Characterization of histamine H3 receptors regulating acetylcholine release in rat entorhinal cortex.

1. The pharmacological properties and location of H3 receptors modulating acetylcholine release have been investigated in non-superfused slices and synaptosomes of rat entorhinal cortex preloaded with [3H]-choline. 2. (R)alpha-methylhistamine, an H3-receptor agonist, potently inhibited the K(+)-evoked tritium release from slices, an effect antagonized by thioperamide, an H3-receptor antagonist, with nanomolar potency. 3. The K(+)-evoked tritium release from synaptosomes remained unaltered in the presence of the potent and selective H3-receptor agonists, imetit and (R)alpha-methylhistamine, suggesting that H3 receptors modulating acetylcholine release are not presynaptically located on cholinergic nerve terminals. 4. Phenylbutanoylhistamine and phenylpropylhistamine, two H3-receptor antagonists of moderate potency, failed to antagonize the inhibitory effects of (R)alpha-methylhistamine observed in slices. Unexpectedly, both compounds when used alone, inhibited tritium release from slices and synaptosomes with micromolar potency and to the same extent (by approximately 50% when added at a final concentration of 200 microM). This inhibitory effect did not involve H1, H2 or H3 receptors and was not mediated by an unknown histamine receptor site, since histamine used at a high concentration neither reproduced nor antagonized the effect of phenylbutanoylhistamine. It remained unaltered in the presence of scopolamine and was neither mimicked nor antagonized by vasoactive intestinal peptide, previously shown to be colocalized with acetylcholine in some neurones. 5. It is concluded that acetylcholine release in rat entorhinal cortex is modulated by H3 receptors presumably not located on cholinergic axon terminals. It remains to be established whether these H3 receptors belong to a receptor subtype different from those previously described since the potency ofphenylbutanoylhistamine and phenylpropylhistamine as H3-receptor antagonists was probably greatly underestimated by additional properties of both drugs.

Cloning of OL1, a putative olfactory receptor and its expression in the developing rat heart.

By using a strategy based on nucleotide sequence homology, we have cloned an intronless DNA encoding a new putative member of G protein-coupled receptors. The deduced amino acid sequence of the rat OL1 receptor, together with its expression at high levels in a small subset of cells in the olfactory neuroepithelium indicate that OL1 is related to the recently discovered olfactory multigene family. PCR and in situ hybridization analyses showed the OL1 transcripts to be not only expressed in the olfactory epithelium, but also in the heart. This unexpected cardiac expression was developmentally regulated, being maximal at early postnatal stages and hardly detectable at adult stages. Moreover, this observation was not restricted to OL1 since it was extended to other putative olfactory receptors. Although its functional significance remains unknown, this transient cardiac expression suggests that receptors belonging to the olfactory superfamily, could be not only involved in odor coding, but also in cardiac morphogenesis. Another olfactory-specific gene transcript encoding PTP NE-3, a recently cloned receptor-type protein-tyrosine phosphatase, could also be detected in heart. The very low levels of expression observed in rat embryo and at early postnatal stages as compared to adult stages, suggest that protein-tyrosine phosphatases, as well as protein-tyrosine kinases, may play a role in the control of cardiac cell growth and morphogenetic processes.

Cloning and selective expression in brain and kidney of ARNT2 homologous to the Ah receptor nuclear translocator (ARNT).

Arnt2, a new member of the basic-helix-loop-helix transcription factor family, was cloned from rat brain cDNAs. Its deduced 712 amino acid sequence displays 63% identity with that of the aryl hydrocarbon receptor nuclear translocator (Arnt1) that was completely established. Whereas Arnt2 gene expression, established by Northern blotting and in situ hybridization histochemistry, occurred selectively in brain and kidney, that of Arnt1 was ubiquitous, suggesting that the two proteins play distinct roles, presumably via dimerization and DNA binding with different partners.

Two splice variants of the hypoxia-inducible factor HIF-1alpha as potential dimerization partners of ARNT2 in neurons.

The hypoxia-inducible factor (HIF-1alpha), a basic helix-loop-helix transcription factor, is known to heterodimerize with ARNT1, a nuclear translocator, to trigger the overexpression in many cells of genes involved in resistance to hypoxia. Although HIF-1alpha and ARNT1 are both expressed in brain, their cellular localization and function therein are unknown. Here, using in situ hybridization and immunocytochemistry, we show that HIF-1alpha is expressed in normoxic cerebral neurons together with not only ARNT1 but also ARNT2, a cerebral translocator homologous to ARNT1 but displaying, unlike ARNT1, a selective neuronal expression. In contrast, other potential partners of the translocators, i.e. the aryl hydrocarbon receptor (AHR) and the single-minded protein 2 (SIM2), are not expressed in the adult brain. We also identify two splice variants of HIF-1alpha in brain, one of which dimerizes with ARNT2 even more avidly than with ARNT1. The resulting heterodimer, in contrast with the HIF-1alpha/ARNT1 complex, does not recognize the HIF-1-binding site of the hypoxia-induced erythropoietin (Epo) gene, suggesting that it controls transcription of a distinct set of genes. We therefore propose that HIF-1alpha and ARNT2 function as preferential dimerization partners in neurons to control specific responses, some of which may not be triggered by hypoxia. In support of this proposal, in nonhypoxic PC12 cells constitutively coexpressing HIF-1alpha, ARNT1 and ARNT2, downregulation of either HIF-1alpha or ARNT2, obtained with selective antisense nucleotides, resulted in inhibition of [3H]thymidine incorporation.

Identification of rat H3 receptor isoforms with different brain expression and signaling properties.

We identified the cDNAs of three functional rat H3 receptor isoforms (H3A, H3B, and H3C) and one nonfunctional truncated H3 receptor (H3T). The H3A, H3B, and H3C receptor isoforms vary in the length of their third intracellular loop; the H3B and H3C receptor lack 32 and 48 amino acids, respectively. Transient expression of the H3A, H3B, and H3C receptors in COS-7 cells results in high affinity binding for the H3 antagonist [125I]iodophenpropit, which is displaced by selective H3 agonists and antagonists. The three isoforms differentially couple to the Gi protein-dependent inhibition of adenylate cyclase or stimulation of p44/p42 mitogen activated protein kinase (MAPK), a new signaling pathway for the H3 receptor. Whereas the H3A receptor was less effective in inhibiting forskolin-induced cAMP production compared with the H3B or H3C receptor, this isoform was more effective in the stimulation of p44/p42 MAPK. The H3 receptor isoforms also displayed differential CNS expression in key areas involved in regulation of sensory, endocrine, and cognitive functions. A differential H3 receptor isoform expression was seen in, for example, hippocampus, where a characteristic dorsoventral distribution was revealed. Differential H3 receptor expression was also characteristic for the cerebellum, indicating possible histaminergic regulation of motor functions. The identification of these new H3 receptor isoforms and their specific signaling properties adds a new level of complexity to our understanding of the role of histamine, and the H3 receptor in brain function. The heterogeneous distribution of the isoforms suggests that H3 receptor isoform-specific regulation is important in several brain functions.

ARNT2, a transcription factor for brain neuron survival?

The processes responsible for the limited ability to divide and long survival of neurons are not well understood but may involve aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a recently identified protein, apparently belonging to the basic helix-loop-helix superfamily of transcription factors, which is expressed almost exclusively in brain during the whole lifetime. In agreement, we show, in the rat, that ARNT2 immunoreactivity could be observed only within nuclei of brain neurons and of dividing and neuronal PC12 cells, a localization consistent with a role in transcription regulation. Cell death elicited either by focal ischaemia in brain or oxidative stress in PC12 cells was largely preceded by an almost complete suppression of ARNT2 expression. In contrast, when PC12 cell cycle progression was impaired, ARNT2 expression was enhanced. Finally, the downregulation of ARNT2 levels induced by antisense oligonucleotides prevented PC12 cell proliferation and induced apoptosis. These observations support the hypothesis that ARNT2 is a neuronal transcription factor, regulating cell cycle progression and preventing cell death, whose sustained expression might ensure brain neuron survival.