ATPase Activity In Homogenates Research Articles

Oxidative Stress and Biochemical Markers of Endothelial Dysfunction and Organ Damage under Conditions of Experimental Nonferrous Metal Intoxication.

Chronic nickel intoxication caused by parenteral nickel chloride administration (0.5 mg/kg of body weight) to Wistar rats led to ROS generation inducing LPO in erythrocyte membranes and homogenates of renal, liver, and myocardial tissue. Superoxide dismutase (SOD) activity was inhibited, while catalase activity and ceruloplasmin concentration increased. LPO and its products disrupted nitric oxide production and reduced its bioavailability, which led to the development of endothelial dysfunction and impaired microcirculatory hemodynamics. At the same time, damage of cytoplasmic membranes of internal organs (kidney, liver, and myocardium) was revealed, which was seen from reduced Na+, K+-ATPase activity in homogenates of these organs and increased serum activity of organ-specific (ALT, AST, and γ-glutamyl transpeptidase) and excretory (alkaline phosphatase) enzymes.

Quantification of Na+,K+ pumps and their transport rate in skeletal muscle: Functional significance

During excitation, muscle cells gain Na+ and lose K+, leading to a rise in extracellular K+ ([K+]o), depolarization, and loss of excitability. Recent studies support the idea that these events are important causes of muscle fatigue and that full use of the Na+,K+-ATPase (also known as the Na+,K+ pump) is often essential for adequate clearance of extracellular K+. As a result of their electrogenic action, Na+,K+ pumps also help reverse depolarization arising during excitation, hyperkalemia, and anoxia, or from cell damage resulting from exercise, rhabdomyolysis, or muscle diseases. The ability to evaluate Na+,K+-pump function and the capacity of the Na+,K+ pumps to fill these needs require quantification of the total content of Na+,K+ pumps in skeletal muscle. Inhibition of Na+,K+-pump activity, or a decrease in their content, reduces muscle contractility. Conversely, stimulation of the Na+,K+-pump transport rate or increasing the content of Na+,K+ pumps enhances muscle excitability and contractility. Measurements of [3H]ouabain binding to skeletal muscle in vivo or in vitro have enabled the reproducible quantification of the total content of Na+,K+ pumps in molar units in various animal species, and in both healthy people and individuals with various diseases. In contrast, measurements of 3-O-methylfluorescein phosphatase activity associated with the Na+,K+-ATPase may show inconsistent results. Measurements of Na+ and K+ fluxes in intact isolated muscles show that, after Na+ loading or intense excitation, all the Na+,K+ pumps are functional, allowing calculation of the maximum Na+,K+-pumping capacity, expressed in molar units/g muscle/min. The activity and content of Na+,K+ pumps are regulated by exercise, inactivity, K+ deficiency, fasting, age, and several hormones and pharmaceuticals. Studies on the α-subunit isoforms of the Na+,K+-ATPase have detected a relative increase in their number in response to exercise and the glucocorticoid dexamethasone but have not involved their quantification in molar units. Determination of ATPase activity in homogenates and plasma membranes obtained from muscle has shown ouabain-suppressible stimulatory effects of Na+ and K+.

The effect of auranofin on the colonic transport of Na+ and fluid in the rat.

Auranofin in the mucosal fluid caused a dose-dependent inhibition of fluid and Na+ absorption by everted sacs of rat colon. Serosal auranofin was without effect. (Na+ + K+)ATPase activity of homogenates of mucosal scrapes of rat colon was inhibited by auranofin in a dose-related manner, while Mg2+-ATPase activity was little affected. These actions of the drug on colonic transport mechanisms could contribute to the diarrhoea associated with auranofin therapy.

Changes in Na +/K +-ATPase activity, unsaturated fatty acids and metallothioneins in gills of the shore crab Carcinus aestuarii after dilute seawater acclimation

We have assessed the activity of Na +/K +-ATPase, cAMP, free fatty acids (FFA) and metallothionein (MT) in the posterior gills of the brackish water shore crab Carcinus aestuarii during acclimation to 10 ppt dilute seawater (DSW). Following 3–18 days acclimation in DSW specific activity of Na +/K +-ATPase in native gill homogenates and partially purified membrane vesicles was progressively increased, from 1.7- to 3.9-fold. After short-term acclimation of crabs in DSW with added sucrose to make media isosmotic with the haemolymph the specific Na +/K +-ATPase activity in homogenates was not increased, relative to SW enzyme activity. Moreover, hyposmotic conditions led to depletion of cAMP in gills. In partially purified membrane vesicles isolated from posterior gills, fatty acids with compositions 16:0, 18:0, 18:1, 20:4 and 20:5 dominated in both SW- and DSW-acclimated Carcinus. During a year in which the metabolic activity of crabs was increased, the arachidonic/linoleic acids ratio (ARA/LA) for DSW-acclimated crabs was markedly increased relative to that in SW. Increased Na + + K +-ATPase activity under hyposmotic stress may be modulated at least partially by the changed proportion of fatty acids in the purified membranes of posterior gills. Long-term acclimation of shore crabs to DSW resulted in a 2.6-fold increase in cytosolic metallothionein (MT) content in posterior gills over those in SW crabs. Assuming an antioxidant role of MT associated with intracellular zinc partitioning, the observed MT induction in posterior gills may be considered an adaptive response of C. aestuarii to hyposmotic stress.

Effect of CDP-choline on hippocampal acetylcholinesterase and Na+,K(+)-ATPase in adult and aged rats.

The aim of this study was to investigate the effect of different cytidine-5'-diphosphocholine (CDP-choline) concentrations (0.1-1 mM) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg(2+)-ATPase activities in homogenates of adult and aged rat hippocampi. Tissues were homogenised, centrifuged at 1000 x g for 10 min and in the supernatant, AChE activity and Na+,K(+)-ATPase and Mg(2+)-ATPase activities were determined according to Ellman's method and Bowler's and Tirri's method, respectively. After an 1-3 h preincubation of the homogenised tissue with CDP-choline, a maximal AChE stimulation of about 25% for both adult and aged rats (p < 0.001) and a Na+,K(+)-ATPase activation of about 50% for adult rats (p < 0.001) and about 60% for aged rats (p < 0.001) were observed, while hippocampal Mg(2+)-ATPase activity was not influenced in either adult or aged animals. It is suggested that: CDP-choline can restore hippocampal AChE and Na+,K(+)-ATPase activities in the aged rat and thus it may play a role in improving memory performance which is impaired by aging and some neuronal disturbances.

EFFECTS OF DIETARY MANIPULATION ON MUSCLE GLYCOGEN AND SARCOPLASMIC RETICULUM FUNCTION DURING PROLONGED EXERCISE

The purpose of this study was to investigate the hypothesis that exercise performed during low glycogen levels in associated with an increased depression in sarcoplasmic reticulum (SR) function. To investigate this hypothesis, untrained males (5) and females (5), with a peak aerobic power (VO2 peak) of 3.5 ± 0.1 and 2.4 ± 0.1 L/min, respectively ( ± SE), cycled (65% VO2 peak), both while on a low carbohydrate (LO CHO) (23 ± 0.5% of energy from CHO) and normal diet (Normal CHO) (53 ± 2.2% of energy from CHO) for 4 days following glycogen depleting exercise. Resting muscle glycogen contents in vastus lateralis (378 ± 12 vs 259 ± 8 glucosyl units kg dry wt−1) were higher (p < 0.05) and times to fatigue (110 ± 37 vs 85 ± 24 min) were longer with Normal CHO. Maximal Ca2+-ATPase activities in homogenates, although higher (P < 0.05) at rest in LO CHO (131 ± 6 vs 119 ± 5 μmol.min g protein −1) were not different at fatigue. Homogenate based Ca2+-uptake, (μmol.min−1-1.g protein) were similar between diets at rest (351 ± 44 vs 369 ± 49). but decreased more rapidly (P < 0.05) on LO CHO compared to Normal CHO (304 ± 44 vs 369 ± 169 ± 29). These results demonstrate that the effects of dietary manipulation on SR function is specific to both the property measured (Ca2+-ATPase activity vs Ca2+-uptake) and the condition (rest vs exercise). Supported by NSERC (Canada)

Interferon-gamma downregulates ion transport in murine small intestine cultured in vitro.

Effects of IFN-gamma on mammalian small intestinal ion transport were studied in vitro using incubated sheets of murine small intestine in Ussing chambers. In oxygenated standard culture medium containing hydrocortisone and antibiotics, they maintained their short-circuit current (I(sc)) responses to glucose and theophylline for 48 h. Histological examination revealed a 50% diminution of villus height over 36 h but no change in crypts. Height was better maintained during a 36-h incubation of small intestine from SCID mice, suggesting a role for B or T lymphocytes in villus atrophy. Exposure of small intestine to 100 U/ml IFN-gamma for 36 h decreased basal I(sc) by 40% and I(sc) responses to glucose and theophylline by approximately 70%; at 1,000 U/ml for 36 h, IFN-gamma inhibited these I(sc) responses by 90%. An inhibitor of inducible NO synthase did not reverse these effects, suggesting that they are not mediated by NO. Tissue resistance, mucosal K(+) content, and epithelial morphology were not affected. Ouabain-sensitive ATPase activity in homogenates was inhibited 60% by IFN-gamma (100 U/ml for 36 h). IFN-gamma inhibition of I(sc) responses to glucose and theophylline also occurred in SCID mouse small intestine. Thus murine small intestinal sheets can be maintained viable in vitro for at least 48 h, although villus blunting develops (but less so in SCID mouse small intestine). Also, prolonged exposure to IFN-gamma downregulates Na(+)-coupled glucose absorption, active Cl(-) secretion, and Na(+)-K(+)-ATPase activity, effects unlikely to be mediated by enhanced NO.

Effect of CDP-choline on brain acetylcholinesterase and Na +,K +-ATPase in adult rats

Objective: The aim of this work was to investigate the effect of different cytidine-5′-diphosphocholine (CDP-choline) concentrations (0.1–1 mM) on acetylcholinesterase (AChE), (Na +,K +)-ATPase and Mg 2+-ATPase activities in homogenates of adult rat whole brain and in pure (nonmembrane bound) enzymes. Design and Methods: Tissues were homogenized, centrifuged at 1000 ×g for 10 min, and in the supernatant AChE activity and Na +,K +-ATPase, Mg 2+-ATPase activities were determined according to Ellman’s method and Bowler’s and Tirri’s, respectively. Results: After a 1–3 h CDP-choline preincubation, this substance induced a maximal stimulation of 20%–25% (p < 0.001) for AChE and 50-55% ( p < 0.001) for Na +,K +-ATPase, but it did not influence Mg 2+-ATPase activity. One mM acetylcholine (ACh) resulted in an approximately 18% ( p < 0.001) AChE inhibition by excess substrate in the brain homogenate, while 0.01 mM noradrenaline did not influence Na +,K +-ATPase activity. Conclusions: CDP-choline can stimulate brain AChE and Na +,K +-ATPase independently of ACh and noradrenaline. This enzymatic stimulation may be due to the transformation of CDP-choline to membrane phophatidylcholine. The above data could explain in part the clinical effects of this substance in some neuronal disturbances.

D-glucose stimulates the Na+/K+ pump in mouse pancreatic islet cells.

To determine the effect of D-glucose on the β-cell Na+/K+ pump, 86Rb+ influx was studied in isolated, -cell-rich islets of Umeå-ob/ob mice in the absence or presence of lmM ouabain. D-glucose (20 mM) stimulated the ouabain-sensitive portion of 86Rb+ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na+/K+ ATPase activity in homogenates of islets of Umeå-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3–20 mM) was observed in either ob/ob or normal islets at the optimal Na+/K+ ratio for the enzyme (135 mM Na+ and 20 mM K+). Neither D-glucose (3–20 mM) nor L-glucose or 3-O-methyl-D-glucose (20 mM) affected the enzyme activity at a high Na+/K+ ratio (175 mM Na+ and 0.7mM K+). Diphenylhydantoin (150 μM) decreased the enzyme activity at optimal Na+/K+ ratio, whereas 50 μM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na+/K+ pump of β-cells in intact islets and that D-glucose does not exert any direct effect on the Na+/K+ ATPase activity.

Modulation of beta-cell ouabain-sensitive 86Rb+ influx (Na+/K+ pump) by D-glucose, glibenclamide or diazoxide.

The activity of the β-cell Na+/K+ pump was studied by using ouabain-sensitive (lmM ouabain) 86Rb+ influx in β-cell-rich islets of Umeå-ob/ob mice as an indicator of the pump function. The present results show that the stimulatory effect of glucose on ouabain-sensitive 86Rb+ influx reached its approximate maximum at 5mM glucose. Pre-treatment of the islets with 20mM glucose for 60 min strongly reduced the glucose-induced stimulation of the Na+/K+ pump. Pre-treatment (60 or 180 min) of islets at 0mM glucose, on the other hand, did not affect the magnitude of the glucose-induced stimulation of 86Rb+ influx dunng the subsequent 5-min incubation. Glibenclamide stimulated the ouabain-sensitive 86Rb+ uptake in the same manner as glucose. The stimulatory effect, showed its apparent maximum at 0.5μM. Pre-treatment (60 min) of islets with 1μM glibenclamide did not reduce the subsequent stimulation of the ouabain-sensitive 86Rb+ influx. The stimulatory effect of glibenclamide and D-glucose were not .additive, suggesting that they may have the same mechanism of action. No direct effect of glibenclamide (0.01-1μM) was observed on the Na+/K+ ATPase activity in homogenates of islets. Diazoxide (0.4mM) inhibited the Na+/K+ pump. This effect was sustained even after 60 min of pre-treatment of islets with 0.4mM diazoxide. The stimulatory effect of glibenclamide and D-glucose were abolished by diazoxide. It is concluded that nutrient as well as non-nutrient insulin secretagogues activate the Na+/K+ pump, probably as part of the membrane repolarisation process.

Seasonal differences in activity of perch ( Perca flavescens) gill Na +/K + ATPase

We measured Na +/K + ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na +/K + ATPase activity was measured at 8, 17, 25, and 37°C. V max values for winter fish increased from 0.48±0.07 μmol P mg −1 protein h −1 at 8°C to 7.21±0.79 μmol P mg −1 protein h −1 at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg −1 protein h −1. The K m for ATP and for Na + at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na +/K + ATPase from summer fish was 10 309 ( μmol P mg −1 h −1) K −1. In winter fish, the K m for ATP and Na + increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The K m values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression.

Regulation of Na(+)-K(+)-ATPase expression in cultured renal cells by incubation in hypertonic medium.

To determine whether alterations in cell volume affect Na(+)-K(+)-adenosinetriphosphatase (ATPase) expression, a subclone of the Madin-Darby canine kidney (MDCK) cell line was incubated in anisotonic serum-free medium and alpha- and beta-subunit mRNA, Na(+)-K(+)-ATPase activity, and active K+ transport were measured. In medium adjusted to 500 mosmol/kgH2O by adding NaCl, the alpha-subunit mRNA concentration was 2.93 +/- 0.14 (SE) times control and beta-mRNA was 1.93 +/- 0.27 times control. When sucrose was added to increase osmolality, alpha-subunit mRNA increased to 1.85 +/- 0.18 times control. Na(+)-K(+)-ATPase activity of homogenates from cells incubated in 500 mosmol/kgH2O medium for 24 h increased to 2.62 +/- 0.52 times control when NaCl was added and 2.31 +/- 0.34 times control when sucrose was added. Active K+ transport increased between 60 and 90% after cells were incubated in 450 mosmol/kgH2O medium with either NaCl or sucrose added. Stimulation of Na(+)-K(+)-ATPase expression in renal cells facing hypertonic stress may represent a long-term mechanism that allows cells to maintain cation gradients in a hypertonic environment.

Inner ester derivatives of gangliosides protect autonomic nerves of alloxan-diabetic rats against Na +, K +-ATPase activity defects

Bovine brain gangliosides have been shown to prevent decay in Na +,K +-ATPase activity in sciatic and optic nerves of alloxan- and streptozotocin-diabetic rats. In the search for a drug with greater bioavailability and increased incorporation into neural tissue, ganglioside inner ester derivatives (AGF 1) were recently developed. We evaluated the effect of AGF 1 treatment on Na +,K +-ATPase activity in homogenates of vagus nerve from alloxan-diabetic rats (100 mg/kg s.c.). Animals were treated with AGF 1: 10 mg/kg 6 days/week i.p., or 30 mg/kg biweekly i.p. Treatment began 10 d post-alloxan and continued for 8 consecutive weeks. Normal age- and sex-matched rats were used as controls. Alloxan intoxication produced a 39% decrease in Na +,K +-ATPase activity of the vagus nerve, which was completely restored (96–97% recovery) by both AGF 1 regimes. Results suggest that ganglioside inner ester derivatives may be used in the clinical setting for the management of diabetic autonomic neuropathy.

Effect of morphine on Na +, K + -ATPase from homogenate of synaptosomes and of synaptic membrane of rat cerebral cortex

Effects of morphine on noradrenaline release from rat cerebrocortical synaptosomes and on the Na +, K +-ATPase activity in homogenates of synaptosomes and of synaptic membranes were examined. Both morphine (10 −3-10 −5 M) and methionine-enkephalin (M-Enk; 10 −5 M) inhibited the enhanced [ 3H]noradrenaline ([ 3H]NA) release evoked by high concentrations of K + from synaptosomes and these inhibitory actions were antagonized by naloxone (10 −4, 10 −5 M). Morphine (10 −3-10 −5 M) and M-Enk (10 −5 M) stimulated the Na +, Na +, K +-ATPase activity in homogenates of synaptosomes but not of synaptic membranes in the incubation medium containing2.2 × 10 −6–4.7 × 10 −7M free Ca 2+ and these stimulatory effects were antagonized by naloxone. In homogenates of synaptic membranes, the same concentrations of morphine and M-Enk stimulated the Na +, K +-ATPase activity suppressed by FeCl 2 (5 × 10 −7M) but not CuCl 2 nor ZnCl 2, and these stimulatory effects were antagonized by naloxone. Significant levels of Fe 2+ were liberated from synaptosomes during the preparation of synaptic membrane using distilled water. These results suggest that both morphine and M-Enk stimulate the suppressed Na +, K + -ATPase activity by interacting with Fe 2+ at opioid receptor sites, and they may play a role in the suppression of membrane depolarization and/or the release of NA through their stimulatory action on the Na +, K + -ATPase activity probably suppressed by Fe 2+ in the rat cerebral cortex.

The Influence of Arginine-specific Reagents on Nitrate Uptake by Corn Seedlings

The effects of three arginine-specific reagents on NO3 uptake were studied using corn seedlings (Zea mays L., Golden Cross Bantam). In the presence of borate, 0-25 mM 2,3-butanedione (BD) and 10 mM 1,2-cyclohexanedione (CHD) inhibited NO3 uptake by 76% and 68%, respectively, compared to the controls. However, in the absence of borate, only 18% and 38% inhibition was observed for 0-25 mM BD and 1-0 mM CHD, respectively. Similarly, 0-5 mM phenylglyoxal (PGO) resulted in 75% inhibition. The degree of inhibition of nitrate uptake exhibited a concentration-dependence with respect to the reagents. Corn seedlings are 2- or 3-fold more sensitive to BD than to PGO and CHD, respectively, presumably due to the unfavourable steric effects of the benzal ring. Uptake of NO3 was partially restored after removal of BD, CHD, and PGO from the uptake medium. No significant differences were observed for the ATPase and plasma membrane-associated vanadate-sensitive H+-ATPase or K+-stimulated ATPase activity in homogenates and microsomal fractions prepared from corn seedlings which had been incubated for 2 h in the presence or absence of 0-5 mM BD or 10 mM PGO. This suggests that inhibition of nitrate uptake by the arginine specific reagents was not caused by the indirect effect of their binding and inhibiting H+-ATPase. The fact that the arginine specific reagents strongly inhibit NOi uptake indicates that the NO^ transport system has arginine residues at or near the active site.

Characterization of a vacuolar proton ATPase in Dictyostelium discoideum

Of the total ATPase activity in homogenates of the ameba, Dictyostelium discoideum, approximately one-third was inhibited at pH 7 by 25 microM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Upon isopycnic sucrose density gradient centrifugation, the bulk of the NBD-CI-sensitive ATPase activity was recovered in a major membrane fraction with a broad peak at 1.16 g/ml, well-resolved from markers for plasma membranes, mitochondria, lysosomes and contractile vacuoles. The gradient peak had a specific activity of 0.5 mumol/min per mg protein. The activity was half-inhibited by 1 microM silicotungstate, 2 microM diisothiocyanatostilbene disulfonate (DIDS), 2.5 microM dicyclohexylcarbodiimide (DCCD), 4 microM NBD-CI and 20 microM N-ethylmaleimide (NEM) but was resistant to conventional inhibitors of mitochondrial and plasma membrane ATPase. That this ATPase activity constituted a proton pump was shown by the MgATP-dependent uptake and quenching of Acridine orange fluorescence by partially purified vacuoles. The Acridine orange uptake was specifically blocked by the aforementioned inhibitors. The generation of proton electrochemical gradients was suggested by the stimulation of enzyme activity by protonophores (fatty acids) and cation exchangers (nigericin). Uncoupling stimulated the ATPase activity as much as 20-fold, revealing an unusually high impermeability of the membranes to protons. ATPase activity was also stimulated by halide ions, apparently through a parallel conductance pathway. Under a variety of sensitive test conditions, the reverse enzyme reaction (i.e., incorporation of 32Pi into ATP) was not detected. We conclude that this major H+-ATPase serves to acidify the abundant prelysosomal vacuoles found in D. discoideum (Padh et al. (1989) J. Cell Biol. 108, 865-874). The finding of a vacuolar H+-ATPase in a protist suggests the ubiquity of this enzyme among the eukaryotic kingdoms.

Effects of 4,4′-di-isothiocyano-2,2′-stilbene disulphonate on iodide uptake by primary cultures of turtle thyroid follicular cells

Iodide uptake by primary cultures of turtle thyroid follicular cells is directly proportional to the Na+ concentration and is inversely proportional to the HCO3- concentration in culture medium, but is not affected by the Cl- concentration. Addition of 4,4'-di-isothiocyano-2,2'-stilbene disulphonate (DIDS; 10 mumol/l and higher doses) to medium containing different concentrations of Na+ (5-140 mmol/l), HCO3- (0-40 mmol/l) and Cl- (120 mmol/l) generally enhanced iodide uptake by the cultured cells; however, there was no significant effect in Na+-free and in low Cl- (90 mmol/l and less) medium. The inhibitory effects on iodide uptake of ouabain, frusemide and perchlorate were attenuated by DIDS which also antagonized the stimulatory effects on iodide uptake of TSH, although both DIDS and TSH increased the 125I- cell/medium ratio when they were given alone. At doses of 100 mumol/l and higher, DIDS lowered the intracellular pH of cultured cells when the pH of the medium was maintained at a constant level. It also increased the intracellular Cl- concentration, but had no effect on intracellular Na+ or K+. The input and specific resistances of cell membranes in cultured thyroid cells and in isolated thyroid slices increased (decreased conductance) after adding DIDS to the perfusion fluids. Both Na+/K+- and HCO3(-)-ATPase activities in homogenates of turtle thyroid tissue were inhibited by DIDS. Results from this investigation demonstrate (1) that in addition to preventing the leak of iodide from thyroid cells, DIDS may act to increase the sensitivity of the Na+-anion carrier to I- and thereby increases iodide uptake, and (2) that a HCO3(-)-Cl- exchange system is present in the thyroid cell membrane and appears to be linked to the transport of iodide into thyroid cells.

Direct effects of diphenylhydantoin (phenytoin) on the ion-transporting ATPases of cultured osteoblast-like cells.

Diphenylhydantoin (phenytoin) added to cultures of osteoblast-like cells at a concentration of 10 microM, at the high end of the therapeutic range in plasma of unbound phenytoin (4-8 microM), caused reductions in Na+, K+-ATPase and alkaline phosphatase activities and increases in Ca2+-ATPase and HCO3--ATPase activities in homogenates of whole cells and in subcellular fractions of cultured osteoblast-like cells. These data suggest that, if these changes occur in vivo, the osteomalacia associated with treatment with diphenylhydantoin may be mediated in part by direct effects on the ability of bone cells to transport ions.

Effect of prolactin on 86Rb+ uptake, potassium content and [G-3H]ouabain binding of lactating rabbit mammary tissue.

1. Ouabain-sensitive 86Rb+ uptake in slices of lactating rabbit mammary gland significantly increased after 20 min or 1 hr of incubation with ovine prolactin (NIH-P-S12; 1 microgram/ml.). 2. Total K+ content of the tissue significantly increased at 20 min of incubation with prolactin. 3. Neither vasopressin nor oxytocin, at concentrations of 2,20 or 40 mui.u./ml., had a significant effect on ouabain-sensitive 86Rb+ uptake or total K+ of the tissue after 30 min or 1 hr of incubation. 4. Tissue slices incubated in cycloheximide at 10 micrograms/ml. for up to 260 min showed a reduction in ouabain-sensitive 86Rb+ uptake and total K+ content, with half-lives of 115 and 63 min, respectively. 5. No consistent in vitro effect of prolactin on (Na+ + K+)-activated ATPase activity in homogenates, crude microsomal fractions or NaI-activated membrane fractions from lactating rabbit mammary gland was found. 6. After 3 hr of incubation of tissue slices in the presence or absence of prolactin (5 micrograms/ml.), no significant differences in the number of [G-3H]ouabain molecules bound per cell (5.2 X 10(4) and 6.2 X 10(4), respectively) or in the dissociation constant (KD) for ouabain binding (5.4 X 10(-7) M and 5.9 X 10(-7) M, respectively) were observed. 7. Incubation of the tissue with cycloheximide (10 micrograms/ml.) for 1-6 hr caused an exponential decrease in [G-3H]ouabain binding with a half-life of 3 hr. 8. It is concluded that prolactin stimulates the activity of the (Na+ + K+)-activated ATPase in slices of lactating rabbit mammary gland within one hour but over this period does not affect the number of ouabain-binding sites, which are apparently turned over with a half-life of 1-3 hr.

ATPases of the cat carotid body and of the neighbouring ganglia.

The average ATPase activity in homogenates of the cat carotid body is found to be 20 nmol Pi liberated per minute per mg fresh tissue weight at 25 degrees C. ATPase activity in the nodose ganglion and the superior cervical ganglion is found in the same range. Most of the activity remains in the supernatant after removal of the mitochondria by centrifugation. The activity is inhibited by ouabain only marginally (ganglia) or not at all (carotid body). In all these organs up to 80% of the activity is seen in the absence of Mg2+ and the presence of increasing concentrations of Ca2+.