Increased brain histamine in an alcohol-preferring rat line and modulation of ethanol consumption by H(3) receptor mechanisms.
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Alcohol Drinking↗
Biomedical subjects
R Leurs
Publications and source records attributed to R Leurs.
Histamine H(1)-receptor activation of nuclear factor-kappa B: roles for G beta gamma- and G alpha(q/11)-subunits in constitutive and agonist-mediated signaling.
Nuclear factor kappa B (NF-kappa B) is an important transcription factor in inflammation that has obtained a great interest as a drug target for the treatment of various allergic conditions. In this study, we show that the histamine H(1) receptor, which is also an important player in allergic and inflammatory conditions, activates NF-kappa B in both a constitutive and agonist-dependent manner. Moreover, the observed constitutive NF-kappa B activation is inhibited by various H(1)-receptor antagonists, suggesting that inverse agonism may account, at least in part, for their ascribed antiallergic properties. Investigation of the H(1) receptor-mediated NF-kappa B activation in transfected COS-7 cells indicates that the level of the observed constitutive activity of the H(1) receptor can be modulated by the expression levels of either G alpha-proteins or G beta gamma-heterodimers. Members of the G alpha(q/11)-family of G alpha-proteins are most effective in increasing H(1) constitutive activity. Also, coexpression of G beta(2) in combination with either G gamma(1) or G gamma(2) results in an increased constitutive activity of the H(1) receptor, whereas scavenging of G beta gamma-subunits by coexpression of G alpha(t) completely neutralizes the constitutive, but not the agonist-induced, NF-kappa B activity. Our data suggest that both G alpha(q/11)- and G beta gamma-subunits play a role in the agonist-induced, H(1) receptor-mediated NF-kappa B activation, but that constitutive NF-kappa B activation by the H(1) receptor is primarily mediated through G beta gamma-subunits.
Animals↗
Identification of rat H3 receptor isoforms with different brain expression and signaling properties.
We identified the cDNAs of three functional rat H3 receptor isoforms (H3A, H3B, and H3C) and one nonfunctional truncated H3 receptor (H3T). The H3A, H3B, and H3C receptor isoforms vary in the length of their third intracellular loop; the H3B and H3C receptor lack 32 and 48 amino acids, respectively. Transient expression of the H3A, H3B, and H3C receptors in COS-7 cells results in high affinity binding for the H3 antagonist [125I]iodophenpropit, which is displaced by selective H3 agonists and antagonists. The three isoforms differentially couple to the Gi protein-dependent inhibition of adenylate cyclase or stimulation of p44/p42 mitogen activated protein kinase (MAPK), a new signaling pathway for the H3 receptor. Whereas the H3A receptor was less effective in inhibiting forskolin-induced cAMP production compared with the H3B or H3C receptor, this isoform was more effective in the stimulation of p44/p42 MAPK. The H3 receptor isoforms also displayed differential CNS expression in key areas involved in regulation of sensory, endocrine, and cognitive functions. A differential H3 receptor isoform expression was seen in, for example, hippocampus, where a characteristic dorsoventral distribution was revealed. Differential H3 receptor expression was also characteristic for the cerebellum, indicating possible histaminergic regulation of motor functions. The identification of these new H3 receptor isoforms and their specific signaling properties adds a new level of complexity to our understanding of the role of histamine, and the H3 receptor in brain function. The heterogeneous distribution of the isoforms suggests that H3 receptor isoform-specific regulation is important in several brain functions.
Adenylyl Cyclases↗
Constitutive activity of histamine h(3) receptors stably expressed in SK-N-MC cells: display of agonism and inverse agonism by H(3) antagonists.
Agonist-independent activity of G-protein-coupled receptor, also referred to as constitutive activity, is a well-documented phenomenon and has been reported recently for both the histamine H(1) and H(2) receptors. Using SK-N-MC cell lines stably expressing the human and rat H(3) receptors at physiological receptor densities (500-600 fmol/mg of protein), we show that both the rat and human H(3) receptors show a high degree of constitutive activity. The forskolin-mediated cAMP production in SK-N-MC cells is inhibited strongly upon expression of the G(i)-coupled H(3) receptor. The cAMP production can be further inhibited upon agonist stimulation of the H(3) receptor and can be enhanced by a variety of H(3) antagonists acting as inverse agonists at the H(3) receptor. Thioperamide, clobenpropit, and iodophenpropit raise the cAMP levels in SK-N-MC cells with potencies that match their receptor binding affinities. Surprisingly, impentamine and burimamide act as effective H(3) agonists. Modification of the amine group of impentamine dramatically affected the pharmacological activity of the ligand. Receptor affinity was reduced slightly for most impentamine analogs, but the functional activity of the ligands varied from agonist to neutral antagonist and inverse agonist, indicating that subtle changes in the chemical structures of impentamine analogs have major impact on the (de)activation steps of the H(3) receptor. In conclusion, upon stable expression of the rat and human H(3) receptor in SK-N-MC cells constitutive receptor activity is detected. In this experimental system, H(3) receptors ligands, previously identified as H(3) antagonists, cover the whole spectrum of pharmacological activities, ranging from full inverse agonists to agonists.
Animals↗
Improgan, a cimetidine analog, induces morphine-like antinociception in opioid receptor-knockout mice.
Improgan is an analog of the H(2) antagonist cimetidine that does not act on known histamine receptors, but induces highly effective analgesia in rodents following intracerebroventricular (icv) administration. Since the mechanism of action of this compound remains unknown, improgan analgesia was characterized presently with the tail immersion nociceptive test in mutant mice lacking either the mu (exon 1 of MOR-1), delta (exon 2 of DOR-1) or kappa (exon 3 of KOR-1) opioid receptor. Improgan (30 microg, icv) induced reversible, maximal analgesia in both sexes of all three genotypes (+/+, +/- and -/-) of MOR-1 mutant mice 10 and 20 min after administration, whereas morphine analgesia was reduced (+/-) or abolished (-/-) in these subjects. In DOR-1 mutant mice, improgan was equally effective in all three genotypes, despite the reduction (+/-) or complete loss (-/-) of delta opioid receptor (3H-[D-Pen(2), D-Pen(5)]enkephalin, DPDPE) binding. Similarly, improgan analgesia was equivalent in all three genotypes of KOR-1 mutant mice, whereas kappa-mediated analgesia (U50,488) and kappa opioid (3H-U69,593) binding were abolished in the homozygous (-/-) mice. These studies demonstrate that improgan analgesia does not require intact MOR-1, DOR-1, or KOR-1 genes, and support the hypothesis that improgan-like analgesics act in the CNS by non-opioid mechanisms.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
Characterization of the binding site of the histamine H(3) receptor. 2. Synthesis, in vitro pharmacology, and QSAR of a series of monosubstituted benzyl analogues of thioperamide.
A series of monosubstituted benzyl analogues of the histamine H(3) receptor antagonist thioperamide were synthesized and evaluated for their histamine H(3) receptor activity on the guinea pig jejunum. Incorporation of Cl, Br, and I at the ortho position of the benzyl moiety led to an increase of the pA(2) value, whereas the same substituents at the para position led to a decrease. However, a fluorine substituent gave a strong decrease in pA(2), regardless of the position. Molecular modeling revealed a QSAR with a correlation (r = 0.93) between the pA(2) and the dihedral angle between the thiourea and the benzyl moiety and the calculated electron density on the substituted carbon atom. To verify whether this QSAR model had a predictive value, the ortho tert-butyl and methyl analogues were synthesized and evaluated. Indeed it was shown that the predicted pA(2) values of these two compounds were in accordance with the measured pA(2) values.
Benzyl Compounds↗
Constitutive activity of the histamine H(1) receptor reveals inverse agonism of histamine H(1) receptor antagonists.
Transient expression of the wild-type human histamine H(1) receptor in SV40-immortalised African green monkey kidney cells resulted in an agonist-independent elevation of the basal levels of the second messenger inositoltrisphospate. Several histamine H(1) receptor antagonists, including the therapeutically used anti-allergics cetirizine, loratadine and epinastine reduced this constitutive histamine H(1) receptor activity. Inverse agonism, i.e., stabilisation of an inactive conformation of the human histamine H(1) receptor, may therefore be a key component of the anti-allergic mechanism of action of clinically used antihistamines.
Animals↗
Antinociceptive activity of derivatives of improgran and burimamide.
Improgan, a compound related to H2 and H3 antagonists, induces antinociception in rodents after intraventricular administration. Characteristics of improgan and its congeners include: (a) morphine-like antinociception on thermal and mechanical tests in two species; (b) no impairment of motor coordination or locomotor activity; (c) evidence for a novel, nonopioid mechanism that is independent of known histamine receptors; (d) lack of tolerance with daily dosing; and (e) unique structure-activity relationships (SARs). Presently, the antinociceptive activity of several new derivatives of improgan was investigated in rats. Among compounds similar to burimamide, VUF4577 (possessing a two-carbon side chain) and VUF4582 (an N-phenyl derivative of VUF4577) induced complete, dose- and time-dependent antinociception on the hot-plate and tail-flick tests with no behavioral side effects. These compounds (with ED50 values of 71-117 nmol) were approximately twice as potent as burimamide itself (a four-carbon derivative). Two other derivatives in which the thiourea group (C=S, known to cause human toxicity) was replaced by either nitroethene (C=CH-NO2, VUF5405) or urea (C=O, VUF5407) also showed effective, potent antinociception on both assays. The latter compound is the most potent improgan-like drug discovered to date (ED50 = 71 nmol). Furthermore, positional isomers of antinociceptive compounds either lacked activity (VUF5394) or induced toxicity (VUF5393), revealing a high degree of pharmacological specificity. Although the mechanism of improgan antinociception remains unknown, the present results show promise for the further development of safe, effective, and potent pain-relieving compounds.
Analgesics, Non-Narcotic↗
Characterization of histamine H3 receptors in mouse brain using the H3 antagonist [125I]iodophenpropit.
We have characterized the binding of the histamine H3 receptor antagonist [125I]iodophenpropit to mouse brain. [125I]Iodophenpropit saturably bound to mouse brain membranes with a pKd-value of 9.31+/-0.04 nM and a receptor binding density of 290+/-8 fmol per mg protein. Saturation binding analysis revealed binding of [125I]iodophenpropit to a single class of sites, showing linear Scatchard plots and Hill coefficients not different from unity (nH=0.98+/-0.02). At a concentration of 0.25 nM [125I]iodophenpropit, specific binding represented about 75% of the total binding. Competition binding curves for H3 receptor antagonists were fitted best to a one-site model, showing pKi-values in general accordance with the pA2-values obtained in mouse cerebral cortex. Displacement of [125I]iodophenpropit by the H3 receptor agonists (R)-alpha-methylhistamine, immepip, imetit and histamine were fitted best to a two-site model. Competition binding curves of (R)-alpha-methylhistamine showed a rightward shift upon incubation with GTPgammaS (10 microM), indicating the involvement of G-proteins in H3 agonist binding. In contrast, competition binding curves of the antagonists iodophenpropit, thioperamide and burimamide were not affected by GTPgammaS (10 microM). Autoradiographic experiments showed that [125I]iodophenpropit binding sites were heterogeneously distributed, similarly to the distribution of histamine H3 receptors reported in rat brain. Highest densities were observed in the cerebral cortex, the striatum, the nucleus accumbens, the globus pallidus and the substantia nigra. In conclusion, we have demonstrated that in mouse brain, [125I]iodophenpropit selectively binds to histamine H3 receptors. We also observed that the mouse brain H3 receptors labelled by [125I]iodophenpropit displayed binding characteristics and a distribution similar to rat brain.
Animals↗
Viral-encoded G-protein coupled receptors: new targets for drug research?
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Animals↗
Constitutive activity of G protein coupled receptors and drug action.
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Animals↗
H3 receptor gene is cloned at last.
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Animals↗
Modulation of histamine H(2) receptor signalling by G-protein-coupled receptor kinase 2 and 3.
To evaluate the role of G-protein-coupled receptor kinases (GRK) in the desensitization of the histamine H(2) receptor, the H(2) receptor was transiently cotransfected with GRK2, 3, 5 or 6 in COS-7 cells and the cyclic AMP levels in response to histamine were studied. Coexpression of the H(2) receptor with GRK2 and 3 significantly decreased both the basal cyclic AMP levels and the cyclic AMP response to 100 microM histamine. Moreover, preincubation with 100 microM histamine desensitized the H(2) receptor response to 53+/-8%. Coexpression of GRK2 and 3 increased the H(2) receptor desensitization to 27+/-4% and 24+/-4% respectively. No effect on either cyclic AMP response or desensitization was found when GRK5, GRK6 or dominant negative mutants of GRK2 or 3 (GRK2K(220)R and GRK3K(220)R) were coexpressed. To study the role of the C-terminal tail in the GRK-mediated desensitization of the H(2) receptor, three truncations of C-tail were constructed: H(2)T295, H(2)T307 and H(2)T341. H(2)T307 and 341 H(2)T341 expressed and responded normally to 100 microM histamine. The interaction of the H(2) receptor with GRK2 and 3 was also not altered upon truncation of the C-terminal tail. These findings strongly suggest a role of GRK2 and 3 in the desensitization of the H(2) receptor. Furthermore, the finding that C-terminal truncations of the H(2) receptor did not abolish the effect of GRK2 and 3 suggests that the C-terminus is not involved in the GRK mediated desensitization of the histamine H(2) receptor.
Amino Acid Sequence↗
A third life for burimamide. Discovery and characterization of a novel class of non-opioid analgesics derived from histamine antagonists.
Burimamide, a histamine (HA) derivative with both H2- and H3-blocking properties, induces antinociception when injected into the rodent CNS. Several related compounds share this property, and structure-activity studies have shown that this new class of analgesics is distinct from known HA antagonists. The prototype, named improgan, shows a preclinical profile of a highly effective analgesic, with activity against thermal, mechanical and inflammatory nociception after doses that do not alter motor balance or locomotor activity. Improgen analgesia is not blocked by opioid antagonists and is observed in opioid receptor knock-out mice. Unlike morphine, improgan does not induce tolerance after daily dosing. Extensive in vitro pharmacology studies have excluded known histaminergic, opioid, serotonergic, GABAergic and adrenergic receptor mechanisms, as well as 50 other sites of action. The improgan-like analgesic activity of some HA congeners suggests an analgesic action on a novel HA receptor, but further studies are required to substantiate this. Studies in progress are characterizing the sites and mechanisms of action of improgan, and developing brain-penetrating derivatives that could be useful for clinical pain.
Analgesics, Non-Narcotic↗
The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H(2) receptor.
In previous studies we showed that the wild-type histamine H(2) receptor stably expressed in Chinese hamster ovary cells is constitutively active. Because constitutive activity of the H(2) receptor is already found at low expression levels (300 fmol/mg protein) this receptor is a relatively unique member of the G-protein-coupled receptor (GPCR) family and a useful tool for studying GPCR activation. In this study the role of the highly conserved DRY motif in activation of the H(2) receptor was investigated. Mutation of the aspartate 115 residue in this motif resulted in H(2) receptors with high constitutive activity, increased agonist affinity, and increased signaling properties. In addition, the mutant receptors were shown to be highly structurally instable. Mutation of the arginine 116 residue in the DRY motif resulted also in a highly structurally instable receptor; expression of the receptor could only be detected after stabilization with either an agonist or inverse agonist. Moreover, the agonist affinity at the Arg-116 mutant receptors was increased, whereas the signal transduction properties of these receptors were decreased. We conclude that the Arg-116 mutant receptors can adopt an active conformation but have a decreased ability to couple to or activate the G(s)-protein. This study examines the pivotal role of the aspartate and arginine residues of the DRY motif in GPCR function. Disruption of receptor stabilizing constraints by mutation in the DRY motif leads to the formation of active GPCR conformations, but concomitantly to GPCR instability.
Amino Acid Motifs↗
Mutational analysis of the antagonist-binding site of the histamine H(1) receptor.
We combined in a previously derived three-dimensional model of the histamine H(1) receptor (Ter Laak, A. M., Timmerman, H., Leurs, H., Nederkoorn, P. H. J., Smit, M. J., and Donne-Op den Kelder, G. M. (1995) J. Comp. Aid. Mol. Design. 9, 319-330) a pharmacophore for the H(1) antagonist binding site (Ter Laak, A. M., Venhorst, J., Timmerman, H., and Donné-Op de Kelder, G. M. (1994) J. Med. Chem. 38, 3351-3360) with the known interacting amino acid residue Asp(116) (in transmembrane domain III) of the H(1) receptor and verified the predicted receptor-ligand interactions by site-directed mutagenesis. This resulted in the identification of the aromatic amino acids Trp(167), Phe(433), and Phe(436) in transmembrane domains IV and VI of the H(1) receptor as probable interaction points for the trans-aromatic ring of the H(1) antagonists. Subsequently, a specific interaction of carboxylate moieties of two therapeutically important, zwitterionic H(1) antagonists with Lys(200) in transmembrane domain V was predicted. A Lys(200) --> Ala mutation results in a 50- (acrivastine) to 8-fold (d-cetirizine) loss of affinity of these zwitterionic antagonists. In contrast, the affinities of structural analogs of acrivastine and cetirizine lacking the carboxylate group, triprolidine and meclozine, respectively, are unaffected by the Lys(200) --> Ala mutation. These data strongly suggest that Lys(200), unique for the H(1) receptor, acts as a specific anchor point for these "second generation" H(1) antagonists.
Animals↗
Characterization of the binding site of the histamine H3 receptor. 1. Various approaches to the synthesis of 2-(1H-imidazol-4-yl)cyclopropylamine and histaminergic activity of (1R,2R)- and (1S,2S)-2-(1H-imidazol-4-yl)-cyclopropylamine.
Various approaches to the synthesis of all four stereoisomers of 2-(1H-imidazol-4-yl)cyclopropylamine (cyclopropylhistamine) are described. The rapid and convenient synthesis and resolution of trans-cyclopropylhistamine is reported. The absolute configuration of its enantiomers was determined by single-crystal X-ray crystallographic analysis. The distinct trans-cyclopropylhistamine enantiomers were tested for their activity and affinity on the histamine H3 receptor. (1S,2S)-Cyclopropylhistamine (VUF 5297) acts as an agonist both on the rat cortex (pD2 = 7.1; alpha = 0.75) and on guinea pig jejunum (pD2 = 6.6; alpha = 0.75). Its enantiomer, (1R, 2R)-cyclopropylhistamine (VUF 5296), is about 1 order of magnitude less active. Both enantiomers show weak activity on H1 and H2 receptors. All synthetic attempts to cis-cyclopropylhistamine were unsuccessful. Nevertheless, the results of this study provide an ideal template for molecular modeling studies of histamine H3 receptor ligands.
Animals↗
Antinociceptive activity of impentamine, a histamine congener, after CNS administration.
The brain neuromodulator histamine induces antinociception when administered directly into the rodent CNS. However, several compounds derived from H2 and H3 antagonists also produce antinociception after central administration. Pharmacological studies have shown that a prototype of these agents, improgan, induces analgesia that is not mediated by actions on known histamine receptors. Presently, the antinociceptive properties of a compound that chemically resembles both improgan and histamine were investigated in rats. Intraventricular (i.v.t.) administration of impentamine (4-imidazolylpentylamine) induced reversible, near-maximal antinociception on the hot plate and tail flick tests (15 microg, 98 nmol). The dose-response function was extremely steep, however, since other doses showed either no effect or behavioral toxicity. On the tail flick test, impentamine antinociception was resistant to antagonism by blockers of H1, H2, or H3 receptors, similar to characteristics previously found for improgan. In contrast, histamine antinociception was highly attenuated by H1 and H2 antagonists. These findings suggest that: 1) the histamine congener impentamine may induce antinociception by a mechanism similar to that produced by improgan, and 2) additional histamine receptors may be discovered that are linked to pain-attenuating processes.
Analgesics↗