Role of calcium in the regulation of sugar transport in the avian erythrocyte: Effects of the calcium ionophore,A23187

The tissue/medium distribution of the nonmetabolized glucose analog [¹⁴C]-3-0-methyl-D-glucose was measured in pigeon erythrocytes and related to changes in ⁴⁵Ca uptake and efflux, total calcium content and ATP levels. Sugar transport was not affected by changes in external Ca²⁺. However, both sugar and ⁴⁵Ca influx were increased by the Ca-ionophore A23187. In the absence of external Ca²⁺, the ionophore caused a delayed increase in sugar transport and net loss of calcium, probably through releasing Ca²⁺ from internal storage sites into the cytoplasm. Increasing internal Na⁺ through Na⁺ pump inhibition or using the sodium ionophore monensin did not alter influx of sugar or ⁴⁵Ca, indicating Na⁺-Ca²⁺ exchange was absent in these cells. The results are consistent with A23187 causing increased Ca²⁺ influx or release from mitochondrial storage and the resulting rise in cytoplasmic Ca²⁺ stimulating hexose transport. Experiments with low Mg⁺⁺ and high K⁺ media and measurements of ATP levels exclude alternative explanations for the action of A23187. We conclude that sugar transport regulation in avian erythrocytes is Ca²⁺-dependent and resembles that in muscle in its basic mechanism. It differs in the response to some modulating agents, largely because of a different pattern of Ca²⁺ fluxes in these cells.     

On the functional use of the membrane compartmentalized pool of ATP by the Na+ and Ca++ pumps in human red blood cell ghosts

Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane''s Na⁺ and Ca²⁺ pumps. The formation of the Na⁺- and Ca²⁺-dependent phosphointermediates of both types of pumps (E_Na-P and E_Ca-P) that conventionally can be labeled with trace amounts of [γ-³P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [γ-³P]ATP in the pool. Running the pumps forward with either Na⁺ or Ca²⁺ removes pool ATP and allows the normal formation of labeled E_Na-P or E_Ca-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10–15 minutes. We observed that when unlabeled “caged ATP” is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of E_Na-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na⁺ (without Mg²⁺) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of ⁴⁵Ca by the Ca²⁺ pump, analogous to the Na⁺ pump uptake of ²²Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na⁺ and Ca²⁺ pumps.     

Kinetic studies on sodium-dependent calcium uptake by myocardial cells and neuroblastoma cells in culture

Kinetic analyses were made on intracellular Na⁺-dependent Ca²⁺ uptake by myocardial cells and neuroblastoma cells (N-18 strain) in culture. Cells loaded with various concentrations of Na⁺ could be prepared by incubating them in Ca²⁺-free medium containing various concentrations of Na⁺. Cells pre-loaded with various concentrations of Na⁺ were incubated in medium containing Ca²⁺ and ⁴⁵Ca. The resulting ⁴⁵Ca uptake by the two types of cell depended greatly on the initial intracellular concentrations of Na⁺. Lineweaver-Burk plots of the initial rate of Ca²⁺ uptake against the external concentration of Ca²⁺ fitted well to straight lines obtained by linear regression (r > 0.95). This result shows that Ca²⁺ uptake by the two types of cell was achieved by a carrier-mediated transport system. This Na⁺-dependent Ca²⁺ uptake was accompanied by Na⁺ release and the ratio of Na⁺ release to Ca²⁺ uptake was close to 3 : 1. A comparison of the kinetic data between myocardial cells and N-18 cells suggested that N-18 cells possess a carrier showing the same properties as that of myocardial cells, i.e.: (1) a similar dependency on the intracellular concentration of Na⁺; (2) the coincidence of the apparent Michaelis constants for Ca²⁺ (0.1 mM); (3) the similarities of the K_i values for Co²⁺, Sr²⁺ and Mg²⁺ (Co²⁺ < Sr²⁺ < Mg²⁺) and (4) a similar dependency on pH. However, the maximal initial rate, V, of N-18 cells was about $\text{1}\text{100}$ that of myocardial cells. The rate of Na⁺-dependent Ca²⁺ uptake by non-excitable cells was much lower than that by myocardial cells.     

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine

The presence of an Na⁺/Ca²⁺ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na⁺ gradient-dependent Ca²⁺ uptake and the inhibition of ATP-dependent Ca²⁺ accumulation by Na⁺. The presence of 75 mM Na⁺ in the uptake solution reduces ATP-dependent Ca²⁺ transport by 45%, despite the fact that Na⁺ does not affect Ca²⁺-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na⁺ inhibition of ATP-dependent Ca²⁺ uptake to 20%, apparently by preventing accumulation of Na⁺ in the vesicles realized by the Na⁺-pump. It was concluded that high intravesicular Na⁺ competes with Ca²⁺ for intravesicular Ca²⁺ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca²⁺ transport shows a sigmoidal dependence on the Na⁺ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na⁺ is between 15 and 20 mM. Uptake of Ca²⁺ in the absence of ATP can be enhanced by an Na⁺ gradient (Na⁺ inside > Na⁺ outside). This Na⁺ gradient-dependent Ca²⁺ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca²⁺ of this system is below 1 μM. In contrast to the ATP-dependent Ca²⁺ transport, there is no significant difference in Na⁺ gradient-dependent Ca²⁺ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca²⁺ uptake is 5-times greater than the Na⁺/Ca²⁺ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na⁺/Ca²⁺ exchange mechanism is not subject to regulation by 1α,25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na⁺/Ca²⁺ exchange system while it doubles the ATP-driven Ca²⁺ pump activity.     

Ca-dependent inhibitory effects of Na and K on Ca transport in sarcoplasmic reticulum vesicles

Effects of Na⁺ and K⁺ on Ca²⁺ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg²⁺ and ATP (2mM) and low Ca²⁺ (0.44μM) concentrations. Under these conditions, Na⁺ and K⁺ inhibit Ca²⁺ uptake. ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca²⁺ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca²⁺ pump of the sarcoplasmic reticulum operates below its maximal capacity.     

Biochemical aspects of the visual process. XXXVI. Calcium accumulation in cattle rod outer segments: Evidence for a calcium-sodium exchange carrier in the rod sac membrane

Accumulation of calcium has been studied in bovine rod outer segments (rods), isolated by sucrose density gradient centrifugation. Calcium-depleted rods are obtained by having ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA) present during isolation.Rods thus isolated have a leaky plasma membrane, as shown by the effects of ionophore A23187 and by their light-induced phosphorylation behaviour. The accumulation of ⁴⁵Ca, determined by incubation followed by a single fast washing-filtration procedure, thus represents translocation across the rod sac membrane.Accumulation in non-depleted rods is independent of the external calcium level and of ATP, suggesting exchange of ⁴⁰Ca by ⁴⁵Ca. In depleted rods in the presence of ATP there is net uptake, sigmoidally increasing with the external calcium concentration to the level attained in non-depleted rods. This net uptake is abolished by omission of ATP, its replacement by β,γ-methylene ATP and lowering the temperature to 0° C, suggesting involvement of enzymatic hydrolysis of ATP.Replacement of KCl by NaCl in the medium causes marked inhibition of ⁴⁵Ca uptake, both net uptake and exchange. Oligomycin, ruthenium red, lanthanum and ouabain do not inhibit accumulation.Efflux of ⁴⁵Ca from pre-loaded rods is slow in a KCl medium ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～30}\text{min}$ at 25° C), but is greatly accelerated by addition of NaCl or Ca²⁺ ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{10}\text{s}$ at 25°C).It is concluded that the rod sac membrane contains a carrier system, which is sensitive towards Ca²⁺ and Na⁺ and which requires ATP for net uptake of Ca²⁺ but not for exchange transport of Ca²⁺ with Ca²⁺ or Na⁺.     

Dual effect of adrenalin on sugar transport in rat diaphragm muscle

The effect of adrenalin on the membrane transport of the non-metabolized sugar, 3-methylglucose, was studied in isolated “intact” rat hemidiaphragms and related to simultaneously occurring changes in the internal levels of Na⁺, ATP, glucose-6-P, glycerol formation and ⁴⁵Ca uptake and loss. Basal sugar transport was inhibited by low (10⁻⁸−10⁻⁵ M) concentrations of adrenalin; this was antagonized by propranolol and practolol. High concentrations (10⁻⁴−10⁻³ M) stimulated sugar transport, and this was blocked by propranolol and butoxamine and was dependent on external Ca²⁺. These results suggest interaction with two different classes of adrenergic receptors, possibly of β₁ and β₂ types. Both low and high concentrations increased Na⁺ and K⁺ gradients by a practolol-sensitive effect. Isoproterenol behaved identically but phenylephrine had only the two practolol-sensitive effects on sugar and ion transport. Insulin did not interfere with inhibition of sugar transport and decrease in internal Na⁺ but prevented stimulation of sugar transport. Under anoxia adrenalin had no effect on sugar transport but led to greater Na⁺ gain by tissue. Addition of 3.0 mM palmitate decreased inhibition of sugar transport without changing receptor specificity. ATP was decreased and lipolysis enchanged by high adrenalin but glucose-6-P was increased by the low concentration as well. Influx of ⁴⁵Ca was decreased by low and increased by high adrenalin; ⁴⁵Ca efflux was also differentially affected. The results indicate that inhibition and stimulation of sugar transport depend on different receptors and that the latter response may override the former. The data are consistent with the earlier postulated regulatory role of sarcoplasmic Ca²⁺ on sugar transport in muscle, with adrenalin affecting Ca²⁺ fluxes and distribution both directly and indirectly.     

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

Summary ATP-dependent Ca²⁺ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca²⁺-specific electrode and⁴⁵Ca²⁺. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na⁺,K⁺(Mg²⁺)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared,⁴⁵Ca²⁺ uptake correlated with the RNA content and not with the Na⁺,K⁺(Mg²⁺)ATPase activity. The characteristics of nonmitochondrial Ca²⁺ uptake into leaky isolated cells and⁴⁵Ca²⁺ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg²⁺, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca²⁺ concentration of 2 and 2 mol/liter, respectively. Calcium uptake decreased at higher free Ca²⁺ concentration.⁴⁵Ca²⁺ uptake was dependent on monovalent cations (Rb⁺>K⁺>Na⁺>Li⁺>choline⁺) and different anions (Cl^–>Br^–>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–>SCN^–) in both preparations. Twenty mmol/liter oxalate enhanced⁴⁵Ca²⁺ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca²⁺ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca²⁺ concentration to 0.4 mol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca²⁺ concentration in pancreatic acinar cells.     

Ca2+-dependent inhibitory effects of Na+ and K− on Ca2+ transport in sarcoplasmic reticulum vesicles

Effects of Na⁺ and K⁺ on Ca²⁺ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg²⁺ and ATP (2mM) and low Ca²⁺ (0.44μM) concentrations. Under these conditions, Na⁺ and K⁺ inhibit Ca²⁺ uptake. ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca²⁺ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca²⁺ pump of the sarcoplasmic reticulum operates below its maximal capacity.     

Sodium-calcium exchange in renal epithelial cells: Dependence on cell sodium and competitive inhibition by magnesium

Summary Kinetic properties of Na⁺–Ca²⁺ exchange in a renal epithelial cell line (LLC-MK₂) were assessed by measuring cytosolic free Ca²⁺ with fura-2 and⁴⁵Ca²⁺ influx. Replacing external Na⁺ with K⁺ produced relatively small increases in free Ca²⁺ and⁴⁵Ca²⁺ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na⁺ and strongly potentiated the effect of replacing external Na⁺ with K⁺ on free Ca²⁺ and⁴⁵Ca²⁺ uptake.⁴⁵Ca²⁺ influx in 140mm K⁺ or N-methyl-d-glucamine minus influx in 140mm Na⁺ was used to quantify Na⁺–Ca²⁺ exchange activity of Na⁺-loaded cells. The dependence of exchange on cell Na⁺ was sigmoidal; theK _0.5 was 26±3 mmol/liter cell water space, and the Hill coefficient was 3.1±0.2. The kinetic features of the dependence of exchange on cell Na⁺ partly account for the small increase in Ca²⁺ influx when all external Na⁺ is replaced by K⁺. Besides raising cell Na⁺ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg²⁺ was 8.2-fold lower with potassium instead of N-methyl-d-glucamine or choline as the replacement for external Na⁺. Potassium also increased theV _max of exchange by 86% and had no effect on theK _m for Ca²⁺. The exchanger does not cause detectable²²Na⁺–Mg²⁺ exchange and does not appear to require K⁺ or transport⁸⁶Rb⁺. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na⁺–Ca²⁺ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.     

Na+-dependence of 45Ca2+ uptake in adult rat isolated cardiac cells

We developed a technique that yields isolated adult rat myocytes, 70% of which are elongated and morphologically similar to intact tissue. Electrophysiological studies showed most of these cells were quiescent, Ca²⁺-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed ⁴⁵Ca²⁺ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca²⁺ was found in the slow compartment; the rest was almost equally divided between the instantaneous and fast compartments. Replacement of extracellular Na⁺ by Li⁺ or Tris increased ⁴⁵Ca²⁺ uptake by the fast compartment; high [K⁺]_o increased this uptake further. These increases appeared to be related also to internal concentrations of Na⁺. This conclusion was supported by experiments with digitonin-treated cells. Our results indicate that the way Na⁺-dependent ⁴⁵Ca²⁺ uptake is affected by [Na⁺]_o, [Na⁺]_i and [K⁺]_o is compatible with the Na⁺-Ca²⁺ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca²⁺ transport in cardiac muscles.     

Effect of extracellular adenosine 5′-triphosphate on Ca2+ efflux from freshly isolated adult rat cardiomyocytes

The effect of extracellular adenosine 5′-triphosphate (ATP) on Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes was examined. ATP stimulated the efflux of ⁴⁵Ca²⁺ from the cells in a concentration-dependent manner (0.01–1 mM). The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by adenosine-5′-O-(3-thiotriphosphate) (ATP-γs) and α,β-methylene-ATP and adenosine 5′-diphosphate, but not by adenosine 5′-monophosphate and adenosine. The ATP-stimulated ⁴⁵Ca²⁺ efflux was not affected by deprivation of the extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. These results indicate that ATP stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane P₂ purinoceptors which may couple to Na⁺/Ca²⁺ exchange.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

An ion exchange chromatographic method for the determination of synaptosomal calcium uptake

A simple, rapid method for determining depolarization-induced⁴⁵Ca influx into synaptosomes is described. Synaptosomes which had been depolarized in the presence of⁴⁵Ca were applied to a small column of Chelex-100 resin to separate free Ca²⁺ from that taken up by the tissue. Approximately 70% of the synaptosomal protein applied to the column was recovered in the initial eluate. The magnitude of⁴⁵Ca uptake was dependent on the amount of Ca²⁺ in the incubation medium and on the KCl concentration. Calcium influx reached a plateau after 90 sec of incubation at 24°C. The Na⁺ channel activator veratridine also produced a substantial influx of⁴⁵Ca, and this effect was blocked by tetrodotoxin. Thus, this ion exchange procedure makes it possible to measure depolarization-induced Ca²⁺ influx in synaptosomes without subjecting them to high vacuum or centrifugation pressures or to EGTA-containing solutions.     

Na+/Ca2+ countertransport in plasma membrane of rat pancreatic acinar cells

Summary The presence of a coupled Na⁺/Ca²⁺ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na⁺/Ca²⁺ exchange was investigated by measuring⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (=) in the presence of sodium. Plasma membranes were prepared by a MgCl₂ precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na⁺+K⁺)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochromec reductase, as well as for mitochondria, the cytochromec oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na⁺/Ca²⁺ countertransport system, the Ca²⁺ uptake after 1 min of incubation was half-maximal at 0.62 mol/liter Ca²⁺ and at 20 mmol/liter Na⁺ concentration and maximal at 10 mol/liter Ca²⁺ and 150 mmol/liter Na⁺ concentration, respecitively. When Na⁺ was replaced by Li⁺, maximal Ca²⁺ uptake was 75% as compared to that in the presence of Na⁺. Amiloride (10^–3 mol/liter) at 200 mmol/liter Na⁺ did not inhibit Na⁺/Ca²⁺ countertransport, whereas at low Na⁺ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10^–2 mol/liter. CFCCP (10^–5 mol/liter) did not influence Na⁺/Ca²⁺ countertransport. Monensin inhibited dose dependently; at a concentration of 5×10^–6 mol/liter inhibition was 80%. A SCN^– or K⁺ diffusion potential (=), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na⁺/Ca²⁺ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na⁺ for 1 Ca²⁺. In the absence of Na⁺, did not promote Ca²⁺ uptake. We conclude that in addition to ATP-dependent Ca²⁺ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & 1. Schulz, 1985,J. Membrane Biol. 84:45–60) the Na⁺/Ca²⁺ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca²⁺ level.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Ca2+ dependency of Na+ transport by rabbit renal brush border membrane

Summary Intracellular Ca²⁺ has been suggested to play an important role in the regulation of epithelial Na⁺ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca²⁺-free medium enhanced Na⁺ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na⁺ entry across the apical membrane by intracellular Ca²⁺. In the present study, we have examined the effect of Ca²⁺-free preincubation on apical membrane Na⁺ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1mm Ca²⁺ medium exhibited higher Na⁺ uptake than those derived from proximal tubules incubated in Ca²⁺-free EGTA medium. Such effect of Ca²⁺ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation, Ba²⁺ (1mm). Ca²⁺ in the preincubation medium did not affect Na⁺-dependent BBM glucose uptake, and its effect on BBM Na⁺ uptake was pH gradient dependent and amiloride (10^–3 m) sensitive, suggesting the involvement of Na⁺/H⁺ antiport system. Addition of verapamil (10^–4 m) to 1mm Ca²⁺ preincubation medium abolished while ionomycin (10^–6 m) potentiated the effect of Ca²⁺ to increase BBM Na⁺ uptake, suggesting that the effect of Ca²⁺ in the preincubation medium is likely to be mediated by Ca²⁺-dependent cellular pathways and not due to a direct effect of extracellular Ca²⁺ on BBM. Neither the proximal tubule content of cAMP nor the inhibitory effect of 8, bromo-cAMP (0.1mm) on BBM Na⁺ uptake was affected by the presence of Ca²⁺ in the preincubation medium, suggesting that Ca²⁺ in the preincubation medium did not increase BBM Na⁺ uptake by removing the inhibitory effect of cAMP. Addition of calmodulin inhibitor, trifluoperazine (10^–4 m) to 1mm Ca²⁺ preincubation medium did not prevent the increase in BBM Na⁺ uptake. The effect of Ca²⁺ was, however, abolished when protein kinase C in the proximal tubule was downregulated by prolonged (24 hr) incubation with phorbol 12-myristate 13-acetate (10^–6 m). In summary, these results show the Ca²⁺ dependency of Na⁺ transport by renal BBM, possibly through stimulation of Na⁺/H⁺ exchanger by protein kinase C.     

Active transport of Calcium ions in the skin of the salamander Ambystoma tigrinum

We measured Ca²⁺ exchanges across the skin of larval and adult Ambystoma tigrinum using the radio-isotope influx method. We found that the skin of both morphs takes up Ca²⁺ in a manner that is proportional to external [Ca²⁺], saturable and oriented against the electrochemical gradient for Ca²⁺. We conclude that this uptake occurs by active transport. Kinetic analysis yields affinities for calcium ions that are similar to the affinities for both Ca²⁺ and Na⁺ in the skin of other amphibians. The capacity for calcium is similar to Ca²⁺ capacity in other amphibians. The capacity for Ca²⁺ is lower than the capacity for Na⁺. Cutaneous Ca²⁺ deposits are lower in this urodele than found in anurans. Adults tend to have higher levels of Ca in their skin than do larvae. Accepted: 25 June 1999