Regulation of the Na+2Cl–K+ cotransporter in isolated rat colon crypts

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.     

Na+–K+ pump location and translocation during muscle contraction in rat skeletal muscle

Muscle contraction may up-regulate the number of Na(+)-K(+) pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na(+)-K(+) pump alpha(2) subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the alpha(2) subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na(+)-K(+) pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the alpha(1) subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the alpha(2), the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the alpha subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.     

Large Na+ influx and high Na+, K+–ATPase activity in mitochondria-rich epithelial cells of the inner ear endolymphatic sac

Fluid in the mammalian endolymphatic sac (ES) is connected to the endolymph in the cochlea and the vestibule. Since the dominant ion in the ES is Na(+), it has been postulated that Na(+) transport is essential for regulating the endolymph pressure. This study focused on the cellular mechanism of Na(+) transport in ES epithelial cells. To evaluate the Na(+) transport capability of the ES epithelial cells, changes in intracellular Na(+) concentration ([Na(+)](i)) of individual ES cells were measured with sodium-binding benzofurzan isophthalate in a freshly dissected ES sheet and in dissociated ES cells in response to either the K(+)-free or ouabain-containing solution. Analysis of the [Na(+)](i) changes by the Na(+) load and mitochondrial staining with rhodamine 123 showed that the ES cells were classified into two groups; one exhibited an intensive [Na(+)](i) increase, higher Na(+), K(+)-ATPase activity, and intensive mitochondrial staining (mitochondria-rich cells), and the other exhibited a moderate [Na(+)](i) increase, lower Na(+), K(+)-ATPase activity, and moderate mitochondrial staining (filament-rich cells). These results suggest that mitochondria-rich ES epithelial cells (ca. 30% of ES cells) endowed with high Na(+) permeability and Na(+), K(+)-ATPase activity potentially contribute to the transport of Na(+) outside of the endolymphatic sac.     

Differences in the protein-kinase-A-dependent regulation of CFTR Cl– channels and Na+–K+ pumps in guinea-pig ventricular myocytes

Protein-kinase-A- (PKA-) dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current (I(CFTR)) and Na+-K+ pump current (Ip) was studied in single guinea-pig ventricular myocytes. Both currents were measured simultaneously by means of whole-cell recording at 30 degrees C. The adenylyl cyclase activator forskolin was used to stimulate PKA activity. At -20 mV, forskolin (4 microM) induced a fast activation of I(CFTR) and a delayed stimulation of Ip. Despite the strikingly different time courses, however, the potency of the drug to regulate both currents was identical. Half-maximal activation of I(CFTR) and stimulation of Ip, respectively, were observed at 9.6 x 10(-8) M and 9.9 x 10(-8) M forskolin. Inclusion of a specific peptide inhibitor of PKA in the pipette solution (PKI, 20 microM) blocked forskolin's effect on Ip. However, regardless of the time allowed for cell dialysis, there still was a marked, transient activation of I(CFTR), which could be prevented by: (1) a short pre-activation of I(CFTR) with forskolin or (2) the additional inclusion in the pipette solution of a synthetic peptide (Ht31 peptide, 60 microM) that interferes with PKA binding to its anchoring proteins. Thus, there is a tight functional coupling between PKA and CFTR Cl- channels in guinea-pig ventricular myocytes. The coupling is probably due to the close physical proximity of channels and kinases mediated by PKA anchoring proteins. Na+-K+ pumps, on the other hand, though also regulated by PKA, appear to be loosely coupled to the kinases.     

The Na+2Cl–K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage

Effects of cAMP on Cl^– secretion, intracellular Cl^– activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (I _sc) showed that cAMP stimulates Na⁺Cl^– secretion in a biphasic manner. The first and rapid phase corresponds to Cl^– exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl^– conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1–2 min) activation of the Na⁺2Cl^–K⁺ cotransporter. During the first phase cytosolic Cl^– activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of I _sc activation. When cytosolic Cl^– activity was increased by exposing the RGT to a high K⁺ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na⁺2Cl^–K⁺ cotransport is transient cell shrinkage. Received: 11 February 1999 / Accepted: 24 March 1999     

Na+,K+-ATP酶信号转导功能的分子机制

近年来研究表明,Na+,K+-ATP酶不仅能主动跨膜转运钠钾离子,还具有信号转导功能。Na+,K+-ATP酶作为受体与其配体（如强心类甾醇）结合后,可激活细胞内多种信号转导通路,从而参与心肌、平滑肌等正常细胞肥大、增殖,肿瘤细胞凋亡等多种生理、病理过程。对Na+,K+-ATP酶信号转导功能的深入研究,可能为心血管疾病、肿瘤等的治疗提供新思路。     

Na+-independent proton secretion in MDCK–C11 cells

In this work we studied the proton secretion mechanisms in recently cloned MDCK-C11 cells. We measured intracellular pH (pHi) in monolayers grown on permeable filters, using the pH-sensitive probe BCECF and an inverted epifluorescence microscope. The cells have a basal pHi of 7.20+/-0.01 (n=136) and after an acid-releasing NH4Cl pulse pHi recovered at a rate (dpHi/dt) of 0.167+/-0.006 pH units/ per minute (n=20). This rate decreased significantly when Na+ was removed from both cell surfaces, and was further reduced when they were both perfused with a solution containing no Na+ and K+. pHi recovery fell again in the presence of concanamycin (at a concentration of 4.6x10(-8) M; a specific inhibitor of the vacuolar H+-ATPase). When Na+ was removed from the apical or the basolateral side, pHi recovery (in pH units per minute) was significantly reduced to 0.099+/-0.008 (n=11) and 0.086+/-0.01 (n=10), respectively. The Na+-independent mechanism of pHi recovery was significantly inhibited by the presence of 5 x 10(-5) M Schering 28080 (an inhibitor of the H+-K+-ATPase) at the apical side (0.065+/-0.01 versus 0.099+/-0.008 pH units per minute, P<0.05), but not at the basolateral side (0.072+/-0.01 versus 0.086+/-0.01 pH units per minute). On the other hand, concanamycin inhibited the Na+-independent pHi recovery when applied apically (0.0304+/-0.005 pH units per minute, n=7) and basolaterally (0.025+/-0.004 pH units per minute, n=7). From these results we conclude that monolayers of MDCK-C11 cells have a Na+/H+ exchanger and a concanamycin-sensitive H+-ATPase on their apical and basolateral membranes; and a K+-dependent, Schering 28080-sensitive H+-K+-ATPase on their apical side.     

两种电解质稀释液对干化学测定尿K+、Na+结果的影响

目的比较两种尿电解质稀释液对干化学电极电位法测定尿K+、Na+结果的差异.方法分别用去离子水和醋酸镁尿电解质稀释液作稀释,采用电极电位法,检测 50例儿童新鲜尿液标本的K+、Na+含量.结果两种尿电解质稀释液,对尿Na+结果的测定差别无统计学意义(p＞0.05).而对尿K+结果的测定差别有统计学意义(p＜0.05).结论用干化学电极电位法测定尿K+、Na+时,选用醋酸镁尿电解质稀释液,所得的结果更真实地反映K+ 、Na+的含量.     

消化道出血患者大量输血后PT、FIB、Na+、K+指标观察

目的 探讨消化道出血患者大量输血后PI、FIB、Na+、K+等指标变化.方法 以2014年1月至2016年1月我院收治的150例消化道出血需进行输血的患者为研究对象.分别抽取输血前、输血后1、3d及5d四个时间段4 mL血,检测患者的PT(凝血酶原时间)、FIB(纤维蛋白原)、Na+、K+等指标的变化.研究短期内大量输血患者血清电解质凝血指标变化.结果 K+在输血后1d较输血前明显升高;输血后3d较输血后1d降低;输血后3d与输血前差异无统计学意义(P＞0.05);输血后5d较输血后1d明显降低,差异均具有统计学意义(P＜0.05);发生高钾血症比例为18.00％;与输血前相比,输血后1d PT显著延长(P＜0.05),FIB显著降低(P＜0.05);输血后3、5d PT、FIB恢复至输血前水平.结论 大量输血是救治消化道出血患者的主要手段,但同时对K+浓度、凝血功能和纤溶系统短期内有较大影响,须及时监测电解质和凝血功能指标变化,及早发现可能的凝血功能障碍,提高输血疗效.     

Der Einfluß von Na+ auf K+- und Ca++-Transporte der isolierten perfundierten Amphibienniere

Zusammenfassung Zur Analyse der Frage, ob für eine tubuläre K⁺-Sekretion — im Sinne der Flußrichtung oder eines aktiven Transportes — Na⁺ essentiell ist, werden Versuche an isolierten künstlich perfundierten Nieren von Rana ridibunda durchgeführt.Dabei wird in der aortalen Perfusionsflüssigkeit, die in die Glomerula gelangt und das Glomerulumfiltrat liefert, Na⁺ steigend durch isosmotische Konzentration von Mannit und Raffinose ersetzt, K⁺ befindet sich — außer in der Kontrollreihe — nicht im Perfusat. In der renoportalen Perfusionsflüssigkeit wird Na⁺ stets durch isosmotische Mannit- oder Raffinosekonzentrationen ersetzt, die K⁺-Konzentration beträgt gleichmäßig 2,75 mMol/l.Gleichgültig, ob Na⁺ durch Mannit oder Raffinose ersetzt wurde, gilt: Bei renoportaler K⁺-Zufuhr ohne Na⁺ in der aortalen Perfusionsflüssigkeit wird K⁺ geringgradig ausgeschieden. Dieses K⁺ entstammt, wie Kontrollversuche mit K⁺-freien Lösungen zeigen, dem Nierengewebe, das unter den vorliegenden Versuchsbedingungen K⁺ verliert. Mit Steigerung der luminalen Na⁺-Konzentration von 2,75 bis 76,0 mMol/l steigt die Menge ausgeschiedenen K⁺ erst schnell, dann langsamer. Demnach erweist sich Na⁺ für den transcellulären K⁺-Transport im Sinne der Sekretion als essentiell, ein Na⁺/K⁺-Austausch in einem molaren 1:1-Verhältnis findet nicht statt.

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To critically study the question as to whether Na⁺ is essential for tubular K⁺ secretion, in the sense of flow direction or active transport, experiments were carried out on isolated and perfused kidneys of Rana ridibunda.In the aortal perfusion fluid reaching the glomeruli and supplying the glomerular filtrate, Na⁺ was increasingly replaced by isoosmotic concentrations of mannitol and raffinose; except in the control series, K⁺ was not present in the perfusate. In the reniportal perfusion fluid, Na⁺ was invariably replaced by isoosmotic mannitol or raffinose concentrations; the K⁺ concentration was kept constant with 2.75 mM/l.Regardless of whether Na⁺ is replaced by mannitol or raffinose, it holds true that with the reniportal supply of K⁺ without Na⁺ in the aortal perfusion fluid, K⁺ is excreted only in a trifling degree. As control experiments showed, with K⁺-free solutions, this K⁺ arises from the renal tissues which lose it under the experimental conditions described. By increasing the luminal Na⁺ concentration from 2.75 to 76.0 mM/l, the excretion of K⁺ is at first rapid and then slows down. Accordingly, Na⁺ proved essential in the secretory sense for transcellular K⁺ transport. A Na⁺/K⁺ interchange in a 1:1 molar ratio does not occur.

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Herrn Prof. Dr. med. Max Schneider, Direktor des Instituts für normale und pathologische Physiologie der Universität Köln, zum 60. Geburtstag in Dankbarkeit und Verehrung zugeeignet.     

Long-term modulation of Na+ and K+ channels by TGF-��1 in neonatal rat cardiac myocytes

Previous work shows that transforming growth factor-β1 (TGF-β1) promotes several heart alterations, including atrial fibrillation (AF). In this work, we hypothesized that these effects might be associated with a potential modulation of Na(+) and K(+) channels. Atrial myocytes were cultured 1-2?days under either control conditions, or the presence of TGF-β1. Subsequently, Na(+) (I(Na)) and K(+) (I(K)) currents were investigated under whole-cell patch-clamp conditions. Three K(+) currents were isolated: inward rectifier (I(Kin)), outward transitory (I(to)), and outward sustained (I(Ksus)). Interestingly, TGF-β1 decreased (50%) the densities of I(Kin) and I(Ksus) but not of I(to). In addition, the growth factor reduced by 80% the amount of I(Na) available at -80?mV. This effect was due to a significant reduction (30%) in the maximum I(Na) recruited at very negative potentials or I(max), as well as to an increased fraction of inactivated Na(+) channels. The latter effect was, in turn, associated to a -7?mV shift in V(1/2) of inactivation. TGF-β1 also reduced by 60% the maximum amount of intramembrane charge movement of Na(+) channels or Q(max), but did not affect the corresponding voltage dependence of activation. This suggests that TGF-β1 promotes loss of Na(+) channels from the plasma membrane. Moreover, TGF-β1 also reduced (50%) the expression of the principal subunit of Na(+) channels, as indicated by western blot analysis. Thus, TGF-β1 inhibits the expression of Na(+) channels, as well as the activity of K(+) channels that give rise to I(Ksus) and I(Kin). These results may contribute to explaining the previously observed proarrhythmic effects of TGF-β1.     

K+-induced swelling of vestibular dark cells is dependent on Na+ and Cl− and inhibited by piretanide

Epithelial cell height was measured in order to estimate the cell volume of dark cells from the ampullae of the semicircular canal of the gerbil. Under control conditions, addition of 10^–4 mol/l piretanide, 10^–5 mol/l 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB), 5 mmol/l barium or 10^–3 mol/l quinidine had no significant effect on cell height. Addition of 10^–4 mol/l NPPB or 10^–3 mol/l ouabain led to a small significant decrease in cell height which was not reversible. Substitution of Na⁺ by N-methyl-d-glucamine or of Cl^– by gluconate led to a significant and reversible reduction in cell height. Isotonic elevation of [K⁺] from 3.6 to 25 mmol/l in a PO₄-buffered, HCO₃-free solution led to an increase in cell height from 5.8±0.1 (SEM) to 8.7±0.2 (n= 62) during the first 40 s. During prolonged exposure to elevated [K⁺] (3–5 min; n=19), some tissue samples underwent a regulatory volume decrease. K⁺-induced swelling was absent in both isotonic Cl^– -free and isotonic Na⁺-free solutions and was inhibited by the loop diuretic piretanide (10^–5 and 10^–4 mol/l) or by the (Na⁺+ K⁺) ATPase inhibitor ouabain (10^–3 mol/l) or by 10^–4mol/l NPPB. After the removal of ouabain or 10^–4 mol/l NPPB, K⁺-induced swelling under control conditions was enhanced and was less reversible as compared to control conditions before the experiment. K⁺-induced swelling was not altered by NPPB (10^–5 mol/l) or barium (5 mmol/l); however, barium slowed shrinking upon return of [K⁺] to control level. In the presence of 10^–3 mol/l quinidine, K⁺-induced swelling was enhanced and not reversible. These data suggest that dark cells from the semicircular canal possess an Na⁺2Cl^–K⁺ cotransporter as a solute uptake mechanism and a solute efflux mechanism which is sensitive to barium and inhibited by quinidine.     

Characterization of the relationship between Na+–Ca2+ exchange rate and cytosolic calcium in trout cardiac myocytes

The whole-cell patch-clamp technique combined with rapid caffeine (CAF) applications was used to measure Na+-Ca2+ exchange (NCX) currents (I(NCX)). The rate of Ca2+ extrusion and the amount of Ca2+ extruded from the cell upon a rapid CAF exposure were obtained from I(NCX) and its time integral, respectively. This gave a maximal NCX rate (V(NCX)) of 151 amol pF(-1) s(-1) or 2.3 mM s(-1) and a half-maximal V(NCX) (K0.5) at a total cellular [Ca2+] ([Ca2+]tot) of 15.4 amol pF(-1). Using the same approach for the tail current induced by repolarization to -80 mV gave a K0.5 of 7.0 amol pF(-1) corresponding to 108 microM total or 2-4 microM free Ca2+. The relationship between [Ca2+]tot and V(NCX) was linear in the physiological range. Inhibition of the SR function with cyclopiazonic acid plus ryanodine reduced the slope significantly from 23.2+/-1.4 to 17.6+/-1.6 s(-1), while ryanodine alone had no effect. The relationship between [Ca2+]tot and V(NCX) was steeper at more negative membrane potentials, and with identical SR Ca2+ loads the maximal VNCX at -10 mV was reduced to 39.7+/-2.7% of the value at -90 mV. Long depolarizations caused SR Ca2+ loading through reverse-mode NCX. Between -30 and +10 mV reverse mode V(NCX)=Vm.0.047 amol pF(-1) s(-1) mV(-1)+2.51 amol pF(-1) s(-1), giving a reversal potential of -54 mV. In conclusion, the relationship between V(NCX) and [Ca2+]tot shows that the NCX is capable of removing a total Ca2+ transient of 60 microM at physiological heart rates, while reverse-mode NCX reloads the sarcoplasmic reticulum (SR) during depolarization. Furthermore, small alterations in the action potential configuration are predicted to change significantly the relative importance of the NCX in the regulation of cytosolic [Ca2+] and SR Ca2+ loading.     

地高辛抑制豚鼠心室肌细胞Na+，K+-ATP酶和人胚肾上皮细胞上HERG钾通道电流

强心苷类药对Na+,K+-ATP酶的作用在低浓度时为兴奋、在高浓度时呈抑制作用[1]。应用膜片钳技术观察地高辛(Digoxin)对豚鼠心室肌细胞Na+,K+-ATP酶电流(Ip)的作用还未见报道。此外,由于很多药物的心脏不良反应是通过抑制人ether-a-go-go-related基因(HERG)编码的快速激活的延迟整流钾通道(IKr)而引起QT间期延长和尖端扭转性室     

PPARγ agonists do not directly enhance basal or insulin-stimulated Na+ transport via the epithelial Na+ channel

Selective agonists of peroxisome proliferator-activated receptor gamma (PPAR) are anti-diabetic drugs that enhance cellular responsiveness to insulin. However, in some patients, fluid retention, plasma volume expansion, and edema have been observed. It is well established that insulin regulates Na⁺ reabsorption via the epithelial sodium channel (ENaC) located in the distal tubule. Therefore, we hypothesized that these agonists may positively modulate insulin-stimulated ENaC activity leading to increased Na⁺ reabsorption and fluid retention. Using electrophysiological techniques, dose–response curves for insulin-mediated Na⁺ transport in the A6, M-1, and mpkCCD_cl4 cell lines were performed. Each line demonstrated hormone efficacy within physiological concentration ranges and, therefore, can be used to monitor clinically relevant effects of pharmacological agents which may affect electrolyte transport. Immunodetection and quantitative PCR analyses showed that each cell line expresses viable and functional PPAR receptors. Despite this finding, two PPAR agonists, pioglitazone and GW7845 did not directly enhance basal or insulin-stimulated Na⁺ flux via ENaC, as shown by electrophysiological methodologies. These studies provide important results, which eliminate insulin-mediated ENaC activation as a candidate mechanism underlying the fluid retention observed with PPAR agonist use.