Klaus-Dieter Willim

BIBP 3226, the first selective neuropeptide Y1 receptor antagonist: A review of its pharmacological properties

Based on the assumption that the pharmacophoric groups interacting with the Y1 receptor are located in the C-terminal part of neuropeptide Y, low molecular weight compounds with high affinity and selectivity for the Y1 receptor were designed and synthesized. The prototype BIBP 3226 possesses affinity for the Y1 receptor in the nanomolar range. In addition, this compound is selective displaying rather low affinity for Y2, Y3, Y4 and a set of 60 other receptors. Both biochemical and pharmacological studies showed that BIBP 3226 behaves as a competitive antagonist. Using BIBP 3226 it was possible to investigate the role of NPY and/or Y1 receptors in blood pressure regulation. The interesting observation was that antagonism to Y1 receptors had no major influence on the basal blood pressure but attenuated stress induced hypertension. This strongly supports the hypothesis that NPY is mainly released during stress involving intense sympathetic nervous system activation. Moreover, BIBP 3226 can be used to characterize NPY receptor subtypes. For instance, we were able to show that presynaptic NPY receptors mediating catecholamine release do not solely belong to the Y2 subtype, but that presynaptic Y1 receptors also exist. In conclusion, BIBP 3226 has been shown to be an important tool for the elucidation of the physiological role of Y1 receptors in the cardiovascular system.

Labeling of neuropeptide Y receptors in SK-N-MC cells using the novel, nonpeptide Y1 receptor-selective antagonist [3H]BIBP3226

The binding of tritium-labelled BIBP3226, N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide, to human neuroblastoma SK-N-MC cells was investigated. [3H]BIBP3226 reversibly binds to neuropeptide Y receptors of the Y1 subtype expressed in SK-N-MC cells with a KD of 2.1 +/- 0.3 nM (mean +/- S.E.M., n = 3) and a Bmax of 58,400 +/- 1100 sites/cell. Non-specific binding did not exceed 30% of the total radioactivity bound at KD. In competition experiments [3H]BIBP3226 is concentration-dependently displaced by neuropeptide Y and its peptide analogues with an affinity pattern neuropeptide Y = [Leu31, Pro34]neuropeptide Y >> neuropeptide Y-(18-36). This rank order of potencies is consistent with the interaction of [3H]BIBP3226 with neuropeptide Y receptors of the Y1 subtype. Therefore, [3H]BIBP3226 can be used as selective ligand to study neuropeptide Y Y1 receptors.

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 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. Ca2+ 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 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 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 Mn2+ ions, yielding a binding profile of receptors with low affinity for [Leu31,Pro34]NPY (IC50 = 50 nM) and for BIBO3304 (IC50 > 10,000 nM). Addition of Mn2+ ions to cortical or of Ca2+ 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, Ca2+ was necessary to detect high amounts of specific binding and Mn2+ 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.