柴胡皂甙和甘草甜素抑制Na+,K+-ATP酶活性的构效关系

研究在离体条件下各种单体柴胡皂甙和甘草甜素抑制Na⁺,K⁺-ATP酶活性的构效关系。实验结果表明,各种柴胡皂甙抑制Na⁺,K⁺-ATP酶活性的作用强度依次为:b₁>d>b₂>b₄>a>b₃>e>c。柴胡皂甙化学结构中的C₂₃-OH,C₁₆-OH及C₁₁和C₁₃的共轭双烯可能对其抑制活性起重要作用。甘草甜素(GL),甘草次酸(GA)和生胃酮(18-β-甘草次酸半琥珀酸双钠盐,CX)抑制Na⁺,K⁺-ATP酶活性的作用强度依次为GA≥CX>GL。研究还证明,柴胡皂甙d对Na⁺,K⁺-ATP酶的抑制为非竟争性抑制。     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

α-青心酮对损伤的脑线粒体Na+,K+-ATPase活性和脑细胞耗氧的作用

目的研究α-青心酮对抗坏血酸和硫酸亚铁诱导脑线粒体Na⁺，K⁺-ATPase活性和脑细胞耗氧的作用。方法采用无机磷法测定Na⁺，K⁺-ATPase活性,分光光度法检测脑线粒体膨胀和脂质过氧化物，氧电极法测定脑细胞耗氧量。结果在抗坏血酸和硫酸亚铁的作用下，鼠脑线粒体Na⁺，K⁺-ATPase活性降低，线粒体膨胀和脑细胞脂质过氧化物升高。α-青心酮抑制其抗坏血酸和硫酸亚铁诱导脑线粒体和细胞的损伤,增加Na⁺，K⁺-ATPase活性，降低脑线粒体膨胀和脑细胞脂质过氧化物生成。α-青心酮还具有减少ADP刺激的脑细胞耗氧的作用。结论α-青心酮通过清除自由基和抗氧化作用保护脑细胞结构和功能的完整。     

KB-R7943对豚鼠心室肌细胞Na+-Ca2+交换电流的作用

目的　观察KB-R7943对豚鼠心室肌细胞Na⁺-Ca²⁺交换电流(I_Na-Ca)的内向电流成分和外向电流成分的影响。方法　采用缺血再灌时胞内Na⁺超载的细胞模型,在同时记录内向、外向电流的双向离子条件下,用膜片钳全细胞技术,记录I_Na-Ca的电流-电压关系曲线。结果　10^-6和10^-5mol·L^-1KB-R7943,在+50mV时,对I_Na-Ca的抑制率分别是29.4%和61.7%;在-80mV时抑制率分别是22.1%和56.9%。结论　KB-R7943对豚鼠心室肌细胞I_Na-Ca有抑制作用,但对外向成分和内向成分的抑制不具选择性。     

CD4+CD25+调节性T淋巴细胞在儿童传染性单核细胞增多症的变化及作用

目的 观察儿童传染性单核细胞增多症（IM）治疗前后外周血T淋巴细胞亚群和CD4⁺CD25⁺调节性T淋巴细胞（Treg细胞）比例变化,分析Treg对IM的影响。方法 选取2017年1月至2018年5月安徽医科大学第二附属医院儿科收治的IM患儿60例作为观察组,以同期于本院儿童保健门诊行体检的40例健康儿童作为对照组,采用流式细胞术检测观察组治疗前、后及对照组的外周血T细胞亚群（CD3⁺T细胞、CD3⁺CD4⁺T细胞、CD3⁺CD8⁺T细胞比例,CD4/CD8比值）、CD4⁺CD25⁺调节性T淋巴细胞比例,对比分析两组患者机体细胞免疫功能的变化。结果 观察组治疗前CD3⁺T细胞比例、CD3⁺CD8⁺T细胞比例显著高于对照组;CD3⁺CD4⁺T细胞比例、CD4/CD8比值低于对照组,差异均有统计学意义（P <0.05）。观察组治疗后CD3⁺T细胞比例、CD3⁺CD8⁺T细胞比例均低于观察组治疗前;CD3⁺CD4⁺T细胞比例、CD4/CD8比值高于治疗前,差异均有统计学意义（P <0.05）。观察组治疗前外周血Treg细胞比例低于正常对照组,差异有统计学意义（P <0.05）;而观察组治疗后外周血Treg细胞比例高于治疗前,差异有统计学意义（P <0.05）。结论 IM患儿存在T细胞亚群比例的变化,Treg细胞水平的降低,可能参与IM患儿疾病的发生发展过程。     

前胡香豆素对肾型高血压大鼠左室肥厚及心肌胞内钙、Na+,K+-ATP酶和Ca2+,Mg2+-ATP酶活性的影响

目的研究前胡香豆素组分对肾型高血压左室肥厚的预防和逆转作用及机制。方法用两肾一夹肾型高血压左室肥厚大鼠(RHR)模型,测定前胡香豆素组分对其血压、左室湿重、心肌细胞面积、胞内静息钙及胞膜和线粒体ATP酶活性的影响。结果前胡香豆素组分(30 mg·kg^-1·d^-1,ig)预防组及逆转组大鼠血压、左室湿重/体重均较肥厚组明显降低;左室心肌细胞面积、胞内静息钙均较肥厚组降低;对KCl致钙浓度升高亦明显低于肥厚组;两组均可增加心肌细胞膜及线粒体Na⁺,K⁺-ATP酶和Ca²⁺,Mg²⁺-ATP酶活性。结论前胡香豆素组分可预防及逆转RHR左室肥厚,减少心肌细胞内钙含量,增加ATP酶活性。     

儿童过敏性鼻炎治疗前后外周血CD4+CD25+CD127lo/-Treg细胞与炎症因子变化分析

目的 探讨儿童过敏性鼻炎（AR）治疗前后外周血CD4⁺CD25⁺CD127^lo/-Treg及白细胞介素2（IL-2）、肿瘤坏死因子α（TNF-α）、人转化生长因子β1（TGF-β1）的水平变化及临床意义。方法 将厦门长庚医院收治的108例AR患儿作为AR组,根据疾病严重程度分为轻度、中重度组,另采用单纯随机抽样法选取年龄、性别匹配的80例健康儿童作为对照组,检测两组对象入院时CD4⁺CD25⁺CD127^lo/-Treg、IL-2、TNF-α、TGF-β1水平;AR患者均给予对症治疗,检测治疗前、治疗1个月、治疗6个月CD4⁺CD25⁺CD127^lo/-Treg、IL-2、TNF-α、TGF-β1水平,采用Pearson相关分析疾病严重程度与外周血CD4⁺CD25⁺CD127^lo/-Treg的相关性及两者与IL-2、TNF-α、TGF-β1相关性。结果 AR患者CD4⁺CD25⁺CD127^lo/-Treg、IL-2、TGF-β1水平分别为（6.264±0.135）%、（50.16±11.67）ng/L、（14.21±5.34）ng/L,均低于对照组;TNF-α为（1.82±0.62）ng/L,高于对照组的（0.34±0.19）ng/L,差异均有统计学意义（P<0.05）。轻度AR患者CD4⁺CD25⁺CD127^lo/-Treg、IL-2、TGF-β1水平分别为（6.305±0.137）%、（59.34±13.05）ng/L、（16.97±4.69）ng/L,均高于中重度AR组患者,TNF-α水平为（1.64±0.57）ng/L,低于中重度AR组患者的（1.93±0.65）ng/L,差异均有统计学意义（P<0.05）。AR组治疗后第1、6个月CD4⁺CD25⁺CD127^lo/-Treg、IL-2、TGF-β1水平高于治疗前,TNF-α水平低于治疗前,差异均有统计学意义（P<0.05）。Pearson相关分析显示：外周血CD4⁺CD25⁺CD127^lo/-Treg、TNF-α与疾病严重程度呈正相关关系（r=0.681、0.581,P<0.05）,IL-2、TGF-β1与疾病严重程度呈负相关关系（r=-0.613、-0.579,P<0.05）;外周血CD4⁺CD25⁺CD127^lo/-Treg与IL-2、TGF-β1呈负相关性（r=-0.675、-0.691,P<0.05）,与TNF-α呈正相关关系（r=0.618,P<0.05）。结论 AR患者存在CD4⁺CD25⁺CD127^lo/-Treg及IL-2、TGF-β1下降,TNF-α上升,且与病情程度有关,治疗后均明显改善。     

芪参胶囊对大鼠局灶性脑缺血再灌注损伤的保护作用

目的研究芪参胶囊对大鼠局灶性脑缺血再灌注损伤的保护作用。方法用线栓法建立大鼠局灶性脑缺血模型,大鼠于缺血1h再灌注2h断头取脑,检测大脑组织Ca²⁺]-ATPase,Na⁺],K⁺]-ATPase,NOS活性和NO,水的含量及大脑皮层神经元内游离钙离子浓度,行为学评价及梗死面积,常规石蜡切片,HE染色,作病理学检查。结果芪参胶囊显著降低缺血再灌注后大脑皮层神经元内游离钙离子浓度及大脑组织NOS活性,NO的含量和水肿程度及梗死面积;显著增强Ca²⁺]-ATPase,Na⁺],K⁺]-ATPase的活性;病理学检查显示芪参胶囊能明显减轻脑水肿及神经元坏死程度。结论芪参胶囊对大鼠局灶性脑缺血再灌注损伤有明显保护作用。     

一种新的钠-钙交换电流记录方法及阿米洛利的作用

目的：研究胞外不同Ca²⁺浓度对豚鼠心室肌细胞钠-钙交换电流(Na⁺-Ca²⁺ exchange current, INa-Ca)的影响和阿米洛利(amiloride)对该电流的作用。方法：建立缺血再灌时胞内Na⁺超载的细胞模型,用膜片钳全细胞技术,记录I_Na-Ca的电流-电压关系曲线。结果：阿米洛利10^-5,3×10^-5和10^-4 mol.L^-1,在+50 mV时,对I_Na-Ca的抑制率分别是15.4%,22.6%和40.9%;在-80 mV时抑制率分别是5.6%,14.6%和23.2%。结论：胞内Na⁺超载确可引起Na⁺-Ca²⁺交换系统激活;阿米洛利对豚鼠心室肌细胞I_Na-Ca有抑制作用,且对I_Na-Ca外向成分的抑制作用大于对内向成分的抑制作用。     

人参二醇皂甙和三醇皂甙对兔纹状体ATP酶的影响

本文报道用体外给药法,观察了PDS和PTS对纹状体ATP酶(Na⁺、K⁺-ATP酶,Ca²⁺-ATP酶及M²⁺-ATP酶)的影响。结果发现PDS和PTS对Na⁺,K⁺-ATP酶都有明显的抑制作用,且随PDS和PTS浓度的高低,其抑制作用增强或减弱;对Ca²⁺-ATP酶,PDS在10^-5g/ml时有激活作用,当浓度增高到10^-3g/mL时则转为抑制,而PTS仅为抑制效应;对于Mg²⁺-ATP酶能被PDS所兴奋,而被PTS所抑制。此结果表明PDS和PTS对中枢神经系统的作用,可能与其影响脑内ATP酶有密切的内在联系。     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

The effect of diuretics of Na+-K+-ATPase and c-AMP levels in toad bladder epithelial cells

Summary Na⁺-K⁺-ATPase was inhibited by 1x10^–4 M ethacrynic acid and mercuderamide, and by 1x10^–3 M hydrochlorothiazide and furosemide. A modification of Gilman's (1970) protein displacement assay has been used to measure c-AMP levels in toad bladder epithelial cells. Vasopressin (50 mU/ml) caused c-AMP levels to rise from 4.27 to 9.27 pmol/mg protein. Ethacrynic acid had no effect on cellular c-AMP levels after 10 min exposure to the drug, but at 90 min caused a reduction of both basal and vasopressin stimulated levels. Furosemide caused an apparent rise in c-AMP levels, dilution ratio measurements indicated interference by this drug in the assay procedure, mecuderamide also caused substantial interference with the c-AMP assay. Hydrochlorothiazide had no effect on basal or hormone stimulated levels of c-AMP.It was concluded that the inhibition of sodium transport produced by ethacrynic acid in toad bladder is probably due to inhibition of adenylate cyclase, an effect not shared by other diuretics.     

Effect of the flame retardant tris (1,3-dichloro-2-propyl) phosphate (TDCPP) on Na+-K+-ATPase and Cl− transport in HeLa cells

Tris (1, 3-dichloro-2-propyl) phosphate (TDCPP) is one of the most diffused phosphorus flame retardants in the environment and is highly persistent and abundant in residential dust samples. To date the cellular targets and mechanisms underlying its toxic effects are not completely understood. The aim of this work was to study the effects of TDCPP on ion transport mechanisms fundamental for the cellular ionic homeostasis, such as Na⁺-K⁺-ATPase and Cl^? transport. HeLa cells were used as experimental model. TDCPP showed a dose-dependent effect on cell viability in cells exposed for 24?h as assessed by MTT test (IC₅₀?=?52.5?µM). The flame retardant was able to exert a dose and time-dependent inhibition on the Na⁺-K⁺-ATPase activity. A short-term exposure (1?h) was able to exert a significant inhibition at 75 and 100?µM TDCPP, suggesting that TDCPP is able to directly interfere with the Na⁺-K⁺-ATPase phosphate catalytic activity. The sensitivity of the pump to lower TDCPP concentrations increased with the increase of the time of exposure. Following 24?h exposure a significant inhibition of about 40% was evident already at 10?µM and the IC₅₀ value observed was 12.8?±?6.0?µM. Moreover, TDCPP was also able to impair the NKCC mediated Cl^? transport in HeLa cells, as assessed in YFP-H148Q/I152L-expressing HeLa cells. Following 1?h exposure TDCPP significantly inhibited the transport by about 30%. The kinetic analysis demonstrated a noncompetitive mechanism of inhibition. In conclusion, results demonstrated the impairment of ion transport mechanisms fundamental for ion homeostasis by TDCPP on HeLa cells.     

Die Haftung verschiedener Cardenolide am Papillarmuskel und einer mikrosomalen ATPase des Meerschweinchenherzens

Summary In experiments on isolated electrically stimulated guinea pig papillary muscles and on isolated cardiac Na⁺-K⁺-activated ATPase preparations the action and the reversibility of action of 3 different cardenolides-digitoxin, k-strophanthidin and strophanthidin-3-bromoacetate (SBA) (supposed to be an irreversible inhibitor of the transport ATPase)-were studied.The equieffective concentrations for maximum positive inotropic effects (around 90%) were 2×10^–6, 2×10^–5 and 4×10^–5 M, respectively. In washout experiments the positive inotropic action of all these substances was found to be completely reversible: the rates of decline of the positive inotropic effects were about 2.7%/min with digitoxin, 24%/min with strophanthidin and 22%/min respectivety 5.7%/0/min (two components) with SBA.The equieffective concentrations for maximum inhibition (90–95%) of the Na⁺-K⁺-activated ATPase by digitoxin, strophanthidin and SBA were 10^–4, 2×10^–4 and 10^–4 M respectively. In washout experiments (repeated centrifugations) different degrees of reversibility of these inhibitory effects were observed depending upon the experimental conditions. Preincubation of the enzyme with the cardenolides in theabsence of Na⁺, Mg²⁺ and ATP resulted in a persisting inhibition of the Na⁺-K⁺-ATPase of 14% with digitoxin, 10% with k-strophanthidin and- significantly higher (p < 0.05)-33% with SBA. Corresponding experiments with preincubation of the enzyme in thepresence of Na⁺, Mg²⁺ and ATP, however, demonstrated a full reversibility of the inhibitory action of all these substances.These results are in contrast, in certain respects, with those obtained in previous experiments on brain ATPase.It is concluded that SBA is able to inhibit irreversibly only the non-phosphorylated form of the cardiac Na⁺-K⁺-activated ATPase, whereas the phosphorylated intermediate of this enzyme seems to be protected against the irreversible inhibition by this substance. Assuming that the latter state of the enzyme is predominant in the intact heart muscle cell, a complete reversibility of the pharmacological action of SBA would be expected if the inotropic effect is mediated by an inhibition of the enzyme. Our results are compatible with this hypothesis.

Wir danken der Deutschen Forschungsgemeinschaft für die Unterstützung durch Sachbeihilfen und der Volkswagenstiftung für die Geräteausstattung.     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

The Action of Thioridazine and Promazine on Biological Membranes: Relationship between ATPase Inhibition and Membrane Stabilization

The effects of promazine and thioridazine on hypotonic haemolysis of human erythrocytes are compared with their effect on Na⁺-K⁺-ATPase of washed human erythrocyte ghosts. Promazine (5 × 10^-5 - 5 × 10^-4 M) and thioridazine (10^-5 - 10^-4 M) stabilize erythrocytes against hypotonic haemolysis, but have lytic effects at higher concentrations. Both drugs inhibit Na⁺-K⁺-ATPase of erythrocyte membranes. This inhibition is slight at drug concentrations which have a membrane-stabilizing action, but is complete and irreversible at lytic concentrations of the drugs. Promazine and thioridazine also inhibit Na⁺-K⁺-ATPase in a membrane fraction prepared from rat hearts. The relative order of potency of the two drugs in this respect does not reflect their relative potency as general cardiac depressants. It is concluded that Na⁺-K⁺-ATPase is not a primary target for the action of promazine and thioridazine in the rat heart. It is suggested that inhibition of Na⁺-K⁺-ATPase by these agents is secondary to more general alterations of the physical properties of the cell membrane.     

Chronic digoxin treatment on canine myocardial Na+, K+-ATPase

Summary In order to determine whether a prolonged inhibition of cardiac Na⁺, K⁺-ATPase causes a compensatory or adaptive change in this enzyme, the relationships among serum digoxin concentration, binding of digoxin to the enzyme and cardiac Na⁺, K⁺-ATPase and sodium pump activity were studied in dogs chronically treated with digoxin. Digoxin was injected intravenously twice daily up to 4 weeks. Two hours after the injection of a single non-toxic dose of digoxin, Na⁺, K⁺-ATPase and sodium pump activities were inhibited quantitatively in a manner corresponding to the binding of digoxin to the enzyme. The magnitude of sodium pump inhibition was reduced 12 h after the digoxin injection, with simultaneous decreases in serum digoxin concentration and the binding of digoxin to the enzyme. After 1 or 4 weeks of digoxin treatment with non-toxic doses, the relationships among serum digoxin concentration, binding of digoxin to cardiac Na⁺, K⁺-ATPase and the degree of cardiac Na⁺, K⁺-ATPase or sodium pump inhibition remained unchanged. The magnitude of the inhibition was related to serum digoxin concentrations and digoxin binding to cardiac Na⁺, K⁺-ATPase, in a manner similar to that observed after a single digoxin injection. After 4 weeks of digoxin treatment with toxic doses, these relationships were also unaffected. It was concluded that prolonged digoxin treatment fails to alter the inhibition of myocardial Na⁺, K⁺-ATPase by this agent.This work was supported by U.S. Public Health Service Grant HL-16052.     

Cardiac glycosides: Correlations among Na+, K+-ATPase,sodium pump and contractility in the guinea pig heart

Summary Relationships among positive inotropic response to cardiac glycosides, Na⁺,K⁺-ATPase inhibition and monovalent cation pump activities were studied using paced Langendorff preparations of guinea-pig heart. Na⁺,K⁺-ATPase activity was estimated from the initial velocity of (³H)-ouabain binding in ventricular homogenates, and cation pump activity from ouabain-sensitive ⁸⁶Rb uptake of ventricular slices. These parameters were assayed in control, ouabain- or digitoxintreated hearts either at the time of inotropic response to the cardiac glycosides or during the course of drug washout. Development and loss of the inotropic response during ouabain or digitoxin perfusion and washout was accompanied by reduction and subsequent recovery of the initial ouabain binding velocity, respectively. If homogenates from glycoside-treated hearts were incubated at 37°C for 10 min during ouabain-binding studies, the levels of binding were not different from those of control hearts, indicating a rapid dissociation of the glycosides from cardiac Na⁺,K⁺-ATPase in this species. Despite differences in the time course of the loss of inotropic responses produced by ouabain or digitoxin, the relationship between Na⁺,K⁺-ATPase inhibition and inotropic responses were similar. Inotropic responses to digitoxin during perfusion, and subsequent los during washout, also were accompanied by a reduction and subsequent recovery of ⁸⁶Rb uptake. A correlation between inhibition of cation pump activity and positive inotropy has hitherto not been demonstrated. Thus, it appears that with cardiac glycosides, a relationship exists among contractility, cardiac Na⁺,K⁺-ATPase and monovalent cation pump activities.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

Inhibition of human colonic (Na++K+)-ATPase by arachidonic and linoleic acid

Summary The sodium pump, (Na⁺+K⁺)-ATPase, which is involved in the transport of cations and water movement by the colonic mucosa, may be decreased in various diarrhoeal states. In this study, we have measured ³H-ouabain binding and (Na⁺+K⁺)-ATPase activity in human colonic biopsy homogenates and the influence of various inflammatory and antiinflammatory compounds on these parameters. ³H-ouabain binds to one site of high affinity (K _D 1.9±0.2×10^–9 mol/l) with a maximal binding capacity of 7.5±0.8×10¹⁴ binding sites/g protein. Both arachidonic and linoleic acid inhibited (Na⁺+K⁺)-ATPase activity (IC₅₀ arachidonic acid: 7.5×10^–5 mol/l, linoleic acid: 6.5×10^–5 mol/l) and Mg²⁺-ATPase activity (IC₅₀ arachidonic acid: 9×10^–5 mol/l, linoleic acid: 4×10^–5 mol/l). Arachidonic acid inhibited ³H-ouabain binding, (IC₅₀ 3.2×10^–5 mol/l). The following antiinflammatory compounds, at concentrations up to 1×10^–3 mol/l, did not influence ATPase activity directly nor reverse the arachidonic acid-induced inhibition: indomethacin (cyclooxygenase inhibitor), nordihydroguaretic acid (lipoxygenase inhibitor), sulphasalazine and its metabolites: 5-aminosalicylic acid, N-acetylaminosalicylic acid and sulphapyridine.These results indicate that human colonic (Na⁺+K⁺)-ATPase is inhibited by the prostanoid precursors, arachidonic and linoleic acid. From a therapeutic point of view (effect on colonic (Na⁺+K⁺)-ATPase and perhaps diarrhoea), the suppression of the production of these prostanoid precursors by drugs may, therefore, be beneficial in the treatment of inflammatory bowel disease.Supported by DFG (Er65/4-4)