Thyrotropin-Releasing Hormone Receptors in the Pituitary of Rainbow Trout (Oncorhynchus mykiss)

Abstract

Binding parameters of radiolabeled pGlu-3-Me-His2-pro-NH2 ([3H]MeTRH) to pituitary TRH receptors of rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) were investigated. Washed pituitary membranes were incubated with [3H]MeTRH in the absence (B0) or presence of TRH or TRH analogs under various paradigms. Specific binding (Bsp) was thermolabile and tissue dependent. Association of Bsp was slow (k+1 = 1.43 × 107M-1 min-1) achieving equilibrium binding after 60 min, and remaining stable for at least 60 min. Thereafter, equilibrium-bound [3H]MeTRH was dissociated by addition of excess MeTRH; estimated dissociation rate constant (k-1) and half-life (t1/2) were 6.74 × 10-2 min-1 and 10.2 min, respectively, with a kinetically derived dissociation constant (k-1/k+1) of 4.71 × 10-9M. [3H]MeTRH binding was differentially displaced by TRH and TRH analogs. LIGAND analysis of multiple homologous (MeTRH) and heterologous (TRH) displacement experiments indicates the presence of a single class of binding sites with nanomolar affinity. Data pooled from multiple heterologous (TRH) displacement experiments suggested the presence of an additional class of higher-affinity (picomolar) binding sites. Additionally, examination of separate preparations of neurointermediate lobes and pars distalis from trout and goldfish suggested that these two classes of binding sites were differentially located between the two lobes. Specificity analysis of the trout pituitary TRH receptor indicates that the rank order of potency for [3H]MeTRH displacement was MeTRH > TRH > pGlu-Phe-Pro-NH2. Analogs with N- or C-terminal substitutions had little competitive potential. This study indicates the presence and binding characteristics of specific TRH receptors in the salmonid and cyprinid pituitary gland.