Studies on the increase in cytosolic free calcium induced by epidermal growth factor, serum, and nucleotides in individual A431 cells.
The response of cytosolic calcium [Ca2+]i to epidermal growth factor (EGF), fetal calf serum, and nucleotides was determined in individual A431 cells, using the fluorescent probe fura-2 and quantitative digital video fluorescence microscopy. In the presence of 1 mM external Ca2+, EGF caused a rapid rise in [Ca2+]i, followed by a slower and variable decrease. The cells responded after a lag that varied from 10 to 30 seconds, and there was considerable cell-to-cell variation in extent of the rise in [Ca2+]i. A second challenge with EGF gave negative results. No response was obtained in nominally Ca2+-free medium supplemented with 100 microM EGTA. Somewhat similar results were obtained with fetal calf serum except that a rise in [Ca2+]i was observed both in the presence and absence of external Ca2+. The A431 cells responded to external ATP with a rise in [Ca2+]i in less than 10 seconds, both in Ca2+-containing and Ca2+-free media. A coverslip with attached cells was mounted on a small chamber, allowing complete change of medium in 2 seconds. A nearly full response was obtained with only 10 seconds of contact of cells with ATP-containing medium. After washing out ATP, there was little or no response to a second addition given 100 seconds after the first. However, a second response was obtained when the concentration of agonist was increased 10-20-fold. These data favor the idea of receptor desensitization. Both homologous and heterologous receptor desensitization was observed. A transient rise in [Ca2+]i was also noted with UTP, while ITP and CTP were inactive.
Gonzalez
F A
FA
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Gross
D J
DJ
Heppel
L A
LA
Webb
W W
WW
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001572:Benzofurans; D001769:Blood; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003470:Culture Media; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D016257:Fura-2; D007700:Kinetics; D012265:Ribonucleotides / Q000494:pharmacology*
D001572:Benzofurans; D003470:Culture Media; D012265:Ribonucleotides; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2