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A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca(2+) Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.

Lee JH, Ha JM, Leem CH - Korean J. Physiol. Pharmacol. (2015)

Bottom Line: With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM.However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+).Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Ulsan College of Medicine/Asan Medical Center, Seoul 138-736, Korea.

ABSTRACT
Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca(2+)]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca(2+)], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca(2+)] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca(2+)], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca(2+)] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM. This 1 µM cytosolic Ca(2+) could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+). Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

No MeSH data available.


Results of NADH and Fura-2-FF interference correction. The left panels show the signals prior to correction and the right panels show the signals after correction. NADH and the ratio of Fura-2-FF signals were changed following correction. The calculated [Ca2+]m was substantially decreased. (A) NADH signals at 450-nm emission. (B) Fura-2-FF signals at 500-nm emission. The figure shows F400,500 (- -), F353,500 (----), and the ratio of Fura-2-FF (-). (C) The mitochondrial calcium concentration calculated from the Fura-2-FF ratio.
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Figure 8: Results of NADH and Fura-2-FF interference correction. The left panels show the signals prior to correction and the right panels show the signals after correction. NADH and the ratio of Fura-2-FF signals were changed following correction. The calculated [Ca2+]m was substantially decreased. (A) NADH signals at 450-nm emission. (B) Fura-2-FF signals at 500-nm emission. The figure shows F400,500 (- -), F353,500 (----), and the ratio of Fura-2-FF (-). (C) The mitochondrial calcium concentration calculated from the Fura-2-FF ratio.

Mentions: Fig. 8 shows the differences in Ca2+ concentrations before and after the correction. The results clearly showed the importance of correction for NADH and Fura-2-FF interference. The mitochondrial resting calcium concentration was 1.03±0.13 µM (mean±S.E., n=32) and the maximum [Ca2+]m at 1 µM cytosolic Ca2+ ([Ca2+]c) was 29.6±1.61 µM (mean±S.E., n=33) (Fig. 9).


A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca(2+) Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.

Lee JH, Ha JM, Leem CH - Korean J. Physiol. Pharmacol. (2015)

Results of NADH and Fura-2-FF interference correction. The left panels show the signals prior to correction and the right panels show the signals after correction. NADH and the ratio of Fura-2-FF signals were changed following correction. The calculated [Ca2+]m was substantially decreased. (A) NADH signals at 450-nm emission. (B) Fura-2-FF signals at 500-nm emission. The figure shows F400,500 (- -), F353,500 (----), and the ratio of Fura-2-FF (-). (C) The mitochondrial calcium concentration calculated from the Fura-2-FF ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499650&req=5

Figure 8: Results of NADH and Fura-2-FF interference correction. The left panels show the signals prior to correction and the right panels show the signals after correction. NADH and the ratio of Fura-2-FF signals were changed following correction. The calculated [Ca2+]m was substantially decreased. (A) NADH signals at 450-nm emission. (B) Fura-2-FF signals at 500-nm emission. The figure shows F400,500 (- -), F353,500 (----), and the ratio of Fura-2-FF (-). (C) The mitochondrial calcium concentration calculated from the Fura-2-FF ratio.
Mentions: Fig. 8 shows the differences in Ca2+ concentrations before and after the correction. The results clearly showed the importance of correction for NADH and Fura-2-FF interference. The mitochondrial resting calcium concentration was 1.03±0.13 µM (mean±S.E., n=32) and the maximum [Ca2+]m at 1 µM cytosolic Ca2+ ([Ca2+]c) was 29.6±1.61 µM (mean±S.E., n=33) (Fig. 9).

Bottom Line: With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM.However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+).Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Ulsan College of Medicine/Asan Medical Center, Seoul 138-736, Korea.

ABSTRACT
Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca(2+)]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca(2+)], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca(2+)] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca(2+)], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca(2+)] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM. This 1 µM cytosolic Ca(2+) could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+). Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

No MeSH data available.