Responses of Na+-K+ ATPase activities from dog olfactory tissue to selected odorants.

Na⁺-K⁺ ATPase activity from nerve ending particle (NEP) fractions of dog olfactory tissue homogenates showed different patterns of response to odorants. Similar turbinal groupings were removed from the right and left sides of the septum in the nasal cavity and NEP preparations were tested with eight different odor compounds, including 2-keto alkane homologs and the optical isomers d- and l-carvone. Odorant stimulation of Na⁺-K⁺ ATPase activity from paired turbinal groupings did not show bilateral symmetry. Different patterns of stimulation were observed for each turbinal grouping and for each odorant. A stimulation of over 200% was observed in one preparation in response to 2-nonanone.A study of the response of Na⁺-K⁺ ATPase activity from individual turbinals showed that the enzyme in each turbinal had a different response pattern to six different odorants. Inhibitory and stimulatory responses were observed for the individual turbinal NEP preparations. These results support the proposal that odor sensing initiation may occur through odorant perturbation of the Na⁺-K⁺ ATPase activity.     

Effects of delta9-tetrahydrocannabinol on ATPases in mouse brain subcellular fractions.

The effect of (?)-Δ⁹-THC on the activities of Mg²⁺?, Na⁺?K⁺? and Mg²⁺Ca²⁺-ATPases were studied in mouse brain subcellular fractions. $\text{In vitro}$treatment with Δ⁹-THC produced a dose dependent stimulation of Mg²⁺ ATPase in the crude mitochondrial fraction and its subfractions and a dose-related inhibition of this activity in the microsomal fraction. Na⁺-K⁺- and Mg²⁺-Ca²⁺-ATPase activities were inhibited in a dose-related manner in all subcellular fractions studied.     

UNILATERAL BRAIN INJURY IN THE RABBIT; REVERSIBLE AND IRREVERSIBLE DAMAGE OF THE MEMBRANAL ATPases

Modifications of some membranal enzymatic activities in rabbit brain edema induced by cold injury were studied. The edema was characterized by the tissue H₂O content and the K⁺/Na⁺ ratio. Comparison of the respiratory rate of isolated mitochondria in the state 3 and 4 and the ADP/O ratio suggested an alteration in the ATP synthesis mechanism. The oligomycin sensitive ATPase activity was severely reduced in mitochondria isolated from edematous cells. The alteration of the ouabain sensitive Na⁺-K⁺-ATPase was first qualitative in the sense where the response of the ATPase to the K⁺/Na⁺ ratio was modified. A loss of the total activity was then observed. Intravenous injection of CDP choline induced a regression of the edema, a restoration of the sensitivity of the mitochondrial ATPase towards oligomycin and a restoration of the sensitivity of the Na⁺-K⁺-ATPase to the K⁺/Na⁺ ratio. These results suggest that the reversible damages of the cells induced by cold injury were due to a disorder at the protein-lipid interaction level.     

Adenosinetriphosphatase and nucleotide metabolism in synaptosomes of rat brain

—In the presence of synaptosomes prepared from rat brain, only ATP, dATP and ADP but not dADP were active as substrates of phosphatase (ATP phosphohydrolase; EC 3.6.1 4) in the presence of 150mm-Na⁺ and 20mm-K⁺. An active adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3.) was demonstrated in the synaptosomal fractions by means of paper chromatography, paper electrophoresis and enzymic reactions, so that the high activity with ADP as substrate could represent an activity of an ATPase. Apparently dADP was not a substrate for the kinase; no dATP was formed when dADP was incubated with the synaptosomal fraction in the presence of Na⁺, K⁺ and Mg²⁺. Small amounts of P₁ were liberated with dADP, IDP, GDP or CDP, but not UDP, as substrates, but none was produced in the presence of mononucleotides. The adenine-deoxyribose bond, but not the adenine-ribose bond, was hydrolysed upon the addition of 5% (w/v) TCA to the reaction mixture. The K_M for the hydrolysis of ATP but not ITP, in the presence of Mg²⁺, or of Na⁺, K⁺ and Mg²⁺, was lower for the synaptosomal ATPase than for the microsomal ATPase, and the values for V_max for synaptosomal ATPase were higher. The activation increment was generally higher for the synaptosomal ATPase and no distinct differences in the properties of the enzyme from either particulate fractions were observed. Mg²⁺ could be partially replaced by Mn²⁺ in the synaptosomal ATPase system, but there was little Na⁺-K⁺-activation observed in the presence of the latter. The effects of ouabain and of homogenization under various conditions suggested localization of the K⁺-sensitive site of the ATPase on the surface of the synaptosomal membrane. Activity of the Na⁺-K⁺-Mg²⁺ ATPase increased after freezing and thawing of the sonicated, sucrose or tris-treated preparations but decreased considerably in the synaptosomes treated with 001 m-deoxycholate. Activity of the Mg²⁺ ATPase in the latter preparation showed little change.     

CHARACTERIZATION OF MEMBRANES OBTAINED FROM ELECTRIC ORGAN OF THE ELECTRIC EEL BY SUCROSE GRADIENT FRACTIONATION AND BY MICRODISSECTION

Abstract— Two membrane fractions were obtained from electric organ tissue of the electric eel by sucrose gradient centrifugation of tissue homogenates. Electron microscopic examination showed that both fractions contained mainly vesicular structures (microsacs). Both the light and heavy fractions had a-bungarotoxin-binding capacity and Na⁺-K⁺ ATPase activity, while only the light fraction had AChE activity. The polypeptide patterns of vesicles derived from both the light and heavy fractions were examined by SDS-polyacrylamide gel electrophoresis and found to be very similar. The ratio of protein to phospholipid in the light vesicles was much lower than in the heavy vesicles, but the relative amounts of individual phospholipids in the two fractions were similar. A marked difference in the permeability of the light and heavy vesicles was observed by measuring efflux of both [¹⁴C]sucrose and ²²Na⁺, and also by monitoring volume changes induced by changing the osmotic strength of the medium. All three methods showed the heavy vesicles to be much more permeable than the light ones. Only the light vesicles displayed increased sodium efflux in the presence of carbamylcholine. The AChE in the light fraction does not appear to be membrane-bound, but is rather a soluble enzyme, detached from the membrane during homogenization, which migrates on the gradient similarly to that of the light vesicles. This is supported by the fact that the bulk of the AChE is readily removed by washing the vesicles. Moreover, under the conditions employed in our sucrose gradient separations,‘native’14 S + 18 S AChE exists in the form of aggregates which migrate very similarly to the major peak of AChE activity of tissue homogenates. Separated innervated and non-innervated surfaces of isolated electroplax were obtained by microdissection. α-Bungarotoxin-binding capacity was observed only in the innervated membrane. About 80% of the AChE was in the innervated membrane, and about 70% of the Na⁺-K⁺ ATPase in the non-innervated membrane. The data presented indicate that the light and heavy vesicle fractions separated by sucrose gradient centrifugation are not derived exclusively from the innervated and non-innervated membranes respectively, as previously suggested by others, but contain membrane fragments from both sides of the electroplax. The separation of two populations on sucrose gradients may be explained both by the differences in permeability and in protein to phospholipid ratios.     

Variations in myocardial CPK and Na+-K+ ATPase following normo- and hypothermic exposure to dimethyl sulfoxide and glycerol.

Exposure of canine myocardial tissue homogenates to Me₂SO glycerol (20 to 60%) for periods up to 8 hr resulted in significant alterations in enzyme activity at 0 °, 18 °, and 37 °C. Both CPK and Na⁺-K⁺ ATPase demonstrate anomalous enhancement of activity at each temperature with glycerol. Me₂SO provides a similar enhancement of Na⁺-K⁺ ATPase activity at hypothermic temperatures up to 40%. Thereafter, nearly complete inhibition resulted. Under normothermic conditions complete Me₂SO inhibition occurred at 40 °. CPK activity diminished in a linear fashion after 4 hr at 18 ° and 37 ° but was unaffected by up to 40% Me₂SO at 0 °C. The results suggest that disruption of the CPK-Na⁺-K⁺ ATPase systems may be minimized by hypothermic perfusion at low cryoprotectant concentrations.     

Cardiac sarcolemmal Na+-Ca2+ exchange and Na+-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na⁺-Ca²⁺ exchange, Na⁺-K⁺ ATPase and Ca²⁺-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na⁺-K⁺ ATPase and Ca²⁺-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg²⁺ ATPase activity was not depressed throughout the experimental periods. Both Na⁺-Ca²⁺ exchange and ATP-dependent Ca²⁺-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na⁺-K⁺ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of _--subunit of Na⁺-K⁺ ATPase and Na⁺-Ca²⁺ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na⁺-K⁺ ATPase _-- subunit mRNA was noted in diabetic heart. A significant increase in the Na⁺-Ca²⁺ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na⁺-K⁺ ATPase, Na⁺Ca²⁺ exchange and Ca²⁺-pump activities may lead to the occurrence of intracellular Ca²⁺ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.     

Studies on the glycoprotein component of (Na+-K+)ATPase from guinea pig brain

In this study an attempt was made to elucidate the possible mechanism of the brain microsomal (Na⁺-K⁺)ATPase inhibition based on the assumption that glycoprotein part of the enzyme is exposed on the outer membrane surface. In our experiments the modification with concanavalin A of sugar end groups exposed by neuraminidase treatment resulted in a significant decrease of the brain (Na⁺-K⁺)ATPase activity. The percentage of the enzyme inhibition by concanavalin A binding to the neuraminidase-treated preparation corresponds to the amount of liberated sialic acids. The modification of the glycoprotein part of the brain (Na⁺-K⁺)ATPase complex by neuraminidase and concanavalin A treatments did not affect K⁺-nitrophenylphosphatase activity.     

Effects of Germination on NA-K-stimulated Adenosine 5'-Triphosphatase and ATP-dependent Ion Transport of Isolated Membranes from Cotyledons

A purified membrane fraction featuring ATPase activity was isolated from cotyledon tissue of Phaseolus vulgaris at different stages of germination. The fraction is enriched in both basal and Na⁺-K⁺-stimulated ATPase and is relatively free of contamination by fragments of mitochondrial membrane and microsomes. The isolated membranes have been tentatively identified as partially purified plasma membrane.     

Sodium-potassium exchange in sea urchin egg. I. Kinetic and biochemical characterization at fertilization

Biochemical and kinetic characteristics of the Na⁺-K⁺ exchange were studied in Paracentrotus lividus eggs. Measurement of the ⁸⁶Rb uptake shows that ouabain-sensitive ⁸⁶Rb uptake is dramatically stimulated within the first minute following fertilization. The Na⁺-K⁺ pump-mediated K⁺ entry presents a maximal rate at 8 min postfertilization and then decreases to reach a plateau within 30 min. We assess that the steep rise in cell K⁺ occurring at fertilization (J.P. Girard, P. Payan, C. Sardet, Exp. Cell. Res. 142:215–221, 1982) does not originate from a net entry of external K⁺. Measured 30 min postfertilization, the half-maximal activation by K⁺ of the ouabain-sensitive Na⁺-K⁺ exchange is 5–6 mM and the ouabain lC₅₀ is 5.10^?5 M. Egg cortices from unfertilized and fertilized eggs show comparable Na⁺-K⁺ ATPase activity with a 50% ouabain-sensitive fraction. V_m and K_m for Na⁺ and K⁺ of the enzyme are of the same order of magnitude in cortices of unfertilized and fertilized eggs. Cortical Na⁺-K⁺ ATPase from unfertilized eggs shows a ten fold increase of activity between pH 6.7 and pH 7.7. The results strongly suggest that the plasma membrane of unfertilized eggs contains a preexisting Na⁺-K⁺ transporting system which is obligatorily stimulated at fertilization.     

Effect of antibody to anisole binding protein on odorant perturbation of Na+-K+ ATPase activity

Odorant perturbation of Na⁺-K⁺ ATPase activity from cow olfactorytissue was strongly affected by ng quantities of antibodiesto anisole binding protein from dog olfactory mucosa. Antibodyprotein (80 ng per ml reaction mixture) prevented odorant perturbationof Na⁺-K⁺ ATPase activity. Antibody effect on odorant perturbationshowed concentration dependence and was active against a numberof different odorous chemicals. Electrophysiological studies(Goldberg et al, 1979) showed that mouse EOG responses due toodorants were inhibited 50% by previous exposure to 0.8 ng antibodies.Thus electrophysiological and biochemical responses showed sensitivityto the antibodies from the anisole binding protein from dogolfactory tissue. It is proposed that NA⁺-K⁺ ATPase may participatein the initiation of nerve signals caused by odorant-enzymecomplex interactions.     

Preliminary studies on rat olfactory tissue: effects of odorants on NA+-K+ atpase activity

Nerve ending particle (B) fractions were prepared from three different rat olfactory endoturbinals. This fraction was tested for Na⁺K⁺ ATPase activity and its response $\text{in vitro}$to low levels of odorants. Quite different differential responses of the enzyme activity were noted between the three tissue preparations for a given odorant and between the three odorants for a given tissue. A possible relationship between enzyme perturbation and initiation of odor sensing mechanism is discussed.     

Tissue specificity of dopamine effects on brain ATPases

Dopamine inhibits Mg²⁺,Na⁺,K⁺- and Na⁺,K⁺-ATPase activities but does not modify Mg²⁺-ATPase activity of nerve ending membranes isolated from rat cerebral cortex. In the presence of the soluble fraction of brain, dopamine activates total, Na⁺,K⁺-, and Mg²⁺-ATPases. Dopamine stimulation of nerve ending membrane ATPases is achieved when soluble fractions of brain, kidney, or liver are used. On the other hand, dopamine effects are not observed on kidney or heart ATPase preparations. These results indicate tissue specificity of dopamine effects with respect to the enzyme source; there is no tissue specificity for the requirement of the soluble fraction to achieve stimulation of ATPases by dopamine.     

EFFECT OF UNILATERAL VISUAL DEPRIVATION AND VISUAL STIMULATION ON THE ACTIVITIES OF ALKALINE PHOSPHATASE, ACID PHOSPHATASE, Na+−K+ ACTIVATED Mg2+ CATALYSED ADENOSINE TRIPHOSPHATASE AND ON THE CONTENT OF SODIUM AND POTASSIUM IONS OF THE OPTIC LOBE OF ADULT PIGEON

—A study was made of the effects of unilateral visual deprivation and stimulation upon the activities of alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), Na⁺-K⁺ activated Mg²⁺ catalysed ATPase (EC 3.6.1.4) and upon the Na⁺ and K⁺ contents of the optic lobe of adult pigeon (Columba livia). Visual deprivation was achieved by eyelid suturing or by enucleation and maintained for 1–9 weeks. Unilateral visual stimulation was maintained for 75 min following 72 h of darkness. A statistically significant increase in the activity of alkaline phosphatase activity was observed in the optic lobe after unilateral visual deprivation whereas unilateral visual stimulation resulted in the opposite effect. Acid phosphatase activity was found to be unchanged under all experimental conditions. Na⁺-K⁺ ATPase activity was found to increase significantly following unilateral visual stimulation and following eyelid suturing in the corresponding optic lobes; unilateral enucleation resulted in a decrease in the Na⁺-K⁺ ATPase activity. An increase in the enzyme activity was found to be associated with an increase in the level of Na⁺-ion and a decrease in the level of K⁺-ion, and vice versa.     

Effect of tissue specificity of brain soluble fractions on Na+, K+-ATPase activity

Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na⁺, K⁺-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na⁺, K⁺-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na⁺, K⁺- and Mg²⁺-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na⁺, K⁺-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg²⁺-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na⁺, K⁺-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.     

Activity-Dependent Adenosine Release May Be Linked to Activation of Na+-K+ ATPase: An In Vitro Rat Study

In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na⁺ influxes associated with neuronal signalling activate the Na⁺-K⁺ ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na⁺. We have simultaneously imaged intracellular Na⁺ and measured adenosine release. The accumulation of intracellular Na⁺ during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca²⁺, and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na⁺-K⁺ ATPase in the release of adenosine. Under conditions which caused a Na⁺ influx, brief applications of ouabain increased the accumulation of intracellular Na⁺ but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na⁺-K⁺ ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na⁺-K⁺ ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep.     

Distinguishable ATPase activities of cell wall and plasma membrane

Basal and Na⁺-K⁺ stimulated ATPase (ATP phosphohydrolase, E.C. 3.6.1.3) are both present in isolated preparations of purified cell wall and plasma membrane from cotyledon tissue of Phaseolus vulgaris. A comparison of the enzymes in the two fractions has revealed that the specific activities of basal and cation-sensitive ATPase are markedly higher in isolated cell wall than in the plasma membrane fraction. In addition, enrichments of both enzymes calculated on a protein basis relative to corresponding homogenates were considerably higher for cell wall than for plasma membrane. Thus, while part of the ATP-hydrolyzing activity of the wall may be attributable to the enzymatic properties of imbedded plasma membrane, there must also be additional non-membranous ATPase in the protein complement of the wall itself.     

Response to environmental salinity of Na-KATPase activity in individual gills of the euryhaline crab Cyrtograpsus angulatus

The occurrence, localization and response to environmental salinity changes of Na⁺-K⁺ATPase activity were studied in each of the individual gills 4-8 of the euryhaline crab Cyrtograpsus angulatus from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). Na⁺-K⁺ATPase activity appeared to be differentially sensitive to environmental salinity among gills. Upon an abrupt change to low salinity, a differential response of Na⁺-K⁺ATPase activity occurred in each individual gill which could suggest a differential role of this enzyme in ion transport process in the different gills of C. angulatus. With the exception of gill 8, a short-term increase of Na⁺-K⁺ATPase specific activity was observed in posterior gills, which is similar to adaptative variations of this activity described in other euryhaline crabs. However, and conversely to that described in other hyperregulating crabs, the highest increase of activity occurred in anterior gills 4 by 1 day after the change to dilute media which could suggest also a role for these gills in ion transport processes in C. angulatus. The fact that variations of Na⁺-K⁺ATPase activity in anterior and posterior gills were concomitant with the transition to hyperregulation indicate that this enzyme could be a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab. The results suggest a differential participation of branchial Na⁺-K⁺ATPase activity in ionoregulatory mechanisms of C. angulatus. The possible existence of functional differences as well as distinct regulation mechanisms operating in individual gills is discussed.     

The Extrusion of Sodium Ions from Presynaptic Nerve Endings of Rat Cerebral Cortex

(Na⁺-K⁺) ATPase is present in synaptosomal preparations and it is assumed to represent the sodium-potassium pump. 10 μm -noradrenaline activates (Na⁺-K⁺) ATPase approximately 100%, but 50 μm -noradrenaline does not stimulate the rate of ²²Na extrusion from synaptosomes. The results suggest that it is unlikely that the noradrenaline stimulation of (Na⁺-K⁺) ATPase is part of a feedback mechanism whereby released noradrenaline can influence the activity of the presynaptic sodium pump.     

Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: Implications for angiotensin II-induced vascular dysfunction

Glutathionylation of the Na⁺-K⁺ pump’s β₁-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na⁺-K⁺ pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na⁺-K⁺ pump activity is an important determinant of vascular tone through effects on [Ca²⁺]_i, we have examined the role of oxidative regulation of the Na⁺-K⁺ pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β₁-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na⁺-K⁺ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K⁺-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na⁺-K⁺ ATPase and decrease in K⁺-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β₁-subunit. Knock-out of FXYD1 dramatically decreased K⁺-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β₁-subunit glutathionylation, and enhanced K⁺-induced vasorelaxation. Ang II inhibits the Na⁺-K⁺ pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump’s β₁-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na⁺-K⁺ pump and may have an important protective role in the vasculature under conditions of oxidative stress.