GBA mutations in Parkinson disease: earlier death but similar neuropathological features.

BACKGROUND AND PURPOSE: Mutations in the glucocerebrosidase (GBA) gene are known to be a risk factor for Parkinson's disease (PD). Data on clinicopathological correlation are limited. The purpose of this study was to determine the clinicopathological findings that might distinguish PD cases with and without mutations in the GBA gene. METHODS: Data from the Arizona Study of Aging and Neurodegenerative Disorders were used to identify autopsied PD cases that did or did not have a GBA gene mutation. Clinical and neuropathological data were compared. RESULTS: Twelve PD cases had a GBA mutation and 102 did not. The GBA mutation cases died younger (76 vs. 81 years of age) but there was no difference in disease duration or clinical examination findings. No neuropathological differences were found in total or regional semi-quantitative scores for Lewy-type synucleinopathy, senile plaques, neurofibrillary tangles, white matter rarefaction or cerebral amyloid angiopathy scores. CONCLUSIONS: In longitudinally assessed, autopsied PD cases, those with GBA mutations had a younger age at death but there was no evidence for clinical or neuropathological differences compared to cases without GBA mutations. Due to the small GBA group size, small differences cannot be excluded.

Reducing the incidence of twins from IVF treatments: predictive modelling from a retrospective cohort.

BACKGROUND: IVF treatments carry a high risk of twin pregnancy which confers a higher risk to the mother and child than singletons. Increased use of elective single embryo transfer (eSET) can reduce this twin rate. We aimed to utilize a previously published data set and statistical model based on routinely collected clinical data to predict the outcomes of policies that increase the proportion of eSET. METHODS: The models allow simultaneous prediction of outcomes from double embryo transfer (DET) and SET. These models were used to predict outcomes for different scenarios using SET in both the initial (fresh) transfer and over a complete cycle (transfer of all embryos created, with cryopreservation). A total of 16 096 cycles (12 487 fresh and 3609 frozen) from 9040 couples treated between 2000 and 2005 were included in the final analyses. RESULTS: For any transfer, SET has about a one-third lower live birth rate relative to DET: this can be partially mitigated by appropriate patient and treatment cycle selection, with several realistic policies performing similarly. However, if we consider complete cycles with embryo cryopreservation, it is possible for repeat SET to produce more live births per egg retrieval than repeat DET. CONCLUSIONS: All patients receiving SET would have a higher chance of successful treatment in that cycle if they received DET. The selection of appropriate patients for SET can partially ameliorate the overall loss. For complete cycles, repeat SET could produce more live births per egg retrieval than repeat DET. All treatments involving SET will increase the number of treatments required to achieve a successful outcome and this extra treatment burden will be a significant barrier to the implementation of such treatments.

Prognostic factors influencing fresh and frozen IVF outcomes: an analysis of the UK national database.

National registry data provides a valuable resource to quantify factors which influence the outcomes of IVF treatments. Multilevel logistic regression analyses for live birth and multiple births given a live birth were undertaken using data from the UK Human Fertilization and Embryology Authority's registry of treatments conducted between 2000 and 2005. This study analysed 119,930 fresh and 19,918 frozen transfers from 85,349 patients in 84 centres. As well as quantifying the effects of a range of previously identified prognostic factors, the analyses showed that embryo cryopreservation reduced the live birth rate substantially (odds ratio (OR) 0.60, 95% confidence interval (CI) 0.57-0.64) compared with typical fresh cycles. Prognostic factors for transfers following cryopreservation were largely similar to those for fresh cycles, with some evidence that female age is less important, and there were small differences in the effects of the numbers of embryos transferred and previous IVF attempts that can be ascribed to selection effects. No factors were identified which specifically predicted twin outcomes; patients with a high twin risk were those with a high chance of treatment success. After allowing for all prognostic factors, there remained clinically important variability between centres (median OR 1.4) and between patients (median OR 1.8). National registry data provides a valuable resource which can be used to determine to what extent clinical and patient characteristics influence the outcomes of IVF treatments. We fitted complex statistical models for live birth and multiple births to data from the UK national registry of treatments conducted between 2000 and 2005. The analysis included 119,930 fresh and 19,918 frozen transfers from 85,349 patients in 84 centres. As well as quantifying the effects of a range of previously identified prognostic factors, we were able to estimate the magnitude of the losses due to embryo freezing and thawing. The effects of clinical and patient characteristics on the outcomes for frozen transfers were largely similar to those for fresh cycles. No factors were identified which specifically predicted twin outcomes; patients with a high twin risk were those with a high chance of success. After allowing for all the identified prognostic factors, there remained clinically important variability between centres and between patients which may suggest that the patients and clinics differ in their chances of success due to characteristics which are not currently being measured.

Embryo and uterine influences on IVF outcomes: an analysis of a UK multi-centre cohort.

BACKGROUND: In order to optimize IVF strategies, particularly with the use of single embryo transfer, good predictive models are required. Here, we develop a model to allow such prediction, and the structure of the models point to more general conclusions about the mode of action of prognostic factors. METHODS: Anonymized data from consecutive embryo transfers in five IVF centres in the UK for the 2000-2005 period were extracted and the morphological grade based on common scoring criteria was included. There were 16 096 (12 487 fresh and 3609 frozen) transfers, for 8775 couples, available for analysis. Live birth data were fitted to a model with separate sub-models for embryo and recipient effects [the 'Embryo-Uterus' (EU) model]. All covariates were included, with sub-model selection using Akaike's information criterion. RESULTS: Age, number of embryos created, attempt number, previous history of pregnancy, duration of infertility, day of transfer and tubal diagnosis were all identified as significant prognostic factors, along with embryo grade and growth rate. Frozen transfers were substantially less likely to lead to a live birth with odds ratios of 1/3 to 1/2 compared with fresh transfers, with no evidence of differential loss for any particular patient group. Age acts predominantly through the embryo component with only a weak effect on the uterus. The embryo number, attempt number, previous pregnancies and duration of infertility act predominantly through the uterine environment. Both sub-models show significant heterogeneity between centres. CONCLUSIONS: The EU modelling framework has generated a model for predicting outcomes of embryo-transfer procedures, subject to the limitations of routinely collected data. With this large data set, the model allows identification of factors that act specifically on embryo viability or maternal receptivity. Variability in the two components between centres with similar overall outcomes suggests scope for further optimization of IVF treatment.

Decision Framework for the environmental management of explosive contaminated land.

The environmental risks from explosive manufacturing and testing activities are usually evaluated using a qualitative process such as environmental impact prioritisation as recommended by legislation and guidance. However, standard environmental management system (EMS) guidance rarely provides detailed information on how to objectively assess the significance of the environmental impacts based on a rational scientific evidence. Quantitative exposure and eco-toxicity assessments are frequently used in combination with environmental threshold limit guidelines, but these omit important environmental impacts such as physical damage to land, nuisance and contribution to climate change. These impacts are particularly relevant to the explosives industry where noise nuisance and physical damage are given high priority. In addition, contamination from explosive compositions may comprise mixtures of multiple legacy and new generation explosives such as 1,3,5-trinitro-1,3,5-triazinane (RDX), 2,4,6-trinitrotoluene (TNT), 5-nitro-1,2,4-triazol-3-one (NTO), 2,4-dinitroanisole (DNAN) and nitroguandine (NQ), which may have combined impacts not captured by conventional eco-toxicity assessments. Further, threshold limits for energetic materials in soil and water have not been established for most nations. Additionally, in the explosive industry wider concerns such as legislative compliance and stakeholder concerns may help to provide a more broadly applicable assessment of environmental impact. Therefore in this study a novel decision framework was developed to integrate empirical data with business risks to enable rational decision making for the environmental management of explosive manufacturing facilities. The application of the framework was illustrated using three case studies from the explosive manufacturing industry to demonstrate how the framework can be used to justify environmental management decision making. By linking the environmental impacts to business risks, we demonstrate that manufacturers are able to assess a wide spectrum of issues that might not be identified in the initial environmental assessment such as non-toxic pollution incidents, breaches in legislation and stakeholder perceptions.

Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

GABA(B2) is essential for g-protein coupling of the GABA(B) receptor heterodimer.

GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

Are speed enforcement cameras more effective than other speed management measures? An evaluation of the relationship between speed and accident reductions.

In this paper, models are developed which enable a prediction of how the impact of speed management schemes on accidents varies both with speed changes and with site and scheme characteristics. It was found that, the impact of schemes with vertical deflections is independent of the change in mean speed: an accident reduction of 44% is predicted by the model irrespective of the impact on speed. For cameras and other types of engineering schemes, a simple relationship between the change in mean speed and the consequent change in accidents is available. For the range of mean speeds typically found on 30 mph roads, the percentage accident reduction per 1 mph speed reduction is around 4% for cameras and 7-8% for schemes with horizontal features. While larger percentage accident reductions are achieved per 1 mph speed reduction on lower speed roads, larger speed reductions and larger overall percentage accident reductions are obtained on roads with higher before mean speeds. It is possible to predict both changes in speeds and accidents before treatment using the models derived from this study and these models confirm that schemes with vertical deflections are most effective in reducing both speeds and accidents.

Are speed enforcement cameras more effective than other speed management measures? The impact of speed management schemes on 30 mph roads.

This paper presents the results of an evaluation of the impact of various types of speed management schemes on both traffic speeds and accidents. The study controls for general trends in accidents, regression-to-mean effects and migration, separately estimating the accident changes attributable to the impact of the schemes on traffic speed and on traffic volume. It was found that, when judged in absolute terms, all types of speed management scheme have remarkably similar effects on accidents, with an average fall in personal injury accidents of about 1 accident/km/year. In terms of the percentage accident reduction, however, engineering schemes incorporating vertical deflections (such as speed humps or cushions) offer the largest benefits: at 44%, the average reduction in personal injury accidents attributable to such schemes, is twice that at sites where safety cameras were used to control speeds (22%) and they were the only type of scheme to have a significant impact on fatal and serious accidents. Other types of engineering scheme (with a fall of 29% in personal injury accidents) were on average less effective in reducing accidents than schemes with vertical features but more effective than cameras. All types of scheme were generally effective in reducing speeds, with the largest reductions tending to be obtained with vertical deflections and the smallest with other types of engineering schemes.

Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors.

Previous studies have shown that expression of the immune co-stimulator B7.1 reduces the tumorigenicity of some, but not all, malignant cell lines. However, B7.1-expressing tumor cells are not very effective in inducing the rejection of established tumors. This may in part be due to induction of anergy in the potentially reactive T cells. Previous studies have shown that IL-2 can reverse the anergic state both in vitro and in vivo. Therefore, we have examined the effect of retrovirus-mediated delivery and expression of murine B7.1 and interleukin-2 on tumor formation and rejection of established MHC class I+/II- NC adenocarcinomas. Neither the expression of B7.1 nor IL-2 alone had a significant effect on NC tumorigenicity. In contrast, combined expression of B7.1 and IL-2 substantially decreased the tumorigenicity of these cells in the immunecompetent syngeneic hosts. T-cell depletion studies show this to be dependent primarily on the activation of CD4+ cells. Furthermore, distant subcutaneous injection of irradiated NC/IL-2/B7.1 can induce, much more effectively than NC/B7.1 or NC/IL-2, the rejection of small NC tumors, and prevent the recurrence of large surgically resected tumors. Together, these results suggest that tumor cells genetically modified to express B7.1 and IL-2 can induce the immune-mediated rejection of established class II- tumors by a mechanism involving CD4+ cells.

The characterization of an amnesic syndrome following hypoxic ischemic injury.

We used clinical analysis and criteria derived from the experimental studies of classic amnesic syndromes to characterize the amnesia that follows hypoxic ischemic brain injury from cardiac or respiratory arrest. The results show that patients with hypoxic ischemic amnesia have several neuropsychological features in common with other amnesics, including intact short-term memory, severely depressed free recall, and less depressed recognition of visual and verbal material. Unlike amnesics with alcoholic Korsakoff's syndrome, they are oriented and do not confabulate. Hypoxic ischemic brain injury is a common hospital occurrence, and a study of the characteristics of the amnesia that can occur after such injury should enlarge our understanding of the amnesic syndromes.

Further characterization of patients with amnesia after cardiac arrest: preserved recognition memory.

We observed that patients with amnesia after cardiac arrest had preserved recognition memory despite profound loss of recall memory. In the present study, rate of forgetting was measured in six amnesic subjects for both recall and recognition memory of verbal material. The data show that recall decayed significantly faster for the amnesic subjects than for controls, whereas the rate of forgetting for recognition memory was comparable in both groups. Dissociation between recall and recognition performance is a feature of the amnesic syndrome after cardiac arrest.

Encoding of spatial relations with amnesia.

Incidental and intentional learning of spatial location was studied in amnesics and normals. It was found that spatial encoding of amnesics improved with instruction whereas the spatial encoding of normal adults did not. The finding suggests that spatial encoding in amnesics may be qualitatively different from that of normal adults.

Differential involvement of 5-HT(1B/1D) and 5-HT6 receptors in cognitive and non-cognitive symptoms in Alzheimer's disease.

Growing evidence suggests that a compromised serotonergic system plays an important role in the pathophysiology of Alzheimer's disease (AD). We assessed the expression of 5-HT(1B/1D) and 5-HT(6) receptors and cholinacetyltransferase (ChAT) activity in post-mortem frontal and temporal cortex from AD patients who had been prospectively assessed for cognitive function using the Mini-Mental State Examination (MMSE) and behavioral changes using the Present Behavioral Examination (PBE). 5-HT(1B/1D) and 5-HT(6) receptor densities were significantly reduced in both cortical areas. 5-HT(1B/1D) receptor density was correlated to MMSE decline in the frontal cortex, supporting its implication in memory impairment. The best predictor for lowered 5-HT(6) receptor density in the temporal cortex was the PBE measure of overactivity. The 5-HT(6)/ChAT ratio was related to aggression both in the frontal and temporal cortex. Therefore, antagonists acting at 5-HT(6) receptors could be useful in the treatment of non-cognitive symptoms associated to AD.

Whole blood assay for assessment of the mixed lymphocyte reaction.

When lymphocytes from genetically different individuals are mixed together in tissue culture blast transformation occurs, a reaction known as the mixed lymphocyte reaction (MLR). The MLR is a clinically relevant in vitro assay where lymphocytes from one individual (effector, E) are incubated with the lymphocytes of another individual (stimulator, S) which have been previously rendered incapable of blast transformation by gamma-irradiation. We have standardised a whole blood (WB) MLR assay where the E lymphocytes were provided by 20 microl of WB and the S lymphocytes were provided by irradiated peripheral blood mononuclear cells (PBMCs) either as a mixed pool of 20 donor PBMCs or as single donor PBMC. The optimum number of S lymphocytes needed was comparatively higher than in the standard PBMC MLR: the optimum calculated E:S ratio was 1:20 compared to a E:S ratio of 1:1 or 3:2 in the standard PBMC MLR. In ten normal individuals the WB/PBMC MLR was similar to the standard PBMC/PBMC MLR. As a clinical example, the WB/PBMC MLR proliferative capacity of 13 patients with malignant mesothelioma was no different from the proliferative capacity of their age-sex matched controls. This standardised WB/PBMC MLR assay is a simple and more practical assay than the standard MLR assay and can be incorporated easily in clinical studies with biological end-points.

Gabapentin is not a GABAB receptor agonist.

Recent experiments have demonstrated that formation of functional type B gamma-aminobutyric acid (GABA(B)) receptors requires co-expression of two receptor subunits, GABA(B1) and GABA(B2). Despite the identification of these subunits and a number of associated splice variants, there has been little convincing evidence of pharmacological diversity between GABA(B) receptors comprising different subunit combinations. However, Ng et al. [Mol. Pharmacol., 59 (2000) 144] have recently suggested a novel and important pharmacological difference between GABA(B) receptor heterodimers expressing the GABA(B1a) and GABA(B1b) receptor subunits. This study suggested that the antiepileptic GABA analogue gabapentin (Neurontin) is an agonist at GABA(B) receptors expressing the GABA(B1a) but not the GABA(B1b) receptor subunit. The importance of this finding with respect to identifying novel GABA(B) receptor subunit specific agonists prompted us to repeat these experiments in our own [35S]-GTPgammaS binding and second messenger assay systems. Here we report that gabapentin was completely inactive at recombinant GABA(B) heterodimers expressing either GABA(B1a) or GABA(B1b) receptor subunits in combination with GABA(B2) receptor subunits. In addition, in both CA1 and CA3 pyramidal neurones from rodent hippocampal slices we were unable to demonstrate any agonist-like effects of gabapentin at either pre- or post-synaptic GABA(B) receptors. In contrast, gabapentin activated a GABA(A) receptor mediated chloride conductance. Our data suggest that gabapentin is not a GABA(B)-receptor agonist let alone a GABA(B) receptor subunit selective agonist.

Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes.

1. 5-Hydroxytryptamine (5-HT) elicited a dose-dependent stimulation of intracellular adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 +/- 0.08 (mean +/- s.e. mean). 2. In order to characterize the 5-HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5-carboxamidotryptamine (5-CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5-CT (pEC50 = 7.81 +/- 0.09) > 5-methoxytryptamine (5-MeOT) (pEC50 = 6.86 +/- 0.36) > 5-HT (pEC50 = 6.68 +/- 0.08). The following compounds, at concentrations up to 10 microM, did not affect basal cyclic AMP levels 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was methiothepin (pKi = 7.98 +/- 0.25) > mesulergine (pKi = 7.58 +/- 0.18) > ritanserin (pKi = 7.20 +/- 0.24) > clozapine (pKi = 7.03 +/- 0.19) > mianserin (pKi = 6.41 +/- 0.19). The following compounds, at concentrations up to 10 microM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5-HT7 receptor subtype-mediated effects. 3. The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 +/- 75% and 572 +/- 63% of basal levels for 5-CT and 5-HT, respectively) compared to brainstem (274 +/- 51% and 318 +/- 46%, respectively) and colliculus astrocytes (244 +/- 15% and 301 +/- 24%, respectively). No significant differences in pEC50 (for either 5-HT or 5-CT) values were observed. 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for the 5-HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5-HT6 receptor also amplified a cDNA fragment from the same samples. 5. From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5-HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally-dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5-HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5-HT6 and 5-HT7 receptor messenger RNA.

Characterization of the binding of [(125)I]-human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor.

GPR10 is a novel G-protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor-1 (UHR-1). Human prolactin-releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [(125)I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [(125)I]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with K:(D:) values of 0.026+/-0.006 and 0.57+/-0.14 nM respectively, and B(max) values of 3010+/-400 and 8570+/-2240 fmol mg protein(-1) respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a K:(D:) of 0.012 nM. Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstrated any affinity for GPR10. Human PrRP-20 failed to alter basal or forskolin-stimulated levels of intracellular cyclic AMP in HEK293-GPR10 cells, suggesting that GPR10 does not couple via either G(s) or G(i). Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP-20 and PrRP-31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca(2+) stores. These studies indicate that [(125)I]-PrRP-20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.

Characterization of [(125)I]-SB-258585 binding to human recombinant and native 5-HT(6) receptors in rat, pig and human brain tissue.

SB-258585 (4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzen esulphonamide) is a high affinity ligand at 5-HT(6) receptors. It displays over 100 fold selectivity for the 5-HT(6) receptor over all other 5-HT receptors tested so far. SB-258585 has been radiolabelled, to high specific activity, for its characterization as a 5-HT(6) receptor selective radioligand. [(125)I]-SB-258585 bound, with high affinity, to a single population of receptors in a cell line expressing human recombinant 5-HT(6) receptors. Kinetic and saturation binding experiments gave pK(D) values of 9.01+/-0.09 and 9.09+/-0.02, respectively. In membranes derived from rat or pig striatum and human caudate putamen, [(125)I]-SB-258585 labelled a single site with high levels (>60%) of specific binding. Saturation analysis revealed pK(D) values of 8.56+/-0.07 for rat, 8.60+/-0.10 for pig and 8.90+/-0.02 for human. B(max) values for the tissues ranged from 173+/-23 and 181+/-25 fmol mg(-1) protein in rat and pig striatum, respectively, to 215+/-41 fmol mg(-1) protein in human caudate putamen. The pK(i) rank order of potency for a number of compounds, determined in competition binding assays with [(125)I]-SB-258585, at human caudate putamen membranes was: SB-271046>SB-258585>SB-214111>methiothepin>clozapine>5-Me-OT>5-HT>Ro 04-6790>mianserin>ritanserin=amitriptyline>5-CT>mesulergine. Similar profiles were obtained from pig and rat striatal membranes and recombinant 5-HT(6) receptors; data from the latter correlated well with [(3)H]-LSD binding. Thus, [(125)I]-SB-258585 is a high affinity, selective radioligand which can be used to label both recombinant and native 5-HT(6) receptors and will facilitate further characterization of this receptor subtype in animal and human tissues.