Annexin-mediated Ca2+ influx regulates growth plate chondrocyte maturation and apoptosis

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes.     

Characteristics of Ca2+ transport by corn mitochondria

Mitochondria isolated from corn (Zea mays L.) coleoptiles by an improved procedure which yields functionally intact preparations are much more active in respiration-coupled Ca²⁺ accumulation than those employed in most earlier studies. Ca²⁺ uptake by these mitochondria is phosphate-dependent and is accompanied by decrease in Δψ, H⁺ extrusion and increase in the rate of respiration. A sigmoidal plot with a Hill coefficient of 2.22 was obtained when the rates of Ca²⁺ uptake were plotted as a function of free Ca²⁺ concentration. The K_0.5 for Ca²⁺ influx was about 31 μM and a V_max of 140 nmol Ca²⁺ per min per mg was attained at a free-Ca²⁺ concentration of about 120 μM. Ca²⁺ uptake is sensitive to inhibition by ruthenium red and Mg²⁺. The external free-Ca²⁺ concentration maintained at steady state was about 2 μM and was independent of the respiratory substrate and of external Na⁺, but was increased by exogenous Mg²⁺. In addition, this preparation of corn mitochondria has shown a much higher ability for Ca²⁺ retention in the presence of phosphate and NAD(P)H oxidants than liver mitochondria.     

Ca2+ transport by coupled Trypanosoma cruzi mitochondria in situ

The use of digitonin to permeabilize Trypanosoma cruzi plasma membrane enabled us to study Ca2+ transport and oxidative phosphorylation in mitochondria in situ. Addition of Ca2+ to these preparations evoked a cycle of respiratory stimulation. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by antimycin A, and stimulated by inorganic phosphate. Addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone to digitonin-permeabilized T. cruzi epimastigotes under steady-state conditions was followed by Ca2+ release. Antimycin A- and carbonyl cyanide p-trifluoromethoxyphenylhydrazonein-sensitive Ca2+ uptake was also detected in digitonin-permeabilized epimastigotes. Accordingly, ATP stimulated Ca2+ uptake by preparations de-energized by oligomycin and antimycin A. In conclusion, in contrast to previous reports indicating that a Ca2+ transport system occurs only in mitochondria from vertebrate tissues, T. cruzi epimastigotes also possess a similar system. In addition, these protozoan mitochondria have an extremely high resistance to the deleterious effects of massive Ca2+ loads in comparison with most types of mammalian mitochondria.     

PIM-2 is an independent regulator of chondrocyte survival and autophagy in the epiphyseal growth plate

The overall goal of the investigation was to examine the activity and role of the PIM serine/threonine protein kinases in the growth plate. We showed for the first time that PIM-2 was highly expressed in epiphyseal chondrocytes and that the kinase was required for critical activities linked to cell survival. These activities were independent of those mediated by Akt-1. It was noted that PIM-2 protected chondrocytes from rapamycin sensitized (TOR inhibited) cell death. Since inhibition of mTOR caused autophagy, we examined the autophagic response of PIM-2 silenced cells. We showed that PIM-2 promoted expression and organization of autophagic proteins LC3, and Beclin-1 and enhanced lysosomal acidification. At the same time, PIM-2 modulated the activity of a key regulator of apoptosis, BAD. Since BAD inhibition and Beclin-1 expression activated autophagy, it is likely that induction of the autophagic pathway would serve to inhibit apoptosis and preserve the life of the terminally differentiated chondrocyte. We conclude that PIM-2 regulates a new intermediate stage in the differentiation pathway, the induction of autophagy.     

Characteristics of Ca2+ transport by Trypanosoma cruzi mitochondria in situ

The use of digitonin to permeabilize Trypanosoma cruzi plasma membrane has allowed the study of Ca2+ transport and oxidative phosphorylation in mitochondria in situ (R. Docampo and A. E. Vercesi (1989) J. Biol. Chem. 264, 108-111). The present results show that these mitochondria are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Addition of Ca2+ or ethylene glycol bis (beta-aminoethyl ether) N-N'-tetraacetic acid to these preparations under steady-state conditions was followed by Ca2+ uptake or release, respectively, tending to restore the original Ca2+ "set point" at about 0.9 microM. In addition, large amounts of Ca2+ were retained by T. cruzi mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butyl hydroperoxide, diamide, and the 1,2-naphthoquinone beta-lapachone. However, when ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine in the presence of antimycin A was used as subtrate, beta-lapachone caused pyridine nucleotide oxidation, and Ca2+ accumulation by these mitochondria was considerably lower than in control preparations, this effect being dose-dependent.     

Energy dispersive X-ray elemental analysis of isolated epiphyseal growth plate chondrocyte fragments

Summary Isolated cells of matrix fragments of freeze fractured and freeze dried growth plate from the four species was analyzed by EDX. Cells were removed from the tissue by stereoscopic microdissection using an SEM and mounted on thin film supports on TEM grids: this approach eliminated specimen X-ray background and reduced instrumental and support back-ground levels to insignificant proportions. Chondrocyte and matrix fragments were dissected and analyzed. Cell Ca reaches EDX detectable levels in hypertrophic cells close to the mineralization front. At all stages of maturation, the cells exhibit high P; however, matrix Ca levels are elevated before P. This data suggests that the early cartilage matrix is accumulating Ca and that the cells' role in this process may be to elevate the matrix Ca and P concentration. All cells showed clear S peaks although these are reduced in late hypertrophic cells. With mineralization, matrix S levels fall, indicating a loss of sulfated proteoglycans. Matrix before mineralization contains more K than would be expected from data of previous studies. It is suggested that while this K may be bound to fixed anionic sites in the matrix, reported values for cartilage lymph and extracellular fluid should be reviewed.     

Modulation of Ca2+ efflux and rebounding Ca2+ transport in rat liver mitochondria

The independent pathway for Ca2+ efflux of rat liver mitochondria exhibits a sharp temperature and pH dependence. The Arrhenius plot displays a break at 18 degrees C, activation energy being about 117 kJ/mol below 18 degrees C and 59 kJ/mol above 18 degrees C. The pH profile is bell-shaped, with a broad optimum at pH 7.0. These properties of the efflux pathway, together with the membrane potential modulation recently described (Bernardi, P. and Azzone, G.F. (1983) Eur. J. Biochem. 134, 377-383), suggest an explanation for the phenomenon of rebounding Ca2+ transport. Addition of a Ca2+ pulse to respiring mitochondria causes (i) a phase of rapid Ca2+ uptake, leading to a decrease of extramitochondrial free Ca2+ to a lower level with respect to that maintained before Ca2+ addition, and (ii) a slower phase of net Ca2+ efflux, leading to restoration of the steady-state extramitochondrial free Ca2+ preceeding Ca2+ addition. Evidence is provided that the excess Ca2+ uptake is linked to transient inactivation of the efflux pathway due to membrane depolarization. Conversely, the efflux phase is linked to reactivation of the efflux pathway upon repolarization. The efflux component of the rebound cycle and the isolated efflux pathway exhibit similar dependence on temperature, pH and membrane potential.     

Effect of ebselen on Ca2+ transport in mitochondria

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclosporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.     

Some characteristics of Ca2+ transport in plant mitochondria

Coupled mitochondria isolated from the white leaves of cabbage (Brassica Oleracea, var. capitata) were inactive in respiration-coupled Ca2+ accumulation, in contrast to mitochondria isolated from etiolated corn (Zea mays) which showed the ability to take up Ca2+ from the medium, although with a much lower activity than liver mitochondria. The addition of corn mitochondria to aerobic medium containing succinate as respiratory substrate and a free Ca2+ concentration of 40 microM resulted in Ca2+ uptake with a decrease in free Ca2+ concentration until a steady state of about 2.0 microM was reached and maintained constant for several minutes. Perturbation of this steady state by the addition of Ca2+ or EGTA was followed by Ca2+ uptake or release, respectively, until the steady state was attained at the original extramitochondrial free Ca2+ concentration. These results indicate that corn but not cabbage mitochondria, as with some animal mitochondria, have the ability to buffer external Ca2+ and may be involved in the maintenance of Ca2+ homeostasis in the cell.     

Role of mitochondria in calcification, Mitochondrial activity distribution in the epiphyseal plate and accumulation of calcium and phosphate ions by chondrocyte mitochondria

Histologically homogeneous sections corresponding to resting, columnar and hypertrophic zones of the epiphyseal plate of calf scapula were homogenized and assayed for mitochondrial, lysosomal and others important to calcification activities. These activities were found to be significantly higher in the columnar and hypertrophic zones as compared with those in the resting zone. Mitochondria obtained from the hypertrophic zone of the epiphyseal plate of calf scapula or calf costal chondral junction were resolved into a “light” and a “heavy” population by isopycnic centrifugation presumably due to difference in the content of granule forming calcium phosphate. Finally mitochondria from resting cartilage can give rise only to a “light” mitochondrial population, unless they are allowed to accumulate calcium and phosphate ions from the medium during respiration where a “light” and a “heavy” population results upon centrifugation.     

Passive transport of Ca2+ in a myometrium mitochondria fraction

Na+, pH, prostaglandin F2 alpha are studied for their effect on Ca2+ transport into fractions of cow's myometrium mitochondria. Na+ does not affect a passive release of Ca2+ from mitochondria and its energy-dependent accumulation. A decrease of the incubation medium pH from 7.5 to 6.5 stimulates Ca2+ release from mitochondria and inhibits its energy-dependent pumping into them. Prostaglandin F2 alpha (10(-8)--2 X 10(-4) M) does not affect the activity of Ca2+ accumulation and release systems. A conclusion is made that the Na+-Ca2+-exchange system is absent in mitochondria of smooth muscle cells and Ca2+ release proceeds as a result of H+-Ca2+-antiport system functioning.     

Ca2+ transport in mitochondria from yeast expressing recombinant aequorin

We have expressed aequorin in mitochondria of the yeast Saccharomyces cerevisiae and characterized the resulting strain with respect to mitochondrial Ca(2+) transport in vivo and in vitro. When intact cells are suspended in water containing 1.4 mM ethanol and 14 mM CaCl(2), the matrix free Ca(2+) concentration is 200 nM, similar to the values expected in cytoplasm. Addition of ionophore ETH 129 allows an active accumulation of Ca(2+) and promptly increases the value to 1.2 microM. Elevated Ca(2+) concentrations are maintained for periods of 6 min or longer under these conditions. Isolated yeast mitochondria oxidizing ethanol also accumulate Ca(2+) when ETH 129 is present, but the cation is not retained depending on the medium conditions. This finding confirms the presence of a Ca(2+) release mechanism that requires free fatty acids as previously described [P.C. Bradshaw et al. (2001) J. Biol. Chem. 276, 40502-40509]. When a respiratory substrate is not present, Ca(2+) enters and leaves yeast mitochondria slowly, at a specific activity near 0.2 nmol/min/mg protein. Transport under these conditions equilibrates the internal and external concentrations of Ca(2+) and is not affected by ruthenium red, uncouplers, or ionophores that perturb transmembrane gradients of charge and pH. This activity displays sigmoid kinetics and a K(1/2) value for Ca(2+) that is near to 900 nM, in the absence of ethanol or when it is present. It is furthermore shown that the activity coefficient of Ca(2+) in yeast mitochondria is a function of the matrix Ca(2+) content and is substantially larger than that in mammalian mitochondria. Characteristics of the aequorin-expressing strain appear suitable for its use in expression-based methods directed at cloning Ca(2+) transporters from mammalian mitochondria and for further examining the interrelationships between mitochondrial and cytoplasmic Ca(2+) in yeast.     

Ca 2+ transport activity in mitochondria from some plant tissues

Mitochondria isolated from some 14 different higher plants and fungi were examined for their capacity to carry out respiration-dependent accumulation of Ca²⁺. Additions of Ca²⁺ give little or no stimulation of state 4 respiration of plant mitochondria, although the added Ca²⁺ was largely accumulated. Accumulation of Ca²⁺ required phosphate and, in most cases, was stimulated by Mg²⁺ and ADP or ATP. Ca²⁺ uptake was abolished by respiratory inhibitors and uncoupling agents. The ratio of Ca²⁺ ions taken up per pair of electrons per energy-conserving site was normal at about 2.0 for mitochondria from sweet potato and white potato; mitochondria from other plants showed somewhat lower ratios. Accumulated Ca²⁺ was only very slowly released from previously loaded plant mitochondria. Respiration-inhibited sweet potato mitochondria show both high-affinity and low-affinity Ca²⁺ binding sites sensitive to uncouplers, La³⁺, and ruthenium red and thus resemble animal mitochondria. Most other plant mitochondria lack high affinity sites. In general, mitochondria from sweet potato and white potato tubers resemble those from animal tissues, but mitochondria from carrots, beets, turnips, onions, cabbage, artichokes, cauliflower, avocados, mung bean and corn seedlings, and mushrooms show rather low affinity and activity in accumulation of Ca²⁺, probably due to lack of a specific Ca²⁺ carrier.     

Anti-microtubular herbicides and fungicides affect Ca2+ transport in plant mitochondria

The herbicides amiprophosmethyl (APM) trifluralin, and oryzalin as well as the fungicides methylbenzimidazolyl carbamate (MBC), O-isopropyl N-phenyl carbamate (IPC), and chlorisopropyl N-phenyl carbamate (CIPC), which are known to cause the destruction of microtubules in vivo but do not interfere with tubulin polymerization in vitro, have been examined with respect to their ability to affect Ca²⁺ transport in isolated cell organelles. In contrast to colchicine which has no effect on Ca²⁺ transport in isolated mitochondrial and microsomal fractions, all of the substances investigated caused considerable reduction of ca²⁺ net uptake into mitochondrial but not into microsomal fractions. This reduction has been shown to be due to an increase in passive Ca²⁺ efflux. These results have been extrapolated to in vivo situations where they are postulated to act by raising cytoplasmic Ca²⁺ levels.Abbreviations APM amiprophosmethyl - CIPC chlorisopropyl N-phenyl carbamate - IPC O-isopropyl N-phenyl carbamate - MBC methylbenzimidazolyl carbamate - Mops 3-(N-Morpholino) propanesulfonic acid - DMSO dimethylsulfoxide     

Factors affecting Ca2+ transport in mouse islet and kidney mitochondria

The transport of Ca2+ in islet and kidney mitochondria respiring on succinate was inhibited by atractylate and fluorocitrate, and stimulated by pyruvate, isocitrate, alpha-ketoglutarate, dibutyryl cAMP, oligomycin and bongkrekate, and by in vivo administration of glucagon, glyceraldehyde or glucose. The kidney [beta-hydroxybutyrate]/[acetoacetate] ratio was increased in glyceraldehyde treated mice. The data suggest a relationship, which might be influenced by cAMP, between activity of pyruvate, isocitrate and alpha-ketoglutarate dehydrogenases and transport of Ca2+ in islet and kidney mitochondria. A contributory role of reductive carboxylation for Ca2+ uptake, and a role of citrate for Ca2+ retention are discussed.     

Effect of Mg2+ and spermine on the kinetics of Ca2+ transport in rat-liver mitochondria

Plots relating the initial rate of mitochondrial Ca²⁺ transport to the Ca²⁺ concentration (kinetic plots) have a hyperbolic shape in a Ca²⁺ concentration range of 2.5–100 µM as measured in sucrose or KCl media. In the presence of Mg²⁺ or a polyamine spermine, which both are competitive inhibitors of Ca²⁺ binding to low affinity sites at the membrane surface, the shape of the plots becomes sigmoidal. At higher concentrations of these agents linear kinetic plots are obtained as measured in a sucrose medium. In a KCl medium the sigmoidality of the kinetic plots is enhanced by an increase in the Mg²⁺ or spermine concentration. It is suggested that Mg²⁺ and spermine affect the kinetics of Ca²⁺ transport by interfering with Ca²⁺ binding to low affinity sites of the membrane surface and that the binding of Ca²⁺ to these sites is the first step of the mitochondrial Ca²⁺ transport.     

Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate

The goal of this review is to examine the fate of the hypertrophic chondrocyte in the epiphyseal growth plate and consider the impact of the cartilage microenvironment on cell survival and apoptosis. Early investigations pointed to a direct role of the hypertrophic chondrocyte in osteogenesis. The terminally differentiated cells were considered to undergo a dramatic change in shape, size, and phenotype, and assume the characteristics of an osteoblast. While some studies have supported the notion of transdifferentiation, much of the evidence in favor of reprogramming epiphyseal chondrocytes is circumstantial and based on microscopic evaluation of cells that are present at the chondro-osseous junction. Although these investigations provided a novel perspective on endochondral bone formation, they were flawed by the failure to consider the importance of stem cells in osseous tissue formation. Subsequent studies indicated that many, if not all, of the cells of the cartilage plate die through the induction of apoptosis. With respect to agents that mediate apoptosis, at the chondro-osseous junction, solubilization of mineral and hydrolysis of organic matrix constituents by septoclasts generates high local concentrations of ions, peptides, and glycans, and secreted matrix metalloproteins. Individually, and in combination, a number of these agents serve as potent chondrocyte apoptogens. We present a new concept: hypertrophic cells die through the induction of autophagy. In the cartilage microenvironment, combinations of local factors cause chondrocytes to express an initial survival phenotype and oxidize their own structural macromolecules to generate ATP. While delaying death, autophagy leads to a state in which cells are further sensitized to changes in the local microenvironment. One such change is similar to ischemia reperfusion injury, a condition that leads to tissue damage and cell death. In the growth cartilage, an immediate effect of this type of injury is sensitization to local apoptogens. These two concepts (type II programmed cell death and ischemia reperfusion injury) emphasize the importance of the local microenvironment, in particular pO(2), in directing chondrocyte survival and apoptosis.     

Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump

Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca²⁺ transport function of the Ca²⁺-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca²⁺ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca²⁺/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca²⁺-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca²⁺ translocation function of the Ca²⁺-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.     

Regulation of Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase at limiting [Ca2+

The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 microM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways.