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Determination of intracellular pH of BALBc-3T3 cells using the fluorescence of pyranine
ANALYTICAL
BIOCHEMISTRY
167,362-37
1 (1987)
Determination of Intracellular pH of BALB/c-3T3 Using the Fluorescence of Pyranine KENNETH A. GIULIANOANDROBERT
Cells
J.GILLIES’
Department of Biochemistry, Colorado State University. Ft. Collins, Colorado 80523 Received April 28, 1987 In terms of accuracy and sensitivity, intracellularly trapped, pH-dependent fluorescent probes are appropriate to accurately measure intracellular pH. These probes are commonly introduced into living cells in esterified form, wherein the free acid is produced through enzymatic hydrolysis. The fluorescence characteristics of the ester and the free acid can differ markedly and spectral uncertainty can occur. We describe here the measurement of intracellular pH using I-hydroxypyrene- 1,3,6-trisulfonic acid (pyranine) that has been scrape-loaded into BALB/c-3T3 mouse cells. The excitation spectrum of pyranine is pH sensitive, with an isosbestic point at 415 nm and peaks at 405 and 465 nm which decrease and increase with pH, respectively. The 465/405 ratio can be used to monitor the pH, while the fluorescence at 415 nm indicates the total dye-dependent signal remaining. The scrape-loaded dye persists in cells for periods up to 6 h. We have calibrated this dye in situ using nigericin/high K+, and have found that the pK, of the dye in situ is 7.82, as compared to 7.68 in vitro. We have observed that the cells can slowly equilibrate their intracellular pH to near control levels when presented with either an acute alkaline or acid load. 0 1987 Academic Press, hc. KEY WORDS: pyranine; intracellular pH; fluorescence; BALB/c 3T3.
ft is becoming apparent that changes in intracellular pH (pHi) are associated with a number of cellular processes, such as proliferation (I), insulin secretion (2), and, possibly, differentiation (3,4). The ability to ascribe physiological significance to these pH changes depends on the capability of accurately measuring this parameter. In terms of sensitivity and time resolution, methods employing intracellularly trapped, pH-dependent fluorescence probes compare favorably with the use of NMR (5) or micro-pH electrodes (6). The fluorometric characteristics of many of these probes have been discussed (7,8). Fluorescence ratio imaging is a powerful technique when used to determine the role of intracellular ions in cellular physiology (9). A fluorescence ratio essentially normalizes fluorescence signal variations due to changes in
both cellular thickness and dye content (9). Derivatives of fluorescein are commonly used with a fluorescence excitation ratio method to determine pHi (7,9). The pH-sensitive fluorescent dye 1,4-dihydroxyphtalonitrile has found application in those systems where a fluorescence emission ratio is more appropriate ( 10). Fluorescent pH probes are frequently introduced into a living cell in an esterified form, wherein the free acid is produced through enzymatic hydrolysis (5, IO). This method can be problematic, since the fluorescence spectra of these various esters often differs markedly from that of the free acid (10,ll). In some cases, the intracellular hydrolysis of esters can cause cytoplasmic acidification (12). For these reasons, it is often useful to load only that fluorescent species which reports the pHi. Several methods are used for introducing impermeant molecules into living cells.
’ To whom correspondence should be addressed. 0003-2697/87$3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Determination of intracellular potassium concentration. The intracellular volume was determined as a function of total cellular protein using a modification of the double label method as described by Gillies and Deamer (21). Serum-stimulated cells were washed three times with an ice-cold K-free buffer containing 1.3 mM CaC12, 0.8 1 mM MgS04, 0.44 mM NaH2P04, 115 mM NaCl, 0.35 IIIM Na2HP04, 1.0% (w/v) glucose, 4.2 mM NaHC03, 2 mM glutamine, and 25 mM Hepes with a pH of 7.2 at 25°C. Two mililiters of the K+ free buffer containing 1 PC1 MATERIALS AND METHODS of D-[‘4C]sorbitol (200 mCi mmol-‘; ICN BiCell culture. Balb/c-3T3 mouse embryo omedicals, Inc., Irvine, CA) and 10 &i of cells were obtained from American Type [3H]H20 (100 mCi ml-‘; ICN Biomedicals, Culture Collection (ATCC CCL 163). They Inc., Irvine, CA) were then added to the cells were cultured in Dulbecco’s modified Eagle’s and incubated for 5 min at room temperamedium (DME2; Gibco, Grand Island, NY), ture. As much of this solution as possible was supplemented with 10% NuSerum (Collaboaspirated from the plate. One mililiter of 0.1 rative Research, Inc., Lexington, MA). The N NaOH was then added to solubilize the initial inoculum as received was grown to cells. Aliquots of the radioactive bathing so70% confluency at 37°C in a humidified, 5% lution and the solubilized cells were then CO2 atmosphere in 300 cm2 at which time subjected to liquid scintillation counting. A the cells were frozen in DME supplemented Coomassie blue dye-binding protein assay with 10% (v/v) dimethyl sulfoxide, and 20% (Bio-Rad Laboratories, Richmond, CA) was NuSerum at a density of 1 X lo6 cells per performed on the solubilized cell solution freezing ampule. The cells were recovered using egg white lysozyme as a standard as quarterly in 75-cm2 flasks and passed bi- suggested by Tal et al. (22). The calculations weekly at an inoculation density of 2 X lo5 used to determine the cell volume as a funccells per 75-cm2 flask. Cultures were tested tion of cell protein using this double label monthly for mycoplasma contamination. method have been previously described (2 1). For experiments, cells were plated from these To determine the K+ content as a function stocks at a density of 1 X lo6 per 59-cm2 of total protein, cells washed in IS+-free culture dish in DME plus 10% NuSerum. On buffer as described above were immediately the following day, these cultures were either solubilized with 0.1 N NaOH. One aliquot of scrape-loaded with pyranine (serum-stimuthe solubilized cell solution was assayed for lated cells) or the growth medium was total protein and the other for potassium changed to DME with 0.2% NuSerum. These using atomic absorption spectrometry. To (serum deprived) cultures were incubated determine intracellular K+, the ratio of under low serum for 36-40 h, at which time moles K+ to protein was divided by the ratio they were scrape-loaded with pyranine. of cell volume to protein. Scrape-loading of pyranine dye. Pyranine was scrape-loaded into the cells using the * Abbreviations used: DME, Dulbecco’s modified method described by McNeil et al. (14). Eagle’s medium; Hepes, 4-(2-hydroxyethyl)-l-piperaBriefly, the cells to be loaded were washed zineethanesulfonic acid; Mes, 4-morpholineethanesulthree times with a buffer, at 37°C containing fonic acid; BCECF, 2’,7’-bis(carboxyethyl)-5,6-carboxy 1.3 mM CaC12, 1 mM MgS04, 5.4 mM KCl, fluorescein.
These include microinjection ( 13) scrapeloading ( 14), cell fusion ( 15), electropermeabilization ( 16), and hypertonic ( 17) or ultrasonic shock ( 18). In this report we show that &hydroxypyrene- 1,3,6-trisulfonic acid (pyranine), previously used as a pH probe within vesicles (19,20), can be introduced into adherent BALB/c-3T3 cells via scrapeloading and that the pHi can be sensitively measured for a number of hours afterward using a fluorescence excitation ratio method.
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0.44 mM KH2P04, 110 mM NaCl, 0.35 mM Na2HP04, 5 mM glucose, 4.2 mM NaHC03, 2 mM glutamine, and 25 mM Hepes at a pH of 7.15 at 37°C (cell suspension buffer). The cells were then scraped from the plate after overlaying them with 5 ml of 1 mM pyranine in cell suspension buffer at 37°C. Suspended cells were then washed twice with ice-cold DME plus the same concentration of NuSerum in which the cells were cultured. The final cell pellet was resuspended in this same wash solution at 37°C and 300 ~1 of this suspension (l-3 X lo5 cells) was applied to a glass coverslip (9 X 22 mm). The cells were then allowed to attach to the coverslip for 1 h at 37°C in a humidified, 5% CO* atmosphere. To determine cellular autofluorescence, cells were subjected to the scrapeloading procedure using a buffer without any added pyranine. Spectrofluorometric measurements. An SLM 4800 spectrofluorometer (SLM Instruments, Inc., Urbana, IL) was operated in the ratio mode to correct for changes in lamp intensity as a function of both time and excitation wavelength. The emission monochrometer was set to 5 14 nm with a slit width of 8 nm while the excitation monochrometer slit width was 2 nm. Data were collected through an interface to an IBM personal computer and were analyzed using programs written in PASCAL (Turbo Pascal 3.0, Borland International, Inc., Scotts Valley, CA). The spectrofluorometer does not allow for rapid excitation of the sample with selected wavelengths of light. Instead, this instrument cycles through 5-nm steps which require a total time of 5 s each to allow for acquisition of a fluorescence intensity value (2.5 s) and an extracellular pH value (2 s; see below). In order to derive instantaneous values for an excitation ratio we interpolated between two numerator values to a time at which a denominator was measured, and vice versa, to produce a ratio value each time the fluorescence intensity was measured at either the numerator or denominator wavelength.
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Emission spectra were collected using the same monochrometer slit widths as above with the excitation monochrometer fixed at 405 nm. These spectra were corrected for the wavelength-dependent sensitivity of the photomultiplier tube using a computer program provided by the manufacturer. Samples were maintained at 37°C in a 1-cm* cuvette into which a slide holder manufactured after that of Ohkuma and Poole (23) was inserted to position the glass coverslips that supported the cells. Two coverslips mounted in the holder back to back, were continuously perfused at 0.8 ml min-’ with an appropriate buffer solution. Extracellular pH measurements. The pH of the solution in the cuvette was monitored during an experiment by means of micro-pH and microreference electrodes (Microelectrodes, Inc., Londonderry, NH) which fit through the cap of the perfusion apparatus. The microelectrodes were connected to a Beckman Model 7 1 pH meter which in turn was interfaced to the computer. These electrodes were calibrated at the beginning of each experiment at two known pH values using commercially prepared standards. In situ calibration of pyranine with nigeritin. The pHi of the cells was set equal to the extracellular pH by using the K+/H+ ionophore, nigericin (5,24). The cells on the coverslip were bathed in a buffer containing 5 pg/ml nigericin, 146 mM KCl, 0.1% (w/v) glucose, 2 mM glutamine, 10 mM Hepes, 10 mM Mes, and 10 mM Bicine at a pH between 5 and 9 (nigericin buffer). The cuvette containing freshly inserted coverslips was perfused with nigericin buffer at low pH for 30 min. The pH of the solution in the cuvette was then slowly raised over the next 2 h by means of a linear pH gradient formed by mixing two equal volumes of nigericin buffer of differing pH. This gradient was monitored with the microelectrodes which recorded the extracellular pH every 5 s. The pH response of pyranine was also calibrated in vitro in much the same manner except that the dye was dissolved in the nigericin buffer and the
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FIG. 1. In vitro fluorescence excitation pH titration of pyranine in nigericin buffer. One micromolar pyranine was dissolved in nigericin buffer at various pH values between 6 and 8. Excitation spectra were collected using an emission wavelength of 5 14 nm. The prominent features of the spectra include an isosbestic point at 4 15 nm and peaks near 405 and 465 nm which decrease and increase, respectively, as the solution pH increases.
pH gradient was run in the absence of cells. In another experiment, the pH of the cuvette solution was increased stepwise by perfusing the cells with different buffers of fixed pH for at least 30 min at each pH. To analyze data from these experiments, a number of extracellular pH values, usually 15, were chosen such that their acquisition times were evenly distributed about the acquisition of each excitation ratio value. These pH values were fit to a straight line using a least-squares regression. The parameters of the line were used to interpolate an extracellular pH value at a time coincident with the acquisition time of each excitation ratio.
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intensity at 415 nm was therefore used to measure the intracellular dye concentration whereas the excitation ratio of the intensities at wavelengths 465/405 provided a sensitive, concentration-independent quantitation of pHi * The pH dependence of the emission spectrum of pyranine is shown in Fig. 2. The intensity of the 5 14-nm peak decreases as the solution pH increases. The emission ratio of 465/5 14 rendered a concentration-independent measure of solution pH (Fig. 2, inset). The intracellular dye concentration was found to be approximately 10 PM 1 h after scrape-loading in buffer containing 1 InM pyranine. These intracellular concentration estimates were based on the fluorescence values of known concentrations of dye dissolved in cell suspension buffer at 37”C, the known amount of protein applied to each coverslip, and the relationship of the cellular volume to total cellular protein. Under these conditions, we found that the pHi could be monitored for about 4 h before the cellular autofluorescence signal became nearly 5% that of the fluorescence of the dye. In control experiments, we monitored the autofluorescence of cells which had not been loaded with dye. This fluorescence decreased with
RESULTS Pyranine dye (1 FM) was dissolved in nigericin buffer, without nigericin, at a pH between 6 and 8. This buffer contained K+ at a concentration equal to that of intracellular K+ at 146 mM. Like the absorption spectrum of pyranine (8), the fluorescence excitation spectrum of the dye has prominent features that include a peak at about 405 nm which decreases in intensity with increasing pH, a peak at about 465 nm which increases in intensity with increasing pH, and a region at approximately 4 15 nm where the intensity is pH independent (Fig. 1). The fluorescence
FIG. 2. In vitro fluorescence emission pH titration of pyranine in nigericin buffer. One micromolar pyranine was dissolved in nigericin buffer at various pH values between 6 and 8. These corrected emission spectra were collected using an excitation wavelength of 405 nm. Inset: The in vitro dependence of the 465 nm/514 nm emission ratio on the solution pH.
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first-order kinetics such that half of the signal was present after 3 h of constant perfusion in the cuvette. Similar data were obtained when the protein contents of coverslips were determined after various times of perfusion (data not shown). Both of these experiments indicate that cells are sloughed off of the coverslips with a first-order halflife of approximately 3 h. The type of kinetic report obtained with the excitation ratio procedure is demonstrated in Fig. 3. In this experiment the intracellular fluorescence of pyranine was eval-
AND GILLIES GAAFIENT 5000
““..-...“.......: _._.-.. C”-- “....” . . . .. . .. .
4400 3800 3200 2600 2000 8.50 7.90 7.30 6.70 6.10 5.50 2.00 1.60 1.20 0.80 0.40 0.00 0
25
50 Time
75 lminl
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125
FIG. 4. In situ calibration of pyranine in the presence of nigericin with a linear pH gradient. Pyranine-loaded BALB/c-3T3 cells were equilibrated at a pH of 5.6 for 30 min in the presence of nigericin-containing nigericin buffer. A linear pH gradient was then started and the F4i5, extracellular pH, and excitation ratio (Fti5,& were continuously monitored as in Fig. 2. The top panel indicates that the rate of loss of pyranine signal was greatest as the extracellular pH reached a level greater than 7.2.
0
50
100 150 Time lminl
200
250
FIG. 3. In situ calibration of pyranine in the presence of nigericin-containing nigericin buffer with a stepwise pH gradient. BALB/c-3T3 cells were scrape-loaded with pyranine as described in the text. At the indicated times, the pH of the perfusate was changed as follows: 0 min, pH 5.67; 30 min, pH 6.63; 70 min, pH 7.05; 110 min, pH 7.47; 145 min, pH 7.98; 185 min, pH 8.46. The top panel shows a plot of the fluorescence intensity at 415 nm (F4J as a function of time. This graph indicates the rate of loss of pyranine signal over time. The middle panel is a plot of the extracellular pH as measured by the microelectrodes placed in the fluorescence cuvette. The bottom panel is a graph of the excitation ratio (Fd65,.& of pyranine which, in this experiment, is to be calibrated to the pHi .
uated at various excitation wavelengths while the pHi was fixed at known values using a stepwise pH gradient in the presence of nigericin. The loss of signal over time is a combination of both loss of cells from the coverslip and dye leakage as confirmed by fluorescent microscopic observation as well as analysis of total protein on the coverslips before and after an experiment. The rates of dye leakage and/or cell detachment from the slide were found to increase with increasing pH in the presence of nigericin buffer (Figs. 3 and 4). Below pH 7.2, the fluorescence intensity at an excitation wavelength of 415 nm declined slowly whereas it decreased much more rapidly when the pH was greater than 7.2 (Fig. 4). It was difficult to make measurements of pHi above an extracellular
INTRACELLULAR
pH DETERMINED
‘“l----t y
1.00
.
invitro-
o.ool~~ 5.0 6.0 7.0 Extracellular
OF PYRANINE
.!-insitu
RI12[ 11 where R is the excitation ratio and Rminand R,,, are the limiting values of the excitation PH = PKO + log[(K - &in)l(G,,
0.0
9.0 PH
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mining the inflection points of the calibration curves, we found the pKc, of the dye in vitro (pK, = 7.68) to be less than that in situ (pK, = 7.82). Wolfbeis et al. (8) previously described the dependence of the pK, of pyranine on the nature and ionic strength of the solution that it was dissolved in. Solution pH can be estimated from the excitation ratio of a pH-sensitive fluoroprobe by using the equation (7)
.
2.00.
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FIG. 5. Comparison of in vitro, in situ, and theoretical pyranine calibration curves. In situ kinetic data from Figs. 2 and 3 were transformed into a time-independent curve as described in the text. The data for a stepwise pH gradient are plotted for both serum-stimulated (A) and serum-deprived (I) cells and agree well with those from the continuous pH gradient ( * . *) plotted on the same graph. An in vitro calibration curve (-) was determined by dissolving the pyranine dye (0.1 pM) in the two buffers used to form the pH gradient. The pK, values for pyranine determined from the inflection points of the in vitro and in situ calibration curves are 7.68 and 7.82, respectively. Theoretical values of the intracellular pH (0) were calculated from the excitation ratios using the in situ value of the pKO for pyranine and the minimum (0.0390) and maximum (2.150) values of the excitation ratio employing the method described in the text.
pH of 8.5 because the cells could not tolerate those conditions for a sufficient period of time. Pyranine was also titrated in situ and using a continuous pH gradient (Fig. 4). A continuous pH gradient produced many more extracellular pH values than did the step gradient over the same period of time. To transform kinetic data to a time-independent calibration curve of the excitation ratio versus PHi, a time-averaged extracellular pH value was determined for each excitation ratio (Fig. 5). In situ calibration using either a stepwise or continuous pH gradient were comparable below pH 8.0. Above this pH the continuous pH gradient did not allow enough time for the intracellular pH to equilibrate with the extracellular pH. By deter-
-
ratio at extremes of acid or alkaline pH, respectively. Calculated values of PHi compare well with the in situ calibration data by using values of 7.82, 0.0390, and 2.15 for the pK,, Knin, and Max, respectively (Fig. 5). In order to evaluate the ability of the scrape-loaded cells to regulate PHi, they were perfused with cell suspension buffer containing 10 InM NH&l to induce an acute alkaline load (Fig. 6). This treatment caused a rapid increase in PHi of almost 0.15 pH unit above the basal level with a subsequent stabilization just above the basal level 30 min after the continuous perfusion of ammonium chloride began. The cells were then perfused with cell suspension buffer minus ammonium chloride to induce an acute acid load. Figure 6 indicates that the PHi quickly dropped 0.25 pH unit followed by a realkalinization period of 40 min to a pH slightly below the untreated cell PHi. To determine the intracellular buffering power, an analysis described by Roos and Boron (25) was applied to these experimental data to first yield an intracellular concentration of ammonium ion of 14 mM. This amount of base induces a 0.13 pH unit alkalinization, yielding a buffering power value of 86 mM H+/pH unit. This is within the range of values given by these authors for a variety of cell types. DISCUSSION
A reliable method for quantitating PHi is essential to study its effects on cellular pro-
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cesses. Many cellular responses to hormones take 1-2 days to develop (26). An intracellular pH-sensitive, long-lived fluorescent probe would be extremely useful to investigate the role of pHi in these responses. We describe here the use of the impermeant probe, pyranine, for quantitating pHi for several hours after being scrape-loaded into adherent cells. Pyranine has two excitation peaks which reciprocally vary with changes in solution pH making the ratio of these intensities a sensitive, concentration-independent measure of pHi. Scrape-loading the free acid form of pyranine into the cells precludes the need for
tAC I,
8
-AC I
I
I
NIGERICIN III
I,
60004
0.57 0.45
1 0
30
60 90 Time lminl
120
150
FIG. 6. Effects of acute alkali and acid load on the pHi of BALB/c-3T3 cells. Pyranine-loaded cells were equilibrated in cell suspension buffer (pH 7.2 at 37°C) for 20 min followed by perfusion of the cells for 40 min with 10 mM ammonium chloride (AC) dissolved in the same buffer to induce an alkaline load. At a time of 70 min, the perfusate was changed back to cell suspension buffer thus causing an acid load as unprotonated ammonia left the cells. At a time of 110 min, the perfusate was changed to a nigericin-containing nigericin buffer at pH 7.0 and indicated that the pHr, as calculated from the excitation ratio, equilibrated with the extracellular pH within 15 min.
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intracellular enzymatic liberation of a pHsensitive dye. In situ calibration of pyranine using nigericin gives a pK, of 7.82. Our experimental data show that the relationship of pHi to excitation ratio is not linear near pH 7. The data can instead be described by Eq. [I], thus permitting the accurate measurement of pHi, even below pH 7. At the end of an experiment, we perfuse the cells with nigericin-containing nigericin buffer at a fixed pH and have found that the excitation ratio consistently falls on the in situ calibration curve. The excitation of pyranine with a uv laser can cause the release of protons from the dye (27). We do not see this as a problem in our studies because the potential amount of liberated protons is not significant enough to effect the pHi. In the former report, 20 PM pyranine photoinducibly released l-3 X 1Om6 M H+ when it was excited with intense uv light (353 nm). This would compare to the release of a maximum of ca. 10e6 H+ under our conditions (10 PM pyranine). This amount would have insignificant effect on the pHi of our system since the buffering power of the cytosol is 86 mM H+/pH unit. We believe that the fluorescence characteristics of the dye are not altered during an experiment even though it is continuously exposed to exciting light of varying wavelength. This is supported by the observations that steady-state fluctuations in measured pHi are low (kO.002 pH unit) and that nigericin/high K+ treatment of cells at the end of an experiment yields a point which falls on a previously constructed standard curve. Wolfbeis et al. (8) have indicated that one of the advantages of using pyranine is its relative insensitivity to quenching in the presence of oxygen. The loss of intracellular dye and/or loss of dye-containing cells from the glass coverslip is monitored throughout an experiment by measuring the fluorescence intensity at an excitation wavelength of 4 15 nm. At pH 7.2, the decrease in this signal intensity occurs with a half-life of approximately 45 min (Fig.
INTRACELLULAR
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DETERMINED
6 and data not shown). As discussed above, the loss of cells from the coverslips occurs four times slower. The leakage rate of dye, therefore, can be calculated to have a half-life of about 1 h. This leaked dye will not contribute to our measured signal, since the samples are continuously perfused with cell suspension buffer. However, since there is a constant loss of dye on a per-cell basis, this leakage limits the length of time data can be collected before cellular autofluorescence becomes a significant contributor to the observed signal, thus increasing the measurement error. There are a number of inherent characteristics of fluorescence-based pHi determinations which contribute to the measurement error (28). In the present technique, there are errors due to uncertainties in signal to noise (S/N), temperature, and calibration. Under the loading conditions described, intracellular pyranine fluorescence can be monitored for 4 h before the cellular autofluorescence becomes 5% that of pyranine at its limiting pH values. This error contributes standard errors of 0.022, 0.012, and 0.011, and 0.018 pH unit at pH values of 6.0,6.5,7.0, and 7.5, respectively. If one is interested in measuring pHi between 6.5 and 7.0, and can tolerate estimation errors of 0.05 pH unit (25) then measurements could be made for 12 h under the present conditions before the error due to autofluorescence becomes limiting. Each twofold increase in concentration of intracellular dye will allow for an extra 4 h of acquisition. Errors due to inaccuracies of temperature and calibration are potentially more severe than those due to S/N. The pK, of pyranine is relatively temperature sensitive, increasing by 0.25 unit upon decreasing temperature from 37 to 30°C (data not shown). This sensitivity is therefore 0.036 pH unit/C, indicating that temperature must be accurately measured and maintained at rt 1.5”C for pH measurements to be accurate to kO.05 pH unit. Errors due to inaccuracies in calibration are the most problematic, since no real
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basis for comparison exists. We must assume that the nigericin/high K+ treatment completely breaks down the transmembrane pH gradient, that there is not a significant Donnan potential in the presence of nigericin/ high K+, that the external pH is properly measured, and that the intracellular environment does not significantly affect the behavior of the dye. An estimate of the potential error in calibration can be obtained from Fig. 5, which compares in vitro and in situ titration curves. The difference between these curves is 0.14 pH unit at the pK,, suggesting that errors due to unknown calibration conditions may be the most severe. The above error analysis pertains to determinations of pHi. Errors arise from inaccuracies in calibration, temperature, and S/N, in descending order of importance, and are not significantly different in magnitude from those of other methods (25,28). The technique described here is also similar to other methods, in that it is much more accurate in assigning relative, rather than absolute, pHi. In this case, we can assume that most errors due to temperature, autofluorescence, and calibration are systematic within an experiment, and that the major variable is a possibility of nonpHi-related perturbation of the intracellular environment. It is virtually impossible to accurately estimate the severity of this error, due to the number of potential effecters. However, an estimate of error in the absence of change can be obtained by analyzing the fluctuations in measured pHi that occur in the steady state. Such analyses indicate that the pHi measurements using the current technique are stable to within 0.002 pH unit over time. Kurtz and Balaban ( 10) have reported that the pH-sensitive fluorescent dye 1,4-dihydroxyphthalonitrile can be calibrated in vitro using the ratio of two wavelengths of emission intensity measured simultaneously. We attempted to scrape-load this dye into BALB/c-3T3 cells but found that the cells had no measurable fluorescence after the required l-h cell reattachment time. Although
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data acquisition was slower using the excitation ratio method described here, the loss of pyranine fluorescence intensity was much slower than that of 1,4-dihydroxyphthalonitrile. We have provided evidence that the fluorescence emission ratio of pyranine can be used to determine solution pH in vitro. Because of its intracellular retention, pyranine may find utility as an indicator of pHi in those systems which monitor two wavelengths of emission intensity simultaneously. Dimethyl fluorescein-dextran is a pH-sensitive fluoroprobe that is long lived once trapped within the cell (4,17). Estimation of pHi using dimethyl fluorescein-dextran is accomplished with total fluorescence intensities, meaning that an in situ calibration curve must be constructed for each experiment. It must also be assumed that no change in dye concentration occurs during an experiment. Another derivative of fluorescein, 2’,7’-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF), also reports pHi using total fluorescence intensity (5) or a fluorescence excitation ratio (9). The dye is loaded in an esterified, nonfluorescent form into cells, which need not be adherent, promptly followed by pHi measurements. Even though the pK, of pyranine is relatively high, we feel that it is at least as sensitive as BCECF for measuring pHi. The ratio of the fluorescences of the two reciprocally related excitation peaks of pyranine provides an accurate and stable pHi value which is highly reproducible. The isosbestic point of pyranine also has a significant fluorescence intensity, thus allowing for an equally sensitive evaluation of intracellular dye concentration. A drawback to the use of pyranine as an indicator of pHi is that it is not available in an esterified form. It will be useful in only those cell types in which it can be loaded using a method such as those described above (see introduction). The pyranine-loaded cells were able to demonstrate pHi regulation when acutely presented with both an alkaline and acid load in the same experiment. The pHi of
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BALB/c-3T3 cells reacidified to near control levels during continuous treatment with 10 mM ammonium chloride. These cells realkalinized, again to near control levels, after an acute acid load caused by rapid removal of extracellular ammonium chloride. Therefore, the method described here employs the simple technique of scrape-loading to introduce the free acid of the pH-sensitive fluoroprobe pyranine into adherent cells and permits the continuous evaluation of pHi in physiologically sound cells for a number of hours afterward. ACKNOWLEDGMENTS The authors thank J. M. Sneider for her assistance in preparing cell cultures and J. A. Cook for his helpful discussions. This work was supported by NIH Grant ROl GM-34656.
REFERENCES 1. Moolenaar, W. H. (1986) Annu. Rev. Physiol. 48, 363-376. 2. Lindstrom, P., and Sehlin, J. (1986) Biochem. J. 239, 199-204. 3. Boonstra, J.. Moolenaar, W. H., Harrison, P. H., Moed, P., van der Saag, P. T.. and de Laat, S. W. (1983) J. Cell Biol. 97, 92-98. 4. Chandler, C. E., Cragoe. E. J., Jr., and Glaser, L. (1985) J. Cell. Physiol. 125, 367-378. 5. Rink, T. J., Tsien, R. Y., and Pozzan, T. (1982) J. Cell Biol. 95, 189- 196. 6. Chaillet, J. R., and Boron, W. F. (1985) J. Gen. Physiol. 86, 765-794. 7. Graber, M. L., DiLillo, D. C., Friedman, B. L.. and Pastoriza-Munoz, E. (1986) Anal. Biochem. 156, 202-212. 8. Wolfbeis, 0. S., Furlinger. E., Kroneis, H., and Marsoner, H. (1983). Fresenius’ 2. Anal. Chem. 314, 119-124. 9. Tsien, R. Y., and Poenie, M. (1986) Trends Biothem. Sci. 11,450-459. 10. Kurtz, I., and Balaban, R. S. (1985) Biophys. J. 48, 499-508. 11. Thomas, J. A., Kolbeck, P. C., and Langworthy, T. A. (1982) in Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions (Nuccitelli, R., and Deamer, D. W., Eds.), A. R. Liss, New York. 12. Spray, D. C., Nerbonne, J., Campos de Carvalho, A., Harris, A. L., and Bennet, M. V. L. (1984) J. Cell. Biol. 99, I74- 179. 13. Heiple, J. M., and Taylor, D. L. (1982) in Intracel-
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14. 15. 16. 17. 18. 19. 20.
pH DETERMINED
lular pH: Its Measurement, Regulation, and Utilization in Cellular Functions (Nuccitelli, R., and Deamer, D. W., Eds.), A. R. Liss, New York. McNeil, P. L., Murphy, R. F., Lanni, F., and Taylor, D. L. (1984) J. Cell Biol. 98, 1556-1564. Doxsey, S. J., Sambrook, J., Helenius. A., and White, J. (1985) J. Cell Biol. 101, 19-27. Knight, D. E., and Scrutton, M. C. (I 986) Biochem. J. 234,497-506. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983) J. Biol. Chem. 258, 12,644-12,653. Fechheimer, M., Denny, C., Murphy, R. F., and Taylor, D. L. (1986) Eur. J. Cell Biol. 40, 242-247. Clement, N. R., and Gould, J. M. ( 198 1) Biochemistry 20, 1534-1538. Lee, H. C. (1985) J. Biol. Chem. 260, 10,79410,799.
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21. Gillies, R. J., and Deamer, D. W. (1979) J. Cell. Physiol. 100,23-32. 22. Tal, M., Silberstein, A., and Nusser, E. (1985) J. Biol. Chem. 260,9976-9980. 23. Ohkuma, S., and Poole, B. (1978) Proc. Natl. Acad. Sci. USA 75,3327-333 1. 24. Thomas, J. A., Buchsbaum, R. N., Zimniak, A., and Racker, E. (1979) Biochemistry 18,2210-2218. 25. Roos, A., and Boron, W. F. (1984) Physiol. Rev. 61, 296-434. 26. Baserga, R. (1985) The Biology of Cell Reproduction. Harvard Univ. Press, Cambridge, MA. 27. Politi, M. J., and Fendler, J. H. (1984) J. Amer. Chem. Sot. 106,265-273. 28. Nuccitelli, R., and Deamer, D. W. (1982) in Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions (Nuccitelli, R., and Deamer, D. W., Eds), A. R. Liss, New York.
BIOCHEMISTRY
167,362-37
1 (1987)
Determination of Intracellular pH of BALB/c-3T3 Using the Fluorescence of Pyranine KENNETH A. GIULIANOANDROBERT
Cells
J.GILLIES’
Department of Biochemistry, Colorado State University. Ft. Collins, Colorado 80523 Received April 28, 1987 In terms of accuracy and sensitivity, intracellularly trapped, pH-dependent fluorescent probes are appropriate to accurately measure intracellular pH. These probes are commonly introduced into living cells in esterified form, wherein the free acid is produced through enzymatic hydrolysis. The fluorescence characteristics of the ester and the free acid can differ markedly and spectral uncertainty can occur. We describe here the measurement of intracellular pH using I-hydroxypyrene- 1,3,6-trisulfonic acid (pyranine) that has been scrape-loaded into BALB/c-3T3 mouse cells. The excitation spectrum of pyranine is pH sensitive, with an isosbestic point at 415 nm and peaks at 405 and 465 nm which decrease and increase with pH, respectively. The 465/405 ratio can be used to monitor the pH, while the fluorescence at 415 nm indicates the total dye-dependent signal remaining. The scrape-loaded dye persists in cells for periods up to 6 h. We have calibrated this dye in situ using nigericin/high K+, and have found that the pK, of the dye in situ is 7.82, as compared to 7.68 in vitro. We have observed that the cells can slowly equilibrate their intracellular pH to near control levels when presented with either an acute alkaline or acid load. 0 1987 Academic Press, hc. KEY WORDS: pyranine; intracellular pH; fluorescence; BALB/c 3T3.
ft is becoming apparent that changes in intracellular pH (pHi) are associated with a number of cellular processes, such as proliferation (I), insulin secretion (2), and, possibly, differentiation (3,4). The ability to ascribe physiological significance to these pH changes depends on the capability of accurately measuring this parameter. In terms of sensitivity and time resolution, methods employing intracellularly trapped, pH-dependent fluorescence probes compare favorably with the use of NMR (5) or micro-pH electrodes (6). The fluorometric characteristics of many of these probes have been discussed (7,8). Fluorescence ratio imaging is a powerful technique when used to determine the role of intracellular ions in cellular physiology (9). A fluorescence ratio essentially normalizes fluorescence signal variations due to changes in
both cellular thickness and dye content (9). Derivatives of fluorescein are commonly used with a fluorescence excitation ratio method to determine pHi (7,9). The pH-sensitive fluorescent dye 1,4-dihydroxyphtalonitrile has found application in those systems where a fluorescence emission ratio is more appropriate ( 10). Fluorescent pH probes are frequently introduced into a living cell in an esterified form, wherein the free acid is produced through enzymatic hydrolysis (5, IO). This method can be problematic, since the fluorescence spectra of these various esters often differs markedly from that of the free acid (10,ll). In some cases, the intracellular hydrolysis of esters can cause cytoplasmic acidification (12). For these reasons, it is often useful to load only that fluorescent species which reports the pHi. Several methods are used for introducing impermeant molecules into living cells.
’ To whom correspondence should be addressed. 0003-2697/87$3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Determination of intracellular potassium concentration. The intracellular volume was determined as a function of total cellular protein using a modification of the double label method as described by Gillies and Deamer (21). Serum-stimulated cells were washed three times with an ice-cold K-free buffer containing 1.3 mM CaC12, 0.8 1 mM MgS04, 0.44 mM NaH2P04, 115 mM NaCl, 0.35 IIIM Na2HP04, 1.0% (w/v) glucose, 4.2 mM NaHC03, 2 mM glutamine, and 25 mM Hepes with a pH of 7.2 at 25°C. Two mililiters of the K+ free buffer containing 1 PC1 MATERIALS AND METHODS of D-[‘4C]sorbitol (200 mCi mmol-‘; ICN BiCell culture. Balb/c-3T3 mouse embryo omedicals, Inc., Irvine, CA) and 10 &i of cells were obtained from American Type [3H]H20 (100 mCi ml-‘; ICN Biomedicals, Culture Collection (ATCC CCL 163). They Inc., Irvine, CA) were then added to the cells were cultured in Dulbecco’s modified Eagle’s and incubated for 5 min at room temperamedium (DME2; Gibco, Grand Island, NY), ture. As much of this solution as possible was supplemented with 10% NuSerum (Collaboaspirated from the plate. One mililiter of 0.1 rative Research, Inc., Lexington, MA). The N NaOH was then added to solubilize the initial inoculum as received was grown to cells. Aliquots of the radioactive bathing so70% confluency at 37°C in a humidified, 5% lution and the solubilized cells were then CO2 atmosphere in 300 cm2 at which time subjected to liquid scintillation counting. A the cells were frozen in DME supplemented Coomassie blue dye-binding protein assay with 10% (v/v) dimethyl sulfoxide, and 20% (Bio-Rad Laboratories, Richmond, CA) was NuSerum at a density of 1 X lo6 cells per performed on the solubilized cell solution freezing ampule. The cells were recovered using egg white lysozyme as a standard as quarterly in 75-cm2 flasks and passed bi- suggested by Tal et al. (22). The calculations weekly at an inoculation density of 2 X lo5 used to determine the cell volume as a funccells per 75-cm2 flask. Cultures were tested tion of cell protein using this double label monthly for mycoplasma contamination. method have been previously described (2 1). For experiments, cells were plated from these To determine the K+ content as a function stocks at a density of 1 X lo6 per 59-cm2 of total protein, cells washed in IS+-free culture dish in DME plus 10% NuSerum. On buffer as described above were immediately the following day, these cultures were either solubilized with 0.1 N NaOH. One aliquot of scrape-loaded with pyranine (serum-stimuthe solubilized cell solution was assayed for lated cells) or the growth medium was total protein and the other for potassium changed to DME with 0.2% NuSerum. These using atomic absorption spectrometry. To (serum deprived) cultures were incubated determine intracellular K+, the ratio of under low serum for 36-40 h, at which time moles K+ to protein was divided by the ratio they were scrape-loaded with pyranine. of cell volume to protein. Scrape-loading of pyranine dye. Pyranine was scrape-loaded into the cells using the * Abbreviations used: DME, Dulbecco’s modified method described by McNeil et al. (14). Eagle’s medium; Hepes, 4-(2-hydroxyethyl)-l-piperaBriefly, the cells to be loaded were washed zineethanesulfonic acid; Mes, 4-morpholineethanesulthree times with a buffer, at 37°C containing fonic acid; BCECF, 2’,7’-bis(carboxyethyl)-5,6-carboxy 1.3 mM CaC12, 1 mM MgS04, 5.4 mM KCl, fluorescein.
These include microinjection ( 13) scrapeloading ( 14), cell fusion ( 15), electropermeabilization ( 16), and hypertonic ( 17) or ultrasonic shock ( 18). In this report we show that &hydroxypyrene- 1,3,6-trisulfonic acid (pyranine), previously used as a pH probe within vesicles (19,20), can be introduced into adherent BALB/c-3T3 cells via scrapeloading and that the pHi can be sensitively measured for a number of hours afterward using a fluorescence excitation ratio method.
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0.44 mM KH2P04, 110 mM NaCl, 0.35 mM Na2HP04, 5 mM glucose, 4.2 mM NaHC03, 2 mM glutamine, and 25 mM Hepes at a pH of 7.15 at 37°C (cell suspension buffer). The cells were then scraped from the plate after overlaying them with 5 ml of 1 mM pyranine in cell suspension buffer at 37°C. Suspended cells were then washed twice with ice-cold DME plus the same concentration of NuSerum in which the cells were cultured. The final cell pellet was resuspended in this same wash solution at 37°C and 300 ~1 of this suspension (l-3 X lo5 cells) was applied to a glass coverslip (9 X 22 mm). The cells were then allowed to attach to the coverslip for 1 h at 37°C in a humidified, 5% CO* atmosphere. To determine cellular autofluorescence, cells were subjected to the scrapeloading procedure using a buffer without any added pyranine. Spectrofluorometric measurements. An SLM 4800 spectrofluorometer (SLM Instruments, Inc., Urbana, IL) was operated in the ratio mode to correct for changes in lamp intensity as a function of both time and excitation wavelength. The emission monochrometer was set to 5 14 nm with a slit width of 8 nm while the excitation monochrometer slit width was 2 nm. Data were collected through an interface to an IBM personal computer and were analyzed using programs written in PASCAL (Turbo Pascal 3.0, Borland International, Inc., Scotts Valley, CA). The spectrofluorometer does not allow for rapid excitation of the sample with selected wavelengths of light. Instead, this instrument cycles through 5-nm steps which require a total time of 5 s each to allow for acquisition of a fluorescence intensity value (2.5 s) and an extracellular pH value (2 s; see below). In order to derive instantaneous values for an excitation ratio we interpolated between two numerator values to a time at which a denominator was measured, and vice versa, to produce a ratio value each time the fluorescence intensity was measured at either the numerator or denominator wavelength.
AND GILLIES
Emission spectra were collected using the same monochrometer slit widths as above with the excitation monochrometer fixed at 405 nm. These spectra were corrected for the wavelength-dependent sensitivity of the photomultiplier tube using a computer program provided by the manufacturer. Samples were maintained at 37°C in a 1-cm* cuvette into which a slide holder manufactured after that of Ohkuma and Poole (23) was inserted to position the glass coverslips that supported the cells. Two coverslips mounted in the holder back to back, were continuously perfused at 0.8 ml min-’ with an appropriate buffer solution. Extracellular pH measurements. The pH of the solution in the cuvette was monitored during an experiment by means of micro-pH and microreference electrodes (Microelectrodes, Inc., Londonderry, NH) which fit through the cap of the perfusion apparatus. The microelectrodes were connected to a Beckman Model 7 1 pH meter which in turn was interfaced to the computer. These electrodes were calibrated at the beginning of each experiment at two known pH values using commercially prepared standards. In situ calibration of pyranine with nigeritin. The pHi of the cells was set equal to the extracellular pH by using the K+/H+ ionophore, nigericin (5,24). The cells on the coverslip were bathed in a buffer containing 5 pg/ml nigericin, 146 mM KCl, 0.1% (w/v) glucose, 2 mM glutamine, 10 mM Hepes, 10 mM Mes, and 10 mM Bicine at a pH between 5 and 9 (nigericin buffer). The cuvette containing freshly inserted coverslips was perfused with nigericin buffer at low pH for 30 min. The pH of the solution in the cuvette was then slowly raised over the next 2 h by means of a linear pH gradient formed by mixing two equal volumes of nigericin buffer of differing pH. This gradient was monitored with the microelectrodes which recorded the extracellular pH every 5 s. The pH response of pyranine was also calibrated in vitro in much the same manner except that the dye was dissolved in the nigericin buffer and the
INTRACELLULAR
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450 (nm)
500
FIG. 1. In vitro fluorescence excitation pH titration of pyranine in nigericin buffer. One micromolar pyranine was dissolved in nigericin buffer at various pH values between 6 and 8. Excitation spectra were collected using an emission wavelength of 5 14 nm. The prominent features of the spectra include an isosbestic point at 4 15 nm and peaks near 405 and 465 nm which decrease and increase, respectively, as the solution pH increases.
pH gradient was run in the absence of cells. In another experiment, the pH of the cuvette solution was increased stepwise by perfusing the cells with different buffers of fixed pH for at least 30 min at each pH. To analyze data from these experiments, a number of extracellular pH values, usually 15, were chosen such that their acquisition times were evenly distributed about the acquisition of each excitation ratio value. These pH values were fit to a straight line using a least-squares regression. The parameters of the line were used to interpolate an extracellular pH value at a time coincident with the acquisition time of each excitation ratio.
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intensity at 415 nm was therefore used to measure the intracellular dye concentration whereas the excitation ratio of the intensities at wavelengths 465/405 provided a sensitive, concentration-independent quantitation of pHi * The pH dependence of the emission spectrum of pyranine is shown in Fig. 2. The intensity of the 5 14-nm peak decreases as the solution pH increases. The emission ratio of 465/5 14 rendered a concentration-independent measure of solution pH (Fig. 2, inset). The intracellular dye concentration was found to be approximately 10 PM 1 h after scrape-loading in buffer containing 1 InM pyranine. These intracellular concentration estimates were based on the fluorescence values of known concentrations of dye dissolved in cell suspension buffer at 37”C, the known amount of protein applied to each coverslip, and the relationship of the cellular volume to total cellular protein. Under these conditions, we found that the pHi could be monitored for about 4 h before the cellular autofluorescence signal became nearly 5% that of the fluorescence of the dye. In control experiments, we monitored the autofluorescence of cells which had not been loaded with dye. This fluorescence decreased with
RESULTS Pyranine dye (1 FM) was dissolved in nigericin buffer, without nigericin, at a pH between 6 and 8. This buffer contained K+ at a concentration equal to that of intracellular K+ at 146 mM. Like the absorption spectrum of pyranine (8), the fluorescence excitation spectrum of the dye has prominent features that include a peak at about 405 nm which decreases in intensity with increasing pH, a peak at about 465 nm which increases in intensity with increasing pH, and a region at approximately 4 15 nm where the intensity is pH independent (Fig. 1). The fluorescence
FIG. 2. In vitro fluorescence emission pH titration of pyranine in nigericin buffer. One micromolar pyranine was dissolved in nigericin buffer at various pH values between 6 and 8. These corrected emission spectra were collected using an excitation wavelength of 405 nm. Inset: The in vitro dependence of the 465 nm/514 nm emission ratio on the solution pH.
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first-order kinetics such that half of the signal was present after 3 h of constant perfusion in the cuvette. Similar data were obtained when the protein contents of coverslips were determined after various times of perfusion (data not shown). Both of these experiments indicate that cells are sloughed off of the coverslips with a first-order halflife of approximately 3 h. The type of kinetic report obtained with the excitation ratio procedure is demonstrated in Fig. 3. In this experiment the intracellular fluorescence of pyranine was eval-
AND GILLIES GAAFIENT 5000
““..-...“.......: _._.-.. C”-- “....” . . . .. . .. .
4400 3800 3200 2600 2000 8.50 7.90 7.30 6.70 6.10 5.50 2.00 1.60 1.20 0.80 0.40 0.00 0
25
50 Time
75 lminl
100
125
FIG. 4. In situ calibration of pyranine in the presence of nigericin with a linear pH gradient. Pyranine-loaded BALB/c-3T3 cells were equilibrated at a pH of 5.6 for 30 min in the presence of nigericin-containing nigericin buffer. A linear pH gradient was then started and the F4i5, extracellular pH, and excitation ratio (Fti5,& were continuously monitored as in Fig. 2. The top panel indicates that the rate of loss of pyranine signal was greatest as the extracellular pH reached a level greater than 7.2.
0
50
100 150 Time lminl
200
250
FIG. 3. In situ calibration of pyranine in the presence of nigericin-containing nigericin buffer with a stepwise pH gradient. BALB/c-3T3 cells were scrape-loaded with pyranine as described in the text. At the indicated times, the pH of the perfusate was changed as follows: 0 min, pH 5.67; 30 min, pH 6.63; 70 min, pH 7.05; 110 min, pH 7.47; 145 min, pH 7.98; 185 min, pH 8.46. The top panel shows a plot of the fluorescence intensity at 415 nm (F4J as a function of time. This graph indicates the rate of loss of pyranine signal over time. The middle panel is a plot of the extracellular pH as measured by the microelectrodes placed in the fluorescence cuvette. The bottom panel is a graph of the excitation ratio (Fd65,.& of pyranine which, in this experiment, is to be calibrated to the pHi .
uated at various excitation wavelengths while the pHi was fixed at known values using a stepwise pH gradient in the presence of nigericin. The loss of signal over time is a combination of both loss of cells from the coverslip and dye leakage as confirmed by fluorescent microscopic observation as well as analysis of total protein on the coverslips before and after an experiment. The rates of dye leakage and/or cell detachment from the slide were found to increase with increasing pH in the presence of nigericin buffer (Figs. 3 and 4). Below pH 7.2, the fluorescence intensity at an excitation wavelength of 415 nm declined slowly whereas it decreased much more rapidly when the pH was greater than 7.2 (Fig. 4). It was difficult to make measurements of pHi above an extracellular
INTRACELLULAR
pH DETERMINED
‘“l----t y
1.00
.
invitro-
o.ool~~ 5.0 6.0 7.0 Extracellular
OF PYRANINE
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RI12[ 11 where R is the excitation ratio and Rminand R,,, are the limiting values of the excitation PH = PKO + log[(K - &in)l(G,,
0.0
9.0 PH
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mining the inflection points of the calibration curves, we found the pKc, of the dye in vitro (pK, = 7.68) to be less than that in situ (pK, = 7.82). Wolfbeis et al. (8) previously described the dependence of the pK, of pyranine on the nature and ionic strength of the solution that it was dissolved in. Solution pH can be estimated from the excitation ratio of a pH-sensitive fluoroprobe by using the equation (7)
.
2.00.
USING FLUORESCENCE
10.0
FIG. 5. Comparison of in vitro, in situ, and theoretical pyranine calibration curves. In situ kinetic data from Figs. 2 and 3 were transformed into a time-independent curve as described in the text. The data for a stepwise pH gradient are plotted for both serum-stimulated (A) and serum-deprived (I) cells and agree well with those from the continuous pH gradient ( * . *) plotted on the same graph. An in vitro calibration curve (-) was determined by dissolving the pyranine dye (0.1 pM) in the two buffers used to form the pH gradient. The pK, values for pyranine determined from the inflection points of the in vitro and in situ calibration curves are 7.68 and 7.82, respectively. Theoretical values of the intracellular pH (0) were calculated from the excitation ratios using the in situ value of the pKO for pyranine and the minimum (0.0390) and maximum (2.150) values of the excitation ratio employing the method described in the text.
pH of 8.5 because the cells could not tolerate those conditions for a sufficient period of time. Pyranine was also titrated in situ and using a continuous pH gradient (Fig. 4). A continuous pH gradient produced many more extracellular pH values than did the step gradient over the same period of time. To transform kinetic data to a time-independent calibration curve of the excitation ratio versus PHi, a time-averaged extracellular pH value was determined for each excitation ratio (Fig. 5). In situ calibration using either a stepwise or continuous pH gradient were comparable below pH 8.0. Above this pH the continuous pH gradient did not allow enough time for the intracellular pH to equilibrate with the extracellular pH. By deter-
-
ratio at extremes of acid or alkaline pH, respectively. Calculated values of PHi compare well with the in situ calibration data by using values of 7.82, 0.0390, and 2.15 for the pK,, Knin, and Max, respectively (Fig. 5). In order to evaluate the ability of the scrape-loaded cells to regulate PHi, they were perfused with cell suspension buffer containing 10 InM NH&l to induce an acute alkaline load (Fig. 6). This treatment caused a rapid increase in PHi of almost 0.15 pH unit above the basal level with a subsequent stabilization just above the basal level 30 min after the continuous perfusion of ammonium chloride began. The cells were then perfused with cell suspension buffer minus ammonium chloride to induce an acute acid load. Figure 6 indicates that the PHi quickly dropped 0.25 pH unit followed by a realkalinization period of 40 min to a pH slightly below the untreated cell PHi. To determine the intracellular buffering power, an analysis described by Roos and Boron (25) was applied to these experimental data to first yield an intracellular concentration of ammonium ion of 14 mM. This amount of base induces a 0.13 pH unit alkalinization, yielding a buffering power value of 86 mM H+/pH unit. This is within the range of values given by these authors for a variety of cell types. DISCUSSION
A reliable method for quantitating PHi is essential to study its effects on cellular pro-
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cesses. Many cellular responses to hormones take 1-2 days to develop (26). An intracellular pH-sensitive, long-lived fluorescent probe would be extremely useful to investigate the role of pHi in these responses. We describe here the use of the impermeant probe, pyranine, for quantitating pHi for several hours after being scrape-loaded into adherent cells. Pyranine has two excitation peaks which reciprocally vary with changes in solution pH making the ratio of these intensities a sensitive, concentration-independent measure of pHi. Scrape-loading the free acid form of pyranine into the cells precludes the need for
tAC I,
8
-AC I
I
I
NIGERICIN III
I,
60004
0.57 0.45
1 0
30
60 90 Time lminl
120
150
FIG. 6. Effects of acute alkali and acid load on the pHi of BALB/c-3T3 cells. Pyranine-loaded cells were equilibrated in cell suspension buffer (pH 7.2 at 37°C) for 20 min followed by perfusion of the cells for 40 min with 10 mM ammonium chloride (AC) dissolved in the same buffer to induce an alkaline load. At a time of 70 min, the perfusate was changed back to cell suspension buffer thus causing an acid load as unprotonated ammonia left the cells. At a time of 110 min, the perfusate was changed to a nigericin-containing nigericin buffer at pH 7.0 and indicated that the pHr, as calculated from the excitation ratio, equilibrated with the extracellular pH within 15 min.
AND GILLIES
intracellular enzymatic liberation of a pHsensitive dye. In situ calibration of pyranine using nigericin gives a pK, of 7.82. Our experimental data show that the relationship of pHi to excitation ratio is not linear near pH 7. The data can instead be described by Eq. [I], thus permitting the accurate measurement of pHi, even below pH 7. At the end of an experiment, we perfuse the cells with nigericin-containing nigericin buffer at a fixed pH and have found that the excitation ratio consistently falls on the in situ calibration curve. The excitation of pyranine with a uv laser can cause the release of protons from the dye (27). We do not see this as a problem in our studies because the potential amount of liberated protons is not significant enough to effect the pHi. In the former report, 20 PM pyranine photoinducibly released l-3 X 1Om6 M H+ when it was excited with intense uv light (353 nm). This would compare to the release of a maximum of ca. 10e6 H+ under our conditions (10 PM pyranine). This amount would have insignificant effect on the pHi of our system since the buffering power of the cytosol is 86 mM H+/pH unit. We believe that the fluorescence characteristics of the dye are not altered during an experiment even though it is continuously exposed to exciting light of varying wavelength. This is supported by the observations that steady-state fluctuations in measured pHi are low (kO.002 pH unit) and that nigericin/high K+ treatment of cells at the end of an experiment yields a point which falls on a previously constructed standard curve. Wolfbeis et al. (8) have indicated that one of the advantages of using pyranine is its relative insensitivity to quenching in the presence of oxygen. The loss of intracellular dye and/or loss of dye-containing cells from the glass coverslip is monitored throughout an experiment by measuring the fluorescence intensity at an excitation wavelength of 4 15 nm. At pH 7.2, the decrease in this signal intensity occurs with a half-life of approximately 45 min (Fig.
INTRACELLULAR
pH
DETERMINED
6 and data not shown). As discussed above, the loss of cells from the coverslips occurs four times slower. The leakage rate of dye, therefore, can be calculated to have a half-life of about 1 h. This leaked dye will not contribute to our measured signal, since the samples are continuously perfused with cell suspension buffer. However, since there is a constant loss of dye on a per-cell basis, this leakage limits the length of time data can be collected before cellular autofluorescence becomes a significant contributor to the observed signal, thus increasing the measurement error. There are a number of inherent characteristics of fluorescence-based pHi determinations which contribute to the measurement error (28). In the present technique, there are errors due to uncertainties in signal to noise (S/N), temperature, and calibration. Under the loading conditions described, intracellular pyranine fluorescence can be monitored for 4 h before the cellular autofluorescence becomes 5% that of pyranine at its limiting pH values. This error contributes standard errors of 0.022, 0.012, and 0.011, and 0.018 pH unit at pH values of 6.0,6.5,7.0, and 7.5, respectively. If one is interested in measuring pHi between 6.5 and 7.0, and can tolerate estimation errors of 0.05 pH unit (25) then measurements could be made for 12 h under the present conditions before the error due to autofluorescence becomes limiting. Each twofold increase in concentration of intracellular dye will allow for an extra 4 h of acquisition. Errors due to inaccuracies of temperature and calibration are potentially more severe than those due to S/N. The pK, of pyranine is relatively temperature sensitive, increasing by 0.25 unit upon decreasing temperature from 37 to 30°C (data not shown). This sensitivity is therefore 0.036 pH unit/C, indicating that temperature must be accurately measured and maintained at rt 1.5”C for pH measurements to be accurate to kO.05 pH unit. Errors due to inaccuracies in calibration are the most problematic, since no real
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basis for comparison exists. We must assume that the nigericin/high K+ treatment completely breaks down the transmembrane pH gradient, that there is not a significant Donnan potential in the presence of nigericin/ high K+, that the external pH is properly measured, and that the intracellular environment does not significantly affect the behavior of the dye. An estimate of the potential error in calibration can be obtained from Fig. 5, which compares in vitro and in situ titration curves. The difference between these curves is 0.14 pH unit at the pK,, suggesting that errors due to unknown calibration conditions may be the most severe. The above error analysis pertains to determinations of pHi. Errors arise from inaccuracies in calibration, temperature, and S/N, in descending order of importance, and are not significantly different in magnitude from those of other methods (25,28). The technique described here is also similar to other methods, in that it is much more accurate in assigning relative, rather than absolute, pHi. In this case, we can assume that most errors due to temperature, autofluorescence, and calibration are systematic within an experiment, and that the major variable is a possibility of nonpHi-related perturbation of the intracellular environment. It is virtually impossible to accurately estimate the severity of this error, due to the number of potential effecters. However, an estimate of error in the absence of change can be obtained by analyzing the fluctuations in measured pHi that occur in the steady state. Such analyses indicate that the pHi measurements using the current technique are stable to within 0.002 pH unit over time. Kurtz and Balaban ( 10) have reported that the pH-sensitive fluorescent dye 1,4-dihydroxyphthalonitrile can be calibrated in vitro using the ratio of two wavelengths of emission intensity measured simultaneously. We attempted to scrape-load this dye into BALB/c-3T3 cells but found that the cells had no measurable fluorescence after the required l-h cell reattachment time. Although
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data acquisition was slower using the excitation ratio method described here, the loss of pyranine fluorescence intensity was much slower than that of 1,4-dihydroxyphthalonitrile. We have provided evidence that the fluorescence emission ratio of pyranine can be used to determine solution pH in vitro. Because of its intracellular retention, pyranine may find utility as an indicator of pHi in those systems which monitor two wavelengths of emission intensity simultaneously. Dimethyl fluorescein-dextran is a pH-sensitive fluoroprobe that is long lived once trapped within the cell (4,17). Estimation of pHi using dimethyl fluorescein-dextran is accomplished with total fluorescence intensities, meaning that an in situ calibration curve must be constructed for each experiment. It must also be assumed that no change in dye concentration occurs during an experiment. Another derivative of fluorescein, 2’,7’-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF), also reports pHi using total fluorescence intensity (5) or a fluorescence excitation ratio (9). The dye is loaded in an esterified, nonfluorescent form into cells, which need not be adherent, promptly followed by pHi measurements. Even though the pK, of pyranine is relatively high, we feel that it is at least as sensitive as BCECF for measuring pHi. The ratio of the fluorescences of the two reciprocally related excitation peaks of pyranine provides an accurate and stable pHi value which is highly reproducible. The isosbestic point of pyranine also has a significant fluorescence intensity, thus allowing for an equally sensitive evaluation of intracellular dye concentration. A drawback to the use of pyranine as an indicator of pHi is that it is not available in an esterified form. It will be useful in only those cell types in which it can be loaded using a method such as those described above (see introduction). The pyranine-loaded cells were able to demonstrate pHi regulation when acutely presented with both an alkaline and acid load in the same experiment. The pHi of
AND GILLIES
BALB/c-3T3 cells reacidified to near control levels during continuous treatment with 10 mM ammonium chloride. These cells realkalinized, again to near control levels, after an acute acid load caused by rapid removal of extracellular ammonium chloride. Therefore, the method described here employs the simple technique of scrape-loading to introduce the free acid of the pH-sensitive fluoroprobe pyranine into adherent cells and permits the continuous evaluation of pHi in physiologically sound cells for a number of hours afterward. ACKNOWLEDGMENTS The authors thank J. M. Sneider for her assistance in preparing cell cultures and J. A. Cook for his helpful discussions. This work was supported by NIH Grant ROl GM-34656.
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