DIVALENT-CATIONS INFLUENCING NEUROPEPTIDE-Y RECEPTOR SUBTYPE BINDING IN RAT HIPPOCAMPUS AND CORTEX MEMBRANES AS WELL AS IN RECOMBINANT CELLS

At least six types of neuropeptide Y (NPY) receptors (Y1-Y6) have been pharmacologically distinguished of which only the Y1, Y2, Y4 and Y5 s ubtypes have been thoroughly characterized. In order to further classi fy receptor subtypes in the brain, we performed receptor binding studi es using rat cortical and hippocampal membranes and, in particular, st udied the effects of different ion compositions of the buffer on the b inding behaviour of several NPY agonists and the Y1 receptor antagonis t BIBO3304. Ca2+ was necessary for reliable Y1 receptor subtype classi fication in rat cortical membranes (with Hill coefficients close to un ity) for the peptide agonists. This was further substantiated by the Y 1 selective antagonist BIBO3304 displaying an IC50 value of 0.9+/-0.5 nM for 80% of the total receptors, the remaining sites being BIBO3304 insensitive (IC50 > 10 000 nM). Replacing Ca2+ by Mn2+ resulted in a c omplete loss of BIBO3304 sensitive sites. On the other hand, using hip pocampal membrane preparations, displacement curves with Hill coeffici ents close to unity were only obtained in the presence of Mn2+ ions, y ielding a binding profile of receptors with low affinity for [Leu(31), Pro(34)]NPY (IC50 = 50 nM) and for BIBO3304 (IC50 > 10 000 nM). Addit ion of Mn2+ ions to cortical or of Ca2+ ions to hippocampal membrane p reparations resulted in binding profiles differing from typical recept or classification. Therefore, the influence of divalent cations on Y1 receptors expressed on recombinant cells was studied. In this monorece ptor system, Ca2+ was necessary to detect high amounts of specific bin ding and Mn2+ ions induced a change in the affinity state. These findi ngs indicate that apparent NPY receptor heterogeneity does not only de pend on the brain region examined and that divalent ions modulate liga nd binding properties. (C) 1998 Elsevier Science BN. All rights reserv ed.