H2S对兔离体心脏心率的影响
发表时间：2010-06-02 浏览次数：337次
作者：陈立锋 徐涛 范凌 赵兰平 陈少峰 温晓竞 张晓云 马建伟 尹万斌 作者单位：1.河北北方学院医学院生理教研室，河北 张家口 075000;2.河北北方学院附属第一医院心血管内科，河北 张家口 075000;3.河北北方学院附属第一医院血液科，河北 张家口 075000;4.河北北方学院临床医学本科2006级2班，河北 张家口 075000
【摘要】 目的：探讨硫化氢(H2S)对兔离体心脏心率的影响。方法：用标准微电极细胞内记录技术，观测硫化氢对兔右心房心肌细胞动作电位频率的影响。观测指标：自发放电频率(rate of pacemaker firing，RPF)。结果：(1) 用200μmol/LNaHS 灌流时，和正常对照组相比，RPF显著减小(P<0.01)。(2) 400μmol/L NaHS与对照组比较， RPF显著减小(P<0.01)。和200μmol/LNaHS相比，RPF显著减小(P<0.01)。(3)以上使RPF减小的效应在NaHS灌流3～5min出现，10min时已经有显著减小(P<0.01)，25～30min达到高峰。结论：H2S可浓度依赖性的影响兔离体心脏的心率。
【关键词】 心率;电生理; 硫化氢 ;心脏;兔
Hydrogen sulfide(H2S)has been proved to be the third endogenous signaling gasotransmitter,besides nitric oxide(NO) and carbon monoxide(CO).It shows important physiological functions,involving in the longterm potentiation in the hippocampus in the central nervous system,relaxing gastrointestinal tract smooth muscles[1,2].H2S has been also found to play many regulating functions in cardiovascular system,and play a certain effect in many cardiovascular diseases[3].But the effect on heart rate hasnt been deeply reseached.By using standard intracellular microelectrode technique to record Aps,hydrogen sulfide was used to investigate the rate of pacemaker firing of left atrium.The aim of the study was to observe the effects of hydrogen sulfide on heart rate of rabbit hearts in vitro,elucidate the mechanism(s) involved,provide electrophysiologic proof for the nosogenesis of some cardiovascular diseases and provide research directions for the development of cardiovascular drugs.
1 MATERIALS AND METHODS
1.1 Animal and Reagents
Rabbits of either sex(2.2±0.2)kg,provided by Experiment Animal Center of HeBei North University.NaHS was prepared with HCl and Na2S after the experiment began.
1.2 Specimen Preparation
Rabbits were killed with a single blow on the head and the hearts were quickly removed and placed in warm(36.0±0.5)℃ improved Locke solution(NaCl 0.157mol/L,KCl 0.56×10-2mol/L,CaCl 0.21×10-2mol/L,NaHCO30.18×10-2mol/L and glycose 0.56×10-2mol/L).It was oxygenated with 95%O2 and 5%CO2 and maintained at (36.0±0.5)℃ with pH of 7.40±0.03.The right atrium myocytes(including sinuatrial node) were cut from the right atrium and then pinned on a thin silicon disc at the bottom of a perfusion chamber.The preparations were perfused (5mL/min) with warm (36.0±0.5)℃ improved Locke solution.It was oxygenated with 95%O2 and 5%CO2 and maintained at (36.0±0.5)℃ with pH of 7.40[4].
1.3 Potential Recording
By using standard intracellular microelectrode technique to record Aps,the transmembrane action potentials were recorded by 3mol/L KClfilled glass microelectrode(a tip resistance of 10～20MΩ) coupled to a high input impedence amplifier(made in Chengdu Instrument factory), The amplified signals were fed to a multichannel physiological signal acquisition system (RM6280C,made in Chengdu Instrument Factory) and processed by a microcomputer. The parameters of action potentials were automatically analyzed by the microcomputer.
1.4 Lab Proc and Grouping
Aps were recored after equilibrium for 60min.After recording 3 control Aps,NaHS (,200,400,μmol/L) were applied respectively.Aps were then recorded respectively at 1,2,3,5,10,15,20,25 and 30min after application of NaHS.The preparation was washed for 25min with the improved Locke solution to observe the recovery of Aps.Experiment grouping:(1)control group(n=8);(2) 200μmol/L NaHS(n=8);(3) 400μmol/L NaHS(n=8).
1.5 Electrophysiological Parameters
Rate of pacemaker firing(RPF)
1.6 Statistics
All data were presented as means±SEM(x±s).The differences in the parameters between before and after chemical application were analyzed by paired Students t test.Differences between groups were assessed by oneway ANOVA and unpaired test.Statistical significance was set at P<0.05.
2 RESULTS
Effects of hydrogen sulfide on the rate of action potential of right atrium myocytes:
(1)Perfusion with NaHS(H2S donor,200μmol/L) led to a significant decrease in RPF(P<0.01).
(2)Application of 400μmol/L NaHS resulted in a significant decrease in RPF(P<0.01).Compared with the result of 200μmol/L NaHS group,400μmol/L NaHS showed a significant decrease in RPF(P<0.01).
(3) The abovementioned effects occurred after 3～5min of NaHS superfusion,had a significant decrease(P<0.01)after 10 min and reached the peak within 25～30min.2009年6月CHEN Lifeng et al：Effects of Hydrogen Sulfide on Heart Rate of Rabbit Hearts in Vitro第3期2009年6月河北北方学院学报(医学版)第3期Table 1 Effects of hydrogen sulfide on heart rate of rabbit hearts in vitro( x±s，n=8)
groupingcontrolPerfusion at 10minPerfusion at 20minPerfusion at 30minNaHS 200(μmol/L)176.50±2.71165.33±3.16**153.42±2.36**144.12±3.07**washout144.12±3.07150.76±3.23162.52±2.98170.14±4.04**NaHS 400(μmol/L)176.50±2.71151.07±3.50**115.08±2.77**75.44±2.81**##washout75.44±2.81101.32±2.66132.53±4.60161.36±4.92**
注：vs control*P<0.05,**P<0.01;vs NaHS200(μmol/L)# P<0.05,## P<0.01
A.control;B.200μmol/L NaHS;
C.400μmol/L NaHS;D.washout
Fig.1 Effects of hydrogen sulfide on the rate of action potential of right atrium myocytes of rabbits
3 DISCUSSION
Hydrogen sulfide(H2S),traditionally considered to be a toxic gas in contaminated environments playing a neurotoxic role and inhibiting the respiratory system,has been proved to be the third endogenous signaling gasotransmitter with important physiological functions[5],besides nitric oxide(NO)and carbon monoxide(CO),Endogenous H2S can be generated from Lcysteine catalyzed by two pyridoxal5phosphatedependent enzymes,cystathionine βsynthase(CBS) and cystathionineγlyase(CSE) in mammalian tissues.The expressions of these two enzymes are tissuespecific.CBS could not be found in cardiac atrium,ventricle,internal mammary artery,femoral artery,femoral vein,coronary artery and aorta.H2S was directly produced in myocardial tissues,arterial and venous tissues by CSE[6],Geng et al[6],whose study had demonstrated that H2S contents was (10.04±2.10)μmol·mg-1(protein) in myocardial tissues.
H2S molecules have two forms in vivo,one is gas,another is NaHS.NaHS can be dissociated into Na+ and HS-;HS- and H+ can be combined into H2S.Na+ and HS- not only maintain H2S stabilization,but also do not change pH level of internal environment.So were explored of hydrogen sulfide (H2S) on action potentials(Aps) of rabbit right atrium myocytes by NaHS were explored.
It is clear now that H2S has the vasorelaxant function.In vascular smooth muscle cells(VSMCs),It is reporded that the vasorelaxant function of H2S has been achieved by opening of ATPsensitive potassium(KATP)channels and inhibiting calcium inflow.but the cGMP pathway was not included[2].H2S is the first identified gaseous opener of the KATP channels in VSMCs and the KATP channels are widely distributed in cardiac cells.It is recently reported that hydrogen sulfide(H2S) generated by cardiac tissues has negative inotropic function[7],whose effect has been achieved by opening of ATPsensitive potassium(KATP)channels and inhibiting calcium inflow.
The study showed that H2S could decrease the rate of action potential of left atrium myocytes of rabbits.Because the rate of action potential of left atrium myocytes and the autorhythmicity of pacemaker cells in sinoatrial node were uniformity,the possible reason was that H2S exerted a negative chronotropic action on pacemaker cells in sinoatrial nodes of rabbits,which led to a decrease in RPF of pacemaker cells in sinoatrial node.It is well known that RPF is determined by maximal diastolic potential(MDP),level of threshold and velocity of diastolic depolarization(VDD).The major ionic currents responsible for diastolic depolarization are the decaying potassium currents(IK),increasing slow inward calcium currents(ICaL and ICaT)and increasing inward sodium currents(If).Therefore,any factors promoting potassium efflux or/and inhibiting calcium or/and sodium influx may decrease VDD[8].The possible reason was that H2S decreased VDD by promoting potassium efflux and inhibiting calcium influx [7,9,10],which led to a significant decrease in RPF,and subsequently resulted in a significant decrease in heart rate.
Endogenous H2S as a gaseous opener of the KATP channels,might be of an important physiological and pathophysiological significance to cardiac tissues,whose more knowledge is yet to be further pursued.
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