Search Results (5)

Adenosine 5′ triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease. Full article

Liquidambar styraciflua L. is an aromatic species, popularly used in traditional Chinese medicine to treat diarrhea, dysentery, coughs, and skin sores. The present study was designed to investigate the chemical composition and biological potential of extracts obtained from the fruits of this plant. For the chemical evaluation, it was used mainly liquid and gas chromatography, plus NMR, and colorimetric methods. The aqueous extract (EA) originated two other fractions: an aqueous (P-EA) and an ethanolic (S-EA). The three extracts were composed of proteins, phenolic compounds, and carbohydrates in different proportions. The analyses showed that the polysaccharide extract (P-EA) contained pectic polysaccharides, such as acetylated and methyl esterified homogalacturonans together with arabinogalactan, while the fraction S-EA presented phenolic acids and terpenes such as gallic acid, protocathecuic acid, liquidambaric acid, combretastatin, and atractyloside A. EA, P-EA, and S-EA showed antioxidant activity, with IC₅₀ values of 4.64 µg/mL, 16.45 µg/mL, and 3.67 µg/mL, respectively. The cytotoxicity followed the sequence S-EA > EA > P-EA, demonstrating that the toxic compounds were separated from the non-toxic ones by ethanol precipitation. While the fraction S-EA is very toxic to any cell line, the fraction P-EA is a promising candidate for studies against cancer due to its high toxicity to tumoral cells and low toxicity to normal cells. Full article

Atractylodis Rhizoma is the dried rhizome of Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz and is often processed by stir-frying with wheat bran to reduce its dryness and increase its spleen tonifying activity. However, the mechanism by which the processing has this effect remains unknown. To explain the mechanism based on the pharmacokinetics of the active compounds, a rapid, sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was developed to analyze atractylenolides I, II, and III, and atractyloside A simultaneously in rat plasma after oral administration of raw and processed Atractylodis Rhizoma. Acetaminophen was used as the internal standard and the plasma samples were pretreated with methanol. Positive ionization mode coupled with multiple reaction monitoring mode was used to analyze the four compounds. The method validation revealed that all the calibration curves displayed good linear regression over the concentration ranges of 3.2–350, 4–500, 4–500, and 3.44–430 ng/mL for atractylenolides I, II, and III, and atractyloside A, respectively. The relative standard deviations of the intra- and inter-day precisions of the four compounds were less than 6% with accuracies (relative error) below 2.38%, and the extraction recoveries were more than 71.90 ± 4.97%. The main pharmacokinetic parameters of the four compounds were estimated with Drug and Statistics 3.0 and the integral pharmacokinetics were determined based on an area under the curve weighting method. The results showed that the integral maximum plasma concentration and area under the curve increased after oral administration of processed Atractylodis Rhizoma. Full article

Although beneficial effects of non-secreting intracellular renin (ns-renin) against ischemia have been reported, the precise mechanism remains unclear. In this study, we investigated the roles of ns-renin and mitochondrial extracellular signal-related kinase (ERK) 1/2 on mitochondrial permeability transition pore (mPTP) opening during ischemia in diabetes mellitus (DM) hearts. When isolated hearts from Wistar rats (non-DM hearts) and Goto-Kakizaki rats (DM hearts) were subjected to ischemia for 70 min by left anterior descending coronary artery ligation, DM hearts exhibited higher left ventricular (LV) developed pressure and lower LV end-diastolic pressure than non-DM hearts, suggesting ischemic resistance. In addition, DM hearts showed increased intracellular renin (int-renin, including secreting and non-secreting renin) in the ischemic area, and a direct renin inhibitor (DRI; aliskiren) attenuated ischemic resistance in DM hearts. ERK1/2 was significantly phosphorylated after ischemia in both whole cell and mitochondrial fractions in DM hearts. In isolated mitochondria from DM hearts, rat recombinant renin (r-renin) significantly phosphorylated mitochondrial ERK1/2, and hyperpolarized mitochondrial membrane potential (ΔΨ_m) in a U0126 (an inhibitor of mitogen-activated protein kinases/ERK kinases)-sensitive manner. R-renin also attenuated atractyloside (Atr, an mPTP opener)-induced ΔΨ_m depolarization and Atr-induced mitochondrial swelling in an U0126-sensitive manner in isolated mitochondria from DM hearts. Furthermore, U0126 attenuated ischemic resistance in DM hearts, whereas it did not alter the hemodynamics in non-DM hearts. Our results suggest that the increased int-renin during ischemia may inhibit mPTP opening through activation of mitochondrial ERK1/2, which may be involved in ischemic resistance in DM hearts. Full article

Atractyloside (ATR) is found in many Asteraceae plants that are commonly used as medicinal herbs in China and other eastern Asian countries. ATR binds specifically to the adenine nucleotide translocator in the inner mitochondrial membrane and competitively inhibits ADP and ATP transport. The toxicity of ATR in medical herbs can be reduced by hydrothermal processing, but the mechanisms of ATR degradation are not well understood. In this study, GC-MS coupled with SPE and TMS derivatisation was used to detect ATR levels in traditional Chinese medicinal herbs. Our results suggest that ATR molecules were disrupted by decomposition, hydrolysis and saponification after heating with water (decoction) for a long period of time. Hydrothermal processing could decompose the endogenous toxic compounds and also facilitate the detoxification of raw materials used in the Chinese medicine industry. Full article