硫化氢对离体家兔肾动脉血管环张力调节的研究

发布时间：2018-01-20 07:31

  本文关键词： 硫化氢 肾动脉 CSE ATP敏感钾通道 格列苯脲 Bay K8644　出处：《河北医科大学》2009年硕士论文　论文类型：学位论文

【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。
[Abstract]:Hydrogen sulfide (hydrogen sulfide H2S) has been considered as a colorless, toxic gas with a rotten egg smell, it can inhibit the respiration of.H2S and inhibition of the mitochondrial cytochrome oxidase is the endogenous nitric oxide gas molecules (nitric oxide, NO), carbon monoxide (carbon monoxide, CO) third endogenous gaseous messenger after the discovery (Gasotransmitter). It has been confirmed that H2S can regulate the heart function in physiological and pathological conditions, and in spontaneously hypertensive rats, hypoxic pulmonary hypertension in rats, the incidence of high pulmonary blood flow induced pulmonary hypertension and renal vascular hypertension also plays an important role in the regulation of.H2S by opening KATP the channel capacity of vessels of rat thoracic aorta, but also dependent hyperpolarizing factor through the KATP channel (endothelium-derived hyperpolarizing factor, and EDHF) KCa channel mediated interaction With the relaxation of mesenteric artery vascular resistance. The vasodilatory effect induced by visible H2S is different from other gasotransmitter (such as NO, CO is the guanylate cyclase cGMP pathway).H2S can also inhibit the proliferation of vascular smooth muscle cells and promote its apoptosis, relieve the reconstruction of vascular structure. May indicate that H2S play an important role in the regulation of cardiovascular homeostasis. Renal artery tension is an important factor in the regulation of peripheral vascular resistance, increased renal vascular resistance is an important feature of renal hemodynamics in hypertensive patients. Renal artery lesions lead to luminal stenosis of the renal cortex and renal ischemia stimulate the juxtaglomerular apparatus cells renin secretion excessive, cause renin angiotensin system activation, systemic arterial systolic blood pressure increased, resulting in renal vascular hypertension. Visible renal artery tension in hypertension and renal blood vessels The pathogenesis of high blood pressure plays an important role, and the impact of H2S on it has yet to be reported.
objective
To observe the effect of H2S on the tension of the isolated renal artery ring of rabbit in vitro, the possible mechanism of its possible action was investigated.
Method
1. the content of H2S in normal rabbit plasma was measured.
1% in vitro (mass fraction) of zinc acetate 0.5 ml distilled water, 2.5 ml, 0.1 ml plasma, then mix. Add 7.2 mol/L of hydrochloric acid (containing 20 mmol/L N, N- two - two of benzene methyl amine hydrochloride) 0.5 ml, adding 1.2 mol/L HCl (30 mmol/L FeCl3) 0.4 ml. After incubation at room temperature for 20 min, then adding 10% three 1 ml. the chloroacetic acid content after 5 min centrifuge tube Torikami Kiyo, in the detection wavelength of 665 nm optical absorption spectrophotometer. Calculation of plasma H2S concentration by H2S standard curve, the results were expressed as mol/L.
2. the activity of cystathionine -lyase (CSE) was measured on the renal artery.
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