2 CLASSES OF BINDING-SITES FOR [H-3] SUBSTANCE-P IN RAT CEREBRAL-CORTEX

The binding characteristics of [H-3]substance P ([H-3]SP) were investigated in membranes prepared from rat cerebral cortex. Binding of [H-3]SP reached equilibrium after 50 min at 25-degrees-C and was saturable at 8 nM. Saturation data could be resolved into high affinity (equilibrium dissociation constant, K(d), 0.22 nM) and low affinity sites (K(d), 2.65 nM). The low affinity sites were more numerous than the high affinity sites, with a ratio of 4:1. The non-hydrolyzable GTP analogue GppNHp had no effect on binding, indicating that the high and low affinity sites are not guanine nucleotide-regulated states of the same (NK-1) receptor. The low affinity sites are unlikely to represent NK-3 receptors since coincubation with the selective NK-3 receptor agonist senktide did not alter the biphasic nature of [H-3]SP binding. The rank order of potency for inhibition of [H-3]SP (2 nM) binding was SP greater-than-or-equal-to [Sar9,Met(O2)11]-SP greater-than-or-equal-to physalaemin much greater than SP(3-11) > NPgamma = [Ala3]-Sp greater-than-or-equal-to SP(4-11) greater-than-or-equal-to NPK greater-than-or-equal-to (SP(5-11) greater-than-or-equal-to NKB congruent-to NKA much greater than SP(1-9), compatible with binding to an NK-1 site. N-terminal fragments and non-amidated analogues were ineffective competitors for [H-3]SP binding. However, competition data for several peptides including substance P (SP) and the NK-1 selective agonist [Sar9,Met(O2)11]-SP could be resolved into two components. Using two site LIGAND analysis, SP followed by physalaemin and [Sar9,Met(02)11]-SP were found to be the most potent competitors for the high affinity component of [H-3]SP binding, whereas SP and SP(3-11) were the most potent competitors for the low affinity component. In the presence of 1 muM SP(7-11), competition curves for both SP and [Sar9,Met(O2)11]-SP were shifted to the right and the percentage of sites representing low affinity binding was increased. The data show that [H-3]SP binds to a small population of SP- and physalaemin-prefering high affinity sites, which probably represent classical NK-1 receptors recognising the amidated C-terminus of SP. The structural requirements for binding to the much larger population of low affinity sites are not clear, but Lys3 appears to be important.