Histamine H3-receptor-mediated 35SGTPγS binding: evidence for constitutive activity of the recombinant and native rat and human H3 receptors

摘要

class="enumerated" style="list-style-type:decimal">Constitutive activity of the recombinant and native rat and human H3 receptors (H3Rs) was studied using H3R-mediated [³⁵S]GTPγ[S] binding and [³H]-arachidonic acid release.Ciproxifan, an inverse agonist at the rat H3R (rH3R), decreased [³H]arachidonic acid release from CHO cells expressing moderate densities (∼200–300 fmol mg⁻¹ protein) of the human H3R (hH3R). This effect occurred with the same magnitude than at the rH3R.The expression of the hH3R was associated with an increase in [³⁵S]GTPγ[S] binding to membranes of CHO cells. Ciproxifan decreased [³⁵S]GTPγ[S] binding to membranes of CHO (hH3R) cells. Both effects were correlated to receptor density and revealed that constitutive activity of the hH3R, although lower than that of the rH3R in this assay, was again observed at physiological densities (<500 fmol mg⁻¹ protein). Ciproxifan was less potent at the human than the rat receptor, not only as an antagonist (Ki=45 nM), but also as an inverse agonist (EC50=15 nM).Constitutive activity of the hH3R was also evidenced using inhibition of [³⁵S]GTPγ[S] binding by unlabelled GTPγS. The expression of the hH3R generated a high affinity binding for GTPγS which was increased by imetit, but partially decreased by ciproxifan, therefore acting as a partial inverse agonist.[³⁵S]GTPγ[S] binding to rat brain membranes was decreased in several regions by thioperamide, ciproxifan and FUB 465, three inverse agonists at the H₃R, whose effects were blocked by proxyfan, a neutral antagonist. [³⁵S]GTPγ[S] binding was also decreased by an A₁-adenosine receptor inverse agonist, but remained unchanged in the presence of inverse agonists at D₂/D₃ dopamine, H₁ and H₂ histamine, α₂-adrenergic and δ opioid receptors.In conclusion, the present study shows that the recombinant rat and human H₃ receptors expressed at physiological densities display constitutive activity and suggests that constitutive activity of native H₃Rs is one of the highest among G-protein-coupled receptors present in rat brain.