Calcium homeostasis in the presence of fura-2 in neurons dissociated from rat nucleus basalis: theoretical and experimental analysis of chelating action of fura-2.
Intracellular calcium ions (Ca2+) play important roles in cell functions. Measurements of intracellular calcium ion concentration ([Ca2+]i) are often made with the fura-2 fluorescence recording technique in various preparations including neurons. Fura-2 has, however, a Ca(2+)-chelating action which complicates the interpretation of experimental results. In this report the chelating action of intracellular fura-2 was studied by means of computer simulations. The chelating action of an endogenous Ca(2+)-binding protein, calmodulin, was also estimated. Furthermore, whole-cell patch-clamp recordings of calcium currents (ICa) and fura-2 microfluorimetric recordings of [Ca2+]i were simultaneously made from neurons which were acutely dissociated from the rat nucleus basalis. Since Ca2+ influx can be initiated and terminated by using the voltage-clamp technique, the relationship between Ca2+ influx and rapid [Ca2+]i increase was examined. The present theoretical evaluations and experimental results disclosed the relationship between fura-2 and endogenous Ca(2+)-binding proteins; fura-2 at low concentration (10 microM) did not substantially affect the endogenous Ca2+ buffering mechanisms, but at high concentration (200 microM) effectively buffered cytosolic Ca2+ instead of endogenous Ca2+ buffers. Calcium homeostasis in neurons is furthermore discussed.
Tatsumi
H
H
Department of Autonomic Physiology, Tokyo Medical and Dental University, Japan.
Katayama
Y
Y
D000818:Animals; D001479:Basal Ganglia / Q000187:drug effects / Q000378:metabolism*; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology*; D003470:Culture Media; D004558:Electric Stimulation; D016257:Fura-2 / Q000494:pharmacology*; D006706:Homeostasis / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence
D002021:Buffers; D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D003470:Culture Media; D002118:Calcium; D016257:Fura-2