MCLA hydrochloride

Carteolol hydrochloride protects human corneal epithelial cells from UVB-induced damage in vitro

Purpose: To investigate whether carteolol hydrochloride has protective effects against ultraviolet B (UVB)-induced damage in human corneal epithelial cells (HCECs). Methods: Cultured HCECs were exposed to a single dose of UVB 300 mJ/cm, and the cell viability was measured 12 hours after the UVB irradiation using a cell-counting kit. Test samples at 0.01-1.0 mmol/L (carteolol hydrochloride, timolol maleate, betaxolol hydrochloride, levobunolol hydrochloride, or nipradilol) were added to the HCECs before, during, or after UVB irradiation. UV absorption spectra for each drug sample were determined using a spectrophotometer. Hydrogen peroxide (H2O2) and carteolol hydrochloride were simultaneously added to the HCECs for 10 minutes, and the cell viability was measured 12 hours later. The ability of carteolol hydrochloride to scavenge superoxide anion (O2) and singlet oxygen (O2) was investigated using the MCLA chemiluminescence method. Results: UVB irradiation decreased the number of viable HCECs in a dose-dependent manner. Carteolol hydrochloride at 1 mmol/L attenuated the UVB-induced cell damage when added before, during, or after UVB irradiation (P<0.01). Levobunolol hydrochloride at 1 mmol/L (P<0.01) added during or after irradiation and timolol maleate at 0.1 mmol/L or higher (P<0.05) added during irradiation attenuated the UVB-induced cell damage. Betaxolol hydrochloride and nipradilol had no effect. The UV absorption spectra of timolol maleate and levobunolol hydrochloride overlapped with the UVB wavelength spectrum, while carteolol hydrochloride, betaxolol hydrochloride, and nipradilol showed a partial overlap. Carteolol hydrochloride at 1 mmol/L (P<0.05) significantly inhibited H2O2-induced cell damage and was able to scavenge O2 (EC50 value: 48 mmol/L). Conclusions: These data strongly suggest that carteolol hydrochloride has a protective action against UVB-induced HCEC damage, and its radical scavenging ability may be an important basis for this effect.

Chapter 25 Analysis of electron transfer and superoxide generation in the cytochrome bc1 complex

During the electron transfer through the cytochrome bc(1) complex (ubiquinol-cytochrome c oxidoreductase or complex III), protons are translocated across the membrane, and production of superoxide anion radicals (O(2)(*-)) is observed. The bc(1) complex is purified from broken mitochondrial preparation prepared from frozen heart muscles by repeated detergent solubilization and salt fractionation. The electron transfer of the purified complex is determined spectrophotometrically. The activity depends on the choice of detergent, protein concentration, and ubiquinol derivatives used. The proton translocation activity of 2H(+)/e(-) is determined in the reconstituted bc(1)-PL vesicles. The O(2)(*-) production by bc(1) is determined by measuring the chemiluminescence of the 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazol[1,2-1]pyrazin-3-one hydrochloride (MCLA)-O(2)(*-) adduct during a single turnover of bc(1) complex, with the Applied Photophysics stopped-flow reaction analyzer SX.18MV, by leaving the excitation light source off and registering the light emission. Production of O(2)(*-) by bc(1) is in an inverse relationship to its electron transfer activity. Inactivation of the bc(1) complex by incubating at elevated temperature (37 degrees C) or by treatment with proteinase K results in an increase in O(2)(*-)-generating activity to the same level as that of the antimycin A-inhibited complex. These results suggest that the structural integrity of protein subunits is not required for O(2)(*-)-generating activity in the bc(1) complex.

Study on the generation mechanism of reactive oxygen species on calcium peroxide by chemiluminescence and UV-visible spectra

In the present work, the generation mechanism of reactive oxygen species (ROS) on calcium peroxide (CaO(2)) was studied. A very intense chemiluminescence (CL) signal was observed when adding an aqueous solution of luminol or 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2alpha]-pyrazin-3-one hydrochloride (MCLA) to a suspension of CaO(2). The ROS released on CaO(2) were thought to be oxidizing agents leading to CL, and were characterized by CL, UV-visible (UV-vis) spectra and the effective scavengers of the special ROS. From experimental results, the hydroxyl (.OH) and superoxide (.O(2) (-)) radicals were suggested to exist on the surface of CaO(2). A reaction scheme for the formation of the ROS on CaO(2) was also proposed and discussed. Of more interest was the finding that the CaO(2) which released the .OH and .O(2) (-) on the surface exhibited good transition properties compared with alkaline-earth metal peroxides of the same group (MgO(2), BaO(2)).

Generation of superoxide anion by succinate-cytochrome c reductase from bovine heart mitochondria

Production of superoxide anion (O-2), measured as the chemiluminescence of the 2-methyl-6-(p-methoxyphenyl)-3, 7-dihydroimidazo[1,2-a]pyrazin-3-one hydrochloride (MCLA)-O-2 adduct, was observed during electron transfer from succinate to cytochrome c by reconstituted succinate-cytochrome c reductase-phospholipid vesicles replenished with succinate dehydrogenase. Addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone or detergent to the reconstituted reductase-phospholipid vesicles abolished O-2 production, suggesting that O-2 generation is caused by the membrane potential generated during electron transfer through the cytochrome bc1 complex. Production of O-2 was also observed during electron transfer from succinate to cytochrome c by antimycin-treated reductase, in which approximately 99.7% of the reductase activity was inhibited. The rate of O-2 production was closely related to the rate of antimycin-insensitive cytochrome c reduction. Factors affecting antimycin-insensitive reduction of cytochrome c also affected O-2 production and vice versa. When the oxygen concentration in the system was decreased, the rate of O-2 production and cytochrome c reduction by antimycin-treated reductase decreased. When the concentrations of MCLA and cytochrome c were increased, the rate of O-2 production and cytochrome c reduction by antimycin-treated reductase increased. The rate of antimycin-insensitive cytochrome c reduction was sensitive to Qo site inhibitors such as 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole. These results indicate that generation of O-2 during the oxidation of ubiquinol by the cytochrome bc1 complex results from a leakage of the second electron of ubiquinol from its Q cycle electron transfer pathway to interact with oxygen. The electron-leaking site is located at the reduced cytochrome b566 or ubisemiquinone of the Qo site because addition of MCLA to antimycin-treated cytochrome bc1 complex, in the presence of catalytic amounts of succinate-cytochrome c reductase, delayed cytochrome b reduction by succinate. In the presence of oxidized cytochrome c, purified succinate dehydrogenase also catalyzed oxidation of succinate to generate O-2. When succinate dehydrogenase was reconstituted with the bc1 particles to form succinate-cytochrome c reductase, the production of O-2 diminished. These results suggest that reduced FAD of succinate dehydrogenase is the electron donor for oxygen to produce O-2 in the absence of their immediate electron acceptor and in the presence of cytochrome c.

Beta Blockers Suppress Dextrose-Induced Endoplasmic Reticulum Stress, Oxidative Stress, and Apoptosis in Human Coronary Artery Endothelial Cells

Beta blockers are known to have favorable effects on endothelial function partly because of their capacity to reduce oxidative stress. To determine whether beta blockers can also prevent dextrose-induced endoplasmic reticulum (ER) stress in addition to their antioxidative effects, human coronary artery endothelial cells and hepatocyte-derived HepG2 cells were treated with 27.5 mM dextrose for 24 hours in the presence of carvedilol (a lipophilic beta blockers with alpha blocking activity), propranolol (a lipophilic nonselective beta blockers), and atenolol (a water-soluble selective beta blockers), and ER stress, oxidative, stress and cell death were measured. ER stress was measured using the placental alkaline phosphatase assay and Western blot analysis of glucose regulated protein 78, c-Jun-N-terminal kinase (JNK), phospho-JNK, eukaryotic initiating factor 2α (eIF2α), and phospho-eIF2α and measurement of X-box binding protein 1 (XBP1) mRNA splicing using reverse transcriptase-polymerase chain reaction. Superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride (MCLA) chemiluminescence. Cell viability was measured by propidium iodide staining method. The ER stress, SO production, and cell death induced by 27.5 mM dextrose were inhibited by all 3 beta blockers tested. The antioxidative and ER stress reducing effects of beta blockers were also observed in HepG2 cells. The salutary effects of beta blockers on endothelial cells in reducing both ER stress and oxidative stress may contribute to the cardioprotective effects of these agents.