N HC1 at 4 °C and diluted just before use in 0.1 ~ ascorbic acid.

* Abbreviations used: QNB, quinuclidinyl benzilate; DHE, dihydroergocryptine; DHA, dihydroalprenolol; HAL, haloperidol; GABA, ~'-aminobutyricacid; TRH, thyrotropin releasing hormone; ETO, etorphine; and LOT, lateral olfactory tract.

411

L-[aH]carnosine was synthesized in this laboratory from [3-3H(N)]fl-alanine (36 Ci/mmole) obtained from New England Nuclear and purified immediately prior to use 2° and unlabeled L-histidine using chicken pectoral muscle carnosine synthetase prepared and assayed as described by Horinishi et al. 2°. The reaction conditions for L-[3H]carnosine synthesis were as follows: 100 mM potassium phosphate buffer, pH 7.5; 2 mM dithiothreitol; 4 mM MgC12; 4 mM ATP; 100/tM L-histidine; 2 mCi of [3H]fl-alanine (ll2/zM, 109 cpm); plus an aliquot of enzyme containing 2-3 mg of protein in a total volume of 0.5 ml. After incubation at 37 °C for 2.5 h, an additional 0.5 ml of the above substrate and enzyme mixture, but without additional [3H]flalanine, was added to the tube and incubation was continued for another 2.5 h period at 37 °C. The reaction was stopped by boiling and L-[3H]carnosine was extracted from the reaction mixture with ethanol and purified by ion-exchange column chromatography 2s. Sixty to 65 ~ of the input [aH]fl-alanine was converted to L-[3H]carnosine under these conditions. The purity of the product was determined by reverse-phase chromatography of a fluorescent derivative 41 and had a specific activity of 31 Ci/mmole. It was stored in 50~o ethanol 0.88 pmole//~l) at --20 °C. After 2-3 weeks of storage, unidentified products of radiolysis accumulated that caused high blank binding values. Repurification of the L-[3H]carnosine as previously described 19 eliminated the contaminants. GABA, a-amino acids, diphenhydramine, histamine, dopamine, serotonin, phospholipase A from V. russelli and unlabeled dipeptides, unless otherwise noted, were purchased from Sigma Chemicals (St. Louis, Mo.). Except as indicated, a-amino acids, free or in peptide linkage, were always in the L-configuration. Histidyl-fl-alanine, fl-alanyl-fl-alanine and o-carnosine (fl-alanyl-o-histidine) were products of Vega-FoxBiochemicals (Tucson, Ariz.). The compounds, Ro-22-0419 (ACTH4-10 analogue); L-2-benzamido-3-/< [5-methylimidazol-4-yl]methyl > thio/-propionamide (Ro- 124407); L-l- [hydroxymethyl]-2- [imidazol-4-yl]ethyl > amino/- [methylamino]methylene/ urea (Ro- 12-3997); l-cyano-2- [2-imidazol-4-yl]ethyl-3-methylguanidine (Ro- 12-0924); cimetidine (Ro-12-0105); metiamide (Ro-11-8912); glycylhistamine (Ro-1-3353); 2amino-3-/<[5-methylimidazol-4-yl]methyl>thio/-1-propanol (Ro-12-2891); pyroglutamylhistidine and its O-methyl ester; TRH; glycylhistidyllysine and its amide were provided by the Chemical Research Department of Hoffmann-La Roche (Nutley, N.J.). Pyrilamine maleate, pargyline and amino-oxyacetic acid were purchased from Sandoz (East Hanover, N.J.), Abbott Laboratories (N. Chicago, Ill.) and Eastman Kodak (Rochester, N.Y.), respectively. Haloperidol, QNB, leu-enkephalin, naloxone, amphotericin B, nystatin and filipin were gifts from Dr. A. J. Blume of this Institute. Dr. W. Schlosser (Hoffmann-La Roche, Nutley, N.J.) provided sodium pentobarbital, flurazepam, 1,3-dihydro-5-methyl-2H,1;4-benzodiazepine-2-one (Ro-5-3663) and nipecotic acid. Dr. J. Tarver (Hoffmann-La Roche, Nutley, N.J.) provided (+)-bicuculline and Dr. S. Spector of this Institute provided phentolamine, (--)-propranolol and chlorpromazine.

412 RESULTS

General characteristics of L-[aH]carnoshte binding Stereospecific binding of L-[3H]carnosine at 188 nM increased linearly as a function of the membrane protein concentration up to 540 #g/assay (Fig. ! A). Stereospecific binding of L-pH]carnosine at 188 n M reached a plateau between 30 and 60 min at 23 °C (Fig. 1B). Carnosinase activity was undetectable in these membranes, since after 60 min of incubation no [3H]#-alanine was found in the reaction mixtures. In addition, all of the 3H-labeled material associated with the filters eluted as L-[3H]carnosine when the filters were extracted with ethanol and the eluates subjected to ion-exchange chro-

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Fig. 1. Characteristics of L-[3H]carnosine binding. Specific binding was determined by the filter binding assay described in Materials and Methods. A: carnosine binding as a function of membrane protein concentration. L-[aH]carnosine was present at 188 nM. The data is from one representative experiment performed in triplicate. B : time course of specific binding. L-[aH]carnosine was present at 188 n M and incubation took place at 23 °C. Values are points from two separate experiments performed in triplicate. C: dissociation of L-pH]carnosine from bulb membranes. Membranes were incubated at 23 °C for 40 rain with 188 nML-pH]carnosine. At this time unlabeled L-carnosine was added to a final concentration of 1 mM. Aliquots of the reaction were removed, diluted, filtered and washed as described in Materials and Methods. The data is from one representative experiment performed in triplicate. D : effect of pH on binding. Binding was determined in 20 mM potassium phosphate buffer at the indicated pH. L-[aH]carnosine was present at 188 nM. Data points are the means of two separate experiments performed in triplicate.

413 matography 20. The dissociation of stereospecifically bound carnosine from bulb membranes in the presence of unlabeled L-carnosine was quite rapid (Fig. 1C) and was apparently first-order with a t~ of about 2 min (Fig. 1C, inset). D-Carnosine was ineffective in displacing L-[aH]carnosine, indicating the stereospecificity of the binding. Specific binding decreased with increasing pH and especially above pH 7.0 (Fig. 1D). This was found to be due both to a decrease in total binding and an increase in non-specific binding with increasing pH. At pH 5.5, 60-65 ~o of the total binding was specific, while at pH 9.5 only 10-12 ~ was specific. A pH of 6.8 for routine assays was chosen as a compromise that was still near the physiological range. At this pH 30 ~ of the total binding was stereospecific. When the stereospecific binding of L-[3H]carnosine was evaluated as a function of the L-[3H]carnosine concentration, saturation of binding was observed (Fig. 2). A Scatchard plot a4 of these data (Fig. 3A) yielded a single Ka of 859 n M and a Bmax of 361 fmole/mg protein. A Hill plot of these data (Fig. 3B) yielded a single straight line with a slope (Hill coefficient, nil) of 0.94. The specific binding data was also analyzed by a double reciprocal plot (Fig. 3C), which yielded a Km of 625 n M and a Bm ax of 400 fmole/mg protein. The latter value was used to replot the data as a Hill plot (Fig. 3D). A Hill coefficient of 1.1 again indicated the absence of cooperativity in binding. Scatchard analysis of data obtained from two additional experiments with different preparations of L-[3H]carnosine and olfactory bulb membranes yielded very similar results with single Ka values ranging from 480 to 970 nM and Bmax values of 200-382 fmole/mg protein. Thus, the mean observed Ka and Bm ax values were 770 n M and 314 fmole/mg protein respectively. If uptake of L-[3H]carnosine into membranous vesicles was occurring, increasing sucrose concentrations should reduce the internal volume of these vesicles and reduce the quantity of L-[3H]carnosine trapped inside. Instead, increasing concentrations of sucrose added to the reaction mixtures stimulated specific binding to as much as twofold at 0.8 M (data not shown). The effect of ions on L-[3H]carnosine binding was also tested. Sodium, which is required for GABA and amino acid uptakea9, 43, inhibited binding (75 ~ at 60 mM) as did potassium (45 ~ at 120 raM), while Mg z+, Mn z+ and

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Fig. 3. Analysisof saturable stereospecific binding by Scatchard, Hill and double reciprocal plots. The specificbindingdata plotted was taken from Fig. 2 and all lines weregenerated by computer using a linear regression analysis program. A: Scatchard plot of data from Fig. 2. B : Hill plot of data in Fig. 2 using the Bmaxof 361 fmole/mg protein obtained in A, C: expression of the data in Fig. 2 as a double-reciprocal plot. D : Hill plot of data in Fig. 2 using the Bmax of 400 fmole/mg protein obtained in C. Zn 2+ at 5 m M had no effect on binding. Calcium ion stimulated binding by 50 ~o at 1 m M and 70 ~ at 5 mM, while 0.2 m M E G T A inhibited binding by 45-50 ~ in the absence of added Ca 2+ (data not shown). Pretreatment of the membranes with 0.05 Triton X-100, which improved the [3H]GABA binding assays, was found to markedly inhibit L-[3H]carnosine binding. Triton not only lowered total binding, but destroyed stereospecificity as well. Pretreatment of membranes with trypsin and phospholipases resulted in a 50-90 ~ reduction of specific carnosine binding, while neuraminidase was ineffective. This is similar to several known receptor sites previously characterized in brain 37. Treatment of bulb membranes with the lipid-perturbing drugs nystatin and filipin resulted in 80 ~ inhibition of stereospecific carnosine binding, while amphotericin B caused about a 20 ~ inhibition (data not shown). Thus, these data suggest that both lipids and proteins have some involvement in L-[3H]carnosine binding to membranes. Several other brain regions were tested for stereospecific carnosine binding. Of the

415 TABLE I

L-EZH]Carnosine binding by various mouse brah~ regions Results are mean ± S.E.M. for the number of experiments in parentheses. L-[aH]Carnosine was present at 188 nM, which is below saturation. At saturation the value for bulb extrapolates to about 300 fmole L-[3H]carnosine/mg protein (see Fig. 3).

Region

fmole L-[aHjCarnosine bound/mg protein

Olfactory bulb Spinal medulla Olfactory tubercle and LOT* Cerebellum Cerebral hemispheres**

49 16 15 10 4

± ± + ± ±

5 (7) 4 (4) 1 (4) 3 (4) 1 (3)

* The portion of the lower forebrain surrounding the lateral olfactory tract (LOT) was shaved offthe brain with a scalpel blade. ** Both grey and white matter were included.

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~-~log io E01SPLACER]~M) Fig. 4. Displacement of L-[3H]ca~nosine by peptide analogues. Increasing concentrations of unlabeled L-carnosine or peptides w e r e added to standard binding assays prepared as described in Materials and Methods. L-[aH]carnosine was present at 188 nM. A: L-carnosine ( 0 • ) ; D-carnosine (O ©); anserine ( A A); homocarnosine ( [] [] ). B: histidyl-fl-alanine ( 0 • ); glycylhistidylglycine (O O); glycylhistidyllysine amide (A A); histidylalanine ([] []). C: alanylhistidine (O - - O ) ; glycylhistidyllysine (O O); TRH ( A ~ ) ; glycylglycine ([] []). D : glycylhistidine (Q Q); histidylglycine (O O), glycylphenylalanine (A A); leu-enkephalin ([] []). In B, C and D the dotted line represents the displacement curve for L-carnosine shown in panel A. All results are means of two separate experiments performed in triplicate that varied < l0 700.The following compounds produced no displacement at 0.1 mM: GABA, L-histidine, fl-alanine, glycine, L-aspartic acid, L-glutamic acid, L-proline, taurine, histamine, diketopiperazine, phentolamine, (--)-propranolol, halperidol, chlorpromazine, pyrilamine maleate, diphenhydramine, cimetidine, metiamide, ergothioneine, QNB, dopamine, serotonin, (--)-bicuculline, muscimol, sodium pentobarbital, flurazepam, Ro-5-3663, nipecotic acid, amino-oxyacetic acid, glycylhistamine, 5"-guanylylimidodiphosphate and the imidazole derivatives, Ro-12-4407, Ro-12-3997, Ro-12-0924 and Ro-12-2891. The following peptides were essentially ineffective at 0.1 or 1.0 mM: fl-alanyl-fl-alanine, fl-alanylalanine, fl-alanylglycine, glycylglycylglycylglycine, pyroglutamylhistidine and its O-methyl ester and an analogue of ACTH4-10 (Ro-22-0419).

416 regions tested, olfactory bulb membranes bound the most L-[aH]carnosine followed by the olfactory tubercle-LOT and spinal medulla, the cerebellum and cerebral hemispheres (Table I). No specific binding was detected in liver, muscle or heart membranes. Binding of L-[aH]carnosine to membranes from olfactory epithelium is currently under investigation. A difference in carnosine binding between bulb and epithelium has already been reported 6. The specificity of the L-[3H]carnosine binding site was studied by testing the ability of a wide variety of compounds to displace L-[3H]carnosine fi'om bulb membranes. L-Anserine (fl-alanyl-N'-methylhistidine) inhibited binding, while homocarnosine (GABA-L-histidine) and D-carnosine were essentially ineffective below 1 m M (Fig. 4A). Some, but not all, peptides containing histidine were more potent at inhibiting binding than L-carnosine itself (Figs. 4B, C and D). Leu-enkephalin also inhibited binding as did glycylglycine and glycylphenylalanine (Figs. 4B and C). However, neither the binding of carnosine nor its displacement by leu-enkephalin were blocked by naloxone or levallorphan at 0.1 mM. Many compounds produced no displacement at 0. l m M (see legend to Fig. 4). Binding of other ligands In order to characterize the nature of the neurotransmitter binding sites present in olfactory bulb membranes, the binding of other tritiated ligands was evaluated. Table II gives the amounts of [3H]QNB, DHE, DHA, HAL and ETO (each at 5 nM) and GABA (28 nM) specifically bound per mg of bulb membrane protein. [3H]Quinuclidinyl benzilate was bound to the greatest extent by bulb membranes followed by GABA, DHE, ETO, DHA and HAL. In all cases, we showed that the binding studied was saturable and was similar in characteristics to previously published results 7,8,12A4, 36,38,42,43. For example, the binding of QNB was inhibited 90-98 % by 0.1 # M atropine and 72% by 0.2 mM carbamyl choline, but was inhibited only 12 % by 0.5 m M hexamethonium. The binding of GABA was inhibited by (÷)-bicuculline and muscimol but not by nipecotic acid. All binding sites exhibited Ka values determined by Scatchard plots of 1-5 n M except for GABA which manifested a Ka of 91 nM.

TABLE 1I Receptor binding of other ligands in olfactory bulb membranes

[aH]Ligands were present in the assays at 5 nM(GABA at 28 nM). The method for determination of specificbinding is described in Materials and Methods. Values are mean ± S.E.M. for the number of experiments shown in parentheses. [ZH]Ligand

fmole [ZH]Ligand bound~ragprotein

Quinuclidinyl benzilate 1768 ± 35 (4) 7-Aminobutyric acid 427 ± 31 (4) Dihydroergocryptine 258 ~ 5 (3) Etorphine 177 ± 15 (3) Dihydroalprenolol 55 ± 9 (4) Haloperidol 36 ± 5 (3)

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DAYS AFTER ZnSO4 TREATMENT Fig. 5. Effect of peripheral deafferentation on L-[SH]carnosine and other [3H]ligand binding in the bulb. Lower panel: © and • represent two separate binding experiments performed with bulb membranes at the indicated times after intranasal irrigation with ZnSO4. L-[ZH]Carnosine was present at 188 nM. Control binding was within the range shown in Table I. The open triangles represent separate binding experiments performed in triplicate with the other [3H]ligands. 1 : HAL; 2 : DHA ; 3 : QNB; 4: GABA; 5: DHE; 6: ETO. Control binding was within the ranges shown in Table II. Specific binding was determined as described in Materials and Methods. Upper panel: bulb weight loss with time after ZnSO4 denervation.

Effect of denervation on [3H]ligand binding Peripheral deafferentation of the bulb led to profound changes in L-[SH]carnosine binding in bulb membranes (Fig. 5). Specific binding of L-[3H]carnosine declined rapidly after denervation, reaching a minimum of about 5-10 ~ of the normal level by 4 days after ZnSO4 treatment. The half-time for disappearance of specific binding was 1.5 days. Essentially, no recovery of binding was detected even in animals tested 6 months after denervation. No differences could be detected among normal animals tested at equivalent times after treatment. In contrast to the decline in carnosine binding, the binding of all of the other ligands tested only declined from 0 to 30 ~ and showed little change from 1 to 6 months after denervation. The same conclusion was reached whether the data was calculated per mg of membrane protein or per mg of tissue wet weight. This demonstrates the specificity of the effect of peripheral deafferentation on L-[3H]carnosine binding. The time course of bulb weight loss after denervation (Fig. 5) indicates a progressive weight loss to almost 50 ~ of the normal wet weight subsequent to denervation. Preliminary analysis of the decline in stereospecific binding of L-[3H]carnosine suggests that this occurs in two stages. There is an initial loss of stereospecificity in the absence of any change in total binding in the first two weeks after deafferentation followed by a decline in total binding and the absence of significant displaceable L-[aH]carnosine at the later time points (data not shown). DISCUSSION

The possible role of carnosine as a neurotransmitter or neuromodulator in the glomerular layer of the olfactory bulb has been investigated at a biochemical level in

418 this laboratory z7. We now report a binding site for this dipeptide in membrane fractions prepared from mouse olfactory bulbs. The binding of a variety of known receptor ligands has also been studied in the bulb in order to evaluate receptors present in this brain area and to determine the effects of peripheral deafferentation on the binding of carnosine and these other ligands. The stereospecific binding of L-[3H]carnosine to olfactory bulb membranes was saturable and non-cooperative and appears to occur at a single population of binding sites. The Ka for the binding was about 770 nM. These binding sites are clearly distinct from the non-stereospecific, low-affinity sites detected in olfactory mucosal membranes by Brown et al. using proton magnetic resonance techniques 6. Attempts at evaluating equivalent low-affinity sites in bulb by the techniques described here required very high L-[3H]carnosine concentrations which led to a preponderance of non-specific binding that was not amenable to Scatchard analysis. Stereospecific L-[3H]carnosine binding was readily reversible and it represented about 30 ~ of the total binding observed under standard assay conditions at pH 6.8. The pH at which the binding assays were performed was found to have a profound effect on the fraction of the total binding represented by the specific binding. This may be related to the fact that the decrease of specific binding with increasing pH coincides with the deprotonation of the histidine ring in carnosine that occurs over a similar range 23. At pH 7.4, specific binding represented about 20 ~/o of the total binding. The possibility that uptake processes might account for our data prompted the testing of the effects of sucrose and ions on carnosine binding. If uptake into membranous vesicles was occurring, then shrinking the internal volume of these vesicles should have reduced the amount of L-[3H]carnosine associated with them. In contrast, sucrose stimulated binding, similar to observations reported by Grollman et al. 16. Further evidence against uptake derives from the effects of ions on binding. In contrast to the effects of sodium on the uptake of GABA and amino acid transmitters 39,43, it inhibited carnosine binding as did potassium, while Mg 2+, Mn 2+ and Zn 2÷ had no effect. The stimulation by calcium ion coupled with the inhibitory effect of EGTA suggested that this stimulation was Ca2+-specific. Similar ion effects have been reported for other systems 2,40. The specificity of L-[3H]carnosine binding was demonstrated by the inability of many compounds active at other binding sites in brain tissue to displace L-[aH]carno sine. I n addition, except for glycylglycine, dipeptides lacking histidine or phenylalanine were ineffective inhibitors. The opiate pentapeptide leu-enkephalin inhibited binding, probably due to the presence in its sequence of glycylglycine and glycylphenylalanine, each of which inhibited binding when tested alone. The D-isomer of carnosine, /3alanyl-D-histidine, was ineffective as a binding competitor clearly demonstrating the stereospecificity of the binding site for L-carnosine. However, the reverse peptide of carnosine, histidyl-fl-alanine, did cause substantial inhibition of binding and histidylalanine was the most potent peptide tested, inhibiting binding substantially at concentrations as low as 1 /~M. Several di- and tripeptides containing glycine and alanine in place of fi-alanine also inhibited binding, but none were as effective as histidylalanine. Glycylhistidyllysine and glycylhistidylglycine inhibited binding more than glycyl-

419 histidine. Glycylhistidyllysine amide was inactive, suggesting that a free carboxyterminus is required. Anserine was less effective than L-carnosine, while homocarnosine was ineffective. Carnosinase activity was not detectable in the membranes used in these experiments, and the possibility that L-[3H]carnosine could bind to inactive enzyme trapped in or adsorbed to the membranes is inconsistent with what is known of the substrate and metal ion requirements of the enzyme22,a2 (and unpublished observations). Stereospecific L-[3H]carnosine binding was detected in several other brain regions and in the olfactory epithelium. Since this binding has not yet been fully characterized, one cannot conclude that the binding sites in these other locations and in the bulb are the same. Bulb membranes contain the highest levels of binding detected so far, and this site is clearly different from that studied previously in our laboratory using proton magnetic resonance 6. Prior to an analysis of denervation effects on [3H]ligand binding in the bulb, the binding of 6 other radioligands to bulb membranes was evaluated. As expected from earlier histochemical and electrophysiological studies4,~,ll,15,17,24, al, receptors for GABA, dopamine and norepinephrine were detected. In addition, muscarinic-cholinergic and opiate receptors were also found. Levels and characteristics of ligand binding in bulb membranes were similar to those reported by others 7,8,1z,36.aS,4z,43 for other brain areas in various species. L-carnosine at 1 m M had no effect on the binding of any of the [aH]ligands used except L-[3H]carnosine. Experiments designed to evaluate the effect of peripheral denervation on ligand binding in the bulb yielded several significant findings. Within 4 days after deafferentation stereospecific L-[3H]carnosine binding declined to 5-10 ~ of normal levels and did not return even after 180 days. The loss of binding sites is preceded by a loss of stereospecificity. Binding of other ligands tested decreased at most 30 ~ by 180 days, whether the results were calculated on the basis of fmole [aH]ligand bound/mg protein or per mg tissue. Other studies have shown changes in ligand binding after denervation, but the effects were not as marked or as rapidly appearing as those shown here for carnosine. Rotter et al. 33 reported a loss of binding to muscarinic-cholinergic synapses in the hypoglossal nucleus as early as two days after axotomy, but the maximal decrease in binding (50 ~ ) occurred about one week after the lesion. Viral-induced depletion of cerebellar granule cells caused [aH]GABA binding to decrease by 60-75 ~ after 3-6 weeks aS. In contrast, destruction of cell bodies in the nigrostriatal dopaminergic pathway leads to a 45-55 % increase in [all]HAL binding in striatal membranes 2-7 months after the lesion in some animals 9. The rapid disappearance of carnosine binding in bulbs after peripheral deafferentation is most similar in time course to results obtained with presynaptic a-adrenergic receptors in denervated cat nictitating membranes 21. In that study, the presynaptic actions of phentolamine and clonidine were reduced by l 8 h after denervation, at which time their postsynaptic activities were unchanged. The rapid disappearance of these presynaptic effects and the similarly rapid loss of carnosine binding in the bulb leads us to speculate that the carnosine binding site in the bulb might be presynaptic. How-

420 ever, since u l t r a s t r u c t u r a l data3,13,18 indicated r a p i d a l t e r a t i o n s in p r e s y n a p t i c terminals, as well as in p o s t s y n a p t i c dendrites following olfactory nerve section o r olfactory epithelium ablation, the exact localization o f the carnosine binding site r e m a i n s uncertain. There is as yet no electrophysiological evidence for any role for carnosine as a n e u r o t r a n s m i t t e r or n e u r o m o d u l a t o r . Thus, the m o s t i m p o r t a n t criterion 10 for the carnosine binding site representing a true ' r e c e p t o r ' has n o t yet been fulfilled. However, the previous biochemical d a t a ~7 and the current d e m o n s t r a t i o n that carnosine b i n d i n g to b u l b m e m b r a n e s is saturable, reversible, stereospecific, a n a t o m i c a l l y localized, occurs at physiologically significant c o n c e n t r a t i o n s and shows selective specificity o f displacem e n t argue strongly t h a t this d i p e p t i d e be seriously considered as a c a n d i d a t e neurotransmitt,~r or m o d u l a t o r . Hopefully, these results will stimulate those with electrophysiological capabilities to systematically study the role of carnosine a n d its analogues in n e u r o t r a n s m i s s i o n b o t h in the olfactory p a t h w a y and elsewhere in the nervous system. ACKNOWLEDGEMENTS The a u t h o r s w o u l d like to t h a n k Dr. A r t h u r J. Blume for valuable discussions, and Jill C l u e s m a n n for excellent secretarial assistance.

REFERENCES 1 Von Baumgarten, R., Bloom, F. E., Oliver, A. P. and Salmoiraghi, G. C., Response of individual olfactory nerve cells to microelectrophoretically administered chemical substances, Arch. ges. Physiol., 277 (1963) 125-140. 2 Bennett, J. P., Jr. and Snyder, S. H., Serotonin and lysergic acid diethylamide binding in rat brain membranes: relationship to postsynaptic serotonin receptors, Molec. PharmacoL, 12 (1976) 373389. 3 Berger, B., D6gSn6rescence transsynaptique dans le bulbe olfactif du lapin, apr6s d6safferentation peripherique, Acta nearopath. (BerL), 24 (1973) 128-152. 4 Bloom, F., Costa, E. and Salmoiraghi, G. C., Analysis of individual rabbit olfactory bulb neuron responses to the microelectrophoresis of acetylcholine, norepinephrine and serotonin synergists and antagonists, J. Pharmacol. exp. Ther., 146 (1964) 16-23. 5 Broadwell, R. D. and Jacobowitz, D. M., Olfactory relationships of the telencephalon and diencephalon in the rabbit, lII. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations, J. comp. NeuroL, 170 (1976) 321-346. 6 Brown, C. E., Margolis, F. L., Williams, T. H., Pitcher, R. G. and Elgar, G., Carnc sine in olfaction. Proton magnetic resonance spectral evidence for tissue-specific carnosine binding sites, Neurochem. Res., 2 (1977) 555-579. 7 Butt, D. R., Creese, I. and Snyder, S. H., Properties of [aH]haloperidol and [3H]dopamine binding associated with dopamine receptors in calf brain membranes, Molec. PharmacoL, 12 (1976) 800-812. 8 Bylund, D. B. and Snyder, S. H., Beta adrenergic receptor binding in membrane preparations from mammalian brain, Molec. PharmacoL, 12 (1976) 568-580. 9 Creese, I., Butt, D. R. and Snyder, S. H., Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity, Science, 197 (1977) 596-598. l0 Cuatrecasas, P. and Hollenberg, M. D., Membrane receptors and hormone action. In C. B. Anfinsen, J. T. Edsall and F. M. Richards (Eds.), Advances in Protein Chemistry, Vol. 30, Academic Press, New York, 1976, pp. 251-445. 11 DahlstrSm, A., Fuxe, K., Olson, L. and Ungerstedt, U., On the distribution of monoamine nerve terminals in the olfactory bulb of the rabbit, Life Sci., 4 (1965) 2071-2074. 12 Davis, J. N., Strittmatter, W. J., Hoyler, E. and Lefkowitz, R. J., [aH]Dihydroergocryptine binding in rat brain, Brain Research, 132 (1977) 327-336.

421 13 Estable-Puig, J. F. and DeEstable, R., Acute ultrastructural changes in the rat olfactory glomeruli after peripheral deafferentation, Exp. Neurol., 24 (1969) 592-602. 14 Enna, S. J. and Snyder, S. H., GABA receptor binding in frog spinal cord and brain, J. Neurochem., 28 (1977) 857-860. 15 Graham, L. T., Jr., Distribution of glutamic acid decarboxylase activity and GABA content in the olfactory bulb, Life Sci., 12 (1973) 443-447. 16 Grollman, E. A., Lee, G., Ambesi-lmpiombato, F. S., Meldolesi, M. F., Aloj, S. M., Coon, H. M., Kaback, H. R. and Kohn, L. D., Effects of thyrotropin on the thyroid cell membrane: hyperpolarization induced by hormone-receptor interaction, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 23522356. 17 Hal~isz, N., Jungdahl, L., H6kfelt, T., Johansson, O., Goldstein, M., Park, D. and Biberfeld, P., Transmitter histochemistry of the rat olfactory bulb. I. Immunohistochemical localization ofmonoamine synthesizing enzymes. Support for intrabulbar, periglomerular dopamine neurons, Brain Research, 126 (1977) 455-474. 18 Harding, J., Graziadei, P. P. C., Monti Graziadei, G. A. and Margolis, F. L., Denervation in the primary olfactory pathway. IV. Biochemical and morphological evidence for neuronal replacement following nerve section, Brain Research. 132 (1977) 11-28. 19 Harding, J. and Margolis, F. L., Denervation in the primary olfactory pathway of mice. III. Effect on enzymes of carnosine metabolism, Brain Research, 110 (1976) 351-360. 20 Horinishi, H., Grillo, M. and Margolis, F. L., Purification and characterization of carnosine synthetase from mouse olfactory bulbs, J. Neurochem, (1978) in press. 21 Langer, S. Z., Presynaptic receptors and their role in the regulation ot transmitter release, Brit. J. Pharmacol., 60 (1977) 481-497. 22 Lenney, J. F., Specificity and distribution of mammalian carnosinase, Biochim. biophys. Acta (Amst.), 429 (1976) 214-219. 23 Lenz, G. R. and Martell, A. E., Metal complexes of carnosine, Biochemistry, 3 (1964) 750-753. 24 Lichtensteiger, W., Uptake of norepinephrine in periglomerular cells of the olfactory bulb of the mouse, Nature (Lond.), 210 (1966) 955-956. 25 Limbird, L. E. and Lefkowitz, R. J., Adenylate cyclase-coupled beta adrenergic receptors : effect of membrane lipid-perturbing agents on receptor binding and enzyme stimulation by catecholamines, Molec. PharmacoL, 12 (1976) 559-567. 26 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.,Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 27 Margolis, F. L., Biochemical markers in the primary olfactory pathway: A model neural system. In B. W. Agranoff and M. H. Aprison (Eds.), Advances in Neurochemistry, Vol. 1, Plenum Press, New York, 1975, pp. 193-246. 28 Margolis, F. L. and Grillo, M., Axoplasmic transport of carnosine (fl-alanyl-L-histidine) in the mouse olfactory pathway, Neurochem. Res., 2 (1977) 507-519. 29 Margolis, F. L., Roberts, N., Ferriero, D. and Feldman, J., Denervation in the primary olfactory pathway of mice: biochemical and morphological effects, Brain Research, 81 (1974) 469-483. 30 Peck, E. J., Milter, A. L. and Lester, B. R., Pentobarbital and synaptic high-affinity receptive sites for gamma-aminobutyric acid, Brain Res. Bull., 1 (1976) 595-597. 31 Ribak, C. E., Vaughn, J. E., Saito, K., Barbzr, R. and Roberts, E., Glutamate decarboxylase localization in neurons of the olfactory bulb, Brain Research, 126 (1977) 1-18. 32 R•senberg•A. •The activati•n •f carn•sinase by diva•ent meta• i•ns•Bi•chim, bi•phys. Acta ( Amst. • • 45 (1960) 297-316. 33 Rotter, A., Birdsall, N. J. M., Burgen, A. S. V., Field, P. M. and Raisman, G., Axotomy causes loss of muscarinic receptors and loss of synaptic contacts in the hypoglossal nucleus, Nature (Lond.), 266 (1977) 734-735. 34 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N. Y. Acad. Sci., 51 (1949) 660-672. 35 Simantov, R., Oster-Granite, M. L., Herndon, R. M. and Snyder, S. H., Gamma-aminobutyric acid (GABA receptor) binding selectively depleted by viral induced granule cell loss in hamster cerebellum, Brain Research, 105 (1976) 365-371. 36 Simon, E. J., Hiller, J. M. and Edelman I., Stereospecific binding of the potent narcotic analgesic [aH]etorphine to rat brain homogenate, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 1947-1949. 37 Snyder, S. H., Neurotransmitter and drug receptors in the brain, Biochem. Pharmacol., 24 (1975) 1371-1374.

422 38 Snyder, S. H. and Bennett, J. P., Jr., Neurotransmitter receptors in the brain: biochemical identification, Ann. Rev. Physiol., 38 (1976) 153-175. 39 Snyder, S. H., Yamamura, H. I., Pert, C. B., Logan, W. J. and Bennett, J. P., Jr., Neuronal uptake of neurotransmitters and their precursors: studies with 'transmitter' amino acids and choline. In A. J. Mandell (Ed.), New Concepts of Neurotransmitter Regulation, Plenum Press, New York, 1973, pp. 195-222. 4~) Uhl, G. R., Bennett, J. P., Jr. and Snyder S. H., Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes, Brain Research, 130 (1977) 299-313. 41 Wideman, J., Brink, L. and Stein, S., New automated fluorometric peptide microassay at picomole level: carnosine in mouse olfactory bulb, Analyt. Biochem., 86 (1978) 670-678. 42 Yamamura, H. I. and Snyder, S. H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1725-1729. 43 Zukin, S. R.., Young, A. B. and Snyder, S. H., Gamma-aminobutyric acid binding to receptor sites in the rat central nervous system, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 4802-4807.