[125I]iodobolpyramine, a highly sensitive probe for histamine H1-receptors in guinea-pig brain

European Journal of Pharmacolog},, 120 (1986) 151-160

151

Elsevier

[12Sl]IODOBOLPYRAMINE, A HIGHLY SENSITIVE P R O B E FOR H I S T A M I N E H I - R E C E P T O R S IN GUINEA-PIG BRAIN MARIE KORNER, MARIE-LOUISE BOUTHENET *, C. ROBIN GANELLIN **, MONIQUE GARBARG, CLAUDE GROS. ROBERT J. IFE **, NICOLE SALES * and JEAN-CHARLES SCHWARTZ *** Unitb 109 de Neurobiologie, Centre Paul Broca de I'INSERM, 2ter rue d'Alksia, 75014 Paris. * Lahoratoire de Plo'siologie. Facultb de Pharmaeie, 4 Avenue de I'Observatoire, 75005 Paris, France, and ** Smith Kline and French Research Limited. The Frvthe. Welwvn, A L6 9A R Hertfordshire, United Kingdom

Received 23 JuLy 1985, revised MS received 20 September 1985, accepted 15 October 1985

M. K O R N E R , M.L. B O U T H E N E T , C.R. G A N E L L I N , M. G A R B A R G , C. GROS, R.J. IFE, N. SALES and J.C. S C H W A R T Z , [125I]lodobolpyramine, a highly sensitit,e probe f o r histamine Hi-receptors in guinea-pig brain, European J. Pharmacol. 120 (1986) 151-160. [lzsI]Iodobolpyramine is a novel 125I-ligand for histamine Hi-receptors, synthesised using the ~-'51-Bolton Hunter reagent (2000 C i / m m o l ) for acylation of an aminopentyl analogue of mepyramine. Its specific binding varied linearly with the concentration of guinea-pig cerebellar membranes and represented about 80% of the total. Selective interaction with Hi-receptors was demonstrated by estimation of K i values of known agonists and antagonists and confirmed by the low affinity of histamine H 2- and H3-receptor antagonists and of non-histaminergic agents. At 25°C, [~zsI]iodobolpyramine exhibited a slow association rate (180-240 min to reach equilibrium) and a slow dissociation rate (t~/2 = 201 min). Kinetic and saturation data yielded KI~ values of 0.05 and 0.15 nM, respectively, indicating that it is among the most potent H~-receptor antagonists known. The sensitivity for detecting H~-receptors in guinea-pig cerebellum using [1251]iodobolpyramine was increased 50-fold relative to use of [~H]mepyramine. Well-contrasted autoradiograms of guinea-pig brain, obtained after a short exposure time, confirmed previous H~-receptor localisation established with [3H]mepyramine and revealed new localisations, e.g. in cerebral cortex and nucleus accumbens.

Hi-receptor binding

Autoradiography

Brain

1. Introduction

Several tritium-labelled ligands have been identified for labelling histamine Hi-receptors, namely [3H]mepyramine (Hill et al., 1977, 1978; Chang et al., 1978), [3H]doxepin (Tran et al., 1981; Taylor and Richelson, 1982) and [3H]mianserin (Peroutka and Snyder, 1981). Because of its high selectivity, [3H]mepyramine has been widely used to study H~-receptors in membrane preparations, in tissue sections for autoradiography (Palacios et al., 1979, 1981b,c; Wamsley and Palacios, 1984) and in the brain of living animals (Quach et al., 1979, 1980). High affinity 125I-labelled ligands offer obvious

*** To whom all correspondence should be addressed. 0014-2999/86/$03.50 ':!::1986 Elsevier Science Publishers B.V.

Iodobolpyramine

technical advantages over corresponding ~Hligands since they can be obtained with a 50-100 times higher specific radioactivity and thereby provide increased sensitivity for receptor assays. We now report that [leSl]iodobolpyramine, a mepyramine-derived compound showing good pharmacological specificity for H~-receptors, is a suitable ligand for sensitive assay and localisation of histamine Hi-receptors in the guinea-pig brain.

2. Materials and methods 2.1. Chemicals

[3H]Mepyramine (24.1 Ci/mmol) was from New England Nuclear, N-succinimidyl-3-(4-hy-

152 droxy-5-[ 125l]iodophenyl)propionate (Bolton-Hunter reagent, 2 000 Ci/mmol) was from Amersham. The drugs and their sources were: cimetidine, impromidine, burimamide, 2-pyridylethylamine, 2thiazolylethylamine, S K & F 94461, S K & F 95507 (bolpyramine) from Smith Kline and French (England); triprolidine, ( +)- and ( -)-chlorpheniramine from Burrough-Wellcome; spiperone from Janssen-Lebrun; mianserin from Organon: mepyramine from Specia; (-)-sulpiride from Delagrange; methysergide from Sandoz: doxepin from Merrell Toraude; phentolamine and imipramine from Ciba-Geigy; alprenolol and scopolamine from Sigma. Reagents were from commercial sources.

~H3

MEPYRAMINE

N~CH2CH2NCH3 L CH2

0

OCH3

2.2. Preparation of [l"~I]iodobolpyramine SK & F 94461 dimaleate, N-(5-aminopentyl)-N'(4-methoxybenzyl)-N-methyl-N'-2-pyridinyl-l,2ethanediamine dimaleate, an aminopentyl analogue of mepyramine (synthesis to be described elsewhere) was converted into [~:Sl]iodobolpyramine, i.e. N- { 5-[3-(4-hydroxy-5-[ 1:s I]iodophenyl)propionamido]pentyl }-N'-(4-methoxybenzyl)-N-met hyl-N'-2-pyridinyl- 1,2-ethanediamine, by N-succinimidyl 3-(4-hydroxy, 5-[1-'51] iodophenyl) propionate (fig. 1) based on the method of Bohon and Hunter (1973) as follows: S K & F 94461 (10 /*g in 10 >1 of 0,1 M borate buffer, pH 8.5) was added to the dried [L'sI]-Bolton-Hunter reagent (1 mCi). After 15 min at room temperature, the mixture was spotted onto a thinlayer chromatography (TLC) silica gel plate (60 F 254 Merck) and the chromatogram was developed for 10 h in butanol:acetic acid:water ( 4 : 1 : 1 ) . After being located by autoradiography (AgfaGevaert D7), the [l:51]iodobolpyramine spot (R~ = 0.33) was scraped from the plate and extracted with 500 >1 ethanol. Solutions were stored at - 20°C before use.

CH3 I

2.3. Membrane preparation

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SK&F 94461

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Fig. 1. Chemical structures of mepyramine, SK&F 94461, the Bolton-Hunter reagent and [~25I]iodobolpyramine.

Cerebella of male Hartley guinea-pig (about 300 g, kindly provided by Rh6ne-Poulenc) were homogenised with a Polytron blender in 40 volumes of cold N a J K phosphate buffer (50 mM, pH 7.5). After centrifugation for 1 rain at 260 g, the resulting supernatant was recentrifuged (30 rain at 20000 × g). The final pellet was rinsed with 2 ml of cold phosphate buffer and stored at - 8 0 ° C . For binding assays, pellets were resuspended in the phosphate buffer and the protein concentration of the preparation was determined (Lowry et al., 1951) using bovine serum albumin (BSA) as standard, Membranes from cerebral cortex of male Wistar rats (Iffa Credo, France; about 200 g) were prepared in a similar way.

2.4. [¢H]Mepyramine binding assays [)H]Mepyramine binding assays were performed according to Garbarg et al. (1983)i The

153 pellet suspension (450 ~1 containing 150-200 #g protein) was incubated with the radioactive ligand in a final volume of 500 ~1. Incubations were carried out at 25°C for 30 min. Non-specific binding was determined in the presence of 0.2 #M mianserin. The incubations were terminated by addition of 6 ml cold phosphate buffer, followed by rapid filtration through glass fiber filters ( G F / B ) under reduced pressure. The filters were immediately washed with 2 × 10 ml of cold phosphate buffer. The trapped radioactivity was counted in 5 ml of ACS II (The Radiochemical Center Amersham, U.K.) by liquid scintillation spectrometry at an efficiency of 45%. Binding of the ligand to the filters in the absence of membrane preparation represented 0.5% of the total radioactivity.

2.6. Localisation of [J-"sI]iodobolpyrarnine binding sites by autoradiography

2.5, [ 1:~l]lodobolpyramine binding assays

3. Results

All solutions contained 0.1% BSA to prevent adsorption of the iodinated ligand onto tubes and filters. The pellet suspension (100 /~1 containing 5-50/.tg protein according to the concentration of ligand) was incubated with the [125I]ligand in a final volume of 200 /~1. Incubations were carried out at 25°C for 4 h (unless otherwise indicated). Non-specific binding was determined in the presence of 0.2 ~M mianserin. Incubation was stopped by adding 5 × 3 ml of fresh phosphate buffer containing 0.1% BSA and followed by rapid filtration under reduced pressure through glass fibre filters ( G F / B ) which had been treated previously with 0.3% polyethylenimine (Bruns et al., 1983) to reduce the binding of the ligand. The radioactivity trapped on the filters was measured using an LKB gamma counter, with an efficiency of 82%. Radioactivity bound to the filters in the absence of the membrane preparation represented 0.4% of the total. The concentration of the ligand was corrected for the loss of free radioactivity due to adsorption onto the tubes during the incubation period (5-10% of the initial radioactivity). In saturation experiments, the concentration of free ligand was also corrected for membrane-bound radioactivity.

3.1. Kinetics of [I-"Sl]iodobolpyramine binding

After decapitation of the guinea-pigs, the brains were rapidly frozen in liquid monochlorodifluoromethane ( - 4 0 ° C ) . Sections (10 ~m) were prepared using a Bright-Shandon cryostat, mounted onto microscope slides then incubated for 5 h at 25°C in N a z / K phosphate buffer (50 mM, pH 7.5), containing 0.1% BSA and 0.1 nM [125I]iodobolpyramine. Non-specific binding was obtained by adding 0.2 ~M mianserin to the incubation medium. Following five 12 min rinses at 25°C in the phosphate buffer containing 0.1% BSA, the sections were left on Ultrofilm (LKB) for a 2-day period (Palacios et al., 1981a).

The linearity of [125I]iodobolpyramine with protein concentration was checked at a ligand concentration of 0.12 nM and compared to [3H]mepyramine binding. Specific [I25I]iodobolpyramine binding increased linearly with the membrane concentration from 1 ~g up to 15 #g protein/200 t~l (fig. 2). At 0.55 nM, specific [3H]mepyramine binding increased linearly with the membrane concentration from 25 ~g up to 150 /~g protein/500 /~1. With these ligand concentrations, even at the low amount of 2 ~g of membrane protein, total binding of [125I]iodobolpyramine represented 2 700 dpm 66% of which specific. In comparison, total binding of [3H]mepyramine to 100/~g membrane protein represented 750 dpm over a non-specific binding of 125 dpm. At 25°C, binding of [~25I]iodobolpyramine (0.16 nM, 20 #g of membrane protein) occurred slowly, equilibrium only being reached after about 3 h (fig. 3A). An association rate constant (k~) of 0.0553 min.nM ~ was calculated from these data. Equilibrium was reached after about 5 h at 20°C and after about 2h at 30°C (data not shown). Dissociation followed first-order kinetics with a t~/2 of 201 min, corresponding to a rate constant k 1 = 0.00308 min 1 at 25°C (fig. 3B). The equi-

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Fig. 2. Specific binding of [125I]iodobolpyramine and [3H]mepyramine as a function of guinea-pig cerebellar membrane protein concentration. Increasing amounts of guinea-pig cerebellar membranes were incubated with either 0.12 nM [1251]iodobolpyramine or 0.55 nM [3H]mepyramine (see Methods for experimental conditions). Specific binding was determined as the difference between total and non-specific binding measured in the presence of 0.2 p.M mianserin.

librium dissociation constant, i.e. the ratio of k to k t, was 0.055 nM. When cerebellar membranes (37/zg) were incubated for 4 h at 25°C, non-specific binding increased linearly with the concentration of free ~25I-ligand (0.04-0.5 nM) and represented 15-35% of the total (fig. 4A). Scatchard analysis of the specific binding gave a straight line (fig. 4B) with K D 0.155_+0.016 nM and B...... 2 1 7 + 1 2 f m o l / m g protein.

3.2. Pharmacology of [ I-'51]iodobolpyraminebinding -

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The specific binding of 0.12 nM [125I]iodobolpyramine was completely inhibited by a series of Ht-receptor agonists or antagonists (table 1), and K~ values were calculated from the analysis of the various competition curves. The K~ values are Fig. 3. Kinetics of the specific binding o f [t25 l]iodobolpyramine to guinea-pig cerebellar membranes at 25°C. (A) Membranes were incubated in the presence of 0.16 nM [1251]iodobolpyramine as described in Methods. Samples were filtered through G F / B glass fiber filters at the times indicated, (B) After a 240 min incubation in the presence of the 1251-1igand, dissociation was initiated by a 10 fold dilution with fresh medium containing 0.2 /LM mianserin but no 1251-1igand. Non-specific binding was determined in the presence of 0.2 ~M mianserin. Results shown are from one representative experiment performed in triplicate. The association and dissociation rate constants were calculated according to Weiland and Molinoff (1981).

155 shown together with corresponding published values for inhibition of [3H]mepyramine binding to guinea-pig cerebral (mainly cerebellar) membranes and for inhibition of histamine-stimulated contrac-

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Fig. 4. Saturation of [~25i]iodobolpyraminebinding to guineapig cerebellar membranes. (A) Membranes were incubated at 25°C for 4 h with [1251]iodobolpyramine in increasing concentrations. Specific binding represents the difference between total and non-specific binding (in the presence of 0.2 FM mianserin). The incubation mixture contained 37 #g of protein in a final volume of 200 ,ttl, except for ligand concentrations below 0.1 nM for which the final volume was increased to 500 #1 to reduce the receptor concentration. All values were corrected for the radioactivity retained on filters in the absence of membranes (see Methods). The results shown are from one typical experiment in triplicate. (B) Scatchard transformation of specific binding.

tions of the guinea-pig ileum (table 1). M o n o phasic inhibition of [125I]iodobolpyramine binding was observed in most cases as indicated by the pseudo-Hill coefficients close to unity (except in the case of ( - )-chlorpheniramine). There is a close correlation between the three sets of K, values, the sole exception being mianserin which was apparently 10-fold more potent against the 125I-ligand than in the two other systems (table 1). However, when the inhibition of [3H]mepyramine binding by mianserin was studied under the conditions used for the 125I-ligand (4 h incubation in the presence of 0.1% BSA) a K i value of 0.32 _+ 0.04 n M was obtained (i.e. comparable with the value 0.22 __+0.04 nM found against the 125I-ligand). K B values determined for inhibition of histaminestimulated contractions of the guinea-pig ileum after 8 min equilibration were 32 nM for S K & F 94461, 8 n M for bolpyramine and under similar conditions, 1.3 nM for mepyramine. Antagonists at histamine H 2- or H3-receptors, and at serotonin, catecholamine, or muscarinic receptors displayed a low affinity in inhibiting [125I]iodobolpyramine binding. [125IJIodobolpyramine binding to rat cerebral cortex membranes was tested under conditions similar to those used for guinea-pig cerebellum membranes. At 0.1 nM [125I]iodobolpyramine, total binding was 28 f m o l / m g protein and non-specific binding was 25 f m o l / m g protein. In the same experiment total binding to guinea-pig cerebellum membranes was 104 f m o l / m g protein and nonspecific b i n d i n g was 25 f m o l / m g protein. Bolpyramine inhibited [3 H]mepyramine binding to rat cerebral cortex membranes with a K~ value of 5.4_+ 1.2 nM, while its corresponding value in guinea-pig cerebellum was 1.25 + 0.13 nM (table 1).

3.3. A utoradiographic localisation of [ zz~i]iodobolpyramine binding sites in guinea-pig brain A well-contrasted autoradiographic picture of [125I]iodobolpyramine binding sites was obtained on a medial sagittal section of guinea-pig brain (fig. 5A). The binding was almost eliminated on an adjacent section incubated in the presence of 0.2 /~M mianserin (fig. 5B). The iodinated ligand

156 TABLE 1 Comparison of drug potencies as inhibitors of histamine-stimulated ileum contraction and inhibitors of binding of [~ H]mepyramine or [1251]iodobolpyramine to cerebral membranes from the guinea-pig. [12sl]lodobolpyramine (0.12 nM) was incubated with guinea-pig cerebellar membranes in the presence of 8 to 12 different concentrations of the indicated compounds, except for alprenolol, methysergide, spiperone, sulpiride and scopolamine for which only 3 different concentrations were tested. IC~o values and pseudo-Hill coefficients were determined from pooled data from at least 2 independent experiments, by non-linear regression using a one-site model. IC50 values were converted to K i values using the equation K i = IC50/1 + L / K D, where L = concentration of [125I]iodobolpyramine, K D = equilibrium dissociation constant of [1251iodobolpyramine (0.15 nM). ~' Yeramian et al., 1985; b Hill et al., 1978: Ganellin, 1982; d Tran et al., 1981; c Garbarg el al., 1983; f Vargaftig et al., 1971; ~ Nowak et al., 1983; h Brimbtecombe el al., 1975: i Black et al., 1972; J Figge et al., 1979; k Hill and Young, 1978; i Values determined after 8 min equilibration: '~ Values determined after 4 h incubation at 25°C in the presence of 0.1% BSA corresponding to experimental conditions used for [~2Sl]iodobolpyramine binding. Guinea-pig ileum contraction K B (nM)

Cerebral [ 3H]mepyramine binding K i (nM)

Cerebellar [ 2~ - 1]•odobolpyramine binding K i (nM)

pseudo-Hill coefficient

39000 (a) 86000 (a) 56000 (a)

33 900 -+ 3 800 66000-+7800 58000-+5500

0.90 -+ 0.08 0.91 -+ 0.10 0.94-+0.08

0.6 (e) 0.3 (b) 0.13 (d) 2.4 (g) 0.32 + 0.04 (m) 0.8 (b) 200 (b) 11 -+ t 1.25+0.13 (m) 6.5 (d)

0.33 +0.03 0.22_+0.04 0.12 _+0.01 0.22 + 0.04 0.21 + 0.06 35 -+ 9 8.3+0.3 0.57+_0.09 11 +5

1.07 +_0.10 0.81 +0.11 0.99 + 0.09 0.81 _+0.11 0.72 + 0.20 0.56 -+ 0.06 1.03_+0.03 0.95+0.14 1.21 _+0.81

H / - R e c e p t o r agonists

Histamine 2-Pyridylethylamine 2-Thiazolethylamine H / - Receptor antagonists

Mepyramine Triprolidine Doxepin Mianserin ( + )-Chlorpheniramine ( - )-Chlorpheniramine SK&F 94461 Bolpyramine lmipramine

0.4 (c) 0.1 (c) 0.06 (k) 2.0 (f) 0.5 (c) 14 (c) 32 (1) 8 (1) 13 (j)

O t h e r agents

Cimetidine Burimamide lmpromidine Phentolamine Alprenolol Methysergide Spiperone ( - )-Sulpiride Scopolamine

> 27000 112000 2 000 2 000 140 000 87 000 2 000 2 000 8 000

446000 (h) 228000 (i) 3 400 (c) 825 (d)

11000 (k)

labelled several grey matter areas whereas typical white matter areas such as the corpus cailosum were not labelled. The highest densities of label-

stria terminalis, lateral septum nucleus and nucleus

ling were found in cerebellar cortex (molecular layer), thalamus and nucleus accumbens. A somew h a t l o w e r d e n s i t y o f l a b e l l i n g w a s f o u n d in t h r e e hypothalamic areas (medial mamillary nucleus,

colliculi inferior and superior, central grey matter, lateral habenular nucleus, and several areas of

premamillary nucleus and ventromedial hypot h a l a m i c n u c l e u s ) a n d in f o u r l i m b i c a r e a s ( m o l e c ular layer of the dentate gyrus, bed nucleus of the

of the vertical limb of the diagonal band). A l a b e l l i n g o f l o w e r i n t e n s i t y w a s l o c a l i s e d in t h e

cerebral cortex. In a more lateral sagittal section ( n o t s h o w n ) , s p e c i f i c l a b e l l i n g w a s a l s o o b s e r v e d in various other structures such as the ventral striaturn.

157

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.'%, / Fig. 5. Autoradiograms of sagittal sections from guinea-pig brain generated with [1251]iodobolpyramine. (A) (top) Total binding was achieved by incubation with [i 25 I]iodobolpyramine (0.1 nM) for 5 h at 25°C. (B) (bottom) Non-specific binding was obtained in the presence of 0.2 ,aM mianserin. The structures were identified by comparison with adjacent sections stained with toluidine blue and by comparison with a rat brain stereotaxis atlas (Paxinos and Watson, 1982). Acb: accumbens nucleus; BST: bed nucleus of stria terminalis; CA~: field CA~ of Ammon's horn; CA3: field CA~ of Ammon's horn: CA4: field C A 4 of Ammon's horn; CG: central grey: DG: dentate gyrus; DPG: deep grey layer of superior colliculus; FR: frontal cortex: FRP: fronto parietal cortex; G: granular cell layer of cerebellum; IC: inferior colliculus; ING: intermediate grey layer of superior colliculus; LHb: lateral habenular nucleus; LS: lateral septal nucleus; M: molecular layer of cerebellum; ML: medial mamillary nucleus, lateral part; Mol: molecular layer of dentate gyms; MT: medial thalamic nucleus; Or: oriens layer of hippocampus: PF: parafascicular thalamic nucleus; PM: premamillary nucleus; Pm: pontine nuclei; R: red nucleus; str: striate cortex; SUG: superficial grey layer of superior colliculus; VDB: nucleus of the vertical limb of the diagonal band; VMH: ventromedial hypothalamic nucleus; VTA: ventral tegmental area.

4. Discussion The present study establishes [125I]iodobolpyramine as the first iodinated ligand for selective and highly sensitive labelling of histamine H~receptors. That [~251]iodobolpyramine uniformly labels a single population of sites in cerebellar membranes

is indicated by the linearity of the Scatchard plot (fig. 4B), the first-order kinetics of its dissociation (fig. 3B) and the pseudo-Hill coefficient, not different from unity, in competition experiments with various Hi-receptor agonists or antagonists (table 1). These sites correspond to H~-receptors as shown by: (i) the closely similar capacities (B..... ) of cerebellar binding sites for [125i]iodobolpyramine, i.e. 217 f m o l / m g protein, and the parent compound [3H]mepyramine, a selective Hi-receptor ligand (Hill and Young, 1978; Tran et al., 1978; Garbarg et al., 1983); (ii) their pharmacology which is similar to that of [3H]mepyramine recognition sites; the apparent discrepancy with mianserin was no longer observed when the competition for [-~H]mepyramine binding sites was assessed under the incubation conditions used for [125I]iodobolpyramine, that is a 4 h incubation in a buffer containing 0.1% BSA; (iii) the K~ values of H~-receptor antagonists similar to their corresponding values as inhibitors of histamine-stimulated contractions of the guinea-pig ileum in vitro (table 1); the apparent lower affinity of S K & F 94461 and bolpyramine for Hi-receptors mediating guinea-pig ileum contraction was probably due to the short equilibration time (8 min), since the KR value of bolpyramine decreased by a factor of 2 after 15 rain equilibration. After 30 rain, there was considerable depression of the maximum, however, which complicated the determination of K B values (table 1); (iv) the low affinity of antagonists of other classes of receptors including H 2- and H~-receptors. For instance, cimetidine, a well-known H~-receptor antagonist (with a K B value of 0.8 /~M) (Ganellin, 1982), was very poorly effective. The K i value of burimamide for [125I]iodobolpyramine binding sites (112 /LM) is far from its K B values of 7.8 t~M at H2-receptors (Ganellin, 1982) and 70 nM at H3-receptors (Arrang et al., 1983). The affinity of impromidine for [125i]iodobolpyramine binding sites is 30 times lower than its affinity as an antagonist at H3-receptors and for inhibiting the binding of [3H]tiotidine (Gajtowski et al., 1983). In addition, there is reasonable agreement between the K, values for burimamide and impromidine for [125I]iodobolpyramine binding sites and their K B value for inhibition of histaminestimulated ileum contraction.

158 Interestingly, whereas S K & F 94461, the aminopentyl analogue of mepyramine, displayed about 10 times less affinity for H~-receptors than did mepyramine itself, its acylated derivative, bolpyramine, was approximately as potent as mepyramine, and [125I]iodobolpyramine was even more potent (K D = 0.05 nM from the ratio of K,ff and K ..... and 0.15 nM from saturation data). This suggests that either the free amino group of SK & F 94461 has a detrimental effect on affinity or the 4-hydroxyphenyl propionate region of the bolpyramine molecule contributes to the stability of the ligand-receptor interaction. One striking feature of the interaction of [125I]iodobolpyramine with H~-receptors was its very slow association and dissociation rates, equilibrium being reached only after 3 h at 25°C (fig. 4); at 0°C no significant dissociation could be observed after 2 h (not shown). In contrast, [-~H]mepyramine binding reached equilibrium within 30 rain, a difference possibly attributable, amongst others, to the much larger size of the iodobolpyramine molecule. The main interest in [~25I]iodobolpyramine is that it provides a highly sensitive means of detecting H~receptors due to its high affinity, high specific radioactivity and relatively low non-specific binding. Hence, when used at a concentration close to the K D value, 0.2 fmol of binding sites (corresponding to about 1 ~g protein of guinea-pig cerebellar membranes) can be reliably assayed: this represents a 50-100 fold increase in sensitivity as compared to [3H]mepyramine (fig. 2). Because of its enhanced sensitivity, the new ligand is likely to have several valuable applications, e.g. for the assay of H~-receptors in microdissected brain areas, for tissues with low receptor densities and for H j-receptor purification studies. Receptor autoradiography provides another interesting application for which ~2~I-ligands display distinct advantages of 3H-ligands: shorter exposure times are required and the lower (and more homogenous) tissue absorption facilitates quantification (Kuhar and Unnerstall, 1985). Well-contrasted autoradiographic pictures with low nonspecific binding were obtaiend after only two days' exposure (fig. 5). The highest grain densities were found in the molecular layer of the cerebellum which agrees with both autoradiographic (Palacios

et al., 1979, 1981b) and biochemical (Hill et al., 1978) data with [3H]mepyramine. Functional H 1receptors seem to be present in the cerebellum (Daum et al., 1984) which otherwise contains low L-histidine decarboxylase activity (Schwartz et al., 1970). In the hippocampal formation, a projection field for histaminergic neurons (Barbin et al., 1976; Haas et al., 1978), the distribution of [~25I]iodobolpyramine autoradiographic grains was apparently similar to that of [~H]mepyramine binding sites (Palacios et al.. 1981c). Other areas of very high H~-receptor densities are the nucleus accumbens (not previously reported) and the whole thalamus (Hill et al., 1978). There was more or less uniform labelling of the whole cerebral cortex with, however, some higher densities in superficial layers (laminae II-Ill), whereas in rat brain, [~ H]mepyramine binding sites were mainly located in lamina IV of the temporal cortex (Palacios et al., 1981c). The widespread occurrence of Hl-recetors in the whole cerebral cortex could conceivably account for the sedating ("mental clouding") properties of H~-antihistamine (Quach et al., 1979). Moderate to rather high labelling was found in the colliculi, mesencephalic central grey and ventral tegmental area, as well as in the bed nucleus of the stria terminalis. Very high levels of L-histidine decarboxylase activity have been reported for the three last areas (Pollard et al., 1978; Ben Ari et al., 1977). Several mamillary nuclei in the hypothalamus were distinctly labelled, as observed with [)H]mepyramine (Palacios et al., 1981a). Localisation of H~-receptors in this region, where histamine cell bodies of the ascending pathway have been localised by lesion (Schwartz et al., 1982) and immunohistochemical studies (Watanabe et al., 1984; Steinbusch and Mulder, 1984: Panula et al., 1984; Pollard et al., 1985a,b) raises the possibility (but does not prove) that H~-receptors may be present on histamine cell bodies or dendrites. Hence, in general, the localisation of H~-receptors visualised with [~251]iodobolpyramine in the guinea-pig brain is consistent with that of known histaminergic projections and histamine cell bodies or dendrites and this agrees well with studies using [-~H]mepyramine in rat brain. However, there is also a species difference in regional distribution

159

since the regions with the highest density of H~-receptors in guinea-pig brain, namely cerebellum, thalamus and nucleus accumbens were hardly labelled with [3H]mepyramine in rat brain (Palacios et al., 1981c). Furthermore, mepyramine and several other antagonists display a higher affinity for H~-receptots in the guinea-pig than in rat or mouse brain (Chang et al., 1979; Hill and Young, 1980; Quach et al., 1980). We consistently found bolpyramine to be about 4 times more potent in guinea-pig than in rat cerebral membranes. Also, there was no evidence for significant [125I]iodobolpyramine specific binding displaceable by mianserin at 0.10.15 nM ligand concentration to rat brain membranes. This negative result may be tentatively explained by the combination of a lower affinity of [~25Iliodobolpyramine, a relatively high level of non-specific binding and a limited number of binding sites in rat brain membrane preparations. Specific binding was also not yisualised on tissue sections (not shown). These observations strongly confirm the species differences in Ht-receptor pharmacological specificities but, at the same time, indicate that the new ligand will not be usable for rat tissues.

Acknowledgements We thank W. Tertiuk and C.J. Theobald for help with chemical synthesis and R.C. Blakemore and Dr. M.E. Parsons for testing compounds on the isolated guinea-pig ileum. Studies at the Unit6 de Neurobiologie were supported by a contract from D.R.E.T. and by a grant from SK&F (France).

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