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Intracellular calibration of the fluorescent indicator Fura- L

D. A. WILLIAMS’ and F. S. FAY2

‘Department of Physiology, 2 Department of Physiology, Massachusetts, USA

University of Melbourne, Parkville, Victoria, Australia University of Massachusetts Medical School, Worcester,

calcium

Abstract - We present the techniques we have used and the problems we have encountered in our laboratories in the in vivo calibration of the fluorescent Ga2+-kkator Fura-2. These techniques include the use of potentlometric methods for the precise control and determination of Ga2+ levels in bathing solutions, in association with methods for the equlKbration of internal and external solutions with ionophores (Br4428187, ionomycin, monensin and nigericin). A by-product of these techniques has been the develm of a simple procedure that utilizes Fura- as a general indicator of ionized Ga2’ conce&atfons wfthin the physiological range (pGa 7.5 to 5.8), in other experimental solutions. The major advantages of this relatively simple procedure are that it is (i) rapidly performed, (ii) independent of the total EGTA concentration within each experimental solution, (iii) independent of the absolute EGTA purity, and (iv) unaffected by a large number of potentially interfering cations (i.e. Mg 2t, Ht, Kt, Nap within the test solutions.

Since the initiai report of the synthesis of the fluorescent Ca2+-sensitive indicator Fura- [l], an intensive research effort has been invested in the use of this indicator to measure intracellular Ca2+ concentration in a myriad of cell types (e.g. see other papers in this Issue). However, there has been a disproportionately smaller time committed to the issue of the accurate, in vivo calibration of the fluorescent signals of Fura- and other related dyes. The fast paper dealing with this issue appeared as early (with respect to the lifespan of the ‘new’ generation indicators) as 1985 (see 21). Ionophores

It were used to clamp intracellular [Ca ] to accurately defined and controlled levels in isolated smooth muscle cells. This allowed for the accumte determination of the relationship between relative

excitation ratio (34onm/38Onm) and [Ca2+], and for calculation of the dissociation constant (Kd) for CaFura-2 in the internal environrr~nt of the smooth muscle cell A small population of other researchers has since attempted to calibrate the fluorescence of Fura-2, either in their own cell type [3, 41, or at least with the same optical system inherent errors and idiosynchrosies, and experimental techniques with which the actual experiments were conducted (e.g. P-71).

However, the majority of the literature has opted simply $0 refer to the original in vitro Kd &term&d by Grynkiewicz et al. [l], for an arbritrarily CXXNQllCted experimmtal SOlUticNt. This determir&onwaspmsnmabl madetoserveasa

J+ comparison of the relative Ca affinity of Funk-2 to

75

76

that of the major existing fluorescent Ca2+-probe at that time, Quin-2, rather than to become the staudard value for use in all experimental cixcumstauces.

We present here the experimental techniques which are routinely used in our laboratories to calibrate the fluorescent signals (as [Ca2+] and Kd) measured from the Ca2’-sensitive indicators, and in particular Pura-2. We also discuss some of the difficulties which we have encountered in this process. We do this with the hope that more researchers will be tempted to more precisely deteimine the key factors for the accurate quantification of these indicators. Many of the other papers in this edition have described elegant optical, mechanical and electronic innovations which have been used to visualize and localize fluorescence within single living cells. We hope that this contribution will increase the certainty with which we can interpret the exciting future results obtained with this hardware in terms of actual spatial free calcium ion differences.

Materials and hhethods

Microscopic fluorescence measurements

Pluorescence measurements were made through Nikon Cl? UV-F objectives (magnification x40 and x100) on an Olympus IMT-2 inverted microscope equipped for xenon epifluorescence. The rest of the recording system was similar to that previously described [21. A silicon-intensi&&target camera (Dage MTI SIT-66) or 1 inch diameter photomultiplier tube (Oriel Corporation, MA, USA) were used to record the 51Onm fluorescence intensities excited through narrow band pass falters (340~1, 35Onm 36Onm or 380~1) alternated in the excitation light path. Signals were digitized with boards (PG-100 AT, Imaging Technology; PC-30, Boston Technology) resident in a 80286-based personal computer. The imaging software framework was supplied by Imaging Technology and was modified from source code to provide many of the features essential to these experiments (see other papers in this edition). Data acquisition softwan for aualogue/digital conversion utilizing

CELLCALclIJM

photomultiplier tubes was written in assembly language under the guidance of Mr Cohn Milhuisen, La ‘Robe University, Victoria, Australia.

Spectrofluorometry

Fura- fluorescence was recorded with a modified spex fluorolog (2,1,1) spectrofluorometer (Spex Industries, NJ, USA) in the Department of Physiology, University of Massachusetts, Worcester, MA, USA or a Perkin Elmer system at the Department of Zoology, La Trobe University, Australia. Excitation wavelengths were scanned through a single monochromator (300 to 400 nm, 3.6 run bandpass) or chopped rapidly (15 Hz) between 2 separate monochromators set at fmed wavelengths of 340 and 380 run (each 3.6 nm bandpass) respectively. Emission intensity was monitored at 510 nm (3.6 nm bandpass).

Table 1 Ionized Ca*’ concentrations for experimental solutions measured with the various techniques described in this study

Solution proportions pea

A B Estimad’ Fura-2 Poienttometricc

1.0 0.0 >8.0

0.9 0.1 7.66

0.8 0.2 7.31

0.7 0.3 7.07

0.6 0.4 6.88

0.5 0.5 6.70

0.4 0.6 6.53

0.3 0.7 6.34

0.2 0.8 6.10

0.1 0.9 5.75

0.05 0.95 5.42

0.0 1.0 5.0

-

7.73

7.35

7.05

6.81

6.53

6.35

6.05

5.77

5.63

5.43

-

-

7.66

7.40

7.12

6.81

6.60

6.40

6.22

5.92

5.65

5.45

-

Soh&msamtypicalofthoseusedtobahrheamtrdle apparatus of skinned muscle preprastions (eee text). s-f?cm.xtimwlHoporti~ofCal3GI’~AdK’~- 478 x l$ M-l b-ca&ulatiwithFum-ZMdesaibedinthisstudy (Mmc) c-calculated with potmtiometric techaiqua (Sectian A [13,25])

INTRACELLULAR CALIBRATION OF FURA- 77

Results and Discussion

The two most important requirements of the calibration technique we will describe are. to (i) accurately set and control the [Ca2+l over the range of physiological interest in the solution bathing the cells and then (ii) to completely equilibrate the extracellular and intracellular environments with respect to the clamped [Ca2’l. The first requires precise control of an EGTA-based chemical equilibrium system, while the second requires a process most accurately called ‘guided trial and error’.

A : Control of ionized Ca2+ concentration

Solution composition A series of solutions with different ionized Ca2’ concentrations can be achieved from the mixing of two main stock solutions (denoted A and B), in varied proportions (see Table 1). Both solutions generally contained (n&I): Mg2+ 1.0, Kt 100.0 to 140.0, Nat 10.0 to 30.0, ATP 5.0, HJPES 10.0, creatine phosphate 10.0. In addition solution A contained 10 mM EGTA whilst solution B contained 10 mM nominally equimolar CaEGTA. The absolute concentration of EGTA employed in these solutions was not critical but could be varied to set the ionic strength of these solutions at the level of interest. However, the absolute level of EGTA should be high enough that Fura- represents an insignificant Ca2+-buffering contribution in comparison (EGTA:Fura-2 >> 1OO:l). The exact ionic composition could be varied so as to more closely match the conditions expected to exist within the cell type of interest We aimed to achieve an electroneutral solution similar to that suggested in an extensive review of the ionic composition of skeletal muscle 181. To this end these stock solutions occasionally also contained (mM): inorganic phosphate 5, lactate 2, an amino acid mixture 10, and tiodulin 0 to 0.1 - although these additions had relatively little effect in influencing the calibration curves (see Section C).

Estimation o LF

ee calcium concentration The tree Ca concentration can be estimated from the relative proportion of CaEGTA to EGTA in each

of the solution mixtures (examples shown in Table 1) with an apparent binding constant K’Q and assuming the law of mass action:

[Ca2+] = (l/K’& l ([CaEGTAl/@GTAlti) (Eq. 1)

However, such estimations contain many sources of error. A large amount of recent attention has been focused on the value to be adopted for the apparent aftinity constant (K’c~) for EGTA association with Ca2’ under defined experimental conditions with a large variation in published values. As has recently been pointed out [9], them is even more uncertainty as to the actual total concentration of EGTA in a solution due to impurities and an unknown and variable degree of hydration of EGTA. These factors introduce a large degree of uncertainty into the estimation of the relative CaEGTALEGTA ratio. This uncerurinty propagates into the estimation of fluorescence ratio-[Ca2+] relationship and the Kd for CaFura-2. Ca2+-selective microelectrodes have been used with mixed success to more accurately measure ionized Ca2t-concentrations in bathing solutions [lo]. What is generally overlooked, however, is that even the Ca-electrode methodology requires that the electrical potential of the electrode be calibrated against a series of solutions of known Ca-concentration (e.g. see [ll]). Such standard solutions are generally constructed by mixing stock solutions of CaEGTA and EGTA in varied proportions, or by adding varied amounts of CaCl2 to EGTA to obtain a range of Ca2’ concentrations of physiological interest In these solutions [Ca2’] is calculated much as described above, with an arbritrarily selected binding constant and the estimated proportions of bound to unbound chelator.

More precise determination of Ca2+ concentration can result from the direct experimental measurement of the proportion of CaEGTA and EGTA in each solution with a potentiometric technique. This methodology, described in detail elsewhere [9, 12, 131, takes advantage of the pH change which occurs during the binding of Ca2’ or Cd2’ to EGTA over a wide pH range, and provides for an accuracy of 0.025% in total EGTA determinations [9].

Briefly, the technique involves titration of the

78 CELLCALCIUM

free EGTA in a given 8olution with a high molar&y (250 mM) CaC& solution, while recording the pH (range pH 10 to 7) after each addition of CaCla. The end point occurs when the pH changes very little with further Carla addition. A plot of pH against Ca2+ added (concentration or pmole) will produce a prominent break in the curve at the point whem added Ca2’ equals free EGTA. The total EGTA in the same solution can be determined in a similar fashion except that CdS04 rather than CaCla, and a lower pH range @H 7 to 4) are used in the titration (see

1 131). Cd2+

displacement of Ca + by virtue of a

already bound to EGTA in this pH range, will produce an end-point indicative of the total EGTA concentration. Where solutions contain no added Ca2+ (i.e. no CaBGTA) the two titration methods should produce identical results, providing a good control for the ability of the experimenter to perform these techniques with accuracy. The only major equipment req*nt for this technique is a digital pH meter with accuracy to three decimal places (0.001 pH units).

B : In vivo equilibration of C$’

Choice of ionophores The two major ionophores used to equilibrate Ca2’ between extracellular and intracellular environments are ionomycin [14] and 4-bromo-A23187 [15]. Both ionophores are non-fluorescent in the ultra-violet spectrum and display a high selectivity for Ca2’ over other divalent cations, although in the case of Br-A23187, the Mg2+ rejection is not high, and both ionophores also readily transport Mn2’. Most of our early work was performed with ionomycin (Calbiochem-Behring, La Jolla, CA, USA), but more recently 4-bromo-A23187 has also become commercially available (Calbiochem-Behring or Molecular Probes, OR, USA) and is in standard use. A fact that is not commonly recognized with ionomycin is that although it is more efficient at Ca2+-transport than other ionophores, the trausport process displays a strong pH sensitivity. Peak transport efficiency occurs at high pH (9.5, see [14]) with low transport activity at pH below 7.0. ‘Ibis property has important implications for the use of ionomycin as a Ca2+ -ionophore and is an major factor in defining the optimal concentration for use

in calibration experimenta. In comparison BrX23187 is relatively unaffected by solution PH. Another factor which also influences the optimal concentrations of each of these ionophores is the Ca-ionophore stoichiometry which is 1:l with ionomycin but 1:2 with Br-A23187.

Mauy cell types, including smooth muscle cells [16, 171, are highly efficient in extmdin~ excess Ca2+ resulting from small elevations in Ca + influx, as they possess a high capacity CaATPase in endoplasmic reticuhun or phlsmalemmal membranes. In fact, the activity of these C&pumps in smooth muscle is likely to be directly controlled by the cytosolic calcium with prolonged elevations of [Ca2+] leading to persistent stimulation of the membrane associated Ca-pumps [18], through a Ca2tcalmodulin dependent mechanism [17]. This Ca-pumping capacity presumably will vary between cell types and is probably intricately co$ed to the inherent basal inward leakage of Ca and the fluctuations in &levels that the cell normally faces. This ATPdependent Ca-pump and, to a lesser extent (in smooth muscle), the process of N&ai exchange represent the major pathways of calcium extrusion that must be overcome in driving intracellular Ca2+ to defined levels. The strategy for equilibration therefore involves utilizing a divalent cation ionophore (ionomycin at pH 7.2 to 7.5 or Br-A23 187) to elevate inherent cellular Ca2+ leakage to such an extent that Ca-pump activi

Ps overcome (especially at elevated cytosolic [Ca I), and the intracellular environment can be considered an extension of the extracellular divalent cation content Iu addition, as there are no specific inhibitors of Na&ai exchange, the driving force for calcium extrusion by this process is diminished by dissipating the cellular Nat gradient with the Nat-ionophom monensin (Calbiochem-Behring). Since the NatKt-ATPase may also be highly active in some cell types (e.g. secreting epithelia) it is also advisable to include a Kt-ionophore such as nigericin (Molecular Probes), or a pump inhibitor (ouabaiu), to mimmize the capacity of this pump to be able to restore cellular Na gradients (and hence some Ca-pumping capacity).

The ideal mixture of these ionophores will be a function of the relative activities of each of the pumps or transporters, which are a function of the

INTR4CELLULAR CALIBRATION OF FURA- 79

particular cell type. It might be expected, for example, that secreting or transporting epithelia will require quite different proportions of ionophores to other cell types. This is where the element of trial and error comes into play, guided by any available knowledge of the relative pump activities in the cell type of interest.

Calibration procedure Cells are loaded with Fura- so as to minimim the localization of dye within the internal membrane-bound compartments (e.g. endoplasmic reticulum) as has been described elsewhere ([19] and Fay et al., this Issue). Ideally cytosolic F4rra-2 levels should be kept below 100 @I so as to minimize this contribution to the total Ca2+-buffering of the calibration system. A small concentrated sample <>lO* ml- ) of the cell suspension (with a known absolute number of cells) is placed in a vessel which has as its bottom a disposable glass coverslip (size 1, 1 oz., 0.17mm thickness). We have found it useful to determine the fluorescence ratio of a cross-section of cells at this stage. The techniques for these measurements have been covered in detail elsewhere (see [2] aud other papers in this Issue). Special precautions should be taken to minimim any potential photobleaching of Fura- within both calibration solutions and cells [20]. The cells am then diluted to a density of lo5 ml-’ with a Ca2+-buffered solution selected from the range constructed by mixing solutions A and B (e.g. see Table 1). It is not necessary to have determined the exact [Ca2+] of each mixture prior to this stage of the calibration, although it is essential that it is done later. A mixture of monensin/nigericin (both dissolved in ethanol) is thoroughly dispersed into a volume of the same Ca2+-solution (volume selected so that both ionophoms are at a tinal concentration of 5 pM), and then added to the cell suspension. After equilibration in these ionophores for 30 min, ratios are again determined. If the ratios (and themfore calcium levels) are affected minimally by this procedure (as is the case with amphibian smooth muscle cells) the cells will not have discemibly changed shape, and the same cells as for the initial measurement can be used. The divalent cation ionophore (either Br-A23 187 or ionomycin) is then

addedmasimilarfashion. Throughtrialanderror we have found that a concentration greater than 1 pM is always necessary to cause a noticeable change in fluorescence ratio at the cell density (10’ cells ml-‘) we have used. Ionophore is added progressively and equilibrated until an end-point is reached. The end-point represents attainment of a stable, uniform, cellular fluorescence ratio which is within 5% of the value which is mcorded concomitantl~+~m a neighboring vessel containing the same Ca -buffer mixture and Fura- (5 @I, pentapotassium salt). This SOlUtiOnXell ratio comparison is an integral part of the methodology and obviously places great importance on the composition, ionic strength and even viscosity of the external solutions which should be designed to closely mimic the internal milieu (e.g. see [8]).

This general procedure is repeated with a fresh sample of cells for each of the Ca2’-buffer mixtures that cover the [Ca2+] range of interest Ionophores can be introduced to these solutions through a single addition at concentrations equivalent to those at the end-point of the first determination. Occasionally at the highest Ca2’-levels used it was found that the ionomycin (or Br-A23 187) concentration had to be marginally increased (20%) to achieve a stable end-point Presumably this was due to an increased stimulation of the cellular Ca2+-extrusion processes under these conditions [18]. We favored this method of using ionophom concentrations which just allowed for equilibration of intracellular and extracellular cation concentrations rather than an overdose of ionophom. It minim&d the leakage of Fura- from the cellular environment and as a bonus, also provided information as to the relative importance of the various Ca2+-extrusion processes (Ca-pumps versus Na~-Cai exchange) in these cells.

Our general observations suggest that the efficiency of this calibration procedure relies on the complete dispersion of the ionophores within the cell suspension. A single cell population cau display a heterogenei

2!? in fluorescence ratio at a given

external [Ca ] (as has been report4 in the literature, see [3]), simply because of poor dispersal of ionophore (only some cells in a field being clamped at the selected level). It is also evident that the optimal concentrations of ionophores (defined as the lowest concentration that achieves equilibration

SO CELL CALCIUM

at all Ca2’ levels used), will vary with cell density because at higher densities presumably fewer molecules of ionophore can associate with each cell (a simple probability function). Hence we have discussed our techniques relative to a fuced cell density. Cells that only poorly cleave internalized Fura- (such as human neutrophils [21]), will prove to be difficult to calibrate as they possess fluorescent species which lead to the calculation of erroneous ratios at each clamped [Ca2+]. In this case consideration should be given to correcting the ratio values for the presence of these species. An example of one such correction method is described elsewhere in this issue (Pay et al, this Issue).

The fluorescence ratio of both cells and solutions is plotted a ainst the precisely determined pCa

2g (-log10 1Ca I) of the calibrating solution. These plots should be sigmoidal curves with a steepness reflecting the 1: 1 Ca2+-binding stoichiometry of Fura- (Hill coefficient of 1). The curves for cells and solutions will overlap precisely when endpoints are attainable in all solutions. Where they do not the first step should be to alter the composition of the calibrating solution and repeat the procedure. We recommend that ionic strength and viscosity be varied in the first instance.

Viscosiry and ionic strength It has been reported that solution viscosity influences the fluorescence properties of Fura- and this has been shown to effect the calibration of fluorescent signals in some cell types ([22] and Poenie, this Issue). Fay et al. (this Issue) observed a difference in cell vs solution Fura- fluorescence behavior which is consistent with this reported viscosity effect. The small, 14 nM difference in Kd that existed between solution and cellular values in smooth muscle [2] diminished following small increases in the ionic strength (15 mM) and viscosity (2% as gelatin, due to 4% decrease in absolute value of Rmax) of the in vitro system, or with decreased ionic strength of the solution bathing the cell suspension. We therefore suggest that the influence of viscosity and ionic strength should be investigated in each of the cell types used, as there will undoubtedly be great variation in the absolute viscosity of the cytoplasm of the different cell types. This can be very simply undertaken with the

[K’+Na’] (mM)

Fig. 1 Dissociation constant of 01” from Fura- measured

(open circles) in the presence of different concentrations of Na+

and K’ (similar to the concentratkm of ionic equivalents I, see [9, 241) at 22”C, pH 7.20. The plotted data also includes msults

from earlier studies: (crosses) Gqnkiewicz et al. [l], and (open

circles) Williams et al. [2]

protocol described above by progressively increasing the viscosity (with sucrose or Ca-free gelatin) at any or all of the Ca-levels used. In this way the absolute dependence of the cellular Ca-fluorescence ratio relationship on solution and cellular viscosity can be determined. This strong dependence of CaPura-2 IGt (Fig.1) on the solution ionic strength also underlines the danger of making extrapolations as to the behaviour of Pura-2 in the intracellular environment based simply on observations made in calibrating solutions.

Calibration chamber It is a decided advantage to have the ability to allow for the consecutive comparison of in vivo and in vitro fluorescence ratios at each of the Ca2+-levels investigated, and we feel this is an essential requirement for greatest accuracy of this technique. However, this does place some restrictions on the construction of chambers used for the calibration procedure. There are a large number of potential designs for calibration chambers, including solutions contained within hematocrit tubes or wedged between glass coverslips, or chambers etched or milled into non-fluorescent materials that can be temporarily sealed by glass coverslips. However, we feel the most useful .designs should allow for free access to the calibration solutions during the

INTRACELLULAR CALIBRATION OF FUIU-2 81

procedure (i.e. to increase solution ionic strength or viscosity) and have multiple compartments to allow for consecutive comparisons of cellular and solution fluorescence ratios. They should be able to be temporarily sealed from the atmosphere so as to allow for optional gassing with nitrogen to reduce solution Ga and thereby minimize bleaching of the fluorophore [20]. Obviously, the interface between the chamber and the microscope objective should be thin (size l), ideally replaceable glass coverslips, especially if immersion objectives am to be used. It doesn’t requite a great deal of imagination to design a chamber that fulfiis these basic requirements.

C : A useful by-product of these techniques

A useful technical by-product of the techniques and time that have been invested in critically and accurately calibrating the fluorescence signals of Fura- is a simple procedure for measuring Ca2+ levels in various other buffered physiological solutions used in these laboratories. The importance of accurately measuring ionized Ca2+ in experimental solutions cannot be overemphasized. Suspension media for isolated orgauelles (such as mitochondria, contractiIe proteins and intracellular membrane vesicles), solutions for internal perfusion of axons and muscle fibers, and, of most interest in our laboratories, solutions that bathe shinned muscle preparations [13, 23, 241, all require accurately known ionized Ca2+ concentrations. Measurement of ionized calcium concentrations in experimental solutions has generally been undertaken with Ca-sensitive microelectrodes, or is simply estimated from a knowledge of the ionic species and potential Ca-buffers within the solution as we have already explained (see Section A). Fwa-2 can be used to accurately determine the [Ca2+l in these solutions as long as a Kd appropriate for the experimental conditions is available. To assist in this process we have determined the constant (I?$ for CaFura-2 with the techniques we have described for a range of ionic strengths. These data are plotted in Figure 1 and also include some earlier literature values. A straight line could be well fitted (correlation coefficient 0.993) to all values for Kd when plotted on semilogarithmic axes against the lNa+ + K’] of the experimental solutions. Although the chemical

0 I I I, I I I,, ,

300 350 400

EXCITATION WAVELENGTH (nm)

Fig. 2 Typical excitation spectra generated in the de&mination

of ionized Ca’+ concentration in experimental sohh3ns with Fura-2. Curves (a) wm obtaimd following addition of 2 @vI

Fura- pentapotassium salt to experimental sohhms containing

CaEGTA/EGTA ratios of 0.9 to 0.1 (A) and 0.6 to 0.4 (Et)

nspectively (see Table 1). These cumes were then calibrated by the addition of: (i) sufficient CaCIz (mM) to saturate EGTA and

Fua-2 (curves b); and (ii) an EGTAKOH mixtun: @H j8.0) to

ensure that all Fura- was iu its &-free form (curvea c). All

spectra iere corrected for volume and back ground fluorescence changes following any solution additions

basis of the linear logarithmic relationship between IQ and mat + I?] is not immediately evident, a similar linearity has recently been reported for the dependence of the constant for Ca2’ association of EGTA and the same monovalent cations [24]. With au appropriate knowledge of the approximate ionic strength of experimental solutions it is therefore possible to interpolate from the information in Figure 2 to obtain a &I to be used for the Ca2’ determination for any experimental solution.

The technique involves adding a small (l-5 @!I)

82 CJZLLCALCIUM

concentration of Fura- (pentapotassium salt) from a concentrated stock solution (l-5 mM) to a given solution. The potential Ca-buffering offered and dilution caused by the added Fura- concentration is small (less than 1% of that resident in the solution) and hence overlooked in further calculations. An excitation spectrum (with emission centered at 510 nm) is then obtained with a spectrofluorometer (see Materials and Methods) for each mixture and selected curves for two solutions selected from a range such as might be used in au experiment with skinned muscle preparations are shown in Figure 2 curves (a). To calibrate each separate curve in tears of actual*jY?a2+] it was necessary to perform similar excitation scans on each solution after: (i) addition of enough Ca2’ to saturate the EGTA and added Fura- in the solution (curve b), and (ii) addition of sufficient EGTAKOH mixture (pH 8.5) to ensure that the Fura- was Ca2’ free (curve c). These excitation 8ca1.18 could then be used to calculate [Ca2’] in each solution with the standard calibrating equation (after Grynkiewicz et al.[l]:

[Ca2+] = Kd ((R - Rmin)/(Rmax - R)) B . . . . .Eq. (2)

where R indicates the ratio of intensities measured at 340 and 380 run excitation (read directly from the spectral data in Curve a for a particular solution scan, Rmax and Rmin are the 340/380 ratios obtained for the same solutions where lQra-2 is fully Ca- saturated (curve b) and Ca-free (curve c) respectively. B is the ratio of fluorescence for 380 run ezcitation of Fura-2KaFura-2. All data were corm&d for volume changes occurring with each addition, and for the fluorescence contribution of the solution itself. The Ca2+ concentrations derived from such a calibration of the Fura- signal for a full range of Ca2+-containin solutions are listed in

fi Table 1. The estimated [Ca ] listed in Table 1 was calculated directly from the proportion of CaEGTA/EGTA in each solution with an affinity constant determmed previously 113, 25, 261, to be 4.76 x lo6 MY’ for the conditions used in these experiments. Fii 3 graphically displays the correlation between the [Ca2+] determined for these solutions with the Fura- technique described here and with the previously described potentiometric method (see Section A). A correlation coefficient of

pCa (Potentlometry)

FYg. 3 Conelation between Ca2+ concentrations detumiued with

Fura- snd potentiometric techniquea from a numk of sohtions constructed by the combination of solution B/A (see Table 1). ‘Ihe data are single values obtained for each experimental

sohrtion &tit the hvo methods (correlation coefficht 0.9%7)

0.9967 underlines the very good agreement between the potentiometric and Fura- methods.

The information for this calibration of each fluorescence spectrum could also simply be obtained from fixed wavelength measurements at both 340 and 380 nm excitation wavelengths (e.g. microscope measurements). However, significant additional infomration, which greatly increases the accuracy of Ca2’ measurements, is obtained from the full excitation scans shown in Figure 2. These scans possess characteristics which identify them as representing accurate curves for calibrating [Ca2’]. The peak excitation wavelengths should shift with the binding of Ca2’ from 363 to 335 run and all curves (a,b and c) should intersect at the isosbestic point of around 360 run following volume and background fluorescence corrections [ 11.

The method is independent of [Mg2+] and pH within a broad physiological range, and is not affected by the presence of competitive Cazt buffers such as Al? and calmodnlin (each of which do not affect the spectral characteristics of F%ra-2 when present in the l-20 mM, or l-120 @I range respectively). The method described in this study for Ca2+ determination is independent of the uncertainty as to the purity of EGTA and the value for K’ca [9] as the only parameters that are now being measured, relative Ca-Fura-2JFura-2, are

JNTRACELLULAR CALIBRATION OF FURA- 83

directly obtained from the spec@al data. In a sense, -tion of free [Cal in Ca-EGTA solutions. Am. J.

we have a fluorescent EGTA dire&y indicating the Physiol., 242, c404-408.

bound and unbound proportions of the EGTA in the 11.Yam&&iH.(f986) Reeo&gofintntceJhtIarCa=fmm

solution which is setting the free ionized Ca2’. Bmoodl muscle ctlls by sub-rnkron tip, double-bet&d Gag+” selective micmelectmdes. Cen Cak-itq 7,203.

With a suitably selected Kd (see Fig. 1) a direct and 12. Moisescu DG. Thiekzek R (1978) Calchtm and strotuium

straightforWti [Ca2’] determination is possible. txmcentration changes within sk.imted muscle pmpmttiom

This simple ~h~que is now in con usage in fo~w~a~~~ex~~~~ J. Physiol., 275.241262.

our laboratories and iu coniunction with 13. Smnhenson DG. Williams DA. (1981) Calcium-activated

potentiometric techniques allows Us to precisely determine [Ca2’] in all of the physiological

f& responses in fast- and slow-twitch skimud muscle fibers of the rat at different temperatures. J. Physiol., 317, 281-302.

solutions we use. 14. Lui C. Hermann TE. (1978) characterization of ionomycin as a calcium ionophore. J. Biol. Qmn., 253,5892.

15. Deber CM. Tom-&m J, Mach, E. Grin&in S. (1985)

Acknowledgements Bromo-A23187: a non-fluorescent calcium ionophom for use with fluorescent probes. Anal. B&hem., 146.349-352.

The authors would like to acknowledge the grant support of tbe NIH, MDA and the National Health and Medical Reseamb Council of Australia (NHMRC).

References

1. Gtynkiewicz G. Poenie M. T&n RY. (1985) A new genemtion of Ca” in- with greauy improved fluorescence propeities. J. Biol. Cbem, 360,344@3450.

2. Wilhams DA. Fogarty KE. Tsieu RY. Fay FS. (1985) Calcium gradients in single smooth muscle cells revealed by the digital imaging micmscope using Fura-2. Nature, 3 18, 558-561.

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Please send reprint requests to : h D. A. Williama, Department of Physiology, University of Melbourne, Parkville, 3052, Victoria, Australia.

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