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.     

The binding of arsenazo III to cell components

The Ca²⁺ indicator, arsenazo III, binds to subcellular fractions of rabbit skeletal muscle with sufficient affinity that in living muscle containing 1–2 mM arsenazo III, the estimated free arsenazo III concentration is only 50–200 μM; 80–90% of the bound arsenazo III is associated with soluble proteins.The binding of arsenazo III to soluble proteins decreases the optical response of the dye to Ca²⁺; this is due to a decrease in the affinity of the protein-bound dye for Ca²⁺. Approximately half of the bound arsenazo III is released from the particulate fraction and soluble proteins upon addition of 5 mM Ca²⁺, suggesting that the Ca-arsenazo complex has lower affinity for the protein binding sites than the free dye.The Ca²⁺ binding to the soluble protein fraction of rabbit skeletal muscle is attributable largely to its parvalbumin content.     

Quantitative measurements of the cytosolic Ca2+ activity within isolated guinea pig nerve-endings using entrapped arsenazo III and quin2

The absorbance changes of intrasynaptosomally entrapped arsenazo III have been converted into values of free Ca²⁺ concentration by correcting for the nonlinear response of arsenazo III at different concentrations of the dye as well as for changes in internal pH. An average resting value for free Ca²⁺ concentration around 0.4 μM is obtained. Depolarization with veratridine or gramicidin increases this value to around 3 μM. Measurements of cytosolic free Ca²⁺ with the quin2 method gives much lower values in similar conditions. The release of prelabelled [¹⁴C]noradrenaline from the nerve-endings is maximally activated when the internal free Ca²⁺ concentration rises as measured with arsenazo III to about 4 μM when titrated with increasing concentrations of ionophore A23187.     

The pH Dependence and the Binding Equilibria of the Calcium Indicator — Arsenazo III

The underlying principles of binding equilibria of arsenazo III with Ca²⁺ and Mg²⁺ are presented. Ca²⁺ and Mg²⁺ can bind arsenazo III in several different protonated forms depending on pH. The binding affinities of these different protonated forms of arsenazo III with Ca²⁺ increase in the order of H₄A_4- <H₃A^5- >H₂A^6- and with Mg²⁺, H₄A^4- > H₃A^5- > H₂A^6-. Arsenazo III is not membrane bound. The sensitivity ratio of arsenazo III with Ca²⁺ to arsenazo III with Mg²⁺ is close to two orders of magnitude. Arsenazo III and its complexes are extremely sensitive to pH changes. With 5 μM arsenazo III, the minimum detectable amount of Ca²⁺ can be as low as 0.08 μM. Contrary to current belief, we found that Mg²⁺ can bind to arsenazo III in a slightly acidic medium. Potential applications of arsenazo III to the study of membrane Ca²⁺ transport are also discussed.     

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.     

Affinity and stoichiometry of calcium binding by arsenazo III

Arsenazo III forms a 1:1 complex with calcium. The affinity constant of arsenazo III for calcium (pK_Ca) has been determined by titrating purified arsenazo III with standard calcium solutions. The method of evaluation used allows one to determine correct pK_Ca values even in the presence of micromolar amounts of contaminating calcium. The pK_Ca is influenced by the following factors: (a) in the neutral pH range the apparent pK_Ca increases strongly with pH; (b) alkali ions bind weakly to arsenazo III and millimolar concentrations cause a decrease in the apparent pK_Ca; (c) the magnesium affinity of arsenazo III, although much lower than the calcium affinity, increases strongly with pH in the neutral range (at pH 7.0 the calcium affinity of arsenazo III is not appreciably altered by up to 2 mm magnesium); (d) strontium and barium form weaker complexes with arsenazo III than calcium, but much stronger complexes than magnesium; (e) the apparent pK_Ca decreases with increasing buffer concentration in the millimolar range. The pK_Ca of arsenazo III is so high that, unless the arsenazo III concentration greatly exceeds the calcium concentration, a considerable fraction of the total arsenazo III is in the calcium complexed form. Because of this, arsenazo III responds nonlinearly to all but the lowest calcium concentrations; however, quantitation of the calcium concentration can readily be done from the mass action law provided that the pK_Ca is determined under the actual experimental conditions. Arsenazo III is a reliable calcium indicator if the experimental conditions, particularly pH, are well controlled.     

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.     

Extrusion of calcium from a single isolated neuron of the snailHelix pomatia

Summary Simultaneous optical measurements of extra- and intracellular Ca²⁺ concentrations were carried out on isolated snail neurons injected iontophoretically with Ca²⁺. The fluorescent indicator Fura-2 was used to measure intracellular concentration of free Ca, and the absorbant indicator Antipyrylazo III to measure changes in extracellular calcium concentration in the microchamber containing the cell. The velocity of Ca²⁺ extrusion from a single cell has been shown to be in accordance with the level of free Ca in the neuronal cytoplasm. After an increase in intracellular free Ca by iontophoretic injection from a microeletrode to 0.2–0.5 m, the velocity of Ca²⁺ extrusion from the neuron was approximately 0.3–4.6 m/sec per cell volume. During caffeine-induced calcium-dependent calcium release of Ca²⁺ from intracellular stores a stimulation of calcium extrusion took place, reaching the velocity of 5.0 m/sec per cell volume.     

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.     

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.     

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscle cells in vivo and in vitro

The effect of medium Ca²⁺ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures.There is an easily identifiable class of muscle protein which includes the Ca²⁺-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomyosin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca²⁺ concentration, 0.05–0.3 mM, where fusion was prevented. Similar medium Ca²⁺ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as ‘calcium-modulated proteins’. The increased Ca²⁺ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca²⁺ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca²⁺ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates.The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca²⁺ concentration between 0.06 and 1.8 mM.It is proposed that increase in cytoplasmic free Ca²⁺ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.     

Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase

Light-mediated activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in intact spinach chloroplasts (Spinacia oleracea L.) is enhanced in the presence of 10⁻⁵ molar external free Ca²⁺. The most pronounced effect is observed during the first minutes of illumination. Ruthenium red, an inhibitor of light-induced Ca²⁺ influx, inhibits this Ca²⁺ stimulated activation. In isolated stromal preparations, the activation of fructose-1,6-bisphosphatase is already enhanced by 2 minutes of exposure to elevated Ca²⁺ concentrations in the presence of physiological concentrations of Mg²⁺ and fructose-1,6-bisphosphate. Maximal activation of the enzyme is achieved between 0.34 and 0.51 millimolar Ca²⁺. The Ca²⁺ mediated activation decreases with increasing fructose-1,6-bisphosphate concentration and with increasing pH. The data are consistent with the proposal that the illumination of chloroplasts leads to a transient increase of free stromal Ca²⁺. In dark-kept chloroplasts the steady-state concentration of free stromal Ca²⁺ is 2.4 to 6.3 micromolar as determined by null point titration. These observations support our previous proposal that light-induced Ca²⁺ influx into chloroplasts does not only influence the cytosolic concentration of free Ca²⁺ but also regulates enzymatic processes inside the chloroplast.     

Phosphate dependent,ruthenium red insensitive Ca2+ uptake in mung bean mitochondria

Energy linked Ca²⁺ uptake into mung bean mitochondria has been studied. Using arsenazo III as a monitor of extramitochondrial Ca²⁺, we observe a respiration-linked uptake of Ca²⁺ which requires phosphate and is insensitive to ruthenium red. The rate of uptake is of the order of 5 nmol/mg protein/min. Acetate, sulphate and thiosulphate are unable to support Ca²⁺ uptake. The results suggest that although plant mitochondria accumulate Ca²⁺ in an energy dependent fashion, it is not via a simple electrophoretic uniport mechanism.     

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca²⁺ inside the ER ([Ca²⁺]_ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca²⁺]_ER (EC₅₀) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca²⁺]^ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 μM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 μM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca²⁺. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 107–112, March–April, 2007.     

25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca²⁺ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca²⁺ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca²⁺ permeability. The effect was not specific for Ca²⁺. Other divalent cations were translocated in the order Mn²⁺ > Mg²⁺  Ca²⁺ ? Sr²⁺  Ba²⁺. The permeability of liposomes to the monovalent cation, Na⁺, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.     

Understanding altered contractile properties in advanced age: insights from a systematic muscle modelling approach

Age-related alterations of skeletal muscle are numerous and present inconsistently, and the effect of their interaction on contractile performance can be nonintuitive. Hill-type muscle models predict muscle force according to well-characterised contractile phenomena. Coupled with simple, yet reasonably realistic activation dynamics, such models consist of parameters that are meaningfully linked to fundamental aspects of muscle excitation and contraction. We aimed to illustrate the utility of a muscle model for elucidating relevant mechanisms and predicting changes in output by simulating the individual and combined effects on isometric force of several known ageing-related adaptations. Simulating literature-informed reductions in free Ca²⁺ concentration and Ca²⁺ sensitivity generated predictions at odds qualitatively with the characteristic slowing of contraction speed. Conversely, incorporating slower Ca²⁺ removal or a fractional increase in type I fibre area emulated expected changes; the former was required to simulate slowing of the twitch measured experimentally. Slower Ca²⁺ removal more than compensated for force loss arising from a large reduction in Ca²⁺ sensitivity or moderate reduction in Ca²⁺ release, producing realistic age-related shifts in the force-frequency relationship. Consistent with empirical data, reductions in free Ca²⁺ concentration and Ca²⁺ sensitivity reduced maximum tetanic force only slightly, even when acting in concert, suggesting a modest contribution to lower specific force. Lower tendon stiffness and slower intrinsic shortening speed slowed and prolonged force development in a compliance-dependent manner without affecting force decay. This work demonstrates the advantages of muscle modelling for exploring sources of variation and identifying mechanisms underpinning the altered contractile properties of aged muscle.

     

Sequential occurrence of mitochondrial and plasma membrane alterations, fluctuations in cellular Ca2+ and pH during initial and later phases of cell death

The sequential occurrence of plasma and mitochondrial membrane alterations, intra-cellular pH shifts and changes in intracellular Ca²⁺ concentration after induction of cell death was monitored by flow cytometry in Jurkat and HSB2-cells. Cell death was induced by treatment with anti-Fas antibodies or by irradiation. Phosphatidylserine (PS) exposure and plasma membrane integrity were measured with FITC-Annexin V adhesion and by Propidium Iodide exclusion. Transition of the mitochondrial membrane potential was monitored by the occurrence of decay of DiOC₆ fluorescence. Intracellular pH shifts were monitored by changes in the ratio of fluorescence at 575 nm and at 635 nm of SNARF-1-AM. Fluctuations in intracellular Ca²⁺ concentration were established by changes in Fura red quenching.The Jurkat cells were sensitive to anti-Fas treatment, while HSB-2 cells were not. HSB-2 cells appeared more sensitive to radiation damage than Jurkat cells.In all experiments the transition of mitochondrial membrane potential occurred first, almost immediately followed by PS exposure. Fluctuations in intracellular Ca²⁺ concentration occurred later and were less outspoken. A decrease in intracellular pH occurred not earlier than 24 hours after anti-Fas treatment. Chelation of intracellular Ca²⁺ concentration with BAPTA-AM had no effect on the time sequence of cell death related events.     

Evidence for structural changes in vertebrate thick filaments induced by calcium

Paired sedimentation studies of isolated, native thick filaments at pH 6.8, I = 0.12 and in the presence of 0.3 mm-free Mg²⁺ show that the sedimentation coefficient increases with Ca²⁺ concentration (pCa² midpoint = 5.5), leveling off at pCa 4.7. The addition of ATP or ADP (5 mm) has no effect on the hydrodynamic changes induced by Ca²⁺. At much higher free Mg²⁺ concentrations (5 mm), the midpoint of the transition is shifted to pCa = 5.3. Viscosity measurements of the filament system under comparable conditions reveal a decrease in the relative viscosity over the same range of Ca²⁺ concentration. Synthetic filaments prepared from purified myosin free of C-protein also show the same behavior. Native filaments from which myosin heads have been removed by treatment with papain do not show Ca²⁺ dependence. The dependence of the sedimentation coefficient of filament on protein concentration, as measured by differential sedimentation, is unaffected by Ca²⁺, indicating that the changes in hydrodynamic properties are probably not related to aggregation of the filaments. The Ca²⁺ effects are reversible and are not observed on replacing Ca²⁺ by Mg²⁺. Binding studies carried out at low ionic strength reveal two binding sites for Ca²⁺ (K_a = 1.7 × 10⁵m^?1) per mole myosin within the filament and evidence is presented showing that the DTNB light chain is the site of binding. The combined results are interpreted as indicating that thick filaments of vertebrate muscle undergo conformational changes at physiological levels of Ca²⁺ and provide evidence for a Ca²⁺-sensitive regulatory mechanism at the level of the thick filament.     

Different threshold levels of postsynaptic [Ca2+]i have to be reached to induce LTP and LTD in neocortical pyramidal cells

Changes in [Ca²⁺]_i were measured in layer II–III pyramid cells of the rat visual cortex slices during application of either LTP or LTD inducing stimulation protocols. At dendritic sites activated by the stimulated afferents [Ca²⁺]_i reached higher amplitudes and decayed more slowly with LTP than with LTD inducing stimuli. In the presence of Ca²⁺ chelators, the stimulation protocol that would normally produce LTP induced either LTD or failed to induce synaptic modifications altogether. These results support the hypothesis that the polarity of synaptic gain changes depends on the magnitude of postsynaptic [Ca²⁺]_i reponses, the induction of LTP requiring a more pronounced surge of [Ca²⁺_i than the induction of LTD.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle