Ascorbic acid increases the thyrotropin-releasing hormone content of hypothalamic cell cultures

• Published in: The Journal of neuroscience 1986-06, Vol.6 (6), p.1796-1802
• Main Authors: Glombotski, CC, Manaker, S, Winokur, A, Gibson, TR
• Format: Article
• Language: English
• Subjects: Animals; Applied sciences; Ascorbic Acid - pharmacology; Biological and medical sciences; Cells, Cultured; Exact sciences and technology; Fetus - metabolism; Fundamental and applied biological sciences. Psychology; Hormones and neuropeptides. Regulation; Hypothalamus - drug effects; Hypothalamus - immunology; Hypothalamus. Hypophysis. Epiphysis. Urophysis; Other techniques and industries; Pyrrolidonecarboxylic Acid - analogs & derivatives; Rats; Rats, Inbred Strains; Thyrotropin-Releasing Hormone - analogs & derivatives; Thyrotropin-Releasing Hormone - immunology; Thyrotropin-Releasing Hormone - metabolism; Vertebrates: endocrinology
• ISSN: 0270-6474; 1529-2401
• DOI: 10.1523/jneurosci.06-06-01796.1986

Thyrotropin-releasing hormone (TRH) is one of many COOH-terminal alpha-amidated neuropeptides. Recent work with the intermediate pituitary has indicated that ascorbate is a required cofactor for the COOH-terminal alpha-amidation of alpha-melanotropin. This is consistent with the ascorbate requirement of an enzyme found in pituitary and hypothalamus capable of converting peptides with a COOH-terminal glycine (-X-Gly) to alpha-amidated molecules (-H-NH2). Thus, it has been proposed that COOH-terminal glycine-extended TRH (TRH-Gly) may be the direct precursor to TRH. In the present study, primary hypothalamic cultures supplemented with ascorbate for 7 d contained two- to threefold more TRH immunoactivity (amide-specific) than cultures maintained without ascorbate. A dose-response experiment indicated that 20 microM ascorbate was capable of producing 50% of the maximum observable increase in culture TRH immunoactivity; this concentration is similar to the Km value for ascorbate uptake obtained in adrenal chromaffin and pituitary cells. A stereoisomer of ascorbate, D-isoascorbate, was also capable of producing an increase in TRH immunoactivity, but oxidized ascorbate was not. Recent studies have shown that the amidation enzyme from pituitary is capable of utilizing both L-ascorbate and D-isoascorbate but is incapable of utilizing oxidized ascorbate. The culture extracts were analyzed further by reversed-phase high-performance liquid chromatography; the increased TRH immunoactivity observed in extracts of cultures maintained in ascorbate comigrated with standard synthetic TRH.