Cloning of Rat Histamine H3 Receptor Reveals Distinct Species Pharmacological Profiles
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0022-3565/00/2933-0771$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 293, No. 3
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 293:771–778, 2000 /2394/824789
Cloning of Rat Histamine H3 Receptor Reveals Distinct
Species Pharmacological Profiles
TIMOTHY W. LOVENBERG, JAYASHREE PYATI, HONG CHANG, SANDY J. WILSON, and MARK G. ERLANDER
R. W. Johnson Pharmaceutical Research Institute, San Diego, California
Accepted for publication February 12, 2000 This paper is available online at http://www.jpet.org
ABSTRACT
The recent cloning and characterization of the human histamine tested had low or subnanomolar Ki values similar to that for the
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H3 receptor cDNA marked a significant step toward a greater human recombinant receptor. The antagonists thioperamide
understanding of the role of this receptor in the central nervous and clobenpropit also bound with high affinity (Ki ⫽ 4 and 0.4
system. We now report the cloning of the rat histamine H3 nM, respectively). This is in contrast to the antagonist profile
receptor cDNA and demonstrate distinct pharmacological spe- obtained for the human recombinant receptor that showed Ki
cies differences. The rat cDNA clone encodes a putative 445- values of 58 and 0.6 nM for thioperamide and clobenpropit,
amino acid protein with 93% identity to the human H3 receptor. respectively. These data suggest that the low affinity of thiop-
Northern blot analysis revealed a major single entity of 2.7-kb in eramide for the human H3 receptor represents a species differ-
length expressed only in brain. Transfection of the rat receptor ence in pharmacology and not a unique pharmacological sub-
cDNA into SK-N-MC cells conferred an ability to inhibit forsko- type. It also was found that chloroproxyfan behaved as a full
lin-stimulated cAMP formation in response to histamine and agonist at the rat recombinant receptor. These findings high-
other H3 agonists. N-[3H]methylhistamine saturably bound to light the significance of validating the pharmacology of exper-
transfected cells with high affinity (Kd ⫽ 0.8 nM). All agonists imental compounds at both the rat and human H3 receptors.
The histamine H3 receptor was first identified as a presyn- the human clone that we found represented the putative H3b
aptic autoreceptor on histamine neurons in the brain control- subtype, we cloned and characterized its rat homolog and
ling the stimulated release of histamine (Arrang et al., 1983). determined the pharmacological properties.
It has subsequently been shown to be a presynaptic hetero-
receptor in nonhistamine-containing neurons in both the cen-
tral and peripheral nervous systems (for review, see Hill et
Experimental Procedures
al., 1997). We recently reported the cloning and functional Materials. cDNA synthesis kits were purchased from Life Tech-
characterization of the human H3 receptor cDNA (Lovenberg nologies (Gaithersburg, MD). Gelzyme was from Invitrogen (San
et al., 1999) and showed that the recombinant H3 receptor is Diego, CA) and pCIneo vector was from Promega (Madison, WI).
a G-protein-coupled receptor that signals through the inhi- SK-N-MC cells were obtained from American Type Culture Collec-
tion (Manassas, VA). cAMP flashplates were from NEN (Boston,
bition of adenylate cyclase. Pharmacological comparison be-
MA). G-418 was purchased from Calbiochem (San Diego, CA). All
tween binding potencies of known ligands for the cloned
histamine ligands were purchased from Research Biochemicals
human receptor and previously published rodent data were (Natick, MA). All other reagents were purchased from Sigma Chem-
mostly consistent. However, there were some discrepancies ical Co. (St. Louis, MO).
such as the apparent low affinity of the prototypical H3 Cloning of Rat Histamine H3 Receptor cDNA. To screen for
antagonist thioperamide for the human clone (60 nM), cDNAs encoding the rat ortholog of the human H3 receptor, a cDNA
whereas the H3 antagonist clobenpropit exhibited high affin- library derived from rat hypothalamus (Life Technologies) was con-
ity (1 nM). Thioperamide had been reported by many inves- structed. Briefly, 5 g of poly(A)-selected RNA was synthesized into
tigators to bind with high affinity to rodent H3 receptors from double-stranded cDNA, followed by size selection via a 0.8% low-
various species and tissues (2–5 nM). West et al. (1990) also melting agarose gel. The cDNA in the range of 2.5 to 5 kb was
subsequently ligated (pSport) and transformed into Escherichia coli.
have reported that in rodent tissue, thioperamide binds to
The subsequent cDNA library was screened with a 600-base pair
both high (H3a; 5 nM)- and low (H3b; 68 nM)-affinity sites, 32
P-radiolabeled cDNA fragment (1.5 ⫻ 106 counts/ml). The frag-
thus possibly distinguishing between two distinct popula- ment was derived by polymerase chain reaction (PCR) with primers
tions of receptor (West et al., 1990). To determine whether (identical with the human H3 cDNA sequence) to amplify a fragment
from rat hypothalamus cDNA. Library filters were hybridized over-
Received for publication December 14, 1999. night (buffer from 5⬘ 3 3⬘, Boulder, CO) and then washed twice at
ABBREVIATIONS: bp, base pair; PCR, polymerase chain reaction; SSC, standard saline citrate.
771772 Lovenberg et al. Vol. 293
room temperature in 2⫻ standard saline citrate (SSC)/0.2% SDS for vealed 93% overall sequence identity (Fig. 1). The putative
30 min followed by two washes at 50°C in 0.2⫻ SSC for 30 min. seven transmembrane domains are underlined in Fig. 1 and
Filters were exposed to film (24 h) and developed. Positive clones labeled TM1 to TM7. Analysis within the seven transmem-
were subsequently sequenced (ABI 377; Perkin-Elmer, Norwalk, brane domains revealed 97% identity, corresponding to a
CT). The full-length rat H3 receptor cDNA was subcloned into the total of only five amino acid differences, as denoted by aster-
mammalian expression vector pCIneo (Promega) for recombinant
isks in the figure. There are two amino acid changes found in
expression.
Transfection of Cells with Rat H3 Receptor. SK-N-MC neu-
transmembrane domain 3 and a single change found in each
roblastoma cells were grown to ⬃70 to 80% confluence and removed of transmembrane domains 4, 6, and 7.
from the plate with trypsin and pelleted in a clinical centrifuge. The Rat H3 Receptor Is Selectively Expressed in Brain.
pellets were resuspended in 400 l of complete medium and trans- We have previously demonstrated, via in situ hybridization,
ferred to an electroporation cuvette with a 0.4-cm gap between the that the rat ortholog of human H3 receptor is robustly ex-
electrodes (Bio-Rad 165-2088). One microgram of supercoiled DNA pressed in various regions of the rat brain. To determine
was added to the cells and mixed. The voltage for the electroporation expression in both neural and nonneural tissue, RNA ex-
was set at 0.25 kV, and the capacitance was set at 960 F. After tracted from a variety of rat tissues was probed for H3 recep-
electroporation, the cells were diluted into 10 ml of complete medium tor expression via Northern blot analysis. Similar to the
and plated onto four 10-cm dishes at the following ratios: 1:20, 1:10,
previous findings for the human receptor, we could only
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
identify the brain as the primary site of expression of the
tested for the ability to inhibit forskolin-stimulated cAMP accumu- histamine H3 receptor. Detectable RNA expression was not
present in heart, spleen, lung, liver, skeletal muscle, kidney,
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lation in response to histamine. SK-N-MC cells expressing the hu-
man H3 receptor were derived as previously described (Lovenberg et or testes (Fig. 2).
al., 1999). Rat H3 Receptor-Expressing Cells Inhibit Adenylate
cAMP Accumulation. Transfected cells were plated on 96-well Cyclase in Response to H3 Agonists. We have previously
clear tissue culture plates. Confluent overnight cultures were then shown that the human H3 receptor couples negatively to
incubated with Dulbecco’s modified Eagle’s medium-F12 containing adenylate cyclase. Therefore, cells transfected with the re-
isobutylmethylxanthine (2 mM) for 20 min. Cells were then incu- combinant rat receptor were tested for the same effect. Fig-
bated with test compounds for 5 min, with histamine (various con-
ure 3 shows that histamine, imetit, and R-␣-methylhista-
centrations) for 5 min, and then with forskolin (5 M) for 20 min at
room temperature. The reaction was stopped with one-fifth volume
mine dose dependently inhibit forskolin-stimulated cAMP
0.5 N HCl. Plates were placed at 4°C for at least 2 h and then the cell accumulation with EC50 values of 3, 0.5, and 0.5, respec-
media was tested for cAMP concentration by radioimmunoassay tively. These values are not only similar to those observed for
with cAMP flashplates. When examining agonist potency, cells were the human recombinant H3 receptor but also are consistent
treated with test compound alone before forskolin addition. with literature values for binding and function in rat tissues
N-[3H]Methylhistamine Binding. Cell pellets from SK-N-MC (i.e., nonrecombinant).
cells expressing either the rat or human H3 receptor were homoge- Rat H3 Receptor-Expressing Cells Bind N-[3H]Meth-
nized in 20 mM Tris-HCl/0.5 mM EDTA (for rat tissue analysis, ylhistamine with High Affinity. All binding studies were
frozen rat cortical hemispheres were used instead of cell pellets). done on SK-N-MC cells stably transfected with the rat or
Supernatants from an 800g spin were collected and recentrifuged at
human H3 receptor. Saturation isotherms show that
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]m-
N-[3H]methylhistamine binds saturably to the rat receptor
ethylhistamine plus/minus test compounds for 45 min at 25°C and with an apparent single site with high affinity (Fig. 4). Non-
harvested by rapid filtration over GF/C glass fiber filters (pretreated specific binding was ⬍10% at all concentrations tested. Scat-
with 0.3% polyethylenimine) followed by four washes with ice-cold chard transformation of the saturation data revealed that the
buffer. Nonspecific binding was defined with 10 M histamine. IC50 approximate Kd for N-[3H]methylhistamine binding to the
values were determined by a single site curve-fitting program rat receptor was 0.8 nM (data not shown). N-[3H]methylhis-
(GraphPad, San Diego, CA) and converted to Ki values based on a tamine was used at a concentration of 0.8 nM for the compe-
N-[3H]methylhistamine Kd of 800 pM and a ligand concentration of tition-binding studies. Nontransfected SK-N-MC cells exhibit
800 pM (Cheng and Prusoff, 1973). no specific binding of N-[3H]methylhistamine (data not
RNA Blot. A blot containing poly(A)-selected RNA extracted from
shown).
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 Receptors Show a Different Pharmacological
rat H3 receptor. The blot was hybridized with ExpressHyb (Clontech) Profile Than Human Receptors. We had previously
for 2 h at 68°C and subsequently washed three times at room tem- shown that the recombinant human H3 receptor had a sim-
perature in 2⫻ SSC/0.05% SDS for 30 min followed by two washes at ilar pharmacological profile to that of the rat/mouse/guinea
50°C in 0.1⫻ SSC/0.1% SDS for 30 min each. The blot was exposed to pig receptors, which have been extensively characterized in
X-ray film (36 h) and developed. various tissues over the past decade. We tested the same
compounds in this study for their ability to bind to the re-
combinant rat receptor. For the purposes of this study, bind-
Results ing affinities of the human recombinant receptor, the rat
Rat and Human H3 Receptors Have High Sequence recombinant receptor, and rat cortical tissue were simulta-
Identity. Screening of a size-selected rat hypothalamus neously determined for more accurate cross-comparisons.
cDNA library resulted in the identification of a 2.4-kb cDNA The agonists imetit, immepip, histamine, R-alphamethylhis-
clone. An open reading frame of 1335 bp was identified, tamine, and N-methylhistamine were able to compete for rat
encoding a putative protein of 445 amino acids. Homology recombinant H3 receptor binding with high affinity. The rank
comparison of the rat and human proteins, with Lipman- order of potency was similar to that seen for the human
Pearson pairwise analysis (Lipman and Pearson, 1985), re- recombinant receptor and rat cerebral cortex (Table 1). How-2000 Cloning of Rat H3 Receptor 773
Fig. 1. Amino acid sequence of rat H3 recep-
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tor 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 be-
tween the sequences and differences in the
transmembrane domains are indicated by an
ⴱ above the sequence. Rat DNA and protein
sequences have been deposited with Gen-
Bank (Accession no. AF237919).
affinity between the two recombinant receptors, whereas clo-
benpropit 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 af-
finity (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 (Ki ⬎ 10
M). In contrast, we found that clozapine does effectively
Fig. 2. Northern blot of rat mRNA samples [5 g of poly(A)⫹ RNA/lane]. compete for binding to the recombinant rat H3 receptor,
1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; albeit with low affinity (1.75 M). We performed a correlation
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). analysis comparing the pKi values for rat recombinant recep-
tor binding versus the human recombinant receptor binding.
ever, when we tested the antagonists thioperamide and clo- A highly significant correlation (r2 ⫽ 0.956) was observed
benpropit, we found a distinct species difference between the when thioperamide was left out of the analysis, whereas a
two compounds. Thioperamide showed a clear difference in poor correlation (r2 ⫽ 0.711) was observed when thioperam-774 Lovenberg et al. Vol. 293
Fig. 3. Inhibition of cAMP accumulation in response to H3 agonists. Cells
were treated with 10 M forskolin 5 min after the addition of agonists Fig. 5. Correlation between recombinant rat and human pKi values. Ki
and incubated for an additional 20 min at room tempurature. All values values from Table 1 were converted to pKi values (⫺log(Ki ⫻ 10⫺9;
are determined in triplicate. Error bars represent S.E.M. Data is repre- clozapine excluded) and plotted as rat pKi (ordinate) versus human pKi
sentative of multiple (at least three) experiments. (abscissa). Linear regression was performed with and without thioper-
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amide to determine goodness of fit. The line drawn represents the linear
regression in the absence of the thioperamide data point.
mulation. For the rat receptor, Fig. 6, A and B show the
competitive antagonism by both thioperamide and clobenpro-
pit, respectively, with corresponding Schild regression anal-
yses in Fig. 6, C and D. The rat pA2 values for thioperamide
and clobenpropit were 8.56 and 9.53, respectively. The cor-
responding 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 (pKi) for clobenpropit and thioperamide in
both species. The antagonist potency of the compounds cor-
relate highly with their binding affinities regardless of spe-
cies (r2 ⫽ 0.979). This shows that the compounds are behav-
ing as competitive antagonists and that the binding affinities
Fig. 4. Saturation isotherm of N-[3H]methylhistamine to rat H3-trans- can be predictive of antagonist potency.
fected SK-N-MC cells. Total binding (〫); nonspecific binding (); and Functional Analysis in Recombinant Cells Confirms
specific binding (F). A Kd of 0.8 nM was calculated as ⫺1/slope from a That Chloroproxyfan Is an Agonist. The known H3 ligand
linear Scatchard transformation.
clorproxyfan was tested for its ability to modulate histamine-
TABLE 1 induced inhibition of cAMP accumulation. Our initial analy-
Ki values of known histamine agonists and antagonists sis with chloroproxyfan showed that it could not block the
Values were determined by competition binding with N-[3H]methylhistamine to cell effects of histamine. In fact, chloroproxyfan dose dependently
membranes from either rat recombinant H3-expressing cells, human recombinant
H3-expressing cells, or rat cerebral cortex. Values represent the mean ⫾ S.D. of at
enhanced histamine-induced inhibition of cAMP accumula-
least three experiments. tion (data not shown). When analyzed in the absence of
Rat H3 Ki Human H3 Ki Rat Cortex Ki histamine, chloroproxyfan could dose dependently inhibit
forskolin-stimulated adenylate cyclase with an EC50 of 2 nM
nM
(Fig. 8). This corresponds with its binding affinity of 1 nM at
Imetit 0.2 ⫾ 0.1 0.6 ⫾ 0.1 0.1 ⫾ 0.1
Immepip 1.4 ⫾ 0.5 1.6 ⫾ 0.1 0.5 ⫾ 0.1 both the rat and human recombinant receptors. In addition,
N-Methylhistamine 1.5 ⫾ 0.5 2.4 ⫾ 0.5 1.2 ⫾ 0.2 chloroproxyfan was a full agonist in that it was equally
R-␣-Methylhistamine 1.9 ⫾ 0.6 2.1 ⫾ 0.9 1.5 ⫾ 0.1 efficacious to both histamine and R-␣-methylhistamine at
Clobenpropit 0.4 ⫾ 0.1 0.6 ⫾ 0.1 0.5 ⫾ 0.1
inhibiting ⬎90% of the forskolin-stimulated cAMP accumu-
Thioperamide 4.2 ⫾ 1.8 58.0 ⫾ 19.3 10.2 ⫾ 3.4
Histamine 13.2 ⫾ 1.0 15.2 ⫾ 3.9 12.0 ⫾ 4.2 lation (data not shown).
Clozapine 1,750 ⫾ 350 ⬎10,000 3,000
Discussion
ide was included (Fig. 5). This analysis included all of the The recent cloning of the human histamine H3 receptor
compounds listed in Table 1 except clozapine. cDNA opens up a new area of study for function and phar-
Functional Antagonism in Recombinant H3 Recep- macological intervention of histamine function in the central
tors Correlates with Binding Ki Values. To define the nervous system. Although most of the data reported for the
antagonist potency of various H3 ligands, we measured the human clone were consistent with the known pharmacologi-
pA2 values for competitive antagonism of R-␣-methylhista- cal properties of the rodent H3 receptor characterized in
mine-induced inhibition of forskolin-stimulated cAMP accu- tissue, there were some interesting differences, most notably2000 Cloning of Rat H3 Receptor 775
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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).776 Lovenberg et al. Vol. 293
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 his-
tamine H1 receptor show that the transmembrane domains
are critical for histamine, as well as antagonist binding (Wie-
land 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
Fig. 7. Correlation of antagonist pA2 values to pKi values. Data for both rat and human H3 receptors may account for the differences
rat and human constants for thioperamide (THP) and clobenpropit (CLB) in the pharmacological profiles of thioperamide and clozapine
are plotted.
binding. Results from a site-directed mutagenesis experi-
ment will no doubt lead to a better understanding of how
histamine and synthetic H3 ligands interact with the recep-
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tor 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 in-
nervating 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
Fig. 8. Inhibition of cAMP accumulation in response to chloroproxyfan.
Cells were treated with 5 M forskolin 5 min after the addition of blot (Lovenberg et al., 1999). Our attempts to identify periph-
chloroproxyfan and incubated for an additional 20 min at room temper- eral expression in rat via in situ hybridization have been
ature. All values are determined in triplicate. Data is representative of unsuccessful due to technical difficulties. The question of
multiple (at least three) experiments.
central versus peripheral subtypes of H3 receptors thus
awaits more careful expression analysis of the cDNA we have
the low-affinity binding of the prototypical H3 antagonist cloned versus known sites of action. The presynaptic nature
thioperamide. One possible explanation for the low affinity of the receptor (i.e., distal expression of the mRNA versus
was a simple species difference; however, there is evidence final location of the functional protein) makes it difficult to
reported in the literature to suggest the existence of multiple correlate functional receptors with mRNA expression, partic-
H3 receptor subtypes. To address these two hypotheses, we ularly in the periphery.
cloned and pharmacologically characterized the rat ortholog Functionally, the rat H3 receptor is similar to the human in
of the H3 receptor. that it inhibits adenylate cyclase in response to histamine
We had previously identified, via PCR, a fragment of the and various selective H3 agonists. Inhibition of adenylate
rat ortholog encoding the H3 receptor to map the mRNA cyclase is the mechanism of action shared by the major re-
expression within the central nervous system. In doing so, we lease-inhibiting G-protein-coupled autoreceptors and hetero-
determined that the rat H3 receptor was robustly expressed receptors such as ␣2 (norepinephrine), D2 (dopamine), M2
in the cortex, striatum, thalamus, and hypothalamus. A size- (acetylcholine), 5HT1D (serotonin), and H3 (histamine)
selected cDNA library from rat hypothalamus tissue was (Langer, 1997). Interestingly, the dominant structural ho-
used to identify a full-length clone consisting of 2.4 kb with a mology of the H3 receptor in certain transmembrane domains
1335-bp open reading frame encoding a putative protein of is to ␣2 and M2 receptors, not to H1 or H2 receptors (Loven-
445 amino acids. The cDNA clone showed high sequence berg et al., 1999).
identity to the human receptor with 93% identity at the The EC50 values for agonist activation of the rat H3 recep-
amino acid level. As expected, a majority of the identity is tor were nearly identical with those for activation of the
observed within the transmembrane domains (97%), a result human receptor. In addition, binding of the agonists to the
of only five amino acid substitutions (Fig. 1). These trans- rat receptor showed similar affinity between the rat and the
membrane domains contain the presumed binding pockets human receptors. In contrast, antagonist binding showed
for agonists (e.g., histamine) as well as competitive antago- several pharmacological differences between the rat and hu-
nists (e.g., thioperamide). The binding of various biogenic man receptors. Our previous report of antagonist Ki values at
amines to their respective receptors appears to be highly the human receptor were mostly consistent with published
dependent on specific amino acid residues in transmembrane data for binding to rat tissues. However, one notable distinc-2000 Cloning of Rat H3 Receptor 777
tion was the apparent low affinity of thioperamide for the (Ligneau et al., 1994), were in fact agonists (Schlicker et al.,
human recombinant receptor (58 nM). This finding has re- 1996, Watt et al., 1997). Because the recombinant H3 recep-
cently been confirmed by examining the binding of N-[3H]m- tor systems have allowed an easy determination of agonist
ethylhistamine to human postmortem cerebral cortex where and antagonist potency, we tested one of these compounds,
thioperamide had a reported Ki of 200 nM (West et al., 1999). chloroproxyfan. In both the rat and human recombinant sys-
Earlier functional reports with human saphenous vein (Oike tems, chloroproxyfan behaved as a pure, high-affinity recep-
et al., 1992) and a human gastric cell line (Cherifi et al., 1992) tor agonist. Although it can be argued that that recombinant
had alluded to low Ki values for thioperamide. In this article, systems will not be representative of an intact tissue, these
we show that thioperamide displays high affinity for the systems allow one to easily determine an intrinsic efficacy
recombinant rat H3 receptor and the H3 receptor expressed potential of a compound, particularly against the human
in rat cortex, consistent with literature values for binding to receptor where it is often difficult to obtain fresh, functional
rat tissue. Together, these data suggest that the recombinant tissue preparations.
receptors are representative of the natural binding affinities. The availability of the rat cDNA will now allow for a full
One other difference that was noted was the affinity of analysis of various H3 receptor ligands, and hopefully will
clozapine for the rat receptor. Clozapine had previously been help define the structure-activity requirements for the devel-
reported to bind to the rat H3 receptor (from brain tissue), opment of better agonists and antagonists. There are clear
leading to speculation that some of its antipsychotic effects in pharmacological distinctions that will be critical for the de-
humans may be due to H3 receptor antagonism (Kathmann velopment of novel H3 receptor ligands. Identification of ad-
et al., 1994, Rodrigues et al., 1995, Stark et al., 1996). Our ditional species orthologs of the H3 receptor, such as guinea
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finding (Lovenberg et al., 1999) that clozapine did not signif- pig, also will be extremely useful because much of the liter-
icantly compete for binding to the recombinant human recep- ature discrepancy surrounding H3 subtype identification
tor put serious doubt on the validity of the hypothesis. How- stems from differences between rat and guinea pig H3 func-
ever, our current findings with the rat recombinant receptor tion and pharmacology (Schlicker et al., 1996).
confirm the validity of the original findings in rat tissue
because clozapine effectively competes for binding albeit with Acknowledgments
low affinity (1.75 M). These findings also suggest that much We thank Jose Galindo for his great help in assembling the se-
of the differences in pharmacology reported in the literature quence information, and Drs. Rob Leurs and Kersten Wieland for
may be due solely to species differences and not H3 hetero- assistance in identification and correction of several sequence errors.
geneity (West et al., 1990). It does not however completely We also thank Dr. Nigel Shankley for providing chloroproxyfan.
rule out the existence of multiple H3 receptor subtypes. In- References
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