Ionophore A23187: the effect of H+ concentration on complex formation with divalent and monovalent cations and the demonstration of K+ transport in mitochondria mediated by A23187.

The two-phase extraction technique has been used to study the equilibrium between A23187, metal cations, and H+. Under these conditions the ionophore forms charge neutral isostoichiometric complexes with divalent cations in which both carboxylate groups of the 2:1 A23187:M2+ complexes are deprotonated. In ethanol, however, the methyl ester of A23187 also binds divalent cations indicating that protonated complexes between A23187 and cations should also exist. With monovalent cations, A23187 forms two charge-neutral complexes of stoichiometries and relative stabilities: A2HM greater than AM. Examination of energy utilization K+ and H+ movements, and light scattering capacity of mitochondria in the presence of divalent cation chelators, A23187, and valinomycin demonstrates that A23187 can act as a nigericin type K+ ionophore under appropriate conditions. Formation constants for the A2HM complexes with monovalent cations indicate that with appropriate conditions transport of Li+ and Na+ mediated by A23187 would also be expected. The binding constant data and associated free energies of complex formation are compared as a function of ionic radius and of cation charge. The data indicate that lack of conformational mobility in A23187 is responsible for the high cation size selectivity of this compound. To explain the transport selectivity of A23187 for divalent cations, it is proposed that this ionophore forms a family of five complexes, isostoichiometric between cations of different valence but of which only charge-neutral species are permeant to membranes. The charge of a given complex is in turn determined by that of the cation. The concept is consistent with the divalent cation transport specificity of A23187, explains the observed monovalent cation transport, and is useful in rationalizing the differences in charge selectivity between A23187 and X-537A.     

Fluorescence study of the divalent cation-transport mechanism of ionophore A23187 in phospholipid membranes

The mechanism for transport of divalent cations across phospholipid bilayers by the ionophore A23187 was investigated. The intrinsic fluorescence of the ionophore was used in equilibrium and rapid-mixing experiments as an indicator of ionophore environment and complexation with divalent cations. The neutral (protonated) form of the ionophore binds strongly to the membrane, with a high quantum yield relative to that in the aqueous phase. The negatively charged form of the ionophore binds somewhat less strongly, with a lower quantum yield, and does not move across the membrane. Complexation of the negatively charged form with divalent cations was measured by the decrease in fluorescence. An apparent rate constant (kapp) for transport of the ionophore across the membrane was determined from the rate of fluorescence changes observed in stopped-flow rapid kinetic experiments. The variation of kapp was studied as a function of pH, temperature, ionophore concentration, membrane lipid composition, and divalent cation concentration and type. Analysis and comparison with equilibrium constants for protonation and complexation show that A23187 and its metal:ionophore complexes bind near the membrane-water interface in the lipid polar-head region. The interfacial reactions occur rapidly, compared with the transmembrane reactions, and are thus in equilibrium during transport. The transport cycle can be described as follows: a 1:1 complex is formed between the membrane bound A23187-(Am-) and the aqueous divalent cation with dissociation constant K1 approximately 4.6 x 10(-4) M. This is in equilibrium with a 1:2 (metal:ionophore) complex (K2 approximately 3.0 x 10(-4) [ionophore/lipid]) that is responsible for transporting the divalent cations across the membrane. The rate constant for translocation of the 1:2 complex is 0.1-0.3 s-1. Dissociation of the complex of the trans side and protonation occur rapidly. The rate constant for translocation of H+ . A23187- is 28 s-1. A theory is presented that is capable of reproducing the kinetic data at any calcium concentration. The cation specificity for ionophore complex transport (kapp), determined at low ionophore concentration for a series of divalent cations, was found to be proportional to the equilibrium constant for 1:1 complexation. The order of ion specificity for these processes was found to be Ca2+ greater than Mg2+ greater Sr2+ greater than Ba2+. Interactions with Na+ were not observed. Maximal values of kapp were observed for vesicles prepared from pure dimyristoyl phosphatidylcholine. Inclusion of phosphatidyl ethanolamine, phosphatidic acid, or dipalmatoyl phosphatidylcholine resulted in lower values of kapp. Calcium transport by A23187 is compared with that of X537A, and it is shown that the former is 67-fold faster. The difference in rates is due to differences in the ability of each ionophore to form a 1:2 complex from a 1:1 complex.     

The stoichiometry of A23187- and X537A-mediated calcium ion transport across lipid bilayers

Initial rates of ionophore-mediated Ca2+ transport across egg phosphatidylcholine bilayers of large unilamellar vesicles were measured using the absorbance change of arsenazo III at 650 nm as an indicator of Ca2+ translocation. A23187 induced the movement of Ca2+ in a 2:1 ionophore: Ca2+ complex, whereas its methyl ester (CH3A23187) and X537A mediated Ca2+ movement in a 1:1 ionophore: Ca2+ complex. The relative potencies of these ionophores in transporting Ca2+ across lipid membranes were A23187 much greater than X537A greater than CH3A23187.     

influence of Ca channel blockers on the positive inotropic effect of ionophore A23187 in the myocardium

Calcium ionophore A23187 (10(-5) M) increases the force of contraction of the frog atrium up to 27 + 4.8%. The calcium antagonists d-600 (5 X 10- M), Zn2+ (2 X 10(-5) M), Mn2+ (2 X 10(-4) M) decrease the force of contraction 50, 10 and 20%, respectively, and inhibit the positive inotropic effect of ionophore A23187. Inhibition of the ionophore effect by the blockers is determined by the ability of D-600, Zn2+ and Mn2+ to form complexes with the ionophore. Besides, the affinity of these blockers to the ionophore is higher than that of Ca2+. It is assumed that Zn2+, Mn2+ and D-600 possess higher affinity to ionophore A23187 as compared with myocardial Ca-channels. Fenigidin interacts with Ca-channels to a larger degree than with ionophore A23187.     

General features in the stoichiometry and stability of ionophore A23187-cation complexes in homogeneous solution

Existing literature describing the stoichiometry and stability of complexes between A23187 and divalent cations in solution has been extended to include additional transition series cations, the heavy-metal cations Cd2+ and Pb2+, plus seven lanthanide series trivalent cations. Stability constants of 1:1 complexes between the ionophore and the divalent cations vary by 6.2 orders of magnitude between Cu2+ and Ba2+ which are the strongest and weakest complexes, respectively. Considering alkaline-earth and first-series transition cations together, the pattern of stability constants obeys the extended Irving-Williams series as is seen with many nonionophorous liganding agents. Cd2+ and Pb2+ are bound with an affinity similar to those of Mn2+ and Zn2+, whereas the lanthanides are bound with little selectivity and slightly higher stability. Titration of the ionophore in the 10(-5) M concentration range with di- and trivalent cations gives rise first to complexes of stoichiometry MA2 and subsequently to MA as the metal concentration is increased. The second stepwise stability constants for formation of the MA2 species exceeds the first constant by approximately 10-fold. With lanthanides, heavy metals, and transition-metal cations, OH-, at near physiological concentrations, competes significantly with free ionophore for binding to the 1:1 complexes. This competition is not apparent when Ca2+ or Mg2+ are the central cations. Possible implications of the 1:1 complex selectivity pattern, the ionophore-hydroxide competitive binding equilibria, and potential ternary complexes involving 1:1 ionophore:cation complexes and other anions present in biological systems are discussed with respect to the ionophore's transport selectivity and biological actions.     

Effects of heavy metal ions on phospholipid metabolism in human neutrophils: relationship to ionophore-mediated cytotoxicity

This study examined the effects of 0.1mM heavy metal ions (Au3+, Zn2+, Cr3+, Mn2+, and Cu2+) on ionophore-treated human neutrophils. Treatment of human neutrophils with 5-10 microM ionophore A23187 resulted in phospholipid deacylation and eicosanoid release within 5 min. After approximately 20 min, viability decreased significantly with near total cell death by 50 min. Heavy metal ions altered phospholipid metabolism, eicosanoid synthesis, and cytotoxicity in parallel fashion. Radioimmunoassays for 5-HETE and LTB4 demonstrated that Au3+ and Zn2+ stimulated, Cr3+ had little effect on, and Mn2+ and Cu2+ inhibited eicosanoid release from ionophore-treated neutrophils. Cells prelabelled with [3H]arachidonic acid exhibited similar metal-mediated effects on lipid metabolism. Strong negative correlations between metal effects on viability and the metabolism of arachidonic acid suggest that eicosanoids participate in ionophore-induced cytotoxicity.     

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.     

Intracellular uptake and alpha-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells.

Intracellular uptake of A23187 and the increased release of amylase and lactate dehydrogenase (LDH) accompanying ionophore uptake was studied using dissociated acinar cells prepared from mouse pancreas. Easily detected changes in the fluorescence excitation spectrum of A23187 upon transfer of the ionophore from a Tris-buffered Ringer's to cell membranes were used to monitor A23187 uptake. Uptake was rapid in the absence of extracellular Ca2+ and Mg2+ (t1/2=1 min) and much slower in the presence of Ca2+ or Mg2+ (t1/2=20 min). Cell-associated ionophore was largely intracellular as indicated by fluorescence microscopy, lack of spectral sensitivity to changes in extracellular Ca2+ and Mg2+, and by equivalent interaction of ionophore with membranes of whole and sonicated cells. A23187 (10 micronm) increased amylase release 200% in the presence of extracellular Ca2+ and Mg2+. In the absence of Ca2+ (but in the presence of Mg2+) A23187 did not increase amylase release. A23187 (10 micronm) also produced Ca2+ -dependent cell damage, as judged by increased LDH release, increased permeability to trypan blue, and by disruption of cell morphology. The cell damaging and amylase releasing properties of A23187 were distinguished by their time course and dose-response relationship. A23187 (1 micronm) increased amylase release 140% without increasing LDH release or permeability to trypan blue.     

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.     

Equilibria between ionophore A23187 and divalent cations: stability of 1:1 complexes in solutions of 80% methanol/water

The cation complexation equilibria between ionophore A23187 and several alkaline earth and first transition series divalent cations have been investigated. Absorption and fluorescence spectroscopy were used to monitor the reactions which were studied in solutions of 80% methanol/water, at 25 degrees C, and under conditions of controlled ionic strength and pH. Titration of the ionophore with divalent cations results first in formation of the dimeric species MA2 and subsequently in the formation of MA+ by disproportionation of the first product. With Zn2+, Ni2+, and Co2+ (above pH approximately 6), a third species is detected which is postulated to be MA.OH. The existence of this species with Mn2+ and alkaline earth cations is uncertain. For formation of MA2, the second stepwise stability constant is similar to or exceeds the first value with all cations studied. However, it is possible to isolate the first reaction and determine accurate stability constants by working at an ionophore concentration of 3 X 10(-8) M or less and by employing pH values which preclude interference by the mixed ionophore/hydroxide species. Under these conditions, the relationship between log KMA' and pH is linear and displays a slope of 1.0. pH-independent stability constants were calculated by using pH-dependent stability constants and the known value of the ionophore's protonation constant in this solvent. The logarithms of the values obtained ranged from 7.54 +/- 0.06 for Ni2+ to 3.60 +/- 0.06 for Ba2+. The selectivity sequence and relative affinities (in parentheses) for the species MA+ are as follows: Ni2+ (977) greater than Co2+ (331) greater than Zn2+ (174) greater than Mn2+ (34) greater than Mg2+ (1.00) approximately equal to Ca2+ (0.89) greater than Sr2+ (0.20) greater than Ba2+ (0.11). Data are discussed in comparison to other studies on the complexation properties of A23187 and in terms of their significance to interpreting the transport properties of this ionophore.     

Carboxylic ionophore (lasalocid A and A23187) mediated lanthanide ion transport across phospholipid vesicles

The transport kinetics of three lanthanide ions (viz., Pr3+, Nd3+, and Eu3+) across dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine unilamellar vesicles mediated by the two carboxylic ionophores lasalocid A and A23187 have been studied by proton nuclear magnetic resonance spectroscopy. Time-dependent changes in the chemical shifts of head group choline signals have been measured to calculate apparent rate constants of transport. These experiments have been done at different ionophore concentrations to determine the stoichiometry of the transporting species. The rates of transport have been found to be faster in the absence of intravesicular La3+ compared to those observed in its presence. The stoichiometry of the transporting species has been found to be 2:1 (ionophore:cation) for both lasalocid A and A23187 in dimyristoylphosphatidylcholine vesicles. However, stoichiometries of greater than 2 have been obtained for lasalocid A mediated lanthanide ion transport across dipalmitoylphosphatidylcholine vesicles. Possible reasons for the observations of such noninteger stoichiometries are discussed. Our results also indicated that A23187 is a more efficient carrier ionophore than lasalocid A.     

Lipid peroxidation and 4-hydroxy-2-nonenal formation by copper ion bound to amyloid-beta peptide

The lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is proposed to be a toxic factor in the pathogenesis of Alzheimer disease. The primary products of lipid peroxidation are phospholipid hydroperoxides, and degraded reactive aldehydes, such as HNE, are considered secondary peroxidation products. In this study, we investigated the role of amyloid-beta peptide (A beta) in the formation of phospholipid hydroperoxides and HNE by copper ion bound to A beta. The A beta1-42-Cu2+ (1:1 molar ratio) complex showed an activity to form phospholipid hydroperoxides from a phospholipid, 1-palmitoyl-2-linoleoyl phosphatidylcholine, through Cu2+ reduction in the presence of ascorbic acid. The phospholipid hydroperoxides were considered to be a racemic mixture of 9-hydroperoxide and 13-hydroperoxide of the linoleoyl residue. When Cu2+ was bound to 2 molar equivalents of A beta(1-42) (2 A beta1-42-Cu2+), lipid peroxidation was inhibited. HNE was generated from one of the phospholipid hydroperoxides, 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylcholine (PLPC-OOH), by free Cu2+ in the presence of ascorbic acid through Cu2+ reduction and degradation of PLPC-OOH. HNE generation was markedly inhibited by equimolar concentrations of A beta(1-40) (92%) and A beta(1-42) (92%). However, A beta(1-42) binding 2 or 3 molar equivalents of Cu2+ (A beta1-42-2Cu2+, A beta1-42-3Cu2+) acted as a pro-oxidant to form HNE from PLPC-OOH. These findings suggest that, at moderate concentrations of copper, A beta acts primarily as an antioxidant to prevent Cu2+-catalyzed oxidation of biomolecules, but that, in the presence of excess copper, pro-oxidant complexes of A beta with Cu2+ are formed.     

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187.

The antibiotic A23187 carries Ca2+ across Müller-Rudin membranes made from 1,2-dierucoyl-sn-glycero-3-phosphocholine and n-decane. The conductance of the membranes is not increased by the Ca2+-transport. The flux depends linearly on Ca2+ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4-5 between pH 6 and pH 8. Maximal Ca2+-fluxes of about 10(-10) mol-cm-2-s-1 were found. A counter transport of H+ could not be detected. The complex formation between A23187 and Ca2+ in egg phosphotidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2:1 complex. Optical absorption measurements on single phophatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.     

Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23187 or to prostaglandin E1. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport.

Exposure of 32P-labelled human platelets to ionophore A23187 results in an increased incorporation of 32P into polypeptides with apparent mol.wts. of 47 000 (P47) and 20 000 (P20), whereas exposure to prostaglandin E1 results in increased labelling of polypeptides with apparent mol.wts. of 24 000 (P24) and 22 000 (P22) [Haslam, Lynham & Fox (1979) Biochem. J. 178, 397-406]. Labelled platelets that had been incubated with ionophore A23187 or prostaglandin E1 were sonicated and rapidly separated into three fractions by differential centrifugation. Electron microscopy and measurement of marker enzymes indicated that the 1300-19 000 gav. particulate fraction was enriched in granules, mitochondria and plasma membranes, that the 19 000-90 000 gav. particulate fraction was enriched in both intracellular and plasma membranes and that the 90 000 gav. supernatant contained only soluble proteins. 32P-labelled phosphopolypeptide P47 was present almost exclusively in the 90 000 gav. supernatant, whereas phosphopolypeptide P20 was largely dephosphorylated under fractionation conditions that protected other phosphopolypeptides. 32P-labelled phosphopolypeptide P24 was enriched in both particulate fractions, but particularly in the 19 000-90 000 gav. fraction, and may therefore be present in both the intracellular and plasma membranes. Phosphopolypeptide P22 appeared to be similarly distributed. Both particulate fractions were capable of the ATP-dependent oxalate-stimulated uptake of Ca2+. When the 19 000-90 000 gav. membrane fraction was prepared from platelets that had been incubated with ionophore A23187, active uptake of Ca2+ did not occur, but when this fraction was isolated from platelets that had been exposed to prostaglandin E1, uptake of Ca2+ was significantly greater than observed with the corresponding membranes from control platelets. It is suggested that phosphorylation of polypeptide P24 (or P22) by a cyclic AMP-dependent protein kinase may promote the active transport of Ca2+ out of the platelet cytosol.     

Stimulation of phospholipid hydrolysis and cell death by mercuric chloride: evidence for mercuric ion acting as a calcium-mimetic agent

Mercuric chloride stimulates phospholipid hydrolysis and prostaglandin release in 3T3 mouse fibroblasts. This response is distinctly different from that stimulated by other sulfhydryl-reactive agents, but it exhibits a variety of characteristics similar to the phospholipid hydrolysis response stimulated by Ca2+ plus ionophore A23187. Also, the additivity of phospholipid hydrolytic responses stimulated by Hg2+, Ca2+ and A23187 is consistent with Hg2+ interacting with a Ca2+-dependent enzyme(s). These results are consistent with Hg2+ acting by a novel, Ca2+-mimetic mechanism; i.e., with it entering cells and activating cell processes that are activated by Ca2+ in calcium-dependent cell death.     

Influence of solvent and of cation size on the conformations of lasalocid A-lanthanide(III) ion complexes: circular dichroism and fluorescence studies

The interaction of lanthanide(III) nitrates (La3+ to Lu3+) with the carboxylic ionophore lasalocid A (LS) has been studied by circular dichroism (CD) and fluorescence spectroscopic techniques in acetonitrile and in methanol. Analysis of the CD data in acetonitrile has revealed the coexistence of both 1:1 (ionophore:cation) and 2:1 complexes in solution. For 1.22 A greater than ionic radius greater than 1.13 A, 1:1 complexes are preferred, and for 1.13 A greater than ionic radius greater than 1.03 A, 2:1 complexes are preferred. Induced CD bands for Ln3+ ions have been observed upon binding to LS in acetonitrile. The LS-Ln3+ complexes are less stable in methanol than in acetonitrile. CD spectral changes showed that the conformations of the complexes in methanol are different from those in acetonitrile. The complexes have rather open conformations in methanol compared to those in acetonitrile. The results underscore the importance of ionic radius, solvent environment, and ionization state of LS in determining the conformations of the ionophore-cation complexes.     

Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187

Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current.On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca²⁺ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca²⁺, Mg²⁺, or Mn²⁺ are detected. A23187 produces fluorescent transients under conditions of Ca²⁺ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca²⁺ content. Changes in A23187 fluorescence due to mitochondrial Ca²⁺ accumulation are much smaller by comparison and fluorescence transients are not detected.Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca²⁺ and Mg²⁺, and less than 2 nsec in the presence of excess EDTA.The present results are consistent with a model of ionophore-mediated cation transport in which free M²⁺ binds with A23187 at the membrane surface to form the complex M(A23187)⁺. Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187)₂, which carries the divalent cation through the membrane.These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.     

Conditions limiting the use of ionophore A23187 as a probe of divalent cation involvement in biological reactions. Evidence from the slow fluorescence quenching of type A spinach chloroplasts.

The conditions under which ionophore A23187 can be used as a probe of Mg2+ involvement in the reactions of intact (Type A) spinach chloroplasts have been investigated by monitoring ionophore-induced reversal of slow fluorescence quenching. The following observations were made: (1) A23187-dependent reversal of quenching is a strong function of pH. This is consistent with competition between protons and divalent cations for the carboxylic acid moiety of the ionophore. (2) In the presence of exogenous Mg2+, quenching reversal by A23187 is significantly slowed. It is suggested that formation of the dimeric A23187 . Mg2+ complex delays action of the ionophore at the thylakoid membrane by slowing equilibration of the ionophore among chloroplast membrane phases. (3) In the absence of Mg2+, significant interaction of A23187 with certain monovalent cations--Li+ and Na+, but not K+--is observed. Evaluations of the interaction of ionophore A23187 with specific biological systems and inferences of divalent cation involvement, or lack thereof, must take these limitations into account.     

Ionomycin, a carboxylic acid ionophore, transports Pb(2+) with high selectivity

Studies utilizing phospholipid vesicle loaded with chelator/indicators for polyvalent cations show that ionomycin transports divalent cations with the selectivity sequence Pb(2+) > Cd(2+) > Zn(2+) > Mn(2+) > Ca(2+) > Cu(2+) > Co(2+) > Ni(2+) > Sr(2+). The selectivity of this ionophore for Pb(2+) is in contrast to that observed for A23178 and 4-BrA23187, which transport Pb(2+) at efficiencies that are intermediate between those of other cations. When the selectivity difference of ionomycin for Pb(2+) versus Ca(2+) was calculated from relative rates of transport, with either cation present individually and all other conditions held constant, a value of approximately 450 was obtained. This rose to approximately 3200 when both cations were present and transported simultaneously. 1 microM Pb(2+) inhibited the transport of 1 mM Ca(2+) by approximately 50%, whereas the rate of Pb(2+) transport approached a maximum at a concentration of 10 microM Pb(2+) when 1 mM Ca(2+) was also present. Plots of log rate versus log ionomycin or log Pb(2+) concentration indicated that the transporting species is of 1:1 stoichiometry, ionophore to Pb(2+), but that complexes containing an additional Pb(2+) may occur. The species transporting Pb(2+) may include H.IPb.OH, wherein ionomycin is ionized once and the presence of OH(-) maintains charge neutrality. Ionomycin retained a high efficiency for Pb(2+) transport in A20 B lymphoma cells loaded with Indo-1. Both Pb(2+) entry and efflux were observed. Ionomycin should be considered primarily as an ionophore for Pb(2+), rather than Ca(2+), of possible value for the investigation and treatment of Pb(2+) intoxication.     

Dual effects of manganese on prolactin secretion

The effect of Mn2+ (a commonly used Ca2+ antagonist) on prolactin secretion from pituitary cells was investigated. In the presence of normal extracellular Ca2+ levels (2.5mM), Mn2+ inhibited basal, TRH- and K+- stimulated prolactin secretion. The Ca2+ ionophore, A23187, partially overcame the inhibitory effect of Mn2+. However, in the presence of low extracellular Ca2+ (less than 100 microM), which decreased basal prolactin secretion and abolished any stimulatory effects of TRH or K+, a paradoxical stimulatory effect was observed with Mn2+ in the presence of A23187. In the presence of Ca2+, Mn2+ appeared to be inhibitory due to its Ca2+ antagonistic effects, but at low Ca2+ levels, intracellular stimulatory effects of Mn2+ became apparent.