• The Korean Journal of Physiology and Pharmacology
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• v.9 no.5
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• pp.291-298
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• 2005

To characterize cytosolic $Ca^{2+}$ fluctuations under metabolic inhibition, rat ventricular myocytes were exposed to $200{\mu}M$ 2,4-dinitrophenol (DNP), and mitochondrial $Ca^{2+}$, mitochondrial membrane potential (${\Delta}{\Psi}m$), and cytosolic $Ca^{2+}$ were measured, using Rhod-2 AM, TMRE, and Fluo-4 AM fluorescent dyes, respectively, by Laser Scanning Confocal Microscopy (LSCM). Furthermore, the role of sarcolemmal $Na^+$/$Ca^{2+}$ exchange (NCX) in cytosolic $Ca^{2+}$ efflux was studied in KB-R7943 and $Na^+$-free normal Tyrode's solution (143 mM LiCl ). When DNP was applied to cells loaded with Fluo-4 AM, Fluo-4 AM fluorescence intensity initially increased by $70{\pm}10$% within $70{\pm}10$ s, and later by $400{\pm}200$% at $850{\pm}45$ s. Fluorescence intensity of both Rhod-2 AM and TMRE were initially decreased by DNP, coincident with the initial increase of Fluo-4 AM fluorescence intensity. When sarcoplasmic reticulum (SR) $Ca^{2+}$ was depleted by $1{\mu}M thapsigargin plus $10{\mu}M ryanodine, the initial increase of Fluo-4 AM fluorescence intensity was unaffected, however, the subsequent progressive increase was abolished. KB-R7943 delayed both the first and the second phases of cytosolic $Ca^{2+}$ overload, while $Na^+$-free solution accelerated the second. The above results suggest that: 1) the initial rise in cytosolic $Ca^{2+}$ under DNP results from mitochondrial depolarization; 2) the secondary increase is caused by progressive $Ca^{2+}$ release from SR; 3) NCX plays an important role in transient cytosolic $Ca^{2+}$ shifts under metabolic inhibition with DNP.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.4
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• pp.955-959
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• 2005

We previously demonstrated that the methylene chloride fraction of Scutellaria barbata suppessed human leukemic U937 cell proliferation by inducing apoptosis. In the present study, we have isolated luteolin from Scutellaria barbata and evaluated its apoptotic mechanism in Lewis lung carcinoma cells. Luteolin inhibited the proliferation of Lewis lung carcinoma cells in a concentration-dependent manner. Luteolin effectively increased the portion of $sub-G_1$ DNA content (apoptotic portion) and apoptotic Annexin-V positive cells in a concentration-dependent manner by FACS analysis. Caspase 9 and caspase 3 were activated and PARP was effectively cleaved by luteolin. It also increased the ratio of Bax to Bcl-2 through the decrease of Bcl-2 expression by Western blotting and reduced mitochondrial membrane potential following TMRE staining. These results suggest that luteolin can induce apoptosis through the mitochondrial mediated pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.4
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• pp.373-382
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• 2015

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 ${\Psi}_m$ or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [$Ca^{2+}$] concentration was $1.03{\mu}M$. This $1{\mu}M$ cytosolic $Ca^{2+}$ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [$Ca^{2+}$] increase was limited to ${\sim}30{\mu}M$ in the presence of $1{\mu}M$ 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.