Effect of Toluene and Nutritional Status on Serotonin,Lipid Peroxidation Levels and NA<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase in Adult Rat Brain

The aim was to evaluate the effect of toluene and nutritional status on levels of serotonin (5-HT), 5-hydroxytryptophan (5-HTP), Na⁺/K⁺-ATPase, total ATPase and lipid peroxidation (TBARS) in rat brain. Study was conducted with malnourished (MN), well-nourished (WN) and normal Wistar rats. Three groups were formed for each nutritional status: control group I received 0.9% NaCl; toluene (1 g/kg) was administered to group II, and 1.5 g/kg to group III. Levels of 5-HT decreased (P < 0.05) in WN toluene groups, and 5-HTP decreased (P < 0.05) in the WN 1 g toluene and MN 1.5 g toluene groups. TBARS decreased (P < 0.05) in WN toluene groups. A trend to increase in Na⁺/K⁺-ATPase was found in WN and MN toluene groups, while total ATPase increased (P < 0.05) in the WN 1.5 g toluene group. The results suggest that high concentrations of toluene in single doses induce significant changes in the serotonergic system and alter membrane fluidity more perceptibly in the brain of adult animals with regular diet than in malnourished animals.     

The sodium-plus-potassium ion-activated adenosine triphosphatase of cerebral microsomal fractions: treatment with disrupting agents

1. The Na⁺-plus-K⁺-stimulated adenosine triphosphatase [(Na⁺,K⁺)-ATPase] of microsomal preparations from ox brain was inactivated or diminished in activity by exposure to 2–8m-urea. Similar concentrations of urea diminished the turbidity of the suspensions. 2. Low concentrations (about 2·5mm) of NaATP with the urea gave partial or complete protection of the ATPase, without altering the concomitant change in turbidity. Some protection of the (Na⁺,K⁺)-ATPase was afforded by tris ATP, but the greatest protection was found with NaATP and in its presence the change in (Na⁺,K⁺)-ATPase with 3m-urea included a phase in which activity was enhanced by 40%. 3. The protective effect was specific to NaATP: KATP, NaADP, NaAMP and sodium pyrophosphate were without protective effect and in some cases they augmented the action of urea. 4. The turbidity of cerebral microsomal suspensions was diminished also by ultrasonic irradiation; NaATP did not alter this change. After ultrasonic treatment up to 55% of the protein and of the ATPase activity were no longer deposited by centrifugal forces of 4·5×10⁶g-min. 5. Ultrasonic treatment and centrifugation could be carried out with little or no loss of ATPase and ammonium sulphate flocculation of the supernatant then afforded in the first material precipitated a three- to five-fold enrichment of (Na⁺,K⁺)-ATPase activity. 6. Sodium borohydride and dimethyl sulphoxide also diminished the turbidity of the microsomal fraction but enrichment of the ATPase was not effected by these reagents; ten other compounds were without action on the ATPase. 7. Acetyl phosphate was hydrolysed by the microsomal preparation and this activity was increased by added K⁺. Acetyl-phosphatase activity persisted in the ultrasonically treated and ammonium sulphate-fractionated preparations, which were more exacting in their requirements for K⁺. 8. The findings are discussed in relation to the mechanism of the (Na⁺,K⁺)-ATPase.     

Amino group modification of (Na++K+)-ATPase

The effects of three amino group reagents on the activity of (Na⁺+K⁺)-ATPase³ and its component K⁺-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na⁺ of Mg²⁺ present, the enzyme inactivation process follows complicated kinetics. In the presence of K⁺, Rb⁺, or Tl⁺, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg²⁺ (P ₅₀ 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ and K⁺ have no effects, and ATP only a minor effect, on the degree of modification. The K⁺-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na⁺+K⁺)-ATPase activity. Half-inhibition of the (Na⁺+K⁺)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ has no effect, and ATP has only a slight protecting effect. The K⁺-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na⁺+K⁺)-ATPase activity. Half-inactivation of the (Na⁺+K⁺)-ATPase activity is obtained after 20% modification of the amino groups.This article is No. 52 in the series Studies on (Na⁺+K⁺)-Activated ATPase.     

Kinetic analysis of gill (Na⁺,K⁺)-ATPase activity in selected ontogenetic stages of the Amazon River shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): interactions at ATP- and cation-binding sites

We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄ ⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K _M = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺)-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K _0.5 = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄ ⁺ had no modulatory effect. The affinity for Na⁺ (K _0.5 = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄ ⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄ ⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.     

Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity in the Posterior Gills of the Blue Crab, <Emphasis Type="Italic">Callinectes ornatus</Emphasis> (Decapoda,Brachyura): Modulation of ATP Hydrolysis by the Biogenic Amines Spermidine and Spermine

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ and on inhibition by ouabain of posterior gill microsomal Na⁺,K⁺-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na⁺,K⁺-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na⁺,K⁺-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis–Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na⁺,K⁺-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.     

(Na+,K+)-ATPase: a new assay of Na+-ATPase reveals covert anti-pump antibodies

A new assay is described for rat (Na⁺,K⁺)-ATPase [EC 3.6.1.3] prepared from renal medullary or crude liver membranes. With ATP at 1 μm, initial rates of ouabain-sensitive decreases in substrate concentrations are followed by measuring diminished ATP-driven luciferin-luciferase light production. Under these conditions, using highly purified enzyme preparations, Na⁺ and K⁺ ions stimulate and inhibit initial ATP hydrolysis rates, respectively. Therefore, it is likely that the assay measures Na⁺-ATPase partial reactions of the pump. A monospecific polyclonal rabbit anti-rat pump antiserum blocks Na⁺-dependent ATPase measured with the luciferase-linked ATPase assay, whereas conventional assays of purified pump activity at 3.0 mm ATP fail to reveal immunochemical blockade.     

C-peptide,Na+,K+-ATPase,and Diabetes

Na⁺,K⁺-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na⁺,K⁺-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na⁺,K⁺-ATPase activity was strongly related to blood C-peptide levels in non–insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene.Apolymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na⁺,K⁺-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na⁺,K⁺-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na⁺,K⁺-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na⁺,K⁺-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na⁺,K⁺-ATPase activity. This impairment in Na⁺,K⁺-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetesinduced decrease in Na⁺,K⁺-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na⁺,K⁺-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na⁺,K⁺-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na⁺,K⁺-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

Effects of tricyclohexylhydroxytin on the kinetics of adenosine triphosphatase system and protection by thiol reagents

Tricyclohexylhydroxytin, commonly known as Plictran® inhibited Na⁺, K⁺ -ATPase activity of rat brain synaptosomes in a concentration-dependent manner with median inhibitory concentration (IC-50) of 2 μM. Both K⁺ -stimulated para-nitrophenylphosphatase and [3-H]-ouabain binding to synaptosomes were also inhibited by Plictran with IC-50 values of 11 and 30 μM, respectively. Altered pH and Na⁺, K⁺ -ATPase activity curves demonstrated comparable inhibition in buffered neutral and alkaline pH ranges, and no inhibition was observed in acidic pH. The inhibition of Na⁺, K⁺ -ATPase was independent of temperature. Kinetic studies of substrate (ATP) activation of Na⁺, K⁺ -ATPase indicated uncompetitive inhibition. Results also showed noncompetitive inhibition for p-nitrophenylphosphate and uncompetitive inhibition for K⁺ activations of p-nitrophenylphosphatase. Preincubation of synaptosomes with dithiothreitol, a sulfhydryl (SH) agent, resulted in the complete protection of Plictran inhibition of Na⁺, K⁺ -ATPase, K⁺ -para-nitrophenylphosphatase, and [3-H]-ouabain binding. The protection was specific and concentration dependent since cysteine and glutathione did not afford protection. These results indicate that Plictran inhibited Na⁺, K⁺ -ATPase by interacting with dephosphorylation of the enzyme-phosphoryl complex and exerted a similar effect to that of SH-blocking agents.     

Long lasting effects of intrauterine growth retardation on basal and 5-HT-stimulated Na+/K+-ATPase in the brain of developing rats

Intrauterine growth retardation induced by ligation of the uterine vessels in pregnant rats on the 5th day before delivery was associated with brain and body weights of hypotrophic offspring significantly lower than those of pair-aged control rats, even after 6 weeks of postnatal rearing under normal conditions. In vitro measurements in homogenates indicated that Na⁺/K⁺-ATPase in the forebrain, cerebellum and hippocampus was less active in hypotrophic rats than in pair-aged controls for at least the first month after birth. However, 5-HT and related agonists (RU-24969, bufotenine, and to a lower extent, tryptamine) stimulated Na⁺/K⁺-ATPase activity more efficiently in tissues from hypotrophic rats than in those from control animals. Opposite changes were noted in the brain stem: basal Na⁺/K⁺-ATPase activity was higher in hypotrophic rats during the second half of the first postnatal month but the stimulatory effect of 5-HT was lower than in pair-aged control animals. Since potent 5-HT antagonists such as cinanserin, methiothepin and methysergide, prevented the 5-HT induced-activation of Na⁺/K⁺-ATPase in brain homogenates, these results are discussed in relation with the possible existence of a specific 5-HT receptor controlling Na⁺/K⁺-ATPase activity in the rat brain.     

Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS^?/?) mice hearts that Na⁺/K⁺- and Ca²⁺-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS^?/? mice. Na⁺/K⁺-ATPase activity from crude preparations of adult male eNOS^?/? mice hearts and kidneys was reduced compared with wild-type animals (32 %, p?<?0.05 and 16 %, p?<?0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na⁺/K⁺-ATPase α1 isoform was not significantly changed, there was an important downregulation of the less abundant α2 isoform in the heart (57 %, p?<?0.0001). In addition, although cardiac Ca²⁺-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca²⁺-ATPase 2 protein in eNOS^?/? mice was very high (290 % compared with wild-type animals, p?<?0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p?<?0.01 and 35 %, p?<?0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases.     

Beneficial effects of soy protein in the initiation and progression against dimethylbenz [a] anthracene-induced breast tumors in female rats

Objective: Although recent studies link altered cellular redox state to protein dysfunction in various disease-states, such associations are least studied in clinical diabetes. Therefore, this study assessed the levels of reduced glutathione (GSH) and Na⁺/K⁺ ATPase activities in type 2 diabetic patients with and without microangiopathy. Methods: The study group comprised of a total of 160 subjects, which included non-diabetic healthy controls (n = 40) and type 2 diabetic patients without (n = 60) and with microangiopathy (n = 60), defined as presence of retinopathy with or without nephropathy. Erythrocyte Na⁺/K⁺ ATPase activity and GSH levels were estimated spectrophotometrically and fluorometry was used to determine the plasma thiobarbituric acid reactive substances (TBARS) and serum advanced glycation end products (AGEs). Results: GSH levels in diabetic subjects without (4.8± 0.15 μmol/g Hb) and with microangiopathy (5.2± 0.14 μmol/g Hb) were significantly lower (p < 0.001) compared to control subjects (6.3± 0.14 μmol/g Hb). Erythrocyte Na⁺/K⁺ ATPase activity was significantly reduced (p < 0.001) in diabetes subjects with (272± 7 nmol Pi/mg protein/h) and without microangiopathy (304 ± 8) compared to control (374 ± 6) subjects. TBARS were significantly higher (p < 0.001) in diabetes subjects with (10.65± 0.81 nM/ml) and without microangiopathy (9.90± 0.5 nM/ml) compared to control subjects (5.18± 0.18 nM/ml). Advanced glycation end product levels were also significantly (p < 0.001) elevated in diabetic subjects with microangiopathy (8.2± 1.8 AU) when compared to diabetes subjects without microangiopathy (7.0± 2.0 AU) and control subjects (4.6± 1.9 AU). On multivariate regression analysis, GSH levels showed a positive association with the Na⁺/K⁺ ATPase activity and negative association with TBARS and AGE levels. Conclusion: Hypoglutathionemia and increased oxidative stress appears to be early biochemical aberrations in diabetes, and through protein alterations, oxidative stress and redox modifications may contribute to pathogenesis of diabetic microangiopathy.     

Changes in Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity and Alpha 3 Subunit Expression in CNS After Administration of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitors

The expression of Na⁺, K⁺-ATPase α3 subunit and synaptosomal membrane Na⁺, K⁺-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na⁺, K⁺-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris–HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na⁺, K⁺-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na⁺, K⁺-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na⁺, K⁺-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na⁺, K⁺-ATPase inhibitors modify differentially the expression of Na⁺, K⁺-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.     

5-hydroxytryptamine regulates the (Na++K+)ATPase activity in malpighian tubules of Rhodnius prolixus: Evidence for involvement of G-protein and cAMP-dependent protein kinase

5-Hydroxytryptamine (5-HT, serotonin) acts as a diuretic hormone in Rhodnius prolixus, where it increases to 0.1 μM in the haemolymph during feeding and stimulates the fluid secretion in isolated Malpighian tubules. The ouabain-sensitive (Na⁺+K⁺)ATPase activity present in homogenates of Malpighian tubules from unfed Rhodnius prolixus is inhibited 60% by 0.01 μM 5-HT. This inhibition is reversed by ketanserin, a 5-HT₂ receptor antagonist in mammals, and also by GDPβS, a competitive inhibitor of G-protein GTPase activity. GTPγS, a nonhydrolysable analog of GTP, and cholera toxin, a G_s-protein activator, also inhibit the ouabain-sensitive (Na⁺+K⁺)ATPase activity, while pertussis toxin, a G_i-protein inhibitor, has no effect. The (Na⁺+K⁺)ATPase activity is inhibited 55% by 0.4–100 μM dibutyryl-cAMP in the presence of IBMX, a phosphodiesterase inhibitor, which also potentiates the effect of a low concentration of 5-HT. The cAMP-dependent protein kinase inhibitor peptide abolishes the 5-HT effect. These data suggest that the (Na⁺+K⁺)ATPase activity in Malpighian tubules is inhibited by 5-HT through activation of G_s-protein and a cAMP-dependent protein kinase. Inhibition of the Na⁺+K⁺ pump would contribute to the diuretic effect of 5-HT. Arch. Insect Biochem. Physiol. 36:203–214, 1997. © 1997 Wiley- Liss, Inc.     

Elevation of Different Ion-Specific ATPase Activities by L-Thyroxine (T4) in Different Tissues of Tasar Silkworm, Antheraea mylitta (Lepidoptera: Saturniidae) during Developmental Stages

Tissue-specific age-dependent changes were observed in Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na⁺K⁺- and Ca²⁺-ATPase in testes and only Ca²⁺-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na⁺K⁺-, Ca²⁺- and Mg²⁺-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system.     

Platelet Ca2+ handling in essential hypertension: Role of a plasma ouabain-like factor

A humoral ouabain-like plasma factor has been observed in patients with essential hypertension (EHT). In the present study, we hypothesized that this humoral factor might be responsible for the elevated cytosolic free calcium concentrations [Ca²⁺]_i seen in these patients. Patients with mild to moderate EHT and their normotensive first degree blood relatives (NTBR) participated in the study. Platelet Na⁺, K⁺-ATPase activity was assayed in EHT patients and their NT first-degree relatives. To confirm the ouabain-like activity in plasma from EHT patients, control platelets were incubated with EHT and NTBR plasma and their Na⁺, K⁺-ATPase activity was measured. In addition, the effect of EHT plasma on platelet⁴⁵Ca-uptake was studied. Thein vitro effects of ouabain (10 ΜM) on (i)⁴⁵Ca-uptake and (ii) [Ca²⁺]_i response in control platelets were also observed. A decreased Na⁺K⁺-ATPase activity (P< 0.05) was observed in platelet membranes from EHT patients. Incubation of control platelets with EHT plasma decreased their Na⁺, K⁺-ATPase activity (P< 0.01) and increased their⁴⁵Ca-uptake (P< 0.05). C-18 Sep-Pak filtered hypertensive plasma extracts (containing the ouabain-like fraction) also decreased Na⁺, K⁺-ATPase activity (P< 001) in control platelet membranes.In vitro incubation of control platelets with ouabain increased⁴⁵Ca-uptake (P< 005) and [Ca²⁺]_i response (P< 0.05) in these platelets. Thus it appears that an ouabain-like factor in the EHT plasma may contribute to the elevated platelet [Ca²⁺]_i observed in EHT patients.     

Attenuation of oxidative hemolysis of human red blood cells by the natural phenolic compound,allylpyrocatechol

Abstract

The protecting ability of the Piper betle leaves-derived phenol, allylpyrocatechol (APC) against AAPH-induced membrane damage of human red blood cells (RBCs) was investigated. Compared to control, AAPH (50 mM) treatment resulted in significant hemolysis (55%, p < 0.01), associated with increased malondialdehyde (MDA) (2.9-fold, p < 0.001) and methemoglobin (6.1-fold, p < 0.001) levels. The structural deformation due to membrane damage was confirmed from scanning electron microscopy (SEM) images and Heinz bodies formation, while the cell permeability was evident from the K⁺ efflux (28.7%, p < 0.05) and increased intracellular Na⁺ concentration (8%, p < 0.05). The membrane damage, due to the reduction of the cholesterol/phospholipids ratio and depletion (p < 0.001) of ATP, 2,3-DPG by ?44–54% and Na⁺–K⁺ ATPase activity (43.7%), indicated loss of RBC functionality. The adverse effects of AAPH on all these biochemical parameters and the resultant oxidative hemolysis of RBCs were significantly reduced by pretreating the cells with APC (7 μM) or α-tocopherol (50 μM) for 1 h, prior to incubation with AAPH.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Kinetics of K+-p-Nitrophenyl Phosphatase Stimulation by a Brain Soluble Fraction

We have already described the separation of two brain soluble fractions by Sephadex G-50, one of which stimulates (peak I) and the other inhibits (peak II) Na⁺, K⁺-ATPase and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activities. Here we examine the features of synaptosomal membrane p-NPPase activity in the presence and absence of brain peak I. It was observed that stimulation of Mg²⁺, K⁺-p-NPPase activity by peak I was concentration dependent, The ability of peak I to stimulate p-NPPase activity was lost by heat treatment followed by brief centrifugation. Pure serum albumin also stimulated enzyme activity. K⁺-p-NPPase stimulation by peak I proved dependent on K⁺ concentration but independent of Mg²⁺ and substrate p-nitrophenylphosphate concentrations. Since our determinations were performed in a non-phosphorylating condition reflecting the Na⁺, K⁺-ATPase Na⁺ site, it is suggested that peak I may stimulate the Na⁺-dependent enzyme phosphorylation known to take place from the internal cytoplasmic side.     

Cyclic AMP and calcium modulated ATPase activity in the salivary glands of the lone star tick Amblyomma americanum (L.)

Na⁺,K⁺-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na⁺,K⁺-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na⁺,K⁺-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na⁺,K⁺-ATPase activity exist in the plasma membrane-enriched fraction.ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg²⁺, Ca²⁺ and Na⁺. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg²⁺ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca²⁺-ATPase activity and oligomycin-sensitive Ca²⁺, Mg²⁺-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction.