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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.


Measurement of RN factors. (A) The NADH signal in Fura-2-FF-free myocytes at 361-nm excitation and 450-nm emission was measured in the presence of various mitochondrial substrates. (B) F400,500 (●) and F353,500 (-) were measured simultaneously to monitor the NADH contribution to the Fura-2-FF signal. (C) The graph shows the relationships between F361,450,NADH and F353,500,NADH (●) and between F361,450,NADH and F400,500,NADH (○). Linear relationships are evident in the graph.
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Figure 5: Measurement of RN factors. (A) The NADH signal in Fura-2-FF-free myocytes at 361-nm excitation and 450-nm emission was measured in the presence of various mitochondrial substrates. (B) F400,500 (●) and F353,500 (-) were measured simultaneously to monitor the NADH contribution to the Fura-2-FF signal. (C) The graph shows the relationships between F361,450,NADH and F353,500,NADH (●) and between F361,450,NADH and F400,500,NADH (○). Linear relationships are evident in the graph.

Mentions: If the constants, Rf, RN1, and RN2, were known, the pure NADH and Fura-2-FF signals could be obtained by solving the above equations. The Rf was calculated with the fluorescence intensity observed in Fig. 4A and B. RN1 and RN2 were obtained in myocytes that were not loaded with Fura-2-FF. The NADH signal was changed according to the supply of mitochondrial substrates: malate, pyruvate, or malate plus pyruvate (Fig. 5A and B). The linear relationships of F400,500,NADH vs. F361,450,NADH and F353,500,NADH vs. F361,450,NADH are shown in Fig. 5C, and each slope indicates RN1 and RN2. The equations of each pure signal values are as follows: (10)F361,450,NADH=(A-Rf*B)/(1-Rf*RN2)(11)F353,500,fura/F400,500,fura=Rfura=RN2*A-BRN2*Rf-1/C-RN1A-Rf*B1-Rf*RN2 Rfura is the ratio value of Fura-2-FF that was used to calculate the free calcium concentration.


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)

Measurement of RN factors. (A) The NADH signal in Fura-2-FF-free myocytes at 361-nm excitation and 450-nm emission was measured in the presence of various mitochondrial substrates. (B) F400,500 (●) and F353,500 (-) were measured simultaneously to monitor the NADH contribution to the Fura-2-FF signal. (C) The graph shows the relationships between F361,450,NADH and F353,500,NADH (●) and between F361,450,NADH and F400,500,NADH (○). Linear relationships are evident in the graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Measurement of RN factors. (A) The NADH signal in Fura-2-FF-free myocytes at 361-nm excitation and 450-nm emission was measured in the presence of various mitochondrial substrates. (B) F400,500 (●) and F353,500 (-) were measured simultaneously to monitor the NADH contribution to the Fura-2-FF signal. (C) The graph shows the relationships between F361,450,NADH and F353,500,NADH (●) and between F361,450,NADH and F400,500,NADH (○). Linear relationships are evident in the graph.
Mentions: If the constants, Rf, RN1, and RN2, were known, the pure NADH and Fura-2-FF signals could be obtained by solving the above equations. The Rf was calculated with the fluorescence intensity observed in Fig. 4A and B. RN1 and RN2 were obtained in myocytes that were not loaded with Fura-2-FF. The NADH signal was changed according to the supply of mitochondrial substrates: malate, pyruvate, or malate plus pyruvate (Fig. 5A and B). The linear relationships of F400,500,NADH vs. F361,450,NADH and F353,500,NADH vs. F361,450,NADH are shown in Fig. 5C, and each slope indicates RN1 and RN2. The equations of each pure signal values are as follows: (10)F361,450,NADH=(A-Rf*B)/(1-Rf*RN2)(11)F353,500,fura/F400,500,fura=Rfura=RN2*A-BRN2*Rf-1/C-RN1A-Rf*B1-Rf*RN2 Rfura is the ratio value of Fura-2-FF that was used to calculate the free calcium concentration.

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.