[H-3] LY303870, A NOVEL NONPEPTIDE RADIOLIGAND FOR THE NK-1 RECEPTOR

We synthesized a potent and selective antagonist radioligand for the n eurokinin (NK)-1 receptor and characterized its binding to guinea pig striatal membranes. (R) - N - [2 - [Acetyl[H-3(3)][(2 - methoxyphenyl) methyl]amino] - 1 - (1H - indol - 3 - ylmethyl) ethyl][1,4' - bipiperi dine] - 1'-acetamide ([H-3]LY303870) binds to a single class of sites with an equilibrium K-D of 0.22 nM and a B-max of 723 fmol/mg of prote in. Unlabeled LY303870 potently inhibited the binding with an IC50 of 0.56 nM, whereas the less active (S)-enantiomer (LY306155) was substan tially less potent. The nonpeptide NK-1 antagonists (+/-)-CP96,345 and (+/-)-RP 67580 had IC50 values of 0.74 and 49 nM, respectively. Subst ance P (SP) was also a potent inhibitor with with an IC50 of 3.1 nM. T he inhibition by SP could be separated into two components: a high-aff inity component with a K-i of 0.53 nM and a lower-affinity component w ith a K-i of 155 nM. Addition of 100 mu M guanylyl 5'-imidodiphosphate [Gpp(NH)pl in the incubation increased the relative amount of the low -affinity agonist state of the receptor. Consistent with the antagonis t properties of LY303870, the dissociation rate of [H-3]-LY303870 was not changed by the presence of 100 mu M Gpp(NH)p. The distribution of [H-3]LY303870 binding sites in the guinea pig brain closely matched th e distribution of NK-1 receptors labeled by [H-3]SP. Therefore, [H-3]L Y303870 is a potent and selective antagonist radioligand for NK-1 rece ptors in guinea pig brain. In addition, regulation of NK-1 agonist aff inity by guanine nucleotides is similar to that seen for monoaminergic receptors.