Phillip
Roder
and
Carsten
Hille
*
Physical Chemistry/Applied Laser Sensing in Complex Biosystems (ALS ComBi), Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany. E-mail: hille@uni-potsdam.de
First published on 2nd October 2014
Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+]i), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+]i recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+]i rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl− cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.
The application of Na+-sensitive fluorescent indicators allows the non-invasive and highly sensitive recording of [Na+]i, even in several cells simultaneously.2 The range of appropriate Na+-sensitive fluorescent dyes is limited. Most of these dyes consist of a 15-crown-5 ether, which specifically binds to Na+ and is linked to the fluorophore, such as fluorescein derivatives and benzofuran derivatives. The fluorescence of these compounds is often attenuated by photoinduced electron transfer.3 Most widely used Na+-sensitive fluorescent dyes are currently sodium-binding benzofuran isophthalate (SBFI),4,5 CoroNa Green6,7 and Sodium Green.8,9 Concerning [Na+]i recordings in living cells, these dyes have limitations. SBFI can be used in the dual-excitation ratiometric mode resulting in a more reliable quantification, but it has to be excited in the UV spectral range. Thus, cellular autofluorescence as well as UV-excited fluorescence from pharmacological substances, such as amiloride, a Na+ channel inhibitor, can hamper the recordings. On the other hand, Sodium Green displays improved optical properties with excitation in the visible spectral range, but it is a non-ratiometric dye. Finally, CoroNa Green has been launched for improved dye loading into the cells, since it has less than half the size of the former dyes. However, its dissociation constant is rather high, limiting recordings of relatively low [Na+]i.
A significant improvement in [Na+]i recordings can be realised with the fluorescence lifetime imaging microscopy (FLIM) providing access to the fluorescence decay time of a dye. This nanosecond lifetime is mostly independent of dye concentration and no ratiometric probe is required.5 However, the fluorescence decays of SBFI and Sodium Green display a complex triexponential decay behavior in living cells and their decay times or rather corresponding amplitudes are only weakly Na+-dependent.8–11 A new promising, recently available Na+-sensitive fluorescent dye could be Asante NaTRIUM Green-2 (ANG-2), which has similar spectral properties as Sodium Green. Although some photophysical data of ANG-2 are available by the manufacturer, only for its lower affinity analog ANG-1 experimental data based on two-photon (2P)-excited fluorescence intensity recordings has been published.12 Since no data is available about the application of ANG-2 for FLIM, the purpose of this study is to provide time-resolved fluorescence data and demonstrate the feasibility of ANG-2 for quantitative [Na+]i recordings in living cells using 2P-FLIM. Thereby, FLIM was combined with 2P-excitation, since deeper light penetration into the tissue can be realised and due to lower excitation energy, cells become less damaged and even photobleaching of ANG-2 can be significantly reduced.13 To the end, by applying ANG-2 with 2P-FLIM we were able to record [Na+]i quantitatively in the salivary glands of the American cockroach Periplaneta americana, a well-established model system for aminergically controlled epithelial ion transport. In pharmacological experiments dopamine-induced [Na+]i changes could be unravelled leading to an improved model for salivation in these glands.
For in situ calibration experiments, 0.05–1% (v/v) of the unspecific non-ionic surfactant Triton X-100 (Sigma-Aldrich, Deisenhofen, Germany) was applied, resulting in a dissipation of the [Na+] gradient between the intracellular and extracellular space.
A 10 mM dopamine (Sigma-Aldrich, Deisenhofen, Germany) stock solution in double-distilled water and a 100 mM bumetanide (Sigma-Aldrich, Deisenhofen, Germany) stock solution in DMSO were prepared for physiological experiments. The final concentrations of both substances in physiological saline were 1 μM and 50 μM, respectively.
2P fluorescence excitation action cross-sections ΦFσ2 (with 1 GM = 10−50 cm4 s per photon) were calculated according to eqn (1):
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Measurements of the fluorescence decay times were realised with the MicroTime 200 fluorescence lifetime microscope system (PicoQuant, Berlin, Germany) coupled with the time-correlated single photon counting (TCSPC) module PicoHarp 300 exhibiting a time resolution of 8 ps. For 2P-excitation, a mode-locked fs-fiber laser (C-Fiber A780; Menlo Systems, Martinsried, Germany) was used at wavelength of 780 nm, a pulse width of ∼90 fs and a repetition rate of 50 MHz. The excitation light was coupled via a dichroic mirror (2P-dichroic 725; Chroma, Fürstenfeldbruck, Germany) into the inverted microscope (IX71, Olympus, Hamburg, Germany) equipped with a Plan Apo ×100/NA1.4 oil immersion objective (Olympus). The fluorescence signal passed a 100 μm pinhole and two short-pass filters (SP400-680/OD4, Edmund Optics, Karlsruhe, Germany; SP420-680/OD2, Baader, Mammendorf, Germany) and was detected by a single-photon avalanche diode (SPAD, SPCM-AGR-13, Perkin Elmer, Waltham, USA). Time-resolved fluorescence images were acquired by scanning the sample with a xy-piezoelectric scanner. Thus, full frame images of 80 μm × 80 μm were recorded with a resolution of 150 pixel × 150 pixel and a pixel dwell time of 0.6 ms per pixel.
From the recorded images, the fluorescence decay times were calculated by deconvolution fitting using the SymPhoTime software (ver. 5.3.2.2, PicoQuant, Berlin, Germany). Global multiexponential decay analyses of data sets were conducted with FAST (ver. 2.13, Edinburgh Instruments Ltd., Livingston, Great Britain). The quality of decay fitting was estimated by randomly distributed residuals and small reduced χ2 values. In case of global multiexponential decay analyses, two types of reduced χ2 values were calculated. An individual χ2ind value qualified the goodness of fit to an individual decay curve within the global analysis. A global χ2global value characterized the goodness of fit for the entire set of data and was calculated as the root mean square of all individual χ2ind values. The instrument response function (IRF) was measured daily from the backscattered excitation light with a full width at half maximum (FWHM) of (223 ± 2) ps (N = 35), indicating the detector timing resolution as most critical element. The broadening and temporal shift of the IRF at higher photon count rates as well as color effects are known problems for SPAD modules, but their insignificance in this setup has been already demonstrated recently.21
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Fig. 1 (A) Chemical structure of ANG-2.20 (B) Absorption and normalised fluorescence spectra of Na+-bound (solid lines) and Na+-free (dashed lines) form of ANG-2 with excitation wavelength λex = 517 nm and cdye = 1.3 μM. |
In this context, the fluorescence quantum yield of ANG-2 was determined for the first time. Thus, the fluorescence quantum yield of the Na+-free and the Na+-bound ANG-2 form were ascertained to ΦF = 0.014 ± 0.001 (N = 6) and ΦF = 0.20 ± 0.01 (N = 6), respectively. The increase in the fluorescence quantum yield in the presence of sodium is similar to the determined FEF as expected. Indeed, the relatively low fluorescence quantum yields of ANG-2 hamper fluorescence measurements, but they are in a similar low range as shown for other Na+-sensitive dyes, such as Sodium Green (ΦF = 0.20)22 and SBFI (ΦF = 0.08).4
In order to exploit the advantages of 2P-excitation in live cell imaging, ANG-2 was evaluated on its potential of 2P-excitation for the first time. Thus, 2P fluorescence excitation action cross-sections ΦFσ2 were determined in a 2P-excitation range of 720 nm–900 nm (Fig. 2). For the analysed spectral range, the ΦFσ2-values for the Na+-bound form were always one order of magnitude higher than that for the Na+-free form in aqueous solution. Although these ΦFσ2-values are lower than those of other organic fluorescent dyes (e.g. 10–100 GM23), ANG-2 seems to be 2P-excitable. At 780 nm, the used 2P-excitation wavelength for in situ recordings in this study, an increase in ΦFσ2 from (0.45 ± 0.01) GM for the Na+-free form to (5.91 ± 0.08) GM for the Na+-saturated form was observed. Since spectral properties of dyes can be significantly changed in intracellular environment, measurements were repeated in a cytosol-like buffer solution prepared from salivary gland homogenates.24 Measurements under these conditions showed similar fluorescence quantum yields with ΦF = 0.025 ± 0.001 (N = 6) and ΦF = 0.19 ± 0.01 (N = 3). The resulting ΦFσ2 at 780 nm with (0.84 ± 0.03) GM for the Na+-free form and (5.7 ± 0.1) GM for the Na+-saturated form were similar to those recorded in aqueous solution. From these data it was concluded that 2P-excitability of ANG-2 can be successfully realised in vitro and in living cells.
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Fig. 2 2P fluorescence excitation action cross-sections ΦFσ2 of Na+-free and Na+-saturated ANG-2 buffer solutions (N = 6 for each form). For the reference fluorescein (in NaOH, pH 11) literature data were used;18 1 GM = 10−50 cm4 s per photon; ΦFσ2 is the product of 2P absorption cross-section σ2 and fluorescence quantum yield ΦF. |
Time-resolved fluorescence measurements using the TCSPC technique provided access to the ANG-2 fluorescence decay behaviour for the first time. To investigate the Na+-dependent behaviour of ANG-2 fluorescence decay, 11 different concentrations of sodium ([Na+] + [K+] = 140 mM) were prepared in aqueous solution. ANG-2 displayed a multiexponential decay behaviour (Fig. 3A). Sets of decay curves were examined for a possible mono-, bi- or triexponential decay behaviour. Therefore, curves were globally fitted starting at the peak maximum, in order to avoid influences of possible rise components (Fig. 3C and D). Here, the different ANG-2 forms (at least Na+-free and Na+-bound form) were considered to exhibit different decay time components τi. In the global analysis, the decay time components were linked during the fitting process, to find a set of decay time components optimised for all decay curves. The pre-exponential amplitudes αi were related to the concentration of each ANG-2 form and were fitted independently for each individual decay curve. By increasing the number of decay time components, the χ2global values decreased (109.1 vs. 3.8 vs. 1.2) and residuals alternated more randomly. Thus, a triexponential model was required for appropriate decay fitting (Fig. 3C and D). A triexponential decay behaviour has been already suggested for other Na+-sensitive fluorescent dyes.9 Hence, the decay time components could be calculated to τ1 = 0.3 ns, τ2 = 0.7 ns and τ3 = 2.7 ns. Due to the trend of their normalised amplitudes αi, the decay time components could not clearly assigned to the corresponding ANG-2 forms (Fig. 3B). Whereas the changes in the amplitude of the short decay time component probably correspond to the Na+-free dye form, the changes in the amplitudes of the intermediate and long decay time component could not be related to an ANG-2 form. In addition, one has to point out that the fraction of the Na+-free form of ANG-2 is probably underestimated due to the large difference in the fluorescence quantum yields. A triexponential decay behaviour has been also observed for Sodium Green in vitro, where two shorter decay time components (0.09 ns and 0.40 ns) were associated with the Na+-free form and a longer decay time component (2.53 ns) was associated with the Na+-bound form.9 This decay behaviour of Sodium Green was confirmed by another study with slightly different decay time components (0.22 ns, 0.69 ns, 2.80 ns).22 For SBFI, a complex multiexponential decay behaviour with rather short decay time components (6 ps, 0.25 ns, 0.61 ns) could be found, too.11 Therefore, the amplitude-weighted average fluorescence decay time τamp was calculated according to eqn (2):25
![]() | (2) |
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Fig. 3 In vitro calibration of ANG-2 in different Na+-buffer solutions. (A) Fluorescence decay curves of ANG-2 for different [Na+]: 0, 5, 10, 15, 20, 30, 40, 60, 80, 100 and 140 mM (buffer solutions, pH 7.4; normalised at peak maximum). (B) Trend of normalised amplitudes (αi) and amplitude-weighted average fluorescence decay time (τamp) for the global triexponential analysis of 55 decay curves from A (N = 5 independent preparations) with the Na+-dependent decay time components τ1 = 0.3 ns (black squares), τ2 = 0.7 ns (black circles) and τ3 = 2.7 ns (black triangles), τamp (red squares) and the KD value of (8.3 ± 0.4) mM calculated from the fit of τamp using eqn (3). (C) and (D) Normalised fluorescence decay curves (black) with associated triexponential fit curves (white) and generally used IRF (instrumental response function, grey) of Na+-free and Na+-saturated form of ANG-2. Mono-, bi and triexponential fit results of the global analyses were compared and shown with their corresponding amplitude components, residuals, χ2ind and χ2global values. |
In this, a meaningful Na+-dependent change in τamp could be shown (Fig. 3B). The apparent dissociation constant KD was then calculated fitting the data to eqn (3) using τamp:26
![]() | (3) |
This resulted in an in vitro KD value of (8.3 ± 0.4) mM. However, the in vitro KD value of the manufacturer with KD = 34 mM in the presence of K+ deviates from the experimental value. This discrepancy could be the result of different recording methods, namely steady-state and time-resolved fluorescence measurements. Compared to other Na+-sensitive fluorescent dyes, the in vitro KD of ANG-2 is in a similar range. For SBFI and Sodium Green in vitro KD values of (10 ± 1) mM and (9 ± 1) mM were obtained.11,22
Irreversible dye photobleaching and dye leakage out of the cell can significantly influence long-term fluorescence recordings. To evaluate the dye photobleaching, fluorescence intensity images were recorded during continuous irradiation with a constant laser power (4.3 mW) for 30 min. Thereby, a decrease in the fluorescence intensity to about 50% of the initial intensity was observed after 30 min (data not shown). To evaluate dye leakage, fluorescence intensity images were recorded only every 15 min (laser power 4.5 mW), so that the influence of dye photobleaching could be minimised. After 60 min, the fluorescence intensity dropped to approximately 60% of the initial intensity (data not shown). It was concluded that ANG-2 recordings in living cells seems to be feasible for approx. 40 min as conducted in this study.
Significant differences between in vitro and in situ KD values have been described frequently. In those cases, the in situ KD value is higher than the in vitro KD value.4,9,11,22,27 Reasons for this phenomenon could be lower selectivity to other intracellular ions or interactions with proteins. Consequently, an in situ calibration is required for reliable [Na+]i recordings. At first, a specific calibration procedure with an ionophore/inhibitor combination was performed. Thus, calibration solutions supplemented with 5 μM gramicidin (K+-uniporter28–30), 10 μM nigericin (Na+/K+-antiporter31), 10 μM monensin (Na+/H+-antiporter7) and 50 μM ouabain (Na+/K+-ATPase inhibitor28) were applied for 20 min in order to equilibrate intracellular and extracellular Na+ concentrations. However, the global fluorescence decay analysis of the recorded 2P-FLIM images did not unravel Na+-dependent changes (data not shown). Although this calibration procedure has been successfully applied in living cells previously,7,32 an appropriate drug combination for sufficient control of the Na+ gradient across the plasma membrane could not be established for the cockroach salivary glands.
Nevertheless, the non-ionic surfactant Triton X-100 could be alternatively used for in situ calibration. This surfactant permeabilises the plasma membrane, thus ensuring a complete equilibration of the Na+ concentration gradient.33,34 Salivary gland preparations were perfused for 10 min with Triton X-100 calibration solutions and 2P-FLIM images were acquired every two minutes. From all experiments, these 2P-FLIM images were selected for analysis, which displayed both, the largest effect and no visible cell damage. This procedure was necessary, since the time period for optimal equilibration of the Na+ concentration gradient varied between individual experiments. Similar to the in vitro calibration, the fluorescence decay behaviour of the data set was analysed performing global multiexponential decay fitting starting at the peak maximum. Compared to the monoexponential model, the biexponential model resulted in a smaller χ2global value (3.2 vs. 1.3) and improved residual traces (Fig. 5C and D). Interestingly, a triexponential model as required for the in vitro situation did not further improve the fitting outcomes. The results of the biexponential fit analysis were confirmed, but a rather unreasonable third short decay time component was obtained (10−5 ns). Global analysis of 63 decay curves (Fig. 5A) using the biexponential model revealed a short and a long decay time component of τf = 0.6 ns and τb = 3.3 ns. The changes in their normalised amplitudes unravelled that the short decay time component τf corresponds most probably to the Na+-free ANG-2 form, whereas the long decay time component τb corresponds most probably to the Na+-bound ANG-2 form (Fig. 5B). The apparent dissociation constant was calculated using eqn (3) revealing an in situ KD value of (53 ± 14) mM for ANG-2 in cockroach salivary gland cells. This dissociation constant is about six times higher than the corresponding in vitro KD value. This behaviour has been also observed for other Na+-sensitive dyes. For Sodium Green, the KD value increased in HeLa cells by the factor of about six (9 mM vs. 56 mM).22 In that study, the rather complex triexponential decay behaviour of Sodium Green in vitro did also changed to a simpler biexponential decay behaviour in HeLa cells. As shown in Fig. 5B under Na+-free and Na+-saturated conditions, a significant fraction of the opposite decay time component can be detected. However, from theory one would expect a monoexponential decay behaviour with only one dye form under these conditions. Consequently, the amplitude offset values αf,max = 0.51 ± 0.03 and αb,min = 0.25 ± 0.01 were extracted from the data analysis (Fig. 5B).
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Fig. 5 In situ calibration of ANG-2 in living cells with Triton X-100 and different Na+-buffer solutions. (A) Fluorescence decay curves of ANG-2 for different [Na+]: 0, 10, 20, 40, 70, 100 and 140 mM (buffer solutions, pH 7.4; normalised at peak maximum). (B) Trend of normalised amplitudes (αi) for global biexponential analysis of 63 regions of interest extracted fluorescence decay curves from (A) (N = 9 ducts and acini preparations) with the Na+-free decay time component τf = 0.6 ns (black squares) and the Na+-bound decay time component τb = 3.3 ns (open circles) and the KD value of (53 ± 14) mM calculated from the fit using eqn (3). (C) and (D) Normalised fluorescence decay curves from the extracted FLIM images (black) with associated biexponential fit curves (white) and generally used IRF (instrumental response function, grey) of Na+-free and Na+-saturated form of ANG-2. Mono-, bi- and triexponential fit results of the global analyses were compared and shown with their corresponding amplitude components, residuals, χ2ind and χ2global values. |
For quantitative ion measurements in living cells, a direct correlation between the measured fluorescence decay data and the intracellular ion concentration has been established, when assuming a 1:
1 complexation of the ion with the dye.21,35–37 Briefly, in time-resolved fluorescence recordings the product of a decay time component (τf or τb) and its corresponding normalised amplitude (αb or αf) is equivalent to the fractional fluorescence intensity of the respective dye form. The intensities can be then converted into concentrations of the ion-free and ion-bound dye form by considering the specific 2P-excitabilities (σ2,f and σ2,b) as well as the fluorescence quantum yields (ΦF,f and ΦF,b) of both dye forms. Finally, the following calibration eqn (4) can be derived for quantifying [Na+]i:35,37
![]() | (4) |
The last term of eqn (4) is a constant for a defined experimental setup and could be calculated to S = 0.80 (λ2P-ex = 780 nm) in the present study. Thus, for quantitative [Na+]i recordings using the Na+-sensitive fluorescent dye ANG-2 with the 2P-FLIM technique, only the normalised amplitudes of both dye forms have to be extracted from the recording data. Since the fluorescence decay behaviour of ANG-2 differed between in vitro and in situ conditions, one of the applied models may not exactly describe the photophysics of ANG-2. Although this fact has to be elucidated in further experiments, reliable quantitative [Na+]i recordings in living cells are possible using this calibration procedure.
With the advantages of ANG-2 for 2P-FLIM recordings elucidated in the present study, quantitative [Na+]i recordings were performed in acinar and duct cells. ANG-2 loaded salivary ducts were stimulated with 1 μM dopamine and the recorded 2P-FLIM images were analysed with a biexponential decay model using fixed decay time components τf = 0.6 ns and τb = 3.3 ns. The corresponding normalised amplitudes and the calculated [Na+]i values, according to eqn (4), are shown in Fig. 6A and B. The resting [Na+]i in duct cells was estimated to (18.1 ± 1.6) mM (N = 6). This level fits well to electrophysiological data of (22 ± 19.3) mM.39 About 1 min after dopamine application, a significant [Na+]i increase to (34.1 ± 4.1) mM (N = 6) could be observed (P = 0.0313), which was totally reversible after dopamine washout (Fig. 6A). In addition, a second dopamine stimulus also resulted in a statistically significant reversible [Na+]i rise, indicating the viability of the gland preparations. The [Na+]i changes could be clearly unravelled by the opposite changes of the normalised amplitudes of the Na+-free and Na+-bound ANG-2 form (Fig. 6B). The described dopamine effect could also be identified by false colour-coded 2P-FLIM images (Fig. 6C). The dopamine-induced [Na+]i rise recorded here is markedly lower than that of an electrophysiological study, in which a huge [Na+]i rise up to 92 mM was recorded.39 It should be noted that these [Na+]i changes were recorded with two hardly comparable techniques, because the ion-selective microelectrodes represent an invasive point measurement method and 2P-FLIM is a large-scale non-invasive method. Indeed, this could have an impact on the measured [Na+]i.
It has been already shown that the Na+-K+-2Cl− cotransporter (NKCC) has a significant role in the secretion process in cockroach salivary glands. The NKCC has been localised in the acinar peripheral cells, where it functions as basolateral Na+ uptake mechanism, supporting the secretion of NaCl-rich primary saliva.17 According to this, inhibition of the NKCC can indirectly influence the dopamine-induced effects in the duct cells as shown electrophysiologically.39 Indeed, bath application of 1 μM dopamine together with 50 μM bumetanide, a well-known NKCC inhibitor,40 resulted in a complete suppression of the dopamine-induced [Na+]i rise in duct cells (Fig. 7). In this, only gland preparations were used, which displayed a [Na+]i rise due to a first dopamine-stimulus comparable to that shown before. These control measurements in the well-known duct cells revealed that physiological 2P-FLIM recordings are feasible.
The knowledge about the ion transport processes in the acinar peripheral cells is rather limited. However, original working hypotheses38,41 could be significantly improved due to physiological 2P- and/or FLIM-measurements of Na+ and Cl−.17 In the present study, a so far not quantified dopamine-induced [Na+]i increase in the acinar peripheral cells was analysed quantitatively by using ANG-2 and 2P-FLIM for the first time. Compared to the duct cells, the resting [Na+]i was estimated to a slightly lower value of (8.7 ± 0.5) mM (N = 9). Bath application of 1 μM dopamine induced a reversible [Na+]i rise up to (21.4 ± 1.0) mM, which was statistically significant (P < 0.01) (Fig. 8A). After dopamine washout, a second stimulation period also revealed an increase up to (29.2 ± 1.8) mM, which was however statistically not larger than the first rise (P > 0.05). These [Na+]i changes can be also illustrated by the opposite amplitude behaviour of both ANG-2 forms as well as in 2P-FLIM-images (Fig. 8B and C). Since the cellular volume of an acinar peripheral cell is rather small, it is hard to detect any intracellular Na+ gradients. In addition, in most of the preparations only 2–4 peripheral cells were located in the focal plane and could be observed during an experiment. So, no detailed information can be obtained about the intercellular communication. However, for few preparations it could be observed that not all recorded peripheral cells did response to dopamine (Fig. 8C). The only available quantitative [Na+]i data for acinar peripheral cells were obtained from cryosections using electron-probe X-ray microanalysis.41 Upon treatment with 1 μM dopamine, a [Na+]i increase from 10 mM to 25 mM has been observed, which fits well to the present data. Similar [Na+]i rises have been published for mammalian salivary acinar cells, although in those glands a transepithelial Cl−-transport provides the driving force for saliva secretion.42–44
Since the NKCC is localised in acinar peripheral cells, its pharmacological inhibition with bumetanide should clearly influence [Na+]i. These experiments were conducted as shown for that in duct cells. When observing a [Na+]i rise due to a first dopamine-stimulus, a second one in the presence of 50 μM bumetanide was completely blocked (Fig. 9). The bath application of bumetanide alone seemed to reduce [Na+]i, however this effect was statistically not significant (P > 0.05). Thus, the dopamine-induced [Na+]i rise in acinar peripheral cells is clearly NKCC-dependent.
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