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Originally published In Press as doi:10.1074/jbc.M206768200 on August 23, 2002
J. Biol. Chem., Vol. 277, Issue 44, 41369-41378, November 1, 2002
The General Anesthetic Pentobarbital Slows Desensitization and
Deactivation of the Glycine Receptor in the Rat Spinal Dorsal Horn
Neurons*
Hui
Lü and
Tian-Le
Xu
From the Laboratory of Receptor Pharmacology, Department of
Neurobiology and Biophysics, University of Science and Technology
of China, Hefei 230027, People's Republic of China
Received for publication, July 8, 2002, and in revised form, August 23, 2002
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ABSTRACT |
Although many general anesthetics have been found
to produce anesthetic and analgesic effects by augmenting
GABAA receptor (GABAAR) function, the
role of the glycine receptor (GlyR) in this process is not fully
understood at the neuronal level in the spinal cord. We
investigated the effects of a barbiturate general anesthetic,
pentobarbital (PB), on the glycinergic miniature inhibitory
postsynaptic currents (mIPSCs) and the responses to exogenously applied
glycine, or taurine, a low affinity GlyR agonist, by using the
whole-cell patch-clamp technique in the rat spinal dorsal horn neurons
isolated using a novel mechanical method. Bath application of 30 µM PB significantly prolonged the decay time constant of
the spontaneous glycinergic mIPSC without changing its amplitude and
frequency. Co-application of 0.3 mM PB reduced the peak
amplitude, affected the macroscopic desensitization and deactivation of
the response to externally applied Gly in a
concentration-dependent manner. In addition, the recovery of
Gly response from desensitization was also prolonged by PB. However, PB
did not change the desensitization and deactivation kinetics of the
taurine-induced response. The GABAAR antagonist bicuculline
(10 µM) did not affect the effect of PB on the Gly
response. Thus, PB prolonged the spinal glycinergic mIPSCs by slowing
desensitization and deactivation of GlyR. Two other structurally
different intravenous anesthetics, i.e. propofol (10 µM) and etomidate (3 µM), prolonged the
duration of the glycinergic mIPSC in the rat spinal dorsal horn
neurons. In conclusion, on GlyR-Cl channel
complexes there may exist action site(s) of intravenous general
anesthetics. GlyR and glycinergic neurotransmission may play an
important role in the modulation of general anesthesia in the mammalian
spinal cord.
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INTRODUCTION |
A wide range of chemically diverse compounds, including
barbiturates, steroids, propofol, alcohols, and inhalation agents such
as halothane, isolurane, and enflurane, induce general anesthesia in
animals and humans (1). The mechanisms of general anesthesia still
remain unclear. Biochemical and electrophysiological studies have shown
that all of these drugs mimic and/or potentiate the inhibition mediated
by the GABAA1
receptor (GABAAR) in the brain and that their potency and
efficacy in exerting GABA-like and/or GABA-potentiating actions
correlate with their abilities to induce anesthesia (2). These
observations support the notion that GABAAR plays an
important role in general anesthesia.
The glycine receptor (GlyR), like GABAAR-Cl
channel complexes, is another major inhibitory receptor in the adult
mammalian spinal cord and brain stem (3). Although GlyR and
GABAAR are highly homologous in both structure and
function, we know much less about the effects of general anesthetics on
GlyR than on GABAAR. Recently, several lines of evidence
have indicated that GlyR is positively modulated by volatile ether and
alkanes, n-alcohols and chloral derivatives (4) but is much
less sensitive to the barbiturates, propofol and etomidate (ET)
(2).
One approach to understanding the receptor mechanisms of the general
anesthetics is to investigate the anesthetic-induced modulation of
receptor desensitization and deactivation. The general anesthetic
propofol (5, 6), the volatile anesthetic halothane (7), as well as the
neurosteroid 3 -21-dihydroxy-5-pregnan-20-one (THDOC) (8) and
pregnenolone sulfate (9) have been observed to affect
GABAAR deactivation and/or desensitization. However, little
information is available for the effects of general anesthetics on GlyR
kinetics. On the other hand, a piece of evidence has shown that the
anesthetic targets involved in the suppression of movement in response
to noxious stimuli may lie in the spinal cord (10-12), where glycine
is known to be a key transmitter. In the present study we investigated
the modulation of a hypnotic-anesthetic barbiturate, pentobarbital
(PB), on the glycinergic miniature inhibitory postsynaptic currents
(mIPSCs) as well as GlyR desensitization and deactivation in the
acutely dissociated rat spinal dorsal horn neurons. Knowing that PB
significantly prolonged the duration of the glycinergic mIPSCs and
affected the kinetics of GlyR, we further examined the effects of two
other widely applied intravenous general anesthetics, propofol and ET,
on the glycinergic mIPSCs. The present results suggest that
on the GlyR-Cl channel complexes action site(s) of
intravenous general anesthetics may exist, through which intravenous
general anesthetics augment the spinal inhibitory neurotransmission by
slowing the desensitization and deactivation of the GlyR.
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EXPERIMENTAL PROCEDURES |
Isolation of Neurons--
The care and use of animals used for
these experiments followed the guidelines and protocols approved by our
institutional Animal Care and Use Committee. Wistar rats (2-week-old)
were anesthetized with urethane (1 g/kg, intraperitoneal). The acutely
dissociated rat dorsal horn neurons attached with intact glycinergic
presynaptic terminals were mechanically dissociated as described
previously (13, 14). In brief, rats were sacrificed by decapitation, and the transverse slices (400 µm) of spinal cord were sectioned using a vibrotome tissue slicer (VT1000S, Leica instruments Ltd, Wetzlar, Germany). After incubated at room temperature
(22-25 °C) for 50 min in an incubation solution aerated with 95%
O2 + 5% CO2, the slices were transferred into
standard external solution. A vibration-isolation system (15) was then
used to mechanically dissociate the dorsal horn neurons. Fire-polished
glass pipette mounted on a vibrator touched lightly and vibrated
horizontally at about 5-10 Hz on the surface of the slice under the
control of a pulse generator. The vibration-dissociation lasted for
about 3 min, and then the slices were removed out of the dish. Isolated neurons attached to the bottom of the culture dish and were ready for
electrophysiological experiments within 20 min. These neurons, which
were dissociated without using any enzymes, retained some of their
original morphological features including the proximal dendritic processes.
Electrophysiology--
Whole-cell voltage-clamp recordings were
made at room temperature (22-25 °C). The 35-mm culture dish was
used as the recording chamber, which was perfused at 0.5-2.0 ml/min
with the standard external solution. Patch pipettes were pulled from
glass capillaries (Narishige, Tokyo, Japan) 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 . The liquid junction
potentials were 3-4 mV and were used to calibrate the holding
potential. Data were collected with an Axopatch 200B patch-clamp
amplifier (Axon Instruments, Foster City, CA), which was connected to a Pentium III computer equipped with Digidata 1320A and Clampex and
Clampfit software (Axon Instruments, Foster City, CA) for data
acquisition and analysis. The series resistance, estimated from optical
cancellation of the capacity transient, was 10-20 M. In most
experiments, about 70% series resistance compensation was applied.
Unless otherwise noted, the membrane potential was held at  50 mV in
the voltage-clamp studies.
Solutions and Drugs--
The ionic composition of incubation
solution was (mM): 124, NaCl; 24, NaHCO3; 5, KCl; 1.2, KH2PO4; 2.4, CaCl2; 1.3, MgSO4; 10, glucose; aerated with 95% O2, 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 Na+ channel blocker
tetrodotoxin (TTX) and 0.2 mM Ca2+ channel
blocker CdCl2 for recording glycine-activated current (IGly). For recording miniature inhibitory
postsynaptic currents (mIPSCs), bath solutions routinely contained 0.3 µM TTX, 3 µM CNQX, and 10 µM
D-AP5 to block the glutamatergic responses. The GABAA receptor antagonist bicuculline (BMI, 5 µM) was further added to this solution for recording
glycinergic mIPSCs. The osmolarity of all bath solutions was adjusted
to 325-330 mOsmol/liter with sucrose (3300, Norwood, MA). The patch
pipette solution was (mM): 120, CsCl; 20, TEA-Cl; 2, MgCl2; 1, CaCl2; 10, EGTA; 2, Na2ATP; 10, HEPES. The pH was adjusted to 7.2 with Tris base.
ET was provided by the laboratory of Anesthesia and CPB, Cardiovascular
Institute of Fuwai, CAMS, China. ET was used in an aqueous formulation
containing 35% propylene glycol diluted into normal external standard
solution. Propofol 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 the previous experiments,
did not alter the actions of propofol (5). Other drugs used in the
present experiments were from Sigma and first dissolved with ion-free
water and then diluted to the final concentrations in the standard
external solution just before use. Drugs were applied using a rapid
application technique termed the "Y-tube" method throughout the
experiments (16). This system allows a complete exchange of external
solution surrounding a neuron within 20 ms.
Data Analysis--
Mini Analysis Program (version 4.3.3, Synaptosoft Inc.) was used to analyze mIPSCs. The Kolmogorov-Smirnov
test (K-S test) was used to assess differences in mean values of mIPSCs
from different conditions. Decay kinetics was measured as the time for
the mIPSCs to decay to 37% of its peak amplitude. Current deactivation
and desensitization were fitted by exponential functions, beginning shortly after the peak of response (Clampfit 8.0, Axon Instruments, Foster City, CA). Curve fitting was performed by using simplex algorithm least-squares exponential fitting routines with single or
double exponential equations of the form I(t) = If exp(-t/ f) + Is exp(-t/s), where
If and Is are the
amplitudes of the fast and slow components, and f and
s are their respective time constants. %f was calculated according to the formula:
%f = If/(If + Is). To compare desensitization times between
different exponential conditions, we used a weighted time constant
des = (If/(If + Is) × f + Is/(If + Is) × s). Fitting the
deactivation trace began from the peak of the tail current when the
tail current appeared, otherwise the fit began from the removal of
drug(s). Recovery from desensitization was assessed using a
paired-pulse protocol (8, 17). The percentage recovery, defined as
((peak2 onset2)/(peak1 onset1)) × 100, is plotted as a function of interpulse interval and fitted to a monoexponential function. Origin
(Microcal Software) and Excel (Microsoft, Seattle, WA) were used for
data display and analysis. Statistical comparison was carried out using
Student's t test for two groups' comparison and analysis
of variance (ANOVA) for multiple comparison (as noted) with
p < 0.05 (*) or 0.01(**) considered significant
(n = number of cells). NS indicates no statistical
significance. All values represented the means ± S.E. of the mean
(S.E.).
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RESULTS |
PB Prolongs the Duration of Glycinergic mIPSCs in Dorsal Horn
Neurons--
Neurons were mainly dissociated from the deep dorsal horn
including the sacral dorsal commissural nucleus (13, 16). Most of the
neurons were medium-sized (10-15 µm in diameter) with oval or
triangular soma and 1-3 apical stem dendrites. Whole-cell recordings were obtained from these spinal dorsal horn neurons attached with intact glycinergic presynaptic terminals (13, 14). Stable recordings
could be made for up to 2 h. Most neurons (97 of 121) evaluated
exhibited mIPSCs at a holding potential of 50 mV.
The glycinergic mIPSCs were recorded in the presence of CNQX (3 µM), D-AP5 (10 µM), BMI (10 µM), and TTX (0.3 µM). Superfusing 30 µM PB significantly prolonged the duration of glycinergic
mIPSCs (Fig. 1). The decay time course of
the mIPSCs was markedly increased to 156 ± 5% of the control
(control = 16.7 ± 2.7 ms; PB = 25.7 ± 3.8 ms; n = 9, p < 0.01, Student's paired t test). However, neither the amplitude
nor the frequency of the mIPSC were significantly affected by 30 µM PB (Fig. 1D).
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Fig. 1.
Effect of PB (0.03 mM)
on glycinergic mIPSCs in spinal dorsal horn neurons. A, the
consecutive trace of glycinergic mIPSCs before (top) and 2 min during (bottom) the application of PB. B,
averaged traces of 432 and 268 glycinergic mIPSCs before and
after the addition of PB, respectively. They are superimposed for
comparison. C, cumulative probability distribution of the
decay time of glycinergic mIPSCs in control and during application of
PB. The mIPSCs decay time was significantly prolonged by PB (K-S test,
p < 0.001, n = 9). Inset
illustrates the frequency distribution of mIPSCs decay time in the
corresponding conditions. D, histogram of the mIPSCs decay
time, amplitude, and frequency in the absence (gray) or
presence (black) of PB. Each column represents the mean ± S.E. (n = 9). ** indicates significant difference
from control, p < 0.01 (Student's paired t
test).
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Effect of PB on IGly--
The effect of PB at
clinically relevant concentrations (0.1-0.4 mM) on
Gly-induced current (IGly) was investigated in
the following experiment. In contrast to the observation that the PB
increases the peak amplitude of the GABA-induced current in the same
preparation (13, 18), no increase in the IGly
amplitude was observed in the presence of 0.3 mM PB.
Instead, PB (0.3 mM) inhibited the peak
IGly significantly (Fig.
2). Meanwhile, the IGly desensitization was facilitated in the
presence of PB. Fig. 2 illustrates the ratio of the current amplitude
evoked by the Gly pulse in the presence of PB
(I(Gly+PB)) which was calculated according to
the following formula: ratio = (I(Gly + PB) IPB)/IGly, where
IPB represents the PB-evoked current. In the
present preparation, 0.3 mM PB evoked a small (<100 pA) or
no inward current. The inhibition of the peak
IGly in the presence of 0.3 mM PB
did not show an obvious difference among distinct Gly concentrations
while the ratio of change in IGly
desensitization increased in response to the raise in Gly
concentrations. Alternatively, the alteration of GlyR desensitization
by PB was dependent on the Gly concentrations.
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Fig. 2.
Effect of PB (0.3 mM)
on macroscopic desensitization: influence of Gly concentration.
A, actual currents induced by Gly at various concentrations
in the absence or presence of 0.3 mM PB. B, the
effect of 0.3 mM PB on the Gly-induced current
(IGly). Ordinate represents the ratio
to control. Black and hatched bars represent the
IGly amplitude and the weighted desensitization
time constant (des), respectively. G denotes
Gly with values expressed as micromolar concentration. For each column
n = 6-10. *, p < 0.05 (Student's
paired t test).
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Effect of PB on the IGly Macroscopic
Desensitization--
To determine quantitatively the alteration of the
desensitization kinetics of IGly induced by PB,
we applied 1 mM Gly for 15 s in the absence or
presence of 0.3 mM PB. The co-application of Gly and PB was
always preceded by the 15-s-long pretreatment of PB.
IPB was not shown in the figures. Although the
rate and extent of the IGly desensitization were
quite variable among cells, individual cell responses were consistent
from one application to the next. The desensitization of the 1 mM IGly could be accounted for with
a bi-exponential fit (Fig.
3A). The PB-induced variations of amplitude, fast and slow desensitization time constants, and %f of the IGly desensitizing
along bi-exponential were summarized in Fig. 3B. The changes
in amplitude (14 ± 1% of control, n = 12), and
fast and slow desensitization time constants (30 ± 3 and 41 ± 5%, respectively, n = 12) were statistically
significant (p < 0.05). In contrast, the changes of
either the %f or the ratio between the amplitudes of
the sustained (S) and peak (P) currents (S/P) were not (17 ± 2%
of control, p > 0.05, n = 12; 15 ± 8%, p > 0.05, n = 12).
Concurrently, the current noise was augmented in the presence of PB.
When the IGly was normalized and superimposed
(Fig. 3A), the acceleration of the desensitization by PB
could be identified. In addition, the washout of Gly with PB
co-application induced a transient inward current (rebound current)
(Fig. 3A).
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Fig. 3.
Effect of PB (0.3 mM)
on GlyR desensitization. A, left,
IGly desensitized along a bi-exponential;
middle, IGly in the presence of PB;
right, currents recorded before and after the co-application
of Gly and PB were normalized to peak amplitude for comparison of the
time courses of IGly desensitization.
B, summary of the percentage changes of
IGly produced by PB (n = 12).
Amp is the peak amplitude; is the desensitization time
constant of the IGly. f and
s are the fast and slow desensitization time constants
of the IGly in the presence of PB.
S/P is the ratio between the amplitude at 15 s
(S) and peak amplitude (P), and
%f is the relative contribution of the fast
desensitization component. Each bar represents the mean ± S.E. Arrowhead indicates the "rebound" current that
appears after the washout of the agonist. * indicates significant
difference from control, p < 0.05 (Student's paired
t test).
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We next tested whether the membrane potential influences the action of
PB on IGly desensitization. In the absence of
PB, membrane depolarization was associated with an increase in the time
constant of desensitization (des) (Fig.
4B). des
increased with increasing membrane potentials at a rate of
e-fold/54 mV. PB significantly reduced des at all
tested holding potentials. The change of des induced by
PB increased with the increase in membrane potentials. In the presence
of PB, des did not obviously change when the membrane
potentials increased. The regression slope gave a voltage dependence of
e-fold per 594 mV which was 10-fold slower than that of
IGly in the absence of PB (Fig. 4B),
indicating a significant difference between effects of PB on
desensitization at symmetrical membrane potentials (Fig. 4C)
(the reversal potential of IGly was approximate
0 mV, data not shown). In addition, the amplitude depression of
IGly induced by PB was not influenced by
membrane potential change (p > 0.05, n = 5, one-way ANOVA) (Fig. 4C). The rebound current appeared
at all tested holding potentials.
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Fig. 4.
Voltage-dependence of effect of PB on
IGly. A, currents evoked by Gly
(1 mM, 15 s) at 30 and +30 mV holding potentials are
superimposed. The desensitization was accelerated by PB at both holding
potentials. B, des obtained from 5 different
neurons were averaged and plotted against membrane voltage. A linear
relationship was observed between des and holding
potential (membrane voltage) under control ( ) and in the
presence of 0.3 mM PB ( ). The effect of PB exhibited an
obvious voltage-dependence (n = 5, two-way ANOVA,
p < 0.01). C, summary of the ratio of
amplitude and weighted desensitization time constant of
IGly in the presence of 0.3 mM PB to
control (n = 5). ** indicates significant difference
from control, p < 0.01 (Student's paired t
test).
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Comparison of PB Effects on GlyR in Different Drug Application
Modes--
To investigate the mechanism of PB's effect on GlyR
desensitization, we applied different drug application modes on the
same neurons. As shown in Fig. 5, PB
reduced peak IGly with three different application methods, and the inhibition rates were 0.69 ± 0.08, 0.94 ± 0.01, and 0.86 ± 0.05 (p < 0.05 for
all, n = 8), respectively. The weighted time constant
(des) of IGly macroscopic
desensitization was significantly reduced in the presence of 0.3 mM PB either with (Fig. 5Aa) (0.48 ± 0.05 of control, p < 0.05, n = 8) or
without (Fig. 5Ab) (0.59 ± 0.02 of control,
p < 0.01, n = 8) the preperfusion of
PB. However, with the sequential application of 0.3 mM PB
and 1 mM Gly (Fig. 5Ac), the weighted
des was increased (1.25 ± 0.09 of control,
p < 0.05, n = 8). The rebound current
occurred with the first two application modes but not the third
application mode. Therefore, PB might slow the desensitization of GlyR
(see "Discussion").
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Fig. 5.
Modulation of PB on
IGly with different drug application
modes. A, sample recordings demonstrating PB (0.3 mM) modulation of 1 mM
IGly with three different drug application modes
(a, b, c). B, summary of
the ratio of amplitude and weighted des of
IGly with three application modes of PB and
glycine demonstrated in A (n = 8). ** and *
indicate significant difference from control, p < 0.01 and 0.05 (Student's paired t test), respectively.
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PB Slows the IGly Deactivation--
A previous study
(17) has shown that the rate of GABAA receptor
(GABAAR) deactivation decreased in proportion to the extent of desensitization in the absence of anesthetics. The fact that PB
accelerated the IGly desensitization inspired us
to examine the possible coupling of the IGly
desensitization and deactivation in the presence of PB.
The rates of the IGly desensitization and
deactivation in the presence of PB were found to be quite variable
among cells. However, the change trend of individual cell responses
induced by PB was consistent. As shown in Fig.
6A, preperfusion of PB at high
concentrations (0.3 and 3 mM) evoked a visible current. A
rebound current occurred after the washout of the co-application of 1 mM Gly and PB at high concentrations (0.3 or 3 mM). The inset of Fig. 6 shows representative
superimposed rebound currents in one recording, for PB (0.03, 0.3, and
3 mM) and 1 mM Gly. The relative amplitude of
the rebound current was increased with the raise of the concentrations
of PB. In the presence of PB, the peak current of
IGly was significantly reduced (for three
concentrations of PB, p < 0.05, Student's paired
t test) (Fig. 6B). The ratio of reduction was
increased with the increase of the concentrations of PB. At a low
concentration of PB (0.03 mM), the desensitization of
IGly was not affected (ratio of weighted ,
1.05 ± 0.09, p > 0.05, Student's paired
t test). At higher concentrations, PB significantly
accelerated the desensitization of IGly (for 0.3 mM PB, ratio of weighted , 0.48 ± 0.05, p < 0.05, Student's paired t test; for 3 mM PB, ratio of weighted , 0.41 ± 0.10, p < 0.01, paired t test) (Fig.
6B). The deactivation phase of IGly
in the presence of 0.03 mM PB was fitted by bi-exponential
functions at the completion of the initial sigmoidal onset phase. PB
(0.03 mM) significantly slowed the deactivation of
IGly. The time constants of fast
(f) and slow (s) deactivating components
were increased to 1.34 ± 0.20 (p < 0.05, Student's paired t test) and 1.34 ± 0.23 (p < 0.05, Student's paired t test),
respectively (Fig. 6C).
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Fig. 6.
Effect of PB on GlyR deactivation.
A, sample recordings from the same cell illustrating the
effect of PB on the kinetics of IGly. The
inset shows the tail currents caused by washout of different
concentrations of PB plus 1 mM Gly were normalized to the
amplitude at 15 s for comparison of the tail current amplitude and
the time course of IGly deactivation.
i-iv correspond to the currents caused by the washout of 1 mM Gly, 1 mM Gly plus 0.03 mM, or
0.3 mM, or 3 mM PB, respectively. B,
summary of the ratio of amplitude and weighted desensitization time
constant of IGly in the presence of PB at
different concentrations to control (n = 5).
C, summary of the ratio of deactivation time constant of
IGly at the presence of 0.03 mM PB
(n = 6). Each column represents the mean ± S.E.
** and * indicate significant difference from control,
p < 0.01 and 0.05 (Student's paired t
test), respectively.
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PB Slows the Recovery of IGly from Paired-Pulse
Desensitization--
The recognition of the role of desensitization in
prolonging GABAAR deactivation (17) leads to the hypothesis
that anesthetics, which have slowed deactivation, might do so by
slowing recovery from the desensitized state. To assess this
possibility, previous investigators performed paired-pulse experiments
and found that recovery from the GABAAR desensitization was
delayed by these agents (6-8).
To characterize the recovery of the IGly from
desensitization, we used paired applications of Gly (1 mM,
10 s) on ten cells. The response after the first application of
Gly had an obvious reduction in peak current (Fig.
7A), suggesting that a
fraction of channels may be desensitized during the first application. The recovery of channels from desensitization gradually increased with
the interval increasing between Gly applications, and the recovery time
course of desensitization extended over several seconds. Similar to the
effects of barbiturates on GABAAR, the co-application of PB
with Gly prolonged the interval of paired pulses required for the
recovery of desensitized receptor channels (Fig. 7A). In
Fig. 7B, the average recovery of the response from desensitization plotted against the paired pulse interval illustrated that resensitization of the GlyR was slowed in the presence of 0.3 mM PB. Monoexponential fitting of the recovery showed an
increase from 3.5 to 4.5 s for the recovery time constant in the
presence of PB.
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Fig. 7.
Time-dependent recovery of
IGly from desensitization in
the absence or presence of PB. A, illustration of the
experimental protocol. Conditioning responses were obtained by applying
saturating concentration of Gly (1 mM) or Gly (1 mM) plus PB (0.3 mM) for 15 s.
Conditioning pulses were followed at various time intervals by a 5-s
application of Gly (1 mM) or Gly (1 mM) plus PB
(0.3 mM). Peak conditioning responses were normalized to
minimize the effect of current rundown. Conditioning responses are
superimposed in the left and test responses are shown in the
right. The horizontal lines located above the
current traces indicate the duration of agonist application.
B, percentage recovery, ((peak2 onset2)/(peak1 onset1)) × 100, is plotted as a function of the interpulse interval and fitted to
a monoexponential function. PB depressed the amplitude of the second
response and delayed its recovery (n = 8).
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No Effect of PB on the Desensitization and Deactivation of Low
Affinity Agonist Response--
For the low affinity agonists, such as
taurine (Tau) and  -alanine, the unbinding rate is fast enough that
the channel closing rate becomes the rate-limiting step for
deactivation, which is extremely rapid after agonist withdrawal (7, 8,
19). We took advantage of the very rapid unbinding kinetics of Tau to test whether PB altered the channel closing rate by measuring the
current deactivation rate after a 15-s application of 1 mM Tau in the absence or presence of 0.3 mM PB (Fig.
8). PB caused a decrease in the peak Tau
current, but had no effect on the desensitization (control/PB, 1.01 ± 0.06, n = 6; p > 0.05; Student's paired t test). The influence of PB on rapid deactivation of Tau
current was also neglectable (control/PB,
1.05 ± 0.06, n = 6; p > 0.05; Student's paired t test) (Fig. 8B). Furthermore,
the rebound current disappeared probably due to the rapid unbinding
rate of Tau on GlyR. Similar to that observed for the co-application of
PB and Gly, the noise of the Tau-activated current was augmented in the presence of PB.
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Fig. 8.
Effect of PB on the response to the GlyR
low-affinity agonist Tau. A, left, Tau (1 mM)-induced current (ITau)
desensitized along a single exponential; middle,
ITau of the same neuron as in the left panel to
1 mM Tau in the presence of 0.3 mM PB;
right, currents recorded before and after the co-application
of Tau and PB were normalized to peak amplitude for comparison of the
time courses of ITau desensitization and
deactivation. The inset shows that PB did not affect the
ITau deactivation. B, summary of the
effect of PB on the ITau deactivation and
desensitization (n = 5). PB and
control represent the ITau
deactivation or desensitization time constants in the presence or
absence of 0.3 mM PB, respectively. NS indicates
no statistic significance, p > 0.05 (Student's paired
t test).
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Effect of PB on IGly Is Independent of the Activation
of GABAAR--
To avoid the activation of
GABAAR by PB, the antagonist of GABAAR, BMI (10 µM), was coapplied with Gly and PB, which completely eliminated the PB-induced current. The effect of PB (0.3 mM) on 1 mM IGly,
however, was not significantly influenced by BMI treatment. As shown in
Fig. 9, differences in the ratio to
control of the peak amplitude of IGly (0.85 ± 0.05 to 0.86 ± 0.07, n = 5), the fast and slow
desensitization time constants (0.70 ± 0.11 to 0.71 ± 0.08 and 0.58 ± 0.10 to 0.60 ± 0.07, respectively,
n = 5), as well as the fast and slow deactivation time
constants (1.18 ± 0.05 to 1.16 ± 0.04 and 1.14 ± 0.03 to 1.13 ± 0.03, respectively, n = 5) were not
statistically significant between the two groups treated with or
without BMI (p > 0.05, Student's paired t
test). The variations of the IGly amplitude and
%f were also unaffected after BMI application.
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Fig. 9.
PB's action is not
GABAAR-dependent. A-a, currents
recorded before and after the co-application of Gly (1 mM)
and PB (0.3 mM), or Gly(1 mM) and PB (0.3 mM) plus BMI (0.01 mM) were normalized to peak
amplitude for comparison of the time courses of
IGly desensitization and deactivation.
b, BMI did not abolish the effect of PB on the
IGly deactivation. B, summary of
the effect of BMI on the PB's action of IGly.
Each column represents the mean ± S.E.
(n = 5). NS indicates no statistic
significance. * indicates significant difference from control,
p > 0.05 (Student's paired t test).
|
|
Comparison of PB with Propofol and ET--
The effects of two
other structurally different intravenous anesthetics, propofol and ET
(Fig. 10A), on glycinergic
mIPSCs were also studied. The decay time constants of the mIPSCs were markedly increased by 56 ± 5% (n = 9), 66 ± 20% (n = 7), and 47 ± 20% (n = 9) as compared with the control (p < 0.01, Student's paired t test) in the presence of 30 µM PB, 10 µM propofol, and 3 µM ET, respectively (Fig. 10, B and
C). The frequency of the glycinergic mIPSCs was not
significantly affected by these three intravenous general anesthetics
(Fig. 10C). In addition, propofol augmented the amplitude of
the glycinergic mIPSC.
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Fig. 10.
Effect of three intravenous general
anesthetics on glycinergic mIPSC. A, chemical structures of
three intravenous general anesthetics. B, the consecutive
traces of glycinergic mIPSCs before (left) and 2 min
(right) during the application of anesthetics.
C, changes of decay time, amplitude, and frequency of
the mIPSCs induced by 30 µM PB, 10 µM
propofol, and 3 µM ET. Each column represents
the mean ± S.E. (n = 7-9). ** indicates
significant difference from control, p < 0.01 (Student's paired t test).
|
|
| |
DISCUSSION |
In the present study, we demonstrated that the general anesthetics
PB, propofol and ET all prolonged the duration of mIPSCs in the rat
spinal dorsal horn neurons. Furthermore, PB slowed GlyR desensitization
and deactivation, which might in turn result in the prolongation of the
glycinergic mIPSCs.
Spinal GlyR as An Important Target for General
Anesthetics--
The relevant components of anesthesia (amnesia,
analgesia, unconsciousness, and immobility in response to a noxious
stimulus) have traditionally been thought to be the result of
anesthetic action in the brain. However, recent evidence seems to
provide support for a spinal control of anesthesia (10-12). In
addition, anesthetics could act on the site of brain for amnesia and
unconsciousness by decreasing the transmission of noxious information
from the spinal cord to the brain (20). Thus, the spinal cord is an
important site of anesthetic action in suppression of movement in
response to noxious stimuli (21-23).
Spinal sensory processing and its ascending transmission are under
local tonic inhibitory control which is largely mediated by the
inhibitory amino acid receptors, GABAAR and GlyR. Current available evidence, however, suggests that the potential role of the
GlyR in clinical anesthesia is likely to be restricted to the mediation
of an aspect of volatile anesthetic action (2, 24, 25). The present
results clearly show that three classic intravenous general
anesthetics, PB, propofol, and ET, all prolonged the duration of the
glycinergic mIPSCs in the rat spinal dorsal horn neurons. Previous
studies have shown that the prolongation of IPSCs duration primarily
reflects receptor gating and the unbinding of agonist rather than
diffusion of transmitter within the cleft (17, 26, 27). Therefore, the
anesthetic actions of PB produced expectantly by the reduction in both
unbinding and desensitizing rates as well as slowing the deactivation
and the recovery from paired-pulse depression of the GlyR. These
results provided a strong support for our conclusion that PB prolonged
the glycinergic mIPSCs by slowing GlyR desensitization and Gly
unbinding rate which leads to the prolongation of GlyR deactivation in
spinal dorsal horn neurons. Thus, this study suggests that GlyR is an important target for not only volatile but also intravenous general anesthetic action.
Two Discrete Mechanisms of PB Modulation on GlyR--
The present
results show that PB reduced the peak amplitude of the Gly-induced
current (IGly) and accelerated the
IGly macroscopic desensitization, and that the
extent of the macroscopic desensitization increased with the rise of PB
concentrations (Fig. 6B). These effects may be caused by two
mechanisms: one is the putative channel-blocking effect of PB and the
other is allosteric modulation.
Previous studies have shown that barbiturates at concentrations over
millimolar values can block GABAAR, leading to a reduced peak response (28-30). Molecular biological evidence has shown that
GABAAR and GlyR belong to the same ligand-gated ion channel protein superfamily (31) and that mutations of GlyR subunit (at two
amino acid positions 159 and 161) render the receptor responsive to
GABA (32), suggesting a close relationship between the two receptor
systems. Thus, it is very likely that PB acts as an open-channel
blocker on a block site of GlyR-Cl channel complexes to
accelerate the macroscopic desensitization of
IGly, similar to the effect of MK-801 on NMDA
receptor-mediated current (33) (Fig.
11). The presence of a tail (rebound)
current after washout of agonists is a direct evidence for this
hypothesis. After the washout of PB and Gly, the PB molecule binding to
the block site was removed and the undesensitized GlyR might be
re-activated by the agonist which did not have sufficient time to
unbind, thereby producing the rebound current (Fig. 11). The rebound
current was only induced by the removal of Gly and PB, and did not
occur in the sequential application mode (Fig. 5). Moreover, the
removal of Gly and PB at a low concentration (0.03 mM)
(Fig. 6A) did not induce the rebound current, indicating
that the PB blockage is state- and concentration-dependent.
The result of the voltage-dependence experiment (Fig. 4) is further in
favor of such an open-channel block mechanism. The present experiments
were performed at pH 7.4 at which 20% of PB is in anionic form (30).
When the membrane potential exceeded 0 mV, the driving force for
anionic PB molecule was directed toward intracellular, leading to a
potentiation of the blocking effect. The corollary is a more
significant acceleration of macroscopic desensitization at positive
holding potentials (Fig. 4).
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Fig. 11.
A possible modulation mechanism of PB on
GlyR. 1, the GlyR channel closes before binding the
ligand; 2, the binding of Gly and PB on GlyR leading to
the channel opening allows a large amount of Cl to flux
intracellularly. Subsequently, GlyR desensitizes, resulting in the
decrease of Cl flux. 3, a PB molecule
blocks the opening channel, which accelerates the macroscopic
desensitization of IGly. In this case, only a
small amount of Cl can pass through the channel.
4, the PB molecule is removed, and the channel reopens
which increases the Cl flux. 5, the
agonist, Gly, and the modulator, PB, are removed, resulting in the
closure of GlyR channel and the deactivation of
IGly. The thickness of the line with
arrowhead reflects the amount of Cl
flux.
|
|
On the other hand, PB might allosterically modulate the function of
GlyR, resulting in the slowness but not acceleration of GlyR
desensitization. The result that the preperfusion of PB prior to the
application of Gly caused the prolongation of the time constant of
IGly desensitization (Fig. 5Ac)
indicated the possibility of PB slowing the GlyR desensitization in the
absence of the blockage effect. On the other hand, no effect of low
concentration PB on the amplitude of glycinergic mIPSC and the
macroscopic desensitization of IGly (Fig. 6)
could be caused by the counteraction of the slight blockage effect and
the allosterical modulation due to the low concentration of PB.
According to several previous reports (34-36), the augmentation of the
current noise during co-application of PB and Gly, or PB and taurine,
which reflected channel opening with different conductance or opening
probability, hinted that a modulation site of PB existed on GlyR.
Moreover, the incomplete recovery of desensitization, which appeared in
the wash traces (Figs. 4A and 6A) also indicates
the effect of PB on the desensitization of GlyR. Due to the fact that
the GlyR deactivation induced by low-potency agonist taurine mainly
depends on agonist unbinding, the small effect of PB on GlyR
deactivation after channel activation by taurine (Fig. 7) suggests that
PB reduces the unbinding rate of Gly, leading to a slower deactivation
of GlyR. With regard to the negligible effect of PB on macroscopic
desensitization induced by taurine, a possible interpretation is that
the rapid dissociation of taurine from GlyR impairs the action of PB.
Therefore, although speculating, GlyR-Cl channel
complexes may contain not only block site(s) but also allosteric
modulation site(s) (Fig. 11), and in the presence of low concentrations
of PB, allosteric modulation is dominant (Figs. 1 and 5). Furthermore,
the effect of preperfusion and the slow recovery of
IGly desensitization indicate that other
mechanisms might contribute to the allosterical modulation of PB, which
remain to be studied.
Comparison of GlyR and GABAAR Modulation--
The
anesthetic effect of barbiturates is thought to result, at least in
part, from actions on GABAARs. Depending on the dose, PB
has three effects on GABAAR. At low micromolar
concentrations, PB potentiates the activation of GABAAR
(37). At high micromolar concentrations, PB directly gates the
GABAAR (29, 30, 37-39). At millimolar concentrations PB
blocks the opening channels of GABAAR (29, 30, 37-39). The
actions appear to be mediated via different sites (39, 40).
Similar to GABAAR, PB at high micromolar concentration (300 µM) had a significant effect on the desensitization and
deactivation of IGly by modulating the gating of
GlyR. However, the block effect of PB appeared synchronously, not
needing to reach millimolar concentration, suggesting that PB may have
a stronger block action on GlyR than that on GABAAR. Thus,
similar to GABAAR, PB can act on at least two different
sites of GlyR. Additionally, the present results clearly show that the
peak amplitude of IGly was reduced by PB at
either low or high concentrations. Since there is evidence supporting
that co-transmission between GABAergic and glycinergic transmitter
systems may occur in the spinal dorsal horn (41-43), the different
effects of PB on GABAAR and GlyR would be functionally important. At clinical relevant concentrations, PB enhances the function of GABAAR, but reduces the
IGly. This would provide a mechanism for the
modulation balance of PB, as a clinical anesthetic, between these
two inhibitory transmitters.
With respect to the reduction of macroscopic peak current of
IGly induced by PB, the acceleration of
macroscopic desensitization of IGly cannot
explain it fully due to the fact that PB (0.03 mM)
inhibited the peak IGly significantly but did
not affect the current desensitization at all (Fig. 6B).
Also, the preperfusion effect of PB (Fig. 5Ac) and the
result that the reduction of the peak IGly was
not voltage-dependent whereas the change of the desensitization was voltage-dependent (Fig. 4C)
suggest that the open-channel-block mechanism could not be the rational
explanation. Moreover, the cross-inhibition between
IGly and GABA-induced response recently observed
in the rat spinal dorsal horn neurons (43) was dependent on the agonist
concentrations (44), while the inhibition of the peak
IGly by PB was not Gly
concentration-dependent (Fig. 2B). Additionally,
the effect of PB on IGly is independent of the
activation of GABAAR because BMI, the specific
GABAAR antagonist, failed to alter the effects of PB on the
peak and kinetics of IGly (Fig. 9). Thus the
most likely explanation is the intracellular mechanisms and/or the
lipid solubility. Additional experiments are needed to address the
above mentioned possibilities.
In summary, PB acted on spinal GlyR allosterically modulating or
blocking the IGly, indicating that on GlyR both
modulation and block site(s) may exist (Fig. 11). The results presented
herein provide evidence at the cellular level for the anesthetic action of general anesthetic on inhibitory receptor in spinal cord neurons. The functional change of spinal glycinergic neurotransmission in the
presence of anesthetic may, at least in part, contribute to produce anesthesia.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Lin Xu for advice and
discussion. Drs. Xiao-Min Xu, Xiao-Ping Hu, Qi Cui, and Jian Zuo helped
with English editing. Drs. Xian-Ping Dong and Zhen-Xiong Zhang
contributed partially to Fig. 10.
| |
FOOTNOTES |
*
This study was supported in part by the National Natural
Science Foundation of China (Nos. 3970200, 30125015, 30170247), the National Basic Research Program of China (G1999054000), and the Grant
for Outstanding Young Researchers from the Ministry of Education of
China (to T.-L. Xu).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Neurobiology
and Biophysics, School of Life Sciences, University of Science and
Technology of China, P.O. Box 4, Hefei 230027, P. R. China. Tel.:
86-551-360-3510; Fax: 86-551-360-7014; E-mail:
xutianle@ustc.edu.cn.
Published, JBC Papers in Press, August 23, 2002, DOI 10.1074/jbc.M206768200
| |
ABBREVIATIONS |
The abbreviations used are:
GABA,  -aminobutyric acid;
AP-5, 2- amino-5-phosphonovaleric acid;
BMI, bicuculline;
CNQX, 6-cyano-7-nitroquinoxalne-2,3-dione;
ET, Etomidate;
GABAAR, GABA type A receptor;
GlyR, glycine receptor;
If, the amplitude of the fast component;
IGly, glycine-induced currents;
I(Gly+PB), the current amplitude evoked by the
Gly pulse in the presence of PB;
IPB, PB-evoked
current;
Is, the amplitude of the slow
component;
mIPSCs, miniature inhibitory postsynaptic currents;
K-S test, Kolomogorov-Smirnov test;
PB, pentobarbital;
Tau, taurine;
TEA, tetraethylammonium;
THDOC, 3-21- dihydroxy-5-pregnan-20-one;
TTX, tetrodotoxin.
f, the desensitizing time constant of fast component;
s, the desensitizing time constant of slow
component;
des, time constant of desensitization;
ANOVA, analysis of variance.
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ABSTRACT
INTRODUCTION