First pharmacological characterization of TRH receptors linked to phosphoinositide hydrolysis in GH3 pituitary cells using agonist specificity of eight TRH analogs.

The agonist/antagonist properties of TRH and 8 TRH analogs were ascertained in GH3 cells using accumulation of [3H]inositol phosphates ([3H]IPs) as an index of receptor activation. All TRH analogs, except diketopiperazine (DKP), were full agonists producing similar maximum stimulation (6.5 +/- 1.1-fold) of [3H]IP production. Concentrations of peptides producing half-maximal stimulation of phosphoinositide (PI) hydrolysis were (nM; means +/- SEM): MeTRH (2.4 +/- 0.4); MK-771 (7.3 +/- 0.6); TRH (26.6 +/- 9.2); RX77368 (90.2 +/- 13.9); CG3703 (274.5 +/- 104.4); N-Val2-TRH (2400 +/- 870); CG3509 (16500 +/- 3400); TRH free acid (17.3, 11.0 microM), DKP (greater than 1 mM). The rank order of potency of TRH analogs at inducing PI turnover was similar to that for competition of [3H]MeTRH binding to brain and pituitary homogenates reported previously, thus indicating the identification of functional TRH receptors. These data suggest that while the modifications of the C- and N-termini of the TRH molecule (resulting in MK-771, RX77368, CG3703, CG3509) reduce the apparent affinities of these compounds, the latter still retain considerable agonist activity, and in the case of MK-771 remain equipotent or become slightly more potent than TRH. This study, therefore, constitutes the first to demonstrate the biological activity of the 8 peptide analogs of TRH using the PI turnover technique in cultured clonal cells.