| Endothelium-derived hyperpolarizing factor?EDHF?is the third substance which is produced by vascular endothelial cells and could cause vasodilatation after nitric oxide?NO?and prostacyclin?PGI2?were found.The mechanism of EDHF causing vasorelaxation is activating Ca2+-sensitive K+channel(KCa)on vascular smooth muscle cells?VSMCs?thus mediating VSMCs hyperpolarization.Our previous studies demonstrate that EDHF-mediated vasorelaxation responses in rat cerebral arteries are induced by hydrogen sulfide?H2S?.Total flavones of Rhododendra?TFR?is an effective part which is extracted from Rhododendra flower,and the major constituent of TFR is comprised of rutin,quercetin,hyperin and other flavonoids.We also found that TFR could cause membrane hyperpolarization in vascular smooth muscle cells?VSMCs?and then vasodilatation,but non excitable cells such as endothelial cells have not been found.Therefore,the present study is to investigate the effect of EDHF/H2 S on BKCa channel current and membrane hyperpolarization of TFR.Purpose:1.To investigate the effect of H2 S on membrane potential in rat hippocampal neurons.2.To investigate the effect of EDHF/H2 S on BKCa channel currents in rat hippocampal neurons.3.To investigate the effect of TFR on membrane potential in endothelial cells and rat hippocampal neurons.Methods:1.EA.hy926 cells were cultured by conventional cell culture method.2.Hippocampal neurons were obtained from neonatal SD rats through primary culture method and identified through immunofluorescence.3.The fluorescence intensity of endothelial cells and rat neurons was measured by using Di BAC4?3?fluorescent dyes through the calcium ion imaging system with the excitation wavelength of 488 nm to reflect the membrane potential.?1?Na HS?1×10-91×10-5mol/L?and the large conductance Ca2+-sensitive K+channels(BKCa)-specific inhibitor iberiotoxin?Ib TX?were added into rat hippocampal neurons to observe the membrane potential.?2?TFR?30,90,270,810 mg/L?,Ib TX,co-application of intermediae conductance Ca2+-sensitive K+channels(IKCa)inhibitor charybdotoxin?Ch TX?and small conductance Ca2+-sensitive K+channels(SKCa)inhibitor apamin,CSE inhibitor DL-propargylglycine?PPG?were added into EA.hy926 cells to observe the membrane potential.?3?TFR?30,90,270,810mg/L?and Ib TX were added into rat hippocampal neurons to observe the membrane potential.4.The whole-cell patch-clamp technique was used to record the BKCa channel currents.?1?EA.hy926 cells?ECs?/ rat cerebral vessels?Endo?,acetylcholine?ACh?,the inhibitor of NOS N?G?-nitro-L-arginine methyl ester?L-NAME?,the inhibitor of COX indomethacin?Indo?,Ib TX and PPG were added into rat hippocampal neurons to observe the effect of endogenous EDHF/H2 S on BKCa channel currents.?2?Na HS?1 × 10-91 × 10-5mol/L?and Ib TX were added to observe the effect of exogenous EDHF/H2 S on BKCa channel currents.Results:1.The primary cultured cells were indeed rat hippocampal neurons after the identification of immunofluorescence,cells purity was high,hybrid cells and tissueswere few.2.Na HS?1×10-6and 1×10-5mol/L?could remarkedly cause hyperpolarization of rat hippocampal neurons and this effect could be inhibited by Ib TX.3.The results of patch-clamp to record BKCa channel currents of rat hippocampal neurons were showed that?1?the noise-like outward current evoked by test pulses was in accord with the characteristic of BKCa channel,and this current could be inhibited significantly by Ib TX which indicate that the outward current was a BKCa current carried by the BKCa channel.?2?Neither ACh,nor ECs or Endo could increase BKCa current in rat hippocampal neurons,while the co-application of them could remarkably increase BKCa current,and in the presence of ECs or Endo,LNAME or/and Indo,Ach could also remarkably increase BKCa current which could be inhibited by Ib TX and PPG.?3?Na HS significantly enhanced the neuronal currents at the level of 1×10-6and 1×10-5mol/L and this activation of BKCa channels could be inhibited by Ib TX.4.TFR?270 and 810 mg/L?could remarkedly cause hyperpolarization of EA.hy926 cells and this effect could be inhibited by Ib TX,the co-application of Ch TX and apamin,while PPG couldn't.5.TFR?270 and 810 mg/L?could remarkedly cause hyperpolarization of rat hippocampal neurons and this effect could also be inhibited by Ib TX.Conclusions:1.H2 S could cause membrane hyperpolarization of rat hippocampal neurons in a dosedependent manner.2.EDHF/H2 S could activate BKCa channel and increase BKCa currents of rat hippocampal neurons.3.TFR could cause membrane hyperpolarization in EA.hy926 cells in a dosedependent manner and the possible mechanism was related to activating KCachannels.4.TFR could cause membrane hyperpolarization in rat hippocampal neurons and the possible mechanism was related to activating BKCa channels. |
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