Serotonin is a major component of the inflammatory chemical milieu and contributes to the pain of tissue injury via an action on multiple receptor subtypes. Here we studied mice after genetic or pharmacological disruption of the 5-HT 3 receptor, an excitatory serotonin-gated ion channel. We demonstrate that tissue injury-induced persistent, but not acute, nociception is significantly reduced after functional elimination of this receptor subtype. Specifically, in the setting of tissue injury, the 5-HT 3 receptor mediates activation of nociceptors but does not contribute to injury-associated edema. This result is explained by the localization of 5-HT 3 receptor transcripts to a previously uncharacterized subset of myelinated and unmyelinated afferents, few of which express the proinflammatory neuropeptide substance P. Finally, we provide evidence that central serotonergic circuits modulate nociceptive transmission via a facilitatory action at spinal 5-HT 3 receptors. We conclude that activation of both peripheral and central 5-HT 3 receptors is pronociceptive and that the contribution of peripheral 5-HT 3 receptors involves a novel complement of primary afferent nociceptors.

1 In the presence of (-)-cyanopindolol (1.0 LLM) and sumatriptan (1.0 gM), 0.

1 Full length clones of the human 5-HT2B receptor were isolated from human liver, kidney and pancreas. The cloned human 5-HT2B receptors had a high degree of homology (-80%) with the rat and mouse 5-HT2B receptors. 2 PCR amplification was used to determine the tissue distribution of human 5-HT2B receptor mRNA. mRNA encoding the 5-HT2B receptor was expressed with greatest abundance in human liver and kidney. Lower levels of expression were detected in cerebral cortex, whole brain, pancreas and spleen. Expression was not detected in heart. 3 Northern blot analysis confirmed the presence of 5-HT2B receptor mRNA (a 2.4 kB sized band) in pancreas, liver and kidney. An additional 3.2 kB sized band of hybridization was detected in liver and kidney. This raises the possibility of a splice variant of the receptor or the presence of an additional homologous receptor. On the basis of a higher affinity for ketanserin and a lower affinity for yohimbine the human 5-HT2B receptor also appeared to differ from the rat 5-HT2B receptor. 5 These findings confirm the sequence of the human 5-HT2B receptor and they demonstrate that the receptor has a widespread tissue distribution. In addition, these data suggest that there are differences in ligand affinities between different species homologues of the receptor. Finally, the finding of two distinct bands on the Northern blots of liver and kidney raises the possibility of splice variants or subtypes of 5-HT2B receptors, within these tissues.

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Abnormal accumulation and propagation of the neuronal protein α-synuclein has been hypothesized to underlie the pathogenesis of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Here we report a de novo-developed compound (NPT100-18A) that reduces α-synuclein toxicity through a novel mechanism that involves displacing α-synuclein from the membrane. This compound interacts with a domain in the C-terminus of α-synuclein. The E83R mutation reduces the compound interaction with the 80-90 amino acid region of α-synuclein and prevents the effects of NPT100-18A. In vitro studies showed that NPT100-18A reduced the formation of wild-type α-synuclein oligomers in membranes, reduced the neuronal accumulation of α-synuclein, and decreased markers of cell toxicity. In vivo studies were conducted in three different α-synuclein transgenic rodent models. Treatment with NPT100-18A ameliorated motor deficits in mThy1 wild-type α-synuclein transgenic mice in a dose-dependent manner at two independent institutions. Neuropathological examination showed that NPT100-18A decreased the accumulation of proteinase K-resistant α-synuclein aggregates in the CNS and was accompanied by the normalization of neuronal and inflammatory markers. These results were confirmed in a mutant line of α-synuclein transgenic mice that is prone to generate oligomers. In vivo imaging studies of α-synuclein-GFP transgenic mice using two-photon microscopy showed that NPT100-18A reduced the cortical synaptic accumulation of α-synuclein within 1 h post-administration. Taken together, these studies support the notion that altering the interaction of α-synuclein with the membrane might be a feasible therapeutic approach for developing new disease-modifying treatments of Parkinson's disease and other synucleinopathies.

1 A series of isoquinolines have been identified as 5-HT3 receptor antagonists. One of these, RS 25259-197 [(3aS)-2-[(S)-l-azabicyclo[2.2.2]oct-3-yl]-2,3,3a,4,5,6-hexahydro-l-oxo-lH-benzo [de]isoquinoline-hydrochloride], has two chiral centres. The remaining three enantiomers are denoted as RS 25259-198 (R,R), RS25233-197 (S,R) and RS25233-198 (R,S). 2 At 5-HT3 receptors mediating contraction of guinea-pig isolated ileum, RS 25259-197 antagonized contractile responses to 5-HT in an unsurmountable fashion and the apparent affinity (pKB), estimated at 10 nM, was 8.8 ± 0.2. In this tissue, the -log KB values for the other three enantiomers were 6.7 ± 0.3 (R,R), 6.7 ± 0.1 (S,R) and 7.4 ± 0.1 (R,S), respectively. The apparent affinities of RS 25259-197 and RS 25259-198, RS 25233-197 and RS 25233-198 6 Quantitative autoradiographic studies in rat brain indicated a differential distribution of 5-HT3 receptor sites by [3H]-RS . High densities of sites were seen in nuclear tractus solitaris and area postrema, a medium density in spinal trigeminal tract, ventral dentate gyrus and basal medial amygdala, and a low density of sites in hippocampal CAl, parietal cortex, medium raphe and cerebellum. 7 In conclusion, the functional, binding and distribution studies undertaken with the radiolabelled and non-radiolabelled RS 25259-197 (S,S enantiomer) established the profile of a highly potent and selective 5-HT3 receptor antagonist.

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to de®ne precisely the role of 5-HT 2B receptors in normal and disease processes have been hindered by the absence of selective antagonists. To address this de®ciency, we developed a series of naphthylpyrimidines as potentially useful 5-HT 2B receptor antagonists. 2 RS-127445 (2-amino-4-(4-¯uoronaphth-1-yl)-6-isopropylpyrimidine) was found to have nanomolar a nity for the 5-HT 2B receptor (pK i =9.5+0.1) and 1,000 fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites. 3 In cells expressing human recombinant 5-HT 2B receptors, RS-127445 potently antagonized 5-HTevoked formation of inositol phosphates (pK B =9.5+0.1) and 5-HT-evoked increases in intracellular calcium (pIC 50 =10.4+0.1). RS-127445 also blocked 5-HT-evoked contraction of rat isolated stomach fundus (pA 2 =9.5+1.1) and (+)a-methyl-5-HT-mediated relaxation of the rat jugular vein (pA 2 =9.9+0.3). RS-127445 had no detectable intrinsic activity in these assays. 4 In rats, the fraction of RS-127445 that was bioavailable via the oral or intraperitoneal routes was 14 and 60% respectively. Intraperitoneal administration of RS-127445 (5 mg kg 71 ) produced plasma concentrations predicted to fully saturate accessible 5-HT 2B receptors for at least 4 h. 5 In conclusion, RS-127445 is a selective, high a nity 5-HT 2B receptor antagonist suitable for use in vivo. The therapeutic potential of this molecule is being further evaluated.