Potent and selective tools to investigate neuropeptide Y receptors in the central and peripheral nervous systems: BIBO3304 (Y1) and CGP71683A (Y5)

2000 ◽  
Vol 78 (2) ◽  
pp. 116-125 ◽  
Author(s):  
Yvan Dumont ◽  
Alain Cadieux ◽  
Henri Doods ◽  
Alain Fournier ◽  
Rémi Quirion
Keyword(s):  
Neuropeptide Y ◽  
Rat Brain ◽  
Binding Sites ◽  
Rat Colon ◽  
Receptor Subtypes ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
High Affinity ◽  
293 Cells ◽  
Receptor Cdna

We have evaluated 3 newly developed neuropeptide Y receptor antagonists in various in vitro binding and bioassays: BIBO3304 (Y1), T4[NPY33-36]4 (Y2), and CGP71683A (Y5). In rat brain homogenates, BIBO3304 competes for the same population of [125I][Leu31,Pro34] peptide YY (PYY) binding sites (75%) as BIBP3226, but with a 10 fold greater affinity (IC50 of 0.2 ± 0.04 nM for BIBO3304 vs. 2.4 ± 0.07 nM for BIBP3226),while CGP71683A has high affinity for 25% of specific [125I][Leu31,Pro34]PYY binding sites. Both BIBO3304 and CGP71683A (at 1.0 µM) were unable to compete for a significant proportion of specific [125I]PYY3-36/Y2 sites. The purported Y2 antagonist T4[NPY33-36]4 competed against [125I]PYY3-36 binding sites with an affinity of 750 nM. These results were confirmed in HEK 293 cells transfected with either the rat Y1, Y2, Y4, or Y5 receptor cDNA. BIBO3304, but not CGP71683A, competed with high affinity for [125I][Leu31,Pro34]PYY binding sites in HEK 293 cells transfected with the rat Y1 receptor cDNA, whereas the reverse profile was observed upon transfection with the rat Y5 receptor cDNA. Additionally, both molecules were inactive at Y2 and Y4 receptor subtypes expressed in HEK 293 cells. Receptor autoradiographic studies revealed the presence of [125I][Leu31,Pro34]PYY/BIBO3304-insensitive sites in the rat brain as reported previously for BIBP3226. Finally, the selective antagonistic properties of BIBO3304 were demonstrated in a Y1 bioassay (rabbit saphenous vein; pA2 value of 9.04) while being inactive in Y2 (rat vas deferens) and Y4 (rat colon) bioassays. These results confirm the high affinity and selectivity of BIBO3304 and CGP71683A for the Y1 and Y5 receptor subtypes, respectively, while the purported Y2 antagonist, T4[NPY33-36]4 possesses rather low affinity for this receptor.Key words: NPY receptor antagonist, receptor subtypes, bioassays, receptor binding assays, autoradiographic studies, receptor distribution.

scholarly journals Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization

1993 ◽  
Vol 296 (3) ◽  
pp. 877-883 ◽  
Author(s):  
M Cik ◽  
P L Chazot ◽  
F A Stephenson
Keyword(s):  
Rat Brain ◽  
Biochemical Characterization ◽  
Radioligand Binding ◽  
Fold Increase ◽  
Binding Activity ◽  
Immunological Methods ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Adult Rat ◽  
293 Cells

The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.

Cloning of Trp1β isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein

1999 ◽  
Vol 276 (4) ◽  
pp. C969-C979 ◽  
Author(s):  
Weiching Wang ◽  
Brian O’Connell ◽  
Raymond Dykeman ◽  
Takayuki Sakai ◽  
Christine Delporte ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Protein A ◽  
Protein S ◽  
Polyclonal Antiserum ◽  
Cell Fractionation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Mouse Tissues

The Trp gene product has been proposed as a candidate protein for the store-operated Ca2+channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1β cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1β cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523–536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted with a protein of ∼92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell.

Inhibition of Na+-K+-2Cl−cotransport by mercury

1999 ◽  
Vol 277 (4) ◽  
pp. C684-C692 ◽  
Author(s):  
Steven C. Jacoby ◽  
Edith Gagnon ◽  
Luc Caron ◽  
John Chang ◽  
Paul Isenring
Keyword(s):  
Ion Transport ◽  
Binding Sites ◽  
Essential Role ◽  
Species Differences ◽  
Transmembrane Domain ◽  
Transport Proteins ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH−) groups. The inorganic form (Hg2+) is toxic to epithelial tissues and interacts with various transport proteins including the Na+ pump and Cl− channels. In this study, we determined whether the Na+-K+-Cl−cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg2+ on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg2+inhibits Na+-K+-Cl−cotransport, with inhibitor constant ( K i) values of 25 μM for the shark carrier (sNKCC1) and 43 μM for the human carrier. In further studies, we took advantage of species differences in Hg2+ affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697→Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+binding. We also show that modification of additional SH− groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.

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