Effects of GTP analogs and metal ions on the binding of neurotensin to porcine brain membranes.

Using 125I-labeled neurotensin (NT), porcine brain membranes were found to contain two types of high-affinity receptors, one class (approximately 1/3 of total) with an apparent Kd of 0.12 nM and another with an apparent Kd of 1.4 nM. Nonhydrolyzable analogs of GTP inhibited NT binding in a dose-dependent manner. In the presence of 60 microM guanosine 5'-(3-thio) 5'-(beta, gamma-imino) triphosphate. NT binding was decreased by 35% with an associated decrease in the number of binding sites and little change in the Kd. Cross-linking of 125I-labeled NT to brain membranes using disuccinimidyl suberate was found to specifically label two substances of approximately 120 kDa and approximately 160 kDa, which could represent different binding proteins or complexes. For a series of NT analogs, there was close agreement between the IC50 in the binding assay and the ED50 in a bioassay based on ability to contract the guinea pig ileum. In addition, metal ions inhibited NT binding and the contractile action of NT with the same order of potency (Hg++ > Zn++ > Cu++ > Mn++ > Mg++ > Li++). There was a linear relationship between the standard reduction potential for these ions and the logarithm of the IC50 in the binding assay. The results suggest that porcine brain contains high-affinity, G-protein-linked receptors for NT, the functioning of which depends upon group(s), perhaps sulfhydryl(s), which can interact strongly with certain heavy metal ions.