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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 Ψm. Using Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and Ψm were measured simultaneously. The resting Ψm was assumed to be -150 mV.
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Figure 10: Simultaneous measurement of NADH, [Ca2+]m, and Ψm. Using Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and Ψm were measured simultaneously. The resting Ψm was assumed to be -150 mV.

Mentions: TMRE has a positive charge and is distributed in a membrane potential-dependent manner. If the concentration in each compartment is known, the membrane potential can be calculated using Nernst's equation. TMRE was perfused at 2 nM to record the changes in the TMRE signal in mitochondria. The resting Ψm was assumed to be -150 mV. Based on this, Ψm was calculated. NADH was decreased by the application of Ca2+, but the Ψm remained similar (Fig. 10).


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 Ψm. Using Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and Ψm were measured simultaneously. The resting Ψm was assumed to be -150 mV.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Simultaneous measurement of NADH, [Ca2+]m, and Ψm. Using Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and Ψm were measured simultaneously. The resting Ψm was assumed to be -150 mV.
Mentions: TMRE has a positive charge and is distributed in a membrane potential-dependent manner. If the concentration in each compartment is known, the membrane potential can be calculated using Nernst's equation. TMRE was perfused at 2 nM to record the changes in the TMRE signal in mitochondria. The resting Ψm was assumed to be -150 mV. Based on this, Ψm was calculated. NADH was decreased by the application of Ca2+, but the Ψm remained similar (Fig. 10).

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.