Comparison of the activities of Na(+),K(+)-ATPase in brains of rats at different ages

BACKGROUND: Na(+),K(+)-ATPase is known to be responsible for the ionic homeostasis in excitable tissues. The energy cost of Na(+),K(+)-ATPase activity is increased in the active brain, so it would be important to ascertain whether defects in brain metabolism in aging are associated with changes in the properties of Na(+),K(+)-ATPase. OBJECTIVE: The aim of this study was to investigate the influence of age on the Na(+),K(+)-ATPase activity in developing rat brains from the age of 1 day to 24-28 months. METHODS: Crude microsomal preparations were obtained from the brains of newborn (n = 8), 18-day-old (n = 8), 4- to 5-month-old (n = 12), and 24- to 28-month-old (n = 14) rats. Then the ATPase activity was measured and expressed as micromoles of inorganic phosphorus released per milligram of protein per hour. RESULTS: The increased tendency in brain Na(+),K(+)-ATPase activity from newborn to 18 days of age suggested that the Na pump is mature soon after birth. No significant differences could be detected in the enzyme activity between newborn and adult rats. In contrast, the Na(+),K(+)-ATPase activity in aged rat brains was found to be significantly lower than in the other age groups of rats tested (p < 0.001). CONCLUSION: Our results suggest that aging-induced inhibitions in the brain Na(+),K(+)-ATPase activity may be implicated in the depression of neuronal excitability and in the age-related impairments of cognitive functions.     

Effects of fulminant hepatic encephalopathy on the adult rat brain antioxidant status and the activities of acetylcholinesterase, (Na+,K+)- and Mg2+-ATPase: comparison of the enzymes’ response to in vitro treatment with ammonia

Hepatic encephalopathy can be a life-threatening complication of fulminant hepatic failure. By understanding the pathophysiology involved in the induction of this neuropsychiatric disorder, future therapeutic and/or preventive attempts could be considered. In this study, an attempt has been made in order to shed more light on the mechanisms involved in the effects of thioacetamide (TAA)-induced fulminant hepatic encephalopathy on: (a) the adult rat brain total antioxidant status (TAS) and (b) the activities of acetylcholinesterase (AChE), (Na(+),K(+))-ATPase and Mg(2+)-ATPase. Moreover, in vitro experiments were conducted in order to evaluate the possible role of ammonia (incubated as NH(4)Cl, in a toxic concentration of 3mM) in the observed effects of TAA-induced fulminant hepatic encephalopathy on the examined adult rat brain enzyme activities. Fulminant hepatic encephalopathy caused a significant decrease in TAS (-22%, p < 0.001) and the activity of Na(+),K(+)-ATPase (-26%, p < 0.001), but had non-significant effects on the whole brain AChE and Mg(2+)-ATPase activities. The in vitro experiments (conducted through a 3h incubation with ammonia), showed no significant alterations in any of the examined parameters. Our in vitro and in vivo findings suggest that alterations in AChE and Mg(2+)-ATPase activities are not involved in the pathophysiology of the adult-onset fulminant hepatic encephalopathy, while the observed Na(+),K(+)-ATPase inhibition could be a result of the oxidative stress, neurotransmission deregulation, and/or of the presence of other toxic substances (that appear to act as direct or indirect inhibitors of the enzyme) and not due to the excess accumulation of ammonia in the brain.     

C-Peptide stimulates Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity via PKC alpha in rat medullary thick ascending limb

AIMS/HYPOTHESIS: C-peptide, the cleavage product of proinsulin processing exerts physiological effects including stimulation of Na(+),K(+)-ATPase in erythrocytes and renal proximal tubules. This study was undertaken to assess the physiological effects of connecting peptide on Na(+),K(+)-ATPase activity in the medullary thick ascending limb of Henle's loop. METHODS: Na(+),K(+)-ATPase activity was measured as the ouabain-sensitive generation of (32)Pi from gamma[(32)P]-ATP and (86)Rb uptake on isolated rat medullary thick ascending limbs. The cell-surface expression of Na(+),K(+)-ATPase was evaluated by Western blotting of biotinylated proteins, and its phosphorylation amount was measured by autoradiography. The membrane-associated fraction of protein kinase C isoforms was evaluated by Western blotting. RESULTS: Rat connecting peptide concentration-dependently stimulated Na(+),K(+)-ATPase activity with a threshold at 10(-9) mol/l and a maximal effect at 10(-7) mol/l. C-peptide (10(-7) mol/l) already stimulates Na(+),K(+)-ATPase activity after 5 min with a plateau from 15 to 60 min. C-peptide (10(-7) mol/l) stimulated Na(+),K(+)-ATPase activity and (86)Rb uptake to the same extent, but did not alter Na(+),K(+)-ATPase cell surface expression. The stimulation of Na(+),K(+)-ATPase activity was associated with an increase in Na(+),K(+)-ATPase alpha-subunit phosphorylation and both effects were abolished by a specific protein kinase C inhibitor. Furthermore, connecting peptide induced selective membrane translocation of PKC-alpha. CONCLUSION/INTERPRETATION: This study provides evidence that in rat medullary thick ascending limb, C-peptide stimulates Na(+),K(+)-ATPase activity within a physiological concentration range. This effect is due to an increase in Na(+),K(+)-ATPase turnover rate that is most likely mediated by protein kinase C-alpha phosphorylation of the Na(+),K(+)-ATPase alpha-subunit, suggesting that C-peptide could control Na(+) reabsorption during non-fasting periods.     

Alterations of hepatic Na+,K+-ATPase and bile flow by estrogen: Effects on liver surface membrane lipid structure and function

Administration of the synthetic estrogen ethinyl estradiol (17alpha-ethinyl-1,3,5-estratriene-3,17beta-diol) decreases hepatic Na(+),K(+)-ATPase (ATP phosphohydrolase; EC 3.6.1.3) activity and bile flow to 50% and alters the composition and structure of surface membrane lipid in rats. Although the content of phospholipids was not changed by treatment, free cholesterol (130%) and cholesterol esters (400%) were increased in liver surface membrane fractions. These observations correlate with changes in membrane viscosity, as shown by electron spin resonance probes. Both rotational correlation time, using the isotropic probe methyl (12-nitroxyl)stearate, and the order parameter, determined by the anisotropic probe 5-nitroxylstearic acid, were significantly increased in liver surface membrane fractions from rats treated with ethinyl estradiol. Administration of Triton WR-1339, a nonionic detergent that corrects hepatic and serum lipid changes caused by ethinyl estradiol treatment, restored toward normal elevated membrane lipids and viscosity as well as Na(+),K(+)-ATPase activity and bile flow. Although restoration of normal liver surface membrane structure and function may be due to reversal of abnormal lipid composition, detergents also may directly alter membrane enzyme activity. Addition of Triton WR-1339 in vitro increased Na(+),K(+)-ATPase activity and reduced membrane viscosity of surface membranes from rats treated with ethinyl estradiol. Triton had no effect on either parameter in normal membrane preparations. Studies of membrane structure and function both in vivo and in vitro suggest that alterations in lipid composition may alter Na(+),K(+)-ATPase function and bile flow.     

High-affinity ouabain binding by a chimeric gastric H+,K+-ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the alpha 1-subunit of rat Na+,K+-ATPase

Na(+),K(+)-ATPase and gastric H(+),K(+)-ATPase are two related enzymes that are responsible for active cation transport. Na(+), K(+)-ATPase activity is inhibited specifically by ouabain, whereas H(+),K(+)-ATPase is insensitive to this drug. Because it is not known which parts of the catalytic subunit of Na(+),K(+)-ATPase are responsible for ouabain binding, we prepared chimeras in which small parts of the alpha-subunit of H(+),K(+)-ATPase were replaced by their counterparts of the alpha(1)-subunit of rat Na(+),K(+)-ATPase. A chimeric enzyme in which transmembrane segments 5 and 6 of H(+), K(+)-ATPase were replaced by those of Na(+),K(+)-ATPase could form a phosphorylated intermediate, but hardly showed a K(+)-stimulated dephosphorylation reaction. When transmembrane segments 3 and 4 of Na(+),K(+)-ATPase were also included in this chimeric ATPase, K(+)-stimulated dephosphorylation became apparent. This suggests that there is a direct interaction between the hairpins M3-M4 and M5-M6. Remarkably, this chimeric enzyme, HN34/56, had obtained a high-affinity ouabain-binding site, whereas the rat Na(+), K(+)-ATPase, from which the hairpins originate, has a low affinity for ouabain. The low affinity of the rat Na(+),K(+)-ATPase previously had been attributed to the presence of two charged amino acids in the extracellular domain between M1 and M2. In the HN34/56 chimera, the M1/M2 loop, however, originates from H(+),K(+)-ATPase, which has two polar uncharged amino acids on this position. Placement of two charged amino acids in the M1/M2 loop of chimera HN34/56 results in a decreased ouabain affinity. This indicates that although the M1/M2 loop affects the ouabain affinity, binding occurs when the M3/M4 and M5/M6 hairpins of Na(+),K(+)-ATPase are present.     

Renal aging in WKY rats: changes in Na+,K+ -ATPase function and oxidative stress

It has been suggested that alterations in Na(+),K(+)-ATPase mediate the development of several aging-related pathologies, such as hypertension and diabetes. Thus, we evaluated Na(+),K(+)-ATPase function and H(2)O(2) production in the renal cortex and medulla of Wistar Kyoto (WKY) rats at 13, 52 and 91 weeks of age. Creatinine clearance, proteinuria, urinary excretion of Na(+) and K(+) and fractional excretion of Na(+) were also determined. The results show that at 91 weeks old WKY rats had increased creatinine clearance and did not have proteinuria. Despite aging having had no effect on urinary Na(+) excretion, urinary K(+) excretion was increased and fractional Na(+) excretion was decreased with age. In renal proximal tubules and isolated renal cortical cells, 91 week old rats had decreased Na(+),K(+)-ATPase activity when compared to 13 and 52 week old rats. In renal medulla, 91 week old rats had increased Na(+),K(+)-ATPase activity, paralleled by an increase in protein expression of α(1)-subunit of Na(+),K(+)-ATPase. In addition, renal H(2)O(2) production increased with age and at 91 weeks of age renal medulla H(2)O(2) production was significantly higher than renal cortex production. The present work demonstrates that although at 91 weeks of age WKY rats were able to maintain Na(+) homeostasis, aging was accompanied by alterations in renal Na(+),K(+)-ATPase function. The observed increase in oxidative stress may account, in part, for the observed changes. Possibly, altered Na(+),K(+)-ATPase renal function may precede the development of age-related pathologies and loss of renal function.     

Sodium tanshinone iia sulfonate attenuates seawater aspiration-induced acute pulmonary edema by up-regulating Na(+),K(+)-ATPase activity

Relieving pulmonary edema is the key of a successful treatment to seawater drowning. Sodium tanshinone IIA sulfonate (STS) has been observed to reduce lung edema from lipopolysaccharide (LPS)-induced lung injury. In this study the authors investigated whether STS attenuates seawater aspiration-induced acute pulmonary edema, and examined the effects of sodium-potassium adensosine triphosphatase (Na(+),K(+)-ATPase) on it. Seawater was instilled through an endotracheal tube. The anesthetized and spontaneously breathing rats received STS intraperitoneally after seawater aspiration. Pao(2), lung wet-to-dry weight ratio, and pulmonary microvascular permeability were tested. The authors explored the effects of STS on the expression and activity of Na(+),K(+)-ATPase in vivo and in vitro. Additionally, the authors investigated the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in the stimulation of Na(+),K(+)-ATPase by STS. The results showed that STS significantly improved hypoxemia, attenuated lung edema, and alleviated seawater-induced lung injury in vivo. Both in vivo and in vitro, it was observed that STS up-regulated the expression and activity of Na(+),K(+)-ATPase. ERK1/2 inhibitor partially blocked the effects of STS on Na(+),K(+)-ATPase activity in alveolar type II cells following seawater incubation. These results indicated that STS could improve seawater aspiration-induced acute pulmonary edema by up-regulating Na(+),K(+)-ATPase activity, and the ERK1/2 signaling pathway may be involved in it.     

Platelet Na,K-adenosine triphosphatase as a tissue marker of hyperthyroidism

Platelet Na(+),K(+)-adenosine triphosphatase (ATPase) activity was measured in 34 (15 males, 19 females) healthy subjects, 89 (35 males, 54 females) hyperthyroid patients, and 34 (7 males, 27 females) treated hyperthyroid patients to assess the potential of this measurement as a tissue marker and diagnostic test for hyperthyroidism. Platelet Na(+),K(+)-ATPase activity was measured in platelet lysates by the rate of release of phosphate from adenosine triphosphate (ATP) in the presence and absence of ouabain. Platelet Na(+),K(+)-ATPase activity (median and range) in the hyperthyroid group (271, 169 to 821 pmol/h/g protein) was significantly higher compared with the healthy group (125, 74 to 185 micromol/h/g protein, P <.001 by Mann-Whitney U test). The treated hyperthyroid group had slightly, but significantly higher, free triiodothyronine (FT3) and free thyroxine (FT4), as well as platelet Na(+),K(+)-ATPase activity (147, 98 to 246 micromol/h/g protein, P <.05). If a platelet Na(+),K(+)-ATPase activity of 190 micromol/h/g protein was used as a cut off value, the specificity and sensitivity were 90% and 93%, respectively. We conclude that platelet Na(+),K(+)-ATPase may be a useful tissue marker of hyperthyroidism.     

Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase alpha subunit and regulates its trafficking

Endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I(A) phosphoinositide-3 kinase (PI3K-I(A)) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I(A), through its p85alpha subunit-SH3 domain, binds to a proline-rich region in the Na(+),K(+)-ATPase catalytic alpha subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na(+),K(+)-ATPase alpha subunit and results in increased PI3K-I(A) activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na(+),K(+)-ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I(A) and its activation during Na(+),K(+)-ATPase endocytosis in response to G protein-coupled receptor signals.     

Molecular and functional studies of electrogenic Na(+) transport in the distal colon and rectum of young and elderly subjects

BACKGROUND: Human distal nephron and distal colon both exhibit mineralocorticoid sensitive electrogenic Na(+) absorption and make significant contributions to Na(+) homeostasis. Na(+) resorption in the distal nephron diminishes with age but it is unclear whether a similar change occurs in the distal colon. AIMS: To evaluate the effect of age on expression of apical Na(+) channels and basolateral Na(+), K(+)-ATPase, and on the responsiveness of electrogenic Na(+) absorption to mineralocorticoid stimulation in human distal colon and rectum. MATERIALS AND METHODS: Mucosal biopsies were obtained from healthy sigmoid colon and proximal rectum in "young" (aged 20-40 years) and "old" (aged 70 years or over) patients during routine colonoscopy/flexible sigmoidoscopy. Na(+) channel subunits and Na(+), K(+)-ATPase isoforms were studied at the mRNA level by in situ hybridisation and northern blotting, and at the protein level by immunocytochemistry and western blotting. The mineralocorticoid responsiveness of electrogenic Na(+) absorption was evaluated in the two groups by measuring amiloride sensitive electrical potential difference (PD) in the proximal rectum before and 24 hours after oral administration of 1 mg of fludrocortisone. RESULTS: Na(+) channel subunit and Na(+), K(+)-ATPase isoform expression at the level of mRNA and protein was similar in "young" and "old" patients. Both basal and the fludrocortisone stimulated amiloride sensitive rectal PDs were similar in the two groups. CONCLUSIONS: In contrast with the distal nephron, mineralocorticoid sensitive electrogenic Na(+) absorption in the human distal colon does not diminish with age, and may be particularly important in maintaining Na(+) homeostasis in the elderly.     

Osmoregulatory action of PRL,GH, and cortisol in the gilthead seabream (Sparus aurata L)

The osmoregulatory actions of ovine prolactin (oPRL), ovine growth hormone (oGH), and cortisol were tested in the euryhaline gilthead seabream Sparus aurata. Acclimated to sea water (SW, 40 ppt salinity, 1000 mOsm/kg H(2)O) or brackish water (BW, 5 ppt, salinity, 130 mOsm/kg H(2)O), injected every other day for one week (number of injections, 4) with saline (0.9% NaCl), oPRL (4 microg/g body weight), oGH (4 microg/g body weight) or cortisol (5 microg/g body weight), and transferred from SW to BW or from BW to SW 24h after the last injection. Fish were sampled before and 24h after transfer. Gill Na(+), K(+)-ATPase activity, plasma osmolality, plasma ions (sodium and chloride), plasma glucose, and muscle water moisture were examined. SW-adapted fish showed higher gill Na(+), K(+)-ATPase activity, plasma osmolality, and plasma ions levels than BW-adapted fish. Transfer from SW to BW decreased plasma osmolality and ions levels after 24h, while transfer from BW to SW increased these parameters, whereas gill Na(+),K(+)-ATPase activity was unaffected. oPRL treatment significantly decreased gill Na(+),K(+)-ATPase activity and increased plasma osmolality and ions in SW- and BW-adapted fish. This treatment minimizes loss of osmolality and ions in plasma after transfer to BW and increased these values after transfer to SW. No significant changes were observed in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions in oGH-treated group with respect to saline group before or after transfer from SW to BW or from BW to SW. Treatment with cortisol induced, in SW-adapted fish, a significant increase of gill Na(+),K(+)-ATPase activity and decrease of plasma osmolality and plasma ions. In BW-adapted fish this treatment induced a significant increases in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions. After transfer to SW cortisol-treated fish had higher plasma osmolality than the saline group. Our results support the osmoregulatory role of PRL in the adaptation to hypoosmotic environment in the gilthead seabream S. aurata. Further studies will be necessary to elucidate the osmoregulatory role of GH in this species. Cortisol results suggest a "dual osmoregulatory role" of this hormone in S. aurata.     

Sulfite increases lipoperoxidation and decreases the activity of catalase in brain of rats

The main objective of this study was to investigate the in vitro effects of sulfite, a metabolite accumulated in isolated sulfite oxidase deficiency, on Na ⁺, K ⁺-ATPase activity and on some parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBARS) and catalase activity (antioxidant enzyme) in cerebral cortex, striatum and hippocampus from 10- and 60-day-old rats. Results showed that 500 μM sulfite significantly increased TBARS and reduced catalase activity in the cerebral structures studied from neonates and adults rats; in contrast, sulfite did not alter Na⁺, K⁺-ATPase activity. Our present findings show that sulfite increases lipid peroxidation and decreases antioxidant enzyme defenses in rat brain, suggesting an induction of oxidative stress. We presumed that oxidative stress might be, at least in part, associated with the neuronal dysfunction of patients affected by isolated sulfite oxidase deficiency.     

Equilibrated diet restores the effects of early age choline-deficient feeding on rat brain antioxidant status and enzyme activities: the role of homocysteine, <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine and <Emphasis Type="SmallCaps">l</Emphasis>-alanine

Choline is an essential nutrient that seems to be involved in a wide variety of metabolic reactions and functions, that affect the developing brain. The aim of this study was to: (a)examine the effects of early age choline deficient diet (CDD) administration on the total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), (Na(+),K(+))-ATPase and Mg(2+)-ATPase in the rat brain, (b)investigate the effect of feeding restoration into an equilibrated diet on the above parameters, and (c)study the role of homocysteine (Hcy), L: -phenylalanine (Phe) and L: -alanine (Ala) in certain of the above effects. Male and female Wistar rats were continuously kept off choline (Ch) during their gestational period of life, as well as during the first 6 weeks of their post-gestational life. The animals were sacrificed by decapitation and their whole brains were rapidly removed and homogenated. Their enzyme activities were measured spectrophotometrically. Moreover, in vitro experiments were conducted in order to estimate the effects of Hcy (0.3 mM), Phe (1.2 mM) and/or Ala (1.2 mM) on the above parameters. The administration of CDD led to a statistically significant decrease of the rat brain TAS (-29%, p < 0.001) and to a significant increase of both AChE (+20%, p < 0.001) and (Na(+),K(+))-ATPase (+35%, p < 0.001) activities. Mg(2+)-ATPase activity was found unaltered. Equilibrated diet, administered to early age CDD-treated rats of both sexes for an additional period of 18 weeks, restored the above parameters to control levels. Moreover, the in vitro experiments showed that Hcy could simulate these changes (at least under the examined in vitro conditions), while both Phe and Ala act protectively against the CDD-induced effects on the examined rat brain enzyme activities. The effects of early age CDD-feeding on the examined parameters are proved to be reversible through restoration to equilibrated diet, while our data suggest a role for Hcy (as a causative parameter for the CDD-induced effects) and a possible protective role for Phe and Ala (in reversing the observed CDD-induced effects).     

Synergistically Stimulated (Na+,K+)-Adenosine Triphosphatase from Plasma Membrane of a Marine Diatom

An ATP-hydrolyzing activity with the properties of a Mg(2+)-dependent (Na(+),K(+))-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from a 20-fold purified plasma membrane fraction of the marine diatom, Nitzschia alba is described.The basal activity requires Mg(2+) and further stimulation by Na(+) or Na(+) plus K(+) is dependent on the presence of Mg(2+); Mn(2+) or Co(2+) can partially substitute for the divalent cation requirement but Ca(2+) equimolar with Mg(2+) inhibits the activity by 54%. ATP is the preferred substrate for the Na(+) plus K(+) stimulated activity, while CTP, UTP, and ADP are only slightly hydrolyzed. The apparent K(m) is 8 x 10(-4) M ATP.The ATP hydrolysis-rate is dependent on the relative concentrations of Na(+) and K(+); the K(0.5) for Na(+) and K(+) are 2 mM and 50 mM, respectively. Basal activity is synergistically stimulated by Na(+) plus K(+) only at certain ion concentrations and shows a strong specificity for both cations.In the presence of Na(+) at 5 mM and K(+) at 350 mM, the ATPase is completely inhibited by p-chloromercuric benzoic acid 10(-4) M, N-ethyl maleimide 10(-3) M, and iodoacetamide 10(-2) M, but is insensitive to ouabain at 10(-7) to 10(-3) M.This study demonstrates for the first time that algal plasma membrane contains an ATPase that is synergistically stimulated by Na(+) and K(+).     

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.     

Rat cardiac ventricle has two Na+,K+-ATPases with different affinities for ouabain: developmental changes in immunologically different catalytic subunits

The mechanism of the inotropic effect of cardiac glycosides on the heart has long been controversial. Inotropic effects at low concentrations of cardiac glycosides indicate more than one class of receptor or more than one cellular mechanism. In the brain of the rat, high- and low-affinity cardiac glycoside receptors have been shown to be associated with two structurally different isoforms of the catalytic subunit of the Na+,K+-ATPase, termed alpha and alpha(+). Evidence is presented here that the high- and low-affinity sites in rat cardiac ventricle are associated with Na+,K+-ATPase catalytic subunit forms similar to the alpha(+) and alpha forms in the brain. Membranes from the rat ventricle contained polypeptides with the electrophoretic mobilities of alpha and alpha(+), which could be stained by isoform-specific anti-Na+,K+-ATPase antibodies on electrophoretic blots. Both polypeptides also displayed Na+-stimulated phosphorylation with [gamma-32P]ATP. Inhibition of Na+,K+-ATPase activity by ouabain demonstrated the presence of both high- and low-affinity ATPases proportional to the presence of the alpha(+) and alpha polypeptides. The ratios of the two isoforms changed with postnatal maturation, paralleling known changes in cardiac physiology and cardiac glycoside sensitivity. Cardiac glycoside sensitivity can evidently be regulated at the level of gene expression by developmental signals.     

Sodium pump inhibition and regional expression of sodium pump alpha-isoforms in lens

Both hypertension and cataract formation have been associated with reductions in sodium pump activity, possibly as a result of an endogenous inhibitor. The objective of the present study was to answer 4 closely related questions: (1) Is the lens sodium pump effectively inhibited by a labile, digitalis-like factor we have identified in the peritoneal dialysate from hypertensive patients in end-stage renal failure? (2) How does that inhibition compare to that induced by ouabain? (3) Does sodium pump isoform distribution determine the degree of lens sodium pump inhibition? (This question was precipitated by the unanticipated finding that the labile DLF was more effective in inhibiting lens sodium pump than was anticipated.) (4) Is sodium pump activity altered in lens in response to increased salt intake, a maneuver known to increase endogenous digitalis-like factor? We found that whereas ouabain produced equivalent or significantly less inhibition of lens Na(+), K(+)-ATPase from calf or rabbit, respectively, compared with brain, labile digitalis-like factor preferentially inhibited lens compared with brain. Analysis of whole-lens preparations from rabbit, calf, and normal human lens revealed substantial alpha2- and alpha3-isoforms of the sodium pump but little alpha1-isoform. Ouabain inhibition of whole-lens Na(+),K(+)-ATPase from rabbit and calf were comparable: for rabbit lens, K(i)=5.2x10(-7) mol/L; for calf lens, K(i)=1.0x10(-6) mol/L. Limited quantities of labile digitalis-like factor prohibited similar determinations; however, its concentration-activity profile paralleled that of ouabain. Na(+), K(+)-ATPase activity, measured in the 3 major anatomic regions of lens and normalized to nucleus, was greatest in epithelium (56. 9+/-17.9) compared with cortex (5.8+/-1.4) and nucleus (1.0+/-0.0; P=0.01). Immunohistochemistry of rabbit lens found abundant alpha2- and alpha3-isoforms in epithelium and limited alpha3 but undetectable alpha1 in cortex and nucleus. Finally, rats randomized to a high Na diet showed significantly reduced lens Na(+), K(+)-ATPase activity compared with those on a low Na diet, consistent with the effects of a sodium pump inhibitor. In conclusion, the present study suggests that digitalis-like factor may provide a link between hypertension and cataract formation.