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The Vollum Institute, L474, Oregon Health Sciences University, Portland, Oregon 97201 (S.I., E.W.M., J.T.W.) and Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110 (T.J.W.)
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Summary |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Opioids have been shown to cause a potent inhibition of neurons in the
locus ceruleus (LC) in vivo in brain slices and isolated neurons; however, the kinetics of opioid action have not been described. In this study, we used acutely isolated LC neurons to
examine opioid and 
2-adrenoceptor action on potassium
and calcium currents. [Met]Enkephalin (ME),
[D-Ser2,Leu5,Thr6]enkephalin,
etorphine, and
[D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin
increased potassium conductance, whereas morphine and naloxone
were antagonists. The time constant of potassium channel activation was
~0.7 sec and was the same for each agonist. The amplitude of the
current and the time constant of decay were dependent on the agonist,
suggesting that agonist efficacy and affinity, respectively, determined
these parameters. The amplitude of potassium current induced by the
2-adrenoceptor agonist UK14304 was not significantly
different from that induced by ME, but the time constant of current
activation was half that of ME, and the decline was more rapid. When
potassium conductances were blocked with the combination of internal
cesium and external barium, opioid and 2 agonists had no
effect at potentials more negative than 
50 mV and decreased barium
currents at potentials between 40 and +20 mV. Both morphine and
clonidine caused a small inhibition of barium current. In dorsal root
ganglion cells, morphine alone had small and inconsistent effects on
the calcium current, but it always competitively antagonized the
inhibition caused by
[D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin.
The results in isolated LC neurons suggest 1) the amplitude and time
course of the opioid-induced potassium current depend on agonist
efficacy and affinity and 2) the coupling of both µ-opioid and
2-adrenoceptors to calcium channels seems to be more
efficient than that to potassium channels.
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Introduction |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Opioid inhibition of spontaneous
activity in the LC was first observed using extracellular recording
in vivo (1). In brain slices, the inhibition by both
µ-opioids and 2-adrenoceptor agonists results from an
increase in the same inwardly rectifying potassium conductance (2-4).
Experiments using cell-attached patch recording from acutely
dissociated LC cells indicated that opioids increased potassium channel
activity through a membrane-delimited pathway (5). Measurements of
opioid receptor kinetics are limited in both cell-attached patch
recording from isolated cells and whole-cell recordings in brain slices
by the slow exchange of drugs, either at the tip of patch pipettes or
by diffusion of drugs in brain slices.
Receptors, including the µ-opioid receptor and
2-adrenoceptors, that are linked with
Gi/Go-subtype G proteins are known to activate
inwardly rectifying potassium currents, decrease calcium currents, and
inhibit adenylyl cyclase, all of which have been studied in acutely
dissociated cells (6-9). Part of the opioid-sensitive current in LC
cells in brain slices has also been suggested to result from either
poor voltage control in the dendritic arbor (10) or a sodium-dependent
conductance that is depressed by opioids (11). With acutely isolated
neurons, the issue of voltage control is eliminated, and the control of
the composition of both extracellular and intracellular solutions for
the blockade of specific ion channels is improved compared with
recordings in brain slices. Acutely isolated cells also offer the
distinct advantage for the rapid application and washout of drugs (9).
Although much is known about the pharmacology of the µ-opioid
receptor and 2-adrenoceptor in the LC based on steady
state analysis, nothing is known about kinetics of opioid actions. In
this investigation, rapid applications of opioid receptor and
2-adrenoceptor agonists to acutely isolated LC neurons
were made, and the effects of these agonists on potassium and calcium
currents were measured. The results demonstrate the importance of
agonist affinity and efficacy in sculpting the time course of
opioid-mediated action.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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The method used to isolate LC neurons was based on those described for acutely isolated hippocampal neurons (9). Cells were dissociated from slices taken from animals ranging in age from 5 to 14 days. The region of the LC was microdissected from slices using 18-gauge needles under a dissection microscope. Pieces containing the LC were treated with papain (20 units/ml) at 37° for 2 min, placed in a solution containing trypsin inhibitor and bovine serum albumin for 1 min, washed twice in the dissociation solution, and triturated, and LC cells were plated on uncoated plastic petri dishes. The dissociation solution contained 82 mM Na2SO4, 30 mM K2SO4, 10 mM HEPES, 5 mM MgCl2, and 10 mM glucose, pH adjusted to 7.4 and bubbled with 100% O2. The enzyme solution was made up from the dissociation solution with papain (20 units/ml) and L-cysteine (0.33 mg/ml). Inhibitor solution was also made from the dissociation solution with the addition of trypsin inhibitor and bovine serum albumen (1 mg/ml concentration of each).
Whole-cell recordings were made with a patch-clamp amplifier using
appropriate series resistance compensation. Access resistance of 
10
M
was considered acceptable and was monitored periodically throughout the experiment. Patch pipettes were filled with 115 mM KMeSO4, 20 mM KCl, 1.5 mM MgCl2, 5 mM EGTA, 2 mM ATP, 0.3 mM GTP, and 5 mM HEPES,
pH 7.3. In some experiments, CsCl was substituted for
KMeSO4 and KCl in the internal solution. The control
external solution contained 146 mM NaCl, 5 mM
KCl, 5 mM HEPES, 2 mM CaCl2, and 4 mM MgCl2, pH was adjusted to 7.4 with NaOH, and
the osmolarity was adjusted to 320 mOsM using dextrose.
Unless otherwise stated, agonist-induced currents (inward) were
recorded in a high potassium solution containing 116 mM
NaCl, 30 mM KCl, 5 mM HEPES, 2 mM
CaCl2, and 4 mM MgCl2, pH adjusted
to 7.4, and the osmolarity was adjusted to 320 mOsM using
dextrose.
Drugs were applied by fast perfusion flow pipes. An array of four flow pipes (400-µm o.d.) fixed to a piezoelectric translator was used to change the solution perfusing the cell. Solution exchange using this method has a time constant of ~50 msec measured using the inward current evoked by high potassium solution (Fig. 1A). Each of the flow pipes was connected to a reservoir containing extracellular solution with or without drug. Solutions are warmed in the flow pipes with a heat exchanger just before solutions enter the bath and run continuously through all the pipes during the experiment. Recordings were carried out at 32°. Results are presented as the mean ± standard error and statistical comparisons were made with paired t tests, with significance p < 0.05. Saturating concentrations of agonists (ME, DAMGO, DSLET) were determined by measuring the current induced by at least two concentrations (Table 1).
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Results |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Identification of LC cells. The identification of dissociated LC neurons was based on their size and shape. Although several cell types could be distinguished in the preparation, the microdissection of the nucleus resulted in an enriched population of LC cells. These cells were large (30 × 50 µm) and multipolar with three to six major dendrites that branched into a few secondary processes. There usually were 5-15 neurons/plate that were suitable for recording. Normally, three to six plates were prepared from a single dissection.
Potassium conductance increase.
From a holding potential of
60 mV, inward potassium currents were evoked by changing the
superfusion solution from control to one containing KCl (30 mM). The current induced by changing the extracellular
potassium was used to determine the time course of solution exchange at
the cell (time constant of exchange = 0.054 ± 0.007 sec,
nine experiments; Fig. 1A). In Fig. 1A (top trace), the time
course of solution exchange is shown. All agonist-induced currents were
recorded at a membrane potential of 60 mV in the presence of 30 mM potassium. Fig. 1A (bottom trace) shows the inward current evoked when the solution was changed from the high potassium solution to a high potassium solution plus DAMGO (100 µM). After the application of DAMGO, there was a latency
to the onset of inward current of 0.15 ± 0.02 sec (six
experiments) and the current rose to a peak in 1 ± 0.16 sec (six
experiments). Repeated short applications of DAMGO (1-5 sec) resulted
in reproducible inward currents. Although there was little or no
decline in current during a single application of DAMGO (15 sec), the
current declined with repeated applications of DAMGO over a period of
5-15 min. The inward current caused by DAMGO was observed with
applications that were as short as 20 msec, and the amplitude of the
current was dependent on the duration of the application period (Fig. 1B).
Etorphine, ME, DAMGO, DSLET, and morphine. The increase in potassium current induced by opioids was further investigated by determining the kinetics of activation and inactivation of DAMGO, ME, etorphine, DSLET, and morphine.
Agonists were applied at maximal (saturating) concentrations so that the kinetics of agonist binding would not limit the rate of onset and time to peak of the response. The concentration for each agonist was chosen based on steady state concentration response curves done in brain slices (4) and from preliminary experiments (Table 1). Despite the use of a saturating concentration of agonist, the amplitude of the potassium current was dependent on the period of application (Fig. 1B). The peak amplitude became larger with increasing duration of application. Currents could be detected with a 50-msec application, and a maximum steady state current was observed with application periods of 1-5 sec (DAMGO: 5 sec =490 ± 155 pA, three experiments;
1 sec = 494 ± 156 pA, six experiments; 0.5 sec = 429 ± 149 pA, six experiments; 0.2 sec = 349 ± 115 pA, five experiments; 0.1 sec = 277 ± 98 pA, five experiments). Similar experiments with ME resulted in larger mean currents (1 sec = 817 ± 108 pA, five experiments; 0.5 sec = 723 ± 101 pA, five experiments; 0.2 sec = 610 ± 114 pA, five experiments; 0.1 sec = 539 ± 114 pA, five experiments; 0.05 sec = 375 ± 92 pA, five
experiments).
In experiments in which ME and DAMGO were applied (1 sec) to the same
cell, ME always caused an larger current than DAMGO (DAMGO = 357 ± 32, ME = 482 ± 49, seven experiments;
p < 0.05 paired t test). A more complete
analysis of the current induced by agonists was done using a 5-sec
application of agonist so that the time course of activation (
-on),
the washout (-off), and the current amplitude could be measured in
each cell (Table 2). To compare the effects of different
agonists, ME was tested in each cell along with one of the following:
DAMGO, DSLET, etorphine, or the 2-adrenoceptor agonist
UK14304. The properties of the current induced by ME was used to make
comparisons between each of the other agonists (Table 2). The
activation of potassium current induced by each of the opioid agonists
was the same (0.7 sec, Table 2). The washout, however, was different
for each. The time course of washout was not influenced by rebinding of agonist to the receptor because the recovery on ME washout (1.26 ± 0.16 sec) was not different from that when the solution was stepped
from ME to naloxone (1 µM, 1.12 ± 0.13 sec, five
experiments; p > 0.05 paired t test).
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2-Adrenoceptors.
The
2-adrenoceptor agonist UK14304 evoked a potassium
current that was similar in amplitude to that caused by ME. Unlike all
of the opioid agonists tested, however, the activation of potassium
current induced by UK14304 was significantly faster (Fig. 3B and Table
2). This observation suggests a distinct difference between the
coupling of µ-opioid receptors and 2-adrenoceptors to
potassium conductance. This observation also supports the suggestion that the rate of current activation induced by opioid agonists was not
dependent on agonist receptor binding kinetics. Clonidine has been
recognized as a partial agonist at 2-adrenoceptors on LC
cells in brain slices (12) and did not have any effect on potassium
currents in acutely isolated cells (four experiments).
Calcium conductance decrease.
When potassium conductances were
blocked with the combination internal cesium and the substitution of
BaCl2 (4 mM) for CaCl2 (2.5 mM) in the extracellular solution, ME decreased the current evoked by stepping to 0 mV from 60 mV but did not change the current
at 60 or 80 mV (Fig. 4A). Although the specific
subtype of calcium conductance was not identified, the voltage
dependence indicated that it was a high threshold conductance (Fig.
4B). As has been observed elsewhere (13), the kinetics of current activation was slowed by opioids (Fig. 4A). In addition, the inhibition of the barium current by opioids was relieved by a prepulse to +60 mV
(Fig. 4C), indicating that this action of opioids on LC neurons was
similar to the voltage-dependent inhibition of calcium currents by G
protein-linked receptors that has been observed in many different
neurons (8).
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60 and 80 mV in the presence of 30 mM
KCl in the extracellular solution, and outward potassium currents were
blocked with the use of intracellular solution containing CsCl and no
potassium. Tetrodotoxin (1 µM) blocked sodium channels so
that the step to 0 mV evoked only a calcium current (1.8 ± 0.26 nA, 10 experiments). The results illustrated in Fig. 5,
A and B, show that ME caused an inward potassium current at 60 and
80 mV and decreased the calcium current measured at 0 mV. The time
course of the activation of potassium current and the inhibition of
calcium current were comparable; however, a detailed comparison of
these kinetics was limited by the frequency of steps used to evoke
calcium currents.
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= 0.56 ± 0.11 sec,
five experiments). When the same protocol was used to investigate the
inhibition caused by naloxone (1 µM), the rise time of
the ME current was 0.5-1 min (11 experiments). Thus, the kinetics of
antagonist action seem to be linked to the affinity for the receptor.
Results comparing the action of ME and morphine on calcium, barium, and
potassium currents are summarized in Fig. 5C. Morphine had no effect on
potassium current but decreased the barium current (by 8.8 ± 1.4%) and only partially antagonized the ME-induced inhibition of both
calcium and barium currents. Naloxone (10 µM) completely
blocked all actions of ME (100 µM).
Similar results with morphine were found for the inhibition of calcium
currents in dorsal root ganglion cells. In dorsal root ganglion cells,
morphine was a weak and inconsistent agonist. In a group of 10 cells,
DAMGO (1 µM) caused an inhibition of calcium current in 6 cells (mean inhibition = 22 ± 5%), whereas morphine (50 µM) affected only 2 cells (6% and 3%). In cells that
were affected by morphine, the EC50 value was ~10
µM. Although morphine (1 µM) caused little
or no inhibition of the calcium current itself, it reproducibly
antagonized the inhibition caused by DAMGO (1 µM, Fig.
5D). Antagonism seemed to be competitive because increasing the
concentration of DAMGO 10-fold partially overcame the morphine block
(Fig. 5D). Taken together, the results from both the LC and dorsal root
ganglion indicate that morphine was a weak partial antagonist when
measuring the opioid-induced inhibition of calcium conductance.
2-Adrenoceptors.
The inhibition of barium
current by 2 agonists was examined in another set of
experiments (Fig. 5C). Clonidine caused a small inhibition of barium
current measured at 0 mV (14 ± 1.1%, five experiments), whereas
UK14304 caused a larger inhibition (28 ± 3%, seven experiments).
Thus, as was found with the opioid receptor agonists, clonidine was a
partial agonist at decreasing calcium currents but had no agonist
activity at increasing potassium conductance. These results suggest
that the coupling of both opioid receptor and
2-adrenoceptors to calcium channel inhibition may be
more efficient than to activate potassium conductance.
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Discussion |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Potassium conductance.
The potassium conductance increase
caused by G protein-linked receptors has been studied in several
different preparations. In all studies, rapid application of agonist
was followed by a latency of 50-150 msec before the onset of the
potassium current, and the potassium current rose to a peak over a
0.5-2 sec period (9, 14-17). The latency and activation time course
of potassium current evoked by opioid receptor and
2-adrenoceptor agonists in LC cells were similar to
these other preparations. The time course of the current resulting from
the activation of 2-adrenoceptors was, however, about
twice as fast ( = 300 msec) as that induced by opioid receptor
agonists ( = 700 msec). The difference in activation time seems to
be receptor dependent rather than agonist dependent because the time
course of activation was the same for all opioid agonists (Table 2). In
isolated hippocampal cells, recorded at room temperature, the
-aminobutyric acid B receptor-mediated potassium current
activated with = 225 msec when either -aminobutyric acid or
baclofen was applied in saturating concentrations (9). The latency and
the rise of the current are an indication of the relative turnover rate
of G proteins at receptor, diffusion of active G protein subunits, and
channel activation. Without proposing physical segregation of specific
receptors with potassium channels, the more rapid activation of current
induced by 2-adrenoceptor activation suggests that G
protein turnover at this receptor is greater than that at the
µ-opioid receptor.
1 sec1), then
the time course of recovery (60 sec) can be used to estimate the
affinity (Kd = k 1/k + 1) to be ~1-5 nM. The slow recovery after washout of etorphine also suggests a Kd
value in the low nanomolar range, whereas ME, DAMGO, DSLET, and
morphine would be predicted to have affinities that are 
100-fold
lower. There is a reasonable correspondence of these values with steady
state binding assays done under conditions in which low affinity states can be measured (in the presence of sodium and GTP or GTP analogs) (18,
19). In membrane preparations of C6 cells expressing µ-receptors with
guanosine-5
-O-(3-thio)triphosphate (18) and rabbit
cerebellum with guanosine-5-(
,-imido)triphosphate (19), the
Ki value for naloxone was 0.3 to 4 nM; for etorphine, 25 nM; for morphine, 132 nM to 1.4 µM; for DSLET, 5.4 µM; and for DAMGO, 279 nM to 1.6 µM. Thus, the kinetics of agonist effect observed in the
current study reflect receptor affinity with limited interference caused by diffusion barriers, metabolism, or agonist rebinding that are
present in brain slice experiments.
Subtle differences in the efficacy of agonists were identified based on
the maximum current induced by opioid agonists. The peak potassium
current amplitude was greatest for ME > DAMGO > etorphine = DSLET > morphine = naloxone. The
observation that morphine was a pure antagonist suggests that the
receptor reserve was dramatically diminished in isolated LC cells
compared with brain slice preparations. This effect was not selective
for opioid receptors because clonidine, a well known partial agonist at
2-adrenoceptors, also failed to activate a potassium
current. Thus, the experimental conditions associated with recording
from acutely dissociated cells favor the study of agonists with high
efficacy. The removal of the dendritic arbor, enzymatic or mechanical
disruption, or intracellular dialysis by the patch pipette could all
play a role in the failure to observe the activation of potassium
currents by morphine and clonidine.
The results suggest that ME has the highest efficacy, followed by
DAMGO, etorphine, DSLET, and morphine. Experiments carried out in C6
cells expressing µ receptors indicated that the morphine-stimulated guanosine-5-O-(3-thio)triphosphate binding was 0.83 times
that of DAMGO (18), indicating a reduced efficacy of morphine over other agonists. A similar rank order of agonist efficacy is also known
to occur at the 
-opioid receptor as reported in NG108-15 cells
(22). In that study, most peptide agonists had a similar efficacy
relative to etorphine (1), whereas morphine had a relative efficacy of
0.69. In each of these cell lines, high expression of opioid receptors
seemed to render the effector limiting such that only agonists with
very low efficacy, which do not saturate the effector, are
distinguished. In brain slices, differences in agonist efficacy have
been observed after only acute opioid desensitization (20) or chronic
morphine treatment (21). Morphine normally activates a potassium
current that is >90% of that induced by DAMGO, whereas after acute
desensitization or chronic morphine treatment, the morphine current was
reduced to 40-50% of maximum. Separation of agonists with high
efficacy was not possible. The results in the current study using the
activation of potassium conductance indicate that efficacies of ME,
DAMGO, DSLET, and etorphine can be distinguished in dissociated cells.
Potassium and calcium conductances.
The time course of the
opioid-induced inhibition of calcium current and the activation of
potassium conductance in the present study were similar given that the
measurement of calcium current inhibition was limited to a single point
every 5 sec. A complete study of the time course of opioid-induced
calcium channel inhibition was done using outside-out patches from
primary afferent neurons (7). There was a latency of 150 msec between
the application of DAMGO and the onset of calcium current inhibition,
which rose to a maximum with a time constant of ~1.3 sec (7). The one significant difference between the receptor coupling to potassium and
calcium channels was the fact that morphine and clonidine were partial
agonists at calcium channels and antagonists at potassium channels.
This observation suggests that the coupling of receptors to calcium
current inhibition was more efficient than the activation of potassium
current. Both actions have been shown to result from membrane-delimited
second messenger pathways (5, 7). The partial agonist activity of
morphine on the inhibition of calcium currents in LC neurons was
similar to that observed in dorsal root ganglion cells. The small and
inconsistent action of morphine on calcium currents or action
potentials in primary afferent neurons has been reported but largely
ignored (23). This inconsistency has been suggested to result from
nonselective actions or even a new opioid receptor, the 
-receptor in
rat vas deferens; however, partial agonist properties of morphine may
account for these observations (24, 25). The difference in regulation
of potassium and calcium channels suggested by the partial
agonist/antagonist action of morphine and clonidine may be exploited to
further characterize the steps between G protein-linked receptor and
these two effectors.
2-adrenoceptor agonists increase potassium and decrease calcium conductance. The kinetics of
opioid activation of potassium channels was distinguishable (slower)
from that induced by 2-adrenoceptors. Morphine was a partial agonist when measuring the inhibition of calcium conductance in
both the LC and dorsal root ganglion cells and an antagonist when
measuring the activation of potassium conductance. The rank order of
agonist efficacy to cause potassium currents was ME > DAMGO > DSLET = etorphine > morphine. In addition, recovery after washout of ligand correlated with the affinity of agonists for the
µ-opioid receptor.
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Acknowledgments |
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We thank Vu Dang for preparation of the LC neurons and Drs. Bruce Bean, MacDonald Christie, and Neil Marrion for helpful discussions and comments on the manuscript.
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Footnotes |
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Received January 23, 1997; Accepted April 13, 1997
This work was supported by National Institutes of Health Grants DA01863 (J.T.W.) and DA07415 (E.W.M.).
Send reprint requests to: J. T. Williams, Vollum Institute, L474, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201. E-mail: williamj{at}ohsu.edu
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Abbreviations |
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LC, locus ceruleus;
ME, [Met]enkephalin;
DSLET, [D-Ser2,Leu5,Thr6]enkephalin;
DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
EGTA, ethylene glycol bis(-aminoethyl
ether)-N,N,N,N-tetraacetic
acid.
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References |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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), calcium currents (
), and potassium currents (
). The
inhibition of barium and calcium currents was plotted as the fractional
inhibition of the control current [(Icontrol