A. Rouleau

High constitutive activity of native H3 receptors regulates histamine neurons in brain.

Some G-protein-coupled receptors display ‘constitutive activity’, that is, spontaneous activity in the absence of agonist. This means that a proportion of the receptor population spontaneously undergoes an allosteric transition, leading to a conformation that can bind G proteins. The process has been shown to occur with recombinant receptors expressed at high density, and/or mutated, but also non-mutated recombinant receptors expressed at physiological concentrations. Transgenic mice that express a constitutively active mutant of the β 2-adrenergic receptor display cardiac anomalies; and spontaneous receptor mutations leading to constitutive activity are at the origin of some human diseases. Nevertheless, this process has not previously been found to occur in animals expressing normal levels of receptor. Here we show that two isoforms of the recombinant rat H3 receptor display high constitutive activity. Using drugs that abrogate this activity (‘inverse agonists’) and a drug that opposes both agonists and inverse agonists (‘neutral antagonist’), we show that constitutive activity of native H3 receptors is present in rodent brain and that it controls histaminergic neuron activity in vivo . Inverse agonists may therefore find therapeutic applications, even in the case of diseases involving non-mutated receptors expressed at normal levels.

BF2.649 [1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride], a nonimidazole inverse agonist/antagonist at the human histamine H3 receptor: Preclinical pharmacology.

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5′-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity ∼50% higher than that of ciproxifan. Its in vitro potency was ∼6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

BF2.649, A NON-IMIDAZOLE INVERSE AGONIST/ANTAGONIST AT THE HUMAN HISTAMINE H3 RECEPTOR: PRECLINICAL PHARMACOLOGY

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5′-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity ∼50% higher than that of ciproxifan. Its in vitro potency was ∼6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

Protean agonism at histamine H3 receptors in vitro and in vivo

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.

Histamine H3‐receptor‐mediated [35S]GTPγ[S] binding: evidence for constitutive activity of the recombinant and native rat and human H3 receptors

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.

Cloning and cerebral expression of the guinea pig histamine H3 receptor: evidence for two isoforms.

We cloned the full length guinea pig H3 receptor cDNA using RT-PCR amplification with primers from the human receptor and templates from brain areas. Evidence was obtained for two isoforms, designated H3L and H3S, differing by a 30 amino acid stretch within the third cytosolic loop, presumably generated by alternative splicing. In situ hybridization using a selective cRNA probe showed the gene transcripts to be highly expressed in discrete neuronal populations, e.g. pyramidal cells in the cerebral cortex or cerebellar Purkinje cells, in some instances already known to express other histamine receptor subtypes.

Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms

The existence of mouse H3‐receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH3(445), mH3(413) and mH3(397) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3‐receptor isoforms display different expression patterns in the brain. Following expression in Cos‐1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH3(445), mH3(413) and mH3(397) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40–100‐fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3‐receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.

Expression analysis of the histamine H3 receptor in developing rat tissues

Endogenous histamine is involved in tissue growth and cell proliferation. In accordance with a putative function of the H(3) receptor in this mitogenic effect, we show that H(3)-receptor mRNAs are expressed together with those of the histamine-synthesizing enzyme in the embryonic liver and adipose tissue, and in various epithelia. Finally, we show that activation of recombinant H(3) receptors enhances MAP kinase activity.

Responses of Anterior Pituitary Hormones and Hypothalamic Histamine to Blockade of Histamine Synthesis and to Selective Activation or Inactivation of Presynaptic Histamine H3 Receptors in Stressed Rats

The stress-induced release of anterior pituitary hormones and changes in hypothalamic content of histamine (HA) and its metabolite tele-methylHA (t-meHA) were studied in male rats during inhibition of HA synthesis or activation or blockade of HA H3 receptors. Pretreatment with the HA synthesis inhibitor alpha-fluoromethylhistidine (alpha-FMH; 200 micrograms intracerebroventricularly (icv) at -120 min) or the specific H3 receptor agonist R(alpha)methylhistamine (RmHA; 10 mg/kg intraperitoneally (ip) at -180 and -60 min) inhibited by 30-80% the responses of prolactin (PRL), corticotropin (ACTH) and beta-endorphin (beta-END) immunoreactivity to 1, 2.5 or 5 min of restraint stress (p < 0.05-0.01), but had no effect on basal secretion of the hormones. The inhibitory effect of the H3 receptor agonist RmHA (10 mg/kg x 2) on the hormone response to 5 min of restraint stress was prevented by simultaneous ip administration of the H3 receptor antagonist thioperamide. alpha-FMH reduced the hypothalamic content of HA 60% and that of t-meHA 30%, while RmHA had no effect on the HA content. Restraint stress for 5 min did not affect the HA and t-meHA contents, which may be due to the short duration of stress exposure. Pretreatment with the H3 receptor antagonist thioperamide (5 or 10 mg/kg ip at -120 min) had no effect on basal or restraint stress-induced release of PRL, ACTH or beta-END, although the compound increased the hypothalamic content of t-meHA 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of Pentagastrin-Induced Histamine Release by Histamine H3 Receptors in the Dog

BACKGROUND The histamine H3 receptor has been shown to inhibit pentagastrin-induced gastric acid secretion in dogs. Since pentagastrin releases histamine in dogs, we have now assessed whether the effects of H3-receptor ligands may be indirectly mediated by changes in gastric histamine release. METHODS Pentagastrin infusions (1 or 6 micrograms/kg/h), alone or together with the H3-receptor agonist (R) alpha-methylhistamine (1.2 mumol/kg/h) or the antagonist thioperamide (0.1 mumol/kg/h), were performed in dogs. One group (anaesthetized) was used for enzyme immunoassays of plasma histamine and, when required. (R) alpha-methylhistamine in the gastrosplenic vein, and another group (non-anaesthetized) for measurement of gastric acid secretion. RESULTS Histamine levels were increased five- and eight-fold after 1 and 6 micrograms/kg/h pentagastrin, respectively, whereas acid output was nearly maximal at the lower dosage. (R) alpha-methylhistamine, at a plasma concentration of 0.15 microM, inhibited histamine release by 78% (P < 0.007) and 37% (not significant) and the total acid output by 44% (P < 0.05) and 19% (not significant) after infusion of 1 and 6 micrograms/kg/h pentagastrin, respectively. Thioperamide, together with pentagastrin in low dose, significantly increased histamine release by 212% (P < 0.05), whereas acid output increased by 34% (not significant). CONCLUSIONS The histamine H3 receptor mediates a negative feedback control of pentagastrin-induced release of gastric histamine. It is tonically activated by endogenous histamine after pentagastrin in low dosage. The control of acid secretion by the H3 receptor seems to involve modulation of endogenous histamine release, possibly by means of enterochromaffin-like cells.