The histamine autoreceptor is a short isoform of the H3 receptor.

BACKGROUND AND PURPOSE The histamine H(3) receptor was identified as the autoreceptor of brain histaminergic neurons. After its cloning, functional H(3) receptor isoforms generated by a deletion in the third intracellular loop were found in the brain. Here, we determined if this autoreceptor was the long or the short isoform. EXPERIMENTAL APPROACH We hypothesized that the deletion would affect H(3) receptor stereoselectivity. The effects of the enantiomers of two chiral ligands, N(α)-methyl-α-chloromethylhistamine (N(α) Me-αClMeHA) and sopromidine, were investigated on cAMP formation at the H(3(445)) and H(3(413)) receptor isoforms, common to all species. They were further compared with their effects at autoreceptors. They were also compared on [(35)S]GTPγ[S] binding to membranes of rat cerebral cortex, striatum and hypothalamus, the richest area in autoreceptors. KEY RESULTS The stereoselectivity of N(α) Me-αClMeHA enantiomers as agonists was similar at the H(3(413)) receptor isoform and autoreceptors, but lower at the long isoform. While (S) sopromidine did not discriminate between the isoforms, (R) sopromidine was an antagonist at the H(3(413)) receptor isoform and autoreceptors, but a full agonist at the long isoform. In rat brain, stereoselectivity of N(α) Me-αClMeHA was higher in the hypothalamus than in cerebral cortex or striatum, whereas the opposite pattern was found for sopromidine. CONCLUSIONS AND IMPLICATIONS The pharmacological profiles of H(3) receptor isoforms differed markedly, showing that the function of autoreceptors was fulfilled by a short isoform, such as the H(3(413)) receptor. Development of drugs selectively targeting autoreceptors might enhance their therapeutic efficacy and/or decrease incidence of side effects.

Pharmacological characterization of six trkB antibodies reveals a novel class of functional agents for the study of the BDNF receptor.

BACKGROUND AND PURPOSE: By interacting with trkB receptors, brain-derived neurotrophic factor (BDNF) triggers various signalling pathways responsible for neurone survival, differentiation and modulation of synaptic transmission. Numerous reports have implicated BDNF and trkB in the pathogenesis of various central nervous system affections and in cancer, thus representing trkB as a promising therapeutic target. In this study, we used an antibody-based approach to search for trkB-selective functional reagents. EXPERIMENTAL APPROACH: Six commercially available polyclonal and monoclonal antibodies were tested on recombinant and native, human and rodent trkB receptors. Functional and pharmacological characterization was performed using a modified version of the KIRA-elisa method and radioligand binding studies. Western blot analyses and neurite outgrowth assays were carried out to determine the specificity and selectivity of antibody effects. The survival properties of one antibody were further assessed on cultured neurones in a serum-deprived paradigm. KEY RESULTS: The functional trkB-selective antibodies showed distinct pharmacological profiles, ranging from partial agonists to antagonists, acting on trkB receptors through allosteric modulations. The same diversity of effects was observed on the mitogen-activated protein kinase signalling pathway downstream of trkB and on the subsequent neurite outgrowth. One antibody with partial agonist activity demonstrated cell survival properties by activating the Akt pathway. Finally, these antibodies were functionally validated as true trkB-selective ligands because they failed activating trkA or trkC, and contrary to BDNF, none of them bind to p75(NTR). CONCLUSIONS AND IMPLICATIONS: These trkB-selective antibodies represent a novel class of pharmacological tools to explore the pathophysiological roles of trkB and its potential therapeutic relevance for the treatment of various disorders.

Activation of brain histaminergic neurotransmission: a mechanism for cognitive effects of memantine in Alzheimer's disease.

We previously reported that some N-methyl-D-aspartate (NMDA)-receptor antagonists enhanced histamine neuron activity in rodents. Here, we have investigated the effects of memantine, an NMDA-receptor antagonist used for the treatment of Alzheimer's disease, on histaminergic neurotransmission. In vitro, memantine antagonized native NMDA receptors with a micromolar potency but had no effect at recombinant human histamine receptors. In vivo, a single administration of memantine increased histamine neuron activity, as shown by the 60% increase of tele-methylhistamine (t-MeHA) levels observed in the brain of mice. This increase occurred with an ED(50) of 0.3 ± 0.1 mg/kg, similar to that found on inhibition of ex vivo [(3)H]dizocilpine maleate (MK-801) binding (1.8 ± 1.3 mg/kg). Two days after pretreatment of mice with memantine at 5 mg/kg twice daily for 5 days, t-MeHA levels were enhanced by 50 ± 7% (p < 0.001), indicating a long-lasting activation of histamine neurons. Quantitative polymerase chain reaction analysis was used to explore genes involved in this persistent effect. H(3) receptor mRNAs were strongly increased, but the density of H(3) receptor binding sites was increased solely in hypothalamus (by 141 ± 24%). Up-regulations of brain-derived neurotrophic factor and NMDA-receptor 1 subunit mRNAs were also found but were restricted to hippocampus. mRNA expression of α7-nicotinic receptors remained unchanged in any region. Considering the well established cognitive effects of histamine neurons, the increase in brain t-MeHA levels after single or repeated administration of therapeutic doses of memantine suggests that the drug exerts its beneficial effects on cognitive deficits of Alzheimer's disease, at least partly, by activating histamine neurons.

Effects of betahistine at histamine H3 receptors: mixed inverse agonism/agonism in vitro and partial inverse agonism in vivo.

We previously suggested that therapeutic effects of betahistine in vestibular disorders result from its antagonist properties at histamine H(3) receptors (H(3)Rs). However, H(3)Rs exhibit constitutive activity, and most H(3)R antagonists act as inverse agonists. Here, we have investigated the effects of betahistine at recombinant H(3)R isoforms. On inhibition of cAMP formation and [(3)H]arachidonic acid release, betahistine behaved as a nanomolar inverse agonist and a micromolar agonist. Both effects were suppressed by pertussis toxin, were found at all isoforms tested, and were not detected in mock cells, confirming interactions at H(3)Rs. The inverse agonist potency of betahistine and its affinity on [(125)I]iodoproxyfan binding were similar in rat and human. We then investigated the effects of betahistine on histamine neuron activity by measuring tele-methylhistamine (t-MeHA) levels in the brains of mice. Its acute intraperitoneal administration increased t-MeHA levels with an ED(50) of 0.4 mg/kg, indicating inverse agonism. At higher doses, t-MeHA levels gradually returned to basal levels, a profile probably resulting from agonism. After acute oral administration, betahistine increased t-MeHA levels with an ED(50) of 2 mg/kg, a rightward shift probably caused by almost complete first-pass metabolism. In each case, the maximal effect of betahistine was lower than that of ciproxifan, indicating partial inverse agonism. After an oral 8-day treatment, the only effective dose of betahistine was 30 mg/kg, indicating that a tolerance had developed. These data strongly suggest that therapeutic effects of betahistine result from an enhancement of histamine neuron activity induced by inverse agonism at H(3) autoreceptors.

Histamine potentiates N-methyl-D-aspartate receptors by interacting with an allosteric site distinct from the polyamine binding site.

Histamine potentiates activation of native and recombinant N-methyl-d-aspartate receptors (NMDARs), but its mechanisms of action and physiological functions in the brain remain controversial. Using four different models, we have further investigated the histamine-induced potentiation of various NMDAR-mediated responses. In single cultured hippocampal neurons, histamine potentiated NMDA currents. It also potentiated the NMDA-induced increase in intracellular calcium in the absence, as well as with saturating concentrations, of exogenous d-serine, indicating both glycine-dependent and glycine-independent components of its effect. In rat hippocampal synaptosomes, histamine strongly potentiated NMDA-induced [(3)H]noradrenaline release. The profile of this response contained several signatures of the histamine-mediated effect at neuronal or recombinant NMDARs. It was NR2B-selective, being sensitive to micromolar concentrations of ifenprodil. It was reproduced by tele-methylhistamine, the metabolite of histamine in brain, and it was antagonized by impromidine, an antagonist/inverse agonist of histamine on NMDA currents. Up to now, histamine was generally considered to interact with the polyamine site of the NMDAR. However, spermine did not enhance NMDA-induced [(3)H]noradrenaline release from synaptosomes, and the potentiation of the same response by tele-methylhistamine was not antagonized by the polyamine antagonist arcaine. In hippocampal membranes, like spermine, tele-methylhistamine enhanced [(3)H]dl-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) binding to the glutamate site. In contrast, spermine increased nonequilibrium [(3)H]5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) binding, and suppressed [(3)H]ifenprodil binding, whereas histamine and tele-methylhistamine had no effect. In conclusion, the histamine-induced potentiation of NMDARs occurs in the brain under normal conditions. Histamine does not bind to the polyamine site, but to a distinct entity, the so-called histamine site of the NMDAR.

Autoregulation of McA-RH7777 hepatoma cell proliferation by histamine H3 receptors.

Previous studies have suggested that histamine (HA) acts as an autocrine growth factor. We have explored the modulation of cell proliferation by HA using McA-RH7777 hepatoma cells. High L-histidine decarboxylase (HDC) expression and HA synthesis were found in McA-RH7777 cells. Whereas extracellular HA reached submicromolar concentrations, intracellular levels were very low, indicating that HA was secreted by the cells. McA-RH7777 cells also express H3-receptor (H3R) transcripts and proteins. Reverse transcriptase-polymerase chain reaction analysis detected only transcripts for the long isoform. Immunocytochemistry performed with a selective H3R antibody showed that most cells were immunoreactive. H3R binding sites (Bmax approximately 30 fmol/mg protein) were identified when [125I] iodoproxyfan binding was displaced by the agonist imetit. High-affinity binding also occurred at cytochrome P450 enzymes. This binding was not inhibited by HA, H3R agonists, or by a nonimidazole H3R antagonist but was displaced by imidazole H3R antagonists or by ketoconazole, a imidazole-containing cytochrome inhibitor. HA inhibited proliferation of McA-RH7777 hepatoma cells. The absence of uptake system, its much higher potency at H3Rs, and its low intracellular levels suggested that HA interacted with H3Rs rather than cytochromes. In agreement, both imidazole H3R antagonists, a nonimidazole H3R antagonist, and the HDC inhibitor alpha-monofluoromethyl histidine increased cell proliferation (up to approximately 60%), revealing a H3R-mediated inhibition by endogenous HA. Moreover, exogenous HA inhibited the increase induced by alpha-FMH or H3R antagonists with a nanomolar potency. In conclusion, our findings show that HA regulates proliferation of McA-RH7777 hepatoma cells by interacting with autoinhibitory H3Rs.

Stroke induces histamine accumulation and mast cell degranulation in the neonatal rat brain.

Inflammatory processes are a major cause of hypoxic-ischemic brain damage. The present study focuses on both the cerebral histamine system and mast cells in a model of transient focal ischemia induced by permanent left middle cerebral artery, and homolateral transient common carotid artery occlusion (50 minutes) in the P7 newborn rat. Immunohistochemical analysis revealed that ischemia induces histamine (HA) accumulation in the core of the infarct 6-12 h post-ischemia, and in the penumbra at 24-48 h, although in situ hybridization failed to detect any histidine decarboxylase gene transcripts in these regions. Immunohistochemical co-localization of HA with the MAP2 marker revealed that HA accumulates in neuronal cells before they degenerate, and is accompanied by a very significant increase in the number of mast cells at 12 h and 48 h of reperfusion. In mast cells, histamine immunoreactivity is detected at 2, 6 and 12 h after ischemia, whereas it disappears at 24 h, when a concomitant degranulation of mast cells is observed. Taken together, these data suggest that the recruitment of cerebral mast cells releasing histamine may contribute to ischemia-induced neuronal death in the immature brain.

[The histamine H3 receptor: a new target for the treatment of arousal and cognitive disorders]. / Le récepteur H3 de l'histamine: une cible pour de nouveaux traitements des troubles de l'éveil et de la cognition.

The histamine H3 receptor was identified in the 80's by our group as a presynaptic autoreceptor inhibiting histamine synthesis and release in the rat brain. Sixteen years later, cloning of the related human H3 receptor revealed the existence of isoforms, species pharmacological differences and a high constitutive (spontaneous) activity of the receptor. All these molecular findings have to be taken into account for optimizing aimed at clinical applications ligands. H3 receptor inverse agonists, by increasing histamine neuron activity, promote arousal and enhance cognitive performances. Pharmaceutical firms have shown considerable interest for this new class of drugs and many programmes of clinical development of H3 receptor inverse agonists for the treatment of arousal and cognitive disorders are presently being conducted.

BF2.649 [1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride], a nonimidazole inverse agonist/antagonist at the human histamine H3 receptor: Preclinical pharmacology.

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity approximately 50% higher than that of ciproxifan. Its in vitro potency was approximately 6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

Search for histamine H3 receptor antagonists with combined inhibitory potency at Ntau-methyltransferase: ether derivatives.

With the recent development of new hybrid compounds having histamine H3 receptor antagonist with combined histamine Ntau-methyltransferase (HMT) inhibitory potency an innovative approach was described in the research of novel lead compounds modulating histaminergic neurotransmission. Several compounds containing an ether moiety derived from the recently published 4-(3-piperidinopropoxy)phenylaminoquinoline derivatives (like FUB 836), were synthesized in this study and tested for their affinity at cloned human histamine H3 (hH3) receptors and on the inhibition of rat HMT. Besides different heterocycles, e.g. nitro- or amino-substituted pyridines, quinolines, benzothiazole or pyrroline, three classes of compounds were produced: heteroaromatic 3-piperidinopropyl ethers, keto- or imino-substituted 4-(3-piperidinopropyl)phenyl ethers and 4-(3-piperidinopropyl)phenyl ethers with substituted (alkyl)aminopyridines. Whereas the (3-piperidinopropoxy)heterocycles showed only moderate activities on both test models, the 4-(3-piperidinopropoxy)phenyl derivatives were identified as potent histamine H3 receptor ligands and/or HMT inhibitors. Ki values up to 0.42 nM were found for the affinity to the hH3 receptor. HMT inhibitory potency was identified with IC50 values about 0.3 microM for the most potent compounds in this series. Comparison of the pyridine-containing derivatives to recently published quinoline analogues showed a decrease in potencies for the pyridines. The dual activity, H3 receptor affinity and HMT inhibition, was moderate to good. For all compounds affinities at hH3 receptors were higher than their inhibitory HMT potencies. The described new histamine H3 receptor antagonists with an ether moiety represent a further promising step in our investigations for a dual activity.

Therapeutic implications of constitutive activity of receptors: the example of the histamine H3 receptor.

Some G-protein-coupled receptors display constitutive activity, that is spontaneous activity in the absence of agonist: a proportion of the receptor population adopts a conformation that can bind and activate G proteins. Whereas this was mainly shown to occur with recombinant or pathologically mutated receptors, the physiological relevance of the process has remained debated. We have adressed this question in the case of the histamine H3 receptor, a presynaptic inhibitory receptor regulating histamine release in brain. Having identified a neutral antagonist and inverse agonists with variable intrinsic activity, we show that the native H3 receptor in brain displays high constitutive activity in vitro and, in vivo, controls the release of endogenous histamine. This implies that inverse agonists with high intrinsic activity should be preferred for therapeutic application as "cognitive enhancers" in several psychiatric disorders.

[The histamine H(3) receptor: a target for new drugs]. / Le récepteur H3 de l'histamine: une cible pour de nouveaux médicaments.

The histamine H3 receptor has been identified in the rat brain as a presynaptic autoreceptor inhibiting histamine synthesis and release. Following its recent cloning, more than fifteen years later, the existence of isoforms, species pharmacological differences and the high constitutive activity of the receptor were established. All these molecular findings have to be taken into account for the optimization of the ligands aiming at a clinical use. They show the interest of antagonists/inverse agonists in the symptomatic treatment of schizophrenia and cognitive and attentional deficits.

A detailed mapping of the histamine H(3) receptor and its gene transcripts in rat brain.

The detailed distribution of histamine H(3) receptor mRNAs in rat brain was analyzed by in situ hybridization using a 33P-labelled riboprobe and was combined for the first time with the detailed autoradiographic distribution of the receptor determined in the same animals with [(125)I]iodoproxyfan, a selective radioligand. The signals generated on adjacent brain sections by each probe were quantified and/or rated and were compared in order to identify neuronal populations expressing the receptor. In addition, the cellular localization of the transcripts within various brain structures was analyzed in sections dipped in a photographic emulsion. In the cerebral cortex, the strong mRNA expression in intermediate and deep layers indicates the presence of H(3) receptors on several types of neurons. The binding is dense except in layer V, suggesting that H(3) receptors are located on granule cells and apical dendrites of pyramidal cells. In addition to their localization on monoaminergic afferents, the dense binding in layer IV and strong mRNA expression in thalamic nuclei suggest the presence of heteroreceptors on thalamocortical projections. In the hippocampus, the strong mRNA expression but low binding in pyramidal layers of the CA1 and ventral CA3 fields suggest that H(3) receptors are abundant on efferent projections of pyramidal cells. In the dentate gyrus, some binding sites in the molecular layer may correspond to H(3) receptors synthesized in granule cells and coexpressed with H(1) and H(2) receptors in their dendrites. In the basal ganglia, H(3) receptors are highly expressed in the striatal complex and olfactory tubercles but not in islands of Calleja. Some of the striatal binding sites may correspond to presynaptic receptors present on afferents. The mRNAs in cortical layer V may encode for heteroreceptors on corticostriatal neurons. The presence of mRNAs in the substantia nigra pars compacta suggests that H(3) receptors are located upon nigrostriatal afferents. However, the absence of any signal in the ventral tegmental area indicates that some but not all dopaminergic neurons express H(3) receptors. In addition, the homogeneous mRNA expression within the caudate putamen and nucleus accumbens suggests that many striatal H(3) receptors are present on medium-sized, spiny projection neurons of both the direct and indirect movement pathways. In agreement, a dense binding, but low mRNA expression, is observed in external and internal pallidum and in substantia nigra pars reticulata. In the amygdala, the dense binding and mRNA expression indicate the presence of receptors on both afferents and projections. In the thalamus, the binding in some association nuclei may correspond to receptors present on neurons emanating from the deep cortical layers that strongly express the mRNAs, as well as receptors on the visual systems. However, the low binding and high mRNA expression in most nuclei indicate that many receptors are present upon thalamic projections. In the hypothalamus, the mRNA expression parallels the density of binding sites and is the highest in the tuberomammillary nucleus. Further investigation is needed to know if the dense binding and mRNA expression observed in other nuclei such as the paraventricular, ventromedial and medial tuberal nuclei correspond to pre- and/or postsynaptic receptors. mRNAs are also observed in several areas projecting to the tuberomammillary nucleus, such as the ventrolateral preoptic nucleus. In the lower brainstem, the high mRNA expression and very low binding in the locus coeruleus and raphe nuclei indicate that presynaptic rather than somatodendritic receptors regulate noradrenaline and serotonin release, respectively. A similar pattern in vestibular nuclei suggests that receptors located on projections account for the anti-vertigo properties of H(3) receptor antagonists. In the cerebellum, binding is hardly detectable but a strong mRNA expression is found in most, if not all, Purkinje cells as well as in several central cerebellar nuclei, suggesting the presence of H(3) receptors on efferent projections. The present study reports the first detailed quantification and/or rating of H(3) receptor mRNAs in the brain. The comparison, performed in the same animals, with the distribution of the H(3) receptor protein provides evidence for the presence of H(3) receptors on many neuronal perikarya, dendrites and projections. Although some localizations, mainly as auto- or heteroreceptors, are consistent with previous functional studies, the physiological role, if any, of most of these presynaptic or postsynaptic receptors remains to be established.

Acute and chronic effects of methamphetamine on tele-methylhistamine levels in mouse brain: selective involvement of the D(2) and not D(3) receptor.

We have explored the role of endogenous dopamine in the control of histaminergic neuron activity in mouse brain regions evaluated by changes in tele-methylhistamine (t-MeHA) levels. In vitro, methamphetamine released [(3)H]noradrenaline but failed to release [(3)H]histamine from synaptosomes. In vivo, methamphetamine enhanced t-MeHA levels by about 2-fold with ED(50) values of approximately 1 mg/kg in caudate putamen, nucleus accumbens, cerebral cortex, and hypothalamus. This response selectively involved the D(2) and not the D(3) receptor as indicated by its blockade by haloperidol and by its persistence after administration of nafadotride, a D(3) receptor preferential ligand, or in (-/-) D(3) receptor-deficient mice. The t-MeHA response to methamphetamine was delayed compared with the locomotor-activating effect of this drug, suggesting that it is of compensatory nature. In agreement, ciproxifan, an inverse agonist known to enhance histamine neuron activity, decreased the hyperlocomotion induced by methamphetamine. Repeated methamphetamine administration resulted in the expected sensitization to the hyperlocomotor effect of the drug but did not modify either the ED(50) or the E(max) regarding t-MeHA levels. However, it resulted in an enhanced basal t-MeHA level (+30-40%), which was sustained for at least 11 days after withdrawal in hypothalamus, striatum, and cerebral cortex and suppressed by haloperidol. Hence, both the acute and chronic administration of methamphetamine enhance histamine neuron activity, presumably in a compensatory manner. Repeated methamphetamine administration also resulted in a modified balance in the opposite influences of dopamine and serotonin on histaminergic neurons as revealed by the enhanced response to haloperidol and abolished response to ketanserin, respectively.

Histamine H3-receptor-mediated [35S]GTP gamma[S] binding: evidence for constitutive activity of the recombinant and native rat and human H3 receptors.

Constitutive activity of the recombinant and native rat and human H(3) receptors (H(3)Rs) was studied using H(3)R-mediated [(35)S]GTPgamma[S] binding and [(3)H]-arachidonic acid release. Ciproxifan, an inverse agonist at the rat H(3)R (rH(3)R), decreased [(3)H]arachidonic acid release from CHO cells expressing moderate densities (approximately 200 - 300 fmol mg(-1) protein) of the human H(3)R (hH(3)R). This effect occurred with the same magnitude than at the rH(3)R. The expression of the hH(3)R was associated with an increase in [(35)S]GTPgamma[S] binding to membranes of CHO cells. Ciproxifan decreased [(35)S]GTPgamma[S] binding to membranes of CHO (hH(3)R) cells. Both effects were correlated to receptor density and revealed that constitutive activity of the hH(3)R, although lower than that of the rH(3)R in this assay, was again observed at physiological densities (<500 fmol mg(-1) protein). Ciproxifan was less potent at the human than the rat receptor, not only as an antagonist (K(i)=45 nM), but also as an inverse agonist (EC(50)=15 nM). Constitutive activity of the hH(3)R was also evidenced using inhibition of [(35)S]GTPgamma[S] binding by unlabelled GTPgammaS. The expression of the hH(3)R generated a high affinity binding for GTPgammaS which was increased by imetit, but partially decreased by ciproxifan, therefore acting as a partial inverse agonist. [(35)S]GTPgamma[S] binding to rat brain membranes was decreased in several regions by thioperamide, ciproxifan and FUB 465, three inverse agonists at the H(3)R, whose effects were blocked by proxyfan, a neutral antagonist. [(35)S]GTPgamma[S] binding was also decreased by an A(1)-adenosine receptor inverse agonist, but remained unchanged in the presence of inverse agonists at D(2)/D(3) dopamine, H(1) and H(2) histamine, alpha(2)-adrenergic and delta opioid receptors. In conclusion, the present study shows that the recombinant rat and human H(3) receptors expressed at physiological densities display constitutive activity and suggests that constitutive activity of native H(3)Rs is one of the highest among G-protein-coupled receptors present in rat brain.

Application of genomics to drug design: the example of the histamine H3 receptor.

The histamine H(3) receptor was characterized in the 1980s as an autoreceptor regulating histamine release in brain. Since then, selective drugs have been designed, many of them displaying a high potency in vivo, and used in many studies to delineate the implications of cerebral histaminergic systems in physiological functions such as arousal or cognitive functions. The recent cloning of the H(3) receptor, more than 15 years later, has allowed to start molecular studies that led to important findings for optimization of drug design. In agreement some ligands display distinct affinities for the recombinant rat and human H(3) receptors, a difference that we assign to two amino acids in the third transmembrane domain. In addition, H(3) autoreceptors present in the brain display high constitutive activity including in vivo. As a consequence, inverse agonists enhance histamine neuron activity and constitute a novel potential therapeutic approach to schizophrenia and Alzheimer's disease.

Expression analysis of the histamine H(3) receptor in developing rat tissues.

Endogenous histamine is involved in tissue growth and cell proliferation. In accordance with a putative function of the H(3) receptor in this mitogenic effect, we show that H(3)-receptor mRNAs are expressed together with those of the histamine-synthesizing enzyme in the embryonic liver and adipose tissue, and in various epithelia. Finally, we show that activation of recombinant H(3) receptors enhances MAP kinase activity.

Different antagonist binding properties of human and rat histamine H3 receptors.

Different histamine H3-receptor antagonists have been tested in displacement studies at human and rat H3 receptors in stably transfected cells. Based on an actual rhodopsin structure, models for receptor antagonist interaction were developed for receptors of both species. Similarities and discrepancies in binding profiles can be explained, but not quantified by hydrophilic interactions with Asp114 and an important lipophilic binding pocket modified by two nearby amino acids.

Benzophenone derivatives and related compounds as potent histamine H3-receptor antagonists and potential PET/SPECT ligands.

Para-substituted aromatic ethers with benzophenone or related structural elements and a 3-(1H-imidazol-4-yl)propyloxy moiety were prepared by Mitsunobu-type ether synthesis or SNAr reaction. Most of the title compounds possess high antagonist potency in histamine H3-receptor assays in vitro as well as in vivo in mouse CNS following oral administration. After defining 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl phenyl methanone as a new lead, structure-activity relationships were investigated for this new class of compounds. Substitution of the meta'-position of the benzophenone moiety with halogen atoms (e.g., iodine, fluorine) led to compounds with high antagonist potency in vitro as well as in vivo (Ki = 9.3 and 4.3 nM, ED50 = 0.7 and 0.47 mg/kg p.o., 18 and 12, respectively). A receptor profile of several functional in vitro assays for several biogenic amine receptors for the meta'-iodinated derivative demonstrated high selectivity toward the histamine H3 receptor.

Chromosomal mapping and organization of the human histamine H3 receptor gene.

The histamine H3 receptor (H3R) was recently cloned, and two isoforms, termed H3L and H3S, differing in the third intracytosolic loop, were isolated but the chromosomal mapping and organization of its gene remained unknown. PCR analysis of a human x rodent cell hybrid panel indicated that the H3R gene is located in the telomeric region of chromosome 20q. Alignment of human H3R cDNA sequences with DNA sequences of this chromosome revealed that its coding region comprises three exons interrupted by two introns located in the second transmembrane domain (TM2) and second intracytosolic loop, respectively. Thus the organization of the H3R gene indicates that the H3L and H3S isoforms, that we characterized not only in rodents but also in humans, are generated by retention and deletion, respectively, of a pseudo-intron located in the third intracytosolic loop.