Báo cáo khoa học: Characterization of natural vasostatin-containing peptides in rat heart docx

Characterization of natural vasostatin-containing peptides
in rat heart
Elise Glattard
1
, Tommaso Angelone
2
, Jean-Marc Strub
3
, Angelo Corti
4
, Dominique Aunis
1
,
Bruno Tota
2
, Marie-He
´
le
`
ne Metz-Boutigue
1
and Yannick Goumon
1
1 Inserm U575, Physiopathologie du Syste
`
me Nerveux, Strasbourg, France
2 Laboratory of Cardiovascular Physiology, Department of Cell Biology, University of Calabria, Arcavacata di Rende, Italy
3 CNRS, UMR 7512, Laboratoire de Spectrome
´
trie de Masse BioOrganique, Strasbourg, France
4 Department of Biological and Technological Research, San Raffaele H Scientific Institute, Milan, Italy

Chromogranin A (CGA) is the major member of a
family of acidic glycoproteins, named chromogra-
nins ⁄ secretogranins, originally described in chromaffin
cell granules of bovine adrenal medulla [1,2]. It is pre-
sent in numerous tissues [3], and is stored and released
together with neurotransmitters and hormones in the
nervous, endocrine and diffuse neuroendocrine systems
[2,4–7].
CGA is a well-conserved protein that is widely
distributed, from paramecia to mammals [2,8]. It is
characterized by a high abundance (17%) of glutamic
acid residues and several dibasic sites in its sequence,
representing cleavage sites for endopeptidases (i.e.
prohormone convertases) and carboxypeptidase E ⁄ H
[9–12], giving rise to various derived peptides display-
ing numerous biological effects [7,13].
Post-translational modifications (phosphorylation
and O-glycosylation) of bovine and human CGA have
been identified [14–17]. Phosphorylations are present
all along the sequence, whereas glycosylation sites are
mainly located in the median part of CGA.
CGA and its derived fragments are known to be
released into blood in response to stress, reaching sev-
eral nanomolar concentrations in the peripheral circu-
lation of man [18,19]. Of these fragments, bovine
CGA(4–16) and CGA(47–60) have been shown to
Keywords
chromogranin A; heart; post-translational
modifications; rat; vasostatin
Correspondence

Y. Goumon, Inserm U575, 5, rue Blaise
Pascal, F-67084 Strasbourg Cedex, France
Fax: +33 3 88 60 08 06
Tel: +33 3 88 45 67 24
E-mail:
(Received 17 February 2006, revised 18
May 2006, accepted 24 May 2006)
doi:10.1111/j.1742-4658.2006.05334.x
Chromogranin A (CGA) is a protein that is stored and released together
with neurotransmitters and hormones in the nervous, endocrine and diffuse
neuroendocrine systems. As human vasostatins I and II [CGA(1–76) and
CGA(1–113), respectively] have been reported to affect vessel motility and
exert concentration-dependent cardiosuppressive effects on isolated whole
heart preparations of eel, frog and rat (i.e. negative inotropism and anti-
adrenergic activity), we investigated the presence of vasostatin-containing
peptides in rat heart. Rat heart extracts were purified by RP-HPLC, and
the resulting fractions analyzed for the presence of CGA N-terminal frag-
ments using dot-blot analysis. CGA-immunoreactive fractions were submit-
ted to western blot and MS analysis using the TOF ⁄ TOF technique. Four
endogenous N-terminal CGA-derived peptides [CGA(4–113), CGA(1–124),
CGA(1–135) and CGA(1–199)] containing the vasostatin sequence were
characterized. The following post-translational modifications of these frag-
ments were identified: phosphorylation at Ser96, O-glycosylation (trisaccha-
ride, NAcGal-Gal-NeuAc) at Thr126, and oxidation at three methionine
residues. This first identification of CGA-derived peptides containing the
vasostatin motif in rat heart supports their role in cardiac physiology by
an autocrine ⁄ paracrine mechanism.
Abbreviation
CGA, chromogranin A.
FEBS Journal 273 (2006) 3311–3321 ª 2006 The Authors Journal compilation ª 2006 FEBS 3311

affect microbial growth and nociception [5,20–23].
Vasostatin-I [human CGA(1–76)] and its N-terminal
domain [CGA(1–40)] have also been reported to affect
the dilatation of vessels [24–28] probably via an endo-
thelium-independent vasoinhibitory action [26]. In
addition, circulating CGA concentrations are increased
in cardiac pathology [29]. In patients with chronic
heart failure, these high concentrations are correlated
with the severity of the disease, representing a prognos-
tic indicator of mortality [29]. The recent finding that
CGA-knockout mice show hypertension and cardiac
enlargement [30] further stresses the importance of
CGA in cardiac physiology. Cardiosuppressive effects
(i.e. negative inotropy) of N-terminal CGA-derived
peptides have been well documented in vertebrate heart
[31]. In particular, in the isolated working heart of the
frog Rana esculenta, the concentration-dependent neg-
ative inotropism and inhibition of the b-adrenergic-
dependent positive inotropy elicited by the N-terminal
domain of CGA [i.e. human recombinant vasostatin I
and bovine CGA(7–57)], as well as by the synthetic
peptides corresponding to frog and bovine CGA(4–16),
CGA(47–66), and bovine CGA(1–40), have been ana-
lysed in details [32]. More recently, using the Lange-
ndorff-perfused rat heart model, we have reported that
the two recombinant human vasostatins STA-CGA(1–
76) (vasostatin I) and STA-CGA(1–113) (vasostatin II)
display similar negative inotropic effects and antagon-
ize the b-adrenergic-dependent positive inotropism, the
latter being counteracted by vasostatin I in a noncom-

petitive type of antagonism [33].
An important question arising from these results is
whether cardiac tissues produce vasostatin-containing
peptides. To answer this question and explore the
potential autocrine ⁄ paracrine role of these peptides, we
investigated the presence of endogenous N-terminal
CGA-derived fragments in the rat heart. Our studies
using biochemical techniques (RP-HPLC, dot-blot,
western blot) and MS analysis (TOF-TOF MS)
allowed us to investigate the N-terminal processing of
CGA in rat heart extracts. Our data reveal the pres-
ence of several vasostatin I-containing and vasosta-
tin II-containing peptides in the CGA(1–199) domain,
strongly supporting their role as intracardiac regulators
of cardiac contractile performance.
Results
Western blot analysis of N-terminal CGA-derived
peptides present in rat heart extract
Experiments were carried out to determine whether
acidic or heat treatment of rat heart was able to enrich
and ⁄ or generate artificial CGA fragments (Experimen-
tal procedures). Boiled and unboiled rat heart tissue
extracts were prepared in 40% acetic acid or 0.1%
(v ⁄ v) trifluoroacetic acid and loaded on an SDS ⁄ 15%
polyacrylamide gel to compare the fragmentation pat-
tern of CGA. A rat heart control extracted in the pres-
ence of antiprotease cocktail and not treated with acid
or heat was also used. Gels were electrotransferred to
polyvinylidene difluoride membrane and submitted
to western blot analysis using a rabbit antibody to

CGA(4–16). The electrophoretic profiles of the treated
CGA fragments were identical with that of the non-
treated extract, indicating that no additional fragment
was generated by the treatments used here and that
CGA fragments were enriched using these treatments
(Fig. 1). These results also indicate that 0.1% (v ⁄ v) tri-
fluoroacetic acid in water used for HPLC purification
did not induce further hydrolysis (Fig. 1).
CGA maturation in rat heart was compared
with that in rat adrenal to determine whether CGA
Fig. 1. Western blot analysis of CGA fragments present in different
rat heart preparations. The CGA-immunodetection pattern is shown
for rat heart tissue prepared under five different conditions:
untreated (no acid and no heating but addition of antiprotease cock-
tail); in 40% acetic acid alone; in 40% acetic acid followed by heat-
ing at 100 °C for 3 min; in 0.1% trifluoroacetic acid; in 0.1%
trifluoroacetic acid followed by heating at 100 °C for 3 min.
Samples (20 lg) were separated by SDS ⁄ PAGE (15% gel) and
electrotransferred to a polyvinylidene difluoride membrane. Immuno-
detection was carried out with the rabbit polyclonal antibody to
bovine CGA(4–16).
Vasostatin-containing peptides in rat heart E. Glattard et al.
3312 FEBS Journal 273 (2006) 3311–3321 ª 2006 The Authors Journal compilation ª 2006 FEBS
maturation is tissue-dependent as reported in other
species [20]. Rat heart extract, treated with acetic acid
and heated, was subjected to SDS ⁄ PAGE (15% gel)
(Fig. 2). Rat adrenal gland extract also treated with
acetic acid and heated was used as control. In rat
heart, three groups of bands ranging from 17 to
80 kDa (Fig. 2, lane 2 and labels) were identified: (a)

containing strongly labeled high-molecular-mass bands
(a, b); (b) containing other major bands (c, d, g); (c)
containing weakly labeled bands (e, f, h, i, j, k). Thus,
it appears that the a and b immunoreactive bands, ran-
ging from 80 to 50 kDa, correspond to the apparent
molecular mass of intact rat CGA (with and without
post-translational modifications), as well as to several
large N-terminal-derived fragments. Such a broad pat-
tern is due to the presence of glutamic acid clusters
[e.g. CGA(216–228)], known to modify the electropho-
retic migration of GGA [CGA(1–448), 48 kDa] with
the apparent molecular mass of 80 kDa (Fig. 2, lane 2,
a label) [9]. The second group containing bands c, d
and g at 45, 37 and 27 kDa, respectively, corresponds
to shorter N-terminal CGA-derived fragments. The
third group is composed of weakly labeled bands (e, f,
h, i, j and k labels) which correspond to 32, 30, 24, 23,
18 and 17 kDa apparent molecular mass, respectively.
Interestingly, the 24-kDa and 23-kDa bands (h and i,
respectively) probably correspond to vasostatin II [rat
CGA(1–128)] previously reported to migrate with an
apparent molecular mass of 21–23 kDa [34].
Purification and immunodetection of N-terminal
CGA-derived fragments
Rat heart extract (2 mg) was purified using the
RP-HPLC technique (Fig. 3A). Each peak was col-
lected and dotted on to nitrocellulose sheet before
immunodetection with a rabbit polyclonal antibody to
CGA(4–16) [35]. Immunolabels were observed for frac-
tions 1, 6 and 10–14 (Fig. 3A).

To evaluate the apparent molecular mass of CGA
N-terminal fragments present in these fractions, an ali-
quot (2 : 5, v ⁄ v) of each fraction (1–14) was subjected
to SDS ⁄ PAGE (15% gel), electrotransferred to a
poly(vinylidene difluoride) membrane, and immunode-
tected with the CGA(4–16) antibody (Fig. 3B). High-
molecular-mass CGA-immunoreactive fragments (80–
70 kDa corresponding to the whole CGA with and
without post-translational modifications and long
C-terminal truncated protein) were recovered in the
more hydrophobic fractions as reported for bovine
CGA [9]. Low-molecular-mass fragments (22–25 kDa
in Fig. 3B) representing CGA(1–124 ⁄ 128 ⁄ 130) were
recovered in fractions 1 and 14.
CGA sequence comparison from several species
The rat sequence contains three highly conserved
amino-acid domains on comparison with bovine and
human CGA (Fig. 4, underlined). Thus, the N-ter-
minal rat CGA(1–76) displays 85% similarity and 93%
homology with the bovine CGA sequence, as well as,
respectively, 86% and 94% with the human sequence
(clustalw software [36]). The CGA(94–106) domain
possesses 84% identity and 92% homology with
bovine and human CGA sequences. The C-terminal
rat CGA(334–448) also displays 88% identity and
98% homology with bovine CGA as well as 91% iden-
tity and 96% homology with human CGA.
As CGA is known to have post-translational modifi-
cations, we investigated whether the N-terminal
domain of rat CGA could be modified. The presence

of putative phosphorylations was examined using net-
phos 2.0 software [37]. Phosphorylations could poten-
tially be present on several serine residues located at
position 35, 50, 96, 133, 168, 182 and 185. Among
these, Ser96 in rat CGA corresponds to a previously
M
r
Fig. 2. Comparison of CGA fragments present in rat adrenal gland
and heart. Samples were separated by SDS ⁄ PAGE (15% gel) and
electrotransferred to a poly(vinylidene difluoride) membrane. Lane
1, whole rat adrenal protein extract (50 lg); lane 2, rat heart extract
(20 lg) treated with 40% acetic acid + heating at 100 °C for 3 min.
Western blot analysis, using polyclonal antibody to bovine CGA(4–
16) was performed to detect N-terminal CGA fragments.
E. Glattard et al. Vasostatin-containing peptides in rat heart
FEBS Journal 273 (2006) 3311–3321 ª 2006 The Authors Journal compilation ª 2006 FEBS 3313
described phosphorylated residue (Ser81) in the bovine
CGA sequence (Fig. 5A) [17,38].
In addition, the netoglyc 3.1 software [39] allowed
us to predict potential O-glycosylation on residues
Ser119, Thr126, Thr173, Thr177, Thr181, Thr193 and
Thr194. Consensus N-glycosylation sites (N-X-T ⁄ S)
[40] were also present on Asn107 and Asn175 (netn-
glyc 1.0; Fig. 5A).
Characterization of N-terminal CGA-derived
fragments present in rat heart extract
TOF ⁄ TOF MS was used because it is a direct, highly
sensitive and precise technique validated for the prote-
omic approach of peptides [41]. By direct MS analysis
of the immunoreactive fractions, we determined the

experimental molecular masses of N-terminal CGA-
derived fragments, compared them with the theoretical
molecular mass, and evaluated possible post-transla-
tional modifications (Table 1). Phosphorylations, gly-
cosylations and oxidations were predicted using the
netphos 2.0 and netoglyc 3.1 software, as well as
from previously described post-translational modifica-
tions on CGA from other species (Fig. 5A). Using this
technique, phosphorylation was detected at fragments
CGA(4–113), CGA(1–124), CGA(1–135) and CGA(1–
199), whereas O-glycosylation (trisaccharide, NAcGal-
Gal-NeuAc) was only found at CGA(1–135) (Table 1).
Oxidation could be expected at methionine residues 7,
15, 32, 162 and 163 (Figs 4 and 5A).
With this approach, it has been possible to charac-
terize several fragments starting from residue 1 or 4 of
CGA and ending at residues 113, 124, 135 and 199,
corresponding to potential cleavage sites located at
basic residues (Table 1 and Fig. 5). The cleavage site
Val3 ⁄ Asn4 had previously been reported by us [9].
Discussion
CGA and its derived fragments are present in large,
dense core secretory vesicles of all endocrine and
neuroendocrine tissues. In chromaffin cells, CGA
A
B
M
r
Fig. 3. HPLC purification of CGA fragments
from rat heart extract. (A) 2 mg rat heart

extract was fractionated on a Macherey–
Nagel RP Nucleosil 300–5C-18 column
(4 · 250 mm). Aliquots of each HPLC frac-
tion (1–14) were dotted on to a nitrocellu-
lose sheet and immunodetected with the
polyclonal antibody to bovine CGA(4–16).
Immunoreactive fractions are underlined on
the chromatogram. (B) Eluted fractions 1–14
were separated by SDS ⁄ PAGE (15% gel)
and electrotransferred to a poly(vinylidene
difluoride) membrane before immunodetec-
tion with the polyclonal antibody to bovine
CGA(4–16).
Vasostatin-containing peptides in rat heart E. Glattard et al.
3314 FEBS Journal 273 (2006) 3311–3321 ª 2006 The Authors Journal compilation ª 2006 FEBS
fragments are co-released by exocytosis with catechol-
amines in response to secretagogues [7]. The natural
occurrence of CGA N-terminal peptides in rat endo-
crine tissues is supported by the demonstration of the
presence in the pituitary gland of both CGA and vas-
ostatin II (apparent molecular mass 21–23 kDa) using
gel filtration and western blot analysis with specific
N-terminal CGA-directed antibody [rat CGA(98–106)]
[34]. Previous studies have shown, with no ambiguity,
the presence of CGA in secretory granules of rat atrial
myoendocrine cells [42]. In fact, this immunohisto-
chemical study using electron microscopy has shown
colocalization of CGA and atrial natriuretic peptides
in rat heart cardiomyocytes. In addition, a western
blot analysis performed on the secretory granules isola-

ted from rat atrial myoendocrine cells has revealed the
presence of different CGA-immunoreactive bands.
CGA has also been detected in rat Purkinje fiber cells
of the conducting system, in both rat atrium and vent-
ricle, as well as in H9c2 rat cardiomyocytes [43]. The
possibility that CGA-derived fragments could also ori-
ginate from nerve termini innervating the heart cannot
be excluded [44]. However, direct demonstration of the
intracardiac processing of CGA has so far been lack-
ing. Our data represent the first clear evidence that the
heart produces and processes vasostatin-containing
peptides. This strongly suggests that these fragments
play a role in the autocrine ⁄ paracrine regulation of
cardiac function, being directly involved in the ino-
tropic modulation [33]. CGA-derived peptides are
stored with atrial natriuretic peptides in the secretory
vesicles of rat cardiomyocytes [42]. The actions of
CGA-derived peptides and atrial natriuretic peptides
may be closely integrated under normal [45] and stress-
ful or pathophysiological conditions [46], but this
remains to be clarified.
Our results indicate that, among the CGA frag-
ments, a major broad immunoreactive band at an
apparent molecular mass of 80–50 kDa is present in
rat heart extract (Fig. 2, a and b). This high-molecu-
lar-mass immunoreactive band results from both the
whole CGA protein (with and without post-transla-
tional modifications) and various long C-terminal trun-
cated CGA fragments. This suggests that, compared
with the rat adrenal gland where almost no intact

CGA is found, the maturation process looks incom-
plete and specific to the heart (Fig. 2, lane 1). It
appears also that other low-molecular-mass fragments
differ from those observed in adrenal extract except
for the 27-kDa fragment (Fig. 2, g label). Shorter frag-
ments (Fig. 2, h, i, j, k labels) exist in the heart and
include the cardioactive motif (i.e. the vasostatin I
sequence or a portion of it). Under normal or abnor-
mal (e.g. stressful or pathophysiological) conditions in
response to a specific stimulus-induced proteolytic acti-
vation, an increase in lower-molecular-mass fragments,
Fig. 4. CGA primary structure analysis. Sequence comparison of CGA sequences from rat (r), bovine (b) and human (h). Underlined
sequences correspond to highly conserved regions. Basic residues are indicated in bold.
E. Glattard et al. Vasostatin-containing peptides in rat heart
FEBS Journal 273 (2006) 3311–3321 ª 2006 The Authors Journal compilation ª 2006 FEBS 3315
as well as production of new fragments, could be
induced. Some of these fragments may also result from
extracellular processing, as reported for CGA in the
adrenal gland [9]. In addition, the proteomic approach
using RP-HPLC (Fig. 3) and MS techniques allowed
us to characterize three peptides starting at the first
N-terminal residue and one at the fourth residue of the
protein (Table 1 and Fig. 5B). All these peptides
(Table 1, Figs 4 and 5) ended at a monobasic potential
cleavage site that could be recognized by prohormone
convertases and carboxypeptidases present in rat heart
[47,48]. The difference in the theoretical and experi-
mental molecular mass showed that some of these
fragments are phosphorylated, oxidized, and ⁄ or glycos-
ylated (Table 1). The presence of phosphorylation, pre-

viously reported on Ser81 of bovine CGA, could be
Fig. 5. Characterization of N-terminal CGA
fragments present in rat heart extract. (A)
Sequences alignment of rat (r), bovine (b)
and human (h) N-terminal CGA sequence.
Phosphorylations are marked in bold under-
lined letters and O-glycosylations as a black
round shape. Localization of phosphorylation
and glycosylation sites for bovine and
human CGA are those reported in literature.
Cleavage sites are indicated by arrows. (B)
Schematic representation of the N-terminal
rat CGA fragments,
, phosphorylation;
O, O-glycosylation.
Vasostatin-containing peptides in rat heart E. Glattard et al.
3316 FEBS Journal 273 (2006) 3311–3321 ª 2006 The Authors Journal compilation ª 2006 FEBS
assigned to Ser96 as this site is well conserved
(Fig. 5A) [17,38] and was predicted to be potentially
phosphorylated (netphos 2.0 software). In addition,
we observed molecular mass differences attributable to
O-glycosylation (NAcGal-Gal-NeuAc), probably at
Thr126 (Fig. 5A) according to O-glycosylation site pre-
dictions (netoglyc 3.1 software). This modification
was only observed on CGA(1–135) and not shorter
fragments [i.e. CGA(1–124) and CGA(4–113)], thus
protecting the long fragment from proteolytic degrada-
tion. CGA(1–135) is of particular interest because it is
found in various forms, i.e. unmodified, oxidized,
phosphorylated and ⁄ or O-glycosylated (NAcGal-Gal-

NeuAc; Table 1).
Like other chromogranin family members, CGA is a
protein precursor that is actively processed to low-
molecular-mass bioactive peptides [49]. Rat CGA is a
448-amino-acid protein with a pI of 4.5–5.0 containing a
disulfide bridge and displaying numerous monobasic
(40) and dibasic (nine) residues (Fig. 4) that could repre-
sent potential cleavage sites for proteolytic subtilisin-like
and trypsin-like enzymes including PC1 ⁄ 3, PC2, PC4,
PC5 ⁄ 6, PC7 ⁄ 8, furine ⁄ SPC1 ⁄ PACE [50], as well as
carboxypeptidases [51]. In rat heart, the detected
PC1 ⁄ 3, PC2 and carboxypeptidase H ⁄ E [47,48,52] might
be involved in the CGA maturation process. We have
previously characterized intragranular and extracellular
processing of CGA in chromaffin granules from bovine
adrenal medulla, showing that the processing starts
at both the N-terminus and the C-terminus of the pro-
tein [9]. Among the fragments generated, CGA(1–76)
represents the major product of proteolytic process-
ing in bovine adrenal medulla [9,53], whereas the first
N-terminal cleavage product of rat CGA is b-granin
[rCGA(1–128)], corresponding to vasostatin II, due to
the lack of the first dibasic site [34,54,55] (Fig. 4).
As CGA fragments are likely to be secreted by car-
diomyocytes, extracellular processing can be expected
to occur in the secreted medium, as shown for CGA
and proenkephalin-A secreted from chromaffin cells
[9,56]. The presence of extracellular proteases both on
cardiomyocyte cell membranes and in the extracellular
matrix suggests that extracellular processing does

occur, as proposed for angiotensin II. In the heart, an-
giotensin converting enzyme and ⁄ or renin, which are
present on cell membranes, are involved in the conver-
sion of angiotensinogen into angiotensin II [57].
Because of the presence of extracellular proteases dur-
ing tissue homogenization (for review, see [58]), we
cannot exclude the possibility that CGA-derived frag-
ments are extracellulary processed. Such a mechanism
may be involved in the production of additional N-ter-
minal CGA-derived peptides displaying biological
activity.
Glycosylation of CGA has been reported to be
involved in its 3D folding, its protection against prote-
ase activity [9,15], as well as in trafficking [59]. Glyco-
sylation of the bovine CGA sequence has also been
reported to modulate the antimicrobial activity of the
chromacin [CGA(173–194)] fragment [60]. The present
data reveal that several rat heart CGA-derived pep-
tides possess O-glycosylation sites. The presence of
multiple forms of the CGA(1–135) fragment is intrigu-
ing and suggests an important role for the post-trans-
lational modifications of this peptide, as previously
reported for chromacin [61].
Phosphorylation of bovine and human CGA has
been extensively studied. In the former, phosphoryla-
tion has been found at serine residues 81, 124, 297,
307, 372 and 376, as well as at Tyr173 (Fig. 5B)
[17,60,61]. In the human protein, phosphorylation has
been reported at serine residues 200, 252 and 315
Table 1. MS analysis of HPLC fractions. CGA-derived peptides were identified by comparing the experimental mass obtained by TOF ⁄ TOF

MS analysis with the calculated one. Proteolytic cleavage sites are indicated. O, Oxidation; Na
+
, sodium adducts; O-Gly, O-glycosylation;
P, phosphorylation.
HPLC
fraction
Molecular mass (Da)
Location
Cleavage
site Modifications
Experimental Calculated
1 12 581.4 12 505.4 CGA(4–113) V ⁄ N&K⁄ HP
12 14 009.9 13 864.9 CGA(1–124) K ⁄ D P + 3Na
+
13 14 045.3 P + 2O + 3Na
+
12 15 278.0 15 167.5 CGA(1–135) K ⁄ GP+2O
12 15 866.6 O-Gly + 2Na
+
13 15 868.9
14 15 877.7 O-Gly + 2O + 1Na
+
10 22 092.1 21 948.5 CGA(1–199) R ⁄ G P + 3O + 1Na
+
11 22 097.6
E. Glattard et al. Vasostatin-containing peptides in rat heart
FEBS Journal 273 (2006) 3311–3321 ª 2006 The Authors Journal compilation ª 2006 FEBS 3317
(Fig. 5B) [14]. Phosphorylation of CGA is likely to be
related not only to the type of organ and the physiolo-
gical state, but also to peptide activity. For example,

phosphorylation is known to modulate the antimicro-
bial activity of chromacin [60] and other intragranular
peptides such as enkelytin derived from proenkephalin-
A [bisphosphorylated PEA(209–237)] [62].
Our results also indicate the presence of oxidized
fragments. Oxidation of CGA residues has previously
been described [61]. In chromaffin granules, for
instance, MS analysis has revealed oxidation at methi-
onine residues, which may be related to a redox mech-
anism inside the granules [63].
Taken together, our data clearly indicate that, in rat
heart, CGA is a precursor to numerous peptides that
may control different physiological functions. The con-
cept of the heart as an endocrine organ was firmly
established almost 25 years ago with the discovery of
atrial natriuretic peptides present in the ‘atrial specific
secretory granules’ and identified as homeostatic agents
for protection against plasma volume overload ([64,65]
and references therein). The present study points to the
vasostatin-derived peptides as novel intracardiac mod-
ulators exerting a counter-regulatory inotropic role
particularly against b-adrenergic-elicited positive ino-
tropism [27,32,33,66] through an autocrine and ⁄ or
paracrine mechanism.
Experimental procedures
Rat heart extract
Male Wistar rats (Charles River Laboratories, Les Oncins,
Italy S.p.A) weighing 250–350 g were housed three per cage
in a ventilated cage rack system under standard conditions.
Animals had food and water access ad libitum. Animal care,

killing, and experiments were carried out according to the
European Community guiding principles in the care and
use of animals, and the projects were supervised by the
local ethics committee. Whole hearts (atria and ventricles)
of male Wistar rats were removed just after death and
washed with NaCl ⁄ P
i
buffer to remove blood cells. They
were then homogenized in the presence of 2 mL 40% (v ⁄ v)
acetic acid in water with a Polytron mixer (Richmond
Agencies, Wigan, UK) to give 0.5 g fresh tissue per mL.
The protein extract was centrifuged at 8000 g for 30 min at
4 °C, and the supernatant containing the acid-stable pro-
teins was collected and boiled for 3 min to isolate the ther-
mostable chromogranins. The extract was then centrifuged
at 12 000 g for 30 min to enrich the supernatant in heat-
stable proteins. In some experiments, protease inhibitors
were included, but their presence did not modify the pat-
tern of proteolytic fragments. Control experiments, using
different extraction conditions, were performed to deter-
mine if the acetic acid (40%, v ⁄ v; Arcos, Fairlaw, NJ,
USA), trifluoroacetic acid (0.1% v ⁄ v; HPLC conditions;
Sigma Aldrich, Steinheim, Germany) or heat treatment
(boiling for 3 min) were responsible for the generation of
new CGA fragments. An extraction performed in the pres-
ence of an antiprotease cocktail that inhibits most of the
proteases (except metalloproteases and aspartic proteases;
Complete mini cocktail, Roche Diagnostics, Mannheim,
Germany), in the absence of acid and without heating was
performed as a control.

Rat adrenal glands were homogenized in the presence of
2 mL 40% (v ⁄ v) acetic acid. The protein extract was centri-
fuged at 8000 g for 30 min at 4 °C. The resulting superna-
tant was boiled for 3 min and centrifuged at 12 000 g
(30 min). The supernatant was used as control.
After protein quantification using the Bradford technique
(Protein assay; Bio-Rad, Marnes la Coquette, France), the
extract was diluted in an adequate volume of ultrapure
water, and aliquots of 20 lg protein were stored at )20 °C.
Western blot analysis
Rat heart extract or HPLC fractions were separated by
SDS ⁄ PAGE (15% acrylamide; Euromedex, Souffelweyers-
heim, France). Proteins were electrotransferred (45 min,
75 V) to polyvinylidene difluoride membrane (Amersham
Biosciences, Uppsala, Sweden) and immunodetected with
specific rabbit polyclonal antibodies raised against bovine
CGA(4–16) fragment (1 : 1000 dilution) [53]. A goat anti-
rabbit IgG conjugated to horseradish peroxidase was used
as a secondary antibody (1 : 10 000 dilution; Sigma-Ald-
rich). The poly(vinylidene difluoride) membrane was treated
as previously described [67], and immunodetection was per-
formed with the SuperSignal West Dura Extended Dur-
ation Substrate kit (Pierce, Perbio Science, Brebie
`
res,
France) according to the manufacturer’s instructions.
Dot-blot analysis
To detect the presence of CGA fragments in RP-HPLC
fractions, aliquots (1 : 5, v ⁄ v) were applied to nitrocellulose
membrane (Amersham Biosciences) for dot-blot analysis.

The membrane was then treated as described above for
western blot analysis.
Purification of CGA-derived peptides by RP-HPLC
CGA-derived peptides present in rat heart extracts were
purified using an A
¨
kta Purifier HPLC system (Amersham
Biosciences) and a Nucleosil reverse-phase 300–5C18 col-
umn (4 · 250 mm; particle size 5 lm, porosity 300 A
˚
;
Macherey-Nagel, Hoerdt, France) [53]. Absorbance was
monitored at 214 nm, and the solvent system consisted of
Vasostatin-containing peptides in rat heart E. Glattard et al.
3318 FEBS Journal 273 (2006) 3311–3321 ª 2006 The Authors Journal compilation ª 2006 FEBS
0.1% (v ⁄ v) trifluoroacetic acid in water (solvent A) and
0.09% (v ⁄ v) trifluoroacetic acid in 70% acetonitrile (Carlo
Erba, Rodano Mi, Italy) in water (solvent B). Elutions were
performed at a flow rate of 700 lLÆmin
)1
using the gradient
indicated on the chromatograms.
MS analysis
Mass spectra were acquired using an Ultraflex
TM
TOF ⁄
TOF mass spectrometer (Bruker Daltonik GmbH, Bremen,
Germany) with gridless ion optics under the control of
Flexcontrol 2.0 [41]. This instrument, equipped with
SCOUT

TM
high-resolution optics with X-Y multisample
probe and gridless reflector, was used at a maximum accel-
erating potential of 25 kV and operated in reflector mode
for MS analysis. Ionization was accomplished with a 337-
nm beam from a nitrogen laser with a repetition rate of
20 Hz. The output signal from the detector was digitized
at a sampling rate of 2 GHz. The samples were prepared
by standard dried-droplet preparation on stainless-steel
MALDI targets using 2,5-dihydroxybenzoic acid as matrix.
The external calibration of MALDI mass spectra was per-
formed using singly charged monoisotopic peaks of a mix-
ture of bradykinin(1–7) (m ⁄ z 757.400), human angiotensin
II (m ⁄ z 1046.542), human angiotensin I (m ⁄ z 1296.685),
substance P (m ⁄ z 1347.735), bombesin (m ⁄ z 1619.822),
renin (m ⁄ z 1758.933), ACTH(1–17) (adrenocorticotropic
hormone; m ⁄ z 2093.087) and ACTH(18–39) (m ⁄ z
2465.199).
Acknowledgements
This work was funded by Inserm, the University
Louis-Pasteur of Strasbourg, the French Ministe
`
re
De
´
le
´
gue
´
a

`
la Recherche et a
`
l’Enseignement Supe
´
rieur
(Ph.D. grant to EG), the Fondation pour la Recherche
Me
´
dicale (to MHMB), the Ligue Contre le Cancer (to
DA), the Programme Hospitalier de Recherche Cli-
nique 3150 (to MHMB), the Egide Program (Galile
´
e,
to MHMB) and Vinci Program (to TA). We thank N.
Aslan and R. Lang for technical assistance.
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