Thyroid hormone effects upon Na+K+ dependent adenosine triphosphatase activity of microsomal fraction of liver, muscle and brain of toad Bufo melanostictus

Na+K(+)-ATPase activity in the liver and muscle microsomal membranes have been determined by different doses (0.1, 0.25, 0.5, 1 and 2 micrograms/gm of body weight) of L-triiodothyronine and L-thyroxine in the toad, Bufo melanostictus. The minimum effective dose of T3 was 0.5 microgram/g in case of both liver and muscle to stimulate the enzyme activity and there was dose dependent rise between T3 at the doses of 0.5 and 1 microgram/g. T3 at the doses of 1 and 2 micrograms/g produced more or less the same level of activity. T4 showed an increased activity at 1 and 2 micrograms/g without any dose dependent fashion in the two organs. The doses 0.1 and 0.25 microgram/gm body weight of T3 and 0.1, 0.25 and 0.5 microgram/gm body weight of T4 remained ineffective to elicit any response in both organs. The grain showed no significant change in the enzyme activity at any of the applied doses of T3 and T4. Cycloheximide inhibited T3 induced rise in Na+K(+)-ATPase activity of liver and muscle. Treatment with propylthiouracil caused a significant fall in Na+K(+)-ATPase activity of liver and muscle and the normal value was restored in the two organs after three consecutive injections of T4 at the dose of 1 microgram/g.     

Thyroid hormone-induced changes in different ion-specific adenosine triphosphatase activities in liver of Singi fish Heteropneustes fossilis (Bloch)

A single injection of different doses of T3 (0.5, 5, 20, and 50 micrograms/g) to Singi fish caused an increase in Na+K+-ATPase activity in crude liver homogenate in a dose-dependent non-linear fashion on the 3rd d. Ca++- and Mg++-ATPase activity increased only with 20 and 50 micrograms/g of T3. Lowering the dose of T3 to 0.1 microgram and 0.25 microgram/g in a single injection had not effect on these enzyme activities. TETRAC (1, 2, and 4 micrograms/g) and TRIAC (2 and 4 micrograms/g) in a single injection enhanced the activities of Na+K+-ATPase, but Ca++- and Mg++-ATPase activities remained unchanged on the 3rd d. Immersion of Singi fish in thiourea-containing medium (1 mg/ml) for 30 d caused reduction in Na+K+-ATPase activity, but Ca++- and Mg++-ATPase activity remained unaltered. The reduced level of Na+K+-ATPase activity in the thiourea-treated hypothyroid fish was recovered and even brought above the control level by a single injection of T3 at the dose of 0.5 microgram/g. Differential sensitivity of various ion-specific ATPases to T3 in liver of Singi fish is thus documented.     

Nuclear activation by thyroid hormone in liver of Singi fish: changes in different ion-specific adenosine triphosphatases activities

Various ion-dependent (Na+K+, Ca++ and Mg++) ATPases activities in liver cell nuclear membrane have been determined after a single injection of different doses (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2 and 4 micrograms/g) of L-triiodothyronine (T3) in Singi fish, Heteropneustes fossilis Bloch. Administration of T3 at a minimum effective dose of 0.05 micrograms upto 4 micrograms/g induced a rise (14 to 43% over control value) in the Na+K+-ATPase activity in a dose-dependent fashion maximum upto 1 microgram/g dose, whereas Ca++-ATPase showed a dose-dependent increase (20 to 43% over control) with 0.25-1 microgram/g of T3, although the increase in the respective enzyme activity was maintained upto 4 micrograms/g of T3 dose. Mg++-ATPase activity in liver cell nuclear membrane was found to be increased at 1 microgram-4 micrograms/g of T3 dose, showing a similar magnitude of increase (7% over the control value) with these doses of T3. Other doses of T3 (0.01 and 0.025 micrograms/g) were ineffective in altering the different ion-specific ATPase activity. Treatment of Singi fish with thiourea (1 mg/ml) for 30 days caused a significant fall in Na+K+, Ca++ and Mg++-ATPase activities upto 21%, 17% and 5%, respectively, below the euthyroid control level. A single injection of T3 at the dose of 1 microgram/g in the hypothyroid fish raised the Na+K+ and Ca++-ATPase activities to about 36% over the control value, and the Mg++-ATPase activity was restored to only the control level. Thus a dose-dependent nuclear effect of T3 is evident from the present investigation.     

Demonstration of hepatic cytosolic malic enzyme activity as a thyroid hormone sensitive physiologic parameter in a teleost, Heteropneustes fossilis bloch

Single injections of various doses (0.1, 0.25, 0.5, 5 and 20 micrograms/g) of T3 significantly increased the cytosolic malic enzyme activity (delta OD/min/mg cytosolic protein) in liver of Singi fish Heteropneustes fossilis Bloch, in a dose-dependent nature, maximum up to 5 micrograms/g dose on the 3rd day in comparison to the control. There was no difference in the enzyme activity between 5 and 20 micrograms/g of T3 doses. When the enzyme activity was expressed per mg DNA, the dose-dependent increase in the malic enzyme activity was observed upto 0.5 microgram/g of T3, whereas a fall in the enzyme activity was noticed with 5 and 20 micrograms/g of T3 doses. Lowering the dose of T3 to 0.05 microgram/g was without any effect on the malic enzyme activity (delta OD/min/mg cytosolic protein or DNA). Hepatic cytosolic protein content showed a biphasic nature of variation, significant increase with single injections of 0.05, 0.1, 0.25 and 0.5 microgram/g and a fall with 5 and 20 micrograms/g of T3 doses in comparison to the untreated control. Cycloheximide treatments of the Singi fishes counteracted both the T3-induced rise in the hepatic cytosolic malic enzyme activity (delta OD/min/mg cytosolic protein or DNA) and the hepatic cytosolic protein contents. Thiourea-treated hypothyroid fishes showed significantly decreased level of malic enzyme activity (delta OD/min/mg cytosolic protein or DNA) and cytosolic protein content in liver. A single injection of T3 at 0.25 microgram/g to the thiourea-treated fishes not only recovered but also increased the enzyme activity and cytosolic protein content above the untreated control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of hepatic mitochondrial alpha-glycerophosphate dehydrogenase by L-triiodothyronine in Singi fish (Heteropneustes fossilis Bloch)

A single injection of L-triiodothyronine (T3) in different doses (0.25, 0.5, 5, 20 and 50 micrograms/g) increased the hepatic mitochondrial cytochrome-linked alpha-glycerophosphate dehydrogenase (alpha-GPD) activity and mitochondrial protein content of Singi fish, as observed on the 3rd day. A non-linear dose-response relationship with respect to enzyme activity was observed with different doses of T3. A low dose of 0.1 micrograms of T3 per g failed to cause any change in alpha-GPD activity and mitochondrial protein content of the liver. The enhancement of alpha-GPD activity over the control level with a low and a high dose of T3, viz., 0.5 and 5 micrograms/g, was followed from the 1st to the 7th day, when it was found that enzyme activity reached the maximum level on the 3rd day and then gradually declined to the control value on the 7th day. The percentage increase in enzyme activity with 5 micrograms/g was higher than that with 0.5 microgram/g from the 2nd to 5th day. Compared to the control, these two doses of T3 caused an increase in alpha-GPD activity from the 1st to the 6th day. Cycloheximide inhibited the T3-induced increase in alpha-GPD activity, mitochondrial and total protein content of liver. Immersion of Singi fishes in thiourea-containing (1 mg/ml) medium for 30 days showed a fall in hepatic alpha-GPD activity in comparison to the euthyroid control. A single injection of T3 (0.5 microgram/g) to the hypothyroid fish recovered alpha-GPD activity to more than the euthyroid control level. An increase in mitochondrial protein content in the T3-injected hypothyroid fish has been observed. DNA content of the fish liver remained unchanged in every experimental condition. The results thus showed the significant responsiveness of the fish liver to thyroid hormone.     

Effects of steroid hormones on total brain Na(+)-k+ ATPase activity in Oreochromis mossambicus

The effects of administration of cortisol, corticosterone, testosterone, progesterone and a synthetic estrogen. diethylstilbestrol (DES) on total brain Na(+)-K+- ATPase were investigated in tilapia, O. mossambicus. Exogenous administration of 0.125 and 0.25 microg/g body weight of glucocorticoids and 0.125, 0.25 and 0.5 microg/g body weight of DES for 5 days significantly stimulated Na+(-) K+ ATPase activity by 14-41% in the brain, while 0.5 microg/g body weight of glucocorticoids did not evoke any response on the activity of the enzyme. Progesterone (0.125 and 0.25 microg/g body weight) administration significantly decreased the enzyme activity by 21-36% and high dose (0.5 microg/g body weight) was ineffective. Testosterone exhibited a biphasic effect on Na(+)-K+ ATPase activity--a low dose stimulated by 14% while middle and high doses inhibited it by 19-24%. The results seem to be the first report on the effect of steroids on brain ATPase activity in a teleost. When 0.25microg/g body weight of actinomycin D or puromycin was administered prior to the treatment of similar doses of hormones, the inhibitors significantly inhibited the effect of the hormones by 24-52%. This clearly shows that the effect of the hormones was sensitive to the action of inhibitors suggesting a possible genomic mode of action under long-term treatment. The results suggest that cortisol, corticosterone and DES may possibly stimulate the co-transport of glucose and excitation of membrane potential while progesterone and testosterone inhibit them in the brain of O. mossambicus by regulating the activity of Na(+)-K+ ATPase.     

Mechanism of Na+/K(+)-ATPase activation by trypsin and kallikrein

The mechanism of the Na+/K(+)-ATPase activation by trypsin (from bovine pancreas) and kallikrein (from human plasma) was investigated on enzyme preparations from different sources (beef heart and dog kidney) and at different degrees of purification (beef heart). Kallikrein was effective on both beef and dog enzymes, whereas trypsin stimulated only the beef-heart Na+/K(+)-ATPase. The extent of activation by the proteinases was inversely related to the degree of purification (maximal enzyme activation about 60 and 20% on the partially purified and the more purified enzymes, respectively). Enzyme activation was observed up to 0.5-0.6 microgram/ml of proteinase. At higher concentrations the activation decreased and was converted into inhibition at proteinase concentrations above 1.0 micrograms/ml. Na+/K(+)-ATPase stimulation was due to an increase in the Vmax of the enzyme reaction. Km for ATP remained unaffected. The activating effect was favoured by sodium and counteracted by potassium. Accordingly, Na(+)-ATPase activity was stimulated to a greater extent (up to 350%), whereas K(+)-dependent p-nitrophenylphosphatase activity proved to be insensitive to the actions of the proteinases. The Na+/K(+)-ATPase stimulation by both proteinases was antagonized by either ouabain or canrenone, two drugs that bind on the extracellular side of the Na+/K(+)-ATPase molecule. On the contrary, the enzyme inactivation observed at high proteinase concentrations was not counteracted by these two drugs. The stimulation of either Na+/K(+)- or Na(+)-ATPase activity was shown to be an irreversible effect without any significant protein degradation detectable by SDS gel electrophoresis. The results obtained suggest that proteinases exert their stimulatory effects by interacting preferentially with the E2 conformation of Na+/K(+)-ATPase at site(s) located on the extracellular moiety of the enzyme.     

Quantification of Rat Cerebral Cortex Na+,K+-ATPase: Effect of Age and Potassium Depletion

Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles.     

Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme

Two molecular forms of the (Na+,K+)-ATPase catalytic subunit have been identified in rat adipocyte plasma membranes using immunological techniques. The similarity between these two forms and those in brain (Sweadner, K. J. (1979) J. Biol. Chem. 254, 6060-6067) led us to use the same nomenclature: alpha and alpha(+). The K0.5 values of each form for ouabain (determined by inhibition of phosphorylation of the enzyme from [gamma-32P]ATP) were 3 X 10(-7)M for alpha(+) and 1 X 10(-5)M for alpha. These numbers correlate well with the K0.5 values for the two ouabain-inhibitable components of 86Rb+/K+ pumping in intact cells (1 X 10(-7) M and 4 X 10(-5)M). Quantitation of the Na+ pumps in plasma membranes demonstrated a total of 11.5 +/- 0.2 pmol/mg of membrane protein, of which 8.5 +/- 0.3 pmol/mg, or 75%, was alpha(+). Insulin stimulation of 86Rb+/K+ uptake in rat adipocytes was abolished by ouabain at a concentration sufficient to inhibit only alpha(+)(2-5 X 10(-6)M). Immunological techniques and ouabain inhibition of catalytic labeling of the enzyme from [gamma-32P]ATP demonstrated that alpha(+) was present in skeletal muscle membranes as well as in adipocyte membranes, but was absent from liver membranes. Since insulin stimulates increased Na+ pump activity in adipose and muscle tissue but not in liver, there is a correlation between hormonal regulation of (Na+,K+)-ATPase and the presence of alpha(+). We propose that alpha(+) is the hormonally-sensitive version of the enzyme.     

Vitamin B12-induced alterations in activities of alpha-glycerophosphate dehydrogenase and malic enzyme in brain of singi fish, Heteropneustes fossilis (Bloch)

Different doses of vitamin B12 (0.25, 0.5, 1, 2 and 4 micrograms/g, injected intraperitoneally for three consecutive days) altered the activities of mitochondrial-alpha-glycerophosphate dehydrogenase (alpha-GPD) and NADP-dependent cytosolic malic enzyme (ME) in the brain of singi fish. The alpha-GPD activity increased at doses of 0.5, 1, 2 and 4 micrograms/g vitamin B12. A dose of 0.5 microgram/g vitamin B12 induced less activity than higher doses. ME activity increased with 1, 2 and 4 micrograms/g of vitamin B12/g. The mitochondrial and cytosolic protein content remained unchanged after vitamin B12 administration. Cycloheximide treatment inhibited the vitamin B12-induced increase in alpha-GPD and ME activity. Thus, vitamin B12 is involved in the induction of some enzymes in fish brain.     

Stimulation of rat renal Na+, K+-ATPase activity by thyroid hormones

The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+,K+-ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+,K+-ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+,K+-ATPase activity was observed over T3 concentration range 10 pmol l-1 to 100 nmol l-1; at higher T3 concentrations, Na+,K+-ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l-1 -1 mmol l-1) failed to stimulate Na+,K+-ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.     

Stimulation of the Na+,K(+)-ATPase activity of K562 human erythroleukemia cells by triiodothyronine.

The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity of K562 human erythroleukemic cell was studied to understand why the erythrocyte sodium pump activity is decreased in hyperthyroidism. Na+,K(+)-ATPase activity of K562 cell lysates was assayed by measuring the release of inorganic phosphate (Pi) from ATP. Na+,K(+)-ATPase activity of K562 cell grown in the presence of T3 for 48 hours was significantly higher than that of control (0.98 +/- 0.05 mumol Pi h-1 mg protein-1 vs 0.82 +/- 0.10 mumol Pi h-1 mg protein-1, p < 0.05). The Na+,K(+)-ATPase activity could be stimulated in a time- and concentration-dependent manner; maximum stimulatory effect of T3 was seen at a concentration of 10(-7) mol/L. When an inducer [cytosine-beta-D-arabino-furanoside (ARA-C)] was added to the culture medium, the K562 cells showed signs of differentiation and synthesised haemoglobin. At the same time, the Na+,K(+)-ATPase activity remained high. We conclude that T3 stimulates Na+,K(+)-ATPase activity of K562 cells and in the presence of T3 during differentiation, the enzyme activity remains high.     

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).     

Increased acylphosphatase levels in erythrocytes, muscle and liver of tri-iodothyronine treated rabbits

To explore a possible role of acylphosphatase in the regulation of energy metabolism, we measured this enzyme's activity and content in skeletal muscle, liver and erythrocytes of normal and tri-iodothyronine treated rabbits. Besides acylphosphatase we assayed (Na+ + K+)-ATPase, Ca2(+)-ATPase and several enzymes of carbohydrate metabolism. Acylphosphatase activity in erythrocytes rose steadily during treatment with triiodothyronine (25 micrograms/Kg per day for 5 weeks), and its increase occurred earlier and was much more pronounced than that of other soluble enzymes. In erythrocytes of treated animals (Na+ + K+)-ATPase declined whereas Ca2(+)-ATPase activity increased, in agreement with previously reported findings. In muscle and liver of the treated animals acylphosphatase activity was about twice as high as in the controls; in these tissues we found also increased activities for (Na+ + K+)-ATPase, fructose-1,6-bisphosphatase and glucose-6-phosphatase. In any case, among the enzymes we examined, acylphosphatase was one of the most strongly and regularly stimulated by the treatment. Furthermore we observed, through an immunochemical procedure, that there was a congruence between increases in acylphosphatase activity and content. On the basis of these results we conclude that the rise in acylphosphatase levels in treated animals is probably due to its increased biosynthesis. The possible significance of these findings in the metabolic modifications associated with hyperthyroidism are discussed.     

Changes in lipid composition and some biologically important enzyme activities in the microsomal membranes of developing toad ovary in different seasons

The microsomal membranes isolated by sucrose density gradient centrifugation from developing toad ovary have been found to differ significantly in lipid composition and various enzyme activities in different seasons. All the enzymes studied, viz. Na+, K(+)-ATPase, delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta HSD) and prostaglandin synthetase, exhibited maximum activity during the breeding season (July-September) at all stages of development (a,b,c & d). The activities of Na+, K(+)-ATPase and delta 5-3 beta HSD increased with development while that of prostaglandin synthetase followed the reverse order. The total phospholipid, cholesterol and fatty acid contents also varied with season and development. The increase in Na+, K(+)-ATPase and delta 5-3 beta HSD activities in the microsomal membranes of toad ovary at breeding season is accompanied with concomitant increase in phospholipid and unsaturated fatty acid contents at different stages in this season, thereby suggesting some correlation between them.     

Effects of choline on Na(+)- and K(+)-interactions with the Na+/K(+)-ATPase.

Choline chloride, 100 mM, stimulates Na+/K(+)-ATPase activity of a purified dog kidney enzyme preparation when Na+ is suboptimal (9 mM Na+ and 10 mM K+) and inhibits when K+ is suboptimal (90 mM Na+ and 1 mM K+), but has a negligible effect at optimal concentrations of both (90 mM Na+ and 10 mM K+). Stimulation occurs at low Na+ to K+ ratios, but not at those same ratios when the actual Na+ concentration is high (90 mM). Stimulation decreases or disappears when incubation pH or temperature is increased or when Li+ is substituted for K+ or Rb+. Choline+ also reduces the Km for MgATP at the low ratio of Na+ to K+ but not at the optimal ratio. In the absence of K+, however, choline+ does not stimulate at low Na+ concentrations: either in the Na(+)-ATPase reaction or in the E1 to E2P conformational transition. Together, these observations indicate that choline+ accelerates the rate-limiting step in the Na+/K(+)-ATPase reaction cycle, K(+)-deocclusion; consequently, optimal Na+ concentrations reflect Na+ accelerating that step also. Thus, the observed K0.5 for Na+ includes high-affinity activation of enzyme phosphorylation and low-affinity acceleration of K(+)-deocclusion. Inhibition of Na+/K(+)-ATPase and K(+)-nitrophenylphosphatase reactions by choline+ increases as the K(+)-concentration is decreased; the competition between choline+ and K+ may represent a similar antagonism between conformations selected by choline+ and by K+.     

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.     

The heterogeneity of the Na+, K(+)-ATPase ouabain sensitivity in microsomal membranes of rat colon smooth muscles

The dose dependence of the Na+, K(+)-ATPase ouabain inhibition in the rat colon smooth muscle permeabilized microsomes has been analyzed according to the model of two independent binding sites of inhibitor to determine the activity of separate molecular forms of the enzyme that differ by affinity for cardiac glycosides. The two-phase inhibition curve with moderate content of the high-affinity activity component was revealed. The apparent inhibition constant of the low-affinity component corresponds to the value for the rat kidney microsomal Na+, K(+)-ATPase (alpha1-isoform). The specific role of the alpha2- and alpha1- Na+, K(+)-ATPase catalytic subunit isoforms in colonic smooth muscle electromechanical coupling is considered.     

Hypercortisolemia does not affect the branchial osmoregulatory responses of the marine teleost Sparus sarba

The effect of cortisol treatment on branchial Na(+)-K(+)-ATPase subunit mRNA abundance, enzyme activity, chloride cell number/morphometrics and serum electrolyte levels were investigated for the marine teleost Sparus sarba. Groups of fish received intraperitoneal injections of cortisol at a concentration of 4 micrograms/g body weight, daily, over a seven-day period. This dose of cortisol was sufficiently high enough to maintain a condition of hypercortisolemia as serum cortisol levels in treated fish were eleven fold higher than controls at time of sacrifice. By using branchial Na(+)-K(+)-ATPase alpha- and beta-subunit cDNA clones we were able to demonstrate that cortisol administration to S. sarba caused a significant elevation in the abundance of alpha-mRNA whereas the levels of beta-mRNA were unchanged. In addition Na(+)-K(+)-ATPase activity remained unaltered by cortisol administration. Branchial chloride cell number, exposure, apical area as well as serum Na+ and Cl- levels remained unchanged after cortisol administration. The results of this study suggest that elevated cortisol level may not necessarily translate into modulated branchial Na(+)-K(+)-ATPase activity and chloride cell function in hypo-osmoregulating marine fish.     

Characterization and partial isolation of ouabain-insensitive Na(+) -ATPase in MDCK I cells

We show that MDCK I cells express, besides the classical (Na(+)+K(+))ATPase, a Na(+)-stimulated ATPase activity with the following characteristics: (1) K(0.5) for Na(+) 7.5+/-1.5 mM and V(max) 23.12+/-1.1 nmol Pi/mg per min; (2) insensitive to 1 mM ouabain and 30 mM KCl; and (3) inhibited by furosemide and vanadate (IC(50) 42.1+/-8.0 and 4.3+/-0.3 microM, respectively). This enzyme forms a Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate with molecular weight of 100 kDa. Immunoprecipitation of the (Na(+)+K(+))ATPase with monoclonal anti-alpha(1) antibody reduced its activity in the supernatant by 90%; the Na(+)-ATPase activity was completely maintained. In addition, the formation of the Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate intermediate occurred at the same magnitude as that observed before immunoprecipitation. These data suggest that Na(+)-ATPase and (Na(+)+K(+))ATPase activities are independent, with Na(+)-ATPase belonging to a different enzyme entity.