Effects of lead and a low-molecular-weight endogenous plasma inhibitor on the kinetics of sodium-potassium-activated adenosine triphosphatase and potassium-activated p-nitrophenylphosphatase

1. Lead, ouabain and an endogenous plasma inhibitor were all found to be potent inhibitors of purified hog cerebral cortex sodium-potassium-activated adenosine triphosphatase and potassium-stimulated p-nitrophenyl-phosphatase. 2. The kinetic characteristics of inhibition of both enzymes by lead and the endogenous plasma inhibitor differed in several respects. For sodium-potassium-activated adenosine triphosphatase, lead and the endogenous plasma inhibitor were non-competitive inhibitors with respect to potassium; lead was competitive with respect to sodium, whereas the endogenous plasma inhibitor had no effect; lead was competitive with respect to magnesium adenosine triphosphate, whereas the endogenous plasma inhibitor was uncompetitive. For potassium-activated p-nitrophenylphosphatase, both lead and the endogenous plasma inhibitor were competitive with respect to potassium; lead showed a mixed type of inhibition with respect to p-nitrophenylphosphate, whereas the endogenous plasma inhibitor was non-competitive. 3. Lead and the endogenous plasma inhibitor exhibited synergistic inhibitory activity on sodium-potassium-activated adenosine triphosphatase. 4. These results suggest that lead could play a contributory role in the pathogenesis of essential hypertension via an additive inhibition of vascular smooth muscle sodium-potassium-activated adenosine triphosphatase.     

Effects of aldosterone on rat collecting tubule N-ethylmaleimide-sensitive adenosine triphosphatase

Acidification in the collecting tubule is thought to be mediated by a proton-translocating adenosine triphosphatase (ATPase), and is modulated by aldosterone. Using an enzymatic microassay, we measured N-ethylmaleimide (NEM)-sensitive ATPase activity in isolated tubules obtained from control rats and rats receiving aldosterone by osmotic minipumps (5 micrograms/100 gm/day for 7 days). In control animals, enzyme activity was higher in cortical than in outer medullary collecting tubules (259 +/- 36 vs. 111 +/- 16 pmol/mm/hr, P less than 0.005, respectively). Prolonged aldosterone administration resulted in an increase in enzyme activity in the outer medullary collecting tubule (274 +/- 39 pmol/mm/hr, P less than 0.005), with no change in the cortical collecting tubule (255 +/- 47 pmol/mm/hr). These findings suggest that prolonged enhancement of acidification by mineralocorticoids is mediated by different mechanisms in the two segments of the rat collecting tubule. Whereas in the outer medullary segment an increase in the activity of NEM-sensitive ATPase facilitates the chronic enhancement in acidification, high basal enzyme activity in the cortical segment and institution of favorable voltage condition by mineralocorticoid treatment may obviate the need to increase enzyme activity.     

正常淋巴液对内毒素休克大鼠肺组织髓过氧化物酶活性及膜泵的作用研究

目的观察外源性正常淋巴液对脂多糖（LPS）所致内毒素休克大鼠肺组织匀浆髓过氧化物酶（MPO）活性及肺组织细胞膜泵功能的作用，探讨其干预机制。方法雄性Wistar大鼠40只，按随机数字表法均分为内毒素组、淋巴液组、血浆组和对照组。除对照组外，其他各组应用LPS5mg／kg复制内毒素休克模型，对照组以等量生理盐水代替LPS；15min后，淋巴液组自颈静脉输入仅占全血量1／15的无细胞淋巴液；血浆组以血浆代替淋巴液；内毒素组和对照组以生理盐水代替淋巴液。给予LPS（或相应液体）后4h，制备体积分数为10％的肺组织匀浆，检测肺组织匀浆MPO及膜ATP酶的活性。结果与对照组相比，内毒素组和血浆组大鼠肺组织匀浆MPO活性显著增高、4种膜ATP酶活性均显著下降（P＜0．05或P＜0．01）。淋巴液组肺组织匀浆MPO活性显著高于对照组，Na^＋-K^--ATP酶活性显著低于对照组，两组比较差异均有显著性（P均＜0．05）；而Ca^2＋-ATP酶、Mg^2＋-ATP酶和Ca^2＋-Mg^2＋-ATP酶活性与对照组比较差异均无显著性（P均＞0．05）；淋巴液组肺组织匀浆MPO活性显著低于内毒素组及血浆组（P均＜0．01），4种膜ATP酶活性显著高于内毒素组及血浆组（P＜0．05或P＜0．01）。结论外源性正常淋巴液对内毒素休克所致的肺损伤具有干预作用，其机制可能与减少中性粒细胞激活、提升膜ATP酶活性有关。     

碱性成纤维细胞生长因子对乙醇中毒大鼠ATP酶及抗氧化作用的影响

背景:碱性成纤维细胞生长因子具有多种生物活性,对组织创伤具有修复作用,但其对乙醇中毒的保护作用至今未见报道。目的:观察碱性成纤维细胞生长因子对乙醇中毒大鼠脑和肝组织中ATP酶、超氧化物歧化酶活力和丙二醛水平的影响。方法:选择成年Wistar雄性大鼠,采用白酒灌胃建立乙醇中毒模型,30只造模成功的大鼠分为3组,生理盐水组和碱性成纤维细胞生长因子治疗组分别于造模60d后肌肉注射生理盐水和碱性成纤维细胞生长因子;中毒模型组不作任何干预;另取10只不灌白酒作为正常对照组。结果与结论:与正常对照组相比,乙醇中毒后大鼠肝及脑组织ATP酶活力和超氧化物歧化酶活力显著降低,丙二醛水平明显升高(P<0.05);用碱性成纤维细胞生长因子治疗后ATP酶活力和超氧化物歧化酶活力均显著升高,丙二醛水平显著降低(P<0.05~0.01)。提示碱性成纤维细胞生长因子能提高ATP酶活力且有抗氧化作用,对乙醇中毒所致的脑和肝损伤具有保护作用。     

Calcium transport and adenosine triphosphatase activities of erythrocyte membranes in congenital spherocytosis.

Calcium transport from red cells was measured in seventeen patients with congenital or hereditary spherocytosis (HS). The efflux remained at a lower level in resealed ghost cells of patients than in normal cells both in the presence and absence of adenosine triphosphate (ATP). We studied the activities of Ca2+,Mg2+-ATPase, ouabain-sensitive Na+,K+-ATPase, Mg2+-ATPase and Ca2+-(spectrin-)ATPase in cell membranes prepared by washing the cells with hypotonic medium. The mean +/-SD Ca2+,Mg2+-ATPase/Mg2+-ATPase of HS patients was 3.34 +/- 1.06, and 2.81 +/- 0.42 in control subjects. Na+,K+-ATPase/Mg2+-ATPase was 2.38 +/- 0.38 in HS cells compared to 2.01 +/- 0.41 in normal cells. Ca2+-ATPase/Mg2+-ATPase of HS membranes was 0.57 +/- 0.18 and the control value 0.43 +/- 0.08. These data indicate calcium retention in the erythrocytes of HS patients in spite of increases in Ca2+,Mg2+-ATPase activity in the majority of patients.     

氧自由基对生殖细胞三磷酸腺苷酶和生长抑素的影响

背景：有关细胞自由基的研究,主要集中在动物体内细胞自由基的产生与清除方面。目的：观察氧自由基对体外培养的睾丸生殖细胞三磷酸腺苷酶（ATPase）和生长抑素含量的影响。方法：体外分离、纯化小鼠睾丸生殖细胞,培养24h后,将其分为2组,实验组添加黄嘌呤-黄嘌呤氧化酶建立氧自由基损伤模型;对照添加生理盐水。继续培养24,48h,采用酶组化及免疫组化染色观察各组ATPase和生长抑素表达变化。采用LUZEX-F图像分析仪分别测定其灰度值;用酶联免疫检测仪测定492nm波长的吸光度值。结果与结论：给药后24,48h,实验组ATPase、生长抑素灰度值显著高于对照组（P〈0.01）,吸光度值显著低于对照组（P〈0.01）。提示氧自由基可降低ATPase和生长抑素含量,影响生殖细胞在体外培养过程中的生长与增殖。     

氧自由基对生殖细胞三磷酸腺苷酶和生长抑素的影响

背景:有关细胞自由基的研究,主要集中在动物体内细胞自由基的产生与清除方面.目的:观察氧自由基对体外培养的睾丸生殖细胞三磷酸腺苷酶(ATPase)和生长抑素含量的影响.方法:体外分离、纯化小鼠睾丸生殖细胞,培养24 h 后,将其分为2 组,实验组添加黄嘌呤-黄嘌呤氧化酶建立氧自由基损伤模型;对照添加生理盐水.继续培养24,48 h,采用酶组化及免疫组化染色观察各组ATPase 和生长抑素表达变化.采用LUZEX-F 图像分析仪分别测定其灰度值;用酶联免疫检测仪测定492 nm 波长的吸光度值.结果与结论:给药后24,48 h,实验组ATPase、生长抑素灰度值显著高于对照组(P ＜ 0.01),吸光度值显著低于对照组(P ＜ 0.01).提示氧自由基可降低ATPase 和生长抑素含量,影响生殖细胞在体外培养过程中的生长与增殖.     

雷公藤多甙对局灶性脑缺血再灌注大鼠脑组织三磷酸腺苷酶的影响

目的:观察预应用雷公藤多甙对大鼠局灶性脑缺血再灌注后脑能量代谢和神经运动功能障碍的影响。方法:实验于2001-04/12在郑州大学基础医学院药理学教研室、郑州大学第二附属医院神经内科,郑州市疾病预防控制中心完成。取120只Wistar大鼠随机分为6组:缺血再灌注组,雷公藤多甙45,30,15mg/kg组,尼莫地平组(10mg/kg),假手术组,每组20只。所有动物灌胃给药,1次/d,连灌5d。缺血再灌注组和假手术组灌等体积的生理盐水。末次灌胃后1h,除假手术组不插入尼龙线,其余5组采用线栓法制作大鼠大脑中动脉局灶性缺血再灌注模型,检测各组大鼠脑组织三磷酸腺苷酶活性的变化,并进行神经病学评分(评分越高,功能障碍越重)。结果:120大鼠均进入结果分析。①神经运动功能变化:缺血再灌注组出现明显的神经运动功能障碍,神经病学评分为3.7±0.3;雷公藤多甙15,30,45mg/kg组及尼莫地平组大鼠功能障碍均明显改善,神经病学评分与缺血再灌注组比较差异显著(3.1±0.4,2.7±0.3,2.2±0.2,2.5±0.3,P<0.01)。②三磷酸腺苷酶活性:缺血再灌注组显著低于假手术组(P<0.01),雷公藤多甙各剂量组和尼莫地平组三磷酸腺苷酶活性则高于缺血再灌注组(P<0.05或P<0.01)。结论:在脑缺血再灌注后,脑组织能量代谢障碍,细胞的主动转运功能受损。雷公藤多甙能显著升高三磷酸腺苷酶的活性,改善局灶性脑缺血引起的神经运动功能障碍。     

The distribution of sodium-potassium-activated adenosine triphosphatase in medulla and cortex of the kidney

The activity of sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) is considerably higher in homogenates of outer medulla than in the cortex or papilla of the kidney. The enzyme has similar kinetic characteristics in both cortex and medulla, and binds ouabain in the same proportion. The discrepancy in enzymatic activity is not paralleled by similar change in the activity of adenyl cyclase, 5'nucleotidase, glucose-6-phosphatase, or succinic dehydrogenase. Na-K-ATPase is also higher in distal convoluted tubules (ventral slices) than in the proximal tubules (dorsal slices) of the kidney of Amphiuma. The high concentration of Na-K-ATPase in the red medulla of the kidney is probably related to the presence here of the thick ascending limb of the loop of Henle, and this has important implications with regard to the mechanism of sodium reabsorption by different portions of the nephron.     

Renal adaptation to potassium in the adrenalectomized rabbit. Role of distal tubular sodium-potassium adenosine triphosphatase.

Potassium secretion and sodium-potassium adenosine triphosphatase (Na-K-ATPase) activity in the distal nephron segments are known to be influenced by the dietary intake of K+. This has been attributed to a change in the plasma aldosterone level, which also influences K+ secretion and Na-K-ATPase activity in the distal nephron. To investigate whether or not dietary K+ can modulate Na-K-ATPase activity in the distal nephron independently of aldosterone, we determined Na-K-ATPase activity in four distinct nephron segments of adrenalectomized (adx) rabbits given four specific diets for 1 wk before experimentation. Na-K-ATPase activity was determined by a fluorometric microassay in which ATP hydrolysis is coupled to NADH oxidation. The nephron segments examined were the distal convoluted tubule (DCT), the connecting tubule (CNT), the cortical collecting duct (CCD), and the outer medullary collecting duct (MCD). All diets were similar in composition except for their K+ contents, which were 100, 300, 500, and 700 meq/kg in groups 1-4, respectively. In these adx animals, Na-K-ATPase activity increased greater than 200% in the CCD as the dietary intake of K+ increased. There was a linear relationship between K+ excretion and the enzyme activity in this segment. There was a 50% increase in Na-K-ATPase activity in the CNT as the dietary intake of K+ increased in adx animals. However, there were no significant differences in Na-K-ATPase activities in the DCT and MCD among the four treatment groups. It is concluded that dietary K+ intake can influence Na-K-ATPase activity in the CCD and CNT independently of plasma aldosterone levels.     

Subcellular localization and properties of adenosine triphosphatase in human polymorphonuclear leucocytes

Abstract. Magnesium-dependent adenosine triphosphatase (Mg²⁺-ATPase) activities were studied in human neutrophilic polymorphonuclear leucocytes. Kinetic studies on whole leucocyte homogenates produced curvilinear kinetics suggesting the presence of at least two forms of Mg²⁺-ATPase.
Neutrophils were homogenized in isotonic sucrose and, after low-speed centrifugation, the supernatant was subjected to analytical subcellular fractionation. Gradient fractions were assayed for Mg²⁺-ATPase and for principal organelle marker enzymes. Mg²⁺-ATPase was distributed between the plasma membrane, mitochondrial and cytosol fractions. Kinetic and inhibitor studies on Mg²⁺-ATPase from each localization indicated the presence of three forms of the enzyme. The plasma membrane and mitochondrial activities had a K _m value of 0.2 mmol/1 for ATP, whilst the K _m for the cytosolic enzyme was 1 -8 mmol/I. Inhibitor studies showed further differences between the three enzymes.
Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mUnits/mg protein) of Mg²⁺-ATPase, in contrast to those of alkaline phosphatase, were similar in all three patient groups. This result, together with the fractionation experiments and inhibitor studies, strongly suggest that the ATPase is not attributable to neutrophil alkaline phosphatase.     

Expression and developmental regulation of Na+,K+ adenosine triphosphatase in the rat small intestine.

The Na+,K(+)-ATPase ion pump plays a critical role in fluid and electrolyte physiology of the small intestine. Here we show that, of the three known alpha isotypes (alpha 1, alpha 2, and alpha 3) of the sodium pump found in the rat, only alpha 1 is expressed in the small intestine. The expression of this isotype, considered at the level of mRNA, is under developmental control, with the adult intestine exhibiting approximately a threefold increase in alpha 1 message over the neonate. Cortisone treatment of the neonate results in near-adult levels of alpha 1 mRNA expression. An increase in the abundance of alpha 1 isotype parallels the changes in its mRNA expression. beta subunit mRNA is expressed coordinately with the alpha 1 subunit mRNA. A four- to five-fold rise in the Na+,K(+)-ATPase activity is also developmentally induced.     

Vasoconstrictor and vasodilator effects of adenosine in the mouse kidney due to preferential activation of A1 or A2 adenosine receptors

The present experiments in mice were performed to determine the steady-state effects of exogenous adenosine on the vascular resistance of the whole kidney, of superficial blood vessels, and of afferent arterioles. The steady-state effect of an intravenous infusion of adenosine (5, 10, and 20 microg/min) in wild-type mice was vasodilatation as evidenced by significant reductions of renal and superficial vascular resistance. Resistance decreases were augmented in adenosine 1 receptor (A1AR) -/- mice. Renal vasodilatation by the A2aAR agonist CGS 21680A [2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamido-adenosine hydrochloride] (0.25, 0.5, and 1 microg/kg/min) and inhibition of adenosine-induced relaxation by the A2aAR antagonist ZM-241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol] (20 mg/kg) suggests that the reduction of renovascular resistance was largely mediated by A2aAR. After treatment with Nomega-nitro-L-arginine methyl ester (L-NAME) adenosine was unable to alter superficial blood flow and resistance significantly indicating that adenosine-induced dilatation is NO-dependent. Absence of a dilatory effect in endothelial nitric-oxide synthase (NOS) -/- mice suggests endothelial NOS as the source of NO. When infused into the subcapsular interstitium, adenosine reduced superficial blood flow through A1AR activation. Adenosine (10(-7) M) constricted isolated perfused afferent arterioles when added to the bath but not when added to the luminal perfusate. Luminal adenosine caused vasoconstriction in the presence of L-NAME or the A2AR antagonist 3,7-dimethyl-1-(2-propynyl)xanthine. Our data show that global elevation of renal adenosine causes steady-state vasorelaxation resulting from adenosine 2 receptor (A2AR)-mediated generation of NO. In contrast, selective augmentation of adenosine around afferent arterioles causes persistent vasoconstriction, indicating A1AR dominance. Thus, adenosine is a renal constrictor only when it can interact with afferent arteriolar A1AR without affecting the bulk of renal A2AR at the same time.