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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.


Simultaneous measurement of NADH, [Ca2+]m, and pH. Using carboxy-SNARF- and Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and pH was measured simultaneously. The mitochondrial pH changes were investigated following Ca2+ changes. The increase in [Ca2+]m did not affect mitochondrial pH. Mitochondrial pH was 7.504±0.047 (mean±S.E., n=13).
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Figure 7: Simultaneous measurement of NADH, [Ca2+]m, and pH. Using carboxy-SNARF- and Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and pH was measured simultaneously. The mitochondrial pH changes were investigated following Ca2+ changes. The increase in [Ca2+]m did not affect mitochondrial pH. Mitochondrial pH was 7.504±0.047 (mean±S.E., n=13).

Mentions: Using carboxy-SNARF loaded myocytes, mitochondrial pH changes were determined following Ca2+ changes (Fig. 7). The increase in [Ca2+]m did not affect mitochondrial pH. The mitochondrial pH was 7.504±0.047 (mean±standard error [S.E.], n=13). The Kd value of Fura-2-FF at pH 7.5 was 5.28 µM; this value was used for later Ca2+ calculations.


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)

Simultaneous measurement of NADH, [Ca2+]m, and pH. Using carboxy-SNARF- and Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and pH was measured simultaneously. The mitochondrial pH changes were investigated following Ca2+ changes. The increase in [Ca2+]m did not affect mitochondrial pH. Mitochondrial pH was 7.504±0.047 (mean±S.E., n=13).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Simultaneous measurement of NADH, [Ca2+]m, and pH. Using carboxy-SNARF- and Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and pH was measured simultaneously. The mitochondrial pH changes were investigated following Ca2+ changes. The increase in [Ca2+]m did not affect mitochondrial pH. Mitochondrial pH was 7.504±0.047 (mean±S.E., n=13).
Mentions: Using carboxy-SNARF loaded myocytes, mitochondrial pH changes were determined following Ca2+ changes (Fig. 7). The increase in [Ca2+]m did not affect mitochondrial pH. The mitochondrial pH was 7.504±0.047 (mean±standard error [S.E.], n=13). The Kd value of Fura-2-FF at pH 7.5 was 5.28 µM; this value was used for later Ca2+ calculations.

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.