Anesth Analg 2008; 106:1778-1783
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e318172c3a2
ANESTHETIC PHARMACOLOGY
The Local Anesthetic Butamben Inhibits Total and L-Type Barium Currents in PC12 Cells
Laurentius J.A. Rampaart, MD,
Jeroen P. Beekwilder, MSc,
Gertrudis Th.H. van Kempen, BSc,
Rutgeris J. van den Berg, PhD, and
Dirk L. Ypey, PhD
From the Department of Neurophysiology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
Address correspondence to Dirk L. Ypey, PhD, Department of Cardiology (C5-27), Leiden University Medical Center (LUMC), Postbox 9600, 2300 RC Leiden, The Netherlands. Address e-mail to D.L.Ypey{at}LUMC.NL.
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Abstract
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BACKGROUND: Butamben or n-butyl-p-aminobenzoate is a long-acting experimental local anesthetic for the treatment of chronic pain when given as an epidural suspension. We have investigated whether Cav1.2/L-type calcium channels may be a target of this butamben action.
METHODS: The effect of butamben on these channels was studied in undifferentiated rat PC12-cells with the whole-cell patch-clamp technique in voltage-clamp. Ba2+ ions were used as the charge carriers in the calcium channel currents, whereas K+ currents were removed using K+ free solutions.
RESULTS: Butamben 500 µM reversibly suppressed the total whole-cell barium current by 90% ± 3% (n = 15), whereas 10 µM nifedipine suppressed this barium current by 75% ± 7% (n = 6). Preexposure to butamben followed by washout decreased the inhibition by nifidepine to 47% ± 5% (n = 10). These suppressive effects were not due to the measurement procedure and the drug vehicles in the solutions (<0.1% ethanol; n = 6).
CONCLUSIONS: Butamben inhibits the total barium current through expressed calcium channel types in PC12 cells, including Cav1.2/L-type channels. Because Cav1.2 channels may also occur in human nociceptive C fibers, this result allows the possibility that these L-type channels are involved in the analgesic action of butamben.
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Introduction
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Terminal cancer patients often suffer from severe pain because of tissue damage caused by either the primary tumor or metastasis. Palliation can be achieved by opioids or, if this does not adequately alleviate the pain, sometimes by ablating sensory nerves. These treatments may cause severe side effects, among which motor dysfunction is the most prominent. A current experimental approach to chronic pain treatment is the epidural administration of an aqueous suspension of the local anesthetic n-butyl-p-aminobenzoate, also known as butamben.1,2 The suspension of butamben applied to the spinal dura results in long-lasting (median 29 days) relief from pain, without impairing motor function or other sensory functions. How butamben produces this extraordinary effect is still largely unresolved.
The butamben molecule is an aminobenzoate ester-linked to a butyl group. Its structure is similar to that of other ester-linked local anesthetics such as benzocaine and procaine, which block sodium channels involved in impulse generation and transmission in neurons.3,4 The effects of butamben on sodium currents have been studied in small dorsal root-ganglion (DRG) neurons,5 which are believed to include the cell bodies of nociceptive fibers.6 Butamben (100 µM) had a diverse effect on the various types of sodium channels, ranging from nearly completely blocking fast sodium currents to having no effect on slow sodium currents. The inhibition of DRG fast sodium currents resulted in reduced excitability of DRG neurons, which is likely to contribute to the butamben anesthesia. However, the blocking effect of butamben on the fast sodium currents does not seem to be the only mechanism of butamben analgesia.3
Inward current through calcium channels also plays an important role in action potential generation in sensory neurons7 and possibly also in human impulse transmission in C-type nocifibers.8 Two types of calcium channels that are expressed in neonatal mouse DRG neurons, N-type and T-type, were shown to be suppressed by butamben9,10 with a 50% inhibiting concentration of approximately 200 µM, similar to that for inhibition by butamben of the total calcium or barium current through all the calcium channels. These mouse DRG neurons also express L-type calcium channels (subtype Cav1.2), but in a proportion too small (7% ± 6%, n = 7; unpublished observations) to study with our whole-cell current recording technique. In small adult rat DRG neurons, however, voltage gated L-type calcium channels seem to constitute a significant portion of calcium currents.7 Hence, it is interesting to explore the effect of butamben on L-type calcium currents.
Therefore, we addressed the question of whether butamben inhibits the Cav1.2/L-type current component of whole-cell barium currents through calcium channels. To this end, the patch-clamp technique in whole-cell voltage-clamp configuration was applied to undifferentiated PC12 (pheochromocytoma) cells. These are rat adrenal medullar chromaffin tumor cells that express various types of calcium channels, with a relatively strong expression of the cardiac L-type ( 1C; Refs. 11 and 12), denoted as Cav1.2 in modern terminology.4 By making use of nifedipine, a specific L-type calcium channel blocker,4 we show that butamben, besides blocking the total barium current through calcium channels, at least partly blocks the L-type barium current component in PC12 cells. In the discussion, we consider the implications of the present results for the analgesic action of butamben.
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METHODS
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PC12 Cell Culture
PC12 cells from the Hubrecht Laboratory (Utrecht, the Netherlands) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated horse serum, 5% fetal calf serum (both sera from Invitrogen, Breda, the Netherlands), 100 IU/mL penicillin, and 100 µg/mL streptomycin (both from Sigma-Aldrich, Zwijndrecht, the Netherlands). After cells were grown in a poly-l-lysine (MW 70,000–150,000 D, Sigma-Aldrich) coated culture flask for 7 days and had formed a nearly confluent monolayer, they were dissociated with Versene (Invitrogen) and plated on poly-l-lysine coated cover slips. They were then grown in a culture dish in a humidified 5% CO2 incubator at 37°C to obtain undifferentiated PC12 cells.11 Experiments were conducted 4–7 days after plating.
Whole-Cell Recording
The patch-clamp experiments were performed at room temperature (approximately 23°C). A glass coverslip was mounted in a chamber on the stage of an inverted microscope (Zeiss Axiovert 35). Patch pipettes were fabricated from borosilicate glass (Harvard Apparatus, Edenbridge, Kent, UK) and were giga-sealed to the cells in a microbath (75 µL) continuously perfused with a standard extracellular solution (ECS), containing 125 mM NaCl, 5.5 mM KCl, 0.8 mM MgCl2, 2 mM CaCl2, 10 mM HEPES/NaOH (pH 7.3), 21.8 mM glucose, and 36.5 mM sucrose, which is similar to the solution used by Westerink et al.13 for PC12-cells. The pipette was filled with a CsCl containing intracellular solution (CsICS), consisting of 130 mM CsCl, 1 mM CaCl2, 10 mM HEPES/CsOH (pH 7.2), 10 mM EGTA, 5 mM MgATP, and 0.5 mM TrisGTP. Pipette resistance measured in ECS was 4.2 ± 0.1 M (mean ± sem, n = 23). Flattened adhered polygonal cells were preferred over phase-bright spherical cells, because they exhibited larger calcium channel currents.14 Seal resistances were 1.8 ± 0.2 G (n = 18). After establishment of the whole-cell configuration, the microbath was perfused with a solution containing barium (BaECS), consisting of 140 mM NaCl, 5 mM CsCl, 2 mM MgCl2, 10 mM BaCl2, and 10 mM HEPES/NaOH (pH 7.3). The combined use of BaECS and CsICS enhanced the current through calcium channels and fully removed potassium currents (Fig. 1). Occasional (in <5% of the cells) inward sodium-like currents15 were small and so fast (approximately 3-ms duration) that they did not interfere with our measurements of the slower barium currents. Butamben (OPG Farma, Utrecht, the Netherlands) was added to the BaECS in a concentration of 500 µM, from a stock of 500 mM butamben in ethanol. Nifedipine (Sigma-Aldrich) was used to identify currents through L-type channels. It was added to the BaECS in a concentration of 10 µM, from a stock of 10 mM nifedipine in ethanol. This concentration is close to that for maximal and specific inhibition of Ca1.2/L-type channels under our measurement conditions, i.e., at a holding potential of –80 mV.4 Both for the butamben and nifedipine solution the concentration of ethanol never exceeded 0.1%.
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Figure 1. A, Progress in calcium channel current increase upon exchanging the calcium containing bath solution (ECS) for a barium-containing solution. Current records were evoked by voltage steps from –80 to +10 mV with 15-s intervals, of which here every 4th response is shown. The upper record is taken in the calcium containing ECS, just before infusing the barium solution. The lowest record is an early steady-state record in the barium-containing solution. Note also the absence of the fast inactivation time course in the increased barium current. B, Mean I–V relationship gained by applying depolarizing voltages from a holding potential of –80 mV, after which the barium current's maximum was measured, which was then plotted against the voltage at which it was elicited (mean ± sem, n = 8).
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A PC running Clampex 8 (Axon Instruments, Foster City, CA) and a List EPC 7 amplifier provided voltage protocols. The membrane currents were filtered at 3 kHz. The PC12 cell currents were leak subtracted using the P/4 method. The single exponential capacitive transients revealed the absence of electrical coupling between cells, even when they were visibly in contact. The membrane capacitance of the cells derived from these transients was 24.4 ± 3.1 pF (n = 27). The series resistance was 7.2 ± 0.6 M (n = 9) and was not compensated because of the small size of the recorded currents. Data are presented as mean ± sem for n cells. Means are compared using paired or independent t-tests with the level of significance (P) chosen as 0.05.
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RESULTS
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Upon depolarization, the selected PC12 cells exhibited a small inward calcium current in ECS, consisting of currents through various types of calcium channels.12,14,15 To enhance the current flowing through the calcium channels, barium ions were used as charge carriers (instead of calcium ions) by applying a barium solution (BaECS) to the cells. The cell membrane was held at a voltage of –80 mV and was then step-depolarized to +10 mV for 60 ms at regular intervals of 15 s, until the inward barium current reached its maximal increase (within 4 min, including the arrival– delay of BaECS through the perfusion tubing). Figure 1A shows the inward calcium current of a PC12 cell at 10 mV, as well as the gradual increase in the current at that test potential upon infusion of the barium-containing solution into the bath. The calcium current records in ECS showed a variable composition of a faster and slower inactivating current (Fig. 1A). A current–voltage (I–V) relationship of the barium current was created by applying test potentials between –60 and +40 mV to the PC12 cells at 15 s intervals and by measuring the maximal barium current at each test potential (Fig. 1B). From about –40 mV upwards, the barium current increased in amplitude until it reached its maximum at +10 mV (approximately –200 pA), after which it declined and reversed at potentials extrapolated to >40 mV. To establish whether butamben inhibited the barium current, 500 µM butamben was applied to the cells. This concentration of butamben blocked 90% ± 3% of the control barium current (P < 0.05, n = 15) (Figure 2A), and this level was reached within 4 min, including the butamben arrival delay. This inhibitory effect was largely reversible to 76% ± 6% (n = 8) after approximately 5 min washout.
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Figure 2. A, Addition of 500 µM butamben (BAB) resulted in an almost complete inhibition of the barium current. Note also the increased inactivation rate in the washout record. B, Addition of 10 µM nifedipine resulted in an inhibition of a large part of the barium current. Note the difference in time scales in A and B and Figure 1, indicating variability in calcium channel expression between cells.
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Now that the blocking effect of butamben on the total barium current through calcium channels expressed in PC12 cells was established, 10 µM nifedipine was applied to the cells to prove that at least part of the calcium channels in the PC12 cell membranes was of the L-type. Nifedipine 10 µM blocked 75% ± 7% of the control barium current (P < 0.05, n = 6) (Fig. 2B) within about 3 min. This effect was partly reversible to 47% ± 7% (n = 6) of the initial control current, after approximately 5 min (Fig. 2B). Assuming that the nifedipine only affected the L-type current,4 we conclude that at least 75% of the barium currents generated by the PC12 cells is of the L-type and that since 500 µM butamben blocked 90% of that current, L-type calcium channels are at least partly blocked by butamben.
To gain further support for this conclusion, an experiment was done in which the effects of butamben and nifidepine were determined on the same cell. After an initial barium current control period, a PC12 cell was first exposed to 500 µM butamben for 4 min. Butamben was then washed out for 5.5 min and 10 µM nifedipine was added for 4 min. Finally, nifedipine was washed out for 3.5 min to check for reversibility of the nifedipine effect. During the application and washing-out of the drugs, the cells were step-depolarized from a holding potential of –80 mV to 10 mV with 15-s intervals to elicit the barium current and explore the effect of the drugs on the current. Figure 3A shows example records, and the mean results of 10 of these experiments are shown in Figure 3B. Butamben 500 µM caused a 93% ± 2% inhibition of the inward barium current (P < 0.05, n = 10), reproducing the above results. When butamben was washed out, 74% ± 5% of the original current was regained (P < 0.05, n = 10), also corresponding to the above results. When 10 µM nifedipine was then applied to the cells, we found an inhibition of 47% ± 5% (n = 10) of the preceding butamben washout current peak, corresponding to a remaining 38% ± 4% of the original current (P < 0.05, n = 10). This inhibition is smaller than the fresh exposure inhibition of 75% described earlier. The final barium current amplitude after washout of nifedipine was 43% ± 4% of the original current amplitude (P > 0.05 washout versus nifedipine, n = 7), which corresponds to a recovery of approximately 57% of the preceding butamben washout peak current.
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Figure 3. A, The effect of butamben (BAB) and nifedipine on barium currents elicited in one and the same cell by a voltage step to +10 mV from a holding potential of –80 mV. Barium currents are given for the control condition, after addition of 500 µM butamben, after washout of butamben, after addition of 10 µM nifedipine and after washout of nifedipine, respectively. B, Results of 10 experiments in which the cells were exposed to 500 µM butamben as well as to 10 µM nifedipine, according to the protocol in A. Normalized mean peak barium currents ± sem are given for each condition.
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Since the inward barium current did not completely recover after washout of butamben and nifedipine, the possibility remained that part of the inhibition of the barium current seen during and after the administration of butamben and nifedipine was caused by rundown of the barium current. Therefore, we performed a vehicle/rundown control experiment in which the same drug application protocol was used as in the experiment described earlier. After an initial barium wash-in period of approximately 4 min, we exposed the cells, instead of to butamben and nifedipine, to 0.1% vehicle (ethanol), which also allowed us to check whether the inhibiting effects of butamben and nifedipine were not due to ethanol. Figure 4 shows only a slight decline of the normalized peak barium-current amplitude during the course of the experiment. At the end of the experiment (after approximately 18 min), 85% ± 3% (n = 6) of the original barium current remained. The conclusions that can be drawn from this experiment are that the inhibiting effects seen during the administrations of butamben and nifedipine were not caused by the vehicle (0.1% ethanol) and that the rundown of the barium current may only explain a small percentage ( 15%) of the incomplete recovery after butamben and nifedipine exposure.
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Figure 4. The rundown/vehicle control experiment. Peak barium currents were measured upon depolarizing steps to +10 mV from a holding potential of –80 mV, applied with 15-s intervals. Black horizontal bars indicate the addition of vehicle (0.1% ethanol) according to the protocol in Figure 3. Peak currents were normalized by dividing these currents by the peak current at t = 0 s. All experiments were preceded by a 4-min in-wash period of BaECS (not shown). Mean normalized peak currents ± sem are plotted for six cells. Empty spaces in the plot are interruptions for voltage-clamp protocols to determine I–V curves.
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DISCUSSION
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In the present study, we examined the inhibiting effect of butamben on calcium channels, including Cav1.2/L-type channels, in PC12 cells. These channels are expressed in small rat DRG neurons, and therefore may contribute to pain signal transmission.7 We used undifferentiated PC-12 cells rather than DRG-neurons, because L-type calcium channels constitute in these cells a significant part of the expressed calcium channels,11,14 providing about 75% of the peak barium current in our study. We found that the clinically relevant concentration of 500 µM butamben (close to the maximum solubility concentration of approximately 700 µM in butamben suspensions, see chemical 1504 in The Merck Index, 1989) blocked approximately 90% of the total peak barium current that was mediated by the various types of calcium channels (besides L- probably also N- and T-type) expressed in PC12 cells.14,15 This result is consistent with our earlier studies9,10 on sensory neurons, which showed that butamben inhibits the total barium or calcium current of the smaller neonatal mouse DRG neurons and specifically the barium currents through N- and T-type channels by 80%–90%. Total Kv and isolated Kv1.1 currents were also inhibited for approximately 80% by 500 µM butamben.16 The concentration response curves in all these cases had an IC50 around 200 µM (range, 177–238) and a Hill coefficient of approximately 1.5 (range, 1.1–1.8). We expect, therefore, that Cav1.2 currents have a similar concentration response curve for butamben.
In our preparations of sensory neurons, the L-type currents were too small to be of use for the study of the effect of butamben. We proved here that at least part of the L-type calcium channels in PC12 cells was inhibited by butamben, because the inhibition of the total barium current (approximately 90%) was clearly larger than the percentage of L-type current, which was 75% or 47%, depending on the inhibition protocol used. Our results also show that the inhibiting effect of butamben on calcium channels is not cell type or species-dependent.
It is noteworthy that in the longer protocol, in which butamben was first applied to the cells and washed out, and then nifedipine was administered, nifedipine seemed to inhibit a smaller portion of the barium current (47%) than in the protocol in which nifedipine was the first-exposure drug (75%). One reason may be that the different calcium channel subtype components have differences in run-down and/or run-up time courses, changing the proportions of these components of the total barium current at the observed run-down of approximately 15% over 18 min. Another possibility is that washout of the butamben effect was not complete, and that remaining butamben molecules interfere with nifedipine binding. Nevertheless, it was proven that butamben inhibits L-type calcium channels. Through which mechanism butamben reaches this effect is still unknown. One mechanism might be that butamben causes a relative acceleration of deactivation and inactivation kinetics, which would make it more difficult for the channel to open and stay open in the presence of butamben. This would be consistent with a butamben-induced increase in deactivation and inactivation rate of other calcium channels (N- and T-type) and of Kv1.1 channels as observed by Beekwilder et al.9,10,16 In this respect, it is worth mentioning that incompletely recovered barium currents after butamben washout often showed an increased inactivation rate (Figs. 2A and 3A). Further studies directed at butamben's effect on the gating kinetics of the L-type calcium channel could help clarify butamben's mechanism of action.
The mechanism of butamben analgesia depends on butamben's ability to suppress the generation and/or transmission of action potentials in the neurons that transmit pain signals to the brain. Since L-type calcium channels are possibly present in human nociceptive C fibers,8 butamben's blocking effect on this type of calcium channels might contribute to analgesia when administered epidurally. The present results and those of Beekwilder et al.9,10 add butamben to the list of local anesthetics inhibiting calcium channels.17 They also implicate anesthetic actions of butamben on the peripheral autonomous nervous system. Future studies should be directed at the question of how the integrated effects of butamben on the various types of ion channels in DRG neurons result in analgesia.
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ACKNOWLEDGMENTS
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We thank Prof. Dr. A. van der Laarse (Cardiology, LUMC) for providing lab facilities for several final experiments.
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Footnotes
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Accepted for publication February 11, 2008.
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REFERENCES
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- Beekwilder JP, O'Leary ME, van den Broek LP, van Kempen GThH, Ypey DL, van den Berg RJ. Kv1.1 channels of dorsal root ganglion neurons are inhibited by n-butyl-p-aminobenzoate, a promising anesthetic for the treatment of chronic pain. J Pharm Exper Ther 2003;304:531–8[Abstract/Free Full Text]
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Abstract
Introduction
