ATP-dependent calcium pump and Na+-Ca2+ exchange in plasma membrane vesicles from squid optic nerve

Purified plasma membrane vesicles from the optic nerve of the squid Sepiotheutis sepioidea accumulate calcium in the presence of Mg2+ and ATP. Addition of the Ca2+ ionophore A23187 to vesicles which have reached a steady state of calcium-active uptake induces complete discharge of the accumulated cation. Kinetic analysis of the data indicates that the apparent Km for free Ca2+ and ATP are 0.2 muM and 21 muM, respectively. The average Vmax is 1 nmol Ca2+/min per mg protein at 25 degrees C. This active transport is inhibited by orthovanadate in the micromolar range. An Na+-Ca2+ exchange mechanism is also present in the squid optic nerve membrane. When an outwardly directed Na+ gradient is imposed on the vesicles, they accumulate calcium in the absence of Mg2+ and/or ATP. This ability to accumulate Ca2+ is absolutely dependent on the Na+ gradient: replacement of Na+ by K+, or passive dissipation of the Na+ gradient, abolishes transport activity. The apparent Km for Ca2+ of the Na+-Ca2+ exchange is more than 10-fold higher than that of the ATP-driven pump (app. Km=7.5 muM). While the apparent Km for Na+ is 74 mM, the Vmax of the exchanger is 27 nmol Ca2+/min per mg protein at 25 degrees C. These characteristics are comparable to those displayed by the uncoupled Ca pump and Na+-Ca2+ exchange previously described in dialyzed squid axons.     

Na+-Ca2+ exchange and calcium permeability in canine basolateral membrane vesicles: the effects of dibutyryl cAMP and specific inhibitors

The role of dibutyryl 3',5'-cyclic adenosine monophosphate (dibutyryl cAMP) as putative second messenger for parathyroid hormone (PTH) in regulating canine proximal tubular basolateral membrane Na+-Ca2+ exchange and passive calcium permeability was assessed, as was the nature of this passive calcium permeability. Dibutyryl cAMP (50 mg) infused in vivo over 30 min increased fractional phosphate excretion from 4.9 +/- 1.8% to 20.5 +/- 4.6%, P less than 0.05, n = 6, but had no effect on either passive Ca2+ efflux or sodium-stimulated Ca2+ efflux from Ca2+-preloaded basolateral membrane vesicles (BLMV). Both of these mechanisms have been previously shown to be stimulated by PTH. Further studies were performed to investigate the mechanism of the passive calcium flux. Calcium uptake by BLMV was blocked by lanthanum (La3+) but not by the calcium-channel blocker verapamil. La3+ blocked efflux of Ca2+ from preloaded vesicles when it was placed in the external solution. This La3+-blockable efflux was larger in potassium equivalent BLMV prepared from normal dogs than in BLMV prepared from thyroparathyroidectomized dogs. Benzamil produced 50% inhibition of sodium-stimulated Ca2+ uptake at 250 microM whereas neither amiloride nor diltiazem achieved 50% inhibition at the maximal doses studied. Benzamil, 1 mM, had no effect on passive calcium efflux and neither did the substitution of sucrose for potassium, which has been shown to affect Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. This suggests that the calcium flux under potassium equivalent conditions was not mediated by Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. These results demonstrate that the basolateral membrane of proximal tubular cells possesses both a Na+-Ca2+ exchanger inhibitable by benzamil and a passive calcium permeability not inhibited by benzamil nor by verapamil but by La3+. Neither of these two mechanisms of calcium flux was affected by dibutyryl cAMP whereas both have been shown to be stimulated by PTH.     

Artificial cell based on lipid hollow polyelectrolyte microcapsules: channel reconstruction and membrane potential measurement

The CitM transporter from Bacillus subtilis transports citrate as a complex with Mg2+. In this study, CitM was functionally expressed and characterized in E. coli DH5a cells. In the presence of saturating Mg2+ concentrations, the Km for citrate in CitM was 274 mM, similar to previous studies using whole cells of B. subtilis. CitM has a high substrate specificity for citrate. Other di- and tricarboxylic acids including succinate, isocitrate, cis-aconitate and tricarballylic acid did not significantly inhibit the uptake of citrate in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The highest rate of citrate transport was seen in the presence of Mg2+, followed in order of preference by Mn2+, Ba2+, Ni2+, Co2+ and Ca2+. Citrate transport by CitM appears to be proton coupled. The transport was inhibited in transport buffers more alkaline than pH 7.5 and not affected by pH at acidic values. Transport was also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin. Valinomycin did not affect the uptake by CitM, suggesting that transport is electroneutral. In conclusion, the cloned CitM transporter from B. subtilis expressed in E. coli has properties similar to the transporter in intact B. subtilis cells. The results support a transport model with a coupling stoichiometry of one proton coupled to the uptake of one complex of (Mg2+-citrate)1-.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Role of phosphatidylinositol in cardiac sarcolemmal membrane sodium-calcium exchange

The purpose of this investigation was to study the effects of a distinct type of phospholipase C on sarcolemmal Na+-Ca2+ exchange. With this phospholipase C (Staphylococcus aureus), treatment of cardiac sarcolemmal vesicles resulted in a specific hydrolysis of membrane phosphatidylinositol. This hydrolysis of phosphatidylinositol also released two proteins (110 and 36 kDa) from the sarcolemmal membrane. Phospholipase C pretreatment of the sarcolemma resulted in an unexpected stimulation of Na+-Ca2+ exchange. The Vmax of Na+-Ca2+ exchange was increased but the Km for Ca2+ was not altered. This stimulation was specific to the Na+-Ca2+ exchange pathway. ATP-dependent Ca2+ uptake was depressed after phospholipase C treatment, but passive membrane permeability to Ca2+ was unaffected. Sarcolemmal Na+,K+-ATPase activity was not altered, whereas passive Ca2+ binding was modestly decreased after phospholipase C pretreatment. The stimulation of Na+-Ca2+ exchange after phosphatidylinositol hydrolysis was greater in inside-out vesicles than in a total population of vesicles of mixed orientation. This finding suggests that the cardiac sarcolemmal Na+-Ca2+ exchanger is functionally asymmetrical. The results also suggest that membrane phosphatidylinositol is inhibitory to the Na+-Ca2+ exchanger or, alternatively, this phospholipid may anchor an endogenous inhibitory protein in the sarcolemmal membrane. The observation that a transsarcolemmal Ca2+ flux pathway may be stimulated solely by phosphatidylinositol hydrolysis independently of phosphoinositide metabolic products like inositol triphosphate is novel.     

Site density of the sodium-calcium exchange carrier in reconstituted vesicles from bovine cardiac sarcolemma

The site density of the Na2+-Ca2+ exchanger in bovine cardiac sarcolemma was estimated from measurements of the fraction of reconstituted proteoliposomes exhibiting exchange activity. Sarcolemmal vesicles were solubilized with 1% Triton X-100 in the presence of either 100 mM NaCl or 100 mM KCl; after a 20-40-min incubation period on ice, sufficient KCl, NaCl, CaCl2, and soybean phospholipids were added to each extract to give final concentrations of 40 mM NaCl, 120 mM KCl, 0.1 mM CaCl2, and 10 mg/ml phospholipid. These mixtures were then reconstituted into proteoliposomes, and the rate of 45Ca2+ isotopic exchange was measured under equilibrium conditions. Control studies showed that Na+-Ca2+ exchange activity was completely lost if Na+ was not present during solubilization. The difference in 45Ca2+ uptake between vesicles initially solubilized in the presence or absence of NaCl therefore reflected exchange activity and corresponded to 3.1 +/- 0.3% of the total 45Ca2+ uptake by the entire population of vesicles, as measured in the presence of the Ca2+ ionophore A23187. Assuming that each vesicle with exchange activity contained 1 molecule of the Na+-Ca2+ exchange carrier, a site density of 10-20 pmol/mg of protein for the exchanger was calculated. The Vmax for Na+-Ca2+ exchange activity in the proteoliposomes was approximately 20 nmol/mg of protein.s which indicates that the turnover number of the exchange carrier is 1000 s-1 or more. Thus, the Na+-Ca2+ exchanger is a low density, high turnover transport system.     

Effect of temperature on sodium-calcium exchange in sarcolemma from mammalian and amphibian hearts

We have investigated temperature dependence of Ca2+ uptake by the cardiac sarcolemmal Na(+)-Ca2+ exchanger from dog, rabbit and bullfrog. In native rabbit sarcolemmal vesicles, Ca2+ affinity of the Na(+)-Ca2+ exchanger is unchanged from 7 to 37 degrees C; however, the initial velocity of Ca2+ uptake declines much more steeply below 22 degrees C than above 22 degrees C. In native dog sarcolemma, the temperature dependence of Na(+)-Ca2+ exchange velocity is similar to that of native rabbit. However, in frog heart the velocity of Na(+)-Ca2+ exchange declines much more slowly with decreasing temperature at both temperature ranges. Reconstitution of the Na(+)-Ca2+ exchanger into artificial lipid vesicles consisting of either asolectin or phosphatidylserine, phosphatidylcholine, and cholesterol has little effect on temperature dependence of Na(+)-Ca2+ exchange velocity in any of the three species. We conclude that the lesser temperature sensitivity of the cardiac sarcolemmal Na(+)-Ca2+ exchanger of a poikilothermic species is at least partly an intrinsic property of the transport protein.     

Energy-dependent accumulation of Ca2+ by human embryonic lung fibroblasts.

Human embryonic fibroblasts accumulate Ca2+ in the presence of extracellular ATP and Mg2+, the uptake being maximal at 3 mM ATP. Iodoacetic acid, oligomycin and temperatures of 2 degrees C all inhibit the ATP-potentiated uptake suggesting that an active process may be involved in the transport of Ca2+ into these cells under certain conditions.     

Mechanism for electrosilent Ca2+ transport to cause calcification of spicules in sea urchin embryos

Embryos of the sea urchin, Hemicentrotus pulcherrimus, kept in sea water containing the calcium antagonists, diltiazem and verapamil, or an anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), during a developmental period between the mesenchyme blastula and the pluteus corresponding stage, became abnormal plutei with poorly developed arms and quite small spicules. Treatment with ethacrynic acid and furosemide, inhibitors of chloride transport, during the same period of development yielded quasi-normal plutei with poor spicules and somewhat developed arms. In late gastrulae, the inhibitory effects of these calcium antagonists and DIDS on the uptake of 45Ca2+ in whole embryos were as strong as those on 45Ca deposition in spicules, whereas the effects of chloride transport inhibitors on calcium deposition in the spicules were markedly stronger than on its uptake in whole embryos. Electrosilent uptake of Ca2+ seems to be established mainly by coupled influx of chloride in the cells which mediate spicule calcification, and by concomitant influx of anions in the other cells. In swimming blastulae, 45Ca2+ uptake was inhibited by calcium antagonists and DIDS, but not by chloride transport inhibitors. Ca2+ uptake probably becomes coupled with chloride influx only in embryos in which spicule calcification occurs.     

Alterations by saponins of passive Ca2+ permeability and Na+-Ca2+ exchange activity of canine cardiac sarcolemmal vesicles

Saponins can both permeabilize cell plasma membranes and cause positive inotropic effects in isolated cardiac muscles. Different saponins vary in their relative abilities to cause each effect suggesting that different mechanisms of action may be involved. To investigate this possibility, we have compared the effects of seven different saponins on the passive Ca2+ permeability and Na+-Ca2+ exchange activity of isolated canine cardiac sarcolemmal membranes. Saponins having hemolytic activity reversibly increased the passive efflux of Ca2+ from sarcolemmal vesicles preloaded with 45Ca2+ with the following order of potency: echinoside-A greater than echinoside-B greater than holothurin-A greater than holothurin-B greater than sakuraso-saponin. Ginsenoside-Rd and desacyl-jego-saponin, which lack hemolytic activity, had no significant effect on this variable. The saponins also stimulated Na+-Ca2+ exchange activity measured as Na+-dependent Ca2+ uptake by sarcolemmal vesicles. Ginsenoside-Rd and desacyl-jego-seponin, which did not affect passive Ca2+ permeability, stimulated the uptake, while in contrast, echinoside-A and -B only slightly increased or decreased this latter variable. Thus, the abilities of these compounds to enhance Na+-Ca2+ exchange activity seem to be inversely related to their abilities to increase the Ca2+ permeability. Effects by the echinosides on Na+-Ca2+ exchange may be masked by the loss of Ca2+ from the vesicles due to the increased permeability. These results suggest that the saponins interact with membrane constituent(s) that can influence the passive Ca2+ permeability and the Na+-Ca2+ exchange activity of cardiac sarcolemmal membranes.     

Permeability properties of isolated enterocytes from rat small intestine

Metabolic and permeability properties of enterocytes isolated by treatment of rat small intestine with hyaluronidase or EDTA were compared. No significant difference was observed in the ability of the two types of cell to produce lactate from glucose. However, while cells obtained with hyaluronidase accumulate alpha-methylglucoside, cells obtained with EDTA were unable to accumulate the sugar above the medium concentrations. When resuspended in a medium designed to resemble the intracellular medium, potentiometric measurements showed that cells obtained with hyaluronidase released Ca2+ to the medium while cells obtained with EDTA accumulated it. Using 45Ca transport assays, this was shown to be an ATP-dependent process, the accumulated 45Ca being totally released by the addition of the ionophore A23187. When cells obtained with EDTA were resuspended in a medium containing concentrations of free Ca2+ higher that 10 microM, the uptake was partially inhibited by sodium orthovanadate and also by oligomycin and antimycin. At free Ca2+ concentrations lower than 1 microM, the accumulation was inhibited up to 87% by sodium orthovanadate while mitochondrial inhibitors inhibited only 5%. Thus, it appears that during their preparation cells obtained with hyaluronidase retain their integrity while cells obtained with EDTA become permeable to Ca2+ and other ions. The usefulness of both types of preparation in metabolic and transport studies is discussed.     

Calcium transport into the vacuole of oat roots. Characterization of H+/Ca2+ exchange activity

Calcium (Ca2+) is sequestered into vacuoles of oat root cells through a H+/Ca2+ antiport system that is driven by the proton-motive force of the tonoplast H+-translocating ATPase. The antiport has been characterized directly by imposing a pH gradient in tonoplast-enriched vesicles. The pH gradient was imposed by diluting K+-loaded vesicles into a K+-free medium. Nigericin induced a K+/H+ exchange resulting in a pH gradient of 2 (acid inside). The pH gradient was capable of driving 45Ca2+ accumulation. Ca2+ uptake was tightly coupled to H+ loss as increasing Ca2+ levels progressively dissipated the steady state pH gradient. Ca2+ uptake displayed saturation kinetics with a Km(app) for Ca2+ of 10 microM. The relative affinity of the antiporter for transport of divalent cations was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. La3+ or Mn2+ blocked Ca2+ uptake possibly by occupying the Ca2+-binding site. Ruthenium red (I50 = 40 microM) and N,N'-dicyclohexylcarbodiimide (I50 = 3 microM) specifically inhibited the H+/Ca2+ antiporter. When driven by pH jumps, the H+/Ca2+ exchange generated a membrane potential, interior positive, as shown by [14C]SCN accumulation. Furthermore, Ca2+ uptake was stimulated by an imposed negative membrane potential. The results support a simple model of one Ca2+ taken up per H+ lost. The exchange transport can be reversed, as a Ca2+ gradient (Ca2+in greater than Ca2+out) was effective in forming a pH gradient (acid inside). We suggest that the H+/Ca2+ exchange normally transports Ca2+ into the vacuole; however, under certain conditions, Ca2+ may be released into the cytoplasm via this antiporter.     

Aluminum Inhibits the Fast Phase of Voltage-Dependent Calcium Influx into Synaptosomes

Aluminum has been shown to have neurotoxic effects, but the mechanisms by which it acts are not well understood. Because it has been reported that aluminum can interact with Ca2+-binding sites, the possibility that aluminum might interfere with Ca2+ influx into synaptosomes was examined. At concentrations of 50 microM and greater, aluminum significantly inhibited the fast phase (0-1 s) of the voltage-dependent uptake of 45Ca2+ into synaptosomes. Higher concentrations of aluminum also reduced 45Ca2+ uptake measured at 1 s in nondepolarizing media and inhibited the slow phase of 45Ca2+ uptake into synaptosomes whether they were suspended in either low K or high K media. The possibility that aluminum competitively inhibits the fast phase of Ca2+ influx was investigated. Aluminum (250 microM) increased the apparent KT (concentration of Ca2+ at which Ca2+ transport is half maximal) for 45Ca2+ of fast phase voltage-dependent channels and slightly decreased the maximal influx (Jmax). These effects are characteristic of a mixed type inhibitor, and the apparent Ki for Al3+ is estimated to be 0.64 mM. The interaction of aluminum with the fast phase of voltage-dependent calcium influx may disrupt intraneuronal calcium homeostasis and may also represent a means by which aluminum could accumulate intraneuronally.     

Erythrocyte calcium metabolism. Calcium exchange in normal and sickle-cell-anaemia erythrocytes.

Under exchange conditions (no net increase in calcium), erythrocytes incubated in isoosmotic phosphate-buffered saline have an exchangeable calcium pool comprising about 10% of the total erythrocyte calcium. This pool reaches exchange equilibrium, for either inward-directed or outward-directed transfer of the 45Ca-exchange label, with a half-time of about 20 min. The uptake of Ca2+ requires phosphate, even under hypo-osmotic conditions, where the calcium loading expected as the cells swell is obtained only when phosphate is present. The phosphate requirement is not due to Ca2+ transport as a phosphate salt. This exchangeable-calcium pool is also present in sickle-cell-anemia erythrocytes, and comprises a similar proportion of total cellular calcium.     

ATP-driven calcium transport in membrane vesicles of Streptococcus sanguis.

Calcium transport was investigated in membrane vesicles prepared from the oral bacterium Streptococcus sanguis. Procedures were devised for the preparation of membrane vesicles capable of accumulating 45Ca2+. Uptake was ATP dependent and did not require a proton motive force. Calcium transport in these vesicles was compared with 45Ca2+ accumulation in membrane vesicles from Streptococcus faecalis and Escherichia coli. The data support the existence of an ATP-driven calcium pump in S. sanguis similar to that in S. faecalis. This pump, which catalyzes uptake into membrane vesicles, would be responsible for extrusion of calcium from intact cells.     

Involvement of cellular calcium incorporated from the medium in production of inositol trisphosphate in A-431 cells

The property of intensive 45Ca2+ uptake by A-431 human epidermoidal carcinoma cells was indicated to be an influx, not binding to the cell surface, since the two apparent dissociation constants (Kd) between 45Ca2+ and cells were almost the same when measured in either the presence or absence of 1 mM [ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA); these constants were approximately 5-10 x 10(-6) and 1 x 10(-4) M, respectively, which are much higher than the chelating constant of EGTA for Ca2+ (approximately 10(-11) M). Furthermore, addition of A23187, a calcium ionophore, rapidly released the 45Ca2+ incorporated into cells at both 37 degrees C and 0 degrees C. The 45Ca2+ associated with the cells was slowly released or exchanged when cells were incubated in medium depleted of Ca2+, or in that containing 1 mM non-radioactive Ca2+. The ability of A-431 cells to respond to extracellular ATP by elevating their level of intracellular calcium ions, as well as by producing inositol trisphosphate (InsP3), was suppressed in cells depleted of cellular calcium. These data suggest that calcium ions are extensively incorporated or exchanged with those outside the cells, maintained as stored calcium, and involved in production of InsP3, when A-431 cells are stimulated by ATP to trigger the signal transduction system.     

mRNA-induced expression of the cardiac Na+-Ca2+ exchanger in Xenopus oocytes

Xenopus oocytes were injected with total mRNA isolated from hearts of 1-day-old chicks. After 5 days of incubation the follicular cell layers were removed and the oocytes were loaded with Na+ by incubation in hypertonic EGTA solution at 37 degrees C. The Na+-loaded oocytes accumulated 45Ca2+ from a Na+-free medium at a 3-18-fold higher rate than noninjected oocytes or oocytes injected with control solution containing no mRNA. Oocytes not subjected to the Na+-loading procedure showed no mRNA-dependent 45Ca2+ uptake. Size fractionation of the mRNA using sucrose density gradient centrifugation under denaturing conditions led to the identification of a 25 S fraction competent for induction of the Na+-Ca2+ exchange system.     

Guanosine 3',5'-cyclic monophosphate stimulates release of actively accumulated calcium in purified disks from rod outer segments of bovine retina

Parallel lines of evidence have suggested that light initiates changes in both cGMP metabolism and calcium levels in rod outer segments (ROS). We report that cGMP stimulates release of a pool of Ca2+ actively accumulated within purified ROS disks. Disks were purified and actively loaded with 45Ca2+ by an associated ATP-dependent calcium uptake activity as previously described [Puckett, K.L., Aronson, E.T., & Goldin, S.M. (1985) Biochemistry 24, 390-400]. Spikes of 45Ca2+ released from disks were observed in a rapid superfusion system. The Ca2+ release was specifically stimulated by physiological levels of cGMP (Kapp approximately 20 microM; Hill coefficient = 1.7). 8-Bromo-cGMP could also activate the release mechanism, but cAMP was ineffective. At cGMP levels of greater than or equal to 100 microM, approximately 20% of the loaded Ca2+ was released. The Ca2+ release rate at saturating cGMP levels reached a maximum within the 10-s time resolution of the assay system. In contrast to other recent reports of cGMP activation of ROS ion conductances, the majority of the release activity terminated in a spontaneous manner, suggestive of an intrinsic inactivation process. The amount of Ca2+ released and the release kinetics were similar to the presence or absence of an unbleached pool of rhodopsin. Cyclic nucleotides did not stimulate release from disks passively equilibrated with 45Ca2+, i.e., in the absence of ATP but otherwise under identical conditions. Preincubation of the disks with cGMP also reduced the level of ATP-dependent Ca2+ uptake (approximately 30%); this apparent inhibition may be due to activation of the release mechanism, rather than direct modulation of the uptake activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium transport systems in the LLC-PK1 renal epithelial established cell line

ATP-dependent calcium uptake was measured in membrane vesicles prepared from the renal epithelial LLC-PK1 established cell line. The relative contribution of the nonmitochondrial versus the mitochondrial calcium uptake is larger in LLC-PK1 cell homogenates than in homogenates from renal cortex. Two types of calcium pump, characterized by the formation of calcium-dependent phosphointermediates of 135 kDa and 115 kDa, were found in membrane fractions from LLC-PK1 cells. The 135 kDa calcium pump was also detected by 125I-labelled calmodulin overlay. Although the subcellular localization in LLC-PK1 cell membranes could not be unambiguously determined, it is conceivable that the 135 kDa and the 115 kDa molecules represent the plasma membrane calcium pump and the endoplasmic reticulum calcium pump respectively, in agreement with what was found for renal cortex preparations. Extravesicular sodium partially inhibits ATP-driven calcium uptake in a plasma-membrane-enriched fraction of the LLC-PK1 cells. The effect is potentiated by a vesicle inside-negative membrane potential. Although the effect is less pronounced than in renal cortex basal-lateral membranes, this observation suggests that an Na+-Ca2+ exchange mechanism is also present in LLC-PK1 cells. ATP-dependent calcium uptake in nonmitochondrial intracellular stores was investigated, using saponin-permeabilized cells. Permeabilized LLC-PK1 cells lowered the free calcium concentration in the medium to less than 0.4 microM. More than 60% of the accumulated calcium can be released by addition of inositol 1,4,5-trisphosphate. Our data indicate that the LLC-PK1 cell line can be successfully used as model system for the study of renal calcium handling.     

Magnesium Transport in Bacillus subtilis W23 During Growth and Sporulation

The active transport of magnesium by cells of Bacillus subtilis strain W23 occurs by a highly specific transport system (Mg(2+) is favored over Mn(2+), Co(2+), or Ca(2+)) that is energy dependent (i.e., glucose is required in minimal medium and the system is inhibited by cyanide and m-chlorophenyl carbonylcyanidehydrazone). The rate of magnesium uptake by log-phase B. subtilis cells follows saturation kinetics with a K(m) of 2.5 x 10(-4) M and a V(max) of 4.4 mumol per min per g (dry weight) at 30 C. Manganese is a competitive inhibitor showing a K(i) of 5 x 10(-4) M. During sporulation the rate of magnesium transport declines. This decline in rate is specific for the magnesium system as the manganese and calcium transport rates increase. The residual magnesium transport function in sporulating cells shows both an altered K(m) and an altered V(max). The magnesium content of late sporulating cells is also lower than that for log-phase cells.