糖尿病是一种常见多发的慢性内分泌代谢紊乱综合征，由于体内胰岛素缺乏或相对不足而引起的组织器官的代谢异常功能障碍，并最终可导致一系列合并症出现，如糖尿病性心脑血管疾病、眼病、肾病及神经疾病等，并由此导致截肢、失明甚至死亡等严重后果。糖尿病合并症可涉及体内多个器官和组织，大量研究表明微血管及大血管功能损害是糖尿病合并症产生的重要原因。 近年来的研究提示糖尿病血管合并症与高血糖有着密切关系，慢性高血糖是一个重要的启动因子，在糖尿病血管合并症的发生和发展过程中起着重要作用。如果糖尿病患者血糖控制不好，高血糖本身即可以通过多种中间产物和下游底物诱导细胞功能障碍，则可诱发改变血管的结构和功能的分子机制。高血糖致糖尿病慢性合并症的分子机制是蛋白激酶C活性增强、晚期糖化终产物的产生、氧化应激增强和多元醇通路增强。提示高血糖本身即可通过产生有毒的活性产物或者改变细胞内的信号通路而导致血管内皮细胞或中膜平滑肌细胞的功能障碍。 Ca²⁺是细胞内最普遍和重要的信使物质，不同生物信号的刺激均可引起胞浆内Ca²⁺水平的变化，由此触发一系列的生物效应，广泛参与各种生命活动，如细胞的代谢、分泌、增殖、分化和收缩等。因此研究细胞内Ca²⁺的调控机制具有重要的理论和实际意义。 血管平滑肌细胞胞浆Ca²⁺浓度升高是引起血管收缩的关键环节。胞浆Ca²⁺浓度升高是由胞内Ca²⁺池的Ca²⁺释放和胞外Ca²⁺内流引起。其中胞外Ca²⁺内流是引起胞浆Ca²⁺水平持续性升高的关键因素。目前已知，细胞膜上至少存在三种不同性质的Ca²⁺内流通道，即电压依赖性Ca²⁺通道(voltage-dependent Ca²⁺ channel， VDCC)、受体操纵性 Ca²⁺通道(receptor-operated Ca²⁺ channel， ROCC)和 Ca²⁺池操纵性 Ca²⁺通道(store-operated Ca²⁺ channel， SOCC)。VDCC 随膜电位变化而开放。 ROCC的开放与膜去极化无关，仅与膜受体被激活相关。而SOCC主要是通过耗竭胞内Ca²⁺池，激活SOCC，进而触发Ca²⁺池操纵性Ca²⁺内流(store-operated Ca²⁺ entry， SOCE )。 近年来的研究发现在糖尿病或高血糖状态下多种细胞的Ca²⁺池操纵性Ca²⁺内流发生了异常变化：(1)糖尿病患者的淋巴细胞经TG诱导的SOCE增强。当糖浓度为180㎎/dL， 2型糖尿病患者的血小板的SOCE高于正常对照组，并且近一步发现SOCE的增强可能与酪氨酸激酶的活性增强有关；但是糖尿病患者的中性粒细胞上的SOCE降低。(2)动物实验也发现SOCE的异常变化：Curtis等人在糖尿病大鼠的视网膜微动脉平滑肌上发现SOCE及其Ca²⁺泵的摄取都是降低的。(3)体外高糖实验也同样发现SOCE的异常变化：Tamareille等人通过膜片钳技术发现人脐.静脉内皮细胞经过96小时高糖培养诱导的凋亡与SOCE的升高有关，用SOCC的阻断剂2-APB和La⁽³⁺⁾可降低内皮细胞的凋亡；Mene P等人发现高糖培养的大鼠系膜细胞的充电性Ca²⁺内流是降低；高糖可抑制新生的心肌细胞的充电性Ca²⁺内流。 进一步发现，Ca²⁺池操纵性钙内流又与上述三种发病因素，PKC活性增强、氧化应激和糖化终产物有着密切关系：(1) PKC活性增强：Mene等人发现在高糖培养的大鼠系膜细胞上PKC活性增强参与了充电性Ca²⁺内流的减小；Curtis进一步发现PKC活性增强参与了抑制糖尿病大鼠视网膜微动脉平滑肌的SOCE； (2)氧化应激：氧化剂一氯乙烷在大鼠的结肠细胞上可引起钙释放耗竭钙池激活SOCE。此外，目前认为TRP (transient receptor potential)蛋白可能是SOCC的分子基础，在血管内皮细胞上观察到TRP蛋白是氧化还原反应的传感器。(3)晚期糖化终产物：经过晚期糖化终产物处理的人类系膜细胞，在无Ca²⁺液中，1 μ M angiotensin Ⅱ引起的Ca²⁺释放降低，并且晚期糖化终产物也抑制系膜细胞的Ca²⁺内流。醛糖还原酶与SOCC的关系尚未见相关报道。 血管平滑肌细胞功能异常是导致血管合并症的重要原因。大量的文献报道了糖尿病时血管内皮的损伤和血管平滑肌细胞的收缩，增殖的异常改变。糖尿病血管平滑肌时Ca²⁺信号究竟是如何变化的呢？并且细胞膜上SOCE的异常变化与Ca²⁺池功能的异常变化是否有关呢？细胞内Ca²⁺池按直接介导其释放介质可分为两种，一种为 IP₃敏感 Ca²池(inositol 1，4，5-trisphosphate (IP₃) -sensitive Ca²⁺ store )，另一种为由Ca²⁺诱发其释放的Ca²⁺池，称为Ca²⁺诱导的Ca²⁺释放(calcium-induced calcium release， CICR)，又称 rynaodine/咖啡因敏感的 Ca²⁺池(ryanodine /caffeine-sensitive Ca²⁺ store )。目前，IP₃ 敏感 Ca²⁺池和 rynaodine/咖啡因敏感的Ca²⁺池之间是否有交叉仍存在争议。糖尿病时这两部分Ca²⁺池触发的Ca²⁺释放是否有变化？目前未有相关文献报道。此外，越来越多的研究发现内质网/肌浆网Ca²⁺储存的耗竭和功能紊乱与心血管疾病的的发生和发展过程密切相关。因此，阐明糖尿病发病过程中血管功能损害的产生机制，将为寻找药物作用的新靶点和有效药物的研制具有重要的科学意义和应用前景。 第1章：糖尿病大鼠胸主动脉平滑肌细胞Ca²⁺运动的变化 新鲜分离16周糖尿病大鼠胸主动脉血管平滑肌细胞，采用Fluo-3/AM荧光标记的方法动态监测平滑肌细胞胞浆Ca²⁺运动的变化。探讨糖尿病大鼠血管平滑肌细胞的Ca²⁺调控机制的异常变化。结果显示： 1.Wistar雄性大鼠，体重为228g± 13 g ( n=90)，随机分为正常对照组和糖尿病模型组。建立糖尿病模型72小时后，糖尿病组血糖明显升高(糖尿病组： 23.3±0.5 mmol/L vs 正常组：4.8±0.06 mmol/L，n=45， P＜0.001 )，16 周后血糖仍缓慢升高，血糖分别为(糖尿病组：26.1±0.6mmol/L，n=40 vs正常组： 5.0±0.06 mmol/L，n=45， P＜0.001)。16周时的体重糖尿病组比正常组降低，分别为(糖尿病组：302g±6g，n=40 vs 正常组：481g±4g，n=45，P＜0.001)，并明显出现多饮、多食、多尿和生长迟缓等症状。而正常对照组大鼠体重逐渐增加，从(234±2) g增加到(481±4) g，且血糖无显著性变化。 2.首先观察静息状态下，平滑肌细胞胞浆Ca²⁺的荧光值。16周的糖尿病大鼠和正常组相比(正常组：1995±184，n=7 vs糖尿病组：1565± 173，n=8； P＞0.05)，两组之间胞浆静息Ca²⁺荧光值无显著性差异。 3.16周的糖尿病大鼠KCl触发的胞浆Ca²⁺变化与正常对照组相比，两组之间无显著性差异(正常组：0.59±0.09，n=7 vs糖尿病组：0.65±0.08，n=8； P＞0.05)。结果提示糖尿病主动脉血管平滑肌细胞电压依赖性Ca²⁺运动无显著性变化。 4.与正常组相比，糖尿病组Phenylephrine(Phe)触发的胞浆Ca²⁺变化降低39%，分别为(正常组：0.46±0.06 ， n=7 vs糖尿病组：0.28±0.02，n=6； P＜0.05)，两组之间有显著性差异。结果提示糖尿病时血管平滑肌细胞Phe介导的钙运动显著性降低。 5.CPA是肌浆网上Ca²⁺泵特异性抑制剂，16周的糖尿病病组与正常组相比， CPA触发的Ca²⁺释放和Ca²⁺内流都降低显著性降低。CPA引起的Ca²⁺释放为(正常组：0.45±0.04，n=6 vs 糖尿病组：0.28±0.01，n=6； P ＜0.05)， Ca²⁺内流为(正常组：0.57±0.05，n=6 vs 糖尿病组：0.37±0.01，n=6； P ＜0.05 )，结果提示糖尿病大鼠经SOCC介导的Ca²⁺内流和Ca²⁺释放均是显著性降低。 6.Phe可耗竭IP₃敏感的Ca²⁺池，16周的糖尿病病组与正常组相比，Phe触发的Ca²⁺释放和Ca²⁺内流都降低显著性降低。Phe引起的Ca²⁺释放为(正常组：0.42±0.04，n=7 vs 糖尿病组：0.24±0.04，n=6；P ＜0.05)； Ca²⁺内流为(正常组：0.53±0.04，n=7 vs 糖尿病组：0.34±0.07，n=6；P ＜0.05)，结果提示糖尿病大鼠平滑肌细胞IP₃敏感的钙池的Ca²⁺释放和Ca²⁺内流均是显著性降低。 7.Rynaodine (Ry)可耗竭Ry敏感的Ca²⁺池，16周的糖尿病病组与正常组相比，Ry触发的Ca²⁺释放和Ca²⁺内流都显著性降低。Ry引起的Ca²⁺释放为(正常组：0.26±0.07， n=9 vs 糖尿病组：0.06±0.01，n=5；P ＜0.05)，Ca²⁺内流为(正常组：0.27 ±0.03，n=9 vs 糖尿病组：0.15±0.02，n=5； P ＜0.05 )。结果提示糖尿病大鼠平滑肌细胞Ry敏感钙池的Ca²⁺释放和Ca²⁺内流均是显著性降低。 小结： 1.糖尿病大鼠主动脉血管平滑肌细胞的静息Ca²⁺及电压依赖性Ca²⁺运动无变化；而Phe介导的胞内Ca²⁺升高显著降低。 2.糖尿病大鼠主动脉血管平滑肌细胞的Ca²⁺池操纵性Ca²⁺内流降低，IP₃途径Ca²⁺释放和Ca²⁺内流均呈显著降低，同样ryanodine途径的Ca²⁺释放和Ca²⁺内流也呈显著降低。说明糖尿病血管平滑肌细胞Ca²⁺池功能紊乱。 第2章 糖尿病大鼠主动脉平滑肌IP₃和ryanodine蛋白的表达 为了进一步明确IP₃和Ry受体在蛋白水平上的变化是否与其功能学相一致，我们采用Western blot方法检测了糖尿病平滑肌的IP₃和Ry受体蛋白水平的表达。结果显示： 1.IP₃受体蛋白表达：16周糖尿病大鼠的胸主动脉血管平滑肌层的IP₃受体蛋白表达与对照组相比降低31.8%±7.7%，两组之间相比有显著性差异(n=6， P＜0.05)。本实验进一步从蛋白水平表明血管平滑肌细胞的IP₃受体通道的异常改变。 2.Ry受体蛋白表达：为进一步观察Ry途径的Ca²⁺释放减少的机制，我们检测了16周的糖尿病大鼠的胸主动脉血管平滑肌层的Ry受体蛋白表达，结果发现糖尿病组的Ry受体蛋白与对照组与对照组相比降低69.2 % ± 8.4 %，两组之间相比有显著性差异(n=7，P＜0.05)。结果表明糖尿病时血管平滑肌细胞上Ry受体蛋白水平显著性降低。 小结： 1.主动脉平滑肌层有IP₃受体蛋的表达，分子量约为260kDa；同时表达ryanodine受体蛋白，分子量约为500 kDa。 2.糖尿病时IP₃受体和ryanodine受体在平滑肌上蛋白表达下调。 第3章 糖尿病大鼠主动脉平滑肌细胞IP₃和ryanodine敏感Ca²⁺池Ca²⁺释放对收缩的影响 为了进一步明确糖尿病大鼠IP₃和Ry敏感Ca²⁺池的异常变化对血管平滑肌收缩的影响，我们采用血管环收缩实验来分别观察这两部分Ca²⁺池所触发的Ca²⁺释放引起的相应收缩的变化。结果如下： 1.对Phe的收缩反应： (1)首先，我们在有Ca²⁺的Krebs液中，浓度依赖性的给予Phe (10⁻⁸，3×10⁻⁸， 10⁻⁷， 3×10⁻⁷， 10⁻⁶，3×10⁻⁶，10⁻⁵) mol/l观察16周的糖尿病和正常组的收缩反应。糖尿病病组的最大收缩张力比正常组降低36%，两组之间有显著性差异(正常组：336.95±24.23 vs 糖尿病组：215.85±25.91，n=7，P＜0.01 )。在低浓度时(10⁻⁸和3×10⁻⁸) mol/l，两组没有显著性差异。但是到较高浓度(≥10⁻⁷mol/l)时，两组相比有显著性差异。 (2)为观察IP₃途径的Ca²⁺释放引起的收缩反应，我们在无Ca²⁺等渗透压状态下，加入Phe(10μM)，然后再复Ca²⁺。从IP₃途径的Ca²⁺释放引起的收缩反应，糖尿病组比正常对照组降低41% (正常组：183.31± 14.81 vs糖尿病组：108.44±13.89，n=7； P＜0.01 )，两组之间有显著性差异。随后复Ca²⁺，复Ca²⁺后的收缩也是糖尿病病组比正常对照组降低35.9%，分别为(正常组：318.14 ± 28.02 vs糖尿病组：203.77±37.41， n=7； P＜0.05 )，两组之间有显著性差异。 2.对ryanodine和咖啡因的收缩反应： 为进一步观察Ry途径的释放引起的收缩反应，我们也同样在无Ca²⁺等渗透压状态下，加入ryanodine (1μM)后，发现无收缩反应。我们继续加大ryanodine的剂量(10-100μM)，在正常和糖尿病组均未观察到收缩现象。因咖啡因与其作用的Ca²⁺池的受体相同，我们又加入20 mmol/l的咖啡因，我们仍未观察到血管环的收缩。最后，我们在有Ca²⁺液状态下加入ryanodine，在16个血管环中仅发现一个血管环有1.0 g的收缩张力。因此我们认为16周的wistar大鼠Ry受体途径的Ca²⁺释放触发收缩的信号很微弱，在本实验条件下不易检测。 小结： 1.16周的糖尿病大鼠主动脉的Phe触发的收缩反应显著降低，IP₃途径的Ca²⁺释放所触发的收缩反应显著降低。 2.16周大鼠的Ry受体途径的Ca²⁺释放触发主动脉平滑肌的收缩作用微弱。 关键词：糖尿病；血管平滑肌细胞；Ca²⁺池操纵性Ca²⁺通道；IP₃受体；ryanodine受体；收缩
Diabetes is a common chronic multiple syndrome involved in endocrine metabolic disorder, which is due to relative or absolute deficiency in insulin. In this condition, organisms function abnormally and emerge a series of complications such as diabetic cardiocerebral vessels diseases、ophthalmopathy、nephropathy、 neuropathy and so on, all of which will lead to amputation、acroisa even death. Diabetic complications are involved in many organs. A lot of experiments showed that dysfunction of microvessels and great vessels were the major reason of diabetic complications. In recent years it becomes clearly that diabetic complications are correlated closely with hyperglycemia. Chronic hyperglycemia is an important promoter and plays important role in the development of diabetic complications. If control of the patient's blood glucose is not ideal, hyperglycemia per se can induce cellar dysfunction via many intermediate products and its downstream substrate, accordingly evokes molecule mechanism that will change vassal function and structure. Molecule mechanism of diabetic complications includes augmented activity of protein kinase C, advanced glycosylation end products, activation of oxidative stress and polyalcohol path. It is suggested that hyperglycemia per se can produce toxical active substance or change intracellular signal path, thus, results in dysfunction of endothelial cells and media smooth muscle cells. Ca²⁺ is one of the most popular and important intracellular signal transduction components. Different stimulus can evoke intracellular Ca²⁺ change producing a series of biological effect and then diffusely participate in cell metabolism, secretion, proliferation, differentiation and vasoconstriction. Therefore, it is worth to investigate the mechanism of Ca²⁺ in cellular regulation. The contractile response is depended on elevation of cytoplasmic free Ca²⁺ concentration ( [Ca²⁺]〓) in vascular smooth muscle. The increase in [Ca²⁺]〓 is due to intracellular Ca²⁺ release from the sarcoplasmic reticulum and extracellular Ca²⁺ entry through plasmic membrane Ca²⁺ channels. There are at least three different kinds of Ca²⁺ channels in vascular plasmic membrane, voltage-dependent (VDCC) , receptor-operated (ROCC) Ca²⁺ channels and store-operated Ca²⁺ channel (SOCC). VDCC is activated by membrane depolarization, whereas ROCC is regulated by activation of plasma membrane receptors. The store-operated Ca²⁺ channel (SOCC) whose activation is dependent on the empty of agonist sensitive intracellular Ca²⁺ store. Recent studies have shown that store-operated Ca²⁺ entry (SOCE) is altered in different diabetic or hyperglycemic cells: (1) In diabetic leukomonocyte cells, SOCE is augmented which is induced by TG; In a medium containing 180 mg/dL glucose, platelets from NIDDM patients showed an increased SOCE compared to controls. Further study indicates that SOCE is altered in platelets from NIDDM patients probably due to the increased activity of the tyrosine kinase pp60(src); However, decreased SOCE is found in neutrophilic leukocyte of diabetic patients. (2) The alterations of SOCE are also detected in animal experiments. Curtis etc. find that both of store-operated Ca²⁺ influx and Ca²⁺ uptake are inhibited in retinal arterioles from diabetic rats. (3) The alterations of SOCE are also detected in cellular experiments in vitro. Exposure (96 h) of human umbilical vein endothelial cells to hyperglycemia causes a significant increase in apoptosis. And this increase in apoptosis is associated with an increase in Ca〓 current (by whole cell patch-clamp recorded) resulting from Ca〓 entry mediated by store-operated channels (SOCs). The number of apoptotic cells after prolonged high-glucose exposure was significantly reduced in the presence of the SOC inhibitor 2-APB or of La〓. Furthermore, non-enzymatic glycation, activation of protein kinase C, oxidative stress and high glucose also have close relationships with SOCE in common. (1) activation of protein kinase c: Mene et al. find high glucose level inhibits capacitative Ca²⁺ influx in cultured rat mesangial cells by a protein kinase C-dependent mechanism Further study found diabetes-induced activation of protein kinase C inhibits store-operated Ca²⁺ entry in rat retinal microvascular smooth muscle. (2) oxidative stress: monochloramine (a kind of oxidants) induced accumulation of Ca〓 in the cytoplasm is the result of release from intracellular stores and activate SOCE. In addition, it's generally regard TRP (transient receptor potential) proteins as the molecular basis of SOCC. In endothelial cells, TRP proteins are likely to be a sensing transmitter of oxidation and redox modulation. (3) advanced glycosylation end products: In Ca²⁺ free medium, the Ca²⁺ release is decreased which evoked by 1 μ M angiotensin Ⅱ in human mesangial cells exposed in advanced glycosylation end products. And advanced glycosylation end products inhibit the Ca²⁺ entry in mesangial cells. It's still no report about aldose reductase in relations with SOCC. The alteration of functions in vascular smooth muscle cells is a key cause to lead to diabetic vascular complications. A series of articles have reported that there are impairments in blood vessel endothelium and the changes of contraction and proliferation in diabetic vascular smooth muscle cells. What would be changed of intracellular Ca²⁺ signal in diabetic smooth muscle cells? Are there any changes of SOCE in diabetes and how would Ca²⁺ store change in diabetic smooth muscle cells? According to the mediators released by Ca²⁺ store, Ca²⁺ store could be divided into two types: one is IP₃-sensitive Ca²⁺store and the other is calcium-induced calcium release (CICR), which also be called ryanodine / caffeine-sensitive Ca²⁺ store. Presently, it is still unclear whether IP₃-sensitive and ryanodine-sensitive Ca²⁺ release involve overlapping Ca²⁺ stores or not. Are there some changes in IP₃ receptor and ryanodine receptor expressed in Ca²⁺ stores? In addition, there are more and more articles that showed the closely relationships between the disorder of sarcoplasmic reticulum/endoplasmic reticulum and cardiovascular diseases. To explore the underlying mechanisms of functional impairments in diabetic vascular, there is a promising prospect in discovering a new site and explore an effective drug to prevent diabetic vascular complications. Chapter Ⅰ nvestigation of alteration of Ca²⁺ movement in vascular smooth muscle Cells of diabetic rat To investigate the alteration of Ca²⁺movement in 16 weeks diabetic rats, we record elevation of cytoplasmic free Ca²⁺ concentration ([Ca²⁺]〓) in freshly isolated vascular smooth muscle cells using Fluo-3/AM fluorescence staining technique. The results are showed as followed: 1.Wistar, male rats , which the body weight is 228±13 g (n=90), are divided into two groups randomly including control group and diabetic group. After 72h diabetic model made, the blood glucose increased significantly( diabetes: 23.3±0.5 mmol/L vs control: 4.8±0.06 mmol/L, n=45, P＜0.001). During the procedure, the blood glucose increased progressively. In 16 weeks rats, the blood glucose were (26.1± 0.6)mmol/L in diabetic rats( n=40, P＜0.001) and (5.0± 0.06) mmol/L in control ( n=45).During the procedure, body weight of control rats increased progressively (from 234g±2g to 481g±4g) , and the blood glucose levels remained unchanged. However, body weight of diabetic rats increased slowly (from 229g± 2g to 302g± 6g). And there are a series of symptoms such as polydipsia、 polyphagia、 hyperdiuresis and growth slowly in diabetic rats. 2.First, we investigated the [Ca²⁺]〓 of smooth muscle cells under the resting conditions. The statistical data showed that there were no significant difference between control and diabetic rats (1995±184, n=7 vs 1565±173, n=8, P＞0.05). 3.We examined whether the voltage-dependent calcium channels could founctionally change in diabetic arotic smooth muscle cells. The peak effects initiated by KC1 (60mmol/l) were similar in control group and diabetic group (control: 0.59± 0.09, n=7 vs diabetic: 0.65±0.08, n=8; P＞0.05)。 The data indicated there is no significantly change of voltage-dependent Ca²⁺ movement in diabetic vascular smooth muscle cells. 4.However, the peak elevation caused by Phenylephrine (Phe, 10 μ mol/l) was significantly decreased in diabetic rats compared with control group ( control: 0.46± 0.06, n=7 vs diabetes: 0.28±0.02 , n=6; P＜0.05). The result indicated that the Ca²⁺ movement induced by receptor-operated Ca²⁺ channel was decreased in diabetic vascular smooth muscle cells. 5.CPA, a specific Ca²⁺ sarcoplasmic reticulum (SR) Ca²⁺/ATPase inhibitor, causes depletion of SR Ca²⁺ stores directly. In Ca²⁺-free medium, 10 μ M CPA significantly caused a transient increase(Ca²⁺ release) in [Ca²⁺]〓 and then declined to basal level, subsequent addition 2.0 mM Ca²⁺ to medium evoked a remarkable sustained rise, which was considered to be due to the Ca²⁺ influx through store operated Ca²⁺ entry pathway. The effects of Ca²⁺ release and influx were significantly decreased in diabetic comparing with that of control (Release, 0.28±0.01 in diabetic vs 0.45 ± 0.04 in control; Influx, 0.37±0.01 in diabetic vs 0.57±0.05 in control. P ＜0.05, respectively). The result indicated an alteration of function Ca²⁺ calcium stores in diabetic rats. 6.In Ca²⁺-free medium, phenylephrine (Phe) induced a transient [Ca²⁺]i increase, which was due to the depletion of IP₃-sensitive Ca²⁺ stores. The restoration of extracellular Ca²⁺ evoked a sustained increase in [Ca²⁺]i. The Ca²⁺ release and the Ca²⁺ influx were also significantly decreased in diabetic comparing with that of control(Release, 0.24±0.04 in diabetic, n=6 vs 0.42±0.04 in control ,n=7; Influx, 0.34±0.07 in diabetic, n=6 vs 0.53±0.04 in control, n=7 P＜0.05 respectively) ). The result indicated the Ca²⁺ release and the Ca²⁺ influx evoked by IP₃-sensitive Ca²⁺ stores were significantly decreased in diabetic rats. 7.In Ca²⁺-free medium, ryanodine induced a release [Ca²⁺]i increase, which was considered to be due to the Ca²⁺ depletion from ryanodine-sensitive Ca²⁺ stores. The restoration of extracellular Ca²⁺ also evoked a sustained increase in [Ca²⁺]i. The effects of Ca²⁺ release and influx induced by ryanodine were significantly decreased in diabetes comparing with that of control (Release, 0.06±0.01, n=5 in diabetes vs 0.26±0.07, n=9 in control; Influx, 0.15±0.02, n=5; P＜0.05 in diabetic vs 0.27 ± 0.03.incontrol, P＜0.05 respectively). The result indicated the Ca²⁺ release and the Ca²⁺ influx evoked by Ry-sensitive Ca²⁺ stores were significantly decreased in diabetic rats. Summary: 1.There is no change of resting Ca²⁺ and voltage-dependent Ca²⁺ movement in diabetic vascular smooth muscle cells compared with control. However, the Ca²⁺ movement induced by receptor-operated Ca²⁺ channel was decreased in diabetic vascular smooth muscle cells. 2.The Ca²⁺ movement induced by store-operated Ca²⁺ channel was decreased in diabetic vascular smooth muscle cells. The Ca²⁺ release and the Ca²⁺ influx evoked by IP₃ and Ry-sensitive Ca²⁺ stores were significantly decreased in diabetic rats. Chapter Ⅱ The protein expression of IP₃ receptor and ryanodine receptor in diabetic vascular smooth muscle. To investigate the alteration of IP₃ and ryanodine receptor in 16 weeks diabetic rats, we assayed their protein expression by western bolt. The results are showed as followed: 1.The expression of IP₃ receptor: The protein expression of IP₃ receptor was significantly decreased by 31.8% ±7.7% significantly in diabetes comparing with that of control (n=6, P＜0.05) . The result further confirmed that the IP₃ receptor channel changed in diabetic smooth muscle cells. 2.The expression of Ry receptor: The protein expression of Ry receptor was significantly decreased by 69.2% ±8.4% significantly in diabetes comparing with that of control (n=7, P＜0.05 ) . The result further confirmed that the Ry receptor channel changed in diabetic smooth muscle cells. Summary: 1.IP₃ receptor was expressedin smooth muscle cells, which molecular weight is about 260 kDa. Furthermore, ryanodine receptor was also expressed in smooth muscle cells, which molecular weight is about 500 kDa. 2.The protein expressions of IP₃ receptor and ryanodine receptor were significantly decreased in diabetic smooth muscle. Chapter Ⅲ Effects of contractions evoked by IP₃ and ryanodine pathway in diabetic vascular smooth muscle To investigate effects of contractions evoked by IP₃ and ryanodine pathway in 16 weeks diabetic rats, we assayed their contraction by isometric contraction measurement. The results are showed as followed: 1.The contractile responses to Phe: (1)First, we observed the arotic rings contraction induced by concentration-dependent Phenylephrine (10⁻⁸～10⁻⁵μ mol/1) in containing Ca²⁺ Kreb's solution. The maximal contraction of diabetic group was decreased significantly by 35.9% compared with control group(control: 336.9±24.2 vs diabetes: 215.9±25.9, n=7, P＜0.01). In low concentration of Phe(10⁻⁸ mol/l and 3×10⁻⁸ mol/l), there is no significant difference between the two groups. However, up to higher concentration of Phe (≥10⁻⁷mol/l), there is significant difference between the two groups. The result indicated the contraction induced by receptor-operated calcium channel was decreased in diabetic rats. (2)In Ca²⁺-free medium, phenylephrine (Phe) induced a transient contraction, which was due to the depletion of IP₃-sensitive Ca²⁺ stores. The restoration of extracellular Ca²⁺ evoked a sustained increase in contraction. In Ca²⁺-free solution, the maximal responses induced by Phe(10 u mmo/L) were decreased by 40.9 % in diabetic comparing with that of control (control: 183.3±14.8 vs diabetes: 108.4±13.8, n=7; P＜0.01). In addition, the contractile responses to Ca²⁺ restoration / Ca²⁺ influx was also reduced by 35.9% in diabetic rats (control: 318.1±28.0 vs diabetes: 203.8± 37.4, n=7; P＜0.05). The result indicated that the contraction evoked by IP₃ pathway was decreased in diabetic smooth muscle cells. 2.The contractile responses to ryanodine: However, in Ca²⁺-free solution, ryanodine alone did not cause any contraction in both control and diabetic rings. We continue to enlarge the ryanodine concentration to (10 —100 μ mmo/L) and enven try another agonist of caffeine (2.0 mmo/L),but there still no contraction happened. In Ca²⁺ containing solution (2.0 mmo/L), only 1 of 16 rings developed a slight contraction with ryanodine (0.1g). In addition, the maximal contractile responses to Phe were also unaffected by further addition of ryanodine. Those results suggested that the Ca²⁺ release and influx evoked by ryanodine were very weak in the contractile function of vascular smooth muscle of rats. Summary: 1.The contraction evoked by receptor-operated calcium channel and IP₃ pathway were decreased in diabetic smooth muscle cells. 2.The Ca²⁺ release and influx evoked by ryanodine were very weak in the contractile function of thoracic vascular smooth muscle. Key words: diabetic; vascular smooth muscle cells; store-operated calcium channel; IP₃ receptor; ryanodine receptor; contraction.