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The Journal of Neuroscience, January 15, 2002, 22(2):377-388
Modulatory Mechanism of the Endogenous Peptide Catestatin on
Neuronal Nicotinic Acetylcholine Receptors and Exocytosis
Carlos J.
Herrero1,
Eva
Alés1,
Antonio J.
Pintado1,
Manuela G.
López1,
Esther
García-Palomero1,
Sushil K.
Mahata2,
Daniel T.
O'Connor2,
Antonio G.
García1, and
Carmen
Montiel1
1 Departamento de Farmacología and Instituto
Teófilo Hernando, Facultad de Medicina, Universidad
Autónoma de Madrid, 28029 Madrid, Spain, and
2 Department of Medicine and Center for Molecular Genetics,
University of California at San Diego, San Diego, California 92161
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ABSTRACT |
The catestatin fragment of chromogranin A is the first known
endogenous compound able to inhibit catecholamine release elicited by
the activation of neuronal nicotinic acetylcholine receptors (nAChRs)
of different animal species and catecholaminergic cell types. However,
how catestatin regulates the receptor activity, which subunit
combination of the heteropentameric forms of receptor is better blocked
by the peptide, or how it affects the different stages of the
exocytotic process have not yet been evaluated. To address these
questions, we have assayed the effects of catestatin: (first) on the
inward currents elicited by ACh
(IACh) in voltage-clamped oocytes
expressing different combinations of nAChR subunits; and (second) on
the cytosolic Ca2+ concentration,
[Ca2+]c, and quantal release of
catecholamines simultaneously monitored in single adrenal chromaffin
cells stimulated with ACh. Catestatin potently blocks all the subtypes
of nAChRs studied. Furthermore, it inhibits the
3
4 current in a reversible,
noncompetitive, voltage-, and use-dependent manner, a behavior
compatible with open-channel blockade. In fura-2-loaded single
chromaffin cells, the peptide reduced the
[Ca2+]c signal and the total release
of catecholamines elicited by ACh; however, catestatin did not modify
the kinetics or the last step of the exocytotic process. Our results
suggest that catestatin might play an autocrine regulatory role in
neuroendocrine secretion through its interaction with different native
nAChR subtypes; the extent of receptor blockade by the peptide could be
acutely regulated by the intensity and duration of the presynaptic stimulus.
Key words:
catestatin; chromogranin A; 34 nAChRs; 7 nAChRs; 42 nAChRs; 32 nAChRs; exocytosis; Xenopus
oocytes; chromaffin cells
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INTRODUCTION |
Chromogranin A, the major soluble
protein in endocrine and neuronal storage secretory vesicles (Blaschko
et al., 1967
; Winkler et al., 1986; Huttner et al., 1991), has
encountered considerable speculation regarding its physiological role,
particularly as a precursor of several biologically active peptides.
Clues have emerged about the functions of a few peptides cleaved from
the chromogranin A molecule; i.e., relaxation of blood vessels and inhibition of parathyroid hormone release by vasostatin (Aardal and
Helle, 1992), inhibition of insulin release by pancreastatin (Tatemoto
et al., 1986), and the antibacterial activity of secretolytin and
chromacin (Goumon et al., 1996; Strub et al., 1996).
Recently, Mahata et al. (1997) discovered a novel fragment of
chromogranin A, termed catestatin (bovine chromogranin
A344-364), which specifically inhibited
nicotine-induced norepinephrine release from both rat PC12
pheochromocytoma cells and primary cultures of bovine chromaffin cells.
Several forms of catestatin (bovine, human, rat) showed similar
inhibitory activity and high homology in their amino acid sequences
(Mahata et al., 1997, 2000). These findings raised the hypothesis that
catestatin could serve as a novel autocrine modulator of physiological
neuroendocrine secretion in chromaffin cells and neurons, wherever the
active fragment could be formed and released in vivo. This
seems to be the case, as judged by the extensive processing of
catestatin from chromogranin A found within chromaffin granules and
vesicles of bovine chromaffin cells, rat PC12 cells, and postganglionic
sympathetic nerves; furthermore, catestatin could be released from
stimulated chromaffin cells by an exocytotic mechanism (Taylor et al.,
2000).
Early experiments designed to evaluate the effects of catestatin on
[3H]-norepinephrine release were
performed in cells stimulated with nicotine for several minutes; thus,
such experiments failed to provide a full picture of the mechanism of
action of the peptide at the neuronal nicotinic acetylcholine receptor
(nAChR) or on the secretory process itself, because of poor time
resolution. Additionally, the previous experiments did not provide
information concerning the subtype of nAChR preferentially blocked by
catestatin. This is an important issue, because at least two different
classes of nAChRs, most likely formed by the combination of
3 and 4 subunits or
7 subunits that bind -bungarotoxin, have
been implicated in the control of catecholamine secretion in chromaffin
cells (Criado et al., 1992; Campos-Caro et al., 1997; López et
al., 1998).
In this framework, we planned this study with catestatin to explore the
following: (1) the efficacy of the peptide to block currents generated
by ACh in Xenopus laevis oocytes expressing different combinations of nAChR subunits, (2) the mechanism of the
interaction between catestatin and the expressed nAChR, (3) catestatin
cleavage from chromogranin A within chromaffin granules from human
pheochromocytoma cells, and (4) the actions of catestatin on
[Ca2+]c and the
release of catecholamines, measured simultaneously in single mouse
chromaffin cells stimulated with short pulses of ACh.
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MATERIALS AND METHODS |
Techniques for in vitro transcription of cDNAs
encoding for the nAChR subunits, oocyte injections, and
electrophysiological recordings of the expressed foreign receptors,
using a virtual ground circuit, have been described previously (Miledi
et al., 1989; López et al., 1998; Herrero et al., 1999; Pintado
et al., 2000).
Preparation of cRNA and oocyte injection. The plasmids
containing the cDNA clones encoding for the 3,
4, 4,
2, and 7 rat neuronal
nAChR subunits were linearized with the corresponding restriction
enzyme and transcribed using the mCAP RNA capping Kit (Stratagene, La
Jolla, CA). Mature female Xenopus laevis frogs obtained from a commercial supplier (Centre de Recherches de Biochimie Macromoléculaire du Centre National de la Recherche
Scientifique, Montpellier, France) were anesthetized with a
tricaine solution (0.125%), and ovarian lobes were dissected out.
Then, follicle-enclosed oocytes were manually stripped from the ovary
membranes and incubated overnight at 16°C in a modified Barth's
solution containing (in mM): NaCl 88, KCl 1, NaHCO3 2.4, MgSO4 0.82, Ca(NO3)2 0.33, CaCl2 0.41, HEPES 10, buffered to pH 7.4 and
supplemented with gentamycin (0.1 mg/ml) and sodium pyruvate (5 mM). The next day, healthy follicle-enclosed
oocytes were injected with 50 nl (50 ng) of 7
RNA or 50 nl (25:25 ng) of
3/4,
3/2, or
4/2 cRNAs, using a
Nanoject automatic injector (Drummond Scientific Co., Broomall, PA).
Electrophysiological recordings were performed 2-5 d after RNA injections.
Electrophysiology. Experiments with oocytes were performed
at room temperature (22-25°C) in Ringer's solution containing (in mM): NaCl 115, KCl 2, CaCl2
1.8, HEPES 5, buffered to pH 7.4 with NaOH. Inward currents through the
nAChRs expressed were recorded with a two-electrode voltage-clamp
amplifier (OC-725-B Warner Instrument Corporation, Hamden, CT) using
microelectrodes with resistances of 0.5-5 M
made from borosilicate
glass (GC100TF-15, Clark Electromedical, Pangbourne, UK) and filled
with KCl (3 M). The holding potential in all
experiments was 
60 mV, except in those designed to study the voltage
dependence. Single oocytes were held in a 0.3 ml volume chamber and
constantly superfused with Ringer's solution by gravity (4 ml/min).
The volume in the chamber was maintained constant using the reverse
suction of an air pump. Solutions containing ACh or catestatin were
applied by means of a set of 2-mm-diameter glass tubes located close to the oocyte. Voltage protocols, ACh pulses, and data acquisition were
controlled using a Digidata 1200 Interface and the CLAMPEX software
(Axon Instruments, Foster City, CA).
Flash photolysis of Ca2+ from a
caged-Ca2+ compound injected into the
oocytes. These experiments, designed to directly activate the
native Ca2+-activated chloride current
(ICl(Ca)) expressed in oocytes, were performed as described previously (Montiel et al., 1997; Pintado et
al., 2000). Briefly, oocytes were injected with 41 nl of the caged-Ca2+ solution containing 50 mM DM-nitrophen, 45 mM CaCl2, 5 mM HEPES, at pH 7. After equilibrium in the dark
for at least 30 min, individual oocytes were voltage clamped at 60
mV. ICl(Ca) was activated by
Ca2+ released from the
caged-Ca2+ compound after the application
of repetitive flashes of light using a high intensity Xenon flash lamp
system (Cairn Research Ltd., Faversham, Kent, UK). Ultraviolet light
was focused onto the oocyte surface using a light guide positioned over
the vegetal pole. The capacitance value used was 2000 µF charged to
200 V. Repetitive flashes of constant intensity and duration were given to the oocyte every 4 min; flashes were controlled by a PC computer using the CLAMPEX software. Under these experimental conditions, caged-Ca2+-injected oocytes gave
reproducible ICl(Ca) responses after
repetitive flashes.
Mass spectrometric analysis of immunoprecipitated
catestatin from chromaffin granules. Chromaffin granules from
freshly obtained human pheochromocytoma were prepared by centrifugation
on 0.3 M/1.6 M sucrose
density step gradients, lysed in 1 mM
NaH2PO4 at pH 6.5, and
centrifuged to remove granule membranes (Mahata el al., 1997). Then,
catestatin was immunoprecipitated (IP) from the granule soluble core
lysate using a polyclonal rabbit antibody that recognizes the
catestatin region of human chromogranin A352-372 (SSMKLSFRARAYGFRGPGPQL), according to the protocol described by Taylor
et al. (2000). After immunoprecipitation and washing of the IP product,
immunoprecipitated catestatin was subjected to matrix-assisted laser
desorption ionization (MALDI) mass spectrometry using a Voyager-Elite
mass spectrometer. Samples were embedded in an
-cyano-4-hydroxycinnamic acid matrix and then irradiated with
a nitrogen laser at 337 nm, and the ions produced were accelerated with
a deflection potential of 30,000 V. Ions were then differentiated according to their mass/charge ratio (m/z) using
a time-of-flight mass analyzer. The mass error of this method is
characteristically 
0.1%. Molecular weights from MALDI mass spectra
were interpreted, and peptide fragments within the chromogranin A
primary structure were assigned by the program PAWS (Protein Analysis
WorkSheet, version 8.1.1., for Macintosh), assigning average isotopic
MH+ values for chromogranin A peptides as
described previously (Taylor et al., 2000).
Simultaneous electrochemical detection of
[Ca2+]c and catecholamine release from
single mouse chromaffin cells. Cells were isolated and cultured as
described previously (Hernández-Guijo et al., 1998). Recordings
were made on days 2-5 in culture. Cells attached to glass coverslips
were loaded with the acetoxy-methyl ester form of the fluorescent dye
fura-2 (fura-2 AM) (2.5 µM for 40 min at
37°C, in the dark). Then, cells were washed with Krebs'-HEPES solution (in mM: NaCl 144, KCl 5.9, MgCl2 1.2, CaCl2 2, HEPES 10, glucose 11; pH 7.3, titrated with NaOH) and kept for 10 min at
37°C in an incubator before being placed in a perfusion chamber mounted on the stage of a Nikon Diaphot inverted microscope. The chamber was perfused continuously, at room temperature, with
Krebs'-HEPES. Solutions containing ACh and/or catestatin were changed
using a multi-barreled concentration-clamp device (Gandía et
al., 1993). Only one experimental protocol was run on each single
coverslip. Microelectrodes for the detection of catecholamine release
were prepared by introducing a carbon-fiber electrode (12 mm radius) into a patch pipette (Portex, Kent, UK), insulated as described (Chow
et al., 1992), and sealed into glass micropipettes with epoxy
(CIBA-Geigy). Microelectrodes (20-60 G) were backfilled with 3 M KCl solution and connected to a homemade
amplifier. A constant voltage of 780 mV versus Ag/AgCl reference was
applied to the electrode. The tip of the carbon-fiber electrode was
gently pressed against the cell surface. The amperometric current was filtered at 2 kHz and sampled at 1 kHz. Single-cell fluorescence measurements were performed by exciting the fura-2-loaded cells with
alternating 360 and 390 nm filtered light. The apparent
[Ca2+]c was
calculated from the ratio of the fluorescence signal (Grynkiewicz et
al., 1985): [Ca2+]c = Keff (R R0)/(R1 R), where Keff is an
"effective binding constant," R0 is the
fluorescence ratio at zero Ca2+, and
R1 is the limiting ratio at high
Ca2+. These calibration constants were
experimentally determined as described previously (Almers and Neher,
1985). R is the observed or experimental ratio. An
ADInstrument MacLab/4e interface controlled by a Macintosh G4 running
the MacLab Chart application was used to record, display, and analyze
simultaneously the
[Ca2+]c and
electrochemical data. Individual spike characteristics were analyzed
using the automatic analysis program IGOR (Wavemetrics, Lake Oswego,
OR) and the macros described by Segura et al. (2000).
Materials and solutions. If not specified, all products were
purchased from Sigma (Madrid, Spain). DMEM, fetal calf serum, and
antibiotics were obtained from Invitrogen (Madrid, Spain). Fura-2
AM was from Molecular Probes (Leiden, The Netherlands). Catestatin (bovine chromogranin A derivative peptide A
(RSMRLSFRARGYGFRGPGLQL) was synthesized according to Mahata et al.
(1997).
Fitting procedure and statistical analysis. Values of
agonist concentration eliciting half-maximal current
(EC50), antagonist concentration
eliciting 50% blockade of maximal current
(IC50), and Hill coefficient
(nH) were estimated through nonlinear
regression analysis using the four-parameter logistic equation of the
GraphPad Prism software for a PC computer. To calculate the time
constants (
) for blockade and recovery of the nicotinic responses,
records were fitted to a single exponential curve. Results are
expressed as means ± SE. Differences between groups were compared
by Student's t test using the statistical program
Statworks; a value of p 0.05 was taken as the limit of
statistical significance.
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RESULTS |
Catestatin causes fast and reversible blockade of
ACh-evoked currents
Oocytes expressing
34 nAChRs generated
large inward currents (IACh) after
application of ACh onto their surface. An example of such currents is
shown in Figure 1A.
These original traces were obtained in an oocyte voltage clamped at
60 mV and intermittently stimulated with 20 sec pulses of 100 µM ACh, given at 2 min intervals. Observe that
control IACh peaks had similar
amplitudes after repeated stimulation; currents were reproducible for
>12 pulses (data not shown). To determine the effects of catestatin on
IACh, the peptide (1 µM) was introduced into the superfusion system,
and two successive ACh pulses were applied in its presence; catestatin
was added 1 min before the ACh pulse and maintained throughout the next ACh pulse. Catestatin produced a fast blockade of
IACh; the maximum effect on the peak
current amplitude (~60% blockade) was already observed during the
first ACh pulse. The blocking effect of catestatin was readily
reversible after its washout (Fig. 1A, fifth
trace to the right).
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Figure 1.
Catestatin reduces
34 current
(IACh) expressed in oocytes, with
different potencies according to its mode of application.
A, Typical oocyte expressing
34 nAChRs, voltage clamped at 60 mV,
and continuously superfused with Ringer's solution. Successive pulses
of ACh (100 µM, 20 sec) were applied every 2 min, as
shown by the top horizontal black bars. After 2-3
initial ACh pulses, this protocol gives reproducible signals for >12
pulses. When IACh is stabilized, catestatin
(CAT) was added 1 min before and during the next
two ACh pulses, as indicated by the white horizontal
bar. B, Native Ca2+-activated
chloride currents, ICl(Ca), recruited
by Ca2+ photoreleased from a
caged-Ca2+ compound previously injected into a
typical oocyte. Three light flashes, applied 4 min apart, were used to
induce the photorelease (as indicated by the  ). Catestatin
(CAT) or niflumic acid
(NF), a specific
ICl(Ca) blocker, was applied as indicated by
the top horizontal black bars. Each trace of
ICl(Ca) represents a period of 700 msec.
This experiment was reproduced in three additional oocytes, with
similar results. C, Concentration-response curves for
catestatin blockade on IACh using two
catestatin application protocols. Values were expressed as percentage
of control IACh (in the absence of peptide),
measured at the peak amplitude. The first application protocol
consisted of the addition of catestatin during the ACh pulse
(co, coapplication); in the second, catestatin was
preincubated 1 min before the ACh pulse and maintained during the pulse
(pre+co, preincubation plus coapplication). Each
point represents means ± SE of values obtained in
6-10 oocytes. C, Inset, Original traces
of IACh obtained from one oocyte stimulated
with successive ACh pulses (as in A), in the absence
(Control) or presence of 1 µM
catestatin, using the two application protocols. The maximum blockade
on IACh was achieved with the
pre+co mode.
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Because the nAChR ion pore is permeable to
Ca2+ in addition to
Na+ (Seguela et al., 1993), it was
important to rule out that rather than a direct antagonist action of
catestatin on the nAChR itself, the effect of the peptide on
IACh might have resulted from a
blockade of the current generated by native chloride channels highly
expressed in oocytes (Miledi and Parker, 1984). This chloride current,
ICl(Ca), could be activated by
Ca2+ flowing through the receptor ion
channel. The result shown in Figure 1B proves that
this was not the case. In this experiment, traces of
ICl(Ca) were obtained in one oocyte
injected with DM-nitrophen-caged Ca2+ (see
Materials and Methods). Flashes of light, applied at 4 min intervals,
elicited ICl(Ca) peaks of reproducible
amplitude. Catestatin, at the concentration of 10 µM that fully blocks
IACh through
34 receptors (Fig.
1C), did not affect
ICl(Ca). This result contrasts with
the nearly full current blockade caused by 100 µM niflumic acid, a selective blocker of
ICl(Ca).
Concentration-dependent blockade of
IACh, induced by catestatin when
applied in two different modes
Two modes of catestatin application were assayed in
34-injected oocytes
to determine the mechanism of IACh
blockade by the peptide. The experimental procedure consists of
successive ACh pulses (100 µM, for 20 sec)
applied at regular 2 min intervals. When
IACh responses became stable, after
two or three initial ACh control pulses, catestatin (1 µM) was superfused as follows: (first) in the
coapplication mode, the peptide was given just during the ACh pulse;
(second) in the preincubation plus coapplication mode, catestatin was
given 60 sec before as well as during the ACh pulse. Figure
1C (inset) shows the original traces of
IACh obtained from one oocyte using
the two protocols of catestatin applications; the maximum blockade on
IACh (measuring the peak amplitude or
the net charge) was achieved with the second application mode. With the
two modes of catestatin delivery, the effect of the peptide on
IACh developed gradually along the ACh
pulse. The concentration-response curves plotted in Figure
1C were obtained from different oocytes, and they
represented the blockade of the peak
IACh exerted by increasing
concentrations of catestatin, when the peptide was assayed according to
the two protocols described above. Values were expressed as percentage
of control response evoked by ACh alone (100%). The
IC50 values for catestatin blockade of
IACh obtained from these curves were
4.9 µM when the peptide was present only during
the ACh pulse and 0.4 µM when catestatin was
also preincubated. When calculating the net charge values of
IACh, rather than the peak amplitude,
the IC50 values obtained were 2.1 and 0.2 µM in the absence or presence of catestatin
preincubation, respectively; note that differences in blockade between
the two modes of peptide delivery were still maintained.
Noncompetitive blockade of IACh
by catestatin
To further study the mechanism whereby catestatin blocks nAChRs,
we assayed the effects of the peptide on
34 currents elicited by increasing concentrations of ACh, applied as sequential pulses (20 sec) every 2 min, to avoid receptor desensitization. Figure 2A shows the control
IACh curve; data points represent the
peak amplitude of ACh-evoked currents expressed as percentage of the maximum response to ACh alone (Imax),
which was reached with 1 mM ACh. The threshold
concentration of ACh capable of inducing a measurable current was 10 µM. Figure 2A also represents
the peak amplitudes of the IACh curve
in the presence of 1 µM catestatin (added 1 min
before and during the ACh pulse); data were expressed as percentage of
the Imax obtained from the same
oocyte, in the absence of catestatin. The results from different
oocytes were compiled to generate a plot of mean peak current amplitude
versus ACh concentration. Note that catestatin blockade was
insurmountable by increasing concentrations of ACh; moreover, as shown
in Figure 2A, inset, the peptide
reduced by 59 ± 7% the peak current amplitude evoked by 1 mM ACh, whereas blockade was significantly lower
when IACh was elicited by 10 µM ACh (40 ± 4%; p 0.05). Figure 2B shows two original traces of
IACh obtained in the same
34-injected oocyte
stimulated by ACh (10 µM or 1 mM), in the absence or presence of 1 µM catestatin; note the higher blockade at the
higher ACh concentration. To reinforce the noncompetitive nature of the
effect of catestatin on the nicotinic receptor, a similar experimental design was performed with 10 µM
dihydro--erythroidine (DHE), a well known competitive blocker at
the nAChR. This compound produced a parallel shift to the right of the
concentration-response curve for ACh (Fig. 2A). In
contrast to catestatin, we found that DHE antagonism on
IACh could be overcome by increasing
the ACh concentration (Fig. 2A,
inset).
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Figure 2.
Catestatin blocks 34
receptors in a noncompetitive manner. A, The
concentration-response curve for catestatin (1 µM) and
dihydro--erythroidine (DHE, 10 µM) on IACh elicited by
sequential pulses of increasing concentrations of ACh
(abscissa). Each oocyte was stimulated with pulses of
increasing concentrations of ACh of 20 sec duration, applied at 2 min
intervals. We took advantage of the ready reversibility of catestatin
blockade to assay the effects of two to four different concentrations
of the peptide in each individual oocyte. Each response was normalized
to the response of the same oocyte to 1 mM ACh (100%), a
concentration of ACh that elicited the maximal current
(Imax). Data are means ± SE of
values obtained in the number of oocytes shown in
parentheses. The inset represents the
relative percentage of blockade of control
IACh exerted by the two blockers, at each
concentration of ACh. Blockade of IACh by
catestatin was significantly greater at higher (1 or 3 mM)
than at lower ACh concentrations (10 or 30 µM;
*p < 0.05). B, Original traces of
the 34 currents elicited by two different
concentrations of ACh (10 µM and 1 mM)
assayed in the same oocyte, revealing a higher blockade by catestatin
of IACh elicited by 1 mM Ach.
Note the different calibration bars.
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Maximum peak currents (Imax) induced
by 1 mM ACh, in the absence or presence of
blockers, were obtained from the concentration-response curves for ACh
in Figure 2A. Results show that DHE did not modify control Imax (3.3 ± 0.3 vs
3.0 ± 0.3 µM; n = 7); in
contrast, catestatin significantly reduced it (from 8.6 ± 1.4 to
4.1 ± 1.1 µM; p < 0.001;
n = 7). Moreover, the half-stimulatory concentration of
ACh (EC50 = 94.7 ± 8 µM; n = 14 oocytes) was
significantly reduced in the presence of catestatin (66 ± 5 µM; p 0.01). However, in the
presence of DHE, the calculated EC50 value for
ACh increased to 290 ± 16 µM
(p 0.01). The calculated Hill coefficient
(nH) for the control ACh curve was
1.42 ± 0.16, whereas nH values
in the presence of catestatin and DHE were 1.43 ± 0.34 and
1.34 ± 0.46, respectively.
Blockade of 34 currents by catestatin
exhibits a pronounced voltage dependence
We assayed the voltage dependence of nAChR blockade by the peptide
through its capability to inhibit IACh
at different holding potentials. At the beginning, we used brief ACh
pulses (100 µM, 1 sec), applied at 1 min
intervals, to generate IACh at
different membrane potentials. Under these experimental conditions, 1 µM catestatin that was superfused continuously
onto the surface of the
34-injected oocytes
(during the ACh pulses and intervals between pulses) reduced the
current amplitude more at hyperpolarizing than at depolarizing
potentials. Pooled results from 13 oocytes using these experimental
conditions revealed that at 100 mV, catestatin blocked
IACh by 57 ± 4% and
significantly less at 40 mV (35 ± 4%; p 0.01).
Although the voltage dependence of catestatin in blocking
IACh was clearly demonstrated through
the experiments described above, we attempted to disclose this property
more sharply with another protocol, i.e., using longer periods of ACh
pulses. Figure 3A shows the
IACh trace obtained in an oocyte
expressing 34 receptors, voltage clamped at 40 mV. To prevent current
desensitization during the prolonged stimulation period (160 sec), a
moderate concentration of ACh (10 µM) was used.
When the current became stable (40 sec after starting the ACh
stimulation), catestatin (0.3 µM) was perfused
for 60 sec. The peptide caused a quick relaxation of the current,
reaching a plateau at ~52% of the initial current; washout of
catestatin restored the current amplitude to near its pre-catestatin
value. Figure 3, B and C, shows the results
obtained in the same oocyte using a similar protocol, but this time
voltage clamped at 60 or 100 mV, respectively. Note that now
catestatin produced a faster and higher relaxation of the current,
particularly at more hyperpolarized membrane potential; the removal of
the peptide produced a slower and incomplete recovery of the current. Averaged pooled results from five oocytes showed 54 ± 3% current blockade at 40 mV, 73 ± 4% at 60 mV, 83 ± 4% at 80
mV, and 92 ± 3% at 100 mV. The blockade by catestatin of
IACh was fitted to a single
exponential at each membrane potential. Values of on obtained were 9.8 ± 2.5 sec at 40
mV, 8.8 ± 0.5 sec at 60 mV, and 4.5 ± 0.4 sec at 100
mV; values at 40 mV and 60 mV were significantly different
(p 0.01) from those obtained at 100 mV.
Differences in the time required to remove the blockade after
catestatin washout were also found among the different membrane potentials tested. After catestatin was removed,
IACh blockade recovered faster at 40
mV (off, 13 ± 5 sec) than at 60 mV
(off 24 ± 6 sec; p 0.05); the blockade at 100 mV was hardly removed.
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Figure 3.
Catestatin exhibits a pronounced voltage
dependence in blocking IACh through
34 receptors. A, Original
trace of the 34 current elicited by 3 min
application of 10 µM ACh (top horizontal black
bar) in an oocyte voltage clamped at 40 mV; catestatin was
introduced within the ACh pulse, during the period indicated by the
white bar. B, C, The
original IACh traces obtained in the same
oocyte under similar experimental conditions, but voltage clamped at
60 or 100 mV. D, Results of the maximal inhibition
of IACh reached at the end of the catestatin
application, at different holding potentials. Data, expressed as
percentage of inhibition of peak amplitude before addition of the
peptide, are means ± SE of five oocytes. *p < 0.05; ***p < 0.001; comparing with blockade at
100 mV.
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Blockade by catestatin of IACh through
34 receptors shows use dependence
Two limitations arose while we were planning experiments to define
whether the blockade by catestatin of
IACh exhibited use dependence. Thus,
repeated stimulation with ACh at short intervals could cause receptor
desensitization; additionally, the slow superfusion system precluded
the application of very short ACh pulses. Nevertheless, we assayed
whether the extent of blockade exerted by catestatin was significantly
different using different intervals (5 or 10 sec) between two ACh
pulses. Repeated ACh pulsing (100 µM, 0.5 sec;
applied every 5 sec) generated currents through
34 receptors with
peak amplitudes that remained constant for more than 20 consecutive pulses (Fig. 4A). Also,
when ACh was applied at 10 sec intervals, the current suffered no decay
(data not shown). Note in Figure 4B that during
continuous superfusion of 1 µM catestatin onto an oocyte being stimulated with ACh pulses at 5 sec intervals, the
blockade of IACh developed gradually
and become stable after several pulses, at a current amplitude ~36%
of the initial IACh. The current
recovered partially after catestatin washout (data not shown). Figure
4C shows averaged results of gradual blockade of
IACh caused by 1 µM catestatin, in different oocytes stimulated with successive pulses of ACh, applied at 5 or 10 sec intervals. Note
the faster and higher blockade achieved at 5 sec, with respect to 10 sec intervals.
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Figure 4.
Use-dependent blockade by catestatin of
34 currents. A, Original
IACh traces obtained in one oocyte
expressing 34 nAChRs, voltage clamped at
60 mV, and stimulated with repeated brief ACh pulses (100 µM, 500 msec) applied at 5 sec intervals.
B, Typical 34 current
traces obtained in one oocyte stimulated initially as above, first in
the absence and subsequently in the continuous presence of catestatin,
as shown by the top horizontal bar. C,
Pooled results collected from experiments performed in different
oocytes, assaying the effect of catestatin on
IACh elicited by repeated ACh pulses
applied, first at 5 sec intervals (as above) and subsequently at 10 sec
intervals. The initial pre-catestatin IACh
amplitude was normalized to 1 (IACh max),
and the amplitudes of the currents obtained in the presence of
catestatin were expressed in the ordinate as a fraction of the
IAch max (IACh/IACh
max). Data are means ± SE of eight oocytes from three
different donors. *p < 0.05;
**p < 0.01; comparing the blockade obtained in the
same pulse number at the two intervals.
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Effects of catestatin on nAChRs composed of different
subunit combinations
All experiments described so far were performed on
34 nAChRs expressed
in oocytes. The reason is that these two subunits are highly expressed
in adrenal chromaffin cells (Criado et al., 1992), where catestatin is
synthesized, stored, and co-released together with catecholamines
(Mahata et al., 1997; Taylor et al., 2000). Furthermore, it has been
suggested that nAChRs containing 3 and
4 subunits are dominant in the control of
catecholamine secretion from these cells (Wilson and Kirshner 1977;
Kumakura et al., 1980; Trifaró and Lee, 1980; Kilpatrick et al.,
1981). However, bovine chromaffin cells additionally express
7 nAChRs (García-Guzman et al., 1995),
which are also functional (López et al., 1998). Therefore, it was
of interest to know whether catestatin blocked nAChR subunit
combinations other than the
34.
Different combinations of brain nAChR subunits were expressed in
oocytes, and to obtain reproducible
IACh for each combination assayed,
different periods of ACh pulses and different intervals between
successive pulses were selected. Figure
5A (left) shows IACh traces obtained after application
of 5 sec pulses of 100 µM ACh (applied 2 min
apart) onto an oocyte expressing 7 receptors. The control current relaxed very quickly, likely because of the well
established fast desensitization of this receptor subtype after its
stimulation with agonists (López et al., 1998; Papke and
Thinschmidt, 1998; Herrero et al., 1999). Catestatin (1 µM), added 1 min before and during the ACh
pulse, blocked the current by 66%. Figure 5A
(middle) shows a similar experiment performed in an oocyte
expressing 32
receptors stimulated for 20 sec with pulses of 100 µM ACh (2 min apart). Initially,
IACh through these receptors
desensitized quickly and then much more slowly; catestatin (1 µM) blocked by 60% the peak amplitude of the
32 current. Finally,
Figure 5A (right) shows the original traces
generated by ACh pulses (100 µM, 5 sec) applied
at 4 min intervals in an oocyte expressing
42 receptors; note
that the current also desensitized more slowly than the
7 current and that 1 µM catestatin blocked this current by 40%.
Blockade by catestatin quickly reversed on washout of the peptide, at
each of the three receptor types studied (data not shown).
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Figure 5.
Catestatin potently blocked
IACh generated through
7, 32, and
42 nAChRs. These experiments were
performed in oocytes expressing different combinations of nAChR
subunits, following protocols similar to those described in Figure
2A. A, Original traces of
7, 32, or
42 currents elicited by pulses of ACh
(100 µM), in the absence (Control)
or presence of catestatin (). For each receptor subtype, different
protocols for ACh application were selected to prevent current
desensitization: i.e., for 7 receptors, ACh pulses of 5 sec were applied at 2 min intervals; for
32 receptors, ACh pulses of 20 sec were
applied at 2 min intervals; and for 42
receptors, ACh pulses of 5 sec were applied at 4 min intervals. In all
cases, catestatin (CAT) was added 1 min before
and during the ACh pulse. B, Pooled data of the
concentration-response curves for the inhibitory effects of catestatin
on 7, 32,
and 42 currents expressed in oocytes.
Values represent the amplitude of peak IACh
for each concentration of peptide assayed, and they were normalized in
terms of percentage of control IACh (100%),
considered to be the amplitude of peak IACh
preceding the addition of the peptide. Data are means ± SE of the
number of oocytes shown in parentheses.
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Concentration-response curves for catestatin effects on
IACh were performed in different
oocytes expressing 7,
32, or
42 nAChRs (Fig.
5B). In these experiments, oocytes were stimulated with
successive ACh pulses (100 µM) during the
period and intervals mentioned above for each receptor type. The
IC50 values for catestatin blockade were 0.3 µM for 7, 0.4 µM for
32, and 1.7 µM for
42 receptors. The
calculated EC50 value for ACh in oocytes
expressing 7 nAChRs stimulated with increasing
concentrations of the agonist, applied as brief pulses (5 sec) every 1 min, was 95 ± 16 µM (n = 10); this EC50 value for 7 and the
EC50 value for
34 (98 ± 10 µM) were close to those obtained by Stafford et
al. (1994) in oocytes expressing these two receptor subtypes from rat
brain. These authors also found that the EC50
value for ACh in oocytes expressing rat brain
32 and
42 nAChRs was 43 µM for both receptor subtypes.
Processing of chromogranin A to catestatin in human
pheochromocytoma chromaffin granules
Studies of the mechanism of action of catestatin on nAChRs
expressed in oocytes have been performed here using the synthetic peptide. However, before studying the modulatory effect of catestatin on a physiological response, such as the exocytotic catecholamine secretion elicited by ACh in chromaffin cells, it is important to show
that the catestatin fragment of chromagranin A is indeed formed by
endogenous proteolytic cleavage in vivo. Our results of
MALDI mass spectrometry after anti-catestatin immunoprecipitation of
human pheochromocytoma chromaffin granules confirm previous findings
indicating that the catestatin fragment of chromogranin A is excised
endogenously within the chromaffin granules, at particular amino acid
residues (Taylor et al., 2000). The spectrum presented in Figure
6 reveals two peaks. The major peak (at
m/z = 3771) corresponds to human
chromogranin A340-372
(LEGQEEEEDNRDSSMKLSFRARAYGFRGPGPQL) within the chromogranin A primary
structure; calculated m/z (3771) exactly matches
with the experimental value obtained. This major form of catestatin is
bound on either side by dibasic recognition sites for prohormone
cleavage. We also found a smaller quantity of a form with a higher
molecular weight (m/z = 5377), compatible with the human chromogranin A353-399, which is
bound on its C terminus by a dibasic site. Other MALDI peaks in the
figure could not be unambiguously assigned within the human
chromogranin A primary structure.
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Figure 6.
MALDI mass spectrometry identification of
catestatin in immunoprecipitated human pheochromocytoma chromaffin
granules. Aliquots (200 µl) of pheochromocytoma chromaffin granule
soluble core proteins/peptides were immunoprecipitated (20 µl) with
rabbit anti-human chromogranin A352-372 and then subjected
to MALDI mass spectrometry (1-2 µl). A major form of catestatin
cleaved at dibasic sites was found
(m/z = 3771), as well as a smaller
amount of a form containing a C-terminal flanking peptide
(m/z = 5377).
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Catestatin reduces elevations of
[Ca2+]c and transmitter release evoked
by brief ACh pulses in single chromaffin cells
We demonstrated previously that catestatin exerts a clear and
selective inhibitory effect on ACh-elicited catecholamine secretion from populations of rat PC12 and bovine chromaffin cells (Mahata et
al., 1997). However, in those earlier experiments, the limited time
resolution of the protocols used (i.e.,
[3H]-norepinephrine release from
nicotine-stimulated cells and stimulation periods of 15-30 min) could
not resolve which particular phase of the exocytotic process mediated
by nAChR activation is affected by catestatin. Fura-2 fluorescence
measurements combined with the carbon-fiber amperometry records allow
simultaneous on-line monitoring of
[Ca2+]c signals
and the quantal release of catecholamines in single chromaffin cells.
These techniques and experimental conditions keep intact the
cytoplasmic constituents of the cell and allow the study of the
physiological response induced by brief pulses of ACh. Because robust
amperometric responses to ACh are required for analysis of the
catestatin effects at the single cell level, we selected the mouse
chromaffin cell, which in our hands provides larger and more
reproducible signals during repetitive ACh applications. Nonetheless,
catestatin also blocked, with similar efficacy, the exocytotic and
[Ca2+]c responses
elicited by ACh in single rat and bovine chromaffin cells (experiments
not shown).
A brief pulse of ACh (1 mM, 5 sec) applied to single mouse
chromaffin cells caused a fast and transient increase of the
[Ca2+]c signal
from a resting level of 41 ± 8 nM to 397 ± 65 nM (n = 10). Simultaneously, the evoked
electrochemical current (Iamp) elicited by ACh correlated well with the
[Ca2+]c signal and
exhibited a single secretory component characterized by many
superimposed single secretory spikes that are characteristic of quantal
release of oxidizable neurotransmitter from single vesicles that fused
with the plasma membrane at a high rate while the
Ca2+ signal persisted (Fig.
7, amperometric traces). Between the
first and second
[Ca2+]c or
Iamp responses induced by the first
two pulses of ACh, there is normally a decrease in the signal, but
thereafter the responses during subsequent ACh pulses remained stable
(see how control responses indicated by open symbols in Fig.
8C, normalized to the second
ACh pulse, are practically constants). For this reason, to
calculate the percentage of inhibition induced by catestatin, we took
the [Ca2+]c or
Iamp signals elicited by the second
ACh pulse as 100%. The presence of catestatin (3 µM), added 1 min before and during the ACh
pulse, reduced by 30% (286 ± 58 nM) the
peak amplitude of the
[Ca2+]c signal
(Fig. 7A). Consistently, the secretory response expressed as
Iamp was inhibited proportionally.
Moreover, the inhibitory effect on the
[Ca2+]c signal and
secretion was partially reversible after washout of the peptide. It is
worth noting that when catestatin was present only during the 5 sec of
application of the ACh pulse, blockade of the
[Ca2+]c and
Iamp signals was considerably less
(5-10%). The effect of catestatin was specific for the nicotinic
receptor-mediated responses, because the peptide did not affect the
[Ca2+]c signal or
the amperometric current evoked by cell depolarization (70 mM K+; data not
shown).
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Figure 7.
[Ca2+]c
transients and amperometric signals triggered by ACh in the absence and
presence of catestatin. A, Representative
[Ca2+]c and secretion measured
simultaneously in a single mouse chromaffin cell stimulated with six
sequential pulses of ACh (1 mM, 5 sec) applied at 2 min
intervals. Catestatin was perfused 1 min before the third ACh pulse and
maintained throughout until the end of the fourth ACh pulse.
B, Detail of the exocytotic responses evoked by the
second and third ACh pulse in the absence or presence of catestatin,
respectively; the difference in the current magnitude is appreciable.
In both cases, secretion occurs as an amperometric current burst that
reflects exocytosis of catecholamine-containing vesicles at a high
rate.
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Figure 8.
Catestatin diminishes proportionally
Ca2+ influx and secretion elicited by brief ACh
pulses. A, B,
[Ca2+]c transient integral and
amperometric current integral obtained during the stimulation of
different single cells with six sequential ACh pulses (1 mM, 5 sec) applied at 2 min intervals; catestatin (3 µM) was added following the protocol identical to that
described in Figure 7. Between the first and second ACh pulse, there
was normally a decrease in the [Ca2+]c
and in the exocytotic signal, but thereafter the responses elicited by
successive ACh pulses remained practically constant, as shown in the
control normalized data of C (open
symbols); values are expressed as percentage of the signal
elicited by the second ACh pulse (100%). Filled symbols
represent normalized [Ca2+]c and
secretion values obtained in cells stimulated with ACh as above, but in
the presence of catestatin (added where indicated by the black
bar). The peptide decreased significantly both signals by 45 and 55%, respectively (*p < 0.01).
D, Efficacy of Ca2+ influx to
stimulate exocytosis. Values were obtained from the ratio between the
amperometric current integral and the
[Ca2+]c integral for each ACh pulse.
Values are means ± SE of results obtained in 10 different single
cells.
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Catestatin does not influence the efficacy of
[Ca2+]c to trigger the exocytotic
process
Figure 8, A and B, represents the net values
of inhibition of 3 µM catestatin on the global
[Ca2+]c and the
total Iamp induced by sequential ACh
pulses following the protocol described above. These values were
calculated by integrating the area under the
[Ca2+]c signal and
the amperometric traces obtained in 10 different cells. When both
parameters were normalized (referred to as percentage of the response
obtained during the second ACh pulse) and compared with their
respective control values (obtained in cells stimulated with similar
sequential ACh pulses, but in the absence of catestatin), it was
observed that the effect of catestatin on the release of catecholamines
was proportional to its effect on the global
[Ca2+]c signal
(Fig. 8C). Thus, the reduction of the
[Ca2+]c integral
in the presence of catestatin achieves a proportional reduction of
catecholamine secretion. Under control conditions, in the absence of
catestatin, the efficacy of exocytosis elicited by ACh, defined as the
global catecholamine release per increment in
[Ca2+]c, was
18.9 ± 0.61 pA/µM. In the presence of
catestatin, this value was not significantly modified from the control
(Fig. 8D).
Effects of catestatin on kinetics of
[Ca2+]c and exocytotic responses
To understand how catestatin affects kinetic components of the
[Ca2+]c and
secretory responses, we measured the global
[Ca2+]c increment
and the cumulative charge of amperometric spikes elicited by longer
periods of ACh application. Results obtained in a typical cell are
shown in Figure 9A; the cell
was stimulated with two ACh pulses (1 mM, 30 sec), 2 min apart, in the absence (first pulse, control) or presence of
3 µM catestatin added just during the second
ACh reintroduction. Results obtained in these experiments reveal that
the [Ca2+]c and
the amperometric current elicited by the neurotransmitter persisted
even longer than the duration of the ACh pulse. Moreover, the secretory
pattern consisted of a first rapid phase, similar to that observed with
brief ACh pulses, followed by a second slower-rate secretion phase, in
which individual spikes could be distinguished easily (9A,
inset). The presence of catestatin during the second ACh
pulse modified substantially the amplitude and accelerated the decay of
the [Ca2+]c
signal, which was not modified in the absence of the peptide (data not
shown). Averaged curves of the global
[Ca2+]c decay from
four different cells stimulated as in Figure 9A are shown in
Figure 9B. Control (ACh alone) and experimental (ACh plus
catestatin) decay curves could be fitted to single exponential; catestatin increased by 1.25 times the rate of the
[Ca2+]c decay
( = 11.3 vs 14.2 sec in the control curve).
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Figure 9.
Comparison between
[Ca2+]c signals and catecholamine
secretion in single chromaffin cells stimulated with long ACh pulses,
in the absence or presence of catestatin. A,
[Ca2+]c and electrochemical signals
elicited by two sequential ACh pulses (30 sec), separated by a 2 min
interval, applied to the same cell. Catestatin was introduced
simultaneously to ACh during the second pulse. The inset
shows the pattern of the secretory signal obtained in the presence of
catestatin, at an expanded scale. Amperometric currents consisted of a
first rapid phase followed by a second slow-rate secretion where
individual spikes could be distinguished easily. B,
Average of [Ca2+]c decay obtained from
four chromaffin cells stimulated as described above. Top
and bottom traces show the decay of the
[Ca2+]c signal after the ACh
application, in the absence or presence of the peptide, respectively.
Curves could be fitted to single exponential, giving the control time
constants () shown in the figure. C, Left
panel, The cumulative integrals of spikes obtained from
ACh-treated or ACh + catestatin (ACh + Cat)-treated
cells. After integration of individual responses, the average curve was
obtained from four cells stimulated with pulses of ACh and ACh + catestatin. Right panel shows the normalized data for
kinetic comparisons. Each curve was fitted to a double
exponential.
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The kinetic components of the secretory response were analyzed
measuring the accumulated charge of amperometric spikes in chromaffin
cells stimulated with ACh, in the absence (control) or presence of
catestatin, using a protocol similar to that described in Figure
9A. Results in Figure 9C (left) show
the averaged cumulative integral obtained from several cells.
Continuous secretion induced by ACh, in the presence of catestatin,
showed a marked reduction of the amplitude of the control exocytotic
response (134 ± 25 versus 193 ± 29 pC; n = 4). This difference is probably caused by changes in the
[Ca2+]c levels,
because total
[Ca2+]c influx was
lower in the presence of catestatin, and the kinetics of exocytosis
were very similar. In fact, normalized data shown in Figure
9C (right) indicate that the kinetic curves of
the secretory responses elicited by ACh, in the absence or presence of
catestatin, were almost superimposed; both curves fitted well to a
double exponential, with similar time constants.
The membrane fusion stages of exocytosis were not affected
by catestatin
Detailed analysis of individual amperometric spikes elicited by
ACh pulses (1 mM, 30 sec) applied to chromaffin cells, in the absence or presence of catestatin, is a good approach for studying
whether catestatin could affect the last step of exocytosis, consisting
of the fusion of the vesicle with the plasma membrane, forming an
exocytotic fusion pore with release of catecholamines and other granule
contents (including catestatin) from the vesicle matrix (Alvarez de
Toledo et al., 1993). Figure 10 shows
the frequency histograms of the peak amplitudes
(Imax), the half-width
(t1/2 = duration of the amperometric
signal at 50% of its peak amplitude), cubic root of quantal charge
(Q1/3; considering quantal
charge as the time integral of individual amperometric spikes),
ascending slope (m) and time-to-peak (tP), both
in control conditions (left) or in the presence of
catestatin (right). Results indicate that the mean charge
value for ACh (0.48 pC ± 0.02; n = 250) was
similar to that obtained in the presence of catestatin (0.55 ± 0.04 pC; n = 163). The other kinetic parameters also
remained unaltered when ACh versus ACh plus catestatin were compared:
t1/2 = 19.9 ± 6.1 msec versus
24.6 ± 1.9 msec; Imax = 22.8 ± 1.8 pA versus 28.7 ± 3.2 pA; m = 5.2 ± 0.6 nA/sec versus 7.6 ± 0.1 nA/sec; and
tP = 22.5 ± 1.5 msec versus 25.2 ± 2.7 msec. Furthermore, no significant differences were found in the
occurrence of the foot signals (reflecting transmitter leakage through
an early fusion pore that does not dilate immediately) of the
amperometric events in the presence of catestatin (9.2%) with respect
to control (10.8%).
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Figure 10.
Effect of catestatin on the characteristics of
single fusion events. Frequency histograms were obtained during
analysis of individual amperometric spikes obtained in cells stimulated
with ACh (30 sec), in the absence (left) or presence
(right) of catestatin. On the top, an
example of a typical spike indicates the parameters measured. These
parameters include the peak amplitude
(Imax), half-width
(t1/2; duration of the amperometric
signal at 50% of its peak amplitude), cubic root of quantal charge
(Q1/3; considering quantal charge as
the time integral of individual amperometric spikes), ascending slope
(m; calculated from the linear part of the trace located
between 25 and 75% of the Imax), and
time-to-peak (tP; determined between the point at which
the back-extrapolation of the slope line crossed the base line at the
point of Imax). Data are pooled from
250 (control) or 163 (catestatin) individual secretory spikes.
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DISCUSSION |
Using several combinations of neuronal nAChR subunits expressed in
oocytes, we performed the first electrophysiological study on the
interaction between catestatin and the nicotinic receptor. Our data
indicate that catestatin is a potent and reversible blocker of the
receptor, without significant discrimination among different nAChR
subtypes tested. Moreover, in this paper we present the first detailed
analysis, at the single chromaffin cell level, of how catestatin
affects the kinetics and different stages of the exocytotic process.
The characteristics of catestatin inhibition on
IACh through
34 nAChRs are
compatible with a noncompetitive mechanism, as documented previously in
PC12 cells (Mahata et al., 1997). Molecules known to act as
noncompetitive blockers on muscle, electric organ, and neuronal
nicotinic receptors might inhibit ion translocation on interacting with
two main categories of sites topographically distinct from the
ACh-binding sites: (first) a high affinity site located within the ion
channel pore where the blockers sterically hinder ion flux, and
(second) multiple low-affinity sites, located at the interface of the
receptor with the membrane lipids (Léna and Changeux, 1993; Galzi
and Changeux, 1995). Also, a third possibility exists that
noncompetitive blockers may physically occlude the channel at the
extracellular mouth, outside of the potential field of the membrane.
The following findings are compatible with the hypothesis that
catestatin might be entering and occluding the nAChR channel by a
mechanism compatible with an open channel blockade. (1) The EC50 for ACh became smaller rather than larger in
the presence of catestatin (66 vs 94.7 µM); (2) the
effectiveness of the peptide on IACh
is greater at higher agonist concentrations (Fig.
2B); (3) the strong voltage-dependence of blockade
(Fig. 3) and (4) the more frequently the receptors are activated, the
greater the degree of blockade produced (Fig. 4C). At this
moment, how catestatin is accommodated within the nAChR channel is a
matter of speculation, although on the basis of its predicted
three-dimensional structure, we have proposed a model for the
catestatin docking on the Torpedo nicotinic receptor channel
(Tsigelny et al., 1998). Molecular modeling of the catestatin region
reveals a -strand/loop/-strand structure secured by hydrophobic
interactions, the longitudinal axis of which, at 25 Å, is likely to
occlude the nicotinic cation pore at the pore vestibule. If this is
also true for the neuronal 34 nAChR, and the
electropositive loop structure of catestatin (with three directionally
displayed positive arginine side chains) entered the channel deeply
enough, then the inhibition of IACh by
the peptide should be sensitive to the potential across the membrane.
This is indeed the case, according to the results shown in Figure
3.
Additionally, a striking finding is that the preincubation with
catestatin produces a greater inhibition of the peak
IACh responses than simple
coapplication of the peptide with agonist (Fig. 1C and
inset). These different degrees of blockade could be
interpreted in various ways. One possibility is that catestatin, as
occurs with many other use-dependent inhibitors, has an increased effect with pre-equilibration because it is present at its effective concentration all during the activation phase of the agonist-evoked response. Because solution exchange is relatively slow in oocyte experiments, during a coapplication-evoked response there is a phase
shortly before the peak IACh when
channels are opening in the presence of a relatively low concentration
of antagonist. If this hypothesis is true, much of the preincubation
effect of catestatin on peak IACh
(IC50 values = 4.9 and 0.4 µM, in the absence or presence of
preincubation; data obtained from Fig. 1C) would disappear
if the concentration-response curves for the peptide were calculated
in regard to net charge of current rather than the peak amplitude.
However, our results indicated that this is not the case, because the
differences in blockade between the two modes of catestatin application
were still preserved when IC50 values were referred
to net charge (2.1 and 0.2 µM, in the absence or presence
of preincubation, respectively). The other possibility is that during
the preincubation period catestatin could bind to a second site located
outside the channel. This second site could correspond to the
low-affinity binding sites for the noncompetitive antagonists
(Léna and Changeux, 1993; Galzi and Changeux, 1995). After
binding to high- and low-affinity binding sites, catestatin (like most
of the noncompetitive blockers) might increase the affinity of the
receptor for nicotinic ligands, a phenomenon that may occur in the
absence of agonist and possibly result from the stabilization of the
desensitized state of the receptor (Heidmann and Changeux, 1979a,b;
Boyd and Cohen, 1984).
Our results do not provide precise data about the comparative potency
of the peptide on the four nAChR subunit combinations assayed, because
we have not used equally effective ACh concentrations to construct
TOP
ABSTRACT
INTRODUCTION
