高胆固醇血症病人的红细胞膜ATP酶活性变化

研究表明高胆固醇血症病人的红细胞膜Na⁺-K⁺-ATP酶和Ca²⁺-Mg²⁺-ATP酶活性均降低,并且血浆总胆固醇和低密度脂蛋白-胆固醇浓度与这两种酶活性呈高度负相关,而血浆高密度脂蛋白-胆固醇浓度与Na⁺-K⁺-ATP酶活性呈正相关。这些变化的研究,对于进一步探讨动脉粥样硬化的发生机制及其防治可能具有重要意义。     

The chlorpromazine inhibition of transport ATPase and acetylcholinesterase activities in the microsomal membranes of rat in vitro and in vivo

Chlorpromazine, an antipsychotic drug, is found to inhibit Na⁺,K⁺-ATPase activity in rat brain microsomal membranes in vitro in concentration and time dependent manner but some inconsistency is observed when the effect was studied with respect to different temperatures. Various ligands and/or substrate affect the inhibition by chlorpromazine in different ways. The in vivo study with this drug shows that the activities of Na⁺,K⁺-ATPase, Ca^–2-ATPase and acetylcholinesterase in the microsomal membranes of different organs are inhibit with increases in concentration or lengths of time of treatment and then levels off.     

Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine

Summary The inhibition of Ca^2–-ATPase, (Na⁺+K⁺)-ATPase and Na⁺/Ca²⁺ exchange by Cd²⁺ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven ⁴⁵Ca²⁺ uptake into inside-out membrane vesicles displayed a K _m for Ca²⁺ of 88±17 nm, and was extremely sensitive to Cd²⁺ with an IC₅₀ of 8.2±3.0 pM Cd²⁺, indicating an inhibition via the Ca²⁺ site. (Na⁺+K⁺)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd²⁺, displaying an IC₅₀ of 2.6±0.6 m Cd²⁺. Cd²⁺ ions apparently compete for the Mg²⁺ site of the (Na^– +K⁺)-ATPase. The Na⁺/Ca²⁺ exchanger was inhibited by Cd²⁺ with an IC₅₀ of 73±11 nm. Cd²⁺ is a competitive inhibitor of the exchanger via an interaction with the Ca²⁺ site (K _i = 11 nm). Bepridil, a Na⁺ site specific inhibitor of Na⁺/Ca²⁺ exchange, induced an additional inhibition, but did not change the K _i of Cd²⁺. Also, Cd²⁺ is exchanged against Ca²⁺, albeit to a lesser extent than Ca²⁺. The exchanger is only partly blocked by the binding of Cd²⁺. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na⁺/Ca²⁺ exchanger. We conclude that intracellular Cd²⁺ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.We would like to thank Dr. D. Fackre (University of Alberta, Canada) for stimulating discussions and Mr. F.A.T. Spanings (University of Nijmegen, The Netherlands) for excellent fish husbandry. The fura-2 measurements of intracellular Ca²⁺ concentrations in tilapia enterocytes were carried out in the Department of Physiology, School of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Th.J.M. Schoenmakers and G. Flik were supported by travel grants from the Foundation for Fundamental Biological Research (BION) and the Netherlands Organization for Scientific Research (NWO).     

Roles of transmembrane segment M1 of Na+,K+-ATPase and Ca2+-ATPase, the gatekeeper and the pivot

In this review we summarize mutagenesis work on the structure–function relationship of transmembrane segment M1 in the Na⁺,K⁺-ATPase and the sarco(endo)plasmic reticulum Ca²⁺-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca²⁺ interaction/occlusion in Ca²⁺-ATPase and K⁺ interaction/occlusion in Na⁺,K⁺-ATPase. Leu⁶⁵ of the Ca²⁺-ATPase and Leu⁹⁹ of the Na⁺,K⁺-ATPase, located at homologous positions in M1, function as gate-locking residues that restrict the mobility of the side chain of the cation binding/gating residue of transmembrane segment M4, Glu³⁰⁹/Glu³²⁹. A pivot formed between a pair of a glycine and a bulky residue in M1 and M3 seems critical to the opening of the extracytoplasmic gate in both the Ca²⁺-ATPase and the Na⁺,K⁺-ATPase. All numbering of Na⁺,K⁺-ATPase amino acid residues in this article refers to the sequence of the rat α₁-isoform.     

Elevation of Different Ion-Specific ATPase Activities by L-Thyroxine (T4) in Different Tissues of Tasar Silkworm, Antheraea mylitta (Lepidoptera: Saturniidae) during Developmental Stages

Tissue-specific age-dependent changes were observed in Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na⁺K⁺- and Ca²⁺-ATPase in testes and only Ca²⁺-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na⁺K⁺-, Ca²⁺- and Mg²⁺-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system.     

Methionine Aminopeptidase II: A Molecular Chaperone for Sarcoplasmic Reticulum Calcium ATPase

The monoclonal antibody to the β-subunit of H⁺/K⁺-ATPase (mAbHKβ) cross-reacts with a protein that acts as a molecular chaperone for the structural maturation of sarcoplasmic reticulum (SR) Ca²⁺-ATPase. We partially purified a mAbHKβ-reactive 65-kDa protein from Xenopus ovary. After in-gel digestion and peptide sequencing, the 65-kDa protein was identified as methionine aminopeptidase II (MetAP2). The effects of MetAP2 on SR Ca²⁺-ATPase expression were examined by injecting the cRNA for MetAP2 into Xenopus oocytes. Immunoprecipitation and pulse-chase experiments showed that MetAP2 was transiently associated with the nascent SR Ca²⁺-ATPase. Synthesis of functional SR Ca²⁺-ATPase was facilitated by MetAP2 and prevented by injecting an antibody specific for MetAP2. These results suggest that MetAP2 acts as a molecular chaperone for SR Ca²⁺-ATPase synthesis.     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.     

The Na(+)/Ca(2+)-exchanger: an essential component in the mechanism governing cardiac steroid-induced slow Ca(2+) oscillations

Plasma membrane (PM) Na⁺, K⁺-ATPase, plays crucial roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically bind to the Na⁺, K⁺-ATPase and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds, synthesized in and released from the adrenal gland, are considered a new family of steroid hormones. Previous studies showed that ouabain induces slow Ca²⁺ oscillations in COS-7 cells by enhancing the interactions between Na⁺, K⁺-ATPase, inositol 1,4,5-trisphosphate receptor (IP₃R) and Ankyrin B (Ank-B) to form a Ca²⁺ signaling micro-domain. The activation of this micro-domain, however, is independent of InsP3 generation. Thus, the mechanism underlying the induction of these slow Ca²⁺ oscillations remained largely unclear. We now show that other CS, such as bufalin, can also induce Ca²⁺ oscillations. These oscillations depend on extracellular Ca²⁺ concentrations [Ca²⁺]_out and are inhibited by Ni²⁺. Furthermore, we found that these slow oscillations are Na⁺_out dependent, abolished by Na⁺/Ca²⁺ exchanger1 (NCX1)-specific inhibitors and markedly attenuated by NCX1 siRNA knockdown. Based on these results, a model is presented for the CS-induced slow Ca²⁺ oscillations in COS-7 cells.     

Age-related responses of hepatic ATPases to starvation and cold stress in male garden lizards

Hepatic Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities of male green lizards declined during the maturation phase (juvenile to 1-year-old) and stabilized thereafter. On the other hand, the Ca²⁺-ATPase activity of the liver declined during the later half of the life span (1-year-old to 2–4-year-old). Starvation stress induced a decline in hepatic Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities of juvenile lizards and caused an increase in 1-year-old and 2–4-year-old counterparts. The Ca²⁺-ATPase activity declined only in starved 1-year-old lizards. Following cold stress, the hepatic Na⁺-K⁺-ATPase activity of juvenile lizards showed a higher degree of decline than 2–4-year-old counterparts. The Mg²⁺-ATPase activity declined in cold-stressed juvenile lizards, but the parameter was elevated in similarly treated 1-year-old lizards. On the other hand, the increase in Ca²⁺-ATPase activity in response to cold stress was confined only to 2–4-year-old lizards.     

Identification and characterization of a Mg+2-dependent and an independent Ca+2-ATPase in microsomal membranes of rat testis

Rat testicular microsomal membrane fraction contains both Mg⁺²-dependent and Mg⁺²-independent Ca⁺²-ATPase activity. The latter activity is about two times higher than the former. Calcium ion required for maximum activation of Mg⁺²-independent Ca⁺²-ATPase in 3.0 mM, whereas for the dependent one it is 2.5 mM. Both the enzymes are resistant to cold shock upto seven days. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities, respectively. The pH optima for dependent one is 7.5, whereas for the independent one it is 8.5. Temperature optima for the former is 37°C and for latter one it is 40°C. Among all the nuclestides tested, ATP is found to be the best substrate for both the enzymes. The optimum concentration of ATP for dependent and independent enzyme activities are 3.0 mM and 1.5 mM respectively. Divalent metal ions like Zn⁺², Ba⁺² and Mn⁺² have been found to inhibit Mg⁺²-dependent Ca⁺²-ATPase activity whereas Mg⁺²-independent Ca⁺²-ATPase activity is inhibited by the divalent ions except zinc which is found to stimulate the enzyme activity. Both the enzymes are inhibited by vanadata, EDTA and EGTA. I₅₀, for vanadate is 0.05 and 0.125 mM for dependent and independent activities, respectively. Sulfhydral groups modifying agents e.g., NEM, DTNB and chlorpromazine are found to affect the enzyme activities in different ways. Thus NEM and chlorpromazine are found to inhibit and DTNB stimulate the enzyme activities in both the cases.     

Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

Platelet Ca2+ handling in essential hypertension: Role of a plasma ouabain-like factor

A humoral ouabain-like plasma factor has been observed in patients with essential hypertension (EHT). In the present study, we hypothesized that this humoral factor might be responsible for the elevated cytosolic free calcium concentrations [Ca²⁺]_i seen in these patients. Patients with mild to moderate EHT and their normotensive first degree blood relatives (NTBR) participated in the study. Platelet Na⁺, K⁺-ATPase activity was assayed in EHT patients and their NT first-degree relatives. To confirm the ouabain-like activity in plasma from EHT patients, control platelets were incubated with EHT and NTBR plasma and their Na⁺, K⁺-ATPase activity was measured. In addition, the effect of EHT plasma on platelet⁴⁵Ca-uptake was studied. Thein vitro effects of ouabain (10 ΜM) on (i)⁴⁵Ca-uptake and (ii) [Ca²⁺]_i response in control platelets were also observed. A decreased Na⁺K⁺-ATPase activity (P< 0.05) was observed in platelet membranes from EHT patients. Incubation of control platelets with EHT plasma decreased their Na⁺, K⁺-ATPase activity (P< 0.01) and increased their⁴⁵Ca-uptake (P< 0.05). C-18 Sep-Pak filtered hypertensive plasma extracts (containing the ouabain-like fraction) also decreased Na⁺, K⁺-ATPase activity (P< 001) in control platelet membranes.In vitro incubation of control platelets with ouabain increased⁴⁵Ca-uptake (P< 005) and [Ca²⁺]_i response (P< 0.05) in these platelets. Thus it appears that an ouabain-like factor in the EHT plasma may contribute to the elevated platelet [Ca²⁺]_i observed in EHT patients.     

NhaK,a novel monovalent cation/H<Superscript>+</Superscript> antiporter of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Four Na⁺/H⁺ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na⁺/H⁺ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na⁺(Ca²⁺)/H⁺ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na⁺ and Li⁺. In addition, effects of yvgP expression on a K⁺ uptake-defective mutant of E. coli indicated that YvgP also supported K⁺ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na⁺ (K⁺, Li⁺, Rb⁺)/H⁺ antiport activity was demonstrated. Na⁺ (K⁺, Li⁺)/H⁺ activity was higher at pH 8.5 than at pH 7.5. Mg²⁺, Ca²⁺ and Mn²⁺ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase, increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of added K⁺ and Na⁺.     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

Vanadate inhibition of the Ca-ATPase from human red cell membranes

1. (1) VO₃⁻ combines with high affinity to the Ca²⁺-ATPase and fully inhibits Ca²⁺-ATPase and Ca²⁺-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca²⁺-dependent phosphoenzyme.

2. (2) VO₃⁻ blocks hydrolysis of ATP at the catalytic site. The sites for VO₃⁻ also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca²⁺-ATPase.

3. (3) The sites for VO₃⁻ show positive interactions in affinity with sites for Mg²⁺ and K⁺. This accounts for the dependence on Mg²⁺ and K⁺ of the inhibition by VO₃⁻. Although, with less effectiveness, Na⁺ substitutes for K⁺ whereas Li⁺ does not. The apparent affinities for Mg²⁺ and K⁺ for inhibition by VO₃⁻ seem to be less than those for activation of the Ca²⁺-ATPase.

4. (4) Inhibition by VO₃⁻ is independent of Ca²⁺ at concentrations up to 50 μM. Higher concentrations of Ca²⁺ lead to a progressive release of the inhibitory effect of VO₃⁻.

In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots

The plasmalemma vesicles isolated from cucumber and maize roots were used to study the effect of Cu²⁺ and Cd²⁺ on the hydrolytic and proton pumping activities of ATPase. In vivo application of metal ions to the plant growth solutions resulted in stimulation of the proton transport in maize. In cucumber roots the action of metals was not the same: cadmium stimulated the H⁺ transport through plasmalemma whereas Cu²⁺ almost completely inhibited it. Copper ions decreased the hydrolytic activity of H⁺-ATPase in cucumber, without any effect on this activity in membranes isolated from maize roots. The effect of cadmium on the hydrolytic activities was opposite: ATP-hydrolysis activity in plasmalemma was not altered in cucumber, whereas in maize its stimulation was observed. The amount of accumulated metals was not the main reason of different influence of metals on H⁺-ATPase activity in tested plants. In in vitro experiments Cu²⁺ inhibited H⁺ transport in the cucumber, to a higher degree than Cd²⁺ and both metals did not change this H⁺-ATPase activity of plasmalemma isolated from corn roots. Cu²⁺ added into the incubation medium reduced the hydrolytic activity of ATPase in the plasma membrane isolated from cucumber as well as from corn roots. Cd²⁺ diminished the hydrolytic activity of ATPase in cucumber, and no effect of Cd²⁺ in the plasmalemma isolated from corn roots was found. Our results indicated different in vitro and in vivo action of both metals on H⁺-ATPase and different response of this enzyme to Cu²⁺ and Cd²⁺ in maize and cucumber.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na⁺/Ca²⁺ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca²⁺ reabsorption in the kidney. (1) Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca²⁺ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca²⁺ reabsorption. (2) The vesicle preparation contained high, digitalis-sensitive (Na⁺+K⁺-ATPase activities indicating its origin from the basolateral portion of plasma membrane. (3) The operation of Na⁺/Ca²⁺ antiport device was excluded by the findings that steep Ca²⁺ gradients formed by ATP-dependent Ca²⁺ accumulation in the vesicles were not discharged by extravesicular Na⁺, and did not drive ⁴⁵Ca²⁺ uptake into the vesicles via a Ca²⁺-⁴⁵Ca²⁺ exchange. (4) The ATP-dependent Ca²⁺ uptake into the vesicles became increasingly depressed with time by extravesicular Na⁺. This was not due to an impairment of the Ca²⁺ pump itself, but caused by Na⁺/Ca²⁺ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca²⁺ accumulation in the vesicles. (5) Earlier observations on Na⁺-induced release of Ca²⁺ from vesicles pre-equilibrated with Ca²⁺, seemingly favoring the existence of a Na⁺/Ca²⁺ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na⁺/Ca²⁺ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca²⁺ reabsorption through tubule epithelium essentially depending on the operation of a Na⁺/Ca²⁺ antiport device.     

Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.     

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

Summary In the presence of inhibitors for mitochondrial H⁺-ATPase, (Na⁺+K⁺)- and Ca²⁺-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane (ecto-ATPases). These ATPases accept several nucleotides, are stimulated by Ca²⁺ and Mg²⁺, and are inhibited by N,N-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg²⁺ and Mn²⁺, but not by Ca²⁺, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H⁺ secretion is electrogenic and requires either exit of a permeant anion (Cl^–) or entry of a cation, e.g., Na⁺ via electrogenic Na⁺/d-glucose and Na⁺/l-phenylalanine uptake. In the presence of Na⁺, ATP-driven H⁺ efflux is stimulated by blocking the Na⁺/H⁺ exchanger with amiloride. These data prove the coexistence of Na⁺-coupled substrate transporters, Na⁺/H⁺ exchanger, and an ATP-driven H⁺ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H⁺ pumps with (a part of) the DCCD- and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane.     

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

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