Seymour Reichlin

Thyrotropin-Releasing Hormone: Biosynthesis by Rat Hypothalamic Fragments in vitro

Biosynthesis of thyrotropin-releasing hormone (L-pyroglutamyl-L-histidyl-L-proline amide) in vitro was studied. Rat hypothalamic fragments were incubated in Krebs-Ringer bicarbonate buffer that contained either 14C-labeled proline, histidine, or glutamic acid (the three probable precursor amino acids,) and for control purposes each of 16 other naturally occurring amino acids. A number of labeled peptides were synthesized. With the use of synthetic thyrotropin-releasing hormone, detected by the Pauly reagent or with 1251-labeled thyrotropin-releasing hormone as a marker, thin-layer chromatograms, paper electrophoresis, and carboxymethyl cellulose ion exchange chromatography revealed that only proline, histidine, and glutamic acid were consistently incorporated into peptides associated with the thyrotropin-releasing hormone region. This synthesizing activity was found in stalk median eminence, ventral hypothalamus. and dorsal hypothalamus but not in neural lobe or cerebral cortex. Because the biosynthetic peptide has identical properties with L-pyroglutamyl-L-histidyl-L-proline amide, it is probable that rat thyrotropin-releasing hormone is similar or identical to both bovine and porcine thyrotropin-releasing hormone and that the native material is present in the pyroglutamyl form in tissues.

Thyrotropin Secretion in Rats after Hypothalamic Electrical Stimulation or Injection of Synthetic TSH-Releasing Factor

Plasma thyroid-stimulating hormone (TSH) levels, as measured by radioimmunoassay, begin to rise within 5 minutes after initiation of electrical stimulation of the medial-basal hypothalamus, become significantly elevated at 10 minutes, and reach a peak at 10 to 25 minutes. Intravenous administration of synthetic thyrotropin-releasing factor induces a marked rise in plasma thyroidstimulating hormone which is maximal within 5 minutes after administration. These data are interpreted to indicate that there are neuronal structures within the medial-basal hypothalamus which release preformed thyrotropin-releasing factor. The claim (based on bioassay data) that pyroglutamyl-histidyl-proline amide is a potent thyrotropin-releasing factor has also been confirmed by using a highly specific immunoassay system.

Failure of reserpine to block ether-induced release of prolactin: physiological evidence that stress induced prolactin release is not caused by acute inhibition of PIF secretion.

Abstract Plasma prolactin levels in the rat are elevated by reserpine treatment. This effect has generally been attributed to the inhibition of the monoaminergic component of regulation of secretion of prolactin inhibiting factor (PIF). In this study we report that ether stress which produces prolactin release in normal rats was equally effective in stimulating prolactin release in reserpine treated animals whose initial baseline prolactin levels were elevated. These results indicate that the ether stress induced release of prolactin is not due to inhibition of the secretion of PIF, and are compatible with the view that a prolactin releasing substance mediates this acute secretory response.

Enzymatic Synthesis of Growth Hormone Releasing Factor (GH-RF) by Rat Incubates and by Extracts of Rat and Porcine Hypothalamic Tissue

Summary Incubations of rat hypothalamic fragments and of extracts of rat and porcine hypothalamic tissue found previously to synthesize thyrotropin releasing hormone (TRH), luteinizing hormone releasing factor (LRF) and prolactin releasing factor (PRF) have been used to study the biosynthesis of growth hormone releasing factor (GH-RF). To demonstrate synthesis, GH-RF was measured by the elevation of radioimmunoassayable growth hormone in the estrogen-progesterone-reserpinized male rat. GH-RF is synthesized by rat hypothalamic fragments incubated in Krebs-Ringer bicarbonate buffer under an atmosphere of 95% oxygen and 5% carbon dioxide and by extracts of rat and porcine hypothalamic tissue in the presence of amino acids, ATP and Mg2+. This synthesis is not blocked by puromycin, indicating that the synthesizing system may not be ribosomal dependent. Further proof of a nonribosomal mechanism comes from the fact that a ribosomal-free extract also synthesizes GH-RF. From these observations, it may be concluded that GH-RF is also formed enzymatically by a nonribosomal mechanism.

Enzymatic synthesis of prolactin-releasing factor (PRF) by rat hypothalamic incubates and by extracts of rat hypothalamic tissue: evidence for "PRF synthetase".

Summary Prolactin-releasing factor (PRF), activity of rat hypothalamic fragments was increased after incubation in Krebs–Ringer bicarbonate buffer, an effect observed even when puromycin in a concentration sufficient to block new protein synthesis was added. In the presence of a mixture of twenty naturally occurring amino acids, ATP and MG2+, a dialyzed ribosome-free extract of rat hypothalamic tissue showed an increased amount of PRF activity as compared with extracts incubated in the absence of cofactors and amino acids. These observations suggest that PRF is formed by a mechanism not involving new protein synthesis. Since PRF is formed by soluble hypothalamic extracts in the presence of ATP and amino acids, it is proposed that this hypothalamic hypophysiotropic hormone is a polypeptide and is biosynthesized by a “PRF Synthetase.”