Diagnostic value of combined islet antigen-reactive T cells and autoantibodies assays for type 1 diabetes mellitus.

AIMS/INTRODUCTION: Type 1 diabetes mellitus is a T cell-mediated autoimmune disease. However, the determination of the autoimmune status of type 1 diabetes mellitus relies on islet autoantibodies (Abs), as T-cell assay is not routinely carried out. This study aimed to investigate the diagnostic value of combined assay of islet antigen-specific T cells and Abs in type 1 diabetes mellitus patients. MATERIALS AND METHODS: A total of 54 patients with type 1 diabetes mellitus and 56 healthy controls were enrolled. Abs against glutamic acid decarboxylase (GAD), islet antigen-2 and zinc transporter 8 were detected by radioligand assay. Interferon-γ-secreting T cells responding to glutamic acid decarboxylase 65 and C-peptide (CP) were measured by enzyme-linked immunospot. RESULTS: The positive rate for T-cell responses was significantly higher in patients with type 1 diabetes mellitus than that in controls (P < 0.001). The combined positive rate of Abs and T-cell assay was significantly higher than that of Abs assay alone (85.2% vs 64.8%, P = 0.015). A significant difference in fasting CP level was found between the T+ and T- groups (0.07 ± 0.05 vs 0.11 ± 0.09 nmol/L, P = 0.033). Furthermore, levels of fasting CP and postprandial CP were both lower in the Ab- T+ group than the Ab- T- group (fasting CP 0.06 ± 0.05 vs 0.16 ± 0.12 nmol/L, P = 0.041; postprandial CP 0.12 ± 0.13 vs 0.27 ± 0.12 nmol/L, P = 0.024). CONCLUSIONS: Enzyme-linked immunospot assays in combination with Abs detection could improve the diagnostic sensitivity of autoimmune diabetes.

Synthesis and pharmacology of new psychoactive substance 5F-CUMYL-P7AICA, a scaffold- hopping analog of synthetic cannabinoid receptor agonists 5F-CUMYL-PICA and 5F-CUMYL-PINACA.

Synthetic cannabinoid receptor agonists (SCRAs) are a dynamic class of new psychoactive substances (NPS), with novel chemotypes emerging each year. Following the putative detection of 5F-CUMYL-P7AICA in Australia in 2016, the scaffold-hopping SCRAs 5F-CUMYL-PICA, 5F-CUMYL-PINACA, and 5F-CUMYL-P7AICA were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-quadrupole time-of-flight-MS (LC-QTOF-MS). Since little is known of the pharmacology of 7-azaindole SCRAs like 5F-CUMYL-P7AICA, the binding affinities and functional activities of all compounds at cannabinoid type 1 and type 2 receptors (CB1 and CB2 , respectively) were assessed using tritiated radioligand competition experiments and fluorescence-based plate reader membrane potential assays. Despite CB1 binding affinities differing by over two orders of magnitude (Ki  = 2.95-174 nM), all compounds were potent and efficacious CB1 agonists (EC50  = 0.43-4.7 nM), with consistent rank order for binding and functional activity (5F-CUMYL-PINACA >5F-CUMYL-PICA >5F-CUMYL-P7AICA). Additionally, 5F-CUMYL-P7AICA was found to exert potent cannabimimetic effects in mice, inducing hypothermia (6°C, 3 mg/kg) through a CB1 -dependent mechanism.

The affinity of antipsychotic drugs to dopamine and serotonin 5-HT2 receptors determines their effects on prefrontal-striatal functional connectivity.

One of the major challenges of cross-species translation in psychiatry is the identification of quantifiable brain phenotypes linked to drug efficacy and/or side effects. A measure that has received increasing interest is the effect of antipsychotic drugs on resting-state functional connectivity (FC) in magnetic resonance imaging. However, quantitative comparisons of antipsychotic drug-induced alterations of FC patterns are missing. Consideration of receptor binding affinities provides a means for the effects of antipsychotic drugs on extended brain networks to be related directly to their molecular mechanism of action. Therefore, we examined the relationship between the affinities of three second-generation antipsychotics (amisulpride, risperidone and olanzapine) to dopamine and serotonin receptors and FC patterns related to the prefrontal cortex (PFC) and striatum in Sprague-Dawley rats. FC of the relevant regions was quantified by correlation coefficients and local network properties. Each drug group (32 animals per group) was subdivided into three dose groups and a vehicle control group. A linear relationship was discovered for the mid-dose of antipsychotic compounds, with stronger affinity to serotonin 5-HT2A, 5-HT2C and 5-HT1A receptors and decreased affinity to D3 receptors associated with increased prefrontal-striatal FC (p = 0.0004, r²â€¯= 0.46; p = 0.004, r²â€¯= 0.33; p = 0.002, r²â€¯= 0.37; p = 0.02, r²â€¯= 0.22, respectively). Interestingly, no correlation was observed for the low and high dose groups, and for D2 receptors. Our results indicate that drug-induced FC patterns may be linked to antipsychotic mechanism of action on the molecular level and suggest the technique's value for drug development, especially if our results are extended to a larger number of antipsychotics.

Evolution of microglial activation in ischaemic core and peri-infarct regions after stroke: a PET study with the TSPO molecular imaging biomarker [((11))C]vinpocetine.

Although there is increasing evidence for microglial activation after an ischaemic stroke in the infarct core and the peri-infarct region, the "evolution" of the process in stroke patients is poorly known. Using PET and [((11))C]vinpocetine, we measured the regional changes of TSPO in the brain of nine ischaemic stroke patients up to 14weeks after the insult. Already a week after stroke there was an increased radioligand uptake, indicating the up-regulation of TSPO and the presence of activated microglia, in both the ischaemic core and the peri-infarct zone. This increased activation showed a steady decrease with post stroke time. The proportion between %SUV values in the peri-infarct zone and the ischaemic core increased with time. There were no time-dependent TSPO activity changes in other regions, not affected directly by the stroke. The present observations demonstrate that increased regional microglia activation, as a consequence of stroke, can be visualised with PET, using the TSPO molecular imaging biomarker [((11))C]vinpocetine. The evolution of this microglial activation shows a time dependent decrease the gradient of which is different between the peri-infarct zone and the ischaemic core. The findings indicate an increased microglial activation in the peri-stroke region for several weeks after the insult.

Changes in measurement procedure from a radioassay to a microbiologic assay necessitate adjustment of serum and RBC folate concentrations in the U.S. population from the NHANES 1988-2010.

The NHANES measured serum and RBC folate concentrations by using a radioassay during prefortification (1988-1994) and postfortification (1999-2006) periods followed by the use of a microbiologic assay (MBA) from 2007-2010. The MBA produces higher concentrations than does the radioassay and is considered to be more accurate. To allow for accurate long-term trending (1988-2010), we evaluated different regression models (linear, piecewise linear, and fractional polynomial) to assay-adjust the radioassay results to be comparable to the MBA results. The data used to derive the regression models originated from 2 crossover studies in which the 2 assays were applied to a set of 325 serum and 171 whole-blood samples. Fractional polynomial regression of logarithmically transformed data provided the best fit for serum folate. Linear regression of logarithmically transformed whole-blood data provided an equally good fit compared with the other models and was the simplest to apply for RBC folate. Prefortification serum and RBC folate geometric mean concentrations increased after adjustment from 13.0 to 16.7 nmol/L and from 403 to 747 nmol/L, respectively. Postfortification serum folate concentrations increased from ~30 to ~43 nmol/L, and RBC folate concentrations increased from ~600 to ~1100 nmol/L after adjustment, with some variation across survey cycles. The presented regression equations allow the estimation of more accurate prevalence estimates and long-term trends in blood folate concentrations in the U.S. population by using results that are equivalent to the MBA. This information will be useful to public health officials in the United States who are dealing with folic acid fortification issues.

Triple monoamine inhibitor tesofensine decreases food intake, body weight, and striatal dopamine D2/D3 receptor availability in diet-induced obese rats.

The novel triple monoamine inhibitor tesofensine blocks dopamine, serotonin and norepinephrine re-uptake and is a promising candidate for the treatment of obesity. Obesity is associated with lower striatal dopamine D2 receptor availability, which may be related to disturbed regulation of food intake. This study assesses the effects of chronic tesofensine treatment on food intake and body weight in association with changes in striatal dopamine D2/D3 receptor (D2/3R) availability of diet-induced obese (DIO) rats. Four groups of 15 DIO rats were randomized to one of the following treatments for 28 days: 1. tesofensine (2.0 mg/kg), 2. vehicle, 3. vehicle+restricted diet isocaloric to caloric intake of group 1, and 4. tesofensine (2.0 mg/kg)+ a treatment-free period of 28 days. Caloric intake and weight gain decreased significantly more in the tesofensine-treated rats compared to vehicle-treated rats, which confirms previous findings. After treatment discontinuation, caloric intake and body weight gain gradually increased again. Tesofensine-treated rats showed significantly lower D2/3R availability in nucleus accumbens and dorsal striatum than both vehicle-treated rats and vehicle-treated rats on restricted isocaloric diet. No correlations were observed between food intake or body weight and D2/3R availability. Thus, chronic tesofensine treatment leads to decreased food intake and weight gain. However, this appears not to be directly related to the decreased striatal D2/3R availability, which is mainly a pharmacological effect.

Neurohypophyseal hormones manipulation modulate social and anxiety-related behavior in zebrafish.

RATIONALE: Oxytocin (OT) and arginine-vasopressin (AVP) regulate social behavior in mammals. Zebrafish (Danio rerio) allows higher throughput and ease in studying human brain disorders. OBJECTIVES: This study investigated in zebrafish the effect of non-mammalian homologs isotocin (IT) and vasotocin (AVT) in comparison with OT/AVP on social behavior and fear response to predator. The mechanism was studied using the most human selective OT and AVP receptor antagonists. METHODS: Zebrafish were injected i.m. with increasing doses (0.001-40 ng/kg) of the neuropeptides. DesGly-NH(2)-d(CH(2))(5)-[D-Tyr(2),Thr(4)]OVT) for OT receptor, SR 49059 for V1a subtype receptor, and SSR-149415 for V1b subtype receptor were injected i.m. 10 min before each agonist. RESULTS: All the peptides increased social preference and reduced fear to predator response in a dose-dependent manner interpolated by symmetrical parabolas. AVT/AVP were more potent to elicit anxiolytic than social effect while IT and OT were equally potent. All the antagonists dose-dependently inhibited both the effects induced by the neuropeptides. The ratio between the ED50 obtained for blocking the OT-induced effects on social preference and fear response to predator was very high only for desglyDTTyrOVT (160). SR49059 showed the highest ratio in blocking AVP-induced effects (807). The less selective antagonist appeared to be SSR149415. CONCLUSIONS: For the first time, IT/AVT and OT/AVP were found to modulate in zebrafish, social behavior, unrelated to sex, and fear to predator response through at least two different receptors. Zebrafish is confirmed as a valid, reliable model to study deficit in social behavior characteristic of some psychiatric disorders.

Preclinical pharmacology of TP1, a novel potent triple reuptake inhibitor with antidepressant properties.

Triple reuptake inhibitors (TRIs) that block the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT) are being developed as a new class of antidepressant that may have better efficacy and fewer side effects compared with traditional antidepressants. The purpose of this study was to characterize a new chemical entity, 4-[2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl] phenyl 4-methoxybenzoate hydrochloride (TP1). TP1 was designed as a prodrug of desvenlafaxine. Competitive radioligand binding assays were performed using cells expressing the human dopamine (DA) transporter (hDAT), the human serotonin (5-HT) transporter (hSERT), and the human norepinephrine (NE) transporter (hNET) with K(i) values for TP1 of 190 nM, 2076 nM, and 1023 nM, respectively. Uptake assays were performed with IC(50) values for TP1 of 712 nM, 521 nM, and 628 nM, respectively. TP1 (0.06 mmol/kg, orally) rapidly penetrated rat brain and hypothalamus, translated into desvenlafaxine within 1 h, and demonstrated higher bioavailability and better pharmacokinetic properties than desvenlafaxine succinate (DVS). TP1 (0.06 mmol/kg, orally) significantly increased extracellular levels of DA, NE, and 5-HT compared with baseline in the rat hypothalamus by microdialysis assay. In dose-response assays, oral administration of TP1 reduced the time of immobility in a dose-dependent manner during tail suspension test and forced swimming test (FST). This antidepressant-like effect manifests in the absence of significant increases in motor activity even at doses of up to 32 mg/kg. The ability of TP1 to inhibit the reuptake of three biogenic amines closely linked to the etiology of depression may result in a therapeutic profile different from antidepressants that inhibit the reuptake of serotonin and/or NE.

Serotonin transporter occupancy and the functional neuroanatomic effects of citalopram in geriatric depression.

OBJECTIVES: The functional neuroanatomic changes associated with selective serotonin reuptake inhibitor (SSRI) treatment have been the focus of positron emission tomography (PET) studies of cerebral glucose metabolism in geriatric depression. DESIGN: To evaluate the underlying neurochemical mechanisms, both cerebral glucose metabolism and serotonin transporter (SERT) availability were measured before and during treatment with the SSRI, citalopram. It was hypothesized that SERT occupancy would be observed in cortical and limbic brain regions that have shown metabolic effects, as well as striatal and thalamic regions that have been implicated in prior studies in midlife patients. SETTING: Psychiatric outpatient clinic. PARTICIPANTS: Seven depressed patients who met Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for current major depressive episode were enrolled. INTERVENTION: Patients underwent a 12-week open-label trial of the SSRI, citalopram. MEASUREMENTS: Patients underwent high-resolution research tomography PET scans to measure changes in cerebral glucose metabolism and SERT occupancy by citalopram treatment (after 8-10 weeks of treatment). RESULTS: Three different tracer kinetic models were applied to the [¹¹C]-DASB region-of-interest data and yielded similar results of an average of greater than 70% SERT occupancy in the striatum and thalamus during citalopram treatment. Voxel-wise analyses showed significant SERT occupancy in these regions, as well as cortical (e.g., anterior cingulate, superior and middle frontal, precuneus, and limbic (parahippocampal gyrus) areas that also showed reductions in glucose metabolism. CONCLUSION: The findings suggest that cortical and limbic SERT occupancy may be an underlying mechanism for the regional cerebral metabolic effects of citalopram in geriatric depression.

Opiate-induced dopamine release is modulated by severity of alcohol dependence: an [(18)F]fallypride positron emission tomography study.

BACKGROUND: Preclinical data implicate the reinforcing effects of alcohol to be mediated by interaction between the opioid and dopamine systems of the brain. Specifically, alcohol-induced release of ß-endorphins stimulates µ-opioid receptors (MORs), which is believed to cause dopamine release in the brain reward system. Individual differences in opioid or dopamine neurotransmission have been suggested to be responsible for enhanced liability to abuse alcohol. In the present study, a single dose of the MOR agonist remifentanil was administered in detoxified alcohol-dependent patients and healthy control subjects to mimic the ß-endorphin-releasing properties of ethanol and to assess the effects of direct MOR stimulation on dopamine release in the mesolimbic reward system. METHODS: Availability of D(2/3) receptors was assessed before and after single-dose administration of the MOR agonist remifentanil in 11 detoxified alcohol-dependent patients and 11 healthy control subjects with positron emission tomography with the radiotracer [(18)F]fallypride. Severity of dependence as assessed with the Alcohol Use Disorders Identification Test was compared with remifentanil-induced percentage change in [(18)F]fallypride binding (Δ%BP(ND)). RESULTS: The [(18)F]fallypride binding potentials (BP(ND)s) were significantly reduced in the ventral striatum, dorsal putamen, and amygdala after remifentanil application in both patients and control subjects. In the patient group, ventral striatum Δ%BP(ND) was correlated with the Alcohol Use Disorders Identification Test score. CONCLUSIONS: The data provide evidence for a MOR-mediated interaction between the opioid and the dopamine system, supporting the assumption that one way by which alcohol unfolds its rewarding effects is via a MOR-(γ-aminobutyric acid)-dopamine pathway. No difference in dopamine release was found between patients and control subjects, but evidence for a patient-specific association between sensitivity to MOR stimulation and severity of alcohol dependence was found.

Analysis of variance in neuroreceptor ligand imaging studies.

Radioligand positron emission tomography (PET) with dual scan paradigms can provide valuable insight into changes in synaptic neurotransmitter concentration due to experimental manipulation. The residual t-test has been utilized to improve the sensitivity of the t-test in PET studies. However, no further development of statistical tests using residuals has been proposed so far to be applied in cases when there are more than two conditions. Here, we propose the residual f-test, a one-way analysis of variance (ANOVA), and examine its feasibility using simulated [(11)C]raclopride PET data. We also re-visit data from our previously published [(11)C]raclopride PET study, in which 10 individuals underwent three PET scans under different conditions. We found that the residual f-test is superior in terms of sensitivity than the conventional f-test while still controlling for type 1 error. The test will therefore allow us to reliably test hypotheses in the smaller sample sizes often used in explorative PET studies.

Brain type 1 cannabinoid receptor availability in patients with anorexia and bulimia nervosa.

BACKGROUND: The endocannabinoid system is a possible target in the treatment of eating disorders. We used positron emission tomography to investigate the type 1 cannabinoid receptor (CB1R) in bulimic and anorectic patients. METHODS: We investigated 16 female bulimia nervosa patients (BN) (age = 23.8 ± 7.1 years) and 14 female anorexia nervosa patients (AN) (age = 20.5 ± 3.6 years) using the selective CB1R ligand [(18)F]MK-9470. The control group consisted of 19 age-matched women (age = 25.2 ± 8.5 years). Statistical parametric mapping (p(family-wise error) < .05) and volume-of-interest analyses of CB1R availability were performed. RESULTS: Global CB1R availability was significantly increased in cortical and subcortical brain areas in AN patients compared with healthy control subjects (+24.5%, p = .0003). Regionally, CB1R availability was increased in the insula in both AN and BN patients (p = .01 and p = .0004) and the inferior frontal and temporal cortex in AN patients only (p = .02). CONCLUSIONS: Global CB1R upregulation in AN patients is a possible long-term compensatory mechanism to an underactive endocannabinoid system in anorectic conditions. There is a similarity in CB1R dysregulation both in AN and BN in the insular cortex, which is involved in the integration of interoceptive information, gustatory information, reward, and emotion processing.

Use of the GTPγS ([35S]GTPγS and Eu-GTPγS) binding assay for analysis of ligand potency and efficacy at G protein-coupled receptors.

UNLABELLED: In this review I consider assays for G protein-coupled receptor (GPCR) activity based on the binding of labelled analogues of GTPγS ([(35) S]GTPγS or Eu-GTPγS) to G proteins in tissues (GTPγS binding assays). Such assays provide convenient measures of GPCR activity close to the receptor in the signalling cascade. In order to set up a GTPγS binding assay, the requirements of the assay must be considered. These are tissue source, GTPγS analogue, G protein, GDP, Mg(2+) /Na(+) ions, saponin, incubation time. The assay, once optimized, can be used to generate concentration/response curves for GPCRs signalling via G(i/o) proteins (or to other G proteins with a modified assay) and actions of agonists, inverse agonists and antagonists may, in principle, be assessed. For agonists and inverse agonists, data for the maximal agonist effect, the concentration of ligand giving a half-maximal response and the Hill coefficient may be derived. For antagonists, data for the equilibrium dissociation constant can be obtained. The mechanistic basis of the assay is considered. Although the assay can be used to profile ligands, under the conditions it is used, it may not be measuring the same event that determines GPCR action in cells. LINKED ARTICLES: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6

Reduced alpha4beta2*-nicotinic acetylcholine receptor binding and its relationship to mild cognitive and depressive symptoms in Parkinson disease.

CONTEXT: Cognitive or depressive disorders are frequently noted in patients with Parkinson disease (PD) and may be related to altered signaling through alpha4beta2*-nicotinic acetylcholine receptors (alpha4beta2*-nAChRs). OBJECTIVE: To assess the availability of alpha4beta2*-nAChRs and their relationship to mild cognitive and mild depressive symptoms in vivo in patients with PD. DESIGN: Crossover comparison between patients with PD and healthy volunteers (control group) using the alpha4beta2*-nAChR-specific radioligand 2-[(18)F]fluoro-3-(2[S]-2-azetidinylmethoxy)-pyridine (2-[(18)F]FA-85380) and positron emission tomography. SETTING: Departments of Neurology and Nuclear Medicine, University of Leipzig, Leipzig, Germany. PARTICIPANTS: Twenty-two nonsmoking patients with PD and 9 nonsmoking healthy volunteers. MAIN OUTCOME MEASURES: Level of 2-[(18)F]FA-85380 binding potential (2-FA BP), a measure of alpha4beta2*-nAChR availability. The relationship between severity of cognitive symptoms as rated using the Mini-Mental State Examination and DemTect scale and the level of depressive symptoms as indicated using the Beck Depression Inventory, and 2-FA BP were assessed. RESULTS: In patients with PD compared with healthy volunteers, there was widespread reduced 2-FA BP, especially in the midbrain, pons, anterior cingulate cortex, frontoparietal cortex, and cerebellum. In subgroups of patients with PD with possible depression, reduced 2-FA BP was most pronounced in the cingulate cortex and frontoparieto-occipital cortex, whereas in patients with PD with mild cognitive impairment, 2-FA BP was reduced in the midbrain, pons, and cerebellum. In patients with PD, the strongest associations between depressive symptoms and reduced 2-FA BP were noted in the anterior cingulate cortex, putamen, midbrain, and occipital cortex. In contrast, cognitive symptoms correlated only weakly with reduced 2-FA BP in the thalamus, midbrain, temporal cortex, hippocampus, and cerebellum. CONCLUSIONS: There is a broad reduction of alpha4beta2*-nAChR availability in patients with PD without clinically manifest dementia or depression compared with healthy volunteers. Reduced alpha4beta2*-nAChR binding in patients with PD within the subcortical and cortical regions is associated with the severity of mild cognitive or depressive symptoms. These results provide novel in vivo evidence for a role of the cholinergic neurotransmission in psychiatric comorbidity of PD.

Low dopamine d(2) receptor binding in subregions of the thalamus in schizophrenia.

OBJECTIVE: Several structural and functional brain imaging studies have pointed to a disturbance of thalamic subnuclei in patients with schizophrenia. The dopamine hypothesis of schizophrenia has, however, not been thoroughly examined in terms of this complex structure, which has connections with most brain regions of central interest in schizophrenia research. The aim of the present study was to examine dopamine D(2) receptor binding in subregions of the thalamus in patients with schizophrenia. METHOD: The authors used positron emission tomography and the radioligand [(11)C]FLB457 to examine dopamine D(2) receptor binding in thalamic subregions of 10 drug-naive patients with schizophrenia. Binding potential was calculated by the reference tissue method and used as an index for dopamine D(2) receptor binding. Comparisons were made with 19 healthy subjects. Subregions of interest were defined on individual magnetic resonance images using a percentage-based operational approach. Clinical symptoms were rated by using the Brief Psychiatric Rating Scale (BPRS). RESULTS: The [(11)C]FLB457 binding potential was lower in the central medial and posterior subregions of the thalamus in patients with schizophrenia. At a functional level, there was a significant negative correlation between binding potential and BPRS positive symptom scores. CONCLUSIONS: The subregions with low D(2) receptor binding comprise primarily the dorsomedial nucleus and pulvinar, two important components in circuitries previously suggested in the pathophysiology of schizophrenia. Aberrant dopaminergic neurotransmission in thalamic subregions might be a mechanism underlying positive symptoms in schizophrenia.

High-resolution phosphor imaging: validation for use with human brain tissue sections to determine the affinity and density of radioligand binding.

This study investigated the suitability of high-resolution storage phosphor imaging for the quantitative analysis of radioligand binding to human brain tissue. Hence, the binding of [(3)H]mazindol to the dopamine transporter in caudate-putamen tissue homogenates or frozen tissue sections apposed to either autoradiographic film or phosphor imaging plates was measured. Estimates of binding affinity were similar for homogenate studies and phosphor imaging plates (Kd=6.44+/-0.14 and 6.91+/-0.47 nM, respectively), but higher values were obtained with film autoradiography (Kd=11.31+/-0.82 nM). The density of binding was similar for both autoradiographic techniques (Bmax=371.9+/-30.8 fmol/mg estimated tissue equivalent, ETE (imaging plate) and 425+/-13.77 fmol/mg ETE (film)), although lower values were obtained from tissue homogenates (Bmax=64.27+/-6.74 fmol/mg wet weight). These results suggest that high resolution phosphor imaging can be used to analyse radioligand binding parameters in human brain tissue. Moreover, the reduced exposure time of phosphor imaging plates (e.g. 7 days vs 5 weeks) allows results to be obtained more rapidly than with conventional film autoradiography.

Discovery of recently adopted orphan receptors for apelin, urotensin II, and ghrelin identified using novel radioligands and functional role in the human cardiovascular system.

Using novel synthetic radioligands, we have discovered receptors for the recently paired apelin (APJ orphan receptor), ghrelin (GHS orphan receptor), and urotensin II (orphan GPR14) in the human cardiovascular system and determined their anatomical localisation. In addition, we have established functional vasoactive properties for these three peptides as potential vasoconstrictor/vasodilator mediators and provided evidence for alteration of receptor density in cardiovascular disease. We find that receptors for apelin, ghrelin, and urotensin II are widely distributed in human cardiovascular tissue, suggesting perhaps vasoactive roles for these peptides in human vascular physiology and a potential role in pathophysiology. Apelin and urotensin II are potent vasoconstrictors with low efficacy, consistent with their low receptor density. Ghrelin receptor density was increased (approximately three- to fourfold) with atherosclerosis of coronary artery disease and accelerated atherosclerosis of saphenous vein grafts, compared with normal vessels, highlighting a potentially beneficial role for this novel vasodilator peptide in human vascular disease. Our approach has demonstrated one successful strategy for translating genetic information encoding recently paired orphan receptor ligands into discovery of function. This study has the advantage of focussing on the actual disease processes, which allow the more precise identification of novel therapeutic targets.

Opioid peptide receptor studies. 14. Stereochemistry determines agonist efficacy and intrinsic efficacy in the [(35)S]GTP-gamma-S functional binding assay.

Previous data obtained with the cloned rat mu opioid receptor demonstrated that stereochemistry affects the four parameters of the ligand-receptor interaction: potency (ED(50)), efficacy (maximal stimulation), intrinsic efficacy (effect as a function of receptor occupation), and binding affinity. This study evaluated the activities of structurally diverse opioid receptor ligands in the [(35)S]GTP-gamma-S binding assay, comparing the relationship between receptor binding, activation, efficacy, and intrinsic efficacy. The data, obtained with cloned rat mu receptors, demonstrated that an analgetic, (-)-5-m-hydroxyphenyl-2-methylmorphan (NIH8508), and its (+)-isomer (NIH8509), behave as partial agonists, but had different intrinsic efficacy in the [(35)S]GTP-gamma-S binding assay. Replacement of the methyl group with the phenethyl group on the piperidine nitrogen of NIH8508 and NIH8509 [(1R,5S)-AH019 and (1S, 5R)-AH019] increased affinity for the mu receptor and eliminated any agonist effect, supporting the hypothesis that certain structural features make these compounds antagonists. These study also show that all of the fully efficacious mu agonists studied here had high levels of intrinsic efficacy, producing a 50% response at about 10% receptor occupancy. Comparison of the binding K(i) in competitively inhibiting [(125)I]IOXY binding to the functional K(i) for opioid antagonists [K(i)(IOXY)/K(i)(GTP-gamma-S)] provides more detailed evidence that the [(35)S]GTP-gamma-S binding assay can be used to reliably determine apparent functional antagonist K(i) values in addition to agonist ED(50), efficacy and intrinsic efficacy.

Activation of MT(2) melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock.

The aim of this study was to identify the melatonin receptor type(s) (MT(1) or MT(2)) mediating circadian clock resetting by melatonin in the mammalian suprachiasmatic nucleus (SCN). Quantitative receptor autoradiography with 2-[(125)I]iodomelatonin and in situ hybridization histochemistry, with either (33)P- or digoxigenin-labeled antisense MT(1) and MT(2) melatonin receptor mRNA oligonucleotide probes, revealed specific expression of both melatonin receptor types in the SCN of inbred Long-Evans rats. The melatonin receptor type mediating phase advances of the circadian rhythm of neuronal firing rate in the SCN slice was assessed using competitive melatonin receptor antagonists, the MT(1)/MT(2) nonselective luzindole and the MT(2)-selective 4-phenyl-2-propionamidotetraline (4P-PDOT). Luzindole and 4P-PDOT (1 nM-1 microM) did not affect circadian phase on their own; however, they blocked both the phase advances (approximately 4 h) in the neuronal firing rate induced by melatonin (3 pM) at temporally distinct times of day [i.e., subjective dusk, circadian time (CT) 10; and dawn, CT 23], as well as the associated increases in protein kinase C activity. We conclude that melatonin mediates phase advances of the SCN circadian clock at both dusk and dawn via activation of MT(2) melatonin receptor signaling.

Disparate spinal and supraspinal opioid antinociceptive responses in beta-endorphin-deficient mutant mice.

The role of endogenous opioid systems in the analgesic response to exogenous opiates remains controversial. We previously reported that mice lacking the peptide neurotransmitter beta-endorphin, although unable to produce opioid-mediated stress-induced antinociception, nevertheless displayed intact antinociception after systemic administration of the exogenous opiate morphine. Morphine administered by a peripheral route can activate opioid receptors in both the spinal cord and brain. However, beta-endorphin neuronal projections are confined predominantly to supraspinal nociceptive nuclei. Therefore, we questioned whether the absence of beta-endorphin would differentially affect antinociceptive responses depending on the route of opiate administration. Time- and dose-response curves were obtained in beta-endorphin-deficient and matched wild-type C57BL/6 congenic control mice using the tail-immersion/withdrawal assay. Null mutant mice were found to be more sensitive to supraspinal (i.c.v.) injection of the micro-opioid receptor-selective agonists, morphine and D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin. In contrast, the mutant mice were less sensitive to spinal (i.t.) injection of these same drugs. Quantitative receptor autoradiography revealed no differences between genotypes in the density of mu, delta, or kappa opioid receptor binding sites in either the spinal cord or pain-relevant supraspinal areas. Thus we report that the absence of a putative endogenous ligand for the mu-opioid receptor results in opposite changes in morphine sensitivity between discrete areas of the nervous system, which are not simply caused by changes in opioid receptor expression.