Divalent cations influencing neuropeptide Y receptor subtype binding in rat hippocampus and cortex membranes as well as in recombinant cells

Abstract

At least six types of neuropeptide Y (NPY) receptors (Y1–Y6) have been pharmacologically distinguished of which only the Y1, Y2, Y4 and Y5 subtypes have been thoroughly characterized. In order to further classify receptor subtypes in the brain, we performed receptor binding studies using rat cortical and hippocampal membranes and, in particular, studied the effects of different ion compositions of the buffer on the binding behaviour of several NPY agonists and the Y1 receptor antagonist BIBO3304. Ca 2+ was necessary for reliable Y1 receptor subtype classification in rat cortical membranes (with Hill coefficients close to unity) for the peptide agonists. This was further substantiated by the Y1 selective antagonist BIBO3304 displaying an IC 50 value of 0.9±0.5 nM for 80% of the total receptors, the remaining sites being BIBO3304 insensitive (IC 50>10 000 nM). Replacing Ca 2+ by Mn 2+ resulted in a complete loss of BIBO3304 sensitive sites. On the other hand, using hippocampal membrane preparations, displacement curves with Hill coefficients close to unity were only obtained in the presence of Mn 2+ ions, yielding a binding profile of receptors with low affinity for [Leu 31, Pro 34]NPY (IC 50=50 nM) and for BIBO3304 (IC 50>10 000 nM). Addition of Mn 2+ ions to cortical or of Ca 2+ ions to hippocampal membrane preparations resulted in binding profiles differing from typical receptor classification. Therefore, the influence of divalent cations on Y1 receptors expressed on recombinant cells was studied. In this monoreceptor system, Ca 2+ was necessary to detect high amounts of specific binding and Mn 2+ ions induced a change in the affinity state. These findings indicate that apparent NPY receptor heterogeneity does not only depend on the brain region examined and that divalent ions modulate ligand binding properties.