SPECIAL COMMUNICATION
In situ calibration and [H+]
sensitivity of the fluorescent Na+
indicator SBFI
Abdoullah
Diarra,
Claire
Sheldon, and
John
Church
Department of Anatomy, University of British Columbia,
Vancouver, British Columbia, Canada V6T 1Z3
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ABSTRACT |
Despite the
popularity of Na+-binding benzofuran isophthalate (SBFI) to
measure intracellular free Na+ concentrations
([Na+]i), the in situ calibration techniques
described to date do not favor the straightforward determination of all
of the constants required by the standard equation (Grynkiewicz G,
Poenie M, and Tsien RY. J Biol Chem 260:
3440-3450, 1985) to convert the ratiometric signal into
[Na+]. We describe a simple method in which SBFI ratio
values obtained during a "full" in situ calibration are fit by a
three-parameter hyperbolic equation; the apparent dissociation constant
(Kd) of SBFI for Na+ can then be
resolved by means of a three-parameter hyperbolic decay equation. We
also developed and tested a "one-point" technique for calibrating
SBFI ratios in which the ratio value obtained in a neuron at the end of
an experiment during exposure to gramicidin D and 10 mM Na+
is used as a normalization factor for ratios obtained during the
experiment; each normalized ratio is converted to
[Na+]i using a modification of the standard
equation and parameters obtained from a full calibration. Finally, we
extended the characterization of the pH dependence of SBFI in situ.
Although the Kd of SBFI for Na+ was
relatively insensitive to changes in pH in the range 6.8-7.8, acidification resulted in an apparent decrease, and alkalinization in
an apparent increase, in [Na+]i values. The
magnitudes of the apparent changes in [Na+]i
varied with absolute [Na+]i, and a method was
developed for correcting [Na+]i values
measured with SBFI for changes in intracellular pH.
sodium-binding benzofuran isophthalate; hippocampal neuron; intracellular sodium; intracellular pH
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INTRODUCTION |
THE INTRACELLULAR FREE
CONCENTRATION of Na+ ions
([Na+]i) is an important determinant of
cellular function. In neurons, the electrochemical gradient of
Na+ across the plasma membrane plays a central role in
determining excitability, and changes in
[Na+]i can modulate the activities of ion
channels (40), Na+-coupled transporters and
uptake mechanisms (4, 5, 20), and enzymes
(12). Disturbances in neuronal intracellular
Na+ homeostasis also play a role in pathophysiological
events, including excitotoxic/anoxic injury (7, 9, 11,
39).
Although a variety of methods have been employed to estimate
[Na+]i, Na+-sensitive fluorescent
dyes, especially Na+-binding benzofuran isophthalate
(SBFI), are assuming an increased importance. Despite the many
advantages of SBFI (see Refs. 26 and 32), a number of
difficulties are associated with the calibration procedures required to
convert experimentally derived SBFI ratio values into
[Na+]i. Thus in vitro calibration fails to
take into account the spectral shifts that are introduced when the dye
is present in the cytosol (3, 8, 15, 23, 28). On the other
hand, the in situ techniques described to date do not favor the
straightforward determination of separate values for all of the
constants necessary to convert the ratio of emitted SBFI fluorescence
signals into [Na+] values according to the standard
equation of Grynkiewicz et al. (16)
|
(1)
|
where
= Sf2/Sb2 and is the
ratio of the fluorescence of the free (unbound) dye
(Sf2) to bound dye (Sb2) at the second
excitation wavelength (
2ex),
Kd is the apparent dissociation constant of SBFI
for Na+, R is the fluorescence ratio, Rmin is
the fluorescence ratio at [Na+] = 0 mM, and
Rmax is the fluorescence ratio at saturation. The commonly
employed "three-point" calibration technique (18), for
example, provides only a composite value for
Kd; the accurate determination of a separate
value for
(and, thus the Kd of SBFI for
Na+) is precluded because a value for Sb2
cannot be derived. In the present study, therefore, we developed a
method to determine, from data derived from full in situ calibrations,
all of the constant parameters required to resolve Eq. 1. We
also describe a one-point technique for the in situ calibration of SBFI
ratios and examine the utility of this procedure to calibrate the
changes in SBFI ratios evoked in neurons by veratridine or anoxia.
Another difficulty associated with SBFI is that, in common with many
fluorophores, SBFI fluorescence is sensitive to changes in
[H+] (13, 18, 28, 29, 35, 36). Although the
full pH sensitivity of SBFI has been described in a cell-free in vitro
system (26), the effects of changes in intracellular
H+ concentration ([H+]i)
on measurements of [Na+]i made with SBFI in
situ remain relatively poorly defined. This represents a potential
limitation to the accurate estimation of [Na+]i with SBFI, in part because changes in
[Na+]i may give rise to changes in
intracellular pH (pHi), either directly (e.g., via
alterations in Na+/H+ exchange) or indirectly
[e.g., via alterations in Na+/Ca2+ exchange
and subsequent changes in intracellular Ca2+ concentration
([Ca2+]i)]. Therefore, we used the
procedures developed in the first part of the study to assess the
effect of changes in pHi on the Kd
of SBFI for Na+ in situ. We also characterized the
[H+] sensitivity of SBFI in situ at different values of
[Na+]i and developed a method for correcting,
if necessary, [Na+]i values measured with
SBFI for changes in pHi.
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EXPERIMENTAL PROCEDURES |
Cell Culture
Primary cultures of hippocampal neurons from 4-day postnatal
Wistar rats were prepared as described (14). Briefly, rat
pups were anesthetized with 3% halothane in air, rapidly decapitated, and the hippocampi removed. The hippocampi were enzymatically and
mechanically dissociated, and the resulting cell suspension was
underlain with fetal bovine serum and centrifuged at 150 g at 4°C for 10 min. Cells were then resuspended and plated onto glass
coverslips coated with poly-D-lysine and laminin at a low density of 4 × 105 neurons/cm2. The
initial growth medium was Eagle's minimum essential medium supplemented with 5% horse serum and 5% fetal bovine serum (Life Technologies, Grand Island, NY). After 24 h, this medium was
half-changed with serum-free N2-supplemented medium. Cultures
were then fed every 4-5 days by half-changing the existing medium
with serum-free N2-supplemented medium. Glial proliferation was
inhibited 48 h after initial plating by adding 10 µM cytosine
arabinoside. Neurons were used 6-14 days after plating.
Solutions
The standard perfusion medium contained (in mM) 136.5 NaCl, 3 KCl, 1.5 NaH2PO4, 1.5 MgSO4, 10 D-glucose, 2 CaCl2, and 10 HEPES (titrated to
the appropriate temperature-corrected pH with 10 M NaOH). Calibrating
media contained (in mM) 0.6 MgCl2, 0.5 CaCl2, 10 HEPES, Na+ and K+ such that
[Na+] + [K+] = 130, 100 gluconate, and 30 Cl
(titrated with 10 M KOH to the desired
temperature-corrected pH); gramicidin D, monensin, ouabain, and/or
nigericin (Sigma-Aldrich Canada, Oakville, ON) were added, as indicated
in the text. To limit cross-contamination by ionophores, perfusion
lines were replaced, and the imaging chamber was decontaminated after
each experiment (22). Anoxic media were prepared
immediately before use by adding 1-2 mM sodium dithionite to the
standard perfusion medium and bubbling vigorously with 100%
N2 or Ar (14). During anoxia, the atmosphere
in the recording chamber was switched from room air to 100%
N2 or Ar.
Microspectrofluorometry
The acetoxymethyl esters of SBFI (SBFI-AM) and
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF-AM) were obtained, respectively, from Texas Fluorescence
Labs (Austin, TX) and Molecular Probes (Eugene, OR). Coverslips plated
with neurons were placed in standard perfusion medium containing either
10 µM SBFI-AM (in the presence of 0.15% Pluronic F-127 and 5 mg/ml
of bovine serum albumin) or 2 µM BCECF-AM and incubated at room
temperature for, respectively, 150 and 30 min. Coverslips were
then placed in standard medium for 30 min to ensure deesterification of
the fluorophore and mounted in a temperature-controlled perfusion
chamber to form the base of the chamber. Neurons were superfused at a
rate of 2 ml/min for 15 min with the initial experimental solution at the appropriate temperature before the start of an experiment. Experiments were performed at room temperature (20-22°C) and at 37°C, as indicated in the text.
Measurements of [Na+]i and pHi
were performed using the dual excitation ratio method, employing an
imaging system (Atto Instruments, Rockville, MD) in conjunction with an
Axiovert 10 epifluorescence microscope (Carl Zeiss Canada, Don Mills,
ON). Full details have been provided previously (2, 14,
38). In brief, SBFI- or BCECF-loaded neurons were excited via a
×40 LD Achroplan objective with light provided by a 100-W Hg arc
burner and band-pass filtered alternately at 334 and 380 nm (SBFI) or
at 488 and 452 nm (BCECF). To reduce photobleaching of the
fluorophores, the output of the ultraviolet (UV) light source was
attenuated electronically, neutral density filters were placed in the
light path, and a high-speed shutter was employed to limit UV exposure
to the periods required for data acquisition. Fluorescence emissions,
measured at 510 or 520 nm from neurons loaded with SBFI or BCECF,
respectively, were detected by an intensified charge-coupled device
camera (Atto Instruments) and collected from regions of interest placed
on individual neuronal somata. Experiments were repeated on at least three (usually
5) different coverslips, each allowing collection of
data from up to 99 individual neurons simultaneously. Raw emission intensity data at each excitation wavelength were corrected for background fluorescence before the calculation of a ratio; the intensity of background fluorescence was typically <15% of the total
signal at any given excitation wavelength and remained constant during
the course of a given experiment. Ratio pairs were acquired at 1- to
12-s intervals and analyzed off-line. The one-point
high-[K+]/nigericin technique was employed to convert
background-corrected BCECF emission intensity ratios
(BI488/BI452) into
pHi values, as detailed previously (2, 6, 38).
The procedures employed to calibrate the ratios of the emitted SBFI
signals are detailed in RESULTS.
Data Analysis
Results are reported as means ± SE, with the
accompanying n value referring to the number of cell
populations (i.e., number of coverslips) analyzed. For clarity, values
obtained during the course of the study are presented to two
significant decimal places, although all calculations were performed
using values accurate to four decimal places. Statistical comparisons
were carried out using Student's two-tailed t-test, paired
or unpaired as appropriate, with a 95% confidence limit.
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RESULTS |
Calibration of SBFI Ratio Values
Determination of Rmin, Rmax, and
Kd.
Full calibrations were performed at room temperature in 23 neuronal
populations by exposing neurons sequentially to pH 7.35 media
containing 5 µM gramicidin D and eight different [Na+]
values (range, 0-130 mM). Typical changes in
BI334 and BI380 values
are illustrated in Fig. 1A.
Fluorescence emitted during excitation at 334 nm was
essentially unaffected by changes in [Na+], whereas
during excitation at 380 nm, emitted fluorescence intensity decreased
as [Na+] increased. The lack of an effect of changes in
[Na+] on fluorescence emitted in situ during excitation
at 334 nm in epifluorescence systems has been reported and discussed
previously (3, 13, 15, 21, 23, 24, 28, 29, 34, 35).

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Fig. 1.
In situ full calibration of Na+-binding
benzofuran isophthalate (SBFI) at room temperature and extracellular pH
(pHo) 7.35. A: a full calibration experiment in
which 10 neurons on a single coverslip were exposed to 5 µM
gramicidin D-containing solutions at the [Na+] values
shown above or below the traces. Displayed are changes in emitted
fluorescence intensities during excitation at 334 nm (dotted line) and
380 nm (continuous line); the signals were corrected for background
fluorescence. Whereas the BI380 value displayed
stepwise changes in response to changes in [Na+], the
BI334 value appeared insensitive. The trace is
representative of 23 independent experiments. B:
BI334/BI380 ratio values
were computed from the data shown in A and normalized to
unity at intracellular free Na+ concentration
([Na+]i) = 10 mM. The trace shows the
stepwise changes in normalized
BI334/BI380 ratio values
(Rn) evoked by successive increases in [Na+].
C: curve a (solid line) is a plot of
[Na+] vs. mean normalized
BI334/BI380 ratio values
(Rn) obtained from 4 calibration experiments of the type
shown in B, conducted on sister cultures on the same day.
The curve is the result of a three-parameter hyperbolic fit (Eq. 3) to the data points ( ) indicated. Standard error
bars are contained within each datum point. Curves b and
c (dashed lines) are plots of [Na+] vs. mean
normalized background-subtracted emission intensities during excitation
at 380 nm. The curves are the result of three-parameter hyperbolic
decay fits (Eq. 6) to the respective data points indicated.
The data points employed for curve b ( ) and
curve c ( ) were not drift corrected and were
drift corrected, respectively (see text). In each case, standard error
bars are contained within each datum point. D: Hanes plot of
the calibration data shown in C (plot a). The
continuous line is a linear least-squares regression fit to the data
points indicated (r2 = 0.99).
Rn(max) is drawn from the slope = 1/[Rn(max) Rn(min)], and
 Kd is obtained from the intercept on the
abscissa.
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To determine all the constants (Rmin, Rmax,
, and Kd) necessary to convert the ratio of
emitted SBFI signals into [Na+] values, the standard
equation (Eq. 1) was rearranged to give
|
(2)
|
where Rn is the background-subtracted SBFI
fluorescence ratio
(BI334/BI380)
normalized to [Na+] = 10 mM, and Rn(min) and
Rn(max) are, respectively, the minimum and maximum
obtainable values for the normalized ratio. Background-subtracted SBFI
fluorescence ratios were normalized to the
BI334/BI380 value at
[Na+] = 10 mM (Fig. 1B), and the data points
relating [Na+] to Rn were then fit by a
three-parameter hyperbolic equation having the form
|
(3)
|
where a and b are constants. The data points relating
[Na+] to Rn (Fig. 1C, curve
a) were accurately fitted (r2
>0.99) by Eq. 3, and the resulting fitted
parameters were Rn(min) = 0.74 ± 0.01, a = 1.55 ± 0.02, and b = 51.85 ± 2.12. The value for
Rn(min) obtained in this manner was identical to the value of Rn(min) derived experimentally at [Na+] = 0 mM. The hyperbolic form of curve a in Fig. 1C
underscores the difficulty of experimentally determining accurate
values for Rmax when using SBFI, especially in light of
reports that the dye may not saturate in situ even when
[Na+]i is raised to 150 mM (3,
23). To estimate Rn(max) and a composite value for
Kd, Eq. 3 was rearranged to give
|
(4)
|
It is apparent that Eq. 4 is identical to Eq. 2 when b =
Kd and [a + Rn(min)] = Rn(max). Thus the fitted parameters
of Eq. 3 could be employed to determine those of the
standard equation, Eq. 2. The calculated values of
Rn(max) and
Kd were 2.30 ± 0.03 and 51.85 ± 2.12 mM, respectively.
Hanes plots and related methods have frequently been employed to derive
parameters for the calibration of SBFI ratio values (e.g., Refs.
13, 15, 19, 33).
Therefore, the values of Rn(max) and
Kd obtained via the three-parameter
hyperbolic fit were compared with those resulting from a Hanes plot of
the same data. To derive a Hanes plot, the standard equation (Eq. 2) was rearranged such that
|
(5)
|
Accordingly, a plot of [Na+]/[Rn
Rn(min)] vs. [Na+] gives a straight line,
the slope {1/[Rn(max)
Rn(min)]}
providing an estimate of Rn(max) and the intercept on the
abscissa giving 
Kd (Fig.
1D); Rn(min) is derived from experimental data.
The values of Rn(max) and
Kd
derived in this manner were 2.24 ± 0.03 and 46.75 ± 2.65 mM, respectively, and were not significantly different from those
obtained via the three-parameter hyperbolic fit.
The values of the calibration parameters estimated from hyperbolic fits
to data obtained in different full calibrations showed little
interassay variability (Table 1). In
addition, although it has been suggested that inhibition of the
plasmalemmal Na+-K+-ATPase is required for
optimum transmembrane Na+ equilibration at
[Na+] <5 mM in cardiac myocytes (Ref. 15,
but see Ref. 23), in the present study, in
neurons, the values of Rn(min), Rn(max), and
Kd obtained after the addition of 1 mM
ouabain to gramicidin D-containing media (0.72 ± 0.05, 2.30 ± 0.08, and 47.83 ± 2.09 mM, respectively; n = 3) were not significantly different from those obtained in the absence
of the pump inhibitor. Twelve full calibrations were also performed at
37°C and at extracellular pH (pHo) 7.35;
calculated values of Rn(min), Rn(max), and
Kd were 0.76 ± 0.02, 2.26 ± 0.12, and 50.66 ± 3.92 mM, respectively (P > 0.05 in
each case for the difference to the respective value obtained at room
temperature).
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Table 1.
Values of Rn(min), Rn(max), and
Kd determined by three-parameter hyperbolic fits to
data points obtained in full calibrations
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Determination of
and Kd.
Neither Hanes plots of data derived from full calibrations nor the
commonly employed three-point procedure (18) permits the
straightforward determination of a separate value for
(and, thus a
value for the Kd of SBFI for Na+).
To determine
, one needs to know Sf2 and
Sb2, the intensities of fluorescence emissions when
exciting the fluorophore at the second wavelength
(
2ex = 380 nm) at [Na+] = 0 mM and at saturating Na+, respectively. Although a value
for Sf2 can be obtained experimentally, the
determination of Sb2 requires very high [Na+]
and is compromised by the sensitivity of SBFI to changes in ionic
strength and other factors (3, 23, 26, 28). To derive
Sb2, we employed the BI380 value at
[Na+] = 10 mM as a normalization factor. Normalized
BI380 values (Sn2) at different
[Na+] are shown in Fig. 1C (plot
b). The data points were fitted (r2
>0.99) with a three-parameter hyperbolic decay equation having the
form
|
(6)
|
where
,
, and
are constants. From Eq. 6 it
can be seen that when [Na+] = 0, Sn2 = Sf2 = (
+
), and when [Na+] =
, Sn2 = Sb2 =
. Consequently
|
(7)
|
The values of the parameters for Eq. 6, obtained from
the fit to the data points shown in Fig. 1C (plot
b), were
(i.e., Sb2) = 0.41 ± 0.01,
= 0.94 ± 0.01, and
= 17.96 ± 0.97;
thus Sf2 = 1.34 ± 0.03 and
(Sf2/Sb2) = 3.31 ± 0.03. As
noted above,
Kd = 51.85 ± 2.12 mM;
thus the calculated Kd of SBFI for
Na+ = 15.69 ± 0.15 mM.
A source of error that can arise during in situ calibrations is a
gradual decline ("drift") in emitted fluorescence intensity values
(3, 15, 23, 24). In the present study, declines in
BI334 and BI380 values
were sometimes observed during the latter part of a full calibration
(i.e., at [Na+] >10 mM; Fig.
2). Although this drift will not affect
BI334/BI380 ratio
values (because BI334 and
BI380 decline in parallel; see Fig. 2; also see
Ref. 3), it will decrease Sb2 and increase
, leading to an artifactually low value for
Kd. We were able to correct for drift by
normalizing the BI334 and
BI380 values at [Na+] = 10 mM to
unity (Fig. 2). Because BI334 values are
insensitive to changes in [Na+] under our experimental
conditions, signal changes during excitation at 334 nm are necessarily
due to Na+-independent factors such as dye loss (e.g.,
photobleaching) and/or other nonspecific artifacts. Furthermore,
because BI334 and BI380 decline in parallel, the magnitude of any decline in
BI334 values from unity at any given time point
during a calibration can be employed to correct normalized
BI380 values for drift. Normalized, drift-corrected BI380 values at different
[Na+] are shown in Fig. 1C, where the data
points have been fitted by Eq. 6 (curve c). The
drift-corrected parameters for the fit were Sb2 = 0.47 ± 0.01 and Sf2 = 1.34 ± 0.03;
thus
, corrected for drift, is 2.88 ± 0.05. As noted above,
Kd = 51.85 ± 2.12 mM; thus the
Kd following correction is 17.99 ± 0.31 mM. The latter value is greater (P < 0.05) than the
uncorrected value (15.69 ± 0.15 mM) and is similar to that
reported in vitro in solutions with a combined [Na+] + [K+], which approximates physiological strength
(Kd = 17-19 mM) (26, 28).

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Fig. 2.
Normalized and drift-corrected
BI334 and BI380 values.
Ten neurons on a single coverslip were exposed to 5 µM gramicidin
D-containing solutions at the [Na+] values shown. Changes
in BI334 and BI380 values
were normalized to unity at [Na+] = 10 mM (dotted lines).
The magnitude of the decline in BI334 values
from unity at any given time point was then employed to correct (solid
line) the normalized BI380 values for the drift
that occurred over the course of the experiment.
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One-point calibration.
Full calibrations may be impractical at the end of an experiment, due
to a loss of fluorescence signal with time. We therefore explored the
possibility of applying a one-point procedure to calibrate SBFI ratios
in situ. To illustrate the one-point technique, we measured the changes
in SBFI ratios that occurred in hippocampal neurons at 37°C in
response to 30 µM veratridine (n = 5, a total of 32 neurons) or anoxia (n = 12, a total of 91 neurons). At
the end of an experiment, neurons were exposed to a single calibrating solution containing 5 µM gramicidin D and 10 mM Na+. The
resulting
BI334/BI380 value
at Na+ = 10 mM for a given neuron in the sampled
population was then used as a normalization factor for that neuron.
After dividing experimentally derived
BI334/BI380 values
from a given neuron by the normalization factor for that neuron, each
Rn was converted to [Na+]i using
Eq. 2 and the parameters [Rn(min),
Rn(max),
, and Kd] determined in
a full calibration. Estimated in this manner, resting [Na+]i before veratridine or anoxia was
9.8 ± 0.3 mM (n = 17), a value similar to that
reported by others in hippocampal neurons (30, 34). During
anoxia, [Na+]i increased to 43.1 ± 2.8 mM (n = 12; Fig.
3A), a rise similar to that
observed during energy deprivation or exposure to excitotoxins in a
variety of mammalian central neurons (9, 10, 30). Veratridine (30 µM) evoked an increase in
[Na+]i that failed to recover toward resting
values during the recording period (Fig. 3A).

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Fig. 3.
Changes in [Na+]i evoked by
veratridine and/or anoxia. A: changes in
[Na+]i induced by a 5-min exposure, indicated
by the first bar above the traces, to either 30 µM veratridine
( ) or an anoxic medium (continuous line) in 2 populations of neurons on different coverslips. Each experiment was
performed at 37°C and pHo 7.35. For each selected neuron
in the sampled population, a one-point in situ calibration of the SBFI
ratio signal, indicated by the second bar above the traces, was
performed with a pH 7.35 medium containing 5 µM gramicidin D and 10 mM Na+. The responses to veratridine and anoxia are means
of data obtained simultaneously from 9 and 12 neurons, respectively.
B and C: after a 5-min period of anoxia,
indicated by the first bar above the traces, 5 neurons on a single
coverslip were exposed sequentially to calibrating media (pH 7.35)
containing 5 µM gramicidin D and 6, 10, or 40 mM Na+.
Ratios of the BI334 and
BI380 signals obtained from each neuron during
the course of the experiment were then calibrated separately by the
three-point (B) and one-point (C) methods, the
former using the equations provided by Harootunian et al.
(18) and the latter using Na+ = 10 mM as
the normalization factor and values for Kd,
Rn(min), and Rn(max) obtained from a full
calibration (calibration 3, Table 1).
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In a separate series of experiments, we compared values for
[Na+]i derived via the one-point technique
with those derived via the commonly employed three-point calibration
procedure. Neurons were subjected to 5 min of anoxia and were then
exposed sequentially to calibrating media containing 5 µM gramicidin
D and 6, 10, or 40 mM Na+ (n = 8; Fig. 3,
B and C). The
BI334/BI380 values
obtained from each selected cell in a given experiment were then
calibrated separately, either by the three-point procedure [using the
equations provided by Harootunian et al. (18)] or by the
one-point procedure (using Na+ = 10 mM as the
normalization factor). Similar values for both resting
[Na+]i before anoxia and peak
[Na+]i evoked by anoxia were derived via the
one- and three-point procedures (resting
[Na+]i = 9.9 ± 0.9 and 9.3 ± 1.0 mM, respectively; peak [Na+]i = 45.2 ± 3.4 and 45.2 ± 3.9 mM, respectively). The results support the utility of a one-point procedure for the in situ
calibration of SBFI ratio values.
Effects of Changes in pH on
[Na+]i Measurements With
SBFI In Situ
Effect of calibration media on pHi.
A change in pHi during a calibration might affect the
precision of the procedure. In addition, although SBFI ratios are often calibrated with a combination of gramicidin D, monensin, and ouabain (e.g., see Refs. 10, 13, 29,
34), it was originally suggested that gramicidin D alone
might provide a more accurate calibration for estimating cytosolic
[Na+] (18). Therefore, we assessed the
effects on pHi of exposing neurons to 5 µM gramicidin D
alone or in combination with 10 µM monensin and 1 mM ouabain; in all
cases, pHo was 7.35 at 37°C.
Exposure to a solution containing gramicidin D alone evoked a change in
pHi, the direction and magnitude of which depended on the
resting pHi (Fig. 4,
A and B); overall, the effect of medium containing 5 µM gramicidin D and 10 mM Na+ was to bring
pHi to 7.33 ± 0.01 (n = 5; a total of
46 neurons). Similar results were obtained with medium containing 130 mM Na+ (n = 3; not shown). As illustrated
in Fig. 4A, the addition of nigericin to gramicidin
D-containing medium did not further alter pHi
(n = 5). We also measured the effects on
pHi of altering the pH of media containing 5 µM
gramicidin D. As illustrated in Fig. 4C, at pHo
6.80, steady-state pHi in the presence of gramicidin D was
6.85 ± 0.02, whereas at pHo 7.80, pHi was
7.83 ± 0.03 (n = 5 in each case; a total of 51 neurons in each case). Similar results were obtained when neurons were
exposed to media containing 10 mM Na+ and 5 µM gramicidin
D, 10 µM monensin, and 1 mM ouabain. Examined in six populations of
neurons (a total of 61 cells) at pHo 7.35, pHi
was 7.37 ± 0.04 (a value that was not significantly different compared with the pHi measured in the presence of
gramicidin D alone), and the addition of 10 µM nigericin failed to
further influence pHi. At pHo 6.80 and 7.80, pHi measured in the presence of 5 µM gramicidin D, 10 µM monensin, and 1 mM ouabain was 6.87 ± 0.03 (n = 3) and 7.83 ± 0.04 (n = 3),
respectively (P > 0.05 in each case for the difference
to the pHi measured at the respective pHo in
the presence of gramicidin D alone).

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Fig. 4.
Effect of calibrating media on intracellular pH
(pHi) in neurons loaded with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).
A: 2 different neurons on a single coverslip, one with
resting pHi ~7.48 and the other with resting
pHi ~6.95, during perfusion with standard pH 7.35 medium
at 37°C, were exposed to a pH 7.35 medium containing 10 mM
Na+ and 5 µM gramicidin D for the period indicated by the
bar above the traces. The calibrating medium decreased the
pHi of the neuron with a high initial resting
pHi (continuous line) and increased the pHi of
the neuron with a low initial resting pHi
( ). The subsequent addition of 10 µM nigericin failed
to evoke an additional change in pHi. B: the
changes in steady-state pHi ( pHi) evoked in
22 different neurons by exposure to a pH 7.35, 10 mM Na+
medium containing 5 µM gramicidin D are plotted against the
pHi values measured before application of the calibrating
medium. The line shown is a linear least-squares regression fit to the
data points indicated (r2 = 0.96).
C: a neuron perfused with a standard pH 7.35 medium at
37°C was exposed to a pH 6.80 calibration medium containing 10 mM
Na+ and 5 µM gramicidin D; pHi fell to a new
steady-state value. The pHo was subsequently increased to
7.80, which caused pHi to increase to a new steady-state
level. A one-point BCECF calibration with 10 µM nigericin was
performed immediately after the end of the record shown. The trace is
representative of responses observed in 51 neurons in 5 independent
experiments.
|
|
Effects of changes in pHi on the
Kd of SBFI for
Na+.
To assess whether the Kd of SBFI for
Na+ in situ is sensitive to changes in pH, full
calibrations were performed at pHo 6.80 and pHo
7.80 (n = 8 in each case). The resulting normalized
BI334/BI380 ratios
and normalized drift-corrected fluorescence intensities (at
2ex = 380 nm) were then fitted by the
appropriate equations (Eqs. 3 and 6,
respectively). BI334 values were essentially
unaffected as pHo increased from 6.80 to 7.80 (data not
shown), as was the value for Rn(min) (0.75 ± 0.01 and
0.74 ± 0.03 at pHo 6.80 and 7.80, respectively),
whereas Rn(max) increased slightly from 2.24 ± 0.02 to 2.36 ± 0.05. Values for
Kd also
increased slightly, from 50.04 ± 1.88 mM to 54.67 ± 3.26 mM, as pHo increased from 6.80 to 7.80; this change
reflected an increase in
, and Kd values obtained at pHo 6.80 and 7.80 (17.95 ± 0.03 and
17.55 ± 0.20 mM, respectively) were, in both cases, not
significantly different from the Kd value
established at pHo 7.35 (17.99 ± 0.31 mM; see above).
Effects of changes in pHi on
SBFI ratio values and
[Na+]i.
Initially, we examined the effects on SBFI ratios of a series of
calibrating media containing 5 µM gramicidin D and 0, 10, 40, or 130 mM Na+, at three different pH values (6.80, 7.35, and
7.80). Experiments were performed at room temperature
(n = 6) and 37°C (n = 3); no differences were observed between results at the two temperatures, and
the data were, therefore, pooled. As illustrated in Fig.
5A, Rn values at a
given [Na+] were reduced at pHo 6.80 and
increased at pHo 7.80, compared with pHo 7.35, both effects increasing in magnitude as [Na+] was
increased from 0 to 130 mM. Normalized ratios were converted into
[Na+]i using values for Rn(min),
Rn(max),
, and Kd determined at pHo 7.35 in full calibrations, and the resulting traces of
the effects of changes in pH on [Na+]i at the
different Na+ concentrations are shown in Fig.
5B. Acidification resulted in an apparent decrease and
alkalinization in an apparent increase in
[Na+]i when [Na+]
10 mM.

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|
Fig. 5.
[H+] sensitivity of SBFI in situ.
A: neurons were superfused with a calibration medium
containing 5 µM gramicidin D, and [Na+] was increased
sequentially from 0 to 10 to 40 to 130 mM, as indicated by the bars
beneath the trace. At each [Na+], pHo was
changed from 7.35 to 6.80 to 7.80. BI334/BI380 ratio values
for each cell in the sampled population were normalized to unity at
[Na+] = 10 mM and pHo = 7.35. The trace
is a mean of data obtained simultaneously from 18 neurons on a single
coverslip. B: the normalized
BI334/BI380 ratio values
shown in A were transformed into [Na+] using
the one-point procedure described in the text. C: apparent
changes in [Na+] ( [Na+]) at different
absolute values of [Na+]i (10 mM,
; 40 mM, ; and 130 mM,
) are shown as a function of pHi. For each
absolute [Na+], values of [Na+] were
calculated according to Eq. 8 and were obtained from 9 experiments (3 conducted at 37°C and 6 conducted at room temperature)
of the type illustrated in A; where not shown, standard
error bars are contained within the datum point. The lines shown are
linear least-squares regression fits to the data points indicated for
each absolute value of [Na+]
(r2 >0.99 in each case). D:
[Na+](CF) as a function of
[Na+]i at different pHi values.
[Na+](CF) is the value of
[Na+] (in mM) that is required to correct the apparent
[Na+] obtained at the pHi values indicated
for the pH sensitivity of SBFI in situ. The continuous lines are
linear least-squares regression fits to data points obtained
experimentally at pHi 6.85 ( ) and 7.83 ( ) for [Na+] = 10, 40, and 130 mM in each
case (r2 >0.99 in each case). Each datum point
represents the mean value obtained from 6 to 9 similar experiments;
where missing, standard error bars lie within the point. Dotted lines
show the values for [Na+](CF)
that were calculated for pHi = 6.85 ( ), 7.10 ( ), 7.60 ( ),
and 7.83 ( ) by means of Eq. 9 and
plotted as a function of absolute [Na+]i.
|
|
To quantify the effects of changes in pH on
[Na+]i values measured with SBFI, we
calculated the magnitude of the apparent pHi-induced changes in [Na+]i by taking the measured
[Na+]i at pHi 7.33 (i.e., the
pHi measured in the presence of gramicidin D at
pHo 7.35) as a reference point and calculating the apparent change in [Na+] (
[Na+]) at
pHi = 6.85 and 7.83 (i.e., the pHi values
measured in the presence of gramicidin D at pHo 6.80 and
7.80, respectively) according to
|
(8)
|
where [Na+] = 10, 40, or 130 mM and x = 6.85, 7.33, or 7.83 (pHi-evoked changes in
[Na+]
were very small at [Na+] = 0 mM; these data were
excluded). The results, which are presented in Fig. 5C,
indicate that the effects of changes in pH on
[Na+]i increase as [Na+]
increases and that a linear relationship exists between the effects of
changes in pH on
[Na+] for each [Na+]
examined. The linear nature of the relationship enabled us to construct
an empirical equation by means of which a correction factor could be
calculated and then added to or subtracted from the apparent
[Na+]i to yield a pH-corrected
[Na+]i. The form of this equation was
|
(9)
|
where [Na+](CF) is the
correction factor,
pHi = 7.33
pHi(x) [where pHi(x) corresponds to any
pHi within the tested range of 6.85 to 7.83], and 1/
is
a proportionality factor. Figure 5D shows linear
least-squares regression fits to data points obtained experimentally at
pHi 6.85 and 7.83 and plots obtained using Eq. 9
at four different values of pHi (6.85, 7.10, 7.60, and
7.83) for
= 3.5. The results suggest that Eq. 9
provides a reasonable estimate of
[Na+](CF) at any given
pHi. Furthermore, the linear form of Eq. 9
indicates that the percent error introduced in the estimation of
[Na+]i by a fixed change in pHi
will be constant at all values of [Na+]i.
Thus, for example, if [Na+]i has been
estimated at 10 mM under conditions where pHi is reduced by
0.2 pH units, the corrected value will be 10.57 mM {i.e.,
[Na+](CF) = 2/3.5 = 0.57 mM}. On the other hand, at an estimated [Na+]i of 80 mM, the same reduction in
pHi will give a
[Na+](CF) = 4.57 mM and the
corrected [Na+]i will be 84.57 mM.
 |
DISCUSSION |
The accurate in situ calibration of SBFI is important, not only
because the spectral properties of the dye in situ differ markedly from
those in vitro, but also because physiologically important changes in
neuronal [Na+]i may be small with respect to
resting levels (4, 28). Although full SBFI calibrations
have been fitted by a variety of different means (e.g., see Refs.
3, 13, 15, 19,
24, 29, 37), in this study we
employed a three-parameter hyperbolic equation (Eq. 3) that
not only provided a simple method for estimating Rmin and
Kd, but could also be transformed easily into
the standard equation of Grynkiewicz et al. (16) to
determine Rmax. This is advantageous, given that the high
ionic strength media required for the experimental determination of
Rmax may affect the characteristics of the fluorophore in
situ (3, 23). Because 140 mM Na+ is not the
saturation point for SBFI, the equation employed in the present study
also obviates the approximation made when the ratio of fluorescence
intensities emitted during excitation at 380 nm for [Na+] = 0 mM and ~140 mM are employed to derive values for
and thus the
Kd of SBFI for Na+. We also took
advantage of the insensitivity of the SBFI emission signal during
excitation at 334 nm to changes in [Na+] in situ to
correct the Na+-sensitive 380 nm signal for the drift that
often occurs during the course of a full calibration experiment. The
normalized drift-corrected BI380 signal was
fitted with a three-parameter hyperbolic decay equation (Eq. 6), a procedure that facilitated the determination of
and thus
the Kd of SBFI for Na+. Although
values for Rmax and
Kd obtained
by the aforementioned methods were similar to those derived from Hanes
plots of full calibration data, the equations employed in the present
study offer the advantage over previously described methods (including Hanes plots and the three-point procedure) of allowing the
straightforward in situ determination of separate values for
and
Kd as well as Rmin and
Rmax (i.e., individual values for all the constant parameters of the standard equation). In this way, potential errors introduced by the use of Kd values obtained in
vitro for the calibration of signals from experiments in intact cells
can be avoided, and the effects of experimental maneuvers, such as
changes in pH, on Kd values can easily be determined.
Values for Rmin, Rmax,
, and
Kd estimated from three-parameter hyperbolic
fits to the data points obtained in full calibrations were highly
reproducible. However, a full calibration employing
8 concentrations
of Na+ at the end of an experiment may be impractical
(e.g., see Ref. 13). A frequently employed alternative is
the three-point method introduced by Harootunian et al.
(18). Nevertheless, marked loss of signal may occur even
during this less protracted procedure under some circumstances (e.g.,
following anoxia in mammalian neurons; unpublished observations). Given
these limitations, we examined the possibility of applying a one-point
technique to calibrate SBFI ratio values in situ. In this procedure, a
full calibration curve is constructed (Fig. 1C, curve
a), and the curve is constrained to pass through the points
BI334/BI380 = 1.0, [Na+] = 10 mM. The advantage of this normalization
step is that it permits a one-point calibration for each cell studied.
At the end of every experiment, cell(s) are exposed to a medium
containing 10 mM Na+ and ionophore(s), and
BI334/BI380 values
from the entire experiment for a given cell are divided by the
BI334/BI380 value
at [Na+] = 10 mM for that cell; the normalized
BI334/BI380 values
are then used to calculate [Na+]i, utilizing
Eq. 2 and the appropriate fitted calibration parameters. The
latter are derived from full in situ calibration experiments, which are
required only when the cell type under study or optical equipment is
changed. Not only does a one-point calibration offer the advantages of
being simpler and faster to perform at the end of an experiment than a
full or a three-point calibration, but also the accuracy of the method
appears at least equivalent to that of a three-point procedure. Thus
changes in [Na+]i evoked by anoxia in rat
hippocampal neurons could be estimated as precisely by the one-point
technique as by the three-point procedure (Fig. 3, B and
C). The utility of the one-point calibration procedure is
also illustrated by the data presented in Fig. 5B, where the
solution employed for the one-point calibration contained 10 mM
Na+ at pH 7.35. From this figure, it is apparent that the
calculated values of [Na+]i at
pHo 7.35 closely approximate the values of
[Na+]o employed during the course of the
experiment. Thus although only a single calibration point was employed,
the normalized
BI334/BI380 values
obtained at values of [Na+]o other than 10 mM
were accurately transformed into appropriate values of
[Na+]i. These points having been noted, it is
nevertheless important to state that absolute values for
[Na+]i derived via any calibration procedure
should be held with caution.
The changes in pHi that may occur not only in response to
changes in [Na+]i but also to the ionophores
employed in calibration procedures represent a potential confound to
estimates of [Na+]i made with SBFI. In the
present study, the application of gramicidin D alone was found to
equilibrate pHi and pHo, suggesting that neither monensin nor nigericin are required in calibrating media to
abolish transmembrane H+ gradients. On the other hand,
given the sensitivity of SBFI ratio measurements to
[H+]i in situ (see below), the fact that the
ionophore(s) employed in SBFI calibration procedures cause
pHi to equal pHo reinforce the suggestion
(23, 35) that media employed to calibrate SBFI ratio
signals should be titrated to the normal mean resting pHi of the cell type under study.
Consistent with the reported apparent negative logarithm of the acidic
dissociation constant (6.09) of SBFI in vitro (26; also see
Ref. 17), changes in pH in the range 6.8-7.8 exerted minimal effects on the Kd of SBFI for
Na+ measured in situ. With regard to the [H+]
sensitivity of SBFI ratio measurements at a constant
[Na+], acidification resulted in an apparent decrease and
alkalinization in an apparent increase in
[Na+]i when [Na+]
10 mM,
results that are in broad agreement with those presented previously (13, 28, 29, 33, 34, 36). Although Harootunian et al. (18) failed to observe significant changes in SBFI
ratios when the intracellular compartment of REF52 cells was
alkalinized by 0.4 pH units, Rose and Ransom (34, 35), for
example, found that at [Na+] = 20 mM, 0.4-pH unit changes
induced apparent changes in [Na+] of 3.0 ± 0.6 mM
and 6.1 ± 1.5 mM in hippocampal neurons and astrocytes,
respectively (acidification and alkalinization evoking decreases and
increases, respectively, in apparent [Na+]). Similarly,
Nett and Deitmer (29) observed an apparent 0.77 mM
decrease in [Na+]i for a 0.3-pH unit
reduction in the pH of the calibrating solution at [Na+] = 10 mM in leech giant glial cells. The greater magnitude of the
apparent [Na+] changes evoked by alterations in pH
reported by Rose and Ransom (34, 35), compared with Nett
and Deitmer (29), may in part reflect the observation made
in the present study that the magnitude of the effect of a change in pH
on apparent [Na+]i measured with SBFI is
dependent upon the absolute value of [Na+]i.
However, for a given [Na+], the relationship between the
change in pHi and the change in apparent
[Na+]i is reasonably linear (Fig.
5C), and a simple procedure was developed to correct for
pH-induced changes in apparent [Na+] measured with SBFI.
The correction procedure described may be of use in experiments in
which [Na+]i and pHi are measured
concurrently, under which conditions [Na+]i
could be corrected for pHi on a region-by-region or
pixel-by-pixel basis (as described for simultaneous measurements of
[Ca2+]i and pHi; see Refs.
25 and 27) or in studies where SBFI fluorescence
measurements are made at a known pHi (e.g., following the
application of an H+ ionophore or during combined
patch-clamp and fluorescence ratio imaging, in which the patch pipette
solution contains a high concentration of H+ buffer).
Nevertheless, together, the results of the present study indicate that
the effects of changes in pHi on neuronal
[Na+]i values estimated with SBFI are
relatively small and are unlikely to affect the interpretation of
results under most experimental conditions.
In summary, we developed and tested simplified procedures for
calibrating SBFI ratio measurements in situ. Values for all of the SBFI
calibration constants are drawn from in situ measurements by means of
three-parameter hyperbolic equations, and, once the parameters of the
standard equation have been determined, SBFI ratios measured during the
course of an experiment are transformed into
[Na+]i values by a one-point procedure
applied at the end of the experiment. The results also demonstrate that
the in situ pH sensitivity of SBFI ratio measurements is related
linearly to [Na+]i. Although SBFI is only
weakly sensitive to changes in pH in the range pH 6.8-7.8
(compared, for example, with fura 2 or indo 1; see Refs.
1, 25, and 31), correction factors can
be employed, if required, to correct SBFI-derived measurements of apparent [Na+]i for pHi.
 |
ACKNOWLEDGEMENTS |
We thank S. Atmadja for preparation of the neuronal cultures and
Dr. E. D. W. Moore for helpful comments on an early version of the manuscript.
 |
FOOTNOTES |
Financial support was provided by a Grant-in-Aid from the Heart and
Stroke Foundation of British Columbia and Yukon.
Address for reprint requests and other correspondence:
J. Church, Dept. of Anatomy, Univ. of British Columbia, 2177 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 (E-mail:
jchurch{at}interchange.ubc.ca).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 June 2000; accepted in final form 18 January 2001.
 |
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