Release of Thyrotropin and Prolactin by a Thyrotropin-Releasing Hormone (TRH) Precursor, TRH-Gly: Conversion to TRH Is Sufficient for in vivo Effects

Abstract

The mechanism by which TRH-Gly (pGlu-His-Pro-Gly), a biosynthetic precursor of thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH₂), stimulates pituitary thyrotropin (TSH) and prolactin release has been studied in urethane-anesthetized 2-month-old (250 g) male Sprague-Dawley rats and in vitro with GH<Sub>3</Sub> cells, a rat anterior pituitary tumor cell line. We used specific radioimmunoassays to measure TRH-Gly and TRH levels in rat cortex, hypothalamus, medulla, eyes and whole blood as a function of the intracisternal (IC) dose of TRH and TRH-Gly administered 40 min prior to sacrifice. IC injection of 1.0 mg of TRH-Gly led to a significant (p < 0.005) increase in the TRH levels in hypothalamus, medulla and blood. The relative potency of IC and intracardiac (IK) TRH and TRH-Gly release of rat TSH was compared by radioimmunoassay and further refined using estimates based on in vivo kinetics of TRH-Gly alpha-amidation. The binding of TRH-Gly to the plasma membrane receptors for TRH on GH₃ cells was also investigated. In regard to TSH release, TRH-Gly given IC had only 0.042% of the potency of TRH given IC and was consistent with its rate of IC alpha-amidation. IK TRH-Gly had 0.16% of the potency of IK TRH of TSH release and was also consistent with its rate of intravascular conversion to TRH. The mean peak TSH response occurred at 20 min after IC TRH-Gly or IC TRH injection but the post-peak decline was slower for IC TRH-Gly. The maximum levels of blood TRH-Gly and TRH both occurred at 20 min after IC administration of TRH-Gly and TRH, respectively. The agreement between the directly measured relative potency for TSH release following IC TRH-Gly and that derived from the rate of IC TRH-Gly conversion to TRH suggests that IC TRH-Gly must first be converted to TRH by alpha-amidating enzymes in the cerebrospinal fluid before it transits the blood-brain barrier. TRH-Gly cross-reactivity with the TRH receptor of GH₃ cells was 0.45% of that for TRH while the in vitro potency of prolactin release by GH₃ cells in response to TRH-Gly was 0.41% that for TRH. GH₃ cells incubated for 3 h with or without 50 µg TRH-Gly/ml revealed no significant increase in extracellular or intracellular TRH concentration. We conclude that conversion of TRH-Gly to TRH is the most likely explanation for the stimulation of TSH release in rats but in vitro release of prolactin from GH₃ cells in response to TRH-Gly administration may be mediated via TRH-Gly cross-reaction with the TRH receptor.