吡那地尔对培养大鼠交感神经元烟碱电流的影响

目的　研究ATP敏感性K+ 通道 (KATP)开放剂吡那地尔对大鼠颈上交感神经元烟碱诱发电流的影响。方法　采用培养的交感神经元的全细胞膜片钳技术。结果　吡那地尔在较高浓度时 (10 0 μmol·L-1)对烟碱电流具有一定的抑制作用 ,但并不影响烟碱受体的失敏 ;KATP阻断剂格列苯脲不能对抗吡那地尔对烟碱电流的抑制效应。结论　吡那地尔对大鼠交感神经元烟碱电流具有一定的抑制作用 ,其作用并不通过KATP介导。     

埃他卡林新衍生物激活ATP敏感性钾通道的亚型选择性

目的研究埃他卡林新衍生物对不同亚型KATP通道的选择性作用。方法在特异性表达Kir6.2/SUR1、Kir6.2/SUR2A、Kir6.1/SUR2B3种不同亚型KATP通道的HEK-293细胞模型上,分别给予0.1、1.0、10、100×10-6mol.L-1的埃他卡林新衍生物,研究其作用前后DiBAC4(3)细胞荧光强度的变化,评价其对克隆表达的不同亚型KATP通道的选择性作用。结果7个埃他卡林新衍生物可激活KATP通道,其中衍生物(3)、(7)、(8)、(9)、(12)和(14)对3个亚型的KATP通道均具有激活作用,衍生物(7)对Kir6.2/SUR2A亚型的激活作用最强;衍生物(6)纳他卡林(natakalim)仅激活Kir6.1/SUR2B亚型,对Kir6.2/SUR2A和Kir6.2/SUR1亚型均无激活作用。结论纳他卡林为Kir6.1/SUR2B亚型高选择性开放剂。     

格列苯脲通过激活ROS-JNK通路发挥抗肿瘤作用

目的：探讨ATP敏感性钾通道（KATP）阻断剂格列本脲对胃癌细胞株MGC-803的抗肿瘤作用及相关机制。方法：应用MTT、Hoechst法分别检测格列本脲对细胞活力和凋亡的影响：应用Western．blot、RT-PCR等方法检测MGC-803细胞KATP亚基的表达、线粒体细胞色素C和凋亡诱导因子（AIF）的释放、JNK,Akt的磷酸化,以及NADPH氧化酶催化亚基gp91的表达;应用H2DCFDAROS荧光探针检测ROS和O2-的产生,氧电极法测定线粒体呼吸功能。结果：MGC-803细胞表达由kir6．2和SUR1亚基构成的KATP通道。格列苯脲可降低MGC-803细胞活力、促进其凋亡;格列苯脲能促进NADPH氧化酶源性和线粒体源性ROS的生成,进而活化JNK激酶,并降低线粒体膜电位、促进细胞色素C和凋亡诱导因子（AIF）的释放,触发线粒体通路介导的细胞凋亡;抗氧化剂N-乙酰半胱氨酸（NAC）可抑制格列苯脲降低MGC-803细胞活力、诱发细胞凋亡,以及活化JNK的作用;进一步的研究显示,格列苯脲可显著降低野生型MEF细胞的活力、促进其凋亡,并能抑制抗凋亡的Akt信号通路,但不影响JNK1和JNK2-MEF细胞的存活。结论：KATP,通道阻断剂格列苯脲可通过促进ROS的生成,激活JNK、抑制Akt信号通路,触发线粒体通路介导的细胞凋亡,从而发挥其抗肿瘤作用。研究结果提示,KATP通道可能是发展抗肿瘤药物的有效靶标。     

埃他卡林增强星形胶质细胞摄取谷氨酸的作用

目的研究新型ATP敏感性钾通道开放剂(KATPCO)埃他卡林(iptakalim,Ipt)对星形胶质细胞摄取谷氨酸的影响。方法取新生大鼠脑星形胶质细胞作原代培养，将Ipt直接作用于细胞，观察它对正常和6-羟基多巴胺(6-OHDA)损伤模型细胞摄取谷氨酸的影响；分别加入阳性对照药吡那地尔和非特异性钾通道阻断药格列本脲分析Ipt的作用机制。根据［³H］标记的D,L-谷氨酸摄入量判断细胞谷氨酸摄取作用强度。结果Ipt和吡那地尔都能增强星形胶质细胞的谷氨酸摄取作用、逆转6-OHDA引起的谷氨酸摄取抑制效应，预先加入格列本脲后，上述作用均被取消。结论埃他卡林可能通过促进钾通道开放增强星形胶质细胞摄取谷氨酸的作用。     

吡那地尔对人肺动脉平滑肌细胞KATP通道蛋白及ERK1/2磷酸化的影响

目的 探讨ATP敏感性钾(KA口)通道开放剂吡那地尔(Pin)对内皮素1(ET-1)诱导的人肺动脉平滑肌细胞(PASMCs)KATe通道蛋白表达及细胞外信号调节激酶1和2(ERK1/2)磷酸化的影响.方法 体外培养人PASMCs,用ET-1诱导其增殖,应用Westem blot方法评价Pin对ET-1诱导的人PASMCs KArP通道蛋白磺酰脲受体亚单位(SUR2B)和内向整流性孔区亚单位(Kir6.1)表达的影响以及ERK1/2的磷酸化作用.结果 ET-1显著下调SUR2B的表达,Pin呈浓度依赖性上调SUR2B表达;KATP通道拮抗剂格列本脲阻断Pin的作用,而Kir6.1的表达不受影响.ET-1呈时间依赖性促进人PASMCs ERK1/2磷酸化,10min时最明显,Pin(10μmol/L)拮抗ET-1对ERK1/2磷酸化的影响,格列本脲逆转Pin的作用.结论 KATP通道开放剂Pin通过上调KATP通道蛋白的表达,抑制ET-1诱导的人PASMCs ERK1/2的磷酸化.     

盐酸埃他卡林对心肌ATP-敏感性钾通道的作用

目的　在急性分离的豚鼠心室肌细胞上观察盐酸埃他卡林 (iptakalimhydrochloride ,Ipt)对钾电流的影响 ;研究Ipt对 [3 H]格列本脲 (glibenclamide ,Gli)与心肌ATP敏感性钾通道 (ATP sensitivepotassiumchannel,KATP)的硫脲受体(Sulfonylureareceptor,SUR2A)结合特征以及 [3 H]Gli与心肌膜KATP结合和解离动力学过程的影响 ,以评价Ipt对心肌KATP的作用。方法　分离豚鼠心室肌细胞 ,用全细胞记录技术记录细胞钾电流 ,通过浴槽内灌流给药 ,观察盐酸埃他卡林对钾电流的影响。KATP拮抗剂 [3 H]Gli与大鼠心肌膜特异性结合与解离的动力学试验。结果　①Ipt在浓度为 1和10 0 μmol·L-1时 ,均可明显引起豚鼠心室肌外向钾电流I U曲线上移 ,Ipt作用后 5min内细胞外向钾电流强度分别增强为初始电流强度的 12 4 9%± 9 5 % (n =5 )和 15 1 6 %±11 2 % (n =7) ,与溶媒对照组 (6 9 8%± 3 5 % ,n =7)比较差异均有统计学意义 (P <0 0 1)。在相同条件下 ,KATP开放剂吡那地尔 (pinacidil,Pin)的作用与之相似 ,也可明显引起豚鼠心室肌外向钾电流I U曲线上移 ,显著增强细胞外向钾电流。②非标记Gli与 [3 H]Gli和大鼠心肌膜标本在 2 5℃孵育 6 0min ,可浓度依赖性地抑制 [3 H]Gli与心肌膜SUR2A的特异性结合 ,其IC50 值为 (     

埃他卡林对心肌细胞损伤保护作用的药理学特征

目的研究KATP通道开放剂埃他卡林(iptakalim,Ipt)对心肌细胞氧化应激损伤的保护作用,并探究其作用机制。方法采用200μmol.L-1的过氧化氢(H2O2)损伤体外培养的乳鼠心肌细胞2 h,建立氧化应激心肌细胞损伤模型;以二氮嗪(diazoxide,Dia)作为阳性对照药物,采用生物化学法检测各处理组细胞培养液中乳酸脱氢酶(lactate dehydrogen-ase,LDH)活性、超氧化物歧化酶(superoxide dismutase,SOD)活性及MTT法检测心肌细胞损伤程度,反映埃他卡林对该模型的影响。结果过氧化氢损伤后,细胞存活率降低,LDH释放量明显升高,SOD活力下降;埃他卡林预处理后,细胞培养液中LDH活性较过氧化氢处理组降低(P<0.05),SOD活性升高(P<0.05),细胞存活率浓度依赖性升高(P<0.01)。该保护作用可被线粒体KATP通道阻断剂5-羟基癸酸酯(5-HD)拮抗,也可被KATP通道阻断剂格列本脲(glibenclamide,Gli)阻断。结论埃他卡林对H2O2造成氧化应激损伤的心肌细胞具有明显的保护作用,其保护作用与线粒体膜和细胞膜的KATP通道激活有关。     

埃他卡林对长期低氧大鼠肺动脉平滑肌KATP mRNA的影响

目的 :研究慢性低氧对大鼠肺动脉平滑肌ATP敏感钾通道 (KATP)mRNA表达的影响及新型KATP开放剂盐酸埃他卡林的作用。方法 :SD雄性大鼠 16只随机分成对照组、低氧组、低剂量治疗组 (盐酸埃他卡林 0 .75mg·kg-1·d-1,ig)、高剂量治疗组(盐酸埃他卡林 1.5mg·kg-1·d-1,ig)。将低氧组和治疗组大鼠放入常压低氧舱制备动物模型 ;采用RT PCR技术 ,分析各组肺动脉主干平滑肌KATP mR NA表达。结果 :低氧组SUR2mRNA水平显著低于正常组 ,高剂量治疗组SUR2显著高于低氧组 ,各组Kir 6 .1没有显著差异。结论 :慢性低氧抑制KATP 通道表达 ,而盐酸埃他卡林能提高表达 ,可从肺动脉高压发病机制上理解该药物治疗价值。     

ATP敏感性钾通道开放剂吡那地尔增高Bcl-2表达而抑制PC12细胞缺血性凋亡

目的探讨ATP敏感性钾通道开放剂吡那地尔对缺血缺氧PC12细胞凋亡及对Bcl-2 mRNA和蛋白表达的影响。方法取传代后3d的PC12细胞,分为正常对照组、缺血对照组、吡那地尔处理组、吡那地尔+格列吡嗪处理组共4组。吡那地尔处理组在PC12细胞缺血缺氧前20min加入浓度为100μmol·L-1的吡那地尔;吡那地尔+格列吡嗪处理组则加入浓度100μmol·L-1的吡那地尔和浓度为500μmol·L-1的KATP通道阻断剂格列吡嗪。采用Annexin-V FITC/PI双染流式细胞分析仪检测凋亡率;应用免疫荧光染色和Western blot检测Bcl-2蛋白表达水平;应用RT-PCR检测Bcl-2 mRNA表达水平。结果缺血缺氧后缺血对照组、吡那地尔处理组、吡那地尔+格列吡嗪处理组细胞凋亡率随时间增加而增加,24h达高峰。吡那地尔组与其余组比较差异均有显著性(P<0.01)。缺血对照组、吡那地尔处理组、吡那地尔+格列吡嗪处理组细胞Bcl-2 mRNA及蛋白表达各时间点均增加,12h达高峰。吡那地尔组与其余组比较差异均有显著性(P<0.05,或P<0.01)。缺血对照组和吡那地尔+格列吡嗪处理组比较差异均无显著性(P>0.05)。结论ATP敏感性钾通道开放剂可能通过提高Bcl-2 mRNA及蛋白表达来减轻缺血缺氧后PC12细胞凋亡,发挥保护作用。     

盐酸埃他卡林对大鼠离体工作心脏功能的影响

盐酸埃他卡林是军事医学科学院毒物药物研究所在新药研究中发现的一个结构新颖的抗高血压药物。在清醒自发性高血压大鼠上 ,埃他卡林降压作用确切、平稳、持久、对心率影响轻 ,且具有选择性抗高血压作用 ,同时盐酸埃他卡林的降压作用既可被ＡＴＰ敏感性Ｋ+ 通道拮抗剂格列本脲[1,2 ] 所对抗也可被格列本脲所预防 ,格列本脲的对抗效应具有剂量依赖性 ,提示盐酸埃他卡林的降压作用可能与心血管ＡＴＰ敏感性Ｋ+ 通道有关。在麻醉正常血压大鼠上采用直接插管法观察不同剂量的盐酸埃他卡林对外周血压及心功能的影响 ,以及格列本脲的干扰作用 ,结…     

Characterization of K(ATP)-channels in rat basilar and middle cerebral arteries: studies of vasomotor responses and mRNA expression

Changes in the activity of K+ channels represent a major mechanism that regulates vascular tone. Cerebrovascular adenosine 5'-triphosphate-sensitive K+(K(ATP)) channels were characterized in studies of the molecular expression and vasomotor reactivity to different K(ATP) channel openers in rat basilar and middle cerebral arteries. Both arteries showed strong mRNA expression of the subunits of the pore-forming inward-rectifying K+ channel type 6.1 (Kir6.1), Kir6.2 and the connected sulfonylurea receptor (SUR) subunits, SUR1 and SUR2B, while only weak bands for SUR2A were seen. The K(ATP) channel openers induced relaxation of prostaglalndin F2alpha-precontracted isolated basilar and middle cerebral arteries with the order of potency N-Cyano-N-(1,1-dimethylpropyl)-N'-3pyridylguanidine (P-1075)>levcromakalim>N-(4-Phenylsulfonylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide (ZM226600)>pinacidil>diazoxide. The responses induced by levcromakalim, ZM226600 and diazoxide were significantly more potent in basilar arteries than in middle cerebral arteries, while pinacidil and P-1075 were equipotent. Endothelium removal decreased (P<0.05) the sensitivity (pIC50) of basilar arteries, but not of middle cerebral arteries, to pinacidil, levcromakalim, P-1075 and ZM226600. The maximum relaxant response to P-1075 was stronger (P<0.005) in basilar arteries with endothelium than without endothelium. Correlation of the relaxant potency of K(ATP) channel openers in rat basilar and middle cerebral arteries with historical measurements of affinity obtained in COS-7 cell lines expressing either SUR1, SUR2A or SUR2B showed that vasodilatation by K(ATP) channel openers correlated with binding to either the SUR2A or the SUR2B subunit. Glibenclamide was a blocker of relaxation induced by pinacidil, levcromakalim, P-1075 and ZM226600 in basilar arteries. Only a weak antagonistic effect of glibenclamide on pinacidil-, levcromakalim- and ZM226600-induced relaxations was found in middle cerebral arteries. The subunit profile and the observed pharmacological properties suggest that the K(ATP) channels expressed in rat basilar and middle cerebral artery are likely to be composed of SUR2B co-associated with Kir6.1 or Kir6.2. In basilar arteries, but not in middle cerebral arteries, endothelial K(ATP) channels may be involved.     

Coassembly of different sulfonylurea receptor subtypes extends the phenotypic diversity of ATP-sensitive potassium (KATP) channels

K(ATP) channels are metabolic sensors and targets of potassium channel openers (KCO; e.g., diazoxide and pinacidil). They comprise four sulfonylurea receptors (SUR) and four potassium channel subunits (Kir6) and are critical in regulating insulin secretion. Different SUR subtypes (SUR1, SUR2A, SUR2B) largely determine the metabolic sensitivities and the pharmacological profiles of K(ATP) channels. SUR1- but not SUR2-containing channels are highly sensitive to metabolic inhibition and diazoxide, whereas SUR2 channels are sensitive to pinacidil. It is generally believed that SUR1 and SUR2 are incompatible in channel coassembly. We used triple tandems, T1 and T2, each containing one SUR (SUR1 or SUR2A) and two Kir6.2Delta26 (last 26 residues are deleted) to examine the coassembly of different SUR. When T1 or T2 was expressed in Xenopus laevis oocytes, small whole-cell currents were activated by metabolic inhibition (induced by azide) plus a KCO (diazoxide for T1, pinacidil for T2). When coexpressed with any SUR subtype, the activated-currents were increased by 2- to 13-fold, indicating that different SUR can coassemble. Consistent with this, heteromeric SUR1+SUR2A channels were sensitive to azide, diazoxide, and pinacidil, and their single-channel burst duration was 2-fold longer than that of the T1 channels. Furthermore, SUR2A was coprecipitated with SUR1. Using whole-cell recording and immunostaining, heteromeric channels could also be detected when T1 and SUR2A were coexpressed in mammalian cells. Finally, the response of the SUR1+SUR2A channels to azide was found to be intermediate to those of the homomeric channels. Therefore, different SUR subtypes can coassemble into K(ATP) channels with distinct metabolic sensitivities and pharmacological profiles.     

Troglitazone but not pioglitazone affects ATP-sensitive K(+) channel activity.

We compared the effects of the two thiazolidinedione derivatives, troglitazone and pioglitazone, on ATP-sensitive K(+) (K(ATP)) channel activities. Pancreatic beta-cell type and cardiac type K(ATP) channels were reconstituted in COS-1 cells (SV 40-transformed African green monkey kidney (AGMK) cells) by heterologously expressing sulfonylurea receptor 1 (SUR1) plus Kir6.2 and sulfonylurea receptor 2A (SUR2A) plus Kir6.2, respectively. Troglitazone inhibited [86Rb(+)] efflux in both K(ATP) channel types in the presence of metabolic inhibitors, which was confirmed by electrophysiological techniques. The [86Rb(+)] efflux increased by the channel openers diazoxide and pinacidil was abolished by troglitazone. In contrast, pioglitazone did not affect these channel activities in either type K(ATP) channel. These results suggest that troglitazone modulates the various cellular functions including insulin secretion by inhibiting the K(ATP) channels, while pioglitazone has no effect on K(ATP) channel activity.     

Towards selective Kir6.2/SUR1 potassium channel openers, medicinal chemistry and therapeutic perspectives

ATP sensitive potassium (K(ATP)) channels have important functions in neuroendocrine tissue, in smooth and skeletal muscle and in the heart. In pancreatic beta cells the K(ATP) channels, which are formed by 4 ion channels (Kir6.2) and 4 regulatory sulfonylurea receptors (SUR1), control the glucose stimulated release of insulin. The Kir6.2/SUR1 K(ATP) channels are also present in the brain and in other neuroendocrine tissues. Blockers of Kir6.2/SUR1 channels, e.g. glibenclamide and repaglinide stimulate release of insulin and are used for treatment of type 2 diabetes. Openers of Kir6.2/SUR1 channels, e.g. diazoxide, have in contrast only found limited clinical use in treatment of hypersecretion of insulin associated with certain tumours (insulinoma) and genetic disorders (persistent hyperinsulinemia and hypoglycemia of infancy, PHHI). Recent studies have however, indicated that openers of Kir6.2/SUR1 channels could be useful in treatment of e.g. metabolic disorders and diseases of the CNS. The clinical use of diazoxide has been hampered by its lack of potency and selectivity giving rise to side effects, such as oedema and hirsutism and new selective openers of Kir6.2/SUR1 channels have been pursued. This has provided several structurally diverse series, which include 1,2,4-thiadiazine 1,1-dioxide derivatives, like BPDZ 62, BPDZ 73, NNC 55-0462, NNC 55-0118 and NN414, cyanoguanidines, nitropyrazoles and 4-sulfamoylphenylbenzamides. NN414 has been shown to be a potent and Kir6.2/SUR1 selective K(ATP) channels opener, which inhibits glucose stimulated insulin release in vitro and in vivo and which has beneficial effects on glucose homeostasis in preclinical and clinical studies.     

Effect of metabolic inhibition on glimepiride block of native and cloned cardiac sarcolemmal K(ATP) channels

1. We have investigated the effects of the sulphonylurea, glimepiride, currently used to treat type 2 diabetes, on ATP-sensitive K(+) (K(ATP)) currents of rat cardiac myocytes and on their cloned constituents Kir6.2 and SUR2A expressed in HEK 293 cells. 2. Glimepiride blocked pinacidil-activated whole-cell K(ATP) currents of cardiac myocytes with an IC(50) of 6.8 nM, comparable to the potency of glibenclamide in these cells. Glimepiride blocked K(ATP) channels formed by co-expression of Kir6.2/SUR2A subunits in HEK 293 cells in outside-out excised patches with a similar IC(50) of 6.2 nM. 3. Glimepiride was much less effective at blocking K(ATP) currents activated by either metabolic inhibition (MI) with CN(-) and iodoacetate or by the K(ATP) channel opener diazoxide in the presence of inhibitors of F(0)/F(1)-ATPase (oligomycin) and creatine kinase (DNFB). Thus 10 microM glimepiride blocked pinacidil-activated currents by >99%, MI-activated currents by 70% and diazoxide-activated currents by 82%. 4. In inside-out patches from HEK 293 cells expressing the cloned K(ATP) channel subunits Kir6.2/SUR2A, increasing the concentration of ADP (1 - 100 microM), in the presence of 100 nM glimepiride, lead to significant increases in Kir6.2/SUR2A channel activity. However, over the range tested, ADP did not affect cloned K(ATP) channel activity in the presence of 100 nM glibenclamide. These results are consistent with the suggestion that ADP reduces glimepiride block of K(ATP) channels. 5. Our results show that glimepiride is a potent blocker of native cardiac K(ATP) channels activated by pinacidil and blocks cloned Kir6.2/SUR2A channels activated by ATP depletion with similar potency. However, glimepiride is much less effective when K(ATP) channels are activated by MI and this may reflect a reduction in glimepiride block by increased intracellular ADP.     

Pharmacological comparison of native mitochondrial K(ATP) channels with molecularly defined surface K(ATP) channels

Many mammalian cells have two distinct types of ATP-sensitive potassium (K(ATP)) channels: the classic ones in the surface membrane (sK(ATP)) and others in the mitochondrial inner membrane (mitoK(ATP)). Cardiac mitoK(ATP) channels play a pivotal role in ischemic preconditioning, and thus represent interesting drug targets. Unfortunately, the molecular structure of mitoK(ATP) channels is unknown, in contrast to sK(ATP) channels, which are composed of a pore-forming subunit (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). As a means of probing the molecular makeup of mitoK(ATP) channels, we compared the pharmacology of native cardiac mitoK(ATP) channels with that of molecularly defined sK(ATP) channels expressed heterologously in human embryonic kidney 293 cells. Using mitochondrial oxidation to index mitoK(ATP) channel activity in rabbit ventricular myocytes, we found that pinacidil and diazoxide open mitoK(ATP) channels, but P-1075 does not. On the other hand, 5-hydroxydecanoic acid (5HD), but not HMR-1098, blocks mitoK(ATP) channels. Although pinacidil is a nonselective activator of expressed sK(ATP) channels, diazoxide did not open channels formed by Kir6.1/SUR2A, Kir6.2/SUR2A (known components of cardiac sK(ATP) channels) or Kir6.2/SUR2B. P-1075 activated all the K(ATP) channels, except Kir6.1/SUR1 channels. Glybenclamide potently blocked all sK(ATP) channels, but 5HD only blocked channels formed by SUR1/Kir6.1 or Kir6.2 (IC(50)s of 66 and 81 microM, respectively). This potency is similar to that for block of mitoK(ATP) channels (IC(50) = 95 microM). In addition, HMR-1098 potently blocked Kir6.2/SUR2A channels (IC(50) = 1.5 microM), but was 67 times less potent in blocking Kir6.1/SUR1 channels (IC(50) = 100 microM). Our results demonstrate that mitoK(ATP) channels closely resemble Kir6.1/SUR1 sK(ATP) channels in their pharmacological profiles.     

Binding and effect of K ATP channel openers in the absence of Mg2+

1 Openers of ATP-sensitive K(+) channels (K(ATP) channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs), the regulatory channel subunits. At higher concentrations, some openers activate K(ATP) channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg(2+) and presence of EDTA. 2 Binding of openers to SUR2B was measured using a mutant with high affinity for [(3)H]glibenclamide ([(3)H]GBC). In the absence of Mg(2+), 'typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [(3)H]GBC binding with K(i) values approximately 200 x higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. 3 In the absence/presence of MgATP, N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075) activated the Kir6.2/SUR2B channel in inside-out patches with EC(50)=2000/67nM and E(max)=32/134%. In the absence of Mg(2+), responses were variable with only a small part of the variability being explained by a decrease in channel responsiveness with time after patch excision and to differences in the ATP sensitivity between patches. 4 The rank order of efficacy of the openers was P1075>rilmakalim approximately nicorandil>diazoxide>minoxidil sulphate. 5 The data show that structurally diverse openers are able to bind to, and to activate the Kir6.2/SUR2B channel by a pathway independent of ATP hydrolysis. These effects are observed at concentrations used to define the biochemical mechanism of the openers in the presence of MgATP and allow the openers to be classified into 'typical' and 'atypical' KCOs with diazoxide standing apart.     

Ontogeny of sulphonylurea-binding regulatory subunits of K(ATP) channels in the pregnant rat myometrium

K(ATP) channels are composed of sulphonylurea receptors (SURs) and potassium inward rectifiers (Kir(6.x)) that assemble to form a large octameric channel. This study was designed to examine the expression and role of sulphonylurea-binding regulatory subunits 1 [SUR1 (ABCC8)] and 2 [SUR2 (ABCC9)] of the K(ATP) channels in the pregnant rat myometrium with particular regard to the contractility. RT-PCR and Western blot analysis were performed to detect the presence of SUR1 and SUR2. The SUR1 levels were markedly increased in the early stages of pregnancy. The highest level was detected on day 6 of pregnancy, while in the late stages the levels of SUR1 were significantly decreased. The SUR2 level remained unchanged throughout pregnancy. The SUR-non-selective diazoxide and the SUR2-selective pinacidil inhibited oxytocin-induced contractions. Glibenclamide, a K(ATP) channel blocker, antagonized both pinacidil and diazoxide-induced relaxations. It was established that SURs are responsible for pharmacological reactivity of K(ATP) channel openers. We conclude that, both SURs are involved in the K(ATP) channel in the pregnant rat myometrium. It may further be concluded that "pinacidil-like" K(ATP) channel openers may be of therapeutic relevance as tocolytic agents in the future.     

Interaction of a novel dihydropyridine K+ channel opener, A-312110, with recombinant sulphonylurea receptors and KATP channels: comparison with the cyanoguanidine P1075

1. ATP-sensitive K(+) channels (K(ATP) channels) are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulphonylurea receptors (SURx). Synthetic openers of K(ATP) channels form a chemically heterogeneous class of compounds that are of interest in several therapeutic areas. We have investigated the interaction of a novel dihydropyridine opener, A-312110 ((9R)-9-(4-fluoro-3-iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno [2,3-e]pyridin-8(7H)-one-1,1-dioxide), with SURs and Kir6/SUR channels in comparison to the cyanoguanidine opener P1075. 2. In the presence of 1 mM MgATP, A-312110 bound to SUR2A (the SUR in cardiac and skeletal muscle) and to SUR2B (smooth muscle) with K(i) values of 14 and 18 nM; the corresponding values for P1075 were 16 and 9 nM, respectively. Decreasing the MgATP concentration reduced the affinity of A312110 binding to SUR2A significantly more than that to SUR2B; for P1075, the converse was true. At SUR1 (pancreatic beta-cell), both openers showed little binding up to 100 microM. 3. In the presence of MgATP, both openers inhibited [(3)H]glibenclamide binding to the SUR2 subtypes in a biphasic manner. In the absence of MgATP, the high-affinity component of the inhibition curves was absent. 4. In inside-out patches, the two openers activated the Kir6.2/SUR2A and Kir6.2/SUR2B channels with similar potency (approximately 50 nm). Both were almost 2 x more efficacious in opening the Kir6.2/SUR2B than the Kir6.2/SUR2A channel. 5. The results show that the novel dihydropyridine A-312110 is a potent K(ATP) channel opener with binding and channel-opening properties similar to those of P1075.     

Decreased expression of aortic KIR6.1 and SUR2B in hypertension does not correlate with changes in the functional role of K(ATP) channels

ATP-dependent potassium (K(ATP)) channels are the target of multiple vasoactive factors and drugs. Changes in the functional role of ATP-dependent (K(ATP)) potassium channels in hypertension are controversial. The aim of the present study was to analyze the possible changes of ATP-sensitive potassium channels (K(ATP)) expression and function during hypertension. For this purpose, we used endothelium-denuded aorta segments from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) to analyze the 1) expression of K(ATP) subunits Kir6.1, Kir6.2 and SUR2B by immunohistochemistry and Western blot, 2) the K(ATP) currents recorded in the whole cell configuration of the patch-clamp technique and 3) the vasodilator response to the K(ATP) channel openers, pinacidil and cromakalim. Kir6.1 and SUR2B were expressed in the medial layer of the aorta from WKY rats and SHR rats, while Kir6.2 was not detected in aorta from either strain. Kir6.1 and SUR2B expression were decreased in hypertension. However, the vasodilator responses of pinacidil and cromakalim were similar in WKY rats and SHR rats. Moreover, pinacidil induced increase in K+ currents was also similar in WKY rats and SHR rats and also similarly inhibited by glybenclamide. Our data demonstrate for the first time direct evidence of decreased aortic Kir6.1/SUR2B subunit expression in hypertension, but preserved functional responses to K(ATP) channel openers.