Gill Na(+), K(+)-ATPase in a series of hyper-regulating gammarid amphipods: enzyme characterisation and the effects of salinity acclimation

The enzyme Na(+), K(+)-ATPase was investigated in the gills of selected hyper-regulating gammarid amphipods. Gill Na(+), K(+)-ATPase was characterised with respect to the main cation and co-factor concentrations for the freshwater amphipod Gammarus pulex. The optimum cation and co-factor concentrations for maximal gill Na(+), K(+)-ATPase activity in G. pulex were 100mM Na(+), 15mM K(+), 15mM Mg(2+) and 5mM ATP, at pH 7.2. The effects of salinity acclimation on gill Na(+), K(+)-ATPase activity and haemolymph sodium concentrations was investigated in selected gammarid amphipods from different salinity environments. Maximal enzyme activity occurred in all gammarids when acclimated to the most dilute media. This maximal activity coincided with the largest sodium gradient between the haemolymph and the external media. As the haemolymph/medium sodium gradient decreased, a concomitant reduction in gill Na(+), K(+)-ATPase activity occurred. This implicates the involvement of gill Na(+), K(+)-ATPase in the active uptake of sodium from dilute media in hyper-regulating gammarids.     

Comparison of some minor activities accompanying a preparation of sodium-plus-potassium ion-stimulated adenosine triphosphatase from pig brain

1. An ATPase (adenosine triphosphatase) preparation obtained from pig brain microsomes by treatment with sodium iodide showed four apparently different ouabain-sensitive activities under various conditions. They were (a) ouabain-sensitive Mg(2+)-stimulated ATPase, (b) K(+)-stimulated ATPase, (c) (Na(+),K(+))-stimulated ATPase and (d) Na(+)-stimulated ATPase activities. 2. These activities showed the same substrate specificity, ATP being preferentially hydrolysed and CTP slightly. AMP was not hydrolysed. 3. These activities were inhibited by low concentration of ouabain. The concentration producing 50% inhibition was 0.1mum for ouabain-sensitive Mg(2+)-stimulated ATPase, 0.2mum for K(+)-stimulated ATPase, 0.1mum for (Na(+),K(+))-stimulated ATPase and 0.003mum for Na(+)-stimulated ATPase activity. 4. The ouabain-sensitive ATPase activities were inactivated by N-ethylmaleimide but the insensitive ATPase activity was not. 5. The three ouabain-sensitive ATPase activities were inhibited about 50% by 1mm-Ca(2+), whereas the ouabain-sensitive Mg(2+)-stimulated ATPase activity was activated by the same concentration of Ca(2+). The preparation was treated with ultrasonics at 20kcyc./sec. The 2min. ultrasonic treatment inactivated the ATPase activities by 50%. 7. The temperature coefficient Q(10) was 6.6 for K(+)-stimulated ATPase activity, 3.7 for (Na(+),K(+))-stimulated ATPase and 2.6 for Na(+)-stimulated ATPase. 8. Organic solvents inactivated the ATPase activities, to which treatment the K(+)-stimulated ATPase was the most resistant. 9. The phosphorylation of the enzyme preparation became less dependent on Na(+) with decreasing pH. This Na(+)-independent phosphorylation at low pH was sensitive to K(+) and hydroxylamine as well as the Na(+)-dependent phosphorylation at neutral pH.     

Cation-stimulated Adenosine Triphosphatase Activity and Cation Transport in Corn Roots

ATPase activity of the plasma membrane fraction from primary roots of corn (Zea mays L. WF9 x M14) was activated by Mg(2+) and further stimulated by monovalent cations (K(+) > Rb(+) > Cs(+) > Na(+) > Li(+)). K(+)-stimulated activity required Mg(2+) and was substrate-specific. Maximum ATPase activity in the presence of Mg(2+) and K(+) was at pH 6.5 and 40 C. Calcium and lanthanum (<0.5 mm) were inhibitors of ATPase, but only in the presence of Mg(2+). Oligomycin was not an inhibitor of the plasma membrane ATPase, whereas N,N'-dicyclohexylcarbodiimide was. Activity showed a simple Michaelis-Menten saturation with increasing ATP.Mg. The major effect of K(+) in stimulating ATPase activity was on maximum velocity. The kinetic data of K(+) stimulation were complex, but similar to the kinetics of short term K(+) influx in corn roots. Both K(+)-ATPase and K(+) influx kinetics met all criteria for negative cooperativity. The results provided further support for the concept that cation transport in plants is energized by ATP, and mediated by a cation-ATPase on the plasma membrane.     

Tissue ATPase activity and recovery in the freshwater crab Oziotelphusa senex senex exposed to a sublethal concentration of endosulfan for varying periods of time.

1. Na(+)-K+ and Mg(2+)-tissue ATPases of the freshwater crab Oziotelphusa senex senex showed increasing inhibition when exposed to a sublethal concentration (1.86 mg/l = 0.1 of LC50) of endosulfan for 1-30 days. 2. Na(+)-K(+)-ATPase activity in all tissues (thoracic nerve mass, gill, hepatopancreas and claw muscle) was higher than Mg(2+)-ATPase activity. 3. After 30 days exposure tissue Mg(2+)-ATPase was less affected than Na(+)-K(+)-ATPase. 4. Crabs exposed to endosulfan and then returned to uncontaminated water showed greater recovery of Mg(2+)-ATPase than Na(+)-K(+)-ATPase with 90-95% recovery after 1 day exposure and 60-65% recovery after 30 days exposure. 5. Changes in behaviour of the crabs were noted after 7 days exposure to endosulfan with progressive loss of coordination, decreased activity and increased exudation of mucus.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

ATP steal between cation pumps: a mechanism linking Na+ influx to the onset of necrotic Ca2+ overload

We set out to identify molecular mechanisms underlying the onset of necrotic Ca(2+) overload, triggered in two epithelial cell lines by oxidative stress or metabolic depletion. As reported earlier, the overload was inhibited by extracellular Ca(2+) chelation and the cation channel blocker gadolinium. However, the surface permeability to Ca(2+) was reduced by 60%, thus discarding a role for Ca(2+) channel/carrier activation. Instead, we registered a collapse of the plasma membrane Ca(2+) ATPase (PMCA). Remarkably, inhibition of the Na(+)/K(+) ATPase rescued the PMCA and reverted the Ca(2+) rise. Thermodynamic considerations suggest that the Ca(2+) overload develops when the Na(+)/K(+) ATPase, by virtue of the Na(+) overload, clamps the ATP phosphorylation potential below the minimum required by the PMCA. In addition to providing the mechanism for the onset of Ca(2+) overload, the crosstalk between cation pumps offers a novel explanation for the role of Na(+) in cell death.     

Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.     

Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophytica

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.     

Antioxidant defenses and biochemical changes in the neotropical fish pacu, Piaractus mesopotamicus: Responses to single and combined copper and hypercarbia exposure

This study investigated the potentially detrimental effects of copper and elevated aquatic CO(2) (hypercarbia), alone or in combination, on pacu, Piaractus mesopotamicus. Fish were exposed for 48h to control (no copper addition in normocarbia), to 400μg Cu(2+)L(-1), to hypercarbic (1% CO(2); PCO(2)=6.9mm Hg) water and to 400μg Cu(2+)L(-1)+hypercarbia. In liver the single factors caused an increase in lipid hydroperoxide concentration that was not observed when the factors were combined. Copper exposure elicited increased hepatic superoxide dismutase activity, irrespective of aquatic CO(2) level. On the other hand, the effects of copper on hepatic glutathione peroxidase activity were dependent on water CO(2) levels. The two stressors combined did not affect hepatic catalase activity. Hypercarbic water caused a decline in plasma glucose concentration, but this was not observed when hypercarbia was combined with copper exposure. Copper caused a decrease in branchial Na(+)/K(+)-ATPase activity that was independent of water CO(2) level. Copper caused an increase in branchial metallothionein concentration that was independent of water CO(2) level. Thus, branchial metallothionein and Na(+)/K(+)-ATPase were effective biomarkers of copper exposure that were not affected by water CO(2) level.     

Na+/Mg2+ transporter acts as a Mg2+ buffering mechanism in PC12 cells

Mg(2+) buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na(+)/Mg(2+) transporter by using a newly developed Mg(2+) indicator, KMG-20, and also a Na(+) indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg(2+) concentration ([Mg(2+)](i)). The rate of decrease of [Mg(2+)](i) was slower in a Na(+)-free extracellular medium, suggesting the coupling of Na(+) influx and Mg(2+) efflux. Na(+) influxes were different for normal and imipramine- (a putative inhibitor of the Na(+)/Mg(2+) transporter) containing solutions. FCCP induced a rapid increase in [Na(+)](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg(2+)](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg(2+) depends on the Na(+)/Mg(2+) transporter in PC12 cells.     

Na+-ATPase in the gills of bass (Dicentrarchus labrax)

The authors evidence a Mg2+ dependent ATPase activity stimulated by Na+ in absence of K+ in bass gill microsomes. As this stimulated ATPase shows different features from "baseline" activity measured in the absence of both Na+ and K+ ions (Mg2+-ATPase) and from 1mM ouabain sensitive (Na+ + K+)-ATPase, it has been ascribed to a distinct Na+-ATPase. In the present paper the optimal conditions for bass gill Na+-ATPase assay and the temperature dependence of the enzyme are reported. Moreover the Na+-ATPase appears to be insensitive to 1mM ouabain and 100% inhibited by 2,5mM ethacrynic acid. It is suggested a parallel diffusion of Na+- and (Na+ + K+)-ATPase and a possible physiological role of Na+ATPase in osmoregulation.     

The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1. The intrinsic Na(+), K(+), Mg(2+) and Ca(2+) contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na(+) from 90+/-20 to 24+/-12, the bound K(+) from 27+/-3 to 7+/-2, the bound Mg(2+) from 20+/-2 to 3+/-1 and the bound calcium from 8+/-1 to <1nmol/mg of protein. 3. The activities of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase and the Na(+)-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5mum (ATP/protein ratio 12.5pmol/mug). 4. The Na(+)-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5mum-magnesium chloride and 2mum-potassium chloride. Addition of 2.5mum-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na(+)-dependent ATP hydrolysis was partly restored with 2.5mum-magnesium chloride; addition of K(+) in the range 2-10mum-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0 degrees C with 0.5nmol of K(+)/mg of protein so that the final added K(+) in the reaction mixture was 0.1mum restored the Na(+)-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [(42)K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K(+)/mg of protein was linear over a period of 20min and was inhibited by Na(+). Half-maximal inhibition of (42)K(+)-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na(+)-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K(+) and Mg(2+) of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K(+) from a solution of 0.5mum-potassium chloride.     

Effects of pH on the gill ATPase activity and blood composition of whitefish (Coregonus peled) and trout (Salmo trutta fario)

1. The effects of pH on blood composition and gill (Na+ + Mg2+) ATPase activity were studied in 142 whitefish and trout kept for 3 to 17 hr in tanks at pH 3.0-9.5 in Northern Finland. 2. pH clearly influenced all blood values, except plasma alanine and aspartate aminotransferases. Blood haemoglobin and packed cell volume of whitefish and trout were lower at pH 9.5 but higher at pH 3.0 than those of controls at pH 6.5. 3. Both fishes had lowest plasma sodium and chloride values in groups kept for 9 to 17 hr in tanks with water acidified to pH 3.0 to 3.5. Changes in the plasma chloride concentration were associated with plasma sodium (r = 0.92, n = 102, P less than 0.001). 4. The gill (Na+ + K+ + Mg2+) ATPase of trout showed high activity over wide ranges of pH (6.0-7.5) at 13 degrees C having a distinct optimum at pH 7.0. 5. Blood glucose and lactate concentrations of whitefish and trout increased after exposure to acid and base. High values at pH 3.0 to 3.5 suggested hypoxic stress due to acidaemia.     

Na+/Mg2+ exchange is functionally coupled to the insulin receptor

Regulation of cellular Mg(2+) levels by insulin has been shown in various tissues. However, the mechanisms for hormonal regulation of cellular Mg(2+) have not been well described. We studied the effect of insulin on Na(+)/Mg(2+) exchange in normal human cells, measuring Na(+)/Mg(2+) exchange activity as net total Mg(2+) efflux driven by an inward Na(+) gradient in Mg(2+)-loaded red blood cells (RBCs). Na(+)/Mg(2+) exchange was increased significantly by the addition of 2.4 nmol/L of insulin to the flux medium (from 0.60 +/- 0.06 mmol/L cell x h to 0.75 +/- 0.08 mmol/L cell x h [P = 0.0098, n = 44]). A dose-response curve for the effects of insulin on the exchanger activity gave an estimated EC(50) for insulin of 0.95 +/- 0.31 nmol/L and a V(max) of 0.86 +/- 0.12 mmol/L cell x h (n = 7). Kinetics of the Na(+)/Mg(2+) exchange were characterized by measuring its activity as a function of Mg(2+) and Na(+) concentrations. The K(0.5) for cellular Mg(2+) was not affected by incubation with insulin. However, the K(0.5) for extracellular Na(+) decreased from 69.9 +/- 6.3 to 40.3 +/- 8.4 mol/L (n = 5, P = 0.03) in the presence of insulin. We also studied the effect of wortmannin (WT), a PI 3-kinase inhibitor, on activity of the exchanger. WT significantly blocked the insulin-stimulated Na(+)/Mg(2+) activity (n = 6, P = 0.048), with an IC(50) of 0.5 nmol/L. LY294002, another PI 3-kinase inhibitor, likewise blocked the insulin-stimulated activity of the exchanger. Therefore, insulin regulates cellular Mg(2+) metabolism in part via an increase in the affinity for Na(+) of the Na(+)/Mg(2+) exchange and PI 3-kinase activation, suggesting another role for the PI 3-kinase pathway in insulin-mediated cellular events.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.     

The pathway for spontaneous occlusion of Rb+ in the Na+/K+-ATPase

Occlusion of K (+) in the Na (+)/K (+)-ATPase can be achieved under two conditions: during hydrolysis of ATP, in media with Na (+) and Mg (2+), after the K (+)-stimulated dephosphorylation of E2P (physiological route) or spontaneously, after binding of K (+) to the enzyme (direct route). We investigated the sidedness of spontaneous occlusion and deocclusion of Rb (+) in an unsided, purified preparation of Na (+)/K (+)-ATPase. Our studies were based on two propositions: (i) in the absence of ATP, deocclusion of K (+) and its congeners is a sequential process where two ions are released according to a single file mechanism, both in the absence and in the presence of Mg (2+) plus inorganic orthophosphate (Pi), and (ii) in the presence of Mg (2+) plus Pi, exchange of K (+) would take place through sites exposed to the extracellular surface of the membrane. The experiments included a double incubation sequence where one of the two Rb (+) ions was labeled as (86)Rb (+). We found that, when the enzyme is in the E2 conformation, the first Rb (+) that entered the enzyme in media without Mg (2+) and Pi was the last to leave after addition of Mg (2+) plus Pi, and vice-versa. This indicates that spontaneous exchange of Rb (+) between E2(Rb 2) and the medium takes place when the transport sites are exposed to the extracellular surface of the membrane. Our results open the question if occlusion and deocclusion via the direct route participates in any significant degree in the transport of K (+) during the ATPase activity.     

Increased Na(+)/H(+) exchanger isoform 1 activity in spontaneously hypertensive rats: lack of mutations within the coding region of NHE1

Enhanced Na(+)/H(+) exchange, measured as amiloride derivative-sensitive Na(+) and H(+) fluxes in cells with a preliminary acidified cytoplasm (Deltamu(H+)-induced Na(+)/H(+) exchange), is one of the most prominent intermediate phenotypes of altered vascular smooth muscle cell (VSMC) function in spontaneously hypertensive rats (SHR). Analysis of Na(+)/H(+) exchange in F(2) hybrids of SHR and normotensive rats seems to be the most appropriate approach in the search for the genetic determinants of abnormal activity of this carrier. However, the measurement of Deltamu(H+)-induced Na(+)/H(+) exchange is hardly appropriate for precise analysis of the carrier's activity in VSMC derived from several hundred F(2) hybrids. To overcome this problem, we compared the rate of (22)Na influx under baseline conditions and in Na(+)-loaded (ouabain-treated) VSMC. The dose-dependency of the rate of Deltamu(H+)-induced H(+) efflux as well as of (22)Na influx in control and ouabain-treated cells on ethylisopropylamiloride (EIPA) concentration were not different (K(0.5) approximately 0.3 microM), suggesting that these ion transport pathways are mediated by the same carrier. EIPA-sensitive (22)Na influx in Na(+)-loaded cells was approximately 6-fold higher than in ouabain-untreated VSMC and was increased by 50-70% in two different substrains of SHR. About the same increment of EIPA-sensitive (22)Na influx in Na(+)-loaded VSMC was observed in 5- to 6-week-old SHR (an age at which hypertension has not yet developed) as well as in stroke-prone SHR (SHRSP) with severe hypertension, indicating that the heightened activity of Na(+)/H(+) exchange is not a consequence of long-term blood pressure elevation. To examine whether or not the augmented activity of Na(+)/H(+) exchange in SHR is caused by mutation of NHE1, i.e. the only isoform of this carrier expressed in VSMC, we undertook single-stranded conformational polymorphism analysis of 23 NHE1 cDNA fragments from SHR and SHRSP and sequencing of the 456-2421 NHE1 cDNA fragment. This study did not reveal any mutation in the entire coding region of NHE1. The lack of mutation in the coding region of NHE1 indicates that the augmented activity of the ubiquitous Na(+)/H(+) exchanger in primary hypertension is caused by altered regulation of carrier turnover number or/and its plasma membrane content.     

Influence of cadmium concentration and length of exposure on metabolic rate and gill Na+/K+ ATPase activity of golden shiners (Notemigonus crysoleucas)

Although metabolic rate is considered to be useful as a general indicator of the biological effects of exposure to metals, it is seldom measured in conjunction with specific physiological, biochemical or cellular parameters. The purpose of this investigation was to examine the influence of cadmium (Cd) exposure on metabolic rate and gill Na(+)/K(+) ATPase activity in golden shiners (Notemigonus crysoleucas). Shiners were exposed to six levels of Cd (ranging from control to the maximum sublethal concentration) for 24- and 96-h periods. After 24-h, metabolic rate and Na(+)/K(+) ATPase activity of individual fish were strongly correlated. Shiners exposed to the four highest Cd concentrations (500, 800, 1100, and 1400 μg L(-1)) for 24-h exhibited a shock response that was characterized by mean values for metabolic rate and Na(+)/K(+) ATPase activity that were significantly lower compared to the control. Although results for 96-h exposures reflect a repair/recovery phase, there was no significant correlation between metabolic rate and Na(+)/K(+) ATPase activity. Metabolic rate of shiners was significantly elevated (65-100%) at all concentrations compared to the control after 96-h, whereas Na(+)/K(+) ATPase activity did not differ from the control. Elevated metabolic rate after 96-h likely reflects the influence of a variety of energetically demanding processes associated with repair and recovery.     

Properties of the sarcolemmal calcium ion-stimulated adenosine triphosphatase of hamster skeletal muscle

1. A sarcolemmal fraction was isolated from hamster hind-leg skeletal muscles by successive treatment with lithium bromide and potassium chloride. The membranous fraction was observed to contain a highly active Ca(2+)-stimulated ATPase (adenosine triphosphatase), a Mg(2+)-stimulated ATPase, and an Na(+)+K(+)-stimulated Mg(2+)-dependent ouabain-sensitive ATPase. 2. The Ca(2+)-stimulated ATPase activity was pH-dependent, the optimum being pH7.6. 3. Optimum activation of this enzyme was obtained with 3-4mm-Ca(2+) when 4mm-ATP was present as a substrate, and was not influenced by Na(+), K(+) or ouabain, whereas 2,4-dinitrophenol, sodium azide, oligomycin, sodium fluoride and ethanedioxybis(ethylamine)tetra-acetate were inhibitory. 4. The Ca(2+)-stimulated ATPase was markedly inhibited by thiol-blocking reagents, and cysteine was able to reverse this inhibition. 5. Various bivalent cations stimulated ATP hydrolysis by the sarcolemmal fraction in the following decreasing order of potency: Mg(2+), Ca(2+), Mn(2+), Co(2+), Sr(2+), Ba(2+), Zn(2+), Cu(2+).