p72, a Marker Protein for Melatonin Action in Ovine Pars tuberalis Cells

Peter John Morgan, Perry Barrett, G DAVIDSON, W LAWSON, David Grey Hazlerigg

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The function of the pars tuberalis as a mediator of the action of melatonin remains elusive. As a direct method of assessing the potential role of secretory proteins, ovine pars tuberalis cells have been cultured and radiolabelled with S-35-methionine, and the accumulation of specific radioactive products in the medium, measured after separation by SDS-PAGE and fluorography. The synthesis and secretion of a number of labelled proteins are increased by forskolin (1 muM) and inhibited dose dependently by melatonin (IC50, 300 pM), although consistently a 72-kD protein (p72), is the most intensely labelled of these. Thus, p72 acts as a useful marker of cellular activity for melatonin, whereas prolactin (p23) provides a melatonin non-responsive marker in ovine pars tuberalis cell cultures. The synthesis and secretion of p72 and other melatonin-sensitive proteins is regulated through the cyclic AMP/protein kinase A second-messenger pathway, as analogues of cyclic AMP mimic the action of forskolin, yet 1,9-dideoxyforskolin, a forskolin analogue that is not active on adenylate cyclase, has no effect. However, the phorbol ester, phorbol-12,13-myristate acetate, also regulates the synthesis and secretion of the same profile of proteins as forskolin indicating a potential role for protein kinase C, which occurs through an independent rather than a synergistic pathway. The differential effects of nocadazole (1 muM) and extracellular calcium depletion upon p72 and prolactin secretion indicates that p72 is secreted by a calcium and microtubule independent pathway, in contrast to prolactin. These observations in conjunction with the absence of densecore storage vesicles in melatonin-responsive cells of the ovine PT are consistent with constitutive secretion of p72 from the latter and regulated secretion of prolactin from melatonin non-responsive cells. Using immunoprecipitation de novo synthesis and secretion of either LH or LH-like proteins from ovine pars tuberalis cells could not be detected under the conditions used. The absence of I-125-(Des-Gly10 [D-Ala6]-LHRH-ethylamide) binding over most, but not all, of the ovine pars tuberalis supports the contention that the majority of the cells of the ovine pars tuberalis are not gonadotrophs. These results provide further support for the unique function for the pars tuberalis.

Original languageEnglish
Pages (from-to)325-335
Number of pages11
JournalNeuroendocrinology
Volume59
Issue number4
DOIs
Publication statusPublished - Apr 1994

Keywords

  • melatonin
  • pars tuberalis
  • pituitary
  • secretion
  • protein synthesis
  • luteinizing hormone
  • luteinizing hormone-Rreleasing hormone
  • gonadotropes
  • rat pituitary-cells
  • cyclic-amp
  • binding-sites
  • adenylate-cyclase
  • forskolin
  • localization
  • receptors
  • mechanism
  • camp
  • ultrastructure

Cite this

p72, a Marker Protein for Melatonin Action in Ovine Pars tuberalis Cells. / Morgan, Peter John; Barrett, Perry; DAVIDSON, G ; LAWSON, W ; Hazlerigg, David Grey.

In: Neuroendocrinology, Vol. 59, No. 4, 04.1994, p. 325-335.

Research output: Contribution to journalArticle

Morgan, Peter John ; Barrett, Perry ; DAVIDSON, G ; LAWSON, W ; Hazlerigg, David Grey. / p72, a Marker Protein for Melatonin Action in Ovine Pars tuberalis Cells. In: Neuroendocrinology. 1994 ; Vol. 59, No. 4. pp. 325-335.
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N2 - The function of the pars tuberalis as a mediator of the action of melatonin remains elusive. As a direct method of assessing the potential role of secretory proteins, ovine pars tuberalis cells have been cultured and radiolabelled with S-35-methionine, and the accumulation of specific radioactive products in the medium, measured after separation by SDS-PAGE and fluorography. The synthesis and secretion of a number of labelled proteins are increased by forskolin (1 muM) and inhibited dose dependently by melatonin (IC50, 300 pM), although consistently a 72-kD protein (p72), is the most intensely labelled of these. Thus, p72 acts as a useful marker of cellular activity for melatonin, whereas prolactin (p23) provides a melatonin non-responsive marker in ovine pars tuberalis cell cultures. The synthesis and secretion of p72 and other melatonin-sensitive proteins is regulated through the cyclic AMP/protein kinase A second-messenger pathway, as analogues of cyclic AMP mimic the action of forskolin, yet 1,9-dideoxyforskolin, a forskolin analogue that is not active on adenylate cyclase, has no effect. However, the phorbol ester, phorbol-12,13-myristate acetate, also regulates the synthesis and secretion of the same profile of proteins as forskolin indicating a potential role for protein kinase C, which occurs through an independent rather than a synergistic pathway. The differential effects of nocadazole (1 muM) and extracellular calcium depletion upon p72 and prolactin secretion indicates that p72 is secreted by a calcium and microtubule independent pathway, in contrast to prolactin. These observations in conjunction with the absence of densecore storage vesicles in melatonin-responsive cells of the ovine PT are consistent with constitutive secretion of p72 from the latter and regulated secretion of prolactin from melatonin non-responsive cells. Using immunoprecipitation de novo synthesis and secretion of either LH or LH-like proteins from ovine pars tuberalis cells could not be detected under the conditions used. The absence of I-125-(Des-Gly10 [D-Ala6]-LHRH-ethylamide) binding over most, but not all, of the ovine pars tuberalis supports the contention that the majority of the cells of the ovine pars tuberalis are not gonadotrophs. These results provide further support for the unique function for the pars tuberalis.

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KW - pituitary

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KW - luteinizing hormone

KW - luteinizing hormone-Rreleasing hormone

KW - gonadotropes

KW - rat pituitary-cells

KW - cyclic-amp

KW - binding-sites

KW - adenylate-cyclase

KW - forskolin

KW - localization

KW - receptors

KW - mechanism

KW - camp

KW - ultrastructure

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JO - Neuroendocrinology

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