Introduction

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The histamine H₃ receptor was first identified as a presynaptic autoreceptor on histamine neurons in the brain controlling the stimulated release of histamine (Arrang et al., 1983). It has subsequently been shown to be a presynaptic heteroreceptor in nonhistamine-containing neurons in both the central and peripheral nervous systems (for review, see Hill et al., 1997). We recently reported the cloning and functional characterization of the human H₃ receptor cDNA (Lovenberg et al., 1999) and showed that the recombinant H₃ receptor is a G-protein-coupled receptor that signals through the inhibition of adenylate cyclase. Pharmacological comparison between binding potencies of known ligands for the cloned human receptor and previously published rodent data were mostly consistent. However, there were some discrepancies such as the apparent low affinity of the prototypical H₃ antagonist thioperamide for the human clone (60 nM), whereas the H₃ antagonist clobenpropit exhibited high affinity (1 nM). Thioperamide had been reported by many investigators to bind with high affinity to rodent H₃ receptors from various species and tissues (2-5 nM). West et al. (1990) also have reported that in rodent tissue, thioperamide binds to both high (H3a; 5 nM)- and low (H3b; 68 nM)-affinity sites, thus possibly distinguishing between two distinct populations of receptor (West et al., 1990). To determine whether the human clone that we found represented the putative H3b subtype, we cloned and characterized its rat homolog and determined the pharmacological properties.

	Experimental Procedures

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Materials. cDNA synthesis kits were purchased from Life Technologies (Gaithersburg, MD). Gelzyme was from Invitrogen (San Diego, CA) and pCIneo vector was from Promega (Madison, WI). SK-N-MC cells were obtained from American Type Culture Collection (Manassas, VA). cAMP flashplates were from NEN (Boston, MA). G-418 was purchased from Calbiochem (San Diego, CA). All histamine ligands were purchased from Research Biochemicals (Natick, MA). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Cloning of Rat Histamine H₃ Receptor cDNA. To screen for cDNAs encoding the rat ortholog of the human H3 receptor, a cDNA library derived from rat hypothalamus (Life Technologies) was constructed. Briefly, 5 µg of poly(A)-selected RNA was synthesized into double-stranded cDNA, followed by size selection via a 0.8% low-melting agarose gel. The cDNA in the range of 2.5 to 5 kb was subsequently ligated (pSport) and transformed into Escherichia coli. The subsequent cDNA library was screened with a 600-base pair ³²P-radiolabeled cDNA fragment (1.5 × 10⁶ counts/ml). The fragment was derived by polymerase chain reaction (PCR) with primers (identical with the human H₃ cDNA sequence) to amplify a fragment from rat hypothalamus cDNA. Library filters were hybridized overnight (buffer from 5'  3', Boulder, CO) and then washed twice at room temperature in 2× standard saline citrate (SSC)/0.2% SDS for 30 min followed by two washes at 50°C in 0.2× SSC for 30 min. Filters were exposed to film (24 h) and developed. Positive clones were subsequently sequenced (ABI 377; Perkin-Elmer, Norwalk, CT). The full-length rat H₃ receptor cDNA was subcloned into the mammalian expression vector pCIneo (Promega) for recombinant expression.

Transfection of Cells with Rat H₃ Receptor. SK-N-MC neuroblastoma cells were grown to ~70 to 80% confluence and removed from the plate with trypsin and pelleted in a clinical centrifuge. The pellets were resuspended in 400 µl of complete medium and transferred to an electroporation cuvette with a 0.4-cm gap between the electrodes (Bio-Rad 165-2088). One microgram of supercoiled DNA was added to the cells and mixed. The voltage for the electroporation was set at 0.25 kV, and the capacitance was set at 960 µF. After electroporation, the cells were diluted into 10 ml of complete medium and plated onto four 10-cm dishes at the following ratios: 1:20, 1:10, 1:5, and the rest. The cells were allowed to recover for 24 h before adding G-418. Colonies that survived selection were grown and tested for the ability to inhibit forskolin-stimulated cAMP accumulation in response to histamine. SK-N-MC cells expressing the human H₃ receptor were derived as previously described (Lovenberg et al., 1999).

cAMP Accumulation. Transfected cells were plated on 96-well clear tissue culture plates. Confluent overnight cultures were then incubated with Dulbecco's modified Eagle's medium-F12 containing isobutylmethylxanthine (2 mM) for 20 min. Cells were then incubated with test compounds for 5 min, with histamine (various concentrations) for 5 min, and then with forskolin (5 µM) for 20 min at room temperature. The reaction was stopped with one-fifth volume 0.5 N HCl. Plates were placed at 4°C for at least 2 h and then the cell media was tested for cAMP concentration by radioimmunoassay with cAMP flashplates. When examining agonist potency, cells were treated with test compound alone before forskolin addition.

N-[³H]Methylhistamine Binding. Cell pellets from SK-N-MC cells expressing either the rat or human H₃ receptor were homogenized in 20 mM Tris-HCl/0.5 mM EDTA (for rat tissue analysis, frozen rat cortical hemispheres were used instead of cell pellets). Supernatants from an 800g spin were collected and recentrifuged at 30,000g for 30 min. Pellets were rehomogenized in 50 mM Tris/5 mM EDTA (pH 7.4). Membranes were incubated with 0.8 nM N-[³H]methylhistamine plus/minus test compounds for 45 min at 25°C and harvested by rapid filtration over GF/C glass fiber filters (pretreated with 0.3% polyethylenimine) followed by four washes with ice-cold buffer. Nonspecific binding was defined with 10 µM histamine. IC₅₀ values were determined by a single site curve-fitting program (GraphPad, San Diego, CA) and converted to K_i values based on a N-[³H]methylhistamine K_d of 800 pM and a ligand concentration of 800 pM (Cheng and Prusoff, 1973).

RNA Blot. A blot containing poly(A)-selected RNA extracted from various rat tissues was obtained (Clontech, Palo Alto, CA) and probed with a ³²P-labeled 600-bp cDNA encoding a fragment of the rat H₃ receptor. The blot was hybridized with ExpressHyb (Clontech) for 2 h at 68°C and subsequently washed three times at room temperature in 2× SSC/0.05% SDS for 30 min followed by two washes at 50°C in 0.1× SSC/0.1% SDS for 30 min each. The blot was exposed to X-ray film (36 h) and developed.

	Results

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Rat and Human H₃ Receptors Have High Sequence Identity. Screening of a size-selected rat hypothalamus cDNA library resulted in the identification of a 2.4-kb cDNA clone. An open reading frame of 1335 bp was identified, encoding a putative protein of 445 amino acids. Homology comparison of the rat and human proteins, with Lipman-Pearson pairwise analysis (Lipman and Pearson, 1985), revealed 93% overall sequence identity (Fig. 1). The putative seven transmembrane domains are underlined in Fig. 1 and labeled TM1 to TM7. Analysis within the seven transmembrane domains revealed 97% identity, corresponding to a total of only five amino acid differences, as denoted by asterisks in the figure. There are two amino acid changes found in transmembrane domain 3 and a single change found in each of transmembrane domains 4, 6, and 7. 

View larger version (59K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence of rat H3 receptor compared with the human histamine H3 receptor. Putative transmembrane domains are stated below the sequence and indicated by a solid line. Residues that are identical among all three receptors are repeated between the sequences and differences in the transmembrane domains are indicated by an * above the sequence. Rat DNA and protein sequences have been deposited with GenBank (Accession no. AF237919).

Rat H₃ Receptor Is Selectively Expressed in Brain. We have previously demonstrated, via in situ hybridization, that the rat ortholog of human H₃ receptor is robustly expressed in various regions of the rat brain. To determine expression in both neural and nonneural tissue, RNA extracted from a variety of rat tissues was probed for H₃ receptor expression via Northern blot analysis. Similar to the previous findings for the human receptor, we could only identify the brain as the primary site of expression of the histamine H₃ receptor. Detectable RNA expression was not present in heart, spleen, lung, liver, skeletal muscle, kidney, or testes (Fig. 2).

View larger version (70K): [in this window] [in a new window]   Fig. 2.   Northern blot of rat mRNA samples [5 µg of poly(A)+ RNA/lane]. 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; and 8, testis. The probe was a 600-bp fragment of rat H3 coding sequence. Exposure time to film was 1.5 days (80°C).

Rat H₃ Receptor-Expressing Cells Inhibit Adenylate Cyclase in Response to H₃ Agonists. We have previously shown that the human H₃ receptor couples negatively to adenylate cyclase. Therefore, cells transfected with the recombinant rat receptor were tested for the same effect. Figure 3 shows that histamine, imetit, and R--methylhistamine dose dependently inhibit forskolin-stimulated cAMP accumulation with EC₅₀ values of 3, 0.5, and 0.5, respectively. These values are not only similar to those observed for the human recombinant H₃ receptor but also are consistent with literature values for binding and function in rat tissues (i.e., nonrecombinant).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Inhibition of cAMP accumulation in response to H3 agonists. Cells were treated with 10 µM forskolin 5 min after the addition of agonists and incubated for an additional 20 min at room tempurature. All values are determined in triplicate. Error bars represent S.E.M. Data is representative of multiple (at least three) experiments.

Rat H₃ Receptor-Expressing Cells Bind N-[³H]Methylhistamine with High Affinity. All binding studies were done on SK-N-MC cells stably transfected with the rat or human H₃ receptor. Saturation isotherms show that N-[³H]methylhistamine binds saturably to the rat receptor with an apparent single site with high affinity (Fig. 4). Nonspecific binding was <10% at all concentrations tested. Scatchard transformation of the saturation data revealed that the approximate K_d for N-[³H]methylhistamine binding to the rat receptor was 0.8 nM (data not shown). N-[³H]methylhistamine was used at a concentration of 0.8 nM for the competition-binding studies. Nontransfected SK-N-MC cells exhibit no specific binding of N-[³H]methylhistamine (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Saturation isotherm of N-[3H]methylhistamine to rat H3-transfected SK-N-MC cells. Total binding (); nonspecific binding (); and specific binding (). A Kd of 0.8 nM was calculated as 1/slope from a linear Scatchard transformation.

Rat H₃ Receptors Show a Different Pharmacological Profile Than Human Receptors. We had previously shown that the recombinant human H₃ receptor had a similar pharmacological profile to that of the rat/mouse/guinea pig receptors, which have been extensively characterized in various tissues over the past decade. We tested the same compounds in this study for their ability to bind to the recombinant rat receptor. For the purposes of this study, binding affinities of the human recombinant receptor, the rat recombinant receptor, and rat cortical tissue were simultaneously determined for more accurate cross-comparisons. The agonists imetit, immepip, histamine, R-alphamethylhistamine, and N-methylhistamine were able to compete for rat recombinant H₃ receptor binding with high affinity. The rank order of potency was similar to that seen for the human recombinant receptor and rat cerebral cortex (Table 1). However, when we tested the antagonists thioperamide and clobenpropit, we found a distinct species difference between the two compounds. Thioperamide showed a clear difference in affinity between the two recombinant receptors, whereas clobenpropit displayed high affinity for both receptors (~0.5 nM). Specifically, thioperamide displayed high affinity for the recombinant rat clone (4.2 nM), whereas it had low affinity (58 nM) for the human recombinant receptor. The profile of the recombinant rat receptor was identical with that seen with rat cerebral cortex as a receptor source (Table 1). In addition, we had previously reported that clozapine did not compete for binding to the human H₃ receptor (K_i > 10 µM). In contrast, we found that clozapine does effectively compete for binding to the recombinant rat H₃ receptor, albeit with low affinity (1.75 µM). We performed a correlation analysis comparing the pK_i values for rat recombinant receptor binding versus the human recombinant receptor binding. A highly significant correlation (r² = 0.956) was observed when thioperamide was left out of the analysis, whereas a poor correlation (r² = 0.711) was observed when thioperamide was included (Fig. 5). This analysis included all of the compounds listed in Table 1 except clozapine.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Correlation between recombinant rat and human pKi values. Ki values from Table 1 were converted to pKi values (log(Ki × 109; clozapine excluded) and plotted as rat pKi (ordinate) versus human pKi (abscissa). Linear regression was performed with and without thioperamide to determine goodness of fit. The line drawn represents the linear regression in the absence of the thioperamide data point.

Functional Antagonism in Recombinant H₃ Receptors Correlates with Binding K_i Values. To define the antagonist potency of various H₃ ligands, we measured the pA₂ values for competitive antagonism of R--methylhistamine-induced inhibition of forskolin-stimulated cAMP accumulation. For the rat receptor, Fig. 6, A and B show the competitive antagonism by both thioperamide and clobenpropit, respectively, with corresponding Schild regression analyses in Fig. 6, C and D. The rat pA₂ values for thioperamide and clobenpropit were 8.56 and 9.53, respectively. The corresponding data for the human receptor is shown in Fig. 6, E through H, where the human pA₂ values were 7.06 and 9.11 for thioperamide and clobenpropit, respectively. Figure 7 shows the correlation between antagonist potency (pA₂) and binding affinity (pK_i) for clobenpropit and thioperamide in both species. The antagonist potency of the compounds correlate highly with their binding affinities regardless of species (r² = 0.979). This shows that the compounds are behaving as competitive antagonists and that the binding affinities can be predictive of antagonist potency.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Determination of antagonist potency for thioperamide and clobenpropit. Dose-response curves for R--methylhistamine (RAMH)-induced inhibition of cAMP accumulation were generated in the presence of absence of various concentration of antagonists for rat (A, B) and human (E, F). The log of the (dose ratio 1) at each antagonist concentration was plotted versus log antagonist concentration. These Schild regression plots are shown for rat (C, D) and human (G, H). The dose ratio is determined by dividing "the histamine EC50 in the presence of the antagonist" by "the histamine EC50 in the absence of the antagonist". pA2 values were determined where the regressed line crossed the x-axis at Y = 0. Y = 0 when the dose ratio equals 2 (pA2), which is theoretically equivalent to the antagonist pKi (Schild, 1949).

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Correlation of antagonist pA2 values to pKi values. Data for both rat and human constants for thioperamide (THP) and clobenpropit (CLB) are plotted.

Functional Analysis in Recombinant Cells Confirms That Chloroproxyfan Is an Agonist. The known H₃ ligand clorproxyfan was tested for its ability to modulate histamine-induced inhibition of cAMP accumulation. Our initial analysis with chloroproxyfan showed that it could not block the effects of histamine. In fact, chloroproxyfan dose dependently enhanced histamine-induced inhibition of cAMP accumulation (data not shown). When analyzed in the absence of histamine, chloroproxyfan could dose dependently inhibit forskolin-stimulated adenylate cyclase with an EC₅₀ of 2 nM (Fig. 8). This corresponds with its binding affinity of 1 nM at both the rat and human recombinant receptors. In addition, chloroproxyfan was a full agonist in that it was equally efficacious to both histamine and R--methylhistamine at inhibiting >90% of the forskolin-stimulated cAMP accumulation (data not shown).

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Inhibition of cAMP accumulation in response to chloroproxyfan. Cells were treated with 5 µM forskolin 5 min after the addition of chloroproxyfan and incubated for an additional 20 min at room temperature. All values are determined in triplicate. Data is representative of multiple (at least three) experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The recent cloning of the human histamine H₃ receptor cDNA opens up a new area of study for function and pharmacological intervention of histamine function in the central nervous system. Although most of the data reported for the human clone were consistent with the known pharmacological properties of the rodent H₃ receptor characterized in tissue, there were some interesting differences, most notably the low-affinity binding of the prototypical H₃ antagonist thioperamide. One possible explanation for the low affinity was a simple species difference; however, there is evidence reported in the literature to suggest the existence of multiple H₃ receptor subtypes. To address these two hypotheses, we cloned and pharmacologically characterized the rat ortholog of the H₃ receptor.

We had previously identified, via PCR, a fragment of the rat ortholog encoding the H₃ receptor to map the mRNA expression within the central nervous system. In doing so, we determined that the rat H₃ receptor was robustly expressed in the cortex, striatum, thalamus, and hypothalamus. A size-selected cDNA library from rat hypothalamus tissue was used to identify a full-length clone consisting of 2.4 kb with a 1335-bp open reading frame encoding a putative protein of 445 amino acids. The cDNA clone showed high sequence identity to the human receptor with 93% identity at the amino acid level. As expected, a majority of the identity is observed within the transmembrane domains (97%), a result of only five amino acid substitutions (Fig. 1). These transmembrane domains contain the presumed binding pockets for agonists (e.g., histamine) as well as competitive antagonists (e.g., thioperamide). The binding of various biogenic amines to their respective receptors appears to be highly dependent on specific amino acid residues in transmembrane domains 3, 5, and 6 (for reviews, see Jackson, 1991; Beck-Sickinger, 1996). For example the conserved aspartic acid residue in transmembrane 3 is thought to interact with the primary amine function of catecholamines (Gros et al., 1998) and serotonin (Boess et al., 1998). Likewise, the serine and threonine residues in transmembrane domain 5 are thought to interact with the hydroxyl groups on the catechol ring or indole ring of dopamine or serotonin, respectively (Lee et al., 1994, Liggett, 1999). In fact, mutagenesis studies of the histamine H₁ receptor show that the transmembrane domains are critical for histamine, as well as antagonist binding (Wieland et al., 1999). Because the H₃ receptor is highly related structurally, these previous studies suggest that the five amino acid differences in the transmembrane domains of the rat and human H₃ receptors may account for the differences in the pharmacological profiles of thioperamide and clozapine binding. Results from a site-directed mutagenesis experiment will no doubt lead to a better understanding of how histamine and synthetic H₃ ligands interact with the receptor binding pocket as well as aid in the development of more potent, selective ligands.

The tissue distribution of the rat H₃ receptor, by Northern blot analysis, was identical with that of the human in that it was detected only in brain tissue. There is ample evidence in the literature to suggest peripheral expression of the H₃ receptor, particulary on adrenergic and cholinergic cells innervating the heart, lung, intestine, and spleen among others (Cardell and Edvinsson, 1994; Dimitriadou et al., 1994; Imamura et al., 1995; Coruzzi et al., 2000). Indeed, we noted expression of human H₃ receptor mRNA (via PCR) from human small intestine, prostate, and testis but could not detect mRNA expression in peripheral tissue via Northern blot (Lovenberg et al., 1999). Our attempts to identify peripheral expression in rat via in situ hybridization have been unsuccessful due to technical difficulties. The question of central versus peripheral subtypes of H₃ receptors thus awaits more careful expression analysis of the cDNA we have cloned versus known sites of action. The presynaptic nature of the receptor (i.e., distal expression of the mRNA versus final location of the functional protein) makes it difficult to correlate functional receptors with mRNA expression, particularly in the periphery.

Functionally, the rat H₃ receptor is similar to the human in that it inhibits adenylate cyclase in response to histamine and various selective H₃ agonists. Inhibition of adenylate cyclase is the mechanism of action shared by the major release-inhibiting G-protein-coupled autoreceptors and heteroreceptors such as ₂ (norepinephrine), D₂ (dopamine), M₂ (acetylcholine), 5HT_1D (serotonin), and H₃ (histamine) (Langer, 1997). Interestingly, the dominant structural homology of the H₃ receptor in certain transmembrane domains is to ₂ and M₂ receptors, not to H₁ or H₂ receptors (Lovenberg et al., 1999).

The EC₅₀ values for agonist activation of the rat H₃ receptor were nearly identical with those for activation of the human receptor. In addition, binding of the agonists to the rat receptor showed similar affinity between the rat and the human receptors. In contrast, antagonist binding showed several pharmacological differences between the rat and human receptors. Our previous report of antagonist K_i values at the human receptor were mostly consistent with published data for binding to rat tissues. However, one notable distinction was the apparent low affinity of thioperamide for the human recombinant receptor (58 nM). This finding has recently been confirmed by examining the binding of N-[³H]methylhistamine to human postmortem cerebral cortex where thioperamide had a reported K_i of 200 nM (West et al., 1999). Earlier functional reports with human saphenous vein (Oike et al., 1992) and a human gastric cell line (Cherifi et al., 1992) had alluded to low K_i values for thioperamide. In this article, we show that thioperamide displays high affinity for the recombinant rat H₃ receptor and the H₃ receptor expressed in rat cortex, consistent with literature values for binding to rat tissue. Together, these data suggest that the recombinant receptors are representative of the natural binding affinities.

One other difference that was noted was the affinity of clozapine for the rat receptor. Clozapine had previously been reported to bind to the rat H₃ receptor (from brain tissue), leading to speculation that some of its antipsychotic effects in humans may be due to H₃ receptor antagonism (Kathmann et al., 1994, Rodrigues et al., 1995, Stark et al., 1996). Our finding (Lovenberg et al., 1999) that clozapine did not significantly compete for binding to the recombinant human receptor put serious doubt on the validity of the hypothesis. However, our current findings with the rat recombinant receptor confirm the validity of the original findings in rat tissue because clozapine effectively competes for binding albeit with low affinity (1.75 µM). These findings also suggest that much of the differences in pharmacology reported in the literature may be due solely to species differences and not H₃ heterogeneity (West et al., 1990). It does not however completely rule out the existence of multiple H₃ receptor subtypes. Indeed, a recent article by Harper et al. (1999b) demonstrates a lack of correlation between agonist pK_i values in the guinea pig cerebral cortex versus guinea pig longitudinal muscle myenteric plexus, whereas good correlation was seen between antagonist pK_i values. This could be suggestive of multiple subtypes of binding sites or may be reflective of complex agonist binding. The development of radiolabeled antagonists may help address this issue (Harper et al., 1999a).

One of the difficult aspects of studying presynaptic receptors is the determination of the functional potency of agonists and antagonists. Approaches such as neurogenic ileum twitch and neurotransmitter release are effective, but tend to be both labor-intensive and indirect. In addition, a recent report highlighted that certain classes of compounds behave as antagonists in the guinea pig ileum twitch assay, but actually behave as agonists in neurotransmitter release assays (Sasse et al., 1999). These potential assay inconsistencies led us to evaluate the use of the recombinant system to determine ligand affinities simultaneously with the determination of agonist/antagonist function. This current report demonstrates that the recombinant receptor systems can be used to predict antagonist potency because the pA₂ values derived from Schild regression analysis highly correlate with binding pK_i values regardless of compound or species. In addition, the data demonstrate that the antagonism is competitive because it is surmountable by high agonist concentrations.

In this report, we show that one can accurately determine agonist/antagonist potency of various H₃ ligands. Previous reports have suggested that a series of substituted "proxyfans," which were originally thought to be antagonists (Ligneau et al., 1994), were in fact agonists (Schlicker et al., 1996, Watt et al., 1997). Because the recombinant H₃ receptor systems have allowed an easy determination of agonist and antagonist potency, we tested one of these compounds, chloroproxyfan. In both the rat and human recombinant systems, chloroproxyfan behaved as a pure, high-affinity receptor agonist. Although it can be argued that that recombinant systems will not be representative of an intact tissue, these systems allow one to easily determine an intrinsic efficacy potential of a compound, particularly against the human receptor where it is often difficult to obtain fresh, functional tissue preparations.

The availability of the rat cDNA will now allow for a full analysis of various H₃ receptor ligands, and hopefully will help define the structure-activity requirements for the development of better agonists and antagonists. There are clear pharmacological distinctions that will be critical for the development of novel H₃ receptor ligands. Identification of additional species orthologs of the H₃ receptor, such as guinea pig, also will be extremely useful because much of the literature discrepancy surrounding H₃ subtype identification stems from differences between rat and guinea pig H₃ function and pharmacology (Schlicker et al., 1996).