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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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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)
[Na<SUP>+</SUP>]<IT>=&bgr;K</IT><SUB>d</SUB>[(R<IT>−</IT>R<SUB>min</SUB>)<IT>/</IT>(R<SUB>max</SUB><IT>−</IT>R)] (1)
where beta  = Sf2/Sb2 and is the ratio of the fluorescence of the free (unbound) dye (Sf2) to bound dye (Sb2) at the second excitation wavelength (lambda 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 beta Kd; the accurate determination of a separate value for beta  (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
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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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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Calibration of SBFI Ratio Values

Determination of Rmin, Rmax, and beta 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 (black-triangle) and curve c (triangle ) 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 -beta Kd is obtained from the intercept on the abscissa.

To determine all the constants (Rmin, Rmax, beta , and Kd) necessary to convert the ratio of emitted SBFI signals into [Na+] values, the standard equation (Eq. 1) was rearranged to give
[Na<SUP>+</SUP>]<IT>=&bgr;K</IT><SUB>d</SUB>[(R<SUB>n</SUB><IT>−</IT>R<SUB>n(min)</SUB>)<IT>/</IT>(R<SUB>n(max)</SUB><IT>−</IT>R<SUB>n</SUB>)] (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
R<SUB>n</SUB><IT>=</IT>R<SUB>n(min)</SUB><IT>+</IT>[a([Na<SUP>+</SUP>])<IT>/</IT>(b+[Na<SUP>+</SUP>])] (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 beta Kd, Eq. 3 was rearranged to give
[Na<SUP>+</SUP>]<IT>=</IT>b{(R<SUB>n</SUB><IT>−</IT>R<SUB>n(min)</SUB>)<IT>/</IT>[(a<IT>+</IT>R<SUB>n(min)</SUB>)<IT>−</IT>R<SUB>n</SUB>]} (4)
It is apparent that Eq. 4 is identical to Eq. 2 when b = beta 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 beta 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 beta 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
[Na<SUP>+</SUP>]<IT>/</IT>(R<SUB>n</SUB><IT>−</IT>R<SUB>n(min)</SUB>)<IT>=</IT>{[Na<SUP>+</SUP>]<IT>/</IT>(R<SUB>n(max)</SUB><IT>−</IT>R<SUB>n(min)</SUB>)}<IT>+</IT>[<IT>&bgr;K</IT><SUB>d</SUB><IT>/</IT>(R<SUB>n(max)</SUB><IT>−</IT>R<SUB>n(min)</SUB>)] (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 -beta Kd (Fig. 1D); Rn(min) is derived from experimental data. The values of Rn(max) and beta 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 beta 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 beta 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 beta Kd determined by three-parameter hyperbolic fits to data points obtained in full calibrations

Determination of beta  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 beta  (and, thus a value for the Kd of SBFI for Na+). To determine beta , one needs to know Sf2 and Sb2, the intensities of fluorescence emissions when exciting the fluorophore at the second wavelength (lambda 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
S<SUB>n<IT>2</IT></SUB><IT>=&dgr;+</IT>[<IT>&egr;</IT>(<IT>&phgr;</IT>)<IT>/</IT>(<IT>&phgr;+</IT>[Na<SUP>+</SUP>])] (6)
where delta , epsilon , and phi  are constants. From Eq. 6 it can be seen that when [Na+] = 0, Sn2 = Sf2 = (delta  + epsilon ), and when [Na+] = infinity , Sn2 = Sb2 = delta . Consequently
&bgr;=S<SUB>f<IT>2</IT></SUB><IT>/</IT>S<SUB>b<IT>2</IT></SUB><IT>=</IT>(<IT>&dgr;+&egr;</IT>)<IT>/&dgr;</IT> (7)
The values of the parameters for Eq. 6, obtained from the fit to the data points shown in Fig. 1C (plot b), were delta  (i.e., Sb2) = 0.41 ± 0.01, epsilon  = 0.94 ± 0.01, and phi  = 17.96 ± 0.97; thus Sf2 = 1.34 ± 0.03 and beta  (Sf2/Sb2) = 3.31 ± 0.03. As noted above, beta 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 beta , 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 beta , corrected for drift, is 2.88 ± 0.05. As noted above, beta 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.

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), beta , 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 (open circle ) 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 beta Kd, Rn(min), and Rn(max) obtained from a full calibration (calibration 3, Table 1).

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 (open circle ). The subsequent addition of 10 µM nigericin failed to evoke an additional change in pHi. B: the changes in steady-state pHi (Delta 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 lambda 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 beta 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 beta , 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), beta , 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+] (Delta [Na+]) at different absolute values of [Na+]i (10 mM, ; 40 mM, ; and 130 mM, black-triangle) are shown as a function of pHi. For each absolute [Na+], values of Delta [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 (open circle ), 7.10 (triangle ), 7.60 (down-triangle), 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+] (Delta [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
&Dgr;[Na<SUP>+</SUP>]<IT>=</IT>[Na<SUP>+</SUP>]<SUB>pH<SUB>i</SUB>(x)</SUB><IT>−</IT>[Na<SUP>+</SUP>]<SUB>pH<SUB>i</SUB>7.33</SUB> (8)
where [Na+] = 10, 40, or 130 mM and x = 6.85, 7.33, or 7.83 (pHi-evoked changes in Delta [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 Delta [Na+]i increase as [Na+] increases and that a linear relationship exists between the effects of changes in pH on Delta [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
[Na<SUP>+</SUP>]<SUB>(CF)</SUB><IT>=</IT>[Na<SUP>+</SUP>](<IT>&Dgr;</IT>pH<SUB>i</SUB><IT>/&sfgr;</IT>) (9)
where [Na+](CF) is the correction factor, Delta pHi = 7.33 - pHi(x) [where pHi(x) corresponds to any pHi within the tested range of 6.85 to 7.83], and 1/sigma 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 sigma  = 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 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 beta  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 beta  and thus the Kd of SBFI for Na+. Although values for Rmax and beta 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 beta  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, beta , 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Baker, AJ, Brandes R, Schreur JHM, Camacho SA, and Weiner MW. Protein and acidosis alter calcium-binding and fluorescence spectra of the calcium indicator Indo-1. Biophys J 67: 1646-1654, 1994[Abstract].

2.   Baxter, KA, and Church J. Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones. J Physiol 493: 457-470, 1996[Abstract].

3.   Borzak, S, Reers M, Arruda J, Sharma VK, Sheu SS, Smith TW, and Marsh JD. Na+ efflux measurements in ventricular myocytes: measurement of [Na+]i with Na+-binding benzofuran isophthalate. Am J Physiol Heart Circ Physiol 263: H866-H874, 1992[Abstract/Free Full Text].

4.   Bouron, A, and Reuter H. A role of intracellular Na+ in the regulation of synaptic transmission and turnover of the vesicular pool in cultured hippocampal cells. Neuron 17: 969-978, 1996[ISI][Medline].

5.   Bouvier, M, Szatkowski M, Amato A, and Attwell D. The glial cell glutamate uptake carrier countertransports pH-changing anions. Nature 360: 471-474, 1992[ISI][Medline].

6.   Boyarsky, G, Ganz MB, Sterzel RB, and Boron WF. pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Am J Physiol Cell Physiol 255: C844-C856, 1988[Abstract/Free Full Text].

7.   Breder, J, Sabelhaus CF, Opitz T, Reymann KG, and Schröder UH. Inhibition of different pathways influencing Na+ homeostasis protects organotypic hippocampal slice cultures from hypoxic/hypoglycemic injury. Neuropharmacology 39: 1779-1787, 2000[ISI][Medline].

8.   Bright, GR, Fisher GW, Rogowska J, and Taylor DL. Fluorescence ratio imaging microscopy. Methods Cell Biol 30: 157-192, 1989[ISI][Medline].

9.   Calabresi, P, Marfia GA, Centonze D, Pisani A, and Bernardi G. Sodium influx plays a major role in the membrane depolarization induced by oxygen and glucose deprivation in rat striatal spiny neurons. Stroke 30: 171-179, 1999[Abstract/Free Full Text].

10.   Chen, WH, Chu KC, Wu SJ, Wu JC, Shui HA, and Wu ML. Early metabolic inhibition-induced intracellular sodium and calcium increase in rat cerebellar granule cells. J Physiol 515: 133-146, 1999[Abstract/Free Full Text].

11.   Chinopoulos, C, Tretter L, Rozsa A, and Adam-Vizi V. Exacerbated responses to oxidative stress by an Na+ load in isolated nerve terminals: the role of ATP depletion and rise of [Ca2+]i. J Neurosci 20: 2094-2103, 2000[Abstract/Free Full Text].

12.   Cooper, DMF, Schell MJ, Thorn P, and Irvine RF. Regulation of adenylyl cyclase by membrane potential. J Biol Chem 273: 27703-27707, 1998[Abstract/Free Full Text].

13.   David, G, Barrett JN, and Barrett EF. Spatiotemporal gradients of intra-axonal [Na+] after transection and resealing in lizard peripheral myelinated axons. J Physiol 498: 295-307, 1997[Abstract].

14.   Diarra, A, Sheldon C, Brett CL, Baimbridge KG, and Church J. Anoxia-evoked intracellular pH and Ca2+ concentration changes in cultured postnatal rat hippocampal neurons. Neuroscience 93: 1003-1016, 1999[ISI][Medline].

15.   Donoso, P, Mill JG, O'Neill SC, and Eisner DA. Fluorescence measurements of cytoplasmic and mitochondrial sodium concentration in rat ventricular myocytes. J Physiol 448: 493-509, 1992[Abstract].

16.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

17.   Haigney, MCP, Miyata H, Lakatta EG, Stern MD, and Silverman HS. Dependence of hypoxic cellular calcium loading on Na+-Ca2+ exchange. Circ Res 71: 547-557, 1992[Abstract].

18.   Harootunian, AT, Kao JPY, Eckert BK, and Tsien RY. Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes. J Biol Chem 264: 19458-19467, 1989[Abstract/Free Full Text].

19.   Jung, DW, Apel LM, and Brierley GP. Transmembrane gradients of free Na+ in isolated heart mitochondria estimated using a fluorescent probe. Am J Physiol Cell Physiol 262: C1047-C1055, 1992[Abstract/Free Full Text].

20.   Kiedrowski, L, Brooker G, Costa E, and Wroblewski JT. Glutamate impairs neuronal calcium extrusion while reducing sodium gradient. Neuron 12: 295-300, 1994[ISI][Medline].

21.   Kiedrowski, L, Wroblewski JT, and Costa E. Intracellular sodium concentration in cultured cerebellar granule cells challenged with glutamate. Mol Pharmacol 45: 1050-1054, 1994[Abstract].

22.   Leem, CH, and Vaughan-Jones RD. Out-of-equilibrium pH transients in the guinea-pig ventricular myocyte. J Physiol 509: 471-485, 1998[Abstract/Free Full Text].

23.   Levi, AJ, Lee CO, and Brooksby P. Properties of the fluorescent sodium indicator "SBFI" in rat and rabbit cardiac myocytes. J Cardiovasc Electrophysiol 5: 241-257, 1994[ISI][Medline].

24.   Maier, LS, Pieske B, and Allen DG. Influence of stimulation frequency on [Na+]i and contractile function in Langendorff-perfused rat heart. Am J Physiol Heart Circ Physiol 273: H1246-H1254, 1997[Abstract/Free Full Text].

25.   Martínez-Zaguilán, R, Parnami G, and Lynch RM. Selection of fluorescent ion indicators for simultaneous measurements of pH and Ca2+. Cell Calcium 19: 337-349, 1996[ISI][Medline].

26.   Minta, A, and Tsien RY. Fluorescent indicators for cytosolic sodium. J Biol Chem 264: 19449-19457, 1989[Abstract/Free Full Text].

27.   Morris, SJ. Simultaneous multiple detection of fluorescent molecules. In: Optical Microscopy: Emerging Methods and Applications, edited by Herman B, and Lemasters JJ.. New York: Academic, 1993, p. 177-212.

28.   Negulescu, PA, and Machen TE. Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes. Methods Enzymol 192: 38-81, 1990[Medline].

29.   Nett, W, and Deitmer JW. Intracellular Ca2+ regulation by the leech giant glial cell. J Physiol 507: 147-162, 1998[Abstract/Free Full Text].

30.   Pinelis, VG, Segal M, Greenberger V, and Khodorov BI. Changes in cytosolic sodium caused by a toxic glutamate treatment of cultured hippocampal neurons. Biochem Mol Biol Int 32: 475-482, 1994[ISI][Medline].

31.   Ralenkotter, L, Dales C, Delcamp TJ, and Hadley RW. Cytosolic [Ca2+], [Na+], and pH in guinea pig ventricular myocytes exposed to anoxia and reoxygenation. Am J Physiol Heart Circ Physiol 272: H2679-H2685, 1997[Abstract/Free Full Text].

32.   Robertson, MA, and Foskett JK. Fluorescence measurements of cytosolic sodium concentration. Methods Neurosci 27: 274-288, 1995.

33.   Rose, CR, Kovalchuk Y, Eilers J, and Konnerth A. Two-photon Na+ imaging in spines and fine dendrites of central neurons. Pflügers Arch 439: 201-207, 1999[ISI][Medline].

34.   Rose, CR, and Ransom BR. Regulation of intracellular sodium in cultured rat hippocampal neurones. J Physiol 499: 573-587, 1997[Abstract].

35.   Rose, CR, and Ransom BR. Intracellular sodium homeostasis in rat hippocampal astrocytes. J Physiol 491: 291-305, 1996[Abstract].

36.   Rose, CR, Waxman SG, and Ransom BR. Effects of glucose deprivation, chemical hypoxia, and simulated ischemia on Na+ homeostasis in rat spinal cord astrocytes. J Neurosci 18: 3554-3562, 1998[Abstract/Free Full Text].

37.   Satoh, H, Hayashi H, Noda N, Terada H, Kobayashi A, Yamashita Y, Kawai T, Hirano M, and Yamazaki N. Quantification of intracellular free sodium ions by using a new fluorescent indicator, sodium-binding benzofuran isophthalate in guinea pig myocytes. Biochem Biophys Res Commun 175: 611-616, 1991[ISI][Medline].

38.   Smith, GAM, Brett CL, and Church J. Effects of noradrenaline on intracellular pH in acutely dissociated adult rat hippocampal CA1 neurones. J Physiol 512: 487-505, 1998[Abstract/Free Full Text].

39.   Urenjak, J, and Obrenovitch TP. Pharmacological modulation of voltage-gated Na+ channels: a rational and effective strategy against ischemic brain damage. Pharmacol Rev 48: 21-67, 1996[ISI][Medline].

40.   Yu, XM, and Salter MW. Gain control of NMDA-receptor currents by intracellular sodium. Nature 396: 469-474, 1998[ISI][Medline].


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