Simultaneous determination of catecholamines and unconjugated 3,4-dihydroxyphenylacetic acid in brain tissue by ion-pairing reverse-phase high-performance liquid chromatography with electrochemical detection

A rapid, sensitive method was developed for the simultaneous assay of catecholamines and 3,4-dihydroxyphenylacetic acid in rat brain tissue. The method is simple, involving only tissue disruption, adsorption of the catechols onto alumina, desorption, and injection into a reverse-phase high-performance liquid chromatography system. Selectivity and high sensitivity are obtained using electrochemical detection. The addition of 3,4-dihydroxyphenylacetic acid determination to assays for catecholamines allows one to observe effects of pharmacological maniqulations on in vivo monoamine oxidase activity and/or turnover of dopamine as well as effects on catecholamine concentrations.     

NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment

Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1⁺) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.