The pharmacology of Malo maxima jellyfish venom extract in isolated cardiovascular tissues: A probable cause of the Irukandji syndrome in Western Australia.

Abstract

The in vitro cardiac and vascular pharmacology of Malo maxima, a newly described jellyfish suspected of causing Irukandji syndrome in the Broome region of Western Australia, was investigated in rat tissues. In left atria, M. maxima crude venom extract (CVE; 1-100μg/mL) caused concentration-dependent inotropic responses which were unaffected by atropine (1μM), but significantly attenuated by tetrodotoxin (TTX; 0.1μM), propranolol (1μM), Mg(2+) (6mM) or calcitonin gene-related peptide antagonist (CGRP(8-37); 1μM). CVE caused no change in right atrial rate until 100μg/mL, which elicited bradycardia. This was unaffected by atropine, TTX, propranolol or CGRP(8-37). In the presence of Mg(2+), CVE 30-100μg/mL caused tachycardia. In small mesenteric arteries CVE caused concentration-dependent contractions (pEC(50) 1.03±0.07μg/mL) that were unaffected by prazosin (0.3μM), ω-conotoxin GVIA (0.1μM) or Mg(2+) (6mM). There was a 2-fold increase in sensitivity in the presence of CGRP(8-37) (3μM). TTX (0.1μM), box jellyfish Chironex fleckeri antivenom (92.6U/mL) and benextramine (3μM) decreased sensitivity by 2.6, 1.9 and 2.1-fold, respectively. CVE-induced maximum contractions were attenuated by C. fleckeri antivenom (-22%) or benextramine (-49%). M. maxima CVE appears to activate the sympathetic, but not parasympathetic, nervous system and to stimulate sensory nerve CGRP release in left atria and resistance arteries. These effects are consistent with the catecholamine excess thought to cause Irukandji syndrome, with additional actions of CGRP release.

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Toxicology Letters 201 (2011) 221–229
Contents lists available at ScienceDirect
Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
The pharmacology of Malo maxima jellyfish venom extract in isolated
cardiovascular tissues: A probable cause of the Irukandji syndrome
in Western Australia
Ran Lia,b,1, Christine E. Wrighta,∗,1, Kenneth D. Winkelb, Lisa-Ann Gershwinc, James A. Angusa
aCardiovascular Therapeutics Unit, Department of Pharmacology, The University of Melbourne, Grattan Street, Parkville, Victoria 3010, Australia
bAustralian Venom Research Unit, Department of Pharmacology, The University of Melbourne, Victoria 3010, Australia
cQueen Victoria Museum, Launceston, Tasmania 7250, Australia
a r t i c l ei n f o
Article history:
Received 16 September 2010
Received in revised form 5 January 2011
Accepted 6 January 2011
Available online 13 January 2011
Keywords:
Irukandji
Malo maxima
Carukia barnesi
Catecholamines
Calcitonin gene-related peptide
Jellyfish venom
a b s t r a c t
The in vitro cardiac and vascular pharmacology of Malo maxima, a newly described jellyfish suspected of
causing Irukandji syndrome in the Broome region of Western Australia, was investigated in rat tissues. In
leftatria,M.maximacrudevenomextract(CVE;1–100?g/mL)causedconcentration-dependentinotropic
responses which were unaffected by atropine (1?M), but significantly attenuated by tetrodotoxin (TTX;
0.1?M), propranolol (1?M), Mg2+(6mM) or calcitonin gene-related peptide antagonist (CGRP8–37;
1?M). CVE caused no change in right atrial rate until 100?g/mL, which elicited bradycardia. This
was unaffected by atropine, TTX, propranolol or CGRP8–37. In the presence of Mg2+, CVE 30–100?g/mL
caused tachycardia. In small mesenteric arteries CVE caused concentration-dependent contractions
(pEC50 1.03±0.07?g/mL) that were unaffected by prazosin (0.3?M), ?-conotoxin GVIA (0.1?M) or
Mg2+(6mM). There was a 2-fold increase in sensitivity in the presence of CGRP8–37(3?M). TTX (0.1?M),
box jellyfish Chironex fleckeri antivenom (92.6U/mL) and benextramine (3?M) decreased sensitivity by
2.6, 1.9 and 2.1-fold, respectively. CVE-induced maximum contractions were attenuated by C. fleckeri
antivenom (−22%) or benextramine (−49%). M. maxima CVE appears to activate the sympathetic, but not
parasympathetic, nervous system and to stimulate sensory nerve CGRP release in left atria and resistance
arteries.TheseeffectsareconsistentwiththecatecholamineexcessthoughttocauseIrukandjisyndrome,
with additional actions of CGRP release.
© 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
The Irukandji syndrome is a jellyfish-related envenomation
that has been attributed to the sting of certain species of small,
four-tentacled box jellyfish (Barnes, 1964; Fenner et al., 1988;
Flecker, 1952; Gershwin, 2007, 2008; Gershwin and Alderslade,
2005; Little et al., 2006; Southcott, 1967). The syndrome has three
recognised clinical components consisting of (i) acute muscular
chest and back pain; (ii) catecholamine-like effects notably sweat-
ing, anxiety, nausea, vomiting, headache, tachycardia, potentially
life-threatening hypertension with supraventricular tachyarrhyth-
mias; and (iii) cardiopulmonary decompensation. Most patients
presentwithmilder,non-lifethreateningsymptomsincludinggen-
eralised pain, hypertension, nausea, vomiting and distress. About
∗Corresponding author. Tel.: +61 3 8344 8219; fax: +61 3 8344 0241.
E-mail addresses: ranli01@gmail.com (R. Li), cewright@unimelb.edu.au
(C.E. Wright), kdw@unimelb.edu.au (K.D. Winkel), lisa.gershwin@qvmag.tas.gov.au
(L.-A. Gershwin), jamesaa@unimelb.edu.au (J.A. Angus).
1These authors contributed equally to this work.
half require hospital admission and a small number need advanced
life support, usually because of cardiac failure (Huynh et al., 2003;
Macrokanis et al., 2004). The deaths in 2002 of two tourists in
Far North Queensland, Australia, highlighted the potential risk and
severity of this syndrome (Fenner and Hadok, 2002; Huynh et al.,
2003). It is now appreciated to occur elsewhere in the Indo-Pacific
andtheCaribbean(dePenderetal.,2006;GradyandBurnett,2003;
Pommier et al., 2005; Yoshimoto and Yanagihara, 2002).
Only a single species – Carukia barnesi – has been definitively
confirmed as a cause of Irukandji syndrome, however many dif-
ferent species have been linked to it (Barnes, 1964; Burnett et al.,
1996; Fenner et al., 1985; Gershwin, 2005, 2007; Little et al., 2001,
2006; Little and Seymour, 2003; O’Reilly et al., 2001; Southcott,
1967; Tibballs et al., 2001). One species suspected of causing
severe Irukandji syndrome near the Broome region of Western
Australia is the recently described Malo maxima (Gershwin, 2005).
The syndrome is a major occupational health and safety hazard for
Broome’s pearling industry and a concern for locals and tourists
(Macrokanis et al., 2004). However, aside from the original taxo-
nomical description of M. maxima, no research has been conducted
on this species. Consequently, treatment of Irukandji syndrome
0378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2011.01.003

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R. Li et al. / Toxicology Letters 201 (2011) 221–229
in Western Australia is based purely on the assumption that the
venom of M. maxima has similar, if not identical, mechanisms of
action to that of C. barnesi (Macrokanis et al., 2004).
Current understanding of the pharmacological mechanisms
underlying Irukandji syndrome is rather limited. Amongst sus-
pected Irukandji species, only the venoms of C. barnesi and Alatina
mordens, both thought to be limited to Queensland waters, have
been studied to any extent (Ramasamy et al., 2005; Tibballs et al.,
2001; Winkel et al., 2005; Winter et al., 2008). Clinically, some fea-
tures of Irukandji syndrome resemble that of adrenal medullary or
catecholamine excess, such as seen in phaeochromocytoma and in
acute autonomic overactivity conditions (Baguley, 2008; Burnett
et al., 1998). Indeed, elevated serum catecholamine levels have
been found in vivo when animals were injected with C. barnesi
venom (Ramasamy et al., 2005; Tibballs et al., 2001; Winkel et al.,
2005; Winter et al., 2008). The most effective therapy for Irukandji
syndrome to date seems to be an intravenous infusion of magne-
sium(MgSO4orMgCl2)(Corkeronetal.,2004;Corkeron,2003).The
rationale for the use of magnesium therapy is based on the in vivo
evidence of catecholamine excess, sympathetic and parasympa-
thetic involvement in Irukandji syndrome, combined with the
apparent utility of magnesium in reducing evoked release of sym-
pathetic neurotransmitters (Ohtsuka et al., 2002; Tibballs et al.,
2001). Anecdotal evidence suggests that it is useful in decreasing
both the hypertension and severe pain of the syndrome (Corkeron
et al., 2004; Corkeron, 2003). Therefore, the aims of this study were
to assess whether a species other than C. barnesi can cause effects
consistent with Irukandji syndrome in cardiac and vascular tis-
sues in vitro, and to determine the specific properties of M. maxima
venom. Specifically, the roles of sympathetic and parasympathetic,
aswellasofthesensory,neurotransmittersintheactionsofM.max-
ima venom were ascertained. Further, the effects of magnesium on
the responses to the venom extract were assessed with a view to
treatment of such a potential Irukandji syndrome.
2. Methods
2.1. Collection of specimens
All specimens of M. maxima were caught offshore from 80 Mile Beach, in the
pearlinggroundssouthofBroome,Kimberleycoast,WesternAustralia,fromAprilto
May,2004.Thespecimenswerecapturedatnight,usinglightstoattracttheanimals
and collected either with a custom-designed hand-net or drift-net (Uninet Enclo-
sureSystems;Cairns,QLD,Australia).Uponcollection,specimenswereimmediately
placed in small containers of seawater, pending identification via light microscopy.
Specimens were examined by microscopy and formally identified, prior to pro-
cessing, as M. maxima according to Gershwin’s description of bell, tentacle and
nematocyst morphology (Gershwin, 2005).
Thereafter they were individually placed into plastic zip-lock bags and imme-
diately frozen on dry ice (−80◦C). The frozen specimens were then transported
at −80◦C to the Australian Venom Research Unit (Department of Pharmacology,
The University of Melbourne, Victoria, Australia) and stored at −70◦C as whole
specimens prior to processing.
2.2. Extraction of venom and protein determination
The protocol used to obtain M. maxima crude venom extract (CVE) was adapted
from that used for C. barnesi CVE (Tibballs et al., 2001; Wiltshire et al., 2000; Winkel
et al., 2005).Briefly, thawed specimens of M. maxima were manually ground for
30mininasiliconizedglassmortarandpestlesurroundedbyaniceslurry.Theresul-
tant mixture was centrifuged at 2292×g for 15min at 4◦C (Sigma Laborzentrifugen
3K15; Osterode am Herz, Germany). The supernatant was decanted, separated into
1mL aliquots and frozen at −70◦C until needed.
Protein concentration of CVE was determined using the Bradford protein assay
and compared to a bovine serum albumin (BSA) standard curve of known con-
centration (0–0.2mg/mL) (Bradford, 1976). Bio-Rad®Protein Assay Dye Reagent
Concentrate (Hercules, CA, USA) was added to both the CVE sample and the BSA
standard and the absorbance at 590nm was measured with a spectrophotometer
(Wallac 1420 Victor; Perkin Elmer, Boston, MA, USA).
2.3. Biochemical analysis
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was
used to characterise the protein content of M. maxima CVE. All CVE samples were
first diluted to 5?L aliquots of 2mg/mL CVE. Non-reducing sample buffer (0.125M
Tris–HCl, pH 6.8, 4% SDS, 20% glycerol) was then added and the samples were boiled
for 1min and centrifuged for 20s at 17,968×g. BenchMarkTMPre-Stained Protein
Ladder (InvitrogenTM; Carlsbad, CA, USA) was used as a molecular weight marker
for the range of 181.8–6kDa.
Electrophoresis gels (12.5% acrylamide; 85×60×0.75mm) were prepared at
least a day in advance from 40% acrylamide/bis solution, 37.5:1 (Bio-Rad Labora-
tories, Hercules, CA, USA), stock solutions of resolving gel buffer (1.5M Tris–HCl,
pH 8.8), stacking gel buffer (0.5M Tris–HCl, pH 6.8) and 10% SDS (Sigma–Aldrich
Pty. Ltd., Castle Hill, NSW, Australia), TEMED (Sigma–Aldrich) and freshly made 10%
ammoniumpersulphate(Sigma–Aldrich).Thegelswerestoredat4◦Cuntilrequired.
Thegelswereruninastocksolutionoftankbuffer(0.025MTris,pH8.3,0.192M
glycine, 0.1% SDS), at 50V, 200mA (constant voltage) for 30min, then resolved at
200V, 10mA (constant current) for 120min. Visualisation of the protein bands was
achieved by a 30min stain with Brilliant Blue R concentrate (Sigma–Aldrich), then
treatment with stock Coomassie destain solution (10% acetic acid, 20% methanol)
until satisfactory protein banding visualisation was achieved.
2.4. Catecholamine assays
CVE catecholamine levels were determined with alumina adsorption, separated
by HPLC and the amounts quantified by electrochemical detection, as described
previously (Lambert and Jonsdottir, 1998). Analysis was performed at 24◦C with
the operating potentials set at +0.35V for the guard cell and −0.35 and +0.29V for
detectors 1 and 2, respectively. All measurements were made using the oxidizing
potential applied at detector 2 and compounds in M. maxima CVE were identified
by their retention behaviour compared with that of authentic standard solutions.
2.5. In vitro pharmacological analysis
Animal tissue experiments were approved by The University of Melbourne Ani-
mal Ethics Committee in accordance with the Australian code of practice for the
care and use of animals for scientific purposes (7th edition, 2004, National Health
and Medical Research Council, Australian Government). Male Sprague–Dawley rats
(250–450g) were anaesthetised via spontaneous inhalation of 5% halothane (Vet-
erinary Companies of Australia Pty. Ltd.; Artarmon, NSW, Australia) and 95% O2
for 5min before exsanguination. Tissues were immediately excised and placed in
a beaker of Krebs’ physiological salt solution (PSS) with the following composi-
tion (mM): NaCl 119; KCl 4.69; MgSO4·7H2O 1.17; KH2PO41.18; glucose 11 (5.5 for
mesenteric artery); NaHCO325; CaCl2·6H2O 2.5; EDTA 0.026. The PSS was continu-
ously saturated with carbogen (95% O2; 5% CO2) at pH 7.4.
2.6. Rat left and right atria
Theleftatriumandthespontaneouslybeatingrightatriumweredissectedwhile
submerged in PSS (Nakashima et al., 1982). Each atrium was pierced at opposing
ends by 30G stainless steel hooks; one attached to a platinum loop embedded in the
organ bath leg and the other to an isometric force transducer (FT03C, Grass Instru-
ments; Warwick, RI, USA). Data were recorded via a transducer amplifier and Chart
v.5.5.6forMac(ADInstrumentsPty.Ltd.;BellaVista,NSW,Australia).Mountedatria,
in35–37◦CPSS-filledjacketedorganbaths, werestretchedtoapassiverestingforce
equivalentto1g(9.81mN),lefttoequilibratefor10minandthenre-stretchedto1g.
Theleftatriumwasthenstimulatedelectrically(1Hz,0.3ms,approx.120%threshold
voltage) via two punctate platinum electrodes connected to a Grass S88 stimulator
(Winkel et al., 2005). After 30min stabilisation, tissues were then pre-treated for
60min with either PSS (vehicle), atropine (1?M), propranolol (1?M), tetrodotoxin
(TTX;0.1?M),calcitoningenerelatedpeptide8–37(CGRP8–37;1?M)ormagnesium
sulphate (6mM). M. maxima CVE was added to each bath in increasing cumulative
concentrations (1–100?g/mL), allowing sufficient time between additions for any
change in response to reach a plateau (approx. 5–10min).
2.7. Rat mesenteric arteries
A 2mm long segment of mesenteric artery was mounted on a pair of 40?m
diameter stainless steel wires in a Mulvany-Halpern isometric myograph (610M;
Danish Myo Technology; Aarhus, Denmark) connected to an amplifier (Mulvany
and Halpern, 1977). The tissues were stretched to a passive tension equivalent
to a transmural pressure of 100mmHg, as generated by a passive length-tension
curve, and allowed to equilibrate for 30–60min (Angus and Wright, 2000). Ves-
sel viability was tested by simultaneous exposure to 10?M noradrenaline and
potassium-depolarising solution (KPSS; PSS with an equimolar substitution of KCl
for NaCl; K+124mM) for 2min.
ThemyographwaswashedoutwithPSSandthetissueexposedtoKPSSasecond
time to provide a reference contraction. After a second washout with PSS the ves-
sel was treated for 10min with one of PSS (vehicle), prazosin (0.3?M), ?-conotoxin
GVIA (?-CTX GVIA; 0.1?M), TTX (0.1?M), CGRP8–37(3?M) or magnesium sulphate
(6mM). For benextramine pre-treatment, the arteries were first incubated for 5min
with0.3?Mprazosintobindto?1-adrenoceptors,then3?Mbenextramine,anirre-
versible non-selective ?1- and ?2-adrenoceptor antagonist, was added for a further
5min. The myograph was then washed out 3 times at 5min intervals. This pro-

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223
tocol effectively prepares a tissue with ?2-adrenoceptors irreversibly blocked but
?1-adrenoceptors fully functional (Angus et al., 1988).
The vessel was then actively precontracted with sufficient arginine vasopressin
(AVP; approximately 0.1–0.2nM) to elicit a small contraction equivalent to 5–10%
of the maximum contraction to KPSS. Pre-contraction with AVP was necessary to
unmask contractile effects of any weak vasoconstrictor agents.
2.8. Drugs
Drugs (and suppliers) were as follows: atropine SO4 (Sigma, St. Louis, MO,
USA); box jellyfish antivenom (CSL, Parkville, Vic., Australia); CGRP8–37(rat; Aus-
pep,Parkville,Vic.,Australia);?-conotoxinGVIA(Auspep);noradrenalinebitartrate
(Sigma);prazosinHCl(Sigma);propranololHCl(Sigma);andtetrodotoxin(TTX;Sap-
phire Bioscience, Sydney, NSW, Australia). CVE aliquots were stored at −70◦C until
use and 0.1mmol/L aliquots of CGRP8–37, TTX and ?-conotoxin GVIA at −20◦C.
2.9. Statistical analyses
Data in text, tables and figures are the mean±standard error of the mean
(S.E.M.) of n experiments. For rat isolated mesenteric artery data, sigmoidal
concentration–response curves were fitted by Prism 5 (GraphPad Software, San
Diego, California, USA). The negative logarithm of the CVE concentration required to
cause 50% of the maximal response (pEC50) was compared in the absence (control)
and presence of pre-treatments using one-way analysis of variance (ANOVA), with
Dunnett’s post test for multiple comparisons as appropriate (Prism 5). Rat isolated
left atrial force, or right atrial rate, data were compared within and between groups
by repeated measures ANOVA with Greenhouse–Geisser correction for correlation
(Ludbrook, 1994), using SuperANOVA 1.11 for Mac (Abacus Concepts, Berkeley, CA,
USA). Atrial baselines or maximal changes in force or rate were compared between
groups using one-way ANOVA with Dunnett’s post test for multiple comparisons.
Theeffectofpre-treatmentonbaselineatrialvalueswerecomparedwithingroupby
paired Student’s t test. In all cases, P<0.05 was accepted as statistically significant.
3. Results
3.1. Jellyfish morphology, CVE yield, protein content and
catecholamines
For these experiments, 5 batches of M. maxima CVE were pre-
pared. The overall appearance of a typical M. maxima jellyfish is
shown in Fig. 1a. A close-up of the dominant tentacle nematocysts
Fig. 1. (a) Photograph of a Malo maxima jellyfish in situ offshore Broome, Western Australia, showing the characteristic tall, narrow and robust body with a flattened apex
of the medusa or bell (5cm diameter at base), and the four retracted tentacles. (b) A close-up of the dominant nematocysts along the length of the tentacles. Original
magnification 40× objective (i.e. 400× magnification). (c) Two isolated examples of these tentacular nematocysts showing the spines orientated away from the capsule (the
nematocyst is approximately 40×15?m wide; lower left white scale bar represents 10?m). (d) Another isolated nematocyst, without obvious spine, from a tentacle squash
using the same magnification (lower left white scale bar represents 10?m).

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R. Li et al. / Toxicology Letters 201 (2011) 221–229
Fig. 2. SDS-PAGE (non-reducing conditions) comparison of each batch of Malo maxima and Chironex fleckeri venom extract used in these experiments. Lane M, BenchMarkTM
Pre-Stained Protein Ladder; Lanes 1–5, M. maxima CVE corresponding to batches 1–5; Lane 6, C. fleckeri nematocyst extract.
is shown in Fig. 1b (photographed using a Leica DMLB compound
microscope and Leica IM-50 Image Manager v.1.20 for Windows),
with the morphology of isolated nematocysts presented at higher
magnification (Fig. 1c and d). The protein yield of the typical batch
of5pooledM.maximajellyfishwas11mg/mLpreparedin5mLPBS.
SDS-PAGE analysis revealed that the CVE contained at least 6
major constituent proteins and a large number of minor protein
bands(Fig.2).Themajorproteinspeciesoccurredatapproximately
40, 50, 60 and 80kDa, with at least one major band between 115.5
and181.8kDa.Minorproteinbandswerepresentat20–30kDaand
35kDa; less well differentiated minor protein bands occurred at
<14.8kDa. Each of the 5 batches of M. maxima CVE had consistent
SDS-PAGE profiles and contained similar proportions of each of the
protein components.
Concerning catecholamines, neither noradrenaline nor its
methylation product adrenaline were detected by electrochemical
assessment of the M. maxima CVE itself, but the precursor, DOPA
(dihydroxyphenylalanine)wasdetectedatalevelofapproximately
20ng/mL venom extract (data not shown; personal communica-
tion, Dr Gavin Lambert, Baker IDI Heart and Diabetes Institute).
3.2. Rat isolated left atria
None of the pre-treatments significantly affected baseline force
of contraction in rat isolated electrically driven left atria and values
were similar between groups prior to addition of M. maxima CVE
(P>0.05); the pooled baseline was 0.48±0.03g (n=34). M. maxima
CVE caused concentration-dependent positive inotropic responses
in the vehicle group (n=6; Fig. 3a); contractile force increased
from baseline by 0.46±0.07g at 100?g/mL. Atropine (1?M; n=6)
did not affect CVE-induced inotropic responses (P>0.05; Fig. 3a).
However, pre-treatment with either propranolol (1?M; n=7), TTX
(0.1?M; n=6), an increased concentration of magnesium (6mM
compared to 1.2mM in normal PSS; n=4) or CGRP8–37(1?M; n=5)
significantly attenuated the inotropic responses to CVE (P=0.0001
compared with the vehicle group; Fig. 3).
3.3. Rat isolated right atria
Sixty min pre-treatment with propranolol (1?M) caused
a decrease in rat spontaneously beating right atrial rate of
13 1030100
0.0
0.2
0.4
0.6
a
*
*
Leftatrialforce g
1310 30100
0.0
0.2
0.4
0.6
b
**
Malomaxima CVE (µg/mL)
Leftatrialforce g
Fig.3. Concentration-dependenteffectsofMalomaximacrudevenomextract(CVE;
?g/mL) on rat left atrial force. Data are shown as change (?) from baseline force of
contractionafter60minpre-treatmentwith(a)vehicle(?,PSS,n=6);1?Matropine
(?, n=6); 1?M propranolol (?, n=7); or 1?M CGRP8–37(?, n=5); or (b) vehicle (?,
PSS, n=6); 0.1?M TTX (?, n=6); or 6mM Mg2+(♦, n=4). Error bars are ±1 S.E.M.;
where no error bar is visible the S.E.M. is within the symbol representing the mean.
*P<0.05comparedtovehiclecontrolgroup(repeatedmeasuredANOVA).n,number
of atria.

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13 1030 100
-40
-30
-20
-10
0
10
20
30
*
Malomaxima CVE (µg/mL)
Rat right atrialrate (b/min)
Fig.4. Concentration-dependenteffectsofMalomaximacrudevenomextract(CVE;
?g/mL)onratrightatrialrate.Valuesarechanges(?)frombaselinerateafter60min
pre-treatment with vehicle (?, PSS, n=5); 1?M atropine (?, n=5); 1?M propra-
nolol (?, n=5); 1?M CGRP8–37(?, n=4); 0.1?M TTX (?, n=6); or 6mM Mg2+(♦,
n=6). Error bars are±1 S.E.M.; where no error bar is visible the S.E.M. is within
the symbol representing the mean. *P<0.05 compared to vehicle control group
(repeated measured ANOVA). n, number of atria.
18±4b/min (n=5; P=0.008); TTX (0.1?M) decreased the rate
by 6±2b/min (n=6; P=0.03); and Mg2+(6mM) caused a fall of
22±6b/min (n=6; P=0.01). Nonetheless, prior to addition of CVE,
baseline atrial rate was similar in all treatment groups (P=0.16;
data not shown). M. maxima CVE at concentrations of 1–30?g/mL
caused no significant change in atrial rate in the presence of
vehicle, 1?M atropine, 1?M propranolol, 0.1?M TTX or 1?M
CGRP8–37; Fig. 4; P>0.05). However, at the highest concentration
of 100?g/mL, M. maxima CVE caused significant bradycardia in
each of these treatment groups; compared with baseline, at this
concentration atrial rate fell (in b/min) by 29±11 in the vehicle
group (n=5); 23±21 with atropine (n=5); 23±7 with propra-
nolol (n=5); 18±9 with TTX (n=6); and 19±6 with CGRP8–37
(n=4). In contrast, right atria pre-treated with 6mM Mg2+demon-
strated significant tachycardia of 18±6 and 23±4b/min (n=6)
in response to M. maxima CVE 30 and 100?g/mL, respectively
(P<0.01; Fig. 4).
3.4. Rat isolated mesenteric arteries
In rat isolated mesenteric arteries with no pre-treatment (other
than 5–10% pre-contraction with AVP), M. maxima CVE caused
concentration-dependent contraction (Fig. 5) with a pEC50 of
1.03±0.07?g/mL (n=10; Table 1). CVE pEC50values were unaf-
fectedbythepresenceofanN-typeCa2+channelantagonist(?-CTX
GVIA 0.1?M), ?1-adrenoceptor antagonist (prazosin 0.3?M) or
6mM Mg2+(P>0.05; Table 1 and Fig. 5). Pre-treatment with
TTX (0.1?M) significantly decreased the sensitivity to M. max-
ima CVE in mesenteric arteries, with a 2.6-fold rightward shift
of the curve, whereas CGRP8–37(3?M) pre-treatment caused an
almost 2-fold increase in sensitivity compared to the vehicle con-
trol group (P<0.05; Table 1 and Fig. 5a). Irreversible inhibition of
?2-adrenoceptors by benextramine (3?M; with ?1-adrenoceptors
protected, see Section 2) attenuated the maximum contractile
responsetoCVEby49%andcauseda2.1-folddecreaseinsensitivity
to CVE (P<0.05; Fig. 5a).
Vessels pre-treated with box jellyfish (Chironex fleckeri)
antivenom (92.6U/mL) demonstrated decreases in both maximum
AVP131030 100
0
25
50
75
100
a
*
*
*
†
% KPSS contraction
AVP131030 100
0
25
50
75
100
b
*
†
Malomaxima CVE ( g/mL)
% KPSS contraction
Fig. 5. Malo maxima crude venom extract (CVE; ?g/mL) concentration-contractile
response curves in rat isolated mesenteric arteries. Data are shown as percentage of
KPSS contraction after 10min pre-treatment with (a) vehicle (?, PSS, n=10); 3?M
CGRP8–37(?, n=6); 0.1?M TTX (?, n=5) or 3?M benextramine (?, n=5); and (b)
vehicle (?, PSS, n=10); 6mM Mg2+(♦, n=4); or 92.6U/mL box jellyfish antivenom
(?, n=6). Vertical bars are ±1 S.E.M.; where no error bar is visible the S.E.M. is
within the symbol. Horizontal error bars represent the EC50±1 S.E.M. AVP, arginine
vasopressin tone to precontract to 5–10% of KPSS contraction, immediately prior
to the addition of venom CVE. *P<0.05, EC50compared to control group;†P<0.05,
maximum response compared to control group (one-way ANOVA with Dunnett’s
post test for multiple comparisons). n, number of arteries.
contraction (−22%) and sensitivity (1.9-fold less) to CVE (P<0.05;
Fig. 5b and Table 1). In pilot experiments (data not shown), box
jellyfish antivenom at the same concentration completely abol-
ished the contractile response of mesenteric vessels to box jellyfish
nematocyst extract (Winkel et al., 2005).
Table 1
Malo maxima CVE pEC50for contraction of rat isolated mesenteric arteries in the
presence of various pre-treatments.
Pre-treatment (concentration)
pEC50±S.E.M.
1.03 ± 0.07
1.44 ± 0.06*
0.74 ± 0.09*
1.36 ± 0.13*
1.15 ± 0.05
1.16 ± 0.11
1.30 ± 0.04*
1.21 ± 0.12
n
PSS (vehicle control)
Tetrodotoxin (0.1?M)
CGRP8–37(3?M)
Benextramine (3?M)
Prazosin (0.3?M)
?-Conotoxin GVIA (0.1?M)
Chironex fleckeri antivenom (92.6U/mL)
Mg2+(6mM)
10
5
6
5
4
5
6
4
CVE, crude venom extract. pEC50, negative logarithm of the EC50(concentration of
CVE ?g/mL required to cause 50% of the maximal response).
*P<0.05 compared with the PSS vehicle group, one-way ANOVA with Dunnett’s
post test for multiple comparisons. n, number of vessels.

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Table 2
Comparison of Malo maxima and Carukia barnesi CVE in vitro.
Pre-treatment
Malo maxima CVE
Carukia barnesi CVE
Rat isolated right atria
Vehicle (control)
Atropine (1?M)
Propranolol (1?M)
TTX (0.1?M)
CGRP8–37(1?M)
Mg2+(6mM)
Rat isolated left atria
Vehicle (control)
Atropine (1?M)
Propranolol (1?M)
TTX (0.1?M)
CGRP8–37(1?M)
Mg2+(6mM)
Rat isolated mesenteric arteries
Vehicle (control)
Prazosin (0.3?M)
TTX (0.1?M)
CGRP8–37(3?M)
Mg2+(6mM)
?-Conotoxin GVIA (0.1?M)
Chironex fleckeri antivenom (92.6U/mL)
Benextramine (3?M)
Nil or bradycardiaa
No change from control
No change from control
No change from control
No change from control
Tachycardiab
Tachycardia (++)
Tachycardia (+++)
Bradycardia
Inhibition (+)
Not tested
Not tested
Inotropic (++)
Inotropic (++)
Inotropic (+)
Inotropic (+)
Inotropic (+)
Inotropic (+)
Inotropic (++)
Inotropic (+++)
Not tested
Not tested
Not tested
Not tested
Contraction
No change from control
2.6-fold rightward shift
2.0-fold leftward shift
No change from control
No change from control
1.9-fold rightward shift; 22% decrease in maximum contraction
2.1-fold rightward shift; 49% decrease in maximum contraction
Contraction
No change from control
No change from control
Not tested
Not tested
No change from control
No change from control
Not tested
The relative degree of chronotropic or inotropic response is represented by ‘+’, where (++) is the response in the control group, (+++) represents a more pronounced response
and (+) represents a less pronounced response.
aBradycardia only occurred at the highest concentration of M. maxima CVE (100?g/mL).
bTachycardia only occurred at 30–100?g/mL. C. barnesi results are from (Winkel et al., 2005).
4. Discussion
ThisstudyprovidesthefirstexperimentalevidencethatM.max-
ima may be responsible for Irukandji syndrome in the Broome
region of Western Australia. Specifically these data support the
hypothesis that M. maxima CVE causes catecholamine release
in vitro, a characteristic effect of the other Irukandji venoms exam-
ined to date – C. barnesi and A. mordens (Ramasamy et al., 2005;
Winkel et al., 2005; Winter et al., 2008). As such it is the first
experimental evidence implicating a jellyfish species from outside
Queensland as a cause of the Irukandji syndrome.
Further, the findings of this first biochemical and pharmacolog-
ical analysis of the venom from the jellyfish M. maxima allow for
a comparison of its effects on cardiac and vascular tissues in vitro
with those of the archetype Irukandji jellyfish, C. barnesi (Table 2).
Considering the findings in detail, it was observed that the M. max-
ima CVE-induced positive inotropic response in rat left atria was
partially inhibited by the ?1-adrenoceptor antagonist propranolol,
suggesting release of neuronal noradrenaline, as only DOPA was
detected in the venom sample itself and this precursor of nora-
drenaline had no effect on either ?- or ?-adrenoceptors (data not
shown).Thispositiveinotropicresponsewasmoremarkedlyatten-
uated by the voltage-gated sodium channel inhibitor tetrodotoxin
and sensory peptide antagonist CGRP8–37. So, in left atrial tissues,
the concentration-dependent increase in the force of contraction
appears to be the net result of the positive inotropic effects of
both noradrenaline and CGRP. Mg2+(6mM) was also very effec-
tive at attenuating the positive inotropic effects of M. maxima CVE.
However,thecurrentguidelinesfortheemergencymanagementof
the Irukandji syndrome recommend the use of the ?-adrenoceptor
antagonist, phentolamine, rather than propranolol (Pereira et al.,
2007). Further, in an examination of the cardiovascular effects
of the other Queensland Irukandji jellyfish, A. mordens, ?- but
not ?-adrenoceptor blockade significantly attenuated the pressor
response to intravenous administration of the venom in anaes-
thetised rats (Winter et al., 2008). M. maxima CVE does not appear
tostimulatethereleaseofsignificantamountsofacetylcholine,dif-
fering from the effects of C. barnesi CVE which demonstrated clear
parasympathetic involvement in atrial tissues (Table 2) (Winkel
et al., 2005).
Given that the concentration–response curve to M. maxima CVE
wasattenuated(orslopedepressed)bypropranololaloneandmore
strongly by the CGRP antagonist (alone), it could be assumed that
the toxin is causing the release of neural transmitter noradrenaline
and neural peptide CGRP. If we consider the force response as 100%
at CVE 100?g/mL (with PSS pre-treatment), then crudely speaking
noradrenaline release might be responsible for 40% of the maxi-
muminotropicresponseandCGRPreleasefor60%ofthemaximum
response. How the toxin causes the release of transmitter nora-
drenaline from sympathetic nerves and CGRP from sensory nerve
endings is uncertain, but the TTX and Mg2+experiments show that
much of the inotropic response to the toxin (≈80%) is decreased
by these treatments. Thus, a working hypothesis may suggest that
M. maxima CVE causes a Na+channel-opening response on the
adrenergic and peptidergic sensory varicosities releasing endoge-
nous noradrenaline and CGRP (and possibly neuropeptide Y; not
tested). The residual (≈20%) inotropic response to the M. maxima
CVE in the presence of TTX or Mg2+could be explained by some
direct post-junctional stimulation of ?1-adrenoceptors or CGRP
receptors.
In the right atrial tissue, the weak chronotropic response to
M. maxima CVE (30?g/mL) of about 15beats/min contrasts with
the ≈80beats/min tachycardia caused by 10?g/mL C. barnesi CVE
(Winkel et al., 2005). This weak tachycardic response to M. maxima
CVE (30?g/mL) was inhibited by TTX pre-treatment. Importantly,
M.maximaCVEshowedasignificantbradycardiaat100?g/mLthat
was not significantly affected by TTX. However, this bradycardia
was completely blocked by Mg2+suggesting that in severe enven-
omation with M. maxima, Mg2+administration would protect the
heart rate and thus support cardiac output.
Insmall mesenteric arteries
sympatheticandsensorynervescausevasoconstrictionandvasodi-
latation, respectively, in contrast to the similar inotropic responses
from these neural networks in the heart. With this in mind, if M.
maximaCVEcausesreleaseofbothsympathetic(noradrenalineand
neuropeptide Y) and sensory transmitters (CGRP) as in the heart,
the neuralinnervation of

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227
the “control” curve to the toxin will be the algebraic sum of both
constrictor and dilator stimuli noradrenaline and neuropeptide Y
competing with CGRP, respectively. This hypothesis is borne out
when the “control” curve to M. maxima CVE in mesenteric arter-
ies was significantly shifted to the left by the CGRP antagonist,
CGRP8–37. This curve represents the effects (without CGRP) of con-
strictor stimuli (noradrenaline and neuropeptide Y) where 80% of
the maximum effect to the toxin occurs at 10?g/mL. Further, TTX
abolishes the contraction of 10?g/mL M. maxima CVE suggesting
a neural prejunctional Na+channel release-dependency. Similarly,
Mg2+wouldblockthereleaseofCGRPand,toadegree,thereleaseof
noradrenaline. This interpretation of a nil effect of Mg2+compared
with PSS pretreatment must bear in mind the functional antag-
onism of the dilator and constrictor stimuli. The neural release
of both noradrenaline and CGRP in vascular tissues is dependent
upon N-type calcium channels (Brain and Grant, 2004; De Mey
et al., 2008; Pruneau and Angus, 1990). Inhibition of this channel
with ?-conotoxin GVIA appeared to equally inhibit the respec-
tive agonist contractile and relaxant effects, resulting in the same
concentration-dependent M. maxima CVE contraction of mesen-
teric arteries as observed in the control group. Thus, mesenteric
artery contraction by M. maxima CVE seems to be N-type calcium
channel-independent.
To elucidate the role of noradrenaline we tested prazosin to
antagonise ?1-adrenoceptors with no effect while the irreversible
?2-adrenoceptor antagonist benextramine was effective in shift-
ing the mesenteric artery M. maxima CVE concentration–response
curve to the right and markedly attenuating the maximum plateau
response. These experiments suggest that perhaps the major com-
ponent to sympathetic vasoconstrictors is via ?2-adrenoceptors
with neuropeptide Y co-release making up the powerful contrac-
tion response. We used a specific pretreatment regimen to protect
?1-adrenoceptors when applying the benextramine so that the
antagonist only irreversibly blocked ?2-adrenoceptors. In anec-
dotal experiments, we have since tested benextramine (without
prazosin protection of ?1-adrenoceptors, i.e. to block both ?1- and
?2-adrenoceptors irreversibly) alone, as well as in combination
with CGRP8–37; the contractile responses to M. maxima CVE (data
not shown) were similar to those observed with ?2-adrenoceptor
blockade (Fig. 5a).
This result reconfirms the potential role for ?-adrenoceptor
antagonists in the clinical management of this syndrome. How-
ever, given severe cases can be associated with a late phase of
cardiac failure, hypotension and pulmonary oedema, a reversible
?-adrenoceptor antagonist, with a short half-life, phentolamine,
rather than phenoxybenzamine, an irreversible ?1 and ?2-
adrenoceptor antagonist, is currently recommended for the
treatment of Irukandji syndrome-induced persistent hypertension
that is otherwise uncontrolled by nitrates (Pereira et al., 2007).
Phentolamine, a non-specific ?1- and ?2-adrenoceptor antagonist,
has also been used successfully in the treatment of sympathetically
mediated pain syndromes (Hord et al., 2001; Phillips et al., 2006).
This characteristic may contribute an anti-nociceptive function in
its role in the medical management of the Irukandji syndrome
(Pereira et al., 2007).
In comparison to the efficacy of box jellyfish (C. fleckeri)
antivenom on C. fleckeri venom, it appears that M. maxima CVE
is poorly neutralised by C. fleckeri antivenom. Similarly, C. fleckeri
antivenom has little, if any, effect on C. barnesi CVE (Winkel et al.,
2005) nor on A. mordens venom in vitro (Winter et al., 2008) or in
treating Irukandji stings (Fenner et al., 1986; Winkel et al., 2003).
The decreased maximum and sensitivity of contraction observed
in rat mesenteric arteries pre-treated with C. fleckeri antivenom
may be due to non-specific binding of M. maxima CVE to the ovine
proteins constituent in C. fleckeri antivenom, rather than specific
homology between M. maxima and C. fleckeri venom.
Due to the absence of an Irukandji antivenom, and the evidence
of catecholamine excess, magnesium therapy has been used as a
therapeutic approach to treating Irukandji syndrome (Corkeron
et al., 2004; Corkeron, 2003). These results support the anecdo-
tal clinical evidence that intravenous magnesium infusion may be
effective in treating the pain and hypertension that occurs in this
syndrome. Specifically, the in vitro evidence suggests that mag-
nesium acts to decrease cardiac contractility, rather than cause
vasodilatation, at a clinically relevant concentration (6mM).
These results also offer a plausible explanation for the anal-
gesic effects of magnesium infusion in Irukandji syndrome. These
studies are the first to specifically investigate the role of sensory
neurotransmitters in an experimental model of the cardiovascu-
lar effects of the syndrome. A logical rationale would suggest that
magnesium, as a divalent cation, acts as a competitive antago-
nist to calcium ions, decreasing pre-synaptic calcium influx in
sensory nerves, thereby attenuating the release of CGRP. Addition-
ally, CGRP itself increases post-synaptic l-type calcium channel
current through an adenylate cyclase and cyclic AMP pathway,
a process that may be attenuated by an increased concentration
of magnesium ions (Bell and McDermott, 1994). Influx of Ca2+
throughTRPV1channelsmayalsocauseCGRPrelease,providingyet
another mechanism that might be antagonised by magnesium ions
(Docherty et al., 1996). Indeed recent studies have provided evi-
dence for desensitization-dependent TRPV1 activation caused by
somecnidarianvenoms,includingthecubozoanC.fleckeri(Cuypers
et al., 2006).
The extraction technique used to produce the crude venom
is relevant. Grinding whole jellyfish specimens may produce an
extract that includes components with clinically irrelevant, but
pharmacologically interesting effects, such as those from tenta-
cles.PreviousstudieshavefoundthatalthoughC.barnesitentacular
extracts appear to have distinct pharmacological actions, the main
pharmacological activity present in tentacle extracts reflects that
of nematocyst contents (Calton and Burnett, 1986; Ramasamy
et al., 2005). However, anecdotal evidence suggests that C. barnesi
bell nematocyst venom appears to have at least local effects and
obtaining venom from tentacular nematocysts ignores the possible
involvementofbellnematocystswhicharepresentonalldescribed
Irukandji species (Barnes, 1964; Gershwin, 2005, 2007; Gershwin
and Alderslade, 2005; Southcott, 1967). Clearly future studies of M.
maxima should assess whether all of the activities described here
are attributable to pure nematocyst-derived venom.
The M. maxima SDS-PAGE analysis revealed many similarities
with the Irukandji species C. barnesi. SDS-PAGE protein profiles of
C. barnesi CVE showed a number of major protein bands, occur-
ring at approximately 40, 45, 50, 80, 106 and >106kDa (Wiltshire
et al., 2000). M. maxima CVE contains major bands coinciding with
all major proteins in C. barnesi CVE, except at 50kDa where a minor
protein species exists. However, since these two venoms have
not been simultaneously run on the same gel these comparisons
are qualitative and SDS-PAGE alone is insufficient to determine
whether chemical or functional similarities exist. These results are
consistent with the complexity and spread of proteins extracted by
others from A. mordens and C. fleckeri venoms (Winter et al., 2008).
The potential significance of the detection of DOPA in the
M. maxima extract is unclear as this amino acid is very widely
distributed in the tissues and fluids of invertebrates, includ-
ing cnidarians (Waite, 1992). Interestingly, only DOPA and
related metabolites (5-hydroxy-DOPA and cysteinyl-DOPA) have
been identified in the nervous systems of some cnidarians and
ctenophores (Carlberg, 1988; Carlberg and Elofsson, 1987). Cer-
tainlynopharmacologicalactivitywasattributabletol-DOPAwhen
investigated using the rat isolated right and left atria and the
mesenteric artery at the concentrations detected in the M. maxima
venom extract (data not shown).

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R. Li et al. / Toxicology Letters 201 (2011) 221–229
While the in vitro assays in this study utilised mammalian car-
diac and vascular tissues, the results would be further validated by
extendingthisinvestigationtomoreclinicallyrelevanttissuessuch
as human atrial trabeculae preparations in vitro or anaesthetised
laboratory animals in vivo. The clinical significance of the appar-
entlylowerinvitropotencyofM.maximavenomextract,compared
to that of C. barnesi, on cardiac and vascular tissues in vitro, remains
to be determined. A similar, lesser potency of A. mordens venom,
compared with that of C. barnesi, was seen in studies of anaes-
thetised rats by others (Winter et al., 2008).
Current evidence suggests that Irukandji syndrome occurring in
theBroomeregionofWesternAustraliamaydifferfromthatseenin
north Queensland (Macrokanis et al., 2004). For example, clinical
audits of the presenting features of the syndrome in both local-
ities suggest a higher rate of narcotic analgesia requirements for
the Broome patients (Little and Mulcahy, 1998; Macrokanis et al.,
2004). This also appeared to be accompanied by the highest pre-
sentation rate, in Broome, for Irukandji syndrome yet defined in
Australia. On the other hand, the same audit found a near identi-
cal rate of hypertension (around 50% of patients) and pulmonary
oedema (around 3%) in both localities and a higher rate of admis-
sion for the Cairns hospital cases (28%) compared with Broome
(17%) (Little and Mulcahy, 1998; Macrokanis et al., 2004). Hence,
until there are high quality, long-term prospective clinical studies
using similar outcome measures, it cannot be said that Western
Australian Irukandji syndrome differs significantly from that seen
in north Queensland. Likewise our studies that appear to confirm
the probability that M. maxima is a cause of Irukandji syndrome in
WesternAustraliaandthatsimilartreatmentprotocolsascurrently
recommended for the syndrome, based on Queensland experience
(Pereira et al., 2007), should apply to cases presenting to Broome
hospital. Future studies of all Irukandji venoms should focus on
definingtheprecisemolecularnatureofthetoxinsandtherelevant
effector pathways underlying the syndrome.
Conflict of interest statement
None.
Funding
Lisa-Ann Gershwin is primarily funded by Environment
Australia (ABRS Grant # 208-82) for taxanomy. These specimens
were collected in association with the Pearl Producers Association
& Paspaley Pearling Co.
Acknowledgements
The authors wish to thank Mr Mark Ross-Smith for his technical
expertise in the isolated mesenteric artery myograph experiments.
We also thank Dr Gavin Lambert and Ms Reena Chopra of the
Human Neurotransmitters Laboratory, Baker IDI Heart and Dia-
betes Institute, Melbourne, for undertaking the catecholamine
assays. We thank Dr Angel Yanagihara, Bekesy Laboratory of Neu-
robiology,andAsiaPacificInstituteofTropicalMedicine,University
of Hawaii at Manoa, Hawaii, for the photographs of M. maxima iso-
lated nematocysts as well as Mark Alexander, Paspaley Pearling
Company, for his photograph of M. maxima. The Australian Venom
ResearchUnitisfundedbytheAustralianGovernmentDepartment
of Health and Ageing.
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