Actions of substance P on membrane potential and ionic
currents in guinea pig stellate ganglion neurons
Robert
Gilbert1,
Jennifer S.
Ryan1,
Magda
Horackova2,
Frank M.
Smith3, and
Melanie E. M.
Kelly1
Departments of 1 Pharmacology,
2 Physiology and Biophysics, and
3 Anatomy and Neurobiology,
Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada
B3H 4H7
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ABSTRACT |
Neuropeptides are known to modulate the excitability of
mammalian sympathetic neurons by their actions on various types of K+ and
Ca2+ channels. We used whole cell
patch-clamp recording methods to study the actions of substance P (SP)
on dissociated adult guinea pig stellate ganglion (SG) neurons. Under
current-clamp conditions, SG neurons exhibited overshooting action
potentials followed by afterhyperpolarizations (AHP). The
K+ channel blocker
tetraethylammonium (1 mM), the
Ca2+ channel blocker
Cd2+ (0.1-0.2 mM), and SP
(500 nM) depolarized SG neurons, decreased the AHP amplitude, and
increased the action potential duration. In the presence of
Cd2+, the effect of SP on membrane
potential and AHP was reduced. Under voltage-clamp conditions, several
different K+ currents were
observed, including a transient outward
K+ conductance and a delayed
rectifier outward K+ current
(IK) consisting
of Ca2+-sensitive
[IK(Ca)]
and Ca2+-insensitive components.
SP (500 nM) inhibited
IK. Pretreatment with Cd2+ (20-200
µM) or the high-voltage-activated
Ca2+ channel blocker
-conotoxin
(10 µM) blocked SP's inhibitory effects on
IK. This suggests
that SP reduces
IK primarily
through the inhibition of
IK(Ca) and that
this may occur, in part, via a reduction of
Ca2+ influx through
voltage-dependent Ca2+ channels.
SP's actions on
IK were mediated
by a pertussis toxin-insensitive G protein(s) coupled to
NK1 tachykinin receptors.
Furthermore, we have confirmed that 500 nM SP reduced an inward
Cd2+- and
-conotoxin-sensitive
Ba2+ current in SG neurons. Thus
the actions of SP on
IK(Ca) may be due
in part to a reduction in Ca2+
influx occurring via N-type Ca2+
channels. This study presents the first description of ionic currents
in mammalian SG neurons and demonstrates that SP may modulate
excitability in SG neurons via inhibitory actions on K+ and
Ca2+ currents.
G protein; patch clamp; tachykinin;
-conotoxin
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INTRODUCTION |
THE INVESTIGATION of sympathetic efferent
projections in a number of different mammalian species has revealed
that sympathetic postganglionic neurons innervating the heart arise
primarily from the middle cervical-stellate ganglion (SG) complex (29).
In contrast to autonomic neurons from other paravertebral ganglia, such
as the superior cervical ganglion (SCG) (1, 34, 36) and enteric
ganglion (1, 37, 41), information on the electrophysiological properties of SG neurons and their modulation by neuroactive agents is
sparse. The SG is an important site for modulation of transmission of
the sympathetic cardiomotor drive; therefore, information on the signal
transduction pathways involved in the control of ganglionic transmission is essential for our understanding of autonomic regulation of the heart.
The purpose of the present study was to examine the effects of
substance P (SP) on guinea pig SG neurons. SP is an undecapeptide that
acts as a neurotransmitter and a neuromodulator in the central and
peripheral nervous systems (27, 28). SP is found within the cell bodies
and nerve fibers of a number of different mammalian autonomic ganglia,
including the neurons of the enteric plexus (9, 27) and the neurons of
the SG in situ (13, 20). Immunohistochemical studies have indicated
that nerve terminals in the guinea pig SG may contain SP or other
peptides, including calcitonin gene-related peptide (13, 17). These
peptide-containing fibers are believed to originate from preganglionic
sympathetic neurons of the spinal cord or from sensory neurons (13).
The actions of SP in the mammalian nervous system are typically
associated with slow depolarization (26, 27, 37, 39, 41). In rat
sympathetic SCG neurons, SP-coupled signaling pathways appear to be
involved in the modulation of Ca2+
channels (35), whereas in guinea pig submucosal and celiac neurons a
decreased K+ conductance (the M
current) is primarily responsible for the slow excitatory postsynaptic
potential and depolarization produced by SP (37, 41). This
depolarization also included a component that was due to SP activation
of a nonspecific cation current (37). Although the different ion
channels underlying the modulation of action potential (AP) threshold
and excitability in mammalian SG neurons have not been resolved, SP was
found to depolarize ganglion neurons through a decreased conductance,
possibly a K+ conductance (25).
Our data provide evidence that SP depolarizes dissociated guinea pig SG
neurons via a decrease in outwardly rectifying
K+ current
(IK), primarily
the Ca2+-sensitive component
[IK(Ca)].
We show that the peptide-signaling pathway involves a pertussis toxin
(PTX)-insensitive G protein and that the inhibition of
IK(Ca) may be
mediated in part through SP inhibition of voltage-dependent
Ca2+ influx. Furthermore, we also
demonstrate the occurrence of a transient
K+ current
(IA) in guinea
pig SG neurons that was not affected by SP. Although M current has been
shown to be blocked by SP in other autonomic neurons (37), an M-type
current was rarely observed in guinea pig SG neurons in this study and
is therefore unlikely to contribute significantly to the observed
reduction in K+ conductance.
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MATERIALS AND METHODS |
Ganglion dissection and cell culture.
SG were isolated from adult guinea pigs (250-300 g) according to
Horackova et al. (18). All procedures conformed to guidelines of the
Canadian Council on Animal Care. Briefly, animals were anesthetized
with halothane (Fluothane) and decapitated. The right and left SG were
exposed rostral to the first rib, removed, and placed in bubbled PBS
solution. After removal of excess connective tissue, ganglia were
dissociated by incubation at 37°C in 5 mg/ml collagenase and 1 mg/ml trypsin. The trypsin action was terminated after 50 min by the
addition of 1 ml of newborn calf serum. Cells were then centrifuged
(1,000 g) and resuspended in 2 ml of
DMEM (GIBCO BRL, Burlington, ON, Canada) and gently triturated with a
fire-polished Pasteur pipette. Suspensions of isolated SG somata were
seeded in growth medium on collagen-coated (Boehringer Mannheim, Laval,
PQ, Canada) glass coverslips and placed in a 37°C incubator with an
atmosphere of 5% CO2-95% air.
Growth medium consisted of DMEM containing 10% newborn calf serum, 1%
penicillin-streptomycin, 10 ng/ml nerve growth factor, and 5 µg/ml
cytosine
1-
-D-arabinofuranoside, used
to inhibit the growth of nonneuronal cells (17). Neurons were
maintained in primary culture for 24-72 h and identified by
morphological and electrophysiological criteria (see
RESULTS). For experiments examining
G protein subtypes, pretreatment with PTX was accomplished by the
addition of PTX (500 ng/ml) to the culture medium at the time of cell
plating. SG neurons were exposed to PTX for at least 24 h before
electrophysiological recording. Control SG neurons were plated from the
same ganglion culture as the PTX-treated cells, and recordings of
control and PTX-treated neurons were obtained on the same experimental
day to ensure consistent experimental conditions.
Superfusion and solutions.
Cultured SG neurons grown on glass coverslips were placed in a shallow
recording chamber (2 ml) and superfused at 1-2 ml/min with
standard recording solution containing (in mM) 140 NaCl, 5 KCl, 20 Na+-HEPES, 1 MgCl2, 2 CaCl2, and 10 glucose. In
experiments examining Ca2+
currents, the external solution was (in mM) 150 tetraethylammonium chloride (TEA), 5 BaCl2, 10 HEPES,
0.8 MgCl2, and 10 glucose. In
experiments examining K+ and
Ca2+ currents, 1 µM TTX was
present in the external solution, unless otherwise stated. Standard
internal pipette solutions used in whole cell recordings contained (in
mM) 140 KCl, 0.4 CaCl2, 1 MgCl2, 20 HEPES, 1 EGTA, 5 MgATP,
and 0.3 NaGTP. For recording Ca2+
currents the internal pipette solution was composed of (in mM) 125 CsCl2, 4.5 MgCl2, 10 HEPES, 9 EGTA, 5 MgATP,
and 0.3 Tris · GTP. All solutions were maintained at pH
7.3-7.4, and osmolarity of solutions was 324-329
mosM. Ca2+
concentration was calculated using a software program based on the
algorithm of Goldstein (11). Free
Ca2+ in the standard pipette
solution was estimated at 100 nM, and in the 125 mM CsCl pipette
solution it was <20 nM.
Chemicals.
All drugs and chemicals were obtained from Sigma Chemical (Mississauga,
ON, Canada), except PTX, neurokinin A (NKA), and neurokinin B (NKB),
which were obtained from Rose Scientific (Edmonton, AB, Canada) and
Calbiochem (San Diego, CA).
Electrophysiological recording techniques.
Membrane potential
(Vm) and
currents were recorded using standard whole cell patch-clamp recording
procedures (12). Currents were recorded with an Axopatch 1-D amplifier
(Axon Instruments, Foster City, CA), filtered with a four-pole low-pass
Bessel filter (3 dB at 1 kHz), and digitized at a sampling frequency of
5 kHz. Micropipettes were prepared from borosilicate glass (Sutter
Instruments, Novato, CA) using a two-stage vertical electrode puller
(model PP83, Narishige, Tokyo, Japan). Pipette resistances were
2-3 M
when filled with standard electrophysiological recording
solutions. The tips of the electrodes were coated with beeswax to
reduce capacitance. Voltage commands and acquisition of membrane
current were accomplished using pCLAMP software (Axon Instruments).
Except where indicated, leak and capacitance subtraction were routinely employed. Series resistance compensation (90%) was used in all recordings. Liquid junction potentials (LJP) between the bath and
patch-clamp electrode were measured experimentally and determined as
the potential of the bath solution with respect to the pipette solution
(2). For whole cell recording, the
Vm of the cell was then calculated as
Vm = Vp
LJP,
where Vm is
corrected membrane potential and
Vp is pipette
potential. The LJP was 3 mV for the standard internal and external
solutions used to record K+
currents and was not corrected for in the data shown. For solutions used to record Ca2+ currents, the
LJP was 11 mV and
Vm was corrected
in the current-voltage (I-V) plots
shown. Values for cell capacitance and series resistance were obtained
from the amplifier. Series resistance was always <10 M
.
Current and voltage signals were stored on computer disk as well as on
videotape with the aid of a digital data converter (Medical Systems,
Greenvale, NY).
All experiments were conducted at room temperature (20-22°C).
Values are means ± SE. Student's unpaired
t-test was used to compare differences
between data groups unless otherwise noted. Differences between groups
were considered significant when P < 0.05.
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RESULTS |
Membrane properties of SG neurons.
Details of cell morphology, survival, and the neuropeptide content of
adult guinea pig SG neurons grown in culture for up to 3 mo have been
described previously (18). The average resting Vm
of cultured (1-3 days) SG neurons determined under current-clamp conditions was
49 ± 2 mV (n = 40). Cell capacitance averaged 42 ± 2 pF
(n = 114). Input resistance was 577 ± 212 M
(n = 5, range
52-1,350 M
). These values are similar to mean values described for dissociated rat SCG neurons and rabbit celiac ganglion neurons (1).
Injection of depolarizing current under current-clamp conditions
elicited a fast overshooting AP in SG neurons that showed adaptation (a
slow decrease in frequency of AP elicited by maintained depolarization; Fig.
1A).
Previous investigations of neurons from isolated intact rat and guinea
pig SG and from cultured SG neurons have demonstrated that, in response
to sustained depolarization, the majority of neurons fire phasically
(show adaptation), whereas a smaller proportion show little or no
adaptation and fire tonically (18, 25). Although we did not investigate
firing properties in detail in isolated cultured guinea pig SG neurons,
we found that ~80% of neurons (32 of 40) from which APs were
recorded could best be described as phasic (Fig.
1A); the remaining 20% (8 of 40) exhibited tonic properties (data not shown).

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Fig. 1.
Action potential (AP) generation and firing properties of stellate
ganglion (SG) neurons. APs were recorded in control recording solution
and after addition of 1 µM TTX
(A), 1 mM tetraethylammonium
chloride (TEA, B), or 0.2 mM
Cd2+
(C). AP traces have been
superimposed for comparison. Resting membrane potential is indicated at
left of each trace and by dashed line.
Presence of drug.
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The minimum current required to generate an AP in 22 neurons examined
was 20 ± 2 pA. The average AP amplitude, measured from the resting
Vm to the peak of
the AP, was 79 ± 6 mV (n = 38); the peak afterhyperpolarization (AHP) amplitude was
14 ± 1 mV (n = 34); the AHP duration
(measured at half-maximal AHP amplitude) was 42 ± 8 ms
(n = 27), and the AP duration measured
at resting Vm and
at 0 mV was 20 ± 3 (n = 24) and 5 ± 0.3 ms (n = 36), respectively. These values are comparable to those reported for neurons in in vitro
rat SG and SCG preparations (1, 25). The amplitude of the APs generated
by depolarizing current injection was diminished by exposure to 1 µM
TTX (Fig. 1A, n = 4). For the cell
shown in Fig. 1B, when 1 mM TEA was
added to the superfusate the resting Vm depolarized by
2 mV, the AP duration increased by 13%, and the AHP amplitude was
reduced by 41%. Similar effects were observed in two other cells
exposed to TEA and are consistent with block of voltage-dependent
K+ current. Exposure to the
Ca2+ channel blocker
Cd2+ (0.2 mM) depolarized the
Vm (11 ± 5 mV) in four of seven neurons sampled, whereas in the remaining three
neurons, Cd2+ produced no effect
or a slight hyperpolarization (1-5 mV). However, in all neurons
examined, Cd2+ consistently
decreased the AHP amplitude and duration by 43 ± 13 and 25 ± 2% (n = 7), respectively (Fig.
1C), suggesting the contribution of
a Ca2+-activated
K+ current to AP repolarization.
Actions of SP on Vm and AHP in SG
neurons.
SP (500 nM) was applied to individual SG neurons by a single pressure
ejection from a glass pipette with its tip positioned ~50 µm from
the cell. Application of 500 nM SP produced a small depolarization (7 ± 3 mV) of the
Vm in seven of
seven neurons tested, in which the mean resting
Vm was
50 ± 2 mV (Fig.
2Aa). Pressure application of standard external solution alone did not evoke
a response in SG neurons. However, when neurons were current clamped to
more depolarized potentials of
30 mV, a second SP application
produced a repeatable depolarization of 16 ± 5 mV (n = 7; Fig. 2,
Ab and
Ac), which was associated with a 27 ± 8% (n = 7) decrease
in membrane conductance. In four neurons current clamped at potentials
of
30 mV, an initial pressure application of SP evoked a 25 ± 5 mV depolarization associated with a 42 ± 8% decrease in
membrane conductance. When these same four neurons were superfused with
0.2 mM Cd2+, the SP-evoked
depolarization and conductance decrease were significantly less than
values observed with SP alone (P < 0.01, Student's paired t-test), with
SP now producing a mean depolarization of only 18 ± 5 mV
accompanied by a 19 ± 9% decrease in conductance (Fig. 2Ad).

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Fig. 2.
Effects of substance P (SP) and
Cd2+ on membrane properties of
cultured SG neurons. A: membrane
depolarization measured at 50 mV
(a) and 30 mV
(b and
c) in same neuron in response to
sequential 10-s pressure applications of 500 nM SP; in presence of 0.2 mM Cd2+, depolarizing effects of
500 nM SP are reduced (d).
B and
C: decrease in AP
afterhyperolarization (AHP) induced by 500 nM SP in another neuron in
which membrane potential was continuously clamped at resting membrane
potential (B); in presence of 0.2 mM
Cd2+, effects of 500 nM SP on AHP
are attenuated (C). Resting membrane
potential is indicated at left of each
trace and by dashed line.
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The effect of SP application on depolarization-elicited APs was
examined in Fig. 2B. Pressure
application of 500 nM SP decreased the AHP amplitude from
14.5
to
9 mV and decreased the AHP duration from 35 to 28 ms, with an increase in the AP duration (measured at the resting
Vm) from 11 to
14 ms. In six other neurons, SP produced a mean decrease in AHP
amplitude of 54 ± 15% and reduced the time to one-half AHP
inactivation by 59 ± 13% (n = 6).
Subsequent application of 500 nM SP in the presence of
Cd2+ in the neuron shown in Fig.
2C and three other neurons failed to
significantly affect the AHP.
Thus the depolarizing actions of SP on
Vm, which are
associated with a decrease in the total membrane conductance and the inhibition of AHP in SG neurons, are consistent with inhibition of
K+ current. Because the actions of
SP on Vm and AHP
were reduced in the presence of
Cd2+, which blocks
voltage-dependent Ca2+ influx,
this further suggests that the actions of SP on SG neurons may include
inhibition of
IK(Ca).
Actions of SP on whole cell
K+ currents in
SG neurons.
Figure 3 illustrates representative whole
cell currents recorded in guinea pig SG neurons using standard
recording solutions. The protocol shown in Fig. 3 consisted of 500-ms
depolarizations from holding potentials
(Vh) of
100 or
35 mV followed by test potential steps from
40 to +60 mV in 20-mV increments. In SG neurons, step
depolarizations from a
Vh of
100 mV evoked a transient outward current followed by a
sustained component (Fig. 3Aa). Depolarization of
Vh to
35
mV inactivated the transient outward current component, leaving the
sustained component intact (Fig. 3Ab). Figure
3B shows the
I-V relationship for the peak outward currents recorded at
Vh of
100
and
35 mV.

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Fig. 3.
Whole cell K+ currents in a
cultured SG neuron. A: representative
currents elicited by a series of step depolarizations from 40 to
+60 mV from a holding potential
(Vh) of
100 mV (a) or 35 mV
(b).
B: current-voltage
(I-V) relationships for peak outward
currents measured in neuron shown in A
over a full range of voltage potentials from 100 to +60 mV from
Vh = 100
mV ( ) or 35 mV ( ). C:
conductance (g)-voltage curve for transient
outward current. Conductance was obtained by subtracting currents
measured from Vh = 35 mV from currents measured from
Vh = 100
mV and calculated according to Eq. 1,
with conductance expressed as a percentage of maximum conductance
(gmax) measured at 0 mV.
Inset: representative subtracted
currents measured at 20, 0, and +20 mV.
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The K+ selectivity of the
transient outward current component was confirmed by examination of
current tails, obtained by stepping the
Vm to potentials
negative and positive to the
Vh after 10-ms step depolarizations to +10 mV (data not shown). Outward current tails
reversed at a Vm
of
71.0 ± 5.0 mV [n = 14, calculated equilibrium potential for outward
K+ current
(EK) =
84 mV] in standard recording solutions. The
reversal potential of the tails shifted positively to
48.0 ± 7.0 mV (n = 3, calculated
EK =
41.0
mV) and
24.0 ± 1.0 mV (n = 4, calculated EK =
26.0 mV) when the external
K+ concentration was increased to
20 and 50 mM, respectively. This shift of 47 mV per 10-fold change in
extracellular K+ concentration
approaches the theoretical value of 58 mV predicted by the Nernst
equation.
Figure 3C shows the
conductance-voltage plot for the peak transient outward current,
obtained by subtracting the whole cell currents activated by step
potentials of
100 to +60 mV in 20-mV increments from
Vh of
35
mV from currents activated from
Vh of
100
mV. Figure 3C
(inset) shows representative
subtracted current traces elicited with step depolarizations to
20,
0, and +20 mV. Conductance was calculated as follows
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(1)
|
where
g is the transient outward
K+ conductance,
Ip(
100) is
the peak outward current measured from a
Vh of
100
mV, and
Ip(
35) is
the peak outward current measured from a Vh of
35
mV. V is the membrane potential, and
EK is the
equilibrium potential for outward
K+ currents measured in SG neurons
(see above). The transient K+
conductance activated at a
Vm of
50
mV, and the conductance increased with incremental depolarization to 0 mV and then decreased. The decrease in conductance at potentials
positive to 0 mV primarily reflects inactivation of the transient
current (Fig. 3C, inset) but may
also reflect a small reduction in
IK(ca), resulting
from Ca2+ channel inactivation at
the depolarized
Vh of
35
mV (7, 30, 31).
In mammalian sympathetic neurons, the voltage-gated
K+ channels that mediate
IA exhibit
greater sensitivity to 4-aminopyridine (4-AP) than do
other K+ currents (3). Figure
4A shows
representative current recordings in an SG neuron. The neuron was held
at a Vh of
60 mV, and 500-ms duration steps from potentials of
100
to +20 mV were applied in 20-mV increments. The outward current evoked
by depolarization from a
Vh of
60
mV consisted of an initial transient component and a sustained current
component. Both components of the outward current were reduced by 4-AP.
However, 4-AP was more selective for the transient current in guinea
pig SG neurons, and 2 mM 4-AP essentially eliminated the transient
outward current component over the
Vm range of
50 to
20 mV (those
Vm where the
transient current activates in isolation from the sustained current).
The I-V relationship shown in Fig.
4B was constructed from the peak current traces shown in Fig. 4A and
demonstrates the reduction in transient
K+ current in the presence of 2 mM
4-AP.

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Fig. 4.
Pharmacological block of outward
K+ currents in SG neurons.
A: currents elicited by a series of
step depolarizations to +20 mV from
Vh = 100
mV recorded in absence (control) and presence of 2 mM 4-aminopyridine
(4-AP). B:
I-V relationships for peak component
of outward K+ current measured
from current traces shown in A ()
in absence (control, ) and presence of 4-AP ( ).
C: whole cell currents elicited by a
series of step depolarizations to +140 mV from
Vh = 100
mV in absence (control) and presence of 0.2 mM
Cd2+.
D:
I-V relationships for sustained
outward current component measured from traces shown in
C () in absence (control, )
and presence of 0.2 mM Cd2+ ( ).
Cd2+-sensitive current ( ) was
obtained by subtracting current records measured in presence of
Cd2+ from control currents.
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Figure 4C shows current recordings
obtained from a representative SG neuron in the presence and absence of
the Ca2+ channel blocker
Cd2+. The neuron was stepped for
500 ms from a Vh
of
100 mV to potentials of +140 mV in 20-mV increments.
Depolarization of
Vm in SG neurons elicited sustained outward K+
current, the amplitude of which showed some decline at potentials positive to +80 mV. The I-V
relationship (Fig. 4D) obtained for the current traces in Fig. 4C
demonstrates that in the presence of 0.2 mM
Cd2+ the sustained outward current
was reduced at all
Vm positive to
30 mV. The Cd2+ difference
current was obtained by subtracting currents recorded in the presence
of Cd2+ from control currents. The
Cd2+-sensitive current activated
around
30 mV and increased with depolarization to +75 mV, which
approaches the calculated equilibrium potential for
Ca2+ current (+125 mV) for these
neurons under the recording conditions used, then the current declined.
In 21 neurons tested, 0.2 mM Cd2+
reduced the outward current by 35 ± 6 and 46 ± 5% at 0 and +60 mV, respectively. In four other neurons exposed to nominally
Ca2+-free extracellular solution,
the outward K+ current was reduced
by 41 ± 11% at +60 mV, thus indicating that ~30-50% of
IK activated at
depolarized potentials in SG neurons may be due to
IK(Ca).
Actions of SP on IK in SG neurons.
Figure 5 shows current recordings from an
SG neuron before and during a 30-s pressure application of SP measured
at two different Vh. The current
records in Fig. 5Aa demonstrate the
lack of sensitivity of
IK to SP. Current
records were obtained from a neuron at a Vh of
100
mV, and the Vm
was stepped in 20-mV increments from
100 to +60 mV.
Control recordings indicate that transient and sustained
IK are activated
by depolarization in this neuron (Fig. 5Aa, top
traces). Application of 500 nM SP reduced the
sustained component of
IK but did not
appear to affect the transient component of outward conductance (Fig.
5Aa, bottom traces). The
I-V relationship for the peak
IK recorded in
the presence and absence of 500 nM SP is shown in Fig.
5B. At
Vm where the
transient component of IK was activated
in relative isolation from the sustained component (
50 to
30 mV), SP had little effect. Depolarizing
Vh to
30 mV in the same neuron produced a voltage-dependent inactivation of the
transient component of
IK yet left the
sustained IK
relatively unaffected (Fig. 5Ab, top
traces). Application of 500 nM SP reduced IK (Fig.
5Ab, bottom traces). The
I-V relationship shown in Fig. 5C for current measured at the end of
the voltage pulse demonstrates that SP now reduced
IK over the range
of potentials (
30 to +60 mV) at which sustained
IK would be
activated. For the cell shown in Fig. 5, the inhibition of
IK, measured at
+60 mV, by SP was greater at
Vh of
100
mV (31%) than at
Vh of
30
mV (20%) and may reflect some inactivation of
Ca2+ and
K+ channels at depolarized
potentials (1, 30, 42).

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Fig. 5.
Inhibition of outward K+ current
(IK) by SP.
A:
IK elicited by a
series of step depolarizations to +60 mV from
Vh = 100
mV (a) or 30 mV
(b) recorded in absence (control)
and presence of 500 nM SP. B and
C:
I-V relationships for current records
shown in A measured for peak
(B; ) and sustained
(C; #) components of
IK in absence
(squares) and presence (circles) of 500 nM SP.
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Figure 6A
shows a dose-response curve for inhibition of
IK at +60 mV by
SP. IK was
measured at the end of a 500-ms voltage pulse to +60 mV in cells held
at Vh of
60 mV. SP at 5 nM had little effect on
IK, with maximal
inhibition obtained at 500 nM SP.
IK was reduced 29 ± 5 and 33 ± 4% (n = 31) by
500 nM SP at 0 and +60 mV, respectively.

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Fig. 6.
SP inhibits IK
through interaction with NK1
tachykinin receptors. A:
concentration-response curves for inhibition of
IK by SP.
[SP], SP concentration. Each point is mean; error bars, SE;
numbers in parentheses, number of cells tested.
B: SP-induced inhibition of
IK is attenuated
in presence of NK1- selective
antagonist CGP-49823 (1 µM). Bars represent mean inhibition of
IK by SP in
neurons incubated in absence and presence of 1.0 and 0.25 µM
CGP-49823; error bars, SE. C: mean
inhibition of IK
by 500 nM SP, neurokinin A (NKA), and neurokinin B (NKB); error bars,
SE. NKA and NKB produced significantly less inhibition of
IK than SP:
* P < 0.05, ** P < 0.01 compared with SP.
Currents in B and
C were measured at end of 500-ms step
depolarization to +60 mV from
Vh = 60
mV.
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SP inhibits IK through an
NK1 tachykinin receptor.
The receptors for SP belong to the tachykinin receptor family and
include receptors classified as
NK1,
NK2, and
NK3. The preferred endogenous
ligands for these receptor types are SP, NKA (substance K), and NKB,
respectively (22). We investigated which receptor type might be
involved in SP's inhibition of
IK in SG neurons by using the nonpeptide NK1
selective antagonist CGP-49823 (33). Figure
6B summarizes the effects of CGP-49823
(0.25 and 1 µM) on the response to 500 nM SP. In the absence of the
antagonist, SP reduced
IK by 44 ± 8% (n = 11, see also Fig.
6A). However, when neurons were
exposed to 1 µM CGP-49823, the inhibition of IK by SP was
reduced to 3 ± 1.5% (n = 5). At
0.25 µM, CGP-49823 did not significantly affect SP's ability to
inhibit IK (24 ± 12%, n = 3, P > 0.05).
We also compared the effects of the agonists SP, NKA, and NKB on the
inhibition of IK
in guinea pig SG neurons. The potency order has been reported to be
SP
NKA > NKB for the
NK1 receptor, NKA > NKB
SP for the NK2 receptor,
and NKB > NKA > SP for the NK3
receptor (22, 35). Figure 6C shows the
mean inhibition of
IK produced by
500 nM SP, NKA, or NKB in 27 separate cells stepped to +60 mV for 500 ms from a Vh of
60 mV. Whereas inhibition by SP was 44 ± 8.0%
(n = 11), NKA and NKB reduced
IK by only 6.0 ± 1.0% (n = 8) and 16.0 ± 4.0% (n = 8), respectively
(P < 0.1 and
P < 0.05 vs. control). These data
indicate that SP effects on
IK are mediated
via NK1 receptors, since the
potency order for the inhibition of
IK was SP
NKA
NKB.
Actions of SP on IK(Ca) in SG neurons.
The inhibition of
IK by SP was
significantly diminished in neurons exposed to
Cd2+. Figure
7A shows
current traces recorded before and after SP application in a neuron
superfused with standard extracellular recording solution and
subsequently with solution containing 0.2 mM
Cd2+. Depolarizing voltage steps
to +60 mV were applied from a
Vh of
100
mV for 500 ms. In the presence of
Cd2+, the inhibitory actions of SP
on IK were
reduced.

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Fig. 7.
Effects of SP on Ca2+-activated
K+ current
[IK(Ca)].
A: outward current traces elicited by
a 500-ms depolarizing pulse to +60 mV from
Vh = 100
mV in an SG neuron before (control) and after a 30-s pressure
application of 500 nM SP (a); in
same neuron superfused with 0.2 mM
Cd2+, SP has no effect
(b).
B: mean inhibition of
IK by SP (500 nM)
in absence and presence of 0.2 mM
Cd+2 and 10 µM -conotoxin
(CgTx) in external recording solution; error bars, SE.
IK was measured
at end of 500-ms voltage step to 0 (open bars) or +60 mV (hatched bars)
from Vh = 60 mV. * P < 0.05, ** P < 0.01 compared with
inhibition with SP in absence of
Cd2+ or -conotoxin.
|
|
Figure 7B represents the mean
inhibition of IK
by 500 nM SP in the presence of 0.2 mM
Cd2+ and 10 µM
-conotoxin. SG
neurons were held at a
Vh of
60
or
100 mV and stepped to 0 and +60 mV for 500 ms.
IK was measured at the end of the voltage pulse. Under control conditions, SP reduced
IK by 34 ± 6 and 37 ± 5.0% at 0 and +60 mV, respectively (n = 15); however, when the neurons
were superfused with 0.2 mM Cd2+,
IK was reduced by
only 8 ± 2 and 1.4 ± 0.9% (n = 8), respectively (P < 0.01 and
P < 0.05 vs. control). Similarly, in
the presence of
-conotoxin, inhibition of
IK by SP at 0 and
+60 mV was only 12 ± 6 and 3.0 ± 3.0%
(n = 4).
Actions of SP on IK are mediated by a
PTX-insensitive G protein.
The involvement of G proteins in the signaling pathway of SP is well
established (14). To confirm that SP modulation of IK is mediated
through a G protein-coupled pathway in SG neurons, we compared cells
dialyzed with a GTP-free pipette solution containing 2 mM guanosine
5'-O-(2-thiodiphosphate) (GDP
S), which is an
antagonist of G protein activation, with results obtained using
standard pipette solution (0.3 mM GTP) (16, 35). Neurons were dialyzed for at least 10 min before pressure application of 500 nM SP. Figure
8A
summarizes the effects of GDP
S dialysis on the responsiveness of SG
neurons to SP. SP inhibited
IK (measured at
the end of a 500-ms voltage step to +60 mV from
Vh of
60
mV) by only 9.0 ± 4.0% (n = 5) in
neurons dialyzed with 2 mM GDP
S compared with 33 ± 4.0% (n = 31) in control cells
dialyzed with GTP (Fig. 8A). These
data confirm that the SP-mediated inhibition of
IK is via a G
protein-coupled pathway. To identify the type of G protein involved in
SP-mediated inhibition of
IK, we incubated
SG neurons with PTX. PTX inactivates G proteins of the
Gi,
Gz, or
Go class by catalyzing
NAD-dependent ADP ribosylation of the
-subunit (24). Overnight
(24-h) treatment with PTX (500 ng/ml) was ineffective in blocking SP
inhibition of IK
(40 ± 8.0%, n = 5; Fig.
8B) compared with control untreated
cells (43 ± 9%, n = 6). Thus we
conclude that the G protein(s) involved in SP modulation of
IK in SG neurons is not of the Gi,
Gz, or
Go class.

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Fig. 8.
Inhibition of IK
by SP is mediated by a pertussis toxin (PTX)-insensitive G protein.
A: inclusion of guanosine
5'-O-(2-thiodiphosphate) (GDP S) in pipette
attenuates SP response. Bars represent mean inhibition of
IK by 500 nM SP
with standard pipette solution (0.3 mM GTP) or with 2 mM GDP S (0 GTP); error bars, SE. In all experiments, 10 min were allowed after
break in and before recording to allow for sufficient guanine
nucleotide dialysis. B: inhibition of
IK by SP is PTX
insensitive. Bars represent mean inhibition of steady-state
IK at +60 mV by
SP; error bars, SE. Cells were cultured overnight with 500 ng/ml PTX.
Current inhibition in control neurons was recorded with standard
recording solutions. * P < 0.05.
|
|
Actions of SP on
Ca2+ channel
currents in SG neurons.
The reduction of the inhibitory effect of SP on
IK in SG neurons
pretreated with Cd2+ or the
high-voltage-activated (HVA) N-type
Ca2+ channel blocker
-conotoxin
(40) suggests that SP may act to inhibit
IK(Ca) in SG
neurons via the reduction of voltage-dependent Ca2+ influx. SP has been reported
to act on NK1 receptors inhibiting N-type Ca2+ channels in
dissociated rat SCG neurons (35). Therefore, we investigated the
effects of SP on Ca2+ channel
currents in SG neurons. Ion substitutions used to isolate Ca2+ channel currents are
described in MATERIALS AND METHODS. In
K+- and
Na+-free solutions with external 5 mM Ba2+ as the current carrier,
all K+ and
Na+ currents were blocked and on
membrane depolarization an inward current
(IBa) was
observed. Figure
9A shows
inward IBa
recorded after step depolarization to +10 mV from a
Vh of
70
mV. The inward current in this neuron was reduced by >88% by 0.2 mM
Cd2+. Similar results were
observed in all cells examined, with a mean reduction in
IBa amplitude
measured at 0 mV by Cd2+ of 96.0 ± 6.0% (n = 5). Figure
9B shows
IBa recorded
after a step depolarization to 0 mV in the absence and presence of
10 µM
-conotoxin. In the presence of
-conotoxin, >95% (97 ± 1.5, n = 9) of the inward current at 0 mV
was inhibited and showed no recovery during a 15-min postdrug recovery
period. In five other cells,
IBa at 0 mV was
inhibited 96 ± 3.0% after a 15-min exposure to
-conotoxin.
Subsequent application of 0.2 mM
Cd2+ to these cells resulted in no
further reduction in
IBa in four cells
and an 11% reduction in one cell, suggesting that current through
-conotoxin-sensitive Ca2+
channels accounts for most of the
Ca2+ channel current in SG neurons
under the conditions used in this study. Figure
9D shows the
I-V plot for the
-conotoxin-sensitive current. Inward current, from a
Vh of
90
mV, activates at around
30 mV, with peak inward current
occurring around a potential of
10 mV. However, voltage steps
from
90 mV to
Vm positive to +30 mV elicited outward
-conotoxin-sensitive currents, which may
represent Cs+ moving out of the
cell via
-conotoxin-sensitive
Ca2+ channels (7, 8).

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Fig. 9.
Pharmacological block of voltage-dependent
Ba2+ currents
(IBa) in
cultured SG neurons. A:
IBa elicited with
a step depolarization to +10 mV from
Vh = 70 mV
in absence (control) and presence of 0.2 mM
Cd2+.
B and
C: superimposed
IBa elicited with
a step depolarization to 0 mV from
Vh = 90 mV
in absence (control) and presence of 10 µM -conotoxin
(B) and 500 nM SP
(C).
D:
I-V relationship for
-conotoxin-sensitive current obtained by subtracting currents
recorded in presence of -conotoxin from control currents (neuron in
B).
E:
I-V relationship for SP-sensitive
currents obtained by subtracting currents recorded in presence of SP
from control currents (neuron in
C).
|
|
Figure 9C shows
IBa recorded at 0 mV in an SG neuron before and during application of 500 nM SP. In this
cell, SP decreased the amplitude of
IBa by 33%.
Similar results were observed in eight cells examined with an average
inhibition of IBa
by SP of 49 ± 7.7%. The I-V plot
in Fig. 9E represents the SP-sensitive inward current measured from peak inward currents recorded in the
presence of 500 nM SP. As shown for the
-conotoxin-sensitive current
in Fig. 9B, the inward SP-sensitive
current activates at
30 mV, with peak current around 0 mV. These
data indicate that
IBa in SG neurons
is carried primarily via
-conotoxin-sensitive Ca2+ channels and that SP
inhibition of Ca2+ current may
account for the observed SP-induced decrease in
IK(Ca).
 |
DISCUSSION |
These results describe the cellular mechanisms by which the
neuropeptide SP modulates ionic conductances in adult mammalian sympathetic neurons. We have used patch-clamp recording to investigate the membrane properties and whole cell
K+ and
Ca2+ currents in guinea pig SG
neurons. We have not investigated the voltage-dependent characteristics
of the TTX-dependent Na+ current
that was observed in these SG neurons. Details of
K+ and
Ca2+ currents recorded in SG
neurons are discussed below and compared with ionic conductances
described for sympathetic neurons of other vertebrate ganglia and
species.
We also report that SP depolarized guinea pig SG neurons, decreased
membrane conductance at potentials positive to resting Vm, and inhibited
the AP AHP. These actions of SP were reduced by the
Ca2+ channel blocker
Cd2+ and are consistent with
inhibition of a Ca2+-activated
K+ conductance. Voltage-clamp
analysis of currents in SG neurons revealed that SP inhibited at least
two ionic conductances: a Ca2+-sensitive
K+ conductance and an inward
Ca2+ conductance. Evidence for the
modulation of these currents by SP and the results of an investigation
of the molecular signaling pathways underlying the action of SP on
IK(Ca) are
discussed.
Voltage-dependent
K+ and
Ca2+ currents in
guinea pig SG neurons: comparison with other autonomic neurons.
The outward current in guinea pig SG neurons was selective for
K+ and consisted of several
distinct voltage-dependent K+
conductances. These conductances were identified on the basis of
voltage dependence and pharmacological blockade and consisted of a
transient outward K+ conductance,
which resembled the A-type conductance described in mammalian autonomic
neurons (3), and a delayed rectifier outward
K+ current consisting of
Ca2+-activated and
Ca2+-insensitive components.
In guinea pig SG neurons the transient outward
K+ current
(IA)
demonstrated voltage-dependent activation and was significantly inactivated at potentials near the measured resting
Vm. This current typically activated within 1 ms of step depolarizations to
Vm positive to
50 mV and reached a maximum amplitude within 10 ms. Peak
conductance was followed by a rapid phase of current decay. The
transient outward conductance was maximal after hyperpolarization and
decreased sharply as the membrane voltage, before the depolarizing steps, became less negative. The voltage dependence and kinetics of the
IA in guinea pig
SG neurons resembled the
IA described in
other mammalian sympathetic neurons (1, 3, 6). Consistent with the
pharmacological properties observed for
IA in neurons of
other mammalian sympathetic ganglia,
IA in guinea pig
SG neurons was also sensitive to 4-AP.
The predominant current expressed in all guinea pig SG neurons studied
was a sustained outward K+
current. This sustained outward current activated with a brief delay
after the onset of membrane depolarization to potentials positive to
30 mV and persisted while the depolarization was maintained. The
voltage dependence and kinetics of the sustained K+ current in guinea pig SG
neurons closely resembled delayed rectifier K+ currents described in neurons
from several different sympathetic ganglia, including the rat SCG (1,
3, 34) and guinea pig enteric ganglia (41). The delayed rectifier
outward current in guinea pig SG neurons was reversibly decreased by
the external application of 4-AP and abolished by internal dialysis
with Cs+. This is consistent with
the pharmacological properties described for delayed rectifier
K+ currents in neurons of other
mammalian autonomic ganglia (1, 41, 42). Recent voltage-clamp studies
of mammalian sympathetic neurons (1, 19), together with data from other
neuronal types (21, 42), have provided support for the existence of
several classes of Ca2+-dependent
K+ currents contributing to AP
repolarization and spike AHP. In the present study we observed a
reduction of a portion of the sustained outward
K+ current after superfusion with
Ca2+ channel blockers or nominally
Ca2+-free solution. Our data
indicate that ~35-50% of the delayed rectifier
K+ current in guinea pig SG
neurons can be attributed to the activation of
Ca2+-activated
K+ channels.
In addition to the
IK(Ca), we
occasionally recorded a small noninactivating outward current in guinea
pig SG neurons that exhibited voltage- and time-dependent properties
similar to the M current described in several other sympathetic
neurons, including the SCG neuron (5, 23), and guinea pig inferior
mesenteric ganglia (10). Because of the infrequent occurrence (<15%
of neurons) and the small amplitude of current relaxations (10 ± 5.6 pA, n = 15 with voltage steps from
Vh =
30 to
70 mV) of M-like current in cultured guinea pig SG neurons, it
is unlikely to make a significant contribution to the actions of SP
described in this study. In support of this, the difficulty in studying the M current in sympathetic neurons has been reported previously in
neurons where the M current is evident using intracellular recording
techniques (41) but was not observed in the same cell type under whole
cell patch-clamp recording techniques. This may reflect washout of the
cytosolic constituents that are required for the regulation of the M
current, a common unavoidable problem in the use of the whole cell
patch-clamp configuration (12).
We have also identified an inward
Ca2+ current in cultured SG
neurons. Guinea pig SG neurons exhibited a voltage-dependent
Ca2+ current that activated on
step depolarization to voltages more positive than
30 mV and was
abolished by 0.2 mM Cd2+ or 10 µM
-conotoxin. This current resembles the descriptions of HVA,
-conotoxin-sensitive Ca2+
currents reported in a number of mammalian autonomic neurons, including
the rat SCG neurons (30, 31, 34, 40) and the rat myenteric and guinea
pig submucosal neurons (15, 37). In mammalian sympathetic neurons the
major component (85-90%) of whole cell inward
Ca2+ current has been shown to be
carried by dihydropyridine-insensitive, high-threshold N-type
Ca2+ channels (30, 31, 34). The
remainder of the conductance has been attributed to current carried
through dihydropyridine-sensitive L-type
Ca2+ channels. The block of
>95% of the Ca2+ current by
-conotoxin in guinea pig SG neurons and the voltage dependence of
the
-conotoxin-sensitive current indicate that the whole cell
Ca2+ current observed in these
neurons is due primarily to the activation of a high-threshold N-type
Ca2+ conductance.
Actions of SP on voltage-dependent
K+ and
Ca2+ currents in
guinea pig SG neurons.
A variety of peptides including SP, somatostatin, luteinizing
hormone-releasing hormone, angiotensin II, atrial natriuretic factor,
and neuropeptide Y have been demonstrated to modulate neuronal ionic
conductances (4, 15, 18, 26, 33, 35-37, 41). In light of the
potential importance of such modulation to neuronal excitability, much
attention has been placed on understanding the underlying mechanisms of
the actions of these neuropeptides.
Under our recording conditions, SP reversibly depolarized
Vm and decreased
membrane conductance. These actions are consistent with inhibition of
an outward K+ conductance by SP in
SG neurons. In the presence of the
Ca2+ channel blocker
Cd2+, a reduction in SP's ability
to elicit membrane depolarization and a conductance decrease were
observed, suggesting that SP may exert its effects, in part, via
inhibition of
IK(Ca). Our
current-clamp studies also revealed a residual depolarization and small
conductance decrease still elicited by SP in the presence of 0.2 mM
Cd2+, which may reflect an
incomplete block of
IK(Ca) by
Cd2+ in those neurons tested
and/or actions on K+
currents distinct from the
IK(Ca). For
example, SP inhibition of a background (leak)
K+ conductance has been described
in other autonomic neurons, including guinea pig celiac and submucosal
neurons (37, 39, 41).
Our subsequent voltage-clamp studies focused on
IK(Ca) and
confirmed that the actions of SP on outward
K+ current were primarily mediated
via inhibition of IK(Ca), since SP's inhibitory actions measured at 0 and +60 mV were reduced in the
presence of 0.2 mM Cd2+ or the
N-type Ca2+ channel blocker
-conotoxin (10 µM). There was a slightly greater reduction in
SP's inhibitory action on
IK(Ca) at more
positive potentials in the presence of
Ca2+ channel inhibition, which may
reflect alterations in the driving force for
Ca2+ and decreased
Ca2+ availability at these
potentials (32). The
Ca2+-insensitive component of the
sustained outward K+ current,
evident in the presence of Cd2+,
was not significantly affected by SP application.
The identity of the receptor believed to mediate SP-induced modulation
of IK(Ca) in
guinea pig SG neurons was investigated. Three tachykinins, SP, NKA, and
NKB, each possess a defined order of potency for the three known
mammalian tachykinin receptors: NK1,
NK2, and
NK3 (22, 35). The rank order of
potency for inhibition of
IK in this study
was SP > NKA
NKB, which demonstrates an
NK1 subtype profile for tachykinin
inhibition of
IK(Ca) by SP.
We have also examined the effects of SP on the
Ca2+ channel current
(ICa) in guinea
pig SG neurons. Bath application of SP produced an inhibition of
ICa similar to
that described in frog and rat sympathetic neurons (4, 35). Because
<5% of the whole cell ICa in SG neurons
is likely to be carried via L-type
Ca2+ channels, we did not attempt
to determine the effectiveness of SP on L-type currents in isolation.
Our data indicate that SP inhibits ~50% of
ICa in SG
neurons, in which the majority of inward ICa represents
activation of
-conotoxin-sensitive HVA
Ca2+ channels. Although these
findings suggest that the effects of SP on
IK(Ca) may
represent a secondary consequence of the inhibition of
Ca2+ influx, this does not exclude
any additional direct effects of SP on
K+ currents.
Transduction mechanisms underlying IK(Ca)
inhibition.
A series of experiments were designed to further examine the potential
signaling pathway underlying SP's actions on
IK in guinea pig
SG neurons. We expected that the modulation of
IK by SP was
mediated by G protein(s), and therefore we used GDP
S and PTX to test
this hypothesis. In the presence of GDP
S, a G protein blocker, SP's
ability to inhibit
IK was
significantly diminished, thereby indicating G protein involvement. We
found no effect of PTX pretreatment on the ability of SP to inhibit
IK in SG neurons, indicating that the receptors responding to SP are not coupled to G
proteins of the Gi,
Go, and
Gz family. These findings are consistent with other reports of receptor-mediated SP actions that are
PTX insensitive. These include the inhibition of N-type Ca2+ channels in rat SCG neurons
and frog sympathetic neurons (4, 35) and the inhibition of inwardly
rectifying K+ channels in the
nucleus basalis of the rat forebrain (26), both of which occur through
interaction with an NK1 tachykinin receptor.
In summary, this study presents the first description of ionic currents
in mammalian SG neurons and demonstrates that SP, acting via
NK1 receptors coupled to a
PTX-insensitive G protein pathway, may increase excitability in
mammalian SG neurons via inhibitory actions on
K+ and
Ca2+ currents.
 |
ACKNOWLEDGEMENTS |
We thank Dr. P. S. Pennefather for valuable discussions and
suggestions, Z. Byczko for excellent technical assistance, and C. Jollimore for editing the manuscript.
 |
FOOTNOTES |
This study was funded by Medical Research Council of Canada Grants
MT-13484 to M. E. M. Kelly, MT-4128 to M. Horackova, and MT-11622 to F. M. Smith. F. M. Smith was a Heart and Stroke Foundation of Canada
Research Scholar.
Address for reprint requests: M. E. M. Kelly, Dept. of Pharmacology,
Sir Charles Tupper Medical Bldg., Dalhousie University, Halifax, NS,
Canada B3H 4H7.
Received 31 March 1997; accepted in final form 16 December 1997.
 |
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