Effectors of the frequency of calcium oscillations in HEK-293 cells: wavelet analysis and a computer model.
Oscillations of the intracellular concentration of Ca(2+) in cultured HEK-293 cells, which heterologously expressed the calcium-sensing receptor, were recorded with the fluorophore Fura-2 using fluorescence microscopy. HEK-293 cells are extremely sensitive to small perturbations in extracellular calcium concentrations. Resting cells were attached to cover slips and perifused with saline solution containing physiologically relevant extracellular Ca(2+) concentrations in the range 0.5-5 mM. Acquired digitized images of the cells showed oscillatory fluctuations in the intracellular Ca(2+) concentration over the time course, and were processed as a function of the change in Fura-2 excitation ratio and frequency at 12-37 degrees C. Newly developed data processing techniques with wavelet analysis were used to estimate the frequency at which the rectified sinusoidal oscillations occurred; we estimated ~4 min(-1) under normal conditions. Temperature variations revealed an Arrhenius relationship in oscillation frequency. A critical Ca(2+) concentration of ~2 mM was estimated, below which oscillations did not occur. These data were used to develop a kinetic model of the system that was simulated using Mathematica; kinetic parameter values were adjusted to match the experimentally observed oscillations of intracellular Ca(2+) concentration as a function of extracellular Ca(2+) concentration, and temperature; and from these, limit cycles were obtained and control coefficients were estimated for all parameters.
Szekely
David
D
School of Molecular and Microbial Biosciences, University of Sydney, Building G08, Sydney, NSW, 2006, Australia.
Brennan
Sarah C
SC
Mun
Hee-Chang
HC
Conigrave
Arthur D
AD
Kuchel
Philip W
PW
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D003198:Computer Simulation; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D044169:Receptors, Calcium-Sensing / Q000378:metabolism; D013696:Temperature
D044169:Receptors, Calcium-Sensing; D002118:Calcium