Specific binding of [ 3H]pGlu-3-MeHisProNH 2 ([ 3H]MeTRH) to hypothalamic membranes of juvenile rainbow trout, Oncorhynchus mykiss

Abstract

We investigated the existence and nature of specific [ 3H]pGlu-3-MeHisProNH 2 ([ 3H]MeTRH) binding sites in juvenile rainbow trout ( Oncorhynchus mykiss) hypothalamus. Washed hypothalamic membranes were incubated with [ 3H]MeTRH in the absence (B 0) or presence of pGluHisProNH 2 (TRH) or MeTRH under various experimental paradigms; incubations were terminated by filtration and bound radioactivity was determined by liquid scintillation spectroscopy. Specific binding (B sp) was tissue dependent, associable, dissociable, and thermolabile. Estimated rates of association (k +1) and dissociation (k −1) were 1.64 × 10]su7 M −1 min −1 and 1.98 × 10 −2 min −1, respectively, providing a kinetically derived dissociation rate constant (K d) of 1.21 × 10 −9 M. [ 3H]MeTRH binding was displaceable; LIGAND-analysis of three independent homologous displacement experiments consistently indicated a single class of binding sites with an average K d = 6.91 (4.32) × 10 −9M and average maximum binding capacity (B max) of 8.84 (±2.72) × 10 −15mol/mg protein. Native TRH also displaced the radiolabel in a dose dependent manner; LIGAND-estimates for K d and B max were 1.52 (±0.12) × 10 −9M and 3.79 (±0.99) × 10 −15mol/mg protein (n = 3 experiments), respectively. Our data indicate the presence of a single class of specific high-affinity TRH-binding sites in the rainbow trout hypothalamus; these findings suggest a role for TRH in regulating the release of hypophysiotrophic factors in the teleost hypothalamus.