The effect of (o-phenanthroline)2-cupric sulfate on several properties associated with (N-A+ + K+)-dependent adenosine triphosphatase.

Previous studies have shown that the large polypeptide of purified (Na⁺ + K⁺)-dependent adenosine triphosphatase (NaK ATPase) reacts to form a dimer and other higher oligomeric structures of the enzyme as a result of cross-linking with (o-phenanthroline)₂-cupric sulfate (CP). In the present communication, I show that both NaK ATPase activity and p-nitrophenylphosphatase (NPPase) activity decline rapidly and nearly in parallel when the enzyme is reacted with CP. Similarly, ATP binding is lost with kinetics close to those of ATPase activity and NPPase activity. The loss of ATPase activity, NPPase activity, and ATP binding occurs at a considerably faster rate than cross-linking of the large polypeptide, suggesting that CP may also be forming intrachain disulfide bonds. The binding of ouabain to NaK ATPase is also altered as a result of reacting the enzyme with CP. In marked contrast to ATP binding, however, ouabain binding is lost at a slower rate which closely parallels the rate of reaction of the large polypeptide to form cross-linked oligomeric structures.     

Characterization of the stimulation of neuronal Na+, K+-ATPase activity by low concentrations of ouabain

Low concentrations (< 10^?7 M) of ouabain stimulate the activity of Na⁺, K⁺-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10^?9 to 10^?7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na⁺, K⁺-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na⁺, K⁺-ATPase.     

Effects of ecdysone and juvenile hormone on DNA metabolism of imaginal disks of Drosophila melanogaster

Imaginal disks of Drosophila melanogaster isolated en masse and incubated in Robb's tissue culture medium incorporate ³H-thymidine into nuclear DNA. Both α- and β-ecdysone stimulate the rate of ³H-thymidine incorporation into disk DNA. Concentrations of ecdysone that induce complete evagination of disks in vitro cause the initiation of DNA synthesis in some disk cells. Juvenile hormone has no effect on DNA synthesis in control disks. However, juvenile hormone blocks the ecdysone stimulation of DNA synthesis. It is proposed that juvenile hormone and ecdysone act in a balanced fashion to regulate DNA synthesis in imaginal disks.     

Transport ATPase: further studies on the properties of the thimerosal-treated enzyme.

Our previous studies showed that when ethylmercurithiosalicylate (thimerosal) interacts with the transport ATPase of the guinea pig kidney under specified conditions, the Na⁺ + K⁺-dependent ATPase activity is inhibited, while the Na⁺-dependent ATPase, the Na⁺ + ATP-dependent phosphorylation of the enzyme, and the K⁺-dependent discharge of the phosphoenzyme seem to be unaffected. Here we describe other properties of the thimerosal-treated enzyme: Na⁺-dependent ADP-ATP exchange, Na⁺-dependent UTPase, and K⁺-dependent p-nitrophenylphosphatase activities of the modified enzyme are not inhibited. Kinetics of the Na⁺ effect on the UTPase activities of the native and the modified enzyme are the same. However, K⁺ has a greater inhibitory effect on the Na⁺-UTPase of the modified enzyme than on the Na⁺-UTPase of the native enzyme. The increase in the apparent affinity of the thimerosal-treated enzyme for K⁺ is also evident from the kinetics of the K⁺ effect on p-nitrophenylphosphatase. Neither the native enzyme nor the modified enzyme catalyzes a P₁-ATP exchange. The uninhibited activities of the thimerosal-treated enzyme are sensitive to ouabain. These data provide further support for those reaction mechanisms in which the existence of two ATP sites within the enzyme is assumed.     

The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.     

Effects of alpha-ecdysone, beta-ecdysone and inokosterone on the in vitro evagination of Drosophila leg discs and the subsequent differentiation of imaginal integumentary structures

The morphogenetic activity of three hormonal substances—α-ecdysone, β-ecdysone, and inokosterone—has been studied in vitro on isolated imaginal leg discs of third-instar larvae of Drosophila melanogaster.In the presence of α-ecdysone (0.3–3 μg/ml) and also of the phytohormone inokosterone (0.3–3 μg/ml), the discs underwent metamorphosis, as characterized by complete evagination (in less than 24 hr), secretion, and shedding (48 hr after explanation) of the pupal cuticle, secretion, and structural differentiation of the imaginal cuticle, namely pigmentation and formation of claws, bristles, and hairs (during days 3–6).In the presence of β-ecdysone (10, 6, 3, 0.3, 0.03, 0.003 μg/ml), evagination was always abnormal and incomplete. With all concentrations but the lowest, the partially everted legs had a swollen appearance and, at all concentrations, the subsequent development was inhibited. No imaginal differentiation occurred at any of the concentrations tested.Larval fat body or larval epidermis added to the isolated discs had no influence on their response to either α-ecdysone or β-ecdysone.Changing the osmotic pressure of the β-ecdysone containing medium likewise did not alter the noxious effect of β-ecdysone.Discs cultured first in the presence of β-ecdysone (for 24 hr), then transferred to fresh medium containing α-ecdysone were unable to undergo normal development. The inhibitory effect of β-ecdysone thus appears to be irreversible.Discs cultured first in the presence of α-ecdysone (for 24, 48 or 72 hr), then transferred to β-ecdysone containing medium, were unable to continue their normal differentiation. Further development was blocked within a few hours after the transfer.Results are discussed in view of results obtained with other in vitro and in vivo cultivation techniques. In conclusion, isolated leg discs of Drosophila are unable to respond physiologically to exogenous β-ecdysone. Only α-ecdysone and inokosterone will induce complete and normal metamorphosis in leg discs cultured in vitro.     

Properties and nature of (Na+ + Mg2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na⁺ + Mg²⁺)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase in both cases.(Na⁺ + Mg²⁺)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI₂, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na⁺ + Mg²⁺)-dependent ATPase activity of gills arises from the presence of a (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase.     

Coupled Na+ -K+ transport in vesicles containing a purified (NaK)-ATPase and only phosphatidyl choline.

Endogenous phospholipids of a purified (NaK)-ATPase were displaced by exogenous phosphatidyl choline. If vesicles were made from phosphatidyl choline and enzyme containing only phosphatidyl choline, coupled Na⁺K⁺ transport could be demonstrated. This transport was inhibitable by ouabain. Therefore, the number of components necessary for Na⁺K⁺ transport has been reduced to the purified (NaK)-ATPase and one phospholipid.     

Induction by ouabain of hemoglobin synthesis in cultured friend erythroleukemic cells

Induction of erythroid differentiation in ouabain-resistant murine erythroleukemia cells by ouabain is reported. Ouabain induction results in the appearance of hemoglobin-containing cells 12–24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide. The concentration of ouabain required to induce hemoglobin synthesis depends upon the K⁺ ion levels in the culture medium. Lowering the extracellular K⁺ ion concentration 2–4 fold reduced by 10–40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis. In low K⁺ medium (1.8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0.02 mM. This K⁺ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K⁺ ions in the culture medium. These results suggest that the binding of ouabain to the plasma membrane enzyme, $\text{Na}\text{K}$ ATPase, is required for the induction of erythroid differentiation by ouabain. A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells. The observation that the binding of ouabain to the $\text{Na}\text{K}$ ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K⁺ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells.     

Interactions of ouabain and vanadate with (Na+,K+)ATPase and isolated cardiac muscle

The interactions of ouabain and vanadate with (Na⁺,K⁺)ATPase were investigated at different potassium concentrations. Also, the contractile effects of a mixture of these two inhibitors were compared to those produced by ouabain or vanadate alone. The results from the enzyme and contractile studies suggested that inhibition of sarcolemmal (Na⁺,K⁺)ATPase was involved in mediating the positive inotropic effect of vanadate.     

The synthetic and minimal culture requirements for evagination of imaginal discs of Drosophila melanogaster in vitro

Imaginal discs are induced by β-ecdysone to evaginate and undergo imaginal differentiation in completely defined culture medium (Robb's). The minimal nutritional requirements for evagination are salts, glucose, and 6 or 7 amino acids. Concentrations of β-ecdysone which cause evagination also produce increases in RNA and protein synthesis. Inhibitors of RNA and protein synthesis and amino acid starvation block evagination. Inhibitors of DNA synthesis do not inhibit evagination. The effects of β-ecdysone are concentration dependent. To produce complete evagination, discs must be exposed to low concentrations (0.1 μg/ml) of β-ecdysone for a longer time than to high concentrations (10 μg/ml). However, high concentrations of hormone reduce the rate, and under some conditions, the degree of evagination.     

Lead ion activates phosphorylation of electroplax Na+- and K+-dependent adenosine triphosphatase ((NaK)-ATPase) in the absence of sodium ion.

PbCl₂ in micromolar concentrations stimulates phosphorylation of electroplax microsomal protein in the absence of Na⁺. Other divalent cations showed little or no such effect. The (Mg²⁺ + Pb²⁺)- and (Mg²⁺ + Na⁺)-dependent membrane-bound protein kinase activities in electroplax particulate preparations exhibit properties in common, including their acid stability, ouabain sensitivity, ATP specificity, and molecular size. It is concluded that the (Mg²⁺ + Pb²⁺)-dependent phosphoprotein is part of the Na⁺-, K⁺-dependent adenosine triphosphatase [(NaK)ATPase]. The Pb²⁺-dependent product, in contrast to the Na⁺-dependent one, is insensitive to K⁺ and the hydrolysis of ATP is thus inhibited.     

Transport ATPase of erythrocyte membrane: sensitivities of Na plus, K, plus-ATPase and Kplus-phosphatase activities to ouabain.

Cardiac glycosides are inhibitors of Na⁺,K⁺-ATPase, and K⁺-phosphatase activities of the transport enzyme. Previous studies have shown that when the sensitivities of these two activities to ouabain are compared by the addition of varying concentrations of the drug to the assay media, the K⁺-phosphatase is significantly less sensitive than Na⁺,K⁺-ATPase. This work was done to seek an explanation for this phenomenon. 3-O-Methyl-fluorescein phosphate was used as substrate for the continuous fluorimetric assay of K⁺-phosphatase obtained from human red cells. When ouabain was added to the assay medium, a time-dependent inhibition of K⁺-phosphatase was observed. The rate of inhibition was also influenced by the order of additions of K⁺ and ouabain. In view of these results, several enzyme samples exposed to ouabain for varying lengths of time were prepared, and their Na⁺,K⁺-ATPase and K⁺-phosphatase activities were then determined. A good correlation between the extent of inhibition of the two activities was obtained. These results prove that the previously observed discrepancies between the sensitivities of Na⁺,K⁺-ATPase and K⁺-phosphatase to ouabain are due to the different kinetics of drug interaction with the enzyme under the different conditions of the two assays and that once a certain level of ouabain binding to the enzyme is achieved, both activities are equally inhibited.     

Relationship of Na+-K+-activated ATPase to fluid production by Malpighian tubules of Locusta migratoria.

The presence of a Na⁺K⁺-activated, Mg²⁺-dependent ATPase (E.C. 3.6.1.3) has been demonstrated in microsomal preparations from the Malpighian tubules of Locusta. The effects of sodium and potassium ions, and different concentrations of ouabain, have been studied in relation to the activity of this enzyme and the ability of in vitro Malpighian tubule preparations to secrete fluid. From these studies it seems highly likely that a Na⁺K⁺ activated ATPase ‘pump’ is involved in fluid transport across the walls of the tubules.     

Sodium plus Potassium-Activated, Ouabain-Inhibited AdenosineTriphosphatase from a Fraction of Rat Skeletal Muscle, and Lack of Insulin Effect on It

An ATPase, activated by Na⁺ plus K⁺ in the presence of Mg⁺⁺ and inhibited by ouabain, has been obtained from rat skeletal muscle. Unlike ATPase's with similar properties obtained from other preparations, this ATPase was found only in the fraction containing fragmented sarcoplasmic reticulum. It is suggested that in rat skeletal muscle this ATPase may reside in sarcoplasmic reticulum and not in sarcolemma. This ATPase differed in its pH optimum and in its cation sensitivity from that of rat brain and from that of human muscle reported by Samaha and Gergely (1965, 1966). Because insulin accelerates Na⁺ efflux from muscle, efforts were made to determine whether or not this effect of insulin could be attributed to increased Na⁺ + K⁺-activated ATPase activity. Insulin, administered either in vivo or in vitro, had no demonstrable effect on the enzyme system, nor did it protect against inhibition by ouabain.     

Fluid and cation secretion by the Malpighian tubules of Locusta

In vitro preparations of Locusta Malpighian tubules are able to transport K⁺ against its concentration gradient. The ‘urine’ is slightly hyper-osmotic with respect to the bathing solution and the rate of secretion is inversely dependent on the osmotic pressure of the latter. The rate of fluid secretion increases with increasing temperature; being maximal at approx 40°C. The ionic composition of the secreted fluid, as indicated by Na⁺/K⁺ ratios, is altered by the presence of 1 mM ouabain in the bathing solution. Fluid secretion is inhibited by 1 mM ouabain. In addition, oxygen consumption by the Malpighian tubules is inhibited by either the presence of 1 mM ouabain or the absence of K⁺ in the bathing solution. The relationship between respiration, active transport and the Na⁺K⁺-activated ATPase is discussed.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

The appearance of dopa decarboxylase activity in imaginal discs of Sarcophaga bullata, undergoing development in vitro

During the postembryonic development of Sarcophaga bullata, two large peaks of dopa decarboxylase activity were observed. These were associated with the sclerotization (hardening) of the puparium and the adult cuticle, respectively. A small peak of activity 5.5–6.5 days after pupariation was possibly associated with the sclerotization of the prothoracic spiracles.A premature increase in enzyme activity was observed in young, third-instar larvae injected with 20 μg of β-ecdysone. However, the advantage of studying the effect of the hormone on enzyme activity in vitro led to an attempt to induce² dopa decarboxylase in cultured wing discs.In the presence of β-ecdysone, wing discs underwent evagination and a substantial increase in dopa decarboxylase activity was observed in these discs. The enzyme activity began to appear after the rupture of the peripodial membrane and reached a maximum about the time disc evagination ceased. We suggest that this enzyme activity was responsible for the slight sclerotization of a fine cuticle secreted by the discs. The cultured imaginal discs underwent changes that are very similar to those which occur in intact animals. Therefore, this system appears promising for further studies on the role in differentiation of the hormonal control of enzyme activity.     

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.