高钾饮食激活肾远曲小管肾外髓钾通道

肾外髓钾通道(renal outer medullary potassium channel, ROMK)是肾脏重要的排钾通道,经ROMK通道分泌的K+是尿钾大部分或全部的来源。既往在肾小管离子转运机制调控研究中,学者多将ROMK通道的研究靶点集中于髓袢和集合管,对肾远曲小管末段(late distal convoluted tubule, DCT2) ROMK通道参与机体K+排泄的研究较少。本研究旨在应用单通道及全细胞膜片钳技术,在肾DCT2顶端膜记录ROMK通道电流并观察高钾饮食对该通道活性的影响。结果显示,在肾DCT2顶端膜可记录到一种电导为39 pS的小电导通道电流,且该电流可被ROMK通道特异性阻断药抑制。与正常钾饮食组相比,高钾饮食能够显著增加肾DCT2顶端膜ROMK通道电流出现的概率,增强该通道活性(P <0.01)。Western blot结果显示,高钾饮食能够显著上调肾脏ROMK通道及上皮钠通道(epithelial sodium channel, ENaC)的蛋白表达,下调肾脏钠-氯同向转运体(Na+-Clcotransporter, NCC)的蛋白表达,且高...     

小檗碱激活人结肠癌细胞容积敏感的氯通道

本文旨在研究小檗碱对人结肠癌细胞(SW480)氯通道的作用.采用膜片钳技术记录小檗碱激活的SW480全细胞氯电流,并用高渗和低渗灌流液、以及氯通道阻断剂研究该电流的生理学和药理学特性.结果显示,当细胞处在等渗液中,可在SW480细胞膜上记录到微弱且稳定的背景电流;小檗碱(10 nmol/L)可诱发SW480细胞迅速产生...     

细胞外Ba^2＋对内向整流钾通道的阻断作用

实验采用双微电极电压箝（ＴＥＶ）法研究Ｂａ＾２＋对非洲爪蟾卵母细胞表达的内向整流钾通道（ＩＲＫ１）的阻断作用。细胞外Ｂａ＾２＋浓度分别为０,１,３,１０和１００μｍｏｌ／Ｌ,Ｋ＾＋浓度分别为１０和９０ｍｍｏｌ／Ｌ,可见快速开通道阻断剂Ｂａ＾２＋对ＩＲＫ１的瞬间电流（施加电压后１ｍｓ）的阻断作用依赖Ｂａ＾２＋、Ｋ＾＋、时间和电压;但对ＩＲＫ１的开关特性几乎无影响,ＩＲＫ１对之不通透。三级指数拟合的结     

味觉传导有不同的机制

细胞的分离及培养技术的发展,加上电生理的斑片钳(patch-clamp)技术的进展,对味觉传导的细胞机制有了更深入的了解。大量研究表明,不同的味觉(酸、甜、咸、苦等)有不同的传导路径。酸味一般由酸即氢离子产生,氢离子能阻断味觉细胞顶膜的电压依赖性K~ 通道,引起细胞的去极化,产生酸味觉。用K~ 通道阻断剂TEA~ (四乙铵)作用于顶膜,可产生同样效果,而作用于基膜则无此效果,说明只有顶膜的K~ 通道传导     

人乳腺MCF10A细胞系K~+通道的表达和特性及其与增殖的关系

近年来发现,K+通道与乳腺癌细胞的增殖和转化密切相关,但机制尚不清楚。本研究室前期报道了K+通道阻断剂4-氨基吡啶(4-aminopyridine,4-AP)能够抑制人乳腺上皮细胞的增殖,本文则进一步检测几种电压门控K+通道(voltage-gatedK+channel,Kv)在人乳腺上皮细胞系MCF10A中的表达,运用全细胞膜片钳技术,初步研究了该细胞K+通道的特性,观察K+通道阻断剂对细胞增殖以及信号通路蛋白活性的影响。结果显示,MCF10A细胞均有Kv1.1、Kv1.2、Kv1.3和Kv1.5基因mRNA的表达,其中Kv1.5表达量明显高于乳腺癌细胞MCF7。全细胞膜片钳钳制细胞于-60mV,给予持续时间800ms、范围从-60mV到+60mV的去极化刺激电压,步幅为10mV,然后给予持续150ms的-60mV的刺激,刺激频率为1Hz,可记录到一种跨膜电流,该电流具有电压依赖、外向整流的特性,并且能被Kv通道阻断剂4-AP阻断,证实该细胞膜存在Kv通道。此外,4-AP阻断K+通道10min后,与增殖相关的有丝分裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号通路ERK1/2蛋白活性增强而p38蛋白活性减弱;5mmol/L4-AP处理细胞48h后,MCF10A的生长抑制率为25.29%。以上结果提示,在人乳腺上皮细胞系MCF10A细胞膜上存在不同亚型的Kv通道,该通道可被4-AP阻断,并且4-AP能够抑制MCF10A细胞的增殖,其机制可能与细胞增殖信号通路不同成员的活性调节有关。     

多不饱和脂肪酸对成年雪貂心肌钾通道的作用

本研究是在成年雪貂的心肌上研究多不饱和脂肪酸（PUFA）对电压门控钾通道的效应。我们观察到,n-3 PUFA能抑制短时性外向钾电流（Ito)和延迟整流钾电流（IK),而对内向整流钾电流（IK1）则没有明显影响。二十二碳六烯酸（DHA）对Ito和Ik能产生浓度依赖性的抑制作用,其IC50分别为7.5和20μmol/L,但不影响IK1。二十碳五烯酸（EPA）对这三种钾通道的作用与DHA相似。花生四烯酸（5或10μmol/L)先引起IK的抑制,然后引起IK,AA的激活;用环氧合酶抑制剂消炎痛可以阻断花生四烯酸激活IK,AA的作用。不具有抗心律失常作用的单不饱和脂肪酸和饱和脂肪酸都不明显影响这些钾通道的活性。上述实验结果证明,n-3 PUFA能抑制心肌细胞的Ito和IK,但和我们以前报道的PUFA对心肌钠电流和钙电流的作用相比,其对Ito和IK抑制作用的效能较低。n-3 PUFA的抗心律失常效应可能与它们抑制心肌钠、钙、钾通道的作用有关。     

藿香正气水对猪远端气道完整上皮HCO3-分泌的作用

本文应用短路电流技术检测了cAMP激动剂forskolin/IBMX和中成药藿香正气水(Huoxiang.zhengqi liquid,HZL)对猪远端气道完整上皮HCO3-分泌的作用.新鲜分离的气道上皮组织可测得(94.9±8.2)μtA/cm2的跨上皮基础电流,其中的16.6%和62.7%可分别被amiloride(上皮钠离子通道阻断剂,100 Ixmol/L)和NPPB(囊性纤维化跨膜电导调节体CI-通道阻断剂,100μmol/L)所阻断.用葡萄糖酸根替代浴液中的CI-,跨上皮基础电流降低为(54.0±6.7)laA/cm2,当进一步替代掉浴液中HCO3-时,此电流可被去除,提示在末受刺激条件下存存HCO3-分泌.forskolin/IBMX可刺激HCO3-依赖的电流增加(7.3±0.5)μA/cm2.值得注意的是,HZL也能引起HCO3-电流增加(7.4±1.9)μA/cm2,而这种刺激作用不受forskolin/IBMX预处理的影响,提示一种不依赖于cAMP的信号通路.以上结果提示,无论是否受刺激,猪远端气道上皮都分泌HC03.HZL对远端气道上皮HC03-分泌的刺激作用,提示其有希望成为一种新的、有治疗意义的远端气道HCO3-分泌刺激剂.     

肾小管管周膜Kir4.1及Kir4.1/Kir5.1通道的功能及调控

远端肾小管管周膜内向整流钾通道(inwardly-rectifying potassium channel, Kir)可控制细胞膜静息电位和跨膜电压,从而参与水和电解质转运的调控。Kir4.1及Kir4.1/Kir5.1异源四聚体高表达于髓袢升支粗段、远曲小管、连接小管和集合管管周膜,是调控Na~+重吸收和K~+分泌的重要转运蛋白。编码Kir4.1的KCNJ10功能缺失性突变可导致EAST/SeSAME综合征的发生,主要表现为:癫痫、共济失调、神经性耳聋及以盐丢失、低镁血症、低钾血症和代谢性碱中毒为主要表现的水盐代谢紊乱。而靶向敲除KCNJ16编码的Kir5.1除了引起低血钾以外,还表现为严重的高氯性代谢性酸中毒和高钙尿。另外,KCNJ10敲除抑制钠氯同向转运体在远曲小管管腔膜的表达及活动,同时,可增强连接小管和集合管上皮钠通道的表达。细胞内的pH值、多巴胺、胰岛素和胰岛素样生长因子等因素均可调控Kir4.1和Kir4.1/Kir5.1通道活动,涉及的机制包括PKC、PI3K、Src家族以及WNK-SPAK等信号转导途径。本综述对近年肾小管管周膜Kir的研究进展加以总结,重点阐述Kir4.1和Kir4.1/Kir5.1通道功能及其活性调控。     

Kappa 阿片受体的抗缺血性心脏保护作用--信息机制

有证据表明,心脏细胞产生强腓肽和强腓肽类多肽,它们是kappa阿片受体(κ-0R)的激动剂。κ-0R是心脏一种优势的阿片受体,其激活可改变在体和离体心脏的功能。在正常和病理情况下,内源性κ-阿片肽可能通过自分泌或旁分泌的方式调节心脏功能。心肌缺血是导致心脏功能紊乱的一个常见原因,主要表现为心肌功能减弱,心律失常及心肌梗塞等。心肌缺血时,交感神经发放增强,从而增加作功负荷及氧消耗量;而这又使缺血引发的状况更为恶化。机体抵抗缺血引发心肌损害／心律失常的保护机制之一是抑制β-肾上腺素受体(β—AR)的兴奋。κ-0R确实能抑制β-AR的激动。这种抑制主要是由于GS蛋白受到抑制,也在较小程度上由于信息通路的腺苷酸环化酶的抑制。因为该种酶能通过对百日咳毒素敏感的G蛋白转导β—AR的激动。另一保护心肌对抗缺血性损害的机制是预处理。预处理是指预先受到缺血等损伤使心脏对随后更严重的损伤产生较强的耐受能力。这种保护作用可以在预处理后即时产生,也可延至预处理后1—3天。在采用缺血或其产生的后果之一——代谢抑制作为预处理而致的心脏保护中,κ-OR参与媒介预处理的作用。用κ—OR的特异性激动剂U50488H激活κ—OR(U50488H药理性预处理,UP)可激活蛋白激酶C(PKC),开放ATY敏感的钾通道(KATP channels)及增加热休克蛋白(HSP)的产生。阻断PKC的作用,关闭KATP通道或抑制HSP的合成,均可消除UP的心脏保护作用。这些发现表明,PKC、KATP通道和HSP在UP的心脏保护中均具重要作用。此外,UP也能减低缺血造成心肌损害的因素之一,即Ca^2 的超负荷。这个事实表明UP发挥心脏保护作用至少部分地是通过减低Ca^2 的超负荷。最有趣的是,以阻断剂阻塞KATP通道,在消除UP的延迟性心脏保护作用的同时也降低了UP对Ca^2 超负荷的抑制作用。这个事实揭示了KATP通道开放所致的心脏保护作用至少部分地可能是由于防止或减低了Ca^2 的超负荷。     

Effects of carbon monoxide on ion transport across rat distal colon

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current (I(sc)). A maximal response was achieved at a concentration of 2.5·10(-4) mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl(-) and HCO(3)(-) underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the I(sc) induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na(+)-K(+)-2Cl(-) cotransporter, combined with 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid sodium salt, an inhibitor of the basolateral Cl(-)/HCO(3)(-) exchanger, inhibited the CORM-2-induced I(sc). Membrane permeabilization experiments indicated an activation of basolateral K(+) and apical Cl(-) channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca(2+) concentration. This increase depended on the influx of extracellular Ca(2+), but not on the release of Ca(2+) from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.     

Purinergic P2Y6 receptors induce Ca2+ and CFTR dependent Cl- secretion in mouse trachea.

In airways Cl- secretion is activated and Na+ absorption is inhibited when P2Y2 receptors are stimulated by ATP or UTP. Both nucleotides are subject to degradation to ADP and UDP by ecto-nucleotidases. Here we show that these metabolites change electrolyte transport by stimulation of P2Y6 receptors in mouse trachea. Immunohistochemistry confirmed luminal and basolateral expression of P2Y6 receptors. In Ussing chamber experiments luminal ADP, UDP or the P2Y6 receptor agonist INS48823 induced both transient and persistent increase in short circuit currents (ISC). Activation of ISC was inhibited by the P2Y6 receptor blocker PPADS. The transient response was inhibited by DIDS, whereas the persistent ISC was inhibited by glibenclamide and by the protein kinase A (PKA) blocker H-89. Moreover, sustained activation of ISC by luminal UDP was inhibited by blocking basolateral K+ channels with 293B. Possible effects of diphosphates on P2Y1 or adenosine receptors were excluded by the inhibitors MRS2179 and 8-SPT, respectively. Inhibition of amiloride sensitive Na+ absorption was only seen after blocking basolateral K+ channels with 293B. In contrast, Cl- secretion activated by basolateral ADP or UDP was only transient and was blocked by the sk4 K+ channel blocker clotrimazole. In summary, activation of luminal P2Y6 receptors in the airways shifts electrolyte transport towards secretion by increasing intracellular Ca+ and activation of PKA.     

TNF-alpha down-regulates the Na+-K+ ATPase and the Na+-K+-2Cl-cotransporter in the rat colon via PGE2

TNF-alpha is believed to play a pivotal role in the pathogenesis of inflammatory bowel diseases which have diarrhea as one of their symptoms. This work studies the effect of the cytokine on electrolyte and water movements in the rat distal colon using an intestinal perfusion technique and attempts to determine its underlying mechanism of action. TNF-alpha inhibited net water and chloride absorption, down-regulated in both surface and crypt colonocytes the Na+-K+-2Cl- cotransporter, and reduced the protein expression and activity of the Na+-K+ ATPase. Indomethacin up-regulated the pump and the cotransporter in surface cells but not in crypt cells, and in its presence, TNF-alpha could not exert its effect, suggesting an involvement of PGE2 in the cytokine action. The effect of TNF-alpha on the pump and symporter was studied also in cultured Caco-2 cells in isolation of the effect of other cells and tissues, to test whether the cytokine acts directly on intestinal cells. In these cells, TNF-alpha and PGE2 had a similar effect on the pump expression and activity as that observed in crypt cells but were without any effect on the Na+-K+-2Cl- cotransporter. It was concluded that the effect of the cytokine on colonocytes is mediated via PGE2. By inhibiting the Na+-K+ ATPase, it reduces the Na+ gradient needed for NaCl absorption, and by down-regulating the expression of the Na+-K+-2Cl- symporter, it reduces basolateral Cl- entry and luminal Cl- secretion. The inhibitory effect on absorption is more significant than the inhibitory effect on secretion resulting in a decrease in net electrolyte uptake and consequently in more water retention in the lumen.     

Cyclic AMP-induced K⁺ secretion occurs independently of Cl⁻ secretion in rat distal colon

cAMP induces both active Cl(-) and active K(+) secretion in mammalian colon. It is generally assumed that a mechanism for K(+) exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl(-) secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K(+) secretion and whether cAMP-induced K(+) secretion provides the driving force for Cl(-) secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K(+) fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na(+) orthovanadate (P-type ATPase inhibitor) inhibited active K(+) absorption normally present in rat distal colon. In the presence of mucosal Na(+) orthovanadate, serosal FSK induced both K(+) and Cl(-) secretion. FSK-induced K(+) secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172). By contrast, FSK-induced Cl(-) secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTR(inh)-172. These results indicate that cAMP-induced colonic K(+) secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K(+) losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl(-) secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.     

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.     

Site-specific regulation of ion transport by prolactin in rat colon epithelium

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.     

Prolactin, an activator of epithelial Na+ channel, inhibits basolateral K+ channels in adult tree frog skin

Adult amphibian skin actively transports Na+ from its apical to basolateral side while in turn, K+ is recycled through Na+, K+-ATPase and K+ channels located in the basolateral membrane. We previously found that PRL stimulates Na+ transport in the skin of the adult tree frog (Hyla arborea japonica) via an increase in the open-channel density of the epithelial Na+ channel (ENaC). If PRL also activates basolateral K+ channels, this activation would help to stimulate Na+ transport, too. Whether PRL does indeed stimulate basolateral K+ channels in the adult tree frog was examined by measuring the short-circuit current across nystatin-treated skin. Both tolbutamide, a K(ATP) channel blocker, and tetrapentylammonium (TPA), a KCa channel blocker, blocked the current, the effect of TPA being more powerful than that of tolbutamide. Contrary to expectation, PRL inhibited the basolateral K+ channels in this skin. In the presence of basolateral amiloride, PRL still inhibited the basolateral K+ current, suggesting that the (Na+)-H+ exchanger located in the basolateral membrane does not mediate the inhibitory effect of PRL on the basolateral K+ channels in Hyla.     

Electrolyte Transport in the Mouse Trachea: No Evidence for a Contribution of Luminal K+ Conductance

Recent studies on frog skin acini have challenged the question whether Cl(-) secretion or Na(+) absorption in the airways is driven by luminal K(+) channels in series to a basolateral K(+) conductance. We examined the possible role of luminal K(+) channels in electrolyte transport in mouse trachea in Ussing-chamber experiments. Tracheas of both normal and CFTR (-/-) mice showed a dominant amiloride-sensitive Na+ absorption under both, control conditions and after cAMP-dependent stimulation. The lumen-negative transepithelial voltage was enhanced after application of IBMX and forskolin and Cl(-) secretion was activated. Electrolyte secretion induced by IBMX and forskolin was inhibited by luminal glibenclamide and the blocker of basolateral Na(+2)Cl(-)K(+) cotransporter azosemide. Similarly, the compound 293B, a blocker of basolateral KCNQ1/KCNE3 K(+) channels effectively blocked Cl(-) secretion when applied to either the luminal or basolateral side of the epithelium. RT-PCR analysis suggested expression of additional K(+) channels in tracheal epithelial cells such as Slo1 and Kir6.2. However, we did not detect any functional evidence for expression of luminal K(+) channels in mouse airways, using luminal 293B, clotrimazole and Ba(2+) or different K(+) channel toxins such as charybdotoxin, apamin and a-dendrotoxin. Thus, the present study demonstrates Cl(-) secretion in mouse airways, which depends on basolateral Na(+2)Cl(-)K(+) cotransport and luminal CFTR and non-CFTR Cl(-) channels. Cl(-) secretion is maintained by the activity of basolateral K(+) channels, while no clear evidence was found for the presence of a luminal K(+) conductance.