I. Introduction and Historical Perspective

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The classification of histamine receptors has to date been based on rigorous classical pharmacological analysis, and as yet, the classification of the three histamine receptors that have been defined by this process, (i.e., the H₁-, H₂-, and H₃-receptors) have not been added to because of more recent molecular biological approaches (Schwartz et al., 1991, 1995; Hill, 1990; Leurs et al., 1995b). The scant number of known histamine receptors, compared with the plethora of receptors for some other endogenous substances, probably reflects the relative neglect of histamine rather than a paucity of its receptors. There is some preliminary evidence of heterogeneity of the known histamine receptors (which will be reviewed later in this article), but the acceptance of additional subtypes still awaits the identification of "sequence differences" within a single species and the development of selective agonists and antagonists providing the structural, recognition, and transductional information necessary for reliable classification.

The first histamine receptor antagonists (popularly referred to as the classical antihistamines but now called H₁-receptor antagonists) were synthesized (Bovet and Staub, 1936; Bovet, 1950) over 20 years after the discovery (Barger and Dale, 1910) and descriptions of some of the physiological effects (Dale and Laidlaw, 1910) of histamine. These accomplishments had been preceded, as for some other endogenous biogenic amines, by its synthesis as a chemical curiosity (Windaus and Vogt, 1907). Early studies of the antihistamines were qualitative, for example, the demonstration of their effectiveness in protecting against bronchospasm produced in guinea pigs by anaphylaxis or administration of histamine (Bovet and Staub, 1936). Though qualitative, these studies yielded compounds, e.g., mepyramine (pyrilamine), that remain major ligands to define histamine receptors.

These antagonists were shown to reduce the effects of histamine on many tissues, notably vascular and extravascular smooth muscle (e.g., guinea pig ileum), but it became apparent that some of the effects of histamine were refractory to these classical antihistamines (Loew, 1947). For example, histamine-stimulated gastric secretion was shown to be unresponsive to three different antihistamines (Ashford et al., 1949). The vasodilator response to histamine in the cat was shown to be only partly sensitive to an antihistamine, leading to the suggestion that histamine causes vasodilatation by combining with more than one receptor (Folkow et al., 1948). The application of the method of Schild (Arunlakshana and Schild, 1959) to the classification of receptors revealed that the pA₂ (log K_B) value of mepyramine for antagonism of the positive chronotropic effect of histamine on the right atrium of the guinea pig differed from mepyramine's pA₂ value for antagonism of the contractile response to histamine in guinea pig ileum, implying that the receptors involved were distinct (Arunlakshana and Schild, 1959; Trendelenburg, 1960). The histamine receptor in guinea pig ileum and in other tissues that showed the same or similar pA₂ value for these early antihistamines was then named the H₁-receptor (Ash and Schild, 1966). As the relative potencies of these histamine antagonists and histamine agonists on gastric acid secretion, relaxation of rat uterus, and chronotropy of the guinea pig right atrium differed from those on the H₁-receptor, it was concluded that a separate histamine receptor was involved in these responses.

The development of specific antagonists (H₂-antagonists) for this novel receptor represents a classic example of rational drug design (Black et al., 1972; Black, 1989) and showed the "practical value" (Green and Maayani, 1987; Jenkinson, 1987) of a quantitative approach to the analysis of receptor antagonism (Arunlakshana and Schild, 1959). Burimamide was the first compound to be described (Black et al., 1972) that had a higher pA₂ for antagonism of the histamine-mediated responses on guinea pig atrium and rat uterus than the pA₂ determined for antagonism of the contractile response to histamine in guinea pig ileum. Burimamide was also able to reduce gastric acid secretion in dogs and humans and to reduce the blood pressure response of the cat to histamine (Black et al., 1972). A large number of more potent and selective H₂-receptor antagonists have since been developed (Cooper et al., 1990), although further quantitative investigations of the antagonist potency of burimamide on other histamine-mediated responses contributed to the definition and classification of the histamine H₃-receptor (Arrang et al., 1983).

The third histamine receptor was also defined by a functional assay. Histamine was found to inhibit its own synthesis and release in rat cerebral cortical slices, and the effects of H₁- and H₂- receptor agonists and antagonists indicated a distinct receptor (Arrang et al., 1983, 1987b). A highly selective agonist, R-()-methylhistamine, and antagonist, thioperamide, clearly defined the H₃-receptor (Arrang et al., 1987). Since that time, considerable efforts have been made to develop other H₃-receptor-selective agonists and antagonists (Garbarg et al., 1992; Jansen et al., 1992; Van der Goot et al., 1992; Vollinga et al., 1994; Ganellin et al., 1995; Ligneau et al., 1995; Stark et al., 1996b,c).

Table 1 summarizes some of the operational characteristics used to define the nature of the histamine receptor involved in different tissue responses. Histamine derivatives are numbered according to the system given in figure 1 (Black and Ganellin, 1974).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Numbering for histamine derivatives.

	II. Histamine H1-Receptor

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The study of the distribution of histamine H₁-receptors in different mammalian tissues has been greatly aided by the development of selective radioligands for this particular histamine receptor subtype. [³H]mepyramine was originally developed in 1977 (Hill et al., 1977) and since that time has been used successfully to detect H₁-receptors in a wide variety of tissues including: mammalian brain; smooth muscle from airways, gastrointestinal tract, genitourinary system, and the cardiovascular system; adrenal medulla; and endothelial cells and lymphocytes (Hill, 1990). In some tissues and cells, however, it is notable that [³H]mepyramine additionally binds to secondary non-H₁-receptor sites (Chang et al., 1979a; Hill and Young, 1980; Hadfield et al., 1983; Mitsuhashi and Payan, 1988; Arias-Montano and Young, 1993; Dickenson and Hill, 1994; Leurs et al., 1995b). In rat liver, in which [³H]mepyramine predominantly binds to a protein homologous with debrisoquine 4-hydroxylase cytochrome P450 (Fukui et al., 1990), quinine can be used to inhibit this nonspecific binding. This observation has led Liu et al. (1992) to suggest that quinine may be used to inhibit binding to other lower affinity sites. However, it is clear that not all secondary binding sites for [³H]mepyramine are sensitive to inhibition by quinine (Dickenson and Hill, 1994). Thus, in DDT₁MF-2 cells, a 38 to 40 kDa protein has been isolated, which binds H₁-receptor antagonists with K_D values in the micromolar range (Mitsuhashi and Payan, 1988, Mitsuhashi et al., 1989) but which is not sensitive to inhibition by quinine (Dickenson and Hill, 1994). Nevertheless, DDT₁MF-2 cells can be shown to additionally possess [³H]mepyramine binding sites that have the characteristics of H₁-receptors (i.e., K_D values in the nanomolar range) and to mediate functional responses, which are clearly produced by histamine H₁-receptor activation (Dickenson and Hill, 1992; White et al., 1993; Dickenson and Hill, 1994).

Other radioligands that have been used to study histamine H₁-receptors are [³H]mianserin (Peroutka and Snyder, 1981), [³H]doxepin (Tran et al., 1981; Kamba and Richelson, 1984; Taylor and Richelson, 1982), [¹²⁵I]iodobolpyramine (Bouthenet et al., 1988), [¹²⁵I]iodoazidophenpyramine (Ruat et al., 1988), and [³H](+)-N-methyl-4-methyldiphenhydramine (Treherne and Young, 1988b). [¹²⁵I]Iodobolpyramine has been used for autoradiographic localization of H₁-receptors in guinea pig brain, although less success has been achieved in rat brain (Körner et al., 1986; Bouthenet et al., 1988). The very slow dissociation of [³H]mepyramine from H₁-receptors at low temperatures (e.g., 4°C) does, however, mean that this ligand can also be used for autoradiography (Palacios et al., 1981a,b; Rotter and Frostholm, 1986). [¹²⁵I]Iodoazidophenpyramine is a very potent H₁-receptor antagonist that can bind irreversibly to H₁-receptors following irradiation with ultraviolet light (Ruat et al., 1988). [¹¹C]Mepyramine and [¹¹C]doxepin have also proved useful for imaging histamine H₁-receptors in the living human brain (Villemagne et al., 1991; Yanai et al., 1992, 1995).

H₁-receptors have been extensively studied in blood vessels (Barger and Dale, 1910; Dale and Laidlaw, 1910; Folkow et al., 1948; Black et al., 1972) and other smooth muscle preparations (Ash and Schild, 1966; Black et al., 1972; Marshall, 1955; Hill, 1990). In smooth muscles, such as the guinea pig ileum, which freely generate muscle action potentials, modulation of action-potential discharge by low concentrations of histamine is an important mechanism by which tension is increased (Bolton, 1979; Bolton et al., 1981; Bülbring and Burnstock, 1960). In guinea pig ileum, there is also evidence that a component of the contractile response to histamine is mediated by inositol 1,4,5-trisphosphate-induced mobilization of intracellular calcium (Morel et al., 1987; Bolton and Lim, 1989; Donaldson and Hill, 1986b). In nonexcitable smooth muscles, such as airway and vascular smooth muscle, contractile responses to H₁-receptor stimulation primarily involve mobilization of calcium from intracellular stores as a consequence of inositol phospholipid hydrolysis (Matsumoto et al., 1986; Kotlikoff et al., 1987; Takuwa et al., 1987; Hall and Hill, 1988; Paniettieri et al., 1989; Van Amsterdam et al., 1989).

In vascular endothelial cells, H₁-receptor stimulation leads to several cellular responses including: (a) changes in vascular permeability (particularly in postcapillary venules) as a result of endothelial cell contraction (Majno and Palade, 1961; Majno et al., 1968; Meyrick and Brigham, 1983; Grega, 1986; Killackey et al., 1986; Svensjo and Grega, 1986); (b) prostacyclin synthesis (McIntyre et al., 1985; Brotherton, 1986; Carter et al., 1988; Resink et al., 1987); (c) synthesis of platelet-activating factor (McIntyre et al., 1985); (d) release of Von Willebrand factor (Hamilton and Sims, 1987); and (e) release of nitric oxide (Van De Voorde and Leusen, 1993; Toda, 1984). The H₁-receptor has also been characterized on human T lymphocytes using [¹²⁵I]iodobolpyramine (Villemain et al., 1990) and shown to increase [Ca²⁺]_i (Kitamura et al., 1996).

Histamine H₁-receptors have long been established to be present in the adrenal medulla and to elicit the release of catecholamines (Emmelin and Muren, 1949; Staszewska-Barczak and Vane, 1965; Robinson, 1982; Livett and Marley, 1986; Noble et al., 1988). Thus, histamine can induce the release of both adrenaline and noradrenaline from cultured bovine adrenal chromaffin cells (Livett and Marley, 1986). In these cells, histamine can also stimulate phosphorylation of the catecholamine biosynthesis enzyme tyrosine hydroxylase via a mechanism that involves release of intracellular calcium (Bunn et al., 1995). In addition to its effects on catecholamine synthesis and release from adrenal chromaffin cells, histamine can also elicit the release of leucine- and methionine-enkephalin (Bommer et al., 1987). Furthermore, after prolonged exposure to histamine, there is a marked increase in messenger ribonucleic acid-encoding proenkephalin A (Bommer et al., 1987; Kley, 1988; Wan et al., 1989).

In human atrial myocardium and guinea pig ventricle, histamine produces negative inotropic effects (Guo et al., 1984; Genovese et al., 1988; Zavecz and Levi, 1978). In human myocardium, this response is associated with inhibitory effects on heart rate and can be unmasked when the positive effects of histamine on the rate and force of contraction (mediated via H₂-receptors) are attenuated by conjoint administration of adenosine or adenosine A₁-receptor agonists (Genovese et al., 1988). However, in guinea pig left atria (Reinhardt et al., 1974, 1977; Steinberg and Holland, 1975; Hattori et al., 1983, 1988a) and rabbit papillary muscle (Hattori et al., 1988b), histamine produces a positive inotropic response via a mechanism that is not associated with a rise in adenosine 3c,5c-cyclic monophosphate (cAMP^b) levels (see Hill, 1990).

Histamine H₁-receptors are widely distributed in mammalian brain (Hill, 1990; Schwartz et al., 1991). In human brain, higher densities of H₁-receptors are found in neocortex, hippocampus, nucleus accumbens, thalamus, and posterior hypothalamus, whereas cerebellum and basal ganglia show lower densities (Chang et al., 1979b; Kamba and Richelson, 1984; Martinez-Mir et al., 1990; Villemagne et al., 1991; Yanai et al., 1992). The distributions in rat (Palacios et al., 1981a) and guinea pig (Palacios et al., 1981b; Bouthenet et al., 1988) are similar to each other and to humans with the exception that the guinea pig cerebellum shows high density (Ruat and Schwartz, 1989; Chang et al., 1979b; Hill and Young, 1980; Palacios et al., 1981b; Bouthenet et al., 1988). In most brain areas, there was overlap of H₁-receptor binding sites and messenger ribonucleic acid levels except in hippocampus and cerebellum in which the discrepancy is likely to reflect the presence of abundant H₁-receptors in dendrites of pyramidal and Purkinje cells, respectively (Traiffort et al., 1994). Histamine H₁-receptor activation causes inhibition of firing and hyperpolarization in hippocampal neurons (Haas, 1981) and an apamine-sensitive outward current in olfactory bulb interneurons (Jahn et al., 1995), effects most likely produced by intracellular Ca²⁺ release. However, many other notably vegetative ganglia (Christian et al., 1989), hypothalamic supraoptic (Haas et al., 1975), brainstem (Gerber et al., 1990; Khateb et al., 1990), thalamic (McCormick and Williamson, 1991), and human cortical neurons (Reiner and Kamondi, 1994) are excited by histamine H₁-receptor activation through a block of a potassium conductance.

Although a large number of compounds have been synthesized as selective and competitive antagonists of the histamine H₁-receptor (see for example Casy, 1977; Ganellin, 1982), chemical effort directed at the generation of highly potent and selective H₁-receptor agonists has not achieved the same success. Modification of the ethylamine side chain of histamine is not favorable for H₁-receptor agonism (Leurs et al., 1995b). Furthermore, resolution of the enantiomers of the chiral compounds generated by methylation of the - or -positions did not reveal any stereoselectivity of the side chain for the H₁-receptor (Arrang et al., 1987; Leurs et al., 1995). Alkylation of the side chain amine group does not drastically reduce H₁-receptor activity, but N- and N,N-dimethylhistamine are also potent agonists for the H₃-receptor (table 2; fig. 2; Arrang et al., 1983). Modification of the imidazole moiety of histamine has been the most successful approach for obtaining agonists with selectivity for the H₁-receptor. Replacement of the imidazole moiety of histamine by other aromatic heterocyclic ring structures in 2-pyridylethylamine and 2-thiazolylethylamine yields two compounds with selectivity for the H₁-receptor (table 2; fig. 2). Both compounds act as full agonists in producing contraction of guinea pig ileum (Donaldson and Hill, 1986c), but in other tissues (e.g., guinea pig cerebral cortical slices or DDT₁MF-2 cells), 2-pyridylethylamine behaves as a low-efficacy agonist (Donaldson and Hill, 1986a; White et al., 1993). Substitutions in the 2-position of the imidazole ring of histamine have produced compounds that are the most selective H₁-agonists available (Zingel et al., 1995). Thus, 2(3-bromophenyl)histamine and 2[3-(trifluoromethyl)phenyl]histamine are both relatively potent and highly selective H₁-agonists (table 2; fig. 2; Leschke et al., 1995). Both compounds appear to be potent H₁-agonists in guinea pig ileum (Leschke et al., 1995), although some of the halogenated 2-phenylhistamines are low-efficacy agonists in DDT₁MF-2 cells (Zingel et al., 1990; White et al., 1993) and in guinea pig aorta (Leschke et al., 1995) and can exhibit partial agonist properties.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Histamine receptor agonists (H1, H2, and H3).

Mepyramine (also known as pyrilamine) is the reference selective and high-affinity H₁-receptor antagonist (table 3; Hill, 1990). Other classical H₁-antagonists that have been used for characterization purposes include chlorpheniramine, tripelennamine, promethazine, and diphenhydramine (fig. 3). Some of these, however, possess marked muscarinic receptor antagonist properties (Hill, 1990, 1987), and consequently the selectivity of these compounds between the three different histamine receptors (table 3) does not guarantee an unambiguous characterization. This can only be achieved by appropriate quantitative assessment of receptor antagonism, preferably with a range of compounds of very different chemical structure. The stereoisomers of chlorpheniramine are particularly useful in this regard (table 3). The enantiomers of 4-methyl-diphenhydramine and brompheniramine also differ by two orders of magnitude in their affinity for the H₁-receptor (Chang et al., 1979b; Treherne and Young, 1988b). The geometric isomer trans-triprolidine is three orders of magnitude more potent than its cis counterpart and is one of the most potent H₁-antagonists available for the guinea pig H₁-receptor (tables 3 and 4; Ison et al., 1973). The tricyclic antidepressants amitriptyline and doxepin are also very potent H₁-receptor antagonists (K_D 0.6 and 0.1 nM respectively; Figge et al., 1979; Aceves et al., 1985).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Histamine H1-receptor antagonists.

At therapeutic dosages, many of the classical H₁-antihistamines give rise to sedative side effects that have been attributed to occupancy of H₁-receptors in the central nervous system (CNS) (Schwartz et al., 1981; Nicholson et al., 1991; Leurs et al., 1995b). Most of the classical H₁-antihistamines, including promethazine and (+)-chlorpheniramine, readily cross the blood-brain barrier. However, several compounds that penetrate poorly into the CNS and appear to be devoid of central depressant effects are now available (fig. 4). These include terfenadine (Rose et al., 1982; Wiech and Martin, 1982), astemizole (Laduron et al., 1982; Niemegeers et al., 1982), mequitazine (Uzan and Le Fer, 1979), loratadine (Ahn and Barnett, 1986), acrivastine (Leighton et al., 1983; Cohen et al., 1985), cetirizine (Timmerman, 1992b), and temelastine (Brown et al., 1986; Calcutt et al., 1987). The pK_i values for these agents are given in table 5 (Ter Laak et al., 1994).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   "Nonsedating" H1-receptor antagonists. , The pharmaceutical product may be a mix of (E) and (Z) forms.

Photoaffinity binding studies using [¹²⁵I]iodoazidophenpyramine and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis have indicated that the H₁-receptor protein has a molecular weight of 56 kDa under reducing conditions in rat, guinea pig, and mouse brain (Ruat et al., 1988, 1990b; Ruat and Schwartz, 1989). Similarly, studies in bovine adrenal medullar membranes with another photoaffinity ligand [³H]azidobenzamide (Yamashita et al., 1991b) found labeled peptides in the size range 53 to 58 kDa. Interestingly, the specifically labeled H₁-receptor (with [¹²⁵I]iodoazidophenpyramine) in guinea pig heart was found to have a substantially higher molecular weight, although there is no obvious difference in the pharmacological characteristics of the H₁-receptor in this tissue (Ruat et al., 1990a).

The bovine adrenal medulla H₁-receptor was cloned in 1991 by expression cloning in the Xenopus oocyte system (Yamashita et al., 1991a). The deduced amino acid sequence represents a 491 amino acid protein with a calculated molecular weight of 56 kDa (table 6). The protein has the seven putative transmembrane (TM) domains expected of a G-protein-coupled receptor and possesses N-terminal glycosylation sites. A striking feature of the proposed structure is the very large third intracellular loop (212 amino acids) and relatively short (17 amino acids) intracellular C terminal tail. The availability of the bovine sequence and lack of introns has enabled the H₁-receptor to be cloned from several species (table 6) including rat (Fujimoto et al., 1993), guinea pig (Horio et al., 1993; Traiffort et al., 1994), mouse (Inove et al., 1996), and human (De Backer et al., 1993; Fukui et al., 1994; Moguilevsky et al., 1994; Smit et al., 1996c). The human histamine H₁-receptor gene has now been localized to chromosome 3 bands 3p14-p21 (Le Coniat et al., 1994).

At the present time, these different clones should be regarded as true species homologues of the histamine H₁-receptor, even though there are notable differences between them in some antagonist potencies (table 4). Unfortunately, the number of H₁-receptor antagonists evaluated in binding studies in cells transfected with the different recombinant receptors is rather limited. Nevertheless, it is clear that the stereoisomers of chlorpheniramine show marked differences between species. For example, the guinea pig H₁-receptor has a K_D of 0.9 nM for (+)-chlorpheniramine, whereas for the rat H₁-receptor, the value is nearer 8 nM (table 4). Similar differences for this compound and others (notably mepyramine and triprolidine) have been reported for the native H₁-receptors in guinea pig and rat brain, respectively (table 4; Chang et al., 1979b; Hill and Young, 1980; Hill, 1990). Such species differences may also explain why [¹²⁵I]iodobolpyramine can label guinea pig CNS H₁-receptors but is unable to detect H₁-receptors in rat brain (Körner et al., 1986; Bouthenet et al., 1988). The native H₁-receptor protein has been solubilized from both guinea pig and rat brain membranes (Toll and Snyder, 1982; Treherne and Young, 1988a), and the solubilized receptor retains the same differences in H₁-antagonist potency for (+)-chlorpheniramine as that observed in membranes (Toll and Snyder, 1982). What is not clear, however, is why mepyramine appears to be more potent as an antagonist of the recombinant rat H₁-receptor (expressed in C6 cells) than it is of the native H₁-receptor in rat brain membranes (table 4; Chang et al., 1979b; Hill and Young, 1980; Fujimoto et al., 1993). The recombinant study performed in rat C6 cells (Fujimoto et al., 1993) is complicated by the presence of a low level of endogenous H₁-receptors (Peakman and Hill, 1994), but a high affinity for mepyramine (K_D = 1 nM) has been deduced from functional studies in untransfected C6 cells (table 4; Peakman and Hill, 1994).

Site-directed mutagenesis has begun to shed some light on the binding domains for H₁-agonists and -antagonists. Amino acid sequence alignment of the cloned histamine H₁- and H₂-receptors (see fig. 5) has led to the suggestion that the third (TM3) and fifth (TM5) transmembrane domains of the receptor proteins are responsible for binding histamine (Birdsall, 1991; Timmerman, 1992a). Aspartate (107) in TM3 of the human H₁-receptor, which is conserved in all aminergic receptors, has been shown to be essential for the binding of histamine and H₁-receptor antagonists to the H₁-receptor (Ohta et al., 1994). In the ₂- and ₂-adrenoceptors, two serine residues in TM5 accept the phenolic hydroxyl groups of the catechol ring of noradrenaline. In the H₁-receptor, the residues corresponding to asparagine (198) and threonine (194) are in corresponding positions in TM5 of the human H₁-receptor. However, substitution of an alanine for threonine (194) did not influence either agonist or antagonist binding (Ohta et al., 1994; Moguilevsky et al., 1995). Substitution of alanine (198) for asparagine (198) substantially decreased agonist, but not antagonist affinity (Ohta et al., 1994; Moguilevsky et al., 1995). Similar mutations to the corresponding residues (threonine (203) and asparagine (207) in the guinea pig H₁-receptor sequence produce very similar results (Leurs et al., 1994a). It is interesting to note, however, that whereas 2-methylhistamine is similarly affected by the asparagine²⁰⁷ alanine mutation, the H₁-selective agonists 2-thiazolylethylamine, 2-pyridylethylamine, and 2-(3-bromophenyl)histamine are much less affected by this mutation (Leurs et al., 1994a). These data suggest that asparagine (207) interacts with the N-nitrogen of the imidazole ring of histamine. Furthermore, Leurs et al. (1995a) have recently shown that lysine (200) interacts with the N-nitrogen of histamine and is important for the activation of the H₁-receptor by histamine and the nonimidazole agonist, 2-pyridylethylamine. Interestingly, however, the lysine (200) alanine mutation did not alter the binding affinity of 2-pyridylethylamine to the guinea pig H₁-receptor (Leurs et al., 1995).

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Alignment of amino acid sequences of the human histamine H1- and H2-receptors. Residues that are identical in the two sequences are shown in bold. Lines show the putative transmembrane spanning domains.

The primary mechanism by which histamine H₁-receptors produce functional responses in cells is the activation of phospholipase C via a pertussis toxin-insensitive G-protein that is probably related to the G_q/11 family of G-proteins (Hill, 1990; Leurs et al., 1995b). The number of tissues and cell types in which a histamine H₁-receptor-mediated increase in either inositol phosphate accumulation or intracellular calcium mobilization has been described is extensive, and further details are provided in several comprehensive reviews (Hill, 1990; Hill and Donaldson, 1992; Leurs et al., 1995b). Stimulation by histamine of [³H]inositol phosphate accumulation and calcium mobilization has also been observed in Chinese hamster ovary (CHO) cells transfected with the human, bovine, and guinea pig H₁-receptor complementary deoxyribonucleic acid (cDNA) (Leurs et al., 1994c; Smit et al., 1996c; Iredale et al., 1993; Megson et al., 1995). It is worth noting, however, that in some tissues, histamine can stimulate inositol phospholipid hydrolysis independently of H₁-receptors. Thus, in the longitudinal smooth muscle of guinea pig ileum and neonatal rat brain (Donaldson and Hill, 1985, 1986b; Claro et al., 1987), a component can be identified in the response to histamine that is resistant to inhibition by H₁-receptor antagonists. It remains to be established, however, whether these effects are due to "tyramine-like" effects of histamine on neurotransmitter release (Bailey et al., 1987; Young et al., 1988a) or direct effects of histamine on the associated G-proteins (Seifert et al., 1994).

In addition to effects on the inositol phospholipid signaling systems, histamine H₁-receptor activation can lead to activation of several other signaling pathways, many of which appear to be secondary to changes in intracellular calcium concentration or the activation of protein kinase C. Thus, histamine can stimulate nitric oxide synthase activity (via a Ca²⁺/calmodulin-dependent pathway) and subsequent activation of soluble guanylyl cyclase in a variety of different cell types (Schmidt et al., 1990; Leurs et al., 1991a; Yuan et al., 1993; Casale et al., 1985; Duncan et al., 1980; Hattori et al., 1990; Sertl et al., 1987). Arachidonic acid release and the synthesis of arachidonic acid metabolites such as prostacyclin and thromboxane A₂ can also be enhanced by H₁-receptor stimulation (Carter et al., 1988; Resink et al., 1987; Leurs et al., 1994c; Muriyama et al., 1990). Interestingly, in CHO-K1 cells transfected with the guinea pig H₁-receptor, the histamine-stimulated release of arachidonic acid is partially inhibited (approximately 40%) by pertussis toxin, whereas the same response in HeLa cells possessing a native H₁-receptor was resistant to pertussis toxin treatment (Leurs et al., 1994c). The reason for this difference remains to be established, but it does caution against the use of signal transduction pathways in highly expressed recombinant cell systems as a primary receptor classification tool. This point is best illustrated by the fact that in intact cellular systems, H₁-receptor activation can produce substantial changes in the intracellular levels of cAMP. In most tissues, histamine H₁-receptor activation does not activate adenylyl cyclase directly but acts to amplify direct cAMP responses to histamine H₂-, adenosine A₂-, and vasoactive intestinal polypeptide receptors (Palacios et al., 1978; Al-Gadi and Hill, 1987, 1985; Donaldson et al., 1989; Garbarg and Schwartz, 1988; Magistretti and Schorderet, 1985; Marley et al., 1991). In many of these cases, a role for both intracellular Ca²⁺ ions and protein kinase C has been implicated in this augmentation response (Al-Gadi and Hill, 1987; Schwabe et al., 1978; Garbarg and Schwartz, 1988). In CHO cells transfected with the bovine or guinea pig H₁-receptor, H₁-receptor activation can also lead to both direct cAMP responses and to an enhancement of forskolin-stimulated cAMP formation (Leurs et al., 1994c; Sanderson et al., 1996).

	III. Histamine H2-Receptor

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Unlike the situation with H₁-selective radioligands, attempts to map the distribution of H₂-receptors by using radiolabeled H₂-receptor antagonists have met with variable success (Hill, 1990). Thus, [³H]cimetidine and [³H]ranitidine have proved unsuitable as H₂-radioligands, and in the case of cimetidine, the binding to sites specifically labeled with the radioligand is potently inhibited by imidazoles that have very low H₂-receptor binding affinities (Burkard, 1978; Kendall et al., 1980; Smith et al., 1980; Bristow et al., 1981; Warrender et al., 1983). More success has been achieved with [³H]tiotidine, which has a higher affinity for the H₂-receptor (table 7) in guinea pig brain, lung parenchyma, and CHO-K1 cells transfected with the human H₂-receptor cDNA (Gajtkowski et al., 1983; Norris et al., 1984; Sterk et al., 1986; Foreman et al., 1985a; Gantz et al., 1991a), although studies in rat brain were not successful (Maayani et al., 1982). At the present time, [¹²⁵I]iodoaminopotentidine is the most successful H₂-radioligand (Hirschfeld et al., 1992). It has high affinity (K_D = 0.3 nM) for the histamine H₂-receptor in brain membranes (Martinez-Mir et al., 1990; Ruat et al., 1990b; Traiffort et al., 1992a) and CHO-K1 cells expressing the cloned rat H₂-receptor (Traiffort et al., 1992b). The compound has also been used for autoradiographic mapping of H₂-receptors in mammalian brain (Ruat et al., 1990a; Traiffort et al., 1992a). In human brain, histamine H₂-receptors are widely distributed with highest densities (measured using [¹²⁵I]iodoaminopotentidine) in the basal ganglia, hippocampus, amygdala, and cerebral cortex (Traiffort et al., 1992a). Lowest densities were detected in cerebellum and hypothalamus (Traiffort et al., 1992a). A similar distribution has been observed in guinea pig brain (Ruat et al., 1990b). [¹²⁵I]Iodoazidopotentidine has successfully been used for irreversible labeling (Ruat et al., 1990b; Hirschfeld et al., 1992).

Most information to date on the distribution of histamine H₂-receptor, however, has been provided by functional studies in different tissues (Hill, 1990). Histamine H₂-receptor-stimulated cAMP accumulation or adenylyl cyclase activity has been demonstrated in a variety of tissues including brain (Hegstrand et al., 1976; Green et al., 1977; Kanof et al., 1977; Palacios et al., 1978; Gajtkowski et al., 1983; Al-Gadi and Hill, 1985, 1987), gastric cells (Soll and Wollin, 1979; Gespach et al., 1982), and cardiac tissue (Johnson et al., 1979a,b; Kanof and Greengard, 1979a; Johnson, 1982). Histamine H₂-receptors have a potent effect on gastric acid secretion, and the inhibition of this secretory process by H₂-receptor antagonists has provided evidence for an important physiological role of histamine in the regulation of gastric secretion (Black et al., 1972; Black and Shankley, 1985; Soll and Berglindh, 1987). High concentrations of histamine are also present in cardiac tissues of most animal species and can mediate positive chronotropic and inotropic effects on atrial or ventricular tissues via H₂-receptor stimulation (Black et al., 1972; Inui and Imamura, 1976; Levi et al., 1982; Hattori et al., 1983; Hattori and Levi, 1984; Hescheler et al., 1987; Levi and Alloatti, 1988). H₂-receptor-mediated smooth muscle relaxation has also been documented in airway, uterine, and vascular smooth muscle (Black et al., 1972; Reinhardt and Ritter, 1979; Gross et al., 1981; Eyre and Chand, 1982; Edvinsson et al., 1983; Foreman et al., 1985b; Ottosson et al., 1989). Finally, histamine H₂-receptors can inhibit a variety of functions within the immune system (Hill, 1990). H₂-receptors on basophils and mast cells have been shown to negatively regulate the release of histamine (Bourne et al., 1971; Lichtenstein and Gillespie, 1975; Lett-Brown and Leonard, 1977; Ting et al., 1980; Plaut and Lichtenstein, 1982). Furthermore, there is increasing evidence that H₂-receptors on lymphocytes can inhibit antibody synthesis, T-cell proliferation, cell-mediated cytolysis, and cytokine production (Bourne et al., 1971; Melmon et al., 1974, 1981; Griswold et al., 1984; Khan et al., 1985, 1986; Sansoni et al., 1985; Melmon and Khan, 1987). In the CNS, histamine H₂-receptor activation can inhibit nerve cells (Haas and Bucher, 1975; Haas and Wolf, 1977), but the most intriguing action is a block of the long-lasting after-hyperpolarization and the accommodation of firing, an effect with a remarkably long duration leading to potentiation of excitation in rodents (Haas and Konnerth, 1983; Haas and Greene, 1986) and human brain (Haas et al., 1988). A slow excitation is also common (Greene and Haas, 1989; Phelan et al., 1990). Synaptic transmission in the hippocampus is profoundly enhanced (Kostopoulos et al., 1988), and synaptic plasticity is induced or enhanced (Brown et al., 1995). An increase of the hyperpolarization-activated current has also been described in thalamic relay neurons (McCormick and Williamson, 1991). Indications for non-cAMP mediated actions of H₂-receptor activation are given by Haas et al. (1978) and Jahn et al. (1995).

The initial definition of the H₁- and H₂-subclasses of histamine receptor by Ash and Schild (1966) and Black and colleagues (1972) led to a successful search for H₂-receptor selective antagonists with clinical relevance for the treatment of peptic ulcer. Burimamide was the first compound developed that showed selectivity for the H₂-receptor (Black et al., 1972), but more recent work has shown that this compound is a more potent H₃-receptor antagonist (Arrang et al., 1983). Cimetidine and metiamide were developed directly from burimamide (Black et al., 1974; Brimblecombe et al., 1975; Ganellin, 1978). Since then, a large number of compounds have been developed with H₂-receptor antagonist properties [see Ganellin (1992) for review]. These include ranitidine (Bradshaw et al., 1979), tiotidine (Yellin et al., 1979), nizatidine (Lin et al., 1986), famotidine (Takeda et al., 1982), and mifentidine (Donetti et al., 1984), which have been extensively used for characterization purposes (table 3; fig. 6). Iodoaminopotentidine (K_D = 2.5 nM) is one of the most potent H₂-receptor antagonists available, and, as mentioned above, this compound has been used as a successful radioligand (Hirschfeld et al., 1992). Most H₂-receptor antagonists are polar compounds and penetrate poorly into the CNS. Although this property is of great use for selective actions on peripheral tissues (e.g., gastric mucosa), it does limit the use of the compounds for the in vivo evaluation of H₂-receptor function within the CNS. However, one compound (zolantidine) is a potent and selective brain-penetrating histamine H₂-receptor antagonist (table 3; Calcutt et al., 1988; Young et al., 1988b). Both cimetidine and ranitidine have been shown to demonstrate inverse agonism on histamine H₂-receptors transfected into CHO cells (Smit et al., 1996a). Thus, in CHO cells expressing high levels of H₂-receptors, in which a considerable constitutive activation of H₂-receptors was demonstrated, cimetidine and ranitidine inhibited basal adenylyl cyclase activity (Smit et al., 1996a). In contrast, burimamide behaved as a neutral antagonist (Smit et al., 1996a).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Histamine H2-receptor antagonists.

4-Methylhistamine was the first agonist described that had any selectivity for the H₂-receptor (Black et al., 1972), although more potent and selective H₂-agonists are now available (table 2). It is noteworthy that many of the selective H₂-agonists exhibit H₁- or H₃-antagonist properties (see table 2); consequently the demonstration of H₂-agonism in a given tissue or cell type needs confirming with H₂-antagonists. Impromidine is approximately 48 times more potent than histamine in mediating atrial chronotropic responses, but in several other H₂-receptor-containing tissues, its relative potency and efficacy are lower (Durant et al., 1978; Leurs et al., 1995b). A large number of impromidine analogues have been synthesized and evaluated for H₂-agonism. These studies have led to the development of the potent H₂-agonists, sopromidine and arpromidine (table 2; Timmerman, 1992c). Arpromidine and analogues are potential candidates for treatment of congestive heart failure (Buschauer, 1989; Buschauer and Baumann, 1991; Mörsdorf et al., 1990). Another potent H₂-agonist has been derived as an analogue of dimaprit by considering cyclic forms of the isothiourea group (Eriks et al., 1992).

Photoaffinity binding studies using [¹²⁵I]iodoazidopotentidine and sodium dodecyl sulfate-polyacrylamide gel electrophoresis have suggested that the H₂-receptor in guinea pig hippocampus and striatum has a molecular weight of 59 kDa (Ruat et al., 1990b). However, comparison with the calculated molecular weights (40.2 to 40.5 kDa) for the recently cloned H₂-receptors (table 6) suggests that the native H₂-receptor in guinea pig brain is glycosylated. Consistent with this proposal, it is noteworthy that all of the cloned H₂-receptor proteins possess N-glycosylation sites in the N-terminus region (Gantz et al., 1991a,b; Ruat et al., 1991; Traiffort et al., 1995). Removal of these glycosylation sites by site-directed mutagenesis, however, has shown that N-glycosylation of the H₂-receptor is not essential for cell surface localization, ligand binding, or coupling via G_s to adenylyl cyclase (Fukushima et al., 1995).

The H₂-receptor was first cloned by Gantz and colleagues using the polymerase chain reaction to amplify a partial length H₂-receptor sequence from canine gastric parietal cDNA using degenerate oligonucleotide primers (Gantz et al., 1991b). This sequence was then used to identify a full length H₂-receptor clone following screening of a canine genomic library (Gantz et al., 1991b). Rapid cloning of the rat, human, guinea pig, and mouse H₂-receptors followed (Gantz et al., 1991a; Ruat et al., 1991; Traiffort et al., 1995; Kobayashi et al., 1996). These DNA sequences encode for a 359 (canine, human, guinea pig) or 358 (rat) receptor protein that has the general characteristics of a G-protein-coupled receptor. The most notable difference between the structure of the cloned H₂- and H₁- receptors is the much shorter 3^rd intracellular loop of the H₂-receptor and the longer H₂-receptor C terminus. Expression of the rat and human H₂-receptor proteins in CHO cells has revealed the expected pharmacological specificity of H₂-receptors as judged by radioligand binding studies using [¹²⁵I]iodoaminopotentidine (Traiffort et al., 1992b; Leurs et al., 1994c). Recent chromosomal mapping studies have assigned the H₂-receptor gene to human chromosome 5 (Traiffort et al., 1995).

Comparison of the H₂-receptor sequence with other biogenic amine G-protein-coupled receptors has indicated that an aspartate in TM3 and an aspartate and threonine residue in TM5 are responsible for binding histamine (Birdsall, 1991). Replacement of aspartate (98) by an asparagine residue in the canine H₂-receptor results in a receptor that does not bind the antagonist tiotidine and does not stimulate cAMP accumulation in response to histamine (Gantz et al., 1992). Similarly, changing the aspirate (186) of TM5 to an alanine resulted in complete loss of tiotidine binding without affecting the EC₅₀ for histamine-stimulated cAMP formation (Gantz et al., 1992). Changing the threonine (190) to an alanine, however, resulted in a lower K_D for tiotidine and a reduction in both the maximal cAMP response and histamine EC₅₀ value (Gantz et al., 1992). Mutation of Asp (186) and Gly (187) in the canine H₂-receptor (to Ala (186) and Ser (187), however, produces a bifunctional receptor that can be stimulated by adrenaline and inhibited by both propranolol and cimetidine (Delvalle et al., 1995). Thus, these data suggest that the pharmacological specificity of the H₂-receptor resides in only a few key amino acid residues.

Other site-directed mutagenesis studies on the H₂-receptor have been very limited. However, Smit et al. (1996) have identified a residue in the second intracellular loop [leucine (124)] of the rat H₂-receptor, which appears necessary for efficient coupling to G_s.

It is generally accepted that histamine H₂-receptors couple to adenylyl cyclase via the GTP-binding protein G_s (Johnson, 1982; Hill, 1990; Leurs et al., 1995b). Histamine is a potent stimulant of cAMP accumulation in many cell types (Johnson, 1982), particularly those of CNS origin (Daly, 1977). Thus, H₂-receptor-mediated effects on cAMP accumulation have been observed in brain slices (Al-Gadi and Hill, 1985; Palacios et al., 1978), gastric mucosa (Soll and Wollin, 1979; Chew et al., 1980; Batzri et al., 1982; Gespach et al., 1982), fat cells (Grund et al., 1975; Keller et al., 1981), cardiac myocytes (Warbanow and Wollenberger, 1979), vascular smooth muscle (Reinhardt and Ritter, 1979), basophils (Lichtenstein and Gillespie, 1975), and neutrophils (Busse and Sosman, 1977). Furthermore, H₂-receptor-mediated cAMP accumulation has been demonstrated in CHO cells transfected with the rat, canine, or human H₂-receptor cDNA (Gantz et al., 1991a,b; Leurs et al., 1994b; Fukushima et al., 1995).

Direct stimulation of adenylyl cyclase activity in cell-free preparations has been detected in both brain and cardiac muscle membranes (Hegstrand et al., 1976; Green et al., 1977; Green and Maayani, 1977; Kanof et al., 1977; Johnson et al., 1979a,b; Kanof and Greengard, 1979a,b; Newton et al., 1982; Olianas et al., 1984). However, caution is required regarding the interpretation of receptor characterization studies using histamine-stimulated adenylyl cyclase activity alone (Hill, 1990). A striking feature of studies of histamine H₂-receptor-stimulated adenylyl cyclase activity in membrane preparations is the potent antagonism observed with certain neuroleptics and antidepressants (table 8; Spiker et al., 1976; Green et al., 1977; Green and Maayani, 1977; Kanof and Greengard, 1978; Green, 1983). It is notable, however, that most of the neuroleptics and antidepressants are approximately 2 orders of magnitude weaker as antagonists of histamine-stimulated cAMP accumulation in intact cellular systems (table 8; Tuong et al., 1980; Kamba and Richelson, 1983; Hill, 1990). One potential explanation of these differences resides within the buffer systems used for the cell-free adenylyl cyclase assays. Some differences in potency of some antidepressants and neuroleptics have been observed when membrane binding of H₂-receptors has been evaluated using [¹²⁵I]iodoaminopotentidine (table 8; Traiffort et al., 1991). However, invariably the differences observed in the K_i values deduced from ligand binding studies in different buffers are not as large as the differences in K_B values obtained from functional studies (table 8). For example, in the case of amitriptyline, no difference was observed in binding affinity in Krebs and Tris buffers (Traiffort et al., 1991).

In addition to G_s-coupling to adenylyl cyclase, there are reports of H₂-receptors coupling to other signaling systems. For example, in gastric parietal cells, H₂-receptor stimulation has been shown to increase the intracellular free concentration of calcium ions (Chew, 1985, 1986; Chew and Petropoulos, 1991; Malinowska et al., 1988; Delvalle et al., 1992a). A similar calcium response to histamine H₂-receptor stimulation has also been observed in HL-60 cells (Mitsuhashi et al., 1989; Seifert et al., 1992) and in hepatoma-derived cells transfected with the canine H₂-receptor cDNA (Delvalle et al., 1992b). In these latter cells, the influence on [Ca²⁺]_i was accompanied by both an increase in inositol trisphosphate accumulation and a stimulation of cAMP accumulation (Delvalle et al., 1992b). Interestingly, the H₂-receptor-stimulated calcium and inositol trisphosphate responses in these cells were both inhibited by cholera toxin treatment (but not by pertussis toxin), whereas cholera toxin produced the expected increase in cAMP levels (Delvalle et al., 1992a,b). In single parietal cells, H₂-receptors have been shown to release calcium from intracellular calcium stores (Negulescu and Machen, 1988). It should be noted, however, that no effect of H₂-agonists was observed on inositol phosphate accumulation or intracellular calcium levels in CHO cells transfected with the human H₂-receptor (Leurs et al., 1994a). Thus, the effect of H₂-receptor stimulation on intracellular calcium signaling may be very cell-specific.

In CHO cells transfected with the rat H₂-receptor, H₂-receptor stimulation produces both an increase in cAMP accumulation and an inhibition of P_2u-receptor-mediated arachidonic acid release (Traiffort et al., 1992b). Interestingly, however, the effect on phospholipase A₂ activity (i.e., arachidonic acid release) was not mimicked by forskolin, PGE₁, or 8-bromo-cAMP, suggesting a mechanism of activation that is independent of cAMP-mediated protein kinase A activity (Traiffort et al., 1992b). However, in CHO cells transfected with the human H₂-receptor, no inhibitory effects of H₂-receptor stimulation were observed on phospholipase A₂ activity (Leurs et al., 1994b). This observation suggests that these cAMP-independent effects might depend on the level of receptor expression or subtle differences between clonal cell lines.

	IV. Histamine H3-Receptor

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The high apparent affinity of R-()-methylhistamine for the histamine H₃-receptor has enabled the use of this compound as a radiolabeled probe (Arrang et al., 1987). This compound has been successfully used to identify a single binding site in rat cerebral cortical membranes, which in phosphate buffer has the pharmacological characte