Effect of subchronic treatment with mercury chloride on NTPDase, 5′-nucleotidase and acetylcholinesterase from cerebral cortex of rats

The aim of the present investigation was to evaluate the effect of a subchronic treatment (30 days/30 doses) with subcutaneous injections (0.1 mg/kg) of HgCl₂ on NTPDase (E.C. 3.6.1.5), 5′-nucleotidase (E.C. 3.1.3.5) and acetylcholinesterase (AChE, E.C. 3.1.1.7) activities in brain from adult rats. NTPDase and 5′-nucleotidase were measured in cortical synaptosomal fraction and AChE was measured in the homogenate of cerebral cortex and hippocampus. After the subchronic treatment (30 days), NTPDase activity was enhanced approximately 35% (p < 0.05) with ATP and ADP as substrates and no difference was observed in 5′-nucleotidase activity (AMP hydrolysis). In addition, AChE activity was enhanced in the cerebral cortex (22%, p < 0.05) and hippocampus (26%, p < 0.05) after the subchronic treatment. Mercury deposited in brain was measured by cold vapor (atomic absorption spectrometry) and no difference between the control and the subchronically treated group was observed. Here we showed for the first time that exposure to low levels of Hg²⁺, which resembles occupational exposure to low levels of mercury, caused a marked increase in NTPDase and AChE activities. The relationship of these alterations with the neurotoxicity of inorganic mercury deserves further studies.     

Phosphorylation of the Na,K-ATPase and the H,K-ATPase

Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na⁺,K⁺- and H⁺,K⁺-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations.     

In vivo modification of Na,K-ATPase activity in Drosophila

We have constructed and characterized transgenic Drosophila lines with modified Na⁺,K⁺-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type α subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na⁺,K⁺-ATPase α subunit hypomorphic mutant, 2206. In contrast, a mutant α subunit (α^D369N) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled α transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of α subunit transgene. Using the Gal4-UAS system we have limited expression of α transgenes to cell types that normally express a particular Drosophila Na⁺,K⁺-ATPase β (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.     

Digitalis-induced cell signaling by the sodium pump: On the relation of Src to Na/K-ATPase

In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as ouabain. Based mainly on cell-free studies with mixtures of purified Src kinase and Na⁺/K⁺-ATPase, a well-advocated hypothesis on how ouabain initiates the activation of signaling pathways is that there is a preexisting physiological complex of inactive Src bound to the α-subunit of Na⁺/K⁺-ATPase, and that ouabain binding to this subunit disrupts the bound Src and activates it. Because of the published disagreements of the results of such cell-free experiments of two other laboratories, our aim was to attempt the resolution of these discrepancies. We reexamined the effects of ouabain, vanadate, and oligomycin on mixtures of Src, Na⁺/K⁺-ATPase, Mg²⁺, and ATP as specified in prior studies; and assayed for Src-418 autophosphorylation as the measure of Src activation. In contrast to the findings of the proponents of the above hypothesis, our results showed similar effects of the three inhibitors of Na⁺/K⁺-ATPase; indicating that Src activation in such experiments is primarily due to the ATP-sparing effect of the ATPase inhibitor on the mixture of two enzymes competing for ATP. We conclude that there is no solid evidence for direct molecular interaction of Src with Na⁺/K⁺-ATPase under physiological conditions.     

Dual mechanism of laminin modulation of ecto-5′-nucleotidase activity

The myoblast cell surface activity of ecto-5′-nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto-5′-nucleotidase. This increase was related to a higher expression of ecto-5′-nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E′₁ and E₈ modified the AMPase activity of the ecto-5′-nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E₈, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto-5′-nucleotidase. By contrast, the large fragment derived from the short arms, designated E′₁, inhibited the AMPase activity. Furthermore, the monoclonal anti-ecto-5′-nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E₈ on the AMPase activity of ecto-5′-nucleotidase but did not reverse the inhibitory effect of fragment E′₁. In conclusion, laminin stimulates the AMPase activity of ecto-5′-nucleotidase by two mechanisms: inducing the expression of ecto-5′-nucleotidase to the cell surface and direct modulation of the enzymatic activity.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-subunit expression and K-phosphatase activity

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg^− 1 and K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

Inhibition of Na+,K+-ATPase Activity from Rat Hippocampus by Proline

Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na⁺,K⁺-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg²⁺-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na⁺,K⁺-ATPase, but not Mg²⁺-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na⁺,K⁺-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia.     

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na/K-ATPase

Background

Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na⁺/K⁺-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na⁺/K⁺-ATPase expression and activity in rats injected with Bothrops alternatus snake venom.

Methods

Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na⁺/K⁺-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively.

Results

Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na⁺/K⁺-ATPase α₁ subunit were increased 6 h post-venom, whereas Na⁺/K⁺-ATPase activity increased 6 h and 24 h post-venom.

Conclusions

Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na⁺/K⁺-ATPase expression and activity in the early phase of renal damage.

General significance

Enhanced Na⁺/K⁺-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.     

Nitrophenylphosphate as a tool to characterize gill Na, K-ATPase activity in hyperregulating Crustacea

The kinetic properties of a gill Na(+), K(+)-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na(+), K(+)-ATPase hydrolyzed PNPP (K(+)-phosphatase activity) obeying Michaelis-Menten kinetics with K(M)=1.72+/-0.06 mmol l(-1) and V(max)=259.1+/-11.6 U mg(-1). ATP was a competitive inhibitor of K(+)-phosphatase activity with a K(i)=50.1+/-2.5 micromol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K(0.5)=3.62+/-0.18 mmol l(-1); n(H)=1.5) and magnesium ions (K(0.5)=0.61+/-0.02 mmol l(-1), n(H)=1.3) was found. Sodium ions had no effect on K(+)-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K(+)-phosphatase activity by potassium ions. Ouabain (K(i)=762.4+/-26.7 micromol l(-1)) and orthovanadate (K(i)=0.25+/-0.01 micromol l(-1)) completely inhibited the K(+)-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K(+)-phosphatase activity of the Na(+), K(+)-ATPase alone and suggest that the use of PNPP as a substrate to characterize K(+)-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na(+), K(+)-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.     

Restoration of phosphorylation capacity to the dormant half of the α-subunits of Na, K-ATPase

Purified kidney Na⁺,K⁺-ATPase whose α-subunit is cleaved by chymotrypsin at Leu²⁶⁶-Ala²⁶⁷, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of α-cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the α-subunits of oligomeric membrane-bound enzyme are dormant and that interaction of the N-terminal domain of α-subunit with its phosphorylation domain causes this half-site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all α-subunits.     

Inhibition of Na(+),K(+)-ATPase activity in hippocampus of rats subjected to acute administration of homocysteine is prevented by vitamins E and C treatment

In the present study we evaluated the effect of acute homocysteine (Hcy) administration on Na⁺,K⁺-ATPase activity, as well as on some parameters of oxidative stress such as total radical-trapping antioxidant potential (TRAP) and on activities of antioxidant enzymes catalase (CAT), superoxide dismutase and glutathione peroxidase in rat hippocampus. Results showed that Hcy significantly decreased TRAP, Na⁺,K⁺-ATPase and CAT activities, without affecting the activities of superoxide dismutase and glutathione peroxidase. We also verified the effect of chronic pretreatment with vitamins E and C on the reduction of TRAP, Na⁺,K⁺-ATPase and CAT activities caused by Hcy. Vitamins E and C per se did not alter these parameters, but prevented the reduction of TRAP, Na⁺,K⁺-ATPase and CAT activities caused by Hcy. Our results indicate that oxidative stress is probably involved in the pathogenesis of homocystinuria and that reduction of Na⁺,K⁺-ATPase activity may be related to the neuronal dysfunction found in homocystinuric patients.     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

Erythrocyte pyrimidine 5′-nucleotidase inhibition by acute lead exposure in neonatal rats

Inhibition by lead of erythrocyte pyrimidine 5′-nucleotidase (P5N) is thought to contribute to morphological abnormalities observed in red blood cells (RBC) of lead-exposed subjects. However, neither the mechanism of lead inhibition of P5N nor the relationship of this inhibition to blood lead levels attained in exposed subjects is known. In the present investigation, acute in vivo and in vitro lead acetate effects on erythrocyte P5N from 21-day-old rat pups were determined and were related to blood lead concentrations ascertained by atomic absorption spectrophotometry. Acute lead administration to rat pups resulted in a 16% to 21% reduction in erythrocyte P5N, with mean blood lead levels ranging from 77 to 108 μg/dl 24 hours later. Inhibition of erythrocyte P5N was linearly related to blood lead level (r = ?0.67, P < 0.05) following acute lead administration. Lead acetate addition to RBC preparations from 21-day-old rats resulted in concentration-dependent P5N inhibition which was comparable to that produced following acute in vivo exposure. The results indicate that acute P5N inhibition in lead-treated neonatal rats is due to noncompetitive P5N inhibition by lead. The inhibition of P5N produced by acute lead treatment is linearly related to blood lead concentrations.     

Effect of Cd and Hg on the Activity of Na/K-ATPase and Mg-ATPase Adsorbed on Polystyrene Microtiter Plates

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4°C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na⁺/K⁺-ATPase and Mg²⁺-ATPase, retained 70–80% activity after the adsorption. In addition, adsorption stabilizes Na⁺/K⁺-ATPase and Mg²⁺-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na⁺/K⁺-ATPase and Mg²⁺-ATPase and their sensitivity to and mechanism of Cd²⁺- or Hg²⁺-induced inhibition. The only exception is the “high affinity” Mg²⁺-ATPase moiety, whose affinity for ATP and sensitivity toward Cd²⁺ were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Effect of water temperature,salinity, and their interaction on growth,plasma osmolality,and gill Na,K-ATPase activity in juvenile GIFT tilapia Oreochromis niloticus (L.)

We used a central composite rotatable experimental design and response surface methodology to evaluate the effects of temperature (18–37 °C), salinity (0–20‰), and their interaction on specific growth rate (SGR), feed efficiency (FE), plasma osmolality, and gill Na⁺, K⁺-ATPase activity in GIFT tilapia juveniles. The linear and quadratic effects of temperature and salinity on SGR, plasma osmolality, and gill Na⁺, K⁺-ATPase activity were statistically significant (P<0.05). The interactive effects of temperature and salinity on plasma osmolality were significant (P<0.05). In contrast, the interaction term was not significant for SGR, FE, and gill Na⁺, K⁺-ATPase activity ⁽P>0.05). The regression equations for SGR, FE, plasma osmolality, and gill Na⁺, K⁺-ATPase activity against the two factors of interest had coefficients of determination of 0.944, 0.984, 0.966, and 0.960, respectively (P<0.01). The optimal temperature/salinity combination was 28.9 °C/7.8‰ at which SGR (2.26% d¹) and FE (0.82) were highest. These values correspond to the optimal temperature/salinity combination (29.1 °C/7.5‰) and the lowest plasma osmolality (348.38 mOsmol kg⁻¹) and gill Na⁺, K⁺-ATPase activity (1.31 µmol Pi. h⁻¹ g⁻¹ protein), and resulted in an energy-saving effect on osmoregulation, which promoted growth.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca/Mg)-ATPase, and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

1,3-dihydroxy-5-(tridec-4′,7′-dienyl)benzene: a new cytotoxic compound from Lithraea molleoides

A dichloromethane extract from the leaves of Lithraea molleoides (Anacardiaceae), an argentine medicinal plant, showed cytotoxicity on human hepatocellular carcinoma cell line. Bioassay guided fractionation of this extract led to the isolation of a new active 5-alkyl resorcinol: 1,3-dihydroxy-5-(tridec-4',7'-dienyl)benzene. Chemical structure was established based on spectroscopic data (UV, IR, MS, 1H-NMR, 13C-NMR, COSY). This compound presented cytotoxic activity on 3 human tumoral cell lines: hepatocellular carcinoma cell line-Hep G2 (IC50 +/- SD of 68 +/- 2 microM), mucoepidermoid pulmonary carcinoma cell line-H292 (IC50 +/- SD of 63 +/- 5 microM) and mammary gland adenocarcinoma cell line -MCF7 (IC50 +/- SD of 147 +/- 5).