Ionophore-mediated calcium exchange diffusion in liposomes

The exchange diffusion of ⁴⁵Ca in multilamellar liposomes containing the antibiotic ionophore A23187 is enhanced in a dose-related fashion at increasing concentrations of external Ca²⁺ or at increasing A23187/lipid molar ratios. An increase in fluidity of the lipid bilayer augments the permeability to Ca by facilitating both the formation and mobility of the Ca-ionophore complexes.     

An ionophoretic model for the study of calcium-calcium exchange

A Pressman cell was used to study the Br-X537A-mediated translocation of ⁴⁵Ca from one aqueous phase into another across an immiscible organic phase. In this system, an increase in the concentration of ⁴⁰Ca in one chamber (mimicking the extracellular compartment) resulted in an increased efflux of ⁴⁵Ca added in trace amount to the other chamber (simulating the cytosolic compartment). Such a Ca-Ca exchange process was also observed when the concentration of ionophore was increased, and could be blocked by the organic Ca-antagonist suloctidil. However, no Ca-Ca exchange occurred when the downhill rate of ⁴⁰Ca translocation was increased by use of a pH gradient. This ionophoretic model also suggests that a low intracellular concentration of Ca tends to maintain Ca influx at a low rate, and that the asymetrical distribution of Ca facilitates rather than impeeds the ionophoretic extrusion of intracellular Ca against its chemical gradient.     

Ionomycin-mediated calcium transport in rigid and fluid liposomes

The antibiotic ionophore ionomycin translocates Ca from an aqueous medium into or across an organic immiscible phase. At pH 8.0, ionomycin translocates less Ca than A23187, the effects of these ionophores being additive to one another. The capacity of ionomycin to translocate Ca across the organic phase is dramatically decreased when the pH of the aqueous media is reduced from 8.0 to 7.5 or lower values. Ionomycin also mediates Ca exchange-diffusion in liposomes, the magnitude of such a process being greater in fluid than in rigid liposomes. At a physiological pH (7.4), ionomycin is unexpectedly as potent as A23187 in mediating Ca transport in fluid liposomes. These findings suggest that the capacity of ionophores to translocate Ca across model membranes depends on both the transverse and lateral mobility of the ionophoretic molecules. The relative importance of the latter phenomenon itself largely depends on the stoichiometry of the Ca-ionophore complex.     

Movement of calcium through artificial lipid membranes and the effects of ionophores

The calcium efflux from multi-layered vesicles (liposomes) of different lipid composition has been studied. Liposomes composed of lipids extracted from cattle retinas are compared with liposomes which consist of phosphatidylcholine or a 1 : 1 phosphatidylcholine/phosphatidylserine mixture. The percentages of ⁴⁵Ca capture by these three types of liposomes are 10, 1 and 4% respectively.The efflux rates are 2.5 · 10^?6, 2 · 10^?6 and 4 · 10^?5 s^?1 respectively. The semilogarithmic efflux curves for phosphatidylcholine and phosphatidylcholine/phosphatidylserine liposomes are linear with time, but those for the retinal lipid liposomes are discontinuous. The activation energy for the calcium efflux from the latter liposomes is about 10.5 kcal/mol, both before and after the discontinuity.The ionophores X537A and A23187 enhance the calcium leakage from retinal lipid liposomes, the latter ionophore being much more effective than the former. At high concentrations both ionophores seem to transport calcium as a 1 : 2Ca · ionophore complex. At low ionophore concentrations, however, X537A appears to transport calcium as a 1 : 1 complex, but A23187 as a 2 : 1 complex.     

Continuous fluorometric assay of Ca2+ transport by liposomes with Quin 2 entrapped: effect of phospholipase A2 and unsaturated long-chain fatty acids

The amount of free calcium in the cytoplasm is important in stimulation coupled with a number of cellular functions. The putative ionophoretic action of membrane lipid metabolites on Ca2+ offers convenient explanation of the stimulation-coupled mobilization of cytoplasmic Ca2+. To analyze the ionophoretic action of the lipid metabolites, we devised a sensitive method to study Ca2+ transport that uses liposome-entrapped Quin 2. A calcium ionophore, A23187, increased the fluorescence intensity of the Ca2+-Quin 2 complex as a function of Ca2+ transport into liposomes. A similar Ca2+ flux into the liposomes was induced by phospholipase A2 (PLA2) and by various long-chain fatty acids in liposomes that consist of phospholipids containing unsaturated fatty acids. The potencies of the fatty acids for Ca2+ transport is inversely correlated with their melting points. The oxidized products of the unsaturated fatty acids increased the Ca2+ and nonspecific permeability of the biological membranes. These results suggest that stimulation-coupled PLA2 activation might mediates the mobilization of cytoplasmic Ca2+.     

Low root temperature,calcium, and nitrate ion interactions on nonexchangeable rubidium,cesium, and sodium absorption by bush beans

Summary When nonexchangeable absorption of Rb⁸⁶, Na²², and Cs¹³⁷ by bush bean (Phaseolus vulgaris L. var. Improved Tendergreen) was determined at different root temperatures and with and without Ca additions or pretreatments, a strong interaction between temperature and Ca was observed. Ca inhibited Rb⁸⁶ absorption markedly at low temperatures but had less effect on Cs¹³⁷. Absorption of Na²² was inhibited by Ca at both low and high temperatures. Little effect for Ca with sometimes a Viets effect was observed at high temperature for Rb but not for Cs or Na. Ratio pairs of Rb, Ca, and Na were used as an index of similarity of absorption mechanisms. Cs and Rb, and Na and Rb appeared to be absorbed by different mechanisms at 10⁻³ M as indicated by temperature and Ca responses. Nitrate-N stimulated uptake of Rb only at high temperature with or without Ca but not at low temperature. Ca in the pretreatment tended to result in greater long distance transport to shoots of Rb⁸⁶ and Cs¹³⁷ for the high temperature but Ca in the test solution slightly decreased the long distance transport. The data are discussed in terms of the Viets effect and of a possible role of Ca in synthesis of transport proteins.     

Effect of sodium on cellular calcium transport in rat kidney

Summary The influence of extracellular Na (Na _o ) on cellular Ca transport and distribution was studied in rat kidney slices. Calcium efflux from prelabeled slices was depressed when Na _o was completely replaced by choline or tetraethylammonium (TEA) ions and it was markedly stimulated when Na was reintroduced in a Na-free medium. However, reducing Na _o (with choline or TEA as substituting ions) did not increase the total slice⁴⁰Ca, their total exchangeable Ca pool, or the⁴⁰Ca or⁴⁵Ca of mitochondria isolated from these slices. Kinetic analyses of steady-state⁴⁵Ca desaturation curves showed that reducing Na _o depressed the exchange of Ca across the plasma membrane, slightly decreased the cytosolic Ca pool, but did not significantly affect the mitochondrial Ca pool and Ca cycling. Ouabain (10^–3 m) which should reduce the Na gradient across the plasma membrane had no effect on calcium distribution and transport. These results suggest that in kidney cells low Na _o depresses Ca influx as well as Ca efflux; there may be an interaction between Na and Ca at a possible carrier located in the plasma membrane, but there is no Na/Ca exchange as described in several excitable tissues.     

Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes

Summary Osmotic swelling of human and rat erythrocytes does not induce regulatory volume decrease. Regulatory volume increase was observed in shrunken erythrocytes of rats only. This reaction was blocked by the inhibitors of Na⁺/H⁺ exchange. Cytoplasmic acidification in erythrocytes of both species increases the amiloride-inhibited component of²²Na influx by five- to eight-fold. Both the osmotic and isosmotic shrinkage of rat erythrocytes results in the 10- to 30-fold increase of amiloride-inhibited²²Na influx and a two-fold increase of furosemide-inhibited⁸⁶Rb influx. We failed to indicate any significant changes of these ion transport systems in shrunken human erythrocytes. The shrinking of quin 2-loaded human and rat erythrocytes results in the two- to threefold increase of the rate of⁴⁵Ca influx, which is completely blocked by amiloride. The dependence of volume-induced²²Na influx in rat erythrocytes and⁴⁵Ca influx in human erythrocytes on amiloride concentration does not differ. The rate of⁴⁵Ca influx in resealed ghosts was reduced by one order of magnitude when intravesicular potassium and sodium were replaced by choline. It is assumed that the erythrocyte shrinkage increases the rate of a nonselective Ca _o ²⁺ (Na _i ⁺ , K _i ⁺ ) exchange. Erythrocyte shrinking does not induce significant phosphorylation of membrane protein but increases the³²P incorporation in diphosphoinositides. The effect of shrinkage on the³²P labeling of phosphoinositides is diminished after addition of amiloride. It is assumed that volume-induced phosphoinositide response plays an essential role in the mechanism of the activation of transmembrane ion movements.     

Ca2+ translocation across liposomal membranes enhanced by unsaturated long-chain fatty acids

The putative ionophoretic action of phosphatidic acid or arachidonic acid metabolites for Ca2+ has offered an attractive explanation for stimulation-coupled mobilization of cytoplasmic Ca2+. We have examined the effects of Ca2+ ionophore and long-chain unsaturated fatty acids on the translocation of Ca2+ across the liposomal membrane by using Quin II-entrapped liposomes, a sensitive assay system for ionophoresis of Ca2+. A23187 increased Quin II fluorescence intensity corresponding to the translocation of Ca2+ into liposomes. Similar translocation was observed with unsaturated long-chain fatty acids but not with saturated fatty acids. Thus, when phospholipases of cell membrane are activated by certain stimuli, unsaturated long-chain fatty acids are liberated and might mediate the mobilization of cytoplasmic Ca2+.     

Studies of the cation binding properties of an oligomeric derivative of prostaglandin B1

The ionophoretic activity of PGBx, an oligomeric mixture synthesized from 15-dehydro PGB1, with different cations was measured using arsenazo III-entrapped liposomes. The order of ionophoretic activity was Zn2+ greater than Co2+ greater than Mn2+ greater than Cu2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. The intrinsic fluorescence of PGBx was quenched by the binding of divalent cations as well as by La3+ and H+. Quenching by K+ and Na+ was minimal. The order of quenching strength of divalent cations was Zn2+ greater than Co2+ greater than Cu2+ = Mn2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. Binding affinities of these cations determined by a murexide indicator method were in good agreement with that determined by the fluorescence quenching reaction. The cation binding affinity of PGBx in aqueous solutions correlates with the ionophoretic activity in liposomes. The binding affinity for K+ was estimated from the inhibition by K+ of Ca2+ binding by PGBx. Although PGBx has a lower selectivity for divalent cation binding than the ionophore A23187, the characteristics of the binding affinity of these two compounds for various ions were similar. The pK of PGBx as determined by fluorescence quenching was 6.7. The molecular weight of the divalent cation binding unit was estimated to be about 680, with each PGBx molecule having three such binding sites. The binding of Ca2+ to such a site is one-to-one.     

Effects of monensin on metabolism of isolated rat islets of Langerhans.

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.     

Calcium efflux associated with encystment of Phytophthora palmivora zoospores

Zoospores of the fungus $\text{Phytophthora palmivora}$, pre-labeled with ⁴⁵Ca, excreted up to 30% of their total ⁴⁵Ca when stimulated to encyst. Excretion was essentially completed within 90 sec of the application of the stimulus. Encystment of the population was completed within 5 min. Four different stimuli were used: pectin addition (420 μg ml^?1), Sr²⁺ addition (5 mM), cyclic AMP addition (6.7 mM) and mechanical agitation. The kinetics and amount of Ca excretion were essentially the same in each case. The calcium ionophore A23187 increased the rate of ⁴⁵Ca uptake by motile zoospores, incubated in 100 μM CaCl₂, but did not induce encystment under these conditions. The ionophore did not induce ⁴⁵Ca efflux from pre-labeled zoospores. Incubation in EGTA and in K⁺ failed to induce either encystment or ⁴⁵Ca excretion. We conclude that rapid excretion of a significant proportion of the zoospore calcium is linked to the early stage of stimulus-induced encystment, and that this comes from an intracellularly located, non-cytoplasmic source, such as the peripheral vesicles, but that changes in cellular Ca²⁺ are not necessarily the single controlling factor in the induction of encystment.     

Heat-induced stimulation of 3-O-methylglucose transport in rat thymocytes.

Cells incubated at 41–46 °C show a gradual increase in the initial rate of 3-O-methylglucose uptake when subsequently assayed at 37 °C. Cellular ATP levels remain constant throughout this temperature range, but at temperatures higher than 46 °C, ATP levels decline as does the extent of transport stimulation. Cells incubated at 45 °C for 5 min continue to show a gradual increase in transport activity throughout a subsequent 25-min incubation period at 37 °C. The increase in transport activity is characterized by an increase in the proportion of the rapid phase of 3-O-methylglucose uptake, with little or no change in the half-time of either the rapid phase or the slow phase. Transport stimulation at high temperatures is blocked by inhibitors of oxidative phosphorylation. Cells depleted of intracellular exchangeable Ca²⁺ by treatment with the ionophore A23187 in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid show nearly the same degree of stimulation at high temperatures as untreated cells, suggesting that exchangeable Ca²⁺ ions do not play an obligatory role in the mechanism of transport stimulation. It is suggested that structural changes occur at 41–46 °C in the membrane proteins controlling glucose transport activity.     

Na-Ca interactions in barley seedlings: relationship to ion transport and growth

Abstract Salt-stressed plants often show Ca deficiency symptoms. The effects of NaCl salinity (1 to 150 mol m^-3) and supplemental Ca (10 mol m^-3) on Na and Ca transport in barley (Hordeum vulgare L.) and their relationship to growth were investigated. The adjustment of Na and Ca transport was investigated by examining young seedlings exposed to short-term (immediate) and long-term (7 d) exposure to salinity. When the plants were exposed to long-term treatments of salinity, the rate of sodium accumulation in roots was approximately 10 to 15% of short-term treatments. No significant adjustment in the transport to the shoot was observed. Rates of tracer (²²Na) transport were compared to calculated rates based on relative growth rates and tissue element concentrations. Comparisons between measured tracer and calculated rates of transport indicate that ²²Na transport may underestimate transport to the shoot because of dilution of the tracer in the root cytoplasm. Calcium uptake showed only minor adjustment with time. Measured rates of tracer transport to the shoot correlated well with calculated values. The transport and tissue concentrations of Na were significantly affected by supplemental Ca. Calcium transport and tissue concentrations were markedly inhibited by salinity. Supplemental Ca increased Ca transport and accumulation at all NaCl treatments above that of control plants without supplemental Ca. Salinity inhibited plant growth at 150 mol m ^-3NaCl, but not at 75 mol m^-3. Supplemental Ca significantly improved root length but not fresh weight after 7d of salinity, although differences in fresh weight were detected after 9d. There were significant Na-Ca interactions with ion transport, ion accumulation, and growth. The effects of salinity on Na and Ca transport to the shoot do not appear to play a major role in shoot growth of barley.     

Stimulation of glucose transport in skeletal muscle by the sodium ionophore monensin

The tissue/medium distribution of the nonmetabolized glucose analog 3-O-methyl-D-glucose was measured in mouse diaphragm muscle and related to changes in 45Ca influx, Na+ content and Na+-pump activity. In the presence of external Ca2+ the sodium ionophore monensin greatly increased cellular Na+ content (and decreased K+ content) although 86Rb uptake, reflecting Na+-pump activity was increased. Concomitantly, 45Ca influx was stimulated, presumably through activation of Na+-Ca2+ exchange. In parallel to the rise in Ca2+ influx sugar transport was also increased. Sugar transport was also increased by monensin in the nominal absence of external Ca2+, when Ca2+ influx was minimal. To test if monensin releases Ca2+ from intracellular storage sites in the absence of external Ca2+, the ionophore was added to medium perfusing rat hind limb preparations and the total Ca content of muscle mitochondria was determined. When Ca2+ was present in the perfusate, monensin increased the mitochondrial Ca content. In the absence of Ca2+, the mitochondrial Ca content was lower and was further depressed by monensin, suggesting that elevation of internal Na+ by monensin may increase mitochondrial Ca2+ loss via activation of Na+-Ca2+ exchange across the mitochondrial membrane. The above results are consistent with the effect of monensin on sugar transport being due to alterations in Ca2+ distribution. They support the earlier conclusion that regulation of sugar transport in muscle is Ca2+ dependent.     

Role of calcium in the regulation of sugar transport in the avian erythrocyte: Effects of the calcium ionophore,A23187

The tissue/medium distribution of the nonmetabolized glucose analog [¹⁴C]-3-0-methyl-D-glucose was measured in pigeon erythrocytes and related to changes in ⁴⁵Ca uptake and efflux, total calcium content and ATP levels. Sugar transport was not affected by changes in external Ca²⁺. However, both sugar and ⁴⁵Ca influx were increased by the Ca-ionophore A23187. In the absence of external Ca²⁺, the ionophore caused a delayed increase in sugar transport and net loss of calcium, probably through releasing Ca²⁺ from internal storage sites into the cytoplasm. Increasing internal Na⁺ through Na⁺ pump inhibition or using the sodium ionophore monensin did not alter influx of sugar or ⁴⁵Ca, indicating Na⁺-Ca²⁺ exchange was absent in these cells. The results are consistent with A23187 causing increased Ca²⁺ influx or release from mitochondrial storage and the resulting rise in cytoplasmic Ca²⁺ stimulating hexose transport. Experiments with low Mg⁺⁺ and high K⁺ media and measurements of ATP levels exclude alternative explanations for the action of A23187. We conclude that sugar transport regulation in avian erythrocytes is Ca²⁺-dependent and resembles that in muscle in its basic mechanism. It differs in the response to some modulating agents, largely because of a different pattern of Ca²⁺ fluxes in these cells.     

Calcium and pancreatic β-cell function: 3. Validity of the La3+-wash technique for discriminating between superficial and intracellular 45Ca

Exposure to La³⁺ has been proposed as a tool for discriminating between superficially and intracellularly located ⁴⁵Ca. Two different pools of glucose-stimulated ⁴⁵Ca were identified in β-cell-rich pancreatic islets microdissected from ob/ob mice using a standard procedure of washing with 2 mM La³⁺ at 37°C. The results were not critically dependent on the concentration of La³⁺ within a range of 1–20 mM. However, the distribution of ⁴⁵Ca was markedly influenced by the temperature of the washing medium. Reduction of the temperature to 1°C abolished the glucose effect on the La³⁺-displaceable ⁴⁵Ca and considerably increased the amounts of ⁴⁵Ca not displaceable with La³⁺. Separate studies in a perifusion system revealed that La³⁺ in itself was less effective than temperature reduction in depressing the efflux of ⁴⁵Ca from the isolated islets. The data indicate that washing in cold La³⁺ solution gives a better estimate of the intracellular islet calcium than the original procedure. It is suggested that the glucose-stimulated part of the ⁴⁵Ca, which is displaced by washing with La²⁺ at 37°C, is derived from the interior of the β-cells.     

Effects of the calcium ionophore A-23187 on the regulation of sugar transport in muscle

The distribution of (¹⁴C)-3-0-methyl-D-glucose and of (⁴⁵Ca) was followed in perifused left atria and intact hemidiaphragms of the rat. The carboxylic calcium ionophore A-23187 affected sugar and Ca²⁺ influx in parallel, with low concentrations inhibiting and higher ones stimulating influx under basal conditions. The stimulation of sugar transport by insulin, high concentrations of adrenaline or ouabain, or by K⁺-free medium was antagonized by the calcium ionophore. Likewise, A-23187 counteracted the depression of sugar transport caused by low concentrations of ouabain or adrenaline. These results support a role of Ca²⁺ in the regulation of sugar transport in muscle. However, increased influx of Ca²⁺ cannot explain all the effects of A-23187. It is suggested that the ionophore may also act by releasing Ca²⁺ from intracellular storage and binding sites.     

Sarcoplasmic reticulum. XVI. The permeability of phosphatidyl choline vesicles for calcium

The Ca permeability of phosphatidyl choline vesicles of diverse fatty acid composition was measured. The rate of ⁴⁵Ca release from liposomes equilibrated with 1 mm⁴⁵CaCl₂ was found to be about 8 × 10⁻¹⁸ moles of Ca/cm²/sec for egg lecithin and about 5.3 × 10⁻¹⁷ moles of Ca/cm²/sec for dioleyllecithin at 30 °. Incorporation of cholesterol into dioleyllecithin micelles reduced the rate of Ca release. The Ca permeability of the phosphatidyl choline micelles was insensitive to changes in the pH, calcium or sodium concentration of the medium but increased with increasing temperature. The effect of temperature was most marked with dioleyl lecithin dispersions, but was clearly apparent with dipalmitoyl, plant, bovine, and egg lecithins as well. The activation energy of Ca release fell in the range of 4.2–9.6 kcal/mole. Macrocyclic antibiotics (valinomycin, tyrocidin, and gramicidin) at relatively high concentration increased the rate of Ca release similarly to their effects on fragmented sarcoplasmic reticulum membranes.     

Serosal Na/Ca exchange and H+ and Na+ transport by the turtle and toad bladders

Summary A Na/Ca exchange system has been described in the plasma membrane of several tissues and seems to regulate the concentration of calcium in cytosol. Replacement of extracellular Na by sucrose increases calcium uptake into and decreases calcium efflux from the cell, leading to an increase in cytosolic calcium. The effect of an increase in cytosolic calcium mediated by the Na/Ca exchange system on H⁺ and Na transport in the turtle and toad bladder was investigated by replacing serosal Na isosmotically by sucrose or choline. Replacement of serosal by sucrose was associated with a significant inhibition of H⁺ secretion or Na transport which was reversible by addition of NaCl. Replacement of mucosal Na by sucrose failed to alter H⁺ secretion. Removal of serosal Na was associated with a significant increase in⁴⁵Ca uptake which could be blocked by pretreatment with lanthanum chloride. Pretreatment with lanthanum chloride blunted the inhibitory effect of replacement of serosal Na by sucrose on H⁺ and Na transport, thus suggesting that the increase in calcium uptake and the inhibition of transport are causally related. Under anaerobic conditions the rate of H⁺ or Na transport are linked to the rate of lactate production. The inhibition of Na or H⁺ transport by removal of serosal Na was accompanied by a proportional decrease in lactate production, thus suggesting that an increase in cytosolic calcium does not inhibit transport by uncoupling glycolysis from transport. Replacement of serosal Na by sucrose did not alter the force of the H⁺ or Na pump but led to an increase in resistance of the active pathway of H⁺ and Na transport. The inhibition of Na transport by replacement of serosal Na with sucrose could be reversed by addition of amphotericin B, an agent which increases luminal permeability to Na, thus suggesting that decreased Na entry across the apical membrane is the mechanism responsible for the inhibition of Na transport. The results of the present studies strongly suggest that an increase in cytosolic calcium through the serosal Na/Ca exchange system inhibits H⁺ and Na transport in the turtle and toad bladder probably by increasing the resistance of the luminal membrane.