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INTRODUCTION |
The mechanism underlying spinal and regional anesthesia by local anesthetics generally is explained by a blockade of tetrodotoxin-sensitive (TTXs) Na+ channels. Less clear, however, is the mechanism of differential anesthesia, a clinically observed phenomenon of different onsets of suppressions of various sensory and motor functions (Raymond and Gissen 1987
). Explanations have been suggested as varying access of the drug molecules to their receptors at the ion channels that may be subject to varying caliber and myelination of the nerve fibers. Furthermore, differential blockade of neurons may be due to their equipment with different types of ion channels that are differently sensitive to local anesthetics. Of the two groups of Na+ conductances, sensitive and resistant to TTX, the latter has been observed in dorsal root ganglion cells (Elliott and Elliott 1993
; Kostyuk et al. 1981
; Ogata and Tatebayashi 1993
; Roy and Narahashi 1992
), in C fibers contributing to compound action potentials (Jeftinija 1994
) and in human peripheral nerve (Quasthoff et al. 1995
). In myelinated fibers of peripheral nerve, only TTXs Na+ channels could be studied directly with the patch-clamp technique (Scholz et al. 1993
), but it is not possible to use this method for investigation of thin unmyelinated fibers, which also contain TTX-resistant (TTXr) Na+ channels (Quasthoff et al. 1995
). Both Na+ channel types, however, can be studied at the somata of those sensory fibers. About the blockade of TTXr Na+ currents by local anesthetics, however, little is known so far (Akopian et al. 1996
; Roy and Narahashi 1992
). The local anesthetics lidocaine and bupivacaine, both frequently used for spinal anesthesia, have been investigated in their blocking effects on TTXr Na+ currents. Two preparations were used in our study. Pharmacological and voltage-clamp experiments were performed with enzymatically isolated dorsal root ganglion (DRG) neurons with the advantage of easy solution exchange and proper space-clamp condition. Current-clamp experiments were performed with A
- and C-type neurons visually identified in thin DRG slices (Safronov et al. 1996
), avoiding enzymatic treatment and with the advantage of good physiological condition.
Some of these results have been presented to the German Physiological Society (Scholz et al. 1996
).
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METHODS |
Materials
Young Wistar rats (8-19 days) of both sexes were killed, the vertebral column was removed quickly and opened in a cooled dish (2-4°C) containing the preparation solution. DRGs (10-20) from lower thoracic (>Th8) and lumbar levels were dissected. Ganglia were transferred into a petri dish containing preparation solution with 0.7-1.5 mg/ml trypsin (type I, Sigma) and 4-6 mg/ml collagenase (type II Worthington, Biochrome) and incubated for 25-35 min at 37°C in a shaking closed chamber (110 min
1) with 5% CO2-95% O2. After washing four to five times at room temperature in preparation solution, the softened ganglia were dissociated by gentle titration with a small pipette in a petri dish the bottom of which was covered by silicone elastomer (Sylgard Blue, Dow Corning). The cells were stored for
15 h in preparation solution bubbled with a 95% O2-5% CO2 mixture and humidified atmosphere at 24°C and finally were transferred by a small pipette into the experimental petri dish.
In current-clamp experiments, thin 150-µm slices of DRGs (without enzymatic treatment) from young Wistar rats (6-9 days) have been used as described in more detail by Safronov et al. (1996)
.
Solutions
Preparation solution: mixture of 50% F12 and 50% Dulbecco's modified Eagle's medium (D 8900, Sigma) with 1.8 g/l sodium bicarbonate, bubbled with a 95% O2-5% CO2 mixture, 100 U penicillin, and 0.1 mg/ml streptomycin (P 0781 Sigma), adjusted to pH 7.4 with NaOH.
The Ringer bicarbonate solution contained (slice preparation, in mmol/l) 115 NaCl, 5.6 KCl, 2 CaCl2, 1 MgCl2, 11 glucose, 1 NaH2PO4, and 25 NaHCO3 (pH 7.4 by bubbling with a 95% O2-5% CO2 mixture), final Na 141.
The Ringer-tetraethylammonium (TEA; +TTX) solution contained (in mmol/l) 134 NaCl, 4.5 KCl, 20 TEA, 0.1 CaCl2, 5 MgCl2, and 10 N-[2-hydroxyethyl]-piperazine-N'[2-ethanesulfonic acid] (HEPES), adjusted with NaOH to pH 7.4, final Na 144, 0.0003 TTX (Latoxan).
The Ringer 42 Na solution (+TTX) was as the Ringer-TEA solution except that NaCl was 32 mmol/l and cholineCl (102 mmol/l) was added; final Na 42.
The high-K internal solution contained (in mmol/l) 144.4 KCl, 1 MgCl2, 5 NaCl, 10 HEPES, and 3 ethylene glycol-bis(
-aminoethyl ether)N,N,N',N',-tetraacetic acid (EGTA), adjusted with KOH to pH 7.3, final K 155.
The CsCl internal solution contained (in mmol/l) 105 CsCl, 10 NaCl, 2 MgCl2, 10 EGTA, 4 Na2-adenosine 5'-triphosphate (ATP), and 10 HEPES, adjusted with CsOH to pH 7.2, final Cs 135.
The CsF internal solution contained (in mmol/l) 40 CsF, 67.5 CsCl, 6 NaCl, 2 MgCl2, 10 EGTA, 4 Na2-ATP, and 10 HEPES, adjusted with CsOH to pH 7.2, final Cs 135, final Na 14.
Lidocaine was dissolved and stored as stock solution (1 mol/l) in aqua bidest and bupivacaine (0.1 mol/l) in dimethyl sulfoxide. All reagents were purchased from Sigma.
Application system
Control solution and local anesthetics containing solutions were applied by a modified six-barrel perfusion system. Continuous and equal flows out of the six glass barrels were produced by a syringe pump delivering pressure through a tubing to the six syringes. A complete exchange of solutions was accomplished in a few seconds by moving the tip of the pipette with the neuron from one barrel into another one.
Electrophysiology
Voltage-clamp experiments were carried out on isolated cells using an EPC 7 (List) patch-clamp amplifier. Current-clamp experiments were done using a thin slice preparation with an Axopatch 200A (Axon Instruments) patch-clamp amplifier. Data were digitized with a 12-bit AD/DA converter TL/1 (Scientific Solutions) and filtered at least at two times lower frequency with an eight-pole, low-pass Bessel filter (Physiologisches Institut). Voltage and current steps and data acquisition were controlled on-line by a PC/AT computer with pCLAMP software (Axon Instruments). Electrodes were fabricated from borosilicate glass tubings (GC 150, Clark Electromedical Instruments) using a David Kopf clone two-stage vertical puller (Physiologisches Institut) and fire-polished on a microforge (workshop Physiologisches Institut) to a final resistance of 0.6-2.5 M
when filled with internal solution. Whole cell recordings were performed as described by Hamill et al. (1981)
. Additionally series resistance was compensated (55-80%). Experiments were rejected when the voltage error exceeded 5 mV after compensation of the series resistance. Voltage-dependent currents were corrected off-line for unspecific leak currents and transients using averaged recordings evoked by hyperpolarizing impulses. All values presented are means ± SE.
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RESULTS |
Na+ currents were separated on the basis of their different sensitivities to TTX (Elliott and Elliott 1993
; Kostyuk et al. 1981
). TTXr Na+ currents were recorded in the presence of 300 nmol/l TTX. Na+ currents were considered to be TTX sensitive if they demonstrated fast kinetics and if
95% of them could be blocked by 100 or 200 nmol/l TTX at the end of the experiment. K+ currents were suppressed by internal Cs+ (Ogata and Tatebayashi 1993
) and external TEA (Safronov et al. 1996
). Currents through six different types of Ca2+ channels known so far (Mintz et al. 1992
; Scholz et al. 1997
) were abolished by Mg2+ ions added to internal and external solutions (Hess et al. 1986
) and in some experiments by F
ions in internal solutions (Kostyuk et al. 1977
). A low Ca2+ concentration in the external solutions of 100 µmol/l prevented the Ca2+ channels from becoming Na+ conducting (Carbone and Lux 1988
; Hess et al. 1986
).
In general, with increasing cell diameter, we observed a decreasing proportion of TTXr Na+ channels, although a clear correlation between current proportion and neuron diameter could not be established. In neurons >40 µm, TTXs Na+ currents prevailed, and in some cases current amplitude exceeded 10 nA reaching the limits of the amplifier. Therefore Na+ ions in the external solution were substituted partially by choline+ ions, and Na+ concentration was reduced to 42 mmol/l in some of our experiments. Cells <25 µm mainly displayed TTXr Na+ currents. In ~85% of medium sized cells, there was a mixture of TTXs and TTXr Na+ currents in varying proportions with
80% from one type, but the remaining 15% of these cells showed only one type of Na+ current.
Blockade of peak Na+ currents
Medium- and large-sized cells, 14 of 134 cells in this study, displayed TTXs Na+ currents only that showed typical fast activation with a time to peak of 0.65 ± 0.25 ms (mean ± SE, n = 14) and fast inactivation with a time constant of 0.84 ± 0.32 ms at 0 mV (Fig. 1, C and D). These currents showed a reversal potential close to the calculated equilibrium potential of Na+ ions and were abolished completely at the end of the experiment by 100 or 200 nmol/l TTX and were thus identified as TTXs Na+ currents. A complete exchange of the external solution was accomplished by moving the cell under investigation from one barrel into the next (see METHODS). The local anesthetics lidocaine and bupivacaine reversibly blocked TTXs Na+ currents without modifying kinetics of activation and inactivation significantly as shown in Fig. 1, C and D.

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| FIG. 1.
Tetrodotoxin-sensitive (TTXs) and -resistant (TTXr) Na+ channels blocked by lidocaine and bupivacaine, respectively. Whole cell currents recorded at 0 mV from 4 different dorsal root ganglion (DRG) neurons of rat. A: fits to the decaying parts of TTXr Na+ currents (···) showed a of 3.6 ms in Ringer-TTX solution and 3.2 ms in the presence of 300 µmol/l lidocaine. Time to peak was 1.7 and 1.5 ms, respectively. Pipette resistance: 0.9 M . B: TTXr Na+ current decayed with of 1.3 ms in Ringer-TTX and 1.2 ms in the presence of 50 µmol/l bupivacaine. Time to peak was 0.7 and 0.6 ms, respectively. Pipette resistance: 0.6 M . C: TTXs Na+ currents decayed faster with of 0.4 ms in Ringer solution and 0.5 ms in the presence of 50 µmol/l lidocaine. Time to peak was 0.3 and 0.4 ms, respectively. Currents were blocked completely by 200 nmol/l TTX. Pipette resistance: 0.7 M , pipette solution: CsF, bath solution: Ringer with reduced 42 mmol/l Na+. D: another neuron displayed TTXs Na+ currents decaying with of 0.6 ms and 0.7 ms in control and in the presence of 10 µmol/l bubivacaine. Time to peak was 0.4 and 0.5 ms, respectively. Less than 2% of the current remained in the presence of 200 nmol/l TTX. Pipette resistance: 1.4 M . Pulse protocol is shown above the original recordings, durations of prepulses to 110 mV were 50 ms for TTXs Na+ channels and 100 ms for TTXr Na+ channels. - - -, zero level. Holding potential was 80 mV. Current traces shown are corrected for leakage and capacity currents as described in METHODS. Except mentioned, pipette solution, CsCl; bath solution: Ringer or Ringer-TTX; temperature: 22-24°C.
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In contrast, TTXr Na+ channels activated at more positive potentials (E >
30 mV) with two times slower kinetics (time to peak 1.62 ± 0.64 ms) and inactivated with a time constant of 3.5 ± 1.8 ms at 0 mV, showing wide variations (n = 29). These currents were not impaired by 300 nmol/l TTX. As shown by the recordings in Fig. 1, A and B, lidocaine and bupivacaine reduced the amplitude of the TTXr Na+ current. Here, and in another 23 experiments, inactivation kinetics were not changed significantly, and therefore a slow open channel block could be excluded. Kinetics of wash-in and -out were faster than the manoeuvre of switching from one barrel to another (see METHODS).
Half-maximal inhibitory concentrations (IC50) for tonic blockade were derived from fits to data plotted as relative block of peak Na+-current (INa,peak) versus concentration of local anesthetics (Fig. 2). With an IC50 of 42.3 ± 5.4µmol/l, the TTXs Na+ current was five times more sensitive to lidocaine than the TTXr Na+ current with an IC50 of210 ± 31 µmol/l (Fig. 2A). The more lipophilic bupivacaine demonstrated a higher affinity to the TTXs Na+ channel with an IC50 of 12.5 ± 1.4 µmol/l (Fig. 2B) than to the TTXr Na+ channel with an IC50 of 31.7 ± 5.2 µmol/l. Thus the relation of potencies of bupivacaine and lidocaine were higher for TTXr Na+ channels with 6.6 than for TTXs Na+ channels with 3.4. For action of both local anesthetics on both Na+ channel types, the Hill coefficients were near one, indicating an 1:1 stoichiometry of interaction.

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| FIG. 2.
Concentration-block-curves for lidocaine and bupivacaine. A: relative block was calculated from peak Na+ currents in the presence of lidocaine related to those under control conditions at depolarizations to +10 mV. Data points were fitted by Hill equation with IC50 values of 42.3 ± 5.4 µmol/l for TTXs ( , 6 cells) and 210 ± 31 µmol/l for TTXr Na+ channels ( , 8 cells). Hill coefficients were 1.06 and 1.02, respectively. B: blockade by bupivacaine was obtained as described in A. IC50 values for TTXs (4 cells) and for TTXr Na+ currents (5 cells) were 12.5 ± 1.4 and 31.7 ± 5.2 µmol/l, respectively. The Hill coefficients were 1.05 and 0.96, respectively. Frequency of stimulation was 0.33 Hz for TTXs and 0.25 Hz for TTXr Na+ currents. Error bars give SE.
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Blockade of TTXr Na+ channels in inactivated and resting state by lidocaine
It has been shown that TTXs Na+ channels in the inactivated state are more sensitive to lidocaine than in the resting state (Hille 1977
; Ragsdale et al. 1994
). Because this channel characteristic has some impact on its physiological function concerning conducting capacity, we have tested whether different states of TTXr Na+ channels are also differently sensitive to this local anesthetic.
Data for drug binding in the resting state were taken from experiments in Fig. 2 that had been obtained without inactivating prepulse. The protocol for investigation of channel block in the inactivated state shown in Fig. 3 consisted of a 2.5-s prepulse to
35 mV (Epre), a 5-ms hyperpolarizing step to
100 mV (Ehyp), and a 30-ms test pulse to +10 mV (Etest). Epre inactivated most of the channels but caused little activation (Elliott and Elliott 1993
) and allowed channels to reach an equilibrium for binding of local anesthetics to the inactivated state. Ehyp allowed recovery from inactivation, but it was chosen to be short to prevent drug dissociation from most of the blocked channels. The following test pulse activated unblocked channels having recovered from inactivation. The protocol was applied every 10 s. In control solution, INa,peak evoked by the pulse protocol for the inactivated state was 50-60% compared with the current evoked by the pulse protocol for the resting state. This reduction was due to slow inactivation during the long impulse from which channels did not recover during the short pulse to
100 mV (Chandler and Meves 1970
).

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| FIG. 3.
TTXr Na+ channels are more sensitive to lidocaine in the inactivated than in the resting state. Sensitivity of TTXr Na+ channels in the inactivated state was investigated using a 2.5 s prepulse to 35 mV followed by a short gap of 5 ms to 100 mV and a subsequent test impulse to +10 mV. Concentration-block data and curve fit were obtained as in Fig. 2. IC50 values were 59.5 ± 11.7 µmol/l lidocaine for inactivated TTXr Na+ channels ( , 4 cells). Corresponding data for the blockade in the resting state are taken from Fig. 2A and are given as - - -. Bath solution: Ringer-TTX; pipette solution: CsF.
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Thus INa,peak of TTXr channels obtained in control solution and in different concentrations of lidocaine were plotted as relative block in Fig. 3 revealing an IC50 of 59.5 ± 11.7 µmol/l, 3.6 times lower than the blockade of channels in the resting state with 210 µmol/l.
Modulation of recovery from inactivation
It was shown in preceding sections that the time to peak of Na+ current and the time constant of inactivation reflecting the transitions from closed to open and from open to inactivated states remained unaffected in the presence of local anesthetics. As the next step in investigating the effects of local anesthetics on gating of TTXr Na+ channels, we studied the modulation of transition of the channel from the inactivated to the resting state, i.e., on its recovery from inactivation. In these experiments, DRG neurons were stimulated with a double-pulse protocol at
80 mV with intervals varying between 1 ms and 2 s (Fig. 4). The recovery could be fitted adequately with two exponential functions, a fast one with
1 = 2.1 ms comprising 75% of the amplitude, and a slow one with
2 = 514 ms (25%). In the presence of 10 µmol/l bupivacaine, the fast component
1 was slowed to 5.4 ms, whereas the slow component was nearly as well unaffected as the relative amount of the components was nearly unchanged. Thus a major portion of current recovered slower from inactivation already at a concentration of bupivacaine as low as 10 µmol/l (IC50 = 32 µmol/l); this was seen in five experiments. This could limit conduction abilities of a neuron at high firing frequencies as the following experiments will show.

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| FIG. 4.
Modulation of time of recovery from inactivation by bupivacaine. Time constants for recovery from inactivation of TTXr Na+ channels were measured with a double pulse protocol. A first pulse (P1) for 50 ms to +10 mV caused complete inactivation, and INa,P2 evoked by the test pulse (P2) to +10 mV after variable intervals was compared with INa,P1 of the same episode. Resulting data points could be fitted with a double exponential function giving a major fast component with 1 of 2.1 ms (75%) and a slow component with 2 of 514 ms (25%) under control condition ( ). In the presence of 10 µmol/l bupivacaine ( ), the kinetics of 1 was slowed to 5.4 ms (71%), whereas 2 was unaffected with 568 ms (29%). Inset: data graphed of the fast component. Pulse protocol was repeated with 0.1 Hz at a holding potential of 80 mV. Bath solution: Ringer TTX; pipette solution: CsCl; temperature: 22°C.
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Frequency-dependent blockade by bupivacaine and lidocaine
To test whether the TTXr Na+ channel showed a use-dependent (phasic) block as did the TTXs Na+ channel (Hille 1977
; Ragsdale et al. 1994
; Roy and Narahashi 1992
), repetitive impulses to +10 mV were applied at different frequencies. The amplitude of currents evoked by the nth impulse was normalized to that of the current evoked by the first impulse. In contrast to TTXs Na+ channels (Hille 1977
; Schmidtmayer and Ulbricht 1980
), TTXr Na+ channels already in control solution showed a reduction in amplitude that depended on frequency of stimulation (Figs. 5A and 6A,
). This reduction could be explained by the slow recovery of TTXr Na+ channels from inactivation (
2 = 514 ms, see Fig. 4). The effect became stronger with increasing frequency of stimulation from 0.4 to 5 and 20 Hz. In the presence of 10 µmol/l bupivacaine, an additional reduction of the current due to the development of the use-dependent block could be observed with faster kinetics. The use-dependent block was measured as a ratio between the normalized amplitudes of currents evoked by the 20th impulses in the presence and absence of bupivacaine. From these data, the availability of TTXr Na+ currents as a function of stimulation frequency is shown in Fig. 5B. It was calculated by normalizing the current of the 20th pulse (INa,P20) to that of the first pulse (INa,P1). Connecting lines were drawn by eye revealing half-maximal availabilities of TTXr Na+ current at frequencies of 4.9 and 2.1 Hz in control and in the presence of 10 µmol/l bupivacaine, respectively. The effect of 100 µmol/l lidocaine on the relative reduction of peak current amplitude is shown in Fig. 6A. Again, kinetics of reduction of the relative amplitude was faster in presence of the local anesthetic than in control. The more pronounced use-dependent effect of lidocaine also could be seen in Fig. 6B where half-maximal availability of TTXr Na+ currents is shifted from 7.7 Hz in control to 2.2 Hz in the presence of 100 µmol/l lidocaine.

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| FIG. 5.
Use-dependent block of TTXr Na+ channels by bupivacaine. A: to demonstrate a use-dependent block of TTXr Na+ channels, a pulse protocol with repetitive impulses to +10 mV (10 ms long) was used at different stimulating frequencies. Amplitudes of the currents (after correction for leakage and capacity currents) were normalized to the current amplitude of the first impulse under control condition ( ) and in the presence of 10 µmol/l bupivacaine ( ). In this plot, the tonic component of the block by the local anesthetic has been eliminated by normalization and only the use-dependent component is demonstrated. B: relative amplitudes of TTXr Na+ current under steady-state condition at 20th impulse of experiment in A (*) were plotted as depending on frequency of stimulation. Data points were fitted by eye. In the presence of 10 µmol/l bupivacaine, the curve was shifted from 4.9 Hz under control conditions to 2.1 Hz measured at half-maximal inhibition. C: relative phasic blockade of TTXr Na+ currents by 10 µmol/l bupivacaine depending on frequency. Additional relative phasic block was measured at the 20th impulse as relative reduction of the amplitudes of TTXr Na+ currents in A (*). Bath solution: Ringer TTX; pipette solution: CsF; temperature: 24°C.
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| FIG. 6.
Use-dependent block of TTXr Na+ channels by lidocaine. Same type of experiment as in Fig. 5 at another neuron except Ringer-TTX solution containing 100 µmol/l lidocaine. A: relative peak current amplitude at stimulating frequencies of 0.4, 5, and 20 Hz in control condition ( ) and in the presence of 100 µmol/l lidocaine ( ). B: relative peak current amplitudes of TTXr Na+ current from the 20th impulse marked in A (*) were plotted as depending on frequency of stimulation. In the presence of 100 µmol/l lidocaine, the curve was shifted from 7.7 Hz under control conditions to 2.2 Hz measured at half-maximal inhibition. C: relative phasic blockade of TTXr Na+ currents by 100 µmol/l lidocaine at the 20th impulse (* in A) depending on frequency. Bath solution: Ringer TTX; pipette solution: CsCl; pipette resistance: 1 M ; temperature: 23°C.
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Thus the frequency dependent decay of the TTXr Na+ current functions like a filter. A fairly low concentration of a local anesthetic shifts this decay to a lower frequency value. The relative phasic blockade of TTXr Na+ currents by 10 µmol/l bupivacaine depending on frequency is shown in Fig. 5C and revealed the increasing phasic block from 8.6% at 0.2 Hz to a considerable amount of 52.6% at 20 Hz. In presence of 100 µmol/l lidocaine, the phasic block ranged from 5.6% at 0.4 Hz to 81.2% at 20 Hz (Fig. 6C). It adds to the tonic blockade of the channels (shown earlier, Figs. 2 and 3).
Modulation of firing behavior in a slice preparation
The effect of local anesthetics on action potentials (AP) was tested on neurons in a thin slice preparation. This preparation provided access to small cells identified as A
-and C-type neurons (Safronov et al. 1996
) based on soma diameter and duration of AP (Harper and Lawson 1985b
). The resting potentials were below
60 mV and the input resistances were as high as 0.7-3 G
. These neurons mostly contained TTXr Na+ channels. Figure 7A demonstrates the blocking effects of 30 and 300 µmol/l bupivacaine on trains of action potentials elicited by injection of a sustained depolarizing current. In control the neuron responded with a train of action potentials showing some adaptation within the first 500 ms. Firing frequency at the beginning of the train calculated from the time interval between the first two action potentials was 35 Hz. The peak potential of the first AP was +49 mV and decayed slightly to +44 mV at the end of the train.

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| FIG. 7.
A: blockade by bupivacaine of action potentials of an A - or C-type neuron in a slice preparation. Resting potentials was between 61 and 64 mV and was not influenced by application of the local anesthetic. Cell diameter: 23 µm. B: blocking effects by lidocaine of action potentials of another neuron in a slice preparation. Cell diameter: 21 µm. Protocol of current injected under current clamp is given on the top row for both neurons. C and D: part of traces indicated by boxes and symbols in A and B of action potential recordings in control and in presence of 30 µmol/l local anesthetics are shown superimposed on an expanded time scale. Symbols are corresponding to the registrations in A and B. Bath solution: Ringer bicarbonate with TTX; pipette solution: high-K; temperature: 23°C.
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After changing to an external solution containing additional 30 µmol/l bupivacaine, the same stimulus elicited only two action potentials showing a decrease of the peak potentials from +41 to +31 mV and a reduced calculated firing frequency of 22 Hz, which can be seen in detail in Fig. 7C. In the presence of 300 µmol/l bupivacaine, the neuron did not respond with action potentials any more and was unexcitable also with stronger stimuli. After wash out of the local anesthetic the blocking effects were fairly reversible showing a train of six action potentials, a peak potential of +51 mV and a calculated firing frequency of 28 Hz at the beginning.
The blocking effects of 30 and 300 µmol/l lidocaine on trains of action potentials of another neuron are shown in Fig. 7B. In control the calculated firing frequency at the beginning of the train was 35 Hz. The peak potential of the first AP was +46 mV and decayed to +38 mV at the end of the train. In presence of 30 µmol/l lidocaine, the peak potential was unchanged and the calculated firing frequency at the beginning was reduced to 21 Hz. The shape of the first AP was nearly unchanged compared with control, which can be seen on the superimposed recordings in Fig. 7D. The number of APs during the train was reduced from 12 to 6. In contrast to the result with 300 µmol/l bupivacaine, in the presence of 300 µmol/l lidocaine, a single AP could be elicited with a peak potential of +29 mV. At a concentration of 1 mmol/l lidocaine, it was impossible to elicit an AP (data not shown). After wash out of lidocaine a train of nine APs reappeared with a peak potential of +49 mV and a calculated firing frequency of 38 Hz at the beginning. In four other neurons displaying trains of APs, there was a similar reduction observed in calculated firing frequency and number of APs in presence of lidocaine.
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DISCUSSION |
It is shown that native TTXr and TTXs Na+ channels in rat DRG neurons are blocked by lidocaine and more potently by bupivacaine, both frequently used for spinal anesthesia. The distribution of TTXr and TTXs Na+ currents was found to correlate with the diameter of the neurons although there was no close correlation especially for medium-sized neurons. The expression of TTXr Na+ currents in ~60% of all investigated neurons, a great number of medium-sized cells expressing
80% and a smaller population of small diameter neurons expressing mainly TTXr Na+ currents were found in older animals (15-19 days). Roy and Narahashi (1992)
observed only <10% neurons expressing TTXr Na+ current in rats 12 days of age.
The sensitivities of tonic blockade of the TTXs compared with the TTXr Na+ channels were higher by factors of 5 for lidocaine and 2.5 for bupivacaine (Fig. 2). Our IC50 value of 42 µmol/l for lidocaine is somewhat lower than reported for TTXs Na+ currents of amphibian node of Ranvier (Hille 1977
; Schmidtmayer and Ulbricht 1980
) but comparable to the result of Roy and Narahashi (1992)
. For a cloned TTXr Na+ channel, the sensitivity to lidocaine was six times lower (Akopian et al. 1996
) than the one found in this study. The difference might be explained by different
subunits present at the Na+ channel protein or by follicular tissues in Xenopus oocytes, which reduce drug effects on ion channels (Madeja et al. 1997
). In general, the IC50 values found here for both types of Na+ channels were below the concentrations used in whole nerve preparations to block the conduction (Gissen et al. 1980
; Wildsmith et al. 1989
). This can be due to diffusion barriers existing in nerve and in surrounding tissues (Raymond and Gissen 1987
). Another reason could be the safety factor still allowing to elicit action potentials with a reduced number of Na+ channels. Concentrations of bupivacaine measured in cerebrospinal fluid during spinal anesthesia in man were between 50 and 300 µmol/l (Dennhardt and Konder 1983
). Under our conditions these concentrations blocked 60-95% of the TTXr Na+ currents (Fig. 2) and were effective enough to suppress generation of TTXr action potentials (Fig. 7).
During local anesthesia of peripheral nerves, it is general experience that fast conducted sensory fibers are blocked at lower concentrations, and other slow conducting sensory fibers, especially pain transmission, are suppressed at higher concentrations (Gissen et al. 1980
; Wildsmith et al. 1989
). An obvious explanation could be the presence of TTXr Na+ channels underlying TTXr action potentials in small fibers of spinal and peripheral nerve (Jeftinija 1994
; Quasthoff et al. 1995
). These channels activating more slowly and at a higher threshold may be another reason for slower conduction velocities in these fibers
besides small diameter. Thus TTXr Na+ channels exist in both somata of small DRG neurons and axons of small peripheral nerve fibers. Interestingly, they both are parts of A
- and C-type DRG neurons (Harper and Lawson 1985a
,b
). The lower sensitivity of TTXr Na+ channels to lidocaine and bupivacaine compared with TTXs Na+ channels gives an additional explanation of why at higher concentrations of both local anesthetics only C fibers of the nerve were conducting (Gissen et al. 1980
). These authors showed that the difference in sensitivities was two times more pronounced with lidocaine than with bupivacaine; this is in good agreement with our data.
The higher sensitivity of TTXr Na+ channels to lidocaine in the inactivated than in the resting state (Fig. 3) is probably due to the same mechanism as already published for TTXs Na+ channels. A molecular model by Hille (1977)
suggested two different access routes to the binding sites, and a molecular counterpart was shown by Ragsdale et al. (1994)
. In cloned TTXs Na+ channels, they demonstrated two different binding regions for local anesthetics, one of them causing a use-dependent blockade. The higher affinity of lidocaine to the inactivated than to the resting state of the TTXr Na+ channel may in reality be much higher than the one observed in our study
factor 4 (Fig. 3)
because measurement of the real affinity of a local anesthetic to the inactivated channel required a compromise: theoretically, the portion of inactivated Na+ channels not blocked by the local anesthetic should be measured with a test pulse at the end of a long inactivating prepulse. However, a short period for recovery from inactivation at a negative potential has to precede the test pulse to obtain a measurable amount of Na+ current. With increasing duration of the period preceding the test pulse, an increasing fraction of previously blocked channels will be unblocked, leading to an underestimation of the sensitivity of the channel. Practically, we applied a prepulse of 5 ms assuming that the time constant of dissociation of the drug from its receptor would be longer as described for TTXs Na+ channels (Schmidtmayer and Ulbricht 1980
; Ulbricht 1981
). For cloned TTXs Na+ channels blocked by etidocaine, this factor of sensitivities of inactivated to resting channels was ~100 (Ragsdale et al. 1994
).
Our observation of a slowed fast time constant of recovery (Fig. 4) differs from the reported lidocaine-induced slowly recovering fraction (
, ~1-2 s) and additionally from the unchanged time constant of the normally recovering fraction of Na+ channels (Bean at al. 1983). Therefore our finding might be explained by local anesthetic molecules binding to inner parts of the channels. This local anesthetic molecule may impede the recovery from inactivation because the inactivation gate cannot be released until the blocking molecule has dissociated from the receptor. The time needed for this process is reflected by an increase in the fast time constant by a factor of three. Almost the complete fraction of the fast time constant is affected already at a low concentration of bupivacaine. This lends further support to the argument of higher sensitivity of the inactivated state of the channel to local anesthetics.
A low concentration of local anesthetics blocked trains of action potentials at higher frequency in C fibers better than at lower frequencies (Wildsmith et al. 1989
). This observation can be explained by a use-dependent blockade as seen in our experiments with TTXr Na+ currents (Figs. 5 and 6). TTXr Na+ currents even without local anesthetics (Figs. 5A and 6A) at frequencies of 0.4 and more clearly at 5 and 20 Hz showed a reduction of peak amplitudes. It may be due to the slow recovery from inactivation (
2 = 514 ms; Fig. 4). Roy and Narahashi (1992)
reported a lower reduction of amplitude at all frequencies, the remaining relative amplitude of TTXr Na+ currents in the presence of lidocaine was up to three times larger. A possible explanation of the observed differences might be a heterogeneity in kinetics of TTXr Na+ channels (Elliott and Elliott 1993
; Ogata and Tatebayashi 1993
).
The faster kinetics of reduction of the relative peak amplitudes in the presence of local anesthetic (Figs. 5A and 6A) could be explained by adding up the two mechanisms in control and the use-dependent effect of the local anesthetic on the channel.
The shift in E50 for the frequency-dependent reduction of TTXr Na+ currents in the presence of local anesthetics (Figs. 5B and 6B) may work like a filter for higher frequencies. It may explain why at the beginning of spinal anesthesia only a reduced sensory input but no complete insensitivity is reported. This behavior becomes also evident in our observations with trains of action potentials elicited under current-clamp conditions (Fig. 7) showing this decrease of frequency and reduction of number and amplitude of action potentials even at a low concentration of bupivacaine. Single action potentials are blocked on the basis of a resting block, whereas during trains of APs, some additional blockade may develop due to use-dependent blocking mechanisms of TTXr Na+ channels.
This effects also may explain an increase of the threshold for sensory input. The clinical observation of an incomplete anesthesia named Wedensky block may be explained by such a reduction of frequency leading to an increase of the threshold for sensory input.
We suggest that the blockade of TTXr Na channels by local anesthetics has to be considered more seriously as a mechanism of pain suppression in addition to blockade of TTXs Na channels. These mechanisms described above may give an additional explanation to the well known differential block observed during spinal anesthesia for different classes of neurons differently equipped with TTXr and TTXs Na+ channels.
Future experiments will have to show whether other types of ion channels are involved in conduction block by local anesthetics.