BACKGROUND
antagonist affinity measurements have always played a key role in pharmacology  <cit> .
they have been vital in the initial determination of different receptor sub-types and have been a major tool in determining which receptor subtypes are present within a given tissue .
initially this led to the suggestion of the existence of a β4-adrenoceptor, however many studies since, including those using knockout animals  <cit> , have shown that the different antagonist affinities determined are from one receptor, the β1-adrenoceptor, but that this receptor exists in at least two different agonist states  <cit> .
several gpcrs have now been shown to exist in more that one agonist state or conformation.
for some receptors, this results in signalling via more than one class of g-protein  and are therefore considered to exist in a single agonist conformation with a constant antagonist affinity  <cit> .
the histamine h <dig> receptor is a constitutively active gpcr that is involved in neurotransmitter release within the cns  <cit>  and references therein.
the histamine receptor subtypes h <dig> and h <dig> were first characterised in the 1960's and 1970's  <cit> .
then in the 1970's and early 1980's, arrang, schwartz and colleagues observed that not all histamine actions in rat neurones were inhibited by h <dig> and h <dig> antagonists in a manner that fitted the profile for either histamine receptor subtype and therefore proposed a third histamine receptor subtype  <cit> .
the existence of the histamine h <dig> receptor was finally confirmed by the demonstration that r-α-methylhistamine was more potent than s-α-methylhistamine in reducing histamine release from rat brain slices and the identification of thioperamide as a selective h <dig> antagonist  <cit> .
the h <dig> receptor was cloned in  <dig>  <cit>  and many selective and very high potency h <dig> agonists and antagonists have been identified  <cit> .
the h <dig> receptor couples to heterotrimeric gi/o-proteins, activation of which causes a decrease in camp production and pka activation  <cit> .
its other known actions  are all thought to occur via the various subunits of the heterotrimeric g-protein following gi/o activation  <cit> .
recently, several unusual properties for the histamine h <dig> receptor and its ligands have been demonstrated.
of particularly interest here is that the h <dig> receptor exists in different agonist-induced states.
proxyfan has been demonstrated to be a protean agonist  <cit>  of the h <dig> receptor – thus the efficacy observed depends upon the receptor expression level and constitutive activity in the system under study  <cit> .
proxyfan stabilises a medium-efficacy state of the h <dig> receptor and so appears as a full or partial agonist in systems with less constitutive activity, a neutral antagonist of higher efficacy ligands in lower receptor expression systems, and an inverse agonist in highly constitutively active system, where it stabilises a receptor conformation of lower efficacy than the constitutively active state  <cit> .
thus proxyfan has conclusively demonstrated that the histamine h <dig> receptor exists in more than one agonist state or conformation.
the aim of this study was therefore to examine the antagonist affinity measurements made at the human h <dig> histamine receptor stably expressed in cho cells using a cre-reporter gene system.
this study examines, in detail, antagonist affinity measurements made at the different states of the h <dig> receptor including those induced by partial agonists, and at the medium-efficacy proxyfan induced state as distinct from the full-agonist histamine induced state of the receptor.
methods
materials
fetal calf serum was from paa laboratories .
3h-adenine and 14c-camp were from amersham international .
n-α-methylhistamine, r-α-methylhistamine, s-α-methylhistamine, 4-methylhistamine, immepip, imetit, immethridine, impentamine, amthamine, dimaprit, vuf  <dig>  vuf  <dig>  htmt, 2-pyridylethylamine, iodophenpropit, clobenpropit, proxyfan, zolatidine, conessine, thioperamide, dimethindene, triprolidine, clemastine and burimamide were obtained from tocris cookson .
pertussin toxin was from calbiochem .
histamine, cimetidine, ranitidine, 1-methylhistamine, 3-methylhistamine, chlorpheniramine, ibmx and forskolin were from sigma chemicals  who also supplied all other reagents.
cell culture
a stable clonal cho-k <dig> cell line expressing a cre-spap reporter gene  was secondarily transfected with the full length human histamine h <dig> receptor  using lipofectamine and optimem as per manufacturer's instructions.
the transfected cells were selected for neomycin resistance  and hygromycin resistance  for  <dig> weeks and passaged twice during in this period.
a single clone was then isolated by dilution cloning to give a clonal line .
the parent cell cre-spap was also used for control experiments .
cells were grown in dulbecco's modified eagles medium/nutrient mix f <dig>  containing 10% fetal calf serum and  <dig> mm l-glutamine at 37°c in a humidified 5% co2: 95% air atmosphere.
3h-r-α-methylhistamine whole cell binding
cells were grown to confluence in white-sided 96-well view plates.
the media was then removed and replaced with  <dig> μl ice cold pbs  per well  or  <dig> μl ice cold pbs  containing  <dig> μm iodophenpropit .
<dig> μl 3h-r-α-methylhistamine was then immediately added to the wells to give concentrations in the range of  <dig> – <dig> nm.
cells were then maintained a 4°c for  <dig> hours.
all pbs and drugs were removed and the cells washed twice by the addition and removal of  <dig> ×  <dig> μl 4°c pbs.
a white bottom and clear sealant top was added to the wells, the plates left overnight in the dark at room temperature and the plates counted on a topcount  at 21°c  <dig> minute count per well.
cre-spap gene transcription
cells were grown to confluence in 96-well tissue culture plates.
the cells were then serum starved by removing the media and replacing it with  <dig> μl serum free media .
the cells were then incubated for a further  <dig> hours .
where used, pertussis toxin, at a final concentration of  <dig> ng/ml was added to the serum-free media and thus incubated with the cells for  <dig> hours.
on the day of experimentation, the serum-free media was removed and replaced with  <dig> μl serum-free media or  <dig> μl serum-free media containing an antagonist at the final required concentration and the cells incubated for  <dig> hour.
agonist in  <dig> μl  was then added to each well and the cells incubated for  <dig> minutes.
forskolin was then added to all but the basal wells to give a final well concentration of  <dig> μm and the plates incubated for  <dig> hours.
after  <dig> hours, the media and all drugs were removed.
<dig> μl serum-free media was added to each well and the cells incubated for a further  <dig> hour .
the plates were then incubated at 65°c for  <dig> minutes to destroy any endogenous phosphatases.
after cooling to 37°c,  <dig> μl  <dig> mm pnpp in diethanolamine buffer was added to each well and the plates incubated at 37°c in a normal atmosphere until the yellow colour developed.
the plates were then read on a dynatech mrx plate reader at  <dig> nm.
3h-camp accumulation
cells were grown to confluence in 24-well plates.
the media was removed and the cells pre-labelled with 3h-adenine by incubation with  <dig> μci/ml 3h-adenine in serum-free media  for  <dig> hours.
the 3h-adenine was then removed and the cells washed by the addition then removal of  <dig> ml serum-free media.
<dig> ml serum-free media containing  <dig> μm ibmx with or without the final required concentration of antagonist was then added to each well and the cells incubated for  <dig> minutes.
agonist  was added to each well and the plates incubated for  <dig> minutes.
forskolin  was then added to all but the basal wells and the plates incubated for  <dig> minutes.
the reaction was terminated by the addition of  <dig> μl concentrated hcl per well and the plates were then frozen.
later, the plates were thawed and 3h-camp separated from other 3h-nucleotides by sequential dowex and alumina column chromatography, as previously described  <cit> .
when the intrinsic efficacy of the antagonist ligands was examined, the antagonists were incubated for  <dig> hours at 37°c in order to maximise the chance of detecting any changes in 3h-camp accumulation.
data analysis
binding studies
to determine the expression level in the cho-h3-spap cells, the saturation curves for specific 3h-r-α-methylhistamine binding were fitted to the following equation using graphpad prism 2:
specific binding=bmax⁡
however, as the specific binding curves appeared to contain a linear component, the data were also fitted to the following expression:
specific binding=bmax⁡+m×
bmax is the maximum specific binding, kd is the dissociation constant of 3h-r-α-methylhistamine,  is the concentration of 3h-r-α-methylhistamine and m is the slop of the linear component of binding.
functional data
sigmoidal concentration-response curves were fitted to the data using graphpad prism  <dig> and the following equation:
response=emax×ic50+
where emax is the maximal response,  is the agonist concentration and ic <dig> is the concentration of agonist that produces 50% of the maximal response.
antagonist kd values were then calculated from the shift of the agonist concentration responses in the presence of a fixed concentration of antagonist using the following equation:
dr=1+kd
where dr  is the ratio of the agonist concentration required to stimulate an identical response in the presence and absence of a fixed concentration of antagonist .
in experiments where three different fixed concentrations of the same antagonist were used, schild plots were constructed using the following equation:
log  = log  - log
these points were then fitted to a straight line.
a slope of  <dig> then indicates competitive antagonism  <cit> .
when vuf  <dig> was used as an antagonist , the partial agonist nature of this ligand was seen.
partial agonist dissociation constants were therefore estimated according to the method of stephenson  <cit>  using the following equation:
kd partial agonist=x.1−xwhere x=−
where  in the concentration of the partial agonist vuf  <dig>   in the concentration of the agonist at the point where the fixed partial agonist causes the same response,  in the concentration of agonist causing a given response above that achieved by the partial agonist and  the concentration of the agonist, in the presence of the partial agonist, causing the same stimulation as .
in figure 6a, the concentration-response curve is best fitted to a 2-component response, the following equation was used:
% maximal stimulation=.n+.
where n is the percentage of site  <dig>   is the concentration of agonist and ic <dig> and ic <dig> are the respective ic <dig> values for the two agonist sites.
all data are presented as mean ± s.e.m.
of triplicate determinations where n is the number of separate experiments.
RESULTS
determination of cho-h3-spap cell line h3-receptor expression level
from the saturation binding studies, the kd value for 3h-r-α-methylhistamine was  <dig>  ±  <dig>  nm .
the receptor expression level of the human h <dig> receptor as determined by 3h-r-α-methylhistamine was  <dig>  ±  <dig>  fmol/mg protein .
identification of h <dig> agonists
histamine agonists were first screened for inhibition of forskolin-stimulated cre-spap production in cho-h3-spap cells.
in addition to the many ligands traditionally considered to be h <dig> agonists, several others were found to have partial agonist actions – e.g.
burimamide and impentamine in keeping with previous studies .
vuf  <dig> was found to also have agonist properties.
in addition the h <dig> agonists amthamine and dimaprit stimulated agonist responses  as well as htmt .
1-methylhistamine , 3-methylhistamine  and 4-methylhistamine  also stimulated low potency responses.
the h <dig> agonist 2-pyridylethylamine  <cit>  stimulated a response, however the ic <dig> for this was greater than  <dig> μm.
pic <dig> values for 3h-camp accumulation and cre-spap production for the ligands found to be agonists at the histamine h <dig> receptor in cho-h3-spap cells.
values represent mean ± s.e.mean of n separate experiments.
* the cre-spap response to impentamine appeared to consist of  <dig> components .
of this  <dig>  ±  <dig> % was occurring via site 1
investigation of gi and gs coupling
all agonist responses were also examined following  <dig> hours pre-incubation with pertussis toxin .
agonist responses to all of the ligands mentioned above and listed in table  <dig> were abolished by ptx .
none of the antagonist ligands used in this study stimulated any response either in the absence or following  <dig> hours pre-incubation with ptx.
finally all ligands used in the study  were examined in the absence of forskolin  in order to look for any gs-stimulatory responses than might otherwise have been masked by the presence of  <dig> μm forskolin.
no responses were seen to any of the ligands .
determination of antagonist affinity
measurements of antagonist affinity were them made in the presence of agonists of different efficacies, including full agonists, partial agonists and proxyfan .
where possible, the response to each agonist was assessed in the presence of at three different concentrations of antagonists thus allowing a schild plot to be constructed .
however, for many antagonist ligands, the affinity was relatively poor and hence antagonist affinity was assessed from parallel shifts in the presence of one or two concentrations of antagonist.
the ability of the partial agonist vuf  <dig> to inhibit the more efficacious agonists was also assessed  however, here the affinity of vuf  <dig> was calculated by the partial agonist method of stephenson  <cit> .
pkd values for  <dig> antagonists as determined from measurements of cre-spap production from cho-h3-cells made in the presence of the  <dig> different agonists.
values represent mean ± s.e.mean of n separate experiments.
the pkd values quoted are for antagonism of the first component of the impentamine response.
* it was not possible to determine a pkd value for vuf  <dig> in the presence of impentamine due to the partial agonist nature of both responses.
slope of the schild plot from antagonism of those compounds where  <dig> different concentrations of antagonists were possible as determined from measurements of cre-spap production from cho-h3-spap cells.
values represent mean ± s.e.mean of n separate experiments.
* schild plots were not obtained.
schild plots were also not obtained when impentamine and vuf  <dig> were agonists
the ligands were then examined in the 3h-camp accumulation assay and again, the agonist actions of the ligands was clearly observed .
the antagonist affinity measurements of two of the antagonists, clobenpropit and thioperamide, were then examined in the 3h-camp accumulation assay .
given that vuf  <dig> and burimamide were more potent  in the 3h-camp accumulation assay, pkd values were also able to be determined when these ligands were the agonists .
pkd values for clobenpropit and thioperamide as determined from 3h-camp accumulation measurements made in the presence of the  <dig> different agonists.
values represent mean ± s.e.mean of n separate experiments.
impentamine
impentamine stimulated a cre-gene transcription response that was best described by a two-component concentration response curve pic <dig> =  <dig>  ±  <dig> , n =  <dig> and pic <dig> =  <dig>  ±  <dig> , n =  <dig>  figure 6a).
when examined in the presence of any of the antagonists used in this study, only the first component was inhibited and this yielded a pkd value for each antagonist the same as those obtained in the presence of all other agonists .
when the impentamine response was examined following pre-incubation with ptx, the first component was abolished, and only the second component remained .
furthermore, this low potency component was seen in the absence of forskolin in cho-h3-spap cells both with  and without ptx pre-incubation  and in cho-spap cells  both with  and without forskolin .
following  <dig> hours incubation with impentamine, cho-h3-spap cells did not show any intracellular uptake of trypan blue.
finally, the 3h-camp accumulation response is best described by a one-component sigmoidal concentration response curve, the low potency component not being seen at all.
thus at high concentrations, impentamine appears to have been inhibiting the cre-spap transcription or translation rather than appearing toxic to the cells, as they did not take up trypan blue following  <dig> hours incubation with impentamime and the 3h-camp accumulation response remained intact .
taken together, this suggests that the first component of the impentamine response  is due to partial agonism via the h <dig> receptor and the second component due to a non-specific non-receptor mediated inhibition of the cre-spap response downstream from camp, and therefore unlike the two-component responses seen at either the β1-adrenoceptor  or the β3-adrenoceptor .
investigation of the intrinsic activity of the h3-antagonists
finally, as the histamine h <dig> receptor is known have constitutive activity , none of the  <dig> other antagonist ligands stimulated a change in cre-spap production in cho-h3-spap cells.
however, previous studies have however demonstrated that small changes may be more clearly seen in the 3h-camp accumulation assay due to the larger response window.
all antagonist ligands were therefore examined in the 3hc-amp accumulation assay following  <dig> hours incubation with ligands in order to maximise any changes seen.
in this assay, the ic <dig> values for the partial agonist vuf  <dig> remained unchanged .
inverse agonist responses were seen for conessine , thioperamide , ranitidine  and cimetidine .
no responses were seen to zolatidine, dimethindine, iodophenpropit, clobenpropit, triprolidine, clemastine or chlorpheniramine .
lack of gene transcription responses in cho-spap cells
the response to all ligand used in this study was examined in the parent cho-spap cells .
full 7-point concentration response curves were constructed, in the presence and absence of  <dig> μm forskolin.
no responses were seen in response to any ligand , up to concentrations of  <dig> μm .
a small decrease from maximum was seen only at the highest concentration  of vuf  <dig>
discussion
several gpcrs have now been shown to exist in several different conformations, stablised by different agonists, that either couple to different g-proteins, signal via different pathways or to which ligands bind with different affinities  <cit> .
the histamine h <dig> receptor is a gi-coupled receptor with a large range of ligands of varying efficacies thus making a detailed pharmacological study of the receptor possible.
several newer ligands have also been found for the h <dig> receptor and their structure is becoming increasingly diverse , other ligands were also found to have agonist properties e.g.
the h <dig> agonists amthamine and dimaprit.
vuf  <dig> was previously reported to be a neutral antagonist of the histamine h <dig> receptor  <cit> .
however, here it was found to have agonist activity in both the 3h-camp accumulation assay and the cre-spap assay.
as all of these responses did not occur in cre-spap cells  and their responses were sensitive to pre-incubation with pertussis toxin , they too were occurring via the histamine h <dig> receptor.
in view of the fact that the human histamine h <dig> receptor is known to be constitutively active  <cit> , evidence for this was sought in terms of demonstrating inverse agonist action of the antagonist ligands.
given the low expression level of this cell line very little constitutive activity would be expected to be seen, and indeed this was the case.
only conessine, thioperamide, ranitidine and cimetidine were found to be inverse agonists in this relatively low expressing cell system.
however, this nonetheless confirmed the constitute nature of this receptor.
as there was relatively little constitutive activity, this should mean that a protean agonist would appear as a more efficacious agonist, as indeed proxyfan did.
because of this, antagonist affinity measurements from parallel shifts of an agonist response were more easily determined.
it is interesting to note that several histamine ligands were found to have significant agonist activity in this cell system that has not been previously reported e.g.
vuf  <dig>  it could therefore be that vuf  <dig> is also a protean agonist and if examined in systems of different receptor expression and constitutive activity  this might be demonstrated.
proxyfan appears as a full agonist in this cell system and vuf  <dig> as a partial agonist.
if vuf  <dig> is indeed a protean agonist, it stabilises a different, lower efficacy protean state of the receptor to that stabilised by proxyfan.
all agonists at the h <dig> receptor appeared more potent in the 3h-camp accumulation assay than in the cre-gene transcription assay.
this is in contrast to a recent study of the gs-coupled human histamine h <dig> receptor , but is similar to that seen in the gi-coupled adenosine a <dig> receptor  <cit> .
potencies between assays appear similar at the β <dig> and β3-adrenoceptor  but vary depending on the efficacy of the agonist at the β2-adrenoceptor.
the reason for the change in potency between assays, that clearly varies with different gpcrs, remains unknown but may be related to different patterns of phosphorylation, internalisation and desensitisation that occur at different gpcrs  <cit> .
another major conclusion that can be drawn from this study is that not all gi-coupled receptors behave alike in a recombinant cell system.
the gi-coupled adenosine a1-receptor, expressed in the same cho-reporter cell back ground as used in this study, clearly showed the receptor coupling to gi and gs-proteins  <cit> .
in this h <dig> study, all ligands were first assessed for their agonist activity in the presence and absence of forskolin, with and without ptx pre-incubation.
inhibitory responses were only ever seen.
all of these responses were only seen in the presence of forskolin and all responses were abolished by pre-incubating the cells with ptx.
the h <dig> receptor therefore did not demonstrate any gs-coupled stimulatory cre-gene transcription responses, even at high agonist concentrations, and thus has a different agonist receptor activation profile to the a1-adenosine receptor.
the affinity for the antagonists, as measured from parallel shifts of the agonist concentration response curve, remained constant at the human h <dig> receptor.
where values are available, the antagonist affinity values obtained here are similar to those previously published e.g.
<cit> .
this was also true for proxyfan stimulated responses.
conessine, a steroidal alkaloid and a natural product from the stem bark of funtumia elastica, was a high affinity h <dig> inverse agonist and the pkd values for its antagonism of different h3-agonists obtained here were very similar to that reported by cowart et al.,  <cit> .
thus, regardless of which competing agonist was used, the antagonist affinity of a given antagonist remained the same.
furthermore, the schild plots, where possible, all had a slope of  <dig> confirming competitive antagonism.
again, where possible, this was true for the proxyfan stimulated responses.
this was true even when examining the antagonism of partial agonist responses .
this also held even when the antagonism of full agonists by a partial agonist  was examined.
finally, the antagonist affinity values obtained were the same in the 3h-camp accumulation assay as the cre-gene transcription assay.
therefore, the histamine h <dig> receptor is unlike any of the β-adrenoceptors in terms of simple antagonist affinity measurements.
antagonist affinity measurements remain constant at the h <dig> receptor, regardless of the competing agonist's efficacy, the cellular response measured, the time of incubation of agonist or the presence of a pde inhibitor.
CONCLUSIONS
in conclusion, the human histamine h <dig> receptor is a gi-coupled receptor that, in contrast to the human a1-receptor, has no evidence of coupling to gs-proteins in this low receptor expressing recombinant cell system.
several ligands were identified as having agonist activity, including some ligands previously considered to be antagonists .
given the low constitutive activity of this cell system, these ligands might yet turn out to be protean agonists.
conessine was shown to be a high affinity inverse agonist.
finally, the competitive nature of the antagonists was demonstrated and antagonist affinity measurements were constant for each antagonist, including at the proxyfan-induced medium-efficacy state of the h <dig> receptor, in contrast to all three subtypes of the β-adrenoceptors, but in keeping with the traditional pharmacological dogma.
abbreviations
camp, adenosine-3',5'-cyclic monophosphate; cho, chinese hamster ovary; cre, cyclic amp response element; dmem/f <dig>  dulbecco's modified eagles medium/nutrient mix f12; gpcr, g-protein coupled receptor; htmt, histamine trifluoromethyl toluidide; nαmh, nα-methylhistamine; pbs, phosphate buffered saline; pea, 2-pyridylethylamine; pnpp 4-nitrophenyl phosphate; ptx, pertussis toxin; rαmh, r-α-methylhistamine; sαmh, s-α-methylhistamine; spap, secreted placental alkaline phosphatise; vuf  <dig>  4-piperidine dihydrobromide; vuf  <dig>  2-ethyl carbamimidothioic acid ester dihydrobromide.
authors' contributions
jgb conceived the study, carried out the experiments, data analysis and preparation of the manuscript.

 antagonist affinity measurements have always played a key role in pharmacology. they have been vital in the initial determination of different receptor sub-types and have been a major tool in determining which receptor subtypes are present within a given tissue. initially this led to the suggestion of the existence of a β4-adrenoceptor, however many studies since, including those using knockout animals, have shown that the different antagonist affinities determined are from one receptor, the β1-adrenoceptor, but that this receptor exists in at least two different agonist states. several gpcrs have now been shown to exist in more that one agonist state or conformation. for some receptors, this results in signalling via more than one class of g-protein and are therefore considered to exist in a single agonist conformation with a constant antagonist affinity. the histamine h receptor is a constitutively active gpcr that is involved in neurotransmitter release within the cns and references therein. the histamine receptor subtypes h and h were first characterised in the 1960's and 1970's. then in the 1970's and early 1980's, arrang, schwartz and colleagues observed that not all histamine actions in rat neurones were inhibited by h and h antagonists in a manner that fitted the profile for either histamine receptor subtype and therefore proposed a third histamine receptor subtype. the existence of the histamine h receptor was finally confirmed by the demonstration that r-α-methylhistamine was more potent than s-α-methylhistamine in reducing histamine release from rat brain slices and the identification of thioperamide as a selective h antagonist. the h receptor was cloned in and many selective and very high potency h agonists and antagonists have been identified. the h receptor couples to heterotrimeric gi/o-proteins, activation of which causes a decrease in camp production and pka activation. its other known actions are all thought to occur via the various subunits of the heterotrimeric g-protein following gi/o activation. recently, several unusual properties for the histamine h receptor and its ligands have been demonstrated. of particularly interest here is that the h receptor exists in different agonist-induced states. proxyfan has been demonstrated to be a protean agonist of the h receptor – thus the efficacy observed depends upon the receptor expression level and constitutive activity in the system under study. proxyfan stabilises a medium-efficacy state of the h receptor and so appears as a full or partial agonist in systems with less constitutive activity, a neutral antagonist of higher efficacy ligands in lower receptor expression systems, and an inverse agonist in highly constitutively active system, where it stabilises a receptor conformation of lower efficacy than the constitutively active state. thus proxyfan has conclusively demonstrated that the histamine h receptor exists in more than one agonist state or conformation. the aim of this study was therefore to examine the antagonist affinity measurements made at the human h histamine receptor stably expressed in cho cells using a cre-reporter gene system. this study examines, in detail, antagonist affinity measurements made at the different states of the h receptor including those induced by partial agonists, and at the medium-efficacy proxyfan induced state as distinct from the full-agonist histamine induced state of the receptor. methods materials fetal calf serum was from paa laboratories. 3h-adenine and 14c-camp were from amersham international. n-α-methylhistamine, r-α-methylhistamine, s-α-methylhistamine, 4-methylhistamine, immepip, imetit, immethridine, impentamine, amthamine, dimaprit, vuf vuf htmt, 2-pyridylethylamine, iodophenpropit, clobenpropit, proxyfan, zolatidine, conessine, thioperamide, dimethindene, triprolidine, clemastine and burimamide were obtained from tocris cookson. pertussin toxin was from calbiochem. histamine, cimetidine, ranitidine, 1-methylhistamine, 3-methylhistamine, chlorpheniramine, ibmx and forskolin were from sigma chemicals who also supplied all other reagents. cell culture a stable clonal cho-k cell line expressing a cre-spap reporter gene was secondarily transfected with the full length human histamine h receptor using lipofectamine and optimem as per manufacturer's instructions. the transfected cells were selected for neomycin resistance and hygromycin resistance for weeks and passaged twice during in this period. a single clone was then isolated by dilution cloning to give a clonal line. the parent cell cre-spap was also used for control experiments. cells were grown in dulbecco's modified eagles medium/nutrient mix f containing 10% fetal calf serum and mm l-glutamine at 37°c in a humidified 5% co2: 95% air atmosphere. 3h-r-α-methylhistamine whole cell binding cells were grown to confluence in white-sided 96-well view plates. the media was then removed and replaced with μl ice cold pbs per well or μl ice cold pbs containing μm iodophenpropit. μl 3h-r-α-methylhistamine was then immediately added to the wells to give concentrations in the range of – nm. cells were then maintained a 4°c for hours. all pbs and drugs were removed and the cells washed twice by the addition and removal of × μl 4°c pbs. a white bottom and clear sealant top was added to the wells, the plates left overnight in the dark at room temperature and the plates counted on a topcount at 21°c minute count per well. cre-spap gene transcription cells were grown to confluence in 96-well tissue culture plates. the cells were then serum starved by removing the media and replacing it with μl serum free media. the cells were then incubated for a further hours. where used, pertussis toxin, at a final concentration of ng/ml was added to the serum-free media and thus incubated with the cells for hours. on the day of experimentation, the serum-free media was removed and replaced with μl serum-free media or μl serum-free media containing an antagonist at the final required concentration and the cells incubated for hour. agonist in μl was then added to each well and the cells incubated for minutes. forskolin was then added to all but the basal wells to give a final well concentration of μm and the plates incubated for hours. after hours, the media and all drugs were removed. μl serum-free media was added to each well and the cells incubated for a further hour. the plates were then incubated at 65°c for minutes to destroy any endogenous phosphatases. after cooling to 37°c, μl mm pnpp in diethanolamine buffer was added to each well and the plates incubated at 37°c in a normal atmosphere until the yellow colour developed. the plates were then read on a dynatech mrx plate reader at nm. 3h-camp accumulation cells were grown to confluence in 24-well plates. the media was removed and the cells pre-labelled with 3h-adenine by incubation with μci/ml 3h-adenine in serum-free media for hours. the 3h-adenine was then removed and the cells washed by the addition then removal of ml serum-free media. ml serum-free media containing μm ibmx with or without the final required concentration of antagonist was then added to each well and the cells incubated for minutes. agonist was added to each well and the plates incubated for minutes. forskolin was then added to all but the basal wells and the plates incubated for minutes. the reaction was terminated by the addition of μl concentrated hcl per well and the plates were then frozen. later, the plates were thawed and 3h-camp separated from other 3h-nucleotides by sequential dowex and alumina column chromatography, as previously described. when the intrinsic efficacy of the antagonist ligands was examined, the antagonists were incubated for hours at 37°c in order to maximise the chance of detecting any changes in 3h-camp accumulation. data analysis binding studies to determine the expression level in the cho-h3-spap cells, the saturation curves for specific 3h-r-α-methylhistamine binding were fitted to the following equation using graphpad prism 2: specific binding=bmax⁡ however, as the specific binding curves appeared to contain a linear component, the data were also fitted to the following expression: specific binding=bmax⁡+m× bmax is the maximum specific binding, kd is the dissociation constant of 3h-r-α-methylhistamine, is the concentration of 3h-r-α-methylhistamine and m is the slop of the linear component of binding. functional data sigmoidal concentration-response curves were fitted to the data using graphpad prism and the following equation: response=emax×ic50+ where emax is the maximal response, is the agonist concentration and ic is the concentration of agonist that produces 50% of the maximal response. antagonist kd values were then calculated from the shift of the agonist concentration responses in the presence of a fixed concentration of antagonist using the following equation: dr=1+kd where dr is the ratio of the agonist concentration required to stimulate an identical response in the presence and absence of a fixed concentration of antagonist. in experiments where three different fixed concentrations of the same antagonist were used, schild plots were constructed using the following equation: log = log - log these points were then fitted to a straight line. a slope of then indicates competitive antagonism. when vuf was used as an antagonist, the partial agonist nature of this ligand was seen. partial agonist dissociation constants were therefore estimated according to the method of stephenson using the following equation: kd partial agonist=x.1−xwhere x=− where in the concentration of the partial agonist vuf in the concentration of the agonist at the point where the fixed partial agonist causes the same response, in the concentration of agonist causing a given response above that achieved by the partial agonist and the concentration of the agonist, in the presence of the partial agonist, causing the same stimulation as. in figure 6a, the concentration-response curve is best fitted to a 2-component response, the following equation was used: % maximal stimulation=.n+. where n is the percentage of site is the concentration of agonist and ic and ic are the respective ic values for the two agonist sites. all data are presented as mean ± s.e.m. of triplicate determinations where n is the number of separate experiments.  determination of cho-h3-spap cell line h3-receptor expression level from the saturation binding studies, the kd value for 3h-r-α-methylhistamine was ± nm. the receptor expression level of the human h receptor as determined by 3h-r-α-methylhistamine was ± fmol/mg protein. identification of h agonists histamine agonists were first screened for inhibition of forskolin-stimulated cre-spap production in cho-h3-spap cells. in addition to the many ligands traditionally considered to be h agonists, several others were found to have partial agonist actions – e.g. burimamide and impentamine in keeping with previous studies. vuf was found to also have agonist properties. in addition the h agonists amthamine and dimaprit stimulated agonist responses as well as htmt. 1-methylhistamine, 3-methylhistamine and 4-methylhistamine also stimulated low potency responses. the h agonist 2-pyridylethylamine stimulated a response, however the ic for this was greater than μm. pic values for 3h-camp accumulation and cre-spap production for the ligands found to be agonists at the histamine h receptor in cho-h3-spap cells. values represent mean ± s.e.mean of n separate experiments. * the cre-spap response to impentamine appeared to consist of components. of this ± % was occurring via site 1 investigation of gi and gs coupling all agonist responses were also examined following hours pre-incubation with pertussis toxin. agonist responses to all of the ligands mentioned above and listed in table were abolished by ptx. none of the antagonist ligands used in this study stimulated any response either in the absence or following hours pre-incubation with ptx. finally all ligands used in the study were examined in the absence of forskolin in order to look for any gs-stimulatory responses than might otherwise have been masked by the presence of μm forskolin. no responses were seen to any of the ligands. determination of antagonist affinity measurements of antagonist affinity were them made in the presence of agonists of different efficacies, including full agonists, partial agonists and proxyfan. where possible, the response to each agonist was assessed in the presence of at three different concentrations of antagonists thus allowing a schild plot to be constructed. however, for many antagonist ligands, the affinity was relatively poor and hence antagonist affinity was assessed from parallel shifts in the presence of one or two concentrations of antagonist. the ability of the partial agonist vuf to inhibit the more efficacious agonists was also assessed however, here the affinity of vuf was calculated by the partial agonist method of stephenson. pkd values for antagonists as determined from measurements of cre-spap production from cho-h3-cells made in the presence of the different agonists. values represent mean ± s.e.mean of n separate experiments. the pkd values quoted are for antagonism of the first component of the impentamine response. * it was not possible to determine a pkd value for vuf in the presence of impentamine due to the partial agonist nature of both responses. slope of the schild plot from antagonism of those compounds where different concentrations of antagonists were possible as determined from measurements of cre-spap production from cho-h3-spap cells. values represent mean ± s.e.mean of n separate experiments. * schild plots were not obtained. schild plots were also not obtained when impentamine and vuf were agonists the ligands were then examined in the 3h-camp accumulation assay and again, the agonist actions of the ligands was clearly observed. the antagonist affinity measurements of two of the antagonists, clobenpropit and thioperamide, were then examined in the 3h-camp accumulation assay. given that vuf and burimamide were more potent in the 3h-camp accumulation assay, pkd values were also able to be determined when these ligands were the agonists. pkd values for clobenpropit and thioperamide as determined from 3h-camp accumulation measurements made in the presence of the different agonists. values represent mean ± s.e.mean of n separate experiments. impentamine impentamine stimulated a cre-gene transcription response that was best described by a two-component concentration response curve pic = ±, n = and pic = ±, n = figure 6a). when examined in the presence of any of the antagonists used in this study, only the first component was inhibited and this yielded a pkd value for each antagonist the same as those obtained in the presence of all other agonists. when the impentamine response was examined following pre-incubation with ptx, the first component was abolished, and only the second component remained. furthermore, this low potency component was seen in the absence of forskolin in cho-h3-spap cells both with and without ptx pre-incubation and in cho-spap cells both with and without forskolin. following hours incubation with impentamine, cho-h3-spap cells did not show any intracellular uptake of trypan blue. finally, the 3h-camp accumulation response is best described by a one-component sigmoidal concentration response curve, the low potency component not being seen at all. thus at high concentrations, impentamine appears to have been inhibiting the cre-spap transcription or translation rather than appearing toxic to the cells, as they did not take up trypan blue following hours incubation with impentamime and the 3h-camp accumulation response remained intact. taken together, this suggests that the first component of the impentamine response is due to partial agonism via the h receptor and the second component due to a non-specific non-receptor mediated inhibition of the cre-spap response downstream from camp, and therefore unlike the two-component responses seen at either the β1-adrenoceptor or the β3-adrenoceptor. investigation of the intrinsic activity of the h3-antagonists finally, as the histamine h receptor is known have constitutive activity, none of the other antagonist ligands stimulated a change in cre-spap production in cho-h3-spap cells. however, previous studies have however demonstrated that small changes may be more clearly seen in the 3h-camp accumulation assay due to the larger response window. all antagonist ligands were therefore examined in the 3hc-amp accumulation assay following hours incubation with ligands in order to maximise any changes seen. in this assay, the ic values for the partial agonist vuf remained unchanged. inverse agonist responses were seen for conessine, thioperamide, ranitidine and cimetidine. no responses were seen to zolatidine, dimethindine, iodophenpropit, clobenpropit, triprolidine, clemastine or chlorpheniramine. lack of gene transcription responses in cho-spap cells the response to all ligand used in this study was examined in the parent cho-spap cells. full 7-point concentration response curves were constructed, in the presence and absence of μm forskolin. no responses were seen in response to any ligand, up to concentrations of μm. a small decrease from maximum was seen only at the highest concentration of vuf discussion several gpcrs have now been shown to exist in several different conformations, stablised by different agonists, that either couple to different g-proteins, signal via different pathways or to which ligands bind with different affinities. the histamine h receptor is a gi-coupled receptor with a large range of ligands of varying efficacies thus making a detailed pharmacological study of the receptor possible. several newer ligands have also been found for the h receptor and their structure is becoming increasingly diverse, other ligands were also found to have agonist properties e.g. the h agonists amthamine and dimaprit. vuf was previously reported to be a neutral antagonist of the histamine h receptor. however, here it was found to have agonist activity in both the 3h-camp accumulation assay and the cre-spap assay. as all of these responses did not occur in cre-spap cells and their responses were sensitive to pre-incubation with pertussis toxin, they too were occurring via the histamine h receptor. in view of the fact that the human histamine h receptor is known to be constitutively active, evidence for this was sought in terms of demonstrating inverse agonist action of the antagonist ligands. given the low expression level of this cell line very little constitutive activity would be expected to be seen, and indeed this was the case. only conessine, thioperamide, ranitidine and cimetidine were found to be inverse agonists in this relatively low expressing cell system. however, this nonetheless confirmed the constitute nature of this receptor. as there was relatively little constitutive activity, this should mean that a protean agonist would appear as a more efficacious agonist, as indeed proxyfan did. because of this, antagonist affinity measurements from parallel shifts of an agonist response were more easily determined. it is interesting to note that several histamine ligands were found to have significant agonist activity in this cell system that has not been previously reported e.g. vuf it could therefore be that vuf is also a protean agonist and if examined in systems of different receptor expression and constitutive activity this might be demonstrated. proxyfan appears as a full agonist in this cell system and vuf as a partial agonist. if vuf is indeed a protean agonist, it stabilises a different, lower efficacy protean state of the receptor to that stabilised by proxyfan. all agonists at the h receptor appeared more potent in the 3h-camp accumulation assay than in the cre-gene transcription assay. this is in contrast to a recent study of the gs-coupled human histamine h receptor, but is similar to that seen in the gi-coupled adenosine a receptor. potencies between assays appear similar at the β and β3-adrenoceptor but vary depending on the efficacy of the agonist at the β2-adrenoceptor. the reason for the change in potency between assays, that clearly varies with different gpcrs, remains unknown but may be related to different patterns of phosphorylation, internalisation and desensitisation that occur at different gpcrs. another major conclusion that can be drawn from this study is that not all gi-coupled receptors behave alike in a recombinant cell system. the gi-coupled adenosine a1-receptor, expressed in the same cho-reporter cell back ground as used in this study, clearly showed the receptor coupling to gi and gs-proteins. in this h study, all ligands were first assessed for their agonist activity in the presence and absence of forskolin, with and without ptx pre-incubation. inhibitory responses were only ever seen. all of these responses were only seen in the presence of forskolin and all responses were abolished by pre-incubating the cells with ptx. the h receptor therefore did not demonstrate any gs-coupled stimulatory cre-gene transcription responses, even at high agonist concentrations, and thus has a different agonist receptor activation profile to the a1-adenosine receptor. the affinity for the antagonists, as measured from parallel shifts of the agonist concentration response curve, remained constant at the human h receptor. where values are available, the antagonist affinity values obtained here are similar to those previously published e.g.. this was also true for proxyfan stimulated responses. conessine, a steroidal alkaloid and a natural product from the stem bark of funtumia elastica, was a high affinity h inverse agonist and the pkd values for its antagonism of different h3-agonists obtained here were very similar to that reported by cowart et al.,. thus, regardless of which competing agonist was used, the antagonist affinity of a given antagonist remained the same. furthermore, the schild plots, where possible, all had a slope of confirming competitive antagonism. again, where possible, this was true for the proxyfan stimulated responses. this was true even when examining the antagonism of partial agonist responses. this also held even when the antagonism of full agonists by a partial agonist was examined. finally, the antagonist affinity values obtained were the same in the 3h-camp accumulation assay as the cre-gene transcription assay. therefore, the histamine h receptor is unlike any of the β-adrenoceptors in terms of simple antagonist affinity measurements. antagonist affinity measurements remain constant at the h receptor, regardless of the competing agonist's efficacy, the cellular response measured, the time of incubation of agonist or the presence of a pde inhibitor.  in conclusion, the human histamine h receptor is a gi-coupled receptor that, in contrast to the human a1-receptor, has no evidence of coupling to gs-proteins in this low receptor expressing recombinant cell system. several ligands were identified as having agonist activity, including some ligands previously considered to be antagonists. given the low constitutive activity of this cell system, these ligands might yet turn out to be protean agonists. conessine was shown to be a high affinity inverse agonist. finally, the competitive nature of the antagonists was demonstrated and antagonist affinity measurements were constant for each antagonist, including at the proxyfan-induced medium-efficacy state of the h receptor, in contrast to all three subtypes of the β-adrenoceptors, but in keeping with the traditional pharmacological dogma. abbreviations camp, adenosine-3',5'-cyclic monophosphate; cho, chinese hamster ovary; cre, cyclic amp response element; dmem/f dulbecco's modified eagles medium/nutrient mix f12; gpcr, g-protein coupled receptor; htmt, histamine trifluoromethyl toluidide; nαmh, nα-methylhistamine; pbs, phosphate buffered saline; pea, 2-pyridylethylamine; pnpp 4-nitrophenyl phosphate; ptx, pertussis toxin; rαmh, r-α-methylhistamine; sαmh, s-α-methylhistamine; spap, secreted placental alkaline phosphatise; vuf 4-piperidine dihydrobromide; vuf 2-ethyl carbamimidothioic acid ester dihydrobromide. authors' contributions jgb conceived the study, carried out the experiments, data analysis and preparation of the manuscript. 
measurements of cre-gene transcription and 3h-camp accumulation were made. the purpose of this study was to examine the antagonist affinity measurements at the gi-coupling human h receptor, paying particular attention to measurements made in the presence of full agonists, partial agonists and the proxyfan protean agonist-induced state of the receptor. cho cells stably expressing the human histamine h receptor and a cre-spap reporter were used. several gpcrs are now known to exist in different states. the beta-adrenoceptors, the antagonist affinity measurements vary considerably depending on which agonist-dependent state is being identified. antagonist affinity measurements were constant, whether the measurements were made in the presence of a full agonist, a partial agonist or the protean agonist proxyfan. in contrast to all three subtypes of the beta-adrenoceptors, but in keeping with the traditional pharmacological dogma, antagonist affinity measurements remained constant at the human h receptor, including the medium-efficacy proxyfan-induced state of the receptor and the vuf5681-induced state of the receptor. a range of ligands of different agonist efficacies were determined, including some partial agonists e.g.
 the h histamine receptor is a gi-coupled gpcr that has been proven to exist in different agonist-induced states, including that defined by the protean agonist proxyfan. several gpcrs are now known to exist in different states. for some of these, antagonist affinity measurement remain constant regardless of the state of the receptor, for others e.g. the beta-adrenoceptors, the antagonist affinity measurements vary considerably depending on which agonist-dependent state is being identified. the purpose of this study was to examine the antagonist affinity measurements at the gi-coupling human h receptor, paying particular attention to measurements made in the presence of full agonists, partial agonists and the proxyfan protean agonist-induced state of the receptor.  cho cells stably expressing the human histamine h receptor and a cre-spap reporter were used. measurements of cre-gene transcription and 3h-camp accumulation were made. a range of ligands of different agonist efficacies were determined, including some partial agonists e.g. vuf unlike other gi-coupled receptors, no gs-coupled state of the receptor was detected with these ligands. antagonist affinity measurements were constant, whether the measurements were made in the presence of a full agonist, a partial agonist or the protean agonist proxyfan.  in contrast to all three subtypes of the beta-adrenoceptors, but in keeping with the traditional pharmacological dogma, antagonist affinity measurements remained constant at the human h receptor, including the medium-efficacy proxyfan-induced state of the receptor and the vuf5681-induced state of the receptor. 