Visual Overview
Abstract
Among the four G protein–coupled receptors (H1–H4) identified as mediators of the biologic effects of histamine, the H3 receptor (H3R) is distinguished for its almost exclusive expression in the nervous system and the large variety of isoforms generated by alternative splicing of the corresponding mRNA. Additionally, it exhibits dual functionality as autoreceptor and heteroreceptor, and this enables H3Rs to modulate the histaminergic and other neurotransmitter systems. The cloning of the H3R cDNA in 1999 by Lovenberg et al. allowed for detailed studies of its molecular aspects. In this work, we review the characteristics of the H3R, namely, its structure, constitutive activity, isoforms, signal transduction pathways, regional differences in expression and localization, selective agonists, antagonists and inverse agonists, dimerization with other neurotransmitter receptors, and the main presynaptic and postsynaptic effects resulting from its activation. The H3R has attracted interest as a potential drug target for the treatment of several important neurologic and psychiatric disorders, such as Alzheimer and Parkinson diseases, Gilles de la Tourette syndrome, and addiction.
Introduction
In 1910, Sir Henry Dale and colleagues (Dale and Laidlaw, 1910) isolated histamine from ergot and later found that it had a stimulant effect on smooth muscle from the gut and the respiratory tract, caused vasodepression, stimulated cardiac contractility, and induced a shock-like syndrome when injected into animals. In 1920, Popielski demonstrated that histamine stimulated gastric acid secretion, and in 1927, the amine was isolated from the liver and the lung, evidencing that it was a natural constituent of the body (reviewed by Parsons and Ganellin, 2006).
Although histamine was detected in the brain in 1919 by John J. Abel, its role as a neuromodulator became evident only several decades later; using antibodies against the amine and its synthesizing enzyme, histidine decarboxylase (HDC), the morphologic characterization of histamine-producing neurons proved the existence of a histaminergic system in the mammalian brain (Fig. 1).
The histaminergic system in the rat brain. Histamine-synthesizing neurons are located in the hypothalamus tuberomamillary nucleus, and these neurons send projections to the CNS through three major pathways, two ascending bundles that innervate the forebrain structures, and one descending bundle reaching the spinal cord. Thal, thalamus; Str, striatum.
The Brain Histaminergic System
Histamine-synthesizing neurons are located in the hypothalamus tuberomamillary nucleus (TMN), with ∼4500 neurons found in the rat TMN and ∼64,000 neurons in the human TMN. These neurons send diffuse projections to the entire central nervous system (CNS) through three major pathways, two ascending bundles that innervate the forebrain structures and one descending bundle reaching the spinal cord (Watanabe et al., 1984; Airaksinen and Panula, 1988; Haas et al., 2008).
Histaminergic neurons have a resting membrane potential of about −50 mV and spontaneously fire action potentials at 2.1 ± 0.6 Hz with a marked circadian rhythmicity, that is, more active during wakefulness. A noninactivating Na+ current, active even at −70 mV, appears responsible for spontaneous firing, with low-threshold depolarizing Ca2+ currents contributing to the repetitive firing. The action potential is broad with a significant contribution from Ca2+ currents followed by a pronounced (15–20 mV) after hyperpolarization, which activates a depolarizing cationic current (Ih) and two A-type K+ currents that delay the return to resting potential. The Ca2+ currents mediate dendritic histamine release and are the target of the autoreceptor-mediated negative feedback on action potential firing (Haas and Reiner, 1988; Haas et al., 2008).
The behavioral state–dependent activity of the histaminergic neurons is influenced by several neuronal, humoral, and paracrine signals, and the activity is regulated mainly by excitatory glutamatergic inputs from the cerebral cortex and the hypothalamus and by inhibitory GABAergic afferents from the hypothalamic ventrolateral preoptic nucleus (Ericson et al., 1991; Haas et al., 2008; Panula et al., 2015).
Histamine Synthesis, Release, and Catabolism.
Histamine is synthesized from the amino acid (aa) l-histidine by the enzyme HDC, which is expressed in both the neuronal bodies and terminals, and the bioavailability of the precursor is the rate-limiting factor. Histamine is stored in vesicles in neuronal cell bodies and axon varicosities by the vesicular monoamine transporter 2, VMAT-2 (Merickel and Edwards, 1995) and is released by exocytosis upon the arrival of action potentials (Haas et al., 2008). The synthesis and release of histamine are regulated by H3 autoreceptors (Arrang et al., 1983, 1987b; Morisset et al., 2000).
Most histaminergic fibers do not make typical synaptic contacts (Takagi et al., 1986), and the amine is released from several points along the fibers, allowing its action on a large number of cells. In the brain, histamine is also produced by mast cells, which contribute modestly to the total amine levels in the adult brain, but during early postnatal development represent the principal source of the amine (Molina-Hernández et al., 2012; Panula et al., 2015).
Inactivation of histamine in the brain is due primarily to the action of histamine-N-methyltransferase producing telemethylhistamine that is transformed to telemethyl-imidazolacetic acid by monoamine oxidase B. Diamine oxidase is the main histamine-metabolizing enzyme in the peripheral tissues, but its activity in the brain is considerably lower under basal conditions (Barnes and Hough, 2002; Maldonado and Maeyama, 2015). In contrast to most other aminergic neuronal cells, histaminergic neurons lack a specific reuptake transporter, although astrocytes take up histamine with low affinity (Km 0.56 mM and 4.0 mM) through the activity of the plasma membrane monoamine transporter and, to a lesser extent, the organic cation transporter 3 (Yoshikawa et al., 2013).
Histamine Receptors.
The differing potency of antagonists in blocking histamine action to increase the contraction rate in isolated mouse atria and gastric acid secretion or to induce smooth muscle contraction in isolated guinea pig ileum led to the first classification of histamine receptors into the H1 and H2 subtypes by Ash and Schild (1966), supported by the subsequent development of selective H2 receptor (H2R) antagonists (Parsons and Ganellin, 2006). The bovine H1 receptor (H1R) and the canine H2R were cloned in 1991 (Gantz et al., 1991; Yamashita et al., 1991), allowing for the classification of these receptors into the class A, rhodopsin-like, of G protein-coupled receptors (GPCRs). H1Rs and H2Rs are expressed in the brain and the former signals primarily through Gαq/11 proteins, whereas the latter activates mainly Gαs proteins (Panula et al., 2015).
A third receptor (H3R) was pharmacologically identified by Arrang et al. (1983) and cloned in 1999 by Lovenberg et al.; the H3R activates Gαi/o proteins and is expressed almost exclusively by neuronal cells of the CNS and the peripheral nervous system (Panula et al., 2015). Soon after, a fourth receptor was cloned by several groups (e.g., Nakamura et al., 2000; Oda et al., 2000). The H4R is mainly expressed by cells of the immune system and, like the structurally related H3R, activates Gαi/o proteins (Panula et al., 2015).
The Histamine H3 Receptor
In 1983 Arrang et al. reported that in rat cerebro-cortical slices labeled with [3H]-histidine, the depolarization-evoked, Ca2+-dependent release of [3H]-histamine was reduced by exogenous histamine (IC50 41 nM, maximal inhibition 61%). The effect was insensitive to tetrodotoxin, which prevents the generation and propagation of action potentials, mimicked by Nα-methylhistamine, and antagonized by impromidine and burimamide with potencies significantly different from those reported for H2R blockade. More potent H2R antagonists and selective H1R antagonists also showed low potencies. It was therefore proposed that autoinhibition of histamine release was mediated by a novel class of receptor (H3R). In a later study (Arrang et al., 1985a), autoinhibition of depolarization-evoked [3H]-histamine release was also shown for isolated nerve terminals (synaptosomes) from rat cerebral cortex (−30%) and slices of striatum (−49%), hippocampus (−47%) and hypothalamus (−64%). The same group reported that histamine also inhibited its own synthesis evoked by depolarization in rat cerebrocortical slices (IC50 340 nM, maximal inhibition 70%; Arrang et al., 1987b). This effect was competitively antagonized by burimamide and impromidine with potencies similar to those observed for the autoinhibition of release (Arrang et al., 1983), supporting the function of the H3R as an autoreceptor.
In 1999, Lovenberg et al. identified a partial clone (GPCR97) and used it to probe a human thalamus cDNA library. This resulted in the isolation of a full-length clone encoding a 445–amino-acid protein with the characteristics of class A GPCRs and with 20%–27% homology to biogenic amine receptors and 22% and 21.4% homology to the human H1R and H2R, respectively. Upon transfection into human embryonic cell line HEK-293, rat C6 glioma cells, and human SK-N-MC neuroblastoma cells, GPCR97 displayed a pharmacologic profile practically indistinguishable from that of the native H3R. Soon after, the receptor was cloned by sequence similarity from various other species, namely, rat, guinea pig, mouse, and monkey, with a high level of homology (≥93%) across these species (see Fig. 2).
Alignment of the amino acid sequence of the human, monkey, guinea pig, rat, and mouse H3Rs. Sequences were obtained from Uniprot (Washington, DC) and correspond to the full-length H3R (445 aa). Alignment was performed with the Clustal Omega server from the European Bioinformatic Institute (Cambridge, UK). The highly conserved sequences in the seven transmembrane domains are shown in gray. The DRF and NPVLY motifs and the glycosylation site (Asn11) are well conserved among species (green). Residues in blue are responsible for the interspecies differences in H3R pharmacology. Residues in red are likely to be involved in the receptor high constitutive activity on the basis of their identity with a mutated β2-adrenoceptor. The third intracellular loop (ICL3) possesses a region (residues 236–300; italics) that is highly variable among species, followed by a highly conserved sequence. The hH3R has ≥93% sequence identity with the other species, and the same identity can be found in guinea pig, mouse, and rat sequences, with 99% identity between mouse and rat, and among the primate proteins. *, conserved residues; :, conservative mutations; •, nonconservative mutations.
Expression in the CNS
The H3R is expressed mainly by neurons and in very low density by glial cells (Arrang et al., 1987a; Ferreira et al., 2012). The distribution of the H3R in the CNS has been studied via in situ hybridization (mRNA), reverse transcription polymerase chain reaction (RT-PCR) (mRNA), and autoradiography (binding sites) in rodents, humans, and monkeys. In situ hybridization studies report very high levels of H3R mRNA in the cortex (mainly in the V layer, with lower expression in the superficial layers), hippocampus (CA1 and ventral CA3 pyramidal layers of Ammon’s horn), caudate, and putamen. Strong mRNA expression is also observed in the anterior olfactory nucleus, amygdala, bed nucleus of the stria terminalis, cerebellum, thalamus (mostly in the sensory and intralaminar nuclei), and some hypothalamic nuclei, particularly the TMN, where histaminergic neurons are located. Low to moderate mRNA expression is detected in the habenula and zona incerta; the signals are very low in the globus pallidus, substantia nigra (SN), and substantia innominata and are not detected in the islands of Calleja (Tardivel-Lacombe et al., 2000; Pillot et al., 2002; Sallmen et al., 2003).
Analysis by RT-PCR of the expression of the H3R isoforms of 445 and 365 aa in the human brain (see description of the receptor isoforms later herein) indicates high levels in cerebellum and caudate; moderate in the hypothalamus and thalamus; low expression in the SN, hippocampus, prefrontal cortex, corpus callosum, and amygdala; and very low levels in the spinal cord (Bongers et al., 2007b).
Binding studies show a similar H3R distribution in primate and rodent brains. The receptor is widely expressed but with heterogeneous density, and high levels are found in cerebral cortex (except in layer V), tenia tecta, nucleus accumbens, striatum, hippocampus, bed nucleus of the stria terminalis, olfactory nuclei, some hypothalamic nuclei (mainly the TMN), amygdala, and pyriform cortex. In contrast to mRNA expression, dense binding is found in the globus pallidus and the substantia nigra pars reticulata (SNr). A low density of binding sites is found in locus coeruleus and raphe nuclei, and the cerebellum and the pituitary gland are scarcely labeled (Martinez-Mir et al., 1990; Pollard et al., 1993; Anichtchik et al., 2000; Pillot et al., 2002).
The H3R location is mainly presynaptic, either as autoreceptor or as heteroreceptor, but there is also evidence for a postsynaptic location of the H3R in striatum, cerebral cortex, hippocampus, nucleus accumbens, lateral hypothalamus and zona incerta (Pillot et al., 2002; González-Sepúlveda et al., 2013; Parks et al., 2014).
Structure
With more than 800 genes encoding GPCRs, these proteins constitute the largest family of membrane proteins contained in the human genome. These receptors share a common seven-transmembrane (TM) domain structure that forms the core, an extracellular amino terminus (NT), an intracellular carboxyl terminus (CT), three extracellular (ECL), and three intracellular (ICL) loops.
The H3R possesses a DRF motif in the interface of TM3 and ICL2 (instead of the DRY sequence common to most class A GPCRs), a NPVLY motif in TM7 (corresponding to the NPXXY motif present in all GPCRs), and a palmitoylation site in the CT (Cys428) that allows for the formation of helix 8 (Fig. 3). A disulfide bond is formed by Cys107 and Cys188 on ECL1 and ECL2, respectively. The H3R has a short NT (39 aa), with a glycosylation site on Asn11, and a long ICL3 (142 aa). These segments are the loci of the naturally occurring mutations D19E and A280V, respectively (Wiedemann et al., 2002), and cleavage through splicing of the ICL3 leads to several H3R isoforms as discussed below.
Structure of the human H3R. As a GPCR, the H3R contains seven spanning transmembrane domains, an extracellular amino terminus (NT), an intracellular carboxyl terminus (CT), three extracellular (ECL), and three intracellular (ICL) loops, with a long ICL3 (142 aa). Potential residues for phosphorylation are shown in green. The conserved DRF and NPVLY motifs are shown in yellow. The naturally occurring mutations D18E and A280V are depicted in blue. The glycosylation site (Asn11) is shown in red, the palmitoylation site (C228) in gray. Residues Thr119 and Ala122 (purple) are responsible for the interspecies pharmacologic differences. Residues indicated in black in ECL1 and ECL2 correspond to the cysteins forming a disulfide bond important for receptor trafficking. Residues important for agonist recognition are colored in pink.
Since 2000, the rat, guinea pig, monkey, and mouse genes encoding the H3R have been cloned (Lovenberg et al., 2000; Tardivel-Lacombe et al., 2000; Chen et al., 2003; Yao et al., 2003), and this work revealed a homology of ≥93% in the H3R protein sequence among these species (Fig. 2). The interspecies differences in the pharmacologic profiles rely on a double change in residues 119 and 122 located near Asp114 in TM3. Human and monkey receptors have Thr119 and Ala122 at these species-variant sites, whereas rodents have Ala119 and Val122, with the exception of the guinea-pig receptor (Thr119 and Val122). Compared with the human H3R (hH3R), a single-residue change (V362I) in TM6 is found in the rat, mouse, and guinea pig. No differences exist between human and monkey H3Rs in the transmembrane domains (Hancock et al., 2003). In contrast to the small changes in the helical domains, a significant number of differences among species are found in a 65–66 aa-length sequence in the ICL3 (aa 236–300 in the hH3R), but this does not affect the presumed Ser and Thr substrates of phosphorylation, suggesting a conserved kinase modulation profile.
Determinants of G Protein–Coupling and Activation
Structurally and functionally, two faces can be identified on all GPCRs: the extracellularly accessible orthosteric binding site and the internal region responsible for G-protein activation. The crystallization of GPCRs coupled to a G protein has provided insight into the mechanisms of receptor-G protein interaction and activation of the latter. These studies show that the α-CT and the helixes 4 and 5 of the Gα subunit dock to a cavity formed mainly by TM5, TM6, and ICL2 in the GPCR, which adopts an “open” conformation upon receptor activation. The Arg of the DRY motif plays a key role in docking the α-CT of the G protein (Rasmussen et al., 2011) and residues in ICL3, helix 8, and the CT appear to contribute to stabilizing the receptor-G protein interaction because a truncation close to the NPXXY motif prevents G protein activation (van Rijn et al., 2013; Flock et al., 2015).
Computational models of the H3R-G protein interaction have not yet been published, but functional studies suggest that ICL3 plays a role in G-protein activation. Alternative splicing generates several H3R isoforms (see later), and the hH3R of 445 aa (hH3R445) inhibited forskolin-induced cAMP formation in Chinese hamster ovary (CHO) cells, but the hH3R365 (lacking 80 aa in ICL3) failed to do so (Cogé et al., 2001). In rat C6 glioma cells, the hH3R365 was less efficacious in a calcium mobilization assay (Esbenshade et al., 2006), but agonists were 3- to 20-fold more potent in the Receptor Selection and Amplification Technology assay, based on β-galactosidase activity (Wellendorph et al., 2002). The rat H3R413 and H3R397 isoforms that lack 32 and 48 aa in ICL3 showed similar efficacy and higher agonist potency for inhibition of forskolin-induced cAMP formation, but for 42/44-mitogen-activated protein kinase (MAPK) phosphorylation, the H3R445 coupled considerably better (Drutel et al., 2001). The naturally occurring A280V mutation on ICL3 also modifies the functionality of the hH3R445 to inhibit cAMP accumulation and stimulate 42/44-MAPK phosphorylation (Flores-Clemente et al., 2013).
Altogether, the previous information indicated that there is not a single structural determinant for the H3R functionality but that the interplay of several regions provides the appropriate physiologic output.
Constitutive Activity and Determinants
The term constitutive activity refers to a ligand-independent state of the receptor that spontaneously adopts the active conformation. The H3R possesses high constitutive activity as indicated by the reduction of basal G-protein activation induced by inverse agonists (Wieland et al., 2001; Rouleau et al., 2002). Constitutive activity has been shown mainly for transfected human and rat H3Rs but also for native rodent receptors with inverse agonists reducing basal [35S]-GTPγS binding to membranes from the cerebral cortex and hippocampus, enhancing depolarization-induced [3H]-histamine release from cerebrocortical synaptosomes and increasing the levels of the histamine metabolite tele-methylhistamine in homogenates from the cerebral cortex (Morisset et al., 2000; Sallmen et al., 2003).
The DRY motif is responsible for the equilibrium between the inactive and active receptor conformations and therefore plays a pivotal role in constitutive activity. A salt bridge between the Arg in the DRY motif (TM3) and an Asp or Glu in TM6 stabilizes or “locks” the inactive state. Upon receptor activation, this interaction is broken and the Arg residue in TM3 rotates toward TM5 to form a hydrogen bond with a Tyr residue to establish an “active lock” (Valentin-Hansen et al., 2012). Accordingly, the R112A mutation in the hH4R prevents G-protein activation (Schneider et al., 2010), and the R116A mutation in the rat H2R resulted in a highly structurally instable receptor whose expression could be detected only after stabilization with either an agonist or inverse agonist; furthermore, this receptor showed increased agonist affinity and reduced efficacy (Alewijnse et al., 2000). There is no direct evidence for a role of the DRF motif in H3R constitutive activity, however.
As pointed by Morisset et al. (2000), the CT of ICL3 in the rat H3R has a stretch of 8 aa that is similar (six identical and two conserved residues) to the corresponding sequence of a mutated human β2-adrenoceptor with high constitutive activity (Lefkowitz et al., 1993). This region is critical for constitutive activity in other native or mutated GPCRs. Although the sequence is conserved in all H3R isoforms and species (see Fig. 2), constitutive activity varies among isoforms and can differ between species and cell lines (Arrang et al., 2007).
H3R Isoforms
The existence of H3R isoforms was initially suggested by the pharmacologic heterogeneity of the receptor expressed in several brain regions in binding and functional assays (West et al., 1990). The molecular cloning of the receptor indicated that the structure of the hH3R gene comprised three exons and two introns (Tardivel-Lacombe et al., 2001; Wiedemann et al., 2002) or four exons and three introns (Cogé et al., 2001), which allows for the generation of isoforms via RNA splicing. Alternatively, for humans and rodents, the last intron is proposed to be a pseudointron located in the region coding for ICL3, which is retained in the hH3R445 but deleted in the hH3R413 (Tardivel-Lacombe et al., 2001; Bongers et al., 2007a). In both proposed structures, the two first introns are located in the same position in the human and rodent genes, suggesting the existence of a variety of functional and nonfunctional isoforms with certain similarity among species (Tardivel-Lacombe et al., 2000; Drutel et al., 2001; Morisset et al., 2001; Wellendorph et al., 2002).
RT-PCR analysis identified several isoforms that differ in the length of their NT or CT, deletions in the ICL3 or shorter sequences in TMs. To date, the pharmacologic and functional characteristics of the H3R isoforms have been evaluated only in heterologous systems (Table 1).
Brain expression and signaling of H3R isoforms from different species
The human H3R329a and H3R329b isoforms possess the same number of amino acids but differ in the region where alternative splicing occurs.
Some of the identified isoforms lack regions critical for agonist binding (TM3 and 5–7) or signaling (ICL2, ICL3, and CT) and therefore do not trigger the signaling pathways typically associated with H3R activation (Wess, 1997; Uveges et al., 2002; Oldham and Hamm, 2007; Ishikawa et al., 2010; Kim et al., 2011; Kuramasu et al., 2011); however, three rat isoforms (rH3R497, rH3R465, and rH3R449) that do not possess TM7, but do have an extracellular CT of 105 aa without homology to the functional isoforms, reduce the cell surface expression of the rat H3R445 upon coexpression in COS-7 cells (Bakker et al., 2006). The expression and signaling of functional H3Rs could thus be regulated by isoforms incapable of triggering the signaling pathways described to follow. These isoforms could also directly interact with other GPCRs and affect their binding or signaling properties. Furthermore, noncanonical signaling, either agonist-independent or mediated by alternative pathways, cannot be discarded for the truncated or longer H3R isoforms and merits further investigation.
Signaling Pathways
The coupling of the H3R to Gαi/o proteins was first suggested by the inhibitory effect of pertussis toxin (PTX) activity, which decreased the affinity for [3H]-NMHA of the H3R endogenously expressed by murine pituitary AtT cells (Clark et al., 1993) and prevented H3R-stimulated [35S]-GTPγS binding in rat cerebrocortical membranes (Clark and Hill, 1996). As for other GPCRs, both the Gα subunits and the Gβγ complexes mediate the Gαi/o protein-dependent signaling of the H3R (Fig. 4).
H3R signaling pathways. H3R activation triggers or modulates several pathways through the Gα subunits and Gβγ complexes of Gαi/o proteins. AA, arachidonic acid; AC, adenylyl cyclases; cAMP, 3′,5′-cyclic adenosine monophosphate; MAPK, mitogen-activated protein kinases; NHE, Na+/H+ exchanger; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PLA2, phosholipase A2; PLC, phosholipase C.
Inhibition of Adenylyl Cyclases.
In the study that reported the cloning of the hH3R (Lovenberg et al., 1999), receptor activation inhibited forskolin-induced cAMP accumulation, and this effect was disrupted by PTX (Shi et al., 2012). Likewise, for other Gαi/o protein-coupled GPCRs, a direct interaction between the Gαi/o subunit and sensitive adenylyl cyclases (1, 3, 5, 6, and 8) appears to be the mechanism that mediates this effect (Cooper and Crossthwaite, 2006).
Inhibition of the Na+/H+ Exchanger.
The activity of the Na+/H+ exchanger (NHE) represents one of the key mechanisms for restoring the intracellular pH after ischemia-induced acidosis by extruding protons concomitantly with Na+ influx (Karmazyn, 1999). A rise in intracellular Na+ may reverse the Na+/Cl--dependent noradrenaline transporter and induce carrier-mediated neurotransmitter release. H3R activation reduces NHE activity in sympathetic nerve terminals, leading to inhibition of noradrenaline release in myocardial ischemia (Silver et al., 2001); the mechanism is not fully understood, although a direct Gαi/o subunit/NHE interaction appears likely (van Willigen et al., 2000).
Inhibition of N- and P/Q-Type Voltage-Gated Ca2+ Channels.
The inhibitory effect of H3Rs on neurotransmitter release (see later discussion) is most likely linked to the reduction in depolarization-induced Ca2+ entry via the binding of Gβγ complexes to the pore-forming α1-subunit of N- and P/Q-type voltage-gated Ca2+ channels (Zamponi and Currie, 2013). Accordingly, H3R activation reduces depolarization-induced Ca2+ entry in dissociated hypothalamic histaminergic neurons (Takeshita et al., 1998), striatal synaptosomes (Molina-Hernández et al., 2001), transfected human neuroblastoma SH-SY-5Y cells (Silver et al., 2002), and transfected rat pheochromocytoma PC12 cells (Morrey et al., 2008). The inhibition of voltage-gated Ca2+ currents by H3Rs in histaminergic neurons was abolished by PTX (Takeshita et al., 1998), and both a Gβγ scavenger and a phosphoducin-like anti-Gβγ peptide prevented the H3R-mediated reduction in depolarization-induced Ca2+ entry and neurotransmitter exocytosis in pheochromocytoma PC12 cells (Morrey et al., 2008), supporting the participation of Gβγ dimers in these processes.
Activation of G Protein–Gated Inwardly Rectifying K+ Channels.
Gβγ subunits bind and activate G protein–gated inwardly rectifying K+ channels (GIRK) (Bünemann et al., 2001) and transfected hH3R445 and hH3R365 activate channels formed by the GIRK1 (Kir3.1) and GIRK4 (Kir3.4) subunits expressed in Xenopus oocytes (Sahlholm et al., 2012). GIRKs can inhibit synaptic transmission (Meneses et al., 2015), and activation of presynaptic GIRKs would thus represent an additional mechanism for H3Rs to modulate neurotransmitter release. Activation of GIRK channels by H3Rs has also been observed at the postsynaptic level in neurons producing melanin-concentrating hormone (MCH), where the effect was prevented by GDPβS, an inhibitor of G-protein signaling (Parks et al., 2014).
Phospholipase C Activation.
In transfected CHO and SK-N-MC cells, activation of the hH3R445 induces a significant increase in the intracellular concentration of Ca2+ ions ([Ca2+]i) as a result of phospholipase C (PLC) activation and release of Ca2+ from intracellular stores via inositol-1,4,5-trisphosphate (IP3) formation (Cogé et al., 2001; Bongers, 2008). The H3R-mediated increase in [Ca2+]i was prevented by PTX, which implicates the participation of Gαi/o proteins (Bongers, 2008). The PLC/IP3/Ca2+ pathway is most often triggered by the Gα subunits of Gαq/11 proteins, but Gβγ complexes can also activate PLCβ by binding to a region (PH and Y domains) different from the Gαq/11 subunit binding domain (C2 domain and carboxyl terminal region; Rebecchi and Pentyala, 2000; Rhee, 2001).
Activation of the MAPK Pathway.
H3R activation stimulates 42/44-MAPK phosphorylation both in heterologous systems and native tissues (Drutel et al., 2001; Giovannini et al., 2003; Flores-Clemente et al., 2013). Gβγ complexes appear to play a central role in this action because pharmacologic inhibition and Gα-transducin, a Gβγ scavenger, prevent H3R-mediated 42/44-MAPK phosphorylation (Lai et al., 2016). Other mechanisms, however, such as the binding to activated receptors of β-arrestins, that act as a signaling scaffold (Gutkind, 2000) or the transactivation of the epidermal growth factor receptor (Lai et al., 2016) could also contribute to MAPK activation.
Activation of the Phosphatidylinositol 3-Kinase (PI3K) Pathway.
In transfected cells, primary cultures of rat cerebrocortical neurons, and rat striatal slices, H3R activation stimulates the phosphorylation of Akt or protein kinase B, which subsequently phosphorylates and thereby inhibits the action of glycogen synthase kinase 3-β (GSK3β) (Bongers et al., 2007c). This action on the PI3K/Akt pathway was prevented by PTX and probably depends on Gβγ complexes, which are known to activate PI3K (Murga et al., 1998).
Stimulation of Phospholipase A2.
The activation of phospholipase A2 by the H3R induces the release of arachidonic acid, docosahexaenoic acid, and lysophospholipids, inducing functional consequences such as the relaxation of guinea pig bronchioles by enhanced release of the endothelium-derived relaxing factor, a metabolite of arachidonic acid (Burgaud and Oudart, 1993).
Regulation of H3R Signaling
Desensitization.
This process, which leads to changes in signaling efficacy and receptor expression at cell surfaces, represents a major mechanism to regulate GPCR functional responses. Homologous desensitization is triggered by the phosphorylation of activated receptors by GPCR kinases, whereas in the heterologous process, activation of one GPCR leads to the desensitization of one or more unrelated receptors in the same cell. The latter process often involves GPCR phosphorylation by second messenger–activated kinases, in particular, protein kinases A (PKA) and C (PKC). GPCR phosphorylation results in conformational changes that impair G-protein activation and triggers receptor internalization (Gainetdinov et al., 2004; Gurevich et al., 2012).
In CHO-K1 cells stably transfected with the hH3R445, exposure to agonists results in functional desensitization, as well as reduced receptor expression in the cell surface owing to the action of GPCR kinases 2/3 and clathrin-dependent endocytosis (Osorio-Espinoza et al., 2014). In the same expression system, the hH3R445 also experiences PKC-mediated heterologous desensitization upon activation of a second GPCR coupled to the PLC/IP3/DAG pathway (Montejo-López et al., 2016).
RGS Proteins.
The regulator of G-protein signaling (RGS) proteins modulates the intracellular effects of activated GPCRs. Four RGS subfamilies (RZ, R4, R7, R12) act as GTPase-activating proteins to increase the rate of GTP hydrolysis by Gα subunits (Hollinger and Hepler, 2002; Sjögren et al., 2010). Whereas there are no reports on the regulation by RGS of the H3R signaling, RGS proteins are coexpressed with H3Rs in the CNS, for example, in cerebral cortex (RGS 4–8, 10), nucleus accumbens and striatum (RGS 4, 8, 9), hippocampus (RGS 7, 8, 10), and hypothalamus (RGS 4, 6, 7, 8) (Gold et al., 1997), making the RGS-mediated regulation of H3R signaling an aspect deserving attention.
Pharmacology
The H3R shows a complex pharmacology, with drugs acting as full agonists, partial agonists, neutral antagonists, inverse agonists, and protean ligands. H3R affinity for ligands has been determined by radioligand binding assays (Ki or pKi, Table 2) and efficacy (Emax) and potency (pEC50, EC50, pKB, pA2, pD2) through the analysis of [35S]-GTPγS binding, cAMP formation, 42/44-MAPK phosphorylation, calcium mobilization, and PI3K activation (Table 3).
Affinities of the H3R for agonists and antagonists
Data reported as Ki were converted to pKi. For recombinant receptors all values refer to the H3R of 445 aa.
Potencies of H3R agonists and antagonists
For recombinant receptors all values refer to the full-length (445 aa) receptor.
In 2008, the crystal structure of the GPCR opsin was resolved (Scheerer et al., 2008), leading to a revolutionary change in the study of GPCR-drug interactions. Although the crystal structure of the H3R has yet to be elucidated, homology models and point-mutation studies allow for the assessment of ligand recognition by its binding pocket.
Determinants of Histamine Binding
Histamine (2-[4-imidazolyl]ethylamine) is a hydrophilic nonchiral molecule consisting of an imidazole ring and an ethylamine side chain, and its binding to the H3R depends on features shared by other biogenic amine receptors. Although the ligand binding site comprises residues in TM3–TM7, two main residues are essential for histamine binding. One is the negatively charged Asp114 in TM3 that interacts with the protonated amine group of histamine, and the other is Glu206 in TM5, which forms a hydrogen bond with the nitrogen present in the imidazole ring.
In addition to Glu206, other residues important for histamine binding are Trp196, Thr201, Ala202, Thr204, and Phe208 in TM5. Mutations in these residues decrease binding affinity, with the E206A mutation resulting in the most marked change. Interestingly, variations in binding affinity induced by mutations are not always matched in functional assays, with the W196A, T204A, E206A, and F208A mutations increasing ligand potency to inhibit cAMP formation, indicating that conformational changes in the binding pocket do not necessarily translate into receptor-driven G-protein activation (Uveges et al., 2002). Other residues involved in histamine binding are Tyr115 and Cys118 in TM3; Trp371, Tyr374, and Leu401in TM6; and Tyr189 in ECL2 and Trp402 in TM7 (Ishikawa et al., 2010). The high affinity of the H3R for the endogenous agonist does not rely on Asp114 (conserved in all four histamine receptors and essential for histamine binding) but on Glu206, only present in H3Rs and H4Rs, explaining the nonselective actions of several classic H3R ligands at the H4R (Axe et al., 2006). The increased affinity of H3Rs and H4Rs for histamine may be due, at least in part, to the stronger interaction of the imidazole ring with Glu206 compared with the interaction with the Asn or Thr residue found on the same position in H1Rs or H2Rs, respectively (Uveges et al., 2002).
The previous information allows a pharmacophore structure to be drawn for the H3R (Fig. 5), where Glu206 anchors the imidazole moiety of histamine and the other residues form a lipophilic bed in which histamine lays, favoring the interaction of the basic amine moiety with Asp114 in TM3 (de Esch et al., 2000).
Binding sites of agonists and antagonists at the human H3R of 445 aa (hH3R445). (A) Molecular docking of the endogenous agonist histamine in a model of the hH3R445 in the active state. Residues Glu206 and Asp114 are pivotal for agonist binding. (B) Planar view of the histamine binding pocket in the hH3R445. (C) Proposed binding site of a protean ligand (proxyfan) in the inactive hH3R445 as observed by molecular docking, depicting the three functional groups of the ligand important for binding (nitrogen-containing ring, central electronegative group and aromatic ring). (D) Planar view of the suggested binding residues for an antagonist at the hH3R445.
Binding of Synthetic Imidazole-Containing and Non-imidazole Ligands
Imidazole-Containing Agonists.
So far, all agonists at the H3R are based on the structure of histamine and therefore contain the imidazole moiety, which is critical for agonist activity (Leurs et al., 1995). Modifications of the imidazole ring have been unsuccessful. By contrast, modifications of the histamine side chain that preserve the basic amino group or other proton donor moieties have allowed for the design of more selective and potent agonists. Like histamine, these molecules bind to the H3R by using Glu206 as anchor for the imidazole moiety and establishing hydrogen bonds with Asp114 (Fig. 5).
The first highly selective and potent H3R ligands were the agonist RAMH (R-α-methylhistamine) and the antagonist thioperamide, and the differences in the affinity and potency of the ligand enantiomers demonstrated the stereoselectivity of the H3R (Arrang et al., 1987a; De Esch et al., 1999; Kovalainen et al., 1999). The replacement of the histamine amine group by an isothiourea group led to the very potent and selective H3R agonist imetit (Garbarg et al., 1992), and other histamine analogs with a piperidine ring in the side chain, such as immepip, showed improved affinity and efficacy at the H3R (Fig. 6; Table 2). Modifications in the piperidine nitrogen, like VUF-5681, resulted in decreased affinity and functional activity (Shih et al., 1998; Kitbunnadaj et al., 2003, 2005). The H4R has high homology to the H3R and also high affinity for H3R agonists, such as imetit and immepip. The search for more selective ligands led to methimepip (N-methyl-substituted immepip), which retains high affinity and efficacy (Tables 2 and 3) and shows 2000-fold selectivity for the hH3R over the hH4R (Kitbunnadaj et al., 2003, 2005).
Structure of H3R agonists.
Imidazole-Containing Antagonists.
In addition to the imidazole moiety, most of these compounds possess a central electronegative group and a cyclic moiety (Fig. 7). These groups form a conserved pharmacophore with three interactions: 1) a hydrogen bond of the imidazole ring with Glu206 in TM5; 2) a salt bridge of the central electro-negative group with either Tyr374 in TM6 or Asp114 in TM3, which may dictate the torsion of the antagonist; and 3) a π-stacking of the hydrophobic cyclic group with Phe198 in TM5, Tyr189 in ECL2, or Trp110 in TM3 (Levoin et al., 2008 (Fig. 5).
Structure of H3R antagonists.
Extension of the ethylamine side chain, derived from the imidazolyl piperidine compounds by varying the piperidin-substituting groups, resulted in drugs devoid of agonist activity and led to the antagonist thioperamide (Arrang et al., 1987a). The more potent antagonist clobenpropit was derived from imetit through the benzylation of the isothiourea group and other modifications, and its iodinated analog (iodophenpropit) is also a potent antagonist (van der Goot and Timmerman, 2000). Other imidazole-containing antagonists are referred to in Table 3.
The discovery of H3R constitutive activity led to the reclassification of some antagonists as inverse agonists or protean ligands. Thioperamide and ciproxifan, classically considered antagonists, also behave as inverse agonists. Proxyfan acts as agonist, inverse agonist, or neutral antagonist, depending on the level of H3R constitutive activity, and is therefore considered a protean ligand (Morisset et al., 2000; Rouleau et al., 2002; Arrang et al., 2007).
Non-imidazol Antagonists.
The effects of H3R activation on physiologic and pathologic conditions are the main focus of research into the therapeutic potential of selective H3R ligands; however, the imidazole ring reduces penetration of the blood-brain barrier (Young et al., 1988), and imidazole-based drugs inhibit the hepatic cytochrome P450 (Ishikawa et al., 2010). To avoid these side effects, antagonists lacking the imidazole ring, but still capable of forming a hydrogen bond with Glu206, were synthesized (Fig. 7). The first two non-imidazole antagonists were UCL1972 and VUF5391, where the imidazole ring was replaced for a piperidine or pyrrolidine group, respectively. The heterocyclic nitrogen interacts with Glu206, the central oxygen forms a salt bridge with a hydroxyl-containing residue, and the heterocyclic moiety establishes π-stacking interactions (Ganellin et al., 1998, Menge et al., 1998; Levoin et al., 2008).
By exploration of the structure-activity relationship, a series of highly potent and selective non-imidazole antagonists were developed, including ABT-239, JNJ-5207852, NNC 38-1049, and GSK189254, which show antiobesity properties, promote the wake state, and have procognitive effects (Barbier et al., 2004; Esbenshade et al., 2005; Fox et al., 2005; Medhurst et al., 2007).
The imidazole-containing compounds display similar affinities for the H3R across species (see Tables 2 and 3). Nevertheless, some antagonists, such as thioperamide or ciproxifan, are less potent at the hH3R than at the rat H3R, and as mentioned previously, these differences have been attributed to changes in two residues located in TM3 (T119A and A122V). In contrast, the non-imidazole compounds are more potent and selective at the hH3R (West et al., 1999; Ligneau et al., 2000; Parsons and Ganellin, 2006). Furthermore, binding studies with thioperamide and burimamide in rat, human, and monkey tissues indicate the presence of high- and low-affinity sites, which could be related to either different states of the same receptor or to the expression of more than one isoform (West et al., 1990, 1999).
Function
Presynaptic Effects
The best-known function of the H3R is the presynaptic regulation of neurotransmitter release in the CNS and peripheral nervous system. This regulation has been reported for histamine itself, noradrenaline, dopamine, acetylcholine (ACh), GABA, glutamate, serotonin (5-HT), and some neuropeptides. Whereas the evidence for a direct action of H3Rs is consistent for most of these neurotransmitters, this is not established for ACh and dopamine.
Histamine.
The observation that histamine inhibited its own release led to identification of the H3R by Arrang et al. (1983). Control by presynaptic H3Rs of histamine release has been shown for slices of rat cerebral cortex, striatum, hippocampus, and hypothalamus, as well as for rat cerebrocortical synaptosomes (Arrang et al., 1983, 1985a,b). In vivo microdialysis experiments have shown that H3R agonists reduce histamine release in the rat hypothalamus and cerebral cortex, whereas antagonists increased the release in the hypothalamus, nucleus basalis magnocellularis, and cerebral cortex (Jansen et al., 1998; Lamberty et al., 2003; Giannoni et al., 2009).
Acetylcholine.
Modulation of cholinergic transmission by H3Rs was first reported for the peripheral nervous system; histamine and H3R agonists inhibited the electrically evoked contraction of guinea pig ileum strips but had no effect on the action of exogenous ACh (Trzeciakowski, 1987). It was later shown that in the longitudinalis smooth muscle/mienteric plexus preparation, release of [3H]-ACh induced by electrical stimulation was reduced by H3R activation (Poli et al., 1991).
In the CNS, H3R activation inhibited K+-induced [3H]-ACh release from rat entorhinal cortex slices, but this effect was not detected in synaptosomes from the same region or in slices from the hippocampus (Arrang et al., 1995; Alves-Rodrigues et al., 1998), questioning the presence of the receptor on cholinergic nerve terminals. In vivo microdialysis shows that H3R activation reduces ACh release in the rat frontoparietal cortex, hippocampus, nucleus accumbens, and basolateral amygdala (Blandina et al., 1996; Prast et al., 1999; Passani et al., 2001; Bacciottini et al., 2002). These studies also suggest involvement of trans-synaptic effects; for example, H3R-mediated inhibition of GABA or dopamine release could subsequently relieve the inhibitory control by these transmitters of ACh release (Schlicker et al., 1993; Garcia et al., 1997; Prast et al., 1999).
Noradrenaline.
H3R-mediated inhibition of noradrenaline release has been reported for both the CNS and the peripheral nervous system. In cardiac sympathetic terminals, H3R stimulation reduces noradrenaline release by various mechanisms, including inhibition of voltage-activated Ca2+ and Na+ channels, reduction of NHE activity, and transactivation of prostanoid receptors (Endou et al., 1994; Imamura et al., 1995, 1996; Hatta et al., 1997; Mazenot et al., 1999; Silver et al., 2002; Seyedi et al., 2005; Levi et al., 2007).
For the CNS, in vitro experiments indicated that H3R activation reduces depolarization-induced [3H]-noradrenaline release in rodent cerebral cortex, spinal cord, cerebellum, hippocampus, hypothalamus and olfactory bulb, and human cerebral cortex (Schlicker et al., 1989, 1992, 1994, 1999; Celuch, 1995; Timm et al., 1998; Aquino-Miranda et al., 2012). In vivo microdialysis experiments showed that H3R activation reduces noradrenaline release in rat hippocampus and cerebral cortex (Di Carlo et al., 2000; Medhurst et al., 2007).
5-Hydroxytryptamine.
In rat cerebral slices and synaptosomes as well as in SNr slices, H3R activation inhibits 5-HT release evoked by electrical or chemical stimulation (Schlicker et al., 1988; Fink et al., 1990; Threlfell et al., 2004). By contrast, in rat olfactory bulb slices, H3R activation had no effect on depolarization-induced [3H]-5-HT release (Aquino-Miranda et al., 2012).
Dopamine.
In mouse striatum and rat substantia nigra pars reticulata (SNr), depolarization-evoked [3H]-dopamine release is inhibited by H3R activation (Schlicker et al., 1993; Garcia et al., 1997), suggesting that the receptor resides on the terminals and dendrites of the dopaminergic nigrostriatal neurons; however, H3R activation had no effect on depolarization-evoked [3H]-dopamine release from rat and rabbit striatal slices (Smits and Mulder, 1991; Schlicker et al., 1993), indicating differences between species and brain regions.
In microdialysis studies, H3R activation increases the extracellular dopamine levels in rat prefrontal cortex, but not in the striatum (Fox et al., 2005; Medhurst et al., 2007). The systemic or local administration of H3R antagonists augments dopamine release induced by methamphetamine in the nucleus accumbens; however, the effect of the local perfusion of the antagonists was lower than that produced by their systemic administration (Munzar et al., 2004), casting doubt on the presence of H3Rs on the terminals of ventral tegmental area neurons, the main source of dopaminergic innervation to nucleus accumbens (Wise, 2004).
Glutamate.
Presynaptic H3Rs inhibits glutamatergic transmission in the rat hippocampus, striatum, basolateral amygdala, thalamus, and globus pallidus (Doreulee et al., 2001; Jiang et al., 2005; Garduño-Torres et al., 2007; Osorio-Espinoza et al., 2011). In rat striatal and thalamic synaptosomes, H3R stimulation reduces both glutamate release and an increase in the intracellular Ca2+ concentration induced by depolarization (Molina-Hernández et al., 2001; Garduño-Torres et al., 2007). Optogenetic experiments confirmed the H3R-mediated inhibition of corticostriatal and thalamostriatal glutamatergic transmission, and the increased paired-pulse ratio supports the presynaptic location of H3Rs in corticostriatal and thalamostriatal synapses. Of note, H3R activation had no effect on the plasticity of corticostriatal synapses, but the thalamostriatal synapses became significantly facilitatory (Ellender et al., 2011).
GABA.
In slices from rat SNr and striatum, the activation of dopamine D1 receptors, coupled to Gαs proteins, enhances depolarization-evoked [3H]-GABA release, and this effect is selectively counteracted by H3R-mediated inhibition of P/Q-type voltage-activated Ca2+ channels (Garcia et al., 1997; Arias-Montaño et al., 2001, 2007). Likewise, in the nerve terminals of striatopallidal neurons, H3R activation counteracts the facilitatory action of adenosine A2A receptors (A2ARs), which are also coupled to Gαs proteins (Morales-Figueroa et al., 2014).
H3R-mediated inhibition of GABA release has been observed in vivo in rat medial vestibular nucleus and in vitro in primary cultures of rat cerebrocortical neurons and dissociated neurons from the rat hypothalamus ventromedial nucleus (Jang et al., 2001; Bergquist et al., 2006; Dai et al., 2007). In contrast, H3R activation does not modify depolarization-evoked [3H]-GABA release from rat thalamus and olfactory bulb slices (Garduño-Torres et al., 2007; Aquino-Miranda et al., 2012), indicating that not all GABAergic neurons express presynaptic H3Rs.
Neuropeptides.
Histamine inhibits nonadrenergic/noncholinergic contraction of the bronchial and intestinal smooth muscle (Ichinose and Barnes, 1989; Taylor and Kilpatrick, 1992), and H3R activation reduces substance P release from sensorial nerve terminals of the rat hindpaw (Ohkubo et al., 1995) and the release of the calcitonin gene-related peptide from the periarterial nerve terminals of the rat mesenteric artery (Sun et al., 2011).
Postsynaptic effects
There is evidence for postsynaptic H3Rs in some areas of the brain, namely, striatum, cerebral cortex, hippocampus, nucleus accumbens, lateral hypothalamus, and zona incerta (Pillot et al., 2002; González-Sepúlveda et al., 2013; Panula and Nuutinen, 2013; Parks et al., 2014).
Activation of the MAPK and Akt Pathways.
The detection of 42/44-MAPK phosphorylation upon the activation of the rat H3R445 led to the discovery of the link between H3Rs and the MAPK pathway (Drutel et al., 2001), which was also observed for native receptors (Mariottini et al., 2009). In rat hippocampus slices, H3R activation stimulates the MAPK pathway in CA3 pyramidal cells, and MAPK activation seems to be required for H3R-induced memory improvement and consolidation in rats after contextual fear conditioning (Giovannini et al., 2003).
In transfected SK-N-MC cells, hH3R activation stimulates the activity of the Akt/GSK3β axis (Bongers et al., 2007c), and in rat cortical neurons, H3R stimulation results in the phosphorylation of Akt at Ser473 and GSK3β at Ser9, via PI3K and MAPK activation (Mariottini et al., 2009). Akt regulates the expression of apoptosis inhibitors such as Bcl-2 and Bcl-x and thus promotes neuronal cell survival (Song et al., 2004), and in rat cortical neurons, H3R activation increases the expression of Bcl-2 in an Akt-dependent manner. The Akt/GSK3β pathway plays a relevant role in regulating several important cellular processes, including cell plasticity and survival, proliferation, and metabolism, and H3R activation exerts a protective effect against serum deprivation-induced cell death in rat cortical neurons and NMDA-induced neurotoxicity in mixed cultures of mouse cortical cells through the activation of the PI3K-Akt pathway (Mariottini et al., 2009).
GIRK Activation.
In MCH-producing neurons located in the rodent lateral hypothalamus, H3R activation stimulates GIRKs and inhibits neuronal firing (Parks et al., 2014).
H3R Heterodimerization and Functional Consequences
GPCR dimerization is now a well-accepted phenomenon that can be defined as a transitory state in which, through protein-protein interactions, a GPCR can alter the binding, signaling, or desensitization of the second GPCR that it is dimerized with. Although this phenomenon has been described as temporarily, short-lived, and the result of stochastic interactions, it has sufficient impact to cause or modify a physiologic output (Milligan, 2006; Calebiro et al., 2013).
Because of its heteroreceptor nature, the H3R is coexpressed with a large number of GPCRs in different neuronal populations. For example, in striatal medium-sized spiny neurons (MSNs), H3Rs coexist with dopamine D1 (D1R) or D2 (D2R) receptors (González-Sepúlveda et al., 2013), allowing for heterodimer formation. In both interactions, H3R acts as a negative modulator of agonist binding to and signaling of dopamine receptors.
The first insight into H3R/D1R interactions came from functional studies where the H3R inhibited D1R-mediated facilitation of GABA release in SNr and striatum slices (Garcia et al., 1997; Arias-Montaño et al., 2001). In a heterologous expression system, H3R activation induces a shift from cooperative to a noncooperative binding of D1R agonists, which indicates that an intramembrane crosstalk occurs between these receptors. In contrast, D1R stimulation did not modify agonist binding to H3Rs. A change in the D1R signaling pathway from Gαs to Gαi/o was also observed in cAMP formation assays. Furthermore, H3R activation did not induce 42/44-MAPK phosphorylation but did so when the D1R was coexpressed. H3R antagonist prevented the effect of a D1R agonist and vice versa (Ferrada et al., 2009), and cross-antagonism in rat striatal slices of wild-type and D1R-KO mice suggests that the interaction also occurs in vivo. The phosphorylation of 42/44-MAPKs appears to be a particular print of the H3R/D1R interaction, as this effect was not observed in D2R-MSNs. In addition, behavioral analysis showed that a H3R antagonist enhanced D1R-induced locomotor activity (Ferrada et al., 2008; Moreno et al., 2011).
The H3R/D2R interaction is supported by Förster Resonance Energy Transfer analysis in transfected cells and by binding studies in which H3R activation reduced agonist affinity of the D2R high- and low-affinity sites in sheep striatal membranes (Ferrada et al., 2008). The lack of synergism in D2R- and H3R-stimulated [35S]-GTPγS binding in rat striatal membranes (Humbert-Claude et al., 2007), however, argues against a direct H3R/D2R interaction.
The Gαs-coupled A2AR is highly expressed and distinctively limited to D2R-MSNs. A2AR activation inhibits GABA release from MSN collaterals (Kirk and Richardson, 1994), but in the striatopallidal projections, facilitates the release of the neurotransmitter (Mayfield et al., 1993). The latter effect is functionally opposed by H3R activation, which also decreases A2AR affinity for the agonist CGS-21680 in membranes from globus pallidus synaptosomes, suggesting a direct, protein-protein interaction between A2ARs and H3Rs (Morales-Figueroa et al., 2014).
H3Rs and Neurologic and Psychiatric Disorders
The H3R is a potential drug target for the treatment of several important neurologic and psychiatric disorders, and this aspect will be discussed briefly in reference to AD, attention-deficit hyperactivity disorder, PD, GTS, schizophrenia, addiction, and sleep disorders.
Parkinson Disease.
PD is a progressive neurodegenerative movement disorder that primarily results from the death of substantia nigra pars compacta (SNc) dopaminergic neurons. In addition, the noradrenergic, cholinergic, and serotonergic systems are also affected and may contribute to the disorder. Parkinsonian symptoms include bradykinesia, muscular rigidity, rest tremor, and postural and gait impairment (Moore et al., 2005; Kalia and Lang, 2015).
As discussed previously, H3Rs modulate striatal GABAergic, glutamatergic, and dopaminergic transmission at the presynaptic and postsynaptic levels, in addition to controlling the release of GABA, glutamate, and 5-HT in several other nuclei of the basal ganglia. In hemiparkisonian rats, H3R levels increase in the striatum and SNr ipsilateral to the lesioned SNc. Postmortem analysis of PD patients shows increased histaminergic innervation to SN, augmented histamine levels in caudate/putamen, globus pallidus, and SN; higher H3R density in the SNr; and increased H3R mRNA in the external globus pallidus (reviewed in Panula and Nuutinen, 2013). Injection of the H3R agonist immepip into the SNr reverses apomorphine-induced contralateral turning behavior in hemiparkinsonian rats, whereas in naive animals the compound induces ipsilateral turning (García-Ramírez et al., 2004). In hemiparkinsonian rats, apomorphine-induced turning behavior is enhanced by the systemic administration of L-histidine and reduced by inhibiting histamine synthesis (Liu et al., 2008).
Dopamine-replacement therapy has dominated the treatment of PD motor symptoms since the early 1960s; however, chronic administration of the dopamine precursor L-DOPA results in severe side effects, particularly dyskinesias related to excessive D1R-mediated signaling in the basal ganglia (Santini et al., 2007). In parkinsonian marmosets, the systemic administration of immepip exacerbates the symptoms in nontreated animals, but in L-DOPA-treated marmosets, it reduces the total dyskinesia score without affecting the anti-parkinsonian action (Gomez-Ramírez et al., 2006). Altogether, the available information indicates a role for histamine and the H3R in PD pathophysiology and a potential use for drugs acting at the receptor in the treatment of both the disease and the complications of the pharmacologic therapies.
Gilles de la Tourette Syndrome.
GTS is a disorder characterized by motor and phonic tics (echolalia, echopraxia, and coprolalia), sensory and cognitive symptoms, and comorbidities such as obsessive-compulsive and attention-deficit hyperactivity disorders (Shan et al., 2015). GTS is related to the dysfunction of the corticostriatal-thalamic-cortical circuitry and involves the transmitters dopamine, noradrenaline, 5-HT, and histamine (Panula and Nuutinen, 2013; Rapanelli and Pittenger, 2016).
A mutation (W317X) in one of the HDC gene alleles is associated with the familiar occurrence of GTS (Ercan-Sencicek et al., 2010). HDC-knockout mice, with brain histamine levels almost negligible in homozygote animals, exhibit stereotypic movements and spontaneous tic-like symptoms after the intraperitoneal injection of amphetamine (Castellan Baldan et al., 2014), supporting the involvement of the histaminergic system in GTS.
Schizophrenia.
Schizophrenia is a mental disorder defined by positive symptoms (hallucinations, delusions, and thought and movement disorders), negative symptoms (reduced feelings, difficulty to initiate and maintain activities, and reduced speaking), and cognitive symptoms (poor understanding of information, trouble focusing, and problems with working memory) (Flores et al., 2016).
In schizophrenic patients, H3R receptor expression is increased in the prefrontal cortex and decreased in the hippocampus, areas closely related to memory and cognitive processes (Jin et al., 2009). An increase in the histamine metabolite telemethylhistamine was reported for chronic patients (Prell et al., 1995), and antipsychotics drugs affect the activity of the histaminergic neurons by acting at dopamine, 5-HT, and glutamate (NMDA) receptors expressed by these neurons (Javitt and Zukin, 1991; Morisset et al., 1999, 2002) and by antagonizing H1, H2, and H3 receptors (Alves-Rodrigues et al., 1996; Green and Maayani, 1977; Richelson and Souder, 2000). This information suggests that the histaminergic system plays a role in the disease.
Schizophrenia is characterized by the hyperactivity of dopaminergic neurons and the hypoactivity of glutamatergic neuronal cells. H3R antagonists/inverse agonists attenuate dopamine receptor-mediated facilitation of locomotor sensitization (Clapham and Kilpatrick, 1994) and locomotor hyperactivity resultant from blockade of NMDA receptors (Faucard et al., 2006; Mahmood et al., 2012). In DBA/2 mice, administration of a H3R antagonist/inverse agonist reduces the natural deficits in sensorimotor gating (Fox et al., 2005). Although the evidence in animal models supports the antipsychotic properties of H3R antagonists, two clinical trials with ABT-288 and MK-0249 failed to improve the cognitive function in schizophrenic patients (Egan et al., 2013; Haig et al., 2014).
Addiction.
Addiction is a compulsive and persistent dependence on behaviors or substances in which the reward brain circuitry (the dopaminergic mesocortico-limbic system) plays a major role. HDC-KO mice show increased cocaine tolerance and reduction in the place-preference test. Conversely, HDC-KO mice show an increase in alcohol-evoked conditioned place preference and consumption (Zimatkin and Anichtchik, 1999). Substances like alcohol, cocaine, and morphine modulate histamine synthesis, release, and turnover (Nishibori et al., 1985; Ito et al., 1997), suggesting involvement of the histaminergic system in addiction processes. In H3R-KO mice, a reduction in alcohol-induced place preference and consumption is observed, and a similar effect is induced by H3R blockade, indicating the participation of the H3R in alcohol addiction (Brabant et al., 2007; Nuutinen et al., 2010; Galici et al., 2011).
Hyperactivity caused by amphetamine/methamphetamine is attenuated by the H3R antagonists thioperamide, ciproxifan, pitolisant (BF2.649), and ABT-239 (Clapham and Kilpatrick, 1994; Fox et al., 2005; Ligneau et al., 2007a,b; Motawaj and Arrang, 2011); however, other antagonists (GSK-207040, JNJ-5207852, and JNJ-10181457) failed to replicate this response (Komater et al., 2003; Southam et al., 2009). Moreover, thioperamide and clobenpropit increased methamphetamine self-administration (Munzar et al., 2004). On the basis of the previously described cross-talk between the dopaminergic and histaminergic systems and the direct modulation of histaminergic transmission by addictive drugs, targeting the histaminergic neurons is a plausible therapeutic proposal.
Attention-Deficit Hyperactivity Disorder.
Most prevalent in children, attention-deficit hyperactivity disorder is characterized by an ongoing pattern of inattention and/or hyperactivity-impulsivity. This disorder affects several neurotransmitter systems, mainly the dopaminergic, noradrenergic, cholinergic, and serotonergic systems (Vohora and Bhowmik, 2012). H3R antagonists enhance the release of neurotransmitters involved in cognition, including ACh and dopamine in the prefrontal cortex; ACh, dopamine, and noradrenaline in the cingulate cortex; and ACh in the hippocampus (Fox et al., 2005; Medhurst et al., 2007; Ligneau et al., 2007b). In rodents, the proattentional and procognitive actions of these drugs suggest that these compounds have a potential therapeutic use in this disorder (reviewed by Passani and Blandina, 2011; Vohora and Bhowmik, 2012); however, the H3R antagonist bavisant (JNJ-31001074) did not show clinical effectiveness in human adults with attention-deficit hyperactivity disorder, whereas both atomoxetine and methylphenidate induced improvement (Weisler et al., 2012).
Alzheimer Disease.
AD is a progressive neurodegenerative brain disorder resulting in the loss of memory and cognitive functions that is often accompanied by behavioral disturbances like aggression and depression (Querfurth and LaFerla, 2010). The pathologic hallmarks of AD include the accumulation of the protein β-amyloid (Aβ), which leads to the production of extracellular Aβ plaques and hyperphosphorylation of the protein τ. This in turn results in the formation of intracellular neurofibrillary tangles and the massive loss of cholinergic neurons (Brioni et al., 2011).
In AD patients, neurofibrillary tangles occur in the TMN, along with a significant loss (∼50%) of the histaminergic neurons (Nakamura et al., 1993; Shan et al., 2015); but the latter effect is not matched by the reduction (−24%) in HDC mRNA expression, suggesting that enhanced histamine production by the remaining neurons compensates the cell loss. Some reports indicate that brain histamine levels are reduced in hypothalamus, hippocampus, and temporal cortex from AD patients, although others show an increase in the same cerebral regions, together with frontal, parietal, and occipital cortices. Furthermore, H3R mRNA levels seem to be higher in the prefrontal cortex only in female AD patients, indicating a gender-dependent change (Shan et al., 2015). In this regard, differences in histamine levels in AD and normal brains may originate from methodologic issues, genetic background, time-elapsed between death and analysis, and proper diagnosis; however, measurement of histamine content with a sensitive HPLC fluorimetric method in brains from controls and AD patients, with matched age and postmortem time, found a significant decrease in the hypothalamus (−58% of control values), hippocampus (−57%), and temporal cortex (−47%) in AD brains (Panula et al., 1998).
H3R activation could account for the diminished release of noradrenaline and ACh in the prefrontal cortex, both transmitters with an important role in cognition (Blandina et al., 1996; Schlicker et al., 1999). Based on studies with transgenic mice, it may be hypothesized that neurotransmitter release evoked by H3R antagonists leads to postsynaptic effects, such as the phosphorylation of the cAMP response element binding protein, a transcription factor related to cognitive function, or the inhibition of GSK3β, responsible for tau hyperphosphorylation in AD (Hooper et al., 2008; Bitner et al., 2011). These in vivo studies raise the possibility that H3R antagonists indirectly modulate signaling pathways, resulting in symptomatic alleviation in AD patients. H3R antagonists like PF-03654746, GSK189254, MK-0249, ABT-239, and ABT-288 bind the hH3R with high affinity (Ki 0.2 to 3.2 nM), and some have advanced to clinical trials (Brioni et al., 2011; Sadeq et al., 2016).
Sleep Disorders.
The brain circuitry that regulates sleep-wake cycle in mammals comprises cell groups in the thalamus, brainstem, hypothalamus, and basal forebrain. The histaminergic system plays a key role in the maintenance of cortical activation and wakefulness (Lin, 2000). The histaminergic neurons fire tonically, are more active during wakefulness, and send widespread inputs to areas crucially implicated in sleep-wake control, for instance, the cerebral cortex and thalamus. In the latter region, H1R activation decreases a resting leak K+ conductance (IKL), which results in prolonged depolarization of thalamocortical neurons, leading to inhibition of burst firing and the promotion of single-spike firing. This action abolishes sleep-related activity in thalamocortical networks and facilitates the single-spike activity typical of the waking state (McCormick and Bal, 1997).
Impaired wakefulness and increased sleep are observed after the inhibition of histamine synthesis and in HDC-KO mice (Parmentier et al., 2002). The wake-promoting activity of histamine is shared by a variety of H3R antagonists/inverse agonists (Gao et al., 2013). H3Rs presynaptically modulate the release of histamine and other neurotransmitters that participate in the wake-sleep cycle and postsynaptically reduce the excitability of MCH-producing neurons that are also involved in the cycle (Parks et al., 2014). H3R-KO mice present a wide-range of apparently contradictory wake-sleep alterations; the animals show wake deficiency and sleep deterioration but also display signs of enhanced vigilance (Gondard et al., 2013).
Histaminergic neurons are activated by the neuropeptide hypocretin, and type 1 narcolepsy is characterized by excessive daytime sleepiness and cataplexy. The main cause of type 1 narcolepsy is the loss of hypocretinergic neurons; however, an increase in the number of histaminergic neurons has been reported (Valko et al., 2013). Although histamine dysregulation is not central to the pathophysiology, H3R antagonists/inverse agonists such as pitolisant are useful for the treatment of excessive sleepiness disorders, namely, narcolepsy and idiopathic hypersomnia, by improving alertness (Lin et al., 2008; Inocente et al., 2012; Dauvilliers et al., 2013). Furthermore, pitolisant reduces sleepiness in PD patients without interfering with anti-parkinsonian drugs (Schwartz, 2011). On March 2016, pitolisant (Wakix) was approved in the European Union for the treatment of narcolepsy with or without cataplexy in adults, becoming thus the first drug acting at H3Rs that reachs the market (Syed, 2016).
Final Remarks
The histaminergic system exerts a significant modulatory effect on different brain functions, from movement to cognitive processes. The H3R plays a key role in this effect through its wide expression and action as a modulator of several neurotransmitter systems. Although the main effects of H3R activation are known for some brain areas, such as the striatum and the hippocampus, its actions in other areas have yet to be fully explored. Furthermore, determining the precise H3R expression in distinct cells of several neuronal circuits, such as the corticostriato-thalamocortical and mesocortico-limbic circuits, will help understand the participation of the histaminergic system in aspects—such as the control of motor activity, cognition, and the sleep-wake cycle—and certain disorders, including PD, AD, and addiction. A deeper knowledge of the functional and molecular interactions of H3Rs with other receptors is also important for the design of novel therapeutic approaches targeting the histaminergic system, and for this purpose, the elucidation of the H3R crystal structure will represent an important discovery. Finally, more clinical studies are needed to evaluate or confirm the usefulness of H3R ligands in several neuropsychiatric disorders.
Acknowledgments
G. Nieto-Alamilla, R. Márquez-Gómez, A.-M. García-Gálvez, and G.-E. Morales-Figueroa received Conacyt scholarships. G. Nieto-Alamilla is a fellow of Mexico State Council for Science and Technology (Comecyt). The authors offer apologies to all the authors whose work was not included in this review.
Authorship Contribution
Wrote or contributed to the writing of the manuscript: Nieto-Alamilla, Márquez-Gómez, García-Gálvez, Morales-Figueroa, Arias-Montaño.
Footnotes
- Received April 15, 2016.
- Accepted August 24, 2016.
Research in J.-A. A.-M.’s laboratory is supported by Cinvestav and the Mexican Council for Science and Technology (Conacyt).
Abbreviations
- aa
- amino acids
- A2AR
- adenosine A2A receptor
- ACh
- acetylcholine
- AD
- Alzheimer's disease
- CHO
- Chinese hamster ovary
- CNS
- central nervous system
- CT
- carboxyl terminus
- D1R
- dopamine D1 receptor
- D2R
- dopamine D2 receptor
- ECL
- extracellular loop
- 5-HT
- 5-hydroxytriptamine (serotonin)
- GIRK
- G protein–gated inwardly rectifying K+ channel
- GPCR
- G protein–coupled receptor
- GTS
- Gilles de la Tourette syndrome
- H1R
- histamine H1 receptor
- H2R
- histamine H2 receptor
- H3R
- histamine H3 receptor
- H4R
- histamine H4 receptor
- HDC
- histidine decarboxylase
- hH3R
- human H3R
- ICL
- intracellular loop
- IP3
- inositol-1,4,5-trisphosphate
- MAPK
- mitogen-activated protein kinase
- MCH
- melanin-concentrating hormone
- MSN
- striatal medium-sized spiny neuron
- NHE
- Na+/H+ exchanger
- PD
- Parkinson's disease
- PKA
- protein kinase A
- PKC
- protein kinase C
- PLC
- phospholipase C
- PTX
- pertussis toxin
- RT-PCR
- reverse-transcription polymerase chain reaction
- SN
- substantia nigra
- SNc
- substantia nigra pars compacta
- SNr
- substantia nigra pars reticulata
- TM
- transmembrane
- TMN
- tuberomammillary nucleus
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
