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Journal of Neuroscience, Vol 6, 1796-1802, Copyright © 1986 by Society for Neuroscience

ARTICLE

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

CC Glombotski, S Manaker, A Winokur and TR Gibson

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.(ABSTRACT TRUNCATED AT 250 WORDS)

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