scholarly journals Epidermal growth factor-stimulated calcium ion transients in individual A431 cells: initiation kinetics and ligand concentration dependence.

1991 ◽  
Vol 2 (10) ◽  
pp. 827-840 ◽  
Author(s):  
T E Cheyette ◽  
D J Gross
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Calcium Ion ◽  
Cytosolic Calcium ◽  
Ion Concentration ◽  
Lag Phase ◽  
High Dose ◽  
A431 Cells ◽  
Epidermal Growth ◽  
Calcium Ion Concentration

The A431 epidermoid carcinoma cell line responds to epidermal growth factor (EGF) stimulation with a number of rapid changes, including alterations in free cytosolic calcium ion concentration ([Ca2+]i). At the single cell level, these changes in [Ca2+]i are known to proceed after a clear lag phase subsequent to EGF stimulus (Gonzalez et al., 1988). The present study explores the dependence on EGF concentration of this early [Ca2+]i signal. High levels of EGF (9.0-4.3 nM) produce a [Ca2+]i spike followed by an elevation of [Ca2+]i above basal levels. The time of initiation of the spike varies from 5 to 9 s at the high dose and from 8 to 32 s at the low dose in cells that respond. A lower level of EGF (1.5 nM) produces [Ca2+]i oscillations with no prolonged elevation over basal [Ca2+]i. The initiation of response at this [EGF] ranges from 20 to 410 s. Intermediate stimulus levels generate [Ca2+]i responses that are kinetic admixtures of these limiting responses. A simple model based on the enzymatically amplified signal cascade from ligand binding through Ca2+ release or influx is examined. The model predicts a prolonged lag phase followed by a rapid increase in the [CA2+]i signal that compares favorably with the data reported here.

Membrane vesicles of A431 cells contain one class of epidermal growth factor binding sites

1990 ◽  
Vol 1052 (3) ◽  
pp. 453-460 ◽  
Author(s):  
Jos A.M. Berkers ◽  
Paul M.P. van Bergen en Henegouwen ◽  
Arie J. Verkleij ◽  
Johannes Boonstra
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Sites ◽  
Membrane Vesicles ◽  
A431 Cells ◽  
Factor Binding ◽  
Epidermal Growth

Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP

1992 ◽  
Vol 12 (12) ◽  
pp. 5816-5823
Author(s):  
D Park ◽  
S G Rhee
Keyword(s):  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Immunoglobulin M ◽  
Rat Tissues ◽  
A431 Cells ◽  
Sh3 Domains ◽  
Epidermal Growth ◽  
Stimulation Of

The 47-kDa protein coimmunoprecipitated with phospholipase C (PLC)-gamma 1 by anti-PLC-gamma 1 monoclonal antibodies is proved to be Nck, a protein composed almost exclusively of one SH2 and three SH3 domains. Nck and PLC-gamma 1 are recognized by certain anti-PLC-gamma 1 monoclonal antibodies because Nck and PLC-gamma 1 share an epitope that likely is located in their SH3 domains. Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remains unchanged during the development of rat brain, whereas PLC-gamma 1 decreases during the same developmental period. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signalling proteins.

Fura-2 fluorescence is localized to mitochondria in endothelial cells

1987 ◽  
Vol 253 (5) ◽  
pp. C744-C747 ◽  
Author(s):  
S. F. Steinberg ◽  
J. P. Bilezikian ◽  
Q. Al-Awqati
Keyword(s):  
Endothelial Cells ◽  
Calcium Ion ◽  
Cytosolic Calcium ◽  
Ion Concentration ◽  
Homogeneous Distribution ◽  
Aortic Endothelial Cells ◽  
Fura 2 ◽  
Bovine Aortic Endothelial Cells ◽  
Calcium Ion Concentration ◽  
Aortic Endothelial

The new, highly fluorescent, calcium-sensitive dye, fura-2, can be loaded nondisruptively into intact cells by means of its permeant ester and used to measure the free calcium ion concentration in individual cells. For fura-2 to signal cytosolic calcium, it must be distributed homogeneously and exclusively throughout the cytoplasmic space. However, microscopic examination of bovine aortic endothelial cells loaded with fura-2 by exposure to its permeant ester reveals fluorescence associated with discrete intracellular structures rather than the homogeneous distribution expected for a cytosolic stain. Simultaneous labeling of bovine aortic endothelial cells with fura-2 and rhodamine 123 (a mitochondrial fluorescent vital stain) identifies these structures as mitochondria. Subcellular dye localizations are not observed when the cells are loaded with other putative cytosolic stains that gain access to the cytosol by means of a membrane permeant ester. Both carboxyfluorescein and indo-1 (another member of the family of second generation calcium indicators) stain the cytoplasm diffusely. It is suggested that fura-2 fluorescence accumulates in certain cells in association with mitochondria. It is important to assess the intracellular distribution of fura-2 when this indicator is used to measure the free cytosolic calcium ion concentration.

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