在大鼠中15-LO/15-HETE参与缺氧对K_V1.5表达的抑制作用

目的采用分子生物学技术从组织和细胞水平上观察阻断15-LO/15-HETE后,缺氧对KV1.5表达的影响。方法通过酶法分离、培养Wistar大鼠肺动脉血管平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)和大鼠肺动脉,应用Western blot和RT-PCR方法分别从蛋白质水平和mRNA水平上观察在15-LO阻断剂CDC和NDGA作用下,缺氧对KV1.5表达的影响。结果从组织和细胞水平上,用CDC和NDGA阻断15-LO即阻断了内源性15-HETE的产生,KV1.5的表达量在蛋白质水平和mRNA水平与未阻断组比较都增加。在阻断了内源性15-HETE的产生以后,加入外源性15-HETE,KV1.5的表达量减低。说明不仅内源性15-HETE参与诱导缺氧对KV1.5表达的影响,外源性15-HETE也同样能影响缺氧条件下KV1.5的表达量。但在阻断了内源性15-HETE的产生以后,加入外源性15-HETE,KV1.5的表达量减低。说明不仅内源性15-HETE参与诱导缺氧对KV1.5表达的影响,外源性15-HETE也同样能影响缺氧条件下KV1.5的表达量。结论上述结果表明,从大鼠肺动脉组织和细胞水平上,内源性15-HETE介导了缺氧对KV1.5表达的抑制作用。     

亚急性缺氧通过内源性15-HETE抑制大鼠肺动脉Kv1．5通道的表达

目的：缺氧通过抑制Kv通道引起肺血管收缩,但是机制不清楚。我们的研究表明缺氧激活肺动脉远端15-脂氧酶（15-LOX）增加15-羟基二十碳四烯酸（15--HETE）的产量。15-HETE诱导的肺血管收缩是通过抑制Kv通道实现的（Kv1．5,Kv2．1andKv3．4）．然而,目前还没有将缺氧、15-HETE和Kv通道亚型抑制直接联系起来的报道。因此,我们研究了15-LOX／15-HETE通路是否参与了缺氧诱导的Kv下调表达。由于Kv1．5是氧敏感通道,我们首先研究它的作用。结果：我们发现使用NDGA抑制15-LOX的活性显著降低了缺氧的肺血管环对苯肾上腺素的响应。缺氧条件下,脂氧酶抑制剂显著上调了肺动脉和肺动脉平滑肌细胞Kv1．5通道mRNA和蛋白质的表达。抑制15-LOX的活性部分恢复了IKV。结论：15-HETE参与了缺氧条件下Kv1．5通道的下调表达、IKV抑制及肺动脉张力增加。缺氧通过15-LOX／15-HETE通路抑制Kv1．5。     

西地那非通过上调电压依赖性钾通道抗低氧刺激的人肺动脉平滑肌细胞增殖

目的探讨西地那非抗低氧刺激的人肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)增殖的机制与电压依赖性钾通道(voltage-dependent potassium channels,Kv)及环鸟甘酸依赖性蛋白激酶(cGMP-dependent protein kinase,PKG)的关系。方法采用MTT法及细胞免疫荧光法等检测细胞增殖情况;应用膜片钳技术记录低氧刺激PASMCs前后及西地那非或KT-5823干预前后的Kv通道电流,运用Kv1.5抗体透析细胞后的Kv通道电流;应用siRNA干扰技术沉默Kv1.5基因。结果低氧下西地那非组的细胞增殖水平较低氧对照组明显降低;西地那非反转了低氧对细胞K +通道电流,尤其Kv1.5通道电流的降低作用;siRNA组的Kv1.5蛋白表达明显减低,沉默Kv1.5通道基因逆转了西地那非抗低氧刺激的细胞增殖作用;PKG抑制剂KT-5823阻断了西地那非抗低氧刺激的细胞增殖作用,并且反转了西地那非对低氧下的Kv通道电流的上调作用。 结论 西地那非可通过上调Kv通道亚型主要是Kv1.5通道,抑制低氧刺激的人PASMCs增殖,且可能与PKG有关。     

大脑中动脉栓塞模型大鼠的中枢电压依赖性钾通道mRNA表达的改变

目的观察几种代表性电压依赖性钾通道亚型在脑缺血不同时间大鼠海马和皮层mRNA表达水平的变化。方法采用大脑中动脉栓塞模型致大鼠脑缺血损伤，应用RT-PCR方法检测Kv1.4，Kv1.5，Kv2.1和Kv4.2 mRNA表达水平在海马和皮层中的改变。结果大脑中动脉栓塞模型大鼠出现明显的神经损伤症状。缺血2 h时，海马组织的Kv1.4，Kv2.1和Kv4.2 mRNA表达水平分别增加了50%，67%和90%，在缺血24 h时Kv1.4和Kv4.2 mRNA仍保持高水平表达。大鼠皮层组织在缺血2 h后，Kv1.4，Kv1.5，Kv2.1和Kv4.2 mRNA水平均无明显改变，缺血24 h后，Kv2.1和Kv4.2 mRNA水平分别增加了70%和62%。结论大脑中动脉栓塞模型大鼠的海马和皮层组织中电压依赖性钾通道亚型的mRNA表达发生明显上调。     

PKC和血管内皮细胞在15-HETE收缩肺动脉中的作用

目的探讨蛋白激酶C(PKC)、蛋白激酶A(PKA)信号转导途经及血管内皮完整性在15-羟基二十碳四烯酸(15-HETE)收缩肺动脉中的作用。方法采用组织浴槽血管环法与PKC、PKA抑制剂以及除去血管内皮细胞相结合的方法。结果用6·8mmol·L-1hypericin阻断PKC途径可使15-HETE收缩缺氧大鼠肺动脉量效曲线右移(P<0·01);用0·112mmol·L-1KT5720阻断PKA途径使15-HETE收缩缺氧大鼠肺动脉量效曲线右移不明显;除去血管内皮细胞可降低对照组和缺氧组肺动脉血管环对15-HETE的反应性。结论15-HETE收缩肺动脉的作用和PKC信号转导系统以及血管内皮细胞的完整性有关。     

ERK1/2通路在4-AP诱导大鼠肺动脉收缩中的作用

目的探讨细胞外信号调节激酶-1/2(ERK1/2)通路在4-氨基吡啶(4-aminopyridione,4-AP)阻断正常大鼠肺动脉平滑肌细胞(PASMCs)膜上电压依赖性钾通道(KV)所引起的肺动脉收缩中的作用。方法取正常鼠肺动脉制作肺动脉环,分别加入4-AP(KV通道阻断剂),PD98059/U0126+4-AP,比较肺动脉收缩的变化。同时培养肺动脉平滑肌细胞进行Western blot分析4-AP对ERK1/2的影响。结果①在血管环试验中,4-AP引起的肺动脉收缩有浓度依赖性;加入20mmol.L-1PD98059或2μmol.L-1U0126可以抑制4-AP引起的肺动脉收缩。②4-AP可刺激PASMCs ERK1/2蛋白磷酸化;③U0126可抑制4-AP引起的ERK1/2蛋白磷酸化。结论ERK1/2通路参与4-AP阻断正常大鼠肺动脉平滑肌细胞膜上电压依赖性钾通道(KV)引起肺动脉收缩。     

东莨菪碱致记忆障碍大鼠中枢电压依赖性钾通道亚型的表达

目的研究东莨菪碱致记忆障碍大鼠模型中枢电压依赖性钾通道亚型mRNA表达的差异。方法Morris水迷宫实验检验大鼠空间学习记忆能力,用RT-PCR方法检测大脑皮层和海马中5种电压依赖性钾通道Kv1.4,Kv1.5,Kv2.1,Kv4.2及Kv4.3 mRNA的表达。结果注射东莨菪碱大鼠的学习记忆能力明显下降,大脑皮层中Kv4.2的表达比对照组降低28.8%;海马中Kv1.4的表达升高111.7%,Kv2.1的表达升高64.3%,而Kv4.2的表达降低33.9%。其它电压依赖性钾通道表达的变化不大。结论东莨菪碱致大鼠学习记忆障碍的同时可诱发中枢电压依赖性钾通道亚型的表达改变。     

PKA和PKG信号通道对平滑肌细胞 Kv亚型1.5表达的影响

目的 研究PKA(蛋白激酶A)和PKG(蛋白激酶G)信号通道对人气道平滑肌细胞(HASMCs)电压依赖延迟整流钾通道(Kv)亚型Kv1.5表达的影响.方法 采用逆转录聚合酶链反应(RT-PCR)和Western blotting技术,观察PKA激活剂8-brom-cAMP和阻断剂Rp-cAMP及PKG激活剂8-brom-cGMP对HASMCsKv1.5 mRNA和蛋白质表达的影响.结果 PKA激活剂8-brom-cAMP显著增强Kv1.5亚型的表达,该效应可被PKA阻断剂Rp-cAMP显著逆转.PKG激活剂8-brom-cGMP显著增强Kv1.5亚型的表达.结论 活化PKA和PKG促进Kv1.5亚型的表达,抑制HASMCs兴奋性.推测可能为这两条信号通道引起HASMCs舒张反应的作用机制之一.     

15-酮基二十碳四烯酸对大鼠离体肺动脉环的作用

目的用组织浴槽血管环技术研究15-酮基二十碳四烯酸(15-KETE)收缩大鼠离体肺动脉的作用及其离子通道机制。方法16只健康的W istar大鼠,体重为(220±20)g,随机分为两组(n=8);正常组置于正常环境中饲养(F iO2=21%),缺氧组置于缺氧的培养箱中饲养(F iO2=10%),连续饲养9 d后处死,游离直径为0.5~1.0 mm肺内肺动脉(PA)剪成3 mm长的血管环,观察钾离子通道阻断剂、L-型钙离子通道阻断剂和无钙离子Krebs液对15-KETE诱导的正常和缺氧大鼠肺动脉环收缩的影响。结果①15-KETE以浓度(10-8~10-6mol.L-1)依赖的方式收缩大鼠离体肺动脉环;②2 mmol.L-14-氨基吡啶(4-am inopyrid ione,4-AP)可明显降低15-KETE收缩大鼠离体肺动脉环的作用,正常组和缺氧组的结果相似;③10 mmol.L-1四乙胺(tetraeth-ylammon ium,TEA)、10-6mol.L-1格列本脲(glyburide,GLYB)对15-KETE收缩大鼠离体肺动脉环的作用无明显影响;④10-6mol.L-1硝苯地平(n ifed ip ine)和无钙Krebs液对10-6mol.L-115-KETE引起的离体肺动脉环收缩具有显著抑制作用。结论15-KETE收缩离体肺动脉环的作用依赖于Kv通道、细胞外液钙离子和L-型钙离子通道。     

替米沙坦通过直接抑制Kv2.1通道促进离体大鼠的胰岛素分泌

目的 研究替米沙坦促进大鼠胰岛素分泌作用相关的信号通路。方法 (1)分离成年Wistar大鼠胰腺获得胰岛和胰岛细胞,通过胰岛素分泌实验观察药物对胰岛素分泌的影响,通过钙成像实验和全细胞膜片钳技术观察药物对β细胞内Ca²⁺浓度的变化和对离子通道的作用。(2)使用过表达电压门控性钾(voltage-gated potassium channel, Kv)通道2.1亚型(Kv2.1)的慢病毒转染中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞构建CHO-Kv2.1细胞系,使用膜片钳技术观察替米沙坦对Kv2.1通道的直接作用。结果 缬沙坦和厄贝沙坦无类似替米沙坦的高糖浓度下促胰岛素分泌、升高β细胞内Ca²⁺浓度和抑制β细胞的Kv通道等作用。过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor γ,PPARγ)阻断剂GW9662亦未阻断替米沙坦的上述作用。而替米沙坦可以浓度依赖性地抑制CHO-Kv2.1细胞的Kv2.1通道电流。结论 替米沙坦的促胰岛素分泌作用可能与血...     

15-HETE对肺动脉平滑肌细胞钙离子浓度的影响

目的　通过阻断细胞外钙离子 (Ca2+ )内流,观察 15 羟化二十烷四烯酸 ( 15 HETE)对兔肺动脉环收缩张力和肺动脉平滑肌细胞内游离钙浓度 ( [Ca2+ ]i)的影响,探讨缺氧时 15 HETE引起细胞钙动员的机制。方法　采用组织浴槽血管环方法观察L 型钙通道阻断剂硝苯地平和无钙液对15 HETE诱导的兔肺动脉环收缩力的影响;酶法分离培养兔肺动脉平滑肌细胞,激光扫描共聚焦显微镜测定 15 HETE对细胞内 [Ca2+ ]i的作用。结果　在正常组和缺氧组, 10μmol·L-1硝苯地平和无钙液对 1μmol·L-1 15 HETE引起的肺动脉环收缩均没有影响;培养的 15 HETE组细胞 (正常培养的细胞暴露于 1 μmol·L-1 15 HETE下继续孵育 8min)与正常对照组相比,细胞内 [Ca2+ ]i明显增加 (P<0 05)。结论　15 HETE可引起肺动脉平滑肌细胞 [Ca2+ ]i增加,且此钙来源于细胞内贮钙库的释放。     

Subacute hypoxia suppresses Kv3.4 channel expression and whole-cell K+ currents through endogenous 15-hydroxyeicosatetraenoic acid in pulmonary arterial smooth muscle cells

We have previously reported that subacute hypoxia activates lung 15-lipoxygenase (15-LOX), which catalyzes arachidonic acid to produce 15-HETE, leading to constriction of neonatal rabbit pulmonary arteries. Subacute hypoxia suppresses Kv3.4 channel expression and results in an inhibition of whole-cell K(+) currents (I(K)). Although the Kv channel inhibition is likely to be mediated through 15-HETE, direct evidence is still lacking. To reveal the role of the 15-LOX/15-HETE pathway in the hypoxia-induced down-regulation of Kv3.4 channel expression and inhibition of I(K), we performed studies using 15-LOX blockers, whole-cell patch-clamp, semi-quantitative PCR, ELISA and Western blot analysis. We found that Kv3.4 channel expression at the mRNA and protein levels was greatly up-regulated in pulmonary arterial smooth muscle cells after blockade of 15-LOX by CDC or NDGA. The 15-LOX blockade also partially restored I(K). In comparison, 15-HETE had a stronger effect than 12-HETE on the expression of Kv3.4 channels. 5-HETE had no noticeable effect on Kv3.4 channel expression. These data indicate that the 15-LOX pathway via its metabolite, 15-HETE, seems to play a role in the down-regulation of Kv3.4 expression and I(K) inhibition after subacute hypoxia.     

Disappearance of Hypoxic Pulmonary Vasoconstriction and O2-Sensitive Nonselective Cationic Current in Arterial Myocytes of Rats Under Ambient Hypoxia

Acute hypoxia induces contraction of pulmonary artery (PA) to protect ventilation/perfusion mismatch in lungs. As for the cellular mechanism of hypoxic pulmonary vasoconstriction (HPV), hypoxic inhibition of voltage-gated K⁺ channel (Kv) in PA smooth muscle cell (PASMC) has been suggested. In addition, our recent study showed that thromboxane A₂ (TXA₂) and hypoxia-activated nonselective cation channel (I_NSC) is also essential for HPV. However, it is not well understood whether HPV is maintained in the animals exposed to ambient hypoxia for two days (2d-H). Specifically, the associated electrophysiological changes in PASMCs have not been studied. Here we investigate the effects of 2d-H on HPV in isolated ventilated/perfused lungs (V/P lungs) from rats. HPV was almost abolished without structural remodeling of PA in 2d-H rats, and the lost HPV was not recovered by Kv inhibitor, 4-aminopyridine. Patch clamp study showed that the hypoxic inhibition of Kv current in PASMC was similar between 2d-H and control. In contrast, hypoxia and TXA₂-activated I_NSC was not observed in PASMCs of 2d-H. From above results, it is suggested that the decreased I_NSC might be the primary functional cause of HPV disappearance in the relatively early period (2 d) of hypoxia.     

Alterations of voltage‐dependent K+ channels in the mesenteric artery during the early and chronic phases of diabetes

This study investigated the alteration of voltage‐dependent K⁺ (Kv) channels in mesenteric arterial smooth muscle cells from control (Long‐Evans Tokushima Otsuka [LETO]) and diabetic (Otsuka Long‐Evans Tokushima Fatty [OLETF]) rats during the early and chronic phases of diabetes. We demonstrated alterations in the mesenteric Kv channels during the early and chronic phase of diabetes using the patch‐clamp technique, the arterial tone measurement system, and RT‐PCR in Long‐Evans Tokushima (LETO; for control) and Otsuka Long‐Evans Tokushima Fatty (OLETF; for diabetes) type 2 diabetic model rats. In the early phase of diabetes, the amplitude of mesenteric Kv currents induced by depolarizing pulses was greater in OLETF rats than in LETO rats. The contractile response of the mesenteric artery induced by the Kv inhibitor, 4‐aminopyridine (4‐AP), was also greater in OLETF rats. The expression of most Kv subtypes‐ including Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.2, Kv4.1, Kv4.3, Kv5.1, Kv6.2, Kv8.1, Kv9.3, and Kv10.1‐were increased in mesenteric arterial smooth muscle from OLETF rats compared with LETO rats. However, in the chronic phase of diabetes, the Kv current amplitude did not differ between LETO and OLETF rats. In addition, the 4‐AP‐induced contractile response of the mesenteric artery and the expression of Kv subtypes did not differ between the two groups. The increased Kv current amplitude and Kv channel‐related contractile response were attributable to the increase in Kv channel expression during the early phase of diabetes. The increased Kv current amplitude and Kv channel‐related contractile response were reversed during the chronic phase of diabetes.     

Mechanisms of oxygen sensing: a key to therapy of pulmonary hypertension and patent ductus arteriosus

Specialized tissues that sense acute changes in the local oxygen tension include type 1 cells of the carotid body, neuroepithelial bodies in the lungs, and smooth muscle cells of the resistance pulmonary arteries and the ductus arteriosus (DA). Hypoxia inhibits outward potassium current in carotid body type 1 cells, leading to depolarization and calcium entry through L-type calcium channels. Increased intracellular calcium concentration ([Ca+ +]i) leads to exocytosis of neurotransmitters, thus stimulating the carotid sinus nerve and respiration. The same K+ channel inhibition occurs with hypoxia in pulmonary artery smooth muscle cells (PASMCs), causing contraction and providing part of the mechanism of hypoxic pulmonary vasoconstriction (HPV). In the SMCs of the DA, the mechanism works in reverse. It is the shift from hypoxia to normoxia that inhibits K+ channels and causes normoxic ductal contraction. In both PA and DA, the contraction is augmented by release of Ca+ + from the sarcoplasmic reticulum, entry of Ca+ + through store-operated channels (SOC) and by Ca+ + sensitization. The same three 'executive' mechanisms are partly responsible for idiopathic pulmonary arterial hypertension (IPAH). While vasoconstrictor mediators constrict both PA and DA and vasodilators dilate both vessels, only redox changes mimic oxygen by having directly opposite effects on the K+ channels, membrane potential, [Ca(++)]i and tone in the PA and DA. There are several different hypotheses as to how redox might alter tone, which remain to be resolved. However, understanding the mechanism will facilitate drug development for pulmonary hypertension and patent DA.     

缺氧肺动脉高压发病机制研究进展:15-LO/15-HETE的作用

缺氧肺动脉高压(HPH)以急性缺氧肺动脉收缩(HPV)和慢性缺氧导致的肺血管床重构(HPVR)引起的肺动脉压持续升高为特征的临床常见病症。参与HPH的中介因子很多,但没有一种可以完整地阐述其发病机制。本实验室前期工作发现缺氧可使肺血管15-脂酯氧化酶(15-LO)表达升高,后者催化花生四烯酸,生成15-羟基二十碳四烯酸(15-HETE)。研究发现15-LO/15-HETE参与HPV与HPVR的众多过程,提示15-LO/15-HETE可能是HPH发病过程的一个中介因子。对15-LO/15-HETE研究将更好地阐述HPH的发病机制,寻找更有效的临床治疗靶点。