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-Aminobutyric Acid-A and Glycine Receptors in Acutely Dissociated Spinal Dorsal Horn Neurons of the Rat
Laboratory of Receptor Pharmacology, Department of Neurobiology and Biophysics, University of Science and Technology of China, Hefei, People’s Republic of China
Address correspondence and reprint requests to Tian-Le Xu, PhD, MD, Laboratory of Receptor Pharmacology, Department of Neurobiology & Biophysics, School of Life Sciences, University of Science and Technology of China, PO Box 4, Hefei 230027, People’s Republic of China. Address e-mail to xutianle{at}ustc.edu.cn
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Abstract |
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-aminobutyric acid-A receptor (GABAAR) and the glycine receptor (GlyR) in acutely dissociated rat spinal dorsal horn neurons by using whole-cell patch-clamp electrophysiology. Propofol induced Cl- currents (ICl), which were sensitive to bicuculline and, to a lesser extent, to strychnine. The activation, desensitization, and deactivation of propofol-induced ICl were slower than those of GABA- and glycine-induced ICl. In addition, this study revealed similar modulatory actions of propofol on GABAAR and GlyR. Propofol potentiated both GABA- and glycine-induced ICl at small con-centrations and inhibited both GABA- and glycine-induced ICl at large concentrations. The potentiation of propofol on ICl was caused by slowing current desensitization and deactivation, whereas the inhibition actions might be involved in the cross-desensitization between GABA- and propofol-induced ICl and the cross-inhibition between the GABAAR and GlyR. The results suggest that propofol facilitation of GABAAR and GlyR at the spinal level could contribute significantly to general anesthetic-induced analgesia and anesthesia.
IMPLICATIONS: The actions of propofol on the -aminobutyric acid-A receptor (GABAAR) and the glycine receptor (GlyR) were investigated in acutely dissociated rat spinal dorsal horn neurons by using whole-cell patch-clamp electrophysiology. Propofol was found to potentiate the functions of GABAAR and GlyR at the spinal level, which might contribute to propofol-induced analgesia and anesthesia.
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Introduction |
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Recently, considerable research has been directed toward the effects of general anesthetics on the inhibitory -aminobutyric acid-A receptor (GABAAR) and the glycine receptor (GlyR) (2–4). Although GABAAR is generally considered to be the target of propofol action, the effect of propofol on the GlyR is currently controversial. Hara et al. (5,6) and Patten et al. (7) reported that propofol neither activates nor modulates the GlyR in acutely dissociated or cultured rat hippocampal neurons. In contrast, Hales and Lambert (8) found that propofol positively modulated not only GABAAR, but also GlyR, in cultured murine spinal neurons. Molecular biology has shown that GABAAR and GlyR belong to the same ligand-gated ion channel protein superfamily (9) and that mutation of the GlyR subunit renders the receptor responsive to GABA (10), indicating a close relationship between the two receptor systems. We, therefore, suppose that propofol might have actions on GlyR similar to those on GABAAR.
On the basis of the early theory of depression of the brainstem reticular formation, it is believed that general anesthetics act at different levels of the CNS. Recent evidence appears to provide more support for a spinal than a central control of anesthesia (11–13), because anesthetics could decrease the transmission of noxious information from the spinal cord to the brain and indirectly affect the end points of anesthesia (14,15). Therefore, the spinal cord is thought to play an important role in modulating anesthetic-induced suppression of nociceptive transmission (11,12,14).
Although the actions of propofol on GABAAR have been studied in the hippocampus (5,6,16), its effects on the inhibitory amino acid receptors at the spinal level remain to be addressed in detail. Because GABAAR and GlyR in the spinal cord play a crucial role in nociception (17,18) and because the antinociceptive actions of propofol were demonstrated to be involved in the spinal cord (19,20), in these experiments, we explored the effects of propofol on GABAAR and GlyR in acutely dissociated rat spinal dorsal horn neurons. The results indicated that propofol directly activated the GABAAR in a dose-dependent manner, potentiated the GABA-induced current (IGABA) and glycine-induced current (Igly) at small concentrations, and inhibited IGABA and Igly at larger concentrations.
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Methods |
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The ionic composition of incubation solution was (mM) 124 NaCl, 24 NaHCO3, 5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, and 10 glucose, aerated with 95% oxygen/5% CO2 to a final pH of 7.4. The standard external solution contained (mM) 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.4 with tris-hydroxymethyl aminomethane (tris-base). This bath solution contained 0.3 µM tetrodotoxin and 0.2 mM CdCl2 for recording propofol-induced current (Ipro), IGABA, and Igly. The osmolarity of all bath solutions was adjusted to 325–330 mOsm/L with sucrose (3300, Norwood, MA). The patch pipette solution for whole-cell patch recording was (mM) 120 CsCl, 20 tetraethylammonium-Cl, 2 MgCl2, 1 CaCl2, 10 EGTA, 2 Na2 adenosine triphosphate, and 10 HEPES. The internal solution was adjusted to a pH of 7.2 with tris-base.
Drugs used in these experiments were from Sigma (St. Louis, MO) except for propofol, which was prepared from Diprivan (Zeneca Limited, Macclesfield, Cheshire, UK). Each milliliter of Diprivan contains 10 mg of propofol. The vehicle contains glycerol, soybean oil, purified egg phosphatide/egg lecithin, sodium hydroxide, and water. Intralipid, at concentrations equivalent to those used in our experiments, did not affect the actions of propofol (8,16). Other drugs were first dissolved in ion-free water and then diluted to the final concentrations in the standard external solution just before use or were dissolved directly in the standard external solution. Drugs were applied with a rapid application technique, termed the "Y-tube" method, throughout the experiments (18). This system allows a complete exchange of external solution surrounding a neuron within 20 ms.
The electrophysiological recordings were performed in the conventional whole-cell patch recording configurations under voltage-clamp conditions. Patch pipettes were pulled from glass capillaries with an outer diameter of 1.5 mm on a two-stage puller (PP-830; Narishige, Tokyo, Japan). The resistance between the recording electrode filled with pipette solution and the reference electrode was 4–6 M
. Membrane currents were measured with a patch-clamp amplifier (200 B; Axon Instruments, Foster City, CA), sampled, and analyzed with a DigiData 1320A interface and a computer with the pCLAMP system (Version 8.0; Axon Instruments). In most experiments, 70%–90% series resistance was compensated. The holding potential was -50 mV throughout the experiment. All the experiments were performed at room temperature (22°C–25°C). Clampfit software (Version 8.0; Axon Instruments) was used for data analysis. Difference in mean value was tested by Student’s t-test with P < 0.05 considered significant. All values represented the mean ± SEM.
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Results |
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In the following experiments, Ipro was recorded at more than 200 s intervals, in which Ipro can completely recover from inactivation (desensitization). Propofol elicited inward currents in a concentration-dependent manner. The vehicle intralipid induced no noticeable currents. Application of propofol was also accompanied by increased baseline noise (Fig. 1Aa and Fig. 2A), suggesting that the membrane conductance was increased by propofol. At small concentrations, propofol elicited steady-state inward currents with slow onset and offset, whereas at large concentrations, propofol induced a peak and a successive steady-state inward current with faster onset and offset. The threshold concentration for Ipro was approximately 5 µM. The amplitude of Ipro was increased with increasing propofol concentrations in the range between 5 and 300 µM. A further increase in propofol concentration reduced the peak current, leading to a bell-shaped concentration-response curve (Fig. 1Ab). Fitting the data with the Michaelis-Menten equation produced a 50% effective concentration of 53.71 µM. The washout of propofol at all concentrations used induced no noticeable rebound currents, which often appear in many anesthetic-induced currents (2,3,18).
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Bicuculline and Strychnine Sensitivity of Ipro
The following experiments were performed to determine the bicuculline- and strychnine-sensitive components of the Ipro. It was observed that the current induced by 100 µM propofol was reversibly inhibited 90.62% ± 2.24% (n = 4) by 10 µM bicuculline, a selective antagonist of GABAAR, and 12.41% ± 3.03% (n = 4) by 1 µM strychnine, a selective antagonist of GlyR. Coapplication of 10 µM bicuculline and 1 µM strychnine completely inhibited the Ipro. These results suggested that Ipro was sensitive to bicuculline and, to a lesser extent, to strychnine.
Kinetics of Ipro
To further characterize the Ipro, activation (a process that opens ligand-gated channels after application of ligand), desensitization (a process that closes ligand-gated channels during application of ligand) and deactivation (a process that closes ligand-gated channels after removal of ligand) were analyzed by measuring the time constants of activation (
on), desensitization (decay), and deactivation (off) via fitting the current traces monoexponentially. Figure 3A shows the original currents induced by 10 and 100 µM propofol and their normalized currents for a better comparison of the kinetics. The currents induced by 100 µM propofol had much shorter on (9.37 ± 1.07 s for 10 µM; 3.42 ± 0.69 s for 100 µM; n = 5; P < 0.01) and longer decay (10.42 ± 1.80 s for 10 µM; 15.90 ± 1.74 s for 100 µM; P < 0.05) than those by 10 µM propofol, whereas their off values (3.49 ± 0.76 s for 10 µM; 4.17 ± 0.79 s for 100 µM) were not significantly different (Fig. 3A).
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on, decay, and off than those induced by 100 µM GABA (Fig. 3B) and glycine (Fig. 3C), respectively. The amplitude and on, off, and decay induced by 100 µM propofol were also expressed as the relative values to those obtained with the same concentration of GABA and glycine (Fig. 3). The amplitude of Ipro was 44.79% ± 0.05% (n = 7; P < 0.01) of IGABA and 57.17% ± 0.04% (n = 6; P < 0.01) of Igly. The on was 10.47 ± 1.38-fold (n = 6; P < 0.01) of IGABA and 13.96 ± 2.96-fold (n = 5; P < 0.01) of Igly; the decay was 3.67 ± 0.77-fold (n = 6; P < 0.01) of IGABA and 3.60 ± 0.94-fold (n = 5; P < 0.01) of Igly; and the off was 9.65 ± 0.93-fold (n = 6; P < 0.01) of IGABA and 10.37 ± 2.35-fold (n = 5; P < 0.01) of Igly. These results indicated that responses evoked by propofol had a smaller amplitude and a slower rate of onset, decay, and offset compared with those evoked by the same concentrations of GABA and glycine.
Modulation of IGABA and Igly by Propofol
The vehicle control experiments were performed before testing the modulation of propofol on IGABA and Igly. In the presence of vehicle, 10 and 100 µM IGABA were 102.81% ± 2.39% and 97.62% ± 1.36% of control, respectively, and 30 and 100 µM Igly were 101.64% ± 1.08% and 99.48% ± 2.25% of control, respectively (n = 5; P > 0.05). The results indicated that the vehicle did not significantly modulate IGABA or Igly in these experiments.
To evaluate whether the facilitation of the GABA response and the glycine response by propofol was dependent on the agonist concentrations, propofol (5 µM) was coadministered with small (10 µM for GABA and 30 µM for glycine) and subsaturating (100 µM for GABA or glycine) concentrations of the agonists. Figure 4A shows representative current traces generated from the application of 10 and 100 µM GABA, as well as 30 and 100 µM glycine, in the presence of 5 µM propofol and their normalized current traces. The control values used to normalize the currents were induced by GABA and glycine alone. From Figure 4A and 4Ba, it was found that propofol produced a stronger facilitating effect on IGABA than on Igly, and the currents induced by smaller concentrations of agonists were more largely facilitated by propofol. The current induced by 10 µM GABA was increased 6.45 ± 1.58-fold (n = 6), whereas the current induced by 100 µM GABA was increased 1.50 ± 0.10-fold (n = 8). Similar to its effect on IGABA, propofol caused a 1.82 ± 0.20-fold (n = 5) enhancement of the current induced by 30 µM glycine, whereas it caused a 1.19 ± 0.10-fold (n = 8) enhancement of the current induced by 100 µM glycine.
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off by 3.55 ± 0.74-fold for IGABA (P < 0.05; n = 5) and 1.62 ± 0.30-fold for Igly (P < 0.05; n = 10), and it increased decay by 1.87 ± 0.35-fold (P < 0.05; n = 6) for IGABA as compared with those of control but had few effects on the on of IGABA (P > 0.05; n = 9) or on the on and decay (P > 0.05; n = 9) of Igly. The control values used to normalize the currents were induced by GABA and glycine alone. The results, therefore, indicated that propofol might facilitate IGABA by slowing the GABAAR desensitization and deactivation and might facilitate Igly by slowing the GlyR deactivation. In addition, 100 µM propofol inhibited IGABA (100 µM) by 61.67% ± 9.45% (n = 6; P < 0.01) and Igly (100 µM) by 43.50% ± 6.78% (n = 6; P < 0.01). In the presence of 10 µM bicuculline, however, 100 µM propofol induced small currents, and Igly was 100.26% ± 0.76% (n = 8; P > 0.05) of control (Fig. 5).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Direct Activation of Cl- Current by Propofol
The experiments showed that propofol induced inward Cl- currents (Ipro). The concentration-response relationship of the Ipro was bell shaped. The Ipro was highly sensitive to bicuculline, indicating a GABA-mimic action of propofol. The involvement of GlyR could not be completely excluded in this study, because the Ipro was inhibited 12.41% by 1 µM strychnine (Fig. 2), a selective antagonist of GlyR. Although strychnine weakly blocks IGABA in the same preparation (18,23), the strychnine concentration used here was not large enough to inhibit the Ipro to such a degree. The strychnine-sensitive Ipro has been described in dissociated rat hippocampal neurons (5) and in Xenopus laevis oocytes (24).
The Ipro had a smaller amplitude and slower rates of onset, decay, and offset compared with the responses activated by the same concentrations of GABA and glycine, indicating that the affinity of propofol to GABAAR was less than that of GABA. The slow rate of onset might indicate that propofol first accumulates in the cell membrane before it binds to its receptor site. Another interpretation is that the slow onset of Ipro could result from the time required for the drug to partition from intralipid vehicle to the receptors. However, slow onset kinetics were also observed when dimethyl sulfoxide was used as the vehicle (5). Hence, it is more likely that the slow onset of Ipro reflects a slow rate of equilibration with GABAAR. These results indicated that although propofol bound to the GABAAR, it had distinct properties from GABA.
Modulation of GABAAR and GlyR by Propofol
Because propofol is highly lipophilic and binds tightly to serum proteins, the free concentration of propofol in the aqueous phase is much smaller than the total plasma concentration. It is the free concentration that is relevant for studying actions at receptors. The recommended concentration for propofol at physiological pH is 0.4 µM (clinically relevant concentrations) (25). However, in these experiments, 5 µM propofol was used to make the actions of propofol on GABA and glycine response more evident. This concentration was comparable to the concentration used in cultured murine spinal neurons (0.84–16.8 µM) (8), rat acutely dissociated hippocampal neurons (1 or 5 µM) (6), and rat cultured hippocampal neurons (1–10 µM) (7). Furthermore, propofol was evenly distributed in the brain and spinal cord during infusion (26).
Previous studies have indicated that propofol has multiple distinct modulating effects on GABAAR function (2–8,16,27), including potentiation and inhibition of IGABA. Our data demonstrate that propofol influenced GABAAR function in dissociated spinal dorsal horn neurons in similar ways. Moreover, not only IGABA, but also Igly, was modulated by propofol. A clinically relevant concentration of propofol (5 µM) potentiated both IGABA and Igly, whereas a large concentration of propofol (100 µM) inhibited IGABA and Igly. Moreover, 5 µM propofol slowed desensitization and deactivation of 100 µM IGABA. However, 5 µM propofol slowed only deactivation, without altering desensitization of 100 µM Igly. Propofol increased the amplitude of the peak IGABA and Igly, an effect that is consistent with propofol causing a decrease in current desensitization, a prolongation of current deactivation, or both. In summary, the prominent effect of propofol on IGABA and Igly was to slow current deactivation and desensitization, and it is plausible that this effect contributes to its ability to enhance GABAergic (27) and glycinergic neurotransmission (unpublished data) in the spinal cord.
Previous study indicated that the glycine response was not affected by 5 µM propofol in rat dissociated (6) and cultured (7) hippocampal neurons. The discrepancy in the effects on the glycine response between this study and previous ones may be attributable to the different types of 
subunits in the GlyR. Three different -subunit isoforms with different locations in the rat CNS have been identified. The expression of the 2 subunit, which is an embryonic form of GlyR, decreases after birth, whereas 1 and 3 expression increase. The 1 subunit is highly expressed in the spinal cord, whereas the 3 subunit is restricted to the hippocampus. The subunit diversity may serve in assembling GlyR subtypes of different agonist response properties in different regions of the CNS (28–30). These findings suggest that the sensitivity of GlyR to propofol might not be uniform throughout the nervous system.
Inhibition of IGABA by large concentrations of propofol has been demonstrated in cultured hippocampal neurons (16), but the inhibition of Igly by propofol was reported here for the first time. Previous studies have indicated that the Ipro cross-desensitized with the IGABA, but no such interactions were observed with Igly (5,16). Our recent work has demonstrated that GABAAR and GlyR could cross-inhibit each other in rat hippocampal CA1 neurons (31). Because propofol exhibits GABA mimic activity at large concentrations and because the inhibition of Igly by propofol could be eliminated by 10 µM bicuculline (Fig. 5), the inhibitory actions of propofol on Igly might result from the cross-inhibition between GABAAR and GlyR in our preparation.
In conclusion, this study demonstrated that propofol increased GABA and glycine responses in dissociated rat spinal dorsal horn neurons. As an important component of anesthesia, analgesia is involved in the spinal dorsal horn because it is a major site for fine primary afferent input associated with nociceptive transmission. Parallel spinal pathways might relay information to brain circuits that are relevant to either sensory or affective qualities of pain (13,14). Our results suggest that the propofol-induced potentiation of GABAAR and GlyR at the spinal level might contribute to its antinociceptive actions and general anesthesia (19,20).
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Acknowledgments |
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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-Aminobutyric acid-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus. J Auton Nerv Syst 1999; 75: 156–63.[ISI][Medline]
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