Modified kinetics of platelet-derived growth factor-induced Ca2+ increases in NIH-3T3 cells overexpressing phospholipase C gamma 1

Biochem J. 1992 Feb 1;281 ( Pt 3)(Pt 3):775-84. doi: 10.1042/bj2810775.

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 C gamma 1 (PLC gamma 1) to yield a 7-fold overexpression of this enzyme, compared with cells containing normal levels of PLC gamma 1. In a study published recently [Margolis, Zilberstein, Franks, Felder, Kremer, Ullrich, Rhee, Skorecki & Schlessinger (1990) Science 248, 607-610] it was reported that this overexpression of PLC gamma 1 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 PLC gamma 1-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 PLC gamma 1-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 PLC gamma 1-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 PLC gamma 1-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 PLC gamma 1 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 PLC gamma 1-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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bradykinin / pharmacology
  • Calcium / metabolism*
  • Calcium / pharmacology
  • Cattle
  • Cell Line
  • Cell Membrane / metabolism
  • DNA / genetics
  • Gene Expression*
  • Inositol 1,4,5-Trisphosphate / metabolism
  • Inositol Phosphates / metabolism
  • Kinetics
  • Mice
  • Platelet-Derived Growth Factor / pharmacology*
  • Transfection
  • Type C Phospholipases / genetics*
  • Type C Phospholipases / metabolism

Substances

  • Inositol Phosphates
  • Platelet-Derived Growth Factor
  • Inositol 1,4,5-Trisphosphate
  • DNA
  • Type C Phospholipases
  • Bradykinin
  • Calcium