Introduction

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The histamine H₃ receptor was discovered in 1983 by Arrang and coworkers as a presynaptic autoreceptor regulating the release of histamine from histaminergic neurons (Arrang et al., 1983). Since then, the H₃ receptor has been shown to act as heteroreceptor as well, inhibiting the release of important neurotransmitters, e.g., acetylcholine, glutamate, noradrenaline, and serotonin (Leurs et al., 1998). With the availability of a variety of selective and potent H₃ agonists and antagonists (Leurs et al., 1995; Stark et al., 1996), it has become clear that the H₃ receptor is involved in the regulation of several important physiological processes. Consequently, the H₃ receptor is regarded as an interesting target for the modulation of a variety of functions such as cognitive processes, epilepsy, food intake, and sleep-wakefulness (Leurs et al., 1998).

Despite the interest in H₃ receptor ligands for therapeutic application, the actual therapeutic development has for a long time been hampered by the lack of information on the molecular target. Whereas the cloning of the H₁ and H₂ receptor genes was reported in the early 90s (Gantz et al., 1991; Yamashita et al., 1991), it was 1999 before the gene of the human H₃ receptor was cloned finally by Lovenberg et al. (1999) after the identification of a partial sequence of an orphan G-protein-coupled receptor (GPCR) in the Incyte expressed sequence tags database. The H₃ receptor was shown finally to be a GPCR with only limited homology (<30%) with the H₁ and H₂ receptor genes (Lovenberg et al., 1999).

Classical models of GPCRs require agonist occupation of receptors to activate signal transduction pathways. Yet, it is now well-documented that GPCRs can be spontaneously active, and this agonist-independent receptor activity is often referred to as constitutive receptor activity (Costa et al., 1992; Lefkowitz et al., 1993; Milligan et al., 1995). Inverse agonists reduce the constitutive GPCR activity, whereas neutral antagonists do not affect the basal GPCR activity but prevent the action of both agonists and inverse agonists. Constitutive activity has been shown recently for both the histamine H₁ and H₂ receptors (Smit et al., 1996; Bakker et al., 2000), and we reported that the therapeutically important H₁ and H₂ antagonists, in fact, act as inverse agonists. In the present study, we describe that the human and rat histamine H₃ receptors stably expressed in SK-N-MC cells (Lovenberg et al., 1999, 2000) show a high level of constitutive activity, resulting in the identification of several standard H₃ antagonists (thioperamide and clobenpropit) as inverse agonists in this cell system. Moreover, burimamide and impentamine, previously identified as H₃ antagonists (Arrang et al., 1983; Vollinga et al., 1995a, 1995b), behave as H₃ agonists at the recombinant H₃ receptors. The agonistic effects of impentamine could also be demonstrated on the hypothalamic histamine release in the rat brain using in vivo microdialysis. Moreover, in a series of impentamine analogs we were able to manipulate the intrinsic activity. VUF4904, an impentamine analog with an isopropyl group at the amino group of the side chain, bound with a relatively high affinity (12 nM) and acted as a neutral antagonist in the transfected SK-N-MC cells. These data indicate that ligands, previously identified as H₃ antagonists, can cover the whole spectrum of pharmacological activities, ranging from full inverse agonism to agonism, at the recombinant H₃ receptors heterologously expressed in SK-N-MC cells.

	Experimental Procedures

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Materials. (R)--Methylhistamine dihydrobromide was obtained from Sigma Research Biochemicals Inc. (Zwijndrecht, The Netherlands). Burimamide was a kind gift of GlaxoSmithKline (Welwyn Garden City, Hertfordshire, UK). All other H₃ ligands were taken from laboratory stock or (re-)synthesized at the Vrije Universiteit Amsterdam (details will be published elsewhere). Forskolin, 3-isobutyl-1-methylxanthine, cyclic 3',5'-adenosine monophosphate (cAMP), pertussis toxin, and bovine serum albumin were obtained from Sigma. Dulbeccos's modified Eagle's medium, trypsin-EDTA, penicillin, nonessential amino acids, L-glutamine, streptomycin, and sodium-pyruvate were from Invitrogen (Breda, The Netherlands). Eagle's minimal essential medium was from BioWhittaker (Verviers, Belgium), and fetal calf serum was from Integro (Zaandam, The Netherlands). Culture dishes and 24-well plates were from Costar (Haarlemermeer, The Netherlands). G418 was obtained from Calbiochem (Amsterdam, The Netherlands). [³H]cyclic 3',5'-adenosine monophosphate ([³H]cAMP), 40 Ci/mmol, was from Amersham (s'Hertogenbosch, The Netherlands); [³H]N-methylhistamine, 85 Ci/mmol, was from PerkinElmer Life Sciences (Zaventem, Belgium).

Cell Culture. SK-N-MC cells, a human neuroblastoma cell line stably expressing the human histamine H₃ receptor (the 445-amino acid isoform) or the rat histamine H_3A receptor (Lovenberg et al., 1999, 2000), were grown in 10-cm² dishes at 37°C in a humidified atmosphere with 5% CO₂ in Eagle's minimal essential medium, supplemented with 10% v/v fetal calf serum, 50 IU/ml penicillin, nonessential amino acids, 2 mM L-glutamine, 50 µg/ml streptomycin, and 50 µg/ml sodium-pyruvate in presence of 600 µg/ml G418. Cells were detached from the dishes with 0.05% trypsin-EDTA.

[³H]N-Methylhistamine Binding. Confluent 10-cm dishes of SK-N-MC cells stably expressing the rat or human histamine H₃ receptor were harvested using a cell scraper and centrifuged (3 min, 500g), and the pellets were stored at 20°C until the day of the experiment. Before use the pellets were dissolved in distilled water and homogenized for 2 s by sonication (40 Watt, Labsonic 1510). The cell homogenates (30-100 µg) were incubated for 40 min at 25°C with 1 nM [³H]N-methylhistamine (85.0 Ci/mmol) in 50 mM sodium phosphate buffer, pH 7.4, with or without competing ligands. The reaction was terminated by rapid dilution with 3 ml of ice-cold buffer, pH 7.4, and filtration over 0.3% polyethylenimine-pretreated Whatmann GF/C filters with two subsequent washes with 3 ml of buffer. Retained radioactivity was determined by liquid scintillation counting. Nonspecific binding was defined with 100 µM thioperamide as competing ligand.

Measurement of cAMP. SK-N-MC cells stably expressing either the rat or human H₃ receptor were grown overnight in 24-well plates (4.5 × 10⁶ cells/plate), washed once with Dulbeccos's modified Eagle's medium/HEPES (25 mM, pH 7.4 at 37°C), and preincubated in the same medium for 30 min at 37°C. Thereafter the cells were incubated for exactly 10 min with fresh medium supplemented with 0.3 mM 3-isobutyl-1-methylxanthine, 10 µM forskolin, and the respective ligands. To stop the incubation the medium was discarded, 200 µl of ice-cold HCl (0.1 M) was added, and the samples were homogenized for 2 s (40 Watt, Labsonic 1510) and frozen at 20°C.

In Vivo Microdialysis. Male Wistar rats weighing about 250 g were anesthetized with urethane (1.2 g/kg, i.p.) and placed in a stereotaxic apparatus. A dialysis probe (CMA/10; membrane length, 2 mm; CMA/Microdialysis AB, Stockholm, Sweden) was inserted into the anterior hypothalamic area with coordinates of AP, 1.5; L, 0.5; and V, 9.2 mm relative to the bregma, according to the atlas of Paxinos and Watson (1986). The anterior hypothalamic area was perfused with artificial cerebrospinal fluid containing 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, and 5 mM glucose, pH 7.4, through a dialysis probe at 1 µl/min using a microinfusion pump (CMA100, CMA/Microdialysis AB). Two hours after the insertion of a probe, samples were collected every 20 min with a minifraction collector (CMA140, CMA/Microdialysis AB) and frozen immediately at 40°C until analysis. Impentamine and clobenpropit were added to cerebrospinal fluid at the concentration of 10 µM and administered through the dialysis membrane. After the experiment, the brains were removed for histological verification of sites of infusion.

Data Analysis. For the binding studies pIC₅₀ (negative logarithm of the ligand concentration that displaces the radioligand half-maximally) and pK_d values (negative logarithm of the equilibrium dissociation constant of the radioligand, i.e., the concentration, that occupies 50% of the available receptors at equilibrium) were calculated using nonlinear regression analysis using GraphPad Prism (GraphPad Software, San Diego, CA) and converted to pK_i values (negative logarithm of the equilibrium dissociation constant for binding of the unlabeled drug) using the Cheng-Prusoff equation (Cheng and Prusoff, 1973). From the cAMP data pEC₅₀ (negative logarithm of the ligand concentration, that activates the receptor half-maximally) and pIC₅₀ values (negative logarithm of the ligand concentration, that inhibits the receptor half-maximally) were obtained by fitting these data to a sigmoidal relationship using GraphPad Prism. The intrinsic activities were calculated in comparison with the effects of the full agonist (R)--methylhistamine (1 µM) or the full inverse agonist iodophenpropit (10 µM).

	Results

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The generation of the SK-N-MC cells stably expressing either the rat or human histamine H₃ receptor was described previously (Lovenberg et al., 1999, 2000). In the present study, we used SK-N-MC cell lines, expressing 516 ± 23 fmol/mg of protein (n = 3) of the human histamine H₃ receptor or 627 ± 87 fmol/mg of protein (n = 3) of the rat histamine H₃ receptor, as assessed by [³H]N-methylhistamine binding.

As described previously, the H₃ agonists (R)--methylhistamine (pEC₅₀ = 9.26 ± 0.08) and imetit (pEC₅₀ = 9.28 ± 0.04) potently inhibited the 10 µM forskolin-stimulated production of cAMP in human H₃ receptor expressing cells (Fig. 1A; Table 1). In contrast, (R)--methylhistamine had no effect in the parental SK-N-MC cell line (Fig. 1A). As expected for a G_i-coupled receptor, the 1 µM (R)--methylhistamine effects at the human H₃ receptor (reduction to 11 ± 3% of the forskolin-induced cAMP levels) were abolished completely by an overnight pretreatment with 100 ng/ml pertussis toxin (99 ± 10% of forskolin-induced cAMP level, not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Modulation of 10 µM forskolin induced cAMP production in SK-N-MC cells, expressing the human H3 receptor. A, effects of the H3 agonist (R)--methylhistamine on the forskolin response in SK-N-MC cells or SK-N-MC cells expressing the human H3 receptor. B, effects of the H3 inverse agonists clobenpropit and iodophenpropit on the forskolin response in SK-N-MC cells expressing the human H3 receptor. For comparison, the effects of clobenpropit on nontransfected SK-N-MC cells is shown. Cells were incubated for 10 min with the indicated ligands. After termination of the incubation the cAMP levels were determined by a competitive binding assay.

View this table: [in this window] [in a new window]   TABLE 1 Affinities and functional activities of several H3 receptor ligands at the human H3 receptor H3 receptor affinity (pKi) was determined by [3H]-N-methylhistamine binding to membranes of SK-N-MC cells expressing the human H3 receptor. For agonists the pEC50 values were determined by the inhibition of the forskolin-stimulated (10 µM) cAMP production, whereas the inverse agonistic activity was determined by the increase of the forskolin response in SK-N-MC cells, expressing the human H3 receptor. All data shown are the mean ± S.E. of at least three experiments. * indicates a significant difference compared with (R)--methylhistamine. ** indicates a significant difference compared with iodophenpropit.

Interestingly, we noticed an important difference between the forskolin-induced cAMP levels of the parental SK-N-MC cell line and the H₃ receptor expressing cells (Fig. 1B). As this could be an indication of constitutive H₃ receptor activation, we tested a variety of previously identified H₃ antagonists on the SK-N-MC cell line expressing the human H₃ receptor. Standard H₃ antagonists as thioperamide, clobenpropit, and iodophenpropit concentration-dependently increased the forskolin-induced cAMP levels in the transfected SK-N-MC cells (Fig. 1B; Table 1), whereas clobenpropit had no effect on the forskolin response in the parental cell line (Fig. 1B). In our experiments, iodophenpropit and clobenpropit acted as full inverse agonists ( = 1.0-0.9), whereas thioperamide acted as a partial inverse agonist (Table 1). The obtained pEC₅₀ values for the various inverse agonists correspond well with the respective affinities, as obtained in [³H]N-methylhistamine competition experiments (Table 1).

In search of neutral antagonists, we tested a variety of other H₃ antagonists. Surprisingly, the presumed H₃ antagonists burimamide, impentamine, and the imetit homolog VUF 8328 (Van der Goot et al., 1992) all acted as potent H₃ agonists at the human H₃ receptor with intrinsic activities between 0.8 and 0.9 (Fig. 2; Table 1). Comparing the pK_i values of various agonists with their pEC₅₀ values revealed that in general the potencies of the agonists nicely parallel their affinities (Table 1). Only for the full agonists (R)--methylhistamine and perhaps imetit may some sort of receptor reserve be noticed (Table 1).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Agonistic activity of (R)--methylhistamine (RAMH) and burimamide in SK-N-MC cells expressing the human H3 receptor. SK-N-MC cells expressing the human H3 receptor were stimulated with 10 µM forskolin in the presence of increasing concentrations of the H3 ligands for 10 min. After termination of the incubation the cAMP levels were determined by a competitive binding assay.

Based on the identification of agonism of impentamine, we studied several impentamine analogs to identify a neutral H₃ receptor antagonist. In this series of H₃ ligands, the amine function of impentamine was substituted or incorporated in a piperidine ring. Modification of the amine group results in a series of compounds with a wide spectrum of pharmacological activity, including the neutral antagonist VUF4904 (Fig. 3; Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Modulation of 10 µM forskolin induced cAMP production in SK-N-MC cells expressing the human H3 receptor by a variety of impentamine analogs. SK-N-MC cells expressing the human H3 receptor were stimulated with 10 µM forskolin in the presence of increasing concentrations of the H3 ligands for 10 min. For comparison the effects of clobenpropit and (R)--methylhistamine are shown as well. After termination of the incubation the cAMP levels were determined by a competitive binding assay.

View this table: [in this window] [in a new window]   TABLE 2 Affinities and functional activities of several impentamine analogs at the human H3 receptor H3 receptor affinity (pKi) was determined by [3H]-N-methylhistamine binding to membranes of SK-N-MC cells expressing the human H3 receptor. For agonists the pEC50 values were determined by the inhibition of the forskolin-stimulated (10 µM) cAMP production, whereas the inverse agonistic activity was determined by the increase of the forskolin response in SK-N-MC cells, expressing the human H3 receptor. All data shown are the mean ± S.E. of at least three experiments.

Constitutive activity is not restricted to the human H₃ receptor. Whereas the rat and human H₃ receptor were expressed at similar levels (627 fmol/mg of protein versus 516 fmol/mg of protein), the forskolin-induced cAMP levels were always lower in the SK-N-MC cells expressing the human H₃ receptor (Fig. 4). These data indicate that in our experimental model the level of constitutive activity of the human H₃ receptor is more pronounced than that of the rat H₃ receptor. At the recombinant rat receptor, the H₃ ligands burimamide and impentamine also behave as H₃ agonists. In contrast to the human H₃ receptor, both ligands behave as full agonists at the rat H₃ receptor (Table 3). The constitutive activity, displayed by the rat H₃ receptor, can also be inhibited by compounds such as clobenpropit (Fig. 4; Table 3). Especially for the inverse agonists thioperamide and iodophenpropit, we confirmed the reported species differences (Lovenberg et al., 1999, 2000) with respect to their potencies in both receptor binding and functional assays (Table 3).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Modulation of 10 µM forskolin induced cAMP production in SK-N-MC cells expressing the human or rat H3 receptor, by the inverse agonists clobenpropit or iodophenpropit. SK-N-MC cells were stimulated with 10 µM forskolin in the presence of increasing concentrations of H3 ligand for 10 min. After termination of the incubation, the cAMP levels were determined by a competitive binding assay.

View this table: [in this window] [in a new window]   TABLE 3 Affinities and functional activities of several H3 receptor ligands at the rat and human H3 receptor H3 receptor affinity (pKi) was determined by [3H]-N-methylhistamine binding to membranes of SK-N-MC cells expressing the human or rat H3 receptor. For agonists the pEC50 values were determined by the inhibition of the forskolin-stimulated (10 µM) cAMP production, whereas the inverse agonistic activity was determined by the increase of the forskolin response in SK-N-MC cells, expressing the H3 receptor. All data shown are the mean ± S.E. of at least three experiments.

To investigate the predictive value of the data obtained with the recombinant receptors, impentamine and clobenpropit were tested in vivo. Previously, we showed by microdialysis the H₃ receptor-mediated effect on in vivo histamine release in the rat hypothalamus (Jansen et al., 1998). Using the same experimental set-up, we first evaluated the effects of impentamine. The mean values ± S.E.M. of the basal histamine release in the experiments in Fig. 5 were 0.078 ± 0.008 (n = 4) pmol/20 min. This value remained constant throughout the experimental period of 5 h under anesthesia (data not shown). After infusion of impentamine the histamine levels in the hypothalamus rapidly decreased to approximately 40% of the basal levels. Concomitant infusion of clobenpropit reversed the effect of impentamine and even caused an increase (±40%) in the histamine release above basal levels (Fig. 5).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of impentamine and clobenpropit on the in vivo histamine release in the rat hypothalamus as measured by microdialysis. Drugs (10 µM) were infused in the hypothalamus via the microdialysis probe. Fractions were collected every 20 min and the amount of histamine was determined by HPLC, as described under Experimental Procedures. *, a significant (P < 0.05) difference compared with basal levels of histamine release.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The recent cloning of the rat and human H₃ receptor cDNAs by Lovenberg et al. (1999, 2000) has had a great impact in the field of histamine research. The new information has been instrumental in identifying H₃ receptor isoforms (Drutel et al., 2001) and the H₄ receptor (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001) and has also been essential in deriving important information about the signaling properties of the H₃ receptor. Whereas for many years the actual signaling pathways for the H₃ receptor had been unknown, the use of cell lines expressing the H₃ receptor has led to the identification of at least three signal transduction pathways for the H₃ receptor: a G_i-mediated inhibition of adenylate cyclase (Lovenberg et al., 1999, 2000; Drutel et al., 2001), the activation of the MAP kinase pathway (Drutel et al., 2001), and the stimulation of Na⁺/H⁺ exchange (Silver et al., 2001).

Using SK-N-MC cell lines, stably expressing either the human and rat H₃ receptors at physiological receptor densities (500-600 fmol/mg of protein) (Yanai et al., 1994; Brown et al., 1996), we now show that both the rat and human H₃ receptor display a high degree of constitutive activity. The forskolin-mediated cAMP production in SK-N-MC cells is inhibited strongly upon expression of the G_i-coupled H₃ receptor. The cAMP production can be further inhibited upon agonist stimulation of the H₃ receptor and can be enhanced by a variety of H₃ antagonists acting as inverse agonists at the H₃ receptor. Thioperamide, clobenpropit, and iodophenpropit raise the cAMP levels in SK-N-MC cells expressing either the human or rat H₃ receptor with potencies that match their receptor binding affinities. As reported previously (Lovenberg et al., 1999, 2000), an important species difference is noticed for thioperamide in both the binding and cAMP assay.

Burimamide was one of the key compounds used by Arrang et al. (1983) to demonstrate pharmacologically the existence of the H₃ receptor in rat cerebral cortex slices. Remarkably, at recombinant H₃ receptors, the presumed H₃ antagonist burimamide acts as a H₃ agonist. Whereas at the human H₃ receptor burimamide acts as a partial agonist ( = 0.8), full agonism is observed at the rat receptor. It is interesting to note that via the use of heterologous expression systems, burimamide is reclassified for the second time. Previously, we showed that at the human H₂ receptor burimamide acts as a weak partial agonist (Alewijnse et al., 1998). Because burimamide was developed originally as an H₂ antagonist using histamine as a starting point (Black et al., 1972), the discovery of residual agonistic activity at histamine H₂ and H₃ receptors is perhaps not too surprising.

The use of transfected cell lines also suggests a reclassification for impentamine, the histamine homolog previously suggested to differentiate between H₃ receptors in the guinea pig intestine and rat or guinea pig brain (Leurs et al., 1996; Harper et al., 1999). Impentamine is a potent H₃ antagonist in the guinea pig intestine (pA₂ = 8.4), but a partial agonist in the rat brain (pD₂ = 8.2,  = 0.6) (Leurs et al., 1996). Moreover, radioligand binding studies at the H₃ receptor in the guinea pig brain and intestine indicated that impentamine can discriminate between the receptors in the two preparations (Harper et al., 1999). At both the recombinant rat and human H₃ receptors, impentamine behaves as an effective agonist. Moreover, in vivo microdialysis shows that impentamine also acts as an H₃ agonist in the rat hypothalamus, inhibiting the basal release of histamine. Previously we showed that potent H₃ agonists, like immepip (Jansen et al., 1998), inhibit the hypothalamic histamine release to approximately the same extent as observed in this study for impentamine.

Although constitutive GPCR activity is now a widely accepted pharmacological concept, effects due to the presence of the natural agonist cannot be ignored completely. The identification of neutral antagonists has resolved this issue for the histamine H₂ receptor and led to the recognition that the therapeutically important H₂ antagonists are in fact inverse agonists (Smit et al., 1996). To identify a neutral H₃ antagonist we tested a variety of impentamine analogs at the human H₃ receptor. The amine function of impentamine probably interacts with the aspartate residue Asp¹¹⁴ in transmembrane domain 3, which is highly conserved in the family of biogenic amines (De Esch et al., 2000). We hypothesized that modification of the amine function potentially could affect the agonistic properties of impentamine. Indeed, modification of the amine group dramatically affected the pharmacological activity of the ligand. Receptor affinity was reduced slightly for most analogs, unless a p-chloro-benzyl group was used (VUF5205, pK_i = 8.63). Remarkably, introduction of small alkyl groups resulted in reduced agonistic activity (di-methyl substitution, VUF5207,  = 0.7) or neutral antagonism (isopropyl substitution, VUF4904). Substitution of the amine group with a cyclohexyl ring or a p-chlorobenzyl group resulted in (partial) inverse agonists. Our data show that only subtle changes at the amine function alter the pharmacological activity of the ligands. At present, we do not have an explanation for this phenomenon, but this series of ligands may be of great help to understand the mechanism of receptor (in)activation. Detailed studies with receptor mutants, the development of similar, rigid analogs and the generation of a three-dimensional computer model to rationalize receptor-ligand interaction may also be useful in this respect.

In conclusion, in this study we show that both the rat and human H₃ receptors show a considerable level of constitutive activity when expressed at physiological expression levels in SK-N-MC cells. This observation has important consequences for the classification of H₃ receptor ligands, which can now be classified as inverse agonists, neutral antagonists, and agonists.

Constitutive activity of the rat H₃ receptor was also reported very recently by Morisset et al. (2000). Interestingly, the constitutive activity of the rat H₃ receptor was suggested to regulate brain histamine release in both rat and mouse (Morisset et al., 2000). The H₃ receptor is, therefore, one of the few GPCRs for which it is known that they modulate important physiological processes by means of its constitutive activity. In light of the foreseen therapeutic application of H₃ antagonists (Leurs et al., 1998), it remains to be established whether inverse agonists or neutral antagonists will be favored for clinical application.