Dataset Information

Modification of phospholipase C-gamma-induced Ca2+ signal generation by 2-aminoethoxydiphenyl borate.

ABSTRACT: The mechanisms by which Ca(2+)-store-release channels and Ca(2+)-entry channels are coupled to receptor activation are poorly understood. Modification of Ca(2+) signals by 2-aminoethoxydiphenyl borate (2-APB), suggests the agent may target entry channels or the machinery controlling their activation. In DT40 B-cells and Jurkat T-cells, complete Ca(2+) store release was induced by 2-APB (EC(50) 10-20 microM). At 75 microM, 2-APB emptied stores completely in both lymphocyte lines, but had no such effect on other cells. In DT40 cells, 2-APB mimicked B-cell receptor (BCR) cross-linking, but no effect was observed in mutant DT40 lines devoid of inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) or phospholipase C-gamma2 (PLC-gamma2). Like the BCR, 2-APB activated transfected TRPC3 (canonical transient receptor potential) channels, which acted as sensors for PLC-gamma2-generated diacylglycerol in DT40 cells. The action of 2-APB on InsP(3)Rs and TRPC3 channels was prevented by PLC-inhibition, and required PLC-gamma2 catalytic activity. However, unlike BCR activation, no increased InsP(3) level could be measured in response to 2-APB. Also, calyculin A-induced cytoskeletal reorganization prevented 2-APB-induced InsP(3)R and TRPC3-channel activation, but not that induced by the BCR. 2-APB still activated TRPC3 channels in DT40 cells with fully depleted Ca(2+) stores, indicating its action was not via Ca(2+) release. Significantly, 2-APB-induced InsP(3)R and TRPC3 activation was prevented in DT40 knockout cells devoid of the BCR- and PLC-gamma2-coupled adaptor/kinases, Syk, Lyn, Btk or BLNK. The results suggest that 2-APB activates Ca(2+) signals in lymphocytes by initiating and enhancing coupling between components of the BCR-PLC-gamma2 complex and both Ca(2+)-entry and Ca(2+)-release channels.

SUBMITTER: Ma HT

PROVIDER: S-EPMC1223825 | biostudies-other | 2003 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description: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.

Dataset Information

Modification of phospholipase C-gamma-induced Ca2+ signal generation by 2-aminoethoxydiphenyl borate.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets