A study of antagonist affinities for the human histamine H2 receptor

Abstract

Background and purpose: Ligand affinity has been a fundamental concept in the field of pharmacology and has traditionally been considered to be constant for a given receptor-ligand interaction. Recent studies have demonstrated that this is not true for all three members of the G(s)-coupled beta-adrenoceptor family. This study evaluated antagonist affinity measurements at a different G(s)-coupled receptor, the histamine H(2) receptor, to determine whether antagonist affinity measurements made at a different family of GPCRs were constant.

Experimental approach: CHO cells stably expressing the human histamine H(2) receptor and a CRE-SPAP reporter were used and antagonist affinity was assessed in short-term cAMP assays and longer term CRE gene transcription assays.

Key results: Nine agonists and seven antagonists, of sufficient potency at the H(2) receptor to examine in detail, were identified. Measurements of antagonist affinity were the same regardless of the efficacy of the competing agonist, time of agonist incubation, cellular response measured or presence of a PDE inhibitor.

Conclusions and implications: Antagonist affinity at the G(s)-coupled histamine H(2) receptor obeys the accepted dogma for antagonism at GPCRs. This study further confirms that something unusual is indeed happening with the beta-adrenoceptors and is not an artefact related to the transfected cell system used. As the human histamine H(2) receptor does not behave in a similar manner to any of the human beta-adrenoceptors, it is clear that information gathered from one GPCR cannot be simply extrapolated to predict the behaviour of another GPCR. Each GPCR therefore requires careful and detailed evaluation on its own.

Figure 1

Cyclic AMP response element-secreted placental alkaline phosphatase (CRE-SPAP) gene transcription in response to histamine in the absence and presence of (a) ICI 162846, (b) ranitidine, (c) famotidine and (d) cimetidine in Chinese hamster ovary (CHO)-H₂-SPAP cells. Bars in each graph show basal CRE-SPAP gene transcription, that in response to 10 μM histamine, and that in response to the three different concentrations of antagonist used in each experiment. Data points are mean±s.e.mean of triplicate values from a single experiment and are representative of (a) six, (b) five, (c) five and (d) four separate experiments.

Figure 2

Cyclic AMP response element-secreted placental alkaline phosphatase (CRE-SPAP) gene transcription in Chinese hamster ovary (CHO)-H₂-SPAP cells in response to (a) N-α-methylhistamine, (b) 4-methylhistamine, (c) dimaprit and (d) histamine trifluoromethyl toluidide (HTMT) in the absence and presence different concentrations of ICI 162846. Bars show basal CRE-SPAP gene transcription, that in response to 10 μM histamine, and that in response to the various concentrations of ICI 162846 alone as used in each experiment. Data points are mean±s.e.mean of triplicate values from single experiments that are representative of (a) five, (b) three, (c) four and (d) five separate experiments.

Figure 3

Cyclic AMP response element-secreted placental alkaline phosphatase (CRE-SPAP) gene transcription in Chinese hamster ovary (CHO)-H₂-SPAP cells in response to (a) N-α-methylhistamine (Nαmh), (b) 4-methylhistamine (4-mh) and (c) dimaprit in the absence and presence of 1, 3 or 10 μM raniditine and (d) histamine trifluoromethyl toluidide (HTMT) in the absence and presence of 1 μM raniditine. Bars show basal CRE-SPAP gene transcription, that in response to 10 μM histamine, and that in response 1, 3 or 10 μM ranitidine alone. Data points are mean±s.e.mean of triplicate values from single experiments that are representative of (a) five, (b) three, (c) four and (d) five separate experiments.

Figure 4

³H-cAMP accumulation in Chinese hamster ovary (CHO)-H₂-SPAP cells in response to (a) histamine, (b) N-α-methylhistamine (Nαmh), (c) dimaprit and (d) histamine trifluoromethyl toluidide (HTMT) in the absence and presence of 1 μM ranitidine. Bars show basal ³H-cAMP accumulation, that in response to 10 μM histamine, and that in response to 1 μM ranitidine alone. Data points are mean±s.e.mean of triplicate values from single experiments that are representative of (a) four, (b) four, (c) five, and (d) four separate experiments.

Figure 5

³H-cAMP accumulation in (a) Chinese hamster ovary (CHO)-H₂-SPAP and (b) the CHO-SPAP cells (stably expressing the Cyclic AMP response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter but not the transfected receptor) in response to histamine. Bars show basal ³H-cAMP accumulation, that in response to 10 μM forskolin alone. Data points are mean±s.e.mean of triplicate values from a single experiment that is representative of (a) five and (b) four separate experiments. For (a), analysis of the curve fit using the F ratio in Prism 2 to compare the fit of the two-site component concentration–response curve and the traditional one-site sigmoidal concentration–response curve gives a P-value of <0.0001 for the curve fitting best to the two-site component curve.

Figure 6

³H-cAMP accumulation in Chinese hamster ovary (CHO)-H₂-SPAP cells in response to (a) ranitidine, (b) ICI 162846 and (c) famotidine, nizatidine and cimetidine. Bars show basal ³H-cAMP accumulation, that in response to 10 μM histamine alone. Data points are mean±s.e.mean of triplicate values from a single experiment that is representative of (a) four, (b) three and (c) three separate experiments.