Brain Research, 514 (1990) 15-21 Elsevier
15
BRES 15360
Binding of [3H]p-aminoclonidine to two sites, aE-adrenoceptors and imidazoline binding sites: distribution of imidazoline binding sites in rat brain Yoshinori Kamisaki, Tadashi Ishikawa, Yoshiki Takao, Hiroki Omodani, Nobutoshi Kuno and Tadao Itoh Department of Clinical Pharmacology, Tottori University School of Medicine, Yonago (Japan) (Accepted 12 September 1989) Key words: [3HlP-Aminoclonidine; Imidazoline site; Rat brain; Adrenoceptor; Striatum; Histamine receptor
Binding sites labeled by [3H]p-aminoclonidine ([3H]PAC) were investigated by the competitive analysis with imidazoline and non-imidazoline derivatives. Phenylethylamine derivatives displaced only the part of specific sites for [3H]PAC, which was considered as a2-adrenoceptor, whereas imidazoline derivatives, such as clonidine and tolazoline, competed for a further specific binding of [3H]PAC to the non-adrenergic sites, in addition to the a2-adrenoceptor. Because the non-adrenergic sites were specific for the imidazoline structure, they were termed imidazoline sites. The imidazoline sites were not distributed uniformly among rat brain regions. In striatum, hippocampus and medulla oblongata, they occupied 39.6, 33.0 and 36.5% of the specific binding of [3H]PAC, respectively. Saturation isotherms revealed that Kd and Bm~ of imidazoline sites for [3H]PAC were 3.09 + 0.59 nM, 27.4 + 1.7 fmol/mg protein and 2.23 + 0.29 nM, 21.0 + 1.5 fmol/mg protein in striatum and hippocampus, respectively. Because imidazoline binding sites also displayed weak affinities for imidazole compounds, such as histamine and cimetidine, the imidazoline site may be a subtype of histamine H2-receptor.
INTRODUCTION Clonidine, one of the imidazoline derivatives, has been used as an antihypertensive agent. Although the actions of clonidine are complex, its major actions are those of a centrally acting aE-adrenergic agonist. Clonidine impairs sympathetic nerve activity by binding to presynaptic aE-adrenoceptors. [3H]Clonidine and [3H]p-aminoclonidine have been utilized as ligands for aE-adrenoceptors El' 26,28,32,37
Recently, we have revealed that imidazoline derivatives (both a-agonists and antagonists) stimulate the acid secretion of parietal cells isolated from guinea pig stomach. These stimulatory effects were inhibited by HE-antagonists. The binding experiment of [3H]clonidine to the m e m b r a n e fraction suggested the existence of the imidazoline binding sites in parietal cells, which are different from H E- or aE-receptors 17. Since histamine receptors were divided into two subtypes, namely H 1 and HE, many agonists and antagonists have been discovered and investigated to clarify the relationships between their structures and pharma-
cological effects 2'4'19'30. In the case of histamine H 1receptor, the use of [3H]mepyramine allowed direct binding studies in brain and peripheral tissues 6'7,16,36. On the other hand, suitable ligands with high affinities have not been reported to label the HE-receptor. For example, [3H]cimetidine, a HE-antagonist, was used for the studies on the histamine HE-receptor in rat brain m e m b r a n e s 2°. However, the dissociation constant (Kd) of [3H]cimetidine was 400 nM by Scatchard analysis 2°. Although only [3H]tiotidine may meet the criteria for labeling the HE-receptor, the K d was 17 nM in guinea pig cerebral cortex m e m b r a n e 13. Recently, it was observed that histamine regulated its own synthesis and release by a negative feedback process through a new histamine H3-receptor in rat and human brain 1'15'18'31'39. In the present studies, imidazoline binding sites were examined in rat brain using the high-affinity ligand of [3H]PAC to clarify the relation with histamine receptor subtypes. In addition, regional studies were performed in order to determine whether the density of imidazoline site is related to a particular physiological role. The results demonstrate that imidazoline sites locate espe-
Correspondence: Y. Kamisaki, Department of Clinical Pharmacology, Tonori University School of Medicine, 86 Nishimachi, Yonago 683, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
16 cially in the s t r i a t u m
and h i p p o c a m p u s
i m i d a z o l i n e site m a y be a subtype
and that the
of h i s t a m i n e H 2-
receptor. MATERIALS AND METHODS
Materials p-[3,5-3H]Aminoclonidine ([3H]PAC) with a specific activity of 40-50 Ci/mmol was obtained from New England Nuclear. Clonidine HCI and p-aminoclonidine HCI were gifts from BoehringerIngelheim, Ltd. a-Methyl norepinephrine (aMNE) was from the Sterling-Winthrop Research Institute. Tolazoline HCI, naphazoline HCI, histamine diHC1, (-)-norepinephrine HCI, (-)-epinephrine bitartarate, and L-phenylephrine HCI were purchased from Sigma Chemical Co. Cimetidine was from Smith, Kline & French. All other chemicals were of the highest purity available.
Membrane preparations Adult male Wistar rats (240-270 g) were sacrificed by decapitation and brains were rapidly removed. The brains were separated on ice-cold stage into 10 regions (i.e. olfactory bulb, anterior and posterior cerebral cortex, striatum, hippocampus, thalamus + midbrain, hypothalamus, cerebellum, pons, medulla oblongata) essentially as described by Glowinski and Iversen TM. These sections were homogenized in 10 vols. (v/w) of ice-cold 50 mM Tris-HCl buffer (pH 7.5) using a Polytron PT-10 (Kinematica, setting 8, 10 s x 3). The resulting homogenate was centrifuged at 35,000 g (Hitachi 20PR-52) for 45 min at 4 °C. The pellet was washed in 10 vols. of fresh buffer and recentrifuged. These procedures were repeated 3 times. The final pellet was resuspended in 10 vols. of the Tris-HCl buffer. Protein concentrations in aliquots of the suspension were determined by the method of Lowry22.
Receptor binding assay
t5~A o - - O T
RESULTS
Time courses of association and dissociation of [3H]PAC binding A s s h o w n in Fig. 1, the b i n d i n g of [ 3 H ] P A C to brain m e m b r a n e s was s a t u r a b l e with time. [ 3 H ] P A C b i n d i n g to cortex
[3H]PAC binding to brain membranes was measured in 1.0 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 4.0 nM [3H]PAC. Incubation was started by the addition of tissue membrane aliquots containing 0.5-1.0 mg of protein and carried out for 30 min at 25 °C. After the incubation, the content of the assay tubes were immediately filtered over Whatman GF/C filters which had been treated
~:
with 1% polyethylenimine5'37 and rinsed 3 times with 4 ml of ice-cold Tris-HCl buffer 24"26'28'32. Radioactivities of filters were measured with 6 ml of ACS II (Amersham) on a Beckman liquid scintillation counter. Non-specific bindings were defined by parallel incubations containing 10-4 M aMNE and/or p-aminoclonidine (or clonidine). All assays were conducted in duplicate under these conditions. The bindings specific for a2-adrenoceptor and imidazoline sites were calculated from the binding amount of [3H]PAC to the membrane without the reagents minus that in the presence of aMNE, and the binding amount of [3H]PAC in the presence of aMNE minus that in the presence of clonidine, respectively. The non-specific binding, the binding amount of [3H]PAC in the presence of both clonidine (10 -4 M) and aMNE (10 -4 M) was the same as that in the presence of clonidine (10 -4 M) alone. Competition studies were carried out under standard incubation conditions with 10 9-10-3 M of several reagents. The K~ (inhibition constant) for each reagent was obtained from IC5oderived from the inhibition curves. Determination of the binding characteristics with membrane preparations was conducted with concentrations of [3H]PAC ranging from 0.5 to 16 nM. Specific bindings were calculated as described above. A computerized parameter estimation method program 9 was used to resolve the data from the saturation isotherms according to the method of Scatchard 29.
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within 20 min. T h e r e f o r e , 30 rain was c h o s e n as the s t a n d a r d i n c u b a t i o n t i m e for b i n d i n g studies using this radioligand. B i n d i n g was r e v e r s i b l e u p o n a d d i t i o n o f 10 -4 M of a M N E o r clonidine. T h e s e m i l o g a r i t h m i c transfor-
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Fig. 1. Time courses of association of [3H]p-aminoclonidine (PAC) binding to the membrane fractions from anterior cortex (A) or striatum (B). Membranes were incubated with 10-4 M clonidine (A), a-methyl norepinephrine (aMNE, O), or none of them (O) as described in Materials and Methods. At the time indicated by arrowheads, 10-4 M clonidine (A) or aMNE (O) was added to initiate radioligand dissociation. Each value plotted is the mean of duplicated determinations.
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Fig. 2. Competitions for [3H]PAC binding to the membrane from anterior cortex (A) or striatum (B) by phenylethylamines. Membranes were incubated with [3H]PAC (4 nM) and designated concentrations of clonidine (O), aMNE ( ~ ) , norepinephrine (&), epinephrine ( a ) , phenylephrine ( i ) as described in Materials and Methods.
17 mations of association and dissociation were linear, indicating that the equilibrium dissociation constant (Kd) for [3H]PAC binding to cortex and striatum membrane was in the nanomolar range. In the standard condition, specific bindings to cortex and striatum were ca. 60% of the total [3H]PAC binding. Although the non-specific bindings were the same in the anterior cortex (Fig. 1A), the difference was observed in striatum membrane between the non-specific binding defined in the presence of 10-4 M aMNE and that in the presence of 10-4 M clonidine (Fig. 1B).
Properties of [3H] PA C binding sites in cortex and striatum A variety of non- and imidazol(in)e derivatives were examined for their influence on specific [3H]PAC binding to rat cortex and striatum membranes, in order to investigate the discrepancy between the non-specific binding in the presence of aMNE and that in the presence of clonidine. In anterior cortex membrane, the competition curves of these drugs were monophasic, sigmoid and parallel (Figs. 2A and 3A). Imidazoline derivatives (PAC, naphazoline, and clonidine) except tolazoline, were more potent than naturally occurring phenylethylamines, a-adrenergic agonists (aMNE, norepinephrine, epinephrine, and phenylephrine). In striatum membrane, potencies of these compounds competing for the specific binding were similar to those in cortex membrane. However, all imidazoline derivatives, regardless of a-adrenergic agonists and antagonists, inhibited almost completely the specific binding of [3H]PAC (59% of the total binding), whereas phenylethylamines displaced only part of the specific sites (38%
of the total binding) (Figs. 2B and 3B). Moreover, at the high concentration of 10-3 M, imidazole compounds (histamine and cimetidine) also partly competed for the [3H]PAC binding.
Characteristics of aMNE-insensitive [3H]PAC binding sites In striatum, phenylethylamines displaced 60% of the specific binding sites for [3H]PAC, but failed to inhibit the remaining 40%, whereas imidazoline compounds displaced all of specific binding sites. These data suggested that [3H]PAC may interact with more than one class of binding sites in the striatum. In order to clarify properties of the remaining ctMNE-insensitive sites, further competition binding experiments were conducted in the presence of 10-4 M aMNE. Typical examples of such competition binding curves are displayed in Fig. 4. In striatum, the potency series for imidazoline derivatives were as follows: PAC > naphazoline > clonidine > tolazoline. The K i values of PAC, naphazoline, clonidine, and tolazoline were 2.31 nM, 5.24 nM, 23.4 nM, and 91.7 nM, respectively. In addition to imidazoline derivatives, imidazole compounds (histamine and cimetidine) also completely inhibited the [3H]PAC binding to aMNE-insensitive sites with K i of 7.42/~M and 6.11/~M, respectively. However, diphenhydramine, H 1 antagonist and non-imidazole compound, was inactive at 10 -3 M. The other phenylethylamines also showed little or no competition for the [3H]PAC binding to aMNE-insensitive sites at concentrations up to 10-3 M. Non-specific
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Fig. 3. Competitions for [3H]PAC binding to anterior cortex (A) or striatum (B) membranes by imidazol(in)e derivatives. Membranes were incubated with designatedconcentrationsof aMNE (~), PAC (O), clonidine (O), naphazoline (A), tolazoline (&), cimetidine (I), histamine (I-7) as described in Materials and Methods.
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Fig. 4. Competitions for [3H]PAC binding to the imidazoline specific sites in striatum (A) and hippocampus (B) by imidazoi(in)e derivatives. Membranes were incubated with [3H]PAC, aMNE (10-4 M) and varying concentrations of PAC (O), clonidine (O), naphazoline (A), tolazoline (&), cimetidine ( I ) , or histamine (El) as described in Materials and Methods. Each value plotted is the mean of duplicate determinations and plotted figures are representative of 3 separate experiments.
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Fig. 5. Scatchard plots of [3H]PAC binding to membrane from anterior cortex (A), striatum (B) and hippocampus (C). Membranes were incubated with various concentrations of [3H]PAC (0.5-16 nM) and specific bindings for a2-adrenoceptor (0) or imidazoline sites (©) were determined in the presence of 10-4 M aMNE and/or clonidine as described in Materials and Methods. Plotted figures are representative of more than 3 experiments.
bindings (ca. 5100 dpm/mg protein of striatum membrane) which were obtained in the presence of both a M N E and imidazolines (Fig. 4A) were the same as those observed in the presence of imidazoline compounds alone (Figs. 2B and 3B). Therefore, it is suggested that the [3H]PAC binding sites m a y possess, at least, two subpopulations, n a m e l y a2-adrenoceptor and imidazoline sites. Similar results were obtained in aMNE-insensitive sites in h i p p o c a m p u s (Fig. 4B).
Regional distribution of imidazoline-specific binding sites in rat brain Assessment of distribution of a2-adrenoceptor and imidazoline sites was accomplished by dissecting the rat
TABLE I Regional distribution of a2-adrenoceptors and imidazoline sites in rat brain
Standard binding assays were conducted with membrane preparations from 10 brain regions. Specific bindings for az-adrenoceptor and imidazoline sites were calculated from specific activities of [3H]PAC as described in Materials and Methods. Values are expressed as means + S.E.M. from duplicate determinations of 3 separate experiments with percentages in parentheses to the total specific bindings. az-A drenoceptor (fmol/mg prot. )
lmidazoline site (fmol/mg prot. )
Olfactory bulb Anterior cortex
28.3 + 1.9 (79.7) 82.7 + 4.5 (100.7)
7.2 + 2.3 (20.3) - 0.6 + 5.1 (-0.7)
Posterior cortex Striatum Hippocampus Thalamus + midbrain Hypothalamus Cerebellum Pons Medulla oblongata
72.8 __+7.8 (94.3) 41.4 + 1.8 (60.4) 42.1 + 3.1 (67.0) 48.2 + 5.2 (86.5) 41.8 + 7.5 (72.9) 23.4 + 3.5 (79.1) 21.4 + 1.5 (85.9) 30.5 + 3.3 (63.5)
4.4 + 1.0 (5.7) 27.1 + 1.1 (39.6) 20.7 + 1.7 (33.0) 7.5 + 1.6 (13.5) 15.5 + 1.2 (27.1) 6.2 + 1.2 (20.9) 3.5 + 1.4 (14.1) 17.5 + 0.9 (36.5)
brain into 10 regions according to the m e t h o d of Glowinski and Iversen 14. Saturation analysis of [3H]PAC binding to a 2 - a d r e n o c e p t o r and imidazoline sites were p e r f o r m e d in anterior cortex, striatum and hippocampus m e m b r a n e . The bindings specific for a 2- and imidazoline sites were d e t e r m i n e d in the presence of 10 -4 M a M N E or clonidine as a function of increasing concentration of the radioligand (0.5-16 nM). A l t h o u g h both specific bindings were saturable, the non-specific binding, only defined by imidazoline derivatives, was linear in function. As one can see in Fig. 5, Scatchard analysis of the saturation isotherms showed that [3H]PAC binds to a2-adrenoceptor in anterior cortex, striatum, and hippocampus m e m b r a n e with dissociation constants (Kd) of 3.08 + 0.56, 2.69 + 1.29, and 2.76 + 1.00 nM and m a x i m u m n u m b e r of binding sites (Bmax) of 89.0 -- 5.6, 49.5 + 6.5, and 43.8 + 3.4 fmol/mg protein, respectively, and that it binds to imidazoline sites in striatum and hippocampus with K d of 3.09 + 0.58 and 2.23 + 0.29 nM and Bma x of 27.4 + 1.7 and 21.0 + 1.5 fmol/mg protein, respectively. Because the K o values for a 2 - a d r e n o c e p t o r and imidazoline sites were very similar, there was no i m p r o v e m e n t in goodness-of-fit using a m o d e l with two binding sites rather than a m o d e l with one binding site. We failed to investigate these binding sites by saturation experiments in the o t h e r regions, because [3H]PAC binding to imidazoline specific sites was too little in those regions. Thus, the binding results in the standard condition are listed in Table I. If the K d values for a 2a d r e n o c e p t o r and imidazoline sites are similar to each other in the other regions, the binding a m o u n t may reflect the ratio of those r e c e p t o r numbers. Especially in striatum, hippocampus and m e d u l l a oblongata, a M N E insensitive sites occupied m o r e than 30% of the total specific binding. Not only the n u m b e r of imidazoline sites but also the ratio of imidazoline sites to a2-sites were distributed differently a m o n g rat brain regions.
19 DISCUSSION The characteristics and distributions of a2-adrenoce ptots have been investigated using [3H]PAC as a radioligand25'26'28'38. In those studies phenylethylamines (norepinephrine, epinephrine and a-methyl norepinephrine) or imidazoline derivatives (phentolamine, clonidine and tolazoline) were utilized to calculate the non-specific bindings. Recently, it was suggested that [3H]idazoxan, an imidazoline derivative, may bind to non-adrenergic sites with high affinity as well as a2-adrenoceptors s'24. In the present study we have demonstrated that [3H]PAC binding sites consist of two subpopulations, one is aE-adrenoceptor and the other is imidazoline-specific binding site and that the imidazoline sites are not uniformly localized among rat brain regions. According to the density of imidazoline binding sites (Table I), we chose striatum (or hippocampus) and anterior cortex as rich and poor regions in imidazoline sites, respectively. In striatum, phenylethylamine derivatives (aMNE, norepinephrine, epinephrine and phenylephrine) only competed for part of the specific binding of [3H]PAC, although imidazoline derivatives (PAC, clonidine, naphazoline and tolazoline) completely displaced it (Figs. 1-3). However, in anterior cortex, non-specific bindings defined by phenylethylamines and imidazolines were the same. In the further competition experiments, non-adrenergic sites appeared to be specific for the imidazoline structure, because all imidazoline derivatives completely inhibited [3H]PAC binding with high affinities of Ki, 2-100 nM in striatum and hippocampus (Fig. 4). Saturation binding isotherms revealed that K d values (2-3 nM) of [3H]PAC binding to the non-adrenergic sites in striatum and hippocampus were similar to each other (Fig. 5). These K d values were also similar to those to aE-adrenergic sites (2-3 nM), although the reason is unknown. Therefore, in addition to the different distribution of non-adrenergic sites (Table I), these findings confirmed that [3H]PAC also binds to the non-adrenergic sites which are termed as imidazoline sites. It was reported that binding sites labeled by [3H]PAC were characterized in bovine brain membranes prepared from ventrolateral medulla 1°. Norepinephrine and other phenylethylamines displaced [3H]PAC from 70% of the total sites and the remaining 30% were non-adrenergic sites which were termed as imidazole binding sites. Moreover, an endogenous clonidine-like substance was isolated from bovine brain, which binds to the imidazole sites and lowers arterial pressure when it was microinjected into ventrolateral medulla3"11A2'23. Therefore, it was concluded that imidazole binding sites may mediate the hypotensive action of clonidine and participate in the function of an endogenous clonidine-like substance.
Although the total [3H]PAC binding sites were characterized precisely by competition experiments with various reagents including phenylethylamines, imidazolines, and imidazoles, however, characteristics of the imidazolespecific sites were not directly examined 1°. In our experiments, we characterized non-adrenergic parts of [3H]PAC binding sites after a2-adrenergic parts were blocked with aMNE. The imidazol(in)e binding sites may be similar to each other, according to the effects of various compounds on [3H]PAC binding (Fig. 3). However, we termed them imidazoline binding sites because imidazoline derivatives showed 100-fold higher affinities than imidazole compounds (Fig. 4). The imidazoline sites were displaced by imidazole compounds (histamine and cimetidine) with weak affinities (K i values of ca. 10 btM, Fig. 4). However, diphenhydramine, a H~ antagonist, did not inhibit [3H]PAC binding. It is reported that the binding sites for [3H]cimetidine, solubilized from rat brain, were displaced by micromolar concentrations of clonidine34. It was concluded that the [3H]cimetidine binding site is associated with the putative clonidine-sensitive H2-receptor subtype. Therefore, these data suggest that the imidazoline site may be a part or a subtype of the histamine H2-receptor. It was reported that the cell bodies of histaminergic neurons locate in the tuberomammillary nuclei of the posterior hypothalamus and that their nerve fibers spread ascendingly to hypothalamus, cerebral cortex, and olfactory bulb and descendingly to midbrain and pons with synaptic varicosities33,35'4°-42. The distribution of histaminergic nerve terminals was in general consistent with that of histamine Hi-receptor examined by [3H]mepyramine binding6'a6'27. However, several areas such as striatum and lateral hypothalamus were found to contain many histaminergic nerve fibers, but few [3H]mepyramine binding sites 41. Thus, it was predicted that these regions might be rich in non-H~ histamine receptors. On the other hand, by an autoradiographic study with [3H]PAC, it was demonstrated that regions which have a 2 binding sites are innervated by adrenergic neurons 37. However, it was also observed that the densities of a 2 binding sites are not exactly correlated with those of catecholamine innervation. In the present study, we demonstrated that the imidazoline sites may be a part of histamine receptors and that they are localized in striatum and hippocampus. Therefore, especially in striatum, the existence of the imidazoline sites may explain the controversial facts that the density of histamine nerve terminals is higher than that of histamine Hi-receptors , and that the density of adrenergic innervation is relatively lower than that of [3H]PAC binding sites. Similarly, in the other regions,
20 the i m i d a z o l i n e sites m a y also r e s o l v e parts of t h e s e d i s c r e p a n c i e s b e t w e e n distributions of the n e r v e terminals and t h o s e o f the r e c e p t o r sites, a l t h o u g h the f u n c t i o n of t h e i m i d a z o l i n e sites has not b e e n elucidated.
REFERENCES 1 Arrang, J.-M., Garbarg, M., Lancelot, J.-C., Lecomte, J.-M., Pollard, H., Robba, M., Schunack, W. and Schwartz, J.-C., Highly potent and selective ligands for histamine H3-receptor, Nature (Lond.), 327 (1987) 117-123. 2 Ash, A.S.E and Schild, H.O., Receptor mediating some action of histamine, Br. J. Pharmacol., 259 (1966) 427-439. 3 Atlas, D. and Burstein, Y., Isolation and partial purification of a clonidine-displacing endogenous brain substance, Eur. J. Biochem., 144 (1984) 287-293. 4 Black, J.W., Duncan, W.A.M., Durant, G.J., Ganellin, C.R. and Parsons, E.M., Definition and antagonism of histamine H2-receptors, Nature (Lond.), 236 (1972) 385-390. 5 Bruns, R.E, Lawson-Wendling, K. and Pugsley, T.A., A rapid filtration assay for soluble receptors using polyethyleniminetreated filters, Anal. Biochem., 132 (1983) 74-81. 6 Chang, R.S.L., Tran, V.T. and Snyder, S.H., Histamine H lreceptors in brain labeled with [3H]mepyramine, Eur. J. Pharmacol., 48 (1978) 463-464. 7 Chang, R.S.L., Tran, V.T. and Snyder, S.H., Characteristics of histamine H~-receptors in peripheral tissues labeled with [3H]mepyramine, J. Pharmacol. Exp. Ther., 209 (1979) 437-443. 8 Convents, A., Convents, A., De Backer, J.-P., De Keyser, J. and Vauquelin, G., High-affinity binding of 3H Rauwolscine and 3H RX781094 to a 2 adrenergic receptors and non-stereoselective sites in human and rabbit brain cortex membranes, Biochem. Pharmacol., 38 (1989) 455-463. 9 De Lean, A., Hancock, A.A. and Lefkowitz, R.J., Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes, Mol. Pharmacol., 21 (1982) 5-16. 10 Ernsberger, P., Meeley, M.P., Mann, J.J. and Reis, D.J., Clonidine binds to imidazole binding sites as well as a 2adrenoceptors in the ventrolateral medulla, Eur. J. Pharmacol., 134 (1987) 1-13. 11 Ernsberger, P., Meeley, M.P. and Reis, D.J., An endogenous clonidine-like substance binds preferentially to imidazole binding sites in the ventrolateral medulla labelled by 3H-paraaminoclonidine, J. Hypertens., 4 Suppl. 5 (1986) S109-Sl11. 12 Ernsberger, P., Meeley, M.P. and Reis, D.J., An endogenous substance with clonidine-like properties: selective binding to imidazole site in the ventrolateral medulla, Brain Research, 441 (1988) 309-318. 13 Gajtkowski, G.A., Norris, D.B., Rising, T.J. and Wood, T.P., Specific binding of 3H-tiotidine to histamine H a receptors in guinea pig cerebral cortex, Nature (Lond,), 304 (1983) 65-67. 14 Glowinski, J. and Iversen, L.L., Regional studies of catecholamines in rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]dopa in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 15 Hill, S.J., Histamine receptors branch out, Nature (Lond.), 327 (1987) 104-105. 16 Hill, S.J., Emson, P.C. and Young, J.M., The binding of [3H]-mepyramine to histamine H~-receptors in guinea-pig brain, J. Neurochem., 31 (1978) 997-1004. 17 Houi, N., Kamisaki, Y. and Itoh, T., Effects of histamine H 2 receptor antagonists on acid secretion stimulated by imidazoline derivatives in isolated parietal cells, Eur. J. Pharmacol., 144 (1987) 67-76. 18 Ishikawa, S. and Sperelakis, N., A novel class (H3) of histamine receptors on perivascular nerve terminals, Nature (Lond.), 327
Acknowledgements. We would like to thank Dr. H. Kohei (Boehringer-Ingelheim) for generous gifts of drugs and Ms. Y. Yamasaki and R. Kageyama for secretarial assistance. This study was supported by grants from the Japanese Ministry of Education, Science and Culture (63770129) and the Mita Foundation.
(1987) 158-160. 19 Ishikawa, T., Kamisaki, Y. and Itoh, T., Pharmacological properties of the hydroxylated histamine, (+)-4(5)-(2-amino1-hydroxyethyl)-imidazole, Agents Actions, 26 (1989) 261-266. 20 Kendall, D.A, Ferkany, J.W. and Enna, S.J., Properties of 3H-cimetidine binding in rat brain membrane fractions, Life Sci., 26 (1980) 1293-1302. 21 Kobinger, W. and Pichler, L., Evidence for direct alphaadrenoceptor stimulation of effector neurons in cardiovascular centers by clonidine, Eur. J. Pharmacol., 27 (1974) 151-154. 22 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 23 Meeley, M.P., Ernsberger, P.R., Granata, A.R. and Reis, D.J., An endogenous clonidine-displacing substance from bovine brain: receptor binding and hypotensive actions in the ventrolateral medulla, Life Sci., 38 (1986) 1119-1126. 24 Michel, M.C., Brodde, O.-E., Schnepel, B., Behrendt, J., Tschada, R., Motulsky, H.J. and Insel, P.A., [3H]Idazoxan and some other a:-adrenergic drugs also bind with high affinity to a nonadrenergic site, Mol. Pharmacol., 35 (1989) 324-330. 25 Michel, T., Hoffman, B.B. and Lefkowitz, R.J., Differential regulation of the alpha-2 adrenergic receptor by Na ÷ and guanine nucleotides, Nature (Lond.), 288 (1980) 709-711. 26 Mooney, J.J., Home, W.C., Handin, R.I., Schildkraut, J.J. and Alexander, R.W., Sodium inhibits both adenylate cyclase and high-affinity 3H-labeled p-aminoclonidine binding to alpha 2adrenergic receptors in purified human platelet membranes, Mol. Pharmacol., 21 (1982) 600-608. 27 Palacios, J.M., Wamsley, J.K. and Kuhar, M.J., The distribution of histamine Hi-receptors in the rat brain: an autoradiographic study, Neuroscience, 6 (1981) 15-37. 28 Rouot, B.R. and Snyder, H., [3H]Para-amino-clonidine: a novel ligand which binds with high affinity to a-adrenergic receptors, Life Sci., 25 (1979) 769-774. 29 Scatchard, G., The attractions of proteins from small molecules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 660-672. 30 Schild, H.O., pA, a new scale for the measurement of drug antagonism, Br. J. Pharmacol., 2 (1947) 189-206. 31 Schwartz, J.-C., Arrang, J.-M. and Garbarg, M., Three classes of histamine receptors in brain, Trends Pharmacol. Sci., 7 (1986) 24-28. 32 Simmons, R.M.A. and Jones, D.J., Binding of [3H]prazosin and [3H]p-aminoclonidine to a-adrenoceptors in rat spinal cord, Brain Research, 445 (1988) 338-349. 33 Steinbusch, H.W.M. and Mulder, A.H., Immunohistochemical localization of histamine in neurons and mast cells in rat brain. In A. Bj6rklund, T. H6kfelt and M.J. Kuhar (Eds.), Handbook of Chemical Anatomy, Vol. 3, Elsevier, Amsterdam, 1984, pp. 126-140. 34 Subramanian, N. and Slotkin, T.A., Solubilization of a [3H]cimetidine binding site from rat brain: a clonidine-sensitive H-2 receptor subtype?, Mol. Pharmacol., 20 (1981) 240-243. 35 Taguchi, Y., Watanabe, T., Kubota, H., Hayashi, H. and Wada, H., Purification of histidine decarboxylase from the liver of fetal rats and its immunochemical and immunohistochemical characterization, J. Biol. Chem., 259 (1984) 5214-5221. 36 Tran, V.T., Chang, R.S.L. and Snyder, S.H., Histamine H lreceptors identified in mammalian brain with [3H]mepyramine, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 6290-6294. 37 Treherne, J.M. and Young, J.M., Digitonin-solubilized histamine Hi-receptors bind to polyethylenimine-treated glass-fibre filters, J. Pharm. Pharmacol., 40 (1988) 730-733.
21 38 Unnerstall, J.R., Kopajtic, T.A. and Kuhar, M.J., Distribution of a 2 agonist binding sites in rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacological effects of clonidine and related adrenergic agents, Brain Res. Rev., 7 (1984) 69-101. 39 Van der Werf, J.E, Bijloo, G.J., Van der Vleit, A., Bast, A. and Timmerman, H., n 3 receptor assay in electrically-stimulated superfused slices of rat brain cortex; effects of N-a-alkylated histamine and impromidine analogues, Agents Actions, 20 (1987) 239-243. 40 Watanabe, T., Taguchi, Y., Hayashi, H., Tanaka, J. Shiosaka,
S., Tohyama, M., Kubota, H., Terano, Y. and Wada, H., Evidence for the presence of a histaminergic neuron system in rat brain: an immunohistochemical analysis, Neurosci. Lett., 39 (1983) 249-254. 41 Watanabe, T., Taguchi, Y., Shiosaka, S., Tanaka, J., Kubota, H., Terano, Y., Tohyama, M. and Wada, H., Distribution of the histaminergic neuron system in the central nervous system of rats: a fluorescent immunohistochemical analysis with histidine decarboxylase as a marker, Brain Research, 295 (1984) 13-25. 42 Wilcox, B.J. and Seybold, V.S., Localization of neuronal histamine in rat brain, Neurosci. Leg., 29 (1982) 105-110.
15
BRES 15360
Binding of [3H]p-aminoclonidine to two sites, aE-adrenoceptors and imidazoline binding sites: distribution of imidazoline binding sites in rat brain Yoshinori Kamisaki, Tadashi Ishikawa, Yoshiki Takao, Hiroki Omodani, Nobutoshi Kuno and Tadao Itoh Department of Clinical Pharmacology, Tottori University School of Medicine, Yonago (Japan) (Accepted 12 September 1989) Key words: [3HlP-Aminoclonidine; Imidazoline site; Rat brain; Adrenoceptor; Striatum; Histamine receptor
Binding sites labeled by [3H]p-aminoclonidine ([3H]PAC) were investigated by the competitive analysis with imidazoline and non-imidazoline derivatives. Phenylethylamine derivatives displaced only the part of specific sites for [3H]PAC, which was considered as a2-adrenoceptor, whereas imidazoline derivatives, such as clonidine and tolazoline, competed for a further specific binding of [3H]PAC to the non-adrenergic sites, in addition to the a2-adrenoceptor. Because the non-adrenergic sites were specific for the imidazoline structure, they were termed imidazoline sites. The imidazoline sites were not distributed uniformly among rat brain regions. In striatum, hippocampus and medulla oblongata, they occupied 39.6, 33.0 and 36.5% of the specific binding of [3H]PAC, respectively. Saturation isotherms revealed that Kd and Bm~ of imidazoline sites for [3H]PAC were 3.09 + 0.59 nM, 27.4 + 1.7 fmol/mg protein and 2.23 + 0.29 nM, 21.0 + 1.5 fmol/mg protein in striatum and hippocampus, respectively. Because imidazoline binding sites also displayed weak affinities for imidazole compounds, such as histamine and cimetidine, the imidazoline site may be a subtype of histamine H2-receptor.
INTRODUCTION Clonidine, one of the imidazoline derivatives, has been used as an antihypertensive agent. Although the actions of clonidine are complex, its major actions are those of a centrally acting aE-adrenergic agonist. Clonidine impairs sympathetic nerve activity by binding to presynaptic aE-adrenoceptors. [3H]Clonidine and [3H]p-aminoclonidine have been utilized as ligands for aE-adrenoceptors El' 26,28,32,37
Recently, we have revealed that imidazoline derivatives (both a-agonists and antagonists) stimulate the acid secretion of parietal cells isolated from guinea pig stomach. These stimulatory effects were inhibited by HE-antagonists. The binding experiment of [3H]clonidine to the m e m b r a n e fraction suggested the existence of the imidazoline binding sites in parietal cells, which are different from H E- or aE-receptors 17. Since histamine receptors were divided into two subtypes, namely H 1 and HE, many agonists and antagonists have been discovered and investigated to clarify the relationships between their structures and pharma-
cological effects 2'4'19'30. In the case of histamine H 1receptor, the use of [3H]mepyramine allowed direct binding studies in brain and peripheral tissues 6'7,16,36. On the other hand, suitable ligands with high affinities have not been reported to label the HE-receptor. For example, [3H]cimetidine, a HE-antagonist, was used for the studies on the histamine HE-receptor in rat brain m e m b r a n e s 2°. However, the dissociation constant (Kd) of [3H]cimetidine was 400 nM by Scatchard analysis 2°. Although only [3H]tiotidine may meet the criteria for labeling the HE-receptor, the K d was 17 nM in guinea pig cerebral cortex m e m b r a n e 13. Recently, it was observed that histamine regulated its own synthesis and release by a negative feedback process through a new histamine H3-receptor in rat and human brain 1'15'18'31'39. In the present studies, imidazoline binding sites were examined in rat brain using the high-affinity ligand of [3H]PAC to clarify the relation with histamine receptor subtypes. In addition, regional studies were performed in order to determine whether the density of imidazoline site is related to a particular physiological role. The results demonstrate that imidazoline sites locate espe-
Correspondence: Y. Kamisaki, Department of Clinical Pharmacology, Tonori University School of Medicine, 86 Nishimachi, Yonago 683, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
16 cially in the s t r i a t u m
and h i p p o c a m p u s
i m i d a z o l i n e site m a y be a subtype
and that the
of h i s t a m i n e H 2-
receptor. MATERIALS AND METHODS
Materials p-[3,5-3H]Aminoclonidine ([3H]PAC) with a specific activity of 40-50 Ci/mmol was obtained from New England Nuclear. Clonidine HCI and p-aminoclonidine HCI were gifts from BoehringerIngelheim, Ltd. a-Methyl norepinephrine (aMNE) was from the Sterling-Winthrop Research Institute. Tolazoline HCI, naphazoline HCI, histamine diHC1, (-)-norepinephrine HCI, (-)-epinephrine bitartarate, and L-phenylephrine HCI were purchased from Sigma Chemical Co. Cimetidine was from Smith, Kline & French. All other chemicals were of the highest purity available.
Membrane preparations Adult male Wistar rats (240-270 g) were sacrificed by decapitation and brains were rapidly removed. The brains were separated on ice-cold stage into 10 regions (i.e. olfactory bulb, anterior and posterior cerebral cortex, striatum, hippocampus, thalamus + midbrain, hypothalamus, cerebellum, pons, medulla oblongata) essentially as described by Glowinski and Iversen TM. These sections were homogenized in 10 vols. (v/w) of ice-cold 50 mM Tris-HCl buffer (pH 7.5) using a Polytron PT-10 (Kinematica, setting 8, 10 s x 3). The resulting homogenate was centrifuged at 35,000 g (Hitachi 20PR-52) for 45 min at 4 °C. The pellet was washed in 10 vols. of fresh buffer and recentrifuged. These procedures were repeated 3 times. The final pellet was resuspended in 10 vols. of the Tris-HCl buffer. Protein concentrations in aliquots of the suspension were determined by the method of Lowry22.
Receptor binding assay
t5~A o - - O T
RESULTS
Time courses of association and dissociation of [3H]PAC binding A s s h o w n in Fig. 1, the b i n d i n g of [ 3 H ] P A C to brain m e m b r a n e s was s a t u r a b l e with time. [ 3 H ] P A C b i n d i n g to cortex
[3H]PAC binding to brain membranes was measured in 1.0 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 4.0 nM [3H]PAC. Incubation was started by the addition of tissue membrane aliquots containing 0.5-1.0 mg of protein and carried out for 30 min at 25 °C. After the incubation, the content of the assay tubes were immediately filtered over Whatman GF/C filters which had been treated
~:
with 1% polyethylenimine5'37 and rinsed 3 times with 4 ml of ice-cold Tris-HCl buffer 24"26'28'32. Radioactivities of filters were measured with 6 ml of ACS II (Amersham) on a Beckman liquid scintillation counter. Non-specific bindings were defined by parallel incubations containing 10-4 M aMNE and/or p-aminoclonidine (or clonidine). All assays were conducted in duplicate under these conditions. The bindings specific for a2-adrenoceptor and imidazoline sites were calculated from the binding amount of [3H]PAC to the membrane without the reagents minus that in the presence of aMNE, and the binding amount of [3H]PAC in the presence of aMNE minus that in the presence of clonidine, respectively. The non-specific binding, the binding amount of [3H]PAC in the presence of both clonidine (10 -4 M) and aMNE (10 -4 M) was the same as that in the presence of clonidine (10 -4 M) alone. Competition studies were carried out under standard incubation conditions with 10 9-10-3 M of several reagents. The K~ (inhibition constant) for each reagent was obtained from IC5oderived from the inhibition curves. Determination of the binding characteristics with membrane preparations was conducted with concentrations of [3H]PAC ranging from 0.5 to 16 nM. Specific bindings were calculated as described above. A computerized parameter estimation method program 9 was used to resolve the data from the saturation isotherms according to the method of Scatchard 29.
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within 20 min. T h e r e f o r e , 30 rain was c h o s e n as the s t a n d a r d i n c u b a t i o n t i m e for b i n d i n g studies using this radioligand. B i n d i n g was r e v e r s i b l e u p o n a d d i t i o n o f 10 -4 M of a M N E o r clonidine. T h e s e m i l o g a r i t h m i c transfor-
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Fig. 1. Time courses of association of [3H]p-aminoclonidine (PAC) binding to the membrane fractions from anterior cortex (A) or striatum (B). Membranes were incubated with 10-4 M clonidine (A), a-methyl norepinephrine (aMNE, O), or none of them (O) as described in Materials and Methods. At the time indicated by arrowheads, 10-4 M clonidine (A) or aMNE (O) was added to initiate radioligand dissociation. Each value plotted is the mean of duplicated determinations.
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Fig. 2. Competitions for [3H]PAC binding to the membrane from anterior cortex (A) or striatum (B) by phenylethylamines. Membranes were incubated with [3H]PAC (4 nM) and designated concentrations of clonidine (O), aMNE ( ~ ) , norepinephrine (&), epinephrine ( a ) , phenylephrine ( i ) as described in Materials and Methods.
17 mations of association and dissociation were linear, indicating that the equilibrium dissociation constant (Kd) for [3H]PAC binding to cortex and striatum membrane was in the nanomolar range. In the standard condition, specific bindings to cortex and striatum were ca. 60% of the total [3H]PAC binding. Although the non-specific bindings were the same in the anterior cortex (Fig. 1A), the difference was observed in striatum membrane between the non-specific binding defined in the presence of 10-4 M aMNE and that in the presence of 10-4 M clonidine (Fig. 1B).
Properties of [3H] PA C binding sites in cortex and striatum A variety of non- and imidazol(in)e derivatives were examined for their influence on specific [3H]PAC binding to rat cortex and striatum membranes, in order to investigate the discrepancy between the non-specific binding in the presence of aMNE and that in the presence of clonidine. In anterior cortex membrane, the competition curves of these drugs were monophasic, sigmoid and parallel (Figs. 2A and 3A). Imidazoline derivatives (PAC, naphazoline, and clonidine) except tolazoline, were more potent than naturally occurring phenylethylamines, a-adrenergic agonists (aMNE, norepinephrine, epinephrine, and phenylephrine). In striatum membrane, potencies of these compounds competing for the specific binding were similar to those in cortex membrane. However, all imidazoline derivatives, regardless of a-adrenergic agonists and antagonists, inhibited almost completely the specific binding of [3H]PAC (59% of the total binding), whereas phenylethylamines displaced only part of the specific sites (38%
of the total binding) (Figs. 2B and 3B). Moreover, at the high concentration of 10-3 M, imidazole compounds (histamine and cimetidine) also partly competed for the [3H]PAC binding.
Characteristics of aMNE-insensitive [3H]PAC binding sites In striatum, phenylethylamines displaced 60% of the specific binding sites for [3H]PAC, but failed to inhibit the remaining 40%, whereas imidazoline compounds displaced all of specific binding sites. These data suggested that [3H]PAC may interact with more than one class of binding sites in the striatum. In order to clarify properties of the remaining ctMNE-insensitive sites, further competition binding experiments were conducted in the presence of 10-4 M aMNE. Typical examples of such competition binding curves are displayed in Fig. 4. In striatum, the potency series for imidazoline derivatives were as follows: PAC > naphazoline > clonidine > tolazoline. The K i values of PAC, naphazoline, clonidine, and tolazoline were 2.31 nM, 5.24 nM, 23.4 nM, and 91.7 nM, respectively. In addition to imidazoline derivatives, imidazole compounds (histamine and cimetidine) also completely inhibited the [3H]PAC binding to aMNE-insensitive sites with K i of 7.42/~M and 6.11/~M, respectively. However, diphenhydramine, H 1 antagonist and non-imidazole compound, was inactive at 10 -3 M. The other phenylethylamines also showed little or no competition for the [3H]PAC binding to aMNE-insensitive sites at concentrations up to 10-3 M. Non-specific
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Fig. 4. Competitions for [3H]PAC binding to the imidazoline specific sites in striatum (A) and hippocampus (B) by imidazoi(in)e derivatives. Membranes were incubated with [3H]PAC, aMNE (10-4 M) and varying concentrations of PAC (O), clonidine (O), naphazoline (A), tolazoline (&), cimetidine ( I ) , or histamine (El) as described in Materials and Methods. Each value plotted is the mean of duplicate determinations and plotted figures are representative of 3 separate experiments.
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Fig. 5. Scatchard plots of [3H]PAC binding to membrane from anterior cortex (A), striatum (B) and hippocampus (C). Membranes were incubated with various concentrations of [3H]PAC (0.5-16 nM) and specific bindings for a2-adrenoceptor (0) or imidazoline sites (©) were determined in the presence of 10-4 M aMNE and/or clonidine as described in Materials and Methods. Plotted figures are representative of more than 3 experiments.
bindings (ca. 5100 dpm/mg protein of striatum membrane) which were obtained in the presence of both a M N E and imidazolines (Fig. 4A) were the same as those observed in the presence of imidazoline compounds alone (Figs. 2B and 3B). Therefore, it is suggested that the [3H]PAC binding sites m a y possess, at least, two subpopulations, n a m e l y a2-adrenoceptor and imidazoline sites. Similar results were obtained in aMNE-insensitive sites in h i p p o c a m p u s (Fig. 4B).
Regional distribution of imidazoline-specific binding sites in rat brain Assessment of distribution of a2-adrenoceptor and imidazoline sites was accomplished by dissecting the rat
TABLE I Regional distribution of a2-adrenoceptors and imidazoline sites in rat brain
Standard binding assays were conducted with membrane preparations from 10 brain regions. Specific bindings for az-adrenoceptor and imidazoline sites were calculated from specific activities of [3H]PAC as described in Materials and Methods. Values are expressed as means + S.E.M. from duplicate determinations of 3 separate experiments with percentages in parentheses to the total specific bindings. az-A drenoceptor (fmol/mg prot. )
lmidazoline site (fmol/mg prot. )
Olfactory bulb Anterior cortex
28.3 + 1.9 (79.7) 82.7 + 4.5 (100.7)
7.2 + 2.3 (20.3) - 0.6 + 5.1 (-0.7)
Posterior cortex Striatum Hippocampus Thalamus + midbrain Hypothalamus Cerebellum Pons Medulla oblongata
72.8 __+7.8 (94.3) 41.4 + 1.8 (60.4) 42.1 + 3.1 (67.0) 48.2 + 5.2 (86.5) 41.8 + 7.5 (72.9) 23.4 + 3.5 (79.1) 21.4 + 1.5 (85.9) 30.5 + 3.3 (63.5)
4.4 + 1.0 (5.7) 27.1 + 1.1 (39.6) 20.7 + 1.7 (33.0) 7.5 + 1.6 (13.5) 15.5 + 1.2 (27.1) 6.2 + 1.2 (20.9) 3.5 + 1.4 (14.1) 17.5 + 0.9 (36.5)
brain into 10 regions according to the m e t h o d of Glowinski and Iversen 14. Saturation analysis of [3H]PAC binding to a 2 - a d r e n o c e p t o r and imidazoline sites were p e r f o r m e d in anterior cortex, striatum and hippocampus m e m b r a n e . The bindings specific for a 2- and imidazoline sites were d e t e r m i n e d in the presence of 10 -4 M a M N E or clonidine as a function of increasing concentration of the radioligand (0.5-16 nM). A l t h o u g h both specific bindings were saturable, the non-specific binding, only defined by imidazoline derivatives, was linear in function. As one can see in Fig. 5, Scatchard analysis of the saturation isotherms showed that [3H]PAC binds to a2-adrenoceptor in anterior cortex, striatum, and hippocampus m e m b r a n e with dissociation constants (Kd) of 3.08 + 0.56, 2.69 + 1.29, and 2.76 + 1.00 nM and m a x i m u m n u m b e r of binding sites (Bmax) of 89.0 -- 5.6, 49.5 + 6.5, and 43.8 + 3.4 fmol/mg protein, respectively, and that it binds to imidazoline sites in striatum and hippocampus with K d of 3.09 + 0.58 and 2.23 + 0.29 nM and Bma x of 27.4 + 1.7 and 21.0 + 1.5 fmol/mg protein, respectively. Because the K o values for a 2 - a d r e n o c e p t o r and imidazoline sites were very similar, there was no i m p r o v e m e n t in goodness-of-fit using a m o d e l with two binding sites rather than a m o d e l with one binding site. We failed to investigate these binding sites by saturation experiments in the o t h e r regions, because [3H]PAC binding to imidazoline specific sites was too little in those regions. Thus, the binding results in the standard condition are listed in Table I. If the K d values for a 2a d r e n o c e p t o r and imidazoline sites are similar to each other in the other regions, the binding a m o u n t may reflect the ratio of those r e c e p t o r numbers. Especially in striatum, hippocampus and m e d u l l a oblongata, a M N E insensitive sites occupied m o r e than 30% of the total specific binding. Not only the n u m b e r of imidazoline sites but also the ratio of imidazoline sites to a2-sites were distributed differently a m o n g rat brain regions.
19 DISCUSSION The characteristics and distributions of a2-adrenoce ptots have been investigated using [3H]PAC as a radioligand25'26'28'38. In those studies phenylethylamines (norepinephrine, epinephrine and a-methyl norepinephrine) or imidazoline derivatives (phentolamine, clonidine and tolazoline) were utilized to calculate the non-specific bindings. Recently, it was suggested that [3H]idazoxan, an imidazoline derivative, may bind to non-adrenergic sites with high affinity as well as a2-adrenoceptors s'24. In the present study we have demonstrated that [3H]PAC binding sites consist of two subpopulations, one is aE-adrenoceptor and the other is imidazoline-specific binding site and that the imidazoline sites are not uniformly localized among rat brain regions. According to the density of imidazoline binding sites (Table I), we chose striatum (or hippocampus) and anterior cortex as rich and poor regions in imidazoline sites, respectively. In striatum, phenylethylamine derivatives (aMNE, norepinephrine, epinephrine and phenylephrine) only competed for part of the specific binding of [3H]PAC, although imidazoline derivatives (PAC, clonidine, naphazoline and tolazoline) completely displaced it (Figs. 1-3). However, in anterior cortex, non-specific bindings defined by phenylethylamines and imidazolines were the same. In the further competition experiments, non-adrenergic sites appeared to be specific for the imidazoline structure, because all imidazoline derivatives completely inhibited [3H]PAC binding with high affinities of Ki, 2-100 nM in striatum and hippocampus (Fig. 4). Saturation binding isotherms revealed that K d values (2-3 nM) of [3H]PAC binding to the non-adrenergic sites in striatum and hippocampus were similar to each other (Fig. 5). These K d values were also similar to those to aE-adrenergic sites (2-3 nM), although the reason is unknown. Therefore, in addition to the different distribution of non-adrenergic sites (Table I), these findings confirmed that [3H]PAC also binds to the non-adrenergic sites which are termed as imidazoline sites. It was reported that binding sites labeled by [3H]PAC were characterized in bovine brain membranes prepared from ventrolateral medulla 1°. Norepinephrine and other phenylethylamines displaced [3H]PAC from 70% of the total sites and the remaining 30% were non-adrenergic sites which were termed as imidazole binding sites. Moreover, an endogenous clonidine-like substance was isolated from bovine brain, which binds to the imidazole sites and lowers arterial pressure when it was microinjected into ventrolateral medulla3"11A2'23. Therefore, it was concluded that imidazole binding sites may mediate the hypotensive action of clonidine and participate in the function of an endogenous clonidine-like substance.
Although the total [3H]PAC binding sites were characterized precisely by competition experiments with various reagents including phenylethylamines, imidazolines, and imidazoles, however, characteristics of the imidazolespecific sites were not directly examined 1°. In our experiments, we characterized non-adrenergic parts of [3H]PAC binding sites after a2-adrenergic parts were blocked with aMNE. The imidazol(in)e binding sites may be similar to each other, according to the effects of various compounds on [3H]PAC binding (Fig. 3). However, we termed them imidazoline binding sites because imidazoline derivatives showed 100-fold higher affinities than imidazole compounds (Fig. 4). The imidazoline sites were displaced by imidazole compounds (histamine and cimetidine) with weak affinities (K i values of ca. 10 btM, Fig. 4). However, diphenhydramine, a H~ antagonist, did not inhibit [3H]PAC binding. It is reported that the binding sites for [3H]cimetidine, solubilized from rat brain, were displaced by micromolar concentrations of clonidine34. It was concluded that the [3H]cimetidine binding site is associated with the putative clonidine-sensitive H2-receptor subtype. Therefore, these data suggest that the imidazoline site may be a part or a subtype of the histamine H2-receptor. It was reported that the cell bodies of histaminergic neurons locate in the tuberomammillary nuclei of the posterior hypothalamus and that their nerve fibers spread ascendingly to hypothalamus, cerebral cortex, and olfactory bulb and descendingly to midbrain and pons with synaptic varicosities33,35'4°-42. The distribution of histaminergic nerve terminals was in general consistent with that of histamine Hi-receptor examined by [3H]mepyramine binding6'a6'27. However, several areas such as striatum and lateral hypothalamus were found to contain many histaminergic nerve fibers, but few [3H]mepyramine binding sites 41. Thus, it was predicted that these regions might be rich in non-H~ histamine receptors. On the other hand, by an autoradiographic study with [3H]PAC, it was demonstrated that regions which have a 2 binding sites are innervated by adrenergic neurons 37. However, it was also observed that the densities of a 2 binding sites are not exactly correlated with those of catecholamine innervation. In the present study, we demonstrated that the imidazoline sites may be a part of histamine receptors and that they are localized in striatum and hippocampus. Therefore, especially in striatum, the existence of the imidazoline sites may explain the controversial facts that the density of histamine nerve terminals is higher than that of histamine Hi-receptors , and that the density of adrenergic innervation is relatively lower than that of [3H]PAC binding sites. Similarly, in the other regions,
20 the i m i d a z o l i n e sites m a y also r e s o l v e parts of t h e s e d i s c r e p a n c i e s b e t w e e n distributions of the n e r v e terminals and t h o s e o f the r e c e p t o r sites, a l t h o u g h the f u n c t i o n of t h e i m i d a z o l i n e sites has not b e e n elucidated.
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