Calcium dependent ATP losses in intact red blood cells without cellular accumulations of calcium

Summary Intact human red blood cells incubated with ionophore A23187 and calcium develop a depletion of ATP that is dependent upon the concentrations of both A23187 and Ca. Incubations of fresh cells with 0.5 m A23187 and concentrations of Ca at or below 70 m produce a depletion of ATP without a net cellular uptake of Ca. In contrast, ATP-depleted cells display an ionophore-dependent cellular uptake of Ca, under identical conditions. A hypothesis is proposed that relates these ionophore-produced ATP depletions to active Ca extrusion by the Ca ATPase.     

Calcium buffering in presynaptic nerve terminals. II. Kinetic properties of the nonmitochondrial Ca sequestration mechanism

The kinetic properties of the nonmitochondrial ATP-dependent Ca sequestering mechanism in disrupted nerve terminal (synaptosome) preparations have been investigated with radioactive tracer techniques; all solutions contained DNP, NaN3, and oligomycin, to block mitochondrial Ca uptake. The apparent half-saturation constant, KCa, for the nonmitochondrial Ca uptake is approximately 0.4 micrometer Ca; the Hill coefficient is approximately 1.6. Mg is also required for the Ca uptake, and the apparent KMg is approximately 80 micrometer. ATP and deoxy-ATP, but not CTP, GTP, ITP, UTP, ADP, or cyclic AMP, promote Ca uptake; the KATP, is approximately 10 micrometer. ATP analogs with blocked gamma-phosphate groups are unable to replace ATP. Particulate fractions from the disrupted synaptosomes possess Ca-dependent ATPase activity in the presence of Mg; the apparent KCa for this activity is 0.4--0.8 micrometer Ca, and the Hill coefficient is approximately 1.6. The Ca uptake and ATPase kinetic data suggest that the hydrolysis of 1 ATP may energize the transport of two Ca2+ ions into the storage vesicles. The second part of the article concerns the intraterminal distribution of Ca in "intact" terminals. When the terminals are disrupted after 45Ca loading, about one-half of the 45Ca is retained in the particulate material; some of this Ca, presumably stored in mitochondria, is released by the uncoupler, FCCP. Some of the 45Ca is released by A-23187, but not by FCCP; this fraction may be Ca stored in the nonmitochondrial sites described above. The proportion of 45Ca stored in the nonmitochondrial sites is increased when the Ca load is reduced or when the mitochondria are blocked with ruthenium red. These data indicate that the nonmitochondrial Ca storage sites are involved in intraterminal Ca buffering; they may play an important role in synaptic facilitation and post-tetanic potentiation, which result from Ca retention after neural activity.     

Ca++-ionophore-induced dissipation of intracellular proton gradients in rat thymocytes

Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 microM in about 10 minutes at 37 degrees C. The addition of (NH4)2SO4 (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH4+-releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 microM of the Ca++-ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca++-containing physiological saline, the addition of A23187 (at a final concentration of 8 microM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca++, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 microM A23187 in Ca++-containing saline completely abolished NH4+-sensitive dye uptake; less A23187 (0.5 microM) and like or a longer incubation period brought about a striking decrease in NH4+-sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca++, A23187 impaired cell capacity to incorporate dye in a delta pH-dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca++ or also Mg++)-H+ exchange.     

Relations between ion shifting, ATP depletion and lactic acid formation in human red cells during moderate calcium loading using the ionophore A 23187.

Keeping constant cellular magnesium an A 23 187 mediated moderate calcium loading of human red cells causes isoosmotic cell shrinkage, potassium efflux, slight decrease of cellular pH, ATP depletion connected with an increase of AMP, ADP and Pi and enhanced lactic acid formation. The calcium loading and accompanying effects can be abolished by EGTA or by extracellular magnesium, the latter kept more than two orders of magnitude above that of calcium which was 30 micrometer. Inhibition of the (Mg2+ + Ca2+)-dependent ATPase by ruthenium red or lanthanum decreases the calcium stimulated lactic acid formation after a lag phase. However, the ATP depletion proceeds faster and is much more pronounced under these conditions. (Mg+2 + Na+ +K+)-dependent ATPase, hexokinase, phosphofructokinase and cell shrinkage are ruled out, too, as mediators of the ATP depletion. This suggests that an unknown ATP consuming reaction, apparently not being related to the calcium pump, causes the calcium induced ATP depletion.     

The effect of calcium ionophore A23187 on the ATP level of human erythrocytes

Incubation of red cells at 37° with the ionophore A23187 results in a loss of ATP that is dependent on the concentrations of A23187 and Ca²⁺ in the medium. ATP hydrolysis is greatest at micromolar concentrations of Ca²⁺ and decreases as Ca²⁺ in the medium is raised to millimolar levels. The ATP depletion is due to stimulation of calcium ATPase by A23187-mediated Ca²⁺ influx into the cell. The biphasic nature of Ca²⁺-stimulated ATP depletion in whole cells reflects the activity of Ca²⁺-ATPase in membrane preparations at varying Ca²⁺ concentrations. The ionophore can be removed by washing the cells with plasma or bovine serum albumin-containing medium and the ATP levels restored to normal by reincubating with 5 mM adenosine for 1 hr.     

Enzymatic basis for the calcium-induced decrease of membrane protein methyl esterification in intact erythrocytes. Evidence for an impairment of S-adenosylmethionine synthesis

The effect of Ca2+ loading, induced by the ionophore A23187, on methyl esterification of membrane proteins (i.e. bands 2.1, 3, 4.1 and 4.5) has been investigated in intact human erythrocytes. When the cells were incubated with L-[methyl-3H]methionine, 40 microM CaCl2 and 10 microM A23187 induce a 50% inhibition of membrane protein methyl esterification. This effect is selectively due to the increased intracellular Ca2+ concentration, as it is antagonized by 10 mM EGTA, and other divalent cations such as Mn2+ do not exert any inhibition. In order to clarify the mechanism(s) of the reported inhibition, the various events involved in the methyl esterification process in vivo were analyzed. L-Methionine uptake as well as protein methylase II activity are not directly affected by altered intracellular Ca2+ concentrations. Conversely in the Ca2+-loaded erythrocytes the conversion of [3H]methionine into [3H]AdoMet, catalyzed by AdoMet synthetase, decreases up to 25%. When the undialyzed erythrocyte cytosolic fraction is assayed in vitro for AdoMet synthetase the activity of the enzyme from the CaCl2/A23187-treated erythrocytes is significantly lower than the control, up to 5 mM ATP. This result suggests that in the Ca2+-loaded erythrocytes the ATP intracellular concentration is significantly lowered. The direct evaluation of ATP intracellular concentration, by HPLC, confirms a significant drop of ATP level, as a consequence of the Ca2+ loading. The removal of Ca2+ from the cells quantitatively restores both the AdoMet synthesis and the methyl esterification levels. The possible role of altered ATP intracellular concentrations as a regulatory factor in the AdoMet-dependent reactions as well as in post-translational protein methylation related to the ageing process is also discussed.     

Modulation of 32Pi incorporation into phospholipids of polymorphonuclear leukocytes by ionophore A23187.

The effects of ionophore A23187 on the incorporation of 32Pi into phospholipids and on 45Ca2+ uptake and release by polymorphonuclear leukocytes were examined. A23187 increased 32Pi incorporation into phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and the phosphoinositides. It also promoted a rapid burst uptake and release of 45Ca2+ by leukocytes. External Ca2+, but not Mg2+, was required for full stimulation of 32Pi incorporation into phosphatidic acid and the phosphoinositides. In the absence of external Ca2+, the increased radiophosphorus activity of phosphatidic acid, phosphatidylserine and the phosphoinositides was grossly reduced but not eliminated, and the decreased radiophosphorus activity of phosphatidylcholine became pronounced. In addition, the ionophore effect on 32Pi incorporation into leukocyte phospholipids was not abolished by ethyleneglycol bis(beta-amino-ethylether)-N,N'-tetraacetic acid. ATP radiophosphorus activity was also enhanced by the presence of A23187, but the enhancement was much less than that of the acidic phospholipids. Based on these findings, it is suggested that the increased 32Pi incorporation into the acidic phospholipids of leukocytes induced by A23187 was not solely derived from the higher radioactivity of ATP, increased Ca2+ fluxes and perturbation of cellular Ca2+ distribution of leukocytes exposed to A 23187 may trigger part of the altered 32Pi incorporation into phospholipids.     

Assay of the Ca pump ATPase activity of intact red blood cells.

An assay for the Ca pump ATPase of intact human red blood cells (RBCs) was developed. The assay utilized a small volume (typically 10 microliters) of packed RBCs in 1 ml of a buffer of known composition. The assay was based on the exposure of intact RBCs to the ionophore, A23187, in the presence of Ca. Such exposure caused a rapid degradation of ATP in RBCs. This degradation process is modeled in a numerical simulation in a companion paper (Vincenzi, F. F. and Hinds, T. R. (1992) Biochim. Biophys. Acta 1105, 63-70). The loss of ATP followed pseudo-first-order kinetics, and the rate constants for ATP degradation was taken as a measure of the capacity of the Ca pump ATPase. A number of variables were examined to optimize the activity of the ATPase. These variables included the concentrations of Ca and A23187. Because A23187 can promote loss of cellular Mg, it was necessary to include MgCl2 in the incubation medium to optimize ATPase activity. Likewise, it was determined that inclusion of iodoacetic acid optimized the rate of ATP loss, presumably by preventing the resynthesis of ATP from ADP and inorganic phosphate. Cobalt inhibited the ionophore-dependent loss of ATP by apparent competition with Ca for binding to A23187. Results of many assays demonstrated substantial differences in the rate constant for ATP loss in RBCs from different individuals. RBCs were selected according to density. Density associated loss of Ca pump ATPase activity was observed both by the intact RBC assay, and by assay of Ca pump ATPase activity in saponin lysates of RBCs. The correlation coefficient between the two assays was 0.93. It is suggested that the rate constant for ATP loss in intact RBCs exposed to A23187 and Ca can be taken as a measure of the Ca pump ATPase activity. This may be useful when isolated membrane ATPase assays fail (e.g., dog RBCs). The intact cell assay can also be carried out on very small volumes of cells and may be of particular value when RBC volumes are limited.     

The effect of ionophore A23187 on calcium ion fluxes and alpha-adrenergic-agonist action in perfused rat liver.

The effect of ionophore A23187 on cellular Ca2+ fluxes, glycogenolysis and respiration was examined in perfused liver. At low extracellular Ca2+ concentrations (less than 4 microM), A23187 induced the mobilization of intracellular Ca2+ and stimulated the rate of glycogenolysis and respiration. As the extracellular Ca2+ concentration was elevated, biphasic cellular Ca2+ fluxes were observed, with Ca2+ uptake preceding Ca2+ efflux. Under these conditions, both the glycogenolytic response and the respiratory response also became biphasic, allowing the differentiation between the effects of extracellular and intracellular Ca2+. Under all conditions examined the rate of Ca2+ efflux induced by A23187 was much slower than the rate of phenylephrine-induced Ca2+ efflux, although the net amounts of Ca2+ effluxed were similar for both agents. The effect of A23187 on phenylephrine-induced Ca2+ fluxes, glycogenolysis and respiration is dependent on the extracellular Ca2+ concentration. At concentrations of less than 50 microM-Ca2+, A23187 only partially inhibited alpha-agonist action, whereas at 1.3 mM-Ca2+ almost total inhibition was observed. The action of A23187 at the cellular level is complex, dependent on the experimental conditions used, and shows both differences from and similarities to the hepatic action of alpha-adrenergic agonists.     

Analysis of Ca2+ fluxes and Ca2+ pools in pancreatic acini

45Ca2+ movements have been analysed in dispersed acini prepared from rat pancreas in a quasi-steady state for 45Ca2+. Carbamyl choline (carbachol; Cch) caused a quick 45Ca2+ release that was followed by a slower 45Ca2+ 'reuptake'. Subsequent addition of atropine resulted in a further transient increase in cellular 45Ca2+. The data suggest the presence of a Cch-sensitive 'trigger' pool, which could be refilled by the antagonist, and one or more intracellular 'storage' pools. Intracellular Ca2+ sequestration was studied in isolated acini pretreated with saponin to disrupt their plasma membranes. In the presence of 45Ca2+ (1 microM), addition of ATP at 5 mM caused a rapid increase in 45Ca2+ uptake exceeding the control by fivefold. Maximal ATP-promoted Ca2+ uptake was obtained at 10 microM Ca2+ (half-maximal at 0.32 microM Ca2+). In the presence of mitochondrial inhibitors it was 0.1 microM (half-maximal at 0.014 microM). 45Ca2+ release could still be induced by Cch but the subsequent reuptake was missing. The latter was restored by ATP and atropine caused further 45Ca2+ uptake. Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca2+, oxalate and ATP which were absent in intact cells or cells pretreated with A23187. The data suggest the presence of a plasma membrane-bound Cch-sensitive 'trigger' Ca2+ pool and ATP-dependent Ca2+ storage systems in mitochondria and rough endoplasmic reticulum of pancreatic acini. It is assumed that Ca2+ is taken up into these pools after secretagogue-induced Ca2+ release.U     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3'':5''-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.

1. The bivalent cation ionophore A23187 was used to increase the intracellular concentration of Ca2+ in pigeon erythrocytes to investigate whether the increase in cyclic AMP content caused by adrenaline might be influenced by a change in intracellular Ca2+ in intact cells. 2. Incubation of cells with adrenaline, in the concentration range 0.55--55 muM, resulted in an increase in the concentration of cyclic AMP over a period of 60 min. The effect of adrenaline was inhibited by more than 90% with ionophore A23187 (1.9 muM) in the presence of 1 mM-Ca2+. This inhibition could be decreased by decreasing either the concentration of the ionophore or the concentration of extracellular Ca2+, and was independent of the concentration of adrenaline. 3. The effect of ionophore A23187 depended on the time of incubation. Time-course studies showed that maximum inhibition by ionophore A23187 was only observed when the cells were incubated with the ionophore for at least 15 min before the addition of adrenaline. 4. The inhibition by ionophore A23187 depended on the concentration of extracellular Ca2+. In the absence of Mg2+, ionophore A23187 (1.9 muM) inhibited the effect of adrenaline by approx. 30% without added Ca2+, by approx. 66% with 10 muM-Ca2+ and by more than 90% with concentrations of added Ca2+ greater than 30 muM. However, even in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate](0.1--10 mM), ionophore A23187 caused an inhibition of the cyclic AMP response of at least 30%, which may have been due to a decrease in cell Mg2+ concentration. 5. The addition of EGTA after incubation of cells with ionophore A23187 resulted in a partial reversal of the inhibition of the effect of adrenaline. 6. Inclusion of Mg2+ (2 mM) in the incubation medium antagonized the inhibitory action of ionophore A23187. This effect was most marked when the ionophore A23187 was added to medium containing Mg2+ before the addition of the cells. 7. The cellular content of Mg2+ was decreased by approx. 50% after 20 min incubation with ionophore A23187 (1.9 muM) in the presence of Ca2+ (1 mM) but no Mg2+. When Mg2+ (2 mM) was also present in the medium, ionophore A23187 caused an increase of approx. 80% in cell Mg2+ content. Ionophore A23187 had no significant effect on cell K+ content. 8. Ionophore A23187 caused a decrease in cell ATP content under some conditions. Since effects on cyclic AMP content could also be shown when ATP was not significanlty lowered, it appeared that a decrease in ATP in the cells could not explain the effect of ionophore A23187 on cyclic AMP. 9. Ionophore A23187 (1.9 muM), with 1 mM-Ca2+, did not enhance cyclic AMP degradation in intact cells, suggesting that the effect of ionophore A23187 on cyclic AMP content was mediated through an inhibition of adenylate cyclase rather than a stimulation of cyclic AMP phosphodiesterase. 10. It was concluded that in intact pigeon erythrocytes adenylate cyclase may be inhibited by intracellular concentrations of Ca2+ in the range 1-10 muM.     

Uncoupling protein 3 (UCP3) modulates the activity of Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) by decreasing mitochondrial ATP production

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.     

Calcium-dependent inhibition of protein synthesis in rat parotid gland.

1. Protein synthesis in the rat parotid gland in vitro was studied by measuring the incorporation of [3H]phenylalanine into trichloroacetic acid-insoluble proteins. In the unstimulated gland, the rate of incorporation was dependent on the phenylalanine concentration in the medium and proceeded linearly for up to 3h. 2. Adrenaline, carbamoylcholine, phenylephrine and ionophore A23187 inhibited the incorporation of [3H]phenylalanine into acid-insoluble protein; isoprenaline, dibutyryl cyclic AMP and 8-bromo-cyclic GMP were inactive. 3. Inhibition by adrenaline and carbamoylcholine but not by ionophore A23187 required extracellular Ca2+. 4. Both adrenaline and carbamoylcholine increased the magnitude of the acid-soluble [3H]phenylalanine pool at 10 micrometer extracellular phenylalanine, but had no effect if the phenylalanine concentration was increased to 200 micrometer. 5. There was no correlation between cellular ATP content and the observed inhibition of protein synthesis. 6. Our results suggest that both alpha-adrenergic and cholinergic receptors may play a role in the regulation of protein synthesis in the rat parotid gland, and that their effects are mediated by a rise in intracellular free Ca2+.     

The role of calcium in the secretion of surfactant by rat alveolar type II cells

Beta adrenergic agonists, tetradecanoylphorbol acetate, and the ionophore A23187 all stimulate surfactant secretion in type II cells isolated from rats. We found that combinations of these agonists cause augmented secretion, suggesting that the agonists may effect different steps in the secretory process. Previous studies have shown that cAMP is likely to be an intracellular 'second messenger' in type II cells. A23187, which has been reported to increase cAMP in some cell systems, did not increase the cAMP content of type II cells. We investigated the possible role of Ca2+ as another 'second messenger' by studying cellular 45Ca fluxes and the effect of extracellular calcium depletion on secretion. Depletion of extracellular calcium for as long as 3 h did not alter stimulated secretion, although basal secretion was increased. Secretagogues did not stimulate 45Ca influx from extracellular sources. A23187 and, to a lesser extent, terbutaline caused an acceleration of 45Ca efflux from type II cells. The addition of terbutaline or tetradecanoylphorbol acetate to A23187 further accelerated 45Ca efflux, suggesting that these agonists may act on separate calcium pools or by different mechanisms on the same calcium pool. Although secretion from type II cells is not inhibited by extracellular calcium depletion, the studies on 45Ca efflux suggest that Ca2+ plays a role in the regulation of surfactant secretion from isolated type II cells.     

The effects of calcium ions, ionophore A23187 and inhibition of energy metabolism on protein degradation in the rat diaphragm and epitrochlearis muscles in vitro.

1. The effects of external Ca2+, EGTA, ionophore A23187, CN-, dinitrophenol and iodoacetamide on the rate of protein degradation in the rat diaphragm and epitrochlearis muscles in vitro were investigated. 2. External Ca2+ increased protein degradation when compared with external EGTA. Protein degradation was further increased by Ca2+ + ionophore A23187. 3. EGTA and ionophore A23187 decreased ATP and phosphocreatine concentrations and the ATP/ADP ratio. 4. CN-, dinitrophenol and iodoacetamide decreased protein degradation, presumably by interfering with energy metabolism. 5. The effects of EGTA may be caused by disturbances in energy metabolism. The effects of ionophore A23187 cannot be readily explained by disturbances in energy metabolism. 6. Incubation of diaphragms with Ca2+ causes a rapid increase in whole-tissue Ca content. This is further stimulated by ionophore A23187. The uptake of Ca2+ may be, at least in part, into the cytoplasm because an increase in the glycogen phosphorylase activity ratio is observed. 7. A Ca2+-activated proteinase is present in rat heart and diaphragm. This enzyme may mediate in part the effects of Ca2+ described above. The apparent KA of this enzyme for Ca2+ is about 0.25 mM. 8. Because effects of ionophore A23187 cause a large increase in whole-tissue Ca content and because the Ca2+-activated proteinase has a relatively low affinity for Ca2+, it is felt that the effects of Ca2+ upon muscle proteolysis are unlikely to be of importance in steady-state protein turnover in vivo. The mechanism may, however, be important in breakdown of necrotic tissue in the living animal.     

Ca2+ transport studied with arsenazo III in Tetrahymena microsomes. Effects of calcium ionophore A23187 and trifluoperazine

Transport of Ca2+ in microsomal membrane vesicles of the Tetrahymena has been investigated using arsenazo III as a Ca2+ indicator. The microsomes previously shown to carry a Mg2+-dependent, Ca2+-stimulated ATPase (Muto, Y. and Nozawa, Y. (1984) Biochim. Biophys. Acta 777, 67-74) accumulated calcium upon addition of ATP and Ca2+ sequestered into microsomal vesicles was rapidly discharged by the Ca2+ ionophore A23187. Kinetic studies indicated that the apparent Km for free Ca2+ and ATP are 0.4 and 59 microM, respectively. The Vmax was about 40 nmol/mg protein per min at 37 degrees C. The calcium accumulated during ATP-dependent uptake was released after depletion of ATP in the incubation medium. Furthermore, addition of trifluoperazine which inhibited both (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ uptake rapidly released the calcium accumulated in the microsomal vesicles. These observations suggest that Tetrahymena microsome contains both abilities to take up and to release calcium and may act as a Ca2+-regulating site in this organism.     

Lectin- and ionophore-stimulated Ca2+ influx in murine lymphocytes: inhibition by disialoganglioside

Gangliosides suppress lymphocyte mitogenesis when added exogenously to the cells. On the premise that the mechanism of ganglioside action may be an interference with primary induction events, mitogen-induced 45Ca2+ influx in murine lymphocytes was studied. Disialoganglioside (GD1a) at physiopathological concentrations inhibits concanavalin A-induced 45Ca2+ uptake as well as blast transformation. The suppressive action of GD1a is both concentration dependent (50% suppression at 13 microM) and very rapid (within 1 min). GD1a is not cytotoxic nor does it significantly alter the rate of Ca2+ efflux. The uptake studies were extended to A23187, a compound with mitogenic and specific divalent cation ionophore activities. Ca2+ uptake by lymphoid cells from AKR/J, Swiss, and CBA mice is stimulated by A23187; and GD1a, in a dose-dependent manner, inhibits the ionophore-induced 45Ca2+ influx. Pretreatment of thymocytes with GD1a renders the cells greatly insensitive to the subsequent ionophore activity of A23187. The results suggest that exogenous gangliosides may function as an inhibitor of some of the mitogen-triggered early events, including Ca2+ metabolism, and thus influence the immunological behavior of intact lymphoid cells.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition by calcium ions of adenosine cyclic monophosphate formation in sealed pigeon erythrocyte ''ghosts''. A study using the photoprotein obelin.

1. Sealed pigeon erythrocyte 'ghosts' were prepared containing ATP and the Ca2+-activated photoprotein obelin to investigate the relationship cyclic AMP formation and internal free Ca2+. 2. The 'ghosts' were characterized by (a) morphology (optical and electron microscopy), (b) composition (haemoglobin, K+, Na+, Mg2+, ATP, obelin), (c) permeability to Ca2+, assessed by obelin luminescence and (d) hormone sensitivity (the effect of beta-adrenergic agonists and antagonists on cyclic AMP formation). 3. The effect of osmolarity at haemolysis and ATP at resealing on these parameters was investigated. 4. Sealed 'ghosts', containing approx. 2% of original haemoglobin, 150mM-K+, 0.5MM-ATP, 10(3)--10(4) obelin luminescence counts/10(6) 'ghosts', which were relatively impermeable to Ca2+ and in which cyclic AMP formation was stimulated by beta-adrenergic agonists over a concentration range similar to that for intact cells, could be prepared after haemolysis in 6mM-NaCl3mM-MgCl2/50mM-Tes, pH7, and resealing for 30min at 37 degrees C in the presence of ATP and 150mM-KCl. 5. The initial rate of adrenaline-stimulated cyclic AMP formation in these 'ghosts' was 30--50% of that in intact cells and was inhibited by the addition of extracellular Ca2+. Addition of Ca2+ to the 'ghosts' resulted in a stimulation of obelin luminescence, indicating an increase in internal free Ca2+ under these conditions. 6. The ionophore A23187 increased the rate of obelin luminescence in the 'ghosts' and also inhibited the adrenaline-stimulated increase in cyclic AMP. 7. The effect of ionophore A23187 on obelin luminescence and on cyclic AMP formation in the 'ghosts' was markedly decreased by sealing EGTA inside the 'ghosts'. 8. It was concluded that cyclic AMP formation inside sealed pigeon erythrocyte 'ghosts' could be inhibited by more than 50% by free Ca2+ concentrations in the range 1--10 micrometer.