Quantitative determination of free calcium in subcellular compartments, as probed by Indo-1 and confocal microspectrofluorometry.
Confocal UV-microspectrofluorometry has been applied to measure fluorescence emission spectra of Indo-1 for the intracellular determination of free calcium ([Ca2+]i). To perform in situ calibrations of [Ca2+]i in the nucleus and the cytoplasm, Indo-1 has been loaded into living cells under its esterified form Indo-1/AM. For each controlled [Ca2+]i, intranuclear and intracytoplasmic spectra show a systematic blue shift, as compared with spectra in solution at the same [Ca2+]. In the Ca2+ saturated condition, the intranuclear spectra are more blue-shifted than in the cytoplasm. Thus, distinct in situ calibration curves have been distinguished for the nucleus and the cytoplasm. To calculate [Ca2+]i, intracellular spectra of Indo-1 have been characterized by two distinct methods. First, the ratio of emission intensities at 410 and 500 nm has been determined. Secondly, the analyzed spectrum has been decomposed into a linear combination of in situ free and Ca-bound Indo-1 reference spectra from the considered cellular compartment. Satisfactory spectral decompositions have been observed for each [Ca2+]i. The subcellular calibration curves allow one to determine the product of both intracellular constants, i.e. the ratio of quantum yields between free and Ca-bound Indo-1 and the apparent dissociation constant for Ca2+. The product of these constants has been shown to be similar in both subcellular compartments and in a buffered solution. The two methods used for the [Ca2+]i determination lead to equivalent results on unpermeabilized KB cells. They show a 1.3 times higher [Ca2+]i in the cytoplasm than in the nucleus (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Millot
J M
JM
Laboratoire de Spectroscopie Biomoléculaire, UFR de Pharmacie, Reims, France.
Pingret
L
L
Angiboust
J F
JF
Bonhomme
A
A
Pinon
J M
JM
Manfait
M
M
D002118:Calcium / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007211:Indoles / Q000737:chemistry; D007624:KB Cells; D018613:Microscopy, Confocal / Q000379:methods; D015388:Organelles / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
D007211:Indoles; C048960:indo-1; D002118:Calcium