Gradient-dependent Ca2 Research Articles

Unutilized energy reserves and mineral contents of fibroid tissues suggesting perturbed membrane transport processes

Compositions of intramural uterine fibroid (IUF) and subserous uterine fibroid (SUF) tissues were studied. Results showed that IUF and SUF, respectively, contained (%) 77.26 ± 0.05 and 77.49 ± 0.02 moisture and 22.74 ± 0.05 and 22.52 ± 0.02 dry matter, on wet-weight basis. Their respective moisture, crude fat, crude protein, ash and total carbohydrates (%) were 4.67 ± 0.05 and 4.38 ± 0.17, 8.29 ± 0.41 and 5.84 ± 0.43, 72.66 ± 0.72 and 65.16 ± 0.91, 4.39 ± 0.08 and 4.22 ± 0.03 and 10.01 ± 1.18 and 20.40 ± 1.45 while their energy contents (kcal/ 100g) were 405.29 and 394.80, respectively, on dry-matter basis. Their crude fats contained fatty acids, triacylglycerols and cholesterol while their total carbohydrates contained glucose and glycogen. Ca2+, Fe2+, Zn2+, K+, Na+ and Mg2+ contents (mg/ 100g) showed that Na+ was the most abundant mineral amongst them unlike the norm in cells where K+ is the most abundant. The tissues contained high [Ca2+] and [Fe2+], low [Zn2+], [K+] and [Mg2+] and comparable [Na+] relative to cells. These suggested that Na+/ K+-ATPase activity may have been decreased or inhibited by either high [oestrogen] which is a pre-disposing factor to fibroid or low [Mg2+] which may have decreased or inhibited the activity of the electrochemical Na+ gradient-dependent Ca2+transporter. Zn2+ transporter activity seemed to have been decreased or inhibited while cellular influx of Fe2+ seemed to have been increased. The results showed that the IUF and SUF had unutilized energy reserves and mineral contents suggestive of perturbed membrane transport activities. Key words: Energy reserve, fibroid, minerals, membrane transport, proximate composition.

A Cation-regulated and Proton Gradient-dependent Cation Transporter from Chlamydomonas reinhardtii Has a Role in Calcium and Sodium Homeostasis

The CrCAX1 gene encoding a Ca2+/H+ and Na+/H+ exchanger was cloned and characterized from the unicellular green alga Chlamydomonas reinhardtii to begin to understand the mechanisms of cation homeostasis in this model organism. CrCAX1 was more closely related to fungal cation exchanger (CAX) genes than those from higher plants but has structural characteristics similar to plant Ca2+/H+ exchangers including a long N-terminal tail. When CrCAX1-GFP was expressed in Saccharomyces cerevisiae, it localized at the vacuole. CrCAX1 could suppress the Ca2+-hypersensitive phenotype of a yeast mutant and mediated proton gradient-dependent Ca2+/H+ exchange activity in vacuolar membrane vesicles. Ca2+ transport activity was increased following N-terminal truncation of CrCAX1, suggesting the existence of an N-terminal auto-regulatory mechanism. CrCAX1 could also provide tolerance to Na+ stress when expressed in yeast or Arabidopsis thaliana because of Na+/H+ exchange activity. This Na+/H+ exchange activity was not regulated by the N terminus of the CrCAX1 protein. A subtle tolerance by CrCAX1 in yeast to Co2+ stress was also observed. CrCAX1 was transcriptionally regulated in Chlamydomonas cells grown in elevated Ca2+ or Na+. This study has thus uncovered a novel eukaryotic proton-coupled transporter, CrCAX1, that can transport both monovalent and divalent cations and that appears to play a role in cellular cation homeostasis by the transport of Ca2+ and Na+ into the vacuole.

The Putative Amino-terminal Signal Peptide of the Cloned Rat Brain Na+-Ca2+ Exchanger Gene (RBE-1) Is Not Mandatory for Functional Expression

The rat brain Na(+)-Ca2+ exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane alpha helices, the first of which was proposed by Durkin et al. (14) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide (N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site (N26), and the entire putative signal peptide and putative signal peptidase cleavage site (N31). All three mutant clones were transiently expressed in HeLa cells. The average Na+ gradient-dependent Ca2+ transport activity of the mutant exchangers was 108% (N21), 37.2% (N26), and 60.06% (N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 --> Asp, resulted in a decrease in Na(+)-Ca2+ exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1% (N21), 26.75% (N26), 85.36% (N31), and 31% (Ala-32 --> Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-1, N21, N26, N31 and Ala-32 --> Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na(+)-Ca2+ exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 -->Asp. The amino-terminal truncated mutants of the cloned Na(+)-Ca2+ exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na(+)-Ca2+ exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells.

Calcium transport in gill plasma membranes of the crab Carcinus Maenas: Evidence for carriers driven by ATP and A Na+ Gradient

A procedure was developed for the preparation of inside-out vesicles from plasma membranes isolated from the branchial epithelium of the green shore crab Carcinus maenas (L.). Procedures normally applied to fish branchial epithelium required the introduction of an additional hypotonic shock to obtain a preparation containing 22% inside-out vesicles, 33% right-side-out vesicles and 45% leaky membrane fragments. In such membrane preparations, the first direct evidence for uphill (against a [Ca2+] gradient) ATP-dependent and Na(+)-gradient-dependent Ca2+ transport in crustacean gills was found. The affinity for Ca2+ of the ATP-driven Ca2+ transporter was 149 nmol l-1 and that of the Na+/Ca2+ exchanger was 1.78 mumol l-1; the Vmax values were 1.73 and 9.88 nmol min-1 mg-1 protein respectively. The relative importance of these carriers for Ca2+ transport in the branchial epithelium of the crab is evaluated on the basis of their calcium kinetics.

Na+/Ca2+ Exchange, Ca2+ Binding, and Electrogenic Ca2+ Transport in Plasma Membranes of Human Placental Syncytiotrophoblast

To transfer a large amount of Ca2+ to the fetus, the basal (fetal-facing) plasma membrane (BPM) of human placenta must be equipped with various extrusion mechanisms. We studied one such mechanism, Na+/Ca2+ exchange, as well as related membrane potential effects and binding properties of the two membranes. Na+/Ca2+ exchange was present in BPM and absent in microvillous (maternal-facing) membrane. Uptake and efflux of Ca2+ in BPM were enhanced by Na+ when it was present on the opposite side of the membranes. Na(+)-gradient-dependent Ca2+ uptake was saturable with a Km of 19 microM and a Vmax of 0.8 nmol/min/mg. The Na+/Ca2+ exchange in BPM and the facilitated diffusion transporters in both BPM and microvillous membrane are electrogenic processes. Ca2+ binding in both BPM and microvillous membrane was affected by various monovalent cations and enhanced by Na+ more than by K+. In vivo, together with other sequestration mechanisms, Na+/Ca2+ exchange may play an important role in transsyncytial transfer and in regulating intracellular Ca2+, which is essential for a variety of physiologic mechanisms.

A Na+/Ca2+ exchange mechanism in apical membrane vesicles of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lying between the neural retina and the choroid, performs as a transport organ for solutes and water between the choriocapillaries and the subretinal space. It also has the function to maintain the microenvironment of photoreceptors including the regulation of calcium ions during light or dark adaptation. In order to further elucidate the transport functions of the RPE, apical membranes were isolated from RPE by differential precipitation with divalent ions. In this work bovine tissues were used as well as elasmobranch tissues. For the latter, we have already purified and characterized membrane vesicles in a previous paper. Na(+)-K(+)-ATPase, alkaline phosphatase, and 5'-nucleotidase, which are marker enzymes of the apical membrane, were highly enriched in the final membrane fraction. The majority of the fraction consists of right side out vesicles. The fluorescent indicator for sodium, SBFI, or the calcium specific indicator, Fura-2, were pre-loaded into the apical membrane vesicles of RPE of either dogfish eyes or bovine eyes. When an outwardly-directed Ca2+ gradient was formed across the vesicular membranes, the Ca2+ influx was also enhanced by 136% for dogfish RPE and 167% for bovine RPE. This Na+ gradient dependent Ca2+ influx was blocked by bepridil, an antiarrhythmic agent which is a Na+/Ca2+ exchanger inhibitor. These results indicate that a Na+/Ca2+ exchanger is present in the apical membrane of bovine and dogfish RPE.

Calcium efflux in rat lens: Na/Ca-exchange related to cataract induced by selenite.

Sodium gradient-dependent 45Ca2+ transport occurred across the lens membrane both in the direction of Ca2+ uptake by inside-out vesicles and Ca2+ efflux after Ca2+ loading of right-side-out vesicles. Using the calcium ionophore, A23187, greater than 90% of the Na+ gradient-dependent Ca2+ uptake was estimated to be free Ca2+. A normal Na+ gradient was also required to maintain calcium homeostasis in the intact lens. The Na+ gradient contributed to Ca2+ efflux from lenses pre-loaded in medium containing 15 mM CaCl2. Therefore, a Na/Ca-exchange functions to control Ca efflux in rat lens, in addition to the Ca-ATPase. In the preweanling rat mature nuclear cataracts occurred by 96 h after subcutaneous injection of sodium selenite (30 nmol/g animal wt). A 3-5 fold increase of Ca2+ accompanied cataract formation. The loss of Ca2+ homeostasis can be detected by 48 h after treatment selenite treatment. At this time the initial rate of Na+ gradient-dependent Ca2+ uptake was 30% lower in lens vesicles from selenite-treated rats compared to controls. No significant reduction of Na+,K(+)-ATPase activity was detected. Altered Na/Ca-exchange may contribute directly to the loss of Ca2+ homeostasis that leads to nuclear cataract.

Molecular and Mechanistic Heterogeneity of the Na+ ‐ Ca2+ Exchangera

1. Studying the effect of K+ on Na(+)-Ca2+ exchange in rat brain SPMs revealed that a consistent stimulation was obtained. This stimulation persisted also when FCCP was included in the K(+)-containing reaction mixture to minimize the effect of membrane potential on the electrogenic process. 2. Using Rb+ as a K+ analogue revealed that it was cotransported with Ca2+ in a Na+ gradient-dependent manner. The ratio between the amount of Ca2+/Rb+ transported in rat brain SPMs in a Na+ gradient-dependent manner suggests that not all the Na(+)-Ca2+ exchangers in that preparation cotransport Rb+ (K+) with Ca2+. This is supported also by the finding that Na+ gradient-dependent Ca2+ influx can proceed in rat brain SPMs in the complete absence of K+ although to a lesser extent. 3. Protein purification studies and immunological characterization indicate that a 70-kDa protein is consistently detected in rat brain SPMs. Immunological characterization of the proteins expressed in the 14-18 S mRNA-injected Xenopus oocyte in conjunction with Na+ gradient dependent Ca2+ uptake activity or in the same mRNA-fortified reticulocyte lysate suggest that proteins of about 70 kDa are specifically synthesized. 4. Torpedo electric organ Na(+)-Ca2+ exchanger differs at least in two respects from the rat brain Na(+)-Ca2+ exchanger: It has a low affinity to Na+ (K0.5 = 170 mM), and it reaches maximal activity between 15-20 degrees C. Reconstitution studies suggest that the temperature difference might reflect a difference in the proteins themselves rather then a difference in membrane fluidity due to a difference in the membrane lipid composition.

Expression of renal Na(+)-Ca2+ exchange activity in Xenopus laevis oocytes

The expression of a renal Na(+)-Ca2+ exchanger by Xenopus oocytes has been investigated. Each oocyte was injected with 50 ng of poly(A)+ RNA from either rat or rabbit kidney or with an equivalent volume of water. Na(+)-Ca2+ exchange was determined 3 days after injection, by measuring Ca2+ uptake by oocytes in the presence or absence of an outwardly directed Na+ concentration gradient. To manipulate Na+ concentration gradients, oocytes were first loaded with Na+ in Ca(2+)-free medium containing 90 mM Na+ and nystatin. They were then exposed to medium containing 45Ca and either 90 mM or 0 Na+. Na(+)-free media contained (in mM) either 90 K+, 90 choline or 85 choline plus 5 K+. Oocytes injected with rat kidney poly(A)+ RNA showed a Na+ gradient-dependent Ca2+ uptake of 6.6 +/- 0.8 (SE, n = 5) pmol.oocyte-1.30 min-1. This is significantly higher than the value of 3.4 +/- 0.5 (SE, n = 5) pmol.oocyte-1.30 min-1 obtained in water-injected oocytes (P less than 0.001). Similar results were obtained using poly(A)+ RNA from rabbit kidney cortex. Neither 10 microM nifedipine nor 0.5 mM D 600 significantly affected this Ca2+ uptake. However, 90% of the Ca2+ uptake was inhibited in the presence of 0.1 mM La3+. The poly(A)+ RNA-induced Na(+)-Ca2+ exchange activity was stimulated by the presence of 5 mM K+ in the extracellular choline solution compared with choline alone. Fractionation experiments indicate that the rat kidney Na(+)-Ca2+ exchanger was encoded by poly(A)+ RNA of 3-4 kb.

Na(+)-Ca2+ exchangers from rod outer segments and cardiac sarcolemma: comparison of properties

The properties of the Na(+)-Ca2+ exchangers from cardiac sarcolemma (SL) and rod outer segments (ROS) were studied in parallel by measuring the Na+ gradient-dependent Ca2+ uptake into SL or ROS vesicles. The ROS exchanger, but not the SL exchanger, has a striking specific dependence on K+. The ROS exchanger is stimulated by K+ with an apparent concentration at half-maximum of 1 mM. The addition of valinomycin, to produce an inside-positive membrane potential, stimulates the SL exchanger 1.8-fold and the ROS exchanger 1.2-fold. The Michaelis constant for half-maximal transport rate for Ca2+ and the maximal transport rate for the exchangers, in the absence of valinomycin, are estimated to be 20 microM and 8 nmol.mg-1.2.2 s-1 in SL and 5 microM and 1 nmol.mg-1.2.2 s-1 in ROS. Both exchangers are modulated by the same regulatory influences. For example, both are stimulated by proteases, phospholipase D, and intravesicular Ca2+.

The modulation of rat brain Na(+)-Ca2+ exchange by K+

The involvement of potassium ions in the Na(+)-Ca2+ exchange process was studied in rat brain synaptic plasma membrane (SPM) vesicles. Addition of equimolar [K+] to the intravesicular and the extravesicular medium led to a stimulation of the Na+ gradient-dependent Ca2+ influx; this stimulation was noticeable already at 0.5 mM and reached its maximum at 2 mM K+. The magnitude of the K+ stimulation was between 1.3-2.5-fold in different SPM preparations. K+ ions also stimulated the Na(+)-dependent Ca2+ efflux. K+ stimulation of Na(+)-Ca2+ exchange is of considerable specificity, since it is not mimicked by either Li+ or H+. The following lines of evidence suggest that K+ modulation of Na(+)-Ca2+ exchange involves the catalytic moiety of the transporter itself and not an unrelated K+ channel which modulates the membrane potential. 1) K+ stimulation of the transport process was conserved following reconstitution of the transporter into phospholipid-rich liposomes, an experimental condition which presumably separates the native membrane proteins among different vesicular structures. 2) K+ stimulation of Na+ gradient-dependent Ca2+ influx persists also when the build up of negative inside membrane potential is prevented by addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone which renders the membrane highly permeable to protons both in the native and the reconstituted preparation. 3) K+ stimulation of Na+ gradient-dependent Ca2+ influx is obtained also when tetraethylammonium chloride, 2,3-diaminopyridine and Cs+ are added to the Ca2+ uptake medium. Reconstituted SPM vesicles take up 86Rb+ in response to activation of Na+ gradient-dependent Ca2+ influx. The ratio of Ca2+ taken up by SPM vesicles in a Na+ gradient-dependent manner to the corresponding amounts of Rb+ taken up varies between 8-5 in different SPM preparations. If the stoichiometry of the process is 1 Rb+/1 Ca2+, then Rb+ cotransport is mediated by 10-20% of the transporters present in the preparation.

Expression of cardiac sarcolemmal Na+-Ca2+ exchange activity in Xenopus laevis oocytes

Injection of Xenopus laevis oocytes with rabbit heart poly(A)+RNA results in expression of Na+ inside (Nai+)-dependent Ca2+ uptake activity. The activity was measured by first loading the oocytes with Na+ using nystatin and then incubating the oocytes in K+ or Na+ medium containing 45Ca. The expressed Na+ gradient-dependent Ca2+ uptake was five to eight times that observed with water-injected oocytes or with poly(A)+RNA-injected oocytes for which the Na+ load step had been omitted. Induced activity was related to the amount of RNA injected and was insensitive to nifedipine. Fractionation of the poly(A)+RNA on a sucrose gradient determined that the active message had a size range between 3 and 5 kb. The properties of the Na+ gradient-dependent Ca2+ uptake indicated that Na+-Ca2+ exchange activity had been expressed in X. laevis oocytes. The result may be useful for cloning and identifying the molecular component responsible for Na+-Ca2+ exchange.

Stoichiometry of the sodium-calcium exchanger in nerve terminals.

The stoichiometry of the Na+-Ca2+ exchanger from synaptic plasma membranes was studied in both native and reconstituted preparations. In kinetic experiments performed with the native preparation, initial rates of Na+ gradient-dependent Ca2+ influx were compared to Ca2+-dependent Na+ efflux. These experiments showed that 4.82 Na+ ions are exchanged for each Ca2+ ion. A thermodynamic approach in which equilibrium measurements were made with the reconstituted preparation resulted in a similar (4.76) stoichiometry. The effects of membrane potential generated by valinomycin-induced K+ fluxes could be demonstrated in the reconstituted preparation. In addition, the direct contribution of the Na+-Ca2+ exchanger to the membrane potential across the reconstituted vesicle membrane could be demonstrated by using the lipophilic cation tetraphenylphosphonium.

Immunological identification of the synaptic plasma membrane Na+-Ca2+ exchanger.

The protein moiety responsible for Na+-Ca2+ exchange activity was identified in synaptic plasma membranes (SPM). This was done by raising polyclonal antibodies in rabbits against each one of the detectable proteins present in the purified preparation containing the enriched specific transport activity. Two of the antibody preparations bound specifically to native SPM: antibodies which were raised against the 70,000-Da protein (the most prominent species consistently present in the purified preparation) and antibodies raised against a 33,000-Da protein (inconsistently present in variable amounts in the purified preparation). Both antibodies bound exclusively to a protein of 70,000 Da in native SPM. When, however, the purified 33,000- and 70,000-Da proteins were used as antigens, each one of the antibody preparations bound to both proteins. In addition, both antibody preparations immunoprecipitated Na+ gradient-dependent Ca2+ transport activity from detergent-solubilized SPM. This was obtained by incubation of solubilized SPM with a complex containing antibodies bound to Protein A-Sepharose beads, reconstitution of the material excluded from the beads, and determination of the residual transport activity. The decrease in Na+ gradient-dependent Ca2+ transport activity paralleled the amount of antibody bound to Protein A-Sepharose beads and could reach 82% as compared to the activity remaining in control experiments using preimmune sera. In comparison, ATP-dependent Ca2+ transport activity was unimpaired. These results indicate that the 70,000-Da protein in SPM contains the catalytic Na+-Ca2+ antiport activity. The presence of the 33,000-Da protein in some preparations and its properties may be explained by its being either a degradation product or a subunit of the 70,000-Da protein.

Calcium transport in canine renal basolateral membrane vesicles. Effects of parathyroid hormone.

The effects of parathyroid hormone were studied on Ca2+ fluxes in canine renal proximal tubular basolateral membrane vesicles (BLMV). Efflux of Ca2+ from preloaded BLMV was found to be stimulated by an external Na+ gradient, and this was inhibited by the Na+ ionophore, monensin, and enhanced by intravesicular negative electrical potentials, which indicated electrogenic Na+/Ca2+ exchange activity. There was a Na+ gradient independent Ca2+ flux, but membrane binding of Ca2+ was excluded from contributing to the Na+ gradient-dependent efflux. The Na+ gradient-dependent flux of Ca2+ was very rapid, and even 2- and 5-s points may not fully represent absolute initial rates. It was saturable with respect to the interaction of Ca2+ and Na+ with an apparent (5 s) Km for Na+-dependent Ca2+ uptake of 10 microM, and an apparent (5 s) Vmax of 0.33 nmol/mg protein per 5 s. The Na+ concentration that yielded half maximal Ca2+ efflux (2 s) was 11 mM, and the Hill coefficient was two or greater. Both Na+ gradient dependent and independent Ca2+ efflux were decreased in BLMV prepared from kidneys of thyroparathyroidectomized (TPTX) dogs, and both were stimulated by parathyroid hormone (PTH) infusion to TPTX dogs. BLMV from TPTX dogs exhibited significantly reduced maximal stimulation of Na+ gradient-dependent Ca2+ uptake with an apparent (5 s) Vmax of 0.23 nmol/mg protein per 5 s, but the apparent Km was 8 microM, which was unchanged from normal. The Na+ gradient independent Ca2+ uptake was also reduced in BLMV from TPTX dogs compared with normal. Thus, PTH stimulated both Na+/Ca2+ exchange activity and Na+ independent Ca2+ flux. In vivo, the latter could result in an elevation of cytosolic Ca2+ by PTH, and this might contribute to the observed decrease in solute transport in the proximal tubule.

Isolation, purification, and reconstitution of the Na+ gradient-dependent Ca2+ transporter (Na+-Ca2+ exchanger) from brain synaptic plasma membranes.

A [Na+]-gradient-dependent Ca2+ transporter from brain synaptic plasma membranes has been isolated, purified, and reconstituted into brain phospholipid vesicles. The purification was achieved by sucrose-gradient centrifugation after solubilization of the synaptic membranes in cholate in the presence of a 30-fold excess (by weight) of added brain phospholipids and [Na+]-gradient-dependent Ca2+ loading of the reconstituted vesicles. A 128-fold increase in specific activity of [Na+]-gradient-dependent Ca2+ uptake per mg of protein has been obtained. The purified and reconstituted vesicles took up Ca2+ only in response to an outward-oriented [Na+] gradient. The Ca2+ uptake could be inhibited by dissipation of the [Na+] gradient with nigericin. Successful purification was based on the initial [Na+]-gradient dependency of the Ca2+-transport process, the magnitude of the [Na+]-gradient-dependent uptake, and the presence of purified brain phospholipids. Analysis of the sucrose-gradient-purified reconstituted vesicles on NaDodSO4/polyacrylamide gels showed that the activity coincided with enriched appearance of a 70,000-Da protein.

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex.

Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be L:RO:IO = 4:3:1. High-affinity Ca2+-ATPase, ATP-dependent Ca2+ transport and Na+/Ca2+ exchange have been studied with special emphasis on the relative transport capacities of the two Ca2+ transport systems. The kinetic parameters of Ca2+-ATPase activity in digitonin-treated membranes are: Km = 0.11 microM Ca2+ and Vmax = 81 +/- 4 nmol Pi/min X mg protein at 37 degrees C. ATP-dependent Ca2+ transport amounts to 4.3 +/- 0.2 and 7.4 +/- 0.3 nmol Ca2+/min X mg protein at 25 and 37 degrees C, respectively, with an affinity for Ca2+ of 0.13 and 0.07 microM at 25 and 37 degrees C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca2+ transport, a stoichiometry of 0.7 mol Ca2+ transported per mol ATP is found for the Ca2+-ATPase. In the presence of 75 mM Na+ in the incubation medium ATP-dependent Ca2+ uptake is inhibited 22%. When Na+ is present at 5 mM an extra Ca2+ accumulation is observed which amounts to 15% of the ATP-dependent Ca2+ transport rate. This extra Ca2+ accumulation induced by low Na+ is fully inhibited by preincubation of the vesicles with 1 mM ouabain, which indicates that (Na+-K+)-ATPase generates a Na+ gradient favorable for Ca2+ accumulation via the Na+/Ca2+ exchanger. In the absence of ATP, a Na+ gradient-dependent Ca2+ uptake is measured which rate amounts to 5% of the ATP-dependent Ca2+ transport capacity. The Na+ gradient-dependent Ca2+ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na+/Ca2+ exchange system for Ca2+ is between 0.1 and 0.2 microM Ca2+, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca2+ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca2+-ATPase system exceeds the capacity of the Na+/Ca2+ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca2+ homeostasis of rat kidney cortex cells.