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INTRODUCTION |
Nicotinic cholinergic receptors are extracellular ligand-gated
pentameric cation channels activated by the endogenous neurotransmitter acetylcholine or its exogenous analogs such as nicotine (1, 2). The
nicotinic family consists of several distinct but related receptor
subtypes. 10 different nicotinic acetylcholine receptor subunits (
2-
8 and
2-
4) have been identified in vertebrate neurons (2, 3),
and PC12 pheochromocytoma cells express
3,
5,
7,
2, and
4 nicotinic subunits (4, 5).
Desensitization (sometimes referred to tolerance, refractoriness,
subsensitivity, or down-regulation) denotes loss of cell or tissue
responses after repeated or prolonged application of a stimulus.
Nicotinic receptor desensitization by agonist has been known for at
least 40 years, since Katz and Thesleff (6) first characterized the
phenomenon at the neuromuscular junction. Nicotinic desensitization
occurs at both muscle-type and neuronal-type nicotinic receptors (7,
8). Desensitization takes place in cell lines that express nicotinic
functions, such as nicotine-induced release of norepinephrine from
adrenal chromaffin cells (9), or agonist-induced Na+ (10,
11) or other cation (12) fluxes in PC12 pheochromocytoma cells. Cell
lines transfected with defined subunits of nicotinic receptors (such as
2-
2 (13) or
4-
2 (14)) also desensitize upon exposure to
agonist. Recently, nicotinic receptor desensitization has been shown to
occur in midbrain dopamine neurons (15).
Desensitization of the nicotinic acetylcholine receptor may be
modulated by a variety of factors. Noncovalent modulators include the
noncompetitive nicotinic blockers (7), calcium (7), the thymic peptide
hormones thymopoietin and thymopentin (7), substance P (9), and
calcitonin gene-related peptide (7). Covalent modifications of receptor
structure, such as receptor phosphorylation (16), may also play a role.
The neuropeptide substance P modifies the nicotinic response of
chromaffin cells by two distinct actions: (i) substance P inhibits the
secretion of catecholamines evoked by nicotinic agonists (17), and (ii)
substance P protects against desensitization of this nicotinic response
(17). These effects of substance P are selective for the nicotinic
receptor ionophore complex, rather than tachykinin receptors (18).
The catestatin peptide fragment of the catecholamine secretory vesicle
protein chromogranin A (bovine chromogranin A344-364) is a
potent inhibitor of exocytotic catecholamine secretion from PC12 and
chromaffin cells (19, 20). This peptide acts as a noncompetitive
nicotinic cholinergic antagonist, with characteristic inhibitory
effects on nicotinic cationic (Na+, Ca2+)
signal transduction (19, 20). Indeed, catestatin is more potent than
substance P in inhibiting nicotine-induced secretion of catecholamines
(19).
Since catestatin and substance P have similar actions on catecholamine
release, we investigated whether catestatin also affects nicotinic
cholinergic desensitization of catecholamine release from chromaffin
cells. Our results indicate that catestatin specifically and
potently inhibits such desensitization.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Rat PC12 pheochromocytoma cells (Ref. 21, at
passage 8, obtained from Dr. David Schubert, Salk Institute, La Jolla,
CA) were grown at 37 °C, 6% CO2, in 10-cm plates or
6-well plates, in Dulbecco's modified Eagle's/high glucose medium
supplemented with 5% heat-inactivated fetal bovine serum, 10%
heat-inactivated horse serum,, and 1% penicillin/streptomycin (100%
stocks were 10,000 units/ml penicillin G and 10,000 µg/ml
streptomycin sulfate; Life Technologies, Inc.), as described previously
(19).
Secretagogue-stimulated Release of Norepinephrine--
Secretion
of norepinephrine was monitored as described previously (19). PC12
cells were plated on poly-D-lysine-coated 6-well polystyrene dishes (Falcon Labware, Oxnard, CA), labeled for 3 h
with 1 µCi of L-[3H]norepinephrine (71.7 Ci/mmol, NEN Life Science Products Inc., Boston, MA) in 1 ml of PC12
growth medium, washed twice with release buffer (150 mM
NaCl, 5 mM KCl, 2 mM
CaCl2, 10 mM HEPES, pH 7), and then
incubated at 37 °C for 30 min in release buffer with or without
secretagogues, such as nicotine (0.1 to 1000 µM), or cell
membrane depolarization (55 mM KCl). Release buffer for
experiments involving KCl as secretagogue had NaCl reduced to 100 mM, to maintain isotonicity. After 30 min, secretion was
terminated by aspirating the release buffer, and lysing cells into 150 mM NaCl, 5 mM KCl, 10 mM HEPES, pH
7, 0.1% (v/v) Triton X-100. Release medium and cell lysates were
assayed for L-[3H]norepinephrine by liquid
scintillation counting, and results were expressed as % secretion:
(amount released/(amount released + amount in cell lysate)) × 100. Net
secretion is secretagogue-stimulated release minus basal release.
Desensitization of Catecholamine Release, and Its Blockade by
Catestatins, Substance P, or Other Nicotinic Cholinergic
Antagonists--
Cells were preloaded with
L-[3H]norepinephrine and exposed to nicotinic
agonists either alone or in combination with nicotinic antagonists for
periods of 10-60 min (incubation I). Cells were then washed twice (6 min each) in secretion buffer as described above and rechallenged with
nicotinic agonists for a period of 10 min (incubation II) after which
the cells were harvested for measurement of norepinephrine release from
cells and medium.
Desensitization of 22Na+ Uptake by PC12
Cells and Its Blockade by
Catestatin--
22Na+ uptake was performed as
described by Mahata et al. (19). Before experiments, PC12
cells were washed twice with 50 mM Na+-sucrose
media containing 50 mM NaCl, 187 mM sucrose, 5 mM KCl, 2 mM CaCl2 and 5 mM HEPES adjusted to pH 7.4 with NaOH. To measure 22Na+ influx, this medium was then supplemented
with 5 mM ouabain (to prevent active extrusion of newly
taken-up 22Na+ from cells by membrane
Na+/K+-ATPase) and 1.5 µCi/ml
22NaCl (specific activity: 840.21 mCi/mg, NEN Life Scinece
Products Inc.). Incubation was carried out at 22 °C for 5 min, in
the presence or absence of secretagogues, and then the cells were
washed within 10 s with three changes (1 ml each) of 50 mM Na+-sucrose media containing 5 mM ouabain. The cells were lysed (see above) and
22Na+ in the cell lysate was measured in a
-counter (LKB 1274 RIAGAMMA, Wallac Inc., Gaithersburg, MD). The
data were expressed as disintegrations/min/well.
Synthetic Peptides--
Chromogranin A fragment peptides such as
the catestatins (bovine chromogranin A344-364,
RSMRLSFRARGYGFRGPGLQL; human chromogranin A352-372,
SSMKLSFRARAYGFRGPGPQL; and rat chromogranin A367-387,
RSMKLSFRARAYGFRDPGPQL) were synthesized at 10-100 µmol scale by the
solid-phase method (22) using t-boc or Fmoc protection chemistry, and
then purified to >95% homogeneity by reversed phase high pressure
liquid chromatography on C-18 silica columns, monitoring
A280 (aromatic rings) or
A214 (peptide bonds). Authenticity and purity of
peptides were verified by re-chromatography, as well as
electrospray-ionization or MALDI mass spectrometry, or amino acid
composition. Substance P (RPKPQQFFGLM-amide; 40) was obtained from
Peninsula Laboratories (Belmont, CA).
Statistics--
Results are recorded as the mean value ± one S.E. Mean value comparisons were made by t test or by
nonparametric test (Wilcoxon or Mann Whitney), when data were not
normally distributed. Regressions were done by Pearson product moment
correlations, or by Spearman rank order correlations, when data were
not normally distributed. Data were analyzed and plotted with the
programs InStat (GraphPad Software, San Diego, CA), Kaleidagraph
(Abelbeck Software, Reading, PA), or CricketGraph (Cricket Software,
Malvern, PA).
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RESULTS |
Nicotinic Cholinergic Desensitization: Effects on Norepinephrine
Release--
PC12 cells were prelabeled with
[3H]norepinephrine and exposed in an initial incubation
(incubation I) to several doses (0 to 1000 µM) of
nicotine for 10 min. The medium was collected for measurement of
norepinephrine release in incubation I. Cells were then washed twice (6 min each) and rechallenged with a single dose of nicotine (10 µM) during a subsequent incubation (incubation II) for 10 min, after which both medium (supernatant) and cells were harvested for
measurement of norepinephrine release. In the initial period
(incubation I), nicotine released norepinephrine in a
dose-dependent fashion, with the usual biphasic
dose-response curve (Fig. 1A).
Pre-exposure of PC12 cells to nicotine decreased subsequent
norepinephrine release by 57.4 to 69.9% upon agonist rechallenge (Fig.
1B), and the apparent desensitization was nearly maximal for
even the smallest initial exposure (incubation I) dose of nicotine, 10 µM (Fig. 1, A and B).

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Fig. 1.
Nicotinic cholinergic effects on
catecholamine release: initial stimulation of secretion, with
subsequent desensitization upon second exposure to agonist.
A, nicotine effect to release catecholamines. PC12 cells
were prelabeled with L-[3H]norepinephrine and
then treated with ascending log doses of nicotine for 10 min
(incubation I). Supernatants were collected for measurement of
norepinephrine secretion. The data are expressed as net
L-[3H]norepinephrine secretion
(i.e. minus basal (nonstimulated) secretion). Basal release
in incubation I was 437 ± 19 cpm/well (n = 3).
B, nicotinic desensitization of catecholamine release. PC12
cells were washed twice (6 min each) after collection of supernatants
in incubation I and then re-exposed to ascending log doses of nicotine
for 10 min (incubation II). Supernatants were collected for measurement
of L-[3H]norepinephrine secretion. Basal
release in incubation II was 388 ± 12 cpm/well (n = 3).
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Noncompetitive Mechanism of Peptide Antagonists at Nicotinic
Cholinergic Receptors: Inhibition of Catecholamine Release from PC12
Cells--
Catestatin (bovine chromogranin
A344-364) inhibits nicotine-induced release of
norepinephrine, with an IC50 of ~200-300 nM
(19). To demonstrate whether this nicotinic inhibition is noncompetitive, we treated PC12 cells with log10-ascending
doses of nicotine (10 to 1000 µM) either alone or with
ascending doses of catestatin or substance P (0.1 to 10 µM) for 30 min, after which cells were harvested for
measurement of norepinephrine release. Even very high doses of nicotine
(100-1000 µM) could not overcome catestatin or substance
P inhibition of norepinephrine release (Fig.
2), suggesting noncompetitive
inhibition.

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Fig. 2.
Noncompetitive nicotinic cholinergic
inhibition by peptide antagonists: inhibition of catecholamine
release from PC12 cells. Noncompetitive inhibition of
catecholamine release by catestatin or substance P. L-[3H]Norepinephrine preloaded cells were
treated with: A, logarithmically ascending doses
(10-1000 µM) of nicotine; B, 10 µM nicotine; C, 100 µM nicotine;
or D, 1 mM nicotine either alone or in
combination with logarithmically ascending doses (0.1-10
µM) of bovine catestatin or human substance P for 30 min,
before measurement of norepinephrine secretion.
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When the effects of catestatin versus substance P are
studied at several agonist concentrations (Fig. 2), catestatin had
consistently greater potency (lower IC50) than substance P,
especially at higher agonist concentrations (~11.6-fold lower
IC50 for catestatin at 1 mM nicotine). The
competitive nicotinic antagonist
-bungarotoxin (50 nM)
did not affect nicotine (60 µM)-stimulated catecholamine release from PC12 cells: nicotine alone released 18.6 ± 0.62% of
cell norepinephrine stores, while addition of
-bungarotoxin to
nicotine still yielded 18.4 ± 1.1% release.
Peptide Nicotinic Antagonists (Including Catestatin) Block
Agonist-induced Desensitization of Catecholamine Release in a
Dose-dependent, Noncompetitive Effect--
Nicotinic
antagonists may alter agonist desensitization responses (23). Is
nicotinic agonist-induced desensitization of norepinephrine release
(Fig. 1B) influenced by noncompetitive nicotinic antagonists
such as catestatin? PC12 cells were exposed to nicotinic agonists
(nicotine (10 µM) or epibatidine (1 µM)) for 10 min either alone or in combination with
log10-ascending doses (0.1-10 µM) of
catestatin or substance P, then washed twice (6 min each), and finally
rechallenged with nicotine (10 µM, 10 min) for
measurement of norepinephrine release. Pretreatment with catestatin or
substance P caused dose-dependent blockade of nicotinic desensitization of norepinephrine secretion (Fig.
3), whether that desensitization was
elicited by nicotine (Fig. 3A) or epibatidine (Fig.
3B). Even very high doses of nicotinic agonist pre-exposure (10-1000 µM nicotine) failed to overcome the effects of
peptide antagonists (catestatin or substance P; 10 µM) to
block prior agonist desensitization of secretion (Fig. 3C),
suggesting that this action of the peptides is also noncompetitive with
respect to agonist.

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Fig. 3.
Effect of catestatin or substance P to block
nicotinic cholinergic desensitization of catecholamine release: a
dose-dependent, noncompetitive effect. A,
catestatin or substance P protection against desensitization of
catecholamine release induced by prior exposure to the agonist nicotine
(10 µM). L-[3H]Norepinephrine
preloaded cells were treated with nicotinic cholinergic agonist
(nicotine, 10 µM) either alone or in combination with
logarithmically ascending doses (0.1-10 µM) of bovine
catestatin or substance P for 10 min (incubation I), washed twice (6 min each), and rechallenged with nicotine (10 µM) for 10 min (incubation II) before measurement of norepinephrine secretion.
Control cells received nicotine only in incubation II. B,
catestatin or substance P blockade of desensitization of catecholamine
release induced by prior exposure to the agonist epibatidine (1 µM). Same conditions as A, but initial agonist
challenge in incubation I with the nicotinic agonist epibatidine (1 µM). C, nicotinic antagonist peptide blockade
of prior agonist desensitization is noncompetitive with regard to
agonist. L-[3H]Norepinephrine preloaded cells
were treated with ascending doses of nicotinic cholinergic agonist
(nicotine, 10-1000 µM) either alone or in combination
with desensitization blocking doses (10 µM; see
A) of bovine catestatin or substance P for 10 min
(incubation I), washed twice (6 min each), and rechallenged with
ascending doses of nicotine (10-1000 µM) for 10 min
(incubation II) before measurement of norepinephrine secretion. Control
cells received nicotine only in incubation II.
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Relative Potencies of Peptide Nicotinic Antagonists
(Catestatins or Substance P) to Block Agonist-induced Secretion
Versus Desensitization of Catecholamine Release--
For secretion,
PC12 cells were treated with nicotine (60 µM), either
alone or in combination with log10-ascending doses (0.1-10 µM) of catestatins or substance P. Each peptide
inhibited nicotine-stimulated catecholamine release, in a
dose-dependent fashion. IC50 values for
inhibition of nicotinic-stimulated secretion were: human catestatin, ~0.31 µM; bovine catestatin, ~0.3 µM;
rat catestatin, ~1.2 µM; or substance P, ~1.0
µM.
For nicotinic desensitization, PC12 cells were pre-exposed to nicotine
(10 µM, 10 min), either alone or in combination with log10-ascending doses (0.1-10 µM) of
catestatins or substance P in incubation I, then washed twice (6 min
each), and re-exposed to nicotine (10 µM, 10 min) in
incubation II. Each peptide blocked desensitization in a
dose-dependent fashion. IC50 values for
blockade of nicotinic desensitization of release were: human
catestatin, ~0.22 µM; bovine catestatin, ~0.24
µM; rat catestatin, ~0.62 µM; or
substance P, ~0.25 µM.
Lack of Cooperativity in Catestatin's Effect to Inhibit Secretion
or Desensitization: Hill Plots--
Hill plots (Fig.
4) were done for the fractional effect of
catestatin to inhibit nicotinic-stimulated catecholamine secretion (Fig. 4A) versus desensitization of secretion
(Fig. 4B). In each case, the plots were linear over a wide
range of catestatin concentrations (2 orders of magnitude), and the
Hill slopes were near unity (slope = 0.878 for secretion,
slope = 0.958 for desensitization). Thus, in each case the
catestatin effect seems to be noncooperative.

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Fig. 4.
Catestatin effects to inhibit secretion or
block desensitization: Hill plots. Log10 of bovine
catestatin dose (µM) is plotted against log10
of its fractional effect to inhibit nicotinic agonist-induced secretion
(panel A) or to block nicotinic agonist-induced
desensitization (panel B). v, % inhibition by
any given dose of catestatin. V, maximal % inhibition at
the highest dose of catestatin used (10 µM).
A, catestatin inhibition of nicotinic agonist-stimulated
secretion. L-[3H]Norepinephrine preloaded
cells were treated with 60 µM nicotine either alone or in
combination with ascending doses (0.01-10 µM) of bovine
catestatin for 30 min, before measurement of norepinephrine secretion.
n = 6 catestatin concentrations, r = 0.988, p < 0.0001, slope = 0.878. B,
catestatin blockade of nicotinic agonist-induced desensitization.
L-[3H]Norepinephrine preloaded cells were
treated with 10 µM nicotine either alone or in
combination with logarithmically ascending doses (0.01-10
µM) of bovine catestatin for 10 min (incubation I),
washed twice (6 min each), and then secretion was rechallenged with
nicotine (10 µM) for 10 min (incubation II) before
measurement of norepinephrine secretion. Control cells received
nicotine only in incubation II. n = 5 catestatin
concentrations, r = 0.989, p < 0.0001, slope = 0.958.
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Specificity of Catestatin to Block Nicotinic Desensitization of
Norepinephrine Release--
After PC12 cell exposure to the
secretagogues nicotine (10 µM), KCl (55 mM),
or ATP (100 µM), either alone or with catestatin (10 µM) for 10 min, cells were washed twice (6 min each), and then re-exposed to nicotine (10 µM), KCl (55 mM), or ATP (100 µM) for 10 min, before
harvesting for measurement of [3H]norepinephrine release
(Fig. 5). Catestatin blocked apparent desensitization of norepinephrine release only when such
desensitization was induced by nicotine (Fig. 5A), and not
by stimuli which bypass the nicotinic receptor in triggering release of
norepinephrine, such as 55 mM KCl or 100 µM
ATP (Fig. 5, B and C).

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Fig. 5.
Specificity of catestatin effect to block
only nicotinic desensitization of norepinephrine release.
A, catestatin blockade of desensitization of catecholamine
release induced by prior nicotine.
L-[3H]Norepinephrine preloaded cells were
treated with nicotine (60 µM) either alone or in
combination with bovine catestatin (10 µM) for 10 min
(incubation I), washed twice (6 min each), and then secretion was
rechallenged with nicotine (10 µM) for 10 min (incubation
II) before measurement of norepinephrine secretion. Control cells
received nicotine only in incubation II. B, lack of
catestatin blockade of desensitization of catecholamine release induced
by prior membrane depolarization with KCl.
L-[3H]Norepinephrine preloaded cells were
treated with KCl (55 mM) either alone or in combination
with bovine catestatin (10 µM) for 10 min (incubation I),
washed twice (6 min each), and then secretion was rechallenged with KCl
(55 mM) for 10 min (incubation II) before measurement of
norepinephrine secretion. Control cells received KCl only in incubation
II. C, lack of catestatin blockade of desensitization of
catecholamine release induced by prior exposure to the P2x
receptor agonist ATP. L-[3H]Norepinephrine
preloaded cells were treated with ATP (100 µM) either
alone or in combination with bovine catestatin (10 µM)
for 10 min (incubation I), washed twice (6 min each), and then
secretion was rechallenged with ATP (100 µM) for 10 min
(incubation II) before measurement of norepinephrine secretion. Control
cells received nicotine only in incubation II.
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Catestatin Blockade of Nicotinic Desensitization of Extracellular
22Na+ Uptake as the Initial Step in Nicotinic
Signal Transduction--
PC12 cells were exposed to nicotine (10 µM; 10 min), nicotine plus catestatin (10 µM), or vehicle (mock) in incubation I. After washing (6 min twice), the same cell groups were treated again with nicotine (10 µM; 10 min) in the presence of
22Na+. Pre-exposure to nicotine alone caused
82.1% diminution of subsequent 22Na+ uptake,
but initial co-incubation with catestatin dose dependently reversed
this nicotine effect (Fig. 6). Catestatin
IC50 values for blockade of nicotinic desensitization were
indistinguishable for 22Na+ uptake
(IC50 = 0.31 µM; Fig. 6) or catecholamine
release (IC50 = 0.24 µM).

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Fig. 6.
Catestatin blockade of desensitization of
extracellular Na+ uptake, the first step in nicotinic
cholinergic signal transduction. PC12 cells were treated with
nicotine (10 µM) either alone or in combination with
bovine catestatin (10 µM) versus vehicle
(mock) in incubation I, then washed twice (6 min each), and
finally treated with nicotine (10 µM) for 10 min in
incubation II in the presence of extracellular
22Na+. Results of 22Na+
uptake into cells are shown.
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Relative Potencies of Nonpeptide Nicotinic Antagonists to Block
Agonist-induced Secretion Versus Desensitization of Catecholamine
Release--
To see whether the effects of peptide (catestatin and
substance P) nicotinic antagonists is comparable to that exerted by nonpeptide nicotinic antagonists we repeated the experiments described above using several nonpeptide nicotinic antagonists and found that the
IC50 values for inhibition of nicotinic-stimulated
secretion were (Fig. 7A):
trimethaphan, ~8.3 µM; procaine, ~8.45
µM; phencyclidine, ~0.81 µM; cocaine,
~4.83 µM; clonidine, 19.4 µM; or
hexamethonium, ~40 µM. IC50 values for
blockade of nicotinic desensitization of release were (Fig.
7B): trimethaphan, ~18.0 µM; procaine,
~14.4 µM; phencyclidine, >10 µM;
cocaine, ~23.1 µM; clonidine, ~4.91; or
hexamethonium, >1000 µM.

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Fig. 7.
Relative potencies of nonpeptide
noncompetitive nicotinic antagonists to block agonist-induced secretion
versus desensitization of catecholamine release.
A, inhibition of catecholamine release:
[3H]-L-norepinephrine preloaded cells were
treated with nicotine (60 µM) either alone or in
combination with logarithmically ascending doses (0.1 to 1000 µM) of antagonist for 30 min before measurement of
norepinephrine secretion. B, blockade of desensitization of
catecholamine release: L-[3H]norepinephrine
preloaded cells were treated with nicotine (10 µM) either
alone or in combination with logarithmically ascending doses (0.1-10
µM) of antagonist for 10 min (incubation I), washed twice
(6 min each), and then secretion was rechallenged with nicotine (10 µM) for 10 min (incubation II) before measurement of
norepinephrine secretion. Control cells received nicotine only in
incubation II.
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Parallel Potencies for Nicotinic Antagonists to Block
Agonist-induced Secretion Versus Desensitization of Catecholamine
Release: Contrasting Effects of Peptide Versus Nonpeptide
Antagonists--
Nicotinic antagonist IC50 values for
inhibition of nicotinic-stimulated secretion correlated with their
IC50 values for blockade of desensitization
(r = 0.76, n = 10 antagonists,
p = 0.01; Spearman rank-order correlation).
Of note, peptide antagonists as a group had generally lower
IC50 values for both secretion and desensitization than did
nonpeptide antagonists. When peptide versus nonpeptide
antagonists were so stratified (Table I),
peptide antagonists had not only higher molecular weights
(p < 0.0001), and lower secretion IC50
values (p = 0.019) and desensitization IC50
values (p = 0.005), but also increased IC50
ratios of secretion/desensitization (p = 0.03). Thus,
as compared with nonpeptides, peptide antagonists seemed to have a
preferential effect to block desensitization (Table I).
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Table I
Nicotinic antagonist effects on secretion versus desensitization:
effects of peptides (catestatins or substance P) versus nonpeptides
(trimethaphan, procaine, phencyclidine, cocaine, clonidine, or
hexamethonium)
Values are mean ± 1 S.E.
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Reversibility of Nicotinic Antagonist Blockade of Catecholamine
Secretion--
Potential reversibility of secretory inhibition for
both peptide (catestatin and substance P) and nonpeptide (trimethaphan, procaine, phencyclidine, cocaine, clonidine, and hexamethonium) nicotinic antagonists was studied by preincubation with a secretory inhibitory dose of antagonist, followed by extensive washing of the
cells, with subsequent secretory stimulation by the agonist nicotine
(100 µM) (Fig. 8).

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Fig. 8.
Test of reversibility of noncompetitive
nicotinic antagonist effects to block agonist-stimulated catecholamine
release. L-[3H]Norepinephrine preloaded
PC12 cells were treated for 15 min with several nicotinic antagonists,
at a concentration previously shown to exert substantial secretory
inhibition by each antagonist (Fig. 7). Cells were then washed twice (2 min/wash) with secretion buffer, and secretion was then stimulated by
the nicotinic agonist nicotine (100 µM). After 30 min,
medium and cells were harvested for measurement of net % secretion of
norepinephrine.
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Secretion inhibitory effects of the nonpeptide antagonists
phencyclidine and cocaine persisted even after washout, while effects of the other nonpeptide antagonists, as well as the peptide
antagonists, seemed to be readily reversible (Fig. 8).
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DISCUSSION |
In these experiments in pheochromocytoma cells, the chromogranin A
fragment catestatin not only blocked nicotinic-stimulated catecholamine
release (Fig. 2), but also antagonized the effect of prior nicotinic
agonist exposure to desensitize the effect of a subsequent nicotinic
challenge to induce secretion (Fig. 3). Both secretory blockade and
desensitization blockade were noncompetitive with agonist (Figs. 2 and
3C), dose-dependent (Fig. 3) and noncooperative
(Fig. 4).
Noncompetitive nicotinic cholinergic antagonists may modulate agonist
desensitization of the receptor (24). In muscle-type nicotinic
receptors, some noncompetitive nicotinic antagonists, particularly
local anesthetics and some antibiotics, seem to exert their antagonist
effects at least in part by acute desensitization of the receptor
accompanied by an increase in agonist affinity; such effects have been
reported for both the Torpedo californica receptor (25) and
for the muscle-type receptor on mammalian cells (24).
By contrast, in PC12 pheochromocytoma cells we found that
noncompetitive peptide (19) (Fig. 2) neuronal nicotinic antagonists seemed to block nicotinic desensitization (Fig. 3). This blockade of
desensitization seemed to be specific, in that the nicotinic antagonist
catestatin did not affect subsequent secretory responses to other
chromaffin cell secretagogues (Fig. 5), such as membrane depolarization
by KCl, or ATP-triggered opening of P2x cation channels
(19, 26). Furthermore, in dose-dependent fashion, catestatin blocked nicotinic desensitization of the earliest event in
nicotinic signal transduction, extracellular Na+ entry into
the cytosol (Fig. 6), and its potencies were virtually identical for
blockade of desensitization of nicotinic-stimulated catecholamine
release (IC50 = 0.24 µM) and
22Na+ influx (IC50 = 0.31 µM; Fig. 6). Thus, a noncompetitive neuronal nicotinic
antagonist seems to block desensitization in close parallel to its
blockade of agonist-induced cation flux through the nicotinic pore.
These results indicate that catestatin blockade of desensitization of
catecholamine release is highly selective for the nicotinic receptor
ionophore complex, a finding in line with extensive reports for
substance P effects on chromaffin cells (9).
Several species' forms of catestatin (bovine, human, and rat)
exhibited similar effects to block nicotinic desensitization of
catecholamine release, consistent with the sequence conservation of the
catestatin region of chromogranin A across mammalian species (19).
The ability of noncompetitive nicotinic antagonists to block
desensitization paralleled their antagonism of nicotinic-stimulated secretion (r = 0.76, n = 10 antagonists, p = 0.01). Intriguingly, peptide and
nonpeptide antagonists seemed to group into two potency families:
indeed, there was no overlap at all for potency to block desensitization (Table I). Peptide antagonists as a group had superior
potency to block both secretion (p = 0.019) and
desensitization (p = 0.005), as well as higher
secretion/desensitization ratios (p = 0.03) (Table I).
Small, nonpeptide nicotinic antagonists may exert their actions by
lodging deep within the nicotinic cation pore, bounded by the pore's
membrane-spanning
-helical M2 domains (23). By contrast, catestatin
is far too large to effectively enter the membrane-spanning portion of
the cation pore; instead, it is likely to block nicotinic signaling by
occluding the ~25-Å cation pore vestibule at its extracellular
orifice (27). Thus, peptide and nonpeptide nicotinic antagonists differ
not only in size, therefore likely receptor site of action to exert
noncompetitive nicotinic blockade, but also in the potency of such
action. It is conceivable that the superior potencies of peptide
antagonists (Fig. 3, A and B; Table I) arise from
more effective occlusion of the nicotinic cation pore as a result of
vestibule occlusion by the larger antagonists.
Two of the nonpeptide nicotinic antagonists (phencyclidine and cocaine)
also differed from the peptide antagonists in that the smaller
molecules displayed relatively irreversible secretory inhibition (Fig.
8). Such smaller antagonists, with consequent increased ability to
penetrate the nicotinic cation pore, may be more difficult to remove
from the pore with a simple buffer wash. Other properties of such small
antagonists, such as hydrophobicity or charge, may also be important
for relative irreversibility.
Nicotinic receptor subunit composition seems to determine both
activation and desensitization responses to agonists. Expressing neuronal nicotinic receptors in Xenopus oocytes, Fenster
et al. (28) showed that receptors containing
4 subunits
were more sensitive than
3-containing receptors to both activation
and desensitization by nicotine. Fenster et al. (28)
therefore suggested that the
subunit type largely determines the
apparent agonist affinity of active and desensitized states of a
nicotinic receptor, while the
subunit influences the time course of
development of desensitization; other studies (29, 30) suggest that the
subunit also can influence the desensitization time course. Because
homomeric
7 receptors also desensitize, it is likely that the
subunit plays a modulatory rather than a permissive role in
desensitization (28).
3,
5,
7,
2, and
4 nicotinic subunit expression is detectable in PC12 cells (4, 5, 31), although the
precise subunit stoichiometry of any given nicotinic heteropentamer may
be difficult to establish. Lack of effect of
-bungarotoxin on
nicotinic-stimulated PC12 cell catecholamine release weighs against a
role for
7 subunits in the secretion we observed (23).
Although detailed physical models of agonist mechanisms of both channel
gating and desensitization have yet to emerge, electron diffraction
images of the nicotinic acetylcholine receptor trapped in a
channel-open conformation by rapid freezing suggest tilting and
twisting of the M2 transmembrane
-helices accompanied by structural
alterations near the neurotransmitter-binding site (32). During
desensitization, photoactivatable probe incorporation studies suggest
alterations in both the neurotransmitter-binding site and the
pore-lining M2 amino acid side chains (33, 34). Fourier transform
infrared difference spectroscopy and 1H/2H
exchange difference spectroscopy (35) suggest that desensitization causes conformational changes principally in the agonist binding site.
Peptide antagonist fractional dose dependence for secretory inhibition
and blockade of desensitization were parallel, as judged by Hill plots
(Fig. 4), and in each case the Hill slopes were near unity. This
suggests that peptide antagonist effects are noncooperative, and that
such desensitization-blocking peptide antagonists binds to the same
site, at the same affinity, on the nicotinic receptor to exert both
noncompetitive antagonism and inhibition of desensitization.
The catestatin data (Figs. 2-6, Table I) are of principal interest
because this peptide is formed endogenously from chromogranin A, a
protein stored and released with catecholamines under nicotinic cholinergic stimulation (19). Thus, chromaffin cells and sympathetic axons may thereby generate an endogenous antagonist of nicotinic cholinergic desensitization. We have estimated that the concentration of just released chromogranin A in the local extracellular vicinity of
the chromaffin cell may be as high as ~400 µM (19).
Chromogranin A circulates in humans at concentrations ranging from ~1
nM, in healthy controls, to ~1 µM, in
disease states such as pheochromocytoma (36), other neuroendocrine
neoplasia (37), or renal failure (38). Since we found that even 100 nM catestatin caused substantial blockade of nicotinic
desensitization, the potential physiologic consequences of such a
mechanism cannot be ignored.
We speculate that catestatin blockade of nicotinic desensitization of
catecholamine release may be advantageous to an organism, particularly
during circumstances of heightened sympathetic outflow, a setting in
which this action of catestatin might sustain catecholamine release.
Indeed, substance P may augment catecholamine release by protection
against nicotinic desensitization during prolonged stress in an
organism (39).