Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

The measurement of Ca2+ inflow across the liver cell plasma membrane by using quin2 and studies of the roles of Na+ and extracellular Ca2+ in the mechanism of Ca2+ inflow.

1. Rates of Ca2+ inflow across the hepatocyte plasma membrane in the presence of vasopressin were estimated by using quin2. 2. Plots of the rate of Ca2+ inflow as a function of the intracellular quin2 concentration reached a plateau at about 1.7 mM intracellular quin2. Ca2+ inflow was inhibited by 60% in the presence of 400 microM-verapamil. 3. A plot of the rate of Ca2+ inflow as a function of the concentration of extracellular Ca2+ ([Ca2+]o) was biphasic. The second (slower) phase showed no sign of saturation at values of [Ca2+]o up to 5 mM. It is concluded that, in the presence of vasopressin, Ca2+ flows into the liver cell by two different processes, one of which is not readily saturated by Ca2+o. 4. The effect of the replacement of extracellular NaCl by choline or tetramethylammonium chloride on cellular Ca2+ movement was found to depend on the presence or absence of intracellular quin2. 5. In cells loaded with quin2 and incubated in the presence of choline or tetramethylammonium chloride, a small decrease in the basal intracellular free Ca2+ concentration ([Ca2+]i) was observed, and the increase in [Ca2+]i caused by the addition of vasopressin was considerably diminished when compared with cells incubated in the presence of NaCl. In cells loaded with quin2, replacement of NaCl by choline chloride caused a decrease in Ca2+ inflow in the presence of vasopressin, as measured by using quin2 or 45Ca2+ exchange, whereas no change in Ca2+ inflow was observed in the absence of vasopressin. 6. In cells not loaded with quin2, replacement of NaCl by choline chloride did not alter Ca2+ inflow either in the presence or in the absence of vasopressin. 7. It is concluded that (i) Ca2+ inflow through the basal and receptor-activated Ca2+ inflow systems does not involve the inward movement of Ca2+ in exchange for Na+ or the induction of Ca2+ inflow by intracellular Na+, and (ii) the presence of both intracellular quin2 and extracellular choline or tetramethylammonium chloride (in place of NaCl) inhibits Ca2+ inflow through the receptor-activated Ca2+ inflow system but not through the basal Ca2+ inflow system, and inhibits the release of Ca2+ from intracellular stores.     

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.     

Ca2+-activated ATPase of rat liver plasma membrane.

Rat liver plasma membranes hydrolyze ATP in the presence of Ca2+. The rate of hydrolysis is different when Mg2+ions are present in the incubation system. Several parameters differentiate Ca2+-ATPase from Mg2+-ATPase: a) the Km of ATP hydrolysis for Ca2+ (2.25 x 10(-4) M) is lower than for Mg2+ (2.14 x 10(-3) M); b) the shape of the activation curve is hyperbolic in the presence of Ca2+ and sigmoid in the presence of Mg2+; c) Mg2+-ATPase shows two different values of activation energy while Ca2+-ATPase presents only a single value; d) Ca2+-ATPase is inhibited, while Mg2+-ATPase is unaffected by cyclic AMP. Ca2+-ATPase is localized on the plasma membrane and is not inhibited by cysteine. It does not hydrolyze substrates different from nucleotides triphosphate, such as glucose-1-phosphate or alpha-glycero-phosphate. The enzyme is probably related to a mechanism of calcium transport.     

The liver plasma membrane Ca2+ pump: hormonal sensitivity

The liver plasma membrane Ca2+ pump is supposed to extrude cytosolic calcium out of the cell. This system has now been well defined on the basis of its plasma membrane origin, its high affinity Ca2+ -stimulated ATPase activity, its Ca2+ transport activity, its phosphorylated intermediate. The liver calcium pump appears to be a target of hormonal action since it has been shown that glucagon and calcium mobilizing hormones namely alpha 1-adrenergic agonists, vasopressin, angiotensin II inhibit this system. The present review details the mechanism of calcium pump inhibition by glucagon and points out its difference from the inhibition process induced by calcium mobilizing hormones. We conclude that the inhibitory action of the Ca2+ mobilizing hormones and glucagon on the liver plasma membrane Ca2+ pump might play a key role in the actions of these hormones by prolonging the elevation in cytosolic free Ca2+.     

The role of Ca2+ transport across the plasma membrane for cell migration.

Cell migration plays a central role in many physiological and pathophysiological processes. On a cellular level it is based on a highly coordinated restructuring of the cytoskeleton, a continuous cycle of adhesion and de-adhesion as well as on the activity of ion channels and transporters. The cytoplasmic Ca2+ ([Ca2+]i) concentration is an important coordinator of these intracellular processes. Thus, [Ca2+]i must be tightly controlled in migrating cells. This is among other things achieved by the activity of Ca2+ permeable channels, the plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX) in the plasma membrane. Here, we wanted to determine the functional role of these transport proteins in cell migration. We therefore quantified the acute effect of inhibitors of these transport proteins (Gd3+, vanadate, KB-R7943) on migration, [Ca2+]i, and intracellular pH (pHi) of MDCK-F cells. Migration was monitored with computer-assisted time-lapse video microscopy. [Ca2+]i and pHi were measured with the fluorescent indicators fura-2 and BCECF. NCX expression in MDCK-F cells was verified with ion substitution experiments, and expression of PMCA was tested with RT-PCR. All blockers lead to a rapid impairment of cell migration. However, the most prominent effect is elicited by NCX-inhibition with KB-R7943. NCX-blockade leads to an almost complete inhibition of migration which is accompanied by a dose-dependent increase of [Ca2+]i and an intracellular alkalinisation. We show that inhibition of NCX and PMCA strongly affects lamellipodial dynamics of migrating MDCK-F cells. Taken together, our results show that PMCA and in particular NCX are of critical importance for cell migration.     

The Neurospora plasma membrane Ca2+ pump

Plasma membrane vesicles isolated from the eukaryotic microorganism Neurospora crassa by the concanavalin A method catalyze Mg2+-ATP dependent 45Ca2+ accumulation. Since the ATP-responsive vesicles are functionally inverted, the Ca2+ transport system presumably operates as a Ca2+ exit pump in the intact cell. The mechanism of the Ca2+ pump system involves two components: 1) an electrogenic, proton-translocating ATPase (EC 3.6.1.3), which utilizes the chemical energy of ATP hydrolysis to generate a transmembrane electrical potential and pH gradient, and 2) a Ca2+/H+ antiporter, which utilizes the transmembrane pH gradient to energize the active transport of Ca2+. Evidence for this mechanism is presented and the possible implications of these findings for the mechanisms of Ca2+ pumps in other cells are discussed.     

Two Ca2+-dependent ATPases in rat liver plasma membrane. The previously purified (Ca2+-Mg2+)-ATPase is not a Ca2+-pump but an ecto-ATPase

We have shown that the rat liver plasma membrane has at least two (Ca2+-Mg2+)-ATPases. One of them has the properties of a plasma membrane Ca2+-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca2+-Mg2+)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca2+-ATPase and Mg2+-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca2+-ATPase and Mg2+-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca2+-ATPase and Mg2+-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca2+-ATPase and Mg2+-ATPase may be the same protein as the previously purified (Ca2+-Mg2+)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca2+-Mg2+)-ATPase. Both the ecto-ATPase and the (Ca2+-Mg2+)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca2+ and Mg2+ on the ecto-ATPase activity is not additive indicating that both Ca2+- and Mg2+-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca2+-Mg2+)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca2+-Mg2+)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca2+-Mg2+)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca2+ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

A procedure for the rapid isolation from rat liver of plasma membrane vesicles exhibiting Ca2+-transport and Ca2+-ATPase activities.

A technique is described for the isolation of a plasma membrane-enriched preparation from a rat liver post-mitochondrial fraction by using discontinuous Percoll density-gradient centrifugation. The procedure is simple, of high reproducibility and yield and requires a total isolation time of only 90 min. The preparation consists almost exclusively of membrane vesicles and is enriched approx. 26-fold in plasma membrane-localized enzymes with minor contamination (less than 10%) with membranes derived mainly from the endoplasmic reticulum and Golgi apparatus. Approx. 20% of the fraction comprises tightly-sealed vesicles in the inverted orientation which are capable of accumulating calcium ions and exhibiting vanadate-insensitive Ca2+-ATPase activity. The properties of these activities, including insensitivity to vanadate, oxalate, and to p-chloromercuribenzoate as well as a lack of requirement for added Mg2+, contrast markedly with the reported properties of Ca2+ transport by the endoplasmic reticulum isolated from rat liver. The technique may have wide application in the study of plasma membrane-associated activities in rat liver, particularly in relation to sinusoidal membrane surface-related events.     

Ca2+-transporting properties of myometrial plasma cell membrane Ca2+, Mg2+-ATPase reconstituted in liposomes

Purified myometrium cells plasma membrane Ca2+, Mg(2+)-ATPase was reconstitute in liposomes in functionally active state by the method of cholate dialysis: it showed ATP-hydrolase activity increased by 0.8 microM A23187 average 4 times and it showed Mg2+, ATP-dependent Ca(2+)-transporting activity. Reconstituted system transported Ca2+ at an initial rate of 114.4 +/- 16.3 nmol.min-1.mg-1 with the stoichiometry Ca2+: ATP = 1: (3.2-3.7). Calmodulin increased by 30% the initial rate of Ca(2+)-accumulation by the proteoliposomes with reconstituted Ca2+, Mg(2+)-ATPase; 0.1 mM orthovanadate decreased by 80% Ca(2+)-accumulation by this system. Ca2+, Mg(2+)-ATPase reconstituted in liposomes is just Ca(2+)-transporting ATPase of the plasma membrane. Obtained enzyme preparate can be utilised for study of the properties of this important energy-dependent Ca(2+)-transporting system of smooth muscle cell.     

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.     

The binding of divalent metal ions to polyelectrolytes in mixed counterion systems. II. Dextransulfate-Mg2+ and dextransulfate-Ca2+ in solutions containing added NaCl or KCl

Measurements of magnesium and calcium ion activities in solutions of the polyelectrolyte dextransulfate, with added sodium chloride or potassium chloride are presented. A two wavelength dye spectrophotometric method is used. Dextransulfate concentrations Cp (expressed as moles sulfate ion/litre) vary between 0.001 and 0.007, total ionic strengths between 0.005 and 0.08 mole/XXX. Divalent metal ion concentrations are varied between 0 and 1.2 Cp. The results for the metal ion activities are expressed in the form of parameters theta2 = C2/Cp (C(2bp) = bound divalent metal ion concentration) and K2 = theta2/(C2-C2b). For each divalent/univalent counterion pair the values obtained for theta2 and K2 as a function of C2,Cp, and ionic strength are compared to predictions of the "two variable theory" developed for these mixed counterion systems by Manning. This comparison shows that the observed decrease in theta2 with increasing ionic strength at fixed C2 and Cp is generally well predicted by the two variable theory. The extent of divalent ion binding at a given C2, Cp, and ionic strength is largest for the Ca/Na counterion combination, and lowest for the Mg/K combination.     

Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.     

Evidence that guanosine 5''-[gamma-thio]triphosphate stimulates plasma membrane Ca2+ inflow when introduced into hepatocytes.

1. Slowly hydrolysable analogues of GTP were introduced into hepatocytes by incubating the cells in the absence of Mg2+ and in the presence of ATP4-. Experiments using guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])indicated that about 50% of the GTP[S] loaded into the cells was subsequently hydrolysed. 2. In cells loaded with GTP[S] and incubated in the absence of added extracellular Ca2+ (Ca2+o), the rate of activation of glycogen phosphorylase observed after addition of 1.3 mM-Ca2+o was 250% greater than the rate observed in unloaded cells. Smaller effects (130%) were observed in cells loaded with either guanyl-5'-yl imidodiphosphate or guanosine 5-[beta-thio]diphosphate (GDP[S]). Cells loaded with adenosine 5'-[gamma-thio]triphosphate showed no increase in glycogen phosphorylase activity on addition of Ca2+o. 3. The effect of a submaximal concentration of GTP[S] on the Ca2+-induced activation of glycogen phosphorylase was additive with that of a half-maximally effective concentration of vasopressin. GTP[S] did not increase the effect of a maximally effective concentration of the hormone. 4. Cells loaded with GTP[S] exhibited an increased initial rate of 45Ca2+ exchange measured at 1.3 mM-Ca2+o. 5. GTP[S] did not affect the amount of 45Ca2+ exchanged by cells incubated at 0.1 mM-Ca2+o or the ability of vasopressin to release 45Ca2+ from these cells. 6. It is concluded that the introduction of slowly hydrolysable analogues of GTP to the liver cell cytoplasmic space stimulates the inflow of Ca2+ across the plasma membrane through a channel similar to that activated by vasopressin.     

Caloxin: a novel plasma membrane Ca2+ pump inhibitor

Plasma membrane (PM) Ca²⁺ pump is aCa²⁺-Mg²⁺-ATPase that expels Ca²⁺from cells to help them maintain low concentrations of cytosolic Ca²⁺. There are no known extracellularly acting PMCa²⁺ pump inhibitors, as digoxin and ouabain are forNa⁺ pump. In analogy with digoxin, we define caloxins asextracellular PM Ca²⁺ pump inhibitors and describe caloxin2A1. Caloxin 2A1 is a peptide obtained by screening a random peptidephage display library for binding to the second extracellular domain(residues 401-413) sequence of PM Ca²⁺ pump isoform1b. Caloxin 2A1 inhibits Ca²⁺-Mg²⁺-ATPase inhuman erythrocyte leaky ghosts, but it does not affect basalMg²⁺-ATPase or Na⁺-K⁺-ATPase in theghosts or Ca²⁺-Mg²⁺-ATPase in the skeletalmuscle sarcoplasmic reticulum. Caloxin 2A1 also inhibitsCa²⁺-dependent formation of the 140-kDa acid-stableacylphosphate, which is a partial reaction of this enzyme. Consistentwith inhibition of the PM Ca²⁺ pump in vascularendothelium, caloxin 2A1 produces an endothelium-dependent relaxationthat is reversed byN^G-nitro-L-arginine methyl ester.Thus caloxin 2A1 is a novel PM Ca²⁺ pump inhibitor selectedfor binding to an extracellular domain.

     

Ba2+ ions inhibit the release of Ca2+ ions from rat liver mitochondria

The release of Ca2+ from respiring rat liver mitochondria following the addition of either ruthenium red or an uncoupler was measured by a Ca2+-selective electrode or by 45Ca2+ technique. Ba2+ ions are asymmetric inhibitors of both Ca2+ release processes. Ba2+ ions in a concentration of 75 microM inhibited the ruthenium red and the uncoupler induced Ca2+ release by 80% and 50%, respectively. For the inhibition, it was necessary that Ba2+ ions entered the matrix space: Ba2+ ions did not cause any inhibition of Ca2+ release if addition of either ruthenium red or the uncoupler preceded that of Ba2+. The time required for the development of the inhibition of the Ca2+ release and the time course of 140Ba2+ uptake ran in parallel. Ba2+ accumulation is mediated through the Ca2+ uniporter as 140Ba2+ uptake was competitively inhibited by extramitochondrial Ca2+ and prevented by ruthenium red. Due to the inhibition of the ruthenium red insensitive Ca2+ release, Ba2+ shifted the steady-state extramitochondrial Ca2+ concentration to a lower value. Ba2+ is potentially a useful tool to study mitochondrial Ca2+ transport.