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.     

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.     

Reduced external calcium or sodium stimulates calcium influx in Pelvetia eggs

The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca²⁺] greatly increased the permeability of the eggs to Ca²⁺; at 1 mM external Ca²⁺ this permeability was 60 times as great as it was at the normal [Ca²⁺] of 10 mM. Lowering the external [Na⁺] also increased Ca²⁺ influx; at 2 mM Na⁺, the Ca²⁺ influx was 2–3 times as great as it was at the normal [Na⁺] if choline was used as a Na⁺ substitute. Lithium was less effective as a Na⁺ substitute in increasing Ca²⁺ influx. The extra Ca²⁺ influx in low [Na⁺] seemed to be dependent on internal [Na⁺]. The Ca²⁺ efflux increased transiently and then declined in low Na⁺ media.     

Biochemical aspects of the visual process. XXXVI. Calcium accumulation in cattle rod outer segments: Evidence for a calcium-sodium exchange carrier in the rod sac membrane

Accumulation of calcium has been studied in bovine rod outer segments (rods), isolated by sucrose density gradient centrifugation. Calcium-depleted rods are obtained by having ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA) present during isolation.Rods thus isolated have a leaky plasma membrane, as shown by the effects of ionophore A23187 and by their light-induced phosphorylation behaviour. The accumulation of ⁴⁵Ca, determined by incubation followed by a single fast washing-filtration procedure, thus represents translocation across the rod sac membrane.Accumulation in non-depleted rods is independent of the external calcium level and of ATP, suggesting exchange of ⁴⁰Ca by ⁴⁵Ca. In depleted rods in the presence of ATP there is net uptake, sigmoidally increasing with the external calcium concentration to the level attained in non-depleted rods. This net uptake is abolished by omission of ATP, its replacement by β,γ-methylene ATP and lowering the temperature to 0° C, suggesting involvement of enzymatic hydrolysis of ATP.Replacement of KCl by NaCl in the medium causes marked inhibition of ⁴⁵Ca uptake, both net uptake and exchange. Oligomycin, ruthenium red, lanthanum and ouabain do not inhibit accumulation.Efflux of ⁴⁵Ca from pre-loaded rods is slow in a KCl medium ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～30}\text{min}$ at 25° C), but is greatly accelerated by addition of NaCl or Ca²⁺ ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{10}\text{s}$ at 25°C).It is concluded that the rod sac membrane contains a carrier system, which is sensitive towards Ca²⁺ and Na⁺ and which requires ATP for net uptake of Ca²⁺ but not for exchange transport of Ca²⁺ with Ca²⁺ or Na⁺.     

Evidence for a magnesium- and ATP-dependent calcium extrusion pump in dog erythrocytes

Intact dog erythrocytes, whose Ca²⁺ permeability had been increased with A23187 still maintained intracellular Ca²⁺ below electrochemical equilibrium indicating that they could extrude Ca²⁺. This extrusion required no Na⁺ gradient but apparently depended on intracellular ATP and Mg²⁺ suggesting that it was mediated by an ATP-fuelled Ca²⁺ pump.     

ATP-dependent calcium transport in membrane vesicles of the cyanobacterium, Anabaena variabilis

Transport of Ca²⁺ in membrane vesicles of the cyanobacterium Anabaena variabilis has been investigated. The light membranes previously shown to carry a Mg²⁺-dependent, Ca²⁺-stimulated ATPase (Lockau, W. and Pfeffer, S. (1982) Z. Naturforsch. 37C, 658–664) accumulate Ca²⁺ upon addition of ATP, whereas the (heavier) thylakoids do not. A stoichiometry of 0.3 Ca²⁺ taken up per ATP hydrolyzed has been determined from initial rates, which is considered to be an underestimation of the true stoichiometry of the pump. Calcium transport and Ca²⁺-stimulated ATPase activity are both sensitive to Na₃VO₄ (an inhibitor of ATPases forming a phosphorylated intermediate), show the same pH optimum and a comparable dependence on ATP concentration. Calcium transport is also supported by nucleoside triphosphates other than ATP, although at lower rates. Accumulation of calcium is abolished by an ionophore of divalent cations, ionophore A23187, but is resistant to ionophores of monovalent cations and to the inhibitor of F₁-F₀-type ATPases, N,N′-dicyclohexylcarbodiimide. It is concluded that the ATPase is a primary calcium pump.     

Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na/Ca exchange

Fluctuating extracellular Ca²⁺ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca²⁺ from 1.8 to ≤ 0.6 mM by chelation with EGTA induces a marked spike in O₂ consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca²⁺, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na⁺ and the subsequent activation of mitochondrial Na⁺/Ca²⁺ and Na⁺/H⁺ exchange. The mitochondrial Na⁺/Ca²⁺ exchanger (_mNCX) activated by Na⁺ influx reduces Ca²⁺ and increases Na⁺ levels in the mitochondrial matrix. The excess of Na⁺ activates the mitochondrial Na⁺/H⁺ exchanger (NHE) increasing the outward pumping of protons, electron transport and O₂ consumption. Reduction in extracellular Na⁺ and inhibition of Na⁺ influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the _mNCX, L-type voltage gated Ca²⁺ channels or the release of Ca²⁺ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca²⁺ exchange is critical for response development.     

Transcellular calcium transport by midgut of the blowfly, Calliphoravicina

Transcellular calcium transport by the internally perfused $\text{Calliphora}$ midgut has been measured by simultaneously monitoring ⁴⁵Ca removal from the perfusing saline (entry to the cells) and its appearance in the bathing saline (exit from the cells). Reduction of the Na⁺ gradient across the basolateral membranes of midgut epithelial cells by removal of bathing Na⁺ or by addition of monensin or ouabain inhibits calcium transport across the basolateral membranes. Calcium entry at the apical membranes is inhibited in parallel. The calmodulin inhibitors, trifluoperazine or calmidazolium, do not directly affect calcium transport nor do they dissociate the parallel changes in calcium entry and exit when calcium exit is inhibited. Experiments with A23187 are consistent with a role for intracellular calcium in regulating calcium entry at the apical membranes. It is suggested that calcium transport out of midgut epithelial cells is largely by Na⁺-Ca²⁺ countertransport, and that entry may be regulated by cytoplasmic calcium so that the calcium influx never exceeds the capacity of the transport mechanisms to pump it out of the cells.     

Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.     

Mechanisms of calcium transport in the giant axon of the squid and their physiological role

The giant axon of the squid has been extensively used as a model for studying Ca regulation in excitable cells. Different techniques (extrusion, injection and dialysis) have been employed to characterize Ca fluxes across the axon membrane. Since both Ca efflux and influx are markedly dependent on [Ca²⁺]_i, considerable effort has been dedicated to determine the resting value of the [Ca²⁺]_i. Results from different laboratories indicate that the [Ca²⁺]_i, in a normal fibre, range from 20–100 nM. Under dialysis conditions (internal control), with an imposed [Ca²⁺]_i of 80 nM, Ca influx is balanced by an outward Ca movement of about 40 f/CS. Ca extrusion occurs through two parallel transport systems: one having a high affinity for [Ca²⁺]_i, dependent on ATP, not affected by Na_i, Na_o, Ca_o, Mg_o and inhibited by internal vanadate (uncoupled component), the other, more prominent at relatively high [Ca²⁺]_i, does not require ATP, is inhibited by Na_i activated by Na_o and not inhibited by vanadate. (Na_o-dependent component). The existence of these two systems provide the axon with an effective way to maintain in the long term a constant low [Ca²⁺]_i in spite of short term fluctuations due to increased Ca influx during nervous activity.     

Ionophore-mediated Ca2+ countertransport: role of Na+, Li+ or H+ gradient

Summary In an artificial system, the ionophore A23187, which transports Ca²⁺ but not Na⁺, is able to mediate the uphill translocation of Ca²⁺ from one aqueous medium to another across an organic immiscible phase, provided that a Na⁺, Li⁺ or H⁺ gradient is imposed on the system. Therefore, in the process known as Na-Ca countertransport, the downhill influx of Na⁺ may not be necessary for causing Ca²⁺ extrusion against its electrochemical gradient.     

Dual effect of adrenalin on sugar transport in rat diaphragm muscle

The effect of adrenalin on the membrane transport of the non-metabolized sugar, 3-methylglucose, was studied in isolated “intact” rat hemidiaphragms and related to simultaneously occurring changes in the internal levels of Na⁺, ATP, glucose-6-P, glycerol formation and ⁴⁵Ca uptake and loss. Basal sugar transport was inhibited by low (10⁻⁸−10⁻⁵ M) concentrations of adrenalin; this was antagonized by propranolol and practolol. High concentrations (10⁻⁴−10⁻³ M) stimulated sugar transport, and this was blocked by propranolol and butoxamine and was dependent on external Ca²⁺. These results suggest interaction with two different classes of adrenergic receptors, possibly of β₁ and β₂ types. Both low and high concentrations increased Na⁺ and K⁺ gradients by a practolol-sensitive effect. Isoproterenol behaved identically but phenylephrine had only the two practolol-sensitive effects on sugar and ion transport. Insulin did not interfere with inhibition of sugar transport and decrease in internal Na⁺ but prevented stimulation of sugar transport. Under anoxia adrenalin had no effect on sugar transport but led to greater Na⁺ gain by tissue. Addition of 3.0 mM palmitate decreased inhibition of sugar transport without changing receptor specificity. ATP was decreased and lipolysis enchanged by high adrenalin but glucose-6-P was increased by the low concentration as well. Influx of ⁴⁵Ca was decreased by low and increased by high adrenalin; ⁴⁵Ca efflux was also differentially affected. The results indicate that inhibition and stimulation of sugar transport depend on different receptors and that the latter response may override the former. The data are consistent with the earlier postulated regulatory role of sarcoplasmic Ca²⁺ on sugar transport in muscle, with adrenalin affecting Ca²⁺ fluxes and distribution both directly and indirectly.     

Effect of ionophore A23187 upon membrane function and ion movement in human and toad erythrocytes

Summary Addition of 0.1–0.3 m A23187, a divalent cation ionophore, to human erythrocytes suspended in a 1.0mm ⁴⁵Ca²⁺-containing buffer results in a small ( two fold) increase in [Ca²⁺] _i , a significant decrease in osmotic fragility, and a decrease in intracellular K⁺ (100 mmoles/liter of cells to 70 mmoles/liter cells) without significant alteration of intracellular [Na⁺]. This decrease in [K⁺] _i is associated with a significant decrease in packed cell volume and correlates directly with the observed alteration is osmotic fragility. Increasing extracellular K⁺ to 125mm prevents the A23187-induced changes in osmotic fragility, K⁺ content and cell volume, but does not prevent the ionophore-induced uptake of⁴⁵Ca²⁺. Addition of 0.1–0.3 m A23187 to toad erythrocytes leads to an increase in⁴⁵Ca²⁺ uptake comparable to that observed in human erythrocytes, but does not alter osmotic fragility, cell volume or K⁺ content. Higher concentrations of ionophore (3.0–10.0 m) cause a 30- to 50-fold increase in⁴⁵Ca²⁺ uptake and concomitant change in K⁺ content, cell volume and osmotic fragility. These changes in cell properties can be prevented by increasing extracellular [K⁺] to 90mm. The difference in sensitivity of the two cell types to A23187 is attributed to the presence of additional intracellular calcium pools within toad erythrocytes that prevent an increase in cytoplasmic Ca²⁺ until Ca²⁺ uptake is increased substantially at the higher concentrations of A23187.     

Proton countertransport by the reconstituted erythrocyte Ca2+-translocating ATPase: Evidence using ionophoretic compounds

Summary Human erythrocyte Ca²⁺-translocating ATPase was solubilized from calmodulin-depleted membranes using the detergent Triton X-100, and subsequently purified by calmodulin-affinity chromatography. The purified enzyme was reconstituted in artificial phospholipid vesicles using a cholate-dialysis method and various phospholipids. The reconstituted enzyme was able to translocate Ca²⁺ inside the vesicles, both in the absence and in the presence of the Ca²⁺-chelating agent, oxalate, inside the vesicles. The tightness of coupling between ATP hydrolysis and cation translocation was investigated by the use of different ionophoretic compounds. The efficiency of Ca²⁺ translocation was measured by the ability of the ionophores to stimulate ATP hydrolytic activity of the reconstituted enzyme. It was found that the maximum stimulation of the ATP hydrolytic activity was induced by the electroneutral Ca²⁺/2H⁺ ionophore A23187 (9 to 10-fold). A Ca²⁺ ionophore unable to translocate H⁺, CYCLEX-2E, was less efficient in stimulating the activity of the reconstituted enzyme (two- to threefold). However, the combined addition of CYCLEX-2E plus protonophores further increased the ATP hydrolytic activity (around fourfold), whereas, the protonophores did not further stimulate ATP hydrolysis in the presence of A23187. Furthermore, in the absence of Ca²⁺ ionophore, the electroneutral K⁺(Na⁺)/H⁺ ionophoretic exchanger, nigericin, or the electroneutral Na⁺(K⁺)/H⁺ ionophoretic exchanger, monensin, stimulated the rate of ATP hydrolysis in the reconstituted enzyme two- or threefold, respectively. These results suggest that the Ca²⁺-ATPase not only translocates Ca²⁺ but also H⁺ in the opposite direction.     

An ion exchange chromatographic method for the determination of synaptosomal calcium uptake

A simple, rapid method for determining depolarization-induced⁴⁵Ca influx into synaptosomes is described. Synaptosomes which had been depolarized in the presence of⁴⁵Ca were applied to a small column of Chelex-100 resin to separate free Ca²⁺ from that taken up by the tissue. Approximately 70% of the synaptosomal protein applied to the column was recovered in the initial eluate. The magnitude of⁴⁵Ca uptake was dependent on the amount of Ca²⁺ in the incubation medium and on the KCl concentration. Calcium influx reached a plateau after 90 sec of incubation at 24°C. The Na⁺ channel activator veratridine also produced a substantial influx of⁴⁵Ca, and this effect was blocked by tetrodotoxin. Thus, this ion exchange procedure makes it possible to measure depolarization-induced Ca²⁺ influx in synaptosomes without subjecting them to high vacuum or centrifugation pressures or to EGTA-containing solutions.     

Barium Influx Mediated by the Cardiac Sodium-Calcium Exchanger in Transfected Chinese Hamster Ovary Cells

We examined Ba²⁺ influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na⁺/Ca²⁺ exchanger (CK1.4 cells). Ba²⁺ competitively inhibited exchange-me diated ⁴⁵Ca²⁺ uptake with a K _i ∼ 3 mM. Ba²⁺ uptake was stimulated by pretreating the cells with ouabain and by removing extracellular Na⁺, as expected for Na⁺/Ba²⁺ exchange activity. The maximal velocity of Ba²⁺ accumulation was estimated to be 50% of that for Ca²⁺. When the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na⁺, Ba²⁺ influx was negligible in the absence of Na⁺ and increased to a maximum at 20–40 mM Na⁺. At higher Na⁺ concentrations, Ba²⁺ influx declined, presumably due to the competition between Na⁺ and Ba²⁺ for transport sites on the exchanger. Unlike Ca²⁺, Ba²⁺ did not appear to be taken up by intracellular organelles: Thus, ¹³³Ba²⁺ uptake in ouabain-treated cells was not reduced by mitochondrial inhibitors such as Cl-CCP or oligomycin-rotenone. Moreover, intracellular Ca²⁺ stores that had been depleted of Ca²⁺ by pretreatment of the cells with ionomycin (a Ca²⁺ ionophore) remained empty during a subsequent period of Ba²⁺ influx. Ca²⁺ uptake or release by intracellular organelles secondarily regulated exchange activity through alterations in [Ca²⁺]_i. Exchange-mediated Ba²⁺ influx was inhibited when cytosolic [Ca²⁺] was reduced to 20 nM or less and was accelerated at cytosolic Ca²⁺ concentrations of 25–50 nM. We conclude that (a) Ba²⁺ substitutes for Ca²⁺ as a transport substrate for the exchanger, (b) cytosolic Ba²⁺ does not appear to be sequestered by intracellular organelles, and (c) exchange-mediated Ba²⁺ influx is accelerated by low concentrations of cytosolic Ca²⁺.     

A Ca2+-refractory state of the Ca-sensitive K+ permeability mechanism in sickle cell anaemia red cells

Simultaneous measurements of Ca content and ⁴²K⁺ influx in sickle cell anaemia red cells confirm predictions from earlier data in the literature that the increased Ca content of sickle cell anaemia cells which are not metabolically depleted does not cause a quinine-sensitive increase in K⁺ permeability.It is shown that the ionophore, A23187, can cause the Ca contained inside sickle cell anaemia cells to activate the quinine-sensitive K⁺-permeability mechanism. This demonstrates the existence of a Ca²⁺-refractory state of the K⁺ channel in sickle cell anaemia cells and a direct stimulatory effect of the ionophore A23187 on its Ca sensitivity.     

Plasma membrane Ca2+ transport: stimulation by soluble proteins.

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

Inhibition by Quinacrine of Depolarization-Induced Acetylcholine Release and Calcium Influx in Rat Brain Cortical Synaptosomes

The effects of quinacrine on depolarization-induced [³H]acetylcholine (ACh) release and ⁴⁵Ca²⁺ influx were examined in rat brain cortical synaptosomes. Quinacrine significantly reduced the stimulated release of [³H]ACh by high K⁺ and veratridine without affecting the spontaneous efflux from the preloaded synaptosomes. Quinacrine had no effect on ionophore A23187-induced release of [³H]ACh from the synaptosomes. Quinacrine (100 μM) markedly diminished the stimulated Ca²⁺ influx by veratridine and high K⁺ but not that by “Na⁺-free.” Trifluoperazine, a potent calmodulin antagonist, inhibited both Ca²⁺ influx and ACh release induced by the depolarizing agents. Inhibitory potencies of the two drugs on ACh release and Ca²⁺ influx were compared with the antagonism of calmodulin by two drugs, suggesting that the inhibition of depolarization-induced Ca²⁺ influx and ACh release by these drugs could not be explained by the antagonism of calmodulin.