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The spinal cord plays an important role in modulating anesthetic-induced suppression of nociceptive transmission. To gain some insight into the anesthetic mechanisms of propofol at the spinal level, we investigated the direct action of propofol and its modulation on the -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
Propofol (2,6-diisopropylphenol) is structurally unrelated to other anesthetics (1). In recent years, propofol has been used extensively in clinical anesthesia because of its clinical benefits of a rapid onset, clear emergence, and lack of side effects (1). Although considerable information is available on the pharmacokinetic and pharmacodynamic properties of propofol, its cellular mechanism of actions on the central nervous system (CNS) has not yet been entirely elucidated.
Recently, considerable research has been directed toward the effects of general anesthetics on the inhibitory 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 (1113), 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.
The experimental protocol was approved by the institutional care and use committee of our university. Both male and female Wistar rats (2 wk old) were anesthetized with urethane (1 g/kg intraperitoneally). The dorsal horn neurons were mechanically dissociated as described previously (18,21). In brief, rats were then killed by decapitation, and the transverse slices (400 µm) of spinal cord were sectioned by using a vibrotome tissue slicer (Vt1000S; Leica instruments Ltd, Wetzlar, Germany). After being incubated at room temperature (22°C25°C) for 50 min in an incubation solution aerated with 95% oxygen/5% CO2, the slices were transferred into standard external solution. A vibration-isolation system was then used to mechanically dissociate the dorsal horn neurons. Briefly, fire-polished glass pipette mounting on a vibrator touched lightly and vibrated horizontally at approximately 510 Hz on the surface of the slice under the control of a pulse generator. The vibration-dissoci-ation lasted for approximately 3 min, and then the slices were removed from the dish. Isolated neurons would attach to the bottom of the culture dish and be ready for electrophysiological experiments within 20 min. 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 325330 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 46 M
Concentration-Response Relationship of Ipro in the Rat Spinal Dorsal Horn Neurons The neurons were mainly dissociated from the deep dorsal horn, including the sacral dorsal commissural nucleus. In keeping with previous observations, the neurons were morphologically heterogeneous (18,22). Most of the isolated neurons were medium sized (1015 µm in diameter), with oval or triangular soma and one to three apical stem dendrites. Despite morphological heterogeneity, random recordings from these neurons were used for the experiments. 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).
The Reversal Potential of Ipro The ionic basis of the Ipro was investigated by using the ramp voltage-clamp technique. A ramp-voltage command (from a depolarizing pulse of +30 mV to a hyperpolarizing pulse of -80 mV) was applied before and during the application of 100 µM propofol (Fig. 1Ba). The voltage-dependent Na+, K+, and Ca2+ channels were blocked by adding 0.3 µM tetrodotoxin and 0.2 mM Cd2+ in the external solution and by replacing K+ in the internal solution with Cs+ and tetraethylammonium, respectively. Under this condition, the intersection of current-voltage curves directly gave the reversal potential of Ipro (Epro). The measured Epro value was 0.75 ± 2.40 mV, which is close to the Cl- equilibrium potential calculated with the Nernst equation on the basis of the extra- and intracellular Cl- concentrations. The data suggested that Ipro was carried by Cl-.
Bicuculline and Strychnine Sensitivity of Ipro
Kinetics of Ipro
Considering that the Ipro was carried by Cl- (Fig. 1B) and was sensitive to both bicuculline and strychnine (Fig. 2), the kinetics of Ipro were compared with those of IGABA and Igly. The following values were obtained from the currents induced by the same agonist concentration (100 µM). Figure 3Ba and 3Ca shows the original and the normalized currents for a better comparison of the amplitude and the kinetics of Ipro with IGABA and Igly, respectively. The currents induced by 100 µM propofol had a much smaller amplitude and longer 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 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.
Propofol facilitation of IGABA and Igly could result from the enhancement of receptor activation or the slowing of receptor desensitization and deactivation. To test this hypothesis, we studied the kinetics of IGABA (100 µM) and Igly (100 µM) in the absence or presence of propofol (5 µM). As shown in Figure 4Bb, 5 µM propofol increased 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).
The molecular action of propofol on central neuronal receptors and ion channels has received considerable attention over the past few years. Propofol has been reported to act on many kinds of receptors and ion channels in brain neurons (24). However, little information is available about the effect of propofol on the receptors and ion channels in spinal neurons. In this study we investigated the actions of propofol on inhibitory amino acid receptors, GABAAR and GlyR, in acutely dissociated rat spinal dorsal horn neurons.
Direct Activation of Cl- Current by Propofol 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 Previous studies have indicated that propofol has multiple distinct modulating effects on GABAAR function (28,16,27), 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 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).
Supported by the Grant for Outstanding Young Researchers from the Ministry of Education of China, the National Natural Science Foundation of China (Grants 30125015, 39970200, and 30170247), and the National Basic Research Program of China (G1999054000).
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