1. Researchers isolated and characterized a novel peptide from the venom of the Brazilian stingray Potamotrygon gr. orbignyi. 2. Using RP-HPLC and mass spectrometry techniques, they purified a peptide from the venom which was shown to strongly constrict blood vessels when applied to mice cremaster muscle tissue during intravital microscopy experiments. 3. Through de novo amino acid sequencing with mass spectrometry, the researchers determined the peptide's sequence to be HGGYKPTDK, which does not match any known bioactive peptides but aligns with residues 97-105 of creatine kinase, suggesting it may be produced from limited proteolysis of creatine kinase.

1. Orpotrin: A novel vasoconstrictor peptide from the venom of
the Brazilian Stingray Potamotrygon gr. orbignyi
Katia Conceic¸a˜o a,
*, Katsuhiro Konno a
, Robson L. Melo a
, Elineide E. Marques b
,
Cle´lia A. Hiruma-Lima c
, Carla Lima a
, Michael Richardson d
, Daniel C. Pimenta a
,
Moˆnica Lopes-Ferreira a
a
Laborato´rio Especial de Toxinologia Aplicada (LETA), Center for Applied Toxinology (CAT/CEPID), Instituto Butantan,
Avenida Vital Brasil, 1500, Sa˜o Paulo, SP, 05503-900, Brazil
b
Nu´ cleo de Estudos Ambientais (Neamb), Universidade Federal do Tocantins, Campus de Porto Nacional,
Rua 03, Quadra 17, Porto Nacional, TO, 77500-000, Brazil
c
Departamento de Fisiologia, Instituto de Biocieˆncias, Universidade Estadual Paulista Ju´ lio de Mesquita Filho,
Caixa-Postal 510, Botucatu, SP, 18618-000, Brazil
d
Fundac¸a˜o Ezequiel Dias, FUNED, Rua Conde Pereira Carneiro, 80, Belo Horizonte, MG, 30510-010, Brazil
p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 6
a r t i c l e i n f o
Article history:
Received 13 July 2006
Received in revised form
6 September 2006
Accepted 7 September 2006
Published on line 23 October 2006
Keywords:
Orpotrin
Potamotrygon
Venom
Stingrays
Vasoconstriction
De novo sequencing
Natural peptides
Creatine kinase
Abbreviations:
DTT, dithiothreitol
a-CHCA, a-cyano-4-
hydroxycinnamic acid
IAA, iodoacetamide
MALDI, matrix-assisted laser
desorption/ionization
MS, mass spectrometry
a b s t r a c t
Characterization of the peptide content of venoms has a number of potential benefits for
basic research, clinical diagnosis, development of new therapeutic agents, and production
of antiserum. In order to analyze in detail the peptides and small proteins of crude samples,
techniques such as chromatography and mass spectrometry have been employed. The
present study describes the isolation, biochemical characterization, and sequence deter-
mination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The
natural peptide was shown to be effective in microcirculatory environment causing a strong
vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with
mass spectrometry and identified as the novel peptide. Its amino acid sequence,
HGGYKPTDK, aligns only with creatine kinase residues 97–105, but has no similarity to
any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by
limited proteolysis is discussed. Taken together, the results indicate the usefulness of this
single-step approach for low molecular mass compounds in complex samples such as
venoms.
# 2006 Elsevier Inc. All rights reserved.
* Corresponding author. Tel.: +55 11 3726 1024; fax: +55 11 3726 1024.
E-mail address: conceicaokatia@butantan.gov.br (K. Conceic¸a˜o).
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2006.09.002

2. 1. Introduction
Venoms of poisonous animals have been extensively studied
because they are a potential source of pharmacological agents
and physiological tools. During the evolution, venomous
animals developed highly specialized and sophisticated
strategies that basically serve prey capture and/or aggressor
deterrence [14]. While there has been much work characteriz-
ing the biological activity of most terrestrial animals (e.g.
snakes, spiders, and scorpions), comparatively less research
has been undertaken on venomous fish. Even so, fish toxins
represent a vast source of novel pharmacological compounds
that may prove useful for both research tools and therapeutic
agents [7].
Fish constitute almost half the number of vertebrates on
Earth, and approximately 22,000 species of fish are contained
in some 50 orders and 445 families [22]. Although only a
handful of species of venomous fish are thought to be capable
of causing human mortality, many other species of fish can
produce severe envenomation. While not considered life
threatening, envenomation by these fish is associated with
considerable pain induced by many pharmacologically active
components. Therefore, these species still represent sources
of pharmacological compounds that may be useful as research
tools not only for research tools but also for drug leads, and as
such, their pharmacological actions have been the focus of
recent work [7].
South American freshwater stingrays are included in a
single family (Potamotrygonidae) that is comprised of three
valid genera: Plesiotrygon, Paratrygon, and Potamotrygon [1].
Some species of the Potamotrygonidae are endemic to the
most extreme freshwater environment of the Brazil, of the
Parana´ River, Tocantins River and its tributaries, and cause
frequent accidents to humans. Stingrays have one to four
venomous stingers on the dorsum of an elongated, whip-like
caudal appendage. The tapered, vasodentine spines are
bilaterally retroserrated (saw-edged, with the cutting cartilage
pointing away from the apex of the spine). Each spine is
enveloped by an integumentary sheath with a ventrolateral
glandular groove containing venom glands along either edge
[12]. The spine is often covered with a film of venom and
mucus.
Recent study carried out by our group describes the
principal biological and some biochemical properties of the
Brazilian Potamotrygon fish venoms [20]. Potamotrygon gr.
orbignyi venom induced significant edematogenic and noci-
ceptive responses in mice. Increased rolling and adhesion of
leukocytes to the endothelium of cremaster muscle of mice is
seen in response to venom. Our study also presented that the
injection of venom induced necrosis, low level of proteolytic
activity, without inducing hemorrhage. This recent study
provided in vivo evidence of toxic effects on target cells in
microcirculatory environment.
Here, we describe for the first time the isolation,
biochemical characterization, and de novo amino acid
sequencing of a novel peptide, named Orpotrin from the
venom of P. gr. orbignyi. The natural peptide was shown to be
effective in microcirculatory environment causing a strong
vasoconstriction.
2. Material and methods
2.1. Animals
Groups of four Swiss mice weighing 18–22 g were used
throughout. The animals provided by Instituto Butantan
animal house were kept in temperature and humidity-
controlled rooms, and received food and water ad libitum.
All the procedures involving mice were in accordance with the
guidelines provided by the Brazilian College of Animal
Experimentation.
2.2. Reagents
Dithiothreitol (DTT), a-cyano-4-hydroxycinnamic acid (a-
CHCA), sinapic acid, iodoacetamide (IAA), and NaI were
purchased from Sigma–Aldrich (St. Louis, MO, USA). All
solvents were of analytical grade.
2.3. Collection of venom
Specimens of P. gr. orbignyi were collected on Parana´ River and
Tocantins River both in the state of Tocantins, Brazil, and
transferred immediately to laboratory to extract the venom.
The epithelium, that cover the sting, obtained from the
animals were scratched and dissolved in PBS, pH 7.4, and
immediately centrifuged at 6000 Â g for 15 min. Dry venom
was stored at À20 8C until use. Protein content was determined
by the method of Bradford [3] using bovine serum albumin
(Sigma) as standard protein.
2.4. RP-HPLC profiling and peptide purification
A¨ kta binary HPLC system (Amersham Biosciences, Uppsala,
Sweden) was used to perform the venom reversed-phase
chromatography. One milligram of lyophilized venom sample
was dissolved in 1 mL 0.1% TFA and centrifuged at 5000 Â g for
p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 63040
RP-HPLC, reversed-phase
high performance liquid
chromatography
TFA, trifluoroacetic acid
CIF, collision induced fragmentation
CK, creatine kinase

3. 20 min (room temperature). The supernatant was then loaded
onto a Shimadzu C18 column (Shim-Pack 5m, 4.6 mm  250 mm)
and a two-solvent system (A) trifluoroacetic acid (TFA)/H2O
(1:1000) and (B) TFA/acetonitrile (ACN)/H2O (1:900:100) was
employed for the chromatographic separation. The peptides
were eluted at a constant flow rate of 1.0 mL/min with a 10–80%
gradient of solvent B over 40 min. The HPLC column eluates
were monitored by their UV absorbance at 214 nm.
2.5. Liquid chromatography–mass spectrometry
For micro-liquid chromatography–mass spectrometry (LC–MS)
analyses, an Ettan microLC (Amersham Biosciences) was
employed using a mRPC C2/C18 ST 1.0/150 column (Amersham
Biosciences) and a two-solvent system (A1) formic acid 0.1%
and (B1) ACN/H2O/formic acid (900:100:1). The column was
eluted at a flow rate of 60 mL/min with a 5–65% gradient of
solvent B1 over 60 min. The HPLC column eluates were
monitored by their absorbance at 214 nm. The mHPLC was
directly connected to a Q-TOF UltimaAPI (Micromass, Man-
chester, UK) operating under positive ionization mode and the
whole sample was introduced into the mass spectrometer.
External calibration was used employing NaI.
2.6. Mass spectrometry
Molecular mass analyses of the fractions and purified peptides
were performed on a Q-TOF Ultima API (Micromass), under
positive ionization mode and/or by MALDI-TOF mass spectro-
metry on an Ettan MALDI-TOF/Pro system (Amersham
Biosciences), using a-CHCA or sinapic acid as matrices.
2.7. De novo peptide sequencing
Mass spectrometric de novo peptide sequencing was carried
out in positive ionization mode on a Q-TOF Ultima API fitted
with an electrospray ion source (Micromass). Purified lyophi-
lized peptide samples were dissolved in 50 mM ammonium
acetate, reduced with DTT, alkylated by IAA, according to
Westermeier and Naven [29]. The reaction products were then
lyophilized and dissolved into 50% acetonitrile containing
0.1% formic acid and directly infused into the instrument
using a Rheodyne 7010 injector coupled to a LC-10A VP
Shimadzu pump at 20 mL/min, constant flow rate. The
instrument control and data acquisition were conducted by
MassLynx 4.0 data system (Micromass) and experiments were
performed by scanning from a mass-to-charge ratio (m/z) of
50–1800 using a scan time of 2 s applied during the whole
infusion process. The mass spectra corresponding to each
signal from the total ion current (TIC) chromatogram were
averaged, allowing an accurate molecular mass determina-
tion. External calibration of the mass scale was performed
with NaI. For the MS/MS analysis, collision energy ranged from
18 to 45 eV and the precursor ions were selected under a 1-m/z
window.
2.8. Peptide sequencing
For the unequivocal determination of the amino acid
sequence, an HPLC purified sample of Orpotrin was subjected
to Edman degradation using a Shimadzu PPSQ-21 automated
protein sequencer, following the manufacturer’s standard
instructions.
2.9. Peptide synthesis
Synthetic peptide was obtained in automated bench-top
simultaneous multiple solid-phase synthesizer (PSSM 8
system from Shimadzu Co.) using solid phase peptides
synthesis by the Fmoc-Procedure [2]. The peptide was purified
by reversed-phase chromatography (Shim-pack Prep-ODS, 5m,
20 mm  250 mm Shimadzu Co.) semi-preparative HPLC, and
the purity and identity of the peptide confirmed by MALDI-TOF
mass spectrometry and by analytical HPLC, in the same
conditions described above.
2.10. Intravital microscopy
The dynamic of alterations in the microcirculatory network
were determined using intravital microscopy by transillumi-
nation of mice cremaster muscle after topical application the
peptide. Sterile saline was used as control. In three indepen-
dent experiments (n = 5) mice were anaesthetized with
pentobarbital sodium (Hypnol1
Crista´lia; 50 mg/kg, intraper-
itoneal route) and the cremaster muscle was exposed for
microscopic examination in situ as described by Lomonte et al.
[18]. The animals were maintained on a special board
thermostatically controlled at 37 8C, which included a trans-
parent platform on which the tissue to be transilluminated
was placed. Images of the microcirculation were simulta-
neously visualized on a TV monitor and on a computer
monitor using a color video camera (TK-C600, JVC) incorpo-
rated to a triocular microscope (Axioskope, Carl-Zeiss). Images
obtained on the TV monitor were recorded on a video recorder
and digitized images in the computer were analyzed using
standard image analyzer software (KS300, Kontron). The
images were obtained using a Â10/025 longitudinal distance
objective/numeric aperture and Â1.6 optovar.
2.11. Statistical analysis
One-way analysis of variance (ANOVA) followed by Dunnett’s
test was used to determine the levels of difference between all
groups. Differences were considered statistically significant at
p < 0.05. The SPSS statistical package (Release 8.0, Standard
Version, 1997) was employed.
3. Results
3.1. Biochemical characterization and purification of
venom peptides
The chromatographic separation by analytical RP-HPLC of P.
gr. orbignyi venom (Fig. 1) demonstrated that there are several
components evenly distributed along the profile that presents
some 10 clear major peaks. Fractions were pooled along the
profile and tested for effects on the microcirculation as well as
analyzed by MALDI-TOF/MS. MS analyses revealed that this
venom is a rich mixture of peptides and proteins within a
p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 6 3041

4. broad range of molecular masses (Table 1). The major active
fraction over the microcirculatory network, as assessed by the
intravital microscopy, was selected for biochemical charac-
terization. The selected peak, a rather hydrophilic peptide, is
indicated by an arrow in the chromatogram (Fig. 1) and
revealed to contain a single peptide (Fig. 2). This peptide was
selected for ‘de novo’ sequencing (Fig. 3), due to its significant
biological activity. Other peaks presented mild or transient
effects over the microcirculation as well. These minor effects
are probably due to the low peptidic content of the peaks and
are currently undergoing further investigation.
By performing a more accurate LC analysis, under slightly
different conditions (Section 2.5) coupled to an ESI-Q-TOF
mass spectrometer (data not shown), it was possible to
measure the molecular mass of the 38 main molecules present
in this venom (Table 1). Representative peaks (>10% full scale)
were chosen along the TIC profile and the molecular masses
were measured in each peak, as presented in Table 1. In this
table, it is also possible to notice that the LC–MS profiling
conditions were more successful in identifying molecules
individually, for individual ion chromatograms can be
generated from the TIC chromatogram, thus increasing peak
resolution and detection.
3.2. De novo peptide sequencing
The selected active peptide was purified and submitted to de
novo sequencing. Sample was processed according to a
modified protocol of Westermeier and Naven [29]. In order
to assess its cysteine content, the 1001.49 Da peptide was
p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 63042
Fig. 1 – RP-HPLC profile of the venom of P. gr. orbignyi monitored at 214 nm. The arrowed peak contains Orpotrin. Inset: P. gr.
orbignyi.
Table 1 – Representative molecular masses measured in
the major peaks of the HPLC profile of P. gr. orbignyi
venom and putative CK peptide matches
RTa
(min) Molecular mass(es)b
(Da)
24.4 1137.38, 1534.52, 2003.52, 2102.88*
26.3 590.24*
, 658.33*
, 791.34*
, 1786.86*
, 1913.62
28.6 1233.66
28.8 730.35*
, 866.40*
, 973.50*
, 1913.62, 1922.82*
30.6 1192.54*
, 1829.79, 1985.94*
31.4 905.38*
, 987.48*
, 1402.74*
32.3 1072.56*
37.7 1001.52c
55.5 3360.90, 3921.13, 4297.35, 5013.47
60.0 3446.72
64.4 12192.63 Æ 0.19, 12238.62 Æ 0.25, 12311.27 Æ 0.42,
12368.23 Æ 0.98
68.8 11794.63 Æ 0.33
69.8 11794.86 Æ 0.67
72.5 9088.33 Æ 0.94, 12533.19 Æ 1.44, 13434.56 Æ 1.03
The bold data relates to the molecule being described in the paper.
a
LC–MS retention time according to Section 2.5 of a peak (or
peaks) which intensity was, at least, 10% of the full scale.
b
Standard error for molecular mass was calculated when there
were, at least two, different charge states of the same molecule,
otherwise measured molecular mass is presented.
c
Orpotrin.
*
Molecular masses that may correspond to other internal frag-
ments of CK, within the Æ0.03 Da mass.

5. reduced with DTT and alkylated IAA, desalted and subjected to
MALDI-TOF-MS analysis. Absence of alteration in the mole-
cular mass clearly demonstrated that this molecule has no
cysteine residues. The purified peptide, according to sample
preparation described in Section 2.7, was individually selected
for MS/MS analyses and fragmented by collision with argon
(CIF), yielding an ion spectrum as presented in Fig. 3. The MS/
MS spectra were analyzed by the BioLynx software module of
MassLynx 4.0 and manually verified for accuracy in the amino
acid sequence interpretation. The peptide was fully sequenced
by mass spectrometry and identified as the novel peptide
named Orpotrin, whose amino acid sequence, HGGYKPTDK,
aligns only with creatine kinase residues 97–105, but has no
similarity to any bioactive peptide. Moreover, Edman degrada-
tion was performed and successfully confirmed the deduced
amino acid sequence.
p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 6 3043
Fig. 2 – MALDI-TOF spectrum of purified Orpotrin.
Fig. 3 – Representative CIF spectrum of purified Orpotrin performed in a Q-TOF Ultima API (Micromass). b and y series are
presented above the profile and the sequence is annotated using amino acids one-letter code.

6. 3.3. Sequence alignment
Fig. 4 presents the sequence alignment of Orpotrin and CKs
from two other rays. A broader sequence alignment
comprising other CKs from other fish was performed (data
not shown) but, despite the high degree of conservation,
small variations could be observed, including in the region
corresponding to Orpotrin. Since the rays’ CKs are virtually
identical (8 different amino acids out of 381, not considering
analog substitution such as Glu ! Asp or Thr ! Ser), we
chose to compare only these sequences in order to evaluate
whether other putative peptides generated from CK by
limited proteolysis would be present in the venom. Table 1
contains the possible corresponding matching peptides,
indicated by asterisks.
3.4. Orpotrin induces arteriolar constriction
Fig. 5A presents the changes in diameter from the groups of
arterioles in response to the local application of Orpotrin, over
time. A decrease in the diameter of large arterioles of 62 and
40% was observed at times 20 and 30 min, respectively. The
relative magnitude of arteriolar constriction in response to the
peptide was partially restored only after 30 min (Fig. 5B). No
change in rolling leukocyte velocity and diameter of venules
was seen over time in either vehicle- or Orpotrin-treated
animals (Fig. 5B).
4. Discussion
This work reports the purification, characterization and
complete amino acid sequencing of a novel bioactive peptide,
isolated from the venom of the Brazilian Stingray P. gr. orbignyi.
Due to its unique sequence, this peptide was named Orpotrin
and sequenced as HGGYKPTDK by mass spectrometry and
confirmed by Edman degradation.
Interestingly, Orpotrin’s only sequence alignment is with
creatine kinase (CK) residues 97–105 (. . . LL90
DPVIQDRHG-
GYKPTDKHKTDL110
NP . . .). CK is a central controller of cellular
energy homeostasis. By reversible interconversion of creatine
into phosphocreatine, CK builds up a large pool of rapidly
diffusing phosphocreatine for temporal and spatial buffering
of ATP levels. Thus, CK plays a particularly important role in
tissues with large and fluctuating energy demands like muscle
and brain [27], or cells with intermittently high energy
requirements, such as may be the case of venom glands.
Found in all vertebrates, CKs are highly conserved regarding
their amino acid sequences, so Orpotrin aligns with CKs from
different organisms.
Also noteworthy, Orpotrin is comprised between two basic
residues, namely Arg96
and Lys105
, being this Lys residue
Orpotrin’s C-terminal. This may lead one to consider that
Orpotrin may be, indeed, a limited proteolysis product of CK,
in the same way that the well characterized bioactive
hemorphins are derived from hemoglobin by limited proteo-
p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 63044
Fig. 4 – Sequence alignment between Orpotrin and two stingray CKs performed by ClustalW [28]. Proteins—KCRM_TORCA
(P04414): creatine kinase M-type (EC 2.7.3.2) (creatine kinase, M chain) (M-CK), Torpedo californica (pacific electric ray), and
KCRM_TORMA (P00566): creatine kinase M-type (EC 2.7.3.2) (creatine kinase, M chain) (M-CK) (NU-2 protein), Torpedo
marmorata (marbled electric ray).

7. lysis of this gas carrier [10], or in a more closely related
example, the generation of the antimicrobial peptide Parasin I
from Histone H2 by Cathepsin D in the wounded skin of catfish
[6]. Moreover, both Arg and Lys residues are followed by
Histidine residues, which represent a classical di-basic
processing motif. An initial enzyme specificity search per-
formed in the MERPOS Peptidase Database [24] indicates that a
few enzymes (mainly serine, but metallo and aspartic-
proteases as well) are capable of cleaving this particular
peptide bond.
Table 1 presents several other peptide masses and some of
them may correspond to possible peptides derived from CK.
Thorough analyses of these peptides indicate that neither of
them follow a specific cleavage pattern as Orpotrin does
(between a pair of basic amino acids). However, generation by
other enzymes or by combination of enzymes acting sequen-
tially may be possible if one can confirm their CK-origin.
Regardless of the origin of the peptides, it has been clearly
demonstrated that there are peptides present in this venom.
Moreover, these peptides are very likely to be bioactive, as
Orpotrin is, and may be products of limited proteolysis of
larger proteic substrates. The almost perfect similarity
between rays’ CKs justifies this approach, but further
investigation involving peptide purification and sequencing
is required. Also, the isolation and characterization of the
other larger peptides ($3 to $5 kDa) and proteins ($12 kDa)
present in this venom (Table 1) can provide significant new
information regarding these venom pharmacological proper-
ties.
Magalha˜es et al. [20] clearly demonstrated the pro-
inflammatory effects of the crude P. gr. orbignyi venom and
its proteolytic activity but no vasoconstrictor of the crude
venom could be observed. Magalha˜es also presents a SDS-
PAGE analysis of the venom, in which one can clearly see the
presence of several high molecular mass proteins, data not
available in our LC and LC–MS analyses due to column and
solvent choices. So, this fish contains both the enzymes
(secreted in the venom and previously assessed) and the
substrate (CK being ubiquitous throughout the animal king-
dom and being the sole possible described source for
Orpotrin); therefore, Orpotrin production and secretion in
the venom is very likely to a constitutive process for this
animal, mediated by limited proteolysis of CK. Further
investigation is required to demonstrate this hypothesis and
is currently ongoing in our laboratory. Limited proteolysis of
precursor proteins derived either from endogenous or exo-
genous sources are a source for several essential bioactive
peptides. The bioactive peptides and/or hormones may be
generated intra or extra-cellularly, and may act as well in both
the compartments.
Processed-protein derived peptides acting on smooth
muscle contraction in response to bradykinin, oxytocin, and
prostaglandin-F2a have been recently described [4] with
possible implications on vasoconstriction and augmentation
of normal labor through enhancing the action of uterotonins, a
possible effect of Orpotrin, which was able to participate in the
dynamics of the physiologic events happening in the micro-
vessels. These effects, as observed by intravital microscopy,
take into account the figurative elements of the blood,
components of the plasma, hemodinamics variations, and
morphologic alterations of the vascular walls [23].
Moreover, V1a-receptor mediated vasoconstriction [25],
changing arteriolar tonus and contributing to the regulation
of systemic vascular resistance (and thus arterial blood
pressure [8]) may be another physiological target, since there
was a significant decrease in the arteriolar diameter after
Orpotrin administration (Fig. 5B). This effect was mainly over
large arterioles (>50 mm), which ultimately control bloodflow to
the subsequent vessels of the microcirculatory system [9,11].
The mechanism(s) of spasm in arterial conduits has been
an area of intense research and studies by several groups
[5,16,17,26] that have identified the endothelial dysfunction,
specifically the release of endothelium-derived vasoconstric-
tors like thromboxane A2, prostanoids, and endothelin-1 (ET-
1) as significant players in this system. In addition to directly
causing vascular smooth muscle contraction (via interaction
with thromboxane and ET receptors on smooth muscle) these
agents can impair endothelial function and vascular reactivity
through inhibition of NO production/release [19].
The presence of inflammatory cells in venules may have a
major influence on arteriolar constriction [13,30]. In this view,
the arteriolar constriction observed in ischemia–reperfusion
injury model, in which venular adherent leukocytes con-
tributed to the constriction of paired arterioles, was attenu-
ated by the injection of a monoclonal antibody against the
adhesion molecule CD11/CD18 [31]. However, in our model no
change in rolling leukocyte velocity or in diameter of venules
p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 6 3045
Fig. 5 – Intravital micrograph of cremaster muscle (n = 5)
after topical application of 20 mL, 1 mM Orpotrin. (A)
Arteriolar diameter variation over time, and (B) time-
course evaluation of the vasoconstrictor effects.

8. was seen over time in Orpotrin-treated animals, suggesting
that Orpotrin exerts a selective and direct action on arterioles.
In conclusion, a few noteworthy events are described in
this work. First, fish toxins do represent a vast source of novel
pharmacological compounds that may prove useful for both
research tools and therapeutic agents. Second, we report a
novel peptide presenting a major vasoconstrictive effect
isolated from a natural source (P. gr. orbignyi venom) acting
on large arterioles of the microcirculatory network of cre-
master muscle of mice under physiological conditions. Still,
Orpotrin’s mechanism of action is unclear. Further research
will elucidate whether the observed microcirculatory
response to Orpotrin follows a comparable pattern under
pathophysiological conditions such as cardiac arrest [15] and
vasodilatory shock states [21]. Nevertheless, Orpotrin’s unique
origin may represent a novel family of vasoactive peptides.
Acknowledgments
Supported by FAPESP, CAT/CEPID, FAPEMIG (Edital 24000/01),
and CAPES.
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