硫化氢对离体家兔肾动脉血管环张力调节的研究
|
摘要
硫化氢(hydrogen sulfide,H2S)一直被认为是一种无色、具有臭鸡蛋味的毒性气体,它可以抑制细胞色素氧化酶进而抑制线粒体的呼吸作用。H2S是继内源性气体分子一氧化氮(nitric oxide,NO)、一氧化碳(carbon monoxide,CO)后发现的第三个内源性气体信使(Gasotransmitter)。已证实H2S可以调节生理及病理状态下心脏的功能,并在自发性高血压大鼠、低氧性肺动脉高血压大鼠、高肺血流量所致肺动脉高压及肾血管性高血压的发病中也起着重要的调节作用。H2S可通过开放KATP通道使容量血管大鼠胸主动脉舒张,还可通过KATP通道和内依赖性超极化因子(endothelium-derived hyperpolarizing factor,EDHF)介导的KCa通道共同作用使阻力血管肠系膜动脉舒张。可见H2S诱导的舒血管效应有别于其他内源性气体信号分子(如NO、CO是通过鸟苷酸环化酶cGMP通路起作用)。H2S还可以抑制血管平滑肌细胞的增殖和促进其凋亡、缓解血管结构的重建。提示H2S可能在心血管系统稳态调节中发挥着重要作用。肾动脉张力是调节血管外周阻力的重要因素,肾血管阻力增高是高血压病时肾血流动力学改变的重要特征。肾动脉的病变导致管腔的狭窄进而肾脏缺血刺激肾脏皮质内球旁装置细胞分泌肾素过度,引发肾素血管紧张素系统过度激活,全身小动脉收缩血压升高,导致肾血管性高血压。可见肾动脉的张力在高血压病和肾血管性高血压的发病过程中起重要作用,而H2S对它的影响至今尚无人报道。
目的
应用离体血管环张力测定技术观察H2S对家兔离体肾动脉血管环张力的调节作用,探讨其可能的作用机制。
方法
1.测定正常家兔血浆中H2S的含量。
在试管中加入1%(质量分数)醋酸锌0.5 ml,蒸馏水2.5 ml,血浆0.1 ml,混匀。然后加入7.2 mol/L盐酸(含20 mmol/L N,N-二甲基-对苯二胺盐酸盐)0.5 ml,再加入1.2 mol/L HCl(含30 mmol/L FeCl3)0.4 ml,室温孵育20 min,然后加入10%三氯醋酸1 ml。将上述试管内容物离心5 min后取上清,用分光光度计在665 nm波长处检测光吸收度。用H2S标准曲线计算血浆H2S浓度,结果用μmol/L表示。
2.测定肾动脉上硫化氢生成酶(cystathionine -lyase,CSE)的活性。
取家兔的肾动脉在冰冷的磷酸钾缓冲液(50 mmol/L,pH 6.8)中研磨成匀浆。反应在25 ml锥形瓶中进行。反应体积1 ml,含磷酸钾缓冲液(100 mmol/L,pH 7.4)、L-半胱氨酸(10 mmol/L)、5’-磷酸吡哆醛(2 mmol/L)和10%(V/V)组织匀浆。在中央室中加入10%(W/W)醋酸锌0.5 ml,并放入滤纸增加吸收面积。用N2将烧瓶充盈30 s后,石蜡膜封口,转移到37℃水浴摇床中开始反应,90 min后向其中注入50%(W/W)三氯醋酸0.5 ml中止反应,继续水浴60 min。将中央室的内容物转移到含3.5 ml蒸馏水的试管中,加入20 mmol/L对苯二胺盐酸盐0.5 ml和30 mmol/L FeCl3 0.4 ml,20 min后用分光光度计在665 nm波长处检测光吸收度。用H2S标准曲线计算溶液中H2S含量,H2S产出率由(pmol/mg protein/minute)来表示,反映CSE的活性。
3.硫化氢对离体家兔肾动脉血管环张力的调节。
运用离体血管环灌流系统,观察硫化氢作用后血管环张力的变化,由RM6240生物信号采集处理系统进行记录分析,检测血管环张力的变化并探讨其可能的机制。
结果
1.正常兔血浆中H2S的含量为(44.39±4.9)μmol/L。
2.正常家兔肾动脉上H2S生成率为(143.94±4.80)pmol/mg protein/minute。
3. (1)外源性的H2S(NaHS 50,100,200,400,800μmol/L)可以剂量依赖性地舒张由KCl(50 mmol/L)预收缩的肾动脉血管环,其浓度反应曲线的IC50值为281.46±17.26μmol/L,最大舒张率为89.97±2.40%。(2)用KATP通道阻断剂格列苯脲(glibenclamide, 20μmol/L)、钙通道的开放剂Bay K8644(500 nmol/L)、NO合酶的抑制剂L-NAME(100μmol/L)和环氧合酶阻断剂吲哚美辛(10μmol/L)预处理以及去除血管内皮后,H2S的舒张效应均被显著抑制,浓度反应曲线均明显右移,其IC50值分别增大为(299.69±8.64μmol/L,P < 0.05)、(405.32±2.84μmol/L,P < 0.05)、(369.25±0.85μmol/L,P < 0.05)、(387.18±26.26μmol/L,P < 0.01)和(331.15±4.13μmol/L,P < 0.05)。且格列苯脲、Bay K8644和去除血管内皮后使最大舒张率分别减小为(68.65±4.31%,P < 0.01)、(80.10±3.11%,P < 0.05)和(76.03±2.35%,P < 0.01),但应用L-NAME和吲哚美辛并没有改变H2S的最大舒张作用。(3)预先给予鸟苷酸环化酶的抑制剂ODQ(10μmol/L)以及EDHF的抑制剂apamin(50 nmol/L)和charybdotoxin(50 nmol/L)预处理后,对H2S的舒张作用没有显著改变,其IC50值分别为(295.31±16.07μmol/L,P > 0.05)和(261.47±36.57μmol/L,P > 0.05)。(4)做KCl(5,15,25,35,45,55,65 mmol/L)收缩肾动脉环的量效曲线,其IC50值为14.91±0.16 mmol/L。(5)先给予H2S合酶的抑制剂PPG预处理后,KCl的浓度量效曲线左移,其IC50值减小为(11.65±1.08 mmol/L,P < 0.05)。(6)先给予H2S(NaHS 200μmol/L)预处理后,KCl的浓度量效曲线右移,其IC50值增大为(19.16±1.09 mmol/L,P < 0.05)。
结论
1.正常家兔血浆中含有H2S。
2.正常家兔肾动脉含有CSE,且能产生H2S。
3.实验数据表明H2S对肾动脉有舒张作用,此作用是由直接开放血管平滑肌上的KATP通道,同时关闭钙通道来实现的;且此作用是内皮依赖型的,与一氧化氮和前列环素有协同舒张作用,但与鸟苷酸环化酶途径和EDHF途径无关;血管组织生成的内源性H2S可拮抗KCl引起的血管收缩作用。
Hydrogen sulfide (H2S), the colorless gas with a strong odor of rotten eggs, has been generally considered as a toxic gas. The main toxicity effect of H2S is a potent inhibition of mitochondrial cytochrome c oxidase and then inhibits mitochondrial respiration. But recently H2S has been proposed to be the third endogenous gasotransmitter which was similar to two other vasoactive gases, nitric oxide (NO) and carbon monoxide (CO). Some study suggested that heart tissues could endogenously produce H2S as a physiological and pathological cardiac function regulator. It also has an important modulation function on the pathogenesis of spontaneous hypertension, hypoxic pulmonary hypertension, high pulmonary blood hypertension and renovascular hypertension. H2S relaxed volume vascular tissues of rat aortic directly through opening ATP-sensitive potassium channels (KATP) in vascular smooth muscle cells (SMCs), but relaxed resistance vascular tissues of mesentery artery through two targets: opening KATP channels in vascular SMCs and KCa channels which can inhibited by ChTX/apamin in vascular endothelial cells, the target of EDHF. It is thus evident that the pathway of H2S relaxing vascular vessels is different from NO and CO (they mainly activate soluble guanylate cyclase and increase intracellular cGMP concentration). H2S also inhibits SMCs proliferation, enhances SMCs apoptosis and improves pulmonary vascular structural remodeling. The recent study suggested that heart tissues could endogenously produce H2S as a physiological and pathological cardiac function regulator. The pathological renal arteries produced lumens stenosis and then activate rennin-angiotensin system to result in renovascular hypertension. The increscent resistance of renal artery is important character of renal haemodynamics in renovascular hypertension. It is thus evident that renal artery tension has important roles in the pathogenesis of hypertension and renovascular hypertension. The effects of H2S on renal artery have not been reported yet.
Aim
The purpose of the present research was to measure the H2S concentration in normal rabbit plasma, to assay CSE activity in renal artery smooth muscle homogenatestissue, to investigate the effects of H2S on rabbit renal artery and to explore the underlying mechanism.
Methods
1 Measurement of plasma H2S concentration in rabbit.
A sample of plasma (0.1 ml) was added to a test tube containing 0.5 ml of 1% zinc acetate and 2.5 ml of distilled water, then 0.5 ml of 20 mmol/L N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 mol/L HCl and 0.4 ml of 30 mmol/L FeCl3 in 1.2 mol/L HCl were also added to the same test tube for 20 min of incubation at room temperature. The protein in the plasma was removed by adding 1 ml of 10% trichloroacetic acid to the solution and centrifuging it. The optical absorbance of the resulting solution at 665 nm was measured with a spectrometer. H2S concentration in the solution was calculated against the calibration curve of the standard NaHS solution.
2. Assay of renal artery tissue CSE activity.
The renal artery tissues were homogenized in ice-cold 50 mmol/L potassium phosphate buffer (PH 6.8). Reactions were performed in 25 ml Erlenmeyer flasks. The reaction mixture contained (mmol/L): 10 L-cysteine, 2 pyridoxal 5’-phosphate, 100 potassium phosphate buffer (PH 7.4), and 10% (w/v) homogenates. The total volume of the reaction mixture was 1 ml. A small piece of filter paper was put into the central well of the flask and 0.5 ml of 1% zinc acetate was also added in the central well for trapping evolved H2S in the mixture. The flasks were then flushed with N2 before being sealed with a double layer of parafilm. The catalytic reaction was initiated by transferring the flasks from an ice bath to a 37℃shaking water bath. After 90 min at 37℃, the reactions were stopped by injecting 0.5 ml of 50% trichloroacetic acid. Flasks were incubated in the shaking water bath for an additional hour at 37℃to complete trapping of H2S. The content of the central well was transferred to test tubes and mixed with 3.5 ml of distilled water and 0.5 ml of 20 mmol/L N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 mol/L HCl. To each tube, 0.4 ml of 30 mmol/L FeCl3 in 1.2 mol/L HCl was added immediately. After 20 min of incubation at room temperature, the optical absorbance of the resulting solution at 665 nm was measured with a spectrometer. The H2S concentration in the solution was calculated against the calibration curve of the standard H2S solution. For each sample, the measurement was done in duplicate. Protein was determined using the Bradford technique with bovine serum albumin (BSA) as a standard. The H2S production was expressed in a unit of pmol/mg protein/minute.
3. Modulation of hydrogen sulfide on tension of isolate rabbit renal artery and its mechanism.
The functional curve of hydrogen sulfide on rabbit renal artery was measured by recording the changes of H2S concentration and drugs with organ bath system.
Results
1. The plasma level of H2S in normal rabbit was 44.39±4.93μmol/L.
2. The activity of enzymes in renal artery tissue was 143.94±4.80 pmol/mg protein/minute.
3. (1) NaHS (50, 100, 200, 400 and 800μmol/L) induced significant relaxation of renal artery rings with intact endothelium in a concentration-dependent manner preshrunk by KCl (50 mmol/L) as a control. The IC50 of the concentration-response relaxation curve was 281.46±17.26μmol/L. A maximum relaxation of 89.97±2.40% was attained at 800μmol/L of NaHS. (2) Pretreatment with KATP channel blocker glibenclamide (Gli, 20μmol/L), calcium channels agonist Bay K8644 (500 nmol/L), NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100μmol/L), prostaglandin (PGI2) inhibitor indomethacin (10μmol/L) and removal of the endothelium significantly inhibited H2S-induced relaxation. The concentration-response relaxation curve shifted right obviously and the IC50 changed from 281.46±17.26μmol/L to 299.69±8.64μmol/L (P < 0.05), (405.32±2.84μmol/L,P < 0.05), (369.25±0.85μmol/L,P < 0.05), (387.18±26.26μmol/L,P < 0.01) and (331.15±4.13μmol/L,P < 0.05) respectively. Furthermore, pretreatment with glibenclamide, Bay K8644 and removal of the endothelium significantly decrease H2S-induced maximum relaxation from (89.97±2.40%) to (68.65±4.31%, P < 0.01), (80.10±3.11%, P < 0.05) and (76.03±2.35%, P < 0.01) respectively, but L-NAME and indomethacin did not change the maximum relaxation. (3) Pretreatment with the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3a]quinoxalin-1-one (ODQ, 10μmol/L) and co-application of charybdotoxin and apamin (Ca2+-dependent K+ channel blocker, KCa, 50 nmol/L) have no effect on the action of H2S; the IC50 of the concentration-response curve was (295.31±16.07μmol/L,P > 0.05) and (261.47±36.57μmol/L,P > 0.05). (4) After 1 hour equilibration, increasing concentrations of KCl (5, 15, 25, 35, 45, 55 and 65 mmol/L) to study the contraction effect of KCl as compare. KCl induced significant vasoconstriction of renal artery rings in a concentration-dependent manner. The IC50 of the concentration-response curve was 14.91±0.16 mmol/L. (5) Pretreatment with DL-propargylglycine (PPG, CSE inhibitor, 200μmol/L), to inhibit endogenously H2S, for 30 min before the rings were administration with KCl. The vasoconstriction was markedly enhanced; the concentration-response curve shifted to the left and upward, and the IC50 was decreased to 11.65±1.08 mmol/L (P < 0.05). (6) Pretreatment with NaHS (200μmol/L) distinguishably suppressed the vasoconstriction by KCl, the concentration-response curve shifted to the right and downward, and the IC50 was increased to 19.16±1.09 mmol/L (P < 0.05).
Conclusion
1. The plasma concentration of H2S in rabbit was similar to the level of rat (45.6±14.2μmol/L).
2. The activity of enzymes in renal artery tissue was lower than rat pulmonary artery (831.8±70.5) pmol/mg protein/minute.
3. Exogenous H2S could endothelium-dependently relax rabbit renal artery through opening of KATP channels further closing the calcium channels in vascular smooth muscles. NO and PGI2 possible have a synergism with H2S on the effect of vasodilation. After inhibited endogenously H2S induced an increasing contraction effect of the vascular. While H2S relaxed vascular tissue is not dependent on the activation of the cGMP pathway or KCa channels.
目的
应用离体血管环张力测定技术观察H2S对家兔离体肾动脉血管环张力的调节作用,探讨其可能的作用机制。
方法
1.测定正常家兔血浆中H2S的含量。
在试管中加入1%(质量分数)醋酸锌0.5 ml,蒸馏水2.5 ml,血浆0.1 ml,混匀。然后加入7.2 mol/L盐酸(含20 mmol/L N,N-二甲基-对苯二胺盐酸盐)0.5 ml,再加入1.2 mol/L HCl(含30 mmol/L FeCl3)0.4 ml,室温孵育20 min,然后加入10%三氯醋酸1 ml。将上述试管内容物离心5 min后取上清,用分光光度计在665 nm波长处检测光吸收度。用H2S标准曲线计算血浆H2S浓度,结果用μmol/L表示。
2.测定肾动脉上硫化氢生成酶(cystathionine -lyase,CSE)的活性。
取家兔的肾动脉在冰冷的磷酸钾缓冲液(50 mmol/L,pH 6.8)中研磨成匀浆。反应在25 ml锥形瓶中进行。反应体积1 ml,含磷酸钾缓冲液(100 mmol/L,pH 7.4)、L-半胱氨酸(10 mmol/L)、5’-磷酸吡哆醛(2 mmol/L)和10%(V/V)组织匀浆。在中央室中加入10%(W/W)醋酸锌0.5 ml,并放入滤纸增加吸收面积。用N2将烧瓶充盈30 s后,石蜡膜封口,转移到37℃水浴摇床中开始反应,90 min后向其中注入50%(W/W)三氯醋酸0.5 ml中止反应,继续水浴60 min。将中央室的内容物转移到含3.5 ml蒸馏水的试管中,加入20 mmol/L对苯二胺盐酸盐0.5 ml和30 mmol/L FeCl3 0.4 ml,20 min后用分光光度计在665 nm波长处检测光吸收度。用H2S标准曲线计算溶液中H2S含量,H2S产出率由(pmol/mg protein/minute)来表示,反映CSE的活性。
3.硫化氢对离体家兔肾动脉血管环张力的调节。
运用离体血管环灌流系统,观察硫化氢作用后血管环张力的变化,由RM6240生物信号采集处理系统进行记录分析,检测血管环张力的变化并探讨其可能的机制。
结果
1.正常兔血浆中H2S的含量为(44.39±4.9)μmol/L。
2.正常家兔肾动脉上H2S生成率为(143.94±4.80)pmol/mg protein/minute。
3. (1)外源性的H2S(NaHS 50,100,200,400,800μmol/L)可以剂量依赖性地舒张由KCl(50 mmol/L)预收缩的肾动脉血管环,其浓度反应曲线的IC50值为281.46±17.26μmol/L,最大舒张率为89.97±2.40%。(2)用KATP通道阻断剂格列苯脲(glibenclamide, 20μmol/L)、钙通道的开放剂Bay K8644(500 nmol/L)、NO合酶的抑制剂L-NAME(100μmol/L)和环氧合酶阻断剂吲哚美辛(10μmol/L)预处理以及去除血管内皮后,H2S的舒张效应均被显著抑制,浓度反应曲线均明显右移,其IC50值分别增大为(299.69±8.64μmol/L,P < 0.05)、(405.32±2.84μmol/L,P < 0.05)、(369.25±0.85μmol/L,P < 0.05)、(387.18±26.26μmol/L,P < 0.01)和(331.15±4.13μmol/L,P < 0.05)。且格列苯脲、Bay K8644和去除血管内皮后使最大舒张率分别减小为(68.65±4.31%,P < 0.01)、(80.10±3.11%,P < 0.05)和(76.03±2.35%,P < 0.01),但应用L-NAME和吲哚美辛并没有改变H2S的最大舒张作用。(3)预先给予鸟苷酸环化酶的抑制剂ODQ(10μmol/L)以及EDHF的抑制剂apamin(50 nmol/L)和charybdotoxin(50 nmol/L)预处理后,对H2S的舒张作用没有显著改变,其IC50值分别为(295.31±16.07μmol/L,P > 0.05)和(261.47±36.57μmol/L,P > 0.05)。(4)做KCl(5,15,25,35,45,55,65 mmol/L)收缩肾动脉环的量效曲线,其IC50值为14.91±0.16 mmol/L。(5)先给予H2S合酶的抑制剂PPG预处理后,KCl的浓度量效曲线左移,其IC50值减小为(11.65±1.08 mmol/L,P < 0.05)。(6)先给予H2S(NaHS 200μmol/L)预处理后,KCl的浓度量效曲线右移,其IC50值增大为(19.16±1.09 mmol/L,P < 0.05)。
结论
1.正常家兔血浆中含有H2S。
2.正常家兔肾动脉含有CSE,且能产生H2S。
3.实验数据表明H2S对肾动脉有舒张作用,此作用是由直接开放血管平滑肌上的KATP通道,同时关闭钙通道来实现的;且此作用是内皮依赖型的,与一氧化氮和前列环素有协同舒张作用,但与鸟苷酸环化酶途径和EDHF途径无关;血管组织生成的内源性H2S可拮抗KCl引起的血管收缩作用。
Hydrogen sulfide (H2S), the colorless gas with a strong odor of rotten eggs, has been generally considered as a toxic gas. The main toxicity effect of H2S is a potent inhibition of mitochondrial cytochrome c oxidase and then inhibits mitochondrial respiration. But recently H2S has been proposed to be the third endogenous gasotransmitter which was similar to two other vasoactive gases, nitric oxide (NO) and carbon monoxide (CO). Some study suggested that heart tissues could endogenously produce H2S as a physiological and pathological cardiac function regulator. It also has an important modulation function on the pathogenesis of spontaneous hypertension, hypoxic pulmonary hypertension, high pulmonary blood hypertension and renovascular hypertension. H2S relaxed volume vascular tissues of rat aortic directly through opening ATP-sensitive potassium channels (KATP) in vascular smooth muscle cells (SMCs), but relaxed resistance vascular tissues of mesentery artery through two targets: opening KATP channels in vascular SMCs and KCa channels which can inhibited by ChTX/apamin in vascular endothelial cells, the target of EDHF. It is thus evident that the pathway of H2S relaxing vascular vessels is different from NO and CO (they mainly activate soluble guanylate cyclase and increase intracellular cGMP concentration). H2S also inhibits SMCs proliferation, enhances SMCs apoptosis and improves pulmonary vascular structural remodeling. The recent study suggested that heart tissues could endogenously produce H2S as a physiological and pathological cardiac function regulator. The pathological renal arteries produced lumens stenosis and then activate rennin-angiotensin system to result in renovascular hypertension. The increscent resistance of renal artery is important character of renal haemodynamics in renovascular hypertension. It is thus evident that renal artery tension has important roles in the pathogenesis of hypertension and renovascular hypertension. The effects of H2S on renal artery have not been reported yet.
Aim
The purpose of the present research was to measure the H2S concentration in normal rabbit plasma, to assay CSE activity in renal artery smooth muscle homogenatestissue, to investigate the effects of H2S on rabbit renal artery and to explore the underlying mechanism.
Methods
1 Measurement of plasma H2S concentration in rabbit.
A sample of plasma (0.1 ml) was added to a test tube containing 0.5 ml of 1% zinc acetate and 2.5 ml of distilled water, then 0.5 ml of 20 mmol/L N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 mol/L HCl and 0.4 ml of 30 mmol/L FeCl3 in 1.2 mol/L HCl were also added to the same test tube for 20 min of incubation at room temperature. The protein in the plasma was removed by adding 1 ml of 10% trichloroacetic acid to the solution and centrifuging it. The optical absorbance of the resulting solution at 665 nm was measured with a spectrometer. H2S concentration in the solution was calculated against the calibration curve of the standard NaHS solution.
2. Assay of renal artery tissue CSE activity.
The renal artery tissues were homogenized in ice-cold 50 mmol/L potassium phosphate buffer (PH 6.8). Reactions were performed in 25 ml Erlenmeyer flasks. The reaction mixture contained (mmol/L): 10 L-cysteine, 2 pyridoxal 5’-phosphate, 100 potassium phosphate buffer (PH 7.4), and 10% (w/v) homogenates. The total volume of the reaction mixture was 1 ml. A small piece of filter paper was put into the central well of the flask and 0.5 ml of 1% zinc acetate was also added in the central well for trapping evolved H2S in the mixture. The flasks were then flushed with N2 before being sealed with a double layer of parafilm. The catalytic reaction was initiated by transferring the flasks from an ice bath to a 37℃shaking water bath. After 90 min at 37℃, the reactions were stopped by injecting 0.5 ml of 50% trichloroacetic acid. Flasks were incubated in the shaking water bath for an additional hour at 37℃to complete trapping of H2S. The content of the central well was transferred to test tubes and mixed with 3.5 ml of distilled water and 0.5 ml of 20 mmol/L N,N-dimethyl-p-phenylenediamine dihydrochloride in 7.2 mol/L HCl. To each tube, 0.4 ml of 30 mmol/L FeCl3 in 1.2 mol/L HCl was added immediately. After 20 min of incubation at room temperature, the optical absorbance of the resulting solution at 665 nm was measured with a spectrometer. The H2S concentration in the solution was calculated against the calibration curve of the standard H2S solution. For each sample, the measurement was done in duplicate. Protein was determined using the Bradford technique with bovine serum albumin (BSA) as a standard. The H2S production was expressed in a unit of pmol/mg protein/minute.
3. Modulation of hydrogen sulfide on tension of isolate rabbit renal artery and its mechanism.
The functional curve of hydrogen sulfide on rabbit renal artery was measured by recording the changes of H2S concentration and drugs with organ bath system.
Results
1. The plasma level of H2S in normal rabbit was 44.39±4.93μmol/L.
2. The activity of enzymes in renal artery tissue was 143.94±4.80 pmol/mg protein/minute.
3. (1) NaHS (50, 100, 200, 400 and 800μmol/L) induced significant relaxation of renal artery rings with intact endothelium in a concentration-dependent manner preshrunk by KCl (50 mmol/L) as a control. The IC50 of the concentration-response relaxation curve was 281.46±17.26μmol/L. A maximum relaxation of 89.97±2.40% was attained at 800μmol/L of NaHS. (2) Pretreatment with KATP channel blocker glibenclamide (Gli, 20μmol/L), calcium channels agonist Bay K8644 (500 nmol/L), NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100μmol/L), prostaglandin (PGI2) inhibitor indomethacin (10μmol/L) and removal of the endothelium significantly inhibited H2S-induced relaxation. The concentration-response relaxation curve shifted right obviously and the IC50 changed from 281.46±17.26μmol/L to 299.69±8.64μmol/L (P < 0.05), (405.32±2.84μmol/L,P < 0.05), (369.25±0.85μmol/L,P < 0.05), (387.18±26.26μmol/L,P < 0.01) and (331.15±4.13μmol/L,P < 0.05) respectively. Furthermore, pretreatment with glibenclamide, Bay K8644 and removal of the endothelium significantly decrease H2S-induced maximum relaxation from (89.97±2.40%) to (68.65±4.31%, P < 0.01), (80.10±3.11%, P < 0.05) and (76.03±2.35%, P < 0.01) respectively, but L-NAME and indomethacin did not change the maximum relaxation. (3) Pretreatment with the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3a]quinoxalin-1-one (ODQ, 10μmol/L) and co-application of charybdotoxin and apamin (Ca2+-dependent K+ channel blocker, KCa, 50 nmol/L) have no effect on the action of H2S; the IC50 of the concentration-response curve was (295.31±16.07μmol/L,P > 0.05) and (261.47±36.57μmol/L,P > 0.05). (4) After 1 hour equilibration, increasing concentrations of KCl (5, 15, 25, 35, 45, 55 and 65 mmol/L) to study the contraction effect of KCl as compare. KCl induced significant vasoconstriction of renal artery rings in a concentration-dependent manner. The IC50 of the concentration-response curve was 14.91±0.16 mmol/L. (5) Pretreatment with DL-propargylglycine (PPG, CSE inhibitor, 200μmol/L), to inhibit endogenously H2S, for 30 min before the rings were administration with KCl. The vasoconstriction was markedly enhanced; the concentration-response curve shifted to the left and upward, and the IC50 was decreased to 11.65±1.08 mmol/L (P < 0.05). (6) Pretreatment with NaHS (200μmol/L) distinguishably suppressed the vasoconstriction by KCl, the concentration-response curve shifted to the right and downward, and the IC50 was increased to 19.16±1.09 mmol/L (P < 0.05).
Conclusion
1. The plasma concentration of H2S in rabbit was similar to the level of rat (45.6±14.2μmol/L).
2. The activity of enzymes in renal artery tissue was lower than rat pulmonary artery (831.8±70.5) pmol/mg protein/minute.
3. Exogenous H2S could endothelium-dependently relax rabbit renal artery through opening of KATP channels further closing the calcium channels in vascular smooth muscles. NO and PGI2 possible have a synergism with H2S on the effect of vasodilation. After inhibited endogenously H2S induced an increasing contraction effect of the vascular. While H2S relaxed vascular tissue is not dependent on the activation of the cGMP pathway or KCa channels.
引文
1 Doeller JE, Isbell TS, Benavides G, et al. Polarographic measurement of hydrogen sulfide production and consumption by mammalian tissues. Anal Biochem, 2005, 341: 40-51
2 Wang R. Two’s company, three’s a crowd: can H2S be the third endogenous gasous transmitter? FASEB J, 2000, 16: 1792-1798
3 Stipanuk MH. Sulfur amino acid metabolism: pathways for production and removal of homocysteine and cysteine. Annu Rev Nutr, 2004, 24: 539-577
4 Hosoki R, Matsuki N, kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem Biophys Res Commum, 1997, 273: 527-531
5 Chen P, Poddar R, Tipa EV, et al. Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocys-teinemia and cardiovascular disease. Adv Enzyme Regul, 1999, 39: 93-109
6 Bao L, Vlcek C, Paces V, et al. Identification and tissue distribution of human cystathionine beta-synthase mRNA isoforms. Arch Biochem Biophys, 1998, 350: 95-103
7 Wang J, Dudman NP, Wilcken DE, et al. Homocysteine catabolism: levels of 3 enzymes in cultured human vascular endothelium and their relevance to vascular disease.Atherosclerosis, 1992, 97: 97-106
8 Zhao WM, Zhang J, Lu YJ, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J, 2001, 20: 6008-6016
9 Lowicka E, Beltowski J. Hydrogen sulfide (H2S)-the third gas of interest for pharmacologists. Pharmacol Rep, 2007, 59: 4-24
10 Wang R. The gasotransmitter role of hydrogen sulfide. Antioxidants & Redox Sianaling, 2003, 5: 493-501
11 Satoko Kubo, Ichiko Doe, Yuko Kurokawa, et al. Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: Contribution to dual modulation of vascular tension. Toxicology, 2007, 232: 138-146
12 Ali MY, Ping CY, Mok YY, et al. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol, 2006, 149: 625-634
13 Whiteman M, Li L, Kostetski I, et al. Evidence for the formation of a novel nitrosothiol from the gaseous mediators nitric oxide and hydrogen sulphide. Biochem Biophys Res Commun, 2006, 343: 303-310
14 Tang G, Wu L, Liang W, et al. Direct stimulation of KATP channels by exogenous and endogenous hydrogen sulfide in vascular smooth muscle cells. Mol Pharmacol, 2005, 68: 1757-1764
15 Cheng YQ, Joseph FN, Tang GH, et al. Hydrogen sulfide induced relaxation of resistance mesenteric artery beds ofrats. Heart Circ Physiol, 2004, 287: 2316-2323
16 Yang GD,Wu LY,Wang R.Pro-apoptotic effect of endogenous H2S on human aorta smooth muscle cells. FASEB J, 2006, 20: 553-555
17 Li XH, Du JB, Shi L, et al. Down-regulation of endogenous hydrogen sulfide pathway in pulmonary hypertension and pulmonary vascular structural remodeling induced by high pulmonary blood flow in rats. Circ J, 2005, 69: 1418-1424
18 Zhang CY, Du JB, Bu DF, et al. The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in rats. Biochem Biophys Res Commun, 2003, 302: 810-816
19 Huang XL, Zhou XH, Wei P, et al. Role of endogenous hydrogen sulfide in pulmonary hypertension induced by lipopolysaccharide. Acta Physiol Sin, 2008, 60: 211-215
20 Jia-Jia Lim, Yihong Liu, Ester Khin Sandar Win, et al. Vasoconstrictive effect of hydrogen sulfide involves downregulation of cAMP in vascular smooth muscle cells. Am J Physiol Cell Physiol, 2008, 295: C1261-C1270
21 Zhao WM, Wang R. H2S-induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol, 2002, 283: H474-H480
22 Standen NB, Quayle JM, Davies NW, et al. Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science, 1989, 245: 177-180
23 Busse R, Edwards G, Feketou M, et al. EDHF: bring the concepts together. Trends Pharmacol Sci, 2002, 23: 374-380
24 Doughty JM, PlaneF, and Langton PD. Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium. Am J Physiol, 1999, 276: 1107-1112
25 Furchgott RF and Jothianandan D. Endothelium-dependent and-independent vasodilatation involving cyclic GMP: relaxation induced by nitric oxide, carbon monoxide and light. Blood Vessels, 1991, 128: 52-61
26 WU Dai-qin, CHENG You-qin, FANG Ying, et al. Regulatory effect of endogenous hydrogen sulfide system on renovascular hypertension. Chin J Geriatr Heart Brain Vessel Dis, 2006, l8: 748-751
27 Ling Li, Matthew Whiteman, Yan Yi Guan, et al. Characterization of a Novel, Water-Soluble Hydrogen Sulfide-Releasing Molecule (GYY4137). Circulation, 2008, 117: 2351-2360
1 Lowicka E, Beltowski J. Hydrogen sulfide (H2S)-the third gasof interest for pharmacologists. Pharmacol Rep, 2007, 59(1): 4-24
2 Wang R. Two’s company, three’s a crowd:can H2S be the third endogenous gaseous transmitter? FASEB J, 2002, 16(13): 1792-1798
3 Zhao WM, Zhang J, Lu YJ, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J, 2001, 20(21): 6008-6016
4 Zhao WM, Wang R. H2S-induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol, 2002, 283(2): 474-480
5 Wang R. The gasotransmitter role of hydrogen sulfide. Antioxidants & Redox Sianaling, 2003, 5(4): 493-501
6王燕飞,肖慧捷,唐朝枢,等。不同海拔地区健康儿童气体信号因子一氧化氮及硫化氢的变化。中国实用儿科杂志, 2007, 22(4): 254-256
7 Cheng YQ, Joseph FN, Tang GH, et al. Hydrogen sulfide induced relaxation of resistance mesenteric artery beds of rats. Heart Circ Physiol, 2004, 287(5): 2316-2323
8 Tang G, Wu L, Liang W, et al. Direct stimulation of KATP channels by exogenous and endogenous hydrogen sulfide in vascular smooth muscle cells. Mol Pharmacol, 2005, 68(6):1757-1764
9 Junbao D, Hui Y, Yiufan C, et al. The possible role of hydrogen sulfide as a smooth muscle cell proliferation inhibitor in rat cultured cells. Heart Vessels, 2004, 19(2): 75-80
10 Yang G, Cao K, Wu L, et al. Cystathionine gamma- lyase(CSE) overexpression inhibits cell proliferation via a H2S-dependent modulation of ERK1/2 phosphorylation and p21Cip/WAK-1. J Biol Chem, 2004, 279(47): 49199-49205
11陈晓波,杜军保,张春雨,等。硫化氢对低氧性肺动脉高压大鼠肺动脉增殖细胞核抗原及Bcl-2的调节作用。实用儿科临床杂志, 2004, 19(3): 185-187
12 Yang G, Sun X, Wang R. Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen activated protein kinases and caspase-3. FASEB J, 2004, 18(14): 1782-1784
13 Yan H, Du JB, Tang CS. The possible role of hydrogen sulfide on the pathogenesis of spontaneous hypertension in rats. Biochem Biophys Res Commun, 2004, 313(1): 22-27
14李晓惠,杜军保,唐朝枢。硫化氢供体对大鼠高肺血流性肺动脉高压中内皮素- 1及结缔组织生长因子表达的影响。中国病理生理杂志, 2008, 24(3): 446-450
15吴胜英,潘春水,耿彬,等。外源性硫化氢对大鼠血管钙化的影响。基础医学与临床, 2006, 26(5): 466-470
16 Kubo S, Doe I, Kurokawa Y, et al. Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide:Contribution to dual modulation of vascular tension. Toxicology, 2007, 232(1-2): 138-146
17 Ali MY, Ping CY, Mok YY, et al. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol, 2006, 149(6): 625-634
18 Webb GD, Lim LH, Oh VM, et al. Contractile and Vasorelaxant Effects of Hydrogen Sulfide and its Biosynthesis in the Human Internal Mammary. Pharmacol Exp Ther, 2007, 324(2): 876-882
19 Kubo S, Kurokawa Y, Doe I, et al. Hydrogen sulfide inhibits activity of three isoforms of recombinant nitric oxide synthase. Toxicology, 2007, 241(1-2): 92-97
20冯浩楼,崔玉英,魏芳等。硫化氢下调大鼠血小板L-精氨酸/一氧化氮合酶/一氧化氮通路。中国现代医学杂志, 2008, 18(7): 852-855
21 Zhang Q, Du J, Zhang W. Impact of hydrogen sulfide on carbon monoxide/heme oxygenase pathway in the pathogenesis of hypoxic pulmonary hypertension. Biochem Biophys Res Commun, 2004, 317(1): 30-37
22 Huang XL, Zhou XH, Wei P, et al. Role of endogenous hydrogen sulfide in pulmonary hypertension induced by lipopolysaccharide. Acta Physiol Sin, 2008, 60(2): 211-215
23 Ling Li, Matthew Whiteman, Yan Yi Guan, et al. Characterization of a Novel, Water-Soluble Hydrogen Sulfide–Releasing Molecule(GYY4137) New Insights Into the Biology of Hydrogen Sulfide. Circulation, 2008, 117(18):2351-2360
24 Dawe GS, Han SP, Bian JS, et al. Hydrogen sulphide in the hypothalamus causes an ATP-sensitive K+ channel-dependent decrease in blood pressure in freely moving rats. Neuroscience, 2008, 152(1): 169-177
25 Saima Muzaffar, Nilima Shukla, Mark Bond, et al. Exogenous hydrogen sulfide inhibits superoxide formation, NOX-1 expression and Rac1 activity in human vascular smooth muscle cells. Vasc Res, 2008, 45(6): 521-528
2 Wang R. Two’s company, three’s a crowd: can H2S be the third endogenous gasous transmitter? FASEB J, 2000, 16: 1792-1798
3 Stipanuk MH. Sulfur amino acid metabolism: pathways for production and removal of homocysteine and cysteine. Annu Rev Nutr, 2004, 24: 539-577
4 Hosoki R, Matsuki N, kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem Biophys Res Commum, 1997, 273: 527-531
5 Chen P, Poddar R, Tipa EV, et al. Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocys-teinemia and cardiovascular disease. Adv Enzyme Regul, 1999, 39: 93-109
6 Bao L, Vlcek C, Paces V, et al. Identification and tissue distribution of human cystathionine beta-synthase mRNA isoforms. Arch Biochem Biophys, 1998, 350: 95-103
7 Wang J, Dudman NP, Wilcken DE, et al. Homocysteine catabolism: levels of 3 enzymes in cultured human vascular endothelium and their relevance to vascular disease.Atherosclerosis, 1992, 97: 97-106
8 Zhao WM, Zhang J, Lu YJ, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J, 2001, 20: 6008-6016
9 Lowicka E, Beltowski J. Hydrogen sulfide (H2S)-the third gas of interest for pharmacologists. Pharmacol Rep, 2007, 59: 4-24
10 Wang R. The gasotransmitter role of hydrogen sulfide. Antioxidants & Redox Sianaling, 2003, 5: 493-501
11 Satoko Kubo, Ichiko Doe, Yuko Kurokawa, et al. Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: Contribution to dual modulation of vascular tension. Toxicology, 2007, 232: 138-146
12 Ali MY, Ping CY, Mok YY, et al. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol, 2006, 149: 625-634
13 Whiteman M, Li L, Kostetski I, et al. Evidence for the formation of a novel nitrosothiol from the gaseous mediators nitric oxide and hydrogen sulphide. Biochem Biophys Res Commun, 2006, 343: 303-310
14 Tang G, Wu L, Liang W, et al. Direct stimulation of KATP channels by exogenous and endogenous hydrogen sulfide in vascular smooth muscle cells. Mol Pharmacol, 2005, 68: 1757-1764
15 Cheng YQ, Joseph FN, Tang GH, et al. Hydrogen sulfide induced relaxation of resistance mesenteric artery beds ofrats. Heart Circ Physiol, 2004, 287: 2316-2323
16 Yang GD,Wu LY,Wang R.Pro-apoptotic effect of endogenous H2S on human aorta smooth muscle cells. FASEB J, 2006, 20: 553-555
17 Li XH, Du JB, Shi L, et al. Down-regulation of endogenous hydrogen sulfide pathway in pulmonary hypertension and pulmonary vascular structural remodeling induced by high pulmonary blood flow in rats. Circ J, 2005, 69: 1418-1424
18 Zhang CY, Du JB, Bu DF, et al. The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in rats. Biochem Biophys Res Commun, 2003, 302: 810-816
19 Huang XL, Zhou XH, Wei P, et al. Role of endogenous hydrogen sulfide in pulmonary hypertension induced by lipopolysaccharide. Acta Physiol Sin, 2008, 60: 211-215
20 Jia-Jia Lim, Yihong Liu, Ester Khin Sandar Win, et al. Vasoconstrictive effect of hydrogen sulfide involves downregulation of cAMP in vascular smooth muscle cells. Am J Physiol Cell Physiol, 2008, 295: C1261-C1270
21 Zhao WM, Wang R. H2S-induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol, 2002, 283: H474-H480
22 Standen NB, Quayle JM, Davies NW, et al. Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science, 1989, 245: 177-180
23 Busse R, Edwards G, Feketou M, et al. EDHF: bring the concepts together. Trends Pharmacol Sci, 2002, 23: 374-380
24 Doughty JM, PlaneF, and Langton PD. Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium. Am J Physiol, 1999, 276: 1107-1112
25 Furchgott RF and Jothianandan D. Endothelium-dependent and-independent vasodilatation involving cyclic GMP: relaxation induced by nitric oxide, carbon monoxide and light. Blood Vessels, 1991, 128: 52-61
26 WU Dai-qin, CHENG You-qin, FANG Ying, et al. Regulatory effect of endogenous hydrogen sulfide system on renovascular hypertension. Chin J Geriatr Heart Brain Vessel Dis, 2006, l8: 748-751
27 Ling Li, Matthew Whiteman, Yan Yi Guan, et al. Characterization of a Novel, Water-Soluble Hydrogen Sulfide-Releasing Molecule (GYY4137). Circulation, 2008, 117: 2351-2360
1 Lowicka E, Beltowski J. Hydrogen sulfide (H2S)-the third gasof interest for pharmacologists. Pharmacol Rep, 2007, 59(1): 4-24
2 Wang R. Two’s company, three’s a crowd:can H2S be the third endogenous gaseous transmitter? FASEB J, 2002, 16(13): 1792-1798
3 Zhao WM, Zhang J, Lu YJ, et al. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J, 2001, 20(21): 6008-6016
4 Zhao WM, Wang R. H2S-induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol, 2002, 283(2): 474-480
5 Wang R. The gasotransmitter role of hydrogen sulfide. Antioxidants & Redox Sianaling, 2003, 5(4): 493-501
6王燕飞,肖慧捷,唐朝枢,等。不同海拔地区健康儿童气体信号因子一氧化氮及硫化氢的变化。中国实用儿科杂志, 2007, 22(4): 254-256
7 Cheng YQ, Joseph FN, Tang GH, et al. Hydrogen sulfide induced relaxation of resistance mesenteric artery beds of rats. Heart Circ Physiol, 2004, 287(5): 2316-2323
8 Tang G, Wu L, Liang W, et al. Direct stimulation of KATP channels by exogenous and endogenous hydrogen sulfide in vascular smooth muscle cells. Mol Pharmacol, 2005, 68(6):1757-1764
9 Junbao D, Hui Y, Yiufan C, et al. The possible role of hydrogen sulfide as a smooth muscle cell proliferation inhibitor in rat cultured cells. Heart Vessels, 2004, 19(2): 75-80
10 Yang G, Cao K, Wu L, et al. Cystathionine gamma- lyase(CSE) overexpression inhibits cell proliferation via a H2S-dependent modulation of ERK1/2 phosphorylation and p21Cip/WAK-1. J Biol Chem, 2004, 279(47): 49199-49205
11陈晓波,杜军保,张春雨,等。硫化氢对低氧性肺动脉高压大鼠肺动脉增殖细胞核抗原及Bcl-2的调节作用。实用儿科临床杂志, 2004, 19(3): 185-187
12 Yang G, Sun X, Wang R. Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen activated protein kinases and caspase-3. FASEB J, 2004, 18(14): 1782-1784
13 Yan H, Du JB, Tang CS. The possible role of hydrogen sulfide on the pathogenesis of spontaneous hypertension in rats. Biochem Biophys Res Commun, 2004, 313(1): 22-27
14李晓惠,杜军保,唐朝枢。硫化氢供体对大鼠高肺血流性肺动脉高压中内皮素- 1及结缔组织生长因子表达的影响。中国病理生理杂志, 2008, 24(3): 446-450
15吴胜英,潘春水,耿彬,等。外源性硫化氢对大鼠血管钙化的影响。基础医学与临床, 2006, 26(5): 466-470
16 Kubo S, Doe I, Kurokawa Y, et al. Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide:Contribution to dual modulation of vascular tension. Toxicology, 2007, 232(1-2): 138-146
17 Ali MY, Ping CY, Mok YY, et al. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol, 2006, 149(6): 625-634
18 Webb GD, Lim LH, Oh VM, et al. Contractile and Vasorelaxant Effects of Hydrogen Sulfide and its Biosynthesis in the Human Internal Mammary. Pharmacol Exp Ther, 2007, 324(2): 876-882
19 Kubo S, Kurokawa Y, Doe I, et al. Hydrogen sulfide inhibits activity of three isoforms of recombinant nitric oxide synthase. Toxicology, 2007, 241(1-2): 92-97
20冯浩楼,崔玉英,魏芳等。硫化氢下调大鼠血小板L-精氨酸/一氧化氮合酶/一氧化氮通路。中国现代医学杂志, 2008, 18(7): 852-855
21 Zhang Q, Du J, Zhang W. Impact of hydrogen sulfide on carbon monoxide/heme oxygenase pathway in the pathogenesis of hypoxic pulmonary hypertension. Biochem Biophys Res Commun, 2004, 317(1): 30-37
22 Huang XL, Zhou XH, Wei P, et al. Role of endogenous hydrogen sulfide in pulmonary hypertension induced by lipopolysaccharide. Acta Physiol Sin, 2008, 60(2): 211-215
23 Ling Li, Matthew Whiteman, Yan Yi Guan, et al. Characterization of a Novel, Water-Soluble Hydrogen Sulfide–Releasing Molecule(GYY4137) New Insights Into the Biology of Hydrogen Sulfide. Circulation, 2008, 117(18):2351-2360
24 Dawe GS, Han SP, Bian JS, et al. Hydrogen sulphide in the hypothalamus causes an ATP-sensitive K+ channel-dependent decrease in blood pressure in freely moving rats. Neuroscience, 2008, 152(1): 169-177
25 Saima Muzaffar, Nilima Shukla, Mark Bond, et al. Exogenous hydrogen sulfide inhibits superoxide formation, NOX-1 expression and Rac1 activity in human vascular smooth muscle cells. Vasc Res, 2008, 45(6): 521-528