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From the Department of Anesthesiology and Intensive Care, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki, Japan.
Address correspondence and reprint requests to Dr. Onizuka, Department of Anesthesiology and Intensive Care, Faculty of Medicine, University of Miyazaki, Kiyotake-Cho, Miyazaki 889-1692, Japan. Address e-mail to pirotann{at}med.miyazaki-u.ac.jp.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: Intracellular sodium imaging by sodium-binding benzofuran isophthalate and intracellular pH imaging by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein were used to measure [Na+]in and [pH]in. Measurements for [Na+]in were made in normal, Na+ free saline, with modified extracellular pH, and a Na+-H+ exchanger antagonist [(5-N-ethyl-N-isopropyl amiloride, N-methylisopropylamiloride, and 5-(N,N-hexamethylene)-amiloride) pretreatment trials. Furthermore, [Na+]in and [pH]in were recorded simultaneously. From 0.1 to 10 mM, lidocaine, mepivacaine, bupivacaine, prilocaine, and QX-314 were evaluated.
RESULTS: Lidocaine, mepivacaine, and prilocaine increased the [Na+]in in a dose-dependent manner. In contrast, QX-314 did not change the [Na+]in at each dose. In the Na+ free saline or in the presence of each Na+-H+ exchanger antagonist, lidocaine failed to increase [Na+]in. Lidocaine, mepivacaine, and prilocaine induced a significant decrease in [pH]in below baseline with an increase in [Na+]in. In contrast, QX-314 did not change the [pH]in. These results demonstrated that lidocaine increases [Na+]in through Na+-H+ exchanger activated by intracellular acidification, which is induced by the proton trapping of lidocaine. This [Na+]in increase and [pH]in change induces cell toxicity.
CONCLUSION: Lidocaine increases the [Na+] through a Na+-H+ exchanger by proton trapping.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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[Na+]in Imaging
For the measurement of [Na+]in, a ratiometric fluorescent indicator, an acetoxymethyl ester form of sodium-binding benzofuran isophthalate (SBFI-AM) (Molecular probes, Eugene, OR) was used.18,19 1 mM SBFI-AM in dimethyl sulfoxide was mixed with the defined medium containing the neuron at a volume of 0.1%. After incubation at 20°C for 60 min, this defined medium was removed. The intensity of intracellular SBFI fluorescence was measured at two quickly alternating excitation wavelengths (340/390 nm) and then was continuously recorded at 510 nm with an inverted fluorescent microscope (TE-300, Nikon, Japan), a cooled, high-speed charged, coupled device video camera (C-6970, Hamamatu photonics, Tokyo, Japan) and fluorescence imaging system (Aquacosmos, Hamamatu photonics). The exposure time for a single image was 159 ms, resulting in a total time of 600 ms needed for a 340/390 nm pair image. The background fluorescent images were subtracted before analysis.
In Vivo Calibration for [Na+]in
Two fluorescence ratios with SBFI were converted into using the calibration curve for Lymnaea VD neurons in vivo.20 The calibration curve for [Na+]in was constructed by plotting the fluorescence ratio versus Na+ concentration of the calibration solutions. To equilibrate the [Na+]in with extra ([Na+]out), 30 min before the experiment, 10 µM of the Na+ ionophore gramicidin D (ICN Biomedicals, Inc., Costa Mesa, CA) was added, and then the neurons were exposed to free Na+ concentration saline with gramicidin D solution for 30 min. Fluorescent image pairs were taken and neurons were then exposed to saline solutions of increasing sodium concentration (1, 25, 50, 100, and 150 mM) for 10 min each. Image pairs were taken again at the end of each exposure.
Experimental Procedure [Na+]in Imaging
The neurons were divided into five trials: normal saline, QX-314, Na+ free saline, modified pH, and Na+-H+ exchanger antagonist pretreatment trials with EIPA (Sigma Chemical CO), MIA (Sigma Chemical CO), and HMA (Sigma Chemical Co). In the free Na+ saline trials, Na+ free saline [0 mM NaCl, 50 mM N-methyl D-glucamine, 1.7 mM KCl, 4.1 mM CaCl2, 1.5 mM MgCl2, 5.0 mM HEPES pH 8] was used. In the normal saline trials, QX-314 and the Na+-H+ exchanger antagonist pretreatment trials normal saline was used.
In the normal saline trials and the Na+ free saline trials, lidocaine, mepivacaine, bupivacaine, prilocaine, and QX-314 (0.1, 1, and 10 mM) were perfused into the culture dish for 3 min after measuring the baseline values, and [Na+]in were measured at 5 min after lidocaine administration.
In the Na+ free saline trials, normal saline was changed to Na+ free saline at 10 min before the experiment, and [Na+]in were measured at 5 min after lidocaine administration. In the modified pH trials, the normal saline was changed to low pH saline (pH 6) at 1 min and 10 mM lidocaine adjusted to pH 6 was perfused for 3 min and then washed by pH 6 saline for 2 min. Next, the low pH saline (pH 6) was switched to normal saline (pH 8) and then 10 mM lidocaine adjusted to pH 8 was perfused for 3 min and washed by normal saline (pH 8). In each trial, the baseline values were measured 2 min before lidocaine perfusion. In the Na+-H+ exchanger antagonist pretreatment trials, lidocaine (10 mM) was perfused. EIPA, MIA, or HMA (10–1 to 105 nM) were perfused from 5 min before lidocaine perfusion. In these trials, the results were calculated to % inhibition of [Na+]in increase by 10 mM lidocaine, and the plots were fitted by Hille's equation that y = k0 + (k1 – k0)/[1 + (xk3/x)k2], where k0 corresponds to the y intercept, k1 to ymax (maximum of y), k2 to the rate (Hill coefficient), and k3 to the magnitude of half (IC50) in a curve that fit Hille's equation.
The analyses were performed using the Igor pro software program (version 5.01, Wave Metrics Inc., Portland, OR).
BCECF-[pH]in Imaging
For the measurement of [pH]in, the ratiometric fluorescent indicator, an acetoxymethyl ester form of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) (Molecular probes, USA) was used.21 1 mM BCECF-AM in dimethyl sulfoxide was mixed with the defined medium containing the neuron and the final concentration was 1 µM. After incubation at 20°C for 60 min, this defined medium was removed. Measurements were made by exciting this indicator dye at 490 and 440 nm (490/440 nm ratio) and continuously recorded at 520 nm.
In Vivo Calibration for [pH]in
Two fluorescence ratios with BCECF were converted into [pH]in using the calibration curve for each neuron in vivo.22 The calibration curve for [pH]in was constructed by plotting the fluorescence ratio vs pH of the calibration solutions [50 mM KCl, 5 mM NaCl, 1.5 mM MgCl2, 10 mM HEPES, and 10 µM nigericin, adjust each pH with KOH]. To equilibrate the [pH]in with extra ([pH]out), 20 min before the experiment, 10 µM of the K+/H+ exchanger nigericin (Sigma Chemical CO) was added, and then the neurons were exposed to pH calibration solutions with nigericin. Fluorescent image pairs were taken, and then the neurons were exposed to each pH saline solution (pH 6.0, 6.5, 7.0, 7.5, and 8.0) for 20 min each. Thereafter, image pairs were taken again at the end of each exposure.
Experimental Procedure for [pH]in Imaging
[pH]in was measured under lidocaine, mepivacaine, bupivacaine, prilocaine, and QX-314 (10 mM in each) perfusion. Also, [pH]in was measured under normal saline (pH 8), and low pH saline (pH 6). The normal saline was changed to low pH saline (pH 6) at 1 min and 10 mM lidocaine adjusted to pH 6 was perfused for 3 min and then washed by pH 6 saline for 5 min. Next, the low pH saline (pH 6) was switched to normal saline (pH 8) and 10 mM lidocaine, which was adjusted to pH 8 was perfused for 3 min.
Simultaneous Imaging of both [pH]in and [Na+]in
The neurons were incubated in both 50 µM SBFI-AM and 0.5 µM BCECF-AM containing normal saline for 60 min, and then this defined medium was removed.23 Measurements were made by exciting the BCECF at 490 and 450 nm, and also exciting the SBFI at 340 and 390 nm. The images were continuously recorded through the dichroic mirror of 520 ± 20 nm by fluorescence imaging system (Aquacosmos, Hamamatu photonics). The exposure time for a single image was 205 ms. Background fluorescent images were subtracted before analysis. In this trial, lidocaine (10 mM) was perfused into the culture dish after measuring the baseline values. [Na+]in and [pH]in were measured simultaneously before and after lidocaine perfusion.
Statistical Analysis
The results are expressed as the mean ± standard errors of the mean (SEM). The results of repeated measurements in each dose, in each group of trials were analyzed by repeated measurement of one-way analysis of variance, followed by the Scheff
test. Between the normal saline and free Na+ saline, QX-314 and lidocaine, modified pH, and Na+-H+ exchanger antagonist pretreatment trials were analyzed by Student's paired t-test. Stat view (version 4.5, Abacus, Berkeley, CA) was used for these analyses. A value of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1A shows the experimental tracings of [Na+]in before and after 10 mM lidocaine administration. After lidocaine perfusion, [Na+]in was significantly increased by lidocaine, that [Na+]in was from 6 ± 3 in the baseline to 10 ± 4 mM at 0.1 mM, to 18 ± 5 mM at 1 mM, and to 28 mM ± 7 at 10 mM, respectively (Fig. 1B). [Na+]in was also significantly increased by mepivacaine and prilocaine; however, the bupivacaine less increased in [Na+]in than lidocaine. The increases in [Na+]in after lidocaine, mepivacaine, bupivacaine, and prilocaine perfusion were observed in a concentration-dependent manner. In the QX-314 trials, no difference was observed before (control) and after the QX-314 perfusion (Fig. 1B).
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Na+ Free Saline Trials
In the Na+ free saline trials, no difference was observed in the control between the normal saline and Na+ free saline trials; however, the increase in [Na+]in after lidocaine perfusion was significantly lower than that of the normal saline trials, and [Na+]in went from 4 ± 5 at the baseline to 6 ± 6 mM at 0.1 mM, 10 ± 5 at 1 mM, and 11 ± 4 at 10 mM, respectively (Fig. 1B).
Modified pH Trials
In the modified pH trials, the pH was changed from pH 8 to 6. In each pH, after 10 mM lidocaine perfusion, the [Na+]in in pH 6 solution were significantly lower than that of pH 8, that [Na+]in was 6 ± 4 mM at pH 6, and 25 ± 4 at pH 8 (Fig. 2).
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BCECF-[pH]in Imaging
In the [pH]in imaging by BCECF, QX-314 (10 mM) did not induce the [pH]in change; in contrast, lidocaine, mepivacaine, and prilocaine significantly changed the [pH]in that with 10 mM lidocaine perfusion [pH]in was increased at first from pH 6.9 ± 0.2 to 9.2 ± 0.4, and the [pH]in after the washout of lidocaine was significantly decreased below the baseline that 6.1 ± 0.3; however, the bupivacaine less changed in [pH]in than lidocaine (Fig. 3).
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In Figure 4, the pH was changed from pH 8 to 6. This extracellular pH change did not induce [pH]in change. However, at each pH, with 10 mM lidocaine perfusion [pH]in was increased at first. The increase was greater in the high pH (9.0 ± 0.9 mM, pH 8) than in the low pH (7.1 ± 0.4 mM, pH 6, Fig. 4). In addition, the [pH]in after the washout of lidocaine was significantly decreased below the baseline, and was much decreased in the high pH (6.1 ± 0.3 mM, pH 8) than in the low pH (7.0 ± 0.4 mM, pH 6) after washout of lidocaine (Fig. 4).
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Simultaneously Imaging Both [Na+]in and [pH]in
Figure 5A shows the simultaneous imaging of the both [Na+]in by SBFI and [pH]in by BCECF before and after lidocaine perfusion. Lidocaine 10 mM increased [Na+]in and [pH]in, so that [Na+]in rose from 6 ± 3 mM to 27 ± 5 mM and [pH]in went 7.3 ± 0.3 mM at control, to 9.2 ± 0.7 mM, respectively (Fig. 5B). However, after the washout of lidocaine, [pH]in significantly decreased from baseline value 7.3 ± 0.3 to 5.9 ± 0.5 after the washout of lidocaine. In contrast, [Na+]i was still showed an increase after the washout of lidocaine.
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Na+-H+ Exchanger Antagonist Pretreatment Trials
EIPA (1 µM) significantly suppressed the lidocaine-induced [Na+]in increase when compared with normal saline trials, so that [Na+]i increased from 5 ± 4 mM in the baseline to 9 ± 6 mM at 10 mM lidocaine perfusion (Fig. 6A). Each Na+-H+ exchanger antagonist were suppressed the 10 mM lidocaine-induced [Na+]i increase in a concentration-dependent manner (Fig. 6B). The 50% inhibitory concentrations (IC50) of each Na+-H+ exchanger antagonist for 10 mM lidocaine-induced [Na+]i increase were EIPA: 41 nM, MIA: 512 nM, and HMA: 683 nM.
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The [pH]in measurement by BCECF imaging demonstrated that lidocaine, mepivacaine, and prilocaine increased [pH]in; however, QX-314 did not (Fig. 3). The [pH]in was significantly decreased below the baseline after the washout of lidocaine (Figs. 3 and 4). This pH increase, and the following decrease, was significantly greater at the high pH (pH 8) of lidocaine than that of the low pH (pH 6, Fig. 4).
With simultaneous imaging of both [Na+]in and [pH]in, lidocaine increased [pH]in; however, the [pH]in significantly decreased below the baseline with a [Na+]in increase after washout of the lidocaine (Fig. 5).
We demonstrated that some LA including lidocaine show a biphasic effect on intracellular pH before and after washout as does ammonia. Primarily, lidocaine induces intracellular alkalization by H+ trapping. In contrast, after the washout of lidocaine, its base form can cross the lipid bilayer of the cell membrane; as a result, a large amount of H+ is trapped in the cell, and acidification occurs. Thereafter, Na+-H+ exchanger will be promoted from exhaust intracellular H+ to extracellular H+.
This same situation may occur after ammonia perfusion in which ammonia induces intracellular alkalization and, after washout of ammonia, induces intracellular acidification.25,26
With Na+-H+ exchanger antagonist pretreatment, lidocaine did not increase [Na+]in (Fig. 6). These results indicate that the lidocaine-induced enhancement of [Na+]in may result from the activation of Na+-H+ exchanger and that the base form of lidocaine crossing the cell membrane would trap intracellular protons, thus increasing [pH]in. As a result, excess protons would be trapped within the cell. After washout of lidocaine, the protonated form of lidocaine would release protons, resulting in intracellular acidification and the activation of Na+-H+ exchanger via intracellular acidification. These results agree with those of Blanchard et al., Vinnikova et al., and Abdoun et al.,27–29 who showed that the Na+-H+ exchanger was activated by ammonia treatment and increased [Na+]in, and suppressed by antagonists of Na+-H+ exchanger, including EIPA, MIA, and HMA.
Our results suggest that the [Na+]in increase by lidocaine will also cause increases in the intracellular calcium concentration ([Ca2+]in) through Na+-Ca2+ exchanger. Kim-Lee et al. suggested that local anesthetics promote the forward mode of Na+/Ca2+ exchange. Our results also support this notion, because [Na+]in increase by lidocaine will promote Na+/Ca2+ exchanger.30 It had been reported that the [Ca2+]in is related to cell toxicity, including apoptosis by lidocaine.31–33 Therefore, it is probable that intracellular acidification by lidocaine is an important factor in cell toxicity.
The another view is that the mechanism of local anesthetics for their anesthetic effect is due to a Na channel block directly from inside the cell.1,2 In addition, there is another mechanism for the inhibition of sodium currents, so that an increase in [Na+]in will also suppress INa+ according to the Goldman-Hodgkin-Katz equation. This suggests that the ion conductance is determined by the concentration differential between the intra- and extracellular compartments. Therefore, an [Na+]in increase by lidocaine through the Na+-H+ exchanger would also suppress INa+.
In this study, the concentration of each LA was established for clinical use and 10 mM lidocaine showed not only a physiological, but also a toxic effect. Generally, for INa+ measurements, 0.1û1 mM lidocaine has been used. Hill et al. demonstrated on the node of Ranvier that 1 mM lidocaine inhibited the 70% of peak sodium currents. For pH measurements and for measurements Na+ exchangers, Kim-Lee et al. used 2 mM and Bidani et al. used 2.5 mM lidocaine.30,34 In clinical practice, however, from 40 to 80 mM of lidocaine is used; therefore, we should pay attention to this discrepancy between the experimentally effective concentrations for sodium channel actions and for Na+ exchangers, despite the 10 mM lidocaine is thus considered to be a clinically acceptable concentration.
In this study, dorsal ganglion neurons of the fresh water snail Lymnaea stagnalis were chosen. Na+-H+ exchanger and the effects of weak acids and bases, including ammonia and procaine to [pH]in, have been investigated in snail neurons, because these neurons do not require CO2 to retain extracellular pH, thus making it possible to observe the [pH]in and Na+-H+ exchanger activity clearly.35–37
In this study, SBFI-AM and BCECF-AM were used to measure [Na+]in. To investigate the interference of SBFI-AM and BCECF-AM by Lymnaea saline and lidocaine, the fluorescent ratio was measured using the same concentration of SBFI, BCECF, and lidocaine solution, but no interference or fluorescent loss was observed.
To determine how lidocaine changes [Na+]in, more investigations, including the direct stimulation by lidocaine of these exchangers, including the Na+-H+ exchanger or the Na+-K+ exchanger will thus be necessary.
In conclusion, lidocaine increased intracellular sodium concentration through the Na+-H+ exchanger by proton trapping and suggested that [Na+]in and [pH]in change via proton trapping will be one of the factors by which local anesthetics affect cell toxicity.
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Footnotes |
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Supported, in part, by a Grant-in-Aid (No.12770828) for Scientific Research (A) from The Ministry of Education, Science and Technology of Japan.
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REFERENCES |
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