Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells

J. W. Ramey, L. A. Krummen, W. W. Wilfinger, R. F. Highsmith, D. M. Baldwin

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 � 10-10 M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (μM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gin LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/μg iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.

Original languageEnglish (US)
Pages (from-to)1514-1520
Number of pages7
JournalEndocrinology
Volume120
Issue number4
DOIs
StatePublished - Apr 1987

Fingerprint

Luteinizing Hormone
Gonadotropin-Releasing Hormone
Calcium
Glucosamine
Glycosylation
Trichloroacetic Acid
Apoproteins
Methionine
Estradiol

ASJC Scopus subject areas

  • Endocrinology

Cite this

Ramey, J. W., Krummen, L. A., Wilfinger, W. W., Highsmith, R. F., & Baldwin, D. M. (1987). Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells. Endocrinology, 120(4), 1514-1520. https://doi.org/10.1210/endo-120-4-1514

Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells. / Ramey, J. W.; Krummen, L. A.; Wilfinger, W. W.; Highsmith, R. F.; Baldwin, D. M.

In: Endocrinology, Vol. 120, No. 4, 04.1987, p. 1514-1520.

Research output: Contribution to journalArticle

Ramey, JW, Krummen, LA, Wilfinger, WW, Highsmith, RF & Baldwin, DM 1987, 'Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells', Endocrinology, vol. 120, no. 4, pp. 1514-1520. https://doi.org/10.1210/endo-120-4-1514
Ramey, J. W. ; Krummen, L. A. ; Wilfinger, W. W. ; Highsmith, R. F. ; Baldwin, D. M. / Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells. In: Endocrinology. 1987 ; Vol. 120, No. 4. pp. 1514-1520.
@article{8c51e56662014c48a9e47be6d4d49fc3,
title = "Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells",
abstract = "The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 {\~A}� 10-10 M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium ({\^I}¼M) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278{\%}) and cells (290{\%}), as well as total (cells plus medium) 3H- Gin LH (280{\%}) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164{\%}) and total 35S-Met LH (186{\%}) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80{\%} of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/{\^I}¼g iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.",
author = "Ramey, {J. W.} and Krummen, {L. A.} and Wilfinger, {W. W.} and Highsmith, {R. F.} and Baldwin, {D. M.}",
year = "1987",
month = "4",
doi = "10.1210/endo-120-4-1514",
language = "English (US)",
volume = "120",
pages = "1514--1520",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "4",

}

TY - JOUR

T1 - Effects of a low calcium environment on luteinizing hormone biosynthesis in cultured rat anterior pituitary cells

AU - Ramey, J. W.

AU - Krummen, L. A.

AU - Wilfinger, W. W.

AU - Highsmith, R. F.

AU - Baldwin, D. M.

PY - 1987/4

Y1 - 1987/4

N2 - The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 � 10-10 M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (μM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gin LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/μg iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.

AB - The purpose of this study was to investigate the effects of lowering the extracellular calcium concentration on GnRH-stimulated LH glycosylation and LH translation, as measured by the incorporation of [3H]glucosamine (3H-Gln) and [35S]methionine (35S-Met) into immunoprecipitable LH. Cultured anterior pituitary cells, previously exposed to estradiol (5 � 10-10 M) to maximize precursor incorporation were incubated for 4 h in normal calcium (2.5 mM) or low calcium medium (μM) containing radiolabeled precursors with or without 1 nM GnRH. In the presence of normal calcium, GnRH significantly increased 3H-Gln-labeled LH in the medium (278%) and cells (290%), as well as total (cells plus medium) 3H- Gin LH (280%) compared to the control value (no GnRH). GnRH also significantly increased the 35S-Met LH released into the medium (164%) and total 35S-Met LH (186%) over control values. Depletion of extracellular calcium completely inhibited GnRH-stimulated 3H-Gln LH and 35S-Met LH production. Total immunoreactive LH (iLH), as measured by RIA, was also increased significantly by GnRH treatment in the presence of calcium, but this response was prevented by removal of calcium from the medium. Lowering extracellular calcium had no effect on cellular uptake or incorporation of 3H-Gln or 35S-Met into total trichloroacetic acid-precipitable protein. Approximately 80% of newly synthesized LH was released into the medium in all treatment groups independent of whether calcium or GnRH was present. The specific activity (disintegrations per min/μg iLH) of radiolabeled LH released into the medium was significantly reduced by treatment with GnRH due to the large amount of unlabeled iLH released into the medium. However, when the cells were incubated in low calcium, the SA of 3H-Gln LH and 35S-Met LH in the medium was unaltered by GnRH, whereas GnRH-stimulated iLH release was inhibited. We conclude that GnRH stimulation of LH glycosylation and LH apoprotein synthesis involves extracellular calcium-dependent events, and the release of newly synthesized LH is closely coupled to LH biosynthesis and is less dependent on extracellular calcium, whereas the GnRH-stimulated release of previously synthesized, stored LH is dependent on extracellular calcium.

UR - http://www.scopus.com/inward/record.url?scp=0023150608&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023150608&partnerID=8YFLogxK

U2 - 10.1210/endo-120-4-1514

DO - 10.1210/endo-120-4-1514

M3 - Article

C2 - 3549264

AN - SCOPUS:0023150608

VL - 120

SP - 1514

EP - 1520

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 4

ER -