Department of Medicine and Center for Molecular Genetics (S.K.M.,
M.M., D.T.O.) University of California, and Veterans Affairs
San Diego Healthcare System San Diego, California 92161
Department of Pharmacology (A.R.W.) Wayne State University
School of Medicine Detroit, Michigan 48201
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ABSTRACT |
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INTRODUCTION |
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We recently identified a novel peptide, catestatin (bovine chromogranin A344-364), which is a potent inhibitor of nicotinic cholinergic-stimulated catecholamine release from chromaffin and pheochromocytoma cells (7, 8, 9). This peptide is formed within and secreted from chromaffin granules (7, 10) and acts as a nicotinic cholinergic antagonist, with characteristic inhibitory effects on nicotinic cationic (Na+, Ca2+) signal transduction (7). Like substance P (2, 11) catestatin also blocks nicotinic agonist-induced desensitization of catecholamine release (9). This blockade of desensitization appears to be specific to neuronal nicotinic receptors, in that catestatin fails to inhibit desensitization caused by secretagogues, which bypass nicotinic receptors such as membrane depolarization, or ATP-triggered opening of P2x cation channels (9).
To identify the important amino acid residues in the catestatin sequence, we first synthesized serial three-amino acid truncation peptides (amino-terminal, carboxyl-terminal, or bidirectional) and tested their potencies at inhibition of catecholamine release. In a second round of syntheses, we examined the roles of individual amino acid residues. In so doing, we were able to define an active catestatin core as well as the roles of crucial amino acid residues for inhibition of nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and agonist desensitization.
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RESULTS |
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Carboxyl-Terminal Truncations
The catecholamine release inhibitory potency was preserved after
deletion of 6 carboxyl-terminal residues (from
IC50 of 0.41 ± 0.022 µM to
IC50 of 0.18 ± 0.021 µM).
Further deletions of 912 residues resulted in a progressive loss of
potency (P < 0.01), while deletion of 15 residues
abolished potency (IC50, >10
µM; Fig. 1C).
Bidirectional Truncations
Deletion of three residues from each terminus caused an approximately
6.6-fold loss of potency (P < 0.05). Deletion of six
residues resulted in barely detectable potency
(IC50, 10 µM;
P < 0.01), and further deletion abolished the potency
(IC50, >10 µM; Fig. 1D
).
Roles of Individual Amino Acids in the Active Catestatin Core
Sequence (Bovine Chromogranin
A344-358): Effects on
Catecholamine Release
As the results of serial 3-amino acid deletions (Fig. 1) suggested
an active 15 amino acid catestatin core (bovine chromogranin
A344-358;
RSMRLSFRARGYGFR), further deletion and substitution mutagenesis were
conducted on this active core.
Amino-Terminal Residues
Deletion of even the first residue (Arg344) from
the amino-terminus caused a 16-fold loss in catecholamine release
inhibition (P < 0.01). Deletion of the next two
residues (Ser345 and
Met346) caused no further falls (13- to
16-fold; P < 0.01) in potency, whereas deletion of the
next four residues abolished potency altogether
(IC50, >10 µM; Fig. 2A
).
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Interior Amino Acids: Alanine Substitution (Alanine-Scanning)
Mutants
To test the roles of individual residue side-chains, we systematically
replaced each residue in the 15-amino acid catestatin core with
L-alanine (methyl-glycine), the smallest amino acid that
retains chirality. Alanine substitution of bovine chromogranin A
residues 344 (R344A), 345 (S345A), 347 (R347A), 349 (S349A), and 356
(G356A) had little effect on potency (P > 0.05).
Alanine substitution of residues 346 (M346A), 354 (G354A), or 355
(Y355A) caused moderate (4- to 5-fold; P < 0.01)
loss of potency. Substitution of residues 348 (L348A), 350 (F350A), 351
(R351A), 353 (R353A), 357 (F357A), or 358 (R358A) caused more
substantial (
8- to 21-fold; P < 0.01) loss of
potency (Fig. 2C
).
Qualitative Residue Changes: ArginineLysine and Levo
(L)
Dextro (D)
Converting each arginine to lysine in chromogranin
A344-358 caused an
approximately 6-fold (P < 0.01) loss of antisecretory
potency (Fig. 2D). Converting chromogranin
A344-358 from all L
(levorotatory) to all D (dextrorotatory) amino acid isomers resulted in
approximately 9-fold (P < 0.01) loss of antisecretory
potency (Fig. 2D
).
Nicotinic Cholinergic Receptor Cationic Signal Transduction: Role
of Catestatin Domains
We have previously shown that catestatin (bovine
chromogranin
A344-364) dose dependently
blocks nicotine-induced uptake of
22Na+ into PC12 cells, the
initial cationic stage in nicotinic cholinergic signal transduction;
the IC50 of approximately 0.25 µM
paralleled that for blockade of catecholamine release
(IC50, 0.20 µM) (7). Here we
tested the potencies of several catestatin analogs at a
10-µM dose on nicotine-induced uptake of
22Na+; not only full-length
catestatin (bovine chromogranin
A344-364; secretion
IC50, 0.31 ± 0.032 µM), but
also two of its active analogs [bovine chromogranin
A344-358 (secretion
IC50, 0.22 ± 0.023 µM) and
chromogranin A347-358
(secretion IC50, 3.64 ± 0.213
µM)] substantially (>80%; P < 0.01)
inhibited nicotine-induced
22Na+ uptake (Fig. 3
). However, an inactive catestatin
analog (bovine chromogranin
A348-358; secretion
IC50, >10 µM) was
relatively ineffective (
20%, although still P <
0.01) in inhibiting 22Na+
influx triggered by nicotine (Fig. 3
).
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Individual Amino-Terminal Residues
To test the role of amino-terminal amino acids in inhibition of
desensitization, catestatin amino-terminal truncations were tested as
described above; deletion of even the first amino-terminal residue from
the catestatin core (Arg344) caused an
approximately 4.8-fold loss of inhibition (P < 0.01).
Deletion of the next two residues (Ser345 and
Met346) caused modest decrease (6- to 7.5-fold
in potency). Deletion of four to seven additional residues
(Arg347 through Phe350)
resulted in complete loss of detectable potency (Fig. 4B
).
Individual Carboxyl-Terminal Residues
Deletion of even the first carboxyl-terminal residue
(Arg358) caused an approximately 9-fold decrease
in inhibition of desensitization (P < 0.01). Deletion
of the next four residues (Phe357 through
Gly354) resulted in further loss (14-fold) of
potency. Further deletions (past Arg353)
eliminated detectable potency (Fig. 4C
).
Individual Interior Amino Acids: Alanine Substitution
(Alanine-Scanning) Mutants
Alanine substitution of residues 345 (S345A), 347 (R347A), 351 (R351A),
and 357 (F357A) had little effect on the ability of the peptide to
inhibit desensitization (P > 0.05). Alanine
substitution of residue 344 (R344A), 350 (F350A), 353 (R353A), 354
(G354A), 355 (Y355A), 356 (G356A), or 358 (R358A) caused modest
(2.2- to
4.4-fold) loss of inhibition of desensitization
(P < 0.01 to P < 0.05). Substitution
of residue 346 (M346A), 348 (L348A), or 349 (S349A) caused severe
(
4.8- to 7-fold) loss of potency (P < 0.01; Fig. 4D
).
Comparative Potencies for Inhibition of Secretion vs.
Desensitization: Differences in Crucial Individual Amino Acid
Residues
In Fig. 5, we present detailed
dose-response curves for the effects of alanine substitution mutants of
catestatin on secretion (Fig. 5A
) vs. desensitization (Fig. 5B
). Rightward shifts of dose-response curves for crucial mutants are
apparent in each case. Also apparent is a biphasic effect of catestatin
peptides to inhibit desensitization, with a decline in activity at very
high peptide concentrations.
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Catestatin Effects in Vivo
In the in situ perfused rat adrenal gland (Fig. 6), catecholamine release was triggered
by both splanchnic nerve stimulation (10 Hz, 30 sec) and nicotinic
cholinergic agonists (acetylcholine or nicotine). In each
case, catestatin inhibited secretion, although the inhibition was most
efficient (>80%) for secretion caused by nicotine
itself. Of note, neural stimulation can mobilize both acetylcholine and
PACAP as ganglionic transmitters (6), and acetylcholine may trigger
catecholamine secretion by both muscarinic and nicotinic pathways in
the rodent (12); by contrast, catestatin is selective for the nicotinic
cholinergic mechanism (7).
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DISCUSSION |
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Serial three-amino acid deletions (Fig. 1) defined an active core
(chromogranin A344-358)
required for inhibition of nicotine-stimulated
catecholamine release. Serial single amino acid deletions of the active
core (Fig. 2
) revealed the particular importance of amino-terminal
residues; removal of even Arg344 alone reduced
potency by about 12-fold. Within the interior of chromogranin
A344-358, alanine
substitution mutagenesis revealed crucial roles for
Leu348, Phe350,
Arg351, Arg353, and
Arg358. Of note, both hydrophobic
(Leu348, Phe350) and
charged (Arg351, Arg353,
Arg358) residues were important. Although removal
of residue Arg344 caused a substantial
(
16-fold; P < 0.01) decrement in potency (Fig. 2A
),
substitution to Ala344 (in mutant R344A; Fig. 2C
)
did not significantly (P > 0.05) affect potency,
suggesting that although the presence of a residue at position 344 is
important, its qualitative nature may be less important. By contrast,
removal of Arg358 caused only an approximately
5-fold (P < 0.05) decrease in potency (Fig. 2B
),
whereas substitution to Ala358 (in mutant R358A;
Fig. 2C
) caused a more substantial, approximately 21-fold
(P < 0.01) decrement in potency, suggesting that the
precise nature of the residues guanidino side-chain at position 358
is important.
Earlier studies with substance P analogs showed that both the carboxyl- and amino-termini are required for inhibition of nicotine-evoked secretion (13), binding to chromaffin cell membranes (14), and inhibition of agonist-stimulated Na+ influx in pheochromocytoma cells (15).
We also tested the effects of multiple or qualitative changes in
catestatin amino acids (Fig. 2D). Although both Arg and Lys residues
carry a positive charge, conversion of each Arg to Lys decreased
potency about 6-fold (P < 0.01); the guanidino
nitrogens of Arg carry especially high pKa (-log10 Ka
where Ka = dissociation constant) values (pKa 12.48) (16),
perhaps in part accounting for the strong maximum of positive
electrostatic potential at the tip of the catestatin loop (17), which
may be crucial for the docking of catestatin at the electronegative
vestibule of the nicotinic receptor cation pore (17). The nicotinic
antagonist potency of catestatin was also diminished about 9-fold
(P < 0.01) upon inversion of chirality of each amino
acid (all L
D isomers),
suggesting that charge alone does not determine catestatins potency;
instead, particular side chain spatial orientations seem to be
important (Fig. 2D
).
The initial step in nicotinic cholinergic signal transduction is
movement of extracellular Na+ into the
intracellular space through the nicotinic receptor, which is an
extracellular ligand-gated cation pore (18). We have previously shown
that catestatin blocks such nicotinic signaling in parallel with its
blockade of nicotine-stimulated secretion (7). In the present
experiments, we have shown that although active catestatin analogs or
fragments (e.g. bovine chromogranin
A344-358) block nicotinic
signal transduction, fragments that have no effect on secretion
(e.g. bovine chromogranin
A348-358; secretion
IC50, >10 µM) are also
relatively ineffective in blocking signal transduction (Fig. 3). Thus,
blockade of nicotinic signaling seems to be a prerequisite for the
secretory potency of catestatin peptides.
Nicotinic agonist-induced catecholamine release displays desensitization after repeated exposure to agonist (9), and catestatin is a potent inhibitor of this desensitization response (9). Here we show that although active catestatin fragments (e.g. bovine chromogranin A344-358; secretion IC50, 0.22 µM) also inhibit desensitization (IC50, 0.50 µM), inactive catestatin fragments (e.g. bovine chromogranin A344-352; secretion IC50, >10 µM), by contrast, also fail to block desensitization (IC50, >10 µM).
We have recently shown that nicotinic antagonist catestatin inhibits
agonist-induced desensitization of catecholamine release. This blockade
of desensitization was dose dependent (IC50, 0.54
µM; Fig. 4A), noncompetitive with agonist, and
noncooperative (9). In the present study, the catestatin active core
(bovine chromogranin
A344-358) also displayed
dose-dependent inhibition (IC50, 0.50
µM; Fig. 4A
) of desensitization of catecholamine release.
Previous studies revealed that the carboxyl-terminus of substance P is
important for the blockade of nicotinic agonistinduced
desensitization (13).
Within the catestatin core (chromogranin
A344-358), different amino
acid residues seemed to be crucial for the peptides actions to block
nicotinic cholinergic agonist-stimulated catecholamine release (Fig. 2)
compared with agonist-induced desensitization of release (Fig. 4
). At
the amino-terminus, deletion of Arg344 caused an
approximately 16-fold decrement in potency for secretion inhibition
(Fig. 2A
), whereas for desensitization (Fig. 4B
), loss of
Arg344 caused only about 4.8-fold loss of
potency. At the carboxyl-terminus, deletion of
Arg358 caused only about a 5.1-fold decline in
potency for secretion inhibition (Fig. 2B
), but a 9.3-fold fall in
potency for desensitization (Fig. 4C
). Alanine substitution mutagenesis
also suggested differences. For secretion inhibition (Fig. 2C
),
residues Met346, Leu348,
Phe350, Arg351,
Arg353, Gly354,
Tyr355, Phe357, and
Arg358 were crucial (P < 0.01),
whereas for blockade of desensitization (Fig. 4D
), residues
Arg344, Met346,
Leu348, Ser349,
Phe350, Arg353,
Gly354, Tyr355,
Gly356, and Arg358 were
most important (P < 0.01 to P <
0.05). Composite Fig. 5C
(on the effects of alanine scanning mutants on
inhibition of secretion vs. desensitization) illustrates how
particular regions of the active catestatin core were differentially
important for these two functions; alanine substitutions toward the
amino-terminus (especially R344A, M346A, and S349A) had a tendency to
inactivate desensitization blockade, whereas alanine substitutions
toward the carboxyl-terminus (especially R358A, F357A, R353A, R351A,
and F340A) were particularly apt to affect secretion inhibition.
Although we observed dissociation (Fig. 5C) in catestatin domains
important for inhibition of nicotine-stimulated secretion (Fig. 2
)
vs. those involved in inhibiting nicotinic desensitization
(Fig. 4
), the significance of these differences is not completely
apparent; however, the nicotinic heteropentamer itself may occupy
different structural conformations during agonist-stimulated
secretion compared with desensitization (19).
In summary, we provide substantial information about the relationship
between primary structure and function of the catecholamine release
inhibitory peptide catestatin (chromogranin
A344-364). A core of 15
amino acids (chromogranin
A344-358) seems to be
sufficient to exert catestatins typical effects on catecholamine
release (Figs. 1 and 2
), nicotinic cationic signal transduction (Fig. 3
), and blockade of nicotinic agonist-induced desensitization (Fig. 4B
). However, different amino acids within the core seem to be
especially crucial for differentiating the catecholamine release
inhibitory and desensitization inhibitory functions of the peptide.
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MATERIALS AND METHODS |
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Secretagogue-Stimulated Release of Norepinephrine
Norepinephrine secretion from PC12 cells was monitored as
previously described (21). Cells were plated on
poly-D-lysine-coated six-well polystyrene dishes (Falcon
Labware, Oxnard, CA), labeled for 3 h with 1 µCi
L-[3H]norepinephrine (71.7 Ci/mmol;
NEN Life Science Products, Boston, MA) in 1 ml PC12 growth
medium, washed twice with release buffer (150 mM NaCl, 5
mM KCl, 2 mM CaCl2, and
10 mM HEPES pH 7), and then incubated at 37 C for 30 min in
release buffer with or without secretagogues, such as
nicotine (0.11000 µM), or cell membrane
depolarization (55 mM KCl). 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), and 0.1% (vol/vol) Triton X-100. Release medium and cell lysates
were assayed for
L-[3H]norepinephrine by liquid
scintillation counting, and results were expressed as the percent
secretion: [amount released/(amount released + amount in cell
lysate)] x 100. Net secretion is secretagogue-stimulated release
minus basal release.
Desensitization of Catecholamine Release and Its Blockade by
Catestatin
Desensitization of catecholamine release was performed as
described previously (9). Briefly, cells were preloaded with
L-[3H]norepinephrine and then
exposed to nicotine (60 µM) for 10 min
(incubation I). Cells were washed twice (6 min each) in secretion
buffer, as described above, and rechallenged with nicotine
(60 µM) for 10 min (incubation II), after which the cells
were harvested for measurement of norepinephrine release. Control cells
received nicotine (60 µM) only in incubation
II.
Synthetic Peptides
Catestatin 21-mer (bovine chromogranin
A344-364;
RSMRLSFRARGYGFRGPGLQL), catestatin core 15-mer (bovine chromogranin
A344-358;
RSMRLSFRARGYGFR), or their truncation or substitution analogs were
synthesized at 10100 µmol scale by the solid phase method (22)
using t-boc (tertiary butoxycarbonyl) or f-moc
(9-fluorenylmethoxycarbonyl) protection chemistry and then were
purified to more than 95% homogeneity by reverse phase HPLC on
C18 silica columns, monitoring
A280 (aromatic rings) or
A214 (peptide bonds). The authenticity and purity
of wild-type peptides were verified by rechromatography. Peptides were
characterized by ion spray mass spectrometry on a Perkin-Elmer Corp. Sciex API III instrument (Norwalk, CT); in each case, the
molecular mass of the appropriate target peptide was identified. For
some experiments, small (1 µmol) scale peptide syntheses were
accomplished by the pin technology (Chiron Corp., San
Diego, CA), after which peptides were cleaved from the resin and
washed.
Catestatin Effects in Vivo
The in situ perfused rat adrenal gland preparation
was used, as previously described (6, 23). The three stimuli used to
cause exocytotic release of catecholamines were electrical stimulation
of the splanchnic nerve (10 Hz, 30 sec), acetylcholine (10
µM, 2 min), and nicotine (10
µM, 2 min). This degree of electrical field
stimulation selectively activates splanchnic nerves, but not chromaffin
cells (6). Between stimulations, secretion was allowed to return to
basal values over 1 h. Incised left adrenal glands were perfused
through a left renal vein cannula (continuous with the left adrenal
vein) at 37 C with a Krebs-bicarbonate solution at 700 µL/gland·2
min. Two-minute adrenal effluent samples were collected from the
incised glands onto an ice bath. Catecholamines were measured by
spectrofluorometry (23). Experiments were conducted on 3 different
days, and the results were averaged (mean ± 1
SEM).
Statistics
Curve fitting was accomplished in the program Kaleidagraph
(Synergy Software, Reading, PA), using the Stineman
function, which applies a geometric weight ±10% of the data range, to
arrive a smoothed curve. The IC50 value of a
peptide was interpolated as the concentration that achieved 50%
inhibition of nicotinic-stimulated catecholamine release or
desensitization. Experiments were performed in triplicate, with data
(including IC50 values) reported as the mean
± 1 SEM. When only two conditions (e.g. control
and experimental) were compared, the data were evaluated by unpaired
t tests. When multiple conditions (e.g. several
peptides) were compared, one-way ANOVA was used, followed by Dunnetts
multiple comparison post-hoc test, if appropriate.
Statistical significance was concluded at P < 0.05.
Statistics were computed with the program InStat (GraphPad Software, Inc., San Diego, CA).
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FOOTNOTES |
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This work was supported by the Department of Veterans Affairs and the NIH (DA-11311 to S.K.M., and HL-55583 and HL-58120 to D.T.O.C.).
Received for publication November 18, 1999. Revision received June 1, 2000. Accepted for publication June 16, 2000.
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REFERENCES |
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