p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 6

avai lab le at www.sc iencedi rec t .com

journal homepage: www.elsev ier .com/ locate /pept ides

Orpotrin: A novel vasoconstrictor peptide from the venom ofthe Brazilian Stingray Potamotrygon gr. orbignyi

Katia Conceicao a,*, Katsuhiro Konno a, Robson L. Melo a, Elineide E. Marques b,Clelia A. Hiruma-Lima c, Carla Lima a, Michael Richardson d, Daniel C. Pimenta a,Monica Lopes-Ferreira a

a Laboratorio Especial de Toxinologia Aplicada (LETA), Center for Applied Toxinology (CAT/CEPID), Instituto Butantan,

Avenida Vital Brasil, 1500, Sao Paulo, SP, 05503-900, BrazilbNucleo de Estudos Ambientais (Neamb), Universidade Federal do Tocantins, Campus de Porto Nacional,

Rua 03, Quadra 17, Porto Nacional, TO, 77500-000, BrazilcDepartamento de Fisiologia, Instituto de Biociencias, Universidade Estadual Paulista Julio de Mesquita Filho,

Caixa-Postal 510, Botucatu, SP, 18618-000, Brazild Fundacao Ezequiel Dias, FUNED, Rua Conde Pereira Carneiro, 80, Belo Horizonte, MG, 30510-010, Brazil

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. Conceicao).

0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserveddoi:10.1016/j.peptides.2006.09.002

.

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

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 � g for 15 min. Dry venom

was stored at�20 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

Akta 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 � g for

p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 6 3041

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 Cristalia; 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 �10/025 longitudinal distance

objective/numeric aperture and �1.6 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 ofvenom 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 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 inthe major peaks of the HPLC profile of P. gr. orbignyivenom 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 � 0.19, 12238.62 � 0.25, 12311.27 � 0.42,

12368.23 � 0.98

68.8 11794.63 � 0.33

69.8 11794.86 � 0.67

72.5 9088.33 � 0.94, 12533.19 � 1.44, 13434.56 � 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 �0.03 Da mass.

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 6 3043

Fig. 2 – MALDI-TOF spectrum of purified Orpotrin.

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

Fig. 3 – Representative CIF spectrum of purified Orpotrin perfor

presented above the profile and the sequence is annotated usin

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.

med in a Q-TOF Ultima API (Micromass). b and y series are

g amino acids one-letter code.

p e p t i d e s 2 7 ( 2 0 0 6 ) 3 0 3 9 – 3 0 4 63044

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).

Fig. 4 – Sequence alignment between Orpotrin and two stingray

(P04414): creatine kinase M-type (EC 2.7.3.2) (creatine kinase, M

KCRM_TORMA (P00566): creatine kinase M-type (EC 2.7.3.2) (cre

marmorata (marbled electric ray).

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 (. . . LL90DPVIQDRHG-

GYKPTDKHKTDL110NP . . .). 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-

CKs performed by ClustalW [28]. Proteins—KCRM_TORCA

chain) (M-CK), Torpedo californica (pacific electric ray), and

atine kinase, M chain) (M-CK) (NU-2 protein), Torpedo

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.

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.

Magalhaes 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. Magalhaes 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 blood flow 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 63046

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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