Modified kinetics of platelet-derived growth factor-induced Ca2+ increases in NIH-3T3 cells overexpressing phospholipase C gamma 1.
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.
Renard
D C
DC
Thomas Jefferson University, Philadelphia, PA 19107.
Bolton
M M
MM
Rhee
S G
SG
Margolis
B L
BL
Zilberstein
A
A
Schlessinger
J
J
Thomas
A P
AP
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics* / Q000378:metabolism
D007295:Inositol Phosphates; D010982:Platelet-Derived Growth Factor; D015544:Inositol 1,4,5-Trisphosphate; D004247:DNA; D010738:Type C Phospholipases; D001920:Bradykinin; D002118:Calcium