Detection of Ca(2+)-transients elicited by flash photolysis of DM-nitrophen with a fast calcium indicator.
A confocal spot detection optical setup was used to record fluorescence signals in response to calcium pulses, elicited by flash photolysis of DM-nitrophen, with the calcium indicators CaOrange-5N and Fluo-3. Our results yield the following conclusions: [Ca2+] changes are almost perfect spikes at pCa 9 and broader transients followed by a step at pCa 7. The [Ca2+] spikes were used to measure the dissociation rate constant of the Ca2+ dyes. Experiments at pCa 7 were used to verify the kinetic rate constants of the dyes and to obtain those of DM-nitrophen. The association rate constant of this compound was found to be more than one order of magnitude faster than that suggested previously. CaOrange-5N was able to track changes in [Ca2+] more accurately than Fluo-3. This latter dye introduced severe distortions which preclude a quantitative deconvolution of the fluorescence transients into changes in the free [Ca2+].
Escobar
A L
AL
Department of Physiology, UCLA School of Medicine 90024, USA.
Cifuentes
F
F
Vergara
J L
JL
D000085:Acetates / Q000737:chemistry*; D000814:Aniline Compounds; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002614:Chelating Agents; D005029:Ethylenediamines / Q000737:chemistry*; D005456:Fluorescent Dyes; D007700:Kinetics; D009930:Organic Chemicals; D010782:Photolysis; D014966:Xanthenes
D000085:Acetates; D000814:Aniline Compounds; C093686:CaOrange-5N; D002614:Chelating Agents; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D014966:Xanthenes; C057014:DM-nitrophen; C059715:Fluo-3; D002118:Calcium