Ca2+ transport mediated by a synthetic neutral Ca2+ -ionophore in biological membranes.

The effect of a synthetic neutral ligand on the Ca2+ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca2+ -ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca2+ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) are present, presumably to facilitate the diffusion of the charged Ca2+ -ionophore complex across the hydrophobic core of the membrane. In mitochondria, the neutral ionophore promotes the active transport of Ca2+ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.     

Quantitation of Ca2+ uptake by Arsenazo III-loaded liposomes

Arsenazo III-loaded liposomes are in wide use as model systems in the study of Ca2+ transport. The most sophisticated method for quantitation of Ca2+ uptake [E. B. Smaal et al. (1985) Biochim. Biophys. Acta 816, 418] utilizes the absorbance changes (650-700 nm) elicited by sequential additions of EDTA and A23187 to distinguish Ca2+-Arsenazo complexes which are outside and inside the liposomes. In this paper, the analytical approach of Smaal and co-workers is reevaluated and a straightforward treatment that allows calculation both of the concentration of Ca2+ inside liposomes and of total Ca2+ uptake (in moles/mole phospholipid) is developed.     

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.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

Induction of mitochondrial Ca2+ uptake by mersalyl

1. The addition of mersalyl to aged mitochondria from rat kidneys, is followed by induction of an ATP-driven Ca2+ uptake which is sensitive to Ruthenium Red. 2. This Ca2+ influx requires Mg2+, albumin, and is accomplished by membrane energization. 3. The activation of Ca2+ uptake by the mercurial in the presence of ATP can be explained if it is assumed that the inorganic phosphate generated by ATPase activity, and trapped in the matrix by the thiol reagent, provides the negative potential which results in an electrophoresis cation influx.     

Transport of Ca2+ by diltiazem across the lipid bilayer in model liposomes.

The calcium channel antagonist diltiazem was examined for its ability to translocate Ca2+ from an aqueous medium to the nonpolar lipid milieu. We monitored the spectral changes caused by the drug-mediated cation transport at 37 degrees C in unilamellar vesicles made of dimyristoyl phosphatidylcholine (DMPC) and containing the calcium-sensitive dye arsenazo III trapped inside. Vesicle leakage or membrane fusion caused by diltiazem was assessed by the use of vesicles containing fluorescent indicators. These effects were, however, found to be insignificant compared with ion transport. The transport was negligible at temperatures below the liquid crystalline to gel transition temperature of DMPC indicating a carrier mechanism of ion transport. A quantitative analysis of the transport kinetics indicated that a 1:2 Ca(2+)-drug complex is formed inside the lipid. The calcium ionophoretic ability of diltiazem, combined with other related data, suggests a possible role for Ca2+ in the conformation of the drug in the lipid membrane milieu and in its interaction with the calcium channel.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: II. Mechanism of impairment in ATP-dependent Ca2+ transport

The role of the phosphorylation and dephosphorylation of sarcolemma and that of the alteration of membrane lipids in the endotoxin-induced impairment of the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results indicate that the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma was decreased by 30–35% 4h after endotoxin administration. Phosphorylation of sarcolemma by the catalytic subunit of the cAMP-dependent protein kinase or calmodulin stimulated ATP-dependent Ca²⁺ transport in both groups, however, the phosphorylation-stimulated activities remained significantly lower in endotoxic animals. Dephosphorylation of sarcolemma decreased ATP-dependent Ca²⁺ transport in both groups, yet, the time required to reach maximal dephosphorylation was reduced from 120 to 90 min 4 h post-endotoxin. Analysis of sarcolemmal membranes reveals that phosphatidylcholine and phosphatidylethanolamine contents were decreased while their respective lysophosphatide levels were increased significantly after endotoxin injection. Digestion of control heart sarcolemma with phospholipase A₂ inhibited Ca²⁺ transport and the inhibition was reversible by phosphatidylcholine. The inhibition caused by the in vivo administration of endotoxin was completely reversible by the addition of phosphatidylcholine. Based on these data, it is concluded that endotoxin administration impairs ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment may be due to i) a defective phosphorylation of sarcolemma; ii) a reduced number of Ca²⁺ pumps; iii) an accelerated dephosphorylation of sarcolemma; and iv) an alteration in membrane phospholipid profile in response to phospholipase A activation.     

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.     

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.     

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.     

Regulation by Ca2+-calmodulin-dependent phosphorylation of passive transport of Ca2+ in the myometrial sarcolemma

Closed vesiculate preparations of pig myometrium sarcolemma (predominantly with inside-out orientation) are characterized by passive permeability for Ca2+. The kinetics of Ca2+ release from the vesicles is exponential. Using the grapho-analytical subtraction method, the kinetic parameters of this reaction were determined. Myometrium sarcolemma contains endogenous Ca2+-calmodulin-dependent protein kinase and phosphoprotein phosphatase which is inhibited by sodium o-vanadate. The Ca2+-calmodulin-dependent phosphorylation stimulates passive Ca2+ release from sarcolemmal vesicles. In the course of phosphorylation the capacity of the pool providing for rapid Ca2+ release increases by 61%, the initial rate of Ca2+ release showing a 28% increase. Trifluoroperazine, an inhibitor of Ca2+-calmodulin-dependent processes, eliminates the activating effect of phosphorylation on the rate of Ca2+ release from sarcolemmal vesicles.     

Effect of cations on the temperature sensitivity of Ca2+ transport in rat-liver mitochondria and safranine uptake by liposomes

The activation energy of mitochondrial Ca²⁺ transport has been studied in various conditions by Arrhenius plots in the temperature range 6–20°C. In the presence of Mg²⁺ the activation energy is decreased to 18 kJ/mole from that of 40 kJ/mole found in a sucrose medium. In the presence of the polyamine spermine the activation energy is practically 0 kJ/mole. A lanthanide Eu³⁺, which is a potent inhibitor of Ca²⁺ transport, has no significant effect on the activation energy. In a KCl medium the activation energy is increased to 70 kJ/mole. When both K⁺ and Mg⁺ are present the activation energy is nonlinear between 11 and 18°C. In the presence of K⁺ and spermine it is about 0 kJ/mole between 6 and 13°C and at higher temperatures 68 kJ/mole. Neither Mg²⁺ nor spermine affect the slope of the Arrhenius plot for state 4 respiration. Spermine decreases slightly the activation energy of Ca²⁺-stimulated respiration. Spermine also decreases the activation energy of valinomycin- or gramicidin-induced safranine uptake by liposomes from 68 to almost 0 kJ/mole between 17 and 30°C. The results indicate that Ca²⁺ binding to the polar head groups of the phospholipids at the membrane surface is the rate-limiting step of mitochondrial Ca²⁺ transport, because agents that inhibit Ca²⁺ binding to these sites (Mg²⁺, spermine, K⁺) have the most marked effect, whereas Eu³⁺, which, because of the small concentration used, ought to interact mainly with the mitochondrial Ca²⁺ transport system, has no significant effect on the temperature sensitivity of mitochondrial Ca²⁺ transport.     

Ca2+-controlled conformational states of the Ca2+ transport enzyme of sarcoplasmic reticulum.

The fluorescent reagent, S-mercuric N-dansyl-cysteine, reacts specifically with thiols of the purified Ca2+-ATPase of the sarcoplasmic reticulum, producing an increase of fluorescence of fluorescence intensity at 500 nm (lambda ex = 335 nm). The reaction is stoichiometric, and the increase of the fluorescence intensity is proportional to the number of blocked thiols. Twelve reactive thiols per 10(5) daltons of ATPase peptide fall into roughly three classes. Blocking of the most reactive thiol entails little inhibition of enzyme activity. Blocking of the five thiols reacting next (intermediate class) results in almost complete inhibition of both phosphorylated intermediate formation and ATP hydrolysis. The second order rate constants of the reaction of thiols have been determined by stopped flow studies. The most reactive thiol and the six least reactive thiols can each be treated as a single class with respect to the rate constant; five thiols of intermediate reactivity appear to have different rate constants (k2, k3, ..k6). Of these constants, k1, corresponding to the most reactive thiol, does not change with [Ca2+]. Upon increasing [Ca2+] from 10(-9) to 10(-5) M, k2 increase and k7-12 decreases; the changes roughly parallel the activation of ATPase activity and the Ca2+ binding to the high affinity alpha sites (Ikemoto, N. (1975) J. Biol. Chem. 250, 7219-7224). Upon further increase of [Ca2+] k2 decreases and k7-12 increase, in parallel with the inhibition of ATPase activity and with the Ca2+ binding to the low affinity gamma sites.     

Ca2+-dependent K+ transport in lymphocytes

The treatment of rat thymocytes with A23187 + Ca2+, ascorbate-phenazine methosulphate or propranolol induced quinine-sensitive fluxes of K+ (Rb+) suggesting the presence in the cell membrane of Ca2+-dependent K+ channels. Concanavalin A induced K+ channel activation only at very high doses (13 micrograms/ml). Neither quinine nor the increase of the K+ concentration in the medium to 30 mM prevented the stimulation of amino acid transport induced by concanavalin A, suggesting that the Ca2+-dependent K+ channel is not involved in the early phenomena of lymphocyte activation.     

Plasma membrane Ca2+ transport: stimulation by soluble proteins.

Inside-out membrane vesicles were prepared from human red blood cells. In the presence of ATP, these vesicles took up ⁴⁵Ca²⁺ against a chemical gradient. The active transport of Ca²⁺ was increased by addition of an activator protein of (Ca²⁺+Mg²⁺)-ATPase isolated from the membrane-free hemolysate of human red blood cells. A closely related protein, the protein modulator of cyclic AMP phosphodiesterase from bovine brain, also increased the rate of active transport of ⁴⁵Ca²⁺. Addition of the calcium ionophore A23187 caused a rapid efflux of ⁴⁵Ca²⁺ from loaded, inside-out vesicles. When La³⁺ was added to the system in the presence of activator protein, the uptake of ⁴⁵Ca²⁺ was inhibited. Results are compatible with the interpretation that activity of the plasma membrane Ca²⁺ pump may be modulated by certain cytoplasmic proteins.     

Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands.

Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein.