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1.
Oncogene ; 30(33): 3570-84, 2011 Aug 18.
Article in English | MEDLINE | ID: mdl-21399663

ABSTRACT

Existence of humoral immunity has been previously demonstrated in malignant ascitic fluids. However, only a limited number of immunogenic tumor-associated antigens (TAAs) were identified, and few of which are associated with ovarian cancer. Here, we identified salt-inducible kinase 3 (SIK3) as a TAA through screening of a random peptide library in the phage display system. Overexpression of SIK3 markedly promoted cell proliferation, attenuated p21(Waf/Cip1) and p27(Kip) expressions in low-grade OVCAR3 cells, and permitted the cells to grow in mice. Decrease in SIK3 expression in high-grade SK-OV3 cells consistently demonstrated its tumorigenic potency by modulating the protein levels of cell cycle regulators. When the expressions of SIK3 and CA125 were compared in cancer tissues, immunohistochemical (IHC) studies indicated that cytoplasm-localized SIK3 was highly expressed in 55% of the ovarian cancer samples. In contrast, it was rarely detected in adenomyosis, leiomyoma and normal ovary tissues, showing its higher specificity (97%) to CA125 (65%) in ovarian cancer. Moreover, experiments using pharmacological inhibitors to block SIK3-induced p21(Waf/Cip1) expression revealed that activation of c-Src and phosphoinositide-3-kinase were critically required for its biological activity, suggesting that they are the downstream signaling mediators of SIK3. These data were further supported by IHC studies, showing coexpression of c-Src with SIK3 in 85% of the ovarian tumor samples stained positive for SIK3. Collectively, our findings indicate that SIK3 is a novel ovarian TAA. Overexpression of SIK3 promotes G1/S cell cycle progression, bestows survival advantages to cancer cells for growth and correlates the clinicopathological conditions of patients with ovarian cancer.


Subject(s)
Antigens, Neoplasm/physiology , Ovarian Neoplasms/etiology , Protein Kinases/physiology , AMP-Activated Protein Kinases/physiology , Animals , Antigens, Neoplasm/analysis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/genetics , Female , Genes, src , Humans , Male , Mice , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Protein Kinases/analysis
2.
Bioorg Med Chem Lett ; 20(22): 6785-9, 2010 Nov 15.
Article in English | MEDLINE | ID: mdl-20850969

ABSTRACT

We report further expansion of the structure activity relationship (SAR) on the triaryl bis sulfone class of compounds (I), which are potent CB(2) receptor ligands with excellent selectivity over the CB(1) receptor. This study was extended to B ring changes, followed by simultaneous optimization of the A-, B-, and C-rings. Compound 42 has excellent CB(2) potency, selectivity and rat exposure.


Subject(s)
Receptor, Cannabinoid, CB2/drug effects , Sulfones/chemistry , Sulfones/pharmacology , Animals , Ligands , Rats , Receptor, Cannabinoid, CB2/metabolism , Structure-Activity Relationship , Sulfones/metabolism
3.
Am J Pathol ; 159(6): 2303-8, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11733379

ABSTRACT

CD2AP, an adapter protein containing multiple SH3 domains, plays a critical role in kidney function. Mice lacking CD2AP die soon after birth because of kidney failure. In the kidney, CD2AP is expressed in glomerular podocytes, which suggests that it may play a role in a specialized adhesion complex known as the slit diaphragm. One of the major components of the slit diaphragm is nephrin, a podocyte-specific protein. Here we demonstrate that CD2AP localizes to the slit diaphragm in podocytes using immunoelectron microscopy and that nephrin and CD2AP co-immunoprecipitate from a podocyte cell line. The specificity of this interaction was verified by mapping studies, which demonstrated that a novel domain at the C terminus of CD2AP interacts with the C-terminal portion of the nephrin cytoplasmic domain. These studies lend further support to the idea that CD2AP plays a role in the structural integrity of the slit diaphragm.


Subject(s)
Kidney Glomerulus/metabolism , Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Binding Sites/genetics , Cell Line , Cytoskeletal Proteins , Intercellular Junctions/metabolism , Kidney Glomerulus/cytology , Kidney Glomerulus/ultrastructure , Membrane Proteins , Mice , Mice, Knockout , Microscopy, Immunoelectron , Mutation , Precipitin Tests , Protein Binding , Proteins/chemistry , Proteins/genetics
4.
Bioorg Med Chem Lett ; 11(4): 491-4, 2001 Feb 26.
Article in English | MEDLINE | ID: mdl-11229755

ABSTRACT

The NK1 and NK2 receptor activity of a series of 5-[(3,5-bis(trifluoromethyl)phenyl)methoxy]-3-(3,4-dichlorophenyl)-4(Z)-(methoxyimino)pentyl-1-piperidines was evaluated. Compounds 11d, 11e, 11f, 12a, and 12k were found to be our most potent inhibitors.


Subject(s)
Neurokinin-1 Receptor Antagonists , Piperidines/chemical synthesis , Receptors, Neurokinin-2/antagonists & inhibitors , Piperidines/pharmacology , Structure-Activity Relationship
5.
Bioorg Med Chem Lett ; 10(20): 2329-32, 2000 Oct 16.
Article in English | MEDLINE | ID: mdl-11055349

ABSTRACT

Functional probing of the backbone of the Sanofi NK2 antagonist SR 48968 has resulted in the discovery of two new classes of NK1/NK2 dual antagonists: the diamine class and the oxime class. The addition of the amino or the oxime functional group results in the reversal of the stereochemical preference of the NK2 receptor.


Subject(s)
Benzamides/chemistry , Benzamides/chemical synthesis , Diamines/chemical synthesis , Neurokinin-1 Receptor Antagonists , Oximes/chemical synthesis , Piperidines/chemistry , Piperidines/chemical synthesis , Receptors, Neurokinin-2/antagonists & inhibitors , Animals , Benzamides/pharmacology , Diamines/chemistry , Diamines/pharmacology , Drug Design , Kinetics , Molecular Structure , Oximes/chemistry , Oximes/pharmacology , Piperidines/pharmacology , Stereoisomerism , Structure-Activity Relationship
6.
Bioorg Med Chem Lett ; 10(20): 2333-5, 2000 Oct 16.
Article in English | MEDLINE | ID: mdl-11055350

ABSTRACT

A series of 5-[(3,5-bis(trifluoromethyl)phenyl)methoxy]-3-(3,4-dichlorophenyl)-4(Z)- (methoxyimino)pentyl-1-piperazines was prepared and their affinity for the NK1 and NK2 receptors investigated. Compounds 7f, 10o, 10r, and 10s were found to be our most potent inhibitors.


Subject(s)
Neurokinin-1 Receptor Antagonists , Piperazines/chemical synthesis , Receptors, Neurokinin-2/antagonists & inhibitors , Animals , Kinetics , Molecular Structure , Piperazines/chemistry , Piperazines/pharmacology , Structure-Activity Relationship
7.
Science ; 286(5438): 312-5, 1999 Oct 08.
Article in English | MEDLINE | ID: mdl-10514378

ABSTRACT

CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associated with nephrin, the primary component of the slit diaphragm.


Subject(s)
Kidney Glomerulus/metabolism , Kidney Glomerulus/ultrastructure , Nephrotic Syndrome/congenital , Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Basement Membrane/ultrastructure , Cytoskeletal Proteins , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Extracellular Matrix Proteins/metabolism , Glomerular Mesangium/metabolism , Glomerular Mesangium/ultrastructure , Intercellular Junctions/metabolism , Intercellular Junctions/ultrastructure , Kidney Glomerulus/blood supply , Lymphocyte Activation , Membrane Proteins , Mice , Mice, Knockout , Microscopy, Electron , Nephrotic Syndrome/genetics , Nephrotic Syndrome/metabolism , Nephrotic Syndrome/pathology , Proteins/genetics , Recombinant Fusion Proteins/metabolism , T-Lymphocytes/immunology
8.
Arzneimittelforschung ; 48(9): 881-8, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9793613

ABSTRACT

Experiments were performed to characterize the pharmacology of Sch 50971 ((+)-trans-4-(4(R)-methyl-3(R)-pyrolidinyl)-1H-imidazole dihydrochloride, CAS 167610-28-8), a novel histamine H3 receptor agonist. The activity of Sch 50971 was compared with that of (R)-alpha-methylhistamine (CAS 75614-87-8), a potent and moderately selective agonist of histamine H3 receptors, in a series of in vitro and in vivo assays. Sch 50971 is a high affinity, selective H3 receptor agonist in vitro and in vivo. Sch 50971 inhibits [3H]-N-alpha-methylhistamine (CAS 673-50-7) binding to the histamine H3 receptor in human brain (Ki = 5.0 nmol/l) and guinea pig brain (Ki = 2.5 nmol/l). Sch 50971 also inhibits electric field stimulated guinea pig ileum contractions (pD2 = 7.47) and decreases [3H]-norepinephrine (CAS 51-41-2) release (pD2 = 7.48) from guinea pig pulmonary artery by activation of presynaptic inhibitory H3 receptors. The in vitro effects of Sch 50971 are antagonized by low concentrations of a selective H3 antagonist, thioperamide (CAS 106243-16-7). Sch 50971 has low affinity (IC50's > 10 mumol/l) for histamine H1, dopamine D1 and D2, serotonin 5-HT2 and muscarinic cholinergic receptors. It also does not exhibit histamine H2-antagonist activity. In guinea pigs and cats, Sch 50971 exhibits in vivo H3 agonist activity. Sch 50971 inhibits sympathetic hypertension evoked by stimulation of the medulla oblongata in anesthetized guinea pigs (ED30 = 0.3 mg/kg i.v., ED30 = 1.0 mg/kg i.d.). Sch 50971 also inhibits the effects of sympathetic nerve stimulation on nasal resistance in cats. In these assays, Sch 50971 exhibits an efficacy and potency comparable to H3-agonist (R)-alpha-methylhistamine. However, under in vivo conditions, Sch 50971 does not exhibit histamine H1-mediated responses that are seen with (R)-alpha-methylhistamine at doses close to those that produce H3 effects. Therefore, Sch 50971 is a novel, potent and selective agonist of histamine H3 receptors with an improved in vitro and in vivo receptor profile selectivity compared with (R)-alpha-methylhistamine.


Subject(s)
Histamine Agonists/pharmacology , Imidazoles/pharmacology , Pyrrolidines/pharmacology , Airway Resistance/drug effects , Animals , Atrial Function , Blood Pressure/drug effects , Brain/metabolism , Bronchoconstrictor Agents/pharmacology , Cats , Guinea Pigs , Heart Atria/drug effects , Histamine Agonists/pharmacokinetics , Humans , Ileum/drug effects , Ileum/physiology , Imidazoles/pharmacokinetics , In Vitro Techniques , Male , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Muscle, Smooth/physiology , Norepinephrine/metabolism , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , Pyrrolidines/pharmacokinetics , Rats , Receptors, Dopamine/metabolism , Receptors, Histamine H1/metabolism , Receptors, Histamine H3/metabolism , Receptors, Muscarinic/metabolism , Receptors, Serotonin/metabolism , Respiratory Mechanics/drug effects
9.
Mol Cell Biol ; 18(9): 5229-38, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9710607

ABSTRACT

By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. The exact mechanism of their action is, however, still largely unknown. Here we demonstrate a requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation. Expression of dominant negative forms of 14-3-3 resulted in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. Phosphorylation of Raf-1, however, was insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogated kinase activity, whereas addition of 14-3-3 restored it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions.


Subject(s)
Protein Structure, Secondary , Proteins/chemistry , Proteins/metabolism , Proto-Oncogene Proteins c-raf/chemistry , Proto-Oncogene Proteins c-raf/metabolism , Tyrosine 3-Monooxygenase , 14-3-3 Proteins , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Chloramphenicol O-Acetyltransferase/biosynthesis , Cloning, Molecular , Glutathione Transferase , Humans , Mice , Models, Molecular , Mutagenesis, Site-Directed , Phosphorylation , Phosphoserine , Phosphotyrosine , Polymerase Chain Reaction , Protein Biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Spectrum Analysis , Transfection
10.
Bioorg Med Chem Lett ; 8(3): 243-8, 1998 Feb 03.
Article in English | MEDLINE | ID: mdl-9871662

ABSTRACT

Extensive structural modification of immepyr (+)-2 led to the discovery of trans-4-methyl-3-imidazoyl pyrrolidine (+/-)-3a as a potent and highly selective H3 agonist. The pyrroline (+/-)-3a was resolved, and its (+) enantiomer, Sch 50971 [(+)-3a], showed a greater separation of H3 and H1 activities in vivo (H3/H1 ratio >> 330) than (R)-alpha-methylhistamine (+)-1 (H3/H1 ratio = 17), the standard H3 agonist.


Subject(s)
Imidazoles/pharmacology , Pyrrolidines/pharmacology , Receptors, Histamine H3/drug effects , Animals , Guinea Pigs , Ileum/drug effects , Ileum/physiology , Imidazoles/chemistry , In Vitro Techniques , Muscle Contraction/drug effects , Pyrrolidines/chemistry , Stereoisomerism , Structure-Activity Relationship
11.
Bioorg Med Chem Lett ; 8(16): 2263-8, 1998 Aug 18.
Article in English | MEDLINE | ID: mdl-9873525

ABSTRACT

A series of amidine substituted phenyl-, benzyl-, and phenethylimidazoles based on the known H3 agonist SK&F 91606 (4) has been synthesized and tested as ligands for the histamine H3 receptor. Insertion of a phenyl ring between the imidazole ring and the amidine moiety produces antagonists. The benzyl series was found to be the most potent and was further investigated. Compounds 9c and 18 (entries 5 and 12, Table 1) are potent ligands for the H3 receptor with K(i) values of 16 nM and 7.2 nM respectively. In vivo, both compounds were shown to be equipotent to thioperamide (2), the standard H3 antagonist.


Subject(s)
Benzamidines/chemical synthesis , Histamine Antagonists/chemical synthesis , Imidazoles/chemical synthesis , Receptors, Histamine H3/physiology , Animals , Benzamidines/chemistry , Benzamidines/pharmacology , Drug Design , Guinea Pigs , Histamine Antagonists/chemistry , Histamine Antagonists/pharmacology , Imidazoles/chemistry , Imidazoles/pharmacology , In Vitro Techniques , Indicators and Reagents , Kinetics , Ligands , Molecular Conformation , Molecular Structure , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Receptors, Histamine H3/drug effects
12.
J Interferon Cytokine Res ; 17(12): 747-55, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9452362

ABSTRACT

Activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) increased steady-state levels of mRNA encoding the major histocompatibility complex (MHC) class II antigen I-A beta and the class II antigen-associated invariant chain (Ii, CD74) in A20 B lymphoma cells and in normal mouse B cells. The increase in Ii mRNA levels appeared to be due to a slight increase in the rate of gene transcription and an increase in the stability of Ii mRNA. The half-life of Ii mRNA increased from 12 h to >24 h following treatment with TPA, as determined by Northern blot analysis following actinomycin D treatment or by the [3H]-uridine pulse-chase method. Interferon-gamma (IFN-gamma), which has been well characterized as a cytokine that induces class II antigens and the Ii, increased Ii expression slightly in A20 cells. However, cotreatment of cells with TPA and IFN-gamma resulted in a block in the TPA-induced increase in Ii expression. Transcription of the Ii gene was minimally affected following treatment with IFN-gamma alone, and cells treated with both TPA and IFN-gamma had the same transcription rate as the control cells. IFN-gamma did, however, block stabilization of Ii mRNA by TPA. Activation of PKC by TPA, which was previously shown to lead to membrane translocation and downregulation, was not inhibited by IFN-gamma. Therefore, IFN-gamma appeared to block a downstream signal transduction pathway activated by PKC that controls stability of Ii mRNA.


Subject(s)
Antigens, Neoplasm/genetics , Antineoplastic Agents/therapeutic use , Interferon-gamma/therapeutic use , Lymphoma, B-Cell/drug therapy , RNA, Messenger/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Animals , Antigens, Differentiation, B-Lymphocyte/genetics , Enzyme Activation , Histocompatibility Antigens Class II/genetics , Isoenzymes/drug effects , Lymphoma, B-Cell/pathology , Mice , Mice, Inbred BALB C , Protein Kinase C/drug effects , RNA, Messenger/biosynthesis , Tetradecanoylphorbol Acetate/antagonists & inhibitors , Transcription, Genetic , Tumor Cells, Cultured
13.
J Biol Chem ; 271(27): 16040-6, 1996 Jul 05.
Article in English | MEDLINE | ID: mdl-8663197

ABSTRACT

Activation of protein kinase C-delta (PKC-delta) by 12-O-tetradecanoylphorbol-13-acetate (TPA) is followed by a gradual decrease in detectable protein 12-24 h later in the mouse B lymphoma cell line A20. Down-regulation is associated with TPA-induced proteolysis and a 50-86% decrease in PKC-delta mRNA 0.5-24 h post-treatment which is due to both a 50% decrease in transcription and accelerated degradation of PKC-delta mRNA as determined using the pulse-chase method. Destabilization of PKC-delta mRNA is also observed when actinomycin D is added to cells pretreated with TPA for 2 h; however, addition of actinomycin D or cycloheximide prior to TPA treatment blocks destabilization. Addition of PKC inhibitors to TPA-treated cells also blocks destabilization of PKC-delta mRNA. Cells treated with TPA for 4 h contain an activity not found in control cells which destabilizes PKC-delta mRNA but not glyceraldehyde-3-phosphate dehydrogenase mRNA in vitro. Addition of TPA to control extracts fails to increase degradation of PKC-delta mRNA in vitro, suggesting that treatment of intact cells is required to induce the synthesis of a factor(s) that destabilizes PKC-delta mRNA. This factor(s) then acts along with transcriptional and post-translational regulatory mechanisms to down-regulate PKC-delta.


Subject(s)
Gene Expression Regulation, Enzymologic/drug effects , Isoenzymes/biosynthesis , Protein Kinase C/biosynthesis , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic/drug effects , Alkaloids , Animals , Benzophenanthridines , Blotting, Northern , Cell Line , Cell Membrane/enzymology , Cell Nucleus/metabolism , Cytosol/enzymology , Dactinomycin/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Isoenzymes/metabolism , Kinetics , Lymphoma, B-Cell , Mice , Naphthalenes/pharmacology , Phenanthridines/pharmacology , Protein Kinase C/metabolism , Protein Kinase C-delta , RNA Processing, Post-Transcriptional/drug effects , RNA, Messenger/biosynthesis , Time Factors , Tumor Cells, Cultured
14.
Mol Immunol ; 32(9): 643-50, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7643856

ABSTRACT

The invariant chain (Ii, CD74) is a transmembrane glycoprotein that is transiently associated with the MHC class II antigens in the endoplasmic reticulum and in endocytic vesicles. An activator of protein kinase C (PKC), 12-O-tetradecanoyl-1,2-phorbol 13-acetate (TPA), was found to enhance expression of Ii mRNA in the murine B lymphoma cell line, A20, 6-48 hr following treatment. In contrast, TPA did not induce the Ii in NIH 3T3 fibroblasts. TPA addition to either cell line activated PKC. Pretreatment of A20 cells with the PKC inhibitors, staurosporine or chelerythrine chloride, for 5 or 20 min prior to addition of TPA, decreased Ii mRNA levels when compared to cells treated with TPA alone. A 20 min preincubation with the highly specific PKC inhibitor, calphostin C, completely blocked the TPA enhanced expression of the Ii suggesting that activation of PKC was responsible for TPA increased Ii mRNA levels. IFN-gamma also blocked the TPA increased Ii mRNA levels. Constitutive expression of Ii mRNA was decreased by treatment with staurosporine but not chelerythrine chloride or calphostin C, suggesting that non-PKC protein kinases may also be important for maintaining high levels of Ii mRNA in these cells. Western blot analysis using PKC isotype specific antibodies showed that A20 cells express PKC delta abundantly whereas NIH 3T3 cells express primarily PKC alpha. These data suggest that a PKC delta mediated signal transduction pathway plays a crucial role in up-regulation of the Ii.


Subject(s)
Antigens, Differentiation, B-Lymphocyte/genetics , Histocompatibility Antigens Class II/genetics , Isoenzymes/metabolism , Lymphoma, B-Cell/genetics , Protein Kinase C/metabolism , 3T3 Cells , Animals , Base Sequence , DNA Primers/chemistry , Enzyme Activation , Gene Expression Regulation/drug effects , Isoenzymes/antagonists & inhibitors , Mice , Protein Kinase C/antagonists & inhibitors , Protein Kinase C-delta , RNA, Messenger/genetics , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology
15.
J Med Chem ; 38(10): 1593-9, 1995 May 12.
Article in English | MEDLINE | ID: mdl-7752184

ABSTRACT

Employing classical conformational analysis on a known H3 agonist, (R)-alpha-methylhistamine (1), a series of conformationally constrained H3 agonists were proposed and synthesized. Pyrrolidine (+/-)-4a, a compound proposed to mimic the anti-conformation of (R)-alpha-methylhistamine (1), was found to be a potent and selective H3 agonist. The pyrrolidine (+/-)-4a was resolved, and its (+) enantiomer, immepyr [(+)-4a], showed a greater separation of H3 and H1 activities in vivo (H3/H1 ratio >> 550) than (R)-alpha-methylhistamine (1) (H3/H1 ratio = 17), the standard H3 agonist. In fact, no evidence of H1 activity was detected at doses of immepyr [(+)-4a] as high as 100 mg/kg i.v. This pyrrolidine, immepyr [(2R,3S)-(+)-4a], represents, to our knowledge, the first reported cyclic, conformationally restricted analog of histamine to possess selective in vivo H3 agonist activity.


Subject(s)
Histamine Agonists , Histamine/analogs & derivatives , Pyrrolidines/chemistry , Histamine/chemistry , Histamine/pharmacology , Molecular Conformation
16.
Mol Pharmacol ; 38(5): 610-3, 1990 Nov.
Article in English | MEDLINE | ID: mdl-2172771

ABSTRACT

The H3-histamine receptor provides feedback inhibition of histamine synthesis and release as well as inhibition of other neurotransmitter release. We have characterized this receptor by radioligand binding studies with the H3 agonist N alpha-[3H]methylhistamine ([3H]NAMHA). The results of [3H]NAMHA saturation binding and NAMHA inhibition of [3H]NAMHA binding were consistent with an apparently single class of receptors (KD = 0.37 nM, Bmax = 73 fmol/mg of protein) and competition assays with other agonists and the antagonists impromidine and dimaprit disclosed only a single class of sites. In contrast, inhibition of [3H]NAMHA binding by the specific high affinity H3 antagonist thioperamide revealed two classes of sites (KiA = 5 nM, BmaxA = 30 fmol/mg of protein; KiB = 68 nM, BmaxB = 48 fmol/mg of protein). Burimamide, another antagonist that, like thioperamide, contains a thiourea group, likewise discriminated between two classes of sites. In addition to differences between some antagonist potencies for the two receptors, there is a differential guanine nucleotide sensitivity of the two. The affinity of the H3A receptor for [3H] NAMHA was reduced less than 2-fold, whereas [3H]NAMHA binding to the H3B receptor was undetectable in the presence of guanosine 5'-O-(3-thiotriphosphate). The distinction between H3A and H3B receptor subtypes, the former a high affinity and the latter a low affinity thioperamide site, draws support from published in vitro data.


Subject(s)
Methylhistamines/pharmacology , Piperidines/pharmacology , Receptors, Histamine/classification , Animals , Binding Sites/drug effects , Binding, Competitive , Brain Chemistry , Burimamide/pharmacology , Dimaprit , Guanidines/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Histamine/pharmacology , Imidazoles/pharmacology , Impromidine , Male , Radioligand Assay , Rats , Rats, Inbred Strains , Receptors, Histamine/analysis , Receptors, Histamine/drug effects , Receptors, Histamine H3 , Thiourea/pharmacology , Tritium
17.
Biochem Biophys Res Commun ; 168(3): 979-86, 1990 May 16.
Article in English | MEDLINE | ID: mdl-2161226

ABSTRACT

We have used [3H]N alpha-methylhistamine to characterize H3-binding in the guinea pig brain and to study its tissue distribution. Kinetic and equilibrium binding experiments indicate a single class of high affinity sites in membranes isolated from guinea pig brain tissue (Kd = 0.4 nM, Bmax = 41 fmol/mg of protein). Competition binding experiments have confirmed that this ligand associates with H3-receptors and, under the conditions used in these experiments, does not bind to H1- or H2-receptors. Although there was some binding in the ileum and large intestine, H3-binding was found primarily in the central nervous system.


Subject(s)
Methylhistamines/metabolism , Receptors, Histamine/metabolism , Animals , Binding, Competitive , Brain/metabolism , Guinea Pigs , Kinetics , Receptors, Histamine H1/metabolism , Receptors, Histamine H2/metabolism , Receptors, Histamine H3
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