| As a new gaseous transmitter, hydrogen sulfide (H2S) plays an important role in regulating cardiovascular system, it takes part in the pathophysiological procedu-res, like lowering of blood pressure, slowing heart beat and so on. Recent evidence suggests that reactive oxygen species (ROS) mediates cardiovascular effects induced by angiotensin Ⅱ (Ang Ⅱ), while H2S protects neurons through decreasing productio-n of ROS. In addition, ROS also mediates influx of extracellular calcium in microgl-ial cells and astrocyte cells induced by Ang Ⅱ. Our previous experiments demonstra-ted that H2S in the rostral ventrolateral medulla (RVLM) lowered blood pressure and heart rate in vivo. Moreover, we prove that Ang Ⅱ increase ROS, particularly super-oxide (O2·-) production in medullary neurons. On this foundation, in the present study, we aimed to observe the effect of H2S on increase in reactive oxygen species and influx of extracellular calcium in medullary neurons induced by Ang Ⅱ and its possible mechanism.Neurons that involved in cardiovascular system regulation mostly are gluta-matergic neurons, to clarify whether our primary cultured neurons including glutamatergic neurons, immunohistochemical method with fluorescence microscope was used. The result displayed that 90% primary cultured neurons were glutam-atergic neurons. To investigate the effect of Ang Ⅱ on the ROS level in medullary neurons, dihydroethidium (DHE) staining was used and the activity of neurons was detected by CCK-8 assay. The results showed that Ang Ⅱ(1 μmol/L,30 min) significantly increased the level of ROS in medullary neurons (P< 0.05), but had no effect on the activity of neurons (P> 0.05), suggesting that AngⅡhad no toxical effect on medullary neurons and the increased ROS was not induced by neuron damage. Pretreatment with various concentrations of NaHS (exogenous H2S) or NaBu (a CBS agonist), significantly inhibited the increase in ROS induced by Ang Ⅱ in a dose-dependent manner (P< 0.05), while NaHS or NaBu alone did not change the level of ROS in neurons.SOD has special bioactivities, is a common free radical scavenger exist in organisms. To clarify the molecular mechanisms that hydrogen sulfide suppresses the increase in ROS in medullary neurons induced by Ang Ⅱ, we detected total SOD activity of Ang Ⅱ group, Ang Ⅱ+NaHS group, Ang Ⅱ+NaBu group, separately. Ang Ⅱ alone can decrease total SOD activity. Neurons pretreated with NaHS or NaBu, then administrate Ang Ⅱ, total SOD activity increases significantly in comparison with Ang Ⅱ group.To investigate effect of Ang Ⅱ on cytosolic free calcium concentration ([Ca2+]i), fluorescent dye Fura2/AM was used. Results show that Ang Ⅱ alone increases [Ca2+]i, after washing 5 min, neurons were incubated with NaHS or NaBu, then treated with Ang Ⅱ again, [Ca2+]i increases significantly inhibited compared with Ang Ⅱ alone. In other words, exogenous H2S and endogenous H2S both can inhibit increase of [Ca2+]i induced by Ang Ⅱ. To determine the source of [Ca2+], in Ang Ⅱ-stimulated neurons, neurons were bathed either in Ca2+-free medium containing 2 mmol/L DMSO or in normal Ca2+-containing medium. Ang Ⅱ caused an increase in [Ca2+]i after incubation in Ca2+-containing medium. This response was completely abolished in neurons bathed in Ca+-free medium, suggesting that the Ang Ⅱ-induced increase in [Ca2+]i was caused at least partially by an influx of extracellular Ca2+.In conclusion, our present study demonstrates that exogenous and endogenous H2S inhibited ROS production induced by Ang Ⅱ by regulating tatol SOD activity; suppressed influx of extracellular Ca2+ induced by Ang Ⅱ, that ROS may be involved in, providing a new therapeutic target for hypertension. |
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