Molecular cloning of bullfrog corticotropin-releasing factor (CRF): Effect of homologous CRF on the release of TSH from pituitary cells in vitro

Yoichi Ito, Reiko Okada, Hiroshi Mochida, Hiroaki Hayashi, Kazutoshi Yamamoto, Sakaé Kikuyama

    Research output: Contribution to journalArticle

    27 Citations (Scopus)

    Abstract

    Corticotropin-releasing factor (CRF) plays multiple roles in vertebrate species. In non-mammalian vertebrates, CRF has been considered to be the major thyrotropin (TSH)-releasing factor. This notion, however, was derived from experimental data on CRF of mammalian origin. Moreover, in the case of amphibians it has never been directly proved that CRF stimulates the release of TSH from the pituitary. The presently described experiment was conducted to provide direct evidence that homologous CRF enhances the release of TSH from the bullfrog (Rana catesbeiana) pituitary. First, cloning of cDNA encoding bullfrog CRF (fCRF) was accomplished. The cDNA encoding fCRF precursor was isolated from a cDNA library of the bullfrog hypothalamus. The amino acid sequence of fCRF predicted from the amplified cDNA sequence showed 83 and 95% identities with the sequences of ovine and human CRFs, respectively. An antiserum against the fCRF synthesized on the basis of the amino acid sequence was raised and used for immunohistochemical staining of the hypothalamus-hypophyseal region of the bullfrog brain. It stained some of the cell bodies situated mainly in the preoptic area, the nucleus infundibularis dorsalis and nucleus hypothalamicus ventralis and the axons that terminate in the median eminence and neural lobe. The synthetic fCRF was tested for its TSH-releasing activity toward anterior pituitary cells of adult bullfrogs in an in vitro system. As a result, the fCRF caused the release of TSH from the dispersed pituitary cells into the culture medium concentration-dependently, as measured by a specific radioimmunoassay for bullfrog TSH. The potency of the fCRF was almost equivalent to that of ovine CRF. Human urocortin III (hUCN III), a CRF receptor type 2 (CRF-R2) specific agonist enhanced the release of TSH from the pituitary cells in culture, suggesting the involvement of CRF-R2 in the CRF-induced TSH release in the bullfrogs. Culture of pituitary cells in the presence of the hypothalamic extract (HE) and α-helical CRF9-41, a CRF-R antagonist, revealed that the antagonist suppressed the TSH-releasing activity of the HE by approximately 50%, suggesting that endogenous CRF contributes as a TSH-releasing factor.

    Original languageEnglish
    Pages (from-to)218-227
    Number of pages10
    JournalGeneral and Comparative Endocrinology
    Volume138
    Issue number3
    DOIs
    Publication statusPublished - 2004 Sep 15

    Fingerprint

    Thyrotrophs
    corticotropin-releasing hormone
    Rana catesbeiana
    Corticotropin-Releasing Hormone
    Molecular Cloning
    molecular cloning
    cells
    Preoptic Area
    Complementary DNA
    Cell Culture Techniques
    hypothalamus
    Hypothalamus
    Vertebrates
    Amino Acid Sequence
    Sheep
    In Vitro Techniques
    antagonists
    cell culture
    amino acid sequences
    vertebrates

    Keywords

    • Bullfrog
    • Corticotropin-releasing factor (CRF)
    • CRF precursor cDNA
    • Hypothalamus
    • Thyrotropin (TSH)

    ASJC Scopus subject areas

    • Endocrinology

    Cite this

    Molecular cloning of bullfrog corticotropin-releasing factor (CRF) : Effect of homologous CRF on the release of TSH from pituitary cells in vitro. / Ito, Yoichi; Okada, Reiko; Mochida, Hiroshi; Hayashi, Hiroaki; Yamamoto, Kazutoshi; Kikuyama, Sakaé.

    In: General and Comparative Endocrinology, Vol. 138, No. 3, 15.09.2004, p. 218-227.

    Research output: Contribution to journalArticle

    Ito, Yoichi ; Okada, Reiko ; Mochida, Hiroshi ; Hayashi, Hiroaki ; Yamamoto, Kazutoshi ; Kikuyama, Sakaé. / Molecular cloning of bullfrog corticotropin-releasing factor (CRF) : Effect of homologous CRF on the release of TSH from pituitary cells in vitro. In: General and Comparative Endocrinology. 2004 ; Vol. 138, No. 3. pp. 218-227.
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