Modified kinetics of platelet-derived growth factor-induced Ca2+ increases in NIH-3T3 cells overexpressing phospholipase Cγ1

D. C. Renard, M. M. Bolton, S. G. Rhee, B. L. Margolis, A. Zilberstein, J. Schlessinger, Andrew Thomas

Research output: Contribution to journalArticle

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Abstract

The effects of platelet-derived growth factor (PDGF) on cytosolic free Ca2+ concentration ([Ca2+](i)) and inositol phosphates were studied in NIH-3T3 fibroblasts transfected with cDNA for phospholipase Cγ1 (PLCγ1) to yield a 7-fold overexpression of this enzyme, compared with cells containing normal levels of PLCγ1. In a study published recently [Margolis, Zilberstein, Franks, Felder, Kremer, Ullrich, Rhee, Skorecki and Schlessinger (1990) Science 248, 607-610] it was reported that this overexpression of PLCC1 caused a specific potentiation of the inositol phosphate response to PDGF, but this was not associated with an enhancement of the [Ca2+](i) response. In the present study, measurements of the time course and isomeric profile of PDGF-induced inositol phosphate formation demonstrated that the initial rate of Ins(1,4,5)P3 formation was also enhanced in the PLCγ1-overexpressing cells, yielding a 10-fold greater increase at 1 min compared with the parental NIH-3T3 cells. By contrast, bradykinin-induced phosphoinositide metabolism was unchanged in PLCγ1-transfected cells. Measurements of [Ca2+](i) in cell populations and single cells showed a significant latent period following PDGF addition prior to the [Ca2+](i) increases in both cell lines, which decreased in a dose-dependent manner with increasing PDGF concentration. The duration of the latent period was decreased and the maximal rate of [Ca2+](i) rise was increased in the PLCγ1-overexpressing cells at all doses of PDGF examined. In single-cell measurements these cells also responded to PDGF with a greater peak amplitude of [Ca2+](i). Both intracellular Ca2+ mobilization and Ca2+ influx across the plasma membrane were enhanced in the PLCγ1-overexpressing cells. There was no difference between the two cell lines in either the latency or the magnitude of the [Ca2+](i) increases induced by bradykinin. These data provide further evidence that PLCC1 is responsible for the PDGF-induced stimulation of Ins(1,4,5)P3 formation. Moreover, in contrast to earlier conclusions, the modified kinetics of the [Ca2+](i) changes in PLCγ1-overexpressing cells suggest that Ins(1,4,5)P3 does play a predominant second messenger role in the PDGF-induced [Ca2+](i) increases. The data also indicate that the latent period may be a function of the time required to reach a threshold level of Ins(1,4,5)P3, rather than an intrinsic property of the PDGF receptor.

Original languageEnglish (US)
Pages (from-to)775-784
Number of pages10
JournalBiochemical Journal
Volume281
Issue number3
DOIs
StatePublished - Jan 1 1992
Externally publishedYes

Fingerprint

NIH 3T3 Cells
Platelet-Derived Growth Factor
Kinetics
Inositol Phosphates
Cells
Bradykinin
phospholipase C1
Platelet-Derived Growth Factor Receptors
Cell Line
Second Messenger Systems
Fibroblasts
Cell membranes
Phosphatidylinositols
Metabolism
Complementary DNA
Cell Membrane

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Cite this

Renard, D. C. ; Bolton, M. M. ; Rhee, S. G. ; Margolis, B. L. ; Zilberstein, A. ; Schlessinger, J. ; Thomas, Andrew. / Modified kinetics of platelet-derived growth factor-induced Ca2+ increases in NIH-3T3 cells overexpressing phospholipase Cγ1. In: Biochemical Journal. 1992 ; Vol. 281, No. 3. pp. 775-784.
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Modified kinetics of platelet-derived growth factor-induced Ca2+ increases in NIH-3T3 cells overexpressing phospholipase Cγ1. / Renard, D. C.; Bolton, M. M.; Rhee, S. G.; Margolis, B. L.; Zilberstein, A.; Schlessinger, J.; Thomas, Andrew.

In: Biochemical Journal, Vol. 281, No. 3, 01.01.1992, p. 775-784.

Research output: Contribution to journalArticle

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T1 - Modified kinetics of platelet-derived growth factor-induced Ca2+ increases in NIH-3T3 cells overexpressing phospholipase Cγ1

AU - Renard, D. C.

AU - Bolton, M. M.

AU - Rhee, S. G.

AU - Margolis, B. L.

AU - Zilberstein, A.

AU - Schlessinger, J.

AU - Thomas, Andrew

PY - 1992/1/1

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N2 - The effects of platelet-derived growth factor (PDGF) on cytosolic free Ca2+ concentration ([Ca2+](i)) and inositol phosphates were studied in NIH-3T3 fibroblasts transfected with cDNA for phospholipase Cγ1 (PLCγ1) to yield a 7-fold overexpression of this enzyme, compared with cells containing normal levels of PLCγ1. In a study published recently [Margolis, Zilberstein, Franks, Felder, Kremer, Ullrich, Rhee, Skorecki and Schlessinger (1990) Science 248, 607-610] it was reported that this overexpression of PLCC1 caused a specific potentiation of the inositol phosphate response to PDGF, but this was not associated with an enhancement of the [Ca2+](i) response. In the present study, measurements of the time course and isomeric profile of PDGF-induced inositol phosphate formation demonstrated that the initial rate of Ins(1,4,5)P3 formation was also enhanced in the PLCγ1-overexpressing cells, yielding a 10-fold greater increase at 1 min compared with the parental NIH-3T3 cells. By contrast, bradykinin-induced phosphoinositide metabolism was unchanged in PLCγ1-transfected cells. Measurements of [Ca2+](i) in cell populations and single cells showed a significant latent period following PDGF addition prior to the [Ca2+](i) increases in both cell lines, which decreased in a dose-dependent manner with increasing PDGF concentration. The duration of the latent period was decreased and the maximal rate of [Ca2+](i) rise was increased in the PLCγ1-overexpressing cells at all doses of PDGF examined. In single-cell measurements these cells also responded to PDGF with a greater peak amplitude of [Ca2+](i). Both intracellular Ca2+ mobilization and Ca2+ influx across the plasma membrane were enhanced in the PLCγ1-overexpressing cells. There was no difference between the two cell lines in either the latency or the magnitude of the [Ca2+](i) increases induced by bradykinin. These data provide further evidence that PLCC1 is responsible for the PDGF-induced stimulation of Ins(1,4,5)P3 formation. Moreover, in contrast to earlier conclusions, the modified kinetics of the [Ca2+](i) changes in PLCγ1-overexpressing cells suggest that Ins(1,4,5)P3 does play a predominant second messenger role in the PDGF-induced [Ca2+](i) increases. The data also indicate that the latent period may be a function of the time required to reach a threshold level of Ins(1,4,5)P3, rather than an intrinsic property of the PDGF receptor.

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