Binding of [3H](2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl) glycine ([3H]LY341495) to cell membranes expressing recombinant human group III metabotropic glutamate receptor subtypes.
LY341495 is a highly potent and selective antagonist for group II mGlu receptors (mGlu2 and mGlu3). High affinity binding of [3H]LY341495 to recombinant human group II mGlu receptors (mGlu2 and mGlu3), and in rat brain homogenates (Kd approximately 1 nM), has been previously described. Although LY341495 is a very selective nM-potent antagonist for group II mGlu receptors, it is also a relatively potent antagonist for group III mGlu receptors at high nanomolar to low micromolar concentrations. In this study we examined and characterized the binding of [3H]LY341495 to membranes of cells expressing recombinant human group III mGlu receptors. Using up to 100 nM of [3H]LY341495, the level of specific binding in human mGlu4a receptor-expressing cell membranes was not appreciable and binding to this site was not examined further. In contrast, we demonstrated sufficient specific binding of [3H]LY341495 to human mGlu6, mGlu7a and mGlu8a receptor-expressing cell membranes to allow for further characterizations. [3H]LY341495 binding was saturable and rapidly reversible. [3H]LY341495 bound to a single site in each cell line, with Kd and Bmax values of 31.6+/-6.8 nM and 3.3+/-0.7 pmol/mg protein (mGlu6), 72.7+/-22.0 nM and 3.7+/-0.4 pmol/mg protein (mGlu7a), and 14.0+/-1.1 nM and 3.0+/-0.2 pmol/mg protein (mGlu8a). [3H]LY341495 binding to mGlu6, 7a and 8a was displaceable by compounds which interact functionally with group III mGlu receptors. For example, L-AP4 displaced [3H]LY341495 with Ki values of 6.8+/-3.1 microM (mGlu6), 211+/-43 microM (mGlu7a) and 1.6+/-0.3 microM (mGlu8a). With L-glutamate, we obtained Ki values of 12.3+/-3.5, 869+/-154 and 4.5+/-0.83 microM, for mGlu6, mGlu7a and mGlu8a, respectively. Ki values for unlabelled LY341495 were 0.058+/-0.008, 0.22+/-0.05 and 0.029+/-0.008 microM, respectively. These studies demonstrated that [3H]LY341495 is a useful radioligand for studying the pharmacology and expression of recombinant mGlu6, 7a and 8a receptors in cell lines.
Wright
R A
RA
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.
Arnold
M B
MB
Wheeler
W J
WJ
Ornstein
P L
PL
Schoepp
D D
DD
D000596:Amino Acids / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011869:Radioligand Assay; D018094:Receptors, Metabotropic Glutamate / Q000096:biosynthesis / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014316:Tritium; D014966:Xanthenes / Q000378:metabolism* / Q000494:pharmacology
D000596:Amino Acids; D018691:Excitatory Amino Acid Antagonists; C114624:LY 341495; D018094:Receptors, Metabotropic Glutamate; D011994:Recombinant Proteins; D014966:Xanthenes; C113897:metabotropic glutamate receptor 6; C102719:metabotropic glutamate receptor 7; C111435:metabotropic glutamate receptor 8; D014316:Tritium; C108823:metabotropic glutamate receptor 4