Activity and thermal stability of the enzymes of energy support in the common frog

The activity and heat resistance of succinate dehydrogenase (SDG), Na, K-ATPase and Mg-ATPase of the grass frog (39 specimens) have been determined. No correlation was found between individual levels of heat resistance both of either enzyme examined and of the same enzyme but taken from different tissues (SDG of liver and muscles). The average level of heat resistance of SDG in liver is significantly higher than that in m. gastrocnemius. A statistically significant correlation was observed between individual levels of enzyme activity in the internal organs (SDG of liver, Na, K-ATPase and Mg-ATPase of kidney). The activity of SDG of muscles does not correlate with that of either partner.     

Correlations of the individual levels of the activity and heat resistance of energy-support enzymes in the lake frog

A study was made of the correlation between the levels of the activity and heat resistance of Na,K-ATPase, Mg-ATPase and succinate dehydrogenase (SDG) in Rana ridibunda. A correlation was observed between the activities of two ATPases, and between either of them and the activity of liver SDG. The correlation coefficient between the heat resistance levels was statistically significant only in SDG of liver and muscles. The question whether the criterion of T50% inactivation may be good for a comparative appraisal of the heat resistance of enzymes is under discussion.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

Properties of the enzymes of energy supply in the common frog under various physiological states. II. Changes in the heat resistance of adenosine triphosphatases and succinate dehydrogenase

The heat resistance of SDG, Na, K-ATPase and Mg-ATPase of the grass frog was determined in January, March and May, the number of animals examined being 30-40 in either experiment. It was found that the average level of the heat resistance of the enzymes studied shows significant, often differently directed changes, which depend on the physiological state of an organism. Negligible correlation between the thermal sensitivity of different enzymes of an organism during hibernation, completely disappear during the activity state.     

Regulation of Na,K-ATPase function in the lens

The two cell types in the lens, epithelium and fiber, have a very different specific activity of Na,K-ATPase; activity is much higher in the epithelium. However, judged by Western blot, fibers and epithelium express a similar amount of both Na,K-ATPase alpha and beta subunit proteins. Na,K-ATPase protein abundance does not tally with Na,K-ATPase activity. Studies were conducted to examine whether protein synthesis plays a role in maintenance of the high Na,K-ATPase activity in lens epithelium. An increase of cytoplasmic sodium was found to increase Na,K-ATPase protein expression in the epithelium, but not in the fibers. The findings illustrate the ability of lens epithelium to synthesize new Na,K-ATPase protein as a way to boost Na,K-ATPase in response to cell damage or pathological events. Methionine incorporation studies suggested Na,K-ATPase synthesis may also play a role in day to day preservation of high Na,K-ATPase activity. Na,K-ATPase protein in lens epithelial cells appeared to be continually synthesized and degraded. Experiments with cycloheximide suggest that specific activity of Na,K-ATPase in the lens epithelium may depend on the ability of the cells to continuously synthesize fresh Na,K-ATPase proteins. However, other factors such as phosphorylation of Na,K-ATPase alpha subunit may also influence Na,K-ATPase activity. When intact lenses were exposed to the agonist thrombin, Na,K-ATPase activity was diminished, but the response was suppressed by inhibitors of the Src family of non-receptor tyrosine kinases. Thrombin elicited tyrosine phosphorylation of lens epithelium membrane proteins, including a 100 kDa protein band thought to be the Na,K-ATPase alpha 1 subunit. It remains to be determined whether a tyrosine phosphorylation mechanism contributes to the low activity of Na,K-ATPase in lens fibers.     

Therapeutic action of an antioxidant in chronic emotional-pain stress in the rat

Chronic emotional pain stress in rats causes disturbances of the cardiovascular system function (increase in arterial pressure and in heart rate), typical of neuroses-like state, and changes of the vegetative nervous system reactivity tested with functional load by two-hour hypokinesis. Increase in spleen weight is observed as well as a tendency to adrenals weight increase, a decrease of Na, K-ATPase activity and activation of lipid peroxidation in cortical and hippocampal homogenates. Administration of F-801 antioxidant according to therapeutic scheme after the end of stress action, restores normal function of the cardiovascular system, normal reactivity of the vegetative nervous system, decreases adrenals weight and increases the weight of thymus and also normalizes ATPase activity and the level of lipid peroxidation. A backward correlation dependence of the Na, K-ATPase activity on the level of malondialdehyde in the brain tissue has been established.     

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.     

A novel role for protein phosphatase 2A in the dopaminergic regulation of Na,K-ATPase

Stimulation of dopaminergic type 1 (D(1)) receptors increases lung edema clearance by regulating Na,K-ATPase function in the alveolar epithelium. We studied the role of serine/threonine protein phosphatases in the Na,K-ATPase regulation by D(1) agonists in A549 cells. We found that low doses of the type 1/2A protein phosphatase inhibitor okadaic acid as well as SV40 small t antigen transiently transfected into A549 cells prevented the D(1) agonist-induced increase in Na,K-ATPase activity and translocation from intracellular pools to the plasma membrane. This was associated with a rapid and transient increase in protein phosphatase 2A activity. We conclude that D(1) stimulation regulates Na,K-ATPase activity by promoting recruitment of Na,K-ATPases from intracellular pools into the basolateral membranes of A549 cells via a type 2A protein phosphatase.     

Evidence of time-dependent changes in renal medullary Na,K-ATPase activity and expression in diabetic rats.

The medullary thick ascending limb (MTAL) of the kidney displays structural changes during long term diabetes. After twelve weeks of diabetes, there is controversy over the changes in Na,K-ATPase activity. To observe the long-term changes, we studied MTAL Na,K-ATPase activity and protein expression in diabetic animals 6 (6W) and 12 weeks (12W) after induction of diabetes with streptozotocin. Three groups were studied, one control group, one group 6W after, and one group 12W after induction of diabetes. Membrane fractions from the inner strip of the outer medulla representing MTAL were isolated. Na,K-ATPase activity and western blottings of alpha1- and beta1-subunits were carried out. 6W diabetes resulted in an increase, and 12W in a decrease in the MTAL Na,K-ATPase activity versus the control group (respectively 63.3 +/- 21.2; 7.5 +/- 2.4 and 31.6 +/- 11.4; micromol Pi/mg prot/hr +/- SEM). The Na,K-ATPase subunit expression was increased at 6W, and decreased after 12W, resulting in amounts below control values for both alpha1- and beta1-subunits. Our results confirm a diabetes-induced biphasic time-dependent alteration MTAL Na,K-ATPase activity, supported by similar changes in alpha1 and beta1 Na,K-ATPase subunits-expression.     

Kinetic parameters of Na,K-ATPase in the gills of crabs adapted to freshened sea water]

The dependence of Na,K-ATPase activity on concentrations of ATP, Na+, K+, Mg2+ and ouabain in the membrane preparations of crab gills was studied. The first group of crabs was adapted to freshened (25%) and the second one--to normal (100%) sea water. A 40-day adaptation of crabs to the freshened sea water results in an increase of maximal activity of Na,K-ATPase, but does not affect the enzyme affinity for ATP, Na+, K+, Mg2+ and ouabain, as well as its cooperative properties. It is assumed that adaptation of crabs to freshened sea water is accompanied by an accumulation of Na, K-ATPase in the epithelial cell membranes or crab gills without causing any qualitative changes of the enzyme.     

Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures

Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase. Three amiloride analogues were tested within the range of 10(-6) M to 10(-4) M in primary cultures of proximal tubule cells. Only ethylisopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells. The time course study revealed that EIPA (10(-4) M) significantly decreased Na,K-ATPase alpha- and alpha-mRNA abundance within 4 hr and suppressed Na,K-ATPase alpha- and beta-mRNA levels by 76.3 +/- 4.5% and 85.5 +/- 5.8%, respectively, within 24 hr. The decrease in Na,K-ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5 +/- 10.8% and 48.8 +/- 5.9% within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both alpha- and mature beta-protein. The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed. Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising pHi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H+. In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity. Take together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post-translational mechanisms, which confers cytotoxic effects on proximal tubule cells.     

Changes in the Na, K-ATPase of neuronal membranes in penicillin-induced epileptic foci in the cerebral cortex of the rat]

The relationship between electrophysiological changes and Na, K-ATPase activity of neuronal membranes in sodium penicillin-induced epileptic foci was studied. Na,K-ATPase activity is inhibited both in the primary focus and in homotopic contralateral area during latent period and in the stage of forming epileptic activity. In the stage of marked convulsive activity Na, K-ATPase is inhibited only in the primary focus. It is shown that penicillin at a concentration range of 2 x 10(-6)--2 x 10(-3) M does not influence Na,K-ATPase activity of crude synaptosomes of the rat brain cortex. It is suggested that Na,K-ATPase inactivation may serve as a pathogenetic factor in the development of convulsive process.     

A cytophotometric analysis of the activity of oxidative enzymes and Na, K-ATPASE in vertebrate nephrons at different levels of sodium transport in the kidney.]

Using quantitative cytochemistry, activities of Na, K-ATPase, succinate dehydrogenase (SDH) and alpha-keto-glutarate dehydrogenase (alpha-KDH) was investigated in cells of renal tubules at different levels of sodium reabsorption in the kidney. The activity of these enzymes in mammals and birds renal tubule cells was found to be higher than in the cells of corresponding renal tubules of cold-blooded vertebrates. This corresponds to the increased total amount of reabsorbed sodium in the kidney of warm-blooded animals. The summer frogs, as compared to the winter ones, exhibit higher activities of SDH and Na,K-ATPase in the proximal tubule cells where changes in sodium reabsorption are also noted. In the kidney of marine teleosts, a negative correlation between U/PNa and the activity of SDH and Na,K-ATPase in the cells of proximal and distal tubule was observed. Aldosterone was found to stimulate sodium reabsorption and to activate Na,K-ATPase.SDH and alpha-KDH mainly in the distal convoluted tubule. Furosemide was observed to inhibit sodium reabsorption and to reduce SDH and Na,K-ATPase activities in cells of the proximal tubule and Henle's loop. In the kidney of adrenalectomized rats, both sodium reabsorption and activities of Na,K-ATPase, SDH, alpha-KDH decreased in all the segments of the nephron. The data obtained suggest that changes in sodium reabsorption may be coupled with those in the activities of the investigated enzymes.     

Light-induced changes in activity of Na, K-ATPase from the retinal photoreceptors of vertebrates: possible mechanism

The mechanism of light-induced changes in the activity of Na,K-ATPase from plasma membranes (PM) of photoreceptor cells was studied in vitro. Illumination resulted in inhibition of the ATPase activity and an increase of 18O exchange between water and Pi. The maximum light effect was revealed when the PM contained both the inner segments of the rods (RIS) and rod outer segments (ROS) of the photoreceptor cells. Lipid peroxidation stimulated by the FeSO4+ascorbate system induced a decrease of the ATPase activity. Antioxidants (ionol, Na2SeO3, vitamin E) prevented the effect of the lipid peroxidation products on NA,K-ATPase and the photoinduced changes of the enzyme activity. It is supposed that the photoinduced changes of the Na,K-ATPase activity in vitro are due to lipid peroxidation of photoreceptor PM.     

The effect of ionizing radiation on the regulation of the Na,K-ATPase activity of the kidney by univalent cations

The effect of ionizing radiation of 0.206 C/kg on the kinetics of activation of rat kidney Na,K-ATPase preparation by Na and K ions was studied as an index of possible qualitative and quantitative changes in the properties of the enzyme. Ionizing radiation was shown not only to increase the enzyme activity but also to change the optimal rate of ATP hydrolysis by Na,K-ATPase and to induce some differences in the shape of the curve for Na,K-ATPase dependence upon Na-sodium//potassium ion ratio in the incubation medium.     

Activation characteristics of ouabain-sensitive respiration and Na,K-ATPase in the kidney cortex and medullary zone of rats adapted to cold

The endogenous and ouabain-sensitive respiration and Na, K-ATPase activity in the cortex and kidneys medulla of the cold-acclimated male albino rats have been determined. The increase of the respiration rate has been stated to be caused by the Na-pump activation. The obtained changes of Na, K-ATPase activity are supposed to be connected with the regulation of concentration and sodium excretion function of kidneys.     

Na,K-ATPase activity in the myocardial sarcolemma in ischemia

The total time-controlled ischemia (up to 45 min) was studied for its effect on the Na,K-ATPase activity, content of nonesterified fatty acids (NEFA) and intensity of lipid peroxidation (LP) in sarcolemmal (SL) preparations and soluble fractions (SF) from the rat and guinea-pig left ventricles. A strong correlation between Na, K-ATPase inhibition and NEFA accumulation was revealed in the SF. On the contrary, changes in the NEFA content and LP level both in SL and SF did not correlate with a decrease in the enzymic activity. Pretreatment with albumin (0.5 mg/ml) induced equally small increase both in the control and in the ischemic SL preparations. It is suggested that the Na,K-ATPase activity during a short period of myocardial ischemia (up to 45 min) may be due to the NEFA accumulation in the cytosolic and/or extracellular space, but not in SL.     

Postradiation changes of the active ion transport systems of the CNS. Na, K-ATPase of neurons and neuroglia

A study was made of the effect of X-radiation (0.31 C/kg and 3.875 C/kg) on Na, K-ATPase in fractions enriched with neurons and neuroglia. The results show the impairment of the neuronal-glial relationship in Na, K-ATPase activity. The most important differences in the pattern of changes in Na, K-ATPase system of brain cells were followed up after irradiation with lethal and sublethal doses.     

Effect of mineralocorticoids on the Na, K-ATPase activity of the rat brain

The effect of desoxycorticosterone (DOC) on Na, K-ATPase activity was studied in vivo and in vitro on microsomal rat brain fractions. An hour after intramuscular administration of DOC a noticeable increase in the enzyme activity was observed. Preincubation of microsomal brain fractions with 5 and 15 mkg/ml of DOC caused a decrease in Na, K-ATPase activity, with the results evident 3-5 minutes after the addition of the hormone into the incubation medium. The idea of a two-phase hormonal effect is suggested. It is likely that desoxycorticosterone effect is realized both by the direct influence, on Na, K-ATPase of the brain plasma membrane and by the influence on the biosynthesis.     

Intracellular Na+ controls cell surface expression of Na,K-ATPase via a cAMP-independent PKA pathway in mammalian kidney collecting duct cells

In the mammalian kidney the fine control of Na+ reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+ transport. In the cortical collecting duct (CCD), a rise in intracellular Na+ concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4 collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K(+)-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.