Platelet and erythrocyte Mg2+, Ca2+, Na+, K+ and cell membrane adenosine triphosphatase activity in essential hypertension in blacks.

OBJECTIVE: To assess the relationship between intracellular Mg2+, Ca2+, Na+ and K+ and cell membrane adenosine triphosphatase (ATPase) activity in normotensive and hypertensive blacks. DESIGN: Intracellular cations and cell membrane ATPase activity were studied in black patients with untreated essential hypertension and age-, weight- and height-matched normotensive controls. Platelet, erythrocyte and serum Mg2+, Ca2+, Na+ and K+ levels as well as platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities were measured in all subjects. METHODS: Intracellular Na+ and K+ were measured by flame photometry and Mg+ and Ca+ by atomic absorption spectrophotometry. Cell membrane ATPase activity was determined by a colorimetric method. RESULTS: The hypertensive group consistently demonstrated depressed activity of each ATPase studied, with significantly lower serum Mg2+, serum K+, erythrocyte Mg2+ and platelet Mg2+ levels compared with the normotensive group. Platelet Na+ and Ca2+ and erythrocyte Ca2+ were significantly elevated in the hypertensive group. In the hypertensive group, mean arterial pressure (MAP) was inversely correlated with platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase. Serum Mg2+, serum Ca2+ and platelet Mg2+ were negatively correlated with MAP in the hypertensive group whilst erythrocyte and platelet Ca2+ were positively correlated. In the normotensive group, platelet Mg2+ and MAP were negatively, and erythrocyte Ca2+ and MAP, positively correlated. CONCLUSIONS: Black patients with essential hypertension have widespread depression of cell membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities with serum and intracellular Mg2+ depletion and cytosolic Na+ and Ca2+ overload, which may reflect an underlying membrane abnormality in essential hypertension. These cellular abnormalities may be related to the defective transport mechanisms that in turn may be aggravated by Mg2+ depletion.     

Hormonal control on enzymes of osmoregulation in a teleost, Anabas testudineus (BLOCH): an in vivo and in vitro study

Hormonal control of osmoregulation in teleosts is not well understood. Role of cGH, oGH, PRL, T3 and insulin on gill Na+,K+-ATPase, Mg2+ and Ca2+ ATPases was studied in A. testudineus. Short term administration of cGH, PRL or T3 significantly increased Na+,K+-ATPase, Mg2+ and Ca2+ ATPases, while oGH influenced only Mg2+ ATPase, and insulin stimulated Na+,K+-ATPase. Long-term treatment with cGH and PRL also significantly increased Na+,K+-ATPase activity. GH had an additive with T3 on stimulating Na+,K+-ATPase activity. In vitro addition of cGH and oGH also had definite stimulatory effect on gill Na+,K+-ATPase except for 2ng oGH. Bromocryptine treatment caused a significant reduction on Na+,K+-ATPase activity. Both in vivo and in vitro treatments of cGH and PRL independently reversed the action of bromocryptine on Na+,K+-ATPase. Combined treatment of cGH+PRL was more prominent in stimulating Na+,K+-ATPase in bromocryptine treated fish. Present study reveals that GH, PRL and T3 have definite regulatory role on enzymes of osmoregulation in the teleost Anabas testudineus.     

Synergistically Stimulated (Na+,K+)-Adenosine Triphosphatase from Plasma Membrane of a Marine Diatom

An ATP-hydrolyzing activity with the properties of a Mg(2+)-dependent (Na(+),K(+))-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from a 20-fold purified plasma membrane fraction of the marine diatom, Nitzschia alba is described.The basal activity requires Mg(2+) and further stimulation by Na(+) or Na(+) plus K(+) is dependent on the presence of Mg(2+); Mn(2+) or Co(2+) can partially substitute for the divalent cation requirement but Ca(2+) equimolar with Mg(2+) inhibits the activity by 54%. ATP is the preferred substrate for the Na(+) plus K(+) stimulated activity, while CTP, UTP, and ADP are only slightly hydrolyzed. The apparent K(m) is 8 x 10(-4) M ATP.The ATP hydrolysis-rate is dependent on the relative concentrations of Na(+) and K(+); the K(0.5) for Na(+) and K(+) are 2 mM and 50 mM, respectively. Basal activity is synergistically stimulated by Na(+) plus K(+) only at certain ion concentrations and shows a strong specificity for both cations.In the presence of Na(+) at 5 mM and K(+) at 350 mM, the ATPase is completely inhibited by p-chloromercuric benzoic acid 10(-4) M, N-ethyl maleimide 10(-3) M, and iodoacetamide 10(-2) M, but is insensitive to ouabain at 10(-7) to 10(-3) M.This study demonstrates for the first time that algal plasma membrane contains an ATPase that is synergistically stimulated by Na(+) and K(+).     

ATPase activity of rat brain microsomal and synaptosomal fractions in insulin hypoglycemia and its treatment with glucose]

Experiments on albino rats were made to investigate the activity of Na+, K(+)-ATPase and Mg(2+)-ATPase of fractions of microsomes and nerve endings, isolated from the cortex of the cerebral hemispheres, subcortical structures and medulla oblongata. The brain of healthy animals and that of rats in the state of insulin coma (40 units/kg of the hormone intramuscularly) were investigated at various periods after coma arrest with glucose. It has been assumed that an increase in the number of active molecules of Na+, K(+)-ATPase under the influence of structural changes of membranes in hypoglycemia cannot provide the electric activity of neurons in the absence of glycolytic ATP in neuroglycopenia.     

衰竭心肌能量代谢重构及β3肾上腺素能受体、过氧化物酶体增殖物激活受体α表达变化的研究

目的 通过检测衰竭与健康心肌组织代谢底物含量、相关酶活性及β3肾上腺素能受体、过氧化物酶体增殖物激活受体α（PPARα）基因和蛋白表达的变化,探讨衰竭心肌代谢重构的表现及可能机制。方法 选取6例意外伤亡健康者心肌组织和20例心外科瓣膜置换术的心力衰竭患者,检测心肌游离脂肪酸（FFA）、乳酸（LD）含量和ATP酶活力。应用逆转录聚合酶链反应、Western blot和免疫组化法分别检测心肌组织β3受体和PPARα mRNA表达以及蛋白和定位。结果 衰竭心肌FFA和LD含量明显增加（P〈0．01和P〈0．05）。Na^＋K^＋-ATP酶和Ca^2＋Mg^2＋-ATP酶活性则显著降低（P〈0．05和P〈0．01）。衰竭心肌β3肾上腺素能受体mRNA和蛋白表达明显高于正常心肌,而PPARα表达则显著低于正常心肌,但并未发生定位变化。结论心力衰竭时存在代谢重构,表现为心肌代谢底物发生转变,代谢相关酶活性下降等,β3肾上腺素能受体和PPARα在衰竭心肌分别上调和下调,可能参与心肌组织代谢重构。     

The role of cortisol and growth hormone in seawater adaptation and development of hypoosmoregulatory mechanisms in sea trout parr (Salmo trutta trutta)

The role of growth hormone (GH) and cortisol in the development of hypoosmoregulatory mechanisms in sea trout parr, Salmo trutta trutta, was investigated by injecting freshwater (FW) yearlings every second day with saline, ovine growth hormone (oGH, 2.0 micrograms/g), cortisol (hydrocortisone hemisuccinate, 8.0 micrograms/g), or oGH + cortisol for a maximum of 14 days. Subgroups of the treated fish were transferred to three-fourths seawater (SW) after 7 or 15 days of treatment and the effects on plasma Na+, Cl-, muscle water content, gill Na+/K(+)-ATPase activity, and gill interlamellar chloride cell density were examined. In FW, gill Na+/K(+)-ATPase chloride cell density, and chloride cell apical to basal length increased by all hormone treatments, most significant by oGH + cortisol treatment. Plasma ions and muscle water content were unaffected in FW. Both SW transfers resulted in considerable mortality (50%) in control fish, whereas few cortisol-treated and no GH-treated or GH + cortisol-treated fish died. Plasma Na+ and Cl- levels increased dramatically (greater than 50%) in control fish and muscle water content decreased (8%) on Day 2 after both transfers. All hormone-treated groups regulated plasma ions and muscle water significantly better than controls in SW, indicating the physiological significance of the treatment. Notably, the oGH + cortisol-treated fish showed only insignificant changes in ion-osmotic homeostasis after SW transfer, suggesting a synergistic effect of the two hormones. It is concluded that treatment with the two hormones increases the salinity tolerance of sea trout parr at a developmental stage where FW life is obligatory.     

Developmental differences in the responsiveness of gill Na+,K(+)-ATPase to cortisol in salmonids.

The ability of cortisol to increase gill Na+,K(+)-ATPase activity was examined in several salmonid species during development. Coho salmon (Oncorhynchus kisutch) parr were unresponsive to cortisol in vitro (10 micrograms/ml for 2 days) in November. Responsiveness was significant from January to March, peaking in January just prior to seasonal increases in gill Na+,K(+)-ATPase activity. Gill tissue became unresponsive to in vitro cortisol in April when in vivo gill Na+,K(+)-ATPase activity peaked. The ability of cortisol to stimulate gill, Na+,K(+)-ATPase activity in postemergent fry (2-3 months after hatching) was examined in chum (O. keta), chinook (O. tschawytscha), coho, and Atlantic salmon (Salmo salar). Initial levels of gill Na+,K(+)-ATPase activity were elevated in chum salmon, which normally migrate as fry. Cortisol (10 micrograms/ml for 4 days in vitro) increased gill Na+,K(+)-ATPase activity in chum salmon fry (48% above initial levels), had a limited but significant effect in chinook salmon fry, and had no effect in coho and Atlantic salmon fry. In an in vivo experiment, Atlantic salmon previously exposed to simulated natural photoperiod (SNP) and continuous light (L24) received four cortisol injections of 2 micrograms.g-1 every third day. SNP fish responded with increased gill Na+,K(+)-ATPase activity (+66%), whereas L24 fish were not affected. Atlantic salmon presmolts with initially low levels of gill Na+,K(+)-ATPase activity responded to cortisol in vitro, whereas smolts with initially high levels of gill Na+,K(+)-ATPase activity were unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-transport ATPases of vascular smooth muscle

To characterize the Ca2+-transport properties of the plasma membrane and of the endoplasmic reticulum of bovine pulmonary artery, membrane vesicles are subfractionated by a procedure of density-gradient centrifugation that takes advantage of the selective effect of digitonin on the density of plasma-membrane vesicles. The obtained endoplasmic-reticulum fraction contains hardly any plasma-membrane vesicles, whereas the plasma-membrane fraction is still contaminated by a substantial amount of endoplasmic-reticulum vesicles. An adenosine 5'-triphosphate (ATP) energized Ca2+-transport system and a Ca2+-stimulated ATPase activity are present in both subcellular fractions. The Ca2+ transport by the plasma membrane is catalyzed by a (Ca2+,Mg2+)-ATPase of Mr 130,000. It binds calmodulin and it has a low steady-state phosphoprotein intermediate level. The endoplasmic-reticulum vesicles contain a Ca2+-transport ATPase of Mr 100,000 that is characterized by a high steady-state phosphointermediate level. It is antigenically related to the Ca2+-pump protein of cardiac sarcoplasmic reticulum. Phospholamban, the regulatory protein of the Ca2+-transport enzyme of cardiac sarcoplasmic reticulum, is also present in the endoplasmic reticulum of the pulmonary artery. A comparison of these fractions with the previously characterized fractions from porcine gastric smooth muscle reveals important differences in the basal Mg2-ATPase activity, in the ratio of the (Ca2+,Mg2+)-ATPase of the plasmalemma to that of the endoplasmic reticulum, and in the ratio of the (Na+,K+)-ATPase activity to the plasmalemmal (Ca2+,Mg2+)-ATPase activity. These differences can be ascribed in part to the species and in part to the tissue. These data suggest that in the bovine pulmonary artery the Ca2+ extrusion via the ATP-dependent Ca2+ pump may have a less predominant role, and that the Ca2+ uptake by the endoplasmic reticulum, and possibly also the Ca2+ extrusion via the Na+-Ca2+ exchanger could be more important in this tissue than in the porcine stomach.     

The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity, although the activity was lower than that of the wild-type SR Ca(2+)-ATPase. Moreover, this Ca(2+)-sensitive ATPase activity was inhibited by ouabain. The chimera NCC, in which Met1-Gly354 of the SR Ca(2+)-ATPase were replaced with the corresponding portion of the Na+,K(+)-ATPase, lost the thapsigargin-sensitive Ca(2+)-ATPase activity seen in CCC and [n/c]CC. [3H]Ouabain binding to [n/c]CC and NCC demonstrated that the affinity for this inhibitor seen in the wild-type chicken Na+,K(+)-ATPase was restored in these chimeric molecules. Thus, the ouabain-binding domains are distinct from the thapsigargin sites; ouabain binds to the amino-terminal portion (Met1 to Asp200) of the Na+,K(+)-ATPase alpha 1 subunit, whereas thapsigargin interacts with the regions after Asp162 of the Ca(2+)-ATPase. Moreover, the amino-terminal 200 amino acids of the Na+,K(+)-ATPase alpha 1 subunit are sufficient to exert ouabain-dependent inhibition even after incorporation into the corresponding portion of the Ca(2+)-ATPase, and the segment Ile163 to Gly354 of the SR Ca(2+)-ATPase is critical for thapsigargin- and Ca(2+)-sensitive ATPase activity.     

Amiodarone inhibits the 3,5,3'-triiodothyronine-dependent increase of sodium/potassium adenosine triphosphatase activity and concentration in human atrial myocardial tissue.

In animal models the function of the sodium pump (sodium/potassium-adenosine triphosphatase [Na+/K(+)-ATPase]) is enhanced by 3,5,3'-triiodothyronine (T3) and inhibited by the antiarrhythmic agent amio. However, it is still unclear whether the effect of the drug on Na+/K(+)-ATPase depends on the interference with thyroid hormone action. We evaluated the interaction of T3 with amiodarone on Na+/K(+)-ATPase activity and site number in human myocardium. Right atrial slices were cultured with (T3+) and without (T3-) 3 nM T3 in presence and absence of amiodarone at therapeutical dose (1.5 microM). When compared to T3+, T3- preparations showed decreased 3H-ouabain binding (p < 0.004) and lower 20-minute and 45-minute 86Rb-uptake (p < or = 0.004). Amiodarone caused an average 49% reduction of the T3-dependent 3H-ouabain binding and decreased the Western blot signal for the Na+/K(+)-ATPase alpha1 subunit. The drug also inhibited T3-dependent increase in 86Rb-influx at 20 and 45 minutes by 66% and 42%, respectively, without affecting the affinity of the pump for K+. No differences were found in the 3H-ouabain binding and 86Rb-uptake of T3-, T3- amio and T3(+)-amio. In conclusion, T3 stimulates the Na+/K(+)-ATPase in human atrial myocardium by increasing the number of ouabain-binding sites, whereas amiodarone decreases the sodium pump function secondarily to the antagonism with thyroid hormone.     

The effect of thyroxine on (Na+, K+)-ATPase from the heart and the kidney of rabbit.

The effect of thyroxine on membrane bound (Na+, K+)-ATPase isolated as a microsomal fraction from rabbit heart and kidney was investigated. In the heart, thyroxine administration produced an increased Ki value (a concentration of ouabain required for half maximal inhibition of (Na+, K+)-ATPase activity) without alteration of the specific activity of cardiac (Na+, K+)-ATPase, indicating that the digitalis sensitivity of cardiac (Na+, K+)-ATPase was decreased. On the contrary, a significant increase of the specific activity of renal (Na+, K+)-ATPase was observed without change in its digitalis sensitivity. These results suggest that (1) a decreased sensitivity of cardiac (Na+, K+)-ATPase to digitalis glycosides in thyrotoxic animals may contribute to the decrease in the inotropic and toxic effects of the digitalis glycosides in the hyperthyroid state, and that (2) there may be an organ difference in (Na+, K+)-ATPase.     

Erythrocyte membrane lipids and cationic transport systems in men.

OBJECTIVE: The relationship between erythrocyte membrane and plasma lipids and various transmembrane erythrocyte cationic fluxes was examined in 53 normal men. DESIGN: Different measurements of erythrocyte transport systems were obtained: Na(+)-Li+ countertransport activity; Na+, K+ cotransport activity; Na+, K(+)-ATPase pump activity and the ground membrane permeability for Na+ and K+ as well as the intra-erythrocyte Na+, K+ and Mg2+ concentrations. Plasma cholesterol, triglycerides, phospholipids, free fatty acids, low- and high-density lipoprotein cholesterol levels and the erythrocyte membrane contents of cholesterol, phospholipids and free fatty acids were obtained from fasting subjects. RESULTS: In single regression analysis the erythrocyte Na(+)-Li+ countertransport and Na+, K+ cotransport activities were negatively related to the erythrocyte membrane cholesterol, phospholipids and free fatty acids contents. The Na+, K(+)-ATPase pump activity as assessed by the ouabain-sensitive Na+ efflux was also inversely related to the membrane cholesterol and phospholipids contents. In multiple regression analysis the red blood cell Na(+)-Li+ countertransport activity was independently and negatively related to the membrane cholesterol and free fatty acids contents. CONCLUSION: Our data show that an elevated level of erythrocyte membrane lipids in normal men is accompanied by lower Na(+)-Li+ countertransport, Na+, K+ cotransport and Na+, K(+)-ATPase pump activities.     

Osmoregulatory action of PRL,GH, and cortisol in the gilthead seabream (Sparus aurata L)

The osmoregulatory actions of ovine prolactin (oPRL), ovine growth hormone (oGH), and cortisol were tested in the euryhaline gilthead seabream Sparus aurata. Acclimated to sea water (SW, 40 ppt salinity, 1000 mOsm/kg H(2)O) or brackish water (BW, 5 ppt, salinity, 130 mOsm/kg H(2)O), injected every other day for one week (number of injections, 4) with saline (0.9% NaCl), oPRL (4 microg/g body weight), oGH (4 microg/g body weight) or cortisol (5 microg/g body weight), and transferred from SW to BW or from BW to SW 24h after the last injection. Fish were sampled before and 24h after transfer. Gill Na(+), K(+)-ATPase activity, plasma osmolality, plasma ions (sodium and chloride), plasma glucose, and muscle water moisture were examined. SW-adapted fish showed higher gill Na(+), K(+)-ATPase activity, plasma osmolality, and plasma ions levels than BW-adapted fish. Transfer from SW to BW decreased plasma osmolality and ions levels after 24h, while transfer from BW to SW increased these parameters, whereas gill Na(+),K(+)-ATPase activity was unaffected. oPRL treatment significantly decreased gill Na(+),K(+)-ATPase activity and increased plasma osmolality and ions in SW- and BW-adapted fish. This treatment minimizes loss of osmolality and ions in plasma after transfer to BW and increased these values after transfer to SW. No significant changes were observed in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions in oGH-treated group with respect to saline group before or after transfer from SW to BW or from BW to SW. Treatment with cortisol induced, in SW-adapted fish, a significant increase of gill Na(+),K(+)-ATPase activity and decrease of plasma osmolality and plasma ions. In BW-adapted fish this treatment induced a significant increases in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions. After transfer to SW cortisol-treated fish had higher plasma osmolality than the saline group. Our results support the osmoregulatory role of PRL in the adaptation to hypoosmotic environment in the gilthead seabream S. aurata. Further studies will be necessary to elucidate the osmoregulatory role of GH in this species. Cortisol results suggest a "dual osmoregulatory role" of this hormone in S. aurata.     

Inhibition of adenosine triphosphatases by gold

Inhibition of adenosine triphosphatase (ATPase) by chlorauric acid (Au3+) and gold sodium thiomalate (Au+) was studied in dog brain and kidney and in human kidney enzyme preparations. Au3+ indiscriminately affected ouabain-sensitive (Na+ + K+-dependent) ATPase and ouabain-insensitive (Mg2+-dependent) ATPase with concentrations for 50% inhibition (I50) approximately 10(-6) M. The I50 of Au3+ for Na+ + K+ ATPase was several-fold higher in homogenates than in microsomal fractions. The enzyme was protected by bovine serum albumin. Although Au3+ and Au+ were equipotent against Mg2+ ATPase, Au+ inhibited Na+ + K+ ATPase 2 to 3 times more effectively than did Au3+. The inhibitory action of Au3+ (but not Au+) was potentiated by ascorbic acid, suggesting reduction of Au3+ to Au+ by ascorbic acid. The fractional inhibition of Na+ + K+ ATPase by Au3+ or Au+ was not affected by changing concentrations of NaCl, KCl, MgCl2, ATP, and MgATP. Decreasing pH from 8.0 to 6.8 enhanced both Au+ and Au3+ inhibition. We conclude that gold is one of the most potent nonspecific of Na+ + K+ ATPase, with characteristics differing from other metallic inhibitors of this enzyme system.     

IDENTIFICATION OF COMPONENTS OF (NA+ + K+)-ADENOSINE TRIPHOSPHATASE BY DOUBLE ISOTOPIC LABELING AND ELECTROPHORESIS

A microsomal adenosine triphosphatase (ATPase) that requires both sodium and potassium ions is thought to be identical with, or an integral part of, the active cation transport system located in cell membranes. Attempts to isolate and purify (Na(+) + K(+))-ATPase have met with limited success because solubilization of microsomal protein causes partial, if not complete, loss of enzymatic activity. We now report the isolation from rat kidney microsomes of proteins which, though enzymatically inactive, could still be identified as components of the (Na(+) + K(+))-ATPase system.Phosphoproteins known to be intermediates in the hydrolysis of ATP by (Na(+) + K(+))-ATPase were prepared by incubating rat kidney microsomes with gamma-labeled ATP(33) in the presence of sodium or with P(32)-orthophosphate in the presence of ouabain. After the P(32)- and P(33)-labeled microsomes had been dissolved in phenol-acetic acid-urea, the resultant solutions were mixed and subjected to polyacrylamide gel electrophoresis. The radioactivity from both phosphorus isotopes was found almost exclusively in one of the resultant 21 protein bands. In contrast, the radioactive protein from DFP(32)-labeled microsomes moved slightly faster than the radioactive protein from microsomes labeled with P(33)-orthophosphate in the presence of ouabain. DFP inhibits (Na(+) + K(+))-ATPase by reacting with a nucleophilic site at or near the active site. These results suggest that while a single protein component of (Na(+) + K(+))-ATPase accepts the terminal phosphate from ATP, the final splitting of this phosphoprotein intermediate may be catalyzed by nucleophilic sites on a second protein.     

Possible mechanism responsible for mechanical dysfunction of ischemic myocardium: a role of oxygen free radicals

It has been proposed that a major target organelles damaged by the ischemic process, probably by the oxygen free radicals generated, is the portion of the excitation-contraction coupling system that regulates Ca2+ delivery (the sarcoplasmic reticulum and sarcolemma) to the contractile proteins. We tested this hypothesis by studying the effect of in vitro generation of oxygen free radicals from xanthine-xanthine oxidase system or dihydroxyfumarate (DHF)/Fe3+-ADP system on Ca2+ flux behavior of canine cardiac sarcoplasmic reticulum (SR); sarcolemmal (Na+, K+)-ATPase and Na+-Ca2+ exchange activities; and myofibrillar (Ca2+, Mg2+)-ATPase activity. Generation of oxygen free radicals by xanthine oxidase acting on xanthine as a substrate increased the passive Ca2+ efflux and decreased intravesicular Ca2+ with no effect on active Ca2+ influx (Ca2+-ATPase) of SR vesicles. Similar exposure of sarcolemmal vesicles to xanthine plus xanthine oxidase stimulated Na+-Ca2+ exchange activity. When sarcolemmal vesicles were incubated with DHF plus Fe3+-ADP, (Na+, K+)-ATPase activity was decreased. It is postulated that the SR Ca2+ efflux pathways but not catalytic activity of the Ca2+ pump and sarcolemmal (Na+, K+)-ATPase involving Na+-Ca2+ exchange activity are altered by oxygen free radicals, and such changes may partly account for the occurrence of intracellular Ca2+ overload during the course of myocardial ischemia. Interestingly, oxygen free radicals from xanthine-xanthine oxidase system had no effect on myofibrillar pCa-ATPase curve. From this set of observations we would hypothesize that the SR and sarcolemma may be the principal target organelles of oxygen free radicals attack in the ischemic injury and not the contractile proteins per se.     

In vitro effects of E coli endotoxin on K+-activated para-nitrophenylphosphatase activity and ouabain binding in dog hearts

The in vitro effect of E coli endotoxin on the activity of K+-activated paranitrophenylphosphatase (K+-PNPPase), an enzyme which represents the partial activity of (Na+ + K+)-ATPase enzyme system, was studied in isolated adult dog heart myocytes. The results were correlated with ouabain-binding studies. Endotoxin had an inhibitory effect on the Vmax for K+ activation as well as the Vmax for Mg++ and para-nitrophenylphosphate (PNPP) saturation. The inhibition was concentration-dependent and noncompetitive with K+, Mg++, and PNPP and furthermore, reversible. Endotoxin did not displace the bound 3H-ouabain from receptor sites nor did it affect the capacity of ouabain binding, indicating that the total enzyme concentration was not altered. From these findings, it is concluded that endotoxin in vitro affects the myocardial (Na+ + K+)-ATPase enzyme system by decreasing the turnover number of the enzyme molecule. The ability of endotoxin to modify myocyte membrane-associated enzyme activity may be responsible for altered heart metabolism and function in endotoxemia.     

Parallel modulation of brown adipose tissue GDP-binding, substrate uptake and (Na(+)-K+)-ATPase activity in the rat.

Brown adipose tissue (Na(+)-K+)-ATPase activity, in vitro glucose uptake and 2-aminoisobutyric acid uptake, as well as mitochondrial GDP-binding and succinate dehydrogenase activity were determined in order to study the relationship between these parameters in control, cold acclimated and cafeteria-fed rats. GDP-binding, (Na(+)-K+)-ATPase and glucose uptake were increased in interscapular brown adipose tissue from cold-acclimated and cafeteria-fed rats, whereas 2-aminoisobutyric acid uptake was only increased in cafeteria-fed rats. GDP-binding and (Na(+)-K+)-ATPase activity showed a high correlation coefficient suggesting a parallel modulation of both systems, which would probably share a common regulation mechanism.     

Na(+)-, ouabain-, Ca(2+)-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump alpha subunits.

Using the chicken sarcoplasmic/endoplasmic reticulum Ca2+ (SERCA)-ATPase as a parental molecule and replacing various portions with the corresponding portions of the chicken Na+,K(+)-ATPase alpha 1 subunit, Ca2+/thapsigargin- and Na+/ouabain-sensitive domains critical for these P-type ATPase activities were identified. In the chimera, [n/c]CC, the amino-terminal amino acids Met-1 to Asp-162 of the SERCA (isoform 1) (SERCA1) ATPase were replaced with the corresponding portion (Met-1-Asp-200) of the Na+,K(+)-ATPase alpha 1 subunit. In the chimera CC[c/n], the carboxyl-terminal amino acids (Ser-830 to COOH) of the SERCA1 ATPase were replaced with the corresponding segment (Leu-861 to COOH) of the Na+,K(+)-ATPase alpha 1 subunit, and in the chimera CNC, the middle part (Gly-354-Lys-712) of the SERCA1 ATPase was exchanged with the Na+,K(+)-ATPase alpha 1 subunit (Gly-378-Lys-724). None of the chimeric molecules exhibited any detectable ouabain-sensitive Na+,K(+)-ATPase activity, but they did exhibit thapsigargin-sensitive Ca(2+)-ATPase activity. Therefore, the segments Ile-163-Gly-354 and Lys-712-Ser-830 of the SERCA1 ATPase are sufficient for Ca2+ and thapsigargin sensitivity. The SERCA1-ATPase activity of [n/c]CC, but not of CCC, CNC, or CC[c/n], was further stimulated by addition of Na+ in the assay medium containing Ca2+. This additional stimulation of SERCA1-ATPase activity by Na+ was abolished when the amino-terminal region (Met-1-Leu-69) of [n/c]CC was deleted ([delta n/c]CC). In the absence of Na+, the SERCA1-ATPase activity of [n/c]CC was inhibited by ouabain, and, in the presence of Na+, its activity was stimulated by this drug. On the other hand, the ATPase activity of [delta n/c]CC was not affected by ouabain, although [delta n/c]CC can still bind [3H]ouabain. These results suggest that a distinct Na(+)-sensitive domain (Na+ sensor) located within the restricted amino-terminal region (Met-1-Leu-69) of the Na+,K(+)-ATPase alpha 1 subunit regulates ATPase activity. The Na+ sensor also controls ouabain action in concert with the major ouabain-binding region between Ala-70 and Asp-200 of alpha 1 subunit.     

Altered cations and muscle membrane ATPase activity in deoxycorticosterone acetate-salt spontaneously hypertensive rats.

The role of ions and cell membrane function in the pathogenesis of benign and malignant hypertension was investigated in spontaneously hypertensive rats (SHR). Ten-week-old male SHR (n = 50) and SHR treated with deoxycorticosterone acetate (DOCA; n = 70) and 1% NaCl drinking water were studied weekly for 14 weeks. Malignant hypertension developed only in DOCA-salt SHR and was characterised by severe hypertension, failure to thrive and renal fibrinoid necrosis. Fourteen DOCA-salt SHR and one SHR died. Extracellular (serum) and intracellular (erythrocyte and muscle) Na+, K+, Mg2+, Ca2+ and muscle membrane Na+,K(+)-adenosine triphosphatase (ATPase), Ca(2+)-ATPase and Mg(2+)-ATPase were measured at various stages in the development of malignant hypertension. Three developmental phases were defined: benign, premalignant and malignant. DOCA-salt SHR showed persistent hypokalaemia. In the benign phase, there were no differences in Na+, Mg2+ and Ca2+ between SHR and DOCA-salt SHR. In the premalignant phase, serum and erythrocyte Mg2+ and ATPase activity were significantly lower in DOCA-salt SHR compared with SHR. During the late premalignant and malignant phases, intracellular Ca2+ and Na+ were significantly higher in the DOCA-salt SHR compared with SHR. In view of these findings, the abnormalities in DOCA-salt SHR during the early phases of blood pressure elevation could be contributory factors to the development of malignant hypertension.