Assessment of Fura-2 for measurements of cytosolic free calcium

Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular free calcium concentration in lymphocytes of patients with muscular dystrophies

Intracellular free calcium concentration [( Ca2+]i) of human peripheral blood lymphocytes was determined by fluorescence spectroscopic measurements with quin2 in patients with different types of muscular dystrophy and in controls. The [Ca2+]i level in lymphocytes showed a significant increase in adult type (facioscapulohumeral and limb-girdle) muscular dystrophies, while it showed a decrease in Duchenne dystrophy as compared to the values of age- and sex-matched controls. The data obtained suggest an alteration in the effectiveness of the calcium pump in lymphocytes and may represent a sign of generalized membrane damage in these hereditary muscle diseases.     

Intracellular free calcium in rat anterior pituitary cells monitored by fura-2

Rat anterior pituitary cells, loaded with the calcium indicator dye fura-2 after primary culture, were challenged with prolactin and growth hormone secretagogues and inhibitory hormones. To initially validate the technique, the calcium channel activator maitotoxin effectively increased intracellular free calcium [( Ca++]i). Various concentrations of the secretagogues thyrotropin releasing hormone or angiotensin II induced peak increases in [Ca++]i within 15 sec, followed by a lower and prolonged plateau phase. The inhibitory hormones dopamine and somatostatin maximally reduced [Ca++]i by 15-20 sec, followed by a spontaneous return to baseline over 5-10 min. The receptor antagonists saralacin and spiperone blocked the angiotensin II and dopamine effects, respectively. Thus, fura-2 appears to be an adequate probe for resolving second-to-second changes in [Ca++]i induced by hormone receptor activation in anterior pituitary cells.     

Activity-dependent calcium transients in central nervous system myelinated axons revealed by the calcium indicator Fura-2.

Optical measurements from rat optic nerve, loaded with the new Ca2+ indicator Fura-2, provide the first evidence for the presence of activity-dependent fast intracellular [Ca2+] transients in mammalian central nervous system (CNS) myelinated axons. The results suggest that voltage-dependent Ca2+ channels are present in some of the myelinated axons. Optical measurements from axons stained with anterogradely transported voltage-sensitive dye suggest the presence of Ca2+-dependent potassium conductances in these axons. This report also demonstrates that Fura-2 can readily detect changes in [Ca2+] inside cells as a result of electrical activity, and establishes its suitability for measurements of intracellular Ca2+ transients in the millisecond time domain.     

Fura-2 antagonises calcium-induced calcium release

Calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER) takes place through ryanodine receptors (RyRs) and it is often revealed by an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by caffeine. Using fura-2-loaded cells, we find such an effect in bovine adrenal chromaffin cells, but not in cerebellar granule neurones or in HEK-293 cells. In contrast, a caffeine-induced [Ca(2+)](c) increase was clearly visible with either fluo-3 or cytosolic aequorin. Simultaneous loading with fura-2 prevented the [Ca(2+)](c) increase reported by the other Ca(2+) probes. Caffeine-induced Ca(2+) release was also measured by following changes of [Ca(2+)] inside the ER ([Ca(2+)](ER)) with ER-targeted aequorin in HEK-293 cells. Fura-2 loading did not modify Ca(2+) release from the ER. Thus, fura-2, but not fluo-3, antagonises the generation of the cytosolic Ca(2+) signal induced by activation of RyRs. Cytosolic Ca(2+) buffering and/or acceleration of Ca(2+) diffusion through the cytosol may contribute to these actions. Both effects may interfere with the generation of microdomains of high [Ca(2+)](c) near the ER release channels, which are essential for the propagation of the Ca(2+) wave through the cytosol. In any case, our results caution the use of fura-2 to study CICR.     

Regulation of cytosolic free calcium concentration by intrasynaptic mitochondria.

By the use of digitonin permeabilized presynaptic nerve terminals (synaptosomes), we have found that intrasynaptic mitochondria, when studied "in situ," i.e., surrounded by their cytosolic environment, are able to buffer calcium in a range of calcium concentrations close to those usually present in the cytosol of resting synaptosomes. Adenine nucleotides and polyamines, which are usually lost during isolation of mitochondria, greatly improve the calcium-sequestering activity of mitochondria in permeabilized synaptosomes. The hypothesis that the mitochondria contributes to calcium homeostasis at low resting cytosolic free calcium concentration ([Ca2+]i) in synaptosomes has been tested; it has been found that in fact this is the case. Intrasynaptic mitochondria actively accumulates calcium at [Ca2+]i around 10(-7) M, and this activity is necessary for the regulation of [Ca2+]i. When compared with other membrane-limited calcium pools, it was found that depending on external concentration the calcium pool mobilized from mitochondria is similar or even greater than the IP3- or caffeine-sensitive calcium pools. In summary, the results presented argue in favor of a more prominent role of mitochondria in regulating [Ca2+]i in presynaptic nerve terminals, a role that should be reconsidered for other cellular types in light of the present evidence.     

Redox properties of the calcium chelator Fura-2 in mimetic biomembranes

Fura-2 is one of the most commonly used fluorescent dyes to analyze the cytosolic Ca(2+) concentration ([Ca(2+)](i)) of living cells. Fura-2-dependent measurements of [Ca(2+)](i) are susceptible to changes of pH, reactive oxygen species concentration and membrane potential. Fura-2 is often loaded over the lipophilic cell membrane into the cytosol of a cell in its esterified form (Fura-2/AM) which is then cleaved by endogenous esterases. We have analyzed the electrochemical properties of Fura-2/AM and Fura-2 salt by cyclic voltammetry ("three-phase" and "thin-film" electrode methods). Using Fura-2/AM as a redox facilitator, we were able to mimic the transport of various ions across a lipophilic barrier. We show that Fura-2/AM in this biomimetic set-up can be reversibly oxidized in a single electrochemical step. Its redox reaction was highly proton sensitive in buffers with pH< or =6. At physiological pH of around 7.0, the oxidation of Fura-2/AM was coupled to an uptake of mono-anions across the liquid-liquid interface. The voltage-dependence of the redox cycle was sensitive to the free Ca(2+) concentration, either after de-esterification of Fura-2/AM, or when Fura-2 salt was used. The complex between Fura-2 and Ca(2+) ions is ionic (complexation occurs via the dissociated negative groups of Fura forms), while the redox transformations in Fura-2 occurs at the nitrogen atoms of the amino groups. Our results suggest that redox transformations of the Fura-2 forms do not affect the binding ability toward Ca(2+) ions and thus do not interfere with [Ca(2+)](i) measurements.     

Lanthanum increases the rat thymocyte cytoplasmic free calcium concentration by enhancing calcium influx

In the present study, I have examined the effect of lanthanum (La3+) on cytoplasmic free calcium concentration in isolated rat thymocytes employing the quin2 technique. As with its effect on 15Ca accumulation in rat thymocytes (Segal, J. and Ingbar, S.H. (1984) Endocrinology, 115, 160-166), La3+ produced a concentration-related increase in thymocyte cytoplasmic free calcium concentration. This effect of La3+ was very prompt in onset, evident within about 30 s from the time of addition of La3+. The lowest effective concentration of La3+ was 6 microM (+22.7% above control), and it increased progressively to reach maximal values at 25 microM (+100% above control). La3+ added to quin2-loaded thymocytes suspended in a calcium-free medium was without effect. In addition, La3+ had no significant effect on 45Ca efflux, and La3+ did not inhibit calcium-ATPase activity in the rat thymocytes. These results demonstrate that in rat thymocytes La3+ increases cytoplasmic free calcium concentration by increasing the extracellular calcium influx into the cell rather than the release of calcium from an intracellular pool.     

Metal cations as synaptosomal calcium blockers in studies with Fura-2

Metal ions are often used to block calcium channels in various tissues, including synaptosomes. In the present study, Fura-2 was used to determine the effectiveness of various metal ions as calcium channel blockers in rat brain synaptosomes in vitro. In buffer solutions, La³⁺ and Cd²⁺ increased the Fura-2 fluorescence in a manner similar to Ca²⁺. Ni²⁺ and Mn²⁺ appeared to be fluorescence quenching cations, and Sr²⁺ and Co²⁺ had little effect on the fluorescence of Fura-2. In suspensions of synaptosomes under resting conditions, Cd²⁺, Ni²⁺ and Mn²⁺ were found to be not suitable for use in synaptosome studies. On the other hand, La³⁺ and Co²⁺ had little effect on the Fura-2 fluorescence of resting synaptosomes, and under depolarizing conditions, La³⁺ and Co²⁺ decreased the Fura-2 fluorescence. These resuls, therefore, suggest that La³⁺ and Co²⁺ may be suitable as calcium channel blockers in synaptosome studies.     

Elevation of cytoplasmic free calcium concentration by stable thromboxane A2 analogue in human platelets

9, 11-Epithio-11, 12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2, caused a rapid rise in cytoplasmic free Ca2+ concentration ([Ca2+]i) in human platelets as measured with the fluorescent Ca2+ indicator quin2. Concomitantly, this compound induced phosphorylation of myosin light chain which is catalyzed by Ca2+, calmodulin-dependent protein kinase. These reactions were fast enough to trigger serotonin release. 13-Azaprostanoic acid, a receptor level antagonist of thromboxane A2 inhibited STA2-induced elevation of [Ca2+]i, phosphorylation of myosin light chain and serotonin release. These results provide evidence that STA2 interacts with a thromboxane A2 receptor which leads to elevation of [Ca2+]i.     

Fura-2 diffusion and its use as an indicator of transient free calcium changes in single striated muscle cells

Two questions bearing on the use of fura-2 to measure transient changes in intracellular Ca2+ concentration have been addressed. To investigate fura-2 intracellular binding, the amounts of fura-2 and [14C]glycine in Balanus nubilus myofibrillar bundles after loading were determined and their intracellular apparent diffusion constants measured. No significant fura-2 immobilisation occurs under the conditions used. The apparent diffusion constant for fura-2 in aqueous solution was determined. The relationship between half-time for relaxation of force and fura-2 fluorescence transients, and intracellular fura-2 concentration, in voltage-clamped single muscle fibres was examined. Significant buffering of the Ca2+ transient occurred at fura-2 concentrations above approximately 6 microM.     

Measurement of cytoplasmic calcium concentration in cell suspensions: correction for extracellular Fura-2 through use of Mn2+ and probenecid

1321N1 astrocytoma cells loaded with Fura-2 were found to continuously transport Fura-2 to the extracellular medium. To correct for extracellular Fura-2 fluorescence a protocol was developed in which Mn2+ was added to duplicate cuvettes of cells to quench extracellular Fura-2 at the beginning and end of the experimental time course. Since the export of Fura-2 was linear with time, two separate quench determinations allowed the amount of fluorescence from extracellular Fura-2 fluorescence to be estimated at every point in the time course and subtracted from the data. The uncorrected and Mn2+-corrected basal cytoplasmic calcium concentrations averaged 153 nM and 72 nM, respectively. The peak intracellular calcium concentrations following muscarinic stimulation with 300 microM carbachol averaged 1159 nM (uncorrected) and 889 nM (Mn2+-corrected). Probenecid (2.5 mM) was found to block the export of Fura-2 from these cells and did not change the basal calcium concentration or the muscarinic calcium response.     

Free calcium concentrations in bullfrog rods determined in the presence of multiple forms of Fura-2.

We employed the fluorescent calcium indicator Fura-2, loaded into intact retinas of the bullfrog Rana catesbeiana, to measure free calcium concentrations in the rod outer-segment cytosol. We determined that traditional methods of calculation yielded erroneous values of calcium. This error results from the presence of at least two distinct pools of Fura-2 in rod outer segments. Application of manganese quenches each pool, but quenching occurs at different rates. Using this fact, we show that the pools can be isolated by brief exposure to manganese and examined separately. One of these pools has the same fluorescent properties as the free salt of Fura-2 we use in our in vitro calibrations. The other source of fluorescence has more unusual properties. Although insensitive to calcium concentrations in the physiological range, it contributes significant anomalous fluorescence when cytosolic free calcium concentrations are elevated by application of IBMX. Nevertheless, the experimentally isolated, classic pool of Fura-2 is well behaved and allows us to calculate calcium concentrations relative to the Kd of Fura-2 by the usual ratio method. We show that when rods are exposed to saturating light, the free calcium concentration in their outer segments falls to a level not significantly different from zero within 20-30 s.     

Intracellular free Ca2+ concentration in fowl spermatozoa and its relationship to motility and respiration in spermatozoa.

The ability of fowl spermatozoa to accumulate and de-esterify the intracellular fluorescent Ca2+ indicator fura-2 was established. The cytosolic Ca2+ concentrations, measured by this technique, did not change after the addition of 1 mmol EGTA l-1. Subsequently, addition of the calcium ionophore A23187 caused a reduction in cytosolic Ca2+ concentrations, presumably by efflux of Ca2+ from the spermatozoa. Intracellular free Ca2+ concentrations were then significantly increased by the addition of 1 mmol CaCl2 l-1. The motility of demembranated spermatozoa gradually decreased after the addition of EGTA alone or EGTA with A23187, but was instantly restored by the addition of CaCl2 in the presence of both EGTA and A23187. Unlike demembranated spermatozoa, intact spermatozoa maintained their motility, even after the addition of EGTA, but their motility was reduced by the addition of A23187 in the presence of EGTA. The addition of A23187 also reduced the rate of oxygen consumption, but not the ATP concentrations in intact spermatozoa. These results demonstrate that the motility and respiration of fowl spermatozoa are strongly influenced by their intracellular Ca2+ concentrations.     

Spectra of intracellular Fura-2.

In the theory of measurement of calcium ion activity by determination of Fura-2 fluorescence at two excitation wavelengths, the accuracy of the result depends upon the accuracy both of the sample measurements and of the calibration measurements which are made on calcium-bound and free dye. Two factors underlie adequate calibration and accuracy. The first is the elimination of systematic error due to spectral shifts arising from the intracellular environment felt by the dye. To this end, detailed comparisons between complete spectra of both calcium-bound and calcium-free Fura-2 can be used to help separate spectral effects due to light absorption by cellular constituents versus polarity and viscosity of the intracellular milieu. The second major factor which determines accuracy is the experimental uncertainty (in both sample and calibration measurements). For samples in which the ratio of bound to free dye is large, the uncertainty in the ratio is also large, even when it is expressed as a percentage of the ratio itself. The errors in calibration measurements impact on the accuracy of the method primarily through the measurements made at wavelengths which are off the spectral peaks of the bound or free dye, since these are the least accurate. In order to obtain a guide to the choice of wavelengths and estimation of the reliability of results, a mathematical expression is derived for the dependence of the accuracy of the method on the accuracy of both sample and calibration measurements.     

Depolarization-induced calcium release from isolated triads measured with impermeant Fura-2

Summary Depolarization-induced Ca²⁺ release was studied in a mixture of triads and terminal cisternae isolated from rabbit skeletal muscle. The vesicles were actively loaded with known amounts of Ca²⁺ in the absence of precipitating anions in a solution containing 100 mm K propionate buffer. Changes in extravesicular Ca²⁺ were monitored with 10 m Fura-2 (membrane impermeant form). Ca²⁺ release was initiated by diluting an aliquot of the loaded vesicles into a TEACl release solution designed to maintain a constant [K⁺] · [Cl^–] product. Fast release, defined as the percentage of total Ca²⁺ loaded which released in less than 10 sec, occurred when extravesicular free Ca²⁺ was in the submicromolar range and was unaffected by 5 mm caffeine under depolarizing conditions, change in external pH to 6.5, and an increase in external Mg²⁺ concentration from 0.1 to 0.2 mm. Thus, the Ca²⁺ release measured in these studies is distinct from Ca²⁺-induced Ca²⁺ release. The fast release more than doubled when a greater dilution (1 20 versus 1 10) of the loaded vesicles into the release solution, which would produce a larger depolarization, was used. The percentage of loaded Ca²⁺ which released rapidly in a particular triad preparation was similar to the percentage of vesicles structurally coupled as visualized by electron microscopy.We thank Gerry Vaio and Melanie Vander Klok for excellent technical support. This work was supported by the National Institutes of Health program project grant PO1-HL27867, NSF Biological Instrumentation Grant DIR-8812094 and State of Ohio Research Challenge Grant.     

Intracellular binding of ethidium studied by photoaffinity labeling in vivo.

The azide analog of 14C-labeled ethidium bromide was mixed with yeast cells and when photolyzed by visible light, formed covalent complexes with all yeast cell organelles. The 14C counts were found in DNA, RNA and protein of yeast subcellular fractions, illustrating the complexity of binding of a drug which appears highly specific in its actions.     

Activin A increases cytosolic free calcium concentration in rat pituitary somatotropes.

The effect of activin A on the cytosolic free calcium concentration ([Ca2+]i) in normal rat pituitary cells was examined using a calcium sensitive fluorescent dye, indo 1 AM, and a digital imaging fluorescent microscope system. The cells showing an increase in [Ca2+]i in response to activin A were then characterized by comparison with cells responding to growth hormone releasing hormone (GRH), thyrotropin releasing hormone (TRH), corticotropin releasing hormone (CRH), and gonadotropin releasing hormone (GnRH) in monolayer cultures of normal rat pituitary cells. Activin A increased [Ca2+]i in some cells in a mixed population of normal rat pituitary cells. The cells that responded to activin A also responded to GRH. Most of these cells were not affected by other tropic hormones (CRH, TRH, and GnRH), but a few cells responded to both GRH and TRH. None of the activin A-responding cells responded to CRH or GnRH, and none of the CRH- or GnRH-responding cells responded to activin A. In a preparation of somatotropes purified 80-90% by fluorescence-activated cell sorting, activin A increased [Ca2+]i in 30% of the cells that shows a [Ca2+]i-response to GRH. These findings suggest direct involvement of somatotropes in activin A-induced biological events in the rat pituitary gland.