大电导钙激活钾通道对平滑肌的调控作用研究进展

钾通道是组织器官中的一种重要通道,几乎所有的组织中都有该通道的分布,并且它在调节细胞功能方面起着极其重要的作用,例如动作电位的形成和信号传导等。大电导钙激活钾通道(BKca/Maxi K)以其广泛的分布,以及参与调节多种细胞功能吸引了更多研究者的关注。BKca/Maxi K的激活可导致细胞膜的超极化,从而抑制电压依赖性钙通道的激活,抑制钙离子内流,引起平滑肌舒张。近年来研究发现,BKca/Maxi K的激活、失活和变异与多种疾病的发病有关,BKca/Maxi K对心血管平滑肌、子宫平滑肌、呼吸道平滑肌和阴茎勃起等具有调控作用,尤其是其基因疗法对阴茎勃起障碍的治疗逐渐显现出较大的优势。     

Vascular large conductance calcium-activated potassium channels: Functional role and therapeutic potential

Large-conductance Ca²⁺-activated K⁺ channels (BK_Ca or maxiK channels) are expressed in different cell types. They play an essential role in the regulation of various cell functions. In particular, BK_Ca channels have been extensively studied in vascular smooth muscle cells, where they contribute to the control of vascular tone. They facilitate the feedback regulation against the rise of intracellular Ca²⁺, membrane depolarization and vasoconstriction. BK_Ca channels promote a K⁺ outward current and lead to membrane hyperpolarization. In endothelial cells expression and function of BK_Ca channels play an important role in the regulation of the vascular smooth muscle activity. Endothelial BK_Ca channels modulate the biosyntheses and release of various vasoactive modulators and regulate the membrane potential. Because of their regulatory role in vascular tone, endothelial BK_Ca channels have been suggested as therapeutic targets for the treatment of cardiovascular diseases. Hypertension, atherosclerosis, and diabetes are associated with altered current amplitude, open probability, and Ca²⁺-sensing of BK_Ca channels. The properties of BK_Ca channels and their role in endothelial and vascular smooth muscle cells would address them as potential therapeutic targets. Further studies are necessary to identify the detailed molecular mechanisms of action and to investigate selective BK_Ca channels openers as possible therapeutic agents for clinical use.     

Action of a novel potassium channel opener,SR 47063, on human bronchi and on guinea-pig trachea in vitro: comparison with cromakalim

Potassium channels are present on airway smooth muscle cells and their activation results in hyperpolarization and relaxation. Because these effects may have therapeutic relevance to asthma, the aim of our study was to examine the activity of SR 47063, a potassium channel opener (KCO), against a variety of spasmogens or against electrical field stimulation in guinea-pig isolated trachea and in human isolated bronchi in vitro; the effects of SR 47063 were compared with those of cromakalim, isoprenaline, and theophylline. Like cromakalim, SR 47063 reduced the contractility of guinea-pig isolated trachea and the human isolated bronchus in basal tone with pD₂ of 7.79 ± 0.01 and 7.83 ± 0.09, respectively, or during precontractions induced by acetylcholine 10^?4 M, histamine 10^?5 M, or low concentrations of KCl (<30 mM), but not by high KCl concentrations (≥30 mM); these effects were antagonized by glibenclamide 10^?5 M. This spectrum of action is typical of the compounds known as potassium channel openers. Electrical field stimulation (EFS: 16 Hz, 1 ms, 320 mA for 10 sec in the presence of indomethacin 10^?6 M and propranolol 10^?6 M) of guinea-pig isolated main bronchi induced 2 successive contractile responses. Both contractions were reduced significantly by SR 47063 and cromakalim. Although we have not studied the effects of KCOs on exogenous neurokinin A- or substance P-induced contractions, it might be suggested as a hypothesis that this inhibition seems to take place presynaptically and to affect the release of neuromediators produced by electrical field stimulation. In conclusion, SR 47063 exerts in vitro on the bronchial smooth muscle an inhibitory effect which seems to be due to the opening of glibenclamide-sensitive potassium channels. SR 47063 is 3- to 10-fold more potent than cromakalim. © 1993 Wiley-Liss, Inc.     

Acrylamides as potassium channel openers

The acrylamide moiety is widely employed in medicinal chemistry for the design of bioactive molecule and, notably, it has served as a scaffold for a number of potassium channel modulators in recent years. Their synthesis has proven to be an effective approach, especially for the discovery and optimisation of small molecule openers of large-conductance calcium-activated (BK) potassium channels and KCNQ channels. This review provides an overview of the worldwide patent literature surrounding cinnamic amides, with a special focus on their potential medical use and the most salient structural and chemical features involved in the modulation of BK and KCNQ channel activity. Remarkably, such derivatives are among the most potent BK and KCNQ channel openers reported to date.     

钩藤碱对大鼠肺动脉平滑肌细胞钙激活钾通道的影响

用膜片钳单通道记录法研究钩藤碱(Rhy)对大鼠肺动脉平滑肌细胞钙激活钾通道(K_Ca)的影响.结果：Rhy 30, 45和60 μmol·L^-1缩短通道的开放时间, 但浓度依赖性增加K_Ca开放概率,Rhy 15, 30, 45和60 μmol·L^-1使开放概率由加药前的0.085±0.005分别增加到0.176±0.011, 0.315±0.009, 0.485±0.016和0.761±0.012(x±s, 均P<0.01).说明Rhy有增加肺动脉平滑肌细胞K_Ca开放作用.     

Muscarinic stimulation of the mouse isolated whole bladder: physiological responses in young and ageing mice

1 Peripheral autonomous bladder activity is an incompletely understood property that may be important both in normal bladder function and in functional problems of the lower urinary tract. We describe how a muscarinic agonist, arecaidine, influences intravesical pressure and intramural bladder contractions in the isolated mouse and how response varies in ageing mice. 2 A group of 12 mice aged 3-4 months was compared with an 'ageing' group of 12 mice age 28-34 months. Bladders were microsurgically removed and mounted in whole organ tissue baths. The effects of the muscarinic agonist arecaidine on intravesical pressure and intramural contractions were performed at different bladder volumes. 3 In normal mice, arecaidine elicited tonic and phasic contractions, the latter showing a more substantial increase in amplitude with bladder distension. Localized 'micromotion' contractions were seen in the bladder wall, with regional differences arising after exposure to arecaidine. A background release of acetylcholine was inferred from the pressure increase induced by the cholinesterase inhibitor physostigmine. 4 Both micromotion activity and the phasic component of the arecaidine response were substantially reduced in ageing mice; the tonic component was preserved in the same specimens. 5 We conclude that the enhanced pressure fluctuations seen at high bladder volumes may act as a peripheral determinant of bladder capacity, and that changes in such activity may contribute to altered functional capacity and lower urinary tract symptoms in ageing individuals.     

New approaches to the design and discovery of therapies to prevent erectile dysfunction

Introduction: Nitric oxide (NO) is critically involved in erectile function. Since NO synthase (NOS) and arginase compete for the same substrate l-arginine, limiting arginase activity may provide more NO and thus be a beneficial therapeutic approach to erectile dysfunction (ED). In the corpora cavernosa, excessive arginase activity/expression has been implicated through studies of preclinical and clinical models of ED. Further, the inhibition of arginase has shown to increase vascular system relaxation and enhance blood flow in penile circulation. Further studies, therefore, looking at therapies targeting arginase could prove to be clinically useful.

Areas covered: The authors review gene- and cell-based therapies, the involvement of RhoA/Rho-kinase (ROCK), MAPK and arginase in ED.

Expert opinion: Extensive literature supports the view that upregulated arginase activity in cavernosal tissue can reduce NOS function and NO production. Excessive arginase activity has been shown to contribute to the progression of aging-, hypertension- and diabetes-induced vascular dysfunction as well as ED. Earlier studies have shown that RhoA/ROCK and subsequent activation of p38 MAPK mediate elevation of arginase expression/activity in diabetic and hypertensive mice. Reducing corporal arginase activity by gene-based or pharmacological therapy and/or inhibition of upstream regulators of arginase expression may provide novel therapeutic approaches in the management of ED.     

The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system

ATP-sensitive potassium channels (K_ATP) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated K_ATP channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system.     

EMD56431与粉防已碱对犬血管平滑肌作用的比较性研究

目的：研究EMD56431（EMD）和粉防己碱（Tet)对犬血管平滑肌的松弛作用。方法：用20和60mmol/L KCl激动犬冠状动脉环，观察两药的松弛效应及对不同血管平滑肌的半数抑制浓度（IC50）。结果：犬冠脉环无论有无内皮存在，0．7μmol/L EMD和30μmol/L Tet可松弛50％，而．7μmol/L EMD是无松弛反应。给格列本脲（GLB）后，Tet对KCl致冠脉收缩反应无明显影响，而EMD的松弛反应则取消。EMD和Tet均可使KCl(5-30mmol/L)量效曲线非平行右移，但EMD对高钾（≥40mmol/L)则无拮抗作用（最大反应Emax不降低），Tet则仍具有松弛效应，pD'2为4．4．EMD和Tet使苯福林和KCl致标本的收缩产生浓度依赖性的松弛，对肠系膜动脉和冠脉的IC50分别为6．2（EMD）、4．1（Tet）和6．44（EMD），4．1（Tet)。结论：EMD和Tet为非内皮依赖性血管松弛剂。     

Some arylacridine derivatives possessing potassium channel opening activity

In this study, six new 2,2,7,7-tetramethyl-9-aryl-2,3,4,5,6,7,9,10-octahydro-1,8-acridinedione derivatives (1-6) were synthesised and their functional effects on vascular potassium channels and mechanism of induced relaxations on phenylephrine-induced contractile responses in isolated rat mesenteric arteries were investigated. Pinacidil was used as standard potassium channel opener. Compounds 1, 2, 5, 6 and pinacidil induced concentration-dependent relaxation response of vessel rings previously contracted with phenylephrine.     

神经对大鼠骨骼肌小电导钙激活钾通道(SK3)表达的影响

目的小电导钙激活的钾通道(SK3)介导了动作电位后的后超极化电位的产生,在调节可兴奋细胞的膜电位中起关键作用。使去神经支配和肌强直营养不良患者骨骼肌SK3表达显著上调。本实验拟观察神经对骨骼肌SK3钾通道表达的调节作用。方法在Wistar大鼠,分别通过切断坐骨神经和局部注射河豚毒素(TTX)建立比目鱼肌去神经支配模型,和通过钳夹损伤比目鱼肌神经建立比目鱼肌去神经后神经再支配模型。在体给予去神经比目鱼肌频率为30Hz的电刺激(100脉冲/100 s)。实验结束后,免疫荧光法测定比目鱼肌中SK3钾通道的表达和定位;提取比目鱼肌组织总mRNA和蛋白,逆转录PCR和Western Blot分别检测SK3 mRNA和蛋白水平。结果切断坐骨神经能显著上调比目鱼肌SK3 mRNA和蛋白表达,且分别在术后第6 d和第9 d到达稳定。TTX诱导肌肉瘫痪也能显著上调比目鱼肌SK3 mRNA和蛋白表达。钳夹比目鱼肌神经所造成的暂时神经损伤能诱导SK3蛋白表达上调,而随着神经功能的恢复SK3蛋白表达也随之显著下调。在切断除坐骨神经术后6 d,在体给予电刺激6 d能显著下调比目鱼肌SK3的高表达;而且,在去除坐骨神经的同时给予电刺激则能阻止比目鱼肌SK3表达的上调。结论神经对骨骼肌SK3钾通道表达具有抑制作用,其作用与唤起肌肉活性密切相关。在体电刺激能抑制和防止去神经诱导的骨骼肌SK3蛋白表达上调,可能是改善神经损伤后或相关疾病所致的肌强直症状的有效手段之一。     

Methamphetamine inhibits voltage-gated potassium currents in NG108-15 cells: Possible contribution of large-conductance calcium-activated potassium channels

Methamphetamine (MA), a highly abused amphetamine-like psychostimulant, has surged in popularity worldwide in the last decade. Repeated MA exposure has been shown to affect the alternative splice variant expression of large conductance Ca²⁺-activated K⁺ (BK) channels. It remains unclear whether MA affects BK channel activity. The present study investigated the effects of MA on BK channels in NG108-15 mouse neuroblastoma × rat glioma hybrid cells using whole-cell and cell-attached patch clamp techniques. In whole-cell recordings, the macroscopic K⁺ outward currents were inhibited by MA with an EC₅₀ of 146 μM, but not affected by dopamine (DA). It implies that DA is not involved in the effects of MA on K⁺ outward currents. In cell-attached patches, MA significantly decreased BK channel activity. Moreover, MA significantly decreased the BK channel opener NS1619-evoked whole-cell K⁺ outward currents and BK channel activity. Finally, the effect of MA on membrane potential was examined by current-clamp configuration. MA caused membrane depolarization and application of NS1619 returned the depolarized potential to resting value. These findings suggest that MA might act as an inhibitor of BK channels, and thereby increase the neuronal excitability and enhance neurotransmitter release.     

Effects of potassium channel openers on single potassium channels in mouse skeletal muscle

Summary The patch-clamp technique was used to study the effects of the potassium channel openers cromakalim, pinacidil, RP 49356 and diazoxide on single potassium channels in mouse skeletal muscle.In excised patches in the inside-out configuration, one type of potassium channel, the ATP-sensitive potassium channel, could be activated by internally applied RP 49356 even in the absence of internal ATP. At a concentration of 0.4 and 0.8 mmol/l, RP 49356 increased the open-probability of the channels by a factor of 2.7 and 17.4 respectively. The stimulating effect of cromakalim (0.2–0.8 mmol/l) and pinacidil (0.4 mmol/l) depended on the presence of ATP (0.1 mmol/l) at the cytoplasmic side of the patch membrane. The two drugs were able to restore the open-probability of the channels blocked by internal ATP (0.1 mmol/l) to 50–90% of its value in ATP-free solution. No channel reactivation could be observed at a higher ATP concentration (1 mmol/l). Diazoxide (0.4 mmol/l) had almost no effect. None of these channel openers could stimulate the other prominent type of potassium channel in skeletal muscle, the large-conductance Ca²⁺-activated potassium channel.The results show that cromakalim, pinacidil and RP 49356 are specific openers of ATP-sensitive potassium channels in skeletal muscle. It is suggested that the drugs displace the channel blocker ATP and that RP 49356 in addition recruits inactive channels. Send offprint requests to B. Neumcke at the above address     

Muscarinic stimulation of the rat isolated whole bladder: pathophysiological models of detrusor overactivity

1 Hypotheses as to the pathophysiological basis of bladder detrusor muscle overactivity (DO) have identified both central nervous and peripheral mechanisms as likely contributory factors. In this paper, we describe peripheral autonomous bladder activity in two animal models of DO and discuss how the differences observed between the two models support the likelihood that clinical DO has a multifactorial basis. 2 A total of 12 adult female Sprague-Dawley rats underwent obstruction or sham operation for 1 or 4 weeks. Six adult female spontaneously hypertensive rats (SHR) were compared with normal Wistar controls. Bladders were microsurgically removed and mounted in whole organ tissue baths. Recordings of intravesical pressure in response to the muscarinic receptor agonist arecaidine were performed under standardized conditions. 3 In the partially obstructed rat bladder, the amplitude of pressure fluctuations elicited by the muscarinic agonist arecaidine was significantly increased compared with sham-operated animals. The tonic component of the response was no different for the two groups. No difference from controls was apparent in the SHR. 4 We conclude that alterations in autonomous bladder activity in the obstructed rat model suggest that peripheral functional changes contribute to the pathophysiological abnormality. In contrast, the fundamental abnormality in the SHR appears to be at a more central level. The observations support the supposition that lesions at widely separate sites can give rise to apparently similar abnormalities of lower urinary tract function.     

血管平滑肌ATP敏感性钾通道研究进展

ATP敏感性钾通道 (KATP)广泛存在于各类细胞和组织中 ,是药物作用的重要靶点。KATP是由内向整流钾通道Kir和磺酰脲类受体SUR亚基组成。与血管舒缩特性密切相关的是SUR2B/Kir6 1,电导值小 ,对ATP的抑制作用不敏感 ,需要有NDP才能被开放 ,故这类血管平滑肌KATP又被称为NDP依赖性钾通道。内源性和外源性的很多因子引起的血管舒缩反应与血管平滑肌上的KATP有关 ,此信号途径与PKA、PKC等磷酸化激酶有密切联系。不同血管对钾通道开放剂(potassiumchannelopeners,KCO)的反应有差异 ,KCO对血管的选择性作用机制仍不明确。本文就血管平滑肌KATP的分子结构、电生理、药理学特征、信号转导途径和KCO对血管的选择性作用进行综述     

粉防己碱对大鼠肺动脉平滑肌细胞小电导钙激活钾通道的作用

目的　研究粉防己碱 (Tet)对大鼠肺动脉平滑肌细胞小电导钙激活钾通道 (SKCa)的作用。方法　内面朝外膜片箝单通道记录法。结果　Tet 7 5 μmol·L-1对电导值为 10pS的SKCa无明显影响 ,15 μmol·L-1可增加SKCa开放的概率 ,改变通道的开放和关闭模式 ,30 μmol·L-1降低通道的开放概率 ,通道开放以短暂簇状为主。结论　Tet对肺动脉平滑肌SKCa的作用与Tet的浓度有关 ,合适浓度下可增加通道的开放 ,K+ 外流增多 ,与Tet降低肺动脉张力有关。     

Carbon monoxide activates large-conductance calcium-activated potassium channels of human cardiac fibroblasts through various mechanisms

Carbon monoxide (CO) is a cardioprotectant and potential cardiovascular therapeutic agent. Human cardiac fibroblasts (HCFs) are important determinants of myocardial structure and function. Large-conductance Ca²⁺-activated K⁺ (BK) channel is a potential therapeutic target for cardiovascular disease. We investigated whether CO modulates BK channels and the signaling pathways in HCFs using whole-cell mode patch-clamp recordings. CO-releasing molecules (CORMs; CORM-2 and CORM-3) significantly increased the amplitudes of BK currents (IBK). The CO-induced stimulating effects on IBK were blocked by pre-treatment with specific nitric oxide synthase (NOS) blockers (L-N^G-monomethyl arginine citrate and L-N^G-nitroarginine methyl ester). 8-bromo-cyclic GMP increased IBK. KT5823 (inhibits PKG) or ODQ (inhibits soluble guanylate cyclase) blocked the CO-stimulating effect on IBK. Moreover, 8-bromo-cyclic AMP also increased IBK, and pre-treatment with KT5720 (inhibits PKA) or SQ22536 (inhibits adenylate cyclase) blocked the CO effect. Pre-treatment with N-ethylmaleimide (a thiol-alkylating reagent) also blocked the CO effect on IBK, and DL-dithiothreitol (a reducing agent) reversed the CO effect. These data suggest that CO activates IBK through NO via the NOS and through the PKG, PKA, and S-nitrosylation pathways.     

丹参酮ⅡA磺酸钠和丹参素对猪冠状动脉平滑肌细胞钙激活钾通道的激活机制

目的研究丹参酮ⅡA磺酸钠(DS-201)和丹参素(DS-182)的冠状动脉舒张作用除了已知的机制外,是否还与钙激活钾通道(KCa)有关。方法猪冠脉平滑肌细胞贴附式和内面向外式膜片钳单通道电流记录技术。结果DS-201和DS-182均可激活冠脉平滑肌细胞KCa,但DS-201在内面向外膜片方式下激活KCa;而DS-182在细胞贴附膜片方式下激活KCa。结论DS-201和DS-182激活KCa的作用机制不同。DS-201能直接激活KCa,而DS-182则可能需要一系列胞内过程。这种差异可能与二者的结构和溶解性质不同有关。