To evaluate potential mechanisms for neuronal glucosensing, fura-2 Ca 2؉ imaging and single-cell RT-PCR were carried out in dissociated ventromedial hypothalamic nucleus (VMN) neurons. Glucose-excited (GE) neurons increased and glucose-inhibited (GI) neurons decreased intracellular Ca 2؉ ([Ca 2؉ ] i ) oscillations as glucose increased from 0.5 to 2.5 mmol/l. The Kir6.2 subunit mRNA of the ATP-sensitive K ؉ channel was expressed in 42% of GE and GI neurons, but only 15% of nonglucosensing (NG) neurons. Glucokinase (GK), the putative glucosensing gatekeeper, was expressed in 64% of GE, 43% of GI, but only 8% of NG neurons and the GK inhibitor alloxan altered [Ca 2؉ ] i oscillations in ϳ75% of GK-expressing GE and GI neurons. Insulin receptor and GLUT4 mRNAs were coexpressed in 75% of GE, 60% of GI, and 40% of NG neurons, although there were no statistically significant intergroup differences. Hexokinase-I, GLUT3, and lactate dehydrogenase-A and -B were ubiquitous, whereas GLUT2, monocarboxylate transporters-1 and -2, and leptin receptor and GAD mRNAs were expressed less frequently and without apparent relationship to glucosensing capacity. Thus, although GK may mediate glucosensing in up to 60% of VMN neurons, other regulatory mechanisms are likely to control glucosensing in the remaining ones. Diabetes 53:549 -559, 2004

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The generation of dopamine (DA) neurons from stem cells holds great promise in the treatment of Parkinson’s disease and other neural disease associated with dysfunction of DA neurons. Mesenchymal stem cells (MSCs) derived from the adult bone marrow show plasticity with regards to generating cells of other germ layers. In addition to reduced ethical concerns, MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. We have reported on the generation of DA cells from human MSCs using sonic hedgehog (SHH), fibroblast growth factor 8 and basic fibroblast growth factor. Despite the secretion of DA, the cells did not show evidence of functional neurons, and were therefore designated DA progenitors. Here, we report on the role of brain‐derived neurotrophic factor (BDNF) in the maturation of the MSC‐derived DA progenitors. 9‐day induced MSCs show significant tropomyosin‐receptor‐kinase B expression, which correlate with its ligand, BDNF, being able to induce functional maturation. The latter was based on Ca2+ imaging analyses and electrophysiology. BDNF‐treated cells showed the following: increases in intracellular Ca2+ upon depolarization and after stimulation with the neurotransmitters acetylcholine and GABA and, post‐synaptic currents by electrophysiological analyses. In addition, BDNF induced increased DA release upon depolarization. Taken together, these results demonstrate the crucial role for BDNF in the functional maturation of MSC‐derived DA progenitors.

The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for [(3)H]resiniferatoxin (RTX) is diminished by nearly 40-fold. Interestingly, this mutant retains a significant but diminished response to protons, supporting the existence of multiple gating mechanisms for different stimuli. The mutant appears to function by interfering with the gating induced by vanilloids rather than the expression level or permeability of the receptor. In addition, this mutant was found to function as a strong dominant negative mutation when coexpressed with wild-type VR1, providing functional evidence that the VR1 receptor forms a multimeric complex. Analysis of both current density and [(3)H]RTX affinity in cells cotransfected with different ratios of wild-type and mutant VR1 is consistent with tetrameric stoichiometry for the native capsaicin receptor.

This paper describes an extended structure-activity relationships study of aminotetralin-piperazine-based hybrid molecules developed earlier for dopamine D2/D3 receptors. Various analogues as positional isomers have been developed where location of the phenolic hydroxyl group has been varied on the aromatic ring. Between two catechol derivatives, compound 6e with a two methylene linker length exhibited higher affinity and selectivity for D3 over D2 receptors over compound 6f with four methylene linkers (D2/D3 = 50.6 vs 7.51 for 6e and 6f, respectively). In general, the (-)-isomer was more potent than the (+)-isomeric counterpart. Binding results indicated highest selectivity for D3 receptors in compound (-)- 10 ( K i = 0.35 nM; D2/D3 = 71). In the 5-hydroxy series, highest selectivity for D3 receptors was exhibited by compound (-)- 25 ( K i = 0.82 nM; D2/D3 = 31.5). Most potent compounds exhibited binding and functional affinities at the sub-nanomolar level for the D3 receptor. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors by using tritiated spiperone as radioligand for competition studies to evaluate inhibition constants ( K i). A functional assay of selected compounds for stimulating GTPgammaS binding was carried out with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The functional assay results indicated partial to full agonist characteristics of test compounds. Compound (-)- 25 was selected further for in vivo study to evaluate its potency in producing contralateral rotations in rats with unilateral lesion in the nigrostriatal region induced by neurotoxin 6-OHDA, a Parkinsonian animal model. Compound (-)- 25 at 5 micromol/kg exhibited rotational activity that lasted beyond 12 h, whereas at a 1 micromol/kg dose the rotations lasted beyond 8 h.

No abstract

R-(ϩ)-7- 3,4,dro-1H-3-benzazepine hydrochloride (SCH23390) is a widely used, highly selective antagonist of D1 dopamine receptors. While investigating the crosstalk between D1 and D3 dopamine receptor signaling pathways, we discovered that in addition to being a D1 receptor antagonist, SCH23390 and related compounds inhibit G protein-coupled inwardly rectifying potassium (GIRK) channels. We present evidence that SCH23390 blocks endogenous GIRK currents induced by either somatostatin or D3 dopamine receptors in AtT-20 cells (IC 50 , 268 nM). The inhibition is receptor-independent because constitutive GIRK currents in Chinese hamster ovary cells expressing only GIRK channels are also blocked by SCH23390. The inhibition of GIRK channels is somewhat selective because members of the closely related Kir2.0 family of inwardly rectifying potassium channels, as well as various endogenous cationic currents present in AtT-20 cells, are not affected. In addition, in current clamp recordings, SCH23390 can depolarize the membrane potential and induce AtT-20 cells to fire action potentials, indicating potential physiological significance of the GIRK channel inhibition. To identify the chemical features that contribute to GIRK channel block, we tested several structurally related compounds [SKF38393, R-(ϩ)-7-chloro-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (normethyl-SCH23390), and R-(ϩ)-2,3,4,5-tetrahydro-8-iodo-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrochloride (iodo-SCH23390)], and our results indicate that the halide atom is critical for blocking GIRK channels. Taken together, our results suggest that SCH23390 and related compounds might provide the basis for designing novel GIRK channel-selective blockers. Perhaps more importantly, some studies that have exclusively used SCH23390 to probe D1 receptor function or as a diagnostic of D1 receptor involvement may need to be reevaluated in light of these results.We have previously shown that the D3 dopamine receptor couples to and activates G protein-coupled inwardly rectifying potassium (GIRK) channels (Werner et al., 1996;Kuzhikandathil et al., 1998) and inhibits P/Q-type calcium channels (Kuzhikandathil and Oxford, 1999). Given the coexpression of D1 and D3 dopamine receptors in central nervous system neurons, particularly in a subset of nucleus accumbens neurons (Le Moine and Bloch, 1996), we were interested in exploring the functional consequence of D1 receptor activation on D3 receptor-GIRK channel coupling. To address this issue, we used two ligands that are highly selective for D1 receptors and have long served as diagnostic probes of D1 receptor function. One of these ligands, R-(ϩ)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390), is a highly selective antagonist of D1 dopamine receptors with at least a 1000-fold higher affinity for D1 receptors than for D3 receptors (Iorio et al., 1983;Neve and Neve, 1997). SCH23390 is a member of the phenyltetrahydrobenzazepines, a structural class...

Indication Target/marker/ pathway Summary Licensing status Publication and contact information Neurology Parkinson's disease (PD) Dopamine D3 receptor An SAR study characterized a series of heterocyclic analogs of 7-{[2-(4-phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol as dopamine D3 receptor agonists that could help treat PD. The most potent compound had a K i value of 0.92 nM and exhibited higher selectivity for the D3 receptor than for the D2 receptor. In a rat model of PD, the compound inhibited parkinsonian-like movements for up to 10 hours. Ongoing in vitro and in vivo studies are investigating the neuroprotective activity of the most potent compound. Boehringer Ingelheim GmbH and Pfizer Inc. comarket Mirapex pramipexole, a dopamine D2 and D3 receptor agonist, to treat PD. GlaxoSmithKline plc markets Requip ropinirole, also a dopamine D2 and D3 receptor agonist, for PD. Patented by Wayne State University; available for licensing Biswas, S. et al.