Inhibition of the calcium-sequestering activity of liver microsomes in monobromotrichloromethane poisoning: preliminary studies on possible mechanisms

The mechanisms by which the in vivo intoxication with BrCCl3 inhibits the calcium sequestration activity of liver microsomes were studied. The initial rate of Ca2+ transport is inhibited by nearly 50% in the intoxicated rats as compared to the controls; this indicates that the active transport of Ca2+ is markedly affected by the intoxication. The microsomal ATPases activities both in the presence and in the absence of Ca2+ were not decreased at all in the intoxicated animals. However, the Ca2+-dependent extra ATP hydrolysis shows a different kinetics in the BrCCl3-poisoned rats with respect to the controls. The release of Ca2+ from Ca2+ loaded liver microsomes is higher in the intoxicated animals. It seems therefore that the increased permeability of the membrane to Ca2+ contributes to some extent to the haloalkane-induced inhibition of the calcium sequestration activity of liver microsomes.     

Validation of the use of aequorin for cytoplasmic free calcium determination by chemiluminescence in Streptococcus pneumoniae

In the extracellular pathogen Streptococcus pneumoniae, transformable by soluble DNA, calcium transport is shown to play a key role for vegetative growth, developement of competence for genetic transformation and experimental virulence. To get a more precise localisation of Ca2+ in the cell, we cloned the cDNA of apoaequorine in the chromosome of Streptococcus pneumoniae. This allowed the reconstitution of the acquorine system and chemoluminescence measurements of the cytoplasmic free calcium concentration in the bacteria. Intracellular free Ca2+ is 2 microM at the steady state and can reach 14 microM when calcium is added to the bacterial suspension. Increase in free Ca2+ in response to an imposed Ca2+ gradient depends on the initial velocity (Vi) of the DMB-sensitive Ca2+ transport, showing that changes in cytoplasmic Ca2+ involve active transport.     

ATP-dependent calcium accumulation in brain microsomes. Enhancement by phosphate and oxalate.

1. ATP-dependent calcium uptake by a rabbit brain vesicular fraction (microsomes) was studied in the presence of phosphate or oxalate. These anions, which are known to form insoluble calcium salts, increased the rate of calcium uptake and the capacity of the vesicles for calcium accumulation. 2. The degree of activation depended on the concentration of phosphate or oxalate. Under optimal conditions, phosphate promoted a 5-fold increase in the amount of calcium stored at steady state. This level was 200-250 nmol Ca-2+/mg protein. 3. Initial rate of calcium uptake followed Michaelis-Menten kinetics with an apparent Km for calcium of 6.7-10-minus 5 M and a V of 44 nmol/min per mg protein. Optimal pH was 7.0. With 2 mM ATP, optimal Mg-2+ concentration was 2 mM. 4. Dintrophenol and NaN3 inhibited calcium uptake in a mitochondria-enriched fraction but not in the microsomal fraction. 5. Calcium uptake activity was compared in the six subfractions prepared from the whole microsomal fraction by means of a sucrose density gradient fractionation. 6. The Mg-2+-dependent ATPase activity of brain microsomes was activated by calcium. Maximal activation was attained with 100 muM CaCl2. Greater calcium concentrations caused a progressive inhibition. 7. The data suggest that the ATP-dependent calcium uptake in brain microsomes, as in muscle microsomes, is brought about by an active transport process, calcium being accumulated as a free ion inside the vesicles.     

Calcium transport ATPase of canine cardiac sarcoplasmic reticulum. A comparison with that of rabbit fast skeletal muscle sarcoplasmic reticulum.

To define the mechanism responsible for the slow rate of calcium transport by cardiac sarcoplasmic reticulum, the kinetic properties of the Ca2+-dependent ATPase of canine cardiac microsomes were characterized and compared with those of a comparable preparation from rabbit fast skeletal muscle. A phosphoprotein intermediate (E approximately P), which has the stability characteristics of an acyl phosphate, is formed during ATP hydrolysis by cardiac microsomes. Ca2+ is required for the E approximately P formation, and Mg2+ accelerates its decomposition. The Ca2+ concentration required for half-maximal activation of the ATPase is 4.7 +/- 0.2 muM for cardiac microsomes and 1.3 +/- 0.1 muM for skeletal microsomes at pH 6.8 and 0 degrees. The ATPase activities at saturating concentrations of ionized Ca2+ and pH 6.8, expressed as ATP hydrolysis per mg of protein, are 3 to 6 times lower for cardiac microsomes than for skeletal microsomes under a variety of conditions tested. The apparent Km value for MgATP at high concentrations in the presence of saturating concentrations of ionized Ca2+ is 0.18 +/- 0.03 ms at pH 6.8 and 25 degrees. The maximum velocity of ATPase activity under these conditions is 0.45 +/- 0.05 mumol per mg per min for cardiac microsomes and 1.60 +/- 0.05 mumol per mg per min for skeletal microsomes. The maximum steady state level of E approximately P for cardiac microsomes, 1.3 +/- 0.1 nmol per mg, is significantly less than the value of 4.9 +/- 0.2 nmol per mg for skeletal microsomes, so that the turnover number of the Ca2+-dependent ATPase of cardiac microsomes, calculated as the ratio of ATPase activity to the E approximately P level is similar to that of the skeletal ATPase. These findings indicate that the relatively slow rate of calcium transport by cardiac microsomes, whem compared to that of skeletal microsomes, reflects a lower density of calcium pumping sites and lower Ca2+ affinity for these sites, rather than a lower turnover rate.     

The stoichiometry of the exchange catalysed by the mitochondrial calcium/sodium antiporter.

Rat heart mitochondria respiring on succinate in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) released Ca2+ on the calcium/sodium antiporter until a steady state was reached. Addition of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) did not perturb this steady state. Thermodynamic analysis showed that if a Ca2+/nNa+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would cause an obvious change in extramitochondrial free [Ca2+]. Therefore the endogenous calcium/sodium antiporter of mitochondria catalyses electroneutral Ca2+/2Na+ exchange.     

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.     

The calcium mobilizing tumor promoting agent, thapsigargin elevates the platelet cytoplasmic free calcium concentration to a higher steady state level. A possible mechanism of action for the tumor promotion

The ability of the platelet agonists thapsigargin (Tg) and thrombin to elevate the cytoplasmic free calcium level ([Ca2+]i) was examined. Both agonists induced a transient increase of [Ca2+]i with a different time-course, however. Thus, the maximal [Ca2+]i was reached 15 sec and 2 min after stimulation with thrombin and Tg, respectively. The thrombin induced rise of [Ca2+]i was reversible, which indicates that active calcium sequestration and/or extrusion is operating. Tg affected [Ca2+]i in a divergent manner, thus, [Ca2+]i was stabilized on a elevated level without initial formation of a pronounced peak. The decline in [Ca2+]i observed after thrombin stimulation was not impaired by the calmodulin binding drug trifluoperazine but it was strongly reduced by vanadate, which suggests the active calcium transport systems to be insensitive to calmodulin. We put forward the hypothesis that the tumor promoting activity of Tg is attributable to its ability to stabilize [Ca2+]i on a new elevated steady state level.     

Calcium-dependent calcium occlusion in the sarcoplasmic reticulum Ca2+-ATPase. Its enhancement by phosphorylation of the enzyme

45Ca2+-40Ca2+ exchangeability of 45Ca bound to the calcium transport sites of unphosphorylated sarcoplasmic reticulum Ca2+-ATPase at equilibrium has been found to be heterogeneous: Half of the bound calcium is [Ca2+]-dependent in a slowly exchangeable (k less than 0.3 s-1), "occluded" state in the Ca2+-ATPase, and the other calcium is [Ca2+]-independent in a rapidly exchangeable (k approximately 0.3 s-1), "unoccluded" state (Nakamura, J. (1986) Biochim. Biophys. Acta 870, 495-501). In this paper, the two different forms of exchangeable calcium were studied after phosphorylation of the enzyme by ATP without added Mg2+ at pH 7.0 and 0 degree C. By the phosphorylation, the degree of the occlusion became higher (k less than 0.03 s-1). The unoccluded calcium was, however, not significantly affected. The more highly occluded calcium exchanged at the same rate as the decay rate of the phosphoenzyme (EP) in the steady state at a ratio of about 1:1. The occluded calcium was relieved by dephosphorylation of EP by ADP. These results suggest that 1 mol of ADP-sensitive EP more highly occluded 1 mol of calcium, already occluded before phosphorylation. After transformation of ADP-sensitive EP to its ADP-insensitive form by the addition of 20 mM Mg2+ at pH 8.8, the unoccluded calcium was rapidly (k = 0.1-0.3 s-1) released from the transformed EP. However, the occluded calcium was maintained in an occluded state in which the calcium was slowly (k approximately 0.01 s-1) released from the EP without exchange. The results suggest that calcium occlusion in the ADP-sensitive EP is not relieved by the loss of ADP sensitivity of the EP itself.     

Sequential mechanism of calcium binding and translocation in sarcoplasmic reticulum adenosine triphosphatase

Cooperative calcium binding (apparent Kd = 1.04 X 10(-6) M) to the ATPase of sarcoplasmic reticulum vesicles occurs with a maximal stoichiometry of 2 mols of divalent cation/mol of enzyme in the absence of ATP. The bound calcium is distributed into two pools which undergo fast or slow isotopic exchange, respectively. The two pools retain a 1:1 molar ratio under various conditions and are both located within a protein crevice, as suggested by their cooperative interaction and exchange kinetics. Following enzyme phosphorylation by ATP, both pools of bound calcium are "internalized" (cannot be displaced by quench reagents). If following 45Ca2+ binding, isotopic dilution is obtained in the medium by adding 40Ca2+ with ATP, internalization of both pools of bound 45Ca2+ (2 mol/mol of phosphoenzyme) is still observed within the first enzyme cycle. When the cycle is reversed by addition of excess ADP soon after ATP, only half of the internalized 45Ca2+ is released from the enzyme into the medium outside the vesicles, while the other half remains with the vesicles. If half of the bound 45Ca2+ is exchanged (fast exchange) with 40Ca2+ previous to the addition of ATP, none of the remaining 45Ca2+ is released outside the vesicles upon reversal of the enzyme cycle. Therefore, the pool of bound calcium which undergoes slower exchange with the outside medium, is the first to be released inside the vesicles upon enzyme phosphorylation. A sequential mechanism of calcium binding and translocation is proposed, that accounts for binding cooperativity and exchange kinetics, presteady state transients following addition of ATP, and the Ca2+ concentration dependence of ATPase activity in steady state.     

Coordinated regulation of free Ca2+ by isolated organelles from a rat insulinoma

Experiments aimed at the partial reconstitution of the intracellular transport systems regulating the cytosolic free Ca2+ homeostasis are reported. Rat insulinoma subcellular fractions enriched in mitochondria, endoplasmic reticulum (microsomes), and secretory granules were studied. The ambient free Ca2+ concentration maintained by the separate or combined organelles was determined with a Ca2+-selective minielectrode. The data demonstrate that ambient [Ca2+] is established by the microsomes, not by the mitochondria or the secretory granules, in the range of resting cytosolic Ca2+ concentrations (0.1-0.2 microM Ca2+). Furthermore, the microsomes are able to deplete largely the mitochondria of their exchangeable calcium. Nonetheless, both mitochondria and microsomes, but not secretory granules, function as a coordinated unit to restore the previous ambient [Ca2+] following its perturbation. Thus, mitochondria play a major role in bringing down rapidly ambient [Ca2+] to the submicromolar range, whereas the endoplasmic reticulum acts as a relay in the transport mechanisms which lower [Ca2+] to the resting level.     

Phosphorylation and the control of calcium fluxes

Cell activation, e.g. stimulus-contraction or stimulus-secretion coupling, is brought about by a 100-fold increase in cytosolic free Ca2+ concentration from 0.1 to 10 microM, upon release of Ca2+ from intrareticular or extracellular stores along the concentration gradient. A return to steady state is achieved by either Na+-Ca2+ exchange or ATP-dependent Ca2+ transport against the concentration gradient. Both processes, Ca2+ influx and Ca2+ efflux, are regulated by sophisticated covalent mechanisms. The positive inotropic effect of adrenalin is mediated by the cyclic-AMP-dependent phosphorylation of cardiac sarcolemmal proteins, among which calciductin is the major phosphate acceptor. Upon cyclic-AMP-dependent phosphorylation, the slow Ca2+ channel is activated 3.5 time above its basal low-conductance state, and retains its characteristics, competition by divalent metals, inhibition by La3+ and Ca2+ entry blockers. The adrenalin-induced abbreviation of systole is also explained in terms of the dual phosphorylation of the cardiac sarcoplasmic reticulum calcium pump activator, phospholamban, by cyclic-AMP-dependent protein kinase on the one hand and Ca2+-calmodulin-dependent phospholamban kinase on the other. Calciductin and phospholamban are closely similar acidic proteolipids. A phospholamban-like protein is also found in platelet Ca2+-accumulating vesicles, where its cyclic-AMP-dependent phosphorylation doubles the rate of Ca2+ efflux. These observations raise the possibility that calcium fluxes are regulated by phosphorylation of membrane-bound proteolipids. More generally, phosphorylation modulates K+, Na+ and Ca2+ fluxes through membranes, i.e. the general excitability properties of the cell.     

The two calcium ions initially bound to nonphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase can no longer be kinetically distinguished when they dissociate from phosphorylated ATPase toward the lumen.

Using rapid filtration, we investigated the kinetics of release toward the lumen of sarcoplasmic reticulum vesicles of the two Ca2+ ions transported by the Ca(2+)-dependent ATPase of these vesicles. Release rates at 20 degrees C were measured by three methods, with vesicles previously made leaky with an ionophore. First, we measured the rate at which 45Ca2+ bound to ATPase approached its steady-state level after addition of ATP to the 45Ca(2+)-equilibrated ATPase. At pH 6 in the absence of potassium, the observed kinetics did not reveal any very fast phase of 45Ca2+ dissociation from phosphorylated ATPase. Second, we measured the kinetics of 45Ca2+ dissociation from phosphorylated ATPase in a "chase" experiment, by isotopic dilution of calcium under turnover conditions in the presence of potassium. We found that these kinetics were essentially monophasic. Moreover, when they were measured in the presence of a high concentration of calcium, designed to saturate the low-affinity calcium transport sites on the lumenal side of the ATPase, they only departed slightly from monophasic behavior, irrespective of the experimental pH (pH 6, 7, or 9). This small perturbation by high calcium concentrations of the observed dissociation kinetics was attributed to ADP-facilitated rapid exchange of 40Ca2+ for Mg2+ at the catalytic site of phosphorylated ATPase. The third method was based on the fact that phosphorylation-induced 45Ca2+ occlusion occurred faster than 45Ca2+ dissociation from nonphosphorylated ATPase: here, we measured the rate of 45Ca2+ internalization on addition to 45Ca(2+)-saturated ATPase of an unlabeled ATP-containing medium. This method allowed separate observation of the dissociation kinetics of each of the two 45Ca2+ ions bound to phosphorylated ATPase, after either one or the other had been labeled by a preliminary partial isotopic exchange in the non-phosphorylated state of the ATPase. We found that after ATP-induced phosphorylation, the two 45Ca2+ ions dissociated toward the lumenal medium with virtually identical rate constants; this was observed under different ionic and pH conditions and also in the presence of a high Ca2+ concentration. As a control, the same partial isotopic exchange procedure allowed us to confirm that, in contrast, when ATP was absent from the final dissociation medium, the two 45Ca2+ ions dissociated from nonphosphorylated ATPase toward the cytoplasmic medium at different rates, the one bound more deeply only dissociating after a lag period corresponding to dissociation of the superficial one.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mobilization of intracellular calcium by alpha 1-adrenergic receptor activation in muscle cell monolayers

The fluorescent chelating agent quin 2 has been employed to monitor alterations of intracellular free Ca2+ concentrations ([Ca2+]i) in response to alpha 1-adrenergic receptor activation in adherent BC3H-1 cells. To correlate the kinetics of [Ca2+]i changes with transmembrane fluxes of this ion, continuous monitoring of [Ca2+]i has been undertaken on a monolayer of cells. Previous measurements of the transmembrane efflux of Ca2+ show a distinct lag in the response over a range of phenylephrine concentrations. By contrast, the elevation of [Ca2+]i is rapid (t1/2 approximately 2 s) and maintained for 30 s before it begins to decline to basal concentrations. The differences in kinetics indicate that the temporal delay in cellular Ca2+ efflux results from either activation of the transport system for Ca2+ extrusion or translocation of free Ca2+ to the transport site. The decline of [Ca2+]i with continued agonist exposure parallels both the efflux kinetics from the cell and the decline of total cellular Ca2+. At a time when free [Ca2+]i approaches the resting concentration, total cellular Ca2+ is reduced to a steady state value of 60% of that seen prior to stimulation. The Kact for phenylephrine-stimulated elevation in [Ca2+]i on the monolayer is 0.51 microM, which is similar to the Kact of 0.90 microM observed for phenylephrine-activated 45Ca2+ efflux. Addition of phentolamine subsequent to phenylephrine addition immediately reverses the agonist-stimulated Ca2+ mobilization, initiating a rapid return of [Ca2+]i to resting levels. A comparison of the kinetics of Ca2+ mobilization with its transmembrane flux suggests that the agonist augments the rate of recycling of intracellular Ca2+ between the free and bound states rather than causing release as a single bolus from the bound stores.     

Evidence for more than one Ca2+ transport mechanism in mitochondria.

The active transport and internal binding of the Ca2+ analogue Mn2+ by rat liver mitochondria were monitored with electron paramagnetic resonance. The binding of transported Mn2+ depended strongly on internal pH over the range 7.7-8.9. Gradients of free Mn2+ were compared with K+ gradients measured on valinomycin-treated samples. In the steady state, the electrochemical Mn2+ activity was larger outside than inside the mitochondria. The observed gradients of free Mn2+ and of H+ could not be explained by a single "passive" uniport or antiport mechanism of divalent cation transport. This conclusion was further substantiated by observed changes in steady-state Ca2+ and Mn2+ distributions induced by La3+ and ruthenium red. Ruthenium red reduced total Ca2+ or Mn2+ uptake, and both inhibitors caused release of divalent cation from preloaded mitochondria. A model is proposed in which divalent cations are transported by at least two mechanisms: (1) a passive uniport and (2) and active pump, cation antiport or anion symport. The former is more sensitive to La3+ and ruthenium red. Under energized steady-state conditions, the net flux of Ca2+ or Mn2+ is inward over (1) and outward over (2). The need for more than one transport system inregulating cytoplasmic Ca2+ is discussed.     

Equilibrium and steady state thermodynamics of active transport systems studied on simple models simulating Ca2+ transport through sarcoplasmic reticulum membranes

Equilibrium and steady state conditions of primary active transport systems are analyzed in models simulating well known characteristics of calcium transport through sarcoplasmic reticulum membranes. The model for the equilibrium simulations is a closed system with two compartments and a vectorial chemical reaction coupling Ca transport and ATP breakdown. The chemical potential difference for Ca (delta mu Ca) is calculated as a function of the total amount of Ca (Cat) and nucleotides (Nt) in the system. Results are obtained by successive approximations along the thermodynamic pathway of the reaction, up to minimizing free energy of the system, since the solution of the explicit equations cannot be obtained with computers of current precision for data within physiological ranges. delta mu Ca and [Caout] are extremely dependent on Cat and Nt for certain combinations of the variables, i.e. [Caout] can be raised from 10(-8) to 10(-6) M when Cat varies from 0.998 to 1.002 mM, therefore, the running force of the spontaneous reaction is largely shifted by tiny changes in the parameters of the system. For steady state simulations, ATP supply to the system, ADP and Pi drainage, and Ca diffusion through the barrier, are assumed. Again, conditions within physiological ranges can be found where tiny changes in Cat, the rate of ATP supply, diffusion, the ratio between the volumes of the compartments, or a relative uncoupling between the transport and hydrolytic reactions, largely shifts delta mu Ca and [Caout], thus making the steady state highly unstable and therefore well designed to operate as an amplifier of physiological signals. The equilibrium model describes some physicochemical characteristics of the system; the steady state model is more useful to simulate several physiological situations.     

Effects of Mg2+ on calcium accumulation by two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium accumulation by two fractions of sarcoplasmic reticulum presumably derived from longitudinal tubules (light vesicles) and terminal cisternae (heavy vesicles) was examined radiochemically in the presence of various free Mg2+ concentrations. Both fractions of sarcoplasmic reticulum exhibited a Mg2+-dependent increase in phosphate-supported calcium uptake velocity, though half-maximal velocity in heavy vesicles occurred at a much higher free Mg2+ concentration than that in light vesicles (i.e., approx. 0.90 mM vs. approx. 0.02 mM Mg2+). Calcium uptake velocity in light vesicles correlated with Ca2+-dependent ATPase activity, suggesting that Mg2+ stimulated the calcium pump. Calcium uptake velocity in heavy vesicles did not correlate with Ca2+-dependent ATPase activity, although a Mg2+-dependent increase in calcium influx was observed. Thus, Mg2+ may increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles. Analyses of calcium sequestration (in the absence of phosphate) showed a similar trend in that elevation of Mg2+ from 0.07 to 5 mM stimulated calcium sequestration in heavy vesicles much more than in light vesicles. This difference between the two fractions of sarcoplasmic reticulum was not explained by phosphoenzyme (EP) level or distribution. Analyses of calcium uptake, Ca2+-dependent ATPase activity, and unidirectional calcium flux in the presence of approx. 0.4 mM Mg2+ suggested that ruthenium red (0.5 microM) can also increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles, with no effect in light vesicles. These functional differences between light and heavy vesicles suggest that calcium transport in terminal cisternae is regulated differently from that in longitudinal tubules.     

Mechanism of arachidonic acid-induced Ca2+ mobilization from rat liver microsomes

Recent evidence indicates that unesterified arachidonic acid functions as a mediator of intracellular Ca2+ mobilization by inducing Ca2+ release from the endoplasmic reticulum of pancreatic islet beta cells in a manner closely similar to that of inositol 1,4,5-trisphosphate. To test the generality and explore the mechanism of this phenomenon we have examined the effects of arachidonic acid on calcium accumulation and release by hepatocyte subcellular fractions enriched in endoplasmic reticulum (microsomes). At concentrations above 0.017 mumol/mg microsomal protein, arachidonate induced rapid (under 2 min) 45Ca2+ release from microsomes that had been preloaded with 45Ca2+. Arachidonate also suppressed microsomal 45Ca2+ accumulation when present during the loading period, as reflected by reduction both of 45Ca2+ accumulation at steady state and of the rate of uptake. Neither the cyclooxygenase inhibitor indomethacin nor the lipoxygenase/cyclooxygenase inhibitor BW755C suppressed arachidonate-induced 45Ca2+ release, indicating that this effect was not dependent upon oxygenation of the fatty acid to metabolites. The long-chain unsaturated fatty acids oleate and linoleate were less potent than arachidonate in inducing 45Ca2+ release, and the saturated fatty acid stearate did not exert this effect. Albumin prevented 45Ca2+ release by arachidonate, presumably by binding the fatty acid. As is the case for inositol 1,4,5-trisphosphate, the ability of arachidonate to induce 45Ca2+ release was dependent on the ambient free Ca2+ concentration. Arachidonate did not influence microsomal membrane permeability or Ca2+-ATPase activity and may exert its effects on microsomal Ca2+ handling by activation of a Ca2+ extrusion mechanism or by dissociating Ca2+ uptake from Ca2+-ATPase activity.     

Calcium inhibition of rat liver microsomal calcium-dependent ATPase

Measurement of the inward rate of Ca2+ transport by rat liver microsomes under conditions of varying free intravesicular Ca2+ (1 microM to 5 mM) revealed that inward transport rate is maximum at low intravesicular Ca2+, and that transport rate decreases with an apparent inhibition constant of about 250-350 microM as intravesicular Ca2+ accumulates. This relationship is confirmed by measurement of Ca2+-dependent ATPase activity; activity is greatest when intravesicular Ca2+ is 1 microM, is lower when intravesicular Ca2+ is 60 microM, and is minimum when intravesicular Ca2+ is 5 mM. Unexpectedly, the ratio of Ca2+ transport rate to Ca2+-dependent ATP hydrolysis rate appears to be significantly greater than 2:1.     

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.     

Calcium occlusion in plasma membrane Ca2+-ATPase

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca(2+)-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca(2+) in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca(2+) concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μM, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La(III) (inducing accumulation of phosphoenzyme in the E(1)P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E(1)P-phosphorylated intermediate of the PMCA.