THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO₃)₂ resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb²⁺ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P_i on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P_i is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.     

Na-K]ATPase activity in proximal and distal tubules of the rat kidney: modification and application of a quantitative cytochemical technique

A modified cytochemical assay for [Na-K]ATPase in cryostat sections of kidney was further characterized and used to quantify activity in seven functionally distinct sites along the rat nephron. The activity of [Na-K]ATPase was defined as the difference in ATPase activity in specifically identified tubules contained in serial sections incubated with and without ouabain. Preincubation of sections with ouabain was required for maximal inhibition of [Na-K]ATPase activity in several distal sites. The concentration of ouabain necessary for maximal inhibition of activity was 3.0 mM and half-maximal inhibition was obtained in all regions with 30-100 microM ouabain. In distal sites, [Na-K]ATPase formed a higher proportion of total ATPase activity (60-80 per cent) than in proximal sites (20-40 per cent). Enzyme activity was quantified using two different methods. The first measured activity over the basal region of tubules and gave an index of the concentration of [Na-K]ATPase over the basal lateral infoldings of cells composing the tubule. The second read activity over the entire cross section of tubules and provided an estimate of [Na-K]ATPase per length of tubule. The highest activities over the basal basal region were obtained from tubules of the distal nephron including the inner (MALin) and outer (MALout) medullary ascending limb, distal convoluted tubule (DCT) and connecting segment (CS). Lower activities were obtained in proximal convoluted (PCT) tubules, proximal straight (PS) tubules and the papillary collecting duct (PD). Distal convoluted tubules contained the highest activity per length of tubule. Other sites contained lower levels of activity in the following order: MALin greater than MALout greater than PCT greater than PD greater than PS. The modifications introduced increase the sensitivity and precision of this assay and permit the application of this technique to studies of [Na-K]ATPase activity in the major functional regions of the rat nephron.     

Assay of Na-K-ATPase in cultured nephron epithelia

Microenzymatic methods have been utilized in the past to quantify the activity of Na-K-ATPase in tubular segments of the mammalian nephron. The assay reported here measures the precipitated inorganic phosphate liberated by the hydrolysis of gamma-32P-ATP. Activity data in single nephron segments of the cortical collecting tubule (CCT) confirm previous work; specifically, first data on enzyme activity in small cultured cell populations derived from CCT demonstrate that the Na-carrier enzyme can be quantified in nephron cell cultures.     

Proximal tubule function in chronic serum sickness glomerulonephritis of rats

Fatal immune complex glomerulonephritis can be induced in rats by chronic intravenous administration of bovine serum albumin. There are three distinct stages, mild, moderate, and severe, in the development of renal immunopathology and pathophysiology in this model of chronic serum sickness. The work described here was undertaken to evaluate aspects of proximal tubule function in those different stages. Tissue water distribution, oxidative metabolism, and transport of representative organic anions and cations were measured in renal cortical slices. In mild chronic serum sickness all functions were normal except the transport of p-aminohippurate (PAH, organic anion), which was significantly decreased. This decrease appeared to be attributable to immunization with Freund's adjuvant. In the moderate stage of chronic serum sickness, proximal tubule functions and morphology appeared essentially normal. Only Na-K-ATPase activity was somewhat lower than in controls. However, proximal tubule dysfunction was a feature of severe chronic serum sickness. A significant inhibition of anion and cation transport was observed. Reduction in transport functions occurred together with impaired oxidative metabolism and severe reduction in Na-K-ATPase activity. Abnormalities of mitochondrial structure, a decrease in number of mitochondria, and a significant increase in intracellular H2O content provided additional evidence of degenerative changes in proximal tubule cells during the severe stage of chronic serum sickness. It was concluded that decreased transport of organic ions by the basolateral membrane in proximal tubules of rats with severe chronic serum sickness resulted from a breakdown in the metabolic machinery of the tubule epithelium rather than a specific injury to organic ion transport systems.     

Influence of L-thyroxine upon enzymatic activity in the renal tubular epithelium of the rat under normal conditions and in mercury-induced lesions. I. Histochemical studies of alkaline phosphatase, acid phosphatase, adenosine- tri-phosphatase and leucine-aminopeptidase.

HgC12-induced renal tubular lesions in the rat present histochemically with a transitory decrease of alkaline phosphatase, adenosinetriphosphatase (ATPase), and leucine-aminopeptidase activity. The toxic alterations of enzyme activity were more pronounced in the pars recta of the proximal tubule and in the loop of Henle, as compared with the tubulus contortus I. L-thyroxine treatment leads to an accelerated reversal of that enzymatic defect, followinga characteristic pattern, and to a differentiating increase of acid phosphatase and ATPase activity in certain parts of the normal renal tubule. The observations are discussed with reference to the specific mode of action of sublimate and l-thyroxine upon the tubular enzymes and to the well-known metabolic and functional influences of thyroid hormone on the kidney.     

Renal sodium transport in vitamin D resistant hypophosphatemic rickets

To investigate the possible role of a Na transport defect in the pathogenesis of the phosphaturia in vitamin D resistant rickets, we studied the activity of the Na-K ATPase activity along the microdissected segments of the nephron in normal (N) and hypophosphatemic mice (Hyp), the Na uptake by renal brush border membrane (BBM), as well as the interrelationship between Na and phosphate transport through this membrane. In N mice, Na-K ATPase activity was present in decreasing order, in the distal tubule, the ascending branch of the loop of Henle, the proximal tubule, and the collecting tubule. In Hyp mice, the Na-K ATPase activity was comparable to that measured in N mice, except in the granular segment of the distal tubule where a 256% of the control activity was reproducibly observed. In N mice, Na initial uptake by BBM vesicles increased with Na concentration in the incubation medium, according to two kinetic components: one saturable, evident at low substrate concentrations and the other, nonsaturable, corresponding to a passive diffusion. The addition of 5 mM PO4 in the incubation medium did not significantly influence Na transport. In contrast, Na concentration in the incubation medium largely modified the kinetics of PO4 uptake: increasing Na concentration enhanced PO4 uptake and decreased the apparent Km. In Hyp mice, Na uptake by BBM was identical to that observed in N mice, but PO4 uptake was decreased by half. Na concentration in the incubation medium similarly influenced PO4 uptake in N and Hyp mice, and the Km values at each concentration of Na were comparable in the two series of animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of ouabain-insensitive K-ATPase activity in rabbit nephron segments during chronic hyperkalemia

Recently, we demonstrated that an ATPase stimulated by K (and not inhibited by ouabain, Na-K-ATPase inhibitor) is present in the connecting tubule (CNT) and collecting duct segments of the rabbit. In this study, we determined the effects of high- and low-K diet on K-ATPase activity in the CNT and collecting duct segments of rabbit. One group of animals was given a low-K diet (34 mEq/kg diet) and the other group was given a high-K diet (700 mEq/kg diet) for 1 week. K-ATPase activity was measured by a microfluorometric assay in which ATP hydrolysis is coupled to oxidation of NADH. Low-K animals had plasma K = 3.1 +/- 0.2 as compared with 5.5 +/- 0.5 mEq/l in high-K animals. Low-K animals had significant K-ATPase activity in CNT, CCD (cortical collecting duct) and MCD (medullary collecting duct). On the other hand, K-ATPase activity in all 3 segments from high-K animals was not significantly different from zero. These results support a hypothesis that chronic K loading suppresses the ouabain-insensitive K-ATPase in the distal nephron.     

Analytical study on Na-K-ATPase (and cysteine insensitive p-nitrophenylphosphatase) in rat kidney-cortex microsomes subfractioned by zonal centrifugation

The common use of Na-K-ATPase as a marker enzyme for basolateral membranes in the kidney is based on the microscopic localization of the enzyme by the cytochemical assay of Na-K-ATPase as cysteine insensitive p-nitrophenylphosphatase (Ernst S.A., J. Cell Biol. 66, 586-606, 1975). Rat kidney cortex plasma membranes were therefore fractionated by differential pelleting in isotonic sucrose, followed by equilibrium banding in linear sucrose gradients, to compare the distribution of "biochemical" and "cytochemical" assayed Na-K-ATPase. In all fractions, the distribution of Na-K-stimulated Mg-dependent ATPase differed from the distribution of cysteine insensitive p-nitrophenylphosphatase (alkaline phosphatase). Evidence is presented that this difference is not only due to the separation of plasma membranes from different cell types, but simply reflects different membrane location of the enzymic activities.     

拟除虫菊酯对家蝇Na-K-ATPase抑制作用的研究

通过对家蝇神经系统Na-K-ATPase性质的研究,表明Na-K-ATPase反应的适宜pH值为7.0～8.0,适温为35～40℃,K_m为0.22 mmol/L,V_max为555.56 nmol/(mg·min)。比较测定了家蝇敏感品系、Del-R、2Cl-R抗性品系的Na-K-ATPase活性及溴氰菊酯和氯菊酯对该酶的抑制作用。实验证明,敏感与抗性品系间Na-K-ATPase活力无显著的差异,但溴氰菊酯和氯菊酯对不同家蝇品系Na-K-ATPase的抑制作用有明显区别,两种拟除虫菊酯可抑制敏感家蝇品系Na-K-ATPase的活性,而对抗性品系无明显的抑制作用。     

Renal sodium handling and stimulation of medullary Na-K-ATPase during blockade of prostaglandin synthesis

The effect of suppression of prostaglandin synthesis on renal sodium handling and microsomal Na-K ATPase was studied in control and indomethacin treated intact rats maintained on a normal sodium diet (series A) and chronically salt loaded (series B). Indomethacin administration resulted in a decreased GFR and a significantly depressed urinary excretion and an increased fractional reabsorption of sodium in animals fed the normal sodium diet or chronically salt loaded. In rats maintained on a normal Na diet, the activity of the renal medullary Na-K ATPase after indomethacin was 206.3 +/- 6.4 ug Pi/mg protein, i.e. significantly higher as compared with the enzyme activity in the medullary renal fraction from control animals in which it averaged 148 +/- 7.79 ug Pi/mg protein (p less than 0.001). While after chronic salt load a similar increment in the activity of renal medullary Na-K ATPase was observed, no additional stimulation was elicited by subsequent indomethacin administration. The addition of exogenous PGE2, 0.1 mM to microsomal fractions obtained from kidneys of normal rats, was associated with a moderate suppression of the medullary Na-K-ATPase activity, from a basal level of 170 +/- 16 to 151.3 +/- 13 umol Pi/mg protein/hr (p less than 0.005). In isolated segments of medullary thick ascending limb of Henle's loop (MTAL) addition of PGE2 to the incubation medium resulted in a significant inhibition of Na-K ATPase from 37.2 +/- 2 to 21.25 +/- 1.17 x 10(-11) mol/mm/min (p less than 0.0001). These findings suggest that the increased renal Na reabsorption after inhibition of PG synthesis might be related, at least partly, to stimulation of medullary Na-K ATPase. In parallel, the reported natriuretic effect of prostaglandins might imply a direct inhibitory effect of these mediators on renal Na-K ATPase.     

肾脏和肾神经在应激、钠盐所致高血压中的作用

本工作采用电生理、生化、放免、电镜等方法,探讨了慢性应激和盐致高血压大鼠交感神经系统和肾脏功能的改变。实验在雄性ＳＤ大鼠上进行。结果表明：（１）高盐大鼠肾血浆流量（ＲＰＦ）和尿钠排泄明显增加,而应激大鼠ＲＰＦ显著下降。（２）电镜显示高盐大鼠近曲和远曲小管上皮细胞及线粒体变大,应激则使细胞萎缩、线粒体变小。（３）高盐大鼠肾皮质ＮａＫＡＴＰ酶活性下降,应激可使其恢复。（４）频谱分析显示应激大鼠低频波动（０２～０９Ｈｚ）明显增加。（５）应激导致大鼠肾素活性（ＰＲＡ）及血管紧张素Ⅱ（ＡＮＧⅡ）水平升高,并能使高盐大鼠低ＰＲＡ和ＡＮＧⅡ水平升高。（６）大鼠去除双侧肾神经后,应激无法造成血压升高、ＲＰＦ下降和ＰＲＡ、ＡＮＧⅡ上升。上述结果提示：肾交感神经系统兴奋性增加介导的肾脏机制,可能在应激和／或盐致高血压发病过程中具有重要作用。     

Effect of osmolality on renal medullary Na-K-ATPase activity in the postobstructive kidney.

The aim of this study was to determine the effect of changes in osmolality on the reduced renal medullary Na-K-ATPase (EC 3.6.1.3) activity of the postobstructive kidney. The effect of osmolality on renal medullary Na-K-ATPase activity was studied by incubating tissue slices from sham-operated and bilaterally obstructed rats in media with osmolality varied before enzyme isolation using sodium chloride, choline chloride, or sucrose. Both sham-operated and bilaterally obstructed rat renal medullary enzyme showed a similar increase in activity with increased osmolality due to sodium chloride. Medullary Na-K-ATPase from the postobstructive kidney also showed increased activity with osmotic changes induced by choline chloride or sucrose. It is proposed that the decrease of Na-K-ATPase activity observed after bilateral ureteral obstruction is due, at least in part, to the loss of the solute concentration gradient in the kidney.     

Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney.

A study has been made to determine whether renal plasma membranes contain an HCO3 stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney. The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase. The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome c oxidase activity. These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Evaluating the utility of the HTRF Transcreener ADP assay technology: A comparison with the standard HTRF assay technology

The HTRF (homogeneous time-resolved fluorescence) Transcreener ADP assay is a new kinase assay technology marketed by Cis-Bio International (Bagnols-Cèze, France). It measures kinase activity by detecting the formation of ADP using a monoclonal antibody and HTRF detection principles. In this article, we compare this technology with a standard HTRF kinase assay using EGFR [L858R/T790M] mutant enzyme as a case study. We demonstrate that the HTRF Transcreener ADP assay generated similar kinetic constants and inhibitor potency compared with the standard HTRF assay. However, the smaller dynamic window and lower Z′ factor of the HTRF Transcreener ADP assay make this format less preferable for high-throughput screening. Based on the assay principle, the HTRF Transcreener ADP assay can detect both kinase and ATPase activities simultaneously. The ability to probe ATPase activity opens up new avenues for assaying kinases with intrinsic ATPase activity without the need to identify substrates, and this can speed up the drug discovery process. However, caution must be exercised because any contaminating ATPase activity will result in an invalid assay. The inability to tolerate high concentrations of ATP in the assay will also limit the application of this technology, especially in compound mechanistic studies such as ATP competition. Overall, the HTRF Transcreener ADP assay provides a new alternative tool to complement existing assay technologies for drug discovery.     

The polarity of the proximal tubule cell in rat kidney. Different surface charges for the brush-border microvilli and plasma membranes from the basal infoldings

Two different membrane fractions were obtained from a brush-border fraction of rat kidney cortex by using their different electrical surface charges in preparative free-flow electrophoresis. One membrane fraction contained only morphologically intact microvilli and was characterized by a high specific activity of alkaline phosphatase. The other fraction morphologically resembled classical plasma membranes by possessing junctional complexes and a high Na-K-ATPase activity The contamination of the isolated membrane fractions by other cell organelles was extremely low These two fractions represent the apical (luminal) and the basal (interstitial) area of the renal proximal tubule cell membrane and clearly demonstrate the polarity of this cell.     

Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney

A study has been made to determine whether renal plasma membranes contain an HCO₃^? stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney.The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase.The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome $\text{c}$ oxidase activity.These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Predicted location and limited accessibility of protein kinase A phosphorylation site on Na-K-ATPase

Regulation of Na-K-ATPase by cAMP-dependent protein kinase occurs in a variety of tissues. Phosphorylation of the enzyme's catalytic subunit at a classical phosphorylation consensus motif has been observed with purified enzyme. Demonstration of phosphorylation at the same site in normal living cells or tissues has been more difficult, however, making it uncertain that the Na-K-ATPase is a direct physiological substrate of the kinase. Recently, the structure of the homologous sarco(endo)plasmic reticulum Ca-ATPase (SERCA1a) has been determined at 2.6 A resolution (Toyoshima C, Nakasako M, Nomura H, and Ogawa H. Nature 405: 647-655, 2000.), and the Na-K- ATPase should have the same fold. Here, the Na-K-ATPase sequence has been aligned with the Ca-ATPase structure to examine the predicted disposition of the phosphorylation site. The location is close to the membrane and partially buried by adjacent loops, and the site is unlikely to be accessible to the kinase in this conformation. Conditions that may expose the site or further bury it are discussed to highlight the issues facing future research on regulation of Na-K-ATPase by cAMP-dependent pathways.     

Relation of Na-K-ATPase to acute changes in renal tubular sodium and potassium transport

Renal Na-K-ATPase activity changes adaptively in response to chronic alterations in sodium reabsorption or potassium secretion, but the role of this enzyme in rapid adjustments of renal tubular Na and K transport is not known. To evaluate this question, microsomal Na-K-ATPase specific activity and kinetics were determined in the rat and guinea pig kidney after massive but short-term (3 h) sodium or potassium loading. In other experiments renal sodium handling was evaluated in hydropenic and saline-loaded rats in which enzyme synthesis was prevented by the concurrent administration of actinomycin D or cycloheximide. Saline loading increased net sodium reabsorption in both rats and guinea pigs, but microsomal Na-K-ATPase from the outer medulla (where the reabsorptive increment is greatest) did not change significantly in either species. In vitro [3H]ouabain bidint to guinea pig microsomes and apparent Km for sodium of rat microsomal Na-K-ATPase, both from outer medulla, were also unaltered. Actinomycin D and cycloheximide failed to increase sodium excretion and microsomal Na-K-ATPase remained unchanged. KCL loading resulted in a 10-fold increase in K excretion but again Na-K-ATPase specific activity (in cortex, outer medulla, and papilla), and its apparent Km for potassium were not affected. Taken together these results suggest that rapid adjustments in remal tubular Na or K transport are mediated by mechanisms that do not involve the Na-K-ATPase enzyme system.     

Active ion transport in the renal proximal tubule. III. The ATP dependence of the Na pump

The dependence of the Na pump activity of intact renal tubules on the ATP concentration was investigated using a suspension of rabbit cortical tubules. Rotenone (an inhibitor of mitochondrial oxidative phosphorylation) was used in graded fashion to alter the cellular ATP, and the Na pump activity was measured when the pump was stimulated by adding KCl to tubules suspended in a K+-free medium. The K+ uptake into the tubule was measured using an extracellular K+ electrode, and the oxygen consumption (QO2) was measured using a Clark-type oxygen electrode. The Na pump activity was found to have a linear, nonsaturating dependence on the ATP concentration. However, the Na,K- ATPase hydrolytic activity (assayed biochemically) of lysed proximal tubule membranes demonstrated saturation and had a K0.5 value of 0.4 mM ATP. Presumably, unknown cytosolic factors present in the intact renal cell but not normally present in the biochemical assay accounted for the differences between the two measurements. The data suggest that an alteration in the intracellular ATP will result in a proportional change in active ion transport activity. Moreover, additional findings also suggest that the basal (non-transport-related) QO2 may be redirected to support the proximal Na pump activity when transport activity is stressed. Thus, basal respiration is not invariant under all conditions, and ion transport activity appears to be maintained foremost among cellular ATP-dependent processes.     

Mitochondrial ATPase in the gills of the shore crabCarcinus maenas

Posterior gills (No. 7 and 8) of shore crabsCarcinus maenas were homogenized and fractionated by means of differential and density gradient centrifugation. Employment of marker enzymes Na-K-ATPase and carbonic anhydrase for plasma membranes and cytochrome oxidase for mitochondria showed that these structural elements were separated. Ultramicroscopic investigations of combined fractions confirmed the presence of the respective mitochondrial and vesicular plasma membrane structures. An ATPase which did not depend on the presence of sodium (20 mM) ions in the incubation medium but on the presence of potassium (20 mM) ions only was found in the mitochondrial fractions. The mitochondrial ATPase was tightly bound to cellular particulates and activated approximately threefold by bicarbonate (20 mM) ions. The activity of this ATPase was nearly completely inhibited by oligomycin (1 μg ml⁻¹) and greatly inhibited by low levels (5 mM) of thiocyanate and calcium ions, the K_i for Ca²⁺ being ca 4 mM. The results obtained confirm literature data on high mitochondrial densities in crab gills and allow the assumption of significant rates of energy metabolism in these organs. Considering its properties the mitochondrial ATPase is clearly distinct from crab gill Na-K-ATPase and can be measured specifically in samples containing Na-K-ATPase. Mitochondrial ATPase is therefore considered a suitable and reliable marker enzyme for mitochondria.