0022-3565/97/2821-0032$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 282:32–43, 1997
Vol. 282, No. 1
Printed in U.S.A.
Pharmacological Comparison of Transient and Persistent
[3H]Dopamine Release from Mouse Striatal Synaptosomes and
Response to Chronic L-Nicotine Treatment1
SHARON R. GRADY, ELIZABETH U. GRUN, MICHAEL J. MARKS and ALLAN C. COLLINS
Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado
Accepted for publication March 6, 1997
Nicotine-evoked [3H]DA release from striatal synaptosomes is mediated by a receptor exhibiting nicotinic pharmacology (Rapier et al., 1988, 1990; Grady et al., 1992, 1994;
Rowell and Hillebrand, 1994; El-Bizri and Clarke, 1994; Lippiello et al., 1995) and is largely dependent upon calcium
influx through voltage-gated calcium channels (Soliakov et
al., 1995; Turner et al., 1993). NIC-stimulated [3H]DA release from rodent striatal synaptosomes is biphasic with one
component being a transient response with micromolar affinity for L-NIC and substantial initial release, and the other,
a persistent response of low nanomolar affinity for L-NIC but
low initial release (Grady et al., 1994). The persistent response does not require previous transient response; i.e., it is
not a residual or secondary effect of high concentrations of
L-NIC (Rowell, 1995; Grady et al., 1994). The persistent release is dependent on external calcium and is blocked by
DHbE, which indicates that it is a nicotinic response requiring calcium influx (Rowell, 1995).
It is well established that nAChRs exist in multiple forms
with somewhat different pharmacology depending on the
Received for publication September 30, 1996.
1
This work was supported by grants DA-03194 and DA-00197 from the
National Institute on Drug Abuse.
sensitization rates for both phases for agonists, Ki values for
antagonists and Ki values for low concentrations of agonists.
The results are consistent with both phases being mediated by
a single type of receptor. In addition, the effects of chronic
nicotine treatment on transient and persistent [3H]DA release
were measured. For both phases, release was decreased approximately 15% by chronic infusion of 4.0 mg/kg/hr L-nicotine.
Correlation of the results with inactivation of a portion of the
receptors rather than a reversible desensitization is discussed.
subunit composition (McGehee and Role, 1995; Sargent,
1993). Clear evidence of different pharmacology for agonists
and antagonists has been demonstrated for different combinations of subtypes expressed in Xenopus oocytes (Luetje and
Patrick, 1991; Luetje et al., 1993; Cachelin and Jaggi, 1991;
Gross et al., 1991; Cachelin and Rust, 1994; Gerzanich et al.,
1995; McGehee and Role, 1995). Electrophysiological measurements of agonist and antagonist potency for nAChRs in
rat medial habenula, interpeduncular nucleus, superior cervical ganglion and hippocampus have shown that signals can
be differentiated on the basis of pharmacology (Mulle and
Changeux, 1990; Mulle et al., 1991; Covernton et al., 1994;
Alkondon and Albuquerque, 1993, 1995). These potency variations are thought to reflect differences in subtype composition. Functional responses of nAChRs measured by ion flux
and neurotransmitter release from brain slices, cell lines and
synaptosomes also differ pharmacologically (Wonnacott et
al., 1995; Lukas, 1993; Wong et al., 1995; Sacaan et al., 1995,
1996; Gopalakrishnan et al., 1996; Clarke and Reuben, 1996).
For a subtype to form functional presynaptic receptors on
dopaminergic terminals in striatum, the message should be
detectable in substantia nigra. Message for alpha-3, alpha-4,
alpha-5, beta-2 and beta-3 has been shown to exist in sub-
ABBREVIATIONS: ACh, acetylcholine; ATR, atropine; ATX, anatoxin-a; CARB, carbachol; CYT, cytisine; DA, dopamine; DEC, decamethonium;
DFP, diisopropyl fluorophosphate; DHbE, dihydro-b-erythroidine; EPI, (1)-epibatidine; HEPES, N-[2-hydroxyethyl]-piperazine-N9-[2-ethanesulfonate]; HEX, hexamethonium; MEC, mecamylamine; MeCARB, methylcarbachol; MLA, methyllycaconitine; nAChR, neuronal nicotinic acetylcholine receptor; nBTX, neuronal bungarotoxin; NIC, nicotine; dTC, d-tubocurarine; TMA, tetramethylammonium.
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ABSTRACT
3
L-Nicotine stimulates a biphasic release of [ H]dopamine from
mouse striatal synaptosomes which does not persist after agonist is removed. Approximately 80% of the initial release is
transient and disappears with a half-time of less than 1 min; the
other 20% persists for several minutes (t1/2, 5–10 min). Both the
transient and persistent phases were investigated by 10-min
exposures to agonists with an in vitro perfusion technique. A
series of nicotinic agonists and antagonists were used to determine the pharmacological relationship of the two phases.
Parameters measured included EC50 and Vmax values and de-
1997
Transient and Persistent Dopamine Release
Methods
Materials. The following compounds were products of Sigma
Chemical Co., St. Louis, MO: acetylcholine iodide (ACh), cytisine
(CYT), (1)-nicotine-(1)-di-p-toluoyltartrate (D-NIC), carbachol iodide (CARB), tetramethylammonium iodide (TMA), atropine sulfate
(ATR), hexamethonium bromide (HEX), decamethonium bromide
(DEC), d-tubocurarine chloride (dTC), sodium chloride, potassium
chloride, calcium chloride, magnesium sulfate, potassium dihydrogen phosphate, D-(1)-glucose, ascorbic acid, pargyline and diisopropyl fluorophosphate (DFP). Nicotine hydrogen (1)-tartrate (L-NIC)
was obtained from BDH Chemicals Ltd., Poole, England. Anatoxin-a
hydrochloride (ATX), a-bungarotoxin (aBTX), (1)-epibatidine-L-tartrate (EPI), methylcarbamylcholine chloride (MeCARB), dihydro-berythroidine hydrobromide (DHbE) and methyllycaconitine citrate
(MLA) were products of Research Biochemicals International,
Natick, MA. Sucrose and N-[2-hydroxyethyl]-piperazine-N9-[2ethanesulfonate] hemisodium salt (HEPES) were obtained from Boehringer-Mannheim, Indianapolis, IN. Mecamylamine (MEC) was a
gift from Merck Sharp and Dohme Research Laboratory, Rahway,
NJ. Econo-Safe scintillation cocktail was purchased from Research
Products International Corp., Mt. Prospect, IL. [7,8-3H]Dopamine
(40–60 Ci/mmol) and [N-methyl-3H]L-nicotine (75 Ci/mmol) were
obtained from Amersham Corp., Arlington Heights, IL.
Animals. Mice of the C57BL/6J/Ibg strain obtained from the
breeding colony at the Institute for Behavioral Genetics (Boulder,
CO), were maintained on a 12-hr light/12-hr dark cycle (lights on
from 7 A.M. to 7 P.M.), and had free access to food and water. Females
between the ages of 60 and 90 days were used. All animal procedures
were in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the local animal care committee.
For chronic treatment experiments, mice were anesthetized by
injection of pentobarbital (50 mg/kg) and chloral hydrate (100 mg/kg)
and a cannula of silastic tubing was implanted in the right jugular
vein by the method of Barr et al. (1979). Mice were transferred to
individual treatment cages (15 3 15 3 30 cm) after 3 to 5 hr recovery
time. The cannulas were connected to polyethylene tubing attached
to glass syringes mounted on an Infusion Pump (Harvard Instruments, South Natick, MA), and continuous infusion with sterile
saline was started. After 2 days of saline infusion half the mice were
infused with 2 mg/kg/hr L-NIC for a day and then 4 mg/kg/hr L-NIC
for 9 to 10 days. The rest of the mice were infused with saline for the
whole treatment period. Infusion was discontinued 15 hr before
synaptosome preparation. This time period allowed for total metabolism and elimination of nicotine (Petersen et al., 1984).
Synaptosome preparation. A crude P2 synaptosomal pellet was
prepared by homogenization of the striatal tissue from one or two
mice in 1 ml of 0.32 M sucrose buffered with 5 mM HEPES (pH 7.5)
at 4°C with 16 strokes by hand in a glass-Teflon homogenizer. The
homogenate was diluted to 3 ml with the HEPES-buffered sucrose
and centrifuged at 3000 3 g for 10 min. The S1 supernátant was then
centrifuged at 12,000 3 g for 20 min. The resulting P2 pellet was
resuspended in perfusion buffer (128 mM NaCl, 2.4 mM KCl, 3.2 mM
CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4z7H2O, 25 mM HEPES, pH
7.5, 10 mM glucose, 1 mM ascorbate, 0.01 mM pargyline).
3
L-[ H]Nicotine binding. A portion of the P2 synaptosomal preparation from each mouse in the chronic treatment study was frozen
(220°C) for assay after completion of the study. Particulate fractions
were prepared from P2 synaptosomal preparations combined from
two animals. Assays were conducted as described previously (Marks
et al., 1993a) with 20.8 6 1.3 nM L-[3H]NIC purified by the method
of Romm et al. (1990), with a 30 min incubation at 22°C. Nonspecific
binding was determined in the presence of 10 mM L-NIC.
[3H]Dopamine uptake. Synaptosomes from one mouse suspended in 0.8 ml perfusion buffer were incubated at 37°C for 10 min.
[3H]DA was added (4 mCi for an approximate final concentration of
0.1 mM) and the suspension was incubated for an additional 5 min.
Aliquots (0.08–0.09 ml) were collected with mild suction onto 7-mmdiameter type A/E glass-fiber filters cut from larger sheets (Gelman
Sciences, Ann Arbor, MI) and washed once with 0.5 ml perfusion
buffer. These filters were then transferred to the perfusion apparatus. For experiments with ACh as agonist, the synaptosomes were
treated with 100 mM DFP during the uptake procedure.
Perfusion and release. The perfusion apparatus has been described previously (Grady et al., 1992). For these experiments, all
conducted at room temperature, synaptosomes on 7-mm filters were
placed on top of 13-mm-type A/E glass-fiber filters (Gelman Sciences,
Ann Arbor MI) and perfused with buffer at a rate of 0.6 ml/min for 10
min before fraction collection was started. Fractions were collected
for 18 or 30 sec depending on the experiment. Released radioactivity
was primarily [3H]DA (Rapier et al., 1988).
Data analysis. Each sample was plotted as cpm vs. fraction
number or time. An example of such data is shown in figure 1. Basal
release was calculated as a single exponential decay, f 5 aze2bzt, with
fractions collected before and after agonist exposure (first 5 and last
7 fractions of fig. 1A). Curves were fit using the nonlinear least
squares algorithm in SigmaPlot 5.0 (Jandel Scientific, San Rafael,
CA). The calculated base line at time t was subtracted from total cpm
at time t for each fraction and the resulting cpm released above base
line were normalized to base line at time t to give units of release per
fraction ([total cpm 2 base line]/base line). For short agonist exposures (not illustrated here), units of release for fractions with cpm
above base line during the agonist exposure were summed for total
release. For longer exposures of agonist, [3H]DA release is biphasic
(Grady et al., 1994), so data as units per fraction were fit to a
double-exponential equation:
f 5 vT z e2dT z t 1 vP z e2dP z t
(1)
with use of SigmaPlot 5.0 where f is measured release; vT and vP are
maximum release for the first (transient) and second (persistent)
phases, respectively; dT and dP are desensitization rates for the first
and second phases; and t is the time of agonist exposure. Figure 1B
illustrates this curve fit for four experiments where synaptosomes
were exposed to 10 mM L-NIC for 10 min. For purposes of the curve
fit, the first fraction is taken as the peak of response, and the time of
each fraction is taken as the midpoint of the interval of collection.
For experiments in which data for several concentrations of an
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stantia nigra of mice (Marks et al., 1992; Marks, M. J.,
unpublished results); therefore, it is possible that the transient and persistent phases of [3H]DA release from mouse
striatal synaptosomes are the result of stimulation of two
different subtypes of nAChR. In this case, the persistent and
transient responses may express different pharmacologies.
Alternatively, the two responses could be mediated by a
single receptor which exists in two states. Such a two-state
desensitizing model was originally proposed by Katz and
Thesleff (1957) (see also Ochoa et al., 1989; Changeux, 1990),
and subsequently this two-state model was shown to adequately explain the binding properties of L-[3H]NIC to rat
brain membrane (Lippiello et al., 1987). Support for the single-receptor hypothesis comes from the slow onset of the
persistent response (Rowell, 1995), and the ability of low
concentrations of L-NIC to antagonize the transient response
evoked by higher concentrations of the agonist (Grady et al.,
1994; Lippiello et al., 1995). To determine whether it is likely
that the two phases of [3H]DA release are the result of
stimulation of two different receptor populations or kinetic
manifestations of one receptor subtype, the pharmacology of
the transient and persistent phases of agonist-induced
[3H]DA release were compared.
33
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Grady et al.
Vol. 282
Hill equation with double-exponential decay where the Hill coefficients were set to 1:
f 5 ~~An z VT!/~An 1 KTn!!e2~~A
n
z DT!/An1KTn!)t
1 ~~An9 z VP!/~An9 1 KPn9!!e2~~A
n9
z DP!/~An91KPn9!!t
(3)
f 5 ~N z V!/~Ka~1 1 ~I/Ki!! 1 N!
(4)
or noncompetitive inhibition:
f 5 ~N z V!/~Ka~1 1 I/Ki!! 1 N~1 1 I/Ki!
Fig. 1. [3H]DA release stimulated by L-NIC. Graph A represents data
from one filter of striatal synaptosomes exposed to 10 mM L-NIC for 10
min starting at the time indicated. Fractions were collected every 30
sec. The dashed line is the base line calculated from the first five and
the last seven fractions. Graph B presents data from graph A and three
other experiments plotted as units of release per fraction versus time of
L-NIC exposure starting at the peak fraction. The line drawn is the fit to
a double-exponential decay equation (see “Methods” for details of
calculations).
(5)
where n 5 L-NIC concentration (1 mM), I 5 antagonist concentration
V 5 Vmax for L-NIC calculated from agonist parameters setting 1 mM
L-NIC response to 100 (171 and 108.5 for transient and persistent
phases, respectively), Ka 5 activation constant for L-NIC for each
process (KT 5 0.71 and KP 5 0.085, respectively) and Ki 5 the
inhibition constant for the antagonist. The parameters used for
L-NIC were calculated by the simultaneous fit procedure (see fig. 2).
agonist were collected under conditions of long (5–10 min) exposure,
the following method was used to determine activation constants and
maximum initial release for each process. The parameters vT and vP
were calculated as described above for various concentrations of an
agonist. These data were then fit by SigmaPlot 5.0 to the Hill equation:
f 5 ~Vm z An!/~Kmn 1 An!
(2)
where f 5 vT or vP, A 5 agonist concentration, Km 5 agonist concentration at half-maximal release (referred to as KT and KP for transient and persistent release, respectively), Vm 5 maximal release for
each (referred to as VT and VP, respectively) and n 5 the Hill
coefficient (referred to as n for transient release and n9 for persistent
release). All parameters are reported 6 S.E.M.
An alternative method was used which enabled simultaneous
calculation of maximum initial release, activation constants and
desensitization rates for both phases. Units released per fraction
were calculated as described above. Means for several experiments
for 12 to 15 time points were determined for six to nine concentrations of an agonist. These data were then fit by SigmaPlot 5.0 to the
Fig. 2. Simultaneous fit of [3H]DA release data for multiple concentrations of L-NIC. Data points are means of four experiments calculated as
units of release per fraction plotted versus time from the peak fraction.
Lines are the fit by SigmaPlot 5.0 to the Hill equation for two processes,
with both processes decaying exponentially, setting the Hill coefficients
to 1 (see “Methods” for equation).
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where A is the agonist concentration, VT and VP are the maximum
release values for the two processes, KT and KP are the activation
constants for the two processes, DT and DP are the desensitization
rates for the two processes, n and n9 are the Hill coefficients and t is
time in minutes of agonist exposure at the midpoint of each fraction.
The results of this calculation are illustrated in figure 2 with L-NIC
as agonist.
To determine Ki values for nicotinic antagonists for the two phases
of agonist-stimulated [3H]DA release, data were collected by use of 1
mM L-NIC as agonist with an exposure time of 10 min. The concentration of antagonist was varied on different filters within each
experiment and synaptosomes were exposed to antagonist for 12 min
before and during the L-NIC exposure. Base lines were calculated,
and units of release per fraction were determined as above. These
data were fit to the double-exponential decay equation to determine
maximum release for the two processes (vT and vP) in the presence of
various concentrations of inhibitor. These parameters were compared with controls from the same experiments to determine %
control response. To determine Ki values for the two processes, the %
control data were fit by use of SigmaPlot 5.0 to the equations for
competitive inhibition:
1997
Transient and Persistent Dopamine Release
Results
both recover, although in 20 min the transient phase has
recovered to 55%, whereas the persistent phase has recovered to 80% of the original value by use of the base-line
normalizing calculation. Therefore, it is likely that the two
phases of release are dependent upon some aspects of function of the nicotinic receptor or the release mechanism,
rather than any effect of depletion.
To gain information on whether transient and persistent
[3H]DA release may be mediated by different types of nicotinic receptor, the effects of antagonist concentration on initial rates for transient (vT) and persistent (vP) responses were
determined. Synaptosomes were exposed to various concentrations of antagonist for 12 min before and during exposure
to 1 mM L-NIC for 10 min. Data from two to six experiments
per concentration were fit to the double-exponential decay
equation to determine vT and vP. Based on previously published data on inhibition of [3H]DA release at 50 mM L-NIC
(Grady et al., 1992) and inhibition of L-[3H]NIC binding and
86
Rb1 efflux (Marks et al., 1993b) dTC and DHbE appear to
be competitive antagonists. HEX, DEC and MEC are likely
noncompetitive, although DEC may have some competitive
component. MLA is probably competitive in this system
(Marks, M. J., unpublished results). Inhibition constants
were calculated assuming competitive inhibition for dTC,
DHbE and MLA, and noncompetitive inhibition for HEX and
DEC. These results are presented in figure 5. A similar
experiment was conducted with the noncompetitive antagonist MEC. Because this compound is an open-channel
blocker, there is generally some response before the onset of
antagonist action. The curve-fit adds this response to the
transient portion of the release, which leads to an artifactually high Ki value, so this approach with MEC was abandoned. Regression analysis (see fig. 5F) for comparison of Ki
Fig. 3. Effects of calcium, cadmium and
various antagonists on L-NIC-evoked
[3H]DA release. Data presented are from
representative single filters and, for all
graphs, are plotted as total cpm 2 calculated base line for each fraction and
are not normalized to base line. Data for
each filter are offset by 1000 cpm for
clarity. The concentration and duration
of L-NIC exposure are as indicated on
each plot. For graphs A and B, 18-sec
fractions were collected. For graphs C
through F, 30-sec fractions were collected. For graph E, synaptosomes were
incubated at 37°C for 30 min with or
without 1 mM aBTX before the addition
of [3H]DA.
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A preliminary characterization of the effects of nicotinic
antagonists and external calcium and cadmium on both transient and persistent phases was conducted by a long exposure
to L-NIC. The results presented in figure 3, A and B, show
that both transient and persistent responses are dependent
on external calcium and are almost totally inhibited by 200
mM cadmium. Both responses are inhibited by the nicotinic
antagonists DHbE (fig. 3C) and MEC (fig. 3D), and neither
are inhibited by aBTX (fig.3E) or the muscarinic antagonist,
ATR (fig.3F).
In experiments with synaptosomes as a model, depletion of
neurotransmitter and of labeled tracer versus endogenous
compound are always questions. To assess the extent of these
possible problems, synaptosomes were exposed for 1 min to
10 mM L-NIC at various time intervals after uptake was
complete. Figure 4 (inset graph) shows that, although
[3H]DA is being released continually during the perfusion,
the base line normalizing procedure adequately corrects for
any decrease in specific activity over the course of 40 to 50
min, in the absence of prior agonist exposure. The possibility
exists that the two phases of release are representative of two
pools of DA-containing vesicles with differing specific activity. If two such pools exist, the pool with higher specific
activity would be depleted by the transient release, leaving
the pool with lower specific activity to be released as the
persistent phase. This appears to be a change in the amount
of DA released when actually it is only a change in specific
activity. In this case, the transient release would not be
recoverable without uptake of additional [3H]DA. Figure 4
presents a series of experiments in which a 5-min exposure to
10 mM L-NIC was followed by a second 5-min exposure with
varying intervals between the two exposures. The two phases
35
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Grady et al.
Vol. 282
values for inhibition of transient versus persistent response
for five antagonists gave a correlation coefficient of 0.98 (P ,
.005) with a slope of 0.89 and an intercept of 20.30. This
correlation indicates that the effects of these antagonists on
the transient and persistent phases of [3H]DA release were
similar and could be mediated by the same receptor.
The effects of prolonged exposure to nine nicotinic agonists
were also investigated. Dose-response curves for each agonist
were determined by 10-min exposure of aliquots of synaptosomes to various concentrations of agonist. The results obtained at each concentration were fit to the double-exponential decay equation as described under “Methods.” These
curves for the transient phase release (vT) and the persistent
phase (vP) for the nine agonists are presented in figure 6. All
nine nicotinic agonists activated both persistent and transient responses in a concentration-dependent, saturable
manner. Because of the tendency for decreased response at
high agonist concentrations (e.g., 100 mM L-NIC), data were
fit to the Hill equation setting Vmax to the highest actual
release obtained. Parameters 6 S.E.M. for the curve fits are
given in table 1. Comparison of parameters VT and VP indicate that several of these compounds are partial agonists.
Those that have the lowest efficacy for transient release
(CYT and D-NIC) are also partial agonists for persistent
release; however, the difference between the most and least
efficacious agonist is greater for transient release (about
5-fold) than persistent release (about 2-fold). The ratio of
activation concentrations for transient and persistent release
(KT/KP) varied about 6-fold with L-NIC and D-NIC having a
noticeably higher ratio and CYT and ACh a lower ratio than
the other compounds. None of the Hill coefficients calculated
were significantly higher than 1; most tended to be lower
than 1, perhaps because of the curve-broadening effect of
using averaged data points or the uncertainty of Vmax values.
Calculating desensitization rates by an analogous proce-
dure (fitting dT and dP to the Hill equation), proved less
successful because of high errors in the desensitization parameters calculated from lower concentrations of agonists.
To reduce these errors, simultaneous fit of data from all
concentrations of an agonist was performed as described
under “Data Analysis,” setting the Hill coefficients to 1. Parameters 6 S.E.M. calculated by this method for all nine
agonists are given in table 3. The maximum release parameters (VT and VP) calculated by the simultaneous fits (table 2)
were all similar to those estimated previously (table 1). Values for potency of stimulating transient release (KT) for all
nine agonists, as well as values for potency of stimulating
persistent release (KP) for six of the agonists, were not significantly different from the previous calculation. Values of
KP for three agonists, D-NIC, CARB and TMA, were significantly higher by this curve-fitting procedure as indicated in
table 2. Maximal desensitization rates for the transient response varied about 2-fold. The rates measured for ACh,
TMA and CARB were significantly higher than rates of desensitization for the other agonists. Maximal desensitization
rates for the persistent response varied less than 2-fold
among the agonists, and none were significantly different by
one-way analysis of variance.
One of the properties of a desensitizing nAChR is that
response can be inhibited by low concentrations of agonist.
This property has been established for L-NIC-induced
[3H]DA release from mouse striatal synaptosomes (Grady et
al., 1994). To determine inhibition constants for agonists as
inhibitors of transient release, the following protocol was
used. After uptake of [3H]DA, the synaptosomes were perfused with buffer containing varying concentrations of agonist for 15 to 20 min. This interval is approximately ten times
the t1/2 for onset (Grady et al., 1994). Release was then
evoked by exposure to 10 mM L-NIC for 30 sec. The short test
stimulation time was chosen to minimize the persistent re-
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Fig. 4. Recovery of response and effect
of base-line depletion. Data points are
means of three to four experiments. For
the inset graph, fractions were collected
every minute and L-NIC exposure was for
1 min. Base lines have been subtracted
and data normalized to base line for units
of release. Data are offset by 2 units for
clarity. For recovery, all synaptosomes
were exposed to 10 mM L-NIC for 5 min
(initial peak), and the exposure was repeated after 2-, 5-, 10- or 20-min recovery by perfusion with the buffer. Data are
offset by 2 units for clarity. Fractions
were collected every 30 sec and data
calculated as for the inset graph.
1997
Transient and Persistent Dopamine Release
37
sponse component. Not only is the ratio of transient to persistent response highest at early time points, the persistent
phase has a somewhat slower onset (Rowell, 1995; Grady et
al., 1994). The low concentration of agonist present before
and during test exposure stimulated some persistent response which is included in the base line and therefore subtracted from the transient response. As a result, this method
measures inhibition of the transient response only. Units of
release for the L-NIC test exposure were calculated as described under “Data Analysis” and are presented as % control
in figure 7. Figure 8 presents the correlation of low concentration inhibition of transient response (KIA) with activation
of persistent response (KP from table 2). The correlation
coefficient is 0.99 with an intercept of 20.79 and a slope of
0.95, which indicates a strong correlation between these parameters but not identity (slope and correlation coefficient
are close to 1, but intercept is not 0).
To assess whether chronic L-NIC treatment changes the
release profile, [3H]DA release was measured from synaptosomes prepared from mice chronically treated with LNIC or saline (n 5 11 per group) as described under “Methods.” Individual animals were assayed by a 10-min
exposure to 10 mM L-NIC. Data were expressed as units of
release per fraction as means of two to four filters per
animal and were fit to a double-exponential equation (see
“Data Analysis” and fig. 1). Figure 9 shows the data as
mean 6 S.E.M. and curve fits for NIC- and saline-treated
mice. In addition, L-[3H]NIC binding was measured on a
portion of the P2 striatal synaptosomes prepared from
each mouse. For this measurement, pooled tissue from two
mice were assayed (n 5 5 determinations per group) with
20 nM L-[3H]NIC (a concentration close to saturation for
binding). Parameters calculated are presented in table 3.
The parameter vT was significantly different (P , .05)
between groups. Although differences for VP were not
highly significant (P , .20), the decrease to 82% of saline
control value indicated a decrease in function similar to
that for VT. Uptake and base-line release before exposure
to L-NIC during the assay did not differ between groups;
however, [3H]DA remaining in the synaptosomes at the
end of the assay was higher for the L-NIC-treated group,
probably reflecting the decreased release. Binding of
3
L-[ H]NIC for the L-NIC-treated group was not significantly higher than for the saline-treated group.
Discussion
Nicotinic agonist-induced release of [3H]DA from striatal
synaptosomes consists of two distinct phases, one transient and the other persistent. Most of the [3H]DA release
from rodent synaptosomes stimulated by micromolar concentrations of L-NIC is transient, is blocked by nicotinic
antagonists and requires external calcium (Grady et al.,
1992; Soliakov et al., 1995; Rowell et al., 1987; El-Bizri and
Clarke, 1994). The persistent response, remaining after
the transient portion has desensitized, is also calciumdependent and inhibited by DHbE (figs. 3 and 5; cf. Rowell,
1995). In addition, both responses are inhibited by MEC at
approximately the same concentrations (fig. 3D), as expected for a noncompetitive antagonist, but not by atropine
or a-BTX. The absolute dependence of both responses on
external calcium and the total blockade of both transient
and persistent release by 200 mM cadmium (fig. 3) indicate
that both phases are mediated by voltage-sensitive calcium channels (Miller, 1990; Rathouz and Berg, 1994).
External calcium is known to modify the response of some
nAChRs; however, this effect of calcium is not absolute and
in fact may be minimal at high agonist concentrations
(Mulle et al., 1992b). Thus, although nAChRs can flux
calcium (Mulle et al., 1992a; Vernino et al., 1994), it appears that insufficient calcium enters through the nAChR
to directly promote release of [3H]DA.
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 29, 2016
Fig. 5. Inhibition of L-NIC-stimulated
[3H]DA release by nicotinic antagonists.
Data points for graphs A through E are
means 6 S.E.M. or range (n 5 2– 6 experiments) of vT (E) or vP (F) calculated
by double-exponential fit of data expressed as units per fraction for 3H-release evoked by 1 mM L-NIC in the presence of various concentrations of
antagonist expressed as % control response in the absence of antagonist.
Lines are curve fits of the data shown
(see “Methods”). Ki values (in mM) determined from these curve fits for inhibition
of transient response (vT) are: HEX,
11.0 6 3.0; DHbE, 0.038 6 0.018; DEC,
10.1 6 2.2; MLA, 0.036 6 0.013; dTC,
0.54 6 0.17. Ki values (in mM) for inhibition of the persistent response (vP) are:
HEX, 28.8 6 8.7; DHbE, 0.030 6 0.010;
DEC, 29.1 6 10.9; MLA, 0.13 6 0.03;
dTC, 0.84 6 0.39. Graph F shows the
regression analysis comparing these Ki
values. Parameters for the regression
line are: slope, 0.89; intercept, 20.30;
correlation coefficient, 0.98 (P , .005).
38
Grady et al.
Vol. 282
TABLE 1
Parameters calculated by fit to hill equation
Transient Release Parameters
Persistent Release Parameters
Agonist
L-NIC
ACh
CYT
D-NIC
CARB
EPI
MeCARB
ATX
TMA
VT
KT
units
mM
4.28 6 0.75
3.59 6 0.39
1.11 6 0.12
1.20 6 0.45
4.30 6 0.51
2.51 6 0.24
2.36 6 0.14
3.40 6 0.55
5.34 6 1.46
1.05 6 0.74
0.30 6 0.13
0.075 6 0.044
4.81 6 6.28
8.75 6 3.21
0.0027 6 0.0009
0.70 6 0.19
0.092 6 0.063
17.1 6 11.9
n
0.73 6 0.27
0.80 6 0.20
0.61 6 0.17
0.82 6 0.72
0.99 6 0.23
0.78 6 0.23
0.86 6 0.18
0.72 6 0.25
1.15 6 0.57
VP
KP
units
mM
1.15 6 0.12
0.99 6 0.07
0.83 6 0.10
0.61 6 0.02
0.99 6 0.05
0.94 6 0.28
0.87 6 0.11
1.15 6 0.10
1.16 6 0.11
0.13 6 0.07
0.16 6 0.05
0.054 6 0.033
0.52 6 0.07
2.10 6 0.47
0.00065 6 0.00005
0.12 6 0.07
0.024 6 0.009
3.62 6 1.23
n9
0.57 6 0.16
0.89 6 0.19
0.64 6 0.21
1.13 6 0.15
0.97 6 0.18
0.69 6 0.45
0.62 6 0.20
0.92 6 0.28
1.03 6 0.28
TABLE 2
Parameters calculated by simultaneous data fit
Transient Release Parameters
Persistent Release Parameters
Agonist
L-NIC
ACh
CYT
D-NIC
CARB
EPI
MeCARB
ATX
TMA
VT
KT
DT
VP
KP
DP
units
mM
min21
units
min21
1.86 6 0.18
2.95 6 0.28
1.56 6 0.27
1.45 6 0.14
2.66 6 0.20
1.71 6 0.15
1.65 6 0.12
1.28 6 0.12
2.65 6 0.21
1.03 6 0.07
1.10 6 0.04
0.75 6 0.05
0.62 6 0.04
1.06 6 0.06
0.77 6 0.06
0.72 6 0.04
1.14 6 0.10
1.21 6 0.07
mM
3.72 6 0.20
3.31 6 0.23
0.89 6 0.08
1.21 6 0.06
4.25 6 0.21
2.40 6 0.09
2.22 6 0.08
3.09 6 0.13
5.70 6 0.29
0.71 6 0.11
0.22 6 0.03
0.033 6 0.010
2.55 6 0.32
8.82 6 0.99
0.0030 6 0.0004
0.54 6 0.07
0.066 6 0.009
20.8 6 2.4
Asterisks denote significant differences from previously calculated parameters (table 1); * P , .05.
0.085 6 0.010
0.28 6 0.02
0.024 6 0.003
1.83 6 0.13*
5.08 6 0.49*
0.00037 6 0.00005
0.14 6 0.02
0.020 6 0.002
6.84 6 0.71*
0.089 6 0.011
0.099 6 0.008
0.102 6 0.011
0.103 6 0.010
0.143 6 0.011
0.154 6 0.015
0.097 6 0.010
0.079 6 0.013
0.135 6 0.012
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 29, 2016
Fig. 6. Dose-response curves for nicotinic agonist stimulation of transient and persistent [3H]DA release. Data points are means 6 S.E.M. or
range (n 5 2– 6 experiments) for vT (E) or vP (F) calculated by doubleexponential fit of data expressed as units per fraction (see “Methods”
for details). Lines drawn are curve fits by SigmaPlot 5.0 to the Hill
equation setting Vm to the highest actual release achieved and omitting
any decreasing values at high concentrations of agonist from the curve
fits.
The desensitization time course may be a function of the
release mechanism. Desensitization rates were similar for
all the compounds tested for persistent release. Values of
desensitization rates for the transient phase (DT) varied
about 2-fold with three agonists, ACh, CARB and TMA,
having significantly higher rates than the other compounds. Similar desensitization rates for [3H]DA release
from rat striatal synaptosomes stimulated by TMA and
L-NIC have been reported (Lippiello et al., 1995). Experiments with other agents, such as 20 mM K1 or 100 mM
kainate, which promote [3H]DA release via calcium-dependent processes but do not involve the nAChR, indicated
similar rates of desensitization (Grady, S. R., unpublished
results). However, a more direct measure of nAChR
desensitization,86Rb1 flux experiments (Marks et al.,
1996), has shown that neuronal nAChRs do desensitize but
at slightly slower rates than measured by the [3H]DA
release assay. Although these assays may not measure the
same receptor subtype, this result shows that neuronal
nAChRs do desensitize as expected. The observation that
the rate of desensitization of the transient phase of [3H]DA
release does not vary from agonist to agonist and closely
resembles the desensitization rate seen after depolarization could mean that receptor desensitization is not the
rate-limiting step for attenuation of release. For example,
there is evidence for biphasic depletion of cytosolic calcium
concentrations and existence of two calcium sensors (Kao
and Schneider, 1986; Greengard et al., 1993; Geppert et al.,
1994; Littleton and Bellen, 1995). However, in bovine chromaffin cells that exhibit a biphasic pattern of release for
1997
Transient and Persistent Dopamine Release
39
Fig. 7. Agonist inhibition of transient response. Nine agonists were
tested for their ability to inhibit [3H]DA release evoked by a 30-sec
exposure to 10 mM L-NIC. Synaptosomes were exposed to nanomolar
concentrations of agonist as indicated for 15 to 20 min before the test
exposure to L-NIC, also in the presence of nanomolar agonist. Data
were calculated as total units of release and expressed as % control
response in the absence of prior agonist exposure. Each point represents the mean 6 S.E.M. for three to six experiments. Lines drawn are
curve fits for inhibition (see “Methods” for equation). Inhibition constants for agonists (KIA in nM) calculated from these curve fits are:
L-NIC, 19.7 6 4.9; ACh, 38.4 6 8.1; D-NIC, 209 6 28; EPI, 0.060 6
0.013; ATX, 5.1 6 0.8; CYT, 4.2 6 1.5; CARB, 580 6 269; MeCARB,
47.5 6 15.0, TMA, 1081 6 147.
catecholamines, evidence has been presented that replenishment of release granules is not the cause of the biphasic
release pattern (McKay and Schneider, 1984). Several observations indicate that efficacy and potency parameters
measured by the release assay are indicative of nAChR
function. That persistent response is agonist dependent
and can occur without prior transient release shows that
the persistent phase is not simply the direct result of
biphasic calcium depletion. That transient response can be
modulated by treatment with low concentrations of agonists in the absence of significant release argues against
these parameters being affected by calcium or vesicle depletion. In addition agonists show a range of transient
efficacy which would be unlikely if these parameters were
determined by calcium channel activity or some function of
the release mechanism.
Fig. 9. [3H]DA release from striatal synaptosomes of chronically
treated mice. Two groups of mice were chronically infused through
cannulas implanted in the jugular vein, with either saline or L-NIC (4
mg/kg/hr) for 9 to 10 days. Striatal synaptosomes were prepared from
individual mice after a 15-hr withdrawal, and [3H]DA release was measured with a 10-min exposure to 10 mM L-NIC. Data are mean 6 S.E.M.
(n 5 11 mice per group) of agonist-stimulated units of [3H]DA released
per fraction and plotted versus the midpoint of the time of collection of
the fraction (see fig. 1B). Lines are curve fits to a double-exponential
decay equation (see “Methods”).
Data presented here support the concept that both transient and persistent components of L-NIC-evoked [3H]DA
release are mediated by a single receptor. If the two responses are mediated by a single type of receptor, some
predictions can be made assuming the kinetics follow the
desensitization model of Katz and Thesleff (1957).
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 29, 2016
Fig. 8. Correlation of agonist inhibition of transient response with agonist activation of persistent response. Inhibition constants (KIA) for
low-concentration agonist inhibition of transient [3H]DA release by LNIC (see fig. 7) are plotted versus activation constants (KP) for stimulation of persistent [3H]DA release by the agonists (see table 2). The line
is a regression line (SigmaPlot 5.0) with the following parameters: slope,
0.95; intercept, 20.79; correlation coefficient, 0.99 (P , .005).
40
Grady et al.
Vol. 282
TABLE 3
Effects of chronic L-NIC treatment
Saline-treated
3
Uptake of [ H]DA
Base line before
L-NIC exposure
vT
76 6 3%
1,815 6 92 cpm
79 6 2%
1,706 6 178 cpm
3.24 6 0.17 U
dT
1.85 6 0.19 min
vP
1.04 6 0.09 U
dP
L-NIC-treated
21
0.108 6 0.015 min21
cpm on filter after 187,813 6 6,607 cpm
L-NIC exposure
3
L-[ H]NIC binding
69.8 6 4.5 fmol/mg
2.72 6 0.12 U*
(84% control)
1.83 6 0.16 min21
(99% control)
0.85 6 0.06 U
(82% control)
0.099 6 0.012 min21
(92% control)
210,625 6 5,809 cpm*
79.2 6 4.9 fmol/mg
* P , .05.
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If RL is the only opening form of the receptor, the transient
phase of [3H]DA release could be a measure of R 3 RL (rate
constant, k1) which then desensitizes via RL 3 R9L (rate
constant, k3). The persistent phase of release could be a
measure of R9L 3 RL (rate constant, k4). This could occur if
the receptor that mediates dopamine release has a higher
ratio of k4/k3 than previously studied subtypes of the neuronal nicotinic receptor. For example, if the ratio of k7/k8 is
about 1:1 as expected from binding studies (Grady et al.,
1994), the measured Vmax for a functional assay would be
50% of the theoretical Vmax. If the ratio of k3/k4 is 10:1,
persistent release would be 10% of theoretical Vmax, or approximately 20% of the measured Vmax for transient release.
It has been shown for nAChR from Torpedo that Ki values
for competitive antagonists are similar for the desensitized
form, measured by competition binding assays, and the active form, measured by 22Na1 efflux (Popot et al., 1976). The
Ki values for competitive antagonists measured for [3H]norepinephrine release and L-[3H]NIC binding in bovine adrenal
chromaffin cells are also similar (Higgins and Berg, 1988).
Therefore, if the transient and persistent phases of [3H]DA
release are mediated by agonist binding to different forms of
the same receptor, it is likely that Ki values for inhibition by
competitive antagonists as well as noncompetitive antagonists would be similar. If, however, the two phases of [3H]DA
release are mediated by different receptor subtypes, Ki would
likely be different (Alkondon and Albuquerque, 1993; Mulle
et al., 1991; Cachelin and Rust, 1994, 1995; Clarke and Reuben, 1996). The results (fig. 5) for five antagonists show that
the Ki values are similar for the two phases of release. Regression analysis yielded a good correlation (r 5 0.98; slope 5
0.89; intercept 5 20.30) between Ki values for the five an-
tagonists for the two phases of release. The slope and intercept deviate somewhat from expected (1 and 0), which possibly reflects an error in the assumption that DEC and possibly
HEX are purely noncompetitive inhibitors. Inhibition by
these antagonists, however, is similar enough to conclude
that the two processes may be mediated by the same receptor.
Further support for the same receptor hypothesis is derived from a comparison of agonist-induced activation of the
persistent phase and agonist-induced desensitization of the
transient phase. If the transient and persistent responses are
manifestations of the kinetics of a single receptor, as opposed
to responses of two separate types of release, the concentrations required to evoke persistent response (KP) should be
correlated with inhibition of the transient response (KIA),
which is produced by preexposure to low concentrations of
agonist. Such a correlation is expected because both processes require agonist interaction with the desensitized form
of the receptor (the R9 3 R9L step). High correlation between
the activation constants for persistent release (KP) and the
inhibition by low agonist concentrations of the transient response to L-NIC (KIA) was obtained (fig. 8). Whereas KP and
KIA are highly correlated, KP is always larger than KIA (ratio,
6.1 6 0.7; range, 2.9 – 8.8). Calculations based on the Katz
and Thesleff model described by Marks et al. (1996) have
shown that KIA values for inhibition of function are predicted
to be higher than KI values for inhibition of binding. Similar
calculations with ratios of k3:k4 in the range of 10:1 and other
constants set at values determined in experiments reported
here and previously (Grady et al., 1994) indicate that KP is
expected to be larger than KIA.
A range of efficacy was seen for the nine agonists tested in
this study. For the transient release process, initial release
(VT) varied about 5-fold with CYT giving the lowest response
and TMA having the highest transient release. Persistent
release (VP) varied much less (about 2-fold), with D-NIC
having the lowest and TMA having the highest efficacy. In
agreement with these data, CYT has been shown to have
lower efficacy than L-NIC for release of [3H]DA from rat
synaptosomes (El Bizri and Clarke, 1994; Wonnacott et al.,
1995). An initial characterization (Grady et al., 1992) indicated that efficacy was similar for various agonists including
L-NIC, CYT, D-NIC, ACh and CARB. These early experiments with the perfusion system were conducted with Percoll-purified synaptosomes and without current improved
line washing procedures, which resulted in considerably
lower agonist-stimulated release values than are now
achieved. A greater proportion of the response measured for
this initial characterization was possibly of the persistent
phase which varies little in efficacy.
Marks et al. (1993a) showed that chronic treatment of mice
with 4 mg/kg/hr L-NIC produces a functional down-regulation of striatal [3H]DA release as measured by a 1-min exposure to L-NIC. This treatment protocol was repeated in the
experiment reported here, which used a 10-min L-NIC exposure for the [3H]DA assay and a 15-hr withdrawal from
chronic treatment, a length of time sufficient to clear L-NIC
from the bloodstream of mice (Petersen et al., 1984). A small
but statistically insignificant increase in L-[3H]NIC binding
in striatum was seen. Chronic exposure to L-NIC produces a
well-documented up-regulation of L-NIC binding sites in
brain. Regional differences are seen in the amount of up-
1997
41
TABLE 4
Comparison of parameters for [3H]DA release and membrane
binding for nine agonists
Inhibition of
binding
(KI)
Inhibition of
aBTX binding
(KI)
S 5 0.93
S 5 0.80
R 5 0.97
S 5 1.05
R 5 0.82
S 5 0.94
R 5 0.98
S 5 1.00
R 5 0.87
S 5 0.89
R 5 0.98
R 5 0.86
S 5 0.87
L-NIC
Activation of transient
release (KT)
Activation of persistent
release (KP)
Inhibition of transient
release (KIA)
Inhibition of L-NIC
binding (KI)
R 5 0.85
S 5 slope; R 5 correlation coefficient. KT and KP values are from table 2; KIA
values are from fig. 7. KI values for inhibition of [3H]L-NIC binding are from Marks
et al. (1996). KI values for inhibition of [125I]aBTX binding are from unpublished
data (Marks, M.J.).
of receptors expressed in oocytes, the beta-2 subunit is associated with partial agonist activity of cytisine (Luetje and
Patrick, 1991).
It has been established that individual neurons can express multiple subtypes of nAChR (Horch and Sargent, 1995;
Sargent, 1993; McGehee and Role, 1995). In addition, the
existence of receptors containing more than one type of alpha
or beta subunit have been proposed for other systems
(Colquhoun et al., 1993; Vernallis et al., 1993; Mandelzys et
al., 1995). The strong pharmacological correlations between
the transient and persistent phases of [3H]DA release from
striatal synaptosomes presented here indicate that both
phases are likely mediated by one type of receptor. The
correlations of [3H]DA release and L-[3H]NIC binding parameters implicate an a4b2 composition whereas sensitivity to
nBTX favors a3b2 subunit containing receptors. It seems
possible that this nAChR contains both an alpha-3 and an
alpha-4 as well as beta-2 subunits. Although correlations of
pharmacological parameters can suggest that receptors mediating two processes are identical, confirmation of the subunit composition of these receptors must be achieved by other
methods.
Acknowledgments
The authors wish to thank Scott Robinson for assistance with
surgery and chronic treatments, and Jerry Stitzel and Amy Clark for
assistance with L-[3H]NIC binding assays.
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