Non-competitive binding of the nonpeptide antagonist BIBP3226 to rat forebrain neuropeptide Y1 receptors.
[3H]Neuropeptide Y labelled neuropeptide Y receptors in rat forebrain membranes as a homogenous class of high-affinity sites. Between 80 and 85% of these receptors showed high affinity for Y1-selective antagonists such as (R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine amide (BIBP3226). While competitive in functional studies, BIBP3226 produced parallel shifts of the Scatchard plots of [3H]neuropeptide Y saturation binding in rat forebrain membranes. Mechanisms which are routinely invoked to explain non-competitive binding do not apply to BIBP3226. Wash-out experiments, involving successive treatment of the membranes with BIBP3226, buffer (wash-out step) and [3H]neuropeptide Y, argue against irreversible or a pseudo-irreversible binding of the antagonist. Allosteric inhibition is also unlikely since BIBP3226 did not affect the rate of dissociation of [3H]neuropeptide Y in isotope dilution experiments. The non-hydrolyzable guanine nucleotide, 5'-guanylylimidodiphosphate (Gpp(NH)p), abolished the binding of [3H]neuropeptide Y and increased its rate of dissociation in isotope dilution experiments. This suggests that the initial [3H]neuropeptide Y-receptor association is a low affinity process and that the observed binding of [3H]neuropeptide Y is related to the formation of a ternary [3H]neuropeptide Y-receptor-G protein complex. Two- or even multistate models (in which BIBP3226 could potentially behave as an inverse agonist) could therefore be needed to explain the non-competitive antagonism of BIBP3226 in broken cell preparations.
Vanderheyden
P M
PM
Department of Protein Chemistry, Free University of Brussels (VUB), Sint-Genesius Rode, Belgium.
Van Liefde
I
I
De Backer
J P
JP
Vauquelin
G
G
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006207:Half-Life; D066298:In Vitro Techniques; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009478:Neuropeptide Y / Q000378:metabolism; D016548:Prosencephalon / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017476:Receptors, Neuropeptide Y / Q000037:antagonists & inhibitors / Q000378:metabolism*
C092926:BIBP 3226; D009419:Nerve Tissue Proteins; D009478:Neuropeptide Y; D011506:Proteins; D017476:Receptors, Neuropeptide Y; D006165:Guanylyl Imidodiphosphate; D001120:Arginine