The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt 1 and MT 2 , the existence of a novel putative melatonin receptor, MT 3 , was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt 1 and MT 2 . In this report, we present the purification of MT 3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR 2 , EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT 3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR 2 . After transfection of CHO-K1 cells with the human QR 2 gene, not only did the QR 2 enzymatic activity appear, but also the melatonin-binding sites with MT 3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT 3 binding and QR 2 enzymatic activity obtained from samples of Syrian hamster kidney or QR 2 -overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT 3 as the quinone reductase QR 2 and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.Melatonin, a neurohormone produced at night in the pineal gland, is suspected to relay to the peripheral organs the circadian rhythm detected by the central nervous system. Several high affinity melatonin receptors have been identified to date, among which the mt 1 (1) and MT 2 (2) receptors have been cloned from human tissues. The pharmacology of these two receptors is well documented, and several compounds, including melatonin, are ligands with picomolar binding affinity (for review, see Ref.3). Another putative melatonin receptor was identified on pharmacological grounds, with lower melatonin affinity (nanomolar range), very rapid ligand association/dissociation kinetics, and an original pharmacological profile (4 -6). In line with mt 1 and MT 2 receptors, this putative receptor was named MT 3 , according to the nomenclature recommendations of the IUPHAR (7). So far, the known inhibitors of MT 3 hardly reach the nanomolar range and encompass an unusually large structural diversity of highly hydrophobic cyclic or polycyclic compounds (Refs. 5 and 6, and for review, see Ref.3).1 All pharmacological investigations on mt 1 , MT 2 , and MT 3 were performed using the radioligand [125 I]melatonin, a ligand with high affinity for mt 1 and MT 2 (K d ϭ 10 -200 pM) and with lower affinity for MT 3 (K d ϭ 3-9 nM). The hamster kidney, liver, and brain have been used as model tissues for MT 3 pharmacological studies, and our recent data con...

No abstract

Zhang et al . (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein–coupled receptor GPR39. However, we found that I 125 -obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.

No abstract

No abstract

Herein, we evaluate the interaction of the alpha(2)-AR antagonist, yohimbine, as compared to fluparoxan, at multiple monoaminergic receptors and examine their roles in the modulation of adrenergic, dopaminergic and serotonergic transmission in freely-moving rats. Yohimbine displays marked affinity at human (h)alpha(2A)-, halpha(2B)- and halpha(2C)-ARs, significant affinity for h5-HT(1A), h5-HT(1B), h5-HT(1D), and hD(2) receptors and weak affinity for hD(3) receptors. In [(35)S]GTPgammaS binding protocols, yohimbine exerts antagonist actions at halpha(2A)-AR, h5-HT(1B), h5-HT(1D), and hD(2) sites, yet partial agonist actions at h5-HT(1A) sites. In vivo, agonist actions of yohimbine at 5-HT(1A) sites are revealed by WAY100,635-reversible induction of hypothermia in the rat. In guinea pigs, antagonist actions of yohimbine at 5-HT(1B) receptors are revealed by blockade of hypothermia evoked by the 5-HT(1B) agonist, GR46,611. In distinction to yohimbine, fluparoxan shows only modest partial agonist actions at h5-HT(1A) sites versus marked antagonist actions at halpha(2)-ARs. While fluparoxan selectively enhances hippocampal noradrenaline (NAD) turnover, yohimbine also enhances striatal dopamine (DA) turnover and suppresses striatal turnover of 5-HT. Further, yohimbine decreases firing of serotonergic neurones in raphe nuclei, an action reversed by WAY100,635. Fluparoxan increases extracellular levels of DA and NAD, but not 5-HT, in frontal cortex. In analogy, yohimbine enhances FCX levels of DA and NAD, yet suppresses those of 5-HT, the latter effect being antagonized by WAY100,635. The induction by fluoxetine of FCX levels of 5-HT, DA, and NAD is potentiated by fluparoxan. Yohimbine likewise facilitates the influence of fluoxetine upon DA and NAD levels, but not those of 5-HT. In conclusion, the alpha(2)-AR antagonist properties of yohimbine increase DA and NAD levels both alone and in association with fluoxetine. However, in contrast to the selective alpha(2)-AR antagonist, fluparoxan, the 5-HT(1A) agonist actions of yohimbine suppress 5-HT levels alone and underlie its inability to augment the influence of fluoxetine upon 5-HT levels.

Background and purpose: Ivabradine, a specific and use-dependent I f inhibitor, exerts anti-ischaemic activity purely by reducing heart rate. The aim of this work was to characterize its effect on the predominant HCN channel isoform expressed in human sino-atrial nodes (hSAN), to determine its kinetics in HCN channels from multicellular preparations and ratedependency of its action. Experimental approach: RT-PCR analysis of the four HCN channel isoforms was carried out on RNAs from hSAN. Patch-clamp and intracellular recordings were obtained from CHO cells stably expressing hHCN4 and isolated SAN, respectively. Beating rate of rat isolated atria was followed using a transducer. Key results: hHCN4 mRNAs were predominant in hSAN. Ivabradine induced a time-dependent inhibition of hHCN4 with an IC 50 of 0.5 mM. In rabbit SAN, ivabradine progressively reduced the frequency of action potentials: by 10% after 3 h at 0.1 mM, by 14% after 2 h at 0.3 mM and by 17% after 1.5 h at 1 mM. After 3h, ivabradine reduced the beating rate of rat right atria with an IC 30 of 0.2 mM. The onset of action of ivabradine was use-dependent rather than time-dependent with slower effects than caesium, an extracellular I f blocker. Ivabradine 3 mM decreased the frequency of action potentials in SAN from guinea-pig, rabbit and pig by 33%, 21% and 15% at 40 min, respectively. Conclusions and implications:The use-dependent inhibition of hHCN4 current by ivabradine probably contributes to its slow developing effect in isolated SAN and right atria and to its increased effectiveness in species with rapid SAN activity.

Autism spectrum disorders affect millions of individuals worldwide, but their heterogeneity complicates therapeutic intervention that is essentially symptomatic. A versatile yet relevant model to rationally screen among hundreds of therapeutic options would help improving clinical practice. Here we investigated whether neurons differentiated from pluripotent stem cells can provide such a tool using SHANK3 haploinsufficiency as a proof of principle. A library of compounds was screened for potential to increase SHANK3 mRNA content in neurons differentiated from control human embryonic stem cells. Using induced pluripotent stem cell technology, active compounds were then evaluated for efficacy in correcting dysfunctional networks of neurons differentiated from individuals with deleterious point mutations of SHANK3. Among 202 compounds tested, lithium and valproic acid showed the best efficacy at corrected SHANK3 haploinsufficiency associated phenotypes in cellulo. Lithium pharmacotherapy was subsequently provided to one patient and, after one year, an encouraging decrease in autism severity was observed. This demonstrated that pluripotent stem cell-derived neurons provide a novel cellular paradigm exploitable in the search for specific disease-modifying treatments.