JAK2/STAT3 Pathway is Required for α7nAChR-Dependent Expression of POMC and AGRP Neuropeptides in Male Mice.

BACKGROUND/AIMS: Cholinergic signalling mediated by the activation of muscarinic and nicotinic receptors has been described in the literature as a classic and important signalling pathway in the regulation of the inflammatory response. Recent research has investigated the role of acetylcholine, the physiological agonist of these receptors, in the control of energy homeostasis at the central level. Studies have shown that mice that do not express acetylcholine in brain regions regulating energy homeostasis present with excessive weight gain and hyperphagia. However, it has not yet been well-described in the literature which cholinergic receptor subunits are involved in this response; moreover, the signalling pathways responsible for the observed effects are not fully delineated. The hypothalamus is the regulating centre of energy homeostasis, and the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is highly expressed in this region. When active, α7nAChR recruits proteins such as JAK2/STAT3 to mediate its signalling; the same intracellular components are required by leptin, an anorexigenic hormone. The aim of the present study was to evaluate the role of the hypothalamic α7nAChR in the control of energy homeostasis. METHODS: The work was performed on Swiss male mice. Initially, using immunofluorescent staining on brain sections, the presence of α7nAChR in hypothalamic cells regulating energy homeostasis was evaluated. Animals were submitted to stereotaxis in the lateral ventricle and intracerebroventricular stimulation (ICV) was used for the administration of an agonist (PNU) or antagonist (α-bungarotoxin) of α7nAChR. Metabolic parameters were evaluated and the expression of neuropeptides was evaluated in the hypothalamus by real-time PCR and western blot. The expression of hypothalamic neuropeptides was evaluated in mice treated with siRNA or inhibitors of JAK2/STAT3 (AG490 and STATTIC) proteins. We also evaluated food intake in α7nAChR knockout animals (α7KO). Additionally, in mouse hypothalamic cell culture (the mypHoA-POMC/GFP lineage), we evaluated the expression of neuropeptides and pSTAT3 after stimulation with PNU. RESULTS: Our results indicate co-localisation of α7nAChR with α-MSH, AgRP and NPY in hypothalamic cells. Pharmacological activation of α7nAChR reduced food intake and increased hypothalamic POMC expression and decreased NPY and AgRP mRNA levels and the protein content of pAMPK. Inhibition of α7nAChR with an antagonist increased the mRNA content of NPY and AgRP. Inhibition of α7nAChR with siRNA led to the suppression of POMC expression and an increase in AgRP mRNA levels. α7KO mice showed no changes in food intake. Inhibition of proteins involved in the JAK2/STAT3 signalling pathway reversed the effects observed after PNU stimulation. POMC-GFP cells, when treated with PNU, showed increased POMC expression and nuclear translocation of pSTAT3. CONCLUSION: Thus, selective activation of α7nAChR is able to modulate important markers of the response to food intake, suggesting that α7nAChR activation can suppress the expression of orexigenic markers and favour the expression of anorexics using the intracellular JAK2/STAT3 machinery.

Alkaloids Purified from Aristotelia chilensis Inhibit the Human α3ß4 Nicotinic Acetylcholine Receptor with Higher Potencies Compared with the Human α4ß2 and α7 Subtypes.

The alkaloids aristoteline (1), aristoquinoline (2), and aristone (3) were purified from the leaves of the Maqui tree Aristotelia chilensis and chemically characterized by NMR spectroscopy. The pharmacological activity of these natural compounds was evaluated on human (h) α3ß4, α4ß2, and α7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results suggest that these alkaloids do not have agonistic, but inhibitory, activity on each receptor subtype. The obtained IC50 values indicate the following receptor selectivity: hα3ß4 > hα4ß2 ≫ hα7. In the particular case of hα3ß4 AChRs, 1 (0.40 ± 0.20 µM) and 2 (0.96 ± 0.38 µM) show higher potencies compared with 3 (167 ± 3 µM). Molecular docking and structure-activity relationship results indicate that ligand lipophilicity is important for the interaction with the luminal site located close to the cytoplasmic side of the hα3ß4 ion channel between positions -2' and -4'. Compound 1 could be used as a molecular scaffold for the development of more potent noncompetitive inhibitors with higher selectivity for the hα3ß4 AChR that could serve for novel addiction and depression therapies.

Dual effects of a 2-benzylquinuclidinium derivative on α7-containing nicotinic acetylcholine receptors in rat hippocampal interneurons.

Nicotinic acetylcholine receptors (nAChRs) are widely distributed in the brain. Particularly α7-containing nAChRs, associated with several physiological roles and pathologies, are one of the most abundant. Here, we studied 2-(4-hexyloxybenzyl)-1-methylquinuclidin-1-ium iodide (designated as 8d), on ion currents elicited by choline, ICh, (Ch, a selective agonist for α7-containing nAChRs), recorded in interneurons from the stratum radiatum of the rat hippocampal CA1 region by using the whole-cell voltage-clamp technique. The 8d-concentration/Ch-response relationship exhibited high and low inhibitory affinities for α7-containing nAChRs, with IC50 values of 0.59 and 6.80 µM, respectively. Interestingly, 8d in a range of 3-10 µM exerted opposite effects: a short early potentiation and a long late inhibition of the ICh; and 8d alone elicited a non-decaying inward current. Furthermore, potentiation and inhibition of the ICh by 8d depended on the membrane potential, both being stronger at -20 than at -70 mV; indicating that 8d interacts with at least two sites into the ion channel/receptor complex: one for potentiating and another for inhibiting the α7-containing nAChRs. These results suggest that 8d may act as agonist, antagonist and positive modulator of α7-containing nAChRs in hippocampal interneurons.