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Effects of penicillin on procaine-elicited bursts of potential in central neuron of snail, Achatina fulica
Comparative Biochemistry and Physiology, Part C 148 (2008) 128–135
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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p c
Effects of penicillin on procaine-elicited bursts of potential in central neuron of snail, Achatina fulica Yi-Hung Chen a, Kuan-Ling Lu a, Ru-Wan Hsiao a, Ya-Ling Lee b, Hong-Chieh Tsai c, Chia Hsien Lin d, Ming-Cheng Tsai a,⁎ a
Department of Pharmacology, College of Medicine, National Taiwan University, No.1, Sec.1, Jen-Ai Road, Taipei, Taiwan School of Nursing, National Taiwan University, Taipei, Taiwan Department of Neurosurgery, Chang-Gung Memorial Hospital, Taoyuan, Taiwan d Department of Nursing, Ching-Kuo Institute of Management and Health, Keelung, Taiwan b c
A R T I C L E
I N F O
Article history: Received 24 March 2008 Received in revised form 16 April 2008 Accepted 16 April 2008 Available online 22 April 2008 Keywords: Ascorbate Bursts of potential L-NAME Neuron Procaine Penicillin Snail Sodium current
A B S T R A C T Effects of penicillin on changes in procaine-elicited bursts of potential (BoP) were studied in a central neuron (RP4) of snail, Achatina fulica Ferussac. Procaine elicited BoP in the RP4 neuron while penicillin elicited depolarization of the neuron. Penicillin decreased the BoP elicited by procaine in a concentration-dependent manner. The effect of penicillin on the procaine-elicited BoP was not altered in the preparations treated with ascorbate or L-NAME (N-nitro-L-arginine methyl ester). However, the inhibitory effect of penicillin on the procaine-elicited BoP was enhanced with a decrease in extracellular sodium ion. Sodium ion was one of the important ions contributing to the action potential of the neuron. Two-electrode voltage-clamp studies revealed that penicillin decreased the fast sodium inward current of the neuron. It is concluded that penicillin inhibited the BoP elicited by procaine and sodium ion altered the effect of penicillin on procaineelicited BoP. © 2008 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Procaine and penicillin have been long employed as pharmacological agents for life science and clinical therapeutic studies. Procaine is a local anesthetic drug of the amino ester group. It is used primarily to reduce the pain of intramuscular injection of penicillin, and is also used in dentistry. Penicillin is a group of β-lactam antibiotics used in the treatment of bacterial infections caused by susceptible, usually Grampositive, organisms. Procaine penicillin G injection was also used to improved local tolerance to penicillin (Krugman and Ebin, 1958). However, the interaction between procaine and penicillin on the central neuron remained unknown. Our previous study revealed that procaine elicited BoP in the central neuron of the African snail, Achatina fulica Ferussac (Lin and Tsai, 2005a). To elucidate the interactions between procaine and penicillin, their effects on a right parietal ganglia neuron (RP4 neuron) were examined. We found that penicillin inhibited the BoP elicited by procaine and penicillin also decreased the fast sodium inward current of the neuron. The effect may be responsible for the blocking effect of penicillin on procaine-elicited BoP.
Experiments were performed on identified central RP4 neurons from the subesophageal ganglia of the African snail, A. fulica Ferussac 1821 (Achatinidae, Gastropoda). The ganglia were pinned to the bottom of a 3.0-milliliter sylgard-coated perfusion chamber and carefully freed from the connective tissue sheath to allow easy identification and penetration by microelectrodes (Chen and Tsai, 2000; Tsai and Chen, 1995). For intracellular recording, a Gene clamp 500 amplifier (Axon Instruments, Inc., USA) was used. Microelectrodes (5.0–8.0 MΩ) for recording membrane potentials were filled with 3.0 M KCl. The experimental chamber was perfused with control saline, whose composition was (mM) NaCl, 85.0; KCl, 4.0; CaCl2, 8.0; MgCl2, 7.0; Tris–HCl, 10.0 (pH 7.6), at 23.0–24.0 °C (Chen and Tsai, 1996; Lin and Tsai, 2005b). Low-sodium (Na+) saline was partially substituted with sodium ion (20% and 50%) replaced by lithium ion. Low-calcium (Ca2+) saline was also partially substituted with calcium ion (20%) replaced by Tris ion or as described in the text (Chen and Tsai, 2000). Neurons were included for examination only if they had resting membrane potentials exceeding -50 mV with the time constant at 5–8 ms and the rate of increase in action potentials at 5–8 ΔV/s. Penicillin and other drugs were applied by extracellular incubation. All potentials were recorded on tape by a digitalizing unit (Digidata 1200) (Axon Instruments, Inc., USA) and analyzed.
⁎ Corresponding author. Tel.: +886 2 23123456x8314, 0912196786 (mobile); fax: +886 2 23915297. E-mail address: [email protected] (M.-C. Tsai). 1532-0456/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.04.006
Y.-H. Chen et al. / Comparative Biochemistry and Physiology, Part C 148 (2008) 128–135
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Fig. 1. Effects of sodium and calcium ions on the directly elicited action potentials of the RP4 neuron of snails. A and B represented sodium ion was replaced by lithium or Tris buffer, respectively. A1 and B1 were control. A2, B2 and A3, B3 showed the directly elicited action potentials after perfusion with lithium or Tris buffer for 60 or 180 min, respectively. A4 and B4 showed the directly elicited action potential 20 min after further perfusion with lithium or Tris buffer containing cobalt ion and 1 mM EGTA (sodium and calcium free solution) from A3, B3, respectively. A5 and B5 showed the directly elicited action potential after washing off ionic free solution with normal saline from A4 and B4, respectively. The resting membrane potential was set at −70 mV. The horizontal bar on the top left side of each figure represents the potential at 0 mV.
Resting membrane potentials (RMPs), amplitudes and frequency of single spike of action potentials of the RP4 neuron were recorded 60 min after penicillin administration to examine the effects of penicillin on them. To examine the effect of penicillin on the ionic current, voltage clamp methods were employed (Adams et al., 1980). For voltage clamping, the neurons were clamped by a Gene clamp 500 amplifier (Axon Instrument Co.). All potentials and currents were recorded on tape by a digitalizing unit (Digidata 1200) and analyzed using a pCLAMP system. For peak amplitude of total inward current, the currents were elicited by 70 ms of −50 to + 30 mV from a holding potential of −60 mV. For steady-state outward currents, the currents were elicited by 5 s of −100 to + 20 mV from a holding potential of −60 mV (Chen and Tsai, 2000). To examine how ascorbate and L-NAME (nitric oxide synthase inhibitor, N (omega)-nitro-L-arginine methyl ester) influence the effect of penicillin on procaine-elicited BoP in the RP4 neuron, procaine was added first to elicit BoP, followed by application of ascorbate or L-NAME on the chamber with procaine for 60 min or as described in the text. The mean amplitude of the potentials after various treatments was compared with the pre-drug control using Student's two-tailed t-test (Chen et al., 2006; Tsai and Chen, 2007). Statistical data were considered significantly different at p b 0.05. All procedures used in this study were approved by the Animal Ethics Committee of National Taiwan University in accordance with internationally accepted principles for the care and use of experimental animals. All efforts were made to minimize the number of animals used and their suffering.
The RP4 neuron had a resting membrane potential of −58.1±1.1 mV (n=20, mean±S.E.M.), and it showed a spontaneous firing of action potential at a frequency of 42.7±2.3 pulses/min (n=20). The action potentials showed a regularly spaced single spike. No bursts firing of action potential was observed in control RP4 neurons. The mean amplitude of the spontaneously generated action potential was 88.1±1.0 mV (n=20). 3.2. Effects of sodium and calcium ions on directly elicited action potentials of RP4 neuron Effects of sodium and calcium ions on the directly elicited action potentials of the RP4 neuron of snail were tested using two electrodes implanted into the neuron. One electrode is used for recording and the other one is used for injecting depolarization pulses. In the solution perfused with sodium-free buffer containing lithium ion or Tris ion and 1 mM EGTA (sodium and calcium free solution), the directly elicited action potential was abolished. The effects of sodium and calcium ions on the directly elicited action potential were reversible. The directly elicited action potential was restored to control level if normal saline was further perfused (Fig. 1). 3.3. Effects of penicillin on RP4 neuron Effects of penicillin (3–100 mM) on the RP4 neuron are shown in Table 1 and Fig. 2. Penicillin did not alter the RMP, amplitude of and
3. Results
Table 1 Effects of penicillin on the resting membrane potential, amplitude and frequency of spontaneously generated action potential of RP4 neurons
3.1. Identifiable RP4 neuron of A. fulica
Variable
The central right parietal neuron 4 (RP4 neuron) (Tsai and Chen, 1995), the identified neuron in the periodically oscillating neuron, from the subesophageal ganglia of the African snail A. fulica, was sensitive to several neurotransmitters. Glutamic acid (50 μM) induced hyperpolarization of the membrane potential. Serotonin (5-HT) (50 μM), γ-aminobutyric acid (GABA) (50 μM), dopamine (50 μM) or acetylcholine (ACh) (50 μM) increased the frequency of the spontaneous action potential of the neuron (Lin and Tsai, 2003).
Penicillin
Control (n = 20) 3 mM (n = 5) 10 Mm (n = 5) 30 mM (n = 5) 100 mM (n = 5)
RMP (mV)
Amplitudes (mV)
Frequency of single spikes (pulses/min)
−58.1 ± 1.1 −53.8 ± 1.8 −45.0 ± 3.1⁎ −25.0 ± 2.0⁎ −12.7 ± 1.7⁎
88.1 ± 1.0 85.4 ± 2.6 78.4 ± 2.3⁎ 39.7 ± 1.5⁎ –
42.7 ± 2.3 47.3 ± 0.7 38.7 ± 1.8 62.0 ± 5.0⁎ –
The data were recorded after application of penicillin (3–100 mM) for 60 min. Values were expressed as the mean ± S.E.M. (n is the number of neurons tested). ⁎: Statistically significant compared with the data in physiological solution (control), p b 0.05.
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Fig. 2. Effects of penicillin on the central RP4 neurons. Control in A, B, C and D were potentials of control RP4 neurons. At 30, 60 min in A, B, C and D were potentials after 30, 60 min of application of penicillin (3, 10, 30 and 100 mM), respectively. At washing in A, B, C and D were potentials after 120 min of washing with normal saline from preparations incubated with 60 min penicillin of A–D, respectively. The horizontal bar on the top left side is the membrane potential at 0 mV.
frequency of action potential of the RP4 neuron at lower concentrations tested (3 mM); however, penicillin altered the RMP and amplitude of the spontaneous action potential at higher concentrations (10–100 mM) tested. Penicillin (30 mM) depolarized the RMP of the RP4 neuron while it did not elicit BoP after 30 min of incubation. The changes in potential elicited by penicillin were restored to control level after washing off penicillin with normal saline continuously for 120 min (Fig. 2). It appears that higher concentration of penicillin (100 mM) depolarized the RMP
reversibly and it also abolished the spontaneous action potential in the RP4 neuron. 3.4. Effects of procaine on RP4 neuron Procaine elicited BoP in the RP4 neuron in a concentrationdependent manner. At 5 mM, procaine did not elicit BoP in the RP4 neuron even 60 min after procaine administration. However, at 10 mM,
Fig. 3. Effects of procaine on the central RP4 neurons of snails. Controls in A and B were potentials of control RP4 neurons. At 30 and 60 min in A and B were potentials after 30 and 60 min of application of procaine (5 and 10 mM), respectively. At washing in A and B were potentials after 120 min of washing with normal saline from preparations incubated with 60 min penicillin of A and B, respectively. The horizontal bar on the top left side was the membrane potential at 0 mV.
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Fig. 4. Effects of penicillin on the procaine-elicited bursting firing of action potentials on the central RP4 neuron. Control in A, B and C were potentials of control RP4 neurons. Then were the potentials 30 min after addition of penicillin (3, 10 and 30 mM in A, B and C, respectively) from control and 60 min after further addition of procaine (10 mM) in the presence of penicillin. At washing were potentials after 120 min of washing with normal saline. The horizontal bar on the top left side was the membrane potential at 0 mV.
procaine did elicit BoP in the neuron 30 min after procaine administration. The BoP remained even 60 min after drug administration. The effect of procaine on the potential changes was reversible. The spontaneously generated action potential was restored to control level after washing off procaine with normal saline continuously for 120 min (Fig. 3). Similar results were found in five other preparations tested. Two-electrode voltage-clamp studies revealed that procaine decreased the fast inward sodium current by a concentration-dependent manner (Lin, 2005).
3.5. Effects of penicillin on procaine-elicited bursting of action potentials Effect of penicillin on procaine-elicited potential changes was tested and an example is shown in Fig. 4. Penicillin (3, 10 and 30 mM) did not elicit BoP in the RP4 neuron 30 min after incubation; however, the amplitude of the single spike in penicillin (10 and 30 mM)-treated neurons were decreased. Penicillin at 3 mM, did not inhibit the procaineelicited BoP in the RP4 neuron even after 60 min of incubation in
Fig. 5. Effects of sodium ion on penicillin-induced inhibitions on the RP4 neurons. Control in A, B and C were action potentials of RP4 neurons. Then were the potentials 20 min after bathing in normal saline (A), 80% Na+(B) and 50% Na+(C). Thereafter, 60 min after adding 10 mM procaine, and then 20 min after adding penicillin. At wash were potentials after 120 min washing with normal saline. The horizontal bar on the top left is the membrane potential at 0 mV.
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Table 2 Effect of penicillin (10 mM, 30 mM) on the peak of amplitudes of total inward current in the RP4 neurons Test potential (mV)
−50 −40 −30 −20 −10 0 10 20 30
Physiological solution
Penicillin
Control (nA) (n = 9)
10 mM (n = 9)
30 mM (n = 5)
3.4 ± 0.3 −89.3 ± 7.5 −155.6 ± 24.0 −211.5 ± 12.4 −180.8 ± 5.2 −135.8 ± 3.6 −94.2 ± 5.0 −54.4 ± 4.2 − 10.1 ± 3.7
4.8 ± 0.3 −38.4 ± 9.1⁎ −89.2 ± 11.6⁎⁎ −136.5 ± 14.8⁎ − 126.5 ± 10.1⁎ −101.4 ± 6.3⁎⁎ −75.5 ± 3.9⁎ − 32.1 ± 2.7⁎
8.8 ± 2.2 −13.4 ± 5.6# −45.9 ± 3.1 −71.1 ± 11.5## −66.4 ± 19.4 −60.6 ± 17.0 −34.4 ± 13.9 −10.0 ± 10.9 15.6 ± 11.2
−0.41 ± 6.3
The membrane currents were elicited from holding potentials of − 60 mV to test potentials (− 50 to +30 mV) before and after penicillin application. Values were expressed as the mean ± S.E.M. (n is the number of neurons tested). ⁎: Statistically significant compared with physiological solution (control), ⁎p b 0.05, ⁎⁎p b 0.01. #: Statistically significant compared with penicillin (10 mM), #p b 0.05, ##p b 0.01.
procaine (10 mM). However, at 10 mM, penicillin inhibited the procaine (10 mM)-elicited BoP in the RP4 neuron. At 30 mM, penicillin not only inhibited the BoP elicited by procaine, but it also depolarized the RP4 neuron. The effect of penicillin- or procaine-elicited potential changes was restored to control level after washing off penicillin and procaine with normal saline continuously for 120 min. It appeared that penicillin (10 and 30 mM) did reversibly inhibit the elicitation of BoP by procaine.
effect of low-sodium solution on the inhibitory effect of penicillin on bursting of action potential elicited by procaine is shown in Fig. 5. In preparations treated with 20% low-sodium solution, procaine (10 mM) elicited BoP 60 min after procaine administration. If penicillin (10 mM) was further added, the BoP elicited by procaine (10 mM) was decreased, and then shifted to single spike of the potentials. In preparations treated with 50% low-sodium solution, procaine (10 mM) still elicited BoP. If lower concentration of penicillin (3 mM) was further added, the BoP elicited by procaine (10 mM) was decreased, and then shifted to a single spike. It appeared that the concentration of penicillin, which blocked the elicitation of BoP by procaine, was lowered if extracellular sodium ion concentration was decreased to 50% of normal saline. Similar results were found in five other preparations tested. 3.7. Role of calcium ion on inhibitory effect of penicillin on BoP elicited by procaine In order to know the role of extracellular calcium ions on the inhibitory effect of penicillin on BoP elicited by procaine, low-calcium solution (20% of calcium ion replaced by Tris ion) was used (data not shown). In the presence of low-calcium solution, procaine (10 mM) also elicited BoP 60 min after procaine administration. The BoP remained even if penicillin of 3 mM or 5 mM was further added. However, the BoP decreased and the potentials shifted to single spike of action potential if greater concentration of penicillin (10 mM) was added. It appeared that low-calcium medium did not alter the blocking effect of penicillin on procaine-elicited BoP.
3.6. Role of sodium ion on inhibitory effect of penicillin on BoP elicited by procaine
3.8. Effects of penicillin on ionic currents of RP4 neuron
In order to test the role of extracellular sodium ion on the inhibitory effect of penicillin on BoP elicited by procaine, low-sodium solution (20% and 50% of sodium ion replaced by lithium ion) was used. The
The effects of penicillin (10 and 30 mM) on the fast inward currents of the RP4 neuron during shorter steps (70 ms) of voltage clamping are shown in Table 2 and Fig. 6. The effects of penicillin (10–30 mM) on the
Fig. 6. Effects of penicillin on total inward currents of RP4 neurons. Ion currents were elicited by test pulses rang from −50 to +30 mV with 10 mV increments from a holding potential of −60 mV (70 ms). Left: Represented currents of control (A), 30 min after 10 mM penicillin (B) and 30 mM penicillin (C). Lowest trace is the voltage step commands. Right: I–V curves of the total inward currents of control (●), 10 mM penicillin ( ) and 30 mM penicillin (○), respectively. ⁎p b 0.05, ⁎⁎p b 0.01: 10 mM vs. control; #p b 0.05, ##p b 0.01: 30 mM vs.10 mM.
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steady-state outward currents of the RP4 neuron during five-secondlong stepping pulses, holding potential of − 60 mV to stepping potentials of −100 to 20 mV at intervals of 10 mV, were measured at 5 s after voltage stepping. The steady-state current–voltage (I–V) relationships are shown in Fig. 7. Penicillin (10–30 mM) did significantly decrease the fast inward currents in a series of voltage steps from −40 mV to 30 mV (Fig. 6). Note that penicillin (30 mM) significantly decreased the fast inward currents in a concentration manner. Procaine (10 mM) or penicillin (10 mM) decreased the fast inward current of RP1 and RP4 neurons holding at −60 mV. In preparation treated with procaine (10 mM) or penicillin (10 mM), if procaine (10 mM) or penicillin (10 mM) were further added respectively, the fast inward sodium current was further decreased. The decreased inward current was recovered to pre-drug level if drug was washing out with normal saline. It appeared that procaine and penicillin showed additive effect on the fast sodium inward current of the snail RP1 and 4 neurons. Steady-state outward currents were observed if the holding potential was greater than −50 mV. The total outward current was decreased if the command voltage was stepped to 0–20 mV. Penicillin at 10–30 mM did significantly decrease the fast inward sodium current and the steady-state outward potassium currents on the same neuron. 3.9. Effects of ascorbate and L-NAME on inhibitory effects of penicillin on procaine-elicited BoP Effects of ascorbate and L-NAME on the bursts of potential elicited by procaine were shown in Fig. 8. Procaine (10 mM) elicited BoP 60 min after procaine administration. The BoP remained even after 60 min of further addition of ascorbate (1 mM), and still remained even after 30 min of further addition of penicillin (5 mM). However, the procaine-elicited BoP was abolished if penicillin (10 mM) was further added. After washing off ascorbate and penicillin from the solution containing procaine, the elicitation of BoP by procaine was restored. Similarly, BoP elicited 60 min after procaine (10 mM) administration remained even after 60 min of further addition of L-
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NAME (10 mM), and still remained even after 30 min of further addition of penicillin (5 mM). However, the procaine-elicited BoP was abolished if penicillin (10 mM) was further added. After washing off LNAME and penicillin from the solution containing procaine, the elicitation of BoP by procaine was restored. 4. Discussion Our previous studies revealed that extracellular application of procaine (10 mM) reversibly elicited BoP in central neurons (RP1 and RP4) of the giant African snail (A. fulica Ferussac). The procaineelicited BoP was associated with the phospholipase activity and calcium mobilization in the neuron (Lin and Tsai, 2005a,b). In the present study, we found that the action potential of the RP4 neuron depended mainly on extracellular sodium and calcium ions. Our previous study revealed that procaine decreased the fast sodium inward currents by a concentration-dependent manner (Lin, 2005). Procaine (10 mM) or penicillin (10 mM) decreased the fast inward current of RP1 and RP4 neurons. In preparation treated with procaine (10 mM) or penicillin (10 mM), if procaine (10 mM) or penicillin (10 mM) were further added respectively, the fast inward sodium current was further decreased. The decreased inward current was recovered to pre-drug level if drug was washing out with normal saline. It appeared that procaine and penicillin showed additive effect on the fast sodium inward current of the snail RP1 and 4 neurons. In the present study, we also found that penicillin's effect on procaine induced BoP is enhanced by low-sodium medium. The effect may be related to additive effect of procaine and penicillin on sodium current. Penicillin decreased the BoP elicited by procaine in a concentrationdependent manner. At concentration of 10–30 mM, penicillin decreased significantly the BoP elicited by procaine while at 5 mM, penicillin had no inhibitory effect on the BoP elicited by procaine. Ascorbate and nitric oxide have neuroprotective activity against penicillin-induced epileptiform electrocorticogram activity in rats (Ayyildiz et al., 2007a,b). Penicillin (5 mM) had no inhibitory effect on the BoP elicited by procaine even in the presence of ascorbate or L-
Fig. 7. Effects of penicillin on total outward currents of RP4 neurons. Ion currents were elicited by test pulses rang from −100 to +20 mV with 10 mV increments from a holding potential of −60 mV (5 s). Left: Represented currents of control (A), 30 min after 10 mM penicillin (B) and 30 mM penicillin (C). Lowest trace is the voltage step commands. Right: I–V curves of the total outward currents of control (●), 10 mM penicillin ( ) and 30 mM penicillin (○), respectively. ⁎p b 0.05, ⁎⁎p b 0.01: 10 mM or 30 mM vs. control.
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Fig. 8. Effects of L-NAME (10 mM) and Vitamin C (1 mM) on penicillin- and procaine-elicited burst of action potentials on RP4 neuron. A1 and B1 were control. A2 and B2 were potentials after application of procaine (10 mM) for 60 min. A3 and B3 were potentials after application of procaine (10 mM) and further adding of L-NAME (10 mM) or Vitamin C (1 mM) for 60 min, respectively. A4 and B4 were potentials after further incubation of penicillin (5 mM) for 30 min from A3 or B3, respectively. A5 and B5 were potentials after application of further incubation of penicillin (10 mM) for 30 min from A4 or B4, respectively. A6 and B6 were potentials after washing for 3 min from A5 or B5, respectively. A7 and B7 were potentials after 120 min of washing off with normal saline from preparations incubated from A5 or B5, respectively. The horizontal bar at the top left indicated the membrane potential at 0 mV.
NAME, the nitric oxide synthase inhibitor. The results suggested that ascorbate or L-NAME view that there may be no additive effect on the blocking of procaine-elicited BoP by penicillin. It is interesting to note that the concentration of penicillin needed to block BoP elicited by procaine decreased with a decrease in extracellular sodium ion concentration. The ionic requirement for the potentials in RP4 neuron is dependent mainly on sodium ion and calcium ion (Chen and Tsai, 2000). Our voltage-clamped study also
revealed that penicillin decreased the fast sodium inward current of the RP4 neuron, suggesting that sodium ion altered the effect of penicillin on procaine-elicited BoP. Procaine elicited seizures in dogs (Ding et al., 1992). Penicillin also induced epileptiform electrocorticogram activity in rats (Ayyildiz et al., 2007a). However, seizures following the administration of intramuscular penicillin G procaine were rarely found in the literature (Silber and D'Angelo, 1985). Our finding that penicillin inhibited the
Y.-H. Chen et al. / Comparative Biochemistry and Physiology, Part C 148 (2008) 128–135
BoP elicited by procaine supported the view that there may have no additive seizure effects if procaine and penicillin were injected into the CNS simultaneously. Since penicillin does not freely penetrate the CNS, it is not expected to have mutual central drug interaction if procaine and penicillin were injected peripherally. It is concluded that penicillin altered the BoP elicited by procaine. Acknowledgments This work was supported by grant, NSC-96-2320-B-002-063 from National Science Council, Taipei, Taiwan. References Adams, D.J., Smith, S.J., Thompson, S.H., 1980. Ionic currents in molluscan soma. Annu. Rev. Neurosci. 3, 141–167. Ayyildiz, M., Coskun, S., Yildirim, M., Agar, E., 2007a. The effects of ascorbic acid on penicillin-induced epileptiform activity in rats. Epilepsia 48, 1388–1395. Ayyildiz, M., Yildirim, M., Agar, E., 2007b. The involvement of nitric oxide in the anticonvulsant effects of alpha-tocopherol on penicillin-induced epileptiform activity in rats. Epilepsy Res. 73, 166–172. Chen, Y.H., Tsai, M.C., 1996. Bursting firing of action potential in central snail neuron elicited by d-amphetamine: role of the intracellular calcium ions. Comp. Biochem. Physiol. A 115, 195–205.
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Chen, Y.H., Tsai, M.C., 2000. Action potential bursts in central snail neurons elicited by d-amphetamine: roles of ionic currents. Neuroscience 96, 237–248. Chen, Y.H., Lin, C.H., Lin, P.L., Tsai, M.C., 2006. Cocaine elicits action potential bursts in a central snail neuron: the role of delayed rectifying K + current. Neuroscience 138, 257–280. Ding, Z.N., Yoshita, Y., Hirota, K., Yamamoto, K., Kobayashi, T., Murakami, S., 1992. Brainstem auditory evoked potentials during procaine toxicity in dogs. Can. J. Anaesth. 39, 600–603. Krugman, S., Ebin, E.V., 1958. Improved local tolerance to benzathine penicillin G. Pediatrics 21, 243–247. Lin, C.H., 2005. Pharmacological study on bursts activity elicited by procaine and d-amphetamine. Ph.D. thesis. Nastional Taiwan University. Lin, C.H., Tsai, M.C., 2003. d-amphetamine-elicited action potential bursts in central snail neurons: role of second messenger systems. J. Formos. Med. Assoc. 102, 394–403. Lin, C.H., Tsai, M.C., 2005a. Effects of procaine on a central neuron of the snail, Achatina fulica Ferussac. Life Sci. 76, 1641–1666. Lin, C.H., Tsai, M.C., 2005b. The modulation effects of d-amphetamine and procaine on the spontaneously generated action potentials in the central neuron of snail, Achatina fulica Ferussac. Comp. Biochem. Physiol. C 141, 58–68. Silber, T.J., D'Angelo, L., 1985. Psychosis and seizures following the injection of penicillin G procaine. Hoigne's syndrome. Am. J. Dis. Child. 139, 335–337. Tsai, M.C., Chen, Y.H., 1995. Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of the electrogenic sodium pump. Comp. Biochem. Physiol. C 111, 131–141. Tsai, M.C., Chen, Y.H., 2007. (+/−)3,4-Methylenedioxyamphetamine elicits action potential bursts in a central snail neuron. Exp. Neurol. 203, 423–444.
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p c
Effects of penicillin on procaine-elicited bursts of potential in central neuron of snail, Achatina fulica Yi-Hung Chen a, Kuan-Ling Lu a, Ru-Wan Hsiao a, Ya-Ling Lee b, Hong-Chieh Tsai c, Chia Hsien Lin d, Ming-Cheng Tsai a,⁎ a
Department of Pharmacology, College of Medicine, National Taiwan University, No.1, Sec.1, Jen-Ai Road, Taipei, Taiwan School of Nursing, National Taiwan University, Taipei, Taiwan Department of Neurosurgery, Chang-Gung Memorial Hospital, Taoyuan, Taiwan d Department of Nursing, Ching-Kuo Institute of Management and Health, Keelung, Taiwan b c
A R T I C L E
I N F O
Article history: Received 24 March 2008 Received in revised form 16 April 2008 Accepted 16 April 2008 Available online 22 April 2008 Keywords: Ascorbate Bursts of potential L-NAME Neuron Procaine Penicillin Snail Sodium current
A B S T R A C T Effects of penicillin on changes in procaine-elicited bursts of potential (BoP) were studied in a central neuron (RP4) of snail, Achatina fulica Ferussac. Procaine elicited BoP in the RP4 neuron while penicillin elicited depolarization of the neuron. Penicillin decreased the BoP elicited by procaine in a concentration-dependent manner. The effect of penicillin on the procaine-elicited BoP was not altered in the preparations treated with ascorbate or L-NAME (N-nitro-L-arginine methyl ester). However, the inhibitory effect of penicillin on the procaine-elicited BoP was enhanced with a decrease in extracellular sodium ion. Sodium ion was one of the important ions contributing to the action potential of the neuron. Two-electrode voltage-clamp studies revealed that penicillin decreased the fast sodium inward current of the neuron. It is concluded that penicillin inhibited the BoP elicited by procaine and sodium ion altered the effect of penicillin on procaineelicited BoP. © 2008 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Procaine and penicillin have been long employed as pharmacological agents for life science and clinical therapeutic studies. Procaine is a local anesthetic drug of the amino ester group. It is used primarily to reduce the pain of intramuscular injection of penicillin, and is also used in dentistry. Penicillin is a group of β-lactam antibiotics used in the treatment of bacterial infections caused by susceptible, usually Grampositive, organisms. Procaine penicillin G injection was also used to improved local tolerance to penicillin (Krugman and Ebin, 1958). However, the interaction between procaine and penicillin on the central neuron remained unknown. Our previous study revealed that procaine elicited BoP in the central neuron of the African snail, Achatina fulica Ferussac (Lin and Tsai, 2005a). To elucidate the interactions between procaine and penicillin, their effects on a right parietal ganglia neuron (RP4 neuron) were examined. We found that penicillin inhibited the BoP elicited by procaine and penicillin also decreased the fast sodium inward current of the neuron. The effect may be responsible for the blocking effect of penicillin on procaine-elicited BoP.
Experiments were performed on identified central RP4 neurons from the subesophageal ganglia of the African snail, A. fulica Ferussac 1821 (Achatinidae, Gastropoda). The ganglia were pinned to the bottom of a 3.0-milliliter sylgard-coated perfusion chamber and carefully freed from the connective tissue sheath to allow easy identification and penetration by microelectrodes (Chen and Tsai, 2000; Tsai and Chen, 1995). For intracellular recording, a Gene clamp 500 amplifier (Axon Instruments, Inc., USA) was used. Microelectrodes (5.0–8.0 MΩ) for recording membrane potentials were filled with 3.0 M KCl. The experimental chamber was perfused with control saline, whose composition was (mM) NaCl, 85.0; KCl, 4.0; CaCl2, 8.0; MgCl2, 7.0; Tris–HCl, 10.0 (pH 7.6), at 23.0–24.0 °C (Chen and Tsai, 1996; Lin and Tsai, 2005b). Low-sodium (Na+) saline was partially substituted with sodium ion (20% and 50%) replaced by lithium ion. Low-calcium (Ca2+) saline was also partially substituted with calcium ion (20%) replaced by Tris ion or as described in the text (Chen and Tsai, 2000). Neurons were included for examination only if they had resting membrane potentials exceeding -50 mV with the time constant at 5–8 ms and the rate of increase in action potentials at 5–8 ΔV/s. Penicillin and other drugs were applied by extracellular incubation. All potentials were recorded on tape by a digitalizing unit (Digidata 1200) (Axon Instruments, Inc., USA) and analyzed.
⁎ Corresponding author. Tel.: +886 2 23123456x8314, 0912196786 (mobile); fax: +886 2 23915297. E-mail address: [email protected] (M.-C. Tsai). 1532-0456/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.04.006
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Fig. 1. Effects of sodium and calcium ions on the directly elicited action potentials of the RP4 neuron of snails. A and B represented sodium ion was replaced by lithium or Tris buffer, respectively. A1 and B1 were control. A2, B2 and A3, B3 showed the directly elicited action potentials after perfusion with lithium or Tris buffer for 60 or 180 min, respectively. A4 and B4 showed the directly elicited action potential 20 min after further perfusion with lithium or Tris buffer containing cobalt ion and 1 mM EGTA (sodium and calcium free solution) from A3, B3, respectively. A5 and B5 showed the directly elicited action potential after washing off ionic free solution with normal saline from A4 and B4, respectively. The resting membrane potential was set at −70 mV. The horizontal bar on the top left side of each figure represents the potential at 0 mV.
Resting membrane potentials (RMPs), amplitudes and frequency of single spike of action potentials of the RP4 neuron were recorded 60 min after penicillin administration to examine the effects of penicillin on them. To examine the effect of penicillin on the ionic current, voltage clamp methods were employed (Adams et al., 1980). For voltage clamping, the neurons were clamped by a Gene clamp 500 amplifier (Axon Instrument Co.). All potentials and currents were recorded on tape by a digitalizing unit (Digidata 1200) and analyzed using a pCLAMP system. For peak amplitude of total inward current, the currents were elicited by 70 ms of −50 to + 30 mV from a holding potential of −60 mV. For steady-state outward currents, the currents were elicited by 5 s of −100 to + 20 mV from a holding potential of −60 mV (Chen and Tsai, 2000). To examine how ascorbate and L-NAME (nitric oxide synthase inhibitor, N (omega)-nitro-L-arginine methyl ester) influence the effect of penicillin on procaine-elicited BoP in the RP4 neuron, procaine was added first to elicit BoP, followed by application of ascorbate or L-NAME on the chamber with procaine for 60 min or as described in the text. The mean amplitude of the potentials after various treatments was compared with the pre-drug control using Student's two-tailed t-test (Chen et al., 2006; Tsai and Chen, 2007). Statistical data were considered significantly different at p b 0.05. All procedures used in this study were approved by the Animal Ethics Committee of National Taiwan University in accordance with internationally accepted principles for the care and use of experimental animals. All efforts were made to minimize the number of animals used and their suffering.
The RP4 neuron had a resting membrane potential of −58.1±1.1 mV (n=20, mean±S.E.M.), and it showed a spontaneous firing of action potential at a frequency of 42.7±2.3 pulses/min (n=20). The action potentials showed a regularly spaced single spike. No bursts firing of action potential was observed in control RP4 neurons. The mean amplitude of the spontaneously generated action potential was 88.1±1.0 mV (n=20). 3.2. Effects of sodium and calcium ions on directly elicited action potentials of RP4 neuron Effects of sodium and calcium ions on the directly elicited action potentials of the RP4 neuron of snail were tested using two electrodes implanted into the neuron. One electrode is used for recording and the other one is used for injecting depolarization pulses. In the solution perfused with sodium-free buffer containing lithium ion or Tris ion and 1 mM EGTA (sodium and calcium free solution), the directly elicited action potential was abolished. The effects of sodium and calcium ions on the directly elicited action potential were reversible. The directly elicited action potential was restored to control level if normal saline was further perfused (Fig. 1). 3.3. Effects of penicillin on RP4 neuron Effects of penicillin (3–100 mM) on the RP4 neuron are shown in Table 1 and Fig. 2. Penicillin did not alter the RMP, amplitude of and
3. Results
Table 1 Effects of penicillin on the resting membrane potential, amplitude and frequency of spontaneously generated action potential of RP4 neurons
3.1. Identifiable RP4 neuron of A. fulica
Variable
The central right parietal neuron 4 (RP4 neuron) (Tsai and Chen, 1995), the identified neuron in the periodically oscillating neuron, from the subesophageal ganglia of the African snail A. fulica, was sensitive to several neurotransmitters. Glutamic acid (50 μM) induced hyperpolarization of the membrane potential. Serotonin (5-HT) (50 μM), γ-aminobutyric acid (GABA) (50 μM), dopamine (50 μM) or acetylcholine (ACh) (50 μM) increased the frequency of the spontaneous action potential of the neuron (Lin and Tsai, 2003).
Penicillin
Control (n = 20) 3 mM (n = 5) 10 Mm (n = 5) 30 mM (n = 5) 100 mM (n = 5)
RMP (mV)
Amplitudes (mV)
Frequency of single spikes (pulses/min)
−58.1 ± 1.1 −53.8 ± 1.8 −45.0 ± 3.1⁎ −25.0 ± 2.0⁎ −12.7 ± 1.7⁎
88.1 ± 1.0 85.4 ± 2.6 78.4 ± 2.3⁎ 39.7 ± 1.5⁎ –
42.7 ± 2.3 47.3 ± 0.7 38.7 ± 1.8 62.0 ± 5.0⁎ –
The data were recorded after application of penicillin (3–100 mM) for 60 min. Values were expressed as the mean ± S.E.M. (n is the number of neurons tested). ⁎: Statistically significant compared with the data in physiological solution (control), p b 0.05.
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Fig. 2. Effects of penicillin on the central RP4 neurons. Control in A, B, C and D were potentials of control RP4 neurons. At 30, 60 min in A, B, C and D were potentials after 30, 60 min of application of penicillin (3, 10, 30 and 100 mM), respectively. At washing in A, B, C and D were potentials after 120 min of washing with normal saline from preparations incubated with 60 min penicillin of A–D, respectively. The horizontal bar on the top left side is the membrane potential at 0 mV.
frequency of action potential of the RP4 neuron at lower concentrations tested (3 mM); however, penicillin altered the RMP and amplitude of the spontaneous action potential at higher concentrations (10–100 mM) tested. Penicillin (30 mM) depolarized the RMP of the RP4 neuron while it did not elicit BoP after 30 min of incubation. The changes in potential elicited by penicillin were restored to control level after washing off penicillin with normal saline continuously for 120 min (Fig. 2). It appears that higher concentration of penicillin (100 mM) depolarized the RMP
reversibly and it also abolished the spontaneous action potential in the RP4 neuron. 3.4. Effects of procaine on RP4 neuron Procaine elicited BoP in the RP4 neuron in a concentrationdependent manner. At 5 mM, procaine did not elicit BoP in the RP4 neuron even 60 min after procaine administration. However, at 10 mM,
Fig. 3. Effects of procaine on the central RP4 neurons of snails. Controls in A and B were potentials of control RP4 neurons. At 30 and 60 min in A and B were potentials after 30 and 60 min of application of procaine (5 and 10 mM), respectively. At washing in A and B were potentials after 120 min of washing with normal saline from preparations incubated with 60 min penicillin of A and B, respectively. The horizontal bar on the top left side was the membrane potential at 0 mV.
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Fig. 4. Effects of penicillin on the procaine-elicited bursting firing of action potentials on the central RP4 neuron. Control in A, B and C were potentials of control RP4 neurons. Then were the potentials 30 min after addition of penicillin (3, 10 and 30 mM in A, B and C, respectively) from control and 60 min after further addition of procaine (10 mM) in the presence of penicillin. At washing were potentials after 120 min of washing with normal saline. The horizontal bar on the top left side was the membrane potential at 0 mV.
procaine did elicit BoP in the neuron 30 min after procaine administration. The BoP remained even 60 min after drug administration. The effect of procaine on the potential changes was reversible. The spontaneously generated action potential was restored to control level after washing off procaine with normal saline continuously for 120 min (Fig. 3). Similar results were found in five other preparations tested. Two-electrode voltage-clamp studies revealed that procaine decreased the fast inward sodium current by a concentration-dependent manner (Lin, 2005).
3.5. Effects of penicillin on procaine-elicited bursting of action potentials Effect of penicillin on procaine-elicited potential changes was tested and an example is shown in Fig. 4. Penicillin (3, 10 and 30 mM) did not elicit BoP in the RP4 neuron 30 min after incubation; however, the amplitude of the single spike in penicillin (10 and 30 mM)-treated neurons were decreased. Penicillin at 3 mM, did not inhibit the procaineelicited BoP in the RP4 neuron even after 60 min of incubation in
Fig. 5. Effects of sodium ion on penicillin-induced inhibitions on the RP4 neurons. Control in A, B and C were action potentials of RP4 neurons. Then were the potentials 20 min after bathing in normal saline (A), 80% Na+(B) and 50% Na+(C). Thereafter, 60 min after adding 10 mM procaine, and then 20 min after adding penicillin. At wash were potentials after 120 min washing with normal saline. The horizontal bar on the top left is the membrane potential at 0 mV.
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Table 2 Effect of penicillin (10 mM, 30 mM) on the peak of amplitudes of total inward current in the RP4 neurons Test potential (mV)
−50 −40 −30 −20 −10 0 10 20 30
Physiological solution
Penicillin
Control (nA) (n = 9)
10 mM (n = 9)
30 mM (n = 5)
3.4 ± 0.3 −89.3 ± 7.5 −155.6 ± 24.0 −211.5 ± 12.4 −180.8 ± 5.2 −135.8 ± 3.6 −94.2 ± 5.0 −54.4 ± 4.2 − 10.1 ± 3.7
4.8 ± 0.3 −38.4 ± 9.1⁎ −89.2 ± 11.6⁎⁎ −136.5 ± 14.8⁎ − 126.5 ± 10.1⁎ −101.4 ± 6.3⁎⁎ −75.5 ± 3.9⁎ − 32.1 ± 2.7⁎
8.8 ± 2.2 −13.4 ± 5.6# −45.9 ± 3.1 −71.1 ± 11.5## −66.4 ± 19.4 −60.6 ± 17.0 −34.4 ± 13.9 −10.0 ± 10.9 15.6 ± 11.2
−0.41 ± 6.3
The membrane currents were elicited from holding potentials of − 60 mV to test potentials (− 50 to +30 mV) before and after penicillin application. Values were expressed as the mean ± S.E.M. (n is the number of neurons tested). ⁎: Statistically significant compared with physiological solution (control), ⁎p b 0.05, ⁎⁎p b 0.01. #: Statistically significant compared with penicillin (10 mM), #p b 0.05, ##p b 0.01.
procaine (10 mM). However, at 10 mM, penicillin inhibited the procaine (10 mM)-elicited BoP in the RP4 neuron. At 30 mM, penicillin not only inhibited the BoP elicited by procaine, but it also depolarized the RP4 neuron. The effect of penicillin- or procaine-elicited potential changes was restored to control level after washing off penicillin and procaine with normal saline continuously for 120 min. It appeared that penicillin (10 and 30 mM) did reversibly inhibit the elicitation of BoP by procaine.
effect of low-sodium solution on the inhibitory effect of penicillin on bursting of action potential elicited by procaine is shown in Fig. 5. In preparations treated with 20% low-sodium solution, procaine (10 mM) elicited BoP 60 min after procaine administration. If penicillin (10 mM) was further added, the BoP elicited by procaine (10 mM) was decreased, and then shifted to single spike of the potentials. In preparations treated with 50% low-sodium solution, procaine (10 mM) still elicited BoP. If lower concentration of penicillin (3 mM) was further added, the BoP elicited by procaine (10 mM) was decreased, and then shifted to a single spike. It appeared that the concentration of penicillin, which blocked the elicitation of BoP by procaine, was lowered if extracellular sodium ion concentration was decreased to 50% of normal saline. Similar results were found in five other preparations tested. 3.7. Role of calcium ion on inhibitory effect of penicillin on BoP elicited by procaine In order to know the role of extracellular calcium ions on the inhibitory effect of penicillin on BoP elicited by procaine, low-calcium solution (20% of calcium ion replaced by Tris ion) was used (data not shown). In the presence of low-calcium solution, procaine (10 mM) also elicited BoP 60 min after procaine administration. The BoP remained even if penicillin of 3 mM or 5 mM was further added. However, the BoP decreased and the potentials shifted to single spike of action potential if greater concentration of penicillin (10 mM) was added. It appeared that low-calcium medium did not alter the blocking effect of penicillin on procaine-elicited BoP.
3.6. Role of sodium ion on inhibitory effect of penicillin on BoP elicited by procaine
3.8. Effects of penicillin on ionic currents of RP4 neuron
In order to test the role of extracellular sodium ion on the inhibitory effect of penicillin on BoP elicited by procaine, low-sodium solution (20% and 50% of sodium ion replaced by lithium ion) was used. The
The effects of penicillin (10 and 30 mM) on the fast inward currents of the RP4 neuron during shorter steps (70 ms) of voltage clamping are shown in Table 2 and Fig. 6. The effects of penicillin (10–30 mM) on the
Fig. 6. Effects of penicillin on total inward currents of RP4 neurons. Ion currents were elicited by test pulses rang from −50 to +30 mV with 10 mV increments from a holding potential of −60 mV (70 ms). Left: Represented currents of control (A), 30 min after 10 mM penicillin (B) and 30 mM penicillin (C). Lowest trace is the voltage step commands. Right: I–V curves of the total inward currents of control (●), 10 mM penicillin ( ) and 30 mM penicillin (○), respectively. ⁎p b 0.05, ⁎⁎p b 0.01: 10 mM vs. control; #p b 0.05, ##p b 0.01: 30 mM vs.10 mM.
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steady-state outward currents of the RP4 neuron during five-secondlong stepping pulses, holding potential of − 60 mV to stepping potentials of −100 to 20 mV at intervals of 10 mV, were measured at 5 s after voltage stepping. The steady-state current–voltage (I–V) relationships are shown in Fig. 7. Penicillin (10–30 mM) did significantly decrease the fast inward currents in a series of voltage steps from −40 mV to 30 mV (Fig. 6). Note that penicillin (30 mM) significantly decreased the fast inward currents in a concentration manner. Procaine (10 mM) or penicillin (10 mM) decreased the fast inward current of RP1 and RP4 neurons holding at −60 mV. In preparation treated with procaine (10 mM) or penicillin (10 mM), if procaine (10 mM) or penicillin (10 mM) were further added respectively, the fast inward sodium current was further decreased. The decreased inward current was recovered to pre-drug level if drug was washing out with normal saline. It appeared that procaine and penicillin showed additive effect on the fast sodium inward current of the snail RP1 and 4 neurons. Steady-state outward currents were observed if the holding potential was greater than −50 mV. The total outward current was decreased if the command voltage was stepped to 0–20 mV. Penicillin at 10–30 mM did significantly decrease the fast inward sodium current and the steady-state outward potassium currents on the same neuron. 3.9. Effects of ascorbate and L-NAME on inhibitory effects of penicillin on procaine-elicited BoP Effects of ascorbate and L-NAME on the bursts of potential elicited by procaine were shown in Fig. 8. Procaine (10 mM) elicited BoP 60 min after procaine administration. The BoP remained even after 60 min of further addition of ascorbate (1 mM), and still remained even after 30 min of further addition of penicillin (5 mM). However, the procaine-elicited BoP was abolished if penicillin (10 mM) was further added. After washing off ascorbate and penicillin from the solution containing procaine, the elicitation of BoP by procaine was restored. Similarly, BoP elicited 60 min after procaine (10 mM) administration remained even after 60 min of further addition of L-
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NAME (10 mM), and still remained even after 30 min of further addition of penicillin (5 mM). However, the procaine-elicited BoP was abolished if penicillin (10 mM) was further added. After washing off LNAME and penicillin from the solution containing procaine, the elicitation of BoP by procaine was restored. 4. Discussion Our previous studies revealed that extracellular application of procaine (10 mM) reversibly elicited BoP in central neurons (RP1 and RP4) of the giant African snail (A. fulica Ferussac). The procaineelicited BoP was associated with the phospholipase activity and calcium mobilization in the neuron (Lin and Tsai, 2005a,b). In the present study, we found that the action potential of the RP4 neuron depended mainly on extracellular sodium and calcium ions. Our previous study revealed that procaine decreased the fast sodium inward currents by a concentration-dependent manner (Lin, 2005). Procaine (10 mM) or penicillin (10 mM) decreased the fast inward current of RP1 and RP4 neurons. In preparation treated with procaine (10 mM) or penicillin (10 mM), if procaine (10 mM) or penicillin (10 mM) were further added respectively, the fast inward sodium current was further decreased. The decreased inward current was recovered to pre-drug level if drug was washing out with normal saline. It appeared that procaine and penicillin showed additive effect on the fast sodium inward current of the snail RP1 and 4 neurons. In the present study, we also found that penicillin's effect on procaine induced BoP is enhanced by low-sodium medium. The effect may be related to additive effect of procaine and penicillin on sodium current. Penicillin decreased the BoP elicited by procaine in a concentrationdependent manner. At concentration of 10–30 mM, penicillin decreased significantly the BoP elicited by procaine while at 5 mM, penicillin had no inhibitory effect on the BoP elicited by procaine. Ascorbate and nitric oxide have neuroprotective activity against penicillin-induced epileptiform electrocorticogram activity in rats (Ayyildiz et al., 2007a,b). Penicillin (5 mM) had no inhibitory effect on the BoP elicited by procaine even in the presence of ascorbate or L-
Fig. 7. Effects of penicillin on total outward currents of RP4 neurons. Ion currents were elicited by test pulses rang from −100 to +20 mV with 10 mV increments from a holding potential of −60 mV (5 s). Left: Represented currents of control (A), 30 min after 10 mM penicillin (B) and 30 mM penicillin (C). Lowest trace is the voltage step commands. Right: I–V curves of the total outward currents of control (●), 10 mM penicillin ( ) and 30 mM penicillin (○), respectively. ⁎p b 0.05, ⁎⁎p b 0.01: 10 mM or 30 mM vs. control.
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Fig. 8. Effects of L-NAME (10 mM) and Vitamin C (1 mM) on penicillin- and procaine-elicited burst of action potentials on RP4 neuron. A1 and B1 were control. A2 and B2 were potentials after application of procaine (10 mM) for 60 min. A3 and B3 were potentials after application of procaine (10 mM) and further adding of L-NAME (10 mM) or Vitamin C (1 mM) for 60 min, respectively. A4 and B4 were potentials after further incubation of penicillin (5 mM) for 30 min from A3 or B3, respectively. A5 and B5 were potentials after application of further incubation of penicillin (10 mM) for 30 min from A4 or B4, respectively. A6 and B6 were potentials after washing for 3 min from A5 or B5, respectively. A7 and B7 were potentials after 120 min of washing off with normal saline from preparations incubated from A5 or B5, respectively. The horizontal bar at the top left indicated the membrane potential at 0 mV.
NAME, the nitric oxide synthase inhibitor. The results suggested that ascorbate or L-NAME view that there may be no additive effect on the blocking of procaine-elicited BoP by penicillin. It is interesting to note that the concentration of penicillin needed to block BoP elicited by procaine decreased with a decrease in extracellular sodium ion concentration. The ionic requirement for the potentials in RP4 neuron is dependent mainly on sodium ion and calcium ion (Chen and Tsai, 2000). Our voltage-clamped study also
revealed that penicillin decreased the fast sodium inward current of the RP4 neuron, suggesting that sodium ion altered the effect of penicillin on procaine-elicited BoP. Procaine elicited seizures in dogs (Ding et al., 1992). Penicillin also induced epileptiform electrocorticogram activity in rats (Ayyildiz et al., 2007a). However, seizures following the administration of intramuscular penicillin G procaine were rarely found in the literature (Silber and D'Angelo, 1985). Our finding that penicillin inhibited the
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BoP elicited by procaine supported the view that there may have no additive seizure effects if procaine and penicillin were injected into the CNS simultaneously. Since penicillin does not freely penetrate the CNS, it is not expected to have mutual central drug interaction if procaine and penicillin were injected peripherally. It is concluded that penicillin altered the BoP elicited by procaine. Acknowledgments This work was supported by grant, NSC-96-2320-B-002-063 from National Science Council, Taipei, Taiwan. References Adams, D.J., Smith, S.J., Thompson, S.H., 1980. Ionic currents in molluscan soma. Annu. Rev. Neurosci. 3, 141–167. Ayyildiz, M., Coskun, S., Yildirim, M., Agar, E., 2007a. The effects of ascorbic acid on penicillin-induced epileptiform activity in rats. Epilepsia 48, 1388–1395. Ayyildiz, M., Yildirim, M., Agar, E., 2007b. The involvement of nitric oxide in the anticonvulsant effects of alpha-tocopherol on penicillin-induced epileptiform activity in rats. Epilepsy Res. 73, 166–172. Chen, Y.H., Tsai, M.C., 1996. Bursting firing of action potential in central snail neuron elicited by d-amphetamine: role of the intracellular calcium ions. Comp. Biochem. Physiol. A 115, 195–205.
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