Selection of fluorescent ion indicators for simultaneous measurements of pH and Ca2+

Cell Calcium (1996) 19(4), 337-349 Q Pearson Professional Ltd 1996

Research

Selection of fluorescent ion indicators for simultaneous measurements of pH and Ca*+ Raul Martinez-Zaguildnl’, Gaurav Parnamil, Ronald M. Lynch”* Departments Tucson,

of ‘Physiology Arizona, USA

and zPharmacology,

University

of Arizona,

Health

Sciences

Center,

Summary The advent of fluorescent ion sensitive indicators has improved our understanding of the mechanisms involved in regulating pHi and [Ca*+], homeostasis in living cells. However, changes in [Ca*+], can alter pH, regulatory mechanisms and vice versa, making assignment of either ion to a particular physiological response complex. A further complication is that all fluorescent Ca*+ indicators are sensitive to protons. Therefore, techniques to simultaneously measure these two ions have been developed. Although several combinations of pH and Ca*+ probes have been used, few systematic studies have been performed to assess the validity of such measurements. In vitro analysis (i.e. free acid forms of dyes) indicated that significant quenching effects occurred when using specific dye combinations. Fura-2/SNARF-1 and MagFura-2/SNARF-1 probe combinations were found to provide the most accurate pH and [Ca*+] measurements relative to Flue-3/SNARF-1, Ca*+-Green-l/SNARF-1, or BCECFKNARF-1. Similar conclusions were reached when probes were calibrated after loading into cells. The magnitude of interactions between pH and Ca*+ probes could be a factor which may limit the use of certain specific combinations. Loading of probes that exhibit interactions into distinct intracellular compartments (i.e. separated by a biological membrane) abolished the quenching effects. These data indicate that interactions between the probes used to simultaneously monitor pH and Ca*+ must be considered whenever probe combinations are used.

INTRODUCTION

Studies using fluorescent probes have demonstrated that intracellular pH (pHJ and CaZ+ ([Ca2+]J are involved in signal transduction mediating cell growth, secretion, motility, and contraction [ 1,2]. The coordinated activity of both pH, and [Ca”], regulatory mechanisms is required for the maintenance of steady-state levels of these ions,

Received Revised Accepted

16 August 27 December 2 January

1995 1995 7996

Correspondence to: Ronald M. Lynch PhD, Department of Physiology, University of Arizona, Health Sciences Center, Tucson, AZ 85724, USA *Present address: Department of Physiology, Texas Tech University, Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA

since most pH1 regulatory mechanisms are modulated by Ca*+. For example, the activity of Na+/H+ exchange is regulated by Ca*+ [3-51 and insertion of V-H+-ATPases into the plasma membrane is Ca2+ dependent [6]. Moreover, Caz+ regulatory mechanisms are affected by pH; acidic pH can increase the activity of Ca2+-ATPases[7], increase the dissociation constant of calcium binding proteins [8], decrease the opening probability of Ca*+ channels [9], and increase the forward activity of the Na+/Ca*+ exchanger [9]. The interactions between pH and Ca*+ emphasize the need to simultaneously monitor both ions to understand the specific role each plays in the regulation of physiological responses. A further complication when studying [Ca*+], homeostasis using fluorescent probes is that all currently available Ca*+ indicators are sensitive to pH, i.e. the dissociation constant (Kd) of Ca2+ indicators decreases with increasing pH [4, lo- 131. It has been suggested that this is due to a decrease in the association rates of Ca2+with the fluoroprobes [ 141. Since pH, and [Ca*+], are so interrelated, 337

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R Martinez-ZaguilAn, G Parnami, R M Lynch

accurate knowledge of [Ca2+], homeostasis requires correction for H+ binding to the intracellular Ca2+ indicators [4,15]. Fortunately, fluorescent H+ indicators are not affected by Ca2+ [1,4]. We have recently implemented methods to correct for effects of pH on Fura- [4,12,16]. In these experiments, cells were co-loaded with SNARF-1 (a pH indicator) and Fura-2, and the concurrent pH-measurements were used to correct for alterations in the fluorescence properties of Fura- due to H+ binding. Other investigators have also performed simultaneous measurements of pH and Ca2+ using dye combinations such as BCECF/Fura-2 [ 171, Indo-l/SNARF-I [ 151, Ca2+-Green -2/SNARF-1 [ 181, and Fluo-3/SNARF-I [ 191. There are several criteria that have to be taken into account for successful simultaneous measurements of two ions: (i) the fluorescent probes must be sensitive to the ion of interest; (ii) there is no quenching between fluoroprobes; and (iii) there is no spectral overlap between them. Due to the complexity of such effects, it is not surprising that few systematic studies have been performed to study these possibilities before attempting simultaneous measurements of these ions. In the present study, we performed systematic experiments to assess the validity of simultaneous measurements of pHi and [Ca2+li when using several commonly utilized fluoroprobe combinations including Fluo-3/SNARF- 1 [ 191, Ca2+-Green-2/SNARF- 1 [ 181, Fura-2/BCECF [ 171, and MagFura-2/ SNARF-1 [ 131, and Fura-2/SNARF-I [4,12]. MATERIALS

AND

METHODS

Spectrofluorometry

In vitro fluorescence measurements were performed in a SLM 8000C spectrofluorometer (Urbana, IL, USA) using slits set at 4 nm and an external rhodamine standard. Temperature was maintained using a temperature controlled circulating water bath (Lauda Model RM20, Brinkmann Instruments Co). Experiments on single cells were performed using microspectroscopic imaging [ 12,201. Buffers

Standard experimental media (SM) contained: 1.3 mM CaCl,, 1 mM MgSO,, 5.4 mM KCl, 0.44 mM KH,PO,, 110 mM NaCl, 0.35 mM NaH,PO,, 0.1 mM NaHCO,, 0.1 mM glucose, 2 mM glutamine and 25 mM HEPES, at a pH of 715 at 37°C. 0 Ca2+ buffer (KEGTA) contained: 110 mM KCl, 20 mM MOPS, 20 mM NaCI, 10 mM K,H,EGTA. Calcium saturated buffer (CaEGTA) contained: 110 mM KCl, 20 mM MOPS, 20 mM NaCI, 10 mM K,CaEGTA. High K+ buffer (High K+) contained: 146 mM KCI, 5 mM glucose, 2 mM glutamine, 10 mM HEPES, 10 mM MES, 10 mM Bicine. In vitro calibration was carried out using Cell Calcium

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the free acid form of the dyes solubilized in either high KC or in K/Ca-EGTA. Free acid indicators were initially dissolved in methylsulfoxide as a 1 mM stock and maintained at -70°C in the dark until used. For experiments, an aliquot of a dye stock was dissolved in the required buffer at a final concentration as indicated in the text for each specific indicator/combination. Media pH was directly measured with a Beckman model 71 pH meter, using a Corning glass combination electrode. The electrode was calibrated at two known pH values using commercially prepared standards from VWR Scientific (San Francisco, CA, USA). In vitro

Ca*+ determinations

Ca2+-EGTAbuffers of defined composition were used to generate calibration curves with fluorescent Ca2+indicators (Flue-3, Ca*+-Green- 1, Fura- and MagFura-2). Corrections of Ca2+:EGTA association constants for pH, temperature and ionic strength were performed as described elsewhere [4,21]. Once these association constants are calculated, the in vitro and in situ calibration curves with the Ca2+ indicators were generated as described previously [4]. Briefly, the titration was performed starting with 30 ml of KEGTA plus 2 @l of the desired Ca2+ indicator (free acid), adjusting the pH to a desired value at 37°C. A 3.0 ml aliquot from this solution was removed to record a fluorescence spectrum. Then, 3.0 ml of CaEGTA (at the same pH value as the KEGTA buffer) containing 2 PM of the Ca2+ indicator was added to the remaining 27 ml, giving 9 mM KEGT#l mM CaEGTA. Subsequent iterations were performed by removing 3.0 ml and replacing with equal volumes of the Ca*+-containing solution. Fluorescence emission spectra of Fluo-3 and Ca2+-Green-l were monitored using excitation at 480 nm. Excitation fluorescence spectra of Furaand MagFura-2 were monitored at 5 10 nm emission wavelength. Cell culture

Vascular smooth muscle (A7r5) cells and hamster insulinoma (HIT-T1 5) cells were obtained from American Type Cell Culture Collection (ATCC # 1440 and # 1770, respectively). Cells were grown in T-75 culture flasks using Dulbecco’s modified Eagle’s medium (DMEM; Sigma Chemical Co., St Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 10 U/ml penicillin G, 0. 1 mg/ml streptomycin sulfate, and 5 mM glucose, in a 5% CO, atmosphere at 37°C. For experiments on single cells, A7r5 cells were subcultured onto 25 mm round glass coverslips in 10 cm2 culture dishes using the same growing media except phenol red was absent. Cells were plated at an initial density of 0 Pearson

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Simultaneous measurement of pH and C@

approximately 5 x 1O4 cells/ 10 cm2 plate, and used after 2-3 weeks of culture. Loading of fluorescent pH and Ca2+indicators into cytosolic compartments

Cells grown on coverslips were co-loaded with the acetoxymethylester (AM) forms of the pH indicator SNARF-l/AM, and a specific Ca2+ indicator. A coverslip containing cells was washed 3 times with SM, then cells were incubated for 40 min at 37°C with 2 ml SM containing 7 PM SNARF-l/AM and 2 FM of the Ca2+ indicator (MagFura-2, Fura-2, Fluo-3 or Ca2+-Green-l) in their AM forms. After the incubation period, coverslips were washed with SM and further incubated for 30 min in the absence of AM dye to allow for the complete hydrolysis and leakage of the dye. Under these experimental conditions, the dyes distributed uniformly throughout the cytosol as evaluated by single cell imaging (data not shown). Thereafter, the coverslip was transferred to a temperature regulated chamber on the microscope stage for simultaneous pH, and [Ca”], measurements as fully described previously [ 12,201. Loading of Ca*+ indicators in endocytic and cytosolic compartments

For these experiments we took advantage of the fact that Ca2+indicators conjugated to dextran (70,000 MW dextran) are impermeable to the plasma membrane, but the dye can be taken into cells by endocytosis. To load endosomes, the cell culture media was replaced by one containing 50 I.tg/ml Ca2+-Green-DEX. We have previously shown that after 3-24 h, the dye conjugated to dextran is primarily localized in endocytic compartments 1221.In the present study, 12-16 h loading periods were used. Prior to measurements, cells were loaded with 7 uM SNARF-I/ AM for 30 mm at 37°C. Using this approach, SNARF-1 is distributed throughout the cytosol, whereas Ca2+-GreenDEX is localized in endocytic compartments [22]. In situ calibration of pH and [Ca*+],

Since the parameters used to estimate pH1 or [Ca2+],may vary between cell types, possibly due to inherent differences in the intracellular environment, in situ pH and Ca2+calibration curves for the cell line of interest must be performed. Briefly, the cytosol of cells was loaded with SNARF-l/AM and Ca2+ indicators in their AM forms; or by using SNARF-l/AM and Ca2+-Green-DEX to localize the dyes in the cytosolic and endocytic compartment, respectively (vi& supra). In situ calibrations for pH were carried out using the high K+ buffer containing nigericin (6.8 PM), and valinomycin (2 PM). In these experiments, 0 Pearson

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titration started by incubation of cells on the microscope stage for 3 min in high K+ buffer at acidic pH (5.5). Subsequently, pH was increased stepwise using 1 N NaOH and a 3 min incubation was allowed before spectral acquisition. This process was repeated until a pH of 8 was reached. Alternatively, cells were incubated at pH 8, and then pH was decreased stepwise with 1 N HCI. This process was repeated until a pH of 5.5 was reached. The ratio of fluorescence at the ion-sensitive wavelengths of SNARF-1 (6501584) was used to determine pH as described previously [4,16]. In situ calibrations for Ca2+ were performed using the K/CaEGTA buffers containing nigericin (6.8 PM), valinomycin (2 PM) and 4Br-A23187 (5 PM), at selected pH values. In these experiments, cells were preincubated at 0 Ca2+ and a selected pH value, then Ca2+ was increased stepwise by media changes with sequentially increasing Ca2+ concentrations. In all cases, a 3 mm equilibration period was allowed prior to acquisition of spectra. This process was repeated until 10-l 1 different Ca2+ concentrations were obtained. This was followed, in some experiments, by in situ titration of pH (vi& suflrd). The data generated from in situ calibrations similar to those shown in Figures 3 and 5B were evaluated by analysis of the relation between the Ca2+and the ratio values as described by the following equation: [Ca’+] = Kd [(R - R,J/(R,=

- R)]

where Kd is the apparent Fluo-3 or Fura- dissociation constant for Ca2+, Rmaxrepresents the Ca2+-dye chelate, and Rminis the fully Ca2+-free Fluo-3 (or Fura-2) signal intensity. Values from in situ titrations for both pH and Ca2+indicators were routinely fitted using the simplex method and non-linear regression analysis employing commercially available computer software (MINSQ, MicroMath Scientific Software, Salt Lake City, UT, USA). This type of analysis allows the estimation of in situ calibration parameters (i.e. pK, Kd, Rmaxand Rmin)needed to calculate pH, or [Ca2+li [4,12,16]. Data are presented as group means * SE unless otherwise indicated. In situ calibration parameters for pH, measurements obtained in cells co-loaded with Fluo-3 and SNARF-1, fitting data from at least 10 independent experiments, (with at least 6 different pH values each experiment), were as follows: pK = 7583 f 0.063, R,,,, = 2.049 f 0.343, and Rmi, = 0.537 + 0.016 (mean f SEM, n = 7 experiments). Increasing pH also decreases the ratio used to estimate Ca2+by Fluo-3. This effect of pH on the Fluo-3 fluorescence signal is also observed in calibrations performed in the absence of SNARF-1 (data not shown). The magnitude of this effect is smaller for Fura- than for Fluo-3 (see [4,12]). A similar decrease in the Fura- ratio Cell Calcium

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R Martinez-ZaguilAn, G Parnami, R M Lynch

effects of pH on the Fluo-3 Kd are observed from in vitro experiments using Flue-3 in the absence of SNARF-I (Fig. 1, open squares). The pH corrected Fura- Kd values have been reported previously and are expressed as dotted line in Figure 1 [ 121. The parameters that describe the effect of pH on the Fura- Kd parameters are: Kd- = 3586 nM, Kd- = 116 nM, and pK = 5.12. Notice that the magnitude of the effect of pH on the Kd of Fluo-3 is larger than on the Fura- Kd. We have previously shown that pH not only affects the Kd of Fura- but also influences R- and sin [4,12,16]. Similar effects of pH on Flue-3 were observed in the present study and were taken into account for estimation of [Ca2+livalues. Consequently, the effect of pH on the Kd, ~~~ and R,,,,,of either Fluo-3 or Fura- can be predicted by Equations 1 and 2 with the fitting parameters, and used to calculate [Ca’+], by the following equation:

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PH Fig. 1 Effect of pH on the dissociation constant (Kd) of Ca*+ to Flue-3 (solid lines) and to Fura(dashed lines). In situ titration curves were generated as described in Materials and methods. Each point represents the mean f SD of 9-11 different C3+ concentrations, at the indicated pH value, performed in triplicate. H+ and Ca*+ association constants for EGTA were calculated as described elsewhere 1121. Curve fittina was oerformed usina Equation 1 [18,19]. The dashed line represents the effect o?pH on FuraKd obtained from previously published data [19]. These curves were fitted into Equation 2 to obtain the parameters that describe the effect of pH on the Kd (see Materials and methods).

with increasing pH has been previously reported when Fura- is used as a dual excitation Ca2+probe [4,16]. Data from in sitzl titrations have been generated at various pH values, and fitted into Equation 1 to obtain the calibration parameters that describe the effect of pH on Ca2+ dyes. Analysis of the effect of pH on either the Fluo-3 or Fura- signal as Ca2+ concentration is varied provides an estimate of the effect of pH on the Kd for Ca2+ which can be described by the following equation:

KdpHcorr = [Kd,=

+ ~O@“-P~x Kd,i,]/lO@Hi-rq + l]

is the whereKdPHmm

Eq. 2

pH corrected Kd, and Kd,= and Kdmi, are the maximum and minimum Kd, respectively [4,12,16]. The underlying assumptions behind these calculations have been fully described earlier [4,12,16]. By fitting the data from in situ titrations for Caz+ into Equation 1, we can obtain the Kd values which are plotted as a function of pH (Fig. 1, open circles). These data are then fitted into Equation 2, to obtain the following parameters describing the effect of pH on the Fluo-3 Kd: KdmaX = 8840 nM, Kdml”= 2.06 nM, and pK = 4.824. Similar Cell Calcium

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Normalization of signal from Ca*+-Green-l and Flue-3 (single excitation/emission probes) to the ion insensitive wavelength of SNARF-1

Fluo-3 and Ca2+-Green-l can only be used as single emission/excitation probes, therefore, the fluorescence intensity of these probes is altered by changes in either Ca2+ (Fig. 2A), or dye concentration 1231.To partially circumvent the problem inherent to non-ratiometric probes, investigators have compared the fluorescence emission of these indicators to the ion insensitive wavelength of another dye, such as the SNARF-1 ion insensitive (isoemissive) wavelength (Fig. 2B; [ 12,18,19]). Since the isoemissive point at 600 nm does not change with varying pH and the fluorescence properties of SNARF-1 are not affected by Ca*+ [ 1,4], this signal represents a reasonable control for artifactual changes in fluorescence due to changes in cell volume or dye leakage/photobleaching. The assumptions which underlie the use of this normalization procedure include: (i) both dyes are located in the same intracellular compartment; (ii) both dyes leak/photobleach at similar rates during the time frame of an experiment; and (iii) similar dye loading is achieved for both probes. In the present work these data are expressed as 5281600 nm or (l/5 lo)/600 (see [ 121). Measurement of pH and Ca*+ in single cells

Analysis of pH1 and [Ca”], in single cells using microscopic spectral imaging has been described previously [ 12,201. Briefly, an Olympus IMT-2 inverted microscope equipped with a 200 watt Hg lamp as an illumination source was used. Imaging optics included a 60x 1.4 NA 0 Pearson

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Simultaneous measurement of pH and Ca2+ 341

Olympus objective and a 6.7x eyepiece to focus the cell image onto the input slit of a grating monochromator (Aries 250IS/SM spectrograph, Chromex Inc., Albuquerque, NM, USA). The spectral output from the grating was imaged onto a liquid cooled CCD camera (Photometrics Model CH250, Tucson, AZ, USA) equipped with a 512 x 512 element (27 un?/pixel) imaging chip. The digitized output of the CCD camera was stored in a 386 computer. Image analysis was performed on a Silicon Graphics Personal Iris computer, and spectral line analysis was performed on a 486 Gateway computer using Microsoft Excel version 4.0 (Microsoft Co., Redmond, WA, USA) and Sigmaplot for Windows version 2.0 (landel Scientific, San Rafael, CA, USA). Measurements of pH, and [Caz+],in single cells was performed using either Fluo-3/SNARF-1 and Ca2+-Green- 1/SNARF- 1 combinations, with excitation of fluorescence using a 10 nm band pass filter centered at 488 nm (Omega Optics, Brattleborough, VT, USA). Spectra acquired from an entire cell were averaged to improve signal to noise of the measurement. Then, the ratio of fluorescence emissions at 528/600 nm was used to measure [Ca2+] with either Flue-3 or Ca*+-Green-l. The SNARF-I ratio 644/584 was used to monitor pH. For Fura-2/SNARF- 1, a two-wavelength excitation filter was used to provide excitation of Fura- at 380 nm and SNARF-1 at 488 nm. A dichroic filter with low end cut off at 490 mn, 50% transmission at 505 nm and full transmission at 520 mn was used for all experiments (Omega

Optics). Spectra were not corrected for the transmission characteristics of the dichroic mirror. For analysis of pH and Ca*+, fluorescence emission at 5 10 nm (Fura-2) and 584, 600 and 644 nm (SNARF- -1) were analyzed. The ratio (l/510)/600 was used to monitor Ca2+,whereas the SNARF-1 ratio 644/584 was used to monitor PH. This approach has been fully described elsewhere [ 121. Chemicals

Fluorescent ion indicators were obtained from Molecular Probes (Eugene, OR, USA). All other chemicals were of analytical grade and purchased from commercial sources, unless otherwise stated. Statistics

Data are presented as group means + SD unless otherwise indicated. Statistical differences between group means were determined using paired or unpaired Students t-test. A value of P < 0.05 was taken as indicative of a statistically significant difference between group means. RESULTS

Studies using fluorescent probes have demonstrated that alterations in [Ca2+], and pH, are involved in regulating many physiological processes. However, the interactions

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on [Can+], in HIT-T15 cells co-loaded with either Fluo-3 and SNARF-1 (A) or Furaand SNARF-1 (B). cells grown on glass coverslips were loaded with specific dyes and transferred to the stage of a spectral of [Ca*+li and pH, as described previously [12,20]. At the time indicated (arrows), medium KCI concentration [Ca*+], was estimated as described previously, using pH-corrected parameters for Fluo-3 and Fura-2, lines represent the absolute pH-uncorrected Fluo-3 (A) or Fura(B) ratios.

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Fig. 3 (A) Combined emission spectra of Flue-3 and SNARF-1. SNARF-1 (6.6 FM) and Flue-3 (2 FM) were dissolved in Ca-EGTA buffers at various free Ca2+ concentrations. The H+ and Ca2+ association constants for EGTA, used to estimate free Ca2+, were calculated as described previously [4,21]. Spectra were obtained using a spectral imaging microscope as described previously [12,20]. Excitation of fluorescence was carried out using a 10 nm band pass filter centered at 488 nm. The salient characteristics of the spectra are the increase in emission intensity of Flue-3 at 530 nm as Ca2+ concentration increases, and the decrease and increase in the SNARF-1 emission wavelengths at 584 nm and 644 nm, respectively, as pH is increased. Notice that SNARF-1 exhibits an ion insensitive wavelength (isoemissive point). Shown are representative spectra from a full CaZ+ and pH calibration.

between pHi and [Ca2+li complicate assessment of the role each ion has in regulating a specific process. Moreover, all Ca2+ indicators are sensitive to protons. Therefore, techniques to simultaneously monitor these two ions using combinations of fluorescent indicators sensitive to pH and Ca2+have been developed. In the present study, the accuracy of simultaneous pHi and [Ca2+], measurements using several different dye combinations was assessed. Measurements of [Ca2+],with single emission Ca2+ probes using signal-normalization with SNARF-1

Elevation of extracellular K+ to 52 mM induces membrane depolarization and opening of voltage dependent Cell Calcium (1996) 19(4), 337-349

Ca2+channels in many eukaryotic cells. KC1 depolarization of insulin secreting HIT-T1 5 cells loaded with either Flue-3 or Caz+-Green-l, in the absence of SNARF-1, produces large transient increases in the signal from these Ca2+ indicators (data not shown). However, the absence of a normalization signal (SNARF-I isoemission wavelength) obviates correction for potential artifactual signal changes [ 18,191. Combined loading of Fluo-3 with SNARF-1 allows for normalization of the Ca2+ signal to the SNARF-I isoemissive wavelength [ 181, as well as simultaneous pH measurements. The combined fluorescence of these probes trapped in the cytosol of HIT-T 15 cells was monitored by microspectroscopic imaging 112,201. Treatment of HIT-T15 cells with KC1 results in only minor increases in [Ca2+],(50 + 20 nM above resting level; n = 18) when monitored using Fluo-3ISNARF-1 (Fig. 2A). The reported [Ca2+],values were estimated using in situ calibration parameters, and represent pH corrected values (see Fig. 1). A similar small elevation in Ca2+was observed in HIT-T1 5 cells coloaded with CaZ+-Green-l/ SNARF-1. In cells co-loaded with Fura-2/ SNARF-1, KC1 treatment elicited large increases in [Ca2+],(910f 120 nM above resting levels; n = 12; Fig. 2B), similar to those observed when Ca2+ measurements were carried out in the absence of SNARF-1. The apparent attenuation of the Fluo-3 and the Ca2+-Green-l signal responses, when coloaded with SNARF-1, motivated us to characterize the ion sensitivity of these probes while incubated together with SNARF-1 in vitro. Calibration of pH and Ca2+using Flue-3 or Ca2+-Green-l in combination with SNARF-1

The combined emission spectra of Fluo-3 and SNARF-1 using an excitation wavelength of 488 nm is shown in Figure 3. In this experiment, the concentrations of SNARF-1 (2 nM) and Flu03 (6.6 PM) were held constant while either Ca2+or pH was varied. The prominent characteristic of these spectra was an increase in the ion sensitive wavelength of Fluo-3 at 528 nm as Ca2+ was increased (Fig. 3). Increases in fluorescence at 528 mn also were observed when the Fluo-3 concentration was increased from 1 to 7 i.tM at constant Ca2+(tide z&$-u;Fig. 7A). However, the pH sensitive wavelength of SNARF- 1 at 584 nm also was observed to increase with increasing Ca2+ concentration (Fig. 3), or Ca2+ indicator concentration (Fig. 4A, uide infrd). The increased intensity at 584 nm with increasing [Ca2+] resulted in an apparent decrease in pH as measured by the ratio of fluorescence from SNARF-1 H+ sensitive wavelengths (Fig. 4B). Similar qualitative results were obtained for Ca2+-Green- 1/ SNARF-1 combinations. This was not due to an acidification of the buffers by the Ca2+probes (PK of ca 5), since high K+ buffer contains 30 mM of organic buffers (i.e. 0 Pearson Professional Ltd 1996

Simultaneous measurement of pH and Cti

FLUOS

sensitive ratio in agreement with previous studies [4,12,16]. However, similar changes in Ca2+concentration elicit a decrease in the SNARF-1 ratio in the presence of Fluo-3 (Fig. 4B). Similar findings were observed in the presence of Ca2+-Green-l (data not shown). This phenomenon, i.e. a decrease in the fluorescence ratio of SNARF-1 as a function of increasing Ca2+concentration, is not observed when SNARF-1 is used in combination with either Fura- or MagFura-2 [4,12,13,16]. Importantly, in situ titration for Ca2+using Fura- in conjunction with SNARF-1 elicits an increase in the Fura- ratio without change in the fluorescence ratio of SNARF-1 ([12];

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Fig. 4 (A) Effect of increases in Fluo-3 concentration on selected emission wavelengths of SNARF-1. SNARF-1 (6.6 HIM) and Flue-3 at the indicated concentrations were dissolved in high K+ buffer at a pH of 6.5, and their emission spectra were recorded using an SLM8000C spectrofluorometer with excitation set as 488 nm. The Fluo-3 concentration was increased stepwise by adding dye directly into the fluorometer cuvette. (B) Effect of increasing Ca2+ concentration on the SNARF-1 ratio in the absence (open circles) and presence (closed circles) of Fluo-3. Data were obtained from experiments similar to those described for (A), except the concentrations of Fluo-3, SNARF-1, and the pH were held constant while the Caz+ concentration was varied.

HEPES, Bicine, and MES) and direct measurements of pH using electrodes showed that addition of the Ca2+fluoroprobe decreases pH by < 0.02 pH unit. Thus, the dynamic range of the SNARF-1 pH sensitive ratio (in the presence of Flue-3 or Ca2+-Green-l) is decreased as the concentration of Ca2+(Fig. 4B) or the Ca2+probe is increased (Fig. 4A). Figure 4B illustrates that increasing Ca2+ concentration from 0 to 220 nM has no effect on the SNARF-1 pH 0 Pearson

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Ca2+(M) Fig. 5 (A) Effect of pH on SNARF-1 ratios in the presence of Fluo-3 and various [Ca*+]. SNARF-1 (2 PM) and Fluo-3 (2 PM) were dissolved in WCaEGTA buffers at the indicated [Ca*+] and pH values, and emission spectra were recorded using an excitation wavelength of 488 nm. Data are expressed as ratios at 644/584 for SNARFLl (B) Ratio of the Fluo-3 Ca*+ sensitive wavelength (530 nm) and the isoemissive ooint of SNARF-1 (600 nml as a function of Ca2+. These data were’derived from experiments similar to those shown in Figure 2, except that spectra were obtained in a SLM8000C spectrofluorometer using an excitation wavelength of 488 nm and emission from 500-700 nm were collected. Only the 526/600 nm ratio is plotted.

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0.5 z 11 0.4 SNARF-1 (6.6 pM)

The effect of Caz+(or Ca*+ probe) was different on each of the monitored wavelengths of SNARF with the signal at 584 run most sensitive (Fig. 4A). Even in the presence of a Caz+ probe, SNARF-1 can be calibrated, since increases in the 644/584 pH sensitive ratio of SNARF-1 are correlated with increases in pH (Fig. 5A). However, the relative concentration of Fluo-3 and/or the concentration of Ca2+ will influence the absolute value of the SNARF-1 ratio (Fig. 5A) making the accuracy of these measurements questionable. Altogether, these data suggest that interactions between single wavelength Ca2+ indicators and SNARF- 1 occur which not only attenuate 1.5

A

0.1

SNARF-1 -free

0.0 500

550

600

650

700

Emission wavelength (nm)

Ca+2-Green & SNARF-1 Ca?reen-I

(2 pM)

B

0.0

-A 0

2

4

6

8

Flu03 (FM)

B

1.6

550

600

650

700

4

6

0.8

Wavelength

Fig. 6 (A) Effect of increasing Flue-3 concentration at constant SNARF-1 concentration on their emission spectra. Ca*+ and pH fluorescent dyes were dissolved in high K+ buffer at a pH of 6.7, and their emission spectra were recorded using an SLM8000C spectrofluorometer with an excitation wavelength of 488 nm. SNARF-1 concentration was held constant at 6.6 FM while the concentration of Flue-3 was increased stepwise from 0 to 6.9 uM by adding dye directly into the fluorometer cuvette. (B) Effect of increasing SNARF-1 concentration at constant Ca*+-Green concentration on their emission spectra. Dyes were handled as described for (A). Caz+-Green-l concentration was held constant at 2 uM while the concentration of SNARF-1 was increased stepwise from 0 to 1 0 pM by adding dye directly into the fluorometer cuvette. Emission spectra were collected using an excitation wavelength of 488 nm.

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0

2

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6

8

SNARF-1 (FM) Fig. 7 (A) Effect of increasing Flue-3 concentration on fluorescence emission at 528 nm in the presence and absence of SNARF-1. Fluo-3 was excited at 488 nm and its emission was recorded as described in Figure 4, in the absence or presence of 6.6 uM SNARF-1, at a pH of 6.5, and 3 uM Ca2+, in high K+ buffer. (B) Effect of increasing SNARF-1 concentration on the fluorescence emission at 528 nm of either Flue-3 or Cap+-Green-l. Flue-3 or Ca2+-Green-l were dissolved in high K+ buffer at constant pH and Ca*+ as described in the caption to Figure 2, and their fluorescence at 528 nm were monitored using a SLM8000C spectrofluorometer with an excitation wavelength set at 488 nm.

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the observed Ca2+ response (Fig. 2), but also potentially modify the measured pH (Figs 4B & 5). Since the spectra of the two probes overlap substantially (Fig. 3) it was possible that the wavelength specific increases in fluorescence intensity of SNARF-1 with increasing Ca2+ was simply due to an increased contribution of fluorescence from the Ca2+probe as its intensity increased (seeFigs 3 & 5B). On the other hand, this cannot account for the reduced fluorescence observed at longer wavelengths. Assessment of interactions between pH and Ca*+ probes: quenching effects

To rule out spectral overlap issues, the effect of varying SNARF- 1 concentrations on the absolute fluorescence of Ca2+-Green-l or Fluo-3 were determined in vitro. As demonstrated in Figures 6 and 7, increasing the concentration of Flue-3 (Fig. 6A) or Ca2+-Green-l (data not shown), resulted in increases in fluorescence at 528 nm (Eig. 7A, cf Fig. 6A). However, the signal responses were greatly attenuated when the Fluo-3 concentration was increased in the presence of SNARF-1 (see Fig. 7A). Moreover, increasing the concentration of SNARF-1 at constant Fluo-3 or Ca*+-Green-l concentration, resulted in decreased fluorescence at 528 nm (Figs 6B & 7B). These data suggest that SNARF-1 quenches the fluorescence of these specific Ca2+ probes. Since increasing SNARF-1 concentration should increase the total signal

if spectral overlap was a predominant problem, this observation indicates that more complex interactions between these dyes are involved. Quenching requires close proximity of the probes (
0.7

3.01

0.6

0.5

0.7

C? t-8

0.6 ;

$ g

0.5

0 0.4

D

0.8

z 3 -8 ttc cl

0.4

0.6 SNARF-1 (cytosol)

~NARF-I

0.0

I

I

A

(cytosol)

I

I

I

Oe+O 2e-6 4e-6 6e-6 8e-6 le-5 Ca” (M)

B 0.3

I

I

O.Oe+O 4.0e-6

8.0e-6

I

1.2e-5

0.3 1.6e-5

Ca*’ (M)

Fig. 8 (A) Effect of increasing Ca2+ on the ratios of Ca2+-Green-DEX (loaded into endosomes/lysosomes) and SNARF-1 (loaded in the cytosol) in A7r5 cells whose pH and Ca2* gradients have been collapsed. Data were derived using a spectral imaging microscope for analysis of CaZ+ and pH, as described previously [12,20]. Experiments were performed as described for Figures 2 and 5. Cells were titrated in situ for Cap+, in the presence of Ca*+ and H+ ionophores (see Materials and methods). The pH was held constant at 6.7. Data are expressed as the pH sensitive ratio of SNARF-1 (644/584) and the ratio of the Ca2+ sensitive wavelength of Ca2+-Green (528 nm) and the ion insensitive wavelength of SNARF-1 at 600 nm. Notice that the 528/600 ratio increases with increasing Ca2+ while there are no changes in the SNARF-1 pH sensitive ratio at 644/584. (B) Effect of increasing Ca2+ on the ratios of Fluo-3 and SNARF-1 (loaded in the cytosol) in A7r5 cells whose pH and Ca2+ gradients have been collapsed. In situ titrations were performed as described for (A). Data are expressed essentially as described for (A). In these experiments, the pH was maintained constant at 6.7, thus the decrease in the 644/584 ratio of SNARF-1 is not due to pH changes, but rather to quenching between SNARF-1 and Flue-3.

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with increasing Ca*+. Furthermore, in situ titration of Flue-3 and SNARF-1 after loading into the cytosolic compartment of A7r5 cells demonstrated that increasing Ca2+at a constant pH of 6.75 results in an increase in the Flue-3 ratio associated with a decrease in the SNARF-1 ratio. Since pH was maintained constant, the decrease in the SNARF-1 ratio is likely due to quenching effects between Fluo-3 and SNARF-1. In situ titration of cells where SNARF- 1/AM and Fura-Z/AM were co-loaded into the cytosol demonstrates that increasing Ca2+,at constant pH, does not alter the SNARF-1 ratios. Altogether, these data support the idea that dye concentrations and proximity are sufficient to allow quenching between ion indicators located in the cytosolic compartment. Evaluation of quenching between pH and Ca*+ probes

To evaluate the relative usefulness of specific combinations of pH and Ca2+ probes, we performed systematic in vitro experiments using several dye combinations which have been previously used for simultaneous measurement of pH, and [Ca2+]r To empirically evaluate dye interactions, the fluorescence intensity of the acceptor (SNARF-1, or BCECF; IA, is plotted against the fluorescence intensity of the donor (Flue-3, Ca2+-Green-l, Fura-2, or MagFura-2; ID) while donor concentration is changed. As the concentration of the donor was increased, the ratio (I,/$,) decreased (Fig. 9). This type of behavior, i.e. decreases in the ratio 1,/I, with increasing concentration of the donor (IJ was observed at all pH values tested from 6.3 to 77 Similar decreases in the 1,/I, ratios are observed for single emission Ca2+ probes (i.e. Ca2+-Green- 1 or Fluo-3) when the concentration of Ca2+ is varied. The summary of results on dye interactions for different pH and Ca2+indicator combinations is shown in Table 1. For the purpose of comparison, we have related the estimated efficiency of the apparent quenching between indicator combinations to that measured for the well known acceptor/donor pair fluorescein/rhodamine which exhibit quenching [24,25]. Fluorescein/rhodamine was excited at 488 mn and their fluorescence emissions were analyzed at 514 nm (fluorescein) and 526 nm (rhodamine). Since the experiments performed with pH and Ca2+fluoroprobes employed micromolar concentrations of dyes, for the purpose of comparison we also employed a similar range of concentrations for fluorescein/rhodamine. Using this pair of fluoroprobes, we observed that the efficiency of quenching decreases as dye concentration decreases (data not shown). For the purposes of analysis, the maximum 1,/I, value derived from a series of experiments using fluorescein/rhodamine was considered as unity. This type of analysis indicates that quenching is largest for BCECF/SNARF-1, with both Ca2+Green- 1/SNARF-1 and Fluo-3/SNARF-1 combinations Cell Calcium (1996) 19(4), 337-349

exhibiting moderate quenching. The quenching between MagFura-2/SNARF-1 SNARF-1 was minimal.

efficiency of and Fura-2/

DISCUSSION

Primary reasons for monitoring pH, and [Ca2+],simultaneously include: (i) to understand the interrelation between pH, and [Ca2+lihomeostasis, so that the role of either ion in regulating specific physiological responses can be assigned; and (ii) to correct for effects of pH on the properties of fluorescent Ca2+probes. The rationale for selecting a specific combination of pH and Ca2+ probes must include consideration of: (i) ion selectivity; (ii) binding affinities in the physiological range; (iii) spectral overlap between probes; and (iv) possible interactions between probes (e.g. quenching). In the present study, specific dye combinations which have been utilized previously for in situ experiments were assessed to determine if signal responses were altered by probe interactions. Since most cells exhibit nanomolar concentrations of cytosolic Ca2+, the Ca2+ probes that have been used the most are those with dissociation constants (Kd) of ca 200-300 nM (e.g. Fura-2, Ca*+-Green-l, Fluo-3, Indo-1; [26]). On the other hand, BCECF-1 and SNARF-1 often have been used to study pH because they exhibit pK values of ca 70 and 74, within the physiological range of pH, found in most cells (ca 6.8-75). A difficulty in using the fluorescence of Fluo-3 and Ca2+-Green-l at 528 nm to monitor Ca2+is that the measured signal is also proportional to dye-concentration (cf Figs 5, 6A & 7A). This property makes it impossible to determine the exact basis for changes in fluorescence, since the dye concentration can be altered by changes in cell volume or dye leakage. An approach for correction of Fluo-3 or Ca2+-Green-l for these potential artifacts is to normalize the signal to the ion-insensitive wavelength (600 nm) of SNARF-1 (see Fig. 3; [ 12,18,19]). However, as shown in Figure 1, this approach is not exempt from error, since HIT-T15 cells co-loaded with Fluo-3/ SNARF-1 responded with attenuated increases in [Ca2+], in response to KCl-induced membrane depolarization. Clearly, the magnitude of the apparent [Ca’+], response was significantly attenuated when compared to that observed in HIT-T15 cells co-loaded with Fura-2/ SNARF-1 (see Fig. 2) or with Fluo-3 alone. The observation that there are differences in the magnitude of the apparent [Ca2+],response to KC1 treatment when comparing cells co-loaded .with Flue-3/SNARF-1 versus Fura-2/SNARF-1 combinations are somewhat surprising since use of in situ calibration parameters to estimate [Ca2+], would be expected to account for these differences. However, because interactions between Fluo-3 and SNARF-1 occur, the predictions regarding Ca2+ 0 Pearson Professional L td 1996

Simultaneous measurement of pH and Ca2’ 347

magnitude of quenching is related to the relative concentration of probes. Other Implications of this issue have been addressed previously, and were taken into U SNARF-l/FIuo-3 account in the present study [ 161. In order to further evaluate the reasons for this apparent discrepancy in the -cBCECFIFura-2 KCl-induced Ca2+ response between different Ca2+ -ASNARF-IIMag-Fura1.6 probes, a series of systematic in vitro experiments were p performed using the free acid forms of the dyes. r-5 Increasing either dye or Ca2+ concentration resulted in alterations in SNARF-1 fluorescence with the magnitude of this effect larger at shorter wavelengths (i.e. 584 nm) than longer wavelengths (644 run) of SNARF-1 (see Fig. 4A). Consequently, the pH sensitive ratio of SNARF-1 (in the presence of either Fluo-3 or Ca2+-Green-l) decreases when increasing either Ca2+or dye concentration (cf Figs 4B & 5A). Since changes in Ca2+also alter the signal at the SNARF-1 isoemissive wavelength, normalization of the 0.2 1.2 I I I I I fluorescence signal of the Ca2+ probe to that signal can 0 2 4 6 8 10 be problematic. This point is not immediately apparent, Donor (PM) since increasing Caz+resulted in increases in the 5301620 ratio (see Fig. 5B). The observed attenuation of the Ca2+ Fig. 9 Effect of increasing concentration of the donor (Flue-3, signal of either Flue-3 or Ca2+-Green-l in the presence Fura-2, MagFura-2) on the 1,/l, ratios. In these experiments, the of SNARF-1 is likely due to quenching of the Ca2+indicaconcentration of the acceptor (i.e. SNARF-1 or BCECF) was set at 6.6 FM while the concentration of the donor was increased. tor fluorescence by SNARF-1 (cf Fig 6 & 7). Moreover, Throughout the experiment, both the pH and the Ca*+ because of these interactions, the pH, measurements are concentration were held constant at 7.1 and 3 uM, respectively. also altered in the presence of the Ca2+probe (seeFig. 5A). Ca*+ and pH dyes were excited at their optimum excitation wavelengths. Data from pH and Ca*+ probes were also collected at It was possible that dye concentration could limit the their optimum emission wavelength (see Materials and methods). magnitude of the quenching in vivo, since the distance between dyes must be small. In that the relative and absolute dye concentration within a cell can not be controlled easily, experiments to determine quenching between probes was also performed in cells whose cytosol changes using pH corrected parameters are dependent was co-loaded with Ca2+-Green-l/SNARF-1 (Fig. 8B). Our on parameters which cannot be easily controlled. This is data indicated that quenching also can be demonstrated supported by our observations in Figure 2A, which indiin situ. To demonstrate that the in situ observations were cate that, following KC1 treatment, only attenuated dependent on dye proximity, cells were loaded with Ca2+increases in both the measured ratio and the estimated Green-DEX (endosomal compartment) and SNARF- 1/AM [Ca2+] are observed even after signal ‘calibration’. A pri(cytosolic compartment; see Fig. 8A). Under these condimary complication that must be considered when tions, no quenching between probes was observed. analysing this type of data is the issue of relative dye conTherefore, artifacts introduced by dye interactions can be centrations of the pH and Ca2+ fluoroprobes since the 0.8

Table

, I

1.8

1 Combinations

of pH and Ca2+ probes:

summary

of results.

(relative

Efficiency of quenching to fluorescein/rhodamine)

Conditions

Control FluoresceindRodamine,

1

ex 488,

em, 526,

0.85 0.6 0.37 0.16 0.1

ex ex ex ex ex

emA emA emA em, emA

em, 514

Experimental Fura-2dBCECF, Ca2+-Green-l JSNARF-1 Fluo-3JSNAFlF-1 A MagFura-2JSNARF-1 Fura-2$NARF-1,

A A

All data generated in high K+ buffer at pH 7.1, at 37°C. O-10 PM for both donor (D) and acceptor (A) pairs.

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range

of concentrations

used

in these

380, 488, 488, 392, 380,

experiments

529, em, 510 584, em, 528 584, em, 528 584, emD 510 584, em, 510 varied

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avoided if the fluoroprobes are sequestered within independent cellular compartments. The relevance of quenching for several probe combinations was evaluated in an empirical manner, by plotting the ratio of the fluorescence intensity of the acceptor (I,) to that of the donor (I,) versus the fluorescence intensity of the donor. As shown in Figure 9, increasing the concentration of the donor (i.e. Fluo-3, Fura- or MagFura-2), in the presence of a constant concentration of the acceptor (either SNARF-1 or BCECF) resulted in a decrease in the ratio I*/L indicating that quenching occurs between these two probes. However, the magnitude of this effect is different for each probe combination, i.e. being larger for Fura-2/BCECF>Ca*+-Green-l/SNARF-l>Fluo-3/ SNARF- l>MagFura-2/SNARF- 1rFura-2/SNARF- 1 (Table 1). We have previously shown there is no quenching between Fura-2/SNARF-1 when both probes are used in a ratio mode, thus our data are in agreement with these previous observations [4]. Therefore, from in vitro and in situ experiments, we conclude that quenching occurs between Fluo-3/SNARF- 1, Ca2+-Green- 1/ SNARF- 1 and BCECF/Fura-2 combinations (Table 1). In contrast, no significant quenching was observed between MagFura-21 SNARF-1 or Fura-2/ SNARF-1 (Table 1; [4]). Our data indicate that important interactions between certain pH and Ca2+ probes exist that should be considered for appropriate measurement of pH and Ca2+simultaneously. However, if the dyes are loaded in distinct intracellular compartments, these quenching effects are eliminated. To summarize, our in vitro and in situ data indicate that accurate simultaneous measurements of pH and Ca2+ can be made with certain probe combinations (Fura-2/SNARF-1, MagFura-2/SNARF-1), but measurements with other combinations (Fluo-3/SNARF- 1, Ca2+Green-l/SNARF-1 and BCECF/Fura-2) are influenced by dye interactions. Corrections for these interactions are not straightforward, since multiple considerations must be taken, e.g. H+ binding on the Ca2+ indicator and quenching. Moreover, corrections for quenching effects require accurate knowledge of dye concentrations and subcellular distributions of the dyes. Since problems associated with dye interactions are negligible when using Fura-2/SNARF- 1 and MagFura-2/SNARF- 1, these probe combinations are most appropriate for simultaneous measurements of pHi and [Ca2+], ACKNOWLEDGEMENTS

The authors thank MS Juhie Parnami for her technical assistance and Dr Robert J. Gillies for the use of a SLM8000C fluorometer. This work was supported by grants from the American Heart Association/AZ and Arizona Disease Control Research Commission (82-2691) to RML and by training grant support (NIH HL07249-17) to RMZ. Cell Calcium

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REFERENCES 1. Tsien R.Y. Fluorescent indicators of ion concentrations. Methods Cell Bioll989; 30: 127-156. 2. Gillies RJ., Martinez-Zaguilan R., Peterson E.P.,Perona R. Role of intracellular pH in mammalian cell proliferation. Cell Pkysiol Biockem 1992; 2: 159-179. 3. Vicentini L.M., Villereal M.L. Inositol phosphates turnover, cytosolic Ca2+ and pH: putative signals for the control of cell growth. Life Sci 1986; 38: 2269-2276. 4. Martinez-Zaguil&n R., Martinez G.M., Lattanzio F., Gillies RJ. Simultaneous measurement of intracellular pH and Caz+ using the fluorescence of SNARF- 1 and Fura-2. Am J PkysioZl991; 260: C297-C307. 5. Sanchez&mass S.,Martinez-Zaguilan R., Martinez G.M., Gillies R J. Regulation of pH in rat brain synaptosomes. 1. Role of sodium, bicarbonate, and potassium. J Neurophysiol1994; 71: 2236-2248. 6. Van Adelsberg J., Al-Awqati Q. Regulation of cell pH by Caz+mediated exocytotic insertion of H+-ATPases.J Cell Biol1986; 102: 1638-1645. 7 Dixon D.A., Haynes D.H. The pH dependence of the cardiac sarcolemmal Ca*+-transporting ATPase: evidence that the CaZ+ translocator bears a doubly negative charge. Biockim Biophys Acta 1990; 1092: 274-284. 8. Carafoli E. Intracellular calcium homeostasis. Anna Rev Biockem 1987; 56: 395-433. 9. Orchard C.H., Kentish J.C. Effects of changes of pH on the contractile function of cardiac muscle. Am J PhysioZl990; 258: C967-C98 1. 10. Lattanzio F. The effects of pH and temperature on fluorescent calcium indicators as determined with chelex-100 and EDTA buffer systems. Biockem Biopkys Res Commun 1990; 171: 102-10s. 11. Baker A.J., Brandes R., Schreur J.H.M., &macho AS., Weiner M.W. Protein and acidosis alter calcium-binding and fluorescence spectra of the calcium indicator Indo-l . BiopkysJ 1994; 67: 1646-1654. 12. Martinez-Zaguilan R., Gurule W.M., Lynch R.M. Simultaneous measurement of intracellular pH and Caz+in insulin secreting cells by spectral imaging microscopy. Am / PkysioZ1996; 40: In press. 13. Martinez-Zaguilan R., Pamami J., Lynch R.M. Mag-Furaexhibits both low (pM) and high (nM) affinity sites for Caz+ binding. Biophys J 1996; 70: 2 1OA. 14. Lattanzio F., Bartschat D.K. The effect of pH on rate constant, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators. Biockem Biopkys Res Commun 1991; 177: 184-191. 15. Wiegmann T.B., Welling L.W., Beatty D.M., Howard D.E., Vamos S.,Morris SJ. Simultaneous imaging of intracellular [Caz+] and pH in single MDCK and glomerular epithelial cells. Am J Pkysiol 1993;265:C1184-C11190. 16. Martinez-Zaguilan R. Measurements of intracellular [Ca*+],and pH< in cultured cells by fluorescence spectroscopy. In: Watson R.R. (Ed) In Vitro Methods of Toxicology. Boca Raton: CRC Press, 1992; 217-236. 17 Simpson A.W.M., Rink TJ. Elevation of pH, is not an essential step in calcium mobilization in fura- loaded human platelets. FEBS Lett 1987; 222: 144-148. 18. Spencer CL, Berlin J.R. A method for recording intracellular [Ca2+]transients in cardiac myocytes using calcium-green-2. @i&en Arch 1995; 424: In press. 19. Rijkers G.T., Justement L.B., Griffoen A.W., Cambier J.C. 0 Pearson

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20.

2 1. 22.

23.

24.

Improved method for measuring intracellular Ca*+ with FIuo-3. cytometry 1990; 11: 923-927 Martinez-Zaguil&n R., Tompkins L.S.,Lynch R.M. Simultaneous analysis of multiple fluorescent probes in single cells by microspectroscopic imaging. Proc Sot Int Opt Engin SPIE 1994; 2137: 17-28. Harrison S.M., Bers D.M. Correction of proton and CaZ+ association constants of EGTA for temperature and ionic strength. Am JPkysioll989; 256: C1250-C1256. Martinez-Zaguil%n R., Lynch R.M., Martinez G.M., Gillies RJ. Vacuolar type H+-ATPases are functionally expressed in plasma membranes of human tumor cells. Am J PhysioZl993; 265: c1015-c1029. Minta A., Kao J.P.Y.,Tsien R.Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chemical 1989; 264: 8 171-8 178. Lakowicz J.R. F’rinciples of Fluorescence Spectroscopy. New York; Plenum, 1986; 257-339.

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25. Herman B. Resonance energy transfer microscopy. Methods Cell Biol 1989; 30: 219-243. 26. Haugland R.P.In: Larison K.D. (Ed) Handbook of Fluorescent Probes and Research Chemicals. 5th edn. Eugene: Molecular Probes, Inc., 1992; 42 1. 27 Wysynski A-M., Martinez-Zaguikin, R., Alden C., Gandolfi A.J., Gillies R.J.Phenobarbital induces cytosolic acidification in an established liver epithelial cell line. Toxic01Lett 1994; 74: 157-166. 28. Gillies R.J.,Martinez-ZaguiIarr R. Regulation of intracellular pH in BALB/c-3T3 cells: bicarbonate raises pH via NaHCO,/HCl exchange and attenuates the activation of Na+/H+ exchange by serum.J Biol Chemical 1991; 266: 1551-1556. 29. Martinez-ZaguiIan R., Wegner J.A., Gillies RJ., Hoyer P.B. Differential regulation of Caz+homeostasis in ovine large and small luteal cells. Endotinology 1994; 135: 2099-2108. 30. Roos A., Boron W.F. Intracellular PH. Physiol Rev 198 1; 61: 296-434.

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