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R Leurs

Publications and source records attributed to R Leurs.

At least 91 records · Page 5Linked to original sources
PubMed1995

Interaction of clozapine with the histamine H3 receptor in rat brain.

We examined possible interactions between neuroleptics and the histamine H3 receptor and found an interaction of clozapine with this receptor. In competition binding experiments, using the H3 antagonist, [125I]-iodophenpropit, we observed a Ki of 236 +/- 87 nM. Functionally, clozapine was studied on the H3-mediated inhibition of [3H]-5-hydroxytryptamine ([3H]-5-HT) release from rat brain cortex slices. Clozapine acts as an antagonist with an apparent KB value of 79.5 nM.

Animals↗
PubMed1995

Evaluation of the receptor selectivity of the H3 receptor antagonists, iodophenpropit and thioperamide: an interaction with the 5-HT3 receptor revealed.

1. In the present study we evaluated the receptor selectivity of the potent histamine H3 receptor antagonist, iodophenpropit (IPP) in comparison with the prototype antagonist, thioperamide. 2. IPP proved to be a potent competitive H3 receptor antagonist as measured against (R)-alpha-methylhistamine-induced inhibition of electrically-evoked contractions of the guinea-pig jejunum (pA2 = 9.12 +/- 0.06, Schild slope: 1.0 +/- 0.1, n = 8). In the same assay, thioperamide was slightly less potent (pA2 = 8.9 +/- 0.2). 3. In radioligand binding studies, IPP showed a high affinity for the H3 receptor. Displacement of [125I]-IPP binding to rat cortex membranes by unlabelled IPP resulted in a Ki value of 0.97 +/- 0.06 nM (n = 3). In contrast, IPP showed only a weak affinity for the histamine H1- and H2 receptor. Displacement of [3H]-mepyramine and [125I]-iodoaminopotentidine binding to respectively guinea-pig H1- and human H2 receptors by IPP resulted in Ki values of 1.71 +/- 0.32 microM (n = 3) and 2.28 +/- 0.81 microM (n = 3). For thioperamide the affinities for the H1-, H2- and H3 receptor were respectively > 10 microM, > 10 microM and 4.3 +/- 1.6 nM (n = 7). 4. Testing IPP and thioperamide in 39 different receptor binding assays revealed that IPP showed relatively high affinity for the 5-hydroxytryptamine 5-HT3 receptor (Ki = 11 +/- 1 nM, n = 3), the alpha 2-adrenoceptor (Ki = 120 +/- 5 nM, n = 3) and the sigma receptor (Ki = 170 +/- 70 nM, n = 3). Thioperamide showed relatively high affinity for the 5-HT3 receptor (Ki = 120 +/- 30 nM, n = 3) and the sigma receptor (Ki = 180 +/- 90 nM, n = 3). 5. Due to the low density of histamine H3 receptors in the brain, the interaction of IPP with the 5-HT3-, the alpha 2- and the sigma receptor might interfere with [125I]-IPP binding to rat cortex membranes. Yet, in this preparation [125I]-IPP binding was not influenced by ondansetron, yohimbine or haloperidol. The interaction with the 5-HT3 receptor was not restricted to IPP or thioperamide, but was alsofound with other H3 receptor antagonists. The potent H3 receptor agonist imetit, a compound belongingto the same chemical class of IPP, also interacted with the 5-HT3 receptor (Ki = 240 +/- 40 nM). In contrast,histamine or the H3 receptor agonist, (R)-a-methylhistamine showed no affinity for the 5-HT3 receptor.7 In the guinea-pig isolated ileum, imetit evoked concentration-dependent contractions, resulting in apD2 value of 4.72 +/- 0.03 (n = 9). The contractions were antagonized by ondansetron, yielding a pA2 valueof 7.1 +/- 0.1 (n = 9). Similarly ondansetron antagonized the contractions evoked by the 5-HT3 receptoragonist, 2-methyl-5-HT with a pA2 value of 7.3 +/- 0.1 (n = 4). IPP and thioperamide did not mimic 2-methyl-5-HT but non-competitively inhibited the 2-methyl-5-HT-induced contractions of thispreparation.8 In an in vivo model for 5-HT3 activity, the Von Bezold Jarisch reflex, thioperamide showedantagonism in low dosages, which correlated well with the affinity for the 5-HT3 receptor site. Yet, athigher dosages no further 5-HT3 receptor antagonism was observed. For IPP no 5-HT3 receptor activitycould be observed in vivo.9 In the present study we showed that many H3 receptor compounds, that are regarded as highlyselective (including the prototype drug, thioperamide), also interact with the 5-HT3 receptor, albeit athigher drug concentrations.Keywords.: Histamine H3-receptor; iodophenpropit; thioperamide; receptor selectivity; 5-hydroxytryptamine 5-HT3 receptor;guinea-pig intestine; rat brain; Von Bezold Jarisch reflex

Animals↗
PubMed1995

Purification and characterization of [3H]mepyramine (histamine H1 antagonist)-binding protein from rat liver: a highly homologous protein with cytochrome P450 2D.

A protein having a high-affinity binding site for [3H]mepyramine (MBP) was purified to homogeneity from rat liver membranes. The purified MBP has a single type of binding site for [3H]mepyramine with Kd value of 18.5 nM, and its molecular weight was determined to be 56,000 by SDS polyacrylamide gel electrophoresis. Amino acid sequences of twelve tryptic peptides derived from MBP are highly homologous with those of rat debrisoquine 4-hydroxylase (cytochrome P450 2D1) and other rat P450 2D subfamily members. In immunoblotting analysis, an antibody against rat P450 2D1 stained a band corresponding to MBP with Mr of 56,000; its migration position was clearly different from that of rat P450 2D1. Substrates and inhibitors of debrisoquine 4-hydroxylase potently displace [3H]-mepyramine binding to MBP. Quinine and quinidine showed 400 and 80 times, respectively, higher affinity for MBP than for debrisoquine 4-hydroxylase. These results suggest that MBP is a novel P450 2D family member.

Amino Acid Sequence↗
PubMed1994

Rapid desensitization of the histamine H2 receptor on the human monocytic cell line U937.

In the present study we have subjected the histamine H2 receptor on the monocytic cell line U937 to a thorough pharmacological characterization using a series of selective histamine H1, H2 and H3 receptor agonists and antagonists. Recent reports have demonstrated the existence of a histamine H2 receptor on HL-60 and HGT-1 cells with a pharmacological profile distinct from the commonly described histamine H2 receptor. U937 cells, however, seem to express classical histamine H2 receptors. Histamine and dimaprit dose dependently induce the formation of cAMP, whereas dimaprit's inactive analogues, nordimaprit and homodimaprit, show reduced potencies. Histamine H1 and H3 receptor agonists do not show histamine H2 receptor activity. Various histamine H2 receptor antagonists are able to block the histamine induced production of cAMP with an antagonistic profile comparable to that observed in the guinea-pig right atrium. Furthermore, endogenous histamine H2 receptors in U937 cells are found to be susceptible to receptor desensitization, a mechanism which may become apparent under pathophysiological conditions or during drug therapy. A 30-min pre-exposure of U937 cells to histamine (100 microM) results in a 50% attenuation of the production of cAMP to a subsequent application of an agonist. Desensitization of the histamine H2 receptor in U937 cells is found to be homologous as the beta-adrenoceptor mediated response remained unaffected.

Cells, Cultured↗
PubMed1994

Clobenpropit (VUF-9153), a new histamine H3 receptor antagonist, inhibits electrically induced convulsions in mice.

The effect of clobenpropit (VUF-9153), a new histamine H3 receptor antagonist, on electrically induced convulsions was studied in mice. Clobenpropit significantly and dose dependently decreased the duration of each convulsive phase. Its anticonvulsant effects were prevented by pretreatment with (R)-alpha-methylhistamine and imetit (VUF-8325), histamine H3 receptor agonists. These findings suggest that the effect of clobenpropit on electrically induced convulsions is due to an increase in endogenous histamine release in the brain, which is consistent with biochemical results that clobenpropit increased brain histidine decarboxylase activity dose dependently. The anticonvulsive effect of clobenpropit was antagonized by mepyramine, a histamine H1 receptor antagonist, but not by zolantidine, a histamine H2 receptor antagonist, indicating that histamine released by the anticonvulsant effect of clobenpropit interacts with histamine H1 receptors of postsynaptic neurons. The present findings of the effect of clobenpropit on electrically induced convulsions are fully consistent with those of thioperamide as described previously (Yokoyama et al., 1993, Eur. J. Pharmacol. 234, 129), supporting the hypothesis that the central histaminergic neuron system is involved in the inhibition of seizures.

Animals↗
PubMed1994

Site-directed mutagenesis of the histamine H1-receptor reveals a selective interaction of asparagine207 with subclasses of H1-receptor agonists.

In this study we investigated the role of the threonine203 and the asparagine207 residues in the fifth transmembrane domain of the guinea-pig histamine H1-receptor by site-directed mutagenesis to non-functional alanines. Whereas the threonine203 residue is not important for the action of histamine, the asparagine207 residue appears to be involved in the binding of the N tau-nitrogen atom of histamine and its 2-methyl-analogue. For the 2-phenyl-analogue and non-imidazole H1-receptor agonists, this residue is, however, not essential for binding. On the basis of this study we conclude that different histamine H1-receptor agonists interact in different ways with the H1-receptor protein. Moreover, we speculate that the interaction with the N pi-nitrogen atom is essential for H1-receptor activation.

Animals↗
PubMed1994

A new radioligand binding assay for cytochrome P450IID1 (CYP2D1) in rat liver microsomes: a tool to predict sparteine/debrisoquine type polymorphism of drugs.

[3H]-Mepyramine ([3H]-pyrilamine) has a high affinity for cytochrome P450IID1 (CYP2D1) in rat liver microsomes, the isoenzyme involved in the oxidative metabolism of debrisoquine. Drugs known to interact with this enzyme, as either substrate or inhibitor, displaced [3H]-mepyramine. Compounds specific for other P450 isoenzymes did not displace [3H]-mepyramine. Drugs that are positive in this binding assay can be either substrates or inhibitors of CYP2D1 and warrant further research to investigate possible polymorphism. Substrates could reach toxicological concentrations, and inhibitors can have drug interactions with known CYP2D6 substrates. Although care should be taken in the extrapolation from rat to human, since CYP2D1 and CYP2D6 have clear differences, this rapid, easy-to-perform and inexpensive assay has predictive value for the sparteine/debrisoquine type of polymorphic behavior of compounds and could be used at an early stage in drug development.

Animals↗
PubMed1994

Guinea pig histamine H1 receptor. I. Gene cloning, characterization, and tissue expression revealed by in situ hybridization.

An intronless DNA encoding the guinea pig H1 receptor was cloned from a genomic library using probes derived from the bovine H1 receptor. It encodes a protein of 488 amino acids with a calculated molecular mass of 55,619 daltons compared with a size of 56-68 kDa for the photoaffinity-labeled receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The protein displays a 66% homology with the bovine receptor. Stable expression of the H1 receptor, characterized by the appearance of [3H]mepyramine binding sites with a pharmacology similar to that of the native H1 receptor, was obtained following transfection of Chinese hamster ovary cells. Southern blot analysis, using a variety of restriction enzymes, did not provide any evidence of multiple H1 isoreceptors. Northern blot analysis of a variety of guinea pig peripheral or cerebral tissues identified, in most cases, a single transcript of 3.3 kb, but also, in some tissues, a second transcript of 3.7 kb, possibly generated by the use of different promoter or polyadenylation sites or corresponding to a transcript from a distinct gene. In situ hybridization studies showed the highly contrasted cerebral expression of H1-receptor gene transcripts, which was compared with autoradiographic receptor localization. This allowed the identification of some major cell populations expressing the H1 receptor, e.g., Purkinje cells in cerebellum or pyramidal cells in the hippocampal complex.

Amino Acid Sequence↗
PubMed1994

Guinea pig histamine H1 receptor. II. Stable expression in Chinese hamster ovary cells reveals the interaction with three major signal transduction pathways.

A cDNA encoding a guinea pig histamine H1 receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H1), the H1 receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a Gi/Go protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca2+ and of protein kinase C activation by phorbol 12-myristate 13-acetate (PMA). Histamine induced, in a PTX- and PMA-insensitive manner, a biphasic increase in the intracellular Ca2+ level of which only the second sustained phase was dependent on the extracellular Ca2+ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX-insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca2+. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca(2+)- and PMA-sensitive manner, probably through the activation of a cytosolic phospholipase A2, which partly involves coupling to a PTX-sensitive G protein. In comparison, in HeLa cells endowed with a native H1 receptor, the histamine-induced arachidonic acid release was also Ca(2+)- and PMA-sensitive, but totally PTX-insensitive. Finally, in CHO(H1) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca2+, and PMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Pharmacological characterization of the human histamine H2 receptor stably expressed in Chinese hamster ovary cells.

1. The gene for the human histamine H2 receptor was stably expressed in Chinese hamster ovary (CHO) cells and characterized by [125I]-iodoaminopotentidine binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. 2. After cotransfection of CHO cells with pCMVhumH2 and pUT626, a phleomycine-resistant clonal cell line (CHOhumH2) was isolated that expressed 565 +/- 35 fmol kg-1 protein binding sites with high affinity (0.21 +/- 0.02 nM) for the H2 antagonist, [125I]-iodoaminopotentidine. 3. Displacement studies with a variety of H2 antagonists indicated that the encoded protein was indistinguishable from the H2 receptor identified in human brain membranes and guinea-pig right atrium. The Ki-values observed in the various preparations correlated very well (r2 = 0.996-0.920). 4. Displacement studies with histamine showed that a limited fraction (32 +/- 6%) of the binding sites showed a high affinity for histamine (2 +/- 1.2 microM); the shallow displacement curves were reflected by a Hill-coefficient significantly different from unity (nH = 0.58 +/- 0.09). The addition of 100 microM Gpp(NH)p resulted in a steepening of the displacement curve (nH = 0.79 +/- 0.02) and a loss of high affinity sites for histamine. 5. Displacement studies with other agonists indicated that the recently developed specific H2 agonists, amthamine and amselamine, showed an approximately 4-5 fold higher affinity for the human H2 receptor than histamine. 6. Stimulation of CHOhumH2 cells with histamine resulted in a rapid rise of the intracellular cyclic AMP levels. After 10 min an approximately 10 fold increase in cyclic AMP could be measured. TheEC50 value for this response was 7 +/- 1 nM for histamine. This response was effectively blocked by tiotidine and cimetidine, resulting in Ki values of 8 +/- 1 nM and 0.56 +/- 0.24 MicroM respectively.7. Stimulation of CHOhumH2 cells with histamine neither inhibited the A23187-induced release of[3H]-arachidonic acid nor changed the intracellular IP3 levels.8. These results show that the cloned human gene encodes a histamine H2 receptor that is indistinguishable from the H2 receptor identified in human brain tissue. This receptor is functionally coupled to the adenylate cyclase in CHO cells, but does not influence the inositolphosphate turnover or arachidonic acid release.

Animals↗
PubMed1993

Extracellular ATP elevates cytoplasmatic free Ca2+ in HeLa cells by the interaction with a 5'-nucleotide receptor.

In the present study we have characterized the effects of ATP and several other nucleotides on the intracellular Ca2+ levels of HeLa cells. Using fura-2 microscopy fluorescence measurements, the ATP-mediated increase in intracellular Ca2+ was shown to consist of a rapid rise which decreased after a few seconds to a sustained elevated level. In the absence of extracellular Ca2+ or in the presence of 10 microM La3+, only a transient elevation of the Ca2+ concentration was observed. The ATP responses were not altered after treatment of HeLa cells with cholera toxin or pertussis toxin. Pharmacological analysis of this calcium response revealed that this effect was not mediated by the classical P2y purinoceptor but by a 5'-nucleotide receptor.

Adenosine Triphosphate↗
PubMed1993

Molecular cloning, characterization, and localization of a high-affinity serotonin receptor (5-HT7) activating cAMP formation.

By using a strategy based on nucleotide sequence homology, we have cloned a cDNA encoding a functional serotonin (5-HT) receptor. The deduced amino acid sequence of the 5-HT7 receptor displays limited homology with that of other 5-HT receptors. In addition to the seven stretches of hydrophobic amino acids that characterize the superfamily of receptors interacting with guanine nucleotide-binding proteins, the 448-aa sequence of the 5-HT7 receptor contains a hydrophobic domain located at its N-terminal end. Genomic analysis indicated the presence of introns interrupting the coding sequence. The 5-HT7 receptor, stably expressed in transfected CHO cells, bound [3H]5-HT with high affinity (Kd = 1 nM), like receptors of the 5-HT1 subfamily from which, however, it was clearly distinguished by its pharmacology. 5-HT in nanomolar concentrations stimulated cAMP accumulation in these CHO cells by approximately 10-fold, whereas lysergic acid diethylamide displayed low intrinsic agonist activity. These various properties differentiate the 5-HT7 receptor from the four other subfamilies of mammalian 5-HT receptors (i.e., the 5-HT1-, 5-HT2-, 5-HT3-, and 5-HT4-like subfamilies) and, therefore, appear to define another receptor subfamily. Northern blot and in situ hybridization analyses showed the 5-HT7 transcripts to be expressed in discrete areas of the limbic brain (e.g., pyramidal hippocampus cells, tenia tecta, amygdaloid, or mammillary nuclei), suggesting that the receptor mediates serotoninergic controls in functions like mood, learning, or neuroendocrine and vegetative behaviors.

Amino Acid Sequence↗
PubMed1993

A novel rat serotonin (5-HT6) receptor: molecular cloning, localization and stimulation of cAMP accumulation.

Using a strategy based upon nucleotide sequence homology and starting from the sequence of the rat histamine H2 receptor (Ruat et al., Biochem. Biophys. Res. Commun. 1991, 179, 1470-1478), we have cloned a rat cDNA encoding a functional serotonin receptor (5-HT6). Its coding sequence corresponds to a glycoprotein of 436 amino acids displaying significant homology with other cloned monoaminergic receptors, e.g., various serotonin receptors. Genomic analysis of its gene indicated the presence of at least one intron. The major transcript of the 5-HT6 receptor gene has a size of approximately 4.1 kb but another minor 3.2 kb transcript was also evidenced. The highest expression, detected by Northern blot analysis as well as by in situ hybridization occurs in various serotoninergic areas of rat or guinea pig brain such as striatum, olfactory tubercle, nucleus accumbens and hippocampus, but a faint expression is also detectable in rat stomach. When transiently expressed in transfected COS-7 cells the 5-HT6 receptor appears to be positively coupled to cyclic AMP production.

Amino Acid Sequence↗
PubMed1993

Mouse P19 embryonal carcinoma cells express functional histamine H1-receptors.

In the present study, we have investigated the response in P19 embryonal carcinoma (EC) cells to histamine. We show that these cells, that resemble the pluripotent cells of an early mouse embryo, respond to histamine addition by a transient increase in intracellular Ca2+. The response is stereoselectively inhibited by the enantiomers of the H1-receptor antagonists chlorpheniramine and cicletanine. [3H]-mepyramine was found to bind with high affinity (Kd 4 nM) to a membrane preparation of P19 EC cells. The profile of these binding sites corresponded well with the results of the Ca2+ measurements. A high affinity [3H]-mepyramine binding site was also identified on intact cells. These data demonstrate that embryonal carcinoma cells express functional histamine H1-receptors and suggest that histamine might act as a regulatory factor in the early development of the mouse embryo.

Animals↗
PubMed1992

Expression of a cloned rat histamine H2 receptor mediating inhibition of arachidonate release and activation of cAMP accumulation.

A DNA, cloned after screening a rat genomic bank with probes derived from the sequence of a putative dog histamine H2 receptor [Gantz, I., Schäffer, M., Delvalle, J., Logsdon, C., Campbell, V., Uhler, M. & Yamada, T. (1991) Proc. Natl. Acad. Sci. USA 88, 429-433], was used to prepare a probe for Northern blot analysis and to transfect Chinese hamster ovary (CHO) cells. Distribution of the gene transcripts in guinea pig tissues was consistent with that of H2 receptors. Transfected CHO cells expressed a high density of sites binding [125I]iodoaminopotentidine, a selective H2-receptor ligand. These sites were characterized as typical H2 receptors by using a series of competing agents that displayed apparent dissociation constants closely similar to corresponding values at a reference biological system. In transfected cells, histamine stimulated, with high potency and large receptor reserve, the accumulation of cAMP. In addition, in the same cells, histamine potently inhibited the release of arachidonic acid induced either by stimulation of constitutive purinergic receptors or by application of a Ca2+ ionophore. This inhibition was independent of either cAMP or Ca2+ levels. The results suggest that a single H2 receptor may be linked not only to adenylyl cyclase activation but also to reduction of phospholipase A2 activity. Because H1 receptors have been reported to stimulate arachidonic acid release, inhibition of this release, an unexpected signaling pathway for H2 receptors, may account for the opposing physiological responses elicited in many tissues by stimulation of these two receptors subtypes.

Animals↗
PubMed1992

Desensitization of histamine H1 receptor-mediated cyclic GMP production in guinea-pig lung.

Histamine H1 receptor-mediated production of cGMP in guinea-pig lung tissue becomes rapidly desensitized after previous exposure to histamine. This desensitization is clearly concentration dependent and appears to be homologous. Responses to histamine are also inhibited by previous treatment with phorbol 12,13-dibutyrate. Yet, the time course of the inhibition is considerably slower and the maximal inhibition is significantly less compared to receptor desensitization. Moreover, the effects of the phorbol ester are not confined to H1 receptor responses. Since the effects of receptor desensitization are also not prevented by several protein kinase C inhibitors, the development of homologous H1 receptor desensitization is not dependent upon protein kinase C activation, but is caused by a yet unidentified mechanism.

Animals↗