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Anesth Analg 2000;90:1034-1038
© 2000 International Anesthesia Research Society

PEDIATRIC ANESTHESIA

The Effects of Ropivacaine on Sodium Currents in Dorsal Horn Neurons of Neonatal Rats

Bao-Gang Liu, PhD, Xin-Liang Zhuang, MD, Shi-Tong Li, MD, and Guo-Hui Xu, MD

Department of Anesthesiology, Shanghai First People‘s Hospital, Shanghai Medical University, Shanghai, China


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We used a whole cell patch clamp technique to study the effects of ropivacaine on rat dorsal horn neurons. Under voltage clamp, ropivacaine (10–400 µM) produced a dose-dependent inhibition of sodium current. From a holding potential (Vh) of -80 mV, sodium currents evoked by test pulses to 0 mV were inhibited by ropivacaine with a mean drug concentration required to produce 50% current inhibition (IC50) value of 117.3 µM, which was more than the value of the bupivacaine (IC50 53.7 µM). The inhibition effect of ropivacaine was also voltage-dependent. Current evoked from a Vh of -60 mV was inhibited by ropivacaine with a mean IC50 value of 74.3 µM, which was less than that obtained at the Vh of -80 mV. The inhibition effect of ropivacaine on sodium current was use dependent. Repeated activation by a train of depolarizing pulses (5 Hz, 20 ms) increased the inhibitory effects of ropivacaine. The ratio amplitudes of the 20th to the first pulse were 91.2% and 71.1%, respectively, in the absence and presence of ropivacaine (50 µM). Ropivacaine also produced a significant hyperpolarizing shift of 11 mV in the steady-state inactivation curve of sodium current. The inhibition of ropivacaine on the sodium channel may contribute to the mechanism of action of local anesthetics during epidural and spinal anesthesia.

Implications: By using the whole-cell patch technique, ropivacaine is a voltage- and use-dependent inhibitor of the sodium current in dorsal horn neurons; it preferentially acts on steady-state inactivation by the sodium channel. The inhibition of ropivacaine on the sodium channel may contribute to the mechanism of action of local anesthetics during epidural and spinal anesthesia.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The relative blocking potency of ropivacaine and bupivacaine is controversial. It has been shown that the sensory block produced by ropivacaine is similar to that produced by equal concentrations of bupivacaine (1). However, epidural ropivacaine has been found to be less potent than epidural bupivacaine when the two drugs are administered at comparable concentrations (2).

During epidural and spinal anesthesia, local anesthetics act on not only mixed peripheral nerves in the paravertebral spaces and the dorsal root ganglion, but also the spinal cord. Spinal dorsal horn neurons are very important in the transduction of sensory information. The effects of lidocaine and bupivacaine on three sites have been investigated. Several reports (3) described the effect of ropivacaine in the peripheral fibers. The effect of ropivacaine on the spinal dorsal horn neurons has not been determined.

Because local anesthetics block action potential and impulse propagation by interfering with the function of sodium channels in neurons (4), we report the effects of ropivacaine on the voltage-gated sodium channel in acutely dissociated rat dorsal horn neurons for the following purposes: 1) to describe the electrophysiological character of sodium current affected by ropivacaine in the dorsal horn neurons, 2) to further understand the mechanism of local anesthetics during epidural and spinal anesthesia, and 3) to directly compare the blocking potency of ropivacaine and bupivacaine on the sodium current.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
With approval of our animal care committee, Sprague-Dawley rats (2–9 days old) were killed by decapitation. The spinal cord was cut off and incubated in a well-oxygenated (95% O2 and 5% CO2) ice-cold preparation solution in mM (NaCl 124, KCl 5, KH2PO4 1.2, MgSO4 1.3, CaCl2 2.4, NaCO3 26, and glucose 10). After removal of the pial membrane with fine forceps from the lumbar enlargement of the spinal cord, transverse slices (approximately 500 µm) were cut as described (5,6) and incubated in a solution containing pronase (0.25–0.5 mg/mL) at 31°C for 7–20 min and then, containing thermolysin (0.25–0.5 mg/mL) at 31°C for 7–20 min. The slices were nest soaked in a HEPES-buffered solution in mM (NaCl 150, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, and glucose 10). The slices were mechanically dissociated under a stereomicroscope by using a series of fire-polished glass pipettes having a variety of orifice sizes. After the isolated cells adhered to the bottom of a culture dish, a constant superfusion was begun with the oxygenated HEPES-buffered solution fed by gravity (1–2 mL/min).

Patch pipettes were fabricated from 1.5 mm outside diameter pipettes by using a two-stage pipette puller and were polished to final tip resistance of 2–4 m{Omega}. High resistance seals (1–10 G{Omega}) between pipette and neuronal cell membranes were achieved by gentle suction. The whole cell configuration was attained by further suction. Voltage command protocols were generated and sodium currents recorded via a digidata 1200 analog/digital interface controlled by a computer using Clampex 7.0 software (Axon Instruments, Foster City, CA). The capacitance transients and series resistance errors were modified by the amplifier circuitry (Axopatch 200B; Axon Instruments). The series resistance and the whole cell capacitance were taken for the reading of the amplifier. Although it did not change the same in different neurons, the average uncompensated series resistance was 11.2 ± 5.6 M{Omega} and capacitance was 15.3 ± 6.2 pF. Capacitance transients and series resistance errors were modified (75% to 85%) by using the amplifier circuitry. The linear leakage currents were subtracted by using an on-line 'p-5' procedure.

Drugs were applied by using a rapid perfusion system described as the "Y-tube" method (7). The extracellular solution for the sodium channel contained (in mM) NaCl 95, KCl 5.6, CaCl2 0.1, MgCl2 5, glucose 11, NaH2PO4 1, NaHCO3 25, and tetraethylammonium 20 (PH = 7.4 when bubbled with 95% O2, 5% CO2). The pipette solution for sodium current recording contained (in mM) NaCl 5.8, CsCl 134, MgCl2 1, EGTA 3, and HEPES 10, (PH = 7.2 adjusted with 9.2 mM NaOH).

Data were analyzed by using Clampfit 6.0 and Origin 5.0 (Microcal Software). For construction of the concentration-response curve, the peak sodium current was normalized relative to the maximal value. The data were fitted by using a nonlinear least-squares method with the equation f(c) = 1 - C/(C + IC50), in which C is the drug concentration, and IC50 is the drug concentration required to produce 50% current inhibition. For the construction of activation and inactivation curves, the normalized sodium current was plotted against the test pulse potentials for activation and the conditioning pulse potentials for the inactivation. They were fitted to a Boltzmann function according to the equation: I/Imax = 1/[1 + exp(V1/2 - V)/k], in which V1/2 is the membrane potential at which half-maximal channel is activated or inactivated and k is the slope factor.

Data values were expressed as mean ± SD. Statistical comparison was performed by paired and unpaired t-tests. P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
We used several criteria to distinguish between dorsal horn neurons and glial cells. Neurons can generate short action potentials in current clamp mode, the glial cells do not. The sodium current ranged from 0.5 to 4 nA for neurons and 0.01 to 0.3 nA for glial cells (8). Furthermore, the neurons were sensitive to N-methyl-D-aspartate, the glial cell is not (9). In our experiments, the resting potentials were between -60 mV and -70 mV. Neurons were distinguished from glial cells according to the morphology (7) and the amplitude of sodium currents. Only cells in which the amplitude of sodium current exceeded 1 nA were considered to be neurons and useful for experiments. The mean diameter of neurons for experiments was approximately 10–15 µm.

Ropivacaine and bupivacaine both reduced the amplitude of the peak sodium currents in a concentration-dependent manner (Fig. 1). From a holding potential (Vh) of -80 mV, the mean IC50 value of ropivacaine was 117.3 µM, which is significantly larger than the IC50 value of bupivacaine (53.7 µM). The block of sodium current by ropivacaine is incomplete at maximal doses and little ropivacaine-insensitive sodium current remained. The inhibitory effect of ropivacaine was voltage dependent. The IC50 value (mean 74.3 µM), when the sodium current was evoked from a Vh of -60 mV, was lower than that from a Vh of -80 mV.



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  		Figure 1. Voltage- and concentration-dependent inhibition of sodium currents by ropivacaine and bupivacaine in rat dorsal horn neurons. Neurons were voltage-clamped at either -80 mV or -60 mV and stepped to the test potential 0 mV. Currents shown in the absence and presence of increasing concentrations of ropivacaine under holding potential (Vh). A, Vh -80 mV. B, Under Vh -60 mV. C, Effects of bupivacaine on the sodium currents under the Vh -80 mV. D, Concentration-response curves for the inhibition. The abscissa scale represents the micromolar concentration of local anesthetics, the ordinate scale shows the peak current in the presence of the drugs expressed as a percentage of the current obtained in the absence of drug. Data are plotted according to the equation f(c) = 1 - C/(C + IC50) and yielded drug concentration required to produce 50% current inhibition (IC50) values of 117.3 µM for ropivacaine at Vh -80 mV ({blacksquare}), 74.3 µM at Vh -60 mV (•) and 53.7 µM for bupivacaine at Vh -80 mV ({blacktriangleup}). Each point represents mean value and vertical lines represent the SE mean (n = 5)

 
The use-dependent action of ropivacaine was evaluated with trains of repeated pulses (Vh -80 mV, Vtest 0 mV, 5 Hz, 20 ms duration). The ratio of the 20th to the first pulse in the presence of 50 µm ropivacaine was 71.1%, which was significantly lower than that in the control (91.2%, P < 0.05, Fig 2).



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  		Figure 2. Use-dependence of sodium current by ropivacaine in rat dorsal horn neurons. Sodium currents were recorded by trains of depolarizing pulses (Vh -80 mV, Vtest 0 mV, 5 Hz, duration 20 ms) in the absence and the presence of 50 µM ropivacaine. The current amplitudes, which are normalized with respect to the first pulse, are plotted against the pulse number. The ratios of the amplitudes of the 20th to the first pulse were 91.2% and 71.1% in the absence ({blacktriangleup}) and presence ({diamondsuit}) of 50 µM ropivacaine. The greater inhibition of ropivacaine was on repeated current activation (use-dependent). Vh = holding potential.

 
Current:voltage (I:V) relationships were also evaluated in the absence and presence of ropivacaine by using a series of depolarizing pulses from a Vh of -80 mV. Although the I:V curve in our results indicated insufficient space clamp of the neuronal membrane, as in another report (8), the peak current amplitude of sodium current was significantly reduced 72.8% by 50 µM ropivacaine (n = 4). There were no significant changes in either the shape of I:V curve or the pulse potentials at which the peak current was observed in the absence and presence of ropivacaine (50 µM) (Fig. 3). Ropivacaine did not significantly shift the voltage range across which channel activation occurred. The mean half-maximal voltage according to the Boltzmann equation is -59.8 mV and -58.9 mV in the absence and presence of ropivacaine (50 µm), respectively (n = 4). The voltage dependence of steady-state inactivation was also estimated by using two pulse protocols from a Vh of -80 mV. The conditioning pulses of 20 ms to varying potentials between -80 mV and 0 mV were delivered, after a test pulse to 0 mV. By using this protocol, according to the Boltzmann equation, the V1/2 and slope value K were -49.9 mV ± 1.4 and 6.0 ± 1.3 in the control and -60.0 mV ± 1.8 and 7.5 ± 1.4 in the presence of ropivacaine (50 µM). There is significant leftward displacement of the normalized inactivation curve for ropivacaine (n = 4, P < 0.05, Student’s paired t-test, Fig 4).



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  		Figure 3. Effects of ropivacaine on sodium current/voltage (I:V) relationship. The sodium currents were recorded by depolarizing pulses from the Vh of -80 mV to different test potentials in A, the absence and B, the presence of 50 µM ropivacaine. C, Peak currents evoked at each potential are plotted. There was no change in the shape of the I:V curve in the absence and presence of ropivacaine. Vh = holding potential.

 


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  		Figure 4. The effect of ropivacaine on the steady-state inactivation of sodium current. This experiment was conducted from a holding volume (Vh) of -80 mV. A, Currents were recorded by using 20 ms conditioning pulses to different potentials followed immediately by the test pulse to 0 mV. B, The steady-state inactivation curves. The lines of best fit represent Boltmann functions with V1/2 values and the slope parameters are -49.9 mV and 6.0 in the control (•), and -60.0 mV and 7.5 in the presence of 50 µM ropivacaine ({blacksquare}). Points represent the mean values and vertical lines designate the SEM (n = 4). V1/2 = the membrane potential at which half-maximal channel is activated or inactivated.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Local anesthetics block impulses by interfering with the function of the sodium channels (10). The effect of ropivacaine on the sodium channels in dorsal horn neurons has not been reported. Our study provides this information.

Ropivacaine inhibited sodium current in a concentration-dependent manner. In the presence of ropivacaine, the sodium current is decreased, and at a sufficiently large anesthetic concentration, enough sodium channels were impaired and fewer currents were produced. This shows that ropivacaine has the same tonic block on the sodium channel as other local anesthetics (11,12). When comparing the inhibitory potency of ropivacaine and bupivacaine under the Vh of -80 mV, the IC50 of ropivacaine for this tonic channel block is approximately 117.3 µM, which is significantly larger than the value of bupivacaine. This phenomenon in our experiments is at conflict with other reports which found no significant differences in the sensory block between ropivacaine and bupivacaine at the same concentration during epidural anesthesia (13,14). We do not know the reason for these differences. The most likely explanation for our results is that local anesthetic molecules had to diffuse through the neuronal membrane before they reached the binding site on the sodium channel. The lipophilicity of bupivacaine is larger than the value of ropivacaine, so bupivacaine has a faster rate of binding to the sodium channel and is more concentrated in the membrane of neurons and more potent for tonic block than ropivacaine.

The inhibitory effect of ropivacaine on the sodium current in rat dorsal horn neurons is also voltage-dependent. Under the holding potential of -80 mV, there was no steady-state sodium channel inactivation. Thus, the inhibitory effect of ropivacaine reflects ropivacaine’s affinity for the resting sodium channel state. The IC50 value is 117.3 µM, which is more than the value of Vh -60 mV (74.3 µM). The inhibitory effects of ropivacaine were enhanced when the membrane potential was held at relatively positive potentials. This means that ropivacaine inhibits the sodium current in a state-dependent manner. The most likely explanation for this phenomenon is that ropivacaine preferentially interacts with the sodium channels inactivation state. Under depolarized conditions, enhanced ropivacaine activity can be predicted because a considerable proportion of channels are inactivated, and the sodium channel is susceptible to the ropivacaine modulation binding. This explanation is also supported by the effect of ropivacaine on the activation and inactivation curve. Ropivacaine did not shift the voltage range across which channel activation occurred; however, it produced a hyperpolarizing shift in the inactivation curve. When the conditioning pulses were used, the inhibitory effect of ropivacaine was more pronounced, suggesting that ropivacaine acts preferentially with the steady-state inactivation state.

The preferential binding of ropivacaine to the inactivated states can also be explained by the use-dependent inhibition of ropivacaine. The sodium currents in dorsal horn neurons consist of several components. First is the fast component with an inactivation time constant ({tau}f) of 0.6–2.0 ms. The second consisted of a slowly inactivating component ({tau}s) of 5–20 ms and the steady-state component (8). In our experiment, the inactivation state of the sodium channel may have accumulated when repeated pulses were used at a frequency of 5 Hz with the pulse duration of 20 ms. Under these conditions, a significant proportion of channels is converted to the slow inactivation state. The amplitude ratios of the 20th to first pulse were 71.1% in the presence of ropivacaine, which is lower than in the absence of ropivacaine (91.2%). This means that the inactivation state of sodium channels is susceptible to the drug binding; that is, ropivacaine preferentially acts on the inactivation state of sodium channels.

In conclusion, ropivacaine inhibits the sodium channel in a voltage- and use-dependent manner. Ropivacaine preferentially interacts with the inactivation state; and the inhibitory potency of ropivacaine is less than that of bupivacaine.


   Acknowledgments
 
Supported by grants from the Health Institute (99402) of Shanghai, China.


   Footnotes
 
Address correspondence and reprint requests to Dr. Bao-Gang Liu, Department of Anesthesiology, Shanghai First People‘s Hospital, Shanghai, China, 200080. Address e-mail to liubaogang@mailcity.com.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Griffin RP, Reynolds F. Extradural anaesthesia for caesarean section: a double-blind comparison of 0.5% ropivacaine with 0.5% bupivacaine. J Anaesth 1995;74:512–6.
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  4. Butterworth JF, Strichartz GR. Molecular mechanism of local anesthesia: a review. Anesthesiology 1990;72:711.[Web of Science][Medline]
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  8. Safronov BV, Wolff M, Vogel W. Functional distribution of three types of sodium channel on soma and processes of dorsal horn neurons of rat spinal cord. J Physiol 1997;503:371–85.[Abstract/Free Full Text]
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  11. Olschewski A, Hempelmann G, Vogel W, Safronov BV. Blockade of Na+ and K+ currents by local anesthetics in the dorsal horn neurons of the spinal cord. Anesthesiology 1998;88:172–9.[Web of Science][Medline]
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Accepted for publication January 12, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2000 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press