Protean agonism at histamine H3 receptors in vitro and in vivo

Abstract

G protein-coupled receptors (GPCRs) are allosteric proteins that adopt inactive (R) and active (R*) conformations in equilibrium. R* is promoted by agonists or occurs spontaneously, leading to constitutive activity of the receptor. Conversely, inverse agonists promote R and decrease constitutive activity. The existence of another pharmacological entity, referred to as "protean" agonists (after Proteus, the Greek god who could change shape), was assumed on theoretical grounds. It was predicted from the existence of constitutive activity that a same ligand of this class could act either as an agonist or an inverse agonist at the same GPCR. Here, we show that proxyfan, a high-affinity histamine H3-receptor ligand, acts as a protean agonist at recombinant H3 receptors expressed in the same Chinese hamster ovary cells. In support of the physiological relevance of the process, we show that proxyfan also behaves as a protean agonist at native H3 receptors known to display constitutive activity. On neurochemical and behavioral responses in rodents and cats, proxyfan displays a spectrum of activity ranging from full agonism to full inverse agonism. Thus, protean agonism demonstrates the existence of ligand-directed active states LR* different from, and competing with, constitutively active states R* of GPCRs, and defines a pharmacological entity with important therapeutic implications.

Fig. 1.

Effects of H₃ receptor ligands on H₃ receptor-mediated responses in CHO(H₃) cells. The effects of histamine, ciproxifan, and proxyfan were determined in the same CHO(rH₃ (413)) cells (17) expressing 1 pmol per mg protein on MAPK activity (a), [³⁵S]GTPγ[S] binding to membranes (b), forskolin-induced cAMP accumulation (c), and A23187-evoked [³H]arachidonic acid release (d). Results, expressed as the percentage change of the response, are means ± SEM of 3–10 determinations from two to three separate experiments.

Fig. 2.

Effects of H₃ receptor ligands on responses mediated by native H₃ receptors. (a and b) Effects of histamine, ciproxifan, and proxyfan on [³H]histamine release induced by 20 or 55 mM K⁺ from rat (a) or mouse (b) synaptosomes. Means ± SEM of 9–28 values from three to four separate experiments. ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001 vs. control. (c) Changes in brain t-MeHA levels in mice or rats receiving ciproxifan (3 mg/kg) or proxyfan (10 mg/kg). Means ± SEM of 7–16 values. ^*, P < 0.001 vs. control. (d) Effects of H₃ receptor ligands on cyclophosphamide-induced cystitis. Means ± SEM of 10–32 values. ^*, P < 0.01 vs. control; +, P < 0.05 vs. proxyfan. (e and f) Effects of H₃ receptor ligands on the sleep-wake cycle in the mouse (e) or cat (f). (Upper) Representative hypnograms (4 h). (Lower) Cumulative time (in min) spent in each sleep-wake stage (W, wakefulness; SWS1, light slow wave sleep; SWS2, deep slow wave sleep; PS, paradoxical sleep). Means ± SEM of five to nine values. The doses in mg/kg are given between parentheses. ^*, P < 0.05; ^**, P < 0.001 vs. placebo.

Fig. 3.

GPCRs are allosteric proteins that can adopt various conformations in equilibrium. On the contrary to the inactive states (R), the active states can interact with G proteins to initiate response. These active conformations occur either spontaneously (R^*), leading to constitutive activity of GPCRs, or through ligand binding (LR^*). The competition between LR^* and R^* for the G proteins leads to agonism, neutral antagonism, or inverse agonism.