The Na–K–Cl Co-transporter in astrocyte swelling

Ion channels, exchangers and transporters are known to be involved in cell volume regulation. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na⁺–K⁺–Cl⁻ cotransporters has been shown to regulate cell volume in many conditions. The Na⁺–K⁺–Cl− cotransporters (NKCC) are a class of membrane proteins that transport Na, K, and Cl ions into and out of a wide variety of epithelial and nonepithelial cells. Studies have established the role of NKCC1 in astrocyte swelling/brain edema in ischemia and trauma. Our recent studies suggest that NKCC1 activation is also involved in astrocyte swelling induced by ammonia and in the brain edema in the thioacetamide model of acute liver failure. This review will focus on mechanisms of NKCC1 activation and its contribution to astrocyte swelling/brain edema in neurological disorders, with particular emphasis on ammonia neurotoxicity and acute liver failure.     

Na+,K+-ATP酶与脑缺血

Na+,K+-ATP酶的基本功能是维持真核细胞膜内外Na+ - K+电化学梯度平衡,后者为维持细胞渗透压、调节细胞体积和维持可兴奋细胞膜静息电位所必需.Na+,K+ -ATP酶活性的维持在神经元神经递质的摄取和Ca2+外流中起着重要作用.脑缺血后,Na+,K+ -ATP酶活性降低及功能异常参与缺血性脑损伤过程.缺血预处理通过维持缺血后Na+,K+ -ATP酶活性而诱导缺血耐受.强心甾类固醇和胞二磷胆碱可通过提高Na+,K+ -ATP酶活性对脑缺血发挥神经保护效应.     

Na+,K+-ATP酶与脑缺血

Na+,K+-ATP酶的基本功能是维持真核细胞膜内外Na+ - K+电化学梯度平衡,后者为维持细胞渗透压、调节细胞体积和维持可兴奋细胞膜静息电位所必需.Na+,K+ -ATP酶活性的维持在神经元神经递质的摄取和Ca2+外流中起着重要作用.脑缺血后,Na+,K+ -ATP酶活性降低及功能异常参与缺血性脑损伤过程.缺血预处理通过维持缺血后Na+,K+ -ATP酶活性而诱导缺血耐受.强心甾类固醇和胞二磷胆碱可通过提高Na+,K+ -ATP酶活性对脑缺血发挥神经保护效应.     

Na+,K+-ATP酶与脑缺血

Na+,K+-ATP酶的基本功能是维持真核细胞膜内外Na+ - K+电化学梯度平衡,后者为维持细胞渗透压、调节细胞体积和维持可兴奋细胞膜静息电位所必需.Na+,K+ -ATP酶活性的维持在神经元神经递质的摄取和Ca2+外流中起着重要作用.脑缺血后,Na+,K+ -ATP酶活性降低及功能异常参与缺血性脑损伤过程.缺血预处理通过维持缺血后Na+,K+ -ATP酶活性而诱导缺血耐受.强心甾类固醇和胞二磷胆碱可通过提高Na+,K+ -ATP酶活性对脑缺血发挥神经保护效应.     

Altered Na+ transport after an intracellular α-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump

The Na/K pump actively exports 3 Na⁺ in exchange for 2 K⁺ across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the α-subunit diminishes internal Na⁺ (Na_i⁺) affinity ≈25-fold [Morth et al. (2007) Nature 450:1043–1049]. Because external Na⁺ (Na_o⁺) binding is voltage-dependent, we studied the reactions involving this process by using two-electrode and inside-out patch voltage clamp in normal and truncated (ΔKESYY) Xenopus-α1 pumps expressed in oocytes. We observed that ΔKESYY (i) decreased both Na_o⁺ and Na_i⁺ apparent affinities in the absence of K_o⁺, and (ii) did not affect apparent Na_o⁺ affinity at high K_o⁺. These results support a model of strict sequential external release of Na⁺ ions, where the Na⁺-exclusive site releases Na⁺ before the sites shared with K⁺ and the ΔKESYY deletion only reduces Na_o⁺ affinity at the shared sites. Moreover, at nonsaturating K_o⁺, ΔKESYY induced an inward flow of Na⁺ through Na/K pumps at negative potentials. Guanidinium⁺ can also permeate truncated pumps, whereas N-methyl-D-glucamine cannot. Because guanidinium_o⁺ can also traverse normal Na/K pumps in the absence of both Na_o⁺ and K_o⁺ and can also inhibit Na/K pump currents in a Na⁺-like voltage-dependent manner, we conclude that the normal pathway transited by the first externally released Na⁺ is large enough to accommodate guanidinium⁺.     

Crystal structure of the high-affinity Na+,K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site

The Na⁺,K⁺-ATPase maintains electrochemical gradients for Na⁺ and K⁺ that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na⁺,K⁺-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na⁺,K⁺-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg²⁺, and crystallographic studies indicate that Rb⁺ and Mn²⁺, but not Na⁺, bind to this site. Comparison with the low-affinity [K₂]E2–MgF_x–ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742–13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg²⁺. K⁺ binding at the same site unwinds a turn of αM4, dragging residues Ile318–Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K⁺–Mg²⁺ competition and explain the well-known antagonistic effect of K⁺ on CTS binding.     

Na+/K+-ATP酶ATP1A2基因突变与偏头痛

偏头痛是一种常见的反复发作性神经疾病。病因涉及遗传和环境因素,其发病机制尚未完全阐明。家族性偏瘫型偏头痛(familial hemiplegic migraine,FHM)中Na /K ATP酶α2亚基ATP1A2基因突变的发现,是偏头痛遗传学研究的重大突破。目前已鉴定出25种ATP1A2基因突变,且绝大部分为错义突变,这些突变或者引起Na /K -ATP酶α2亚基单个等位基因的功能丧失,或者引起钠钾泵的动力学改变。不过,ATP1A2基因与普通偏头痛的关系尚不明确,有待进一步研究。     

门冬氨酸钾对肝细胞内钾离子浓度及细胞膜Na+·K+-ATP酶活性的影响

目的 了解门冬氨酸钾对肝细胞内钾离子浓度及细胞膜Na+·K+-ATP酶活性的影响.方法 人和大鼠肝细胞株培养传代,通过CCK-8测细胞活力确定门冬氨酸钾和氯化钾分别作用于两株肝细胞的合适浓度,利用该浓度处理细胞,培养0、24和48 h后破碎细胞取上清液测定两株肝细胞内钾离子浓度,提取细胞膜定磷法测定细胞膜Na+· K+-ATP酶活性.统计学处理采用t检验,方差分析及LSD法.结果 与空白组和氯化钾组相比,门冬氨酸钾组K+进入细胞内的量明显增多(P＜0.05或P＜0.01),在24 h、48 h两个时间点,L02细胞内K+浓度比KCl组分别升高了31％和38％,比空白组分别升高了62％和73％;BRL细胞内K+浓度比KCI组分别升高了21％和40％,空白组分别升高了52％和68％.且其细胞膜Na+· K+ -ATP酶活性升高(P＜0.01),但两株细胞间均无明显差异.结论 门冬氨酸钾能促进K+进入细胞内,并提高了肝细胞膜Na+· K+-ATP酶的活性.     

Increased expression and function of the myocardial Na–K–2Cl cotransporter in failing rat hearts

Abstract Recent studies indicate a role of the Na–K–2Cl cotransporter (NKCC) in regulation of myocardial function. However, potential pathophysiological properties of NKCC in conditions like myocardial infarction (MI) and heart failure have not been explored. We investigated the cellular localization of myocardial NKCC and whether myocardial NKCC levels are changed upon induction of post-infarction heart failure in rats. Immunohistochemical analysis demonstrated extensive distribution of NKCC in normal rat myocardium with fairly strong expression in cardiomyocytes, fibroblasts, vascular endothelial cells, as well as smooth muscle cells. Myocardial mRNA levels of NKCC were investigated at 2, 7 and 28 days after induction of MI or sham operation, but no changes were found. Cardiomyocytes and non-cardiomyocytes were isolated 7 days after induction of MI or sham operation. An ∼2-fold increase of the NKCC mRNA levels was found in isolated cardiomyocytes from heart failure rats compared to that of sham-operated rats (P < 0.001), whereas a trend towards decreased mRNA levels of NKCC in isolated non-cardiomyocytes was observed. In addition, we found a bumetanide sensitive ⁸⁶Rb⁺ influx mechanism present in the hearts after induction of MI (P < 0.05). Thus, our data indicate cardiomyocyte specific increase in NKCC mRNA levels and increased NKCC activity in post-infarction heart failure. Our results may indicate a potential role of NKCC during post-infarction remodeling.     

Inhibition of Na+–H+ exchange before resuscitation following hemorrhagic shock is cardioprotective in rats

Background

Stimulation of the Na⁺–H⁺ exchanger during resuscitation following hemorrhagic shock results in myocardial injury and dysfunction. Inhibition of the Na⁺–H⁺ exchanger appears to be a new pharmacological tool for myocardial protection following ischemia–reperfusion. Our lab showed that inhibition of the Na⁺–H⁺ exchanger, using amiloride and dimethyl amiloride, before ex vivo resuscitation of isolated perfused hearts protected the myocardium and improved the post-resuscitation myocardial function. The purpose of the present study was to examine the myocardial protective effects of treating the hemorrhagic shocked rats by intra-arterial injection of 20 μM dimethyl amiloride (DMA), a specific Na⁺–H⁺ exchanger blocker, before in vivo resuscitation.

Methods

Sprague–Dawley rats were assigned to hemorrhagic treated or untreated groups (n = 4 per group). After 60 min of hemorrhagic shock, rats were treated or not by injection of 20 μM 5-(N,N-dimethyl)-amiloride (DMA) intra-arterially. Rats were then resuscitated in vivo and monitored for 30 min. Then hearts were harvested and perfused in the Langendorff system for 60 min for measurements of hemodynamic function.

Results

Administration of DMA before in vivo resuscitation following 60 min of hemorrhagic shock and 30 min of in vivo resuscitation, 20 μM DMA intra-arterially significantly improved post-resuscitation myocardial function.

Conclusion

Our results suggest that DMA protects the heart against post-resuscitation myocardial injury.     

充血性心衰患者尿Na+/K+比值和螺内酯应用的关系

目的：探讨尿Na^ /K^ 比值作为应用醋固酮拮抗剂螺内酯指标的可行性。方法：检测41例心功能Ⅲ-Ⅳ级病人的尿Na^ /K^ 比值，按比值的不同（＜1．0或＞1．0）分两组，比较两组的治疗效果。结果：应用螺内酯后尿Na^ /K^ ＜1．0受益大。结论：尿Na^ /K^ ＜1．0，可以作为使用螺内酯治疗心力衰竭的指标。     

低浓度强心甙对豚鼠心室肌细胞Na+/K+泵电流的激活作用

目的 :解释治疗水平 （低浓度 ）强心甙的正性肌力作用。方法 :用全细胞膜片钳技术 ,在分离的豚鼠心室肌细胞观察到双氢哇巴因 （DHO）对 Na+/ K+泵电流 （Ip ）的激活作用。结果 :DHO在 10 - 8～ 10 - 1 0 m ol· L- 1范围内可增加外向电流。当从细胞外液去除 K+或从电极内液去除 Na+或胞内应用 vanadate抑制了 Ip后 ,外向电流消失。结论 :DHO可使 Na+/ K+泵兴奋 ,产生了外向泵电流。Ip激活是低浓度强心甙对心脏 Na+/ K+泵的直接作用。     

甲状腺激素对大鼠心肌Na+,K+-ATP酶α1亚基表达的影响

目的探讨不同甲状腺激素（TH）水平对大鼠心肌Na^＋，K^＋-ATP酶（钠泵）α1亚基mRNA表达的影响。方法Wistar大鼠随机分为甲状腺功能减退（简称甲减）组和对照组，分别摄入含碘量为50、300mg／kg的饲料，并分别饮用去离子水和自来水，喂养24周后放射免疫法测定血清TH水平，RT—PCR法测定心肌组织钠泵α1亚基mRNA的表达水平。结果甲减组血清TH水平明显低于对照组（P〈0．01）；心肌钠泵α1亚基mRNA表达水平，甲减组（0．59±0．51）与对照组（0．97±0．27）相比明显降低，差异有统计学意义（t=2．57，P〈0．05）。结论低碘饮食可以诱发大鼠甲状腺功能减退，低TH水平导致心肌钠泵α1亚基mRNA表达下降。     

Evidence for a Na+–H+ Exchange Across Human Colonic Basolateral Plasma Membranes Purified from Organ Donor Colons

The mechanism(s) of electrolyte transport across the human colonic contraluminal domain is not well understood. Current studies were undertaken to develop a technique for the isolation and purification of the human colonic basolateral membrane vesicles (BLMV) and to examine the presence of a Na⁺–H⁺ exchange process in these membranes. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll density gradient centrifugation technique, and Na⁺ transport was examined utilizing a rapid filtration, technique. Our data demonstrate that purified basolateral membranes were enriched 10- to 11-fold in Na⁺, K⁺-ATPase activity compared to crude homogenate. Results consistent with the Na⁺–H⁺ exchange in BLMV are as follows: (1) an outwardly directed H⁺ gradient stimulated ²²Na uptake; (2) ²²Na uptake was markedly inhibited by EIPA and amiloride; (3) H⁺-gradient-stimulated ²²Na uptake was not inhibited by bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; (4) ²²Na uptake was voltage insensitive; (5) ²²Na uptake demonstrated saturation kinetics; (6) ²²Na uptake was markedly inhibited by Na⁺ and Li⁺ but was unaffected by N-methyl glucamine⁺, choline⁺, and NH₄ ⁺. Immunoblotting studies demonstrated this Na⁺–H⁺ exchanger isoform to be represented by NHE1. In conclusion, a technique has been established for the purification of functional human proximal colonic BLMV, and an electroneutral Na⁺–H⁺ exchange process has been demonstrated in these membranes.