Photochemically generated cytosolic calcium pulses and their detection by fluo-3

Fluo-3, one member of a family of new fluorescent Ca2+ indicators excitable at wavelengths in the visible (Minta, A., Kao, J. P. Y., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171-8178), has been tested in living cells. We demonstrate that fluo-3 can be loaded into fibroblasts and lymphocytes by incubation with the pentaacetoxymethyl ester of the dye and that the ester is hydrolyzed intracellularly to yield genuine fluo-3 capable of indicating changes in [Ca2+]i induced by agonist stimulation. Fluo-3 can also be microinjected into fibroblasts along with photolabile compounds such as nitr-5 and caged inositol trisphosphate for photorelease experiments. Fluo-3 permits continuous monitoring of [Ca2+]i without interference with use of UV-sensitive caged compounds. A procedure for combined use of ionophore and heavy metal ions in end-of-experiment calibration of fluo-3 intensities to give [Ca2+]i is also described.     

Imaging of calcium transients in skeletal muscle fibers.

Epifluorescence images of Ca2+ transients elicited by electrical stimulation of single skeletal muscle fibers were studied with fast imaging techniques that take advantage of the large fluorescence signals emitted at relatively long wavelengths by the dyes fluo-3 and rhod-2 in response to binding of Ca2+ ions, and of the suitable features of a commercially available CCD video camera. The localized release of Ca2+ in response to microinjection of InsP3 was also monitored to demonstrate the adequate space and time resolutions of the imaging system. The time resolution of the imager system, although limited to the standard video frequency response, still proved to be adequate to investigate the fast Ca2+ release process in skeletal muscle fibers at low temperatures.     

Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths

Free Ca2+ concentrations in the cytosol of individual small cells can be recorded with a new fluorescent Ca2+ indicator, "fura-2", and a fluorescence microscope modified to chop rapidly between two wavelengths of excitation. Both fura-2 and its Ca2+ complex fluoresce strongly, but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca2+-bound dye to free dye and hence cytosolic free Ca2+. This ratio measurement largely cancels out the effects of cell thickness, dye content, or instrumental efficiency, uncertainties that can jeopardize measurements at single wavelengths. We describe instrumentation that supplies rapidly alternating excitation wavelengths to either a standard cuvet or a fluorescence microscope. Its use is illustrated by experiments showing changes in cytosolic [Ca2+] accompanying activation of human platelets in suspension or single mouse thymocytes on the microscope.     

Iminocoumarin-based low affinity fluorescent Ca2+ indicators excited with visible light.

A series of iminocoumarin-based fluorescent Ca2+ indicators were synthesized and the spectral profiles of their free and Ca2+ bound forms were studied. The newly-synthesized compounds incorporate the Ca2+ chelating structure of BAPTA. The chromophore moieties are iminocoumarins substituted at the 3-position with benzothiazolyl, benzoxazolyl and benzimidazolyl groups. These compounds are excited with visible light and their Ca2+ dissociation constants range from 5.4 to 27.5 microM. Fluorescence spectra studies of these probes indicated a clear shift in their excitation wavelength maxima upon Ca2+ binding along with changes in fluorescence intensity that enable the compounds to be used as low Ca2+ affinity, visible excitable probes.     

A new generation of Ca2+ indicators with greatly improved fluorescence properties

A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.     

Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.     

Mitochondrial permeability transition and calcium dynamics in striatal neurons upon intense NMDA receptor activation

Deregulation of the intracellular Ca2+ homeostasis by NMDA receptor activation leads to neuronal cell death. Induction of the mitochondrial permeability transition pore (MPT) by Ca2+ is a critical event in mediating cell death. In this study, we used fluorescent Ca2+ indicators to investigate the effect of high concentrations of NMDA on cytosolic and mitochondrial Ca2+ concentrations ([Ca2+]c and [Ca2+]m, respectively) in cultured striatal neurons. Exposure to NMDA resulted in an immediate, sustained increase in [Ca2+]c followed by a secondary increase in [Ca2+]c. This second increase of [Ca2+]c was prevented by pretreatment with N-methyl-valine-4-cyclosporin (NMV-Cys). Exposure of neurons to NMDA also resulted in an increase in [Ca2+]m that was followed by a precipitous decrease in the rhod-2 signal. This decrease followed the time frame of the secondary increase in [Ca2+]c. Preincubation of the neurons with NMV-Cys prevented the decrease in rhod-2 fluorescence. These dynamic changes in the rhod-2 signal and [Ca2+]m in response to NMDA were confirmed by using confocal microscopy. The presented results indicate that MPT can be detected in living neurons using fluorescent Ca2+ indicators, which would allow the study of the physiological role of MPT in cell death.     

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers.

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.     

Imaging Ca(2+) entering the cytoplasm through a single opening of a plasma membrane cation channel.

Discrete localized fluorescence transients due to openings of a single plasma membrane Ca(2+) permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca(2+) indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375-394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca(2+), suggesting that they are due to Ca(2+) influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca(2+) and Ca(2+)-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca(2+) and Ca(2+)-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a "Ca(2+) spark-like" appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca(2+) current, to study intracellular localized Ca(2+) handling as might occur from single Ca(2+) channel openings, and to localize Ca(2+) permeable ion channels on the plasma membrane.     

Near-membrane iminocoumarin-based low affinity fluorescent Ca(2+) indicators

Two new potential near-membrane iminocoumarin-based fluorescent Ca(2+) indicators were synthesized and the spectral profiles of their free and Ca(2+) bound forms were studied. The probes incorporate in their BAPTA-related structures, the 3-(benzimidazolyl)iminocoumarin or the 3-(benzothiazolyl)iminocoumarin moiety, substituted at the imino nitrogen with an n-dodecyl lipophilic chain. The compounds are excited with visible light and have Ca(2+) dissociation constant values of 5.50 and 4.49 microM, respectively, the highest reported to date in the literature. Fluorescence spectra studies indicated a clear shift in their excitation wavelength maxima upon Ca(2+) binding along with changes in fluorescence intensity that enable the compounds to be used as ratiometric near-membrane, low Ca(2+) affinity probes.     

Functional expression of the human cardiac Na+/Ca2+ exchanger in Sf9 cells: rapid and specific Ni2+ transport

Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.     

Analysis of calcium binding to alpha-lactalbumin using a fluorescent calcium indicator.

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine alpha-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of alpha-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the alpha-lactalbumin--Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25 degrees C, was found to be 123 +/- 2 nM and 103 +/- 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to alpha-lactalbumin did not depend on pH in the range 6.6-8.4 and was differently affected by Na+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 +/- 13 nM and the number of Ca2+ bound/molecule alpha-lactalbumin was lowered to 0.069 +/- 0.006. No interaction between fluo-3 and alpha-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements.     

Kinetics of calcium binding to fluo-3 determined by stopped-flow fluorescence

The kinetics of Ca2+ dissociation from fluo-3 was measured using stopped flow fluorimetry. Analysis of dissociation revealed, in contrast to other commonly used fluorescent Ca2+ indicators, a biexponential behaviour with two distinct dissociation rates of 550 s-1 and 200 s-1 at physiological pH and room temperature. The dissociation rate constant of the fast phase increases to 700 s-1 at physiological temperature, whereas that of the slow phase does not change markedly. While the rate constants do not depend on pH between 6.6 and 7.8, the dissociation turns out to be monoexponential at pH 5.86. The association rate of Ca2+ to fluo-3 could not be measured within the mixing dead time and is estimated to be above 10(9) M-1 s-1. Since the rate constants of fluo-3 are larger than those of other fluorescent Ca2+ indicators, fluo-3 is well suited for investigations of Ca2+ oscillations in biological systems.     

Do we know the absolute values of intracellular free calcium concentration?

More than 20 years ago, it was shown that the addition of EGTA increases the affinity of the plasma membrane Ca2+ pump for Ca2+ by an order of magnitude. The left-hand shift of Ca2+-dependencies in the presence of EGTA has been also documented in studies of the sarcoplasmic reticulum Ca2+ pump, mitochondrial Ca2+-transporter as well as Ca2+-binding by calmodulin and troponin C. These data allow us to hypothesise that this effect is caused by an admixture of di- and trivalent cations possessing high affinity for EGTA and interacting with Ca2+-transporting and binding proteins. Here, we propose that polyvalent cations affect the estimation of absolute values of free intracellular Ca2+ concentration. Indeed, EGTA sharply increases the apparent affinity of the fluorescent Ca2+ indicators quin-2 and fluo-3 for Ca2+. The impact of polyvalent cations on Ca2+ measurement was further confirmed by the study showing the high sensitivity of Ca2+-induced fluo-3 fluorescence to Mn2+, Fe2+, Cu2+, and Co2+ seen in the absence of EGTA.     

Spatial and temporal aspects of Ca2+ signaling mediated by P2Y receptors in cultured rat hippocampal astrocytes

ATP produces a variety of Ca2+ responses in astrocytes. To address the complex spatio-temporal Ca2+ signals, we analyzed the ATP-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal astrocytes using fura-2 or fluo-3 based Ca2+ imaging techniques. ATP at less than 10 nM produced elementary Ca2+ release event "puffs" in a manner independent of extracellular Ca2+. Stimulation with higher ATP concentrations (3 or 10 micro M) resulted in global Ca2+ responses such as intercellular Ca2+ wave. These Ca2+ responses were mainly mediated by metabotropic P2Y receptors. ATP acting on both P2Y1 and P2Y2 receptors produced a transient Ca2+ release by inositol 1,4,5-trisphosphate (InsP3). When cells were stimulated with ATP much longer, the transient [Ca2+]i elevation was followed by sustained Ca2+ entry from the extracellular space. This sustained rise in [Ca2+]i was inhibited by Zn2+ (<10 micro M), an inhibitor of capacitative Ca2+ entry (CCE). CCE induced by cyclopiazonic acid or thapsigargin and Ca2+ entry evoked by ATP share the same pharmacological profile in astrocytes. Taken together, the hierarchical Ca2+ responses to ATP were observed in hippocampal astrocytes, i.e., puffs, global Ca2+ release by InsP3, and CCE in response to depletion of InsP3-sensitive Ca2+ stores. It should be noted that these Ca2+ signals and their modulation by Zn2+ could occur in the hippocampus in situ since both ATP and Zn2+ are rich in the hippocampus and could be released by excitatory stimulation.     

A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes

The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.     

Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes.

We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca2+ uptake and physiological Ca2+ signaling in rat cortical astrocytes. A rise in cytosolic Ca2+ ([Ca2+]cyt), resulting from mobilization of ER Ca2+ stores was followed by a rise in mitochondrial Ca2+ ([Ca2+]m, monitored using rhod-2). Whereas [Ca2+]cyt recovered within approximately 1 min, the time to recovery for [Ca2+]m was approximately 30 min. Dissipating the mitochondrial membrane potential (Deltapsim, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca2+ uptake and slowed the rate of decay of [Ca2+]cyt transients, suggesting that mitochondrial Ca2+ uptake plays a significant role in the clearance of physiological [Ca2+]cyt loads in astrocytes. Ca2+ signals in these cells initiated either by receptor-mediated ER Ca2+ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 +/- 11.2 micrometer/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca2+ uptake into mitochondria as the Ca2+ wave traveled across the cell. Collapse of Deltapsim to prevent mitochondrial Ca2+ uptake significantly increased the rate of propagation of the Ca2+ waves by 50%. Taken together, these data suggest that cytosolic Ca2+ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca2+ signals.     

Distance measurements between metal-binding sites of calmodulin and from these sites to Cys-133 of troponin I in the binary complex

Distances between the four Ca2+-binding sites of calmodulin (CaM) have been measured by fluorescence energy transfer techniques using Eu3+ and Tb3+ as energy donors and a number of other lanthanide ions (Ln3+) as acceptors. It was shown previously that lanthanide ions preferentially bind to sites I and II of CaM with an affinity higher than that for sites III and IV (Kilhoffer, M.-C., Demaille, J. G., and Gerald, D. (1980) FEBS Lett. 116, 269-272; Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., and Gergely, J. (1982) Eur. J. Biochem. 124, 7-12). Thus upon direct excitation with a laser the luminescence lifetimes of Eu1Ln1CaM and Tb1Ln1CaM provide information on the distance between sites I and II. On the other hand, since Tb3+ ions bound to sites III and IV are sensitizable through tyrosine residues, lifetime measurements of Tb2Ln2CaM excited by UV light yield the distance between sites III and IV. Both pairs of sites were found to be separated by a distance of 1.05 +/- 0.07 nm. Binding of Ca2+ to sites III and IV does not alter the distance between sites I and II. We have also attached a chromophoric label, dimethylaminophenylazobenzene, to Cys-133 of skeletal troponin I and carried out distance measurements on its complex with CaM by both direct and indirect excitation. The averaged distances from sites I and II in the N-terminal half and from sites III and IV in the C-terminal half of the CaM molecule to the label on troponin I are 2.7 and 2.5 nm, respectively.     

Measuring zinc in living cells. A new generation of sensitive and selective fluorescent probes

New fluorescent indicators with nanomolar to micromolar affinities for Zn(2+) have been synthesized in wavelengths from UV to the far red. The UV light-excited indicators are ratiometric. The visible wavelength indicators are non-ratiometric and exhibit large and pH-independent fluorescence increases with increasing zinc concentrations, with little to no sensitivity to physiologically relevant Ca(2+) concentrations. Experiments in neuronal and non-neuronal cell cultures show the new indicators to retain their sensitivity to and selectivity for zinc after conversion to cell-permeable forms.