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.     

内皮素B受体对肾近曲小管上皮细胞Na+-K+-ATP酶活性的影响

目的 探讨内皮素B(ETB)受体对肾脏近曲小管(RPT)上皮细胞Na -K -ATP酶活性的影响和机制.方法 以WKY(Wistar-Kyoto)大鼠RPT细胞为研究对象,Na -K -ATP酶活性采用哇巴因法进行测定.结果 ETB受体激动剂BQ3020能明显降低Na -K -ATP酶的活性,这一抑制作用呈浓度和时间依赖性,BQ3020 10-8 mol/L刺激15 min达最大效应,下降幅度达36.1%;用细胞膜钙通道阻断剂尼卡地平预先刺激细胞后,能够阻断ETB受体对Na -K -ATP酶的抑制效应;在无钙状态下,ETB受体激动剂BQ3020对Na -K -ATP酶的抑制效应丧失.结论 ETB受体在RPT处通过降低Na -K -ATP酶活性调节离子转运;细胞外钙内流参与了ETB受体对Na -K -ATP酶活性抑制作用的信号途径.     

替米沙坦对OK细胞Na+-K+ ATP酶活性的影响

目的探讨血管紧张素Ⅱ受体拮抗剂(ARB)替米沙坦和血管紧张素转换酶抑制剂(ACEI)苯那普利对负鼠近端小管上皮细胞(OK细胞)Na+-K+-ATP酶活性的影响.方法培养的OK细胞采用低渗方法制备细胞膜悬液,使用BCA-100蛋白质定量测定试剂盒测定膜蛋白;Na+-K+ ATP酶活性采用孔雀绿比色分析法测定释放的无机磷(Pi)含量,培养液中分别加入血管紧张素Ⅱ(Ang Ⅱ)、Ang Ⅱ+血管紧张素Ⅱ受体拮抗剂替米沙坦(Telmisartan)、Ang Ⅱ+血管紧张素转换酶抑制剂苯那普利(Benazepril),观察它们对OK细胞Na+-K+-ATP酶活性的影响.结果 (1)培养液中加入10-10 mol/L Ang Ⅱ组与对照组相比,OK细胞Na+-K+-ATP酶活性明显上升.(0.0972±0.0080 vs 0.0896±0.0065 μmol·L-1·mg pro-1·h-1, P<0.05)(2) 当培养液中同时加入10{10 mol/L Ang Ⅱ和10-9mol/L Telmisartan,与单加入10-10mol/L AngⅡ组相比,OK细胞Na+-K+-ATP酶活性明显降低.(0.0623±0.0053 vs 0.0972±0.0080 μmol·L-1·mg pro-1·h-1,P<0.05)(3)当培养液中同时加入10-10 mol/L AngⅡ和10-9 mol/L Benazepril,与单加入10-10 mol/L AngⅡ组相比,OK细胞Na+-K+-ATP酶活性无明显变化.(0.1027±0.0166 vs 0.0972±0.0080 μmol·L-1·mg pro-1·h-1, P>0.05).结论血管紧张素Ⅱ作为一种生长因子,不仅能刺激细胞增殖,又能调节近端小管的离子转运,增加Na+-K+-ATP酶活性;替米沙坦能抑制血管紧张素Ⅱ引起的OK细胞Na+-K+-ATP酶活性增加,而苯那普利则无此作用.     

Aβ_(1～42)及二氮嗪预处理对神经细胞Na~+-K~+-ATP酶β亚基蛋白表达的影响

目的探讨Aβ1~42及二氮嗪预干预对神经细胞Na+-K+-ATP酶β亚基蛋白表达的影响。方法原代培养大鼠皮层海马神经细胞,随机分为空白对照组、Aβ1~42组(2μmol/L)、二氮嗪(50μmol/L)预处理1 h后Aβ1~42组、单独二氮嗪预处理组,各组又分为24、72 h两个时间点,采用免疫荧光及免疫印迹法检测干预后不同培养时间细胞Na+-K+-ATP酶β亚基蛋白表达水平的变化。结果免疫荧光显示:Aβ1~42作用细胞72 h降低了Na+-K+-ATP酶β亚基荧光强度;而经二氮嗪预处理后Aβ1~42作用72 h,与单独Aβ1~42组相比较荧光强度有所增强。Western印迹显示:二氮嗪预处理神经细胞1 h协同Aβ1~42作用24 h后及单独二氮嗪组Na+-K+-ATP酶β亚基的蛋白表达明显低于对照组及单独Aβ1~42组(P0.01)。Aβ1~42作用神经细胞72 h后,Na+-K+-ATP酶β亚基蛋白表达较对照组显著降低(P0.05);二氮嗪预处理1h协同Aβ1~42共同作用于神经细胞72 h后,与单独Aβ1~42组相比较,增加了Na+-K+-ATP酶β亚基蛋白表达量(P0.05)。结论 Aβ1~42作用72 h,Na+-K+-ATP酶β亚基蛋白表达量降低,二氮嗪预处理1 h协同Aβ1~42共同作用于神经细胞72 h,Na+-K+-ATP酶β亚基蛋白表达量升高。     

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。     

CGRP对大鼠全脑缺血再灌注ATP酶活性的影响

目的研究降钙素基因相关肽(CGRP)对大鼠全脑缺血再灌注(I/R)脑组织ATP酶活性变化的影响及神经保护机制.方法45只SD大鼠,随机分为假手术组、对照组、CGRP)组.采用四血管阻断方法制备SD大鼠全脑I/R模型,定磷比色法测定全脑I/R及全脑I/R CGRP治疗大鼠海马Na ,K -ATP、Ca2 -ATP酶活性.结果与假手术组比较,大鼠全脑I/R后海马Na ,K -ATP、Ca2 -ATP酶活性降低.CGRP对I/R后海马Na ,K -ATP、Ca2 -ATP酶活性降低有明显的抑制作用.结论CGRP对大鼠全脑I/R脑组织损伤有保护作用.     

二氮嗪对大鼠缺血再灌注损伤脑组织线粒体ATP酶活性的影响

目的 探讨线粒体ATP敏感性钾通道开放剂二氮嗪对大鼠局灶性脑缺血再灌注损伤脑组织线粒体ATP酶活性的影响.方法 采用改良线栓法建立大鼠局灶性大脑中动脉缺血再灌注损伤模型.将21只Wistar雄性大鼠随机分为假手术组(N组)、缺血再灌注组(IR组)、二氮嗪干预组(DZ组).缺血1 h再灌注24 h后留取标本,测定脑组织线粒体Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性的变化.结果 与假手术组比较,缺血再灌注组Na+-K+-ATPase、Ca2+-Mg2+-ATP酶活性明显降低(P<0.05);二氮嗪干预组Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性均较缺血再灌注组有不同程度的提高(P<0.05).结论 二氮嗪预处理能通过提高脑组织线粒体Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性,减轻脑缺血再灌注损伤,保护神经元线粒体的功能,有效维持大脑能量代谢,发挥脑保护作用.     

内皮素B受体对肾近曲小管上皮细胞Na~ -K~ -ATP酶活性的影响

目的探讨内皮素B(ETB)受体对肾脏近曲小管(RPT)上皮细胞Na -K -ATP酶活性的影响和机制。方法以WKY(Wistar-Kyoto)大鼠RPT细胞为研究对象,Na -K -ATP酶活性采用哇巴因法进行测定。结果ETB受体激动剂BQ3020能明显降低Na -K -ATP酶的活性,这一抑制作用呈浓度和时间依赖性,BQ302010-8mol/L刺激15min达最大效应,下降幅度达36.1%;用细胞膜钙通道阻断剂尼卡地平预先刺激细胞后,能够阻断ETB受体对Na -K -ATP酶的抑制效应;在无钙状态下,ETB受体激动剂BQ3020对Na -K -ATP酶的抑制效应丧失。结论ETB受体在RPT处通过降低Na -K -ATP酶活性调节离子转运;细胞外钙内流参与了ETB受体对Na -K -ATP酶活性抑制作用的信号途径。     

药物干预对大鼠急性脑缺血再灌注损伤神经元的保护作用

目的 探讨脑蛋白及银杏叶提取物对大鼠急性脑缺血再灌注损伤后缺血半暗带神经元的保护作用及机制.方法 600只雄性Wistar大鼠随机分为4组(对照组、蛋白组、银杏叶组、联合组),每组大鼠根据缺血后再灌注不同时间又分为缺血2h再灌注6、12、24、72 h、7d五个亚组(30只/亚组).采用线栓法制备大鼠缺血2h再灌注模型,除对照组外,其余三组均采用药物进行干预.分别于再灌注6、24、48、72 h及7d断头取脑,观察缺血半暗带脑组织Na+-K+-ATP酶活性、脑组织水肿程度、脑梗死范围及神经症状评分的变化.结果 对照组大鼠脑缺血半暗带神经元Na+-K+-ATP酶活性于6h开始降低,48 h达最低值,72 h稍有回升,7d趋于稳定.与单一用药组(蛋白组或银杏叶组)比较,联合组能显著提高大鼠缺血半暗带神经元Na+-K+-ATP酶活性(48、72 h亚组,P值均为0.000),降低脑组织含水量(24、48、72 h亚组,P值均为0.000),减小脑梗死面积(24、48、72 h、7d亚组,P值均为0.000),减少神经症状评分(48、72 h、7d亚组,P值均为0.000).结论 联合用药较单一用药能更有效地抑制缺血级联反应,从而最大限度地挽救缺血半暗带神经元的功能.     

丁基苯酞对慢性脑缺血大鼠脑内G-CSF和TNF-α表达的影响

目的 研究丁基苯酞对慢性脑缺血大鼠脑内G-CSF和TNF-α表达的影响.方法 采用双侧颈总动脉永久性结扎(2VO)制备慢性脑缺血模型,治疗组大鼠给予丁基苯酞灌胃,免疫组织化学法多克隆抗体G-CSF和TNF-α标记大鼠脑内梗死区神经元、血管内皮、胶质细胞.持久性2VO 2个月.结果 治疗组大鼠脑内G-CSF明显增多,TNF-α明显减少.结论 丁基苯酞能增加2VO大鼠脑内G-CSF的表达并能减少TNF-α的表达,促进慢性脑缺血的代偿修复.     

Peroxynitrite induced decrease in Na^＋, K^＋-ATPase activity is restored by taurine

AIM: Peroxynitrite (ONOO-) is a powerful oxidant shown to damage membranes. In the present study, the effect of taurine on changes of liver plasma membrane Na+, K+-ATPase induced by ONOO- was investigated. METHODS: Liver plasma membrane was exposed to ONOO-with or without taurine. Na+, K+-ATPase activity and lipid peroxidation as thiobarbituric acid reactive substances (TBARS) levels were measured. RESULTS: Different concentrations of ONOO- (100, 200, 500, and 1 000 μmol/L) were found to decrease liver plasma membrane Na+, K+-ATPase activity significantly. The depletion of enzyme activity was not concentration dependent. Effects of different concentrations of taurine on liver plasma membrane Na+, K+-ATPase activity were also measured. Taurine did not cause any increase in enzyme activity. When plasma membranes were treated with 200 μmol/L ONOO- with different concentrations of taurine, a restoring effect of taurine on enzyme activity was observed. TBARS levels were also measured and taurine was found to decrease the elevated values. CONCLUSION: Taurine is observed to act as an antioxidant of ONOO- to decrease lipid peroxidation and thus affect liver plasma membrane Na+, K+-ATPase by restoring its activity.     

Differential effects of proinsulin C-peptide fragments on Na +, K +-ATPase activity of renal tubule segments

Summary Proinsulin C-peptide has been shown to stimulate the activity of Na ⁺ K ⁺ ATPase of rat renal tubule segments. Thirty-six peptides and amino acids, corresponding to parts of the intact rat C-peptide and suitable controls were screened for capacity to stimulate Na ⁺, K ⁺-ATPase in an attempt to determine potential active sites in the C-peptide molecule. The carboxy-terminal tetra and penta peptides were found to elicit 92–103 % of the intact molecule's activity, and the remaining segment, des-(27–31) C-peptide, did not possess stimulatory activity. Peptides from the middle C-peptide segment, however, centering around a GGPEAG sequence, stimulated Na ⁺, K ⁺-ATPase activity (36–80 % of the intact molecule's effect) but this effect was not balanced by corresponding inactivity of other parts. Furthermore, it was paralleled by activity of a non-native dipeptide d-form. It is concluded that the latter effect and that of the middle segment may represent complex interactions other than the apparently specific effects of the C-terminal segment. [Diabetologia (1998) 41: 287–291] Received: 31 July 1997 and in revised form: 30 October 1997     

不同急性肺损伤大鼠模型Na+-K+-ATP酶活性变化及对地塞米松的反应

目的探讨肺泡上皮细胞膜上Na+-K+-ATP酶在盐酸(HCl)和脂多糖(LPS)两种原因致急性肺损伤大鼠肺水生成中的作用及地塞米松(Dex)治疗肺水肿的意义.方法 96只雄性SD大鼠随机分为6组(每组16只)生理盐水对照组(NS组)、NS+Dex组、HCl模型组、HCl+Dex组、LPS模型组、LPS+Dex组,每组又分为支气管肺泡灌洗(BAL)亚组和非支气管肺泡灌洗(NBAL)亚组.NBAL组观察肺组织病理改变、湿干重比(W/D)、Na+-K+-ATP酶水解活性和哇巴因与其受体Na+-K+-ATP酶特异性结合位点数量的变化;BAL组观察支气管肺泡灌洗液(BALF)中细胞数及蛋白含量的变化.结果①两种模型组Na+-K+-ATP酶水解活性和酶位点数在激素干预前均显著低于干预后水平,并且显著低于对照组水平.②激素干预前两种模型组BALF中细胞总数、PMN分类计数、蛋白含量显著高于干预后水平,显著低于对照组水平.结论 Na+-K+-ATP酶在肺水的转运中起重要作用,ALI时肺泡膜上Na+-K+-ATP酶的活性降低可能是产生肺水肿的重要机制之一,地塞米松可以分别增加两种模型组Na+-K+-ATP酶水解活性和位点数;地塞米松可以减轻LPS组肺水肿(以W/D表示)的程度,但对HCl组无效.     

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.     

Effect of phenylalanine and p-chlorophenylalanine on Na+, K+-ATPase activity in the synaptic plasma membrane from the cerebral cortex of rats

Na⁺, K⁺-ATPase activity was measured in synaptic plasma membrane from cerebral cortex of Wistar rats subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 μmol phenylalanine /g body weight (twice a day) plus 0.9 μmol p-chlorophenylalanine /g body weight (once a day). The treatment was performed from the 6^th to the 14^th postpartum day and rats were killed 12 h after the last injection. Synaptic plasma membrane from cerebral cortex was prepared by a discontinuous density sucrose gradient for Na⁺, K⁺-ATPase activity determination. The results showed that the enzyme activity was decreased by 30% in animals subjected to experimental phenylketonuria when compared to control. Thein vitro effects of the drugs on Na⁺, K⁺-ATPase activity were also investigated. Phenylalanine and p-chlorophenylalanine inhibited the enzyme activity and this inhibition was reversed by alanine. In addition, competition between phenylalanine and p-chlorophenylalanine for binding to the enzyme was observed, suggesting a common binding site for these substances. Our results suggest that reduction of Na⁺, K⁺-ATPase activity may be one of the mechanisms related to the brain dysfunction observed in human PKU.     

肝硬化红细胞钠钾ATP酶、钙镁ATP酶及钠钾钙镁改变

目的:探讨肝硬化时细胞内钠钾钙镁的改变及细胞膜钠钾ATP酶(NKA)、钙镁ATP酶 (CMA)活性改变在细胞内钠钾钙镁改变中的作用．方法:测定了52例肝硬化失代偿期(实验组 A)、36例代偿期(实验组B)患者红细胞及血清钠钾钙镁(RNa、RK、RCa、RMg;SNa、 SK、SCa、SMg)含量和NKA和CMA活性．以 36名健康人为对照组．结果:与对照组比较,实验组A的NKA、 CMA、RK、RMg(t=5．92,P<0．001;t=7．21, P<0．001;t=2．32,P<0．02;t=4．79,P<0．001)和买验组B的NKA、CMA、RK、RMg(t=3．83, P<0．001;t=2．53,P<0．02;t=2．03,P<0．05;t= 3．33,P<0．002)均显著降低;与实验组B比较, 实验组A的NKA、CMA活性(t=2．29,P<0．05; t=4．14,P<0．005)显著降低．与对照组比较, 买验组A的SNa、SK、SCa、SMg(t=8．25, P<0．001;t=5．73,P<0．001;t=9．82,P<0．001; t=6．15,P<0．001)显著降低;与实验组B比较, 买验组A的SNa、SK、SCa、SMg(t=6．94, P<0．001;t=5．00,P<0．001;t=5．57,P<0．001; t=5．73,P<0．001)显著降低．与Child B级组比较,Child C级组的NKA、CMA、RK、RMg、 SNa、SK、SCa、SMg(P<0．05或P<0．01) 显著降低．与非肝性脑病组比较,肝性脑病组NKA、CMA、RK、RMg、SNa、SK、 SMg(P<0．05或P<0．01)显著降低．实验组A中, 低SMg者的NKA和CMA显著低于高SMg者 (16．87±3．19 vs 19．04±3．25;109．83±13．51 vs 120．13±13．27;P均<0．05)．结论:肝硬化患者存在缺钾缺镁,且随病情加重而加重,缺钾缺镁可能为病情加重的原因之一．NKA和CMA活性降低可导致细胞内低钾低镁和钠钙蓄积．缺镁为ATP酶活性在失代偿期进一步降低的原因之一．     

Only limited effects of aminoguanidine treatment on peripheral nerve function, (Na+,K+)-ATPase activity and thrombomodulin expression in streptozotocin-induced diabetic rats

Aims/hypothesis. Aminoguanidine, a potent anti-glycation reagent, is known to be beneficial in experimental diabetic neuropathy. In this study, we explored the mechanisms of how aminoguanidine inhibits neuropathic changes in diabetes and compared its effects with those of insulin treatment. Methods. Wistar rats, aged 8 weeks, were made diabetic by streptozotocin and given aminoguanidine dissolved in drinking water (1 g/l) for 8 weeks. Effects of daily insulin (protamine-zinc) treatment were also examined for comparison. At the end of the 8 weeks, we examined the peripheral nerve function and (Na⁺,K⁺)-ATPase activity and their relation to serum thrombomodulin concentrations that are considered as a marker of endothelial injury. Results. Aminoguanidine treatment reduced the diabetes-induced decrease in tibial nerve conduction velocity by 47 % (p < 0.05 vs untreated diabetic rats) and inhibited the loss of sciatic nerve (Na⁺,K⁺)-ATPase activity by 54 % (p < 0.05 vs untreated diabetic rats). Insulin-treatment of diabetic rats restored these variables by 83 % and 75 %, respectively (both, p < 0.01 vs untreated diabetic rats). Thrombomodulin concentrations were increased (p < 0.01) in diabetic rats compared with those in non-diabetic controls and unaffected by aminoguanidine treatment. In contrast, the concentrations remained within the normal range in the insulin-treated group. Conclusion/interpretation. Although aminoguanidine treatment improved nerve conduction velocity and (Na⁺,K⁺)-ATPase activity, its effects were considerably less than those of insulin and were not apparent in some measures of endothelial cell injury. [Diabetologia (1999) 42: 743–747] Received: 1 December 1998 and in final revised form: 3 February 1999     

Insulin does not regulate vascular smooth muscle Na+, K+-ATPase activity in rabbit aorta

Summary To determine whether insulin regulates vascular smooth muscle Na⁺, K⁺-ATPase activity and if impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity could be a cause of increased vascular reactivity to norepinephrine and angiotensin II in diabetic states, the effects of insulin on Na⁺, K⁺-ATPase activity were examined in normal rabbit aortic intima-media incubated with normal plasma glucose and myo-inositol levels for 30 min. Insulin at 100 U/ml (600 pmol/l) had no effect on Na⁺, K⁺-ATPase activity. At 250 U/ml it caused a 4.2±0.8% increase, and at 500 U/ml insulin caused a 17.7±1.4% increase in Na⁺, K⁺-ATPase activity that was completely inhibited by amiloride (1 mmol/l). Human insulin-like growth factor I (600 pmol/l) caused an 18.0±1.0% increase in Na⁺, K⁺-ATPase activity that was inhibited by amiloride. Insulin does not regulate (stimulate) aortic vascular smooth muscle Na⁺, K⁺-ATPase activity. Supraphysiological insulin concentrations, probably acting through an insulin-like growth factor I receptor, stimulate Na⁺/H⁺ exchange in aortic vascular smooth muscle and cause small secondary increases in Na⁺, K⁺-ATPase activity. In aortic intima-media incubated with normal plasma glucose and myo-inositol levels, endogenously released adenosine stimulates and maintains a component of resting Na⁺, K⁺-ATPase activity and stimulates acute increases in activity when norepinephrine (1 mol/l) or angiotensin II (100 nmol/l) is added. These adenosine-stimulated components of Na⁺, K⁺-ATPase activity are selectively inhibited when the medium glucose is raised to 30 mmol/l during a 30-min equilibration and 30-min incubation. Insulin (100 U/ml) added during the incubation had no effect on the alterations in Na⁺, K⁺-ATPase activity induced by glucose at an elevated plasma level. Impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity is not a possible cause for alterations in vascular reactivity in diabetes.     

Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na⁺, K⁺-and Ca²⁺-ATPase in crude homogenates of rat ventricular myocardium by determination of K⁺-and Ca²⁺-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K⁺-and Ca²⁺-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min^–1×g^–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na⁺, K⁺-and Ca²⁺-ATPase concentrations were estimated to 2 and 10 nmol×g^–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca²⁺-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K⁺-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K⁺-and Ca²⁺-dependentpNPPase activity gradually increased to a plateau. The present assay for Na⁺, K⁺-ATPase quantification has the advantage over [³H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K⁺-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca²⁺-ATPase on crude myocardial homogenates. Age-dependent changes in K⁺-and Ca²⁺-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na⁺, K⁺-and Ca²⁺-ATPase in excitable tissues.Abbreviations Na⁺, K⁺-ATPase sodium, potassium-dependent ATPase - Ca²⁺-ATPase caldium-dependent ATPase - pNP p-nitrophenyl - pNPP p-nitrophenyl phosphate - 3-0-MFP 3-0 methylfluorescein phosphate - DOC sodium deoxycholate     

Inhibition of Na+, K+-ATPase Activity by the Metabolites Accumulating in Homocystinuria

Homocystinuria is an inborn error of sulfur amino acid metabolism characterized predominantly by vascular and nervous system dysfunction. In this study we determined the in vitro effects of homocysteine and methionine, metabolites which accumulate in homocystinuria, on Na⁺, K⁺-ATPase, and Mg²⁺-ATPase activities in synaptic membranes from the hippocampus of rats. The results showed that both metabolites significantly inhibit Na⁺, K⁺-ATPase but not Mg²⁺-ATPase activity at concentrations usually observed in plasma of homocystinuric patients. Furthermore, incubation of hippocampal homogenates with homocysteine also elicited an inhibition of the enzyme activity which was however prevented by the simultaneous addition of cysteine to the medium. In addition, cysteine or methionine per se did not modify the two enzymatic activities. These findings indicate that oxidation of critical groups in the enzyme may possibly be involved in homocysteine inhibitory effect. Moreover, kinetic studies performed to investigate the interaction between homocysteine and methionine on Na⁺, K⁺-ATPase inhibition suggested a common site for the two amino acids in the enzyme. Considering the critical role exerted by Na⁺, K⁺-ATPase in brain, it is proposed that the inhibition provoked by homocysteine and methionine on the enzyme activity may be possibly related to the brain dysfunction characteristic of homocystinuria.