Interaction of metallochromic indicators with calcium sequestering organelles

Examples are presented of the interaction between cell organelles and metallochromic indicators used in the measurement of ionized Ca²⁺. Sarcoplasmic reticulum was found to sequester murexide type indicators along with Ca²⁺ in the presence of ATP, but not to sequester arsenazo III and antipyrylazo III. The presence of a permeable anion suppresses the sequestration of murexide type indicators by the sarcoplasmic reticulum. In the presence of ruthenium red, both rat liver and beef heart mitochondria release sequestered Ca²⁺ with arsenazo III, but not with murexide.     

Oxidation of sulfhydryl groups and inhibition of the (Ca2+ + Mg2+)-ATPase by arsenazo III

In the presence of divalent cations, the metallochromic Ca2+ indicator arsenazo III is reduced by sulfhydryl groups to form an azo anion radical. Reduced arsenazo III is reoxidized back to its original state by oxygen. The formation of the arsenazo III azo anion radical in the presence of sarcoplasmic reticulum vesicles leads to the rapid inhibition of the (Ca2+ + Mg2+)-ATPase. These data indicate that several factors should be considered when arsenazo III is used as a Ca2+ indicator; (1) Functionally important sulfhydryl groups may be oxidized by arsenazo III; (2) the generation of free radicals by arsenazo III reduction may be toxic to the system being studied; (3) the absorbance spectrum of arsenazo III is altered when reduced by sulfhydryl groups.     

The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria.

The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+.     

Spontaneous calcium release from sarcoplasmic reticulum. A re-examination.

A recent study by Blayney and co-workers (Blayney, L., Thomas, H., Muir, J. and Henderson, A. (1977) Biochim. Biophys. Acta 470, 128--133) purported to demonstrate that apparent spontaneous calcium release in sarcoplasmic reticulum is an artifact of the uptake of murexide dye. This report demonstrates that spontaneous calcium release (1) takes place despite equilibration of murexide sarcoplasmic reticulum to a stable baseline; (2) may be reversed by addition of ATP or oxalate after release has begun. The identical phenomenon can be demonstrated utilizing the indicator arsenazo III or Millipore filtration methods. The results suggest that equilibration of the murexide with sarcoplasmic reticulum vesicles must occur prior to ATP addition in order to achieve a stable baseline but that spontaneous calcium release is not an artifact.     

The influence of pH on the absorption spectrum of arsenazo III.

The absorption spectrum of arsenazo III in media containing K+, Mg2+ and Ca2+ is sharply influenced by pH in the range of 7.5--5.0. The effect of pH is particularly pronounced in the wavelength range 532--602 nm due to the large pH dependence of the dissociation constant of Mg-arsenazo III complex. Therefore absorption changes at these wavelengths during muscle contraction cannot be used as reliable indicators of free ionized Ca2+ concentration in the cell. The effect of pH is less pronounced, but still noticeable at the wavelength pairs 575--650 or 660--685 nm. Multiple layers of muscle cells grown on polystyrene coils permit measurement of absorption changes of arsenazo III, introduced into the cells, by equilibration with 0.5 mM arsenazo III under routine culture conditions. The absorbance changes recorded at 660--685 nm are probably related to changes in intracellular free Ca2+ concentration.     

A survey of the available colorimetric indicators for Ca2+ and Mg2+ ions in biological experiments

The binding of Ca²⁺ and Mg²⁺ ions to commercially available and easily synthesizable metallochromic indicators has been systematically examined at pH 7.35, temperature 37°C, ionic strength 0.16, the conditions of blood plasma. The pCa and pMg midpoints of the colour changes of all the useful indicators are reported. In addition to the well-known indicators arsenazo III, chlorophosphonazo III, antipyrylazo III, and murexide for Ca²⁺, and Eriochrome Black T and Eriochrome Blue SE for Mg²⁺, we draw attention to the values of oxyacetazo I, carboxyazo III, tropolone, methylthymol blue, Mordant Black 32, and the tetracyclines.     

How is the cytoplasmic calcium concentration controlled in nerve terminals?

1. The ability of intraterminal organelles to sequester calcium and buffer the cytoplasmic free Ca2+ concentration ([Ca2+]i) has been investigated in isolated mammalian presynaptic nerve terminals (synaptosomes). A combination of biochemical and morphological methods has been used. 2. When the plasmalemma of synaptosomes is disrupted by osmotic shock or saponin, Ca from the medium can be sequestered by two types of intraterminal organelles in the presence of ATP. 2. Typical mitochondrial poisons (e.g., oligomycin, azide and 2,4-dinitrophenol) block the Ca uptake into one type of organelle (mitochondria); the second type of organelle, which has a higher affinity for Ca (half-saturation congruent to 0.35 microM Ca2+) is spared by the mitochondrial poisons. 4. When the "leaky" synaptosomes are incubated in media containing oxalate, and then fixed and prepared for electron microscopy, electron-dense deposits are observed in the intraterminal mitochondria and smooth endoplasmic reticulum (SER). Mitochondrial poisons block the formation of the deposits in the mitochondria, but spare the SER. 5. X-ray microprobe analysis demonstrates that these deposits contain Ca. 6. Experiments with the Ca-sensitive metallochromic indicator, arsenazo III, demonstrate that the intraterminal organelles in the "leaky" synaptosomes can buffer Ca2+ in the medium to below 5 X 10(-7) M. With small (physiological) Ca loads, the Ca2+ is effectively buffered (to < 5 X 10(-7) M) even in the presence of mitochondrial poisons. 7. The data indicate that the SER in presynaptic terminals may play an important role in helping to buffer the Ca that normally enters during neuronal activity.     

The rate of Ca2+ translocation by sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase measured with intravesicular arsenazo III

Release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase into the interior of intact sarcoplasmic reticulum vesicles was measured using arsenazo III, a metallochromic indicator of Ca²⁺. Arsenazo III was placed inside the sarcoplasmic reticulum vesicles by making the vesicles transiently leaky with an osmotic gradient in the presence of arsenazo III. External arsenazo III was then removed by centrifugation. Addition of ATP to the (Ca²⁺ + Mg²⁺)-ATPase in the presence of Ca²⁺ causes the rapid phosphorylation of the enzyme at which time the bound Ca²⁺ becomes inaccessible to external EGTA. The release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase to the interior of the vesicle measured with intravesicular arsenazo III was much slower indicating that there is an occluded from the Ca²⁺-binding site which precedes the release of Ca²⁺ into the vesicle. The rate of Ca²⁺ accumulation by sarcoplasmic reticulum vesicles is increased by K⁺ (5–100 mM) and ATP (50–1000 μM) but the initial rate of Ca²⁺ translocation measured after the simultaneous addition of ATP and EGTA to vesicles that were preincubated in Ca²⁺ was not influenced by these concentrations of K⁺ and ATP.     

Isolation of molecules recognized by monoclonal antibodies and antisera: the solid phase immunoisolation technique

Coupling of Ca2+ transport to ATP hydrolysis in isolated sarcoplasmic reticulum vesicles has been studied following pulsed additions of either ATP or Ca2+. ATP was infused as a pulse into medium, whose free Ca2+ concentration was maintained constant at saturating levels by a calciumstat procedure, using either a Ca2+-selective electrode or the spectrophotometric arsenazo III technique as Ca2+ indicators. The low ATP levels virtually exclude contributions by "basal" ATPase activity. Passive leakage of Ca2+, monitored after an ATP pulse, does not contribute more than 5% to subintegral coupling ratios. Pulsed additions of Ca2+ were made into medium. containing saturating concentrations of ATP, whose hydrolysis was monitored by a pH-stat procedure. Ca2+-stimulated hydrolysis continued until all the Ca2+ was transported into the vesicles. Values for the coupling ratio, Ca2+/ATP, of 1.82 +/- 0.12 and 1.79 +/- 0.15 were obtained by the ATP- and Ca2+-pulse methods, respectively.     

Calcium binding to metallochromic dyes and calmodulin in the presence of combined, AC-DC magnetic fields.

The possibility that weak, ac and dc magnetic fields in combination may affect binding equilibria of calcium-ions (Ca2+) was investigated with two metallochromic dyes as calcium-binding molecules: murexide and arsenazo III. Calcium-dye equilibria were followed by measuring solution absorbances with a fiber-optic spectrophotometer. A Ca(2+)-arsenazo solution was also used indirectly to monitor the binding of Ca2+ to calmodulin. Parallel, ac and dc magnetic fields were applied to each preparation. The ac magnetic field was held constant during each of a series of experiments at a frequency in the range between 50 and 120 Hz (sine wave) or at 50 pps (square wave) and at an rms flux density in the range between 65 and 156 microT. The dc magnetic field was then varied from 0 to 299 microT at 1.3 microT increments. The magnetic fields did not measurably affect equilibria in the binding of metallochromic dyes or calmodulin to Ca2+.     

Essential adaptation of the calcium influx assay into liposomes with entrapped arsenazo III for studies on the possible calcium translocating properties of acidic phospholipids

An adapted version of the Ca2+-influx assay of Weissmann et al. (Weissmann, G., Anderson, P., Serhan, C., Samuelson, E. and Goodman, E. (1980) Proc. Natl. Acad. Sci. USA 77, 1506-1510) is presented for studies on the possible ionophoretic properties of acidic phospholipids. This method is based on the use of the metallochromic dye arsenazo III enclosed in liposomal vesicles, to indicate the Ca2+ influx. An essential control is introduced to discriminate between Ca2+-arsenazo III complex formation inside the vesicles, as a consequence of Ca2+ influx, and outside the vesicles, as a consequence of arsenazo III leakage from the vesicles. Furthermore, some minor improvements are added, like the use of large unilamellar vesicles instead of multilamellar vesicles, and the use of dual wavelength spectrophotometry. Using this method, it was found that dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidylglycerol, were impermeable to Ca2+. In this system a selective Ca2+ permeability could be induced by the addition of the fungal Ca2+ ionophore A23187. In contrast, dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidic acid, incubated in the presence of Ca2+ were permeable to both Ca2+ and arsenazo III.     

Modification of calcium fluxes by dimethyl sulfoxide and 2-butoxyethanol in sarcoplasmic reticulum vesicles: a possible mechanism for skeletal muscle relaxation induced by dimethyl sulfoxide

In order to investigate the mechanism of skeletal muscle relaxation induced by dimethyl sulfoxide, 2-butoxyethanol and dimethyl sulfoxide were examined for their effects on 1) Ca2+ uptake into and efflux from sarcoplasmic reticulum vesicles prepared from rabbit fast skeletal muscle and crayfish tail muscle by the murexide method, 2) ATPase activities of rabbit reticulum vesicles, 3) the isolated phrenic nerve-diaphragm preparation of the rat and 4) crayfish opener muscle preparation. Ca2+ efflux rate from rabbit reticulum vesicles was markedly decreased with increasing concentrations (5-20% v/v) of dimethyl sulfoxide without affecting the maximum Ca2+ uptake by the reticulum. 2-Butoxyethanol showed quite contrary effects. Dimethyl sulfoxide strongly inhibited the activity of basal ATPase rather than of Ca2+-dependent ATPase. 2-Butoxyethanol did not significantly inhibit the activity of basal ATPase, but markedly increased Ca2+-dependent ATPase activity. Antagonisms between dimethyl sulfoxide and caffeine were demonstrated either in contractions of crayfish opener muscles or in the Ca2+ release from crayfish sarcoplasmic reticulum vesicles. These results indicate a possibility that dimethyl sulfoxide reversibly induces skeletal muscle relaxation mainly in the sarcoplasmic reticulum by means of decreasing the rate and the amount of Ca2+ release from the reticulum.     

The effects of Mg2+ and adenine nucleotides on the sensitivity of the heart mitochondrial Na+-Ca2+ carrier to extramitochondrial Ca2+. A study using arsenazo III-loaded mitochondria.

The technique of reversible Ca2+-induced permeabilization [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29, 31-40] has been applied to the preparation of heart mitochondria loaded with the Ca2+ indicator arsenazo III (2 nmol of arsenazo III/mg of mitochondrial protein). The loaded mitochondria ('mitosomes') were used to study the control of the Na+-Ca2+ carrier by extramitochondrial Ca2+ mediated by putative regulatory sites. The Vmax. of the Na+-Ca2+ carrier and the degree of regulatory-site-mediated inhibition were similar to normal heart mitochondria. Ca2+ occupation of the sites in mitosomes yields partial inhibition, which is half-maximal with 0.8 microM external free Ca2+. The inhibition consists of a small decrease in Vmax. and a relatively large increase in apparent Km for internal Ca2+. Mg2+ also appears to interact with the sites, but this is largely abolished by ATP and ADP (but not AMP) under conditions in which the free [Mg2+] is maintained constant. The results indicate that the regulatory sites are effective in controlling the Na+-Ca2+ carrier at physiological concentrations of adenine nucleotides, Mg2+, intra- and extra-mitochondrial free Ca2+.     

Kinetic studies of calcium release from sarcoplasmic reticulum in vitro

Release of Ca2+ from a heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum was triggered by several different methods: (a) increasing extravesicular [Ca2+] [( CaO2+] from about 0.1 microM to 10 microM), (b), adding caffeine, (c) adding quercetin, and (d) substituting a solution containing equimolar choline+ for K+-containing solution (depolarization-induced Ca2+ release). The maximal rate of Ca2+ release triggered by caffeine or quercetin in the presence of 12.5 microM [CaO2+] (21-25 nmol of Ca2+/mg/s) is similar to that of the depolarization-induced Ca2+ release (19 nmol of Ca2+/mg/s), as determined by stopped flow spectrometry of changes in [CaO2+] with arsenazo III. The release is transient and all of the released Ca2+ is reaccumulated. The rates of Ca2+ release triggered by caffeine, quercetin, or membrane depolarization sharply decrease at high [CaO2+], suggesting a negative feedback effect of the released Ca2+. Inhibition of the release pathway allows the sarcoplasmic reticulum to reaccumulate Ca2+. The rate of Ca2+ release triggered by caffeine or quercetin, but not that triggered by membrane depolarization, is also reduced upon decreasing [CaO2+] to the submicromolar range. Passive efflux of intravesicular Ca2+ in solutions containing lower [CaO2+] in the absence of Mg.ATP is attenuated at about the same time (congruent to 1 min) regardless of the amounts of Ca2+ released, indicating that the opened Ca2+ channels close spontaneously. These results suggest that kinetically identical channels are responsible for Ca2+ release independent of the methods of triggering and this in vitro release is consistent with the physiological mechanism both in terms of the rapidity and the reversibility of Ca2+ release.     

Fluorescence and differential light absorption recordings with calcium probes and potential-sensitive dyes in skinned cardiac cells

This report describes an optical system for microspectrophotometry in a single cardiac cell from which the sarcolemma has been removed by microdissection (skinned cardiac cell). This system is attached to the high power inverted microscope used for the microdissection and includes (a) a single variable wavelength microspectrophotometer used to define the spectrum of a given dye or Ca2+ probe; and (b) a dual wavelength, differential microspectrophotometer used to record differentially between the optimum wavelength and a wavelength separated by 25--30 nm. Results are presented using the following optical methods: (a) fluorescence measurements with chlorotetracycline to monitor the amount of Ca2+ bound to the inner face of the sarcoplasmic reticulum (SR) membrane; (b) differential absorption measurements with arsenazo III to measure changes of myoplasmic [Ca2+]free resulting from Ca2+ release from the SR; (c)fluorescence and (or) differential absorption measurements with the potential-sensitive dyes merocyanine 540, NK 2367, and di-S-C3(5) to monitor changes of charge distribution on the SR membrane during Ca2+ accumulation in the SR, as well as before and during Ca2+-induced release of Ca2+ from the SR. A small and rapid signal is observed which precedes the Ca2+-induced release of Ca2+ from the SR. It is detected as an increase of CA2+ binding inside the SR with chlorotetracycline and as a "hyperpolarization" with potential-sensitive dyes, while no transient change of myoplasmic [Ca2+]free is detected with arsenazo III. This small and rapid signal preceding the Ca2+ release may be a first hint to an understanding of the mechanism whereby a small increase of [Ca2+]free outside the SR triggers Ca2+ release from the SR.     

Effects of pH, temperature, and calcium concentration on the stoichiometry of the calcium pump of sarcoplasmic reticulum

Coupling of Ca2+ transport to ATP hydrolysis by isolated skeletal muscle sarcoplasmic reticulum vesicles has been investigated by means of ATP pulse methods. The stoichiometric amounts of Ca2+ transported per pulse of ATP were measured by Ca2+-stat methods, using either a Ca2+ electrode or arsenazo III as end point detectors, or by means of 45CaCl2. Maximum coupling ratios (Ca2+/ATP), of 1.82 +/- 0.13 occurred at pH 6.8, 25 degrees C, and in the presence of saturating Ca2+ concentrations. Ca2+/ATP values decreased at alkaline pH, with an apparent pK alpha of 7.9. The coupling ratio was unaltered between 6 and 30 degrees C, but decreased to 0.4 at 42 degrees C. Uncoupling by alkaline pH and high temperatures was reversible. The coupling process was Ca2+-dependent, with a K0.5 value for Ca2+ of 0.12 microM and a Hill coefficient of 2.0. Ca2+ ions, which were transported into vesicles under conditions resulting in low coupling ratios, were retained as the calcium oxalate precipitate, following complete hydrolysis of substrate. Passive Ca2+ efflux and Ca2+ exchange, were independent of pH. The observed variations in Ca2+/ATP ratio cannot readily be explained on the basis of a pump-leak model. Rather, the Ca2+-ATPase appears to be capable of pumping Ca2+ ions, under physiological conditions, with variable stoichiometry that is dependent upon its thermodynamic loading.     

Diffusion of ions and indicator dyes in neural cytoplasm

The dispersion of dye molecules and small cations injected from a point source in the cytoplasm of molluscan neurons has been measured photometrically and compared with dispersion in aqueous solution. The diffusion of phenol red and arsenazo III was at least a factor of five slower in the cytoplasm than in saline. Movement of both dyes was slowed by about the same factor in a given cell. The dispersion rate of arsenazo III was not significantly affected by preloading the cytoplasm to dye concentrations up to 0.5 mM. Calcium and barium dispersion was measured in neurons and saline droplets preloaded with arsenazo III, while phenol red absorbance changes were used to follow the dispersion of injected protons. Ba2+ and H+ moved very slowly in the cytoplasm compared to aqueous solution. Ca2+ movement in all probability underwent a similar retardation in the neurons but high-affinity buffering of the cytoplasm severely restricted the spread of detectable amounts of this ion away from the injection site.     

A method of estimating the amount of calcium bound to the metallochromic indicator arsenazo III

As a metallochromic indicator for ionized calcium, arsenazo III is approximately 50 times more sensitive than murexide. However, because of the high binding constant for calcium, the following problems may occur: (a) a considerable amount of calcium is bound to arsenazo III, thereby causing an error in estimating the concentration of ionized calcium; (b) the amount of bound calcium varies with the concentrations of calcium, arsenazo III, magnesium ion and monovalent cations; (c) the amount also varies with pH, (d) the relationship between the absorbance change and the concentration of ionized calcium is nonlinear; and (e) the binding constant of arsenazo III for calcium cannot be determined by the conventional double reciprocal plot. A new experimental and theoretical method is presented which copes with these problems.     

Internalization of metallochromic Ca2+ indicators in mammalian cells

Two new techniques for internalizing metallochromic indicators into the cytosol of mammalian cells are described. One method consists of hypertonically treating the cells in the presence of the indicator, followed by a hypoosmotic treatment. The second method consists of incubating the cells at high density in a concentrated indicator solution in physiological saline. Using either method, arsenazo III or antipyrylazo III was internalized into Ehrlich Ascites tumor (EAT) cells at concentrations yielding measurable differential absorbance changes which correspond to changes in the intracellular Ca2+ concentration. In the case of antipyrylazo III, the amount of indicator internalized ranged between 140 and 350 microM, and was dependent on the metabolic state of the cell during loading. Control and loaded cells possessed virtually identical ATP/ADP ratios, as measured by high performance liquid chromatography (HPLC) in cell extracts. Antipyrylazo III was also internalized by rat hepatocytes without detectable cell damage. Treatment of metabolically active EAT cells with the calcium ionophore A23187 results in only a slight increase in the intracellular free Ca2+ concentration, [Ca2+]i, whereas treatment with the calcium ionophore ionomycin induces a substantial but transient increase in the [Ca2+]i. In contrast, metabolically inhibited EAT cells show a large rise in the [Ca2+]i upon addition of A23187. Thus, these techniques offer another way of measuring intracellular free Ca2+ changes in mammalian cells and may prove useful, especially where concentrations of free cytosolic Ca2+ larger than 1 microM are expected.     

Inhibitors of Ca2+ release from the isolated sarcoplasmic reticulum. I. Ca2+ channel blockers

The effects of Ruthenium red and tetracaine, which inhibit Ca2+-induced Ca2+ release from the isolated sarcoplasmic reticulum (e.g., Ohnishi, S.T. (1979) J. Biochem. (Tokyo) 86, 1147-1150), on several types of Ca2+ release in vitro were investigated. Ca2+ release was triggered by several methods: (1) addition of quercetin or caffeine, (2) Ca2+ jump, and (3) replacement of potassium gluconate with choline chloride to produce membrane depolarization. The time-course of Ca2+ release was monitored using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Ruthenium red inhibited all of these types of Ca2+ release with the same concentration for half-inhibition C1/2 = 0.08-0.10 microM. Similarly, tetracaine inhibited these types of Ca2+ release with C1/2 = 0.07-0.11 mM. Procaine also inhibits both types of Ca2+ release induced by method 2 and 3 with C1/2 = 0.67-1.00 mM. These results suggest that Ruthenium red, tetracaine and procaine interfere with a common mechanism of the different types of Ca2+ release. On the basis of several pieces of evidence we propose that Ruthenium red and tetracaine block the Ca2+ channel of sarcoplasmic reticulum.