Pituitary thyrotropin-releasing hormone (TRH) receptors: effects of TRH, drugs mimicking TRH action, and chlordiazepoxide

Binding of TRH to specific cell surface receptors on clonal GH4C1 cells is followed within 10 min by receptor sequestration and over 24 h by receptor down-regulation. These experiments were designed to determine if TRH-activated second messenger systems are responsible for changes in receptor localization or number. BAY K8644 and A23187, which increase intracellular calcium, alone or together with 12-O-tetradecanoyl phorbol acetate (TPA), which activates protein kinase C, did not appear to internalize TRH receptors. Drug treatment did not alter the rate of 3H]MeTRH association or internalization, determined by resistance to an acid/salt wash, or the amount of 3H]MeTRH able to bind at 0 C, where only surface receptors are accessible. TPA (0-100 nM) alone or in combination with BAY K8644 or A23187, also failed to change receptor number or affinity after 48 h when TRH caused a 75% decrease in the density of specific binding sites. Chlordiazepoxide has been reported antagonize TRH binding and TRH-induced phospholipid breakdown. Chlordiazepoxide shifted the dose-response curves for TRH stimulation of PRL release and synthesis to the right, and did not change PRL release alone. The affinity of receptors for chlordiazepoxide was not affected by a nonhydrolyzable analog of GTP whereas affinity for TRH was decreased; these properties are consistent with the classification of chlordiazepoxide as a competitive antagonist. Several experiments tested whether chlordiazepoxide would cause receptor internalization and down-regulation. Chlordiazepoxide did not appear to internalize TRH receptors, because TRH-binding sites became available rapidly and at the same rate after they had been saturated with chlordiazepoxide at 0 or 37 C.(ABSTRACT TRUNCATED AT 250 WORDS)