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AIM:To investigate the role of reactive oxygen species (ROS) in the regulation of intracellular Ca2+induced by angiotensin II ( Ang II) in the primarily cultured medullary neurons .METHODS:Primarily cultured me-dullary neurons were prepared from 14-day-old embryos of Sprague-Dawley rats in the study .The identification of medullary neurons was assessed by double-labeling immunofluorescence .To explore the role of ROS , mainly the superoxide ( O2 ·-) , the O2 ·-generation was measured using the fluorogenic probe dihydroethidium ( DHE) .To determine intracellular free cal-cium concentration ( [ Ca2+] i ) , the neurons were loaded with the Ca 2+-specific dye Fura-2/AM.The cell viability after adding Ang II was also examined using CCK-8 assay.RESULTS:Most of the cultured cells were medullary neurons , more than 80%of which were glutamate positive neurons .Ang II (5 μmol/L) increased the level of ROS within 10 min in the medullary neurons .Ang II at 5μmol/L induced a significant [ Ca2+] i increase in the medullary neurons , and the effect of Ang II occurred rapidly and reached a peak within 20 min after administration.The level of [Ca2+]i started to decline after washout .The Ca2+elevation induced by Ang II was significantly decreased by apocynin or TEMPOL .No significant differ-ence in the cell viability between control group and 5μmol/L Ang II treatment group was observed .CONCLUSION:ROS is involved in the regulation of [Ca2+]i induced by Ang II in the primarily cultured medullary neurons , suggesting a poten-tial intracellular signaling mechanism involved in the Ang II-mediated oxidant regulation of central neural control of blood pressure.
RESUMEN
Aim To establish a method for recording tension changes in small artery in vitro.Methods The vessel segments of intestine were dissected from the mesenteric artery of rats and mounted onto Myograph.The internal circumferences of the segments were normalized with Chart5 software.Then the internal diameters of the segments and initial setting of tension were calculated with self-made Excel file.Following these preparation steps, the tension changes induced by several stimulations were recorded by Myograph.Results Normalization procedure provided the data of lumen diameters of small arteries,which was convenient for initial setting of tension and guaranteed the uniformity of the experimental conditions.Myograph could record the tension changes of small arteries induced by several classic stimulations.Conclusion Precise recording method for tension changes in small arteries in vitro is successfully established.