Radiolabelled Ligand Research Articles

(68)Ga-Pentixafor-PET/CT for Imaging of Chemokine Receptor 4 Expression in Glioblastoma.

Chemokine receptor-4 (CXCR4) has been reported to be overexpressed in glioblastoma (GBM) and to be associated with poor survival. This study investigated the feasibility of non-invasive CXCR4-directed imaging with positron emission tomography/computed tomography (PET/CT) using the radiolabelled chemokine receptor ligand 68Ga-Pentixafor.15 patients with clinical suspicion on primary or recurrent glioblastoma (13 primary, 2 recurrent tumors) underwent 68Ga-Pentixafor-PET/CT for assessment of CXCR4 expression prior to surgery. O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) PET/CT images were available in 11/15 cases and were compared visually and semi-quantitatively (SUVmax, SUVmean). Tumor-to-background ratios (TBR) were calculated for both PET probes. 68Ga-Pentixafor-PET/CT results were also compared to histological CXCR4 expression on neuronavigated surgical samples.68Ga-Pentixafor-PET/CT was visually positive in 13/15 cases with SUVmean and SUVmax of 3.0±1.5 and 3.9±2.0 respectively. Respective values for 18F-FET were 4.4±2.0 (SUVmean) and 5.3±2.3 (SUVmax). TBR for SUVmean and SUVmax were higher for 68Ga-Pentixafor than for 18F-FET (SUVmean 154.0±90.7 vs. 4.1±1.3; SUVmax 70.3±44.0 and 3.8±1.2, p<0.01), respectively. Histological analysis confirmed CXCR4 expression in tumor areas with high 68Ga-Pentixafor uptake; regions of the same tumor without apparent 68Ga-Pentixafor uptake showed no or low receptor expression.In this pilot study, 68Ga-Pentixafor retention has been observed in the vast majority of glioblastoma lesions and served as readout for non-invasive determination of CXCR4 expression. Given the paramount importance of the CXCR4/SDF-1 axis in tumor biology, 68Ga-Pentixafor-PET/CT might prove a useful tool for sensitive, non-invasive in-vivo quantification of CXCR4 as well as selection of patients who might benefit from CXCR4-directed therapy.

Cell‐free synthesis of isotopically labelled peptide ligands for the functional characterization of G protein‐coupled receptors

Cell‐free systems exploit the transcription and translation machinery of cells from different origins to produce proteins in a defined chemical environment. Due to its open nature, cell‐free protein production is a versatile tool to introduce specific labels such as heavy isotopes, non‐natural amino acids and tags into the protein while avoiding cell toxicity. In particular, radiolabelled peptides and proteins are valuable tools for the functional characterization of protein–protein interactions and for studying binding kinetics. In this study we evaluated cell‐free protein production for the generation of radiolabelled ligands for G protein‐coupled receptors (GPCRs). These receptors are seven‐transmembrane‐domain receptors activated by a plethora of extracellular stimuli including peptide ligands. Many GPCR peptide ligands contain disulphide bonds and are thus inherently difficult to produce in bacterial expression hosts or in Escherichia coli‐based cell‐free systems. Here, we established an adapted E. coli‐based cell‐free translation system for the production of disulphide bond‐containing GPCR peptide ligands and specifically introduce tritium labels for detection. The bacterial oxidoreductase DsbA is used as a chaperone to favour the formation of disulphide bonds and to enhance the yield of correctly folded proteins and peptides. We demonstrate the correct folding and formation of disulphide bonds and show high‐affinity ligand binding of the produced radio peptide ligands to the respective receptors. Thus, our system allows the fast, cost‐effective and reliable synthesis of custom GPCR peptide ligands for functional and structural studies.

Synthesis and biological evaluation of 99mTc tricarbonyl complex of O,O′‐diethylethylenediamine‐N,N′‐di‐3‐propanoate as potential tumour diagnostic agent

The extensive development of radiopharmaceuticals towards early tumour detection and treatment has increased the demand for new ligands with higher tumour selectivity. Research has been done on the potential of the novel O,O′‐diethylethylenediamine‐N,N′‐di‐3‐propanoate (L) ligand as a radionuclide vehicle for tumour targeting. Under alkaline conditions, L hydrolyses and produces half ester ligand (L') and diacid ligand (L''), with characteristic donor atom array N,N,O. Ligand L was successfully labelled with 99mTc at pH = 9 by coordination with the octahedral fac‐[99mTc(CO)3(H2O)3]+ intermediate, forming the main radioproduct fac‐[99mTcL′(CO)3] (Tc1). The 99mTc complex showed a low lipophilic character (log P = 0.48) and low binding affinity to human serum albumin (2.51 ± 0.48%). In vitro stability studies in saline and human plasma, as well as challenge studies with cysteine and histidine, revealed high stability of the complex during 24 h. Biodistribution studies of Tc1 in female C57BL/6 mice bearing B16/F1 melanoma metastases showed significant tumour uptake: 9.81 ± 1.19%ID g−1 in the liver, 5.87 ± 0.54%ID g−1 in the lungs and 3.17 ± 0.33%ID g−1 in the ovary at 30 min post‐injection. Favourable physicochemical properties, satisfactory in vitro/in vivo stability and biodistribution profile in the experimental metastatic melanoma model indicate the possible application of the radiolabelled ligand in tumour diagnosis. Copyright © 2015 John Wiley &amp; Sons, Ltd.

PET/CT imaging in prostate cancer

Prostate cancer is the second most common male malignancy worldwide with a wide spectrum of biological behaviour ranging from fairly indolent disease to highly aggressive metastatic castrate resistant tumours. In the past decade there have been advances in therapies for treating advanced and recurrent prostate cancer and improved diagnostic/prognostic tools are required to refine the therapeutic approach at various stages in the management of prostate cancer patients. For tumour staging/lesion localisation multiparametric prostate MRI is the modality of choice. Several PET tracers are proving more sensitive than conventional imaging techniques for early detection of lymph node and bone metastases. However a general limitation of PET is the spatial resolution which limits the reliability for detection of small lesions. The use of F-18 fluoro-deoxyglucose (FDG) PET/CT in prostate cancer has been disappointing largely due to the fact that many prostate cancers have only low level glucose metabolism. C-11/F-18 choline, markers of cell membrane metabolism, are now widely used in Europe and in certain UK centres particularly in the scenario of biochemical relapse and high risk staging with equivocal findings on conventional work-up [1-3]. However, in cases of biochemical relapse with low PSA rise and low PSA velocity, choline PET has low detection rates [4]. Prostate specific membrane antigen (PSMA), a cell surface membrane glycoprotein, is a promising target for the diagnosis and treatment of prostate cancer. Recently Ga-68 radiolabelled ligand targeted to PSMA has been introduced to a few European centres. Early data suggests this may be more sensitive than choline PET/CT in patients with biochemical failure and low PSA [5-7]. In addition it offers the opportunity for Lu-177 or Y-90 targeted radionuclide therapy. F-18 sodium fluoride (NaF) is highly sensitive compared to bone scintigraphy with Tc99m labelled phosphates but only assess skeletal disease [8]. Other PET tracers being explored include C-11 acetate targeting lipogenesis, tracers radiolabelling bombesin and targeting gastrin-releasing peptide receptor (GRPR), amino acid transport with anti-1-amino-3-F18-fluorocyclobutane-1-carboxylic acid (FACBC ) and F-18-fluoro-5α-dihydrotestosterone (FDHT) targeting the androgen receptor [9,10]. This talk reviews the diagnostic utility of PET in prostate cancer.

Molecular imaging of targeted therapies with positron emission tomography: the visualization of personalized cancer care.

Molecular imaging has been defined as the visualization, characterization and measurement of biological processes at the molecular and cellular level in humans and other living systems. In oncology it enables to visualize (part of) the functional behaviour of tumour cells, in contrast to anatomical imaging that focuses on the size and location of malignant lesions. Available molecular imaging techniques include single photon emission computed tomography (SPECT), positron emission tomography (PET) and optical imaging. In PET, a radiotracer consisting of a positron emitting radionuclide attached to the biologically active molecule of interest is administrated to the patient. Several approaches have been undertaken to use PET for the improvement of personalized cancer care. For example, a variety of radiolabelled ligands have been investigated for intratumoural target identification and radiolabelled drugs have been developed for direct visualization of the biodistibution in vivo, including intratumoural therapy uptake. First indications of the clinical value of PET for target identification and response prediction in oncology have been reported. This new imaging approach is rapidly developing, but uniformity of scanning processes, standardized methods for outcome evaluation and implementation in daily clinical practice are still in progress. In this review we discuss the available literature on molecular imaging with PET for personalized targeted treatment strategies. Molecular imaging with radiolabelled targeted anticancer drugs has great potential for the improvement of personalized cancer care. The non-invasive quantification of drug accumulation in tumours and normal tissues provides understanding of the biodistribution in relation to therapeutic and toxic effects.

Synthesis and in vitro evaluation of tetrahydroisoquinolines with pendent aromatics as sigma-2 (σ2) selective ligands

5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxybenzamide 1 is a potent and selective σ2 receptor ligand suitable for further development. A series of new analogues, incorporating a variety of isoquinoline and carboxylic acid moieties, linked together with either a linear or cyclic amine spacer have been synthesised and assessed for their σ1/σ2 binding affinity and selectivity. Compounds with a rigid piperidine spacer gave Ki values for the σ2 receptor between 8.7-845 nM. Changing the configuration of the methoxy groups on the isoquinoline moiety resulted in molecules with σ2Ki values of 4.4-133 nM whereas varying the length and flexibility of the carbon spaces gave σ2Ki values 0.88-15.0 nM, some of the most active, selective σ2 ligands to date. Thus, the flexibility and length of the carbon linker and the carboxylic acid moiety are confirmed to be key to the exceptional binding affinity and selectivity for this active series. Additionally, the incorporation of a halogen on selected carboxylic acid moieties provided a convenient strategy for the introduction of a radiohalogen for applications in pharmacological and imaging studies.

Evaluation of a radiolabelled butyrylcholinesterase ligand, N-methyl-4-piperdinyl-4-[18F]-fluorobenzoate, in human brain tissues

Evaluation of a radiolabelled butyrylcholinesterase ligand, N-methyl-4-piperdinyl-4-[18F]-fluorobenzoate, in human brain tissues

Evaluation of a radiolabelled butyrylcholinesterase ligand, N-methyl-4-piperdinyl-4-[18F]-fluorobenzoate, in human brain tissues

Evaluation of a radiolabelled butyrylcholinesterase ligand, N-methyl-4-piperdinyl-4-[18F]-fluorobenzoate, in human brain tissues

Honokiol blocks store operated calcium entry in CHO cells expressing the M3 muscarinic receptor: honokiol and muscarinic signaling

BackgroundHonokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay.ResultsHonokiol had a potent (EC50 ≈ 5 μmol/l) inhibitory effect on store operated calcium entry (SOCE) that was induced by activation of the M3 receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated calcium release, depletion of ER calcium stores, or disruption of M3 receptor binding.ConclusionsIt is likely that an inhibition of SOCE contributes to honokiol disruption of parasympathetic motor functions, as well as many of its beneficial pharmacological properties.

Molecular requirements for inhibition of the chemokine receptor CCR8 – probe‐dependent allosteric interactions

Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes. The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gα(i) signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148. All compounds were highly potent inverse agonists with EC(50) values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC(50) values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [(125)I]-CCL1 (K(i) 3.4-842 nM), whereas the affinities measured against [(125)I]-MC148 were less widely spread (K(i) 0.37-27 nM), and matched the inverse agonist potencies. Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties.

Abstract 3019: TGFβ3 is a less potent inducer of TGFβ signaling than TGFβ1 in non-small cell lung cancer cells

Abstract Transforming growth factor beta (TGFβ) is an essential cytokine for tissue homeostasis. Its signalling pathway is under intense study as it is commonly dysregulated in many cancers, including pancreatic cancer, non-small cell lung cancer, and colorectal cancer. The TGFβ signalling cascade is propagated by the binding of ligand to cell-surface serine-threonine kinase receptors, which leads to the phosphorylation of receptor regulated Smad (R-Smad) proteins. There are three main ligands capable of activating the classical TGFβ pathway: TGFβ1, TGFβ2 and TGFβ3. These ligands show distinct spatial and temporal expression patterns and have non-overlapping functions in vivo. Extensive studies have been conducted on the role of TGFβ1 in the tumour microenvironment, and the role of TGFβ3 has largely been thought to be the same as TGFβ1 though there are few studies to support this claim. Interestingly in the wound microenvironment, TGFβ1 and TGFβ3 have vastly different signalling outcomes: TGFβ1 promotes the formation of a scar while TGFβ3 induces scar-free wound resolution. The objective of the present study was to evaluate the signalling capacity of TGFβ1 and TGFβ3 in non-small cell lung cancer cells. Our studies show that TGFβ3 is less potent at initiating Smad2 phosphorylation than TGFβ1, resulting in reduced transcriptional activity, as assessed by microarray analysis. We also observed that TGFβ3 is less effective than TGFβ1 at altering cell-cell adhesions. In order to assess the differences in signalling potential, we investigated TGFβ receptor engagement at the cell surface using radiolabelled TGFβ ligands. We observed that the ratio of activated receptors in signalling complexes is altered in the presence of TGFβ3, which may lead to a decreased signalling capacity. Future studies will evaluate the capacity of TGFβ3 to induce cancer cell migration and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3019. doi:1538-7445.AM2012-3019

Growth hormone secretagogues: out of competition

Growth hormone secretagogues (GHS) constitute a new GH deficiency treatment increasing exponentially in number and improved potency and bioavailability over the last decade. The growth hormone releasing activity makes these compounds attractive for the artificial improvement of the human sports skills, now that recombinant human growth hormone (rhGH) administration is effectively detected. The GHS family is extremely diverse both in number and chemical heterogeneity and keeps growing continuously. In this paper, a general screening test is proposed. To develop a universal method, the single common property of growth hormone secretagogues has been targeted: their capacity to bind to the GHS receptor 1a (GHS-R1a). Pretreated urine samples have been tested in a competition assay where eventually the GHS presence detached a radiolabelled ligand from the receptor in a dose-dependent manner. Blank urine samples were processed to determine potential age, gender and exercise effects, and to define a threshold beyond which a specimen is considered positive. Samples from a growth hormone releasing peptide 2 (GHRP-2) excretion study corroborated the screening assay applicability with a detection window of approximately 4.5 h, and results were confirmed by comparison with a dedicated LC-MS quantification of the intact compound.

Synthesis, labelling and evaluation of hydantoin‐substituted indole carboxylic acids as potential ligands for positron emission tomography imaging of the glycine binding site of the N‐methyl‐d‐aspartate receptor

The N‐methyl‐ d‐aspartate (NMDA) receptor as a type of ionotropic glutamatergic receptors is essential for physiological processes such as learning, memory and synaptic plasticity. A glutamate‐induced overactivation of these receptors, accompanied by increased intracellular calcium concentration, causes cell injury and leads to a large number of acute or chronic neurological disorders, such as stroke, trauma, Parkinson's disease and Alzheimer's disease. In an attempt to visualise the glutamatergic neurotransmission in vivo with positron emission tomography, novel fluoroethoxy‐ and methoxy‐substituted reference compounds based on the lead structure of a hydantoin‐substituted indole‐2‐carboxylic acid were synthesised. The affinities towards the glycine binding site of the NMDA receptor showed Ki values between 322 and 11 nM and the lipophilicities ranged from logD values of 1.51 to 2.53. On the basis of these results, precursor compounds were synthesised containing a phenolic hydroxy moiety to obtain the radiolabelled ligands through an alkylation reaction. Radiosynthesis was achieved by labelling the precursor ethyl 4,6‐dichloro‐3‐((3‐(4‐hydroxyphenyl)‐2,4‐dioxoimidazolidin‐1‐yl)methyl)‐indole‐2‐carboxylate with 2‐[18F]fluoroethyl tosylate or [11C]methyl iodide and subsequent cleavage of the ethyl ester moiety. This gave the final products in overall decay‐corrected radiochemical yields of 5–7% and 6–9% and specific activities of 24–67 GBq/µmol and 8–26 GBq/µmol, respectively.

Neurotensin Receptor 1 Is Expressed in Gastrointestinal Stromal Tumors but Not in Interstitial Cells of Cajal

Gastrointestinal stromal tumors (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the KIT or PDGFRA receptor tyrosine kinases are present in the majority of GIST, leading to ligand-independent activation of the intracellular signal transduction pathways. We previously investigated the gene expression profile in the murine Kit K641E GIST model and identified Ntsr1 mRNA, encoding the Neurotensin receptor 1, amongst the upregulated genes. Here we characterized Ntsr1 mRNA and protein expression in the murine Kit K641E GIST model and in tissue microarrays of human GIST. Ntsr1 mRNA upregulation in Kit K641E animals was confirmed by quantitative PCR. Ntsr1 immunoreactivity was not detected in the Kit positive ICC of WT mice, but was present in the Kit positive hyperplasia of Kit K641E mice. In the normal human gut, NTSR1 immunoreactivity was detected in myenteric neurons but not in KIT positive ICC. Two independent tissue microarrays, including a total of 97 GIST, revealed NTSR1 immunoreactivity in all specimens, including the KIT negative GIST with PDGFRA mutation. NTSR1 immunoreactivity exhibited nuclear, cytoplasmic or mixed patterns, which might relate to variable levels of NTSR1 activation. As studies using radio-labeled NTSR1 ligand analogues for whole body tumor imaging and for targeted therapeutic interventions have already been reported, this study opens new perspectives for similar approaches in GIST.

Positron emission tomography imaging in multiple sclerosis-current status and future applications.

Multiple Sclerosis (MS) is traditionally considered as a central nervous system (CNS) white matter inflammatory disease. However, recent studies have focused on the neurodegenerative aspects of the disease, which occur early in the pathological process, providing an opportunity for therapeutic intervention and application of neuroprotective strategies. The relationship between neural inflammation and cell death remains controversial. The recent development of new radiolabelled ligands provides positron emission tomography (PET) imaging with a role for studying early aspects of the MS pathology. We provide an overview of current PET research in MS, particularly focussing on possible applications of new radioligands for studying inflammation and neurodegenerative processes. Pathological aspects of neuroinflammation, axonal degeneration and neuronal repair may be explored in vivo with selective PET tracers. Specific radioligands for the cannabinoid system may be applied in MS research to understand the role of this neurotransmitter system in the pathogenesis of the disease. PET imaging represents a promising tool for elucidating controversial aspects of MS pathology and for the assessment of selective and potentially neuroprotective therapies.

Current In Vitro High Throughput Screening Approaches to Assess Nuclear Receptor Activation

The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluorescently-labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient- and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening.

Gi protein coupling to adenosine A1–A2A receptor heteromers in human brain caudate nucleus

Pharmacological characterization of adenosine A(1) and A(2A) receptors in human brain caudate nucleus membranes led to non-cooperative binding of radiolabelled ligands. In human caudate nucleus but not in cortex, the agonist binding to A(1) receptors was modulated by the agonist binding to A(2A) receptors indicating a functional negative cross-talk. Accordingly, the A(1) receptor-activation-mediated G(i)-dependent guanosine 5'-o-(3-[(35)S]thio-triphosphate) binding was modulated by agonist binding to A(2A) receptors. A(2A) receptors occupation led to a decrease in the potency of A(1) receptor agonists. These results indicate that A(1) but not A(2A) receptors activation, likely occurring at low adenosine concentrations, engages a G(i)-mediated signaling; however, when both receptors are occupied by adenosine, there is an A(2A) receptor-mediated impairment of G(i)-operated transducing units. These findings are relevant to get insight into the complex relationships derived from co-expression of multiple neurotransmitter/neuromodulator receptors subtypes that individually are coupled to different G proteins. A further finding was the demonstration that the A(2A) receptor agonist, CGS 21680, at high concentrations able to significantly bind to the A(1) receptor, behaved as a partial agonist of the later receptor. This fact might be taken into account when characterizing CGS 21680 actions in human cells expressing A(1) receptors when the compound is used at micromolar concentrations.

Muscarinic cholinoceptor agonists and antagonists are modulators of the activity of α1-adrenoceptors in the membranes of rat cerebral cortex

The effects of activation and inhibition of muscarinic cholinoceptors by carbachol and atropine on the binding of specific nonselective α1-antagonist [3H]prazosine in synaptosomal membranes of rat cerebral cortex have been studied. It has been shown that the ligand-receptor interaction of α1-adrenoceptors corresponds to the model suggesting the presence of a single receptor pool and the binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were as follows: Kd = 1.56 ± 0.17 nM, Bmax = 30.25 ± 1.78 fmol/mg protein, n = 2. Upon inhibition of muscarinic cholinoceptors by atropine or their activation by carbachol, the radiolabelled ligand is bound to α1-adrenoceptors according to the same model but at n = 1. In the presence of atropine, the sensitivity of α1-adrenoceptors to [3H]prazosine decreases more than twofold (Kd = 3.52 ± 0.36 nM) and the concentration of the active receptors is 36% lower (Bmax = 19.45 ± 1.46 fmol/mg protein). Carbachol does not reduce the affinity of adrenoceptors to the ligand, while the concentration of active receptors decreases like in the case of atropine. It is supposed that α1-adrenoceptors in the membranes of rat cerebral cortex exist as dimers. The modulating effects of atropine and carbachol on the binding of specific antagonist by α1-adrenoceptors are exhibited as changes in the general character of binding (monomerization of α1-adrenoceptors) and as inhibitory effect on the [3H]prazosine binding parameters.

Endothelial cell-specific ETB receptor knockout: autoradiographic and histological characterisation and crucial role in the clearance of endothelin-1This article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

Inactivation of endothelin B receptors (ETB), either through selective pharmacological antagonism or genetic mutation, increases the circulating concentration of endothelin-1 (ET-1), suggesting ETB plays an important role in clearance of this peptide. However, the cellular site of ETB-mediated clearance has not yet been determined. We have used a novel mouse model of endothelial cell-specific knockout (KO) of ETB (EC ETB(-/-)) to evaluate the relative contribution of EC-ETB to the clearance of ET-1. Phenotypic evidence of EC-specific ETB KO was confirmed by immunocytochemistry and autoradiography. Binding of the radiolabelled selective ETB ligand BQ3020 was significantly and selectively decreased in EC-rich tissues of EC ETB(-/-) mice, including the lung, liver, and kidney. By contrast, ETA binding was unaltered. RT-PCR confirmed equal expression of ET-1 in tissue from EC ETB(-/-) mice and controls, despite increased concentration of plasma ET-1 in EC ETB(-/-). Clearance of an intravenous bolus of [(125)I]ET-1 was impaired in EC ETB(-/-) mice. Pretreatment with the selective ETB antagonist A192621 impaired [(125)I]ET-1 clearance in control animals to a similar extent, but did not further impair clearance in EC ETB(-/-) mice. These studies suggest that EC-ETB are largely responsible for the clearance of ET-1 from the circulation.

The spatial resolution of silicon-based electron detectors in β-autoradiography

Thin tissue autoradiography is an imaging modality where ex-vivo tissue sections are placed in direct contact with autoradiographic film. These tissue sections contain a radiolabelled ligand bound to a specific biomolecule under study. This radioligand emits β − or β+ particles ionizing silver halide crystals in the film. High spatial resolution autoradiograms are obtained using low energy radioisotopes, such as 3H where an intrinsic 0.1–1 µm spatial resolution can be achieved. Several digital alternatives have been presented over the past few years to replace conventional film but their spatial resolution has yet to equal film, although silicon-based imaging technologies have demonstrated higher sensitivity compared to conventional film. It will be shown in this work how pixel size is a critical parameter for achieving high spatial resolution for low energy uncollimated beta imaging. In this work we also examine the confounding factors impeding silicon-based technologies with respect to spatial resolution. The study considers charge diffusion in silicon and detector noise, and this is applied to a range of radioisotopes typically used in autoradiography. Finally an optimal detector geometry to obtain the best possible spatial resolution for a specific technology and a specific radioisotope is suggested.