An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells

J. D. Veldhuis, L. M. Demers

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

We have used primary cultures of swine granulosa cells to investigate the regulatory role of the protein kinase C pathway in the ovary. In this system, we observed the following. (1) Swine granulosa cells bound [3H]phorbol 12,13-dibutyrate ([3H]PDB) specifically with high affinity [apparent K(i) for 12-O-tetradecanoylphorbol 13-acetate (TPA) = 3.1 (2.1-4.7) nM] and low capacity [0.68 (0.34-0.99) pmol/107 cells]. (2) The cytosol of granulosa cells contained functionally active protein kinase C capable of phosphorylating distinct proteins in response to stimulation with active phorbol ester. (3) TPA and PDB induced dose-dependent inhibition (>85%) of follicle-stimulating-hormone (FSH)-stimulated progesterone production. Half-maximally inhibitory concentrations were 0.10 and 0.75 nM for TPA and PDB respectively, whereas phorbol analogues that do not activate protein kinase C were not inhibitory. (4) TPA did not impede cyclic AMP generation in response to FSH, cholera toxin or forskolin acutely (within 48 h), but did inhibit the stimulatory effects of 8-bromo cyclic AMP, insulin and oestradiol on progesterone biosynthesis. (5) In the presence of maximally effective concentrations of 25-hydroxy, 20α-hydroxy- or 22R-hydroxy-cholesterol as exogenous sterol substrates for cholesterol side-chain cleavage, treatment with TPA suppressed pregnenolone, progesterone and 20α-hydroxypregn-4-en-3-one biosynthesis by more than 80%. (6) The inhibitory effects of phorbol esters were not attributable to non-specific cytotoxicity, since prostaglandin F(2α) production increased in the same cultures and aromatization or exogenously supplied testosterone to oestradiol was not suppressed. (7) In intact granulosa cells, the effects of phorbol esters were mimicked by a synthetic non-diterpene diacylglycerol, 1-octanoyl-2-acetylglycerol, and the tumour promoter, mezerein, which specifically activates protein kinase C. We conclude that swine granulosa cells contain specific high-affinity receptors for phorbol esters that are functionally coupled to protein phosphorylation. Moreover, treatment with phorbol esters or non-phorbol activators of protein kinase C results in selective inhibition of cholesterol side-chain cleavage activity without impairing cyclic AMP generation or oestrogen biosynthesis.

Original languageEnglish (US)
Pages (from-to)505-511
Number of pages7
JournalBiochemical Journal
Volume239
Issue number3
DOIs
StatePublished - Jan 1 1986

Fingerprint

Granulosa Cells
Tetradecanoylphorbol Acetate
Protein Kinase C
Phorbol Esters
Acetates
Swine
Biosynthesis
Progesterone
Cholesterol
Follicle Stimulating Hormone
Cyclic AMP
Estradiol
Phorbol 12,13-Dibutyrate
8-Bromo Cyclic Adenosine Monophosphate
Aromatization
Pregnenolone
Phosphorylation
Cholera Toxin
Diglycerides
Prostaglandins F

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{077757205f654cb38eb5371a137cc55d,
title = "An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells",
abstract = "We have used primary cultures of swine granulosa cells to investigate the regulatory role of the protein kinase C pathway in the ovary. In this system, we observed the following. (1) Swine granulosa cells bound [3H]phorbol 12,13-dibutyrate ([3H]PDB) specifically with high affinity [apparent K(i) for 12-O-tetradecanoylphorbol 13-acetate (TPA) = 3.1 (2.1-4.7) nM] and low capacity [0.68 (0.34-0.99) pmol/107 cells]. (2) The cytosol of granulosa cells contained functionally active protein kinase C capable of phosphorylating distinct proteins in response to stimulation with active phorbol ester. (3) TPA and PDB induced dose-dependent inhibition (>85{\%}) of follicle-stimulating-hormone (FSH)-stimulated progesterone production. Half-maximally inhibitory concentrations were 0.10 and 0.75 nM for TPA and PDB respectively, whereas phorbol analogues that do not activate protein kinase C were not inhibitory. (4) TPA did not impede cyclic AMP generation in response to FSH, cholera toxin or forskolin acutely (within 48 h), but did inhibit the stimulatory effects of 8-bromo cyclic AMP, insulin and oestradiol on progesterone biosynthesis. (5) In the presence of maximally effective concentrations of 25-hydroxy, 20α-hydroxy- or 22R-hydroxy-cholesterol as exogenous sterol substrates for cholesterol side-chain cleavage, treatment with TPA suppressed pregnenolone, progesterone and 20α-hydroxypregn-4-en-3-one biosynthesis by more than 80{\%}. (6) The inhibitory effects of phorbol esters were not attributable to non-specific cytotoxicity, since prostaglandin F(2α) production increased in the same cultures and aromatization or exogenously supplied testosterone to oestradiol was not suppressed. (7) In intact granulosa cells, the effects of phorbol esters were mimicked by a synthetic non-diterpene diacylglycerol, 1-octanoyl-2-acetylglycerol, and the tumour promoter, mezerein, which specifically activates protein kinase C. We conclude that swine granulosa cells contain specific high-affinity receptors for phorbol esters that are functionally coupled to protein phosphorylation. Moreover, treatment with phorbol esters or non-phorbol activators of protein kinase C results in selective inhibition of cholesterol side-chain cleavage activity without impairing cyclic AMP generation or oestrogen biosynthesis.",
author = "Veldhuis, {J. D.} and Demers, {L. M.}",
year = "1986",
month = "1",
day = "1",
doi = "10.1042/bj2390505",
language = "English (US)",
volume = "239",
pages = "505--511",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "3",

}

An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells. / Veldhuis, J. D.; Demers, L. M.

In: Biochemical Journal, Vol. 239, No. 3, 01.01.1986, p. 505-511.

Research output: Contribution to journalArticle

TY - JOUR

T1 - An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells

AU - Veldhuis, J. D.

AU - Demers, L. M.

PY - 1986/1/1

Y1 - 1986/1/1

N2 - We have used primary cultures of swine granulosa cells to investigate the regulatory role of the protein kinase C pathway in the ovary. In this system, we observed the following. (1) Swine granulosa cells bound [3H]phorbol 12,13-dibutyrate ([3H]PDB) specifically with high affinity [apparent K(i) for 12-O-tetradecanoylphorbol 13-acetate (TPA) = 3.1 (2.1-4.7) nM] and low capacity [0.68 (0.34-0.99) pmol/107 cells]. (2) The cytosol of granulosa cells contained functionally active protein kinase C capable of phosphorylating distinct proteins in response to stimulation with active phorbol ester. (3) TPA and PDB induced dose-dependent inhibition (>85%) of follicle-stimulating-hormone (FSH)-stimulated progesterone production. Half-maximally inhibitory concentrations were 0.10 and 0.75 nM for TPA and PDB respectively, whereas phorbol analogues that do not activate protein kinase C were not inhibitory. (4) TPA did not impede cyclic AMP generation in response to FSH, cholera toxin or forskolin acutely (within 48 h), but did inhibit the stimulatory effects of 8-bromo cyclic AMP, insulin and oestradiol on progesterone biosynthesis. (5) In the presence of maximally effective concentrations of 25-hydroxy, 20α-hydroxy- or 22R-hydroxy-cholesterol as exogenous sterol substrates for cholesterol side-chain cleavage, treatment with TPA suppressed pregnenolone, progesterone and 20α-hydroxypregn-4-en-3-one biosynthesis by more than 80%. (6) The inhibitory effects of phorbol esters were not attributable to non-specific cytotoxicity, since prostaglandin F(2α) production increased in the same cultures and aromatization or exogenously supplied testosterone to oestradiol was not suppressed. (7) In intact granulosa cells, the effects of phorbol esters were mimicked by a synthetic non-diterpene diacylglycerol, 1-octanoyl-2-acetylglycerol, and the tumour promoter, mezerein, which specifically activates protein kinase C. We conclude that swine granulosa cells contain specific high-affinity receptors for phorbol esters that are functionally coupled to protein phosphorylation. Moreover, treatment with phorbol esters or non-phorbol activators of protein kinase C results in selective inhibition of cholesterol side-chain cleavage activity without impairing cyclic AMP generation or oestrogen biosynthesis.

AB - We have used primary cultures of swine granulosa cells to investigate the regulatory role of the protein kinase C pathway in the ovary. In this system, we observed the following. (1) Swine granulosa cells bound [3H]phorbol 12,13-dibutyrate ([3H]PDB) specifically with high affinity [apparent K(i) for 12-O-tetradecanoylphorbol 13-acetate (TPA) = 3.1 (2.1-4.7) nM] and low capacity [0.68 (0.34-0.99) pmol/107 cells]. (2) The cytosol of granulosa cells contained functionally active protein kinase C capable of phosphorylating distinct proteins in response to stimulation with active phorbol ester. (3) TPA and PDB induced dose-dependent inhibition (>85%) of follicle-stimulating-hormone (FSH)-stimulated progesterone production. Half-maximally inhibitory concentrations were 0.10 and 0.75 nM for TPA and PDB respectively, whereas phorbol analogues that do not activate protein kinase C were not inhibitory. (4) TPA did not impede cyclic AMP generation in response to FSH, cholera toxin or forskolin acutely (within 48 h), but did inhibit the stimulatory effects of 8-bromo cyclic AMP, insulin and oestradiol on progesterone biosynthesis. (5) In the presence of maximally effective concentrations of 25-hydroxy, 20α-hydroxy- or 22R-hydroxy-cholesterol as exogenous sterol substrates for cholesterol side-chain cleavage, treatment with TPA suppressed pregnenolone, progesterone and 20α-hydroxypregn-4-en-3-one biosynthesis by more than 80%. (6) The inhibitory effects of phorbol esters were not attributable to non-specific cytotoxicity, since prostaglandin F(2α) production increased in the same cultures and aromatization or exogenously supplied testosterone to oestradiol was not suppressed. (7) In intact granulosa cells, the effects of phorbol esters were mimicked by a synthetic non-diterpene diacylglycerol, 1-octanoyl-2-acetylglycerol, and the tumour promoter, mezerein, which specifically activates protein kinase C. We conclude that swine granulosa cells contain specific high-affinity receptors for phorbol esters that are functionally coupled to protein phosphorylation. Moreover, treatment with phorbol esters or non-phorbol activators of protein kinase C results in selective inhibition of cholesterol side-chain cleavage activity without impairing cyclic AMP generation or oestrogen biosynthesis.

UR - http://www.scopus.com/inward/record.url?scp=0022828950&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0022828950&partnerID=8YFLogxK

U2 - 10.1042/bj2390505

DO - 10.1042/bj2390505

M3 - Article

C2 - 3103602

AN - SCOPUS:0022828950

VL - 239

SP - 505

EP - 511

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 3

ER -