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R Leurs

Publications and source records attributed to R Leurs.

At least 55 records · Page 3Linked to original sources
PubMed1999

Evaluation of [18F]VUF 5000 as a potential PET ligand for brain imaging of the histamine H3 receptor.

[18F]VUF 5000 was evaluated as a potential PET ligand for the histamine H3 receptor. In the rat a high uptake of [18F]VUF 5000 was observed in liver, lung and kidney and a low uptake in the brain. In order to explain these findings we determined the LogD(oct,7.2) of [18F]VUF 5000, studied the biodistribution in the presence of carrier VUF 5000, modified [18F]VUF 5000 chemically and studied the binding of [18F]VUF 5000 to human serum albumin. From the results of these experiments it was concluded that [18F]VUF 5000 is not suitable as a PET ligand for brain imaging of the histamine H3 receptor, since [18F]VUF 5000 hardly penetrates into the brain.

Animals↗
PubMed1999

Radiosynthesis and biodistribution of 123I-labeled antagonists of the histamine H3 receptor as potential SPECT ligands.

We have synthesized three 123I-labeled histamine H3 receptor ligands, i.e., [123I]GR 190028, [123I]FUB 271, and [123I]iodoproxyfan, in moderate to good radiochemical yields via a Cu+-assisted I-for-123I exchange method. Biodistribution in the rat of these compounds revealed high hepatic and pulmonary uptake. Brain uptake was moderate, but for [123I]iodoproxyfan, brain uptake was high enough for a pilot single photon emission computed tomography (SPECT) study in the rabbit. However, for this compound, the cerebral uptake could not be blocked by a pretreatment with [R]-alpha-methylhistamine, a selective, high-affinity histamine H3 receptor agonist, both in the SPECT study in the rabbit and in the biodistribution study in the rat. Apparently, [123I]iodoproxyfan is binding to a non-H3 receptor binding site. None of the three investigated compounds is suitable for use as a SPECT ligand for the H3 receptor in the brain.

Animals↗
PubMed1999

Human IP-9: A keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3).

Chemokines and their receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-gamma stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 receptor, designated interferon-gamma-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-gamma-inducible protein-9, revealed that interferon-gamma-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-gamma-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as beta-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-gamma-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.

Amino Acid Sequence↗
PubMed1999

Pivotal role for the cytoplasmic carboxyl-terminal tail of a nonmammalian gonadotropin-releasing hormone receptor in cell surface expression, ligand binding, and receptor phosphorylation and internalization.

The gonadotropin-releasing hormone receptor (GnRH-R) of the African catfish couples to phospholipase C and belongs to the large family of G protein-coupled receptors. We recently demonstrated that removal of the carboxyl-terminal tail (S331-Q379) from the catfish GnRH-R results in a loss of agonist binding; the current study sought to define more precisely the role of this region in receptor function. Progressive truncations of the carboxyl-terminal tail decreased cell surface expression detected by either enzyme-linked immunosorbent assay or agonist-binding. The two most truncated receptors (stop331 and stop337) showed no binding but were detected at the cell surface by enzyme-linked immunosorbent assay. All receptors able to bind agonist were also able to activate phospholipase C. The catfish GnRH-R was phosphorylated after agonist-occupation and use of truncated mutants showed this phosphorylation to be within the carboxyl-terminal tail. Furthermore, studies with S356A, S363A and SS356,363AA mutant receptors demonstrated that Ser363 is a major site of agonist-induced phosphorylation. The absence of this phospho-acceptor site markedly impaired agonist-mediated receptor internalization. In addition, both, Ser363 and the last 12 residues of the tail (not containing Ser363) were shown to be important for beta-arrestin-dependent internalization. These observations are relevant to the regulatory function of the carboxyl-terminal tail of G protein-coupled receptors in general and are particularly intriguing given the absence of this region in mammalian GnRH-Rs.

Animals↗
PubMed1999

Platelet adenylyl cyclase activity as a biochemical trait marker for predisposition to alcoholism.

Previous studies demonstrated a reduced Gs-protein stimulated adenylyl cyclase activity in the brain and blood cells of alcoholics. We investigated this phenomenon in platelets of children of alcoholics (COA), i.e., of children at high risk for the acquisition of alcoholism and (as yet) not regularly consuming alcohol. Gs-protein mediated stimulation of adenylyl cyclase by 30 mM NaF and 50 microM forskolin stimulated adenylyl cyclase activity were assessed in platelet membranes of 23 (male and female) COA and 20 control children. Gs-protein stimulated cAMP production by NaF, unlike that induced by direct stimulation of adenylyl cyclase with forskolin, in platelet membranes of COA was profoundly lower than in platelet membranes of control children. Moreover, such a reduced Gs-protein functioning was only observed in platelet membranes of COA with a multigenerational family history of alcoholism. A reduction of Gs-protein stimulated adenylyl cyclase activity in platelets may represent a sensitive and gender-independent trait marker for predisposition to alcoholism, rather than a state marker for alcoholism.

Adenylyl Cyclases↗
PubMed1999

Molecular properties and signalling pathways of the histamine H1 receptor.

With cloning of the gene encoding the histamine H1 receptor, a new area of histamine research has become reality. Finally, it seems feasible to study the target of the therapeutically important clans of antihistamine. Expression of the genes in mammalian cells allows detailed investigations of the various signal transduction routes of the histamine H1 receptor. Moreover, using molecular biological techniques, it is now possible to investigate ligand receptor interaction at the molecular level. Studies with mutant H1 receptors have shown that H1 antagonists bind to a specific amino acid residues in TM3 and 5. It is expected that these new developments will provide much fundamental knowledge on the ligand interaction with the H1 receptor.

Amino Acid Sequence↗
PubMed1998

Absence of antinociceptive tolerance to improgan, a cimetidine analog, in rats.

Improgan, an analog of the histamine receptor antagonist cimetidine, produces highly effective analgesia following intraventricular injection. The present study examined changes in the antinociceptive effects of improgan following once daily intraventricular injections. Improgan (100-150 microg) produced near maximal antinociception 10 and 30 min after daily administration on all 4 test days, whereas comparable morphine treatments (50 microg) induced considerable tolerance. Thus, improgan produced highly effective analgesia without the development of tolerance.

Analgesics, Non-Narcotic↗
PubMed1998

Histamine inhibits the production of interleukin-12 through interaction with H2 receptors.

IL-12 is essential for T helper 1 (Th1) development and inhibits the induction of Th2 responses. Atopic diseases, which are characterized by Th2 responses, are associated with the overproduction of histamine. Here we present evidence that histamine, at physiological concentrations, strongly inhibits human IL-12 p40 and p70 mRNA and protein production by human monocytes. The use of specific histamine receptor antagonists reveals that this inhibition is mediated via the H2 receptor and induction of intracellular cAMP. The inhibition of IL-12 production is independent of IL-10 and IFN-gamma. The observation that histamine strongly reduces the production of the Th1-inducing cytokine IL-12 implies a positive feedback mechanism for the development of Th2 responses in atopic patients.

Dinoprostone↗
PubMed1998

Pharmacological characterisation of the histamine H3 receptor in the rat hippocampus.

The purpose of this report was to pharmacologically characterise the histamine H3 in the rat hippocampus using radioligand binding studies with the H3 receptor antagonist [125I]iodophenpropit and the H3 receptor mediated inhibition of [3H]noradrenaline release. A dissociation constant of 0.33 nM and a maximal number of binding sites of 125 fmol/mg protein were found for [125I]iodophenpropit. Competition studies showed stereoselectivity for the (R) and (S) enantiomers of alpha-methylhistamine and 10 microM of GTPgammaS shifted the curve of (R)-alpha-methylhistamine rightwards. Up to 1 microM, (R)-alpha-methylhistamine displaced only 30% whereas the tested H3-antagonists displaced 50-60% of the total [125I]iodophenpropit bound. This indicates the presence of an additional non-H3 receptor binding site(s) for [125I]iodophenpropit in the rat hippocampus. This secondary site shows low affinity for H3 agonists, but high affinity for the tested H3 antagonists. Electrically evoked [3H]acetylcholine release was shown in slices of rat hippocampus. No H3 receptor modulation of [3H]acetylcholine release from hippocampal slices was detectable. However, H3 receptor activation inhibited 42% of the electrically-evoked [3H]noradrenaline release in rat hippocampal slices. The inhibition of [3H]noradrenaline release was effectively antagonized by the H3 antagonists thioperamide and burimamide. We describe the pharmacological identification of the histamine H3 receptor in the rat hippocampus and its similarities and differences from the cortical H3 receptor. These studies enable us to investigate changes in density and functionality of the hippocampal H3 receptor under (patho)physiological conditions.

Acetylcholine↗
PubMed1998

Therapeutic potential of histamine H3 receptor agonists and antagonists.

The histamine H3 receptor was discovered 15 years ago, and many potent and selective H3 receptor agonists and antagonists have since been developed. Currently, much attention is being focused on the therapeutic potential of H3 receptor ligands. In this review, Rob Leurs, Patrizio Blandina, Clark Tedford and Henk Timmerman describe the available H3 receptor agonists and antagonists and their effects in a variety of pharmacological models in vitro and in vivo. The possible therapeutic applications of the various compounds are discussed.

Animals↗
PubMed1998

Agonist-independent regulation of constitutively active G-protein-coupled receptors.

G-protein-coupled receptors constitute one of the largest protein super-families in mammals. Since the cloning of the encoding genes, these important drug targets have been subjected to thorough biochemical and pharmacological studies. It has become clear that G-protein-coupled receptors not only transmit signals after stimulation by agonists but can also spontaneously couple to signal-transduction pathways. Recent findings show that constitutively active G-protein-coupled receptors can also be regulated in an agonist-independent manner, which has important implications for the interpretation of the actions of (inverse) agonists and the results of site-directed-mutagenesis studies.

Animals↗
PubMed1998

Molecular cloning and characterization of an invertebrate homologue of a neuropeptide Y receptor.

Neuropeptide Y is an abundant and physiologically important peptide in vertebrates having effects on food intake, sexual behaviour, blood pressure and circadian rhythms. Neuropeptide Y homologues have been found in invertebrates, where they are very likely to play similar, important roles. Although five neuropeptide Y-receptor subtypes have been identified in mammals, none has been reported from invertebrates. Here we describe the cloning of a neuropeptide Y-receptor from the brain of the snail Lymnaea stagnalis. The identity of the receptor was deduced by expressing the neuropeptide Y-receptor-encoding cDNA in Chinese Hamster Ovary cells, which were subsequently challenged with size-fractionated Lymnaea brain extracts. An active peptide, selected on the basis of its ability to induce changes in cAMP levels, was purified to homogeneity, analysed by mass spectrometry and amino acid sequence determination, and turned out to be a Lymnaea homologue of neuropeptide Y.

Amino Acid Sequence↗
PubMed1998

Constitutive activity and structural instability of the wild-type human H2 receptor.

Stable expression of the human H2 receptor in Chinese hamster ovary cells resulted in an increase in basal cyclic AMP (cAMP) production, which was inhibited by the inverse agonists cimetidine, famotidine, and ranitidine with potencies similar to those found for the rat H2 receptor. Burimamide, a neutral antagonist at the rat H2 receptor, behaved as a weak partial agonist at the human H2 receptor. Burimamide competitively antagonized both the histamine-induced increase in cAMP and the cimetidine-induced reduction of the basal cAMP level with apparent K(B) values that were similar to its H2 receptor affinity. Investigation of the modulation of receptor expression after long-term drug treatment revealed that at low concentrations histamine induced a significant reduction in H2 receptor expression, whereas at high concentrations receptor expression was slightly increased. The partial agonist burimamide induced, like inverse agonists, an up-regulation of the human H2 receptor after prolonged treatment. These findings suggest a structural instability of the constitutively active human H2 receptor in transfected Chinese hamster ovary cells. Occupation of the H2 receptor by any ligand reduces the instability, thus resulting in higher cellular expression levels.

Animals↗
PubMed1997

Modulation of forskolin-mediated adenylyl cyclase activation by constitutively active G(S)-coupled receptors.

In transfected CHO cells constitutively active histamine H2 receptors not only increase the basal cAMP level, but also enhance forskolin-induced cAMP production. The increased forskolin response was inhibited by inverse H2 agonists with potencies similar to those determined at basal levels. The modulation of the forskolin response was also observed after H2 receptor expression in HEK-293 and Sf9 cells or TSH receptor expression in COS-7 cells. The enhancement of forskolin-induced cAMP production seems to be a general characteristic of constitutively active G(S)-coupled receptors and can be very useful to study inverse agonism at wild-type receptors.

Adenylyl Cyclases↗
PubMed1997

Differences in structure-function relations between nonmammalian and mammalian gonadotropin-releasing hormone receptors.

Mammalian gonadotropin-releasing hormone receptors (GnRH-Rs) differ from other G protein-coupled receptors in lacking the intracellular C-terminus and in showing an exchange of two otherwise highly conserved Asp (D) and Asn (N) residues in transmembrane domains (TMD) 2 and 7, respectively. However, the first GnRH-R characterized from a nonmammalian vertebrate, the African catfish, does contain an intracellular C-terminus and has D residues in TMD 2 and 7. The functional relevance of these structural features was analysed with D90N321, N90D321, N90N321 and C-terminally truncated mutant catfish GnRH-Rs. An antiserum raised against the recombinant extracellular domain of the wild-type catfish GnRH-R detected all mutant receptors at the cell surface of transiently transfected 293T cells. However, only the D90N321 mutant specifically bound GnRHs and activated signal transduction in response to GnRHs; all other mutants were inactive in both respects. We conclude that the catfish GnRH-R differs from the mammalian GnRH-Rs in that both the C-terminal domain and D90 in TMD 2 are important for receptor functioning.

Amino Acid Sequence↗
PubMed1997

Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor.

A cDNA encoding a putative gonadoliberin receptor was cloned from the pituitary of the African catfish. Conceptual translation predicts a protein of 379 amino acids which shows typical characteristics of GTP-binding-protein-coupled receptors. The isolated cDNA was stable expressed in human embryonic kidney (HEK) 293 cells which were used for studies on gonadoliberin-activated second messenger systems (inositol phosphate production; increase in cAMP and/or intracellular Ca2+). The isolated cDNA encoded a functional receptor, designated catfish gonadoliberin receptor (cfGnRH-R), which had an amino acid sequence similarity of 38% with mammalian gonadoliberin receptors. In contrast to its mammalian counterparts which lack an intracellular carboxy-terminal domain, the cfGnRH-R contains an additional 49 amino acid residues. From the two endogenous gonadoliberins in African catfish, chicken gonadoliberin-II had a several hundredfold higher potency than catfish gonadoliberin to activate cfGnRH-R-associated second messenger systems in transfected HEK 293 cells. This is in line with the previously determined higher gonadotropin-release capacity of chicken gonadoliberin-II in catfish. Stimulation of second messenger systems with chicken gonadoliberin-II, but not with catfish gonadoliberin, resulted in a biphasic effect and chicken gonadoliberin-II led to a higher maximum stimulation than catfish gonadoliberin. Challenging cfGnRH-R simultaneously with chicken gonadoliberin-II and catfish gonadoliberin did not lead to additive effects. In contrast, two types of mutual inhibitory effects were recorded. These data indicate that a single cognate cfGnRH-R couples with distinct efficacies to signal transduction systems upon stimulation by the two endogenous gonadoliberins which, in addition, may interact negatively.

Amino Acid Sequence↗
PubMed1997

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells.

1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.

Adenylyl Cyclases↗